observations in clinical and experimental ocular autoimmunity

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Observations in clinical and experimental ocular autoimmunity

de Smet, M.D.

Publication date2000Document VersionFinal published version

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Citation for published version (APA):de Smet, M. D. (2000). Observations in clinical and experimental ocular autoimmunity.

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observations s

onn £(inieal and experimental

£}eu(arr ^Autoimmunit y

OTJnree &. de ^m n

Observationss in Clinicall and Experimental Ocular Autoimmunit y

Printedd by Ponsen & Looijen B.V.

Al ll rigths reserved 20000 J.M.D. He Smet

ISBNN 90-6464-839-5

AMC C Afd.. Oogheelkunde Meibergdreeff 9 11055 AZ Amsterdam Zuid-Oost Tel.+311 20 566 3455

Publicationn of this thesis was financially supported by the University of Amsterdam, Alcon Nederlandd BV, Tramedico BV, Ophtec BV Pharmacia & Upjohn

Observationss in Clinical and Experimental Ocularr Autoimmunit y

ACADEMISCHH PROEFSCHRIFT

terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus

prof.. dr. J.J.M. Franse tenn overstaan van een door het college voor promoties ingestelde

commissie,, in het openbaar te verdedigen in de Aula der Universiteit

opp woensdag 22 november 2000, om 10:00 uur

door r

Josephh Marc Dominique de Smet geborenn te Montreal, Canada

Promotiecommissie e

Promotor:: prof. dr. A. Kijlstra

Overigee leden: prof.dr. P.P. Tak prof.. dr A.F. Deutman prof.. dr. J.J. Weening prof.dr.. S.J.H, van Deventer prof.dr.. J.V. Forrester

Faculteitt der Geneeskunde

Thee research presented in this thesis was carried out in the Laboratory of Immunology, National Eyee Institute, National Institutes of Health, and in the Department of Ophthalmology, Acad-emischh Medisch Centrum, University of Amsterdam.

Vitamm impedere vero

ToTo my parents, Maria Sol, and colleagues whosewhose constant encouragement has kept me going.

Tablee of Content:

Outlin ee of the thesis 9

Introduction :: 11

Chapterr 1: Understandingg Ocular Immunology - What experimental and human studies have taughtt us 13

Sectionn I: Analysis of Immunologic Determinants to Ocular Autoantigens in Experimentall Models 55

Chapterr 2: Analysiss of the pivotal residues of the immunodominant and highly uveitogenic determinantt of interphotoreceptor retinoid-binding protein (IRBP) 57

Chapterr 3: Identificationn of heat shock proteins binding to an immunodominant uveitopathogenic peptidee of IRBP. 67

Chapterr 4: Humann S-antigen: presence of multiple immunogenic and immunopathogenic sites inn the lewis rat. 77

Chapterr 5:

Immunogenicityy and pathogenicity of human s-ag determinants in rat strains 93

Sectionn II : Immunologic Responses to Ocular Autoantigens in Humans 103

Chapterr 6: Cellularr immune responses of uveitis patients to retinal antigens and their fragments. 105 Chapterr 7: AA novel method for the determination of T-cell proliferative responses in patients withh uveitis. 115

Chapterr 8: Prospectivee determination of t cell responses to s-antigen in behcet's disease Patientss and Controls 123

Chapterr 9: Humann S-Ag determinant recognition in uveitis patients 135

7 7

Inhoud Inhoud

Sectionn III : Novel Therapeutic Strategies in Uveitis 147

Chapterr 10: FK506:: treatment of experimental autoimmune uveoretinitis in primates. 149

Chapterr 11: Treatmentt of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocytee growth factor signal transduction. 155

Chapterr 12:

Treatmentt of uveitis with recombinant human interleukin 13. 165

Sectionn IV: Therapeutic Strategies for the Treatment of Human Uveitis 171

Chapterr 13: Clinicall use of cyclosporine in ocular disease. 173 Chapterr 14: Combinedd use of cyclosporine and ketoconazole in the treatment of endogenous uveitis. 191

Chapterr 15: Longg term follow up of patients with endogenous uveitis treated with combination cyclosporinee and ketoconazole. 197

Chapterr 16:

Intraocularr inflammatory disease (uveitis) and the use of oral tolerance: a status report. 207

Conclusionn 219

Summaryy and Conclusions 221

Samenvattingg en conclusies 227

OutlineOutline of the thesis

Despitee the introduction of steroids more than a half century ago, many people still go blind from ocularr inflammation. Uveitis is the third leading cause of blindness in the United States and most off the visual impairment is brought about between the ages of 20 and 50. The social and eco-nomicc impact of visual impairment in this age group is often underestimated. These individuals aree denied the opportunity, through their visual impairment, to contribute actively to the well be-ingg of a nation. They are often a burden on family and friends. You only need to consider how dif-ficultt it is for these patients to visit their eye doctors, how often appointments need to be changed,, how often they require assistance to be seen.

Despitee having such an impact, uveitis is studied by a limited number of individuals. It does not possesss the glamour of many other illnesses for which well organized patient associations raise money,, or evoke the same level of fear as cancer does in our collective ethos. When compared to otherr autoimmune processes which can be life threatening, loss of sight seems so unimportant. Yet,, these patients require care, and new alternatives.

Thee present thesis attempts to address two main issues (1). Understanding the relevance to hu-mann disease of the experimental autoimmune model of uveitis EAU. This is the subject of the firstt two sections of the thesis. The first attempts to define the specific immunopathogenic deter-minantsminants of S-Ag and Interphotoreceptor Retinoid Binding Protein (IRBP), both potent uveito-gens,, in the experimental model EAU. An attempt is also made to determine intracellular bind-ingg mechanisms for these proteins in antigen presenting cells, identifying in the process a novel chaperone.. In the second section, attention is turned to the potential role of these proteins in hu-mans.. Means of identifying relevance in a variety of uveitic conditions, as well as an attempt at devisingg means of following patient responsiveness over time are explored in a series of 4 arti-cles.. The second issue to be addressed is the use of the experimental model to develop novel treatmentt strategies. In section III are presented trials in animal models of novel anti-inflamma-toryy therapies. Section IV discusses treatment modalities used in humans and presents some of thee short and long term risks associated with the implementation of new therapies. Cyclosporine wass first tested in an animal model and found to be 100% effective at preventing disease. Clini-call use demonstrated a number of complications which required the development of treatment algorithmss and dosage adjustments. Attempts were made to reduce toxicity by drug combination ass shown. Finally, an approach using immune modulation by antigen feeding is presented. This promisingg technique has not produced the expected results once the study was completed, but re-mainss an interesting and potentially useful approach. It is included here, as an example of a trial whichh generated much controversy, and stimulated researchers to search for new therapeutic modalities.. It is the forerunner of more sophisticated approaches looking at local immune mod-ulationn in the eye (including the use of gene therapy).

Whilee this thesis does not and cannot hope to prevent blindness in uveitis, it is hoped that some off the techniques and approaches used here may be of benefit in understanding the pathophysi-ologyy of uveitis in humans. Hopefully, it can serve as a foundation for a more systematic study off its course in humans, and lead to judicious therapeutic choices using both existing, and yet to bee discovered drugs.

9 9

Introduction : :

"The"The dramatic changes of opinion over the relatively short period of 20 years are quite startling andand demonstrate how rapidly ideas alter even in one institution when comparison is made withwith the ideas of ophthalmologists in other countries the surprise gives way to bewilderment, and whenwhen it is remembered that even today many of the diagnoses are based on guesswork rather thanthan conclusive evidence, that knowledge is growing in almost geometric progression and that fadsfads and fashions, both of the individual and the herd, have by no means ceased, it will not be surprisingsurprising if 20 years from now what is written here will merely be of historical interest and will possiblypossibly evoke a smile."

Sirr Duke Elder: System of Ophthalmology, Vol IX - on uveitis, 1961

Chapterr 1

Regulationn of Ocular Inflammation Whatt Experimental and Human Studies Have Taught Us

Marcc D. de Smet Chii Chao Chan

Submittedd for publication

Departmentt of Ophthalmology, University of Amsterdam, the Netherlands Laboratoryy of Immunopathology, National Eye Institute, Bethesda, MD

ChapterChapter I

Contents: :

1.. Introduction:

2.. Models of Ocular Autoimmune Disease

2.11 Uveitis mediated through non ocular derived antigens 2.1.11 Endotoxin induced uveitis (EIU) 2.1.22 Cytokine induced inflammation 2.1.33 Adjuvant induced uveitis

2.22 Experimental Autoimmune Uveoretinitis (EAU): 2.2.11 S-Ag

2.2.1.11 T cell recognition sites and the co-stimulatory role of B cells 2.2.1.22 Role of humoral immunity in EAU - abrogation of disease

2.2.22 IRBP 2.2.2.11 T cell recognition sites and the importance of MHC binding 2.2.2.22 Genetic susceptibility to IRBP induced EAU

2.2.33 Rhodopsin and other retinal proteins 2.33 Ocular but non retinal antigen derived autoimmune disease

2.3.11 Lens-induced uveitis 2.3.22 Experimental melanin-protein induced uveitis (EMIU) 2.3.33 Experimental autoimmune encephalitis (EAE) 2.3.44 Other non retinal models of ocular autoimmunity

3.. Modulation of the ocular immune response 3.11 Nature of Class 11+ cells in the retina and uvea tissue 3.22 Immunobiology of dendritic cells

4.. Evidence for ocular autoimmunity in humans 4.11 Cellular responses to retinal proteins 4.22 HLA associations 4.33 Autoantibodies to retinal proteins

4.44 Evidence for immune activation in uveitis patients

5.5. Summary

References s

14 4

REGULATIONN OF OCULAR INFLAMMATIO N

1.. Introductio n

Sincee the eye contains cells derived from all three embryonic layers, which express intracellular, andd cell surface proteins present elsewhere in the body, it is a prime target for a number of sys-temicc autoimmune diseases. Inflammation of connective tissue is manifested as scleritis and is oftenn found in conjunction with rheumatic diseases; intraocular vessels are involved in systemic vasculopathies,, while the retina and optic nerve can be the target of autoimmune diseases of the centrall nervous system. In addition, highly differentiated and specialized cells such as the lens, orr retinal photoreceptors are responsible for specific autoimmune phenomena.

Ocularr inflammation has been recognized as a clinical entity since the earliest ophthalmological recordss (Ebers Papyrus c. 1500 B.C.). The classic clinical description of redness, photophobia, narrowingg of the pupil and pain was first recorded by Charles Saint Yves from St. Lazare in Paris, inn 1722. The term uveitis was introduced around 1850, and from the very beginning, it was used genericallyy to denote any intra-ocular inflammatory condition without reference to an underly-ingg cause. In the 19th and early 20th century, uveitis was felt to be mainly due to infectious causes suchh as tuberculosis and syphilis. In 1910 Elschnig (1), and later Kolmer in 1931 (2) demon-stratedd the role of hypersensitivity to ocular antigens, which in parallel with a generalized de-creasee in the incidence of infection associated with improved sanitation, lead to a progressive shiftt in the cause of ocular inflammation; While AC Woods, in 1941 diagnosed tuberculosis in 80%% of granulomatous cases (3), he made the same diagnosis in only 20% of cases in 1960 (4). Moree recent epidemiologic studies in various parts of the world identified an infectious cause in lesss than 20% of cases (5-12), with the exception of areas with high exposure to pathogens (13, 14).. Uveitis mainly affects individuals in the first four decades of life, and thus, has a major so-ciall impact (15, 16), yet it remains a relatively rare cause for referral to an ophthalmologist (13, 17,, 18). With so many potential sources for ocular inflammation, it is rather surprising how in-frequentlyy the eye is involved.

Speciall anatomical and physiologic features influence the characteristics of the ocular immune response.. Immune privilege was noticed more than a century ago, and is related, as in the brain, testis,, and ovary, to lack of lymphatic drainage and the presence of a blood-tissue barrier (19). In addition,, reduced expression of MHC class I and virtual absence of class II expression in the an-teriorr chamber , vitreous cavity, and subretinal space diminish the chance of generating an im-munee response (20, 21). Fas ligand expression on ocular parenchymal cells helps control the ef-ferentt immune response by causing apoptosis in ocular activated T cells (22, 23). It was thought forr a long time that these conditions maintained the immune system ignorant of ocular antigens. However,, this privilege results from an active process of immune deviation (anterior chamber-associatedd immune deviation : ACAID) (24-26). Rather than being sequestered inside the eye, thee antigenic signal is released into blood vessels and carried to the spleen inside antigen pre-sentingg cells (APC) (26-28). These eye derived APC are themselves deviant, as they were primed byy an intraocular milieu rich in suppressive mediators such as TGFB and vasoactive intestinal peptidee (VIP) (29). A resulting shift to class I antigen presentation occurs, which in the spleen leadss to the production of CD8 suppressor cells (30). The clinical result is a suppression of de-layedd hypersensitivity (DH) and suppression of complement-fixing antibody production.

Itt is clear that immune tolerance is required for optimal vision. This adaptation has obvious ad-vantagess in preventing damage to highly specialized ocular tissues. Delicate ocular structures

15 5

ChapterChapter 1

cannott tolerate intense inflammation without losing integrity and function. Nonetheless ocular inflammationn does occur. Animal models have served as useful templates for dissecting ocular immunee mechanisms and for understanding the immunoregulatory processes which underlie uveitiss in humans. An understanding of these mechanisms is a prerequisite to the development of effectivee - site directed immune therapies.

2.. Models of Ocular Autoimmune Disease

Severall experimental models are widely used to study ocular immunopathology (31-33). Obser-vationss made in each of these models are complementary, and somewhat interchangeable. Not all animall species are equally susceptible, or develop the same pathologic findings. The basis for thesee differences is also being elucidated and explained on an immune basis. In this section, we wil ll attempt to present salient features of each model, as well as some of the key observations withh regards to disease pathogenesis. Ocular inflammation can be induced by exogenous antigen sources,, or by using ocular derived proteins. Key elements of common immunizing protocols are mentionedd in table 1.

2.11 Uveitis mediated through non ocular derived antigens

Directt injection of antigen in the cornea or vitreous induces a progressive immunization reaction (45,, 46). During the first week, one observes an accumulation of lymphocytes and macrophages

Tablee 1: Typical immunizing dose of commonly used antigens

Model l

IC l l

EAU U

EIU U

EMIU U

HAH H

(guinea a

Antigen n

M.. tuberculosis H37RAA Ag(Difco) challenge e S-Agg (bovine)

IRBPP (bovine)

S.tvphimuriumm LPS (Ditto) E.coli055:B444 LPS (Sigma) Melaninn protein (bovine)

forr recurrence LPS Myelinn basic protein

Pig) )

Dose e

100 mg in 0.5mL sc

333 |jtg 10 d later intravitreal 200 jig sc footpad 300 |jLg sc footpad 400 jig/kg sc 1000 jig sc footpad 500 jig sc footpad + 11 jig pertussis toxin ip 0.33 mg sc footpad 0.22 mg sc footpad 255 jig sc footpad + 255 jig ip + live B pertussis ip 55 jig sc footpad 255 jig sc footpad

Adjuvant t

minerall oil

Hunter r CFA(( 1.5 mg) Hunter r CFA(2.5mg) ) CFA(2.5mg) )

PBS S Saline e Hunter r

PBS S CFA(1.5mg) )

Animal l

rabbit t

rat t rat t monkey y rat t mouse e

mouse e rat t rat t

rat t

Reference e

(34) )

(35) ) (36) ) (37) ) (38) )

(39) ) (40) ) (41) ) (42) )

(43) ) (44) )

Abbreviations:: CFA- complete Freund's adjuvant; EAE -experimental autoimmune encephalitis; EAU - experi-mentall autoimmune uveitis; EIU-endotoxin induced uveitis; EMIU -experimental melanin induced uveitis; ICU -- immune complex uveitis: ip- intraperitoneal: IRBP- interphotoreccptor retinal binding protein; LPS -lipopolysaccharide;; PBS- phosphate buffered saline; P35 fragment of S-Ag (see figure °0:R14 fragment of IRBP (seee table 4): sc- subcutaneous Suppliers:: Hunter's adjuvant (TiterMax. CytRx, Norcross. Ga): CFA (Difco. Detroit. Ml)

16 6


A. .

att the site of injection (47). Within about 10 days, a humoral immune response is observed, the intensityy of which depends on the amount of antigen injected, and the capacity of the particular animall to mount an inflammatory response. Soluble antigen injected in the vitreous, slowly dif-fusess out of the eye. The initial immunologic re-sponsee appears to take place in regional lymph nodess and in the spleen (48). Antibody-forming cellss appear in the eye shortly following their appearancee in these other lymphoid tissues at aboutt 10 days, and can be identified in the eye forr several months (49). Once the initial inflam-matoryy response has subsided, systemic re-chal-lengee will lead to a local recurrence, but only in thee previously injected eye. where specific memoryy cells persist (50).

Anotherr model is the immune complex-mediat-edd reaction that occurs within one day after in-traocularr injection of antigen in previously hyperimmunizedd animals, immune complex-triggeredd inflammation appears to be initiated byy cell-bound Fc receptors and amplified by cellularr mediators, activated complement, and neurogenicc factors such as substance P (51-53).

2.1.11 Endotoxin induced uveitis (EIU) Intravenous,, intraperitoneal, or footpad injec-tionn of very low doses of bacterial endotoxin cann induce uveitis without any inflammation in otherr organs (54-57). In the rat. ocular inflam-mationn begins a few hours after lipopolysaccha-ridee (LPS) injection and disappears after a few days.. Repeated LPS injections result in a state off tolerance and the animals do not develop uveitiss (58-60). In the C3H/HeN mouse. LPS inducedd uveitis is characterized by a bi-modal wavee of inflammation (Figure la-c) (40). In the firstfirst wave, polymorphonuclear cells are pre-dominantlyy present, while in the second wave a predominancee of macrophages is seen. Both wavess last for less than 48 hours. They are relat-edd to a difference in the intraocular kinetics of variouss cytokines (40).

Mostt of the effects of EIU are due to the lipid moietyy within the LPS molecule (61). Studies withh radiolabelled LPS show that these mole-culess enter the eye early after systemic injec-

B. .

C. .

FigureFigure I: Endotoxin Uveitis induced in C3H/ HeNHeN mice using 0.3 nig of Salmonella ty-phimurium.phimurium. The inflammatory response is bi-phasic.phasic. (a) At 24 hours, the inflammatory cell infiltrateinfiltrate is predominantly composed of neu-trophils;trophils; (b) by 72 hours, the inflammatory re-sponsesponse has largely subsided; (c)five days after LPSLPS injection, an inflammatory response com-posedposed predominantly of macrophages is ob-served.served. This second phase has largely sub-sidedsided by day 7 (hematoxylin-eosin, original magnificationmagnification 400.x).

17 7

ChapterChapter 1

tion,, but that they are not preferentially bound by ocular tissues (62). In the rat, the effect is prob-ablyy initiated by resident macrophages within the stroma of the ciliary body and iris (63, 64), leadingg to recruitment of T cells and polymorphonuclear cells (PMN) (57, 65, 66). Strain sus-ceptibilityy varies, and appears to be related to the animal's ability to modulate selectin expres-sion,, and produce proinflammatory cytokines, (in particular IL-1, TNF-a, IL-6, and IL-8) (67-72).. Modulation of cytokine expression, or use of TGFp or IL-10 will lead to a decrease in the levell of inflammation (73, 74). Estrogen can also reduce the severity of inflammation (75). Re-centt evidence suggests that pregnancy is also capable of reducing the severity of ocular inflam-mationn in other uveitis models (76-78). Similarly, a reduction in the intensity and frequency of uveitiss flare-ups is felt to occur in man. Apoptosis, which we indicated previously was an im-portantt factor in maintaining ocular homeostasis, helps to clear T cells and macrophages from thee inflamed eye. However, it has littl e effect on infiltrating PMN (41). Through their ability to producee inducible nitric oxide synthase, these may contribute significantly to disease pathogen-esiss in this model.

2.1.22 Cytokine induced inflammation Numerouss cytokines, including TNF-a, IL-1, IL-6, IL-8, IFN -7 , and granulocyte macrophage colony-- stimulating factor (GM-CSF) cause ocular inflammation after intraocular administration (79-85).. The inflammatory reaction occurs readily, starting a few hours after injection. It sets up aa chain of events characterized by the formation of cytokine cascades with either an agonist or an antagonistt activity (86). Of the above mentioned cytokines, TNF-a, and IL-1, both produced by macrophagess are considered to be important initiators of the inflammatory cytokine cascade (87-90). . Thee introduction of transgenic animals over or under expressing cytokine genes has given us ad-ditionall insights on their role in ocular inflammation. Modifying the expression of certain cy-tokiness lead to severe inflammation and disruption of ocular structures as is seen with GM-CSF (91,, 92), others gave a milder inflammatory picture as with IL-4 (93). The picture with IFN-7 wass more complex. IFN -7 is a central cytokine in autoimmune-mediated uveitis. It is primarily producedd by stimulated antigen-specific T cells, and skews the response to a Th 1 phenotype (see sectionn 2.2.2.2). Using an a-crystallin promoter, over expression of IFN-7 in mice induced MHC classs II expression, macrophage infiltration and severe ocular abnormalities, all of which were presentt at birth (94. 95). The same promoter expressed in offspring of transgenic Sprague Daw-Icyy rats back crossed onto Lewis allowed normal ocular development. Upon induction of exper-imentall autoimmune uveitis (EAU discussed in section 2.2), an accelerated onset and severity of diseasee was observed (96). Using IFN-7 deficient mice, the action of IFN-7 and its close rela-tionshipp to IL-12 was further elucidated. When IL-12 is present, Thl cells can develop, and cause EAUU in the absence of IFN-7 (97). IL-12 deficient mice on the other hand do not develop EAU, andd administration of monoclonal antibodies to IL-12 in normal mice, prevents EAU induction (98,, 99). IL-12 is a major differentiation cytokine produced by APC, which plays a crucial role inn the differentiation of naive ThO cells into Thl (100). Much of the modulatory activity of a numberr of other cytokines such as IFN-7, IL-4, IL-10 and IFN-a, appears to be mediated by reg-ulatingg either the production of, or the response to IL-12 (100. 101). However, IL-12 itself, still holdss surprises, since exogenous IL-12 treatment in EAU susceptible mouse strains also prevents thee appearance of disease. This action is likely mediated through induction of apoptosis (102). Understandingg the role of IL-12 and other modulatory cytokines may lead to the development of clinicall applications. The first of these applications, whose mode of action is not yet fully understood iss the use olTFN-a in Behcet disease where it appears to lead to long term remissions (103, 104).

18 8


2.1.33 Adjuvant induced uveitis Bacillee Camette Guerin is known to induce an inflammatory response in patients. It is character-izedd by a reactive arthritis, but in up to 50% of cases can be associated with a transient uveitis (105-107).. Similarly, immunization of Lewis rats with complete Freund's adjuvant causes uveitis inn 22% of cases (108, 109). The inflammation is localized in the anterior segment (iris and cil-iaryy body), and can be recurrent in nature (110-112). Addition of Mycobacterium butyricum in-creasess the incidence of uveitis to over 50% of animals (113).

Inn adjuvant arthritis, mycobacterial heat shock protein (hsp) 65 plays a critical role (114-116). Peptidee sequences derived from hsp65 of Mycobacterium bovis are able to induce inflammation inn the iris, ciliary body and in the anterior chamber. Littl e inflammation is found in the posterior segment,, but extensive photoreceptor loss can at times be observed on histology (117, 118). Hsp655 has also been implicated as the cause of certain human forms of uveitis and has been im-plicatedd in Behcet's disease (119-121).

2.22 Experimental Autoimmune Uveoretinitis (EAU)

EAUU has served for many years as the central model for human ocular inflammation of presumed autoimmunee origin. Specific antigenicity for retinal rods was reported as early as 1906 by Hess andd Romer (31). Next Elschnig suggested that ocular tissue antigens could be responsible for the inflammatoryy reaction in sympathetic ophthalmia (1). For several years, attention was focussed onn uveal pigment, until in 1965, Wacker and Lipton discovered that retinal tissue was much more effectivee than pigment at producing inflammatory eye disease (122, 123). Experimental autoim-munee uveitis was first produced by injecting a single subcutaneous dose of a soluble retinal anti-genn (S-Ag) in adjuvant (31-33). Additional uveitogenic proteins from the photoreceptor layer havee been identified and characterized: interphotoreceptor retinoid binding protein (IRBP), rhodopsin,, phosducin, and recoverin. Clinicall and pathological features of EAU vary according to the animal species and antigen used. However,, in all cases CD4+ cells play a central role in the effector mechanism (124). With S-Ag inducedd EAU, choroiditis and anterior uveitis predominate in the guinea pig, whose retina is avascular.. In the monkey, the mouse, and the rat, retinal vasculitis is prominent. The onset (10 dayss to 2 months), and duration (days to 12 months or more) are dependent on antigen dose, speciess and adjuvant (125-127). Lowering the antigen dose to the minimal amount capable of in-ducingg disease will cause a delayed onset characterized solely by a patchy absence of the pho-toreceptorr cell layer. In the Lewis rat, the disease is acute and self limited, leading within a few dayss to considerable damage to the retina and surrounding tissues (Figure 2a-b). The disease is moree moderate and subacute in less susceptible rat strains, guinea pigs, and in primates. In the latter,, the disease may smolder for months, with progressive destruction of the target tissue (reti-na)) (125, 126, 128, 129). Induction of EAU with IRBP in mice may require the addition of toxins too help increase vascular permeability (such as pertussis toxin) : ' \ Induction with S-Ag is partic-ularlyy difficult and requires pre-treatment of animals with cyclophosphamide to inhibit suppres-sorr cells (130).

2.2.11 S-Antigen Solublee Retinal Antigen (S-Ag), visual arrestin or 48K protein is a major component of rod out-err segments (Figure 3)(32, 131). It is the second most abundant soluble protein within these cells

19 9

ChapterChapter 1

(132).. Its normul physiological role is to quench the visual transduction cascade induced by the lightt activation of rhodopsin (133). This is achieved when S-Ag binds to phosphorylated rhodopsin,, thereby inhibiting binding to a photoreceptor cell specific G protein (transducin) (134.. 135). S-Ag is one of 6 closely related arrestins. B-Arrestins are found distributed through-outt the body (136-139), but rod and cone photoreceptor arrestins are confined to the retina (140). Al ll share a high degree of homology. Five domains within the protein sequence are conserved acrosss all members (135).

Thee amino acid (aa) sequences of S-Antigens from bovine (141). human (142). murine (143), andd rat retinas (144) were deduced from cDNA sequencing. Sequence identity is 97c/c between

FigureFigure 2: Experimental Autoimmune Uveitis in the Lewis rat showing the various phases of the inflammatoryinflammatory response with S-Ag. (a) Early inflammation characterized by retinal perivascular infiltration,infiltration, and patchy inflammatory foci distributed throughout the retina and choroid; (b) Mid-latelate phase with subretinal serous exudation, outer segment loss and partial disruption of inner retinalretinal layers (hematoxylin-eosin, original magnification 200x). Immunohistochemical staining performedperformed on day 11 shows (c) a large number of CD4+ staining cells infiltrating the ciliary body;body; (d) while few CDH+ cells are present in the choroid (original magnification 250.x).

20 0


mousee and rat, and 81 % between bovine and human. Cleavage fragments of S-Ag and synthetic peptidess were used to identify domains involved in immunogenicity, pathogenicity, and lympho-cytee recognition.

2.2.1.12.2.1.1 T cell recognition sites in S-Ag and the co-stimulatory role ofB cells Inn the Lewis rat, S-Ag contains several short amino acid sequences capable of inducing EAU (pathogenicc sites), and several sequences are able to stimulate in vitro proliferation of sensitized lymphocytess (proliferative sites). The major pathogenic sites are located in the C-terminal half of

Tablee 2: Pathogenic Sequences of S-Ag*

Sequencee number

51-70 0 181-200 0 191-210 0 251-270 0 270-289 9 286-305 5 306-325 5 339-352 2 352-364 4

Aminoo Acid Composition

VDPDLVKGKKVYVTLTCAF R R VQHAPLEMGPQPRAEATWQF F QPRAEATWQFFMSDKPLHLA A VVLYSSDYYVKPVAMEEAQ E E PNSSLTKTLTLVPLLANNRE E LLANNRERRG1ALDGKIKHE E DTNLASSTIIKEGIDRTVLG G LGELTSSEVATEVP P PFRLMHPQPEDPD D

References s

(146) ) (146) ) (146) ) (146) ) (149) ) (150) ) (150) ) (148) ) (145) )

** Based on the human sequence except where indicated. =t== Bovine sequence 77 This is the most immunopathogenic sequence

Tablee 3: Pathogenicity of Determinants of Human S-Ag in Various Rat Strains.

Thee pathologic score was evaluated on the 30th day after immunization. Animals were immunized with the pep-tidee determinant in CFA. Each point is an average of 4 animals. Scoring system and peptide numeration is out-linedd inref (146). The RT1 (rat MHC class II) is indicated in superscript by each strain

Tablee 3a: Immunization without intravenous addition of pertussis toxin

Determinant t Number r

19 9 29 9 35 5 36 6

Tablee 3b:

Lewiss '

4.0 0 3.5 5 4.0 0 4.0 0

Immunizationn w

Determinant t Number r

19 9 29 9

35 5 36 6

Lewiss '

4.0 0 4.0 0 4.0 0 3.0 0

F3444 Kl

0 0 0 0

2.0 0 2.0 0

thh addition of intravenou

F3444 M

1.0 0 0 0

3.0 0 3.0 0

C O P" "

0 0 0 0 0 0 0 0

ss pertussis toxin

C O P" "

0 0 0 0 0 0 0 0

WKY1 1

0 0 0 0

2.0 0 2.0 0

WKY ' '

1.0 0 0 0

4.0 0 4.0 0

WF" "

0 0 0 0

3.0 0 3.0 0

WF" "

1.0 0 0 0

2.0 0 4.0 0

BNh h

0 0 0 0

0 0 0 0

BN' '

0 0 0 0

0 0 0 0

21 1

ChapterChapter I

thee protein (Table 2). The most pathogenic site is located around aa 352-364 in a relatively ex-posedd region of the S-Ag molecule (Figure 3) (132, 145, 146). Gregerson and his colleagues foundd a dissociation between proliferation and pathogenicity (145, 147). However, proliferation sitess are often in close proximity to pathogenic ones (148). Pathogenic sequences were found mainlymainly in regions demonstrating a high degree of difference between the bovine and rat se-quencess (149).

Wee analyzed peptide induced EAU in a variety of rat strains. The peptides we studied were derived fromm the major immunopathogenic determinants of human S-Ag identified in the Lewiss rat. Immunopathogenicity was not solely related to sharing the same RT1 (class II antigen) ass the Lewis rat (1). Fisher (F344) rats (Ivl) were poor responders while the Wistar Furth (WF) (u) showedd a pathogenic response to the immunodominant determinants (Table 3). Further-more,, disease susceptibility to certain determinants could be restored in the F344 by the addition off pertussis toxin. Lymphocyte proliferative responses in both F344 and WF were similar, butt lower than that in Lewis rats (Figure 4), indicating that the difference in disease induction was relatedd to a factor provided by the pertussis toxin, and not related to lymphocyte sensitization. Per-tussiss toxin is known to lead to mast cell dcgranulation, which in turn increases the permeability off retinal blood vessels (151). While this may be responsible for part of the observed effect, per-tussiss toxin may also affect the specialization of the T lymphocyte, by favoring the selection of a Thll profile. This issue will be addressed further in the section on IRBP. Ann unusual observation was made in a pathogenic cell line raised to bovine S-Ag sequence 270-289.. Active in vitro proliferation to the bovine sequence was noted, as well as high pathogenici-tyy in the Lewis rat. However, in the presence of the corresponding rat peptide, there was no pro-liferationn in vitro (149, 152. 153). Proliferation to the rat sequence could only be restored by usingg non radiated splenic antigen presenting cells. Further studies revealed that activated B cells weree required for T cell activation to the autologous rat S-Ag sequence (154, 155). Thus, acti-vatedd B cells appeared to provide a distinct co-ligand needed for full activation of auto-reactive TT cells (155, 156). Work done in the pJvlT mouse strain (B cell deficient mouse) using a cy-tochromee c model, has shown a similar B cell activity which is mediated through expression of thee co-ligand B7-2 on the surface of B cells {157). This co-ligand requirement can be seen as an additionall protective mechanism which helps to maintain homeostasis in the eye. It might be pos-siblee to exploit this property to prevent or modulate disease. Blocking co-stimulatory activity be-tweenn APC.T and B cells is an elegant way of by-passing the need to know the specific antigenic stimuluss causing T cell activation. Indeed, blocking B7-L B7-2, CD40L (expressed on T cells) alll reduce or eliminate disease expression in experimental models (158, 159). Onee of the classic mechanisms proposed for the initiation of autoimmune disease suggests that mimicrymimicry occurs between proteins derived from the host and from infectious agents such as virus-es,, bacteria or fungi. The pathogenic epitope in peptide 286-297 of bovine S-Ag, shares sequence homologyy with several bacterial, viral and fungal proteins. A synthetic peptide of yeast histone H33 containing 5 consecutive amino acids identical to peptide M (DTNLA) was found to induce EAUU in Lewis rats (160). Native S. cerevisiae histone displayed a similar pathogenicity, and lymphh node cells from histone H3 peptide-immunized rats adoptively transferred EAU to naive animalss (161). Several viral peptides with homologies to S-Ag limited to only 3 or 4 aa were al-soo pathogenic provided that high doses were injected(162). These results favor the idea that such shortt epitopes may be sufficient for cross reactivity. Another form of homology was found be-tweenn S-Ag sequence 342-355 and HLA-B27 sequence 125-138 (B27PD) (163). The two se-quencess share 5 discontinuous amino acid homologies, located in anchor positions for presenta-

22 2


FigureFigure 3: Stereo Structure of Arrestin. In light green are shown in the immunopathogenic sites. ByBy stereo viewing, it is evident that the immunodominant site is close to the free swinging arm of thethe C terminal domain. (Adapted from Hitch et al (132) and de Smet (175)).

Sharedd Class II Antigen Different Class II Antigen

0011 01 .1 1 10 100 1000 .001 .01 .1 1 10 100 1000

Antigenn Concentration in Culture (|a.g/ml) Antigen Concentration in Culture (p.g/ml)

FigureFigure 4: Variation in Lymphocyte Responsiveness to Human S-Ag Peptide 341-360 in Immu-nizednized Animals ln-vitro proliferation to peptide 341-360 was carried out 14 days after immuniza-tiontion with the same peptide in CFA. No pertussis toxin was given to these animals. Response in WKYWKY and Lewis are virtually the same but significantly different in other species. This difference isis independent of'MHC class II.

23 3

ChapterChapter 1

tionn to HLA class II antigens (164). Immunization of B27PD in Lewis rats leads to a mild re-lapsingg uveitis in 82% of animals. Feeding B27PD to S-Ag immunized animals protected them fromm developing EAU (163). B27PD has homologies with other class I antigens, namely HLA-B55 1, B44, and B45 (165). The latter two are in linkage disequilibrium with HLA-A29. The pos-sibilityy has been raised that these class I antigens may themselves be the source of an aberrant classs II mediated immune response directed against the eye. Further studies will be required to furtherr elucidate the mechanism.

2.2.1,22.2.1,2 Role of humoral immunity in S-Ag mediated EAU - abrogation of disease Whilee cellular immunity is the main component of the efferent immune response in EAU. a hu-morall response is also present. Antibodies to S-Ag are detectable by ELISA in the eye soon af-terr the onset of ocular inflammation, and are present in the serum within the first two weeks af-terr immunization (166). With high dose immunization, immunoglobulin and C3 deposition on outerr retinal segments can be demonstrated (167). Polymorphonuclear (PMN) cell infiltration, whichh is a major component of the immune response in EAU, can be blocked by using cobra ven-omm leading to complement depletion (168). However, the role of antibodies is likely only an ac-cessoryy one, since EAU induced by adoptive transfer of T cells leads to disease induction with-outt any detectable serum antibody in recipients (169, 170). Iff not essential to generate an inflammatory response, antibodies may well play a role in its down regulation.. Injection of monoclonal antibodies directed against specific S-Ag epitopes simulta-neouslyy with the antigen can abrogate the onset of uveitis either partially or completely (171, 172).. These antibodies likely mask the pathogenic epitopes, either directly in the eye or at the site off immunization. In addition, pre-immunization with S2D2. anti-S-Ag antibody, leads to sup-pressionn of uveitis (173). S2D2 binds a site S2 which is situated close to the N terminal of S-Ag, onn peptides 31-50 and 41-60 of the human sequence (174), This site is away from known im-munopathogenicc sequences in thee rat (175), or immunogenic sequences in humans (176). The S2 epitopee also displays sequence homology with an epitope of TNF-a (amino acid sequence 39-45 off human TNF-a). Following immunization with S2D2 and S-Ag. rats remaining disease free havee antibodies directed not only to S2, but also to the homologous S-Ag sequences (31-50, 41-60).. and to TNF-a (174). Generation of these antibodies is felt to occur through stimulation of idiotypicc and anti-idiotypic antibodies responses (177, 178). Healthyy individuals are known to have antibodies to S-Ag (179. 180). Use of pooled human im-munoglobulinn from multiple donors (IV1G) was able to abrogate inflammation in both the EAU andd EIU models (181, 182). Studies in EAE, suggest that the process involves an acquired unre-sponsivenesss to the immunizing antigen associated with a decreased ability to produce IL-2, IFN-77 and TNF-a (183). Activation of Fas and caspases in human lymphocytes and monocytes hass also been implicated (184). As with S2D2, IVIG is felt to lead to a realignment of the natu-rallyy occurring autoantibody networks through stimulation of idiotypic-anti-idiotypic antibodies (185,, 186). IVIG has recently been used to treat Birdshot retinochoroidopathy with significant stabilization,, and even improvement in certain clinical signs of disease (187).

2.2.22 Interphotoreceptor Retinoid Binding Protein (IRBP) IRBPP is a highly conserved glycoprotein of 140 kDa synthesized by photoreceptor cells of the reti-na,, and secreted into the interphotoreceptor space (188, 189). It is the major component of the in-terphotoreceptorr matrix (190), and functions by transporting retinoids between the neural retina andd the RPE (191). IRBP is highly uveitogenic in Lewis rats (192, 193), monkeys (128, 129), rab-bitss (194), and mice (130). However, unlike S-Ag, it is poorly uveitogenic in guinea pigs (195).

24 4


Ass with S-Ag, the pattern of disease as well as the strength of the response varies among species. IRBPP induced EAU is more acute and severe in the Lewis rat. It usually begins 8 to 12 days post immunizationn and subsides within 5 to 10 days (192). The inflammation begins in the anterior segmentt followed by a spread to the retina with minimal involvement of the choroid. By contrast, inn the primate EAU starts after about 3 weeks, and continues for 6 months or more (128). Here, thee greatest infiltration is noted in the choroid. The retina is affected to a lesser extent, and the anteriorr chamber is virtually unaffected (128). Cellular infiltration also differs, being character-izedd by many polymorphonuclear cells in the rat, and granulomas in the primate. The disease phenotypee is more variable in the mouse depending on the strain, the amount of IRBP used for immunizationn and the use of pertussis toxin (196). Animals challenged with a high dose of both agentss developed an acute disease of early onset and short duration (Figure 5). A lower IRBP or pertussiss dose, lead to a later onset and extended duration. On histopathologic examination, at highh dose diffuse retinal damage is noted, while with low dose immunization, focal damage and choroidall thickening is observed (196).

Bovinee (188) and human (197, 198) IRBP genes aree very similar in structure. They encode pro-teinss of 1264 and 1262 residues respectively withh an average of 84.2% sequence identity (197).. The molecule is characterized by a four foldd repeat structure, each repeat consisting of approximatelyy 300 amino acids, with 30-40% sequencee identity between any two of the four repeats.. The rat IRBP model of EAU has been extensivelyy studied in the late 1980's and early 1990ss to characterize the T cell epitopes in-volvedd in pathogenicity. Its immunodominant sitee was particularly well studied from the standpointt of its interaction with MHC.

2.2.2.12.2.2.1 T cell recognition sites in IRBP and thethe importance of MHC binding Twoo different approaches were used to identify thee immunogenic and immunopathogenic sites off IRBP. Donoso and associates investigated humann IRBP by testing 120 overlapping pep-tides,, corresponding to the entire protein se-quencee (199, 200). Nine peptides were uveito-genic,, one of which caused disease at a dose of 0.11 (xg/rat (aa 521-540) (Table 4). The three mostt immunopathogenic peptides, at positions 5211 -540, 821 -840, and 1121-1140. belong to threee of the four homologous domains, and sharee extensive sequence similarity.

Inn the second approach, potential sequences of interestt in the bovine IRBP sequence were cho-

FigureFigure 5: Experimental Autoimmune Uveitis inducedinduced in the mouse. EAU in the mouse can taketake on one of two forms depending on immu-nizingnizing conditions, (a) Mild disease is charac-terizedterized by focal retinal vasculitis with little choroiditis;choroiditis; (b) severe disease leads to diffuse retinalretinal damage. This late stage picture can al-soso be seen following milder disease but after severalseveral relapses have taken place (hema-toxylin-eosin,toxylin-eosin, 400.x).

25 5

ChapterChapter 1

senn based on their amphipaticity: a measure of simultaneous affinity for MHC and TCR (203, 204,, 206, 207). Of 10 selected peptides, 3 were uveitogenic in Lewis rats: 1091-1115 (205) ,1158-1180(203),, 1169-1191 (204). Two of these peptides. 1158-1180, and 1169-1191 were al-soo uveitogenic in monkeys (208). Of particular interest was peptide 1169-1191. This peptide was uveitogenicc in the Lewis rat at a dose of 0.01 nmol/rat (209). This dose is only 5 fold higher than thee minimal dose required for the whole IRBP molecule and is much less than for any other IRBP-derivedd peptide (210, 211). This high immunopathogenic capability was referred to as an immunodominantt response for IRBP in the Lewis rat. Since immunodominance is thought to de-pendd in part on binding affinity to the MHC molecule present on the surface of APC, this partic-ularr determinant was further characterized using truncated peptides (210). The uveitogenic epi-topee was localized to sequence 1179-1191 (201), and the minimal active determinant to 1182-11900 (WEGVGVVPD) (212). A thorough analysis of this antigenic site by testing analogs inn which each of the residues were sequentially substituted with alanine allowed the identifica-tionn of those residues essential for its immunological properties. Two residues, one at each end off the epitope, were pivotal for peptide binding to the MHC molecule, and two other residues for thee interaction with TCR (212). The crucial role played by the MHC binding epitope was demon-stratedd in the surrogate epitope 271 -283. This epitope induces uveitis, but requires high antigen concentrationn (200 nmol/rat). Substitution with the optimal MHC binding amino acids dramati-callyy reduced the antigen concentration needed to cause uveitis (213).

2.2.2.22.2.2.2 Genetic susceptibility to IRBP induced EAU related to Th profile Thee immune response to an antigenic challenge is mediated by two mutually exclusive sets of ef-fectorr cytokines (214). Two different populations of CD4+ T cells promote the corresponding immunee response. T helper l(Thl) cells produce abundant 11-2. IL-12, IFN-7. Cells of this phe-notypee are involved in classic cell mediated functions (215). By contrast T helper 2 (Th2) cells makee the functionally opposite cytokines such as IL-4, IL-5 and ILK) . These activate B cells to

Tablee 4: Pathogenic Sequences of IRBP:

Name e number r

Sequence e Species s Aminoo Acid Composition References

HIRBP715 5 HIRBPP 778 HIRBPP 730 HIRBPP 745 H1RBPP 808 HIRBPP 720 HIRBPP 722 HIRBPP 724 HIRBPP 804

TA12 2

R4I I RR 14 RR 16

R23 3

521-540 0 53!! -550 821-840 0

1121-1140 0 1131-1150 0 6211 -640 661-680 0 701-720 0

1051-1070 0 271-283 3 518-529 9 880-892 2 158-1180 0 1169-1191 1 1177-1191 1

1179-1191* * 1091-1115 5

Human n Human n Human n Human n Human n Human n Human n Human n Human n Bovine e Bovine e Bovine e Bovine e Bovine e Bovine e Bovine e Bovine e

YLLTSHRTATAAEEFAFLMQQ (200) AAEEFAFLMQSLGWATLVGEE (200) KDLYILMSHTSGSAAEAFAHH (200) SKKSMVILTSTVTAGTAEEFF (200)

TVTAGTAEEFTYIMKRLGRAA (200) ALVEGTGHLLRAHYARPEVVV (200) DLESLASQLTADLQEVSGDHH (200)

PAVPSPEELTYUEALFKTEE (200) EHIWKK1MHTDAMIIDMRF NN (200)

SQTWEGSGVLPCVV (201) ALDRAQEVLEFHH (202)

GEAWDLAGVEPDII (201) HVDDTDLYLTIPTARSVGAADGSS (203)

PTARSVGAADGSSWEGVGVVPDVV (204) ADGSSWEGVGVVPDVV (204)

GSSWEGVGVVPDVV (201) PNDSVSELWTLSQLEGERYGSKKSMM (205)

Mostt immunoparhogenie sequence

26 6


producee neutralizing antibodies (IgGl) and IgE (216). (Naive T cells that produce both Thl and Th22 cytokines are called ThO (216).) By the nature of their effects on the opposing cytokine-pro-ducingg subsets, the relative frequencies of these regulatory T cells can determine whether a giv-enn immune response is protective or pathological (217). As we shall see, this concept appears to playy a central role in explaining the difference in disease susceptibility within a given species.

Ass with S-Ag, susceptibility to IRBP induced EAU varies considerably in inbred strains of rats andd mice. While the Lewis rat can develop EAU at a very low immunizing dose, the Fisher rat (F344)) which shares the same class II MHC, are EAU resistant (218). This difference appears to bee related to the cytokine profile which CD4+ cells generate at the time of immunization. Using aa standard immunization protocol using R16 in complete Freund's adjuvant (CFA), lymph node cellss taken from both strains are able to proliferate in the presence of the immunizing antigen, in-dicatingg that primed cells are present in both strains. However, the pattern of cytokine secretion iss very different. Cells derived from the Lewis rats have a strong Th 1 profile while the F344 cells havee a low Thl profile. Adoptive transfer of F344 cells could generate EAU but only when lOx moree cells were transferred than with Lewis CD4+ cells. Addition of pertussis toxin at the time off immunization allowed the F344 to develop severe EAU. Under the additional influence of per-tussis,, the cytokine secretory pattern of lymph node cells had switched to a characteristic strong Thll profile. Conversely Lewis rats immunized with incomplete Freund's adjuvant (which in-ducess a Th2 pattern (219)), became resistant to EAU, and lymph node cells developed a Th2 phe-notype.. Studies in BIO.A (susceptible) and BALB/c (resistant) mice using an identical immu-nizationn protocol demonstrated that T cells start with a similar ThO profile (39, 220). The shift to aa Thl profile in the BALB/c at about 10 days corresponded with the onset of disease. Treatment withh murine IL-4 and IL-10 for the first five days after immunization significantly reduced dis-easee severity in BIO.A indicating that external factors can influence the direction of effector T celll differentiation (221). Several factors are know to influence the direction of effector T cell differentiation,, MHC receptor affinity (or avidity) (222), cytokine milieu (determined in part by innatee immunity) (223), and the presence of appropriate co-factors (type of APC) (224). Indeed systemicc administration of monoclonal antibodies directed against B7-1 or B7-2 co-stimulatory moleculess (224) modulated the effector profile and EAU severity (158).

Additionall factors also appear to play a role.T cells with a low Th 1 profile are able to induce dis-easee in B10.RIII mouse strain (225). The influence of hormonal levels in the adrenal-pituitary axiss (226, 227), the effect of neural immunoregulation (228), and that of other distinct popula-tionss of regulatory cells (229, 230) still require investigation in ocular inflammatory models.

2.2.33 Rhodopsin and other retinal proteins Rhodopsinn when given in the presence of pertussis adjuvant is a potent inducer of EAU in the Lewiss rat, but the immunizing dose is two orders of magnitude greater than with S-Ag or IRBP (231,, 232). Bleaching of the rhodopsin preparation by illumination just before injection decreas-ess pathogenicity, suggesting that the property is conformation-dependent. A 12 residue sequence inn the C-terminus region (aa. 331-342) was identified as the pathogenic site (233, 234).

Recoverin.. a 23Kda calcium-binding protein was recently found to be highly uveitogenic in rats, producingg severe EAU at low doses (235). It is of interest that recoverin was identified as a tar-gett in cancer-associated retinopathy (236). Finally, phosducin, a 33 kDa retinal and pineal phos-phoproteinn involved in visual transduction has a mild uveitogenic capacity (237, 238).

27 7

ChapterChapter I

2.33 Ocular but non retinal antigen derived autoimmune disease

2.3.11 Lens-induced uveitis Lenss induced uveitis is produced by disrupting the lens capsule in animals primed with xenogenicc or allogeneic lens proteins (cryslallins) (239-241). This is an acute granulomatous uveitiss with characteristics of an immune complex mediated inflammation(242) (Figure 6). The clinicall equivalent in man is phacogenic uveitis, a rare disease which can be cured by removing thee lens. A milder inflammation leading to posterior subcapsular cataract formation can be in-ducedd by adjuvant immunization with a bovine lens membrane protein (243).

AA more recent variant makes use of a FVBN/transgenic mouse expressing HEL in the lens. Adoptivee transfer of activated T cells from HEL immunized non transgenic FVBN mice leads to cataractt development within four days (244. 245). An intriguing hypothesis recently proposed, suggestss that posterior subcapsular cataracts may be induced by cross reacting antibodies raised initiallyy against bacterial proteins. The cross reactive response is directed to a-crystallins. The insuingg low grade immune response would be responsible for cataract development (246. 247).

2.3.22 Experimental melanin-protein induced uveitis (EMIU ) Melanin-proteinn induced uveitis is an experimental model induced by a non-soluble melanin-as-sociatedd protein derived from the choroid, iris and ciliary body. Treating this insoluble fraction withh V8 protease, solubilizes the active component, but full characterization of this protein has not yett been achieved (248). By opposition to EAU, the histopathological changes are restricted to the anteriorr segment and the choroid with sparing of the retina (249-255). CD4+ cells are the prima-ryy mediator of disease (250. 256). though histologically accumulation of polymorphonuclear cells iss also observed (Figure 7). Disease onset occurs between 14 to 17 days after immunization and regressess by about 1 month. Spontaneous recurrence in 25% of rats is seen around 50 days fol-lowingg immunization (257). Addition oflow dose endotoxin increases the recurrence rate to 100% (254).. Differences in cytokine secretion profiles, and expression of apoptosis signals, may explain thee limited inflammatory response observed in EMIU, and also the presence of recurrences (257). Inn EMIU by opposition to EAU. the Thl response is less intense, and the expression of Fas and FasLL is weaker. In addition, Bcl-2 expression is higher than in EAU, which may allow a certain numberr of cells to avoid apoptosis and survive in the choroid, facilitating the onset of a recurrence (42,, 257). Since recurrence can be induced, it lends itself to further study. TGF-fól given in-traperitoneallyy can prevent EMIU recurrences by upregulating ocular IL-10 expression (43) There wass no effect on systemic DTH response. Thus, this particular model holds considerable promise inn helping elucidate factors related to ocular recurrence, and means of modulating it.

2.3.33 Experimental autoimmune encephalitis (EAE) EAEE serves as a model for a closely related autoimmune disease, namely multiple sclerosis (MS).. Indeed many of the observations made using this model also apply to EAU. EAE is in-ducedd in susceptible animals using myelin basic protein (MBP) or an immunopathogenic frag-mentt thereof (258-260). It can also be induced by immunizing with fragments of a8-crystallin (261,, 262). EAE is mediated by activated T cells, and can be induced by adoptive transfer of sen-sitizedd T cells. As with MS patients, ocular manifestations are also observed (263). Inn the eye, EAE may manifest itself both in the posterior pole and in the anterior chamber. Pos-teriorr inflammation is centered around the optic nerve. It's severity is dependent on the amount off available myelin in the host animal, strain susceptibility and MBP dose. If severe, the inflam-

28 8


FigureFigure 6: Lens Induced Uveitis. Along the edgeedge of the disrupted lens capsule, a mixed populationpopulation of inflammatory cells is present. A numbernumber of foreign body giant cells can be seen,seen, (hematoxylin-eosin 200x and 400x)

mationn wil l extend into the retina and the vitre-ouss (264). It is characterized by mononuclear celll infiltration, perivasculitis and demyelina-tionn (265-267).

Anteriorr segment inflammation often develops inn Lewis rats with EAE (268, 269). Onset of an-teriorr uveitis coincides with the onset of EAE 111 days after MPB immunization, but the peak off activity in the anterior chamber occurred duringg the recovery phase of the CNS disease, indicatingg that the kinetics of the immune re-sponsee in both sites is somewhat different. Myelinatedd nerve fibers present in the iris may

<f30%00^ -

.. - ' - " ' ' ' - . " . . . - * * '

A. .

^.fs?"" jvr:^...

'i''U*i'

FigureFigure 7: Experimental Melanin Induced Uveitis. In the initial episode, disease is limited to the choroidchoroid (a) and the ciliary body (b). Recurrent disease is very similar in appearance whether or notnot it has been induced by the addition of LPS. Involvement in the posterior pole (c) may be asso-ciatedciated with little retinal inflammation, and is limited to small subretinal foci. Ciliary body in-volvementvolvement is identical (d). (hematoxylin-eosin original magnification 400x retina, 2()()x ciliary

29 29

ChapterChapter 1

bee the initial target for MBP reactive cells (44, 270). Peripheral nerves contain myelin, but the concentrationn is different than in the CNS, leading to a different kinetic response. However, the presencee of an identical T cell receptor motifs in T cells infiltrating the anterior chamber and the CNS,, indicate that both diseases share a common effector mechanism (44). This model was also usedused to demonstrate the importance of the chemokine MCP-1 in the initial recruitment of in-flammatoryy cells into the anterior chamber (271).

2.3.44 Other non retinal models of ocular autoimmunity Corneaa specific autoimmunity can be induced against a 54 kDa protein from corneal epithelium. Thiss particular model has bee used to study several localized disease entities (272, 273). The processs appears to be dependent on complement activation and an intact pathway for antibody synthesiss (274). T cells also appear to play a role, but it only becomes evident when corneal tis-suee is pre-treated with radiation (275). Most studies on corneal inflammation make use of infec-tiouss adjuvants either viral, bacterial or fungal. Inn ocular adnexae, tissue-specific autoimmunity occurs in the lachrymal gland, either as a com-ponentt of Sjogren's syndrome or as isolated chronic dacryoadenitis. Inflammation of the gland iss produced experimentally by immunizing rats with lachrymal gland proteins or by adoptive transferr of in vitro antigen activated lymphocytes from sensitized donors (276-278). Autoimmunityy direct to oculomotor muscles is well known in patients with Grave's disease. Theree is some evidence that the immune response is directed to adipose cells or to muscle anti-genss independent of thyroid antigens (in vitro cytotoxicity and autoantibodies)(279-282). How-ever,, since no animal model has so far been developed, further elucidation of the mechanism is difficult . .

Ann exciting new model for ocular autoimmunity is generated by using tyrosinase related proteins TRP-11 and TRP-2. These are tissue differentiation antigens present in melanocytes. Immuniza-tionn with these proteins causes skin vitiligo, mediated through CD4+ cells (283-285). Using thesee same antigens, inflammation restricted to the choroid and choriocapillary has also been de-scribedd in susceptible pigmented rat species [K. Yamaki: personal communication]. This model appearss to be particularly well suited to the study of Vogt Koyanagi Harada syndrome.

3.00 Modulation of ocular immune response

Inn the experimental models mentioned so far. it is clear that CD4+ T cells, particularly of theThl profile,, play a central role in the effector phase of retinal autoimmunity. B cells appear to have an accessoryy role, possibly even a protective one. Mast cells were briefly mentioned in the context off pertussis toxin, and their role in causing blood ocular barrier disruption. What we have not ad-dressedd so far, is how T cells are recruited into the eye. To activate the local cascade, memory T cellss require local priming, and this function depends on the presence of local APC.

3.11 Nature of Class 11+ cells in the retina and uvea tissue:

Initiallyy local resident cells within the retina: RPR, Muller and migroglial cells were thought to fulfil ll the function of antigen presentation by expressing class II antigens on their surface. How-ever,, RPE and Muller cells are poor presenters and probably function only to down regulate in-

30 0


flammatoryy responses (20, 286, 287). Retinal and CNS mieroglial cells function in similar ways, withh the latter inducing apoptosis of antigen specific T cells (288). In the eye, antigen presenta-tionn to T cells occurs through fairly classic mechanisms, by way of professional APC. Careful histologicc studies of whole mounts of the iris, ciliary body and choroid have shown the presence off dense networks of macrophages and dendritic cells (DC) throughout the uveal tract (289-291). Dendriticc cells derived from the iris and choroid are potent presenters in mixed lymphocyte reactions,, but require the addition of maturational signals such as GM-CSF (292, 293). Similar-lyy to DC elsewhere in the body, these cells have a rather short transit time in the eye of about 2 dayss (294). While it is presumed that the majority of these cells migrate to the spleen due to a lackk of lymphatics within the eye, some of these DC make it to lymph nodes, in the case of the iris,, they migrate to submandibular lymph nodes (295). Thus, the needed mechanisms to drive a cellularr inflammatory response are present.

Inn the early phase of the inflammatory process, EAU may present itself in two ways (Figure 8). Itt may present itself as a pseudo-granuloma with inflammatory cells located around Bruch's membranee and the RPE (presumably the APC in this case are RPE-DC). More commonly it presentss as a vascular cuff surrounding retinal vessels. As the disease establishess itself, accessory cellss are called into the inflammatory focus, and spread to the deeper retinal layers. In this case,, the antigen presenting cell has not been identified, but is likely to be an MHC class 11+ macrophagee located in the perivascular area (287). In EAU and EMIU, marrow derived activated macrophagess are required for an effective induction of inflammation. They are recruited early in thee effector phase of the disease (296-299). Depleting animals of marrow derived macro-phagess significantly reduces the severity of EAU, and absence of the appropriate MHC Class II

FigureFigure 8: Experimental Autoimmune Uveitis induced in the Lewis rat with low dose RI6 of bovinebovine IRBP. (a) Small granuloma at the level of the RPE; (h)Vascular cuffing was noted in a retinalretinal blood vessel (hematoxylin-eosin, original magnification 400x).

31 1

ChapterChapter 1

onn the APC surface effectively prevents EAU induction (296). Surprisingly, in the early phase of EAU.. there is littl e change in the population of resident DC cells either in the iris or choroid. It iss only during the active phase of the inflammatory process that their numbers increase. Once present,, their numbers remain high even 42 days after immunization (299). Phenotypic charac-terizationn of ocular DC cells before and after ocular inflammation has yet to be performed. It is nott known if the infiltrating DC acquire different characteristics during the inflammatory process andd retain these after inflammation subsides.

3.22 Immunobiology of dendriti c cells

Dendriticc cells were only shown not to be responsible for antigen presentation, but are also ca-pablee of influencing the maturation of T cells. Thus, they play an active role in modulating the immunee response. While providing antigenic stimulation to the T cell receptor, DC influence TT cell differentiation into Thl or Th2 cells through co-stimulatory signals (100, 300). The exact repertoiree of co-stimulatory signals provided by mature DC is determined during the DC matu-rationaii process. Shortly after leaving the bone marrow, immature dendritic cells (iDC) migrate too a target organ. There, for a short period of time, they will actively take up antigen, while be-ingg exposed to the local cytokine environment. This micro-environment will determine whether aa given iDC will become pro-inflammatory (DC1) or suppressor (DC2) profile (table 5). Once selected,, this profile remains fixed. As the maturational process nears completion. DC migrate backk to local tissue where upon exposure to the same antigen, they will activate T cells. The cy-tokinee environment provided by ACAID is a prime example of a DC2 inducing environment. A similarr example is provided around chorio-capillaries where PGE2 induces a DC2 phenotype (100.. 301).

Thee functional characteristics of ocular DC have yet to be determined, particularly in the setting off acute inflammation. Recent studies on the effect of ocular inflammation on ACAID, indicate thatt during the acute inflammatory episode, immune deviation is lost, and may even be actively suppressed.. However, once inflammation has subsided immune deviation can be restored at least inn part in the EAU model (302). Modulation of DC function, and active selection of a DC2 pro-filefile may prove to be an effective therapeutic approach in years to come.

4.00 Evidence for ocular autoimmunity in humans

Autoimmunee disease infers a response to a self-antigen. In certain ocular conditions such as lens inducedd uveitis, there is no doubt as to the source of antigenicity. For many other ocular inflam-matoryy conditions, the association is less clear. Although many ocular diseases have been as-cribedd to autoimmunity, the presence of an autoimmune response by itself , does not mean that a diseasee is the result. In many instances, a positive assay may occur as a consequence rather than ass the cause of the disease process. We have shown in previous sections that ocular privilege is nott due to antigen sequestration, but to an active down regulation of a pro-inflammatory re-sponse.. Thus, a response to antigen can be expected under in vitro culture conditions in normal individualss and is often observed (303). It is the intensity of the response, that differentiates pa-tientss from normal individuals (304). Once initiated, the autoimmune response may lead to ex-acerbationn of the inflammatory response and to its perpetuation.


Tablee 5: Features of type-1 and type-2 polarized effector subsets of myeloid DCs (modified from ref. 100)

iDC C Polarizedd effector DC subsets DC11 DC2

Polarizingg factor Surfacee phenotype CD83 3 CD80/86 6 MHC-II I ICAM-1 1 OX40L L CD!! 15 MR R Antigenn uptake Responsivenesss to CD40L L IL-122 modulation Cytokines s IL-12 2 TNF-a a IL-6 6 TT cell priming Stimulation n Polarization n

IFN-7 7

+ + ++ + ++ + ++ +

++ + ++ +

++ + +/--

++ + +/--+ +

++ + Thl l

PGE, ,

++ + ++ + + + + +

++ + +/--

+/--

++ + Th2 2

Abbreviations:: CD40L, CD40 ligand; iDC: immature dendritic cell; MR: mannose receptor

Sincee EAU can be induced in so many different animal species, including the primate, it is temp-ingg to consider that retinal antigens are responsible for a variety of human uveitis conditions off unknown origin. EAU has a very polymorphic phenotype manifesting itself as a fulminant retinitis,, a granulomatous choroiditis, or segmental vasculitis - depending on the immunizing antigen,, its dose, and the chosen animal model. Thus, it can mimic a number of human in-flammatoryy conditions, and as a model, it is very useful to understand clinically manifest disease.. So far, no causal link was made between retinal autoantigens and human retinal in-flammation,, though retinal autoantigens are probably implicated at some point in the pathogen-esiss of a number of uveitic conditions. Over the years, a number of researchers have autoimmu-nizedd themselves with S-Ag or IRBP. None with the possible exception of one individual have hadd ocular sequelae.

4.11 Cellular response to retinal proteins

Numerouss authors have reported positive lymphocyte stimulation responses with crude retinal extracts,, S-antigen, IRBP and a host of other antigens (127). In reviewing the data published up too 1988, Y de Kozak and JP Faure came to the conclusion that cellular responsiveness to ocular antigenss was highest under the following circumstances (127): -- during active disease rather than in remission -- in diffuse forms of uveitis (panuveitis) rather than purely posterior ones -- in subacute or chronic disease states rather than in acute self limited disease -- after the disease process had evolved for some time

33 3

ChapterChapter 1

Sincee then, several studies have been carried out to characterize the response to peptide determi-nantss of S-Ag and ÏRBP in a variety of uveitis conditions (303, 305-309). Response to S-Ag pep-tidess was also seen in a number of other conditions such as retinitis pigmentosa (310), Eale's dis-ease,, (309), and ankylosing spondylitis (307). The response to S-Ag determinants was studied in greaterr detail. Lymphocyte responses to 40 overlapping peptides of human S-Ag were studied in aa number of ocular inflammatory conditions (Figure 9) (176, 303). Behcet's disease and Sar-coidosiss patients gave the most frequent and consistent responses (176). A characteristic of all re-spondingg patient populations was the ability to recognized multiple determinants simultaneous-lyy (Figure 10). In a few patients, the response was examined prospectively over a period of six monthh to one year. The response pattern in these patients was noted to change over time, while a normall individual who was also repeatedly tested, did not demonstrate this variability in re-sponse.. This variation, also called "determinant spread" has been observed in EAE (311, 312) andd in MS patients (313). Each new recurrence of EAE was preceded by the appearance of a re-sponsee to a new determinant (311). Each new determinant appeared in a predictable cascade, and feedingg this determinant prior to disease onset was protective. This phenomenon has not yet been studiedd in the EAU model.

4.22 HLA associations

Strongg HLA associations have been observed in many types of human uveitic diseases (314, 315).. The best known association is between Birdshot retinochoroidopathy and HLA-A29, and moree specifically HLA-A29.2 (316). Other known associations include HLA-B27 for acute an-teriorr uveitis (317, 318), HLA-B51 for Behcet's disease (319-321), HLA-B44 in occlusive vas-culitiss (322). In the case of Birdshot retinochoroidopathy, the S-Ag peptide 349-357 (containing partt of the immunopathogenic determinant for S-Ag) was found to bind efficiently to purified HLA-A29.2(323). .

Itt is striking that in many of the examples mentioned above, the disease appears to be associated withh a class I rather than class II antigens. Thus, one would expect to see a disease mediated by cytotoxicc T cells (CDS) rather than CD4 helper T cells, as predicted by experimental models. To circumventt this anomaly, several alternative explanations have been proposed. In the case of Be-hcet'ss disease, it is felt that the HLA antigen itself is not responsible for disease induction, but thatt it exists in linkage disequilibrium with a neighboring gene located between the locus for B5I andd TNF on chromosome 6 (324). In HLA-B27 disease, a recently proposed theory suggests that HLA-B277 can exist as a dimer on the surface of APC cells, and that this dimer could aberrantly presentt (in a class II fashion) to CD4 cells (325). Another possible explanation was mentioned in ann earlier section (Section 2.2.1.1), namely that it is the HLA antigen itself that is the immuno-genn rather than the antigen presenter. In this regards, a recent study in Behcet disease patients showedd that those with active uveitis did have a significant response to B27PD (a peptide derived fromm HLA-B27 with homology for HLA-B51) as compared to Behcet's disease patients without uveitis,, or normal controls (326).

Certainn conditions causing uveitis are associated with class II antigens. Patients with pars plani-tiss due to MS have a DR2 association (327). Sarcoidosis is associated with HLA-DRB1 or DRB3 (328,, 329), while VKH has an association with HLA-DRB 1 *0405(330-332). A human T cell line restrictedd to HLA DRB1 *0405 derived from a Japanese VKH patient responded to a tyrosinase

34 4


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35 5

ChapterChapter 1

Thiss elevated pool persisted for up to 3 months before returning to the pre-inflammatory level (367).. Prospective studies such as these are still limited in number. In a viral infection model, a 300 fold expansion in the responsive lymphocyte population was seen after viral re-challenge (368,, 369). The elevation also persisted for about 3 months. The regulatory processes underlying thee expansion and contraction of peripheral lymphocyte pools are not well understood. Their studyy should give us some insight on control mechanisms that could potentially be exploited to shortenn inflammatory episodes in patients with active uveitis.

5.. Conclusions:

Thee use of experimental models of uveitis has given us important insights into the mechanisms underlyingg ocular inflammation. Our understanding of many experimental models is quite ex-tensive,, as we have outlined above. However, our knowledge of human disease is much more limited.. We do not know the source of inflammation in the majority of human conditions. We knoww very littl e of the triggers that cause recurrences, which in the human situation is most of-tenn responsible for long term ocular damage. In the coming years, studies will be required to help elucidatee the mechanisms of disease recurrence, as well as to define with even more precision the cascadee of events occurring at the very onset of inflammation. Use of limiting dilution assays, andd culture of ocular specimens from human patients will hopefully provide us with more insight intoo the kinetics of human ocular inflammation, and suggest novel ways of modulating its re-sponse.. Finally, while our goal is to control inflammation to prevent damage to eye tissue, in-flammationflammation may also provide some benefit. Recent experiments in the CNS have shown that au-toimmunityy may help maintain homeostasis, and even promote regeneration following nerve injuryy (370). If this is the case in the eye, we will need to learn not how to control inflammation, butt how to modulate it.

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331.. Goldberg AC. Yamamoto JH. Chiarella JM. Marin MLC. Sibinelli M, Neufeld R, Hirata CE, Olivalves R. Kalii I J. 1998. HLA-DRB1 *0405 is the predominant allele in brazilian patients with Vogt-Koyanagi-Harada disease.. Human Immunol 59: 183-8

332.. Kim MH. Seong MC, Kwak NH. Yoo JS. Huh W. Kim TG. Han H. 2000. Association of HLA with Vogt-Koyanagi-Haradaa syndrome in koreans. Am J Ophthalmol 129: 173-7

333.. KobayashiH. KokuboT. Takahashi M.Sato K. Miyokawa N, Kimura S. Kinouchi R. Katagiri M. 1998. Ty-rosinasee epitope recognized by an HLA-DR-restricted T-cell line from a Vogt-Koyanagi-Harada disease pa-tient.. Immunogenetics 47: 398-403

334.. Charteris DG. Barton K. McCartney ACE. Lightman SL. 1992. CD4+ lymphocyte involvement in ocular Behcet'ss disease. Autoimmunity 12: 201-6

335.. Chan CC. 1988. Relationship between sympathetic ophthalmia, phacoanaphylactic endophthalmitis, and Vogt-Koyanagi-Haradaa disease. Ophthalmol 95: 619-24

336.. Chan CC. Wetzig RP. Palestine AG. Kuwabara T, Nusenblatt RB. 1987. lmmunohistopathology of ocular sarcoidosis.. Report of a case and discussion of immunopathogenesis. Arch Ophthalmol 105: 1398-402

337.. Lightman S, Chan CC. 1990. Immune mechanisms in choroido-retinal inflammation in man. Eye 4; 345-53 338.. Chan CC. Roberge FG. 1996. Sympathetic ophthalmia. In Ocular infection and immunity, ed. JS Pepose.

GNN Holland. KR Wilhelmus, pp. 723-33. St Louis: Mosby 339.. Shah DN. Piacentini MA. Burnier MN. Jr. al e. 1993. Inflammatory cellular kinetics in sympathetic oph-

thalmia:: a study of traumatized (exciting) eyes. Ocul Immunol Inflam 1: 255-63 340.. Kasp E. Graham EM, Stanford MR. al e. 1986, Retinal autoimmunity and circulating immune complexes in

ocularr Behcet disease. In Recent advances in Behcet disease, ed. T Lehner. CG Barnes, pp. 67-72. London: Royall Society of Medicine Services

341.. Nussenblatt RB. Palestine AG, Chan CC. Mochizuki M, Yancey K. 1985. Effectiveness of cyclosporin ther-apyy for Behcet's disease. Arthritis Rheum 20: 671-9

342.. Doekes G. Luyendijk L, Gerritsen MJ, Kijlstra A. 1992. Anti-retinal S-antigen antibodies in human sera: a comparisonn of reactivity in Elisa with human or bovine S-antigen. Int Ophthalmol 16: 147-52

343.. HoekzemaR, Hwan SB. Rothova A. van Haren MA. DonosoLA. Kijlstra A, 1990. Serum antibody response too human and bovine IRBP in uveitis. Curr Eye Res 9: 1177-83

52 2


344.. Chan CC, Palestine AG. Nussenblatt RB. Roberge FG? Benezra D. 1985. Anti-retinal auto-antibodies in Vogt-Koyanagi-Haradaa syndrome, Behcet's disease, and sympathetic ophthalmia. Ophthalmol 92: 1025-8

345.. Goldstein SM, Syed NA, Milam AH, Maguire AM, Lawton TJ. Nichols CW. 1999. Cancer-associated retinopathy.. Arch Ophthalmol 117: 1641-5

346.. Guy J, Aptsiauri N. 1999. Treatment of paraneoplastic visual loss with intravenous immunoglobulin. Arch OphthalmolOphthalmol 117:471-7

347.. Keltner JC, Thirkil l CE. 1999. the 22 kDa antigen in optic nerve and retinal diseases. J Neurophthahnol 19: 71-83 3

348.. Franks WA. Limb GA, Stanford MR, Ogilvie J, Wolstencroft RA, Chignell AH, Dumonde DC. 1992. Cy-tokiness in human intraocular inflammation. Curr Eye Res 1! Suppl: 187-9

349.. Wakefield D, Lloyd A. 1992. The role of cytokines in the pathogenesis of inflammatory eye disease. Cy-tokinetokine 4: 1-5

350.. de Boer JH, van haren MAC, de Vries-Knoppert WAEJ, Baarsma GS, de Jong PVTM, Pstema FJ, Rade-makerss AJJM, KijlstraA. 1992. Analysis of 1L-6 levels inhuman vitreous fli d obtained from uveitis patients, patientss with proliferative intraocular disorders and eye bank eyes. Curr Eye Res 11 (suppl): 181-6

351.. Klok AM, Luyendijk L, Zaal MJ, Rothova A, Hack CE, Kijlstra A. 1998. Elevated serum IL-8 levels are as-sociatedd with disease activity in idiopathic intermediate uveitis. Brii J Ophthalmol 82: 871 -4

352.. el-Shabrawi Y, Livir-Rallatos C, Christen W, Baltatzis S, Foster CS. 1998. High levels of interleukin-12 in thee aqueous humor and vitreous of patients with uveitis. Ophthalmol 105: 1659-63

353.. Petrinovic-Doresic J, Mazuran R. Henc-Petrinovic L. Kuzmanovic B. Jovicic A. 1999. Interleukin 6 and its solublee receptor are elevated in aqueous humor of patients with uveitis. Ocul Immunol Inflamm 7: 75-84

354.. Ongkosuwito JV, Feron FJ, van Doornik CEM, Van der Lelij A, Hoyng CB, La Heij EC, Kijlstra A. 1998. Analysiss of immuno-regulatory cytokines in ocular fluid samples from patients with uveitis. Invest Oph-thalmolthalmol Vis Sci 39: 2659-65

355.. ElnerSG. ElnerVM, JaffeGJ. Stuart A, Kunkel SL, Streiter RM. 1995. Cytokines in proliferative diabetic retinopathyy and proliferative vitreoretinopathy. Curr Eye Res 14: 1045-53

356.. Nishi O, Nishi K, Imanishi M. 1992. Synthesis of interleukin-1 and prostaglandin E, by lens epithelial cells off human cataracts. Brit J Ophthalmol 76: 338-41

357.. Muhaya M, Calder V, Towter HM, Shaer B, McLauchlan M, Lightman S. 1998. Characterization of T cells andd cytokines in the aqueous humour (AH) in patients with Fuchs' heterochromic cyclitis (FHC) and idio-pathicc anterior uveitis (IAU). Clin Exp Immunol 111: 123-8

358.. Klok AM, Luyendijk L, Zaal MJ, Rothova A, Kijlstra A. 1999. Soluble ICAM-1 serum levels in patients withh intermediate uveitis. Brit J Ophthalmol 83: 847-51

359.. Calder VL, Shaer B, Muhaya M, Mclauchlan M, Pearson RV, Jolly G. Towler HMA, Lightman S. 1999. In-creasedd CD4+ expression and decreased IL-10in the anterior chamber in idiopathic uveitis. Invest Ophthal-molmol Vis Sci 40: 2019-24

360.. Kitaichi N, Kotake S, Sasamoto Y, Namba K, Matsuda A, Ogasawara K, Onoe K, Matsuda H, Nishihira J. 1999.. Prominent increase of macrophage migration inhibitory factor in the sera of patients with uveitis. In-vestvest Ophthalmol Vis Sci 40: 247-50

361.. Norose K, Yano A, Wang XC, Tokushima T, Umihira J. Seki A, Nohara M, Segawa K. 1994. Dominance of activatedd T cells and interIeukin-6 in aqueous humor in Vogt-Koyanagi-Harada disease. Invest Ophthalmol VisVis Sci 35: 33-9

362.. Sakaguchi M, Sugita S, Sagawa K, Itoh K, Mochizuki M. 1998. Cytokine production by T cells infiltrating inn the eye of uveitis patients. Jpn J Ophthalmol 42: 262-8

363.. Opremcak EM, Cowans AB, Orosz CG, Adams PW, Whisler RL. 1991. Enumeration of autoreactive helper TT lymphocytes in uveitis. Invest Ophthalmol Vis Sci 32: 2561-7

364.. de Smet MD, Dayan M. Nussenblatt RB. 1998. A novel method for the determination of T-cell proliferative responsess in patients with uveitis. Ocul Immunol Injlam 6: 173-8

365.. Zhang J, Markovic-Plese S. Lacet B, Raus J, Weiner HL, Hafler DA. 1994. Increased frequency of inter-leukinn 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinall fluid of patients with multiple sclerosis. J Exp Med 179: 973-84

366.. Esin S, GUI A, Hodara V, Jeddi-Tehrani M, Dilsen N, Koni^e M, Andersson R. 1997. Peripheral blood T cell expansionss in patients with Behcet's disease. Clin Exp Immunol 107: 520-7

53 3

ChgpttrChgpttr I

367.. de Smet MDVDayan M-2ÖÓ0. if^pspecti^de^wiiatipö of T cell responses to S-antigenin Behcet'sItfs-easee patients and controls. Invest QphihatmoiVis Sci (in press)

368,, Ahmed R, Gray D. 1996. Imittunological inenioiy and protective immunity: understanding their relation, Sdm*?? 272: 54-60,

'WL'WL Meïïeyzer-VVnjiams MG, Davis MM, 1§9& Aastigen-spe lfic development of primary and niemqry T cellss inn Vivo, Séiericè 268; 106-11

37Ö.. SchwiaitzMvGafeéiiIR.2ÖOÖ.Autoimrnun%©an benefi t £mtinmw&.JmmptmtT^y21; 26$-8

54 4


Sectionn I: Analysis of Immunologic Determinants to Ocular Autoantigens inn Experimental Models

"I"I reckon that for all the use it has been to science about four-fifths of my time has been wasted, andand I believe this to be the common lot of people who are not merely playing follow-the-leader inin research... science in its forward motion is not logically propelled. The process by which we comecome to formulate a hypothesis is not illogical, but non-logical, i.e. outside logic. But once we havehave formed an opinion, we can expose it to criticism, usually by experimentation; this episode lieslies within and makes use of logic."

Medawar,, PB: Induction and Intuition in Scientific Thought, American Philosophical Society, Philadelphia,, 1969

55 5

Chapterr 2

Analysiss of the Pivotal Residues of the Immunodominantt and Highly Uveitogenic

Determinantt of Interphotoreceptor Retinoid-Bindingg Protein

Satoshii Kotake, Marc D. de Smet. Barbara Wiggert, T. Michael Redmond, Geraldd J. Chader, Igal Gery

Journall of Immunology 146: 2995-301, 1991 (byy permission © The American Association of Immunologists)

PIVOTALL RESIDUES

' J 'J ÏJ-- I 7*)7 41 Hl jW i O T n M j i UI, u T-.hh .1 . . . H-,». .+ IMM ! V' I l i > ' .."i ' i !>-:i(j!>J. \ i . ' I . \ ! .e . I. r e i ]

( '( ipvrl i ihrr J ! 1.\ TIn- Ami-r i f .111 A w , r l.itl-Ti ! Irr.ir.i i i i.rl. i;i>.!>, f ' r i l fe r i i'l .'' S A

ANALYSI SS OF THE PIVOTAL RESIDUES OF THE IMMUNODOMINAN T AND HIGHLYY UVEITOGENIC DETERMINANT OF INTERPHOTORECEPTOR

RETINOID-BINDINGG PROTEIN1

SATOSHII KOTAKK.* MARC D. iw. SMET,* BARBARA WIGGEKTV T. MIOHAKL RKDMONO.' GERALDD J. CHADER.' AND 1GAL GERY *

Fromm ((if Laborawrirn t>f'Immunology and 'Retinal Crlt and Molvcular Biology, the National /nsrir . Mat tonal InutilitiesInutilities iff Health. Hrtlu-nflu. Ml) 20X92

Interphotoreceptorr retinoid-binding protein (IRBP),, a retinal-specific Ag, induces experimental autoimmunee uveitis in a variety of animals. We have previouslyy shown that sequence 1169-1191 of bo-vinee IRBP is the immunodominant epitope of this proteinn in Lewis rats and is highly immunogenic andd uveitogenic in these rats. The active site of peptidee 1169-1191 was determined by testing its truncatedd forms. The shortest peptide to be immu-nologicallyy active was found to be 1182-1190 (WEGVGVVPD).. To determine the role of individual residuess of this sequence, we have tested the im-munologicc activities of nine analogs of peptide 1181-1191,, in which each of residues 1182-1190 wass substituted with alanine (A). The tested activi-tiess included the capacity to induce experimental autoimmunee uveitis and cellular responses in im-munizedd rats, as well as the capability to stimulate lymphocytess sensitized against IRBP or the parent peptidee 1181-1191. Analogs that did not stimulate thesee lymphocytes were also tested for their capac-ityy to competitively inhibit the proliferative re-sponsee to 1181-1191. Analogs A(1184), A(1186J, andd A(1187) resembled 1181-1191 in their activi-ties,, whereas the other analogs exhibited remarka-blyy reduced activities, with several patterns being noticed.. Analog A(1182) was inactive in all tests. Analogg A(1190) was very weakly uveitogenic and non-immunogenic,, but it stimulated lymphocytes sensitizedd against IRBP or 1181-1191 when added att exceedingly high concentrations. Analogs A(1183)) and A(1185) resembled A(1190] in being weaklyy uveitogenic and A(1185) was also found to bee poorly immunogenic. In addition, relatively high concentrationss of A(l 183) and A(l 185) were needed too stimulate lymphocytes sensitized against IRBP orr 1181-1191. However, a different pattern of activ-itess was exhibited by analogs A(1188) and A[1189). Thesee peptides were uveitogenic and immunogenic, butt failed to stimulate lymphocytes sensitized to IRBPP or 1181-1191. Furthermore. A(1188) and

Kreeivrrll lor [iijhlrcatiori Oelober 18. 1990 AecepterHorpi ihl iei i t im!! i-Vtirimrv 7. IWt] Thee ('(IMS of puhlU'.ltioi i (it thi s artiele were rtf f i; l V(-(i 111 p.ir l hv I lie

pavmeiill <>l page eliaryes This j i r l i r le nuiM ll ieretnre he herein- marked n r f i ' f ii l i scmcnMi i mr . i rd fmiT wi th i m r s . C . Seel ion I 7:*4 solely l o i nd i -ca le th i sh i i ' t . .

' T h i ss work was supported in part hv an Aleim Kesenrrri I r isumie Awardd tl. (',.;.

aa Address eniresporidenre and reprint requests In [)r I C I T Y . MatjonaJ Hyee lusl i lu l t ' . .Nmional Inst i l i i les ol l l r u l i h . Hklu 10. Km IÓN20H. Hr-fiiesda.. MDÏOHöa.

A(1189),, but not A(1182). also inhibited the re-sponsee to 1181-1191 of a cell line specific toward thiss parent peptide. The data are interpreted to showw that residues 1188 and 1189 are involved in thee interaction of the peptide with the TCR, whereas residuess 1182 and 1190 and, perhaps, 1183 and 1185,, are pivotal for the binding of peptide 1181-11900 to the MHC molecules on APC.

Autoimmunityy is assumed to play a major role in the pathogenesiss ol certain intraocular diseases which are groupedd under the term "uveitis" (1, 2). This notion is supportedd by the finding that inflammatory changes sim-ilarr to iho.se in iivritie patients can be induced in experi-mentall animals by immunization with ocular-specific Ag (3,, 4|. The animal disease is designated EAU.:< One of the ocularr Ag that is commonly used to induce EAU is tl ir retinall IRBP. a glyeolipoprotein of -140 kDa, which is though!! to function in the transport of retinoids between thee neural retina and the retinal pigment epithelium (5, 6).. We and other investigators have shown that 1KB!1 is uveitogenicc in a variety of species, including the rat (7, K).. rabbit (9). mouse (10). and primates ( I I ) .

Inn a recent study with Lewis rats we have identified an immunodominantt and highly uveitogenic and immuno-genicc determinant within the sequence 1169-1191 of bovinee IRBP (12). Truncation at the amino terminus of thiss sequence yielded immunologically active peptides, as shortt as the deeapeptide 1182-1191 (12). The present studyy was aimed at further analyzing this antigenic site, too identify the residues that are pivotal for its immuno-logiee activities. By truncation at the earboxyl-terminus of thee site the minimal active peptide was determined to consistt of the nine residues ] 182 I 190. The role of each off these amino acids was further examined by testing the activitiess of peptide analogs in which individual residues weree substituted by alanine. Certain residues were found too be pivotal for the site's activities

VII AT K hi] AT.S AM ) MKTHODS

Af).Af). Movine IRI1F' w; is p t i r i t ied as descr ibed in deta i l e lsewhere ( l . ' i ll The [KHI'-<1rrivt.'c1 peptides tested here were syn i ! i es i /ed ,)n<l ])iiri!it:cll hv Appl ied Biosystems (Koster Ci ty . CA| i i s in i i Itir- r-HOC ehemis t rv ,, on an Appl ied H iosvs l rms mixle l 4.'J0A pepur i r syn lhe s i /er . .

Animfils.Animfils. Male inhrer l Lewis n i l s , S In 12 wk o ld . were suppl ied

11 Abhrevial iui is used in this paper. KALI, experimental ati lranmiune uveitis.. IHlif '. mliTphrilfireei-pLrir r r t in t i id-Miut i i iu proR-m: S I . stimula-ntt >rr mi le*

59 9

http://iho.se

ChapterChapter 2

PIVOTALL RESIDUES WITHIN IMMUNOPATHOGENIC PEPTIDE

byy Charles River Laboratories. Inc., from [he facility in Raleigh. NC fmmuntzarion.. The rats were immunized by a e injection of

1KB!'' or the peptides, emulsified in CFA. containing Mycobacterium tuberculosistuberculosis H37Ra at 2 5 ing'ml (I)ilcu Laboratories. Detroit. Ml|. Thee emulsion was Injected into erne hind footpad in a volume ol 0.1 mll containing various amounts ol the Ag An additional adjuvant, killedd Bordetella pertussis bacteria (Michigan Department of Public Health.. Lansing. Ml. lot 94). was injected i.v., l o " ' organisms per rat.. concurrently with the Immunization.

DiseaseDisease monitoring and assessment. Immunized rats were ex-aminedd daily for clinical ocular changes (7): disease occurrence and severityy were verified by conventional histologic examination (7. 14). Ass mentioned in a previous publication (7). there are no specific featuress to characterize the different grades ol severity ol the IRBP-inducedd ocular inflammation and. therefore, the intensity of changes iss used to grade the disease, using a scale of 0 to 4. The disease inducedd by the short peptides used in this study was relatively mild andd did not exceed the severity grade of 2.0. Tested peptides were alwayss injected al several different doses and their uveitogenicity wass evaluated using lour parameters, i e . the initiiin.i l dose la induce EAU,, incidence ol disease, day of onset and severity. A good corre-lationn was usually observed between these parameters

LymphocyteLymphocyte proliferation assay. Lymph node cells were obtained 122 days after immunization and their proliferative responses were measuredd as described In detail elsewhere (15). with minor modifi-cationss In brief. :i x 10' lymphoid cells were cultured in triplicate Inn flat bottom microplates, with or without the tested Ag. in Kl'MI 16400 medium with HEPES. supplemented with streptomycin (100 ug/ml).. penicillin (100 U/ml), 1'V fresh rat serum, and 2-.VIE at 5 X 100 ' M The cultures wen- incubated fora lotal period ol W0 h. with aa pulse ol 'IMhymidinc (0.5 «Ci/10 «l/well| given for the last Hi h. Thee data are presented as .ST. values (ST. - mean cpm in cultures withh st imulus/mean cpm in control cultures without stimulus).

CellCell line responses A T lymphocyte cell line specific toward peptidee 1181-1191 was established from sensitized lymph node cells,, as described in detail elsewhere 11 6). The line cells were tested lorr proliferation alter incubation lor 4 days with 1L-2. The prolifer-ationn assay was carried out as detailed by I In et al. (16). Briefly. 2 x 10''' line cells were incubated along with öx 10s irradiated syngeneic thymuss cells (serving as APC). with or without stimulants, in a volumee of 0.2 ml of the supplemented Rl'MI 1640 medium. The culturess were incubated for a total of 72 h and pulsed with JH-thymidincc for the last lb' h.

CompetitionCompetition studies. The capacity ol analog pepl ides lo compete withh the parent peptide 1 I HI - I 191 and to inhibit the lymphocyte responsee against it (17. 18) was measured as follows: line cells specificc to 1 181-1 191 (2 x 104| and APC (irradiated thymocytes. 5 xx 10"'). in 100- 1 aliquols. were preincubated for 1 h with the tested analogs,, at differenl concentrations, in 50 „1 Later, peptide 1 181-11911 was added. In aliquots of 50 «1. and the cultures were incubated andd pulsed as described above. The data are expressed as percent inhibition,, calculated as (1 - (mean cpm in cultures with competitor/ meann cpm in cultures without competitor)) x 100.

TruncationTruncation ol carboxyl-terminus. We h a ve p rev ious ly u s edd t he t r u n c a t i on a p p r o a c h, at t he a m i no t e r m i n us of s e q u e n cee 1 1 69 1191 ( P T A R S V G A A D G S S W E G V G V-VPDV) . too local ize t he a c t i ve s i te of t h i s d e t e r m i n a nt l o i l s c a r b o x y l - t e r m i n all p o r t i o n: s e q u e n ce 1 1 8 2 - 1 1 91 w as f o u ndd to e x h i b it all i m m u n o l o g ic a c t i v i t i es of th is de te r-m i n a ntt (12). No s u ch a c t i v i t i es w e re d e t e c t e d, h o w e v e r, i nn s e q u e n ce 1 1 83 1 1 9 1. t h us e s t a b l i s h i ng Ibe c r i t i cal ro lee of t r y p t o p h an at pos i t i on I 182 (12). Da ta co l lec ted byy t r u n c a t i on al t he c a r b o x y l - t e r m i n us a re s u m m a r i z ed i nn T a b le 1 a nd in F i g u r es 1 a nd 2. R e s i d ue 1 191 (va l ine) w ass f o u nd to be n o n - e s s e n t i a l: p e p t i de 1 1 8 2 - 1 1 90 w as f o u ndd to be u v e i t o g e n ic (Tab le I), i m m u n o g e n ic [Fig. 1) a ndd i m m u n o d o m i n a n t, as d e t e r m i n ed by i t s be ing rec-ogn i zedd by l y m p h o c y t es s e n s i t i z ed a g a i n st who le 1RBP (Fig.. 2] (see R e f e r e n c es 19 a nd 20 for de ta i l ed de f in i t i on off " i m m u n o d o m i n a n c e " ). It i s of n o t e, h o w e v e r, t h at s im-i la r l yy to t he o b s e r v a t i on w i t h p e p t i d es t r u n c a t ed al t he a m i noo t e r m i n us (12). t he n o n a p e p t i de exh ib i ted lower i m m u n o l o g iee a c t i v i t i es t h an t he l onger pep t ides. In con-t r a stt to t he r e m o v al of r e s i d ue 1 1 9 1. delet ion of t he

TABLEE I ^pathogenicity^pathogenicity oj truncated pepltdt

Peptide e

11 181 1 191

1 1 8 2 - 1 1 91 1

Dose e (nm.,1 1

Ratj j

100 0 10 0

00 1 100 0

10 0

Inc idence e

4 /4 4 4 /4 4 3 /3 3 0 /3 3 4 /4 4 2 /3 3

Onse e dav v

(mean n

99 0 10.5 5 12.3 3

11 1.(1 177 5

11 182

.. 1 B2 1181 1

11 190

II 189 11 1 89

100 0 10 0

100 0 100 0

0/3 3 0/4 4 0/4 4

11 Sec Materials and Methods lor the a munopathogeniellyy of individual peptide '' NA. Not applicable: tin- in Ikon mal ion v byy histologii

- 4 - 3 - 2 - 11 0 1 2 3 A n t i g e nn ( log u M )

FigureFigure 1. Immunogeniclly ol peptide 1182-1190: proliferative re-sponsess of lymph node eells from a Lewis rat immunized against this peptidee The tested Ag include the immunizing peptide and related mole coless and the data are presented as S I. values The mean cpm SE In thee control cultures, with no stimulant, was 4488 256.

-- 8 - 6 - 4 - 2 0 2 4

Ant igenn (log uM)

Figure2.. Recognition of truncated forms of sequence I 169 1191 by uiplii node cells sensitized against whole IRBP. The proliferative re-alisess are presented as S.1 The mean cpm SE in the unstimulated introll cultures was 4962 270

a s p a r t iee ae id at pos i t i on 1 190 c a u s ed a c o m p l e te l oss of t hee a f o r e m e n t i o n ed ac t i v i t i es, as s h o wn by t he i nac t i v i ty off e i t h er t he o e t a p e p t i d e, 1 1 8 2 -1 189 (Tab le I), or t he n o n a p e p t i d e.. 1 1 8 1 - 1 1 89 (Tab le I, F igs. 1 a nd 2: the n e g a t i vee p ro l i f e ra t i ve r e s p o n s es of r a ts i m m u n i z ed w i t h t h e see t wo p e p t i d es a re no! s h o wn here ).

A / a n i nee s u b s t i t u t ed analogs: uveitogenicity. T o a s-s e sss t he role of e a ch of t he r e s i d u es of s e q u e n ce 1 1 8 2-

60 0

http://initiiin.il

PIVOTALL RESIDUES

PIVOTALL RESIDUES WITHIN [MMUNOt'A I"HCXiENIC PEPTIDE

11900 in the immunologic activities of this determinant, wee have analyzed the activities of' nine analogs of pep-tidess 1181-1191 in which each of residues 1 3 8 2- 11 90 wass substituted by alanine (Table 11). The uveitogenicity off the analog peptides, as compared to that of peptide 1JJ 81 -1191. are summarized in Table 111. In the experi-mentt reeorded here, peptide 1181-1191 produced dis-easee in all rats at the low dose of' 0.2 nmol/rat, ll is assumed,, however, that this dose was the endpoinl of the uveitogenicc activity of" 1 181-1191: data of other experi-mentss (Tahle I] (12] [S. Kotake.M. D. DeSmet.B. Wiggcrt, T.. M, Redmond, G. J. Chadcr, and I. Gery, unpublished data)) indicate that this peptide does not induce EAU at 0,022 nmol/rat. This assumption is also in line with the latee mean onset day of disease (13,7 days) in the rats injectedd with 1181-1191 at 0.2 nmol/rat. In line with thee findings with the truncated peptides recorded above, tryptophann at position 1 182 and aspartic acid at 1 190 weree found pivotal: substitution of residue 1182 com-pletelyy abolished the uveftogenioity of the determinant, whereass substitution at position 1190 drastically reduced thiss activity. Relatively low uveitogenic levels wrcre ob-servedd with analogs A(U83). A( 1 185), and A(1189), whereass the other four substituted peptides showed uvei-togenicc activities that were only slightly or moderately lowerr than that of the unmodified 1 181-1 191.

TAI1L EE II

AkminvAkminv -suhxittmed anulixfs nl' peptide I I SI I )Hl

11811 1191 AA 11182) AA |11H3] AA 11 1 B4) AA I l l S S j AA 11 1B6] AA l l 1N7| AA (1 1HHJ AA (1 189| AA (1 190)

lmnutnopathogeTticlmnutnopathogeTtic pr

rrptidcc cm ln|pi-trrii In m u

1JMT T

SI ' '

m m

SS W K (i V Q V V I' 1) V ss A i: <; v r, v v p n v S W A GG V G V V P D V ss w K A v a v v i' [i v S W F . CC A G V V 1' 1) V ss w K G v A v v r i ) v SS W K G V G A V P L) V ss w F:G V G v A p i ) v S W E I ;; v G v v A n v SS W K G V G V V P A V

TABi. t :: in

ness nl tlip nlfinme-subalUutril "<

KAir r

— — In rr > < . I - —

"" ll '"'" :: ' ' r i t y y

11 I HI l u l l

A ( ll IH2J AA (1 1H3)

A|11M41 1

A| !18.rH H

AA 11 180|

AA [1187)

AA 111 MS)

AA 11 189)

AA [1190)

'200 0 20 0

2 2 0.2 0.2

200 0 '200 0

20 0 200 0

20 0 I I 00 2

200 0 20 0

200 0 20 0

2 2 0.2 2

200 0 20 0

2 2 200 0

20 0 2 2 nn 2

200 0 20 20

2 00 0 20 0

4 /4 4 4 /4 4 ,'i/J J 3/;i i 0 /4 4 4 /4 4 0 /4 4 4/4 4 4/4 4 :v:i i 2 / : Ï Ï 4 /4 4 O,'4 4 4 /4 4 4 /4 4 2 /3 3 2 /3 3

4 4 :t/.i i 0 /3 3 4/4 4 4 /4 4 3 /3 3 11 /3

4 4 1/4 4 11 /4

()/' ! !

10.00 1. 10.55 1 11.00 1. 13.77 1.

11.55 1

9.55 1. 1 088 ] . 11 1 .0 1 . lo.oo l. 12.00 1.

10.55 ] . 1 155 1 12.00 1. 15.55 1 10.88 1 13.33 1.

100 3 1 12.00 1. 12.77 1. 1IÏ.00 1 11 1 H 1. 14.00 1. 15.00 1.

AlanineAlanine substituted analogs- immunogenic it g. The immunogenicityy of the nine analogs, i.e.. their capacity too initiate immune reactions, was assessed by measuring thee proliferative responses of lymph node cells from rats immunizedd with these synthetic peptides. The Ag tcsled inn these cultures were the corresponding immunizing antigen,, as well as peptide 1181-1191. Figure 3 sum-marizess a typical experiment: an identical pattern of specificityy was obtained in two other experiments. A close similarityy was observed between the levels of immuno-genjejtyy and uveitogenicity of the different peptides. In particular,, the two analogs that were poorly- or non-uveitogenic.. A( I 190) and A(l 182], also failed to initiate detectablee lymphocyte responses (namely, S.l, < 2.0). In addition,, very low responses were produced by analog A[ ll I 85), which was weakly uveitogenic. Moderate to high responsess were noted, however, in rats immunized with thee other six analog peptides. All rats with the moderate orr high responses reacted better to the immunizing ana-logg than lo peptide 1 181-1 191; no heteroclitic responses weree detectable in any of these animals. In fact, the responsess to 1181-1191 were just marginally positive in lymphocytess from rals immunized wiih four of the pep-tides,, A( 1 183), A( 1 187), A| 1 188], and A| 1 189).

Alanine-substitutedAlanine-substituted analogs: "immtmodominancc." Thee contribution of each of the residues of sequence 11 182-1 190 to the immunodominancc of this antigenic sitee was evaluated by testing the capacity of the nine analogss to be recognized by and lo stimulate proliferation inn lymph node cells sensitized against whole [RHP (Fig. 4).. In addition, the analogs were tested for their capability too stimulate a cell line specific to the parent peptide, 1181-11911 (Fig. 5). Although the responses of the line cellss were higher than those of the lymph node cells, the patternss of response of the two cell types toward each of thee analogs were similar. Three of the analogs, A(l 182). A(( 1 188). and A( 1189). did not stimulate any significant responsess by either one of the two cell types. It is note-worthy,, however. Ihat two of these non-stimulatory an-alogs,, A( 1 1 88] and A[ 11 89), were found to be both uvei-togenicc (Table III) and immunogenic (Fig. 3], The other sixx analogs were recognized by the lymphocytes sensi-tizedd against whole IRBP or 1181-1191 but I he prolifer-ativee responses they stimulated were usually lower than thosee stimulated by peptide 1 181 1 191. Of particular interestt are the responses stimulated by analog A( 1 190). Thiss peptide, which was found to be very weakly uveito-genicc (Table 111) and failed to initiate a detectable lympho-cytee response (Fig. 3). did stimulate moderate responses inn lymphocytes sensitized against whole IRBP [Fig. 4] and strongg reactions by cells sensitized to 1181-1191 (Fig. 5],, Peptide A( 1 1 90] was active in these cultures, however, onlyy at concentrat ions thai were ~ 1 000-fold higher than thosee of' the parent peptide 1 181-1 191. A pattern some-whatt similar to that of A(l 190| was exhibited by analogs A(1183)) and AJ1185). These two peptides were weakly uveitogenicc and A( I 185) was also poorly immunogenic. A(ll 183) was found to stimulate [RBI'sensitized cells only att a concentration -1000-fold higher than 1181-1191 (Fig.. 4), whereas A( 1 185] stimulated cells sensitized to 1181-11911 at concentrations --100-fold higher than thosee of the parent peptide [Fig. 5).

AnalogsAnalogs compete ivith peptide 1181-1191 and in-hibithibit lymphocyte responses against it. As shown above

61 1

ChapterChapter 2

PIVOTALL RESIDUES WITHIN' IMMUNOPATHOGENIC PEPTIDE

Finn Finn Cellss I

Antigenn (loguM .1.. I inmumigenici l y ol t he a lanlne-subst i tu ted analogs: prol i ferat ive response ol lymph node cel ls fron

nn c.uli ral were tested aga inst the immuniz in g peptide | 0| analog and the parent peptide. I1H1 atedd control c u l t u r es ol each lymph node cell sample were withi n the r a n g ed 271 I 465)1.

I'.. 11 :dagainstt Ih

FigureFigure 4 Kecognll ion ol the a lan ine-subshtu lcd ana-logss by lymph node cel ls sensi t ized against whole IKIiP . Thee response againsl the parent peptide. 1 I M I 191, b includedd lor comparison The mean cpm ! SK In unstitn ulatedd control cul tures was 2 5 27 97

(Fig.. 5| three of the analogs, A(1182), A(1188), and A(ll 189), did not st imulate any significant proliferative responsess by lymphocytes sensitized against peptide 1188 1 1191. These analogs could be tested, therefore, for theirr capacity to compete with 1 181-1 191 and to inhibit lymphocytee responses againsl it |17. 18). As depicted in Figuree 6. the response to 1 181-1 191 was inhibited by twoo ol these analogs. A(l 188) and A| 1 189). whereas an-alogg A(l 182) had virtually no effect. Similar levels ol inhibitionn were produced by A| 1 1 88) and A(1189) and theirr effeel was found to be dose dependent

DISCUSSION N

Dataa recorded here show thai the active site of bovine IRBPP determinant 1 169- 1 191 localizes to the nine resi-duess of sequence 1182-1190. This nonapeptide was foundd to exhibit all immunologic activities ol the parenl determinant,, i.e., immunodominance (Fig. 2). uveilogen-icityy (Table I), and immunogenieity (Fig. 1). As with find-

Antigenn (loguM)

ingss in other systems (21. 22). however, (he levels of activitiess of the nonapeptide were lower than those ol" longerr peptides (Table I; Figs. 1 and 2).

Thee involvement of individual residuesol the nonapep-tidee in each of the immunologic activities of the deter-minantt was determined by testing the activities of pep-tidee analogs in which individual residues were substi-tutedd with alanine. This small and non-charged amino acidd has been the molecule of choice for numerous stud-iess in which peplide residues are substituted, to investi-gatee the residues' functions (23. 24) or the activities of thee analogs (25. 26). It should be underscored, however. thaii the effects of substitution with alanine differ ac-cordingg to the biophysical features of the amino acids beingg substituted. Consequently, the nine substitutions variedd considerably in their level of conservativeness. Despitee this caveat, the data collected in this study, with thee nine analogs, provide information concerning the putativee roles of the individual residues in the various

62 2

PIVOTALL RESIDUES

PIVOTALL RESIDUES WITHIN IMMUNOPATHOGENIC PEPTIDE

Figuree 5. Recognition ol the alanine-subsliluted analogs by . specificc toward Ihe parcnl peptide 1181 1191. The responsi 11 181 1 191 is shown for comparison, The mean rpm SK In l.-iledd com rol cultures was 116 8.

0.3 ..M DD 3 iM

AA (1182) A (1188) A (1189)

Figuree 6. The inhibition of the lymphocyte response to peptide I I hi 11 191 by competition with alanlne-substltuteri analogs. The procedure is describedd in Materials and Methods Peptide 1181 I 191 was used in thesee cultures at the concentration of 0.01 UM. The mean < pin - SK in culturess stimulated with 1181 1191 (without competitors) was 10.508* 306:: the mean cpm SE in unstimulated control cultures was 93 - l I

immunologicc activities of this peptide determinant. Thus, threee of the nine residues (I 184. 1 186, and 1 187) were foundd to be "non-pivotal," as indicated by the immuno-logicc activities of the corresponding analogs, which were similarr or just moderately lower than those of the original peptide.. However, four of the residues were identified to bee "pivotal." with their analogs exhibiting very low or no activityy in at least one of the immunologic assays. The tryptophann at position 1182 was found to be the most prominentt residue and essential for all tested activities off the nonapeptide. The other three residues were found too be pivotal for only one or (wo of the activities. Aspartie acidd at 1 190 was found essential for the uveitogenieity andd immunogenicity. whereas valine al position 1 188 andd proline at 1 189 are apparently pivotal lor the itit-munodominancee of ihis antigenic site: the substituted analogss of these two amino acids were not recognized by lymphocytess sensitized to whole IRBP. The remaining twoo analogs. A( I 183] and A(l 185). resembled to some extentt analog A(1190) in their pattern of deficient im-munologicc activities. Both analogs were weakly uveito-

genicc (Table III) and A( I 185) was also found to be poorly immunogenicc [Fig. 3). In addition, relatively high concen-trationss of these two analogs were needed to stimulate lymphocytess sensitized against IRBI' (Fig. 4) or 1181-11911 (Fig. 5).

Thee substitution with alanine for three of the "pivotal" residues,, valine at positions 1 1 85 and 1 1 88. and proline att 1 189. could be considered conservative. However, sub-stitutionn lor ihe other three residues, tryptophan at I 182, glutamicc acid at 1183. and aspartie acid at 1 190, was non-conservativee and could contribute, therefore, lo the poorr immunologic activities of Ihe corresponding analogs. Thee importance of residues 1182 and 1190 was indi-cated,, however, by our other findings that truncated peptides,, without these amino acids, had no detectable immunologicc activities (12) ('Table I: Figs. 1 and 2).

Thee data collected with analogs A( I 188) and A(1189) alsoo present a finding that deserves to be commented upon,, namely, the discrepancy between the capacity of thesee analogs to induce EAU (Table III) and their poor cross-reactivityy with the native peptide 1181-1 191 (Figs. 33 and 5) or the whole protein (Fig. 4). when tested by the proliferationn assay. This finding closely resembles pre-viouslyy published observations, that the proliferation re-sponsess of lymphocytes toward analogous peptides do nott necessarily correlate with the peptides' capability lo stimulatee uveitogenieity (27. 28) or encephalitogenieity (29.. 30), The reason lor this seeming discrepancy re-mainss obscure, but could be attr ibuted to different levels off sensitivity and/or to different biochemical processes beingg involved in ihe two types ol lymphocyte activation (28.. 30).

"Thee analysis of the activities ol' the pivotal residues of sequencee 1182-1190 makes it possible to learn about theirr putative roles in the interaction of this peptide with Hiee TCR and the MHC molecules on the APC. 'Thus, the dataa are interpreted as demonstrat ing that the valine at positionn 1 188 and the proline at 1 189 are pivotal com-ponentss of the "epitope" that interacts with the TCR. 'This iss indicated mainly by the capacity of analogs A(1188) andd A(l 189) to compete with the native peptide 1 181 II 191 and to inhibit the lymphocyte response against it (Fiü.. 6). This capacity indicates that substitution of resi-duess 1 188 and 1 189 did not affect Ihe binding of the moleculee to the MHC. but prevented its binding to the TCRR specific toward the native molecule (31). The partic-ipationn of residues 1 188 and I 189 in the epitopic activity iss further indicated by the findings that analogs A(l 188) andd A(l 189) l )a re not recognized by lymphocytes sensi-tizedd against Ihe native peptide 1181-1 191 (Fig. 5). but 2)) are immunogenic and lymphocytes sensitized against themm are poorly stimulated by I 181- 1 191 (Fig. 3).

'Twoo of the oilier pivotal residues, tryptophan al I 182 andd aspart ie acid at I 190. are assumed to be essential lorr the pepl ide binding to I he MHC. The activity of residue 11900 is indicated by the finding thai analog A( I 190) stimulatess lymphocytes sensitized against IRBP or pep-tidee 1181 1191 only at concentrat ions -1000-fold higherr than those of the parent peptide (Figs. 4 and 5): thee need lor high concentrat ions of A|1190) in these assayss is interpreted to show (hat this analog binds to thee MHC molecule with a much lower affinity than the patentt peptide 1181 1191 (17. 18. 32). It is proposed. therefore,, that the substitution at position 1 190 affected

63 3

ChapterChapter 2

PIVOTALL HKSIDUES WITHIN IMMÜNOPATHCX1KNK' PEPTIDE

thee b i n d i n g of the pepl ide to the MHC molecule. However,

ana logg A(1182) d id not s t imu la te any detectable re-

sponsess in l ymphocy tes s t imu la ted against 1RRP fir pep-

t idee 1 1 8 1 - 1 1 9 1 a n d t hus , i ts poor h ind ing to MHC could

bee o n l y deduced f r o m the f i n d i n g tha t it d id not compete

w i t hh 1 181 - I 191 (Fig. 6 j . despite the radical subs t i tu t ion

off t r y p t o p h a n to a lan ine . It shou ld be ment ioned here,

however ,, t h a t we cannot ru le out the possibi l i ty tha t

res iduee 1 1H2 is also engaged in the in te rac t ion of" the

pept idee w i t h the TCR molecule We f u r t h e r propose tha t

t h ee poor b i n d i n g to MHC of analogs A ( l 182) a n d A( I 190]

cou ldd be responsib le fo r the i r def ic ient immunogen ic i t y

[Fig,, 3) a n d uve i togenic i ty [Table HI).

T h ee o the r two residues tha t are assumed to cont r ibute

l oo the immuno log i c ac t iv i t ies of pept ide 1181-1191 arc

thee g l u t a m i c acid at 1183 and va l ine at 1 185. As men -

t i onedd above, analogs A[1183) and A f l l S f ) ) resembled

ana logg A ( l Ï 90 ) in the i r ac t i v i t y a n d t hus , it is conceivable

t h a tt these t w o residues also par t i c ipa te to some extent in

thee b i n d i n g of the pept ide to the MHC molecule.

T h ee immuno log i c ac t iv i t ies of t he n ine analogs are

s u m m a r i z e dd in Tab le IV, a long w i t h the deduced involve-

m e n tt of each residue in the pept ide in te rac t ions wi th Ihe

MHCC or (he TCR.

O u rr f i nd i ngs w i t h de te rm inan t 1 1 8 2 - 1 1 9 0 resemble

thosee of M i l i ch et a l . {24] w i t h the immunodom inan t

d e t e r m i n a n tt [120 K i l ) of hepat i t i s l i core Ag in H-S'1

s t r a i n ss of mice. As w i t h the 1 182 1 190 sequence, two

a m i n oo acids were f ound to be p ivo ta l for the in teract ion

off t he hepat i t i s pept ide w i t h the TCR whereas another

twoo res idues were essent ia l for i ts b i n d i n g to the MHC

molecu le . .

A l a n i n ee subs t i t u t i on oi" i nd i v i dua l residues has been

recen t l yy used !o i den t i f y the epi topie and agretopic resi-

duess of de te rm inan t 1 - 9 of mye l i n basic prote in, w h i c h

i sencepha l i t ogen icc in P L / J mice (25, 26). Un l i ke w i th the

IRHPP 1181 1 191 analogs, cer ta in analogs of the mye l i n

bas icc p ro te in pept ide were found to be compet i t ive w i t h -

outt be ing immunnpa thogen i c . These analogs, wh ich b ind

s t rong lyy to the MHC molecule (25), were f ound to i nh ib i t

t hee i n d u c t i o n of disease by the nat ive pept ide (25, 26).

I n a s m u c hh as the analogs of 1 1 8 1 - 1 1 9 1 , A(1188) and

A(( 1 189], were f ound to be uvei togenic. we could not ex-

a m i n e !! he i r effect on E A U i n d u c t i o n . More studies, us ing

s u b s t i t u t i o n ss w i t h a m i n o acids o ther t h a n a lanine, as

we l ll as mu l t i p le subs t i t u t i ons (26. '.i'A). are needed to

TAHI. KK IV urit'urit' turmloq^ ir-Jll n-sldufi TCRTCR or MHC

J 1H) I If-l!

ii pi-liricir inirr

11 1K2 |W) - -11833 IE) - ++ + 11 ]H4 |G) - + + -t * -1 I M 5 | V || * - +

11 IKfi 1(1) * + + + II 1H7 IV) * + + - + II 1HB |V) ++ * * H H S l P )) + +

11 191) [!)) - + nn As assessed I>Y Ihe eapaeilv to st imnlale l vmpb

aâmstt whole IKIip'i.r peptide 11 M I 191.

f u r t h e rr invest igate the in terac t ions between 1 182 -1 190

a n dd the TCR and MHC molecules. It is conceivable that

th iss approach may also yield analogs w i t h the capaci ty

If)) compete w i t h and inh ib i t the responses to 1 182 -1 190

bothh in v i t ro a n d in vivo.

Acknowledgments.Acknowledgments. We are grateful to Barbara Vistica forr invaluable technical assistance and to Noreen Bea-verss for typing the manuscript.

KI- :FERI: \CI :S S

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64 4

PIVOTALL RESIDUES

PIVOTALL RESIDUES WITHIN 1MMUNOPATHOGEN1C PEPTIDE

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65 5

Chapterr 3

Indentificationn of Heat Sock Proteins Binding to ann Immunodominant Uveitopathogenic

Peptidee of IRBP

Kalpanaa Rengarajan, Marc D. de Smet, Gerald J. Chader, Barbara Wiggert

Currentt Eye Research 13: 289-296, 1994 (byy permission © Swets & Zeitlinger Publishers)

67 7

HSPP BINDING TO IRBP

Identificationn of heat shock proteins binding to an immunodominantt uveitopathogenic peptide of IRBP

Kalpanaa Rengarajan, Marc D. de Smei', Gerald J. Chadcr and Barbara Wiggert

Laboratoryy of Retinal Cell and Molecular Biology and 'Laboratory of Immunology, National live Institute, National Institutes off Health, Bethesda, MD 20892. USA

Abstract t

Intracellularr binding proteins have been identified and isolated fromm B cells by their ability to bind to the synthetic peptide (ILL 69 - - 1191). the major immunodominant epitope of bovine inn terp hot o receptor retinoid-binding protein (IRBP) coupled to cyanogenn bromide activated Sepharose 4B, After SDS - PAGE, twoo discrete protein bands of =72 and 74 kDa, were found to bee present in B cells of naive Lewis rats as well as in LBV transformedd B cells from a human patient with ocular Behcet's disease.. Enhanced expression of these peptide-binding proieins wass achieved by incubating the cells with Lipopoly saccharide (LPS)) from 5, thyphimurium. The = 72 and 74 kDa peptide-bindingg proteins reacted in western blot with monoclonal antibodiess specific for both constitutive!)- expressed and inducible 72/744 kDa hsp 70 proteins. The demonstration that these proteins bindd to the immunodominant epitope of IRBP indicates that they mayy play a role in the processing and presentation of antigens byy antigen-presenting cell (APC). Curr. Lye Res, 13: 289 -2%. 1994, ,

Keyy words: chaperone; uveitis; B cell; peptides: interphoto-receptorr retinoid-binding protein (IRBP); Behcet's disease: human:: heat shock proteins

Introduction n

Experimentall autoimmune uveitis (EAL) is a T-cell-mediated autoimmunee disease that serves as a model for several human sight-threateningg ocular inflammatory conditions that mainly affectt the posterior segment of the eye and are immune mediated orr autoimmune in nature (I -8 ). EAU is induced in a variety off animal species by immunization with ocuiar-spccific antigens suchh as S-Antigen (S-Ag) and intcrphotoreceptor retinoid-binding proteinn (IRBP) (1.9). Helper'inducer T cells have been shown too play a predominant role in this experimental disease

Correspondence:Correspondence: Kalpana Rcngaraian. I .ahoratory nl' Kciina! C.di ami Molecularr Biology. Building 6, Rtxim 11X. National Kye Instiiuu-, V annua! Instuuicss ol Htah'h. Hcthcula. Ml) :fW92. USA

(4,10-- 13). T-ccll activation is a complex process that requires thee autoantigen to be processed by circulating or resident antigen-presentingg cells (14.15). This partia! digestion of the antigen generatess fragments that are able to interact with appropriate class Ill molecules on the cell surface (15). This antigen fragmem-MHC complexx then causes selected clones of T cells to become activated throughh their T cell receptor. Even though antigen processing iss recognized as a necessary step for T cell activation, littl e is knownn about the intracellular factors that might favor the productionn of selected immunogenic fragments and allow their translocationn through the cytoplasm to the cell surface. This does nott appear to be a random process as demonstrated by binding experimentss with MHC Class II molecules and T-cell antigenic determinantss in vara (16.17). The affinity of peptides for purified MHCC Class II molecules in detergent solution is of the order of 100 "(' M (16). However, this average equilibrium constant has veryy slow kinetics, i.e.. over a time scale of days at neutral pH. Inn contrast, antigen-presenting cells (APCs) in vivo are able to assemblee completes in 0,5 to 1 hour (17) Moreover, the concentrationn of peptide required for T-cell activation is much towerr in viva by at least 100 (o 1000 fold. These observations andd others suggest the existence of intermediate molecules that aree able to selectively bind processed antigens and concentrate themm (16 . Such a role has been suggested for some members off the chaperone family of' proteins (19).

Molecularr chaperones arc a ubiquitous family of intracellular proteinss which mediate the correct folding of polypeptides and preventt their degradation by cytosolic enzymes (20). Chaperones functionn by binding specifically and non-covalently to interactive proteinn surfaces that arc exposed transiently during cellular processess such as protein synthesis, protein transport across membraness and stress responses. Many chaperones are part of thee heat shock family of proteins, and are among the most highly conservedd and abundant proteins in nature. There is evidence too suggest that these proteins play a role in antigen presentation. Piercee and colleagues, for example, were able to isolate cell-surfacee proteins of approximately 70 kDa from B-lymphoeytes whichh bind the major antigenic determinant of cytochrome c in

Reeoivi-dd im January II). I W . accepted nn K-hniary 4, IW4

Oxfordd University Press

69 9

ChapterChapter 3

thee BIO.A mouse (21). Antibodies raised against this protein

preventedd activation of a cytochrome (.'-specific T cell line In

addition,, these molecules can demonstrate a certain degree of

specificityy in their interactions with oligomeric peptides. BiP.

aa member of the lisp 70 family of proteins, preferentially hinds

7-merr oligopeptides and has both hydrophilic and hydrophobic

bindingg /ones as determined by peptide substitution analysis (22).

AA loose homology has also been noted between the C-temnnal

regionn of a hsp 70 and the probable peptide-hind ing site on the

Xenopuss class I major histocompatibility complex (23).

Inn the present report, we examine the possibility that a similar

proteinn exists within AFCs that is capable of binding to the major

immunodominanii sequence of IRBP. a known ocular autoanligen.

Materialss and methods

Preparatio nn of spleni c ra t lymphocyt e population s

Splenicc lymphocytes were isolated from b to K week old Lewis

ratss (Charles River Laboratories, Raleigh. N O . Contaminating

redd blood cells ( R B O were removed by a 5 minute incuhation

inn ACK I w n g butter. B cells from lymphocytes were purified

byy panning on polystyrene tissue culture plates (Falcon ?3(H)3,

Oxnard.. CA) coated with affinity purified goat anii-rat-lgG (kpl .

Gaithersburg,, M D i . RBC-free splenic cells were incubated at

37 r CC for one hr at a density of 4 x 10" cells per ISO mm plate.

Non-adherentt cells were removed by repeated gentle washing

off the plate with phosphate buffered saline (PBS). The adherent

cellss were collected hy vigorous washing ot' the plate surface with

PBS.. The purified B cells were cxtensivelv washed in Dulbccco's

modifiedd Kagle's medium without cysteine or methionine

( D M H M - C M )) (ICN biochemicals Inc.. Costa Mesa. CAi, The

washedd cells were incubated for three hours at 37"C in 5/Ï C O ;

inn D M H M - C M containing 5ci fetal calf serum (Hyclone, Logan.

Ut)) and 750 /(Ci i "S ) cysteine and (^S) methionine (1143 C i m

mole:: IC i = 37mBq, ICN biochemicals Inc . i . The cells were

thenn collected by low speed ccntrifugation and lysed. These

investigationss were carried out in compliance with Public Health

Servicee Policv on Humane Care and Use of Laboratory Animals

II l.)HLW Publication N IH 80-23) in an accredited animal facility.

Inn another series of experiments, the purified B cells were

culturedd for 72 hr in the presence of 10 ; ig 'ml of lipopoly-

saccharidee (LPS) (D i t to . Detroit. Mi ) in RPM1-1641) medium

withh HEPbS (GIBCO. Grand Island. NY) , supplemented with

glulaminee (2mM), penicill in ( UK) units ni l) , streptomvein I 100

^ g m l ) a n dd 10'v fetal calf serum (Hyclone. Logan. Ut) k 'RPMIi .

Followingg this incubation, the cells were harvested and lysed.

Preparatio nn of LBV- B Ivmphoblastoi d cel l line s

Inn some experiments, a pure population of Fpstein-Barr virus

(F.BV)-transformedd human B cells was used This LBV cell line

wass derived from human mononuclear leukocytes ( P B M O

obtainedd from a patient with Behcet's disease who consented to

participatee in a protocol approved by the Institutional Review

Boardd of the National Hye Institute, N I H . Briefly. PBMC were

separatedd from hepannized whole blood on Isokmph gradients

(Gallard-Schiesinger,, Carle Place. NY) and transformed with

FBVV obtained from the B9S-8 marmoset hmphoblastoid ceil line

l A T C C .. Rockville. MD) in the presence ot CD3 amibodv.

Transformedd B cell lines were clearly evident bv the second week

inn culture, as evidenced b> the presence of tight clumps of various

si/css on phase-contrast microscopy The LBV cell line used in

thiss study was maintained in continuous culture for over 4 months

beforee being tested for the presence of the intracellular binding

protein. .

Antigen ss and affinit y column s

Thee IRBP-derived peptide 1169-1141 (amino acids I 169-1191

inn the bovine IRBP sequence) (2.S) used here was synthesized

andd purified bv Applied Biosystems (Foster City. C A l using t-

BOCC chemistry on an Applied Biosweius model 4.30A peptide

svnthesi/err Peptide 1169 - I 191 was coupled to cyanogen

bromide-activatedd Sepharosc 4B I Pharmacia 1(1.5 mg of peptide

1169-11911 per ml of gel) according to the manufacturers

recommendedd procedure.

AA similar peptide affinity column with amino acids 1135 - 1157

inn the bovine IRBP sequence (25) was constructed. A cvanogen

bromidee activated Sepharose 4B column without ativ peptide

coupledd to it ibut put through the same procedure for coupling.

minuss the peptide) was used as a control column.

Isolatio nn of IRB P peptid e 1169- 1191 bindin g protei n

Thee peptide binding protein was isolated from purified B cells

orr KBV transformed cells after 72 hr of incubation with LPS

1100 / j g n i l i . fol lowing the procedures published by l.akev a ul,

(21)) Si\tv mil l ion cells were incubated with 750 <ci (Ï:*S)-

methioninee and CSl-cvsteine (1143 C i n i mole. IC i = 37mBq.

ICNN Biochemicals Inc.) in D M H M - C M containing 5 ^ fetal calf

scrumm for 3 hr at 37 C. The cells were washed three times in

DMF.M-CM.. followed by incubation in Lysis Buffer ( I *'/<

Nonidett P 40 (proiein grade. Calbiochem-Behring) LSOmM

NuCl/SmMM H D T A 0 . 0 2 f ; NaNVlm.M phenylsulfomllluoride

(PMSF)) in S()mM of T n s - H C l . pH 7.2) tor 1 hr on ice. The

Iyy sate was cenlnfuged at 100.000 j ; for 10 min ai 4 ' C (21). and

thenn passed over the peptide I 169 - 1191 affinitv column that

hadd been equilibrated in Wash Buffer (0.1 % Nonidet P-40/50mM

NiiCl 'SniMM EDTA.O.2';; N a V I m M PMSF in SOmM T n s -

HCII pH 7.2). After extensive washing, bound material was eluted

withh r.Iution Buffer (0. \ri Nonidet P-40;SOOniM NaCISmM

KDTAO.2 ' ' ;; NaNr ImM PMSF in 0.2M acetic acid). Alternat-

ive!;,,, (he bound material was eluted with a solution of 360 ^ M

peptidee in Wash Buffer. The eluate was collected in 5 ml

tractions.. The radioactivity in each fraction was determined bv

dilutingg 1(X) n\ o f each sample in 7 ml of Read) So lv I M HP

countingg cocktail (Beekman. Palo Alto. CA) and measuring

scintillationn in a Beekman I.S 3801 scintillation counter. Fractions

containingg maximal radioactivity were pooled, dialv/ed and

concentratedd by vacuum dialysis using a MicroProDicon

apparatuss (Biomolecular Dynamics, Beaverton. OR). Since in

earlierr studies protein hands could not he detected on a gel. the

eluatee was further concentrated by TCA precipitation before

electrophoresis.. In all subsequent experiments, the eluate fractions

afterr vacuum dialvsis were further concentrated by TCA

70 0

H S PP BINDING TO I R B P

A A B B

kDa a

2000 -

kDa a

2000 -

977 -

688 -

433 -

255 -

188 -144 -

FigureFigure I. Identification of specific peptide 1169 I I'll binding proteins (BPs) in rat H cells incubated with (35S]eysteine and ["SJmcthionine. VV Lanes I -3 are autoradiography Following SDS PAGE of fractions clutcd from peptide 1169 111) I affinity column: < 11 flow-through ( =2(1

/igll i2) cluate ( = 0.5 ng) (3) eluatc (TCA precipitated) ( = I /<g). B: Lanes I - 3 are autoradiography following SOS PAGE of: <11 original lysate (( =30 ng) (2) flow-through cluted with Wash Buffer i =20 ng) (3) Id's eluted with 360 /<M peptide 1169 -1191 in Wash Buffer ( = I fig). <': Laness I 3 are autoradiography following SDS PAGE of fractions eluted from peptide 1135 1157 affinity column: (I) original lysate ( = 30 Hg)Hg) (2) flow-through ( =20 /<g) (3) eluate ( = I ,(g).

precipitation.. In other experiments the cell lysate was passed over peptidee 1135 1157 affinity column or a cyanogen bromide-activatedd Sepharose 4B column without the peptide. The eluatcs fromm both columns were collected as explained earlier, dialyzed andd concentrated. The concentrated (TCA precipitated) sample wass analyzed by SDS -PAGE using a Novex electrophoresis systemm (Novex, Encinitas. CA). The protein concentration of the sampless to be analyzed were determined prior to loading on the gel.. To aliquots of original lysate. flow-through and eluate, an equall volume of Tris Glycine SDS sample buffer containing 59?? mercaptoethanol was added. Samples were incubated at l()0°CC for 5 min. centrifuged. and then applied to X 16'.' Triss —Glycine gels (Novex). The gels were used for autoradio-graphyy or immunoblotiing. The autoradiograms were scanned usingg an L'ltroscan XI. Laser Densitometer and the Gelscan XL softwaree package (Pharmacia LKB Biotechnology AIL Uppsala. Sweden). .

Imimmoblol l

Followingg SDS- PAGE, blotting onto nitrocellulose membrane wass carried out using a Novex Transfer apparatus for 3 hi'. Membraness were blocked with 555 (w/v) Carnation non fat dry milkk in TBS for I hr followed by overnight incubation at 4°C withh specific monoclonal antibodies. Monoclonal antibody (moAb)) a 70 was diluted 1:500 (Affinit y Bioreagents. Neshanic Station,, NJ); moAb a 72 and moAb << 72/73 were diluted 1:1000 respectivelyy (Stressgen, Victoria. Canada); moAb <« 68. a rat IgMM antibody specific for HSP 68 was diluted 1:4 (a kind gift fromm Dr. E. Gunther, Gottingen. Germany). Membranes were furtherr incubated with affinity purified alkaline phosphatase-conjugatcdd goat anti rat IgG or goat anti-mouse IgG respectively

dilutedd 1:1000 in PBS for I hr at 37 C. Alter each incubation. thee membranes were washed with TBS three times for 10 min each.. Color development was carried out using Nitroblue lelra/oliumm Chloride (NBT) and 5-Bromo-4-Chloro-3-indolyl-phosphalee p-Toluidine salt (BCIP).

Results s

Purificationn and specificity of binding proteins (BPs) obtained fromm rat B cells

Inn an initial series of experiments, proteins that bound to peptide 1169-- 1191 were released from purified B cells obtained from ratt spleen labelled with ('5S)-methionine and (,5S)-cysteine by Inn potonicallyy Iv sing the cells using a Lysis Buffer containing I '< nonidett P-40. After lysis, the cell membranes were removed by centrifugationn and the soluble supernatant was applied to the peptidee 1169 1191 affinity column. After extensive washing, thee bound material was eluted and subjected to SDS - PAGE and autoradiography.. The predominant bands visible after elution weree at approximately 70 kDa and 40 kDa (Figure I A. lane 3). Thesee initial experiments indicated that the proteins were producedd in very low amounts which was confirmed by scanning thee autoradiograms using the Densitometer. If the binding proleins weree part of the chaperone family, mitogenic stimulation might enhancee their synthesis (25.26). For this reason, we decided to examinee the effect of LPS. a potent activator of B cells (27). LPSS preferentially induced two major newly synthesized protein bands,, with molecular weights of approximately 70 kDa and 40 kDa.. It is very clear that LPS stimulation resulted in a 45 fold increasee in the expression of these BPs in human LBV trans-formedd B cells when compared to BPs obtained from non-

71 1

ChapterChapter 3

kDa a

2000 -

433 -

255 -

188 -144 -

FigureFigure 2. Autoradiography following SDS PAGE from human EBV transformedd B cells stimulated with LPS following incubation with I^Slcysteincc and [J-\S]methionine; (1) ciuale (from ceils stimulated with LPS—300 ul) (2) eluate (from cells not stimulated with LPS-30 id).

1 22 3 4 5 6 7

11 2 3 S

FigureFigure 3. SDS PAGE and silver Main of human EBV transformed BB cell lysate on a bovine IKBP peptide 1169 I I'll column: (l)eluate ii = 140 ng> i2) flow-through I - 4 pg) (3) original lysate i ~l< /xg) (Si molecularr weight standards < =2 fig).

stimulatedd cells (Figure 2. lanes I and 2). Similar enhancement off BPs was also observed in rat B cells. Ten itg/ml of LPS was foundd to be the optimal dose for stimulation by performing dose kineticss studies (results not shown) Protein synthesis appears too progressively increase fol lowing stimulation since there was lessless protein obtained from cells incubated with LPS tor 18 hr ass compared to 72 hr (results nol shown).

Next,, we tried to determine i f the affinity of the 71) kDa protein handd was specific for peptide 1169—1191. I f the binding is specific,, the protein should be eluted by peptide I 169 1 I ' l l in Washh Buffer. The addition of 360 ;<M peptide 1 169- 111) I to thee Wash Butter led to the recovery of the 70 kDa band whereas thee 40 kDa hand was not observed in this eluate (Figure IB. lane 3).. Another band with an approximate molecular weight o( 120 kDaa which was seen as a minor band with acetic acid elution wass much more prominent when elution was performed with peptidee 1 169 - 1191 in Wash Buffer, l i is possible that the peptide promotess aggregation of either the 70 kDa or the 40 kDa proteins

FigureFigure 4. Autoradiography following SDS —PAGE of proteins from humann EBV transformed B cell lysate incubated with f \S (cysteine and [,5S]methionine.. Lanes 1-4 are SDS PAGE patterns of fractions eluledd from cyanogen bromide activated-Sepharose 4B column withoul anyy peptide coupled to it: ( I ) original lysate ( = 30 (<g) (2) & (3) How throughh fractions with highest absorbance. OD at 280nm ( =20 i<g each) i4)) eluate ( = I /<g) (5) no sample. The flow-through fractions of the abovee column were passed through peptide 1169- 1191 affinity column. Laness 6 8 are SDS PAGE patterns of the fractions eluted from peptide 1169-- 1191 column: (6) & (7) flow-through fractions ( =2(1 II% each) (8)) eluate ( = I jtg).

orr both. It is also possible that the 120 kDa protein is a different proteinn which is more efficiently eluted in the presence of added peptide. .

Thee specificity of the binding proteins with respect to peptide 11699 -1191 was further confirmed using another synthetic peptidee of the bovine IRBP sequence. I 135 - 1157. This peptide whichh is also 23 amino acids in length is a proven nonuveilogenic peptidee in Lewis rats (29). Interestingly the 70 kDa and 40 kDa bindingg proteins were not observed in the eluate obtained from thee peptide 1135 1157 affinity column (Figure IC . lane 3). The 700 kDa and 40 kDa binding proteins are clearly seen in the flow throughh obtained from this column (Figure IC . lane 2). In the flow-throughh other bands are not very prominent. This may be becausee peptide 1135-1157 hinds other proteins very tightly whichh are not eluted by the concentration of acetic acid used in thiss experiment.

Identif icationn of BPs in human B cells

Wee decided to also test a transformed human B cell line to see i ff the binding proteins could be identified in these cells. EBV transformedd B cells are a population of pure B cells that can easily bee grown in large numbers. The cell lysate was passed through thee peptide 1169- 1191 affinity column as explained earlier. The bindingg proteins were present in the EBV transformed B cells (Figuree 3. lane 11. Their isolation was more efficient since the BB cell population was pure. In these experiments, (he intensities

72 2


A A B B

kDa a

2000 -

SS 1 k D a a

!§:: 3

977 -6 8 - | |

4 33 - S

2 55 - " "

188 - -

144 -

FigureFigure 5. SDS - PAGE and immunoblotting follow ing affinity chromatography of human f:.BV transformed B cell lysalc on a bovine IKIi l ' peptide II \M 1191 column: A: (S) molecular weight standards ( = 10 jig) ( I ) Gold-staining profile of BIN ( = 1(1 /<g) from fcBV cells. B: western blot analysiss ofeluicd proteins probed with different monoclonal antibodies: ( i t 70 kDa lisp ( = 10/ig) (Stressgen) probed with moAb or72/73 (constitutive andd inducible form N27) (2) BPs ( = 1? /<g) probed with moAb ((72/73 (constitutive and inducible form N27) (3) BPs ( = 15 ;<g) probed with moAbb a70 (constitutive and inducible form 7.10) (4) BPs ( = 15 ,tgi probed with moAb «72 (inducible form NI5) (5| BPs ( = 15 jig) probed withh moAb a:68 (antibody specific for hsp 68).

off individual bands were measured by scanning the autoradio-gramss as mentioned above.

Nonn specific binding to Sepharose 4B was ruled out by the followingg experiment. Cell lysatc was passed through a cyanogen bromidee activated-Sepharose 4B column that was put through the samee binding procedure, but no peptide was added. The 70 kDa andd 40 kDa BPs were not observed in the eluate obtained from thiss column (Figure 4. lane 4). The flow-through fractions with thee highest absorbance values and radioactivity obtained from thiss column were passed through a peptide 1169 1191 column. Thee eluate obtained from this column clearly showed the 70 kDa andd 40 kDa proteins (Figure 4. lane 8).

Characterizat ionn o f 70 kDa protein band

Too test the putative relationship between the 70 kDa protein band andd the hsp 70 family of proteins, western blotting was performed withh antibodies specific to this family, i.e., moAb N27. moAb N155 and moAb 7.10 (Figure 5B, lanes 2. 3 and 4). The predominantt proteins observed on SDS- PAGE and gold-staining followingg elution from the column were at approximately 74 kDa. 722 kDa and 40 kDa (Figure 5A. lane 1). MoAb N27 and moAb 7.10.. which are specific for both the constitutively expressed and thee inducible 72/74 hsp 70 proteins, bound strongly to 74 kDa andd 72 kDa proteins transferred onto nitrocellulose I Figure 5B. laness 2 and 3). In autoradiography of radiolabeled proteins (figuress 1. 2 and 4) the 72 and 74 kDa proteins appear as a single bandd at = 7 0 kDa because this technique cannot resolve protein handss of such similar molecular size. With moAb N27 the doublet ( = 7 22 and 74 kDa) is clearly observed. Monoclonal antibody N15,, specific for the inducible form of hsp 70 and moAb 8F7. aa rat Ig.M antibody specific for inducible 68 kDa lisp protein showedd no binding (Figure 5B. lanes 4 and ?). The commercial 700 kDa heat shock protein from Stressgen probed with moAb

N277 displayed multiple bands (Figure 5B, lane I ) , probably due

too some degradation of the protein.

Discussion n

Antigenn processing and presentation requires a series of poorly understoodd events involving the internalization o f antigen into intracellularr acidic vesicles where proteolytic cleavage occurs, andd the transport of the resulting peptide fragments to the APC surfacee where they are recognized by the specific T cells along withh class II MHC. The exact mechanisms involved in preventing completee protein degradation and transport of peptide fragments too the cell surface are also not known. Evidence has been mountingg to suggest that intracellular proteins exist that can bind too processed antigen and facilitate its association with MHC class III molecules (21.30,31). To identify potential candidate proteins, ann approach was devised to isolate intracellular peptide-binding proteinss capable of recognizing and binding to a specific antigen (21).. Candidate proteins were first affinity purified f rom detergent-solubilizedd mononuclear cells onto columns that contain thee target antigen: then the bound proteins were isolated under strictt chromatographic conditions including extensive washing inn high salt and detergent buffers. Since Lakey a al. (21) demonstratedd the presence of a mouse intracellular binding protein thatt bound to a fragment of pigeon cytochrome c, we decided too investigate the possibility that similar intracellular binding proteinss might also exist for a uveitopathogenic peptide. Peptide 1169-- 1191 of bovine IRBP was chosen because it contains the immunodominantt and major immunopathological site of IRBP inn the Lewis rat and because it induces a T cell proliferative responsee in patients with uveitis (24.32). These features would alloww us to look for the presence of this intracellular binding proteinn in rat as well as human APCs. Using this approach, we weree able to isolate two bands of == 72 and 74 kDa and another

73 3

ChapterChapter 3

aroundd 40 kDa. The binding proteins proved to he present in bothh rai and human APCs. However. onlv the 72 and 74 kDa proteinss were eiuted from the column using peptide I 169 UlJ! dissolvedd in Wash Butter and did not require the addition of acetic acidd to the Llution Butter. A doublet with similar properties v as foundd for cytochrome c in mouse APC (21). Another prutein nl'' approximate!) 4? kDa was also observed, which was onl> partiallyy eluted using a peptide ot pigeon cytochrome c. Complete elutionn of this protein was possible only using aeid-denattirmg conditionss (21). The 40 KDa protein observed by us binds more tightlyy to the peptide 1169 - 1191 affinity column (since it could bee eluted only with Llution Buffer containing acetic acidl hut itt neither binds to the column without peptide I 169- 119! nor too the peptide 1 135- 1 157 affinity column. In previous work (30.33).. the 70 KDa cytochrome c- binding protein was shown too be pan of the heat shock family ol proteins This was shown byy using monoclonal antibody 7 10 which binds to hoth the constitutivelyy expressed and the inducible hsp 70 proteins. In our study,, we found that the 72 and 74 kDa proteins hound to monoclonall antibodies 7.10 as well as N27. demonstrating that thesee proteins also are part of the hsp 70 family of proteins. However,, the BPs do not bind to moAb 8F7. Monoclonal antibodyy 8F7 detects the inducible 68 kDa hsp in man. mouse. rat,, pig and cattle (34). Monoclonal antibody N27 reach with hspp 6K. and the constitutive form of hsp 70 (35). Monoclonal antibodyy N27 and moAb 8F7 react to different epitopes of hsp 688 (36). The BPs do not bind to Mab NI5. Thus. 72 and 74 kDaa BPs appear to have unique properties as they do not bind too antibodies N15 and 8F7. The appearance of multiple bands whenn the commercial 70 kDa heat shock protein (Stressgen) is probedd with Mab N27 may be due to protein breakdown.

HSPP 70 is a heterogeneous group of proteins consisting of five distinct,, but structurally and immunological!; -related proteins: (1)) a constitutive 73 kDa pi 5.5 protein, (2) a stress inducible 722 kDa'pi 5.6 protein, (3) a stress inducible 73 kDa'pl 6.3 protein.. (4) a constitutive 74- 78 kDa protein present in the endoplasmicc reticulum (glucose- regulated protein) and (5i a 75 kDaa member present in mitochondria. Inducible hsp 70 proteins aree synthesized in response to a range of different stresses includingg heal shock, nutrient deprivation, exposure to mitogens andd oxygen radicals, metabolic disruption, and viral infection (I4J5.26.27.37--39).. The exact nature of the hsp 70 that is synthesizedd depends on the nature of the cellular stress, with some off the hsps 70 being maximally synthesized after stimulation with lectinss (39). The 72 and 74 kDa proteins specific tor bovine IRBP peptidee 1169-1191 could he significantly enhanced in hoth rat andd human B cells following activation with LPS. l.PS is a potent stimulatorr of B cells and LPS-strmulated B cells have been shown too be highly efficient antigen-presenting cells (28). The induction off binding proteins from the hsp 70 family ot' proteins may in factt contribute in part to this increased antigen-presenting ability.

Thee faei that the BPs fail to bind to the noii-uveitopaihogcnic peptidee 1135 - 1157 with 23 amino acids but bind to 1169- 1 191 (233 amino acid length), shows the specificity of binding of these BPss to the uveitopathogenic epitope 1 169- I 191.

Thatt the binding of the 72 kDa. 74 kDa and 40 kDa proteins too the peptide I 169- 1191 affinity column is not the result of nonn specific binding to the Sepharose 4B column is demonstrated

bvv our negative observations obtained hv passing the cell Ivsate throughh an activated Sepharose 4B column lacking peptide 1169-- 1191 (Figure 4. lane 4). Specificity of binding is shown bvv passing the flow-through obtained from this column thiough aa peptide I 169 - 1 191 column and observation ot the protein bandss on the gel (Figure 4. lane H).

Soo far. the role of hsps in autoimmune disease has heen thought too he restricted to their potent antigenic characteristics (37.40), However,, the present study suggests an active role for these proteinss in the induction of the immune response to auioanligens Thee abilnv of fragments trom autoantigens io bind to available hspp 70 proteins might make a given peptide sequence more or lesss immunopathogenic. Its abilit\ to be expressed on the cell surfacee and its abititv to interact with class II MHC may. in fact, bee critically dependent on the interaction with an hsp 70 intermediatee huiding protein. The ease with which members of thee hsp 70 familv are induced under conditions ot cellular activationn might further influence the intensity of the immune responsee to an antigenic challenge.

Futuree studies will include scaled-up experiment in order to isolatee the 72 kDa, 74 kDa and 40 kDa proteins in sufficient quantitiess for partial amino acid sequencing which will identilv thee familv or families of hsp proteins to which these proteins belongg and to determine the identity of the 40 kDa protein.

Acknowledgement t

Wee thank Dr. Igal Gery for the critical review of the manuscript.

Abbreviations s

HSP-heatt shock protein. IRBP—interphotoreceptor retinoid bindingg protein, LPS lipopolysaccharide. RAH—experimental autoimmunee uveitis, PBS—phosphate buffered saline, DMF.M-CM—Dulbecco'ss modified eagle medium-complete medium, LBVV —Rpstein Barr virus, PBMC— peripheral blood mononuclear cells.. PMSF-phenyl sull'unyl fluoride. SDS-PAGF-sodium dodecyll sulfate pol y aery lam idc gel electrophoresis.

References s

1.. Gery. I., Wiggert. B . Redmond. T.M.. Kuwabara. T., Crawford.. M.A., Vistiea. B.P. and Chader, G.J (1986) Cveoretinitiss and pinealitis induced by immunization with interphotoreceplorr reiinoid-binding protein. In vat. Ophthalmol.Ophthalmol. S7.v Set. 27, 1296- 13(H)

2.. Nussenblatt, K.B.. Gerv. I. and Wacker. W.B. (1980) Experimentall autoimmune uveitis: cellular immune responsiveness.. Invest. Ophthalmol. Vis. Sn. 19, 686 690.

3.. Chan. C .C. Mochizuki. M.. Palestine. A,, Ben L/ra. D.. Gery.. I. and Nussenblatt R.B. (1985) Kinetics of T lymphocytee subsets in the eye of lewis rats with experimental autoimmunee uveitis. Cell Immunol. 96. 430 -434.

4.. Gery. I.. Moehi/.uki. M. and Nussenblatt, R.B. (1986) Retinall specific antigen and immunopathogenic processes theyy provoke. In Pmi>res\ and Retinal Resettreh. (Ld Osborne,, N. and Chader, G.J.). Pp. 75 109. Per «anion Press.. Oxford. Ensiland.

74 4


5.. Nusscnblatt, R.B and Palestine, A.G. (1989) Uveitis: Fundamentalss and clinical practice. Year Book Medical Publishers.. Chicago,

6.. Nussenblatt. R.B . Kuwabara, T., DeMonasterio. KM . and Wacker,, W.B. 11981) S-antigen uveitis in primates: A new modell ("or human disease. Arch. Ophthalmol. 99. 10900 1092.

7.. Nussenblatl. R.B.. Mittal. K.K.. Ryan, S.. Green. W.R. andd Maumenee, A.H. (1982) Birdshot retinochoroidopathy associatedd with HLA-A29 antigen and immune responsive-nesss to retinal S-antigen. Am. J. Ophtfuilmot. 94, 147- 158.

8.. Eaure, J.P. (1980) Autoimmunity and the retina. Curr. Top. EyeEye Ren. 2, 215-,102.

9.. Wacker, W.B.. Donoso. L.A., Kalsow. C M .. Yankee!ov Jr.. J. A and Organiseiak. D.T. (1977) Experimental allergic uveitis.. Isolation, characterization and localization of a solublee uveitopathogenic antigen from bovine retina. J. Immunol.Immunol. 119. 149-158.

10.. Mochizuki, M., Kuwabara, T.. McAllister, C, N'ussenblatt, R.B,, and Gery, I. (1985) Adoptive transfer of experimental autoimmunee uvcoreiinitis in rats, invest. Ophthalmol. Vis. Sri.Sri. 26, 1-9.

11.. Rozenszajn, L.A., Muellenbcrg-Coulombre, C, Gery. I.. Saied.. M. HI., Kuwabara. T.. Mochizuki, M., Uando. Z. andd Nussenblatl. R.B. (1986) Induction of experimental autoimmunee uvcoretinitis (HAU) in rats by T lines. immunology,immunology, 57, 559.

12.. Caspi, R.R., Roberge, KG.. McAllister, C.G., El-Saied, M,,, Kuwabara, T,, Gery, I.. Hanna, K and N'ussenblatt, R.B.. (1986) T cell lines mediating experimental autoimmune uveoretinitiss (EAU) in the rat J. Immunol. 136. 928-933.

13.. Cham. B.M.. Kaye, P.M. and Shaw, M.A. (1988) The biochemistryy and cell biology of antigen processing. Immunol.Immunol. Rev. 106, 33 -58.

14.. Unanue. E.R. and Allen, P.M. (1987) The basis for the immunoregulatoryy role of macrophages and other accessory cells.. Science. 236, 551-557.

15.. Unanue. E.R. and Cerottini, J.C. (1989) Antigen presentation.. FASEB J. 3, 2496-2502.

16.. Marsh, E.W.. Dalke, D.P. and Pierce, S.K. (1992) Biochemicall evidence for the rapid assemhly and disassembly off processed antigen-major histocompatibility complex class III complexes in acidic vesicles of B cells. J. Exp. Med. 175. 425-436, ,

17.. Harding. C.V.. Roof, R.W, and Unanue, F..R. (1989) Turnoverr of la-peplide complexes is facilitated in viable antigen-presentingg cells: biosyuthetic turnover of la vs. peptidee exchange. Pmc. Natl. Acad. Sci. USA. 1989. 4230-4234. .

18.. Davidson, H.W., Reid, P.A., Lanzavecchia. A. and Watts.C.. (1991) Processed antigen hinds to newly synthesizedd class II molecules in antigen-specific B lymphocytes.. Cell. 67. 105-116,

19.. Eakey. F.K.. Casten. I..A., Niehling, W.I... Margoliash, K.. and Pierce, S.K. (1988) Time dependence of B cell processingg and presentation of peptide and native protein antigens.. ,/. Immunol. 140. 3309-3314

20.. Ellis, R.J. (1990) The molecular chaperone concept. Semin.

CellCell Biol. 1. 1-9. 21.. Lakey, E.K., Margoliash. E and Pierce. S.K. (1987)

Identificationn of a peptide binding protein that plays a role inn antigen presentation. Pmc. Nail. Acad. Sci. USA. 84, 1659-1693. .

22.. Flynn, (J .C, Pohl, J., Eloeco. M.T. and Rothman, J.E. (1991)) Peptide binding specificity of the molecular chaperone BiP.. Nature, 353, 726-730. '

23.. Hajnik, M.F., Cancl. C . Kramer. J. and Kasahara, M. (1991)) Evolution of the major histocompatibility complex: molecularr cloning of major histocompatibility complex class II from the amphibian Xenopus. Proc. Natl. Acad, Sci. USA. 88.. 537-541.

24.. de Smet, M.D., Yamamoto, J.H., Mochizuki, M., Gery, I.,, Singh, V.K.. Shinohara, T.. Wiggen. B.. Chader, G.J. andd IN'ussenblat. R.B. (1990) Cellular immune responses of patientss with uveitis to retinal antigens and their fragments. AmAm ,/. Ophthalmol. 110, 135-142.

25.. Borst. D.L., Redmond. T.R., Eiser, J.E., Gonda. M.A., B.. Wiggert. Chader, G.J. and Nickerson, J.M. (1989) Inter-photoreceptorr retinoid binding protein-gene characterization, proteinn repeat structure, and its evolution. J Biol. Chem. 264,, II 15 - 1123.

26.. Beckmann, R P.. I.ovett, M, and Welch. W.J. (1992) Examiningg the function and regulation of hsp 70 in cells subjectedd to metabolic stress, J, Cell Biol. 117, 1137-1150.

27.. Ghassemi, M., Heydari. A.R. and Richardson. A. (1991) Inductionn of heat shock proteins inlymphocytes increases withh mitogen stimulation. Immunol. i.ett. 30. 333-338.

28.. Gontijo. C M, and Möller, G. (1991) Antigen processing andd presentation by small and large B cells. Seand. J. Immunol.Immunol. 34. 207-213,

29.. Hu, L.H., Redmond, T.M., Sanui. H., Kuwabara. T.. McAllister.. C C . Wiggcrt. B.. Chader. G.J. and Gery, I. (1989)) Rat T-cell lines specific to a nonimmunodominant deterimantt of a retinal protein (IRBP) produce uveoretinitis andd pinealities. Cell Immunol. 122. 251 - 2 6 1.

30.. Pierce, S.K.. Morris. J.F., Grusby, M.J,, Kaumaya, P.. Vann Buskirk. A.. Srinivasan, M., Crump, B. and Smolenski.L.A.. (1988) Antigen-presenting function of B lymphocytes.. Immtmolog. Rev. 106. 149-180.

31.. Guillet, J.G., Lai, M.Z.. Briner.T.J.. Btius, S., Settc. A., Grey.. H.M.. Smith. J.A. and Getter, M.L . (1987) Immunologicall self, nonse!f discrimination. Science, 235, 865-870 0

32.. Sanui, H.. Redmond. T.M.. Kotake, S., Wiggert. B.. Hu, L.-H-,, Margalit, H., Berzofsky. J.A,. Chader. G.J. and Gery,, I. (1989) Identification of an immunodominant and highlyy immunopalhogenic determinant in the retinal intcrphotoo recept or retinoid-bindtng protein (IRBP). J. Eyt. Med.Med. 169, 1947- I960.

33.. Lindquist, S. (1986) The heal shock response. Annu. Rev. Biochem.Biochem. 55. 1151 - I 191.

34.. Heine. I... Drabent, B., Benecke, B.J. and Gunther. E. (1991)) A novel monoclonal antibody directed against the heatt inducible 68 kl)a heat shock protein. Hybridoma. 10, 722 I - 730,

35.. Minota. S.. Cameron, B., Welch. W.J and Wmfield. J.B.

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(1988)) Autoantibodies to the constitutive 73-kD member of thee hsp 70 hamily of heat shock proteins in systemic lupus erythematosus.. J. Exp. Med. 168. 1475-1480.

36.. Milarski, K.L., Welch, J.W. and Morimoto, R.I. f 1989) Celll cycle-dependent association of hsp 70 with specific cellularr proteins. J. Cell Biol. 96. 286-290.

37.. Young. R,A. (1990). Stress proteins and immunology. Annu RevRev Immunol. 8, 401 -420.

38.. Specior. N.L.. freedman. A.S., Freeman. G., Segil, J., Whitman.. J.F., Welch. W.J. and Nadler. L.M. (1989) Activationn primes human B lymphocytes to respond to heat shock,, J. Exp. Med. 170. 1763-1768.

39.. Haire, R.N.. Peterson. M.S. and O'Leary, J.J. (1988) Mitogenn activation induces the enhanced synthesis of two heat-shockk proteins in human lymphocytes. J. Celt Biol. 106. 883-891. .

40.. Young, D.B. (1992) Heat-shock proteins: immunity and autoimmunity.. Curr. Opin. Immunol. 4. 396 — 400.

76 6

Chapterr 4

Humann S-Antigen: Presence of Multipl e Immunogenicc and Immunopathogenic Sites

inn the Lewis Rat

Marcc D. de Smet, George Bitar, Francois G. Roberge, Igal Gery, Robertt B. Nussenblatt

Journall of Autoimmunity 6: 587-599, 1993 (byy permission © Academic Press Limited)

DETERMINANTSS OK HS-AG IN LEWIS RAT

Humann S-Antigen: Presence of Multipl e Immunogenicc and Immunopathogenic Sites in the

Lewiss Rat

Mar cc D . de S m e t, George B i ta r , F ranco is G. Roberge, Igal Gery andd Robert B. Nussenb la tt

LaboratoryLaboratory of Immunology, National Eye Institute, National Institutes of Health, Betkesda,Betkesda, MD, USA

(Received(Received 30 October 1992 and accepted 9 July 1993)

Too identify the immunogenic and immunopathogenic sites present in humann S-Antigen (S-Ag), 40 overlapping peptides that span the whole lengthh of the S-Ag molecule were synthesized and tested in the Lewis rat modell of experimental autoimmune uveitis. The most pathogenic sequencess were 180-200, 340-360 and 350-370. Ten peptide sequences were identifiedd that induced visible inflammation in the eye. A total of 23 peptidess gave an in-vitro proliferative response following immunization in animals.. The ability to generate an immune response was not linked to the pathogenicc capacity of the sequence. The most pathogenic sequence, 340-360,, was only weakly proliferative. Peptide 180-200 and peptide 340-360 gavee higher T-cell proliferative responses, but these were lower than the maximall proliferative response observed with non-pathogenic sequences. Inn animals immunized with whole S-Ag, the majority of the determinants didd not elicit a proliferative response, indicating that in S-Ag, the majority off the immunogenic determinants are cryptic and are not presented by the APCC located in the lymph nodes.

Introductio n n

Thee term uveitis refers to several different forms of ocular inflammation, all groupedd under the same name. It is a major cause of visual impairment in a productivee segment of the population. T-cell autoimmunity is believed to play a keyy role in the pathogenesis of at least some of these conditions [1-3]. The retinaa contains several organ-specific antigens that can induce T-cell mediated

Reprintt requests to: Mart.' D. de Smet, MI) , Laboratory ol" Immunology, National V.vc Institute, Bldgg 1», Rm ION 11 2, Bethesda, M D 20892, USA.

08966 8411/9-3---050*)87+13 8O8.OÜ/0 < 1993 Academic Press Limited

79 9

ChapterChapter 4

experimentall ocular inflammation in susceptible animal strains. The clinical and histopathologicall appearance of uveitis occurring in these models closely resembles certainn uveitic conditions in man [1, 2, 4, 5]. One uveitogenic retinal antigen which hass been extensively investigated is S antigen (S-Ag), a protein of approximately 488 kD which appears to play a pivotal role in the visual process [6, 7]. S-Ag is a highlyy conserved molecule found in all mammalian species and also in many invertebratess [8], It is found in the retina as well as in the pineal gland [8, 9]. When injectedd into a variety of vertebrate animals, S-Ag induces a severe ocular and pineal inflammatoryy response known as experimental autoimmune uveitis (EAU) and experimentall autoimmune pincalitis (EAP) [1,4]. The possible role of S-Ag in the etiologyy of human uveitis is further supported by the fact that many patients with intermediatee or posterior uveitis have immune responses to the human and bovine sequencess of S-Ag [10-13].

Manyy investigations in recent years have focused on the identification of epitopes responsiblee for the immunogenicity and the immunopathogenicity of S-Ag. Most of thee work has been carried out on bovine S-Ag [14-17]. While there is considerable homologyy between the bovine and the human sequences of S-Ag [18], there are sufficientt differences to suspect that the two antigens contain different immuno-genicc and immunopathogenic epitopes. Only three sites of human S-Ag have been identifiedd so far as being immunopathogenic in Lewis rats: peptides 303-314 and 286-2977 which arc non-dominant [19], and peptide 343 362 which has immuno-dominantt characteristics [20, 21]. In the present article, we have carefully mapped thee immunogenic and immunopathogenic determinants of human S-Ag in the Lewiss rat by using overlapping synthetic peptides. The present study was aimed at determiningg the immunogenicity and the immunopathogenicity of overlapping peptidess of human S-Ag in the EAU model in the Lewis rat. Each peptide consisted off 20 amino acids, thereby minimizing the need for antigen processing by the antigenn presenting cell. The results show that human S-Ag contains several immunopathogenicc epitopes, some of which have not previously been identified.

Material ss and methods

SyntheticSynthetic peptides

Fortyy overlapping oligomeric peptides of human S-Ag, each measuring 20 amino acidss in length, except for the last peptide measuring 15 amino acids, were synthesizedd based on the sequence of human S-Ag reported by Yamaki et al. [22]. Eachh peptide overlapped the previous sequence by 10 amino acids. The exact sequencee as well as the nomenclature used is shown in Fig. 1. All peptides as well ass human M and N peptides were synthesized by solid phase chemistry using t-butyloxycarbonyll derivatives of the amino acids on an automated peptide syn-thesizerr and were purified by Applied Biosystems Inc., Foster City, CA, USA. The sequencee for peptide M, corresponding to positions 303 to 320 of S-Ag, was DTNLASSTIIKEGIDRTV,, while for N peptide, corresponding to positions 281 too 302, the sequence was LPLLANNRERRGIALDGKIKHE. The amino acid compositionn of each peptide was verified using amino acid analysis and automated gas-phasee sequencing. The previously identified immunopathogenic sites are

80 0

DETERMINANTSDETERMINANTS OF HS-AG IN LEWIS RAT

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T a b l ee 1. Clinical grading system for experimental autoimmune uveitisuveitis in the Lewis rat

Scoree Severity of ocular inflammation

0:: No evidence of inflammatory disease

0.5:: Minimal iridocyclitis Conjunctiva!! hyperemia Minimall clouding of the red reflex

1:: Moderate iridocyclitis Irregularr pupil Moderatee clouding of the red reflex

2:: Marked iridocyclitis with evidence of infiltrates Posteriorr synechiae with pupil block Inferiorr hypopyon

3:: Marked obscuration of the red reflex Markedd hypopyon

4:: Pan ophthalmitis with or without hemorrhage Corneall neovascularization Markedd proptosis of the eye

conta inedd in pept ide 29 for 286-297, pept ide 31 for 303 -314 and pept ide 35 for

3 4 3 - 3 6 2. .

ImmunisationImmunisation and disease evaluation

Malee Lewis rats, 8 10 weeks old (Charles River, Raleigh, N C, USA) were immun izedd via the h ind footpad with the various ol igomeric pept ides of h u m an S-Agg emulsif ied in complete Freund 's adjuvant (CFA; Difco, Detro i t, M I , USA) conta in ingg Mycobacterium tuberculosis H37Ra at 2.5 mg/ml. Each animal received 1000 ug of pept ide in 0.1 ml of CFA emulsion. An addit ional adjuvant, Bordetella pertussispertussis (Michigan D e p a r t m e nt of Publ ic Hea l th, Lansing, M I , USA, lot 94), was injectedd intravenously, 101" bacteria per rat. Animals immunized with bovine S-Ag preparedd accord ing to Dorey et al. [23] were used as a positive control. Rats were observedd daily for clinical signs of disease start ing on day 8 until they were killed. Clinicall ocular changes were graded on a scale of 0 -4, based on the intensity of the inflammatory77 changes as indicated in Tab le 1 and modif ied from Gery et al. [3] . Animalss were observed for a total of 30 days; after which they were killed to de terminee the histopathologic grade of disease.

Somee animals were immunized with h u m an S-Ag prepared according to the me thodd of Dorey et al. [2 3]. H u m an cadaver eyes obta ined within 6 hours of death andd shipped on ice by courier were provided by the Nat ional Disease Research In terchangee (Phi ladelphia, PA, USA) according to an approved protocol. T he investigationss were carr ied out in compl iance with Publ ic Heal th Service Policy on H u m a nee Care and Use of Laboratory Animals ( D H EW Publ icat ion, N I H 80-23) in ann accredi ted animal facility.

82 2


LymphocyteLymphocyte proliferation assay

Inn order to determine the immunogenicity of the various fragments, rats were immunizedd according to the same protocol described above. On day 12-14 after immunization,, the draining lymph nodes were collected from individual rats. Single-celll suspensions of the lymph node cells were cultured in 96-well flat bottom microculturee plates at a density of 3 x 105 cells per well in RPMI 1640 supple-mentedd with 5% heat inactivated Fetal Calf Serum (Hyclone, Logan, UT, USA), 22 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 ug/ml) in 5% C 02 .. Cell cultures were set up in triplicate and incubated with antigens for 72 hourss at 37°C, pulse labeled with 0.5 uCi per well of pH]thymidine (New England Nuclear)) for 12-16 hours and harvested on glass fiber filters for scintillation counting.. The data are presented as stimulation index values (S.I. = mean CPM in culturess with antigen/mean CPM in cultures without antigen).

Histopathology Histopathology

Bothh eyes were taken from each animal and fixed for 30 minutes in 4% glutaraldehyde,, followed by 10% buffered neutral formalin. Specimens were routinelyy stained with hematoxylin and eosin for examination. The severity of EAUU in each eye was graded on a scale of 0 to 4+ as shown in Table 2 and modifiedd from Roberge et al. [24]. The scores for the eyes of each animal were averagedd to give a single score per animal.

Results s

DeterminationDetermination of the immunopathogemctty of peptides of human S-Ag

Groupss of Lewis rats were immunized with the tested peptides, as well as bovine S-Agg as a control. We were interested in determining die immunopathogenic potentiall of each oligomeric peptide and in comparing these to die animals immunizedd with bovine S-Ag. Several of the tested peptides were immunopatho-genicc as is shown in Figure 2. While die majority of the immunopathogenic peptidess were located close to the carboxyl terminus, a few peptides were found closerr to the N-terminus of the S-Ag molecule. Peptide 35 contains the immuno-dominantt epitope identified by Gregerson et al. for the Lewis rat [20]. This peptide provedd to be very immunopathogenic. It was the most potent of all peptides in its inductionn of EAU. Other peptides, however, rivaled peptide 35 in the severity and thee speed with which they induced disease. Peptide 19 was particularly pathogenic, withh disease occurring shortly after that induced by peptide 35 and at about the samee time as the control animals immunized with bovine S-Ag (Table 3). Peptide 199 was of particular interest as it had never before been identified as a pathogenic epitopee in the Lewis rat. We also tested M and N peptides which had previously-beenn identified as being immunopathogenic when using the bovine sequence [19]. Onlyy human M peptide was able to induce disease, but the disease was both delayedd and of a lesser intensity as compared with S-Ag. Human N peptide was non-pathogenicc and did not induce an immune response in our hands. We also testedd the strength of the pathogenic response to 3 peptides by varying the

83 3

ChapterChapter 4

T a b l ee 2. Histopathologic grading system for experimental autoimmune uveitis in the LewisLewis rat

Scoree Severity of ocular inflammation

Noo evidence of inflammatory disease

0.5:: Trace inflammation, architecture of the retina is grossly intact Inflammatoryy cell infiltration of the retina without evidence of tissue destruction

orr with outer photoreceptor damage in less than 1/4 of the retina Focall non-granulomatous, monocytic infiltration in the choroid, ciliary body and

retina a

1:: Photoreceptor outer segment damage in > 1 /4 of the retina Focall areas of destruction with marked dropout of photoreceptors Retinall perivascular infiltration and monocytic infiltration in the vitreous

2:: Lesion extending to the outer nuclear layer and in > 1/4 of the retina Smalll exudative retinal detachment Mil dd to moderate number of cells in the vitreous Granulomaa formation in the uvea and retina Occlusivee retinal vasculitis, along with serous retinal detachment and loss of

photoreceptor r

3:: Lesions extending to the inner nuclear layer and in > 1/4 of the retina Retinall architecture beginning to be lost, large exudative retina detachment,

moderatee to large number of cells in the vitreous Formationn of Dalen-Fuchs nodules and the development of subretmal

neovascularization n

4:: Full thickness retinal damage in > l / 4 of the retina Totall destruction of retinal architecture

immunizingg dose. We further tested the peptides overlapping the putative immuno-dominantt site (peptides 35 and 36) as well as the novel highly immunopathogenic peptidee 19 by determining their uveitogenicity at lower doses. Peptide 35 was the mostt pathogenic and was able to induce disease to the lowest level tested (Table 4). Peptidee 36, which also contains the minimal uveitogenic site, was not as pathogenic ass peptide 35 and was in fact comparable to peptide 19.

DeterminationDetermination of the immunogemcity of peptides of human S-Ag

Al ll peptides were also tested for their ability to induce a T-cell proliferative response measuredd by the lymphocyte proliferation assay. The results are demonstrated graphicallyy in Fig. 2. Each peptide was tested for the induction of a response to self andd to bovine S-Ag between days 12 and 14 after immunization. The ability to generatee a strong T-cell proliferative response to either the peptide or to whole bovinee S-Ag did not correlate well with the capacity of the peptide to be pathogenic. Inn fact, peptides inducing a low proliferative response had the greatest pathogenic effect.. Figure 2 also includes a comparison of die rat and human S-Ag sequences. Thee comparison suggested that areas of complete homology were highly immuno-pathogenic,, as demonstrated by peptides 6, 31, 35 and 36. However, immuno-pathogenicityy was also found in areas where complete homology was not present.

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Tablee 3. Paihogenicuy of oligomenc peptides of human S-Ag byby maximal clinical score and date of onset of inflammation

(immunizing(immunizing dose WO/ag/rat)

Maximum m Onsett Rats with clinical

Peptidee no. (Day) EAU/Total score*

01 1 06 6 19 9 20 0 26 6 29 9 31 1 35 5 36 6 MM Peptide S-Ag} }

155 17f 15-20 0 10-12 2

20 0 16 6 12 2 13 3 10 0 11 1 13 3 10 0

2/4 4 3/4 4 8/8 8 4/4 4 2/4 4 3/3 3 4/4 4 4/4 4 4/4 4 4/4 4 4/4 4

1.5 5 1.5 5 4 4 2 2 1 1 3 3 3 3 4 4 4 4 3 3 4 4

*Rcferss to an average score for all eyes of the immunized animals. fDatee of onset refers to when disease was first observed in an animal.

Whenn a range is given, disease was nut seen in some animals until the later date.. The date does not refer to when maximal disease was achieved.

^Animalss were immunized with 30 ug of bovine S-Ag.

Severall peptides were identified, including peptide 19, which induced a strong immunogenicc or an immunopathogenic response despite having three or more non-conservativee amino-acid substitutions from the rat S-Ag sequence.

DeterminationDetermination of the immune response of rats immunized with S-Ag

Finally,, we looked at the lymphoproliferative responses against the various peptides off human S-Ag in animals that were immunized with either bovine or native human S-Ag.. The proliferative responses were determined on day 12 and 14 as was previouslyy done with the various oligomeric peptides. With either S-Ag, multiple sitess of response were observed (Table 5). Immunization with bovine S-Ag gave a patternn of response similar to that seen with the animals immunized with the peptidess and tested against bovine S-Ag. In other words, the bovine S-Ag in vivo elicitedd an immune response to the same peptides that elicited a response to bovine S-Agg in vitro. An exception to this rule were peptides 7 and 33. Immunization with humann S-Ag gave a different pattern of response. In the majority of cases, the immunogenicc peptides corresponded to peptides that had demonstrated immuno-genicityy in culture but no cross reactivity with bovine S-Ag. Some overlap was presentt between the two forms of S-Ag but interestingly there was no overlap with peptidee 35, the putative immunodominant site.

Discussion n

Immunogenicityy and immunopathogenicity of an antigen are a reflection of the abilityy of processed peptides to be presented by antigen presenting cells (APC) in

86 6

DETERMINANTSS OP HS-AG IN LEWIS RAT

associationn with MH C class II [25]. Standard proliferation assays using cleavage productss of a whole molecule arc only capable of eliciting a response to those cleavagee products that are readily produced by peripheral blood APC. However, myelinn basic protein [26], and interphotoreceptor retinoid binding protein [27], containn epitopes that are immunopathogenic but cryptic. They are cryptic because enzymaticc digestion within the lysozomal compartment of the APC does not allow thesee fragments to survive intact, at least not within the APC present in the peripherall blood or in the lymph node. However, these cryptic antigens may in fact playy a determining role in the establishment and the perpetuation of chronicity as wass recently suggested by studies in experimental autoimmune encephalitis [26]. In orderr to evaluate as many cryptic epitopes as possible, we decided to synthesize shortt overlapping peptides that would span the whole S-Ag molecule. Since eight to 100 amino acids appear to be the minimal required number for antigen presentation [28],, it seemed reasonable to construct peptide determinants containing 20 amino acidss each. This should have a good probability of identifying nearly all the epitopes presentt in the human S-Ag molecule. A peptide of this size would require a minimal amountt of antigen processing, and in many cases might be able to interact with both thee APC and the T cell without any enzymatic degradation. Using this set of peptides,, we were able to identify a number of immunogenic and immunopatho-genicc sites. Several of these epitopes had not been previously described. They were previouslyy not detected because of the low proliferative ability of these epitopes. A feww appear to be unique to human S-Ag, peptide 1 and in particular peptides 19 andd 20.

Ourr results suggest that pathogenic epitopes arise preferentially in areas of the S-Agg molecule that are highly conserved. There are no differences between the rat, humann and bovine sequences for peptides 31, 35 and 36 [18, 29], Peptide 19, however,, presents an interesting exception. There are 3 amino-acid substitutions betweenn the human and the rat sequences, and yet it is one of the more pathogenic peptides.. Peptide 19 is comparable in its pathogenicity to peptide 36 and appears to bee more potent than the previously recognized immunopathogenic epitope M peptide. .

Ass has previously been noted in both EAU and EAE, there appears to be a dissociationn between the ability of lymphocytes to proliferate and their ability to causee disease [17, 31-33]. While all pathogenic sites are proliferative, the intensity off the proliferative response is often quite weak as compared to several immuno-genicc but non-pathogenic peptides. This dissociation is most marked for peptides 355 and 36 and to a lesser extent for peptides 6, 19, 20 and 31. The exact reason for thiss dissociation is not known. One possibility is that with tire onset of disease, suppressorr cells appear in the peripheral blood that down regulates the immuno-proliferativee response. However, this dissociation was also seen in established cell liness after several passages where such a mechanism should not be operative [17]. Lymphocytess raised to autologous sequences of S-Ag are generally less proliferative whenn compared to similar but non-autologous sequences [34], Such a protective mechanismm may be operative in peptide 35. While being the most pathogenic determinant,, it is also one of the least immunogenic. We also compared the immunoproliferativee responses of the various peptide determinants in the Lewis rat too those that were published on human patients [10]. The peptides that were most

87 7

ChapterChapter 4

Tabl ee 4. Dose-dependent pathogenicity of oligomeric peptides of humanhuman S-Ag

Maximum m Peptidee Immunizing Onscr* Rats with clinical no.. dose (|ig/rat) (Day) EAUATotal scoret

100 0 50 0 25 5

5 5 100 0 50 0 5 5

100 0 50 0

5 5

12 2 13 3 15 5 — — 12 2 12 2

n n 12 2 13 3

4/4 4 2/2 2 1/2 2 0/2 2 4/4 4 4/4 4 4/4 4 4/4 4 3/4 4 0/4 4

4 4 3 3 2 2 0 0 4 4 4 4 3 3 4 4 3 3 0 0

'Da tee of onset of clinical disease. This does not refer to the date of maximall disease.

ff Refers to the average maximal clinical score for all eyes that were evaluated. .

consistentlyy proliferative from patient to patient were 2, 3, 8, 13, 32 and 35. These peptidess were not particularly immunogenic in the Lewis rat except for peptide 13. Thee pathogenic peptides identified in the Lewis rat were found to be proliferative in relativelyy few patients. Peptide 35 was proliferative (at an S.I. of 2.0 or more) in 28%% of patients, peptide 6 in 20%, while all the remaining pathogenic peptides weree identified in less than 10% of patients.

Animalss immunized with either bovine or human S-Ag had a proliferative responsee to several peptide determinants, with no particular site being particularly dominantt (Table 4). In general, immunization with a whole protein leads to the identificationn of a dominant, often unique, proliferative determinant. The presence off multiple determinants that are nearly equally proliferative indicates that, in the Lewiss rat, S-Ag is a highly immunogenic protein. This degree of immunogenicity is usuallyy not elicited by immunization with adjuvant. It is usually seen after the immunee system has become exposed to an antigen through more normal mech-anismss such as an infection [35]. Under these conditions, it is not unusual to find multiplee immunogenic determinants without any clear immunodominant site. The immunee system might have become exposed to S-Ag or to the immunogenic epitopess of S-Ag prior to immunization through several different mechanisms. S-Ag iss present in the pineal gland which does not have as tight a vascular barrier as the eyee [36], thus allowing some exposure of S-Ag to the immune system. Mimicry betweenn S-Ag epitopes and other proteins that share a similar peptide sequence mightt be another mechanism [37]. Of the immunogenic determinants that were identified,, several were found to be immunopathogenic. It is interesting to note that severall of these determinants were cryptic. Lymphocytes from animals immunized withh either bovine or human S-Ag did not give an immune response when tested in vitro.vitro. The lack of immune response suggests that antigen processing by lymph node

88 8


Tab l ee 5. Stimulation indices to oligomeric pep-tidestides of human S-Ag in Lewis rats immunized

withwith whole S-Ag

Peptide e no.* *

03 3 06 6 07 7 08 8 09 9 10 0 15 5 17 7 18 8 19 9 20 0 21 1 23 3 29 9 33 3 35 5 S-Ag g

Bovine e S-Agf f

4.4 4

6.5 5 3.6 6

13.0 0

2.4 4 9.4 4 6.0 0

29 9

Human n S-Agf f

2.7 7 2.5 5 5.3 3 6.1 1 4.0 0 2.1 1

5.8 8 5.5 5 5.3 3 5.1 1 5.2 2 3.2 2

12 2

*Peptidee concentration and the concentration of bovinee S-Ag was 100 ug/ml,

fOnlyy responses with S.I. above 2.0 arc shown. Valuess represent an average of cells pooled from the ratss of each group.

APCC did not produce these particular epitopes. Processing of the S-Ag molecule by APCC at other sites such as the eye is likely to be different and allow these particular determinantss to cause disease. The use of small synthetic peptides prevents much of thee normal antigen processing that usually occurs and allows hidden determinants too elicit an immune or an immunopathogenic response.

EAUU serves as a model for human uveitis. We have shown in these experiments thatt there are several immunopathogenic determinants within S-Ag, suggesting that thee same may be true in patients. The existence of multiple determinants would placee considerable restrictions on die feasibility of immunotherapy in humans. Immunotherapyy directed against a single determinant is unlikely to be effective. Onlyy approaches that can induce non-specific suppression, even if limited to a specificc inflammatory site, arc likely to be effective.

References s

1.. Faure, J. P. 1980, Autoimmunity and the retina, Curr. Top. Eye Res. 2: 215-302 2.. Gery, I., M. Mochizuki, and R. B. Nussenblatt. 1986. Retinal specific antigen and

immunopathogenicc processes they provoke. Prog. Retinal Res. 5: 75-109 3.. Gery, I., B. Wiggert, T. M. Redmond, T. Kuwabara, M. A. Crawford, B. P. Vistica, and

G.J.. Chader. 1986. Uveoretinitis and pinealitis induced by immunization with inter-photoreceptorr retinoid-binding protein. Invest. Ophthalmol. Vis. Sci. 27: 1296-1300

89 9

ChapterChapter 4

4.. Wackcr, W. B., L. A. Donoso, C. M. Kalsow, J. A. Yankeclov Jr, and D. T. Organisciak. 1977,, Experimental allergic uveitis. Isolation, characterization and localization of a solublee uveitopathogenic antigen from bovine retina. J. Immunol. 119: 149-158

5.. Hirose, S., V. K. Singh, L, A. Donoso, T. Shinohara, S. Kotake, T. Tanaka, T.. Kuwabara, K. Yamaki, I. Gery, and R. Nussenblatt. 1989. An 18-mer peptide derivedd from the retinal S antigen induces uveitis and pinealms in primates. Clin. Exp. Immunol.Immunol. 77: 106 111

6.. Shmohara, T., B. Dietzschold, C. M. Craft, G. Wistow, J. J. Early, L. A. Donoso, J.. Horowitz, and R. Tao. 1987. Primary and secondary structure of bovine retinal S-antigcnn (48-kDa protein). Prac Nad. Acad. Set. USA. 84: 6975 6979

7.. Pfister, C , M. Chabre, J. Plouer, V. V. Tuyen, Y. de Kozak, J. P. Faure, and H. Kahn. 1985.. Retinal S-antigen identified as the 48 k protein regulating light dependent phosphodiesterasee in rods. Science 228: 891-893

8.. Mirshahi, M., C. Boucheix, G. Collcnot, B. Thtllaye, and J. P. Faure. 1985. Retinal S-antigenn epitopes in vertibrate and invertebrate photoreceptors. Invest. Ophthalmol. Vis. Sa.Sa. 26: 1016-1021

9.. van Veen, T., R. Elofsson, H, G. Hartwig, I. Gery, M. Alochizuki, V. Ceha, and D. C. Klein.. 1986. Retinal S-antigen: Immunoeytochemical and immunochemical studies on distributionn in animal photoreceptors and pineal organs. Exp. Biol. 45: 15 25

10.. de Smet, M. D., B. Wiggert, G. J. Chader, M, Mochizuki, I. Gery, and R. B. Nussenblatt.. 1990. Cellular immune responses to fragments of S-antigen in patients withh uveitis. In Ocular Immunology Today. M . Usui, S. Ohno, and K. Aoki, eds. Elsevier Sciencee Publ., Tokyo, Japan, pp. 285-288

11.. de Smet, M. D., J. H. Yamamoto, M. Mochizuki, 1. Gery, V. K. Singh, T. Shinohara, B.. Wiggert, G. J. Chader, and R. B. Nussenblatt. 1990. Cellular immune responses of patientss with uveitis to retinal antigens and their fragments. Am. J. Ophthalmol. 110: 135-142 2

12.. Doekes, G., R. van der Gaag, Y. van Kooyk, L. Broersma, M. J. M. Zaal, G. Dijkman, M .. E. Fortuin, G. S. Baarsma, and A. Kijlstra, 1987. Humoral and cellular immune responsivenesss to human S-antigen in uveitis. Curr. Eye Res. 6: 909-919

13.. Nussenblatt, R, B., K. K. Mittal, S. Ryan, W. R. Green, and A. E. Maumenee. 1982. Birdshott retinochoroidopathy associated with H I J \ - A 2 9 antigen and immune respon-sivenesss to retinal S-antigen. Am. J. Ophthalmol. 94: 147-158

14.. Donoso, L. A., C. E. Merryman, T. W. Sery, T. Shinohara, B. Dietzshold, A. Smith, andd C. M. Kalsow. 1987, S-antigen: characterization of a pathogenic epitope which mediatess experimental autoimmune uveitis and pinealitis in Lewis rats. Curr. Eye Res. 6: 1151-1159 9

15.. Singh, V. K., R. B. Nussenblatt, I.. A. Donoso, K. Yamaki, C. C. Chan, and T.. Shinohara. 1988. Identification of a uveitopathogenic and lymphocyte proliferation sitee in bovine S-antigen. Cell. Immunol. 115: 413 -419

16.. Grcgcrson, D. S., W. F. Obntsch, and S. P. Fling. 1987. Identification of a uveitogenic cyanogenn bromide peptide of bovine S-antigen and preparation of a uveitogenic peptide specificc T cell line. Eur. J. Immunol. 17: 405-411

17.. Grcgcrson, D. S., S. P, Fling, W. F. Obritsch, C. F. Merryman, and L. A. Donoso. 1989.. Identification of T cell recognition sites in S-Antigen: dissociation of proliferative andd pathogenic sites. Cell. Immunol. 123: 427-440

18.. Shinohara, T., L. Donoso, M. Tsuda, K. Yamaki, and V. K. Singh. 1989. S-Antigen: Structure,, function and experimental autoimmune uveitis (EAU). Prog. Retinal Res. 8: 51-65 5

19.. Donoso, L. A., K. Yamaki, C. F. Merryman, T. Shinohara, S. Yue, and T. W. Sery. 1988.. Human S-Antigen: characterization of uveitopathogenic sites. Curr. Eye Res. 7: 1077-1085 5

20.. Gregerson, D. S., C. F. Merryman, W. Obritsch, and L. A. Donoso. 1990. Identifi-cationn of a potent new pathogenic site in human retinal S-antigen which induces experimentall autoimmune uveoretinitis in LEW rats. Cell. Immunol. 128: 209 219

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DETERMINANTSS OF HS-AG IN LEWIS RAT

21.. Merryman, C. F., L. A. Donoso, X. M. Zhang, K. E. Heber, and D. S. Gregerson. 1991.. Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicitss T cells expressing V beta 8 and V alpha 2-like genes, J. Immunol. 146: 75-80

22.. Yamaki, K. and T. Shinohara. 1988. Heterogeneous sequences were found in human retinall S-antigen. Invest. Ophthalmol. Vis. Sci. 29 suppl: 373

23.. Dorey, C , J. Cozette, and J.-P. Faure. 1982. A simple and rapid method for isolation of retinall S-Antigen. Ophthalmic Research 14: 249-255

24.. Roberge, F., H. Loberboum-Galski, P. Le Hoang, M. de Smet, C.-C. Chan, D.. Fitzgerald, and I. Pastan. 1989. Selective immunosuppression of activated T cells withh the chimeric toxin IL-2-PE40. inhibition of experimental autoimmune uveoretini-tis.. J. Immunol. 143: 3498-3502

25.. Lipham, W. J., T. M. Redmond, H. Takahashi, J. A. Berzofsky, B. Wiggert, G. J. Chader,, and I. Gery. 1991. Recognition of peptides that are immunopathogenic but cryptic.. Mechanisms that allow lymphocytes sensitized against cryptic peptides to initiatee pathogenic autoimmune processes. J. Immunol. 146: 3757-3762

26.. Lehmann, P. V., T. Forsthuber, A. Miller , and E. E. Sercarz. 1992. Spreading of T-cell autoimmunityy to cryptic determinants of an autoantigen. Nature 358: 155-157

27.. Hu, L.-H., T. M. Redmond, H. Sanui, T. Kuwabara, C. G. McAllister, B. Wiggert, G.. J. Chader, and I. Gery. 1989. Rat T-cell lines specific to a nonimmunodominant determinantt of a retinal protein (IRBP) produce uveoretinitis and pinealitis. Cell. Immunol.Immunol. 122: 251-261

28.. Sanui, H., T. M. Redmond, S. Kotake, B. Wiggert, L.-H, Hu, H. Margalit, J. A. Berzofsky,, G. J. Chader, and I. Gery. 1989. Identification of an immunodominant and highlyy immunopathogenic determinant in the retinal interphotoreceptor retinoid-bindingg protein (IRBP). J. Exp. Med. 169: 1947-1960

29.. Abe, T. and T. Shinohara. 1990. S-Antigen from the rat retina and pineal gland have identicall sequences. Exp. Eye Res. 51: 111-112

30.. Cornette, J. L., K. B. Cease, H. Margalit, J. L. Spouge, J. A. Berzofsky, and C. DeLisi. 1987.. Hydrophobicity scales and computational techniques for detecting amphipathic structuress in proteins. J. Mol. Biol. 195: 659- 685

31.. Fox, G. M.j T. M. Redmond, B. Wiggert, T. Kuwabara, G. J. Chader, and I. Gery. 1987.. Dissociation between lymphocyte activation for proliferation and for the capacity too adoptively transfer uveoretinitis. J. Immunol. 138: 3242—3246

32.. Mannie, M. D., P. Y. Paterson, D. C, U'Prichard, and G. Flouret. 1989. Encephalito-genicc and proliferative responses of Lewis rats lymphocytes distinguished by position 75-- and 80-substituted peptides of myelin basic protein. J. Immunol. 142: 2608-2616

33.. Merryman, C. F., N. Smith, and L. A. Donoso. 1990. Identification of multiple associativee and dissociative proliferative and uveitogenic T-cell sites in human interstitial retinoid-bindingg protein. Curr. Eye Res. 9: Suppl. 97-102

34.. Fling, S. P., L. A. Donoso, and D. S. Gregerson, 1991. In vitro unresponsiveness to autologouss sequences of the immunopathogenic autoantigen, S-antigen. J. Immunol. 147:: 483-489

35.. Yamashita, K. and E. Hbcr-Katz. 1989. Lack of immunodominance in the T cell responsee to herpes simplex virus. J. Exp. Med. 170: 997-1002

36.. Kalsow, C. M., A. F. Dwyer, A. W. Smith, and J. P. Nifong. 1993. Pinealitis accompanyingg equine recurrent uveitis. Br. J. Ophthalmol. 77: 46-48

37.. Singh, V. K., H. K. Kalra, K. Yamaki, T. Abe, L. A. Donoso, and T. Shinohara. 1990. Molecularr mimicry between a uveitopathogenic site of S-Antigen and viral peptides. J. Immunmal.Immunmal. 144: 1282-1287

91 1

Chapterr 5

Immunogenicityy and Pathogenicity of Humann S-Ag Determinants in Rat Strains

Marcc D. de Smet' +

Sumeett Mainigi+


RATT STRAIN SUSCHPTIBIUTY TO HS-AG DETERMINANTS

Abstract t

Aim:: The present study was initiated to determine rat strain susceptiblity to developing uveitis followingg immunization with immunopathogenic determinants of human S-Antigen (S-Ag), as identifiedd in the LEW rat.

Method:: LEW, F344, COP, WKY, WF and BN rats were immunized with peptides 180-200, 280-300,, 340-360, 350-370 of the human S-Ag sequence. Immunoproliferative responses were de-terminedd at day 14, antibody titers and pathologic score was established at day 30. The influence off pertussis toxin on disease severity was also assessed.

Results:: Strains sharing the same RT-1 (class II antigen) with LEW were most sensitive to dis-easee induction, though COP strain did not develop disease following immunization with any de-terminant.. In this strain, antibody titers were elevated to all determinants at day 30. The WF strainn also developed disease with all determinants except 280-300, though its RT-1 antigen dif-ferss from LEW. Lymphocyte proliferation indices at several determinant concentrations revealed similarr profiles in strains developing disease.

Conclusion:: Susceptibility to uveitis following immunization with determinants of human S-Ag iss not limited to a single RT-1 complex. Additional factors play a role in disease induction, pos-siblyy relating to the induction of a Thl versus a Th2 profile.

Introductio n n

Experimentall autoimmune uveitis (EAU) is a T-cell mediated autoimmune disease that can be in-ducedd in rodents and non-human primates by immunization with antigens such as soluble anti-genn (S-Ag), a retinal arrestin. EAU closely resembles the pathologic changes observed in certain humann forms of uveitis, and serves as a model for these sight-threatening diseases. As with all T-celll mediated processes. EAU is induced after the antigen is processed by antigen presenting cellss (APC) and presented via the major histocompatibility complex (MHC) to the T cell recep-torr (TCR).

Uveitiss susceptibility varies considerably between different strains of rats and mice12. While Lewiss rat are highly susceptible to EAU, Fisher rats, which share the same class II antigen, are muchh more resistant and require the addition of pertussis adjuvant for disease induction2-1. In ad-diton,, whereas most antigens are specifically class II restricted, there exists a small set of anti-genicc determinants which are capable of eliciting an immune and/or pathogenic response from moree than one class II antigen among the various strains of a given species, A subgroup of these antigenss can even elicit a response in several animal species'.

Thee present study aimed at determining the immunogenic and pathogenic potential of certain de-terminantss of human S-Ag in a variety of rat strains. The chosen sequences were all highly path-ogenicc in the Lewis rat, with one corresponding to the immunodominant sequence and capable off inducing EAU at a very low immunizing dose?\ Certain chosen determinants were capable of onlyy recognizing self in lymphocyte proliferation assays, while others were also capable of rec-ognizingg whole S-Ag. The chosen MHC profiles varied from complete homology with Lewis

95 5

ChapterChapter 5

Tablee 1: Peptide sequence, immunologic and pathologic characteristics in the Lewis (LEW) rat:

Determinant t Locationn (hSAg)

180-200 0 280-300 0 340-3600 * 350-370 0

Sequence e

VQHAPLEMGPQPRAEATWQF F

TLTLLPLLANNRERRGIAL D D GFLGELTSSEVATEVPFRLM M VATEVPFRLMHPQPEDPAKE E

Immunogenic c

+ +

++ +

+ +

+ +

ty y Pathogenicity y

++++ +

++++ +

++++ +

++++ +

Bovine e S-Ag--

--++ +

+ +

+ +

Abilit yy of lymphocytes from rats immunized with the determinant to recognize S-Ag in culture, ** Determinant able to induce disease at a dose < 10 u.gm. The immunodominant epitope for human S-Ag is nest-edd in this sequence.

classs II antigen, to partial or total absence of homology. Thus, a response map to human S-Ag de-terminantss in a variety of rat strains could be established.

Methodology: :

Peptidee synthesis and sequence selection: Oligomericc peptides of human S-Ag measuring 20 amino acids in length were synthesized by Appliedd Biosystems (Foster City, CA, USA) using t-butyloxycarbonyl derivatives of amino acids onn an automated solid-phase peptide synthesizer and were purified by HPLC to at least 95% pu-rity.. The amino acid composition of each peptide was verified using amino acid analysis and au-tomatedd gas-phase sequencing. The sequence of each studied peptide as well as its immuno-genicityy and pathogenicity in the Lewis rat is summarized in tabic 1(.

Immunizationn protocol and follow-up: Malee rats between 8 - 10 weeks of age were purchased from Harlan Sprague Dawley Microbio-logicall Associates (Walkerville, MD). The following rat strains were studied: Lew, F344, COP, WKY,, WF, BN. Animals were immunized with a single subcutaneous injection, in the hind foot-padd of complete Freund's adjuvant containing a final concentration of 100(g/rat of peptide de-terminantt and 2.5 mg/mL of Mycobacterium tuberculosis strain 37RA (Difco, MI). Half of the animalss used for the assessment of the pathological response were given an intravenous injection

Tablee 2: Pathologic score at day 30 following immunization with or without Pertussis

Ratt Strain* Bovine 18O-2O0" 280-300 340-360 350-370 S-Ag g

Lew' ' F3441" " COP" " WKY1 1

WF" " BN" "

+* *

4.0 0 3.0 0 0 0 0 0 0 0 0 0

— — 4.0 0 2.0 0 0 0 0 0 0 0 0 0

+ +

4.0 0 1.0 0 0 0 1.0 0 1.0 0 0 0

--3.0 0 0 0 0 0 0 0 0 0 0 0

+ +

4.0 0 0 0 0 0 0 0 0 0 0 0

--3.5 5 0 0 0 0 0 0 0 0 0 0

+ +

4.0 0 3.0 0 0 0

4.0 0 2.0 0 0 0

--4.0 0 2.0 0 0 0

2.0 0 3.0 0 0 0

+ +

3.0 0 3.0 0 0 0

4.0 0 4.0 0 0 0

--4.0 0 2.0 0 0 0

2.0 0 3.0 0 0 0

** In superscript is given the RT-1 (MHC class II) for each rat strain 11 Numbers correspond to the determinant location within the human S-Ag sequence". 11 + corresponds to the animals with additon of pertussis toxin, and - to animals which did not receive pertussis. Eachh data point is the average of 4 animals.

96 6

RATT STRAIN SUSCEPTIBILITY TO HS-AG DETERMINANTS

off Pertussis toxin. Animals used in the lymphocyte proliferation assays were killed on day 1 2 to 14,, while the animals used for evaluation of pathogenicity were killed on day 30. For each assay. 44 animals were used. Pathology was graded based on the degree of cellular infiltration and the degreee of retinal damage as stated elsewhere'1. The study was approved by the Animal Care Com-mitteee of the National Eye Institute and adhered to the policies established by the Public Health Servicee on the Humane Care and Use of Laboratory Animals.

Lymphocytee proliferatio n assay: Mononuclearr cells taken from the draining inguinal nodes of immunized animals were tested on dayy 12 to 14. Cells were cultured at concentration of 1.5 x 10h cells/mL in 96 well flat-bottomed microliterr plates (Costar, Cambridge MA) containing 0.2 mL of RPMT supplemented with glut-aminee and 1% heat inactivated normal rat serum, and 2 mercapto-ethanol. All cultures were per-formedd in quadruplicate against the peptide used for immunization, serially diluted from 100 p-g/mLL down to 0.001(g/mL. Concanavillin A was used as a positive control of proliferation. Culturess were incubated for 4 days at 37 (C in 5% CO,. For the last 12 hours, prior to harvesting att 5 days, each well was pulsed with 'H-thymidine (New England Nuclear, Boston, MA; 2 Ci/mmol,, 0.5 |xCi per 10 plVwell). Results are expressed as a stimulation index (SI - mean c.p.m.. in stimulated cultures/mean c.p.m. in unstimulated control cultures).

ELL ISA assays: Serumm collected on animals killed on day 30 was used to determine the IgG antibody present againstt the immunizing peptide. The peptide was diluted to 0.5 ixg/mL in PBS (pH 7.36) and platedd overnight at 4 °C on 96-weli flat-bottomed Immulon IV plates (Dynatech). Plates were washedd x3 with PBS and incubated with 2% bovine serum albumin (BSA) in PBS for 4 hours at roomm temperature followed by a repeat wash. Fifty microliters of sera diluted to 1:4() in 1 % BS A-PBS-Tweenn 20 (0.05%) (PBS-T) was added to triplicate wells and incubated for 2.5 hours at roomm temperature. Plates were washed three times in PBS-T, left in PBS-T for 10 minutes fol-lowedd by an additional 3 washes. Fifty microliters of the alkaline phosphatase-conjugated sec-ondaryy antibody goat anti-rat IgG (kpl, Gaithersberg, MD) (1:1000) in PBS-T was added, and the platess incubated for 1 hour at room temperature. Plates were washed three times in PBS-T, once inn de-ionized distilled water, and once in p-nitrophenyl phosphate disodium buffer (NPP; Sig-ma),, before adding the chromogenic agent. After further incubation, the optical density (OD) was readd at 490 nm with an ELISA plate reader equipped with the appropriate filters (Molecular De-vices,, Menlo Park, CA).

Tablee 3: Lymphocyte proliferation responses to immunizing antigen between day 12-14 post immunization (anti-genn concentration in culture 10 |ULg/mL)

Ratt Strain*

Lew' ' F3441-' ' COP11 1

WKY1 1

WP P BN" "

Bovinee S

3.7 7 2.0 0 1.8 8 2.2 2 1.7 7 2.3 3

-Ag g 180-200* *

3.7 7 2.0 0 1.8 8 2.2 2 1.7 7 2.3 3

280-300 0

17.0 0 8.0 0 2.9 9

31.0 0 1.9 9 1.6 6

340-360 0

22.5 5 3.6 6

14.5 5 25.7 7 10.7 7 1.6 6

350-370 0

16.5 5 8.0 0 1.2 2

18.0 0 5.7 7 1.5 5

** In superscript is given the RT-! (MHC class II) for each rat strain ** Numbers correspond to the determinant location within the human S-Ag sequence".

97 7

ChapterChapter 5

elicitedd no response in all remaining species, while determinant 180-200. in the presence of per-tussiss did cause a very mild degree of inflammation in F344M, WKY'and WP.

Lymphocytee proliferative responses were studied in two ways. At a fixed antigen dose of 10 u-g/mL,, proliferation responses showed a dissociation between proliferation and pathogenicity (tablee 3). COP"1 which appeared resistant to disease induction nonetheless showed a strong pro-liferativee response to determinant 340-360. The strongest proliferative responses were observed inn WKY1, and were comparable or above those observed with LEW'. This was also evident when studyingg the proliferative response over a range of antigen concentrations {Figure 1 -4). Sharing thee same RT-1 lead to a proliferative response at a lower antigen concentration than when the RT-11 locus was different, with the exception of determinant 180-200, which did not appear to elicit muchh of a proliferative response in any strain except at relatively high concentrations. For im-munoproliferativee determinants, a difference of 10 to 1000 fold in the minimum peptide concen-trationn was noted between strains sharing the "1" locus and those which did not. WKY' gave an equall or higher proliferative response than LEW', though this did not translate in a pathogenic response. .

BB cell stimulation was also studied in the form of serum antibody levels to the immunizing pep-tide.. There was littl e correlation between the presence of an antibody titer and the development off a cellular immune response or the presence of ocular disease. Note that while BN rats showed littl ee proliferative ability to all tested peptides, it did show high antibody titers to determinant 340-360.. Similarly, COP which did not proliferate well to antigenic stimuli in vitro, gave a high antibodyy response to all determinants, either in absolute terms or relative to other tested strains.

Discussion n

Thee immune response in mice has been carefully dissected in a number of autoimmune models'\ Itt appears to begin as a balanced cytokine response which then becomes polarized toward a type 11 T helper cell response (Th 1) in disease-susceptible animals, and a type 2 response (Th2) in dis-ease-resistantt genotypes". The fi nal immune profile can also be influenced by the presence or ab-sencee of adjuvants (incomplete versus complete Freund's adjuvant)7, or the addition of pertussis toxi".. In this study, an attempt was made to determine rat strain susceptibility to uveitis when im-munizedd with immunopathogenic determinants of S-Ag. The influence of pertussis toxin was al-soo assessed with regards to its ability to favor an immunopathogenic response.

Strainss which contained the same class II MHC as the lewis rat were more likely to develop dis-ease,, but this susceptibility was not always present. In the case of COP no disease induction was seen.. In this particular strain, a high antibody titer was observed to all immunized determinants, possiblyy suggesting that a strong Th2 response had favored B cell proliferation. Addition of per-tussiss toxin did help in eliciting a pathologic response to determinant 180-200 and heightened the pathogenicc response in all pathogenic determinants in the WKY rat strain.

Lymphocytee proliferative response profiles in susceptible strains were similar irrespective of the RT-11 locus. A recent theory suggests that the interaction between T-cell receptor, peptide and MHCC can determine Thl/Th2 dominanc. According to this theory. Th phenotype is determined byy the ability of a given MHC to bind antigen. If present in sufficient quantity, a Th 1 response re-

100 0

RATT STRAIN SUSCEPTIBILITY TO HS-AG DETERMINANTS

suits.. Plotting lymphocyte proliferation against a wide range of antigen concentrations, allows onee to determine the proliferative potential of a given antigen, which in a crude way reflects the bindingg affinity to MHC and TCR. An analysis of the cytokines secreted in the presence of the determinantss at each concentration would more clearly define the exact Th profile but was be-yondd the scope of this study. Additional factors also play a important role. In the case of F344, lymphocytee proliferation was low to certain determinants, despite clearly inducing a Thl profile. Expressionn of co-stimulatory factors , fluctuations in the cytokine microenvironment, as well as neuro-endocrinee factors can all influence the level of pathologic response.

Inn summary, we have shown that certain determinants of human S-Ag are capable of causing an immunopathologicc response in a number of rat strains not all of which share the same class II MHCC antigen as the LEW rat. In addition, with the exception of COP, antibody titers were vari-ablee and did not appear to correlate with disease susceptibility. Finally, susceptible rat strains ap-pearedd to generate similar lymphoproliferative response curves. Further studies on cytokines re-leasedd in lymphocyte proliferation assays from these various strains would help to further delineatee the role of Thl and Th2 cells in both susceptible and resistant strains.

References s

1.. Sun B. Rizo LV, Sun SH, Chan CC, Wiggert B. Wilder RL, et al. Genetic susceptibility to experimental au-toimmunee uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2-like re-sponse.. J Immunol 1997: 159: 1004-11.

2.. Caspi RR, Sun B, Agarwal RV, Silver PB, Rizzo LV, Chan CC, et a], T cell mechanisms in experimental au-toSimmunee uveoretinitis: susceptibility is a function of the cytokine response profile. Eye 1997: 11: 209-12.

3.. Stephaniak JA, Gould KE. Sun D. Swanborg RH. A comparative study of experimental autoimmune en-cephalomyelitiss in Lewis and DA rats. J Immunol 1995; 155: 2762-69.

4.. Fukushima A, Lai JC, Chanaud NP. III . Shiloaeh J, Whitcup SM. Nussenblatt RB, et al. Permissive recogni-tionn of immunodominant determinants of the retinal S-antigen in different rat strains, primates and humans. Intt Immunol 1996:9: 169-77.

5.. Gregerson DS, Merryman CF, Obritisch W, Donoso LA. Identification of a potent new pathogenic site in hu-mann retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats. Cell Immunol 1990;; 128:209-19.

6.. de Smet MD. Bitar G, Roberge FG, Gery I, Nussenblatt RB. Human S-antigen: presence of multiple im-munogenicc and immunopathogenic sites in the lewis rat. J Autoimmun 1993; 6: 587-99.

7.. Yip HC, Karulin AY, Tary-Lehmann M, Hesse MD, Radeke H, heeger PS, et al. Adjvant-guided tyupe-1 and type-22 immunity: infectious/noninfectious dichotomy definesthe class response. J IMmunol 1999; 162: 3942-49. .

8.. Sun B. Sun SH, Chan CC, Wiggert B, Caspi RR. Autoimmunty to a pathogenic retinal antigen begins as a bal-ancedd cytokine response that polarizes towards type 1 in a disease-susceptible and towards type 2 in a disease-resistantt genotype. Int Immunol 1999; 11: 1307-12.

9.. Silver PB, Chan CC, Wiggert B. Caspi RR. The requirement for pertussis to induce EAU is strain-dependent: B10.RIII,, but not B10. A mice,, develop EAU and Thl responses to IRBP without pertussis treatment. Invest Ophthalmoll Vis Sci 1999; 40: 2898-905.

10.. Myrray JS. How the MHC selects Thl/Th2 immunity. Immunol Today 1998; 19: 157-63 11.11. Kuchroo VK. Das MP. Brown JA, Ranger AM. Zamvil SS. Sobel RA. et al. B7-1 and B7-2 costimulatory

moleculess activate differentially the Thl/Th2 developmental pathways: application to automimmune disease therapy.. Cell 1995;80:707-18.

12.. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+T cells. Annu Rev Immunol 1994;; 12:635-73.

101 1

Cfl42&eï$ Cfl42&eï$

13.. MacPbee IM/t, Antoai JFAt Mason DW. Spontaneous reepv y of rats frqm ex|>eriiaejnt8l afl*?gte «»-cepbaloinyeKtiss is dependent on regulation &t thé iptótihe system by endogenous adrenal corticosteroids. J ExpMedd 1989; 169:431^5.

1 1

i i

! !

! !

m m

Sectionn II : Immunologic Responses to Ocular Autoantigens in Humans

"..the"..the certainty of real knowledge which men arrived to in these sciences, was not derived from anyany particular advantage they received from two or three general maxims, laid down in the be-ginning;ginning; but from the clear, distinct, complete ideas their thoughts were employed aboul,and the relationrelation of equality and excess so clear between some of them, that they had an intuitive knowl-edge,edge, and by that, a way to discover it in others...."

Johnn Locke: An Essay Concerning Human Understanding, 1689

Chapterr 6

Cellularr Immune Responses of Patients with Uveitiss to Retinal Antigens and their Fragments

Marcc D. de Smet, Joyce H. Yamamoto, Manabu Mochizuki, Igal Gery, Vijayy K. Singh, Tochimichi Shinohara, Barbara Wiggert,

Geraldd J. Chader, Robert B. Nussenblatt

Americann Journal of Ophthalmology 110: 135-142, 1990 (byy permission © Elsevier Science Inc.)

S - AGG AND IRB P IMMUN E RESPONSES IN PATIENTS

Cellularr Immune Responses of Patients Wit h Uveitis to Retinall Antigens and Their Fragments

Mar cc D. de Smet, M.D. , Joyce H. Yamamoto, M.D. , Manabu M o c h i z u k i , M.D. , Igall Gery, Ph .D, Vi ja y K. S i n g h, M.D. , Toch im ichi Sh inohara, Ph .D.,

Barbar aa Wigger t , Ph.D., Gera ld J. Chader, Ph .D., and Robert B. Nussenb la t t, M.D .

Off two patient populations totalin g 82 pa-tients,, one in the United States and the other inn Japan, we studied the cellular immune responsess against S-antigen and interphotore-ceptorr retinoi d bindin g protein as well as to fragmentss of each antigen. Behcet's disease, birdshott retinochoroidopathy, pars planitts , ocularr sarcoid, sympathetic ophthalmia, and thee Vogt-Koyanagi-Harada syndrome were di-agnosedd in these patients. The response pro-filefile of both antigens paralleled each other. Thi ss profil e was more commonly seen in pa-tientss suffering from diseases affecting the retina.. Responders reacting to both antigens orr to several fragments of an antigen were present.. Thi s pattern of response was seen in 266 of the patients tested. Patients wit h uveitis appearedd able to recognize several autoanti-gens.. Thi s might be a consequence of the breakdownn of the blood-retinal barrie r and mayy help perpetuate the inflammator y proc-ess.. Several patients were capable of respond-ingg to more than one epitope of the same antigen,, which indicates that there are major differencess between the experimental model andd human autoimmune diseases in the re-sponsee to autoantigens. Both of these findings mayy to help develop new immunotherapeutic strategiess in the treatment of uveitis.

INTRAOCULARR INFLAMMATOR Y DISEASE (uveitis) is thee cause of about 10% of severe visual loss in

Acceptedd fur publication May 14, 1990. Fromm the Laboratories of Immunology (Drs. de Smet,

Gery,, Singh, Shinohara, and Nussenblatt) and Retinal Celll and Molecular Biology (Drs. Wiggert and Chader), Nationall Eye Institute, National Institutes of Health, Bethesda,, Maryland, Department of Ophthalmology, Tokyoo University (Dr. Yamamoto), and Tokyo University Branchh Hospital (Dr Mochizuki}, Tokyo, Japan,

Reprintt requests Marc D. de Smet, M.D,, Laboratory of Immunology,, National Eve Institute, Bldg. 10, Rm. 10N202,, Bethesda, MÜ 20892.

thee United States.1 The understanding of under-lyingg mechanisms has been increased by the developmentt of an experimental uveitis model inn animals, induced either by the retinal S-antigenn or by the interphotoreceptor retinoid bindingg protein.1'r> Nussenblatt and associ-ates,1'77 Doekes and associates/ and Froebel and associates1** have described patients with uveitis whoo have cell-mediated responses to these an-tigens.. Several fragments of both ant igens have beenn identified as being uveitogenic in ani-mals.1"1-- Observations from experimental auto-immunee uveitis as well as other models of autoimmunee disease have clearly demonstrated thatt the immune systems of different species respondd to different epitopes (that is, frag-ments)) of a given molecule. Cell-mediated and humorall responses are invariably directed againstt different parts of the molecule.

Wee examined the cell-mediated responses of patientss with uveitis to both of these uveito-genicc molecules, as well as to representative fragmentss that have been reported as being uveitogenicc in both lower mammals and in subhumann primates. These responses may en-couragee the development of treatment strate-giess using these molecules and fragments.

Patient ss and Method s

Patientss participating in this study were seen inn the uveitis clinic of the National Eye Insti-tute,, Bethesda, Maryland, and at the Tokyo Universityy Branch Hospital, Tokyo, japan. All patientss gave informed consent before partici-patingg in the study. They were part of an ongoingg protocol approved by each institu-tion'ss committee on human investigation. Al l patientss had active uveitis involving the poster-iorr segment or had a history of active disease involvingg the retina or choroid. The patients

107 7

ChapterChapter 6

testedd had one of the following disorders: Behcet'ss disease, birdshot retinochoroid-opathy,, pars planitis, ocular sarcoid, sympa-theticc ophthalmia, or the Vogt-Koyanagi-Haradaa syndrome.11 Patients with Behcet's diseasee met at least the minimal criteria for incompletee Behcet's set by the Behcet's Disease Researchh Committee of japan14 with all patients havingg ocular disease. Patients with birdshot ret inochoroidopathyy had cream-colored lesions inn the posterior segment, macular edema, and retinall vascular changes. These pat ients were HLAA A-29 positive as well. Patients with sym-patheticc ophthalmia had a history of penetrat-ingg trauma or multiple operations followed by a bilaterall granulomatous uveitis. Patients with Vogt-Koyanagi-Haradaa syndrome were of ei-therr Japanese or American Indian heritage and hadd ocular and systemic changes compatible withh the disorder. The pat ients with ocular sarcoidd had bilateral granulomatous uveitis usuallyy accompanied by either a positive galli-umm scan or a noncaseating granuloma on a biopsyy specimen. Patients were tested irrespec-tivee of their current medical therapy (usually consistingg of cyclosporine, prednisone, or both) orr of their level of activity. Since the antigens testedd were of retinal origin, anterior segment inflammationn was not considered as part of the definitionn of active disease. The presence of retinall infiltrates, perivasculit is, snowbanking, orr vitreous haze were accepted as evidence of activity.. Addit ionally, cystoid macular edema confirmedd by fluorescein angiography was con-sideredd a sign of active disease. Al l the diagnos-ticc categories were based on clinical criteria exceptt ocular sarcoid and birdshot retinocho-roidopathy,, in which confirmation by another testt was required. Control subjects were se-lectedd from either nonresearch staff or from clinicc pat ients not being seen for a uveitic condit ion,, and in whom a retinal or choroidal disorderr had been ruled out.

Ant igenss used in this assay included bovine interphotoreceptorr retinoid binding protein purifiedd to homogeneity, as described by Red-mondd and associates,K ' and bovine S-antigen purifiedd by the method described by Dorey, Cozette,, and Faure.11' Peptides derived from interphotoreceptorr retinoid binding protein weree synthesized and purified by Applied Bio-systemss Inc., Foster City, California, using the t-BOCC chemistry, on a pept ide synthesizer 430A.. The pept ide sequences were derived fromm the sequence of bovine interphotoreceptor ret inoidd binding protein as determined and

reportedd by Borst and associates.17 This consist-edd of sequence 1158-1180 (HVDDTDLYLTIP -TARSVGAADGS)) for R-4 and of sequence 1169-11911 (PTARSVGAADGSSWECVCVVP-DV)) for R-14. Peptides derived from 5-antigen weree based on the bovine 5-antigen sequence reportedd by Shinohara and associates.11" The peptidess were synthesized in accordance with thee method of Donoso and associates,"" on a benzhvdrylaminee resin using an automated peptidee synthesizer (SAM II , Biosearch, Inc., Sann Rafael, California). The sequence for pep-tidee M corresponded to positions 303 to 320 (DTNLAS5T1IKECIDKTV),, and peptide N cor-respondedd to posit ions 281 to 302 (VPLLANN -RERRGIALDGKIKHE)) of the S-antigen.

Proliferationn assays were performed in the samee wav in Japan and the United States, ex-ceptt where indicated. Mononuclear leukocytes fromm heparinized blood samples were separat-edd on Isolymph gradients (Gallard-Schlesinger, Carlee Place, New York) and cultured in Rosweil Parkk Memorial Institute (RPMI) 1640 medium withh HEPES (G1BCO, Grand Island, New York), supplementedd with glutamine (2 mmol/1), pen-icilli nn (100 uni ts /ml), streptomycin (100 u.g/ ml),, and heat inactivated human AB serum. The Nationall Eye Institute used 20% serum from a singlee donor in the cultures, whereas Tokyo Universityy Branch Hospital used 10% commer-ciall serum (lot No. 14510, Pel Freez., Brown Deer,, Wisconsin).

Thee cells were cultured by twro methods. In thee first method, 2 x 10' cel ls/well were incu-batedd in flat-bottom, 96-weIl plates for five days."" In the second method, which under cer-tainn circumstances is believed to increase re-sponsess by increasing cell to cell interactions, 5 xx 10' cel ls/well were incubated in round-bot-tom,, 96-well plates for seven days,"" All cul-turess were in a total volume of 200 u.1 and were sett up in triplicate with or without st imulants. Thee antigen concentration was either 4, 20, 50, orr 100 u.g/ml. The cultures were incubated for thee specified time at 37 C with 100% humidity andd 5% carbon dioxide in air, pulsed for 16 hourss with r iH-thymidine (:fH-TdR, New En-glandd Nuclear, Boston, Massachusetts; 2 C i / mmol,, 0.5 p,Ci per 10 |xl/well) and harvested on glasss fiber filters using a MASH II harvester. Afterr drying, the filter pads were placed in vials withh 3 ml of toluene-based fluor and counted in aa Beekman L3801 liquid scintil lation counter. Severall peptides were tested simultaneously; however,, not all peptides could be tested on eachh patient. Cells from a control subject were

108 8

S-Ac;; AND IRBP IMMUNE RESPONSES IN PATIENTS

usuallyy tested simultaneously with cells from onee or more patients.

Thee mean of the triplicate cultures in counts perr minute was calculated for each set of repli-catee cultures. A stimulation index was derived byy dividing the mean for each of the antigen stimulatedd cultures by the mean for the control culturess in which no antigen was added. For eachh testing center and for each antigen, a meann stimulation index standard deviation wass calculated for the control subjects. A signi-ficantt response in a patient was considered to be presentt when the pat ient 's stimulation index forr a given peptide or determinant was above thee mean for the controls by two S.D.

Thee stimulation indices for each antigen test-edd were also compared by disease category to thee control subjects to determine if any statisti-callyy significant difference was present. Signifi-cancee was assessed by a standard nonpaired Student 'ss f-test. Patients were also assessed by theirr clinical activity. Testing for statistical sig-nificancee was done using chi-square. Results aree given as the mean stimulation indexstandardd error.

Result s s

AA total of 30 control subjects and 82 patients weree tested; 47 patients were from the United

Statess and 35 patients wrere from Japan. The averagee age of the patients in both groups was comparable;; 41 years of age (range, 1 0 to 70) fur thee American patients and 43 years of age (range,, 20 to 70) for the Japanese. The duration off follow-up was also similar in the two groups: 444 months in the United States (range, six to 108)) and 57 months in Japan (range, two to 247).. On average, uveitis had been diagnosed in thee patients for 63 months (range, two to 247). Al ll of the patients examined in Japan were of Japanesee descent, whereas in the American group,, 41 pat ients were white, five were black, andd one patient was Oriental, The number of patientss with clinically active disease varied amongg the various categories (Table 1) and betweenn countries. Overall, half of the pat ients testedd had active ocular disease. The largest discrepancyy was found among pat ients with sarcoid,, in which a greater number of the Japa-nesee patients had active disease. Of all the groups,, the patients with birdshot ret inocho-roidopathyy had the lowest incidence of activity, andd none were tested in Japan, where the dis-easee is extremely rare. The proport ion of pa-tientss with active disease was highest among thosee suffering from Behcet's disease.

Thee various disease entit ies responded differ-entlyy to the uveitogenic antigens (Table 2). A similarr response profile, however, was found forr S-antigen and interphotoreceptor retinoid bindingg protein. Patients with diseases involv-

TA8LEE 1 CHARACTERISTICSS OF AMERICAN AND JAPANESE PATIENTS

TESTINGG MEAN AGE DURATION OF NO OF MALE/ CLINICAL ACTIVITY

CENTERR (YRSf DISEASE (MOS)' PATIENTS FEMALE ACTIVE/IN ACTIVE CYCLOSPORINE PREDNISONE CYTOTOXICS CLINICALL ENTITY

Behcet'ss disease

Vogt-Koyanagi--

HH ara da syndrome

Ocularr sarcoid

Parss planitis

Birdshott retino-

chh oroid opa thy

Sympathetic c

ophthalmia a

Normal l

U.S. .

Japan n

U.S. .

Japan n

U.S. .

Japan n

US . .

U.S. .

US . .

U.S. .

Japan n

344 (28-42)

388 (24-60)

344 (24-60)

477 (29-65)

50(34-61) )

455 (20-70)

311 (16-49)

566 (46-66)

52(10-70) )

355 (26-50)

37(14-67) )

50(10-96) )

54(12-126) )

333 (6-60)

933 (4-247)

322 (7-72)

43(2-180) )

455 (24-60)

67(24-108) )

366 (6-72)

— — — —

8 8

16 6

9 9

10 0

9 9

9 9

6 6

9 9

6 6

20 0

10 0

5/3 3

16/0 0

1/8 8

5/5 5

8/1 1

3/6 6

2/4 4

5/4 4

2/4 4

10/10 0

6/4 4

5/3 3

11/5 5

2/7 7

0/10 0

5/4 4

6/3 3

5/1 1

1/8 8

4/2 2

— — — —

4 4

8' '

2 2

1 1

2 2

0 0

2 2

2 2

1 1

— — — —

8 8

4 4

4 4

7 7

4 4

5 5

3 3

2 2

5 5

** Average, The range is given tn parentheses, ff Nine patients with Behcet's disease also received colchicine.

109 9

ChapterChapter 6

TABL EE 2

RESPONSESS TO PEPTIDE DETERMINANTS OF INTERPHOTORECEPTOR RETINOID BINDING PROTEIN AND S-ANTIGENN UNDER OPTIMAL CULTURE CONDITIONS'

ANTIGENN TESTED

Interphotoreceptor r

retinoidd binding protein

R-4 4

R-U U

S-antigen n

MM peptide

NN peptide

BEHgETS S

DISEASE E

USS JAPAN

3/88 6/16

4/88 —

2/88 2/16

3/88 3/16*

6/88 2/16*

3/4'' 6/16*

VOGT--

KOYANAGI--HARADA A

SYNDROME E

US S

1/9 9

2/9* *

2/9* *

4/9' '

4/9 9

1/4 4

JAPAN N

1/10 0

4/10' '

--3/10 0

2/10* *

1/10' '

5/10* *

OCULAR R

SARCOID D

U.S. .

0/9 9

1/9 9

2/9" "

1/9' '

3/9 9

0/4 4

JAPAN N

0/9 9

3/9' '

— — 2/9 9

0/9 9

3/9* 3/9*

7/9* *

BIRDSHOTT RETINO-

CHOROIDOPATHY Y

U S S

1/9 9

5/9' '

3/9' '

3/9 9

3/9 9

4/9 9

PARSS PLANITIS

US S

2/6 6

1/6' '

1/5' '

1/6 6

1/6 6

0/6 6

SYMPATHETIC C OPHTHALMIA A

U.S. .

2/6 6

2/6' '

2/5 5

1/6 6

0/6 6

1/5' '

** The numerator refers to the number of positive responders The denominator refers to the total number of patients tested. In most

cases,, five-day cultures with 20 ^ig/m) of antigen in each well were found to be optimal except where indicated.

'' Cells were cultured with 4 ^g/ml for seven days.

** Antigen concentration was 100 jug/ml.

** Cells were cultured for seven days with 20 Mg/ml of antigen.

ingg the retina were more likely to have a signifi-cantt proliferative response. In American pa-t ients,, the mean proliferative responses to the S-antigenn (20 u-g/ml) were significant for Behcet'ss disease, 2.8 1.0 (P = .05) and bird-shott ret inochoroidopathy, 2.7 0.7 (P = .02) whereass the control was 1.3 0.2. For the interphotoreceptorr retinoid binding protein (20 jxg/m!),, the proliferative responses were lower, withh a significant difference in Behcet's disease, 1.55 0.4 (P = .04), birdshot retinochoroid-opathy,, 1.4 0.3 (P = .04), and pars planitis, 1.44 0.02 {P = .04) as compared to the control, 0.99 0.1. A similar pattern of response was foundd in the Japanese patients, with the highest numberr of responders found in Behcet's dis-ease.. The patients with ocular sarcoid gave few positivee responders. Their responses to phyto-hemagglut ininn and purified protein derivative, however,, were strong, which indicates that the lackk of response is not caused by a generalized statee of unresponsiveness, but probably reflects ann inability to recognize bovine S-antigen or bovinee interphotoreceptor retinoid binding protein.. Culturing the cells for seven days in round-bot tomm wells increased the number of significantt responses in some groups, but not in all.. There was an increase in sensitivitv mainly forr pat ients with the Vogt-Koyanagi-Harada syndromee where there was an increase in the numberr of significant responders to the S-

antigenn and the interphotoreceptor retinoid bindingg protein. The mean response to the S-antigenn for lymphocytes from patients with Vogt-Koyanagi-Haradaa syndrome in the United Statess was 2.8 1.4, as compared to control subjectss (1.8 + 0.1), which was a statistically significantt difference {P = .03).

Inn addition to testing in vitro responses to the interphotoreceptorr retinoid binding protein andd the S-antigen, the responses to peptide fragmentss of each of these ant igens were deter-minedd (Table 2). There was a correlation be-tweenn the intensity of the proliferative re-sponsee to the interphotoreceptor retinoid bindingg protein or the S-antigen and the exis-tencee of a significant proliferative response to onee or more of the peptide fragments of that antigen.. In five-day cultures, if considering onlyy statistically significant responders to 5-antigen,, seven of nine American patients had a significantt response to one or both peptide fragmentss tested. In seven-day cultures, three off five Japanese patients Tesponsive to S-anti-genn had a response to either M peptide or N peptide,, or to both. In six of the nine American patientss who responded to the interphotore-ceptorr retinoid binding protein, there was a responsee to either R-4 or R-14, or to both. No suchh correlation was seen in the Japanese re-sponderss to interphotoreceptor retinoid bind-ingg protein.

110 0

S-AGG AND IRBP IMMUNE RESPONSES IN PATIENTS

Severall pat ients' lymphocytes had a prolifer-ativee response to a peptide fragment, but did nott recognize the parent antigen. In 12 of 22 Americann patients (54%) responding to one or thee other S-antigen fragment at 20 or 100 p,g/ mll in five-day cultures, there was no cross reactionn with the whole molecule. Under simi-larr condit ions, the responses to interphotore-ceptorr retinoid binding protein fragments gave noo cross reaction in 16 of 20 (84%) American patients.. The Japanese responders to S-antigen fragmentss recognized S-antigen in only six of 199 cases (cells cultured for seven days).

Patientss with Behcet's disease showed a re-sponsee to fragments of both antigens but the responsess were strongest for fragments of S-antigen.. At 20 jxg/ml, M peptide gave the highestt response with a mean stimulation index off 5.3 10 for patients and 1.3 0.2 for controll subjects (P = .0001). The response to N peptidee was also significant with pat ients hav-ingg a mean stimulation index three t imes higher thann control subjects. Patients with birdshot retinochoroidopathyy had similar responses to bothh sets of fragments. The mean stimulation indicess in patients were twice those of control subjectss for both sets of fragments.

Severall patients demonstrated an ability to givee simultaneously a significant proliferative responsee to at least one determinant of each antigen,, but not necessarily to the whole anti-gen.. A total of 32 patients out of the 82 patients testedd (39%) were found to give such responses, 188 among the American patients and 14 among thee Japanese patients. They were found in all diseasee categories but were more frequently foundd among the patients with Behcet's disease orr birdshot retinochoroidopathy. A similar dis-tributionn of patients was found in the two countries.. Of the American patients 11 had activee disease at the time they gave a response too both antigens as compared to six of 18 nonresponderss (P = .02). There is littl e differ-ence,, however, with the number of patients withh active disease (seven of 11 patients) re-spondingg to only one antigen (P = .05). A correlationn between active disease and a signifi-cantt lymphoproliferative response was not foundd among the Japanese patients.

Inn comparing S-antigen to interphotorecep-torr retinoid binding protein, it appears that S-antigenn is more frequently correlated with activee disease (P = .003). As is shown in Table 3,, however, the profile in each disease entity is similarr for the two antigens. An attempt was

TABLEE 3 RELATIONSHIPP BETWEEN CLINICAL ACTIVITY AND THEE PRESENCE OF A SIGNIFICANT LYMPHOCYTE RESPONSEE TO INTERPHOTORECEPTOR RETINOID

BINDINGG PROTEIN AND S-ANTIGEN*

INTER--

PHOTO--

RECEPTOFI I

RETINOID D

CLINICAL L

ENTITY Y

Behcet'ss disease

vogt-- Koy an ag i - H arada

syndrome e

Ocularr sarcoid

Parss plan it is

Birdshot t

retinochoroidopathy y

Sympathetic c

TESTING G CENTER R

U.S. .

Japan n

U.S. .

Japan n

U.S. .

Japan n

U S S

U.S. .

U.S. .

BINDING BINDING

PROTEIN N

2/7 7

6/9 9

0/2 2

0/5 5

1/5 5

2/3 3

2/5 5

0/1 1

1/4 4

S-ANTIGEN N

3/7 7

3/4 4

1/2 2

0/2 2

1/5 5

0/0 0

2/5 5

0/1 1

2/4 4

** Numerator refers to those patients with active disease. The denominatorr refers to all patients with a significant proliferative responsee to the antigen at 20 figjm] or 100 M9/ml T"e data includee all responders with a stimulation index above 2.0 in both five-- and seven-day cultures.

madee to correlate proliferative responses with therapy,, but no correlation was possible. Pa-tientss with active disease were more likely to be treatedd with cyclosporine or prednisone.

Discussion n

Ourr aim in this study was twofold. First, we wantedd to determine the response profile of interphotoreceptorr retinoid binding protein in patientss with uveitis and to compare it to that of S-antigen.. Secondly, we wanted to determine whetherr patients were able to respond to frag-mentss of these antigens, which have been shownn to be uveitopathogenic in animals. Since thee cellular immune response centers around differentt epitopes in different animal species, wee did not know if patients would be able to reactt to any of these fragments in cellular pro-liferationn assays.

Severall previous studies demonstrated that cellularr proliferative responses to S-antigen

111 1

ChapterChapter 6

weree present in inflammatory condit ions affect-ingg the posterior pole, and in particular the retinaa of patients with uveitis.'17 Table 2 sug-gestss that the response profile for interphotore-ceptorr retinoid binding protein is nearly identi-call to that of S-antigen. Patients with Behcet's diseasee gave the highest number of positive responders.. This was true whether the data weree analyzed in terms of the number of pa-t ientss with a stimulation index substantially abovee that of control subjects, as in Table 2, or usingg the Student 's /-test, which compares each groupp as a whole. Using the Student 's f-test, aa significant response to interphotoreceptor ret inoidd binding protein is also found in pa-t ientss with birdshot ret inochoroidopathy, even thoughh Table 2 dues not show this result. The discrepancyy develops from a few control sub-jectss who were able to give a strong prolifera-tivee response to the ant igens tested, which causedd a substantial increase in the value of the standardd deviation.

interphotoreceptorr retinoid binding protein doess not appear to be as predictive of active diseasee as is S-antigen {Table 3). There are, however,, more patients responding to inter-photoreceptorr retinoid binding protein than to S-ant igen.. In the Japanese population, the numberr of pat ients with active disease who respondd to interphotoreceptor retinoid binding proteinn is greater. A much larger number of pat ientss would be required to determine the exactt relat ionship to disease activity. Patients whoo show an ability to proliferate concurrently too both antigens have a slight increase in their probabil i tvv of having active disease, but the differencee did not reach statistical significance. Inn relation to the mechanism of disease induc-tionn and its propagation, it is the existence of a responsee in culture and its greater prevalence in pat ientss with active disease that is significant. Inn a given patient, an in vitro proliferative responsee indicates that lymphocytes sensitized too the antigen tested are circulating in the peripherall blood. In the animal models, sensi-tizedd lymphocytes capable of an in vitro prolif-erativee response were one of the necessary condit ionss for the induction of autoimmune disease.211 -' It is likely, however, that the induc-tionn of uveitis is caused by mult iple factors. Therefore,, it is not surprising to find some controll subjects whose lymphocytes demon-stratee an in vitro response to these antigens. Sensit izat ionn to these ant igens might occur throughh a variety of means such as minor trau-maa or mimicry with other peptides. ' We believe

thatt the difference between a patient and a controll subject lies in the inability of control subjectss to initiate an inflammatory response or too maintain it once initiated. This could occur becausee all of the mechanistic criteria have not beenn met or because suppressor mechanisms aree strong enough to shut down the response.-"

Thee ability of some patients to respond to bothh retinal antigens in culture is a unique finding,finding, which may help to elucidate certain aspectss of the mechanisms involved in chronic uveitis.. We believe that a patient is initiall y sensitizedd to probably only one antigen; how-ever,, after the breakdown of the blood-retinal barrier,"'' the immune system becomes exposed too several new sequestered antigens. These autoant igenss are likely processed by circulating orr resident antigen presenting cel ls.M J: Partial digestionn of the antigen wil l generate frag-mentss that are then able to associate with ap-propriatee class II antigens.2" In the presence of thesee antigens a complex is formed, which whenn expressed on the cell surface is able to interactt and activate T lymphocytes. These acti-vated,, proliferating T lymphocytes can potenti-atee an acute inflammatory episode and possibly helpp perpetuate the inflammatory episode.

Patientss who responded to S-antigen or inter-photoreceptorr retinoid binding protein were ablee to recognize one or both fragments tested, butt none of the responses to the fragments were off a similar magnitude to that of the parent antigen.. This lesser response suggests that thesee fragments do not appear to be the pri-maryy mediators of the cell-mediated immune responsee in humans. Hence, we conclude that theyy are not immunodominant sites.29-10 Some patientss demonstrated an ability to generate an inn vitro response to both fragments of a given antigen.. This capacity to recognize more than onee epitope of S-antigen and interphotorecep-torr retinoid binding protein was not found in thee animal model. In the Lewis rat, immuniza-tionn with interphotoreceptor retinoid binding proteinn generates a strong proliferative in vitro responsee to peptide R-l 4, ' -whereass R-4, which wee also tested, is a nondominant fragment and iss not recognized by animals immunized with thee whole protein.1" In the case of S-antigen, onlyy one immunodominant site has been re-ported/'-- It is likely that this disparity reflects a fundamentall difference between the experi-mentall model and naturally occurring auto-immunee diseases. Future studies are needed too elucidate the importance of the various epitopess of S-antigen and interphotoreceptor

112 2

S - AÜÜ AND ÏRBP IMMUN E RESPONSES IN PATIENTS

retinoidd binding protein. Although most re-sponderss to S-antigen and interphotoreceptor retinoidd binding protein were able to respond too one or both fragments of these antigens, mostt of the responders to the fragments were nott able to recognize the parent antigen.

Thiss study shows that interphotoreceptor ret-inoidd binding protein has a response profile thatt is virtually identical to that of S-antigen. Severall patients were able to mount a response too both antigens in culture. Several patients alsoo showed the ability to respond to more than onee epitope of a given antigen. Both phenome-naa indicate that human disease is much more complexx than suggested by the current autoim-munee models of disease. Several autoant igens appearr to be implicated in chronic uveitis in humans,, and the interactions between each of thesee antigens is unknown. The extent of the plurispecificityy of response to autoant igens also remainss to be determined. It may be a general-izedd characteristic of human class II antigens, andd it is also possible that pat ients possess an enhancedd ability to interact with several differ-entt fragments as compared to normal individ-uals.. Further studies on cell l ines, and by using multiplee fragments of the antigens, may help answerr these questions.

References s

1.. National Institutes of Health: Interim Report of thee National Advisory Eye Council Support for Visu-all Research, U.S. Department of Health, Education, andd Welfare, 1976, pp. 20-22.

2.. Faure, J. P.: Autoimmunity and the retina. Curr. Top.. Eye Res. 2:215, 1980.

3.. Chader, G. J.: Interphotoreceptor ret inoid-bindingg protein (IRBP). A model protein of molecular biologicall and clinically relevant studies. Invest. Ophthalmol.. Vis. Sci. 30:7, 1989.

4.. Gery, I., Mochizuki, M., and Nussenblat t, R. B,: Retinall specific antigen and immunopathogenic processess they provoke. In Osborne, N., and Chader, G.. J. (eds.): Progress in Retinal Research. New York, Pergamonn Press, 1978, pp. 75-109.

5.. Gery, I., Wiggert, B., Redmond, T, M., Kuwa-bara,, T., Crawford, M. A., Vistica, B. P., and Chader, G.. ).: Uveoretinit is and pinealit is induced by immu-nizationn with IRBP. Invest. Ophtha lmol. Vis. Sci. 27:1296,, 1986.

6.. Nussenblatt, R. B., Gery, I., Ballintine, E. J., and Wacker,, W. B,: Cellular immune responsiveness of uveitiss pat ients to retinal S-antigen. Am. J. Ophtha l-mol.. 89:173, 1980.

7.. Nussenblatt, R. B., Mittal , K. K., Ryan, S., jr., Green,, W. R., and Maumenee, A. E.: Birdshot ret ino-

choroidopathyy associated with HLA-A2 9 ant igen andd immune responsiveness to retinal S-ant igen. Am.. J. Ophtha lmol. 94:147, 1982.

8.. Doekes, G., van der Gaag, R., van Kooyk, Y., Broersma,, L., Zaal, M. ). M., Dijkman, G., Fortuin, M,, E., Baarsma, G. S., and Kijlstra, A.: Humoral and cellularr immune responsiveness to human S-antigen inn uveitis. Curr. Eye Res. 6:909, 1987.

9.. Froebel, K. S., Armstrong, S. S., Cliffe, A. M., Urbaniak,, S, ),, and Forrester, J. V.: An investigation off the general immune status and specific responsive-nesss to ret inal-(S)-ant igen in pat ients with chronic posteriorr uveitis. Eye 3:263, 1989,

10.. Donoso, L. A,, Yamaki, K., MeTryman, C. F., Shinohara,, T., Yue, S., and Sery, T. W.: Human S-antigen.. Character izat ion of uvei topathogenic sites.. Curr. Eye Res. 7:1077, 1988.

11.. Sanui, H., Redmond, T. M., Hu, L.-H., Kuwa-bara,, T., Margalit, H,, Cornette, J. L., Wiggert, B., Chader,, G. J., and Gery, I.: Synthetic pept ides de-rivedd from IRBP induce EAU and EAP in Lewis rats. Curr.. Eye Res. 7:727, 1988.

12.. Sanui, H., Redmond, T. M., Kotake, S., Wig-gert,, B„ Hu, L.-H., Margalit, H,, Ber/.ofsky, J. A., Chader,, G. ] . , and Gery, I,: Identification of an im-munodominantt and highly immunopathogenic de-terminantt in the retinal interphotoreceptor ret inoid-bindingg protein (IRBP). J. Exp. Med. 169:1947, 1989.

13.. Nussenblatt, R. B., and Palestine, A. G.: Uvei-tis.. Fundamentals and Clinical Practice. Chicago, Yearr Book Medical Publ ishers, 1989.

14.. Behcet's Disease Research Commit tee of Ja-pan:: Behcet's disease. Guide to diagnosis of Behcet's disease.. Jpn. J. Ophtha lmol. 18:291, 1974.

15.. Redmond, T. M., Wiggert, B., Robey, F. A., Nguyen,, N. Y., Lewis, M. 5., Lee, L,, and Chader, G.. ].: Isolation and characterization of monkey inter-photoreceptorr binding protein, a unique extracellu-larr matrix component of the ret ina. Biochemistry 24:787,, 1985.

16.. Dorey, C , Cozette, ) . , and Faure, J. P.: A sim-plee and rapid method for isolation of retinal S-antigen.. Ophthalmic Res. 14:249, 1982.

17.. Borst, D. E., Redmond, T, M„ Elser, J. E., Gonda,, M. A., Wiggert, B., Chader, G, J,, and Nicker-son,, J. M.: Interphotoreceptor ret inoid-binding pro-tein.. Gene character izat ion, protein repeat structure, andd its evolution. J. Biol. Chem. 264:1115, 1989.

18.. Shinohara, T., Dietzschold, B., Craft, C. M., Wistow,, G., Early, J. J., Donoso, L. A., Horowitz, J., andd Tao, R.: Primary and secondary structure of bovinee retinal S-antigen (48-kDa protein). Proc. Natl.. Acad. Sci. U.S.A. 84:6975, 1987.

19.. Donoso, L. A,, Merryman, C. F., Shinohara, T,,, Dietzschold, B„ Wistow, G„ Craft, C., Morley, W., andd Henry, R. T.: S-antigen. Identification of the MAbA9-C66 monoclonal ant ibody binding site and thee uveitopathogenic sites. Curr. Eye Res. 5:995, 1986. .

20.. Hirose, S., Tanaka, T., Nussenblatt, R. B., Pal-estine,, A. G., Wiggert, B,, Redmond, T. M., Chader, G.. J., and Gery, I,: Lymphocyte responses to ret inal-

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specificc ant igens in uveit is pat ients and healthy sub-jects.. Curr. Eye Res. 7:393, 1988.

211 Fox, G. M., Redmond, T. M.. Wiggert, B., Ku-wabara,, T., Chader, G. J., and Gery, I.: Dissociation betweenn lymphocyte activation for proliferation and forr the capacity to adopt ively transfer uveoretinit is. ] . Immunol.. 138:3242, 1987.

22.. Gregerson, D. S., Fling, S. P., Obri tsch, W. F„ Merryman,, C. F., and Donoso, L. A.: Identification of TT cell recognit ion sites in S-antigen Dissociation of proliferativee and pathogenic sites. Cell Immunol. 123:427,, 1989.

23.. Singh, V. K,, Yamaki, K., Donoso, L. A., and Shinohara,, T.: Molecular mimicry. Yeast histone H3-inducedd exper imental au to immune uveit is. J. Immu-nol.. 142:1512, 1989

24.. lshikura, H,, Kuchroo, V., Abromson-Leeman, S.,, and Dorf, M, E.: Compar ison between helper and suppressorr T-cell induct ion. Immunol. Rev. 106:93, 1988. .

25.. Lightman, S. L., Caspers-Velu, L. E,, Hirose, S.,, Nussenblat t, R. B., and Palestine, A, G,: Angiog-raphyy with f luorescein-labeled dextrans in a primate modell of uveitis. Arch. Ophtha lmol. 105:844, 1987.

26.. Chain, B. M., Kaye, P. M., and Shaw, M. A.: Thee biochemistry and cell biology of ant igen process-ing.. Immunol. Rev. 106:33, 1988.

27.. Unanue, E. R., and Allen, P. M,: The basis for thee immunoregulatory role of macrophages and otherr accessory cells. Science 238:551, 1987.

28.. Unanue, E. R , and Cerott ini, J. C: Antigen presentat ion.. FASEB j . 3:2496, 1989.

29.. Lipham, W J, Sanui, H., Redmond, T. M., Wiggert,, B., de Smet, M. D., Chader, G. J., and Gery, I.:: Immunological features of synthet ic pept ides de-rivedd from the retina) protein 1RBP. Differences be-tweenn immunodominant and non-dominant pep-tides.. Curr. Eye Res. 9:95, 1990.

30.. Berzofsky, J. A.: Immunodominance in T lym-phocytee recognit ion. Immunol. Lett. 18:83, 1988.

31.'' Hu, L.-H., Redmond, T. M„ Sanui, H., Kuwa-bara,, T., McAllister, C. G., Wiggert. B., Chader, G. J., andd Gery, I.: Rat T-cell l ines specific to a nonimmu-nodominantt determinant of a retinal protein (!RBP) producee uveoretinit is and pineali t is. Cell, Immunol. 122:251,, 1989.

32.. Gregerson, D. S., Merryman, C. F., Obri tsch, W.. F., and Donoso, L. A.: Identification of a new uveitopathogenicc site on human S-Ag. A RVO ab-stracts.. Supplement to Invest. Ophtha lmol. Vis. Sci. Phi ladelphia,, J. B. Lippincott, 1990, p. 213.

114 4

Chapterr 7

AA Novel Method for the Determination of T-cell Profliferativ ee Responses in Patients with Uveitis

Marcc D. de Smet, Molly Dayan, Robert B. Nussenblatt

Ocularr Immunology and Inflammation 6: 173-178, 1999 (byy permission © Swets & Zeitlinger Publishers)

MODIFIEDD LDA IN UVEITIS PATIENTS

Researc hh report s

Ocularr Immunology and Inflammation 0927-3948/98/US3;; \2.Of)

OcularOcular Imnutnoli/gy and Inflammation -ty$8.ty$8. Vol. 6, No. i. i>/>. 173-17X ©© A-MÏus Press BurenBuren {The Netherlands) iqyX

Acceptedd i May 1998

AA nove l metho d fo r th e determinatio n of T-cel ll proliferativ e response s in patient s

wi t hh uveiti s

Marcc D. de Smet 12

Moll yy Dayan Rober tt B. Nussenblat t

'Clinicall Immunology Section, Laboratory of Immunology, Nationall Eye Institute, Bethesda, MD, USA

departmentt of Ophthalmology, Academic Medical Center, Universityy of Amsterdam, Amsterdam, The Netherlands

Abs t rac tt Standard proliferation assays using autoantigens such as S-Agg have given erratic responses when studied with human peripheral bloodd mononuclear cells. This erratic response is a reflection of the low numberr of circulating cells in the peripheral blood capable of generating aa response as well as the presence of competing cells for the available cytokiness in culture. The present study compares the standard proliferation assayy with a novel technique in which multiple short-term cell lines are establishedd to S-Ag in medium enriched in helper cytokines. After 12-144 days of culture, these lines were tested for their response to S-Ag. A significantt difference was found between patients and controls in the abilityy to generate responsive cell lines. This translated to a frequency off responsive cells of 0-4 per ioT peripheral blood mononuclear cells (PBMOO in normal individuals and 0-200 per to7 PBMC in patients. Thiss novel technique may provide a means of determining the number off responsive cells to specific autoantigens in the peripheral blood of patientss and the ability to follow the response over lime.

Keyy w o r d s S-antigen; lymphocyte proliferation; uveitis: autoimmunity

In t roduc t io nn Chronic posterior uveitis is an inflammatory disease off unknown origin which may lead to serious visual impairment. It is oftenn seen in the context of systemic inflammation such as sarcoidosis, vasculitiss syndromes such as in Behcet's disease, or in purely ocular inflammatoryy syndromes such as sympathetic ophthalmia. T cells are feltt to play an important role in these disorders.' In addition, a form of uveitiss that is similar to many human conditions can be induced in labo-ratoryy animals by injection of partially purified retinal proteins, such as S-antigcnn (S-Ag) or interphotoreceptor retinoid binding protein (IRBP).'J J

inn patients, the presence of T cell infiltration in pathologic eye specimens

CorrespondenceCorrespondence and reprint requestsrequests to: Marcc de Smet. M.D, Rm.. G2-217, Academic Medical Center r Meihergdreeff 9 11 105 AZ Amsterdam Thee Netherlands Fax;; +31-20-566-9053

L-mail:: m.d.desmetfj* amc.uva.nl

DeterminationDetermination of response to S-Ag

117 7

http://amc.uva.nl

poiniss to the central role played by these cells in the inflammatory process.'" Severall reports have demonstrated that patients with uveitis have a greater cellularr immune reactivity to S-Ag and to IRBP than control individ-uals.. " The presence of a response is indicative of the existence of sensitizedd cells within the peripheral blood that have the ability for in-volvementt in the disease process. However, this assay is qualitative and doess not quantify the number of reactive cells present w ithin the peripheral blood.. One reason is the limited number of cells with the appropriate specificityy circulating at any one Lime. ;

Too circumvent this problem, one can study a statistically significant numberr of wells rather than the limited number usually studied or perform limitingg dilution assays." Previous work has also shown that the magnitude off the response per well has littl e relationship to the actual number of helperr T cells present in a given culture well." This response is a function off the microenvironment in which the T cells are growing. One can partiallyy correel for this phenomenon by establishing a large number of short-termm T cell lines in the presence of cytokines such as IL-2.!1 The proceduree used in these experiments involves culturing human peripheral bloodd mononuclear cells lor two weeks in a medium enriched with T celll growth factors. These short-term lines can then be tested for a proliferativee response to a target antigen. This simple approach shows promisee as a method for the enumeration of autoreactive helper T cells inn the peripheral blood of patients with uveitis.

Material ss and method s

PAiihNii SKl.i-:ri ION Patients with posterior pole uveitis were taken fromm the pool of patients seen in the uveitis clinic of the National Eye Institute.. All patients had a history and evidence of prior disease involving thee retina, choroid, and/or the vitreous. At the time of testing, they were controlledd on a combination of cyclosporine and prednisone. The patients testedd had one of the following diagnosis: Behcet's disease, pars planitis, ocularr sarcoidosis, or the Vogt-Kovanagi-Harada syndrome. The basis forr the diagnosis of each disease is outlined elsewhere.:" Control individuals weree either clinic patients who did not have uveitis or normal volunteers. Controlss were age- and sex-matched with patients. All patients signed ann informed consent approved by the Investigational Review Board of thee National Eye Institute. The study abides by the guidelines set forth inn the Declaration of Helsinki as well as the U.S. Code of Federal Regulationss as it pertains to research on human subjects (45 CFR 4b),

MONONUCLEARR T R U, PREPARATION AND t U L T U R L P e r i p h e r al b l o od

mononuclearr cells (PBMC) were isolated from heparinized whole hlood obtainedd from patients or normal controls. Cells were obtained on standard Isolymphh gradients (Gallard-Schlesinger, Carle Place. NY, USA) by centrifugationn at 400xg. In the standard proliferation assay. 2x10' cells weree incubated in Hat bottom 96-well microliter plates for 5 days in 200 Lill RPMI-1640 medium with HEPES (GIBCO. Grand Island. NY. USA). supplementedd with glutamine (2 mM). penicillin (100 units/ml), strepto-mycinn (100 (.ig/ml) and ioc/< heat-inactivated human AB serum (Biocell Laboratories.. Carson. CA. USA). Bovine S-Ag. prepared according to

MD.MD. de Smet et ai


thee method of Dorey and associates,1" was used as a test antigen at 4 ug/ mll and 20 |iig/m] in triplicate welts; phytohemagglutirtin was used as a positivee control. For the last 16 hours, the celts were pulsed with 3H-thymidinee (3H-TdR. New England Nuclear. Boston. MA, USA; 0,5 iCi perr to u.l/well), then harvested and counted on a scintillation counter. Resultss are reported as a stimulation index (S.I.), which is calculated by dividingg the average count for the wells containing antigen divided by thee average count for the wells containing only medium.

DETERMINATIONN OK THE I-REQUENCY OF RESPONSE IN SHORT-TERM

LINESS In the frequency analysis assay, the cells were incubated at 37°C withh bovine S-Ag for I hour at a cell density of IXIO' . Then, the cells weree diluted to a concentration of ixio'Vml and plated at 200 ul per welll into 480 wells on five 96-weH round-bottom microliter plates (Costar. Cambridge,, MA, USA). Starting on the third day and every three days thereafter,, half of the medium was replaced with fresh medium containing 5%% T cell stimulant (Collaborative Research Inc., Bedford, MA, USA) andd 2 U ml ' human rIL-4 (Genezyme, Boston, MA, USA).

Onn days 12 to 14, each short-term T-cell line (480 in total) was analyzed forr its reactivity to bovine S-Ag. Each microwell was washed and cen-trifugedd twice with medium. The concentration of viable cells was deter-minedd following resuspension using a Neubauer chamber. Aliquots of 10,0000 cells from each T-cell line were placed in presence of 10,000 irradiatedd autologous mononuclear ceils used as antigen-presenting cells (APC).. For each T-cell line, four wells of a 96-well round-bottom microtiter platee were seeded with the appropriate number of T cells; S-Ag-stimulated APCC were added to two wells, while unstimulated APC were added to thee other two. Plates were incubated for 72 hours and pulsed with [3H]-thymidinee during the last 18 hours of culture. The APC were prepared byy pulsing ixio n APC per ml in media with or without bovine S-Ag for onee hour at 37°C. Lines were considered positive for bovine S-Ag if the stimulationn index (S.I.) was above 3.0 and if the coefficient of variation forr each duplicate culture was less than 30%.

R e s u l tss The response to bovine S-Ag was measured in patients and controlss using the standard five-day assay as well as by means of short-termm lines (analysis of frequency). The new assay was tested in five controlss (Table 1) and seven patients (Table 2). The diagnosis in the sevenn patients tested is shown in Table 2. All patients had ocular in-volvementt for at least two years with visible sequelae in the posterior pole.. All patients were on prednisone and/or eyelosporine, but none had beenn treated with cytotoxic agents. All patients had been free of ocular inflammationn for at least two months when they were tested.

Establishingg short-term cell lines in both normal individuals and in patientss was easily performed using the culture conditions described above.. All wells showed signs of strong proliferation at the end of 12 dayss of culture with an apparent doubling or tripling of the number of cellss per well. However, upon challenge with bovine S-Ag, only a limited numberr of wells gave a positive response with an S.I. above 3.0. A lowerr number of responding wells was observed in controls when compared too patients (Tables 1 and 2). No direct correlation was seen between the


119 9

ChapterChapter 7

TABii t i. Stimulation index to bovine S-Agg and percentage of positive wells inn the short-term cell lines in normal subjects. .

ii ABI.K i- Stimulation index to bovine S-Agg and percentage of positive wells inn short-term cell lines in patients with uveitis.' '

StandardStandard proliferation assay fS.L) 2020 \kg/ml 44 \ig/ml

Short-termShort-term cell lines PercentagePercentage of positive \iells

r.6 6 i.o o

1.6 6

I S S

°-5 5 4.0 0

7-? ?

<>-4 4

5-3 3

"Onlyy wells with an S.I. above 3.0 were considered positive and included in the calculation. .

Diagnosis Diagnosis StandardStandard proliferation assay f S.Li Analysis of frequency 2020 \ig/ml 4 \ig/ml L'ercentage of positive wells

Behcet t

Behcet t

Behcet t

Behcet t

V K H H

Parss plaiiitis

Sarcoid d

2-1 2-1

9.0 0

1.7 7 1.1 1

2.4 4

4.0 0

1.8 8

1-5 5 3.0 0

0.7 7

11 ! 2.6 6

3 7 7 1.4 4

40 0

33 3

3 " "

6-5 5 7.6 6

19 9 0.2 2

Alll patients were inactive at the lime of testing. hOnIyy wells with an S.l. above 3.0 were considered positive and included in the calculation. .

intensityy of the response in the standard assay and in the number of respondingg short-term cell lines from cither controls or patients. In fact, ass shown in Table 1, the lowest number of cell lines was isolated from thee control subject who gave the highest response in a standard assay.

D i s c u s s i onn Littl e information is available regarding the number of circulatingg T cells with immune memory for ocular autoantigens. Such knowledgee would be useful to monitor autoreactivity in patients, and is necessaryy if one wants to understand the interplay between the eye and thee peripheral immune system. This is particularly true if one wants to monitorr levels of ocular inflammation by performing in-vitro assays. However,, standard assays are unable to provide such quantitative infor-mationn fur several reasons. The number of circulating cells capable of generatingg a response may be very low, such that responsive celts are nott present in each of the test wells. In addition, the number of T celts requiredd to generate a response is highly variable and dependent on factors suchh as the level of suppressor activity within the culture and competition betweenn cultured cells for those cytokines available in the culture medium.1--"'' Increasing in-vitro concentrations of helper cytokines can compensatee for both of these limiting factors. To compensate for the loww precursor frequency, the easiest approach is to increase the number off cultured cells per well or to increase the number of wells being cultured too a statistically significant number. We decided to use the second approach, particularlyy since it also gave us the possibility of establishing cell lines

120 0

M.D.M.D. de Smetetal.

file:///iells


too S-Ag with a minimum of effort. In past experiments, we had noticed lhatt patients with active disease had a lower proliferative response in thee standard assay.' We had postulated, that in addition to the factors mentionedd above, the inactivity could be due to margination of cells intoo the eye thus decreasing the pool of responsive cells in the peripheral blood.. It is for this reason that we chose to test only those patients who didd not have active ocular disease at the time of the in-vitro study.

Ass shown in Table I, responsive cells to S-Ag can be found in the peripherall blood of normal controls. This is in keeping with previous studiess where patients and controls were found to have both T- and B-celll responses to * In our own study, up to 30% of normal individualss have a proliferative response to bovine S-Ag. However, there appearss to be a quantitative difference in the number of responsive cells betweenn patients and controls, as suggested by our results in Table 2.

Givenn the low number of positive wells that we identified, one can assumee that each well contains only one reactive cell. That this assumption iss true is further supported by work on MBP-responsive lines cultured usingg an analogous method, and by TCR analysis where over 98% of thee wells contained only one rearrangement.-" Thus, the clonal nature of thee cell lines generated by our culture technique allows us to estimate thee number of responsive T cells present in the peripheral blood of both patientss and controls. If one assumes lhat all positive wells contain only onee responsive T cell and that all reactive cells have generated a response, thee number of responsive cells in the peripheral blood is in the range of 0-44 per 107 lymphocytes in normal individuals and 0-200 per to7

lymphocytess in patients. These numbers are comparable 10 that calculated byy Opremcak et al.'J using a limiting dilution assay: they had found a rangee of 0-40 per 1 o'1 lymphocytes for patients with uveitis.

Thiss culture technique shows considerable promise for the future. In additionn to giving an estimate of the number of circulating reactive cells, itt may allow us to determine several characteristics of the responding celll population. Since each positive well represents the clonal expansion off one autoreactive cell, it should allow us to identify the epitopes of S-Agg to which cells are responding as well as the pattern of TCR rear-rangementt present in these autoreactive cells. In addition, by testing patientss on a repeated basis during different stages of disease or immune activation,, it should be possible to gain some insight into the cellular traffickingg of autoreactive cells in peripheral blood.

Reference s s Nussenblattt RB, Palestine AG, Chan CC,, Stevens GJ. Mellow SD. Green SB.. Randomized, double-masked studyy of cyclosporine compared to prednisolonee in the treatment of' endogenouss uveitis. Am J Ophthalmol 1991:112:138-146, , Fauree JP. Autoimmunity and the retina.. Curr Top Eye Res 1980:2:215-302. . Wackerr W B , Donoso LA . Kalsow

CM,, Yankeclov JA Jr, Organisciak DT.. Experimental allergic uveitis. Isolation,, characterization and localizationn of a soluble uveitopathogenicc antigen from bovine retina.. J Immunol 1977:119:149-158. Geryy I. Wiggen B. Redmond TM, Kuwabaraa T. Crawford MA, Vistica BP,, Chadcr GJ. Uveoretinitis and pinealkiss induced by immunization withh interphotorcceptor reiinoid-bindingg protein. Invest Ophthalmol


4 4

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Viss Sci 1986:27:1296-1300 55 Jakobiec FA, Marboe CC, Knowles

DM,, IwamotoT. Harrison W, Chang S,, Coleman DJ. Human sympathetic ophthalmia:: an analysis of the inflammatoryy infiltrate by hybridoma-monoclonall antibodies, immunochemistry,, and correlative electronn microscopy. Ophthalmology 1983:90:76-95. .

66 Chan CC. Nussenblatt RB, Fujikawa LS.. Palestine AG. Steven G Jr, Parver I.M.. Lachcnbach MW, Kuwabaia T. Sympatheticc ophthalmia: imunopathologicc findings. Ophthalmologyy 1986:93:690-695.

77 Nussenblatt RB, Mittal KK, Ryan S, Greenn WR, Maumcnee AH. Birdshol retinochoroidopathyy associated with HLA-A299 antigen and immune responsivenesss to retinal S-antigen, Amm J Ophthalmol [982:94:147-158.

88 de Smet MD, Yamamoto JH. Mochizukii M, Gery I. Singh VK. Shinohamm T, Wiggert B. Chader GJ, Nussenblattt RB. Cellular immune responsess of patients with uveitis to retinall antigens and their fragments. Amm J Ophthalmol 1990:110:135-142.

99 de Smet MD. Wiggen B, Chader GJ. Mochizukii M, Gery 1. Nussenblatt RB, Cellularr immune responses to fragmentss of S-antigen in patients with uveitis.. In: Usui M. Ohno S, Aoki K. editors.. Ocular Immunology Today. Tokyo:: Llsevier Science Publishers. 1990:: 2N5-288.

100 Hirose S. Donoso LA. Shinohara T, Palestinee AG, Nussenblatt RB, Gery 1, Lymphocytee responses to peptide M andd to retinal S-antigen in uveitis patients.. Jpn J Ophthalmol 1990:34:298-305. .

111 Froebel KS, Armstrong SS, Cliff e AM,, Urbaniak SJ. Forrester JV. An investigationn of the general itnmmune statuss and specific responsiveness to retinal-lS)-- antigen in patients with chronicc posterior uveitis Eye 1989:3:263-270. .

11 2 Opremcak EM, Cowans AB. Orosz CG,, Adams PW, Whisler RL. Knumerationn of autoreactive helper T

lymphocytess in uveitis. Invest Ophthalmoll Vis Sci 1991:32:2561-2567, ,

133 Orosz CG, Adams PW, Ferguson RM. Frequencyy of human alloantigen-reactivee T lymphocytes. II . Method for limitingg dilution analysis of alloantigen-reactivee helper T ceils in humann peripheral blood. Transplantationn 1987.43:718.

144 Ota K. Matsui M. Mil ford EL, Maekin GA,, Weiner HL, Hafler DA. T-cell recognitionn of an immunodominant myelinn basic protein epitope in multiplee sclerosis. Nature 1990:346:183-187. .

155 Nussenblatt RB. Palestine AG. Uveitis;; Fundamentals and Clinical Practice.. Chicago: Year Book Medical Publishers,, 19H9.

166 Dorey C, Cozette J. Faure J-P. A simplee and rapid method for isolation off retinal S-Antigcn Ophthalmic Res 1982:14:249-255. .

177 Miller RA, Stutman O. Enumeration off II 2-secreting helper T cells by limitingg dilution analysis, and demonstrationn of unexpectedly high levelss of II 2 production per respondingg cell. J Immunol 1982:128:2258-2264. .

188 Clerici M. Stocks NI. Zajac RA. Boswelll RN. Berstetn DC, Mann DL. Shearerr GM. Berzofsky JA. !nterleukin-22 production used to detect antigenn peptide recognition by T-helperr lymphocytes from a.ssymptomaticc HIV-seropositive individuals.. Nature 1989:339:383-385.

199 Doekes G, van der Gaag R. van Kooyk Y,, Broersma L. Zaal MJM, Dijkman G,, Fortuin ME, Baarsma GS, Kijlstra A,, Humoral and cellular immune responsivenesss to human S-antigen in uveitis.. Curr Eye Res 1987:6:909-919.

200 Wucherpfennig KW, Ota K. Endo N. Seidmann JG, Rosenzwcig A. Weiner HI... Hafler DA. Shared human T cell receptorr Vj} usage to immunodominantt regions of myelin basicc protein Science J 990:248:1016-11 o 19.

M.D.M.D. de Smet et ai

Chapterr 8

Prospectivee Determination of T cell Responses too S-Antigen in Behcet's Disease Patients

andd Controls

Marcc D. de Smet, MD, FRCSC Mollyy Dayan, BSc l

Fromm the 'Laboratory of Immunology, Clinical Immunology Section, National Eye Institute, Bethesda,, MD; : Department of Ophthalmology, Academic Medical Center,

Universityy of Amsterdam, The Netherlands.

Investigativee Ophthalmology and Visual Sciences (In press) (byy permission © Lippincott Williams & Wilkins)

PROSPECTIVEE RESPONSE TO S-AG IN PATIENTS

Abstract t

Purpose:: To prospectively determine, using two different assays, the lymphocyte proliferative re-sponsee to a retinal autoantigen (S-Antigen), in Behcet's disease patients under treatment for oc-ularr inflammation. Methods:: Patients were evaluated at each visit for signs of ocular inflammation. Peripheral blood leukocytess were harvested and cultured in the presence of bovine S-Antigen in a standard culture assay;; as well as by limiting dilution using multiple short term T cell lines. Results:: Five patients were followed for 2 to 10 months. During follow-up, 3 patients developed ann episode of ocular inflammation. No consistent change in proliferative response was observed inn standard proliferation assays. However, an increase in established T cell lines was correlated too the presence of ocular inflammation in all three patients. Ocular activity was associated with aa rise of 9 to 30 fold in the frequency of short term T cell lines. This increase returned to baseline withinn 1 to 3 months. Conclusions:: An increase in S-Antigen responsive lymphocytes is found in the peripheral blood off Behcet's disease patients during episodes of ocular inflammation. This increase cannot be measuredd using standard proliferation assays, but requires the use of techniques exploiting the principless of limiting dilution analysis.

Introductio n n

Behcet'ss disease is characterized by recurrent episodes of severe intraocular inflammation. Sev-erall immunological abnormalities have been reported in this disease suggesting that autoimmu-nityy may play a role in its pathogenesis. Proliferation assays using peripheral blood lymphocytes havee commonly been used to detect cellular immunity to retinal antigens. A heightened response too S-Antigen (S-Ag) has been observed in a variety of uveitis patients including those with Be-hcet'ss disease. However, this assay remains qualitative in nature. While capable of providing ev-idencee of a response, lymphocyte proliferation assays are unable to provide information on the severityy of ocular inflammation and give variable responses over time .

Circumventingg the qualitative nature of the lymphocyte proliferation assay, limiting dilution as-sayss (LDA), allow determination of the number of responding cells to a specific antigen or anti-genss (precursor frequency). LDAs have been used to follow the level of response to the soluble tetanuss toxoid antigen over time. Using a cell culture-based assay that incorporates elements of aa limiting dilution technique, we showed that it is possible to estimate the T cell precursor fre-quencyy to bovine S-Ag in patients with diverse forms of uveitis. Using a similar approach, oth-erss determined the precursor frequency of T cells responding to fragments of myelin basic pro-teinn (MBP) in patients with multiple sclerosis and following spinal cord injury. Adjusting the culturee conditions allowed the demonstration of in vivo clonal expansion of MBP reactive T-cells inn both the blood and the CSF of patients with MS, suggesting active trafficking of these lym-phocytess across the blood-brain barrier. A similar study in Behcet's disease would imply the iso-lationn of lymphocytes from the vitreous of an actively inflamed patient, a procedure which is rarelyy required and not without risk. However, it is possible to study the influence of ocular in-flammationn on lymphocytes within the peripheral blood.

Usingg our modified LDA, we decided to explore the change in S-Ag responsiveness over time in

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ChapterChapter 8

peripherall blood lymphocytes of Behcet's disease patients. We were particularly interested in de-terminingg whether or not fluctuations in responsiveness could be detected following episodes of intraocularr inflammation. Bovine S-Ag was used as the stimulating antigen. Since standard lym-phocytee proliferation assays have not been carefully studied in this setting, we also wanted to studyy the response characteristics of this simpler culture method to ocular inflammation.

Methods s

Patientt selection and assessment of disease: Fivee patients with Behcet disease, seen in the Uveitis clinic of the National Eye Institute were se-lectedd for long term monitoring of their cellular immune responses. All patients met the diag-nosticc criteria established by the International Behcet Disease Study Group. All patients signed ann informed consent approved by the Investigational Review Board of the National Eye Institute. Patientss were tested for immune response at each regularly scheduled patient visit for a minimum off three visits and were monitored until the patient was discharged from the eye clinic, or chose too forego further testing. Prior to drawing blood patients had a complete ocular examination whichh included measurement of visual acuity and examination of the anterior and posterior seg-mentss for evidence of intraocular inflammation. To determine baseline responses, four normal individualss were also tested. An acute inflammatory episode was deemed present when one of thee following events was noted on clinical examination: an increase in vitreous cells associated withh a vision drop of more than two lines on the ETDRS chart, or the appearance of a focus of chorioretinitis,, vascular sheathing or intraretinal hemorrhage. The study abides by the guidelines sett forth in the Declaration of Helsinki as well as the U.S. Code of Federal Regulations as per-tainss to research on human subjects {45 CFR 46).

Lymphocytee isolation and culture: Peripherall blood samples were collected at several time points as indicated in table 1. For each timee point, 60 mLs of peripheral blood was collected. Peripheral blood mononuclear cells (PBMC)) were separated on Isolymph gradient (Gallard-Schlesinger, Carle Place, NY). One frac-tionn was used for both culture assays as indicated below, and were processed immediately. The remainingg PBMC were frozen to be used for antigen presentation. For the standard proliferation assay,, cells were cultured in flat-bottomed 96-weIl microplates, at 2 x 10' cells per well, in 0.2 niLniL RPMI 1640 medium with Hepes (Cellgro, Herndon. VA). supplemented with 10% heat in-activatedd human AB serum (Biocell Laboratories, Carson, CA, USA). Six replicate cultures were stimulatedd with bovine S-Ag at 20 or 100 |xg/mL. Phytohemaglutinin (PHA) at 1 (xg/mL was

Tablee 1: Baseline Patient Characteristics:

Subjectt # Age e (years) )

26 6 28 8 29 9 45 5 32 2

Sex x

F F M M F F F F F F

Durationn of Disease e

3 3 2 2

3 3 8 8 1 1

(yrs) ) Therapy y

Prednisone e (mg/day) )

15 5 30 0

10 0 10 0 2,5 5

Cyc c losporinc c (mg/kg/clay) )

3 3 5 5 2.5 5 2 2

1 1

126 6


usedd as a control of adequate proliferation. Cells were cultured for 5 days, pulsed with pH]-thymidinee fH-TdR, New England Nuclear, Boston, MA; 2 Ci/ mmol, 0.5 JJLCÏ/10 |xl/well), dur-ingg the last 18 hours of culture. Bovine S-Ag was prepared according to the method of Dorey and associates.. The results are expressed in stimulation indices (SI = mean cpm in cultures with stim-ulantt / mean cpm in control cultures without stimulant).

Estimationn of the proportion of circulating T cells reactive to S-Ag:

Inn a previous study, we determined the optimal cell density to detect S-Ag reactive T cells. This wass found to be 2 x 10' cells/well similar to studies in multiple sclerosis patients. At this con-centration,, PCR analysis demonstrated monoclonality of cell lines. Cultures for all subjects were carriedd out in exactly the same manner. PBMCC were incubated at 37 °C, with bovine S-Ag 100 juLg/mL for 1 hour at a cell density of 1 x lOVmL.. These were then washed twice, and resuspended in RPMI 1640 at a concentration of 1 x 107mLL and plated at 2()0p.l per well into 480 wells on five round bottom 96-well microliter platess (Costar, Cambridge, MA ). Starting on the third day and every 3 days thereafter, half of the mediumm was replaced with fresh medium containing 5% T cell stimulant (Collaborative Re-searchh Inc., Bedford, MA) and 2 U/ml of human rIL-4 (Genzyme, Boston, MA). Onn day 12 to 14, each well was analyzed for its reactivity to bovine S-Ag. An aliquot from each culturee well was split into four aliquots (10 000 cells per aliquot), and placed into fresh round bottomedd 96-well plates. Two wells were cultured with 10s PBMC pulsed for 4 hours with S-Ag priorr to irradiation (3000 rads), the two other wells received unpulsed irradiated PBMC. Wells weree considered responsive to bovine S-Ag if the stimulation index (S.I.) was above 3.0, and if thee coefficient of variation for each duplicate culture was less than 30%. Results are expressed as thee percentage of positively responding wells.

Statisticall analysis: Statisticall analysis was performed without assuming a Gaussian distribution using the Mannn Whitney test. Prism by GraphPad Software Inc (San Diego, CA) was used for these analy-ses. .

Results s

Fivee patients with Behcet's disease were included in this study. Four women and one man were followedd fora period of 2 to 10 months (mean 4.5 months) with 3 to 6 sets of analyses being per-formedd per patient. The average patient age was 29 years (range 23 to 37 years). Behcet's disease

Tablee 2: Proliferative Responses in Control Subjects to Bovine S-Ag:

Controll #

1 1 2 2

3 3 4 4

Lymphocyte e 200 n.g

2.4 4 0.7 7 3.9 9 2.1 1

Proliterati i on n

1000 |xg

1.6 6 1.8 8 3.4 4 1.9 9

%% + Short term TT cell lines

4 4 7 7

0.4 4 4 4

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ChapterChapter 8

Tablee 3: Standard Lymphocyte Proliferation Responses to Bovine S-Ag in Behcet's Patients:

Timee Point (months) )

0 0 2 2

2.5 5 3.5 5

Ü Ü

6 6 8 8 10 0 12 2

0 0 ! ! 2 2

0 0 2 2 4 4

0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 9 9

Lymphocyte e 200 ^g

] . ] ]

0.2 2 0.6 6 2,4 4

2.3 3 15 5 0.9 9 0.7 7 0.7 7

2.0 0 1.5 5 3.2 2

1.8 8 1.1 1 1.2 2

5.2 2 3.4 4 9 9 1.1 1 0.7 7 1.0 0 2.5 5 1.6 6 0.9 9

Proliferation n 1000 ixg

0.9 9 1.0 0 1.0 0 1.0 0

3.1 1 9.2 2 0.7 7 0.7 7 0.7 7

2.4 4 0.7 7 1.1 1

2.5 5 1.8 8 1.8 8

10.9 9 5.8 8 24.9 9 0.9 9 1.7 7 0.9 9 2.4 4 1.4 4 1.3 3

** Patient was started on Cytoxan 1 month prior to this assay

hadd been diagnosed for an average of 3.4 years (range; 1 to 8 years) at the time of study inclusion. Alll patients were controlled on medication for a minimum of 3 months, and were being treated withh a combination of low dose prednisone (2.5 to 30 mg/day) and cyclosporine (0.5 to 5 mg/kg/ day).. During the follow-up period, 3 patients developed an ocular flare-up. Upon diagnosing a flare-up,flare-up, the cyclosporine dose was doubled followed by a taper over 1-3 months based on re-sponsee to therapy. The prednisone dose was similarly increased. Baseline data on patients and controlss are summarized in table 1.

Standardd proliferatio n assays: AA comparison of the proliferative response between controls and patients was performed at study entry.. Data on controls is provided in table 2. There was no statistically significant difference be-tweenn the two groups when tested at the 20 |j,g/mL concentration of bovine S-Ag (mean S.I.: 2.3 controls.. 2.5 patients). Similarly there was no statistically significant difference in S-Ag response att 100 |jLg/mL(Mean S.I.: 2.2 controls, 4.3 patients), though a significant coefficient of variation wass present between the two groups. Reducing the difference in this variation by logarithmic transformationn did not change the level of significance.

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Tablee 4: Short Term T cell Line Assay Responses to Bovine S-Ag in Behcet's Patients:

Patientt #

1 1

2 2

3 3

4 4

5 5

Time e (months) )

0 0 2 2

2.5 5 3.5 5

0 0 6 6 8 8 10 0 12 2

0 0 1 1 2 2

0 0 2 2 4 4

0 0 I I 2 2

3 3 4 4 5 5 6 6 7 7 9 9

%% + Short term TT cell lines

0 0 0.5 5 9 9

0.02 2

0.7 7 33 3 1.6 6 0 0 0 0

<0.01 1 <0.01 1

1.6 6

0.8 8 0.8 8 1.0 0

5 5 5.7 7 5 5 30 0 30 0 22 2 0.5 5 2 2

7 7

Disease e Activit y y

----+ +

--

_ _ + +

_* * ----

--

--

_ _ ----

--

--+ +

+ +

--------

Naturee of Ocularr Inflammation

Vascularr sheathing

Papillitis,, branch retinal occlusion,, vitreous haze

Vasculitis,, branch vein occlusionn vitreous haze

** Patient was started on Cytoxan one month prior to this assay

Duringg the follow up period, stimulation indices varied significantly. A raised stimulation index wass correlated with a flare-up in only one of three cases of acute inflammation. In 2 of the 3 pa-tientss who developed a flare-up of uveitis, a significant stimulation index was noted at some time priorr to the flare-up. Significance was defined as a stimulation index above the mean S.I. of con-trolss + 2 standard deviation. In the remaining patient, the stimulation index remained below this levell of significance. Table 3 summarizes the proliferation assay data in patients.

Estimationn of the proportion of circulating S-Ag-reactive T-cells: Inn all patients and controls, it was possible to establish short term T cell lines. Between 80 and 95%% of cultures had a coefficient of variation of less than 30% and thus could be analyzed with respectt to the antigen response. One assay gave a much lower response. In patient 1, assay 3 on-lyy 62% of cultures met the inclusion criteria. At study entry, there was no statistically significant differencee in the number of established lines between the two groups. Out of the five patients tested,, a rise over the base line number of generated T-cell lines was noted in three cases. These weree temporally related to the onset of an episode of intraocular inflammation. The number of establishedd T cell lines returned to baseline within 1 to 3 months following resolution of the in-

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ChapterChapter 8

flammatoryy episode. Interestingly, in one patient, the increase in generated T-cell lines was tem-porallyy associated with a decrease in response observed with the lymphocyte proliferation assay (patientt 5). Results are summarized in table 4.

Discussion n

Patientss with Behcet's disease are known to be sensitized to retinal autoantigens, S-Ag and IRBP. Theyy are also known to be sensitized to 65KD heat shock proteins, and possibly other antigens. Whilee the exact trigger for the onset of disease still needs to be identified, once sensitization to retinall autoantigens has occurred, these T cells will be recruited to the site of inflammation. Adoptivelyy transferred S-Ag sensitized T cells can be found in rat retinas within the first 24 hours off transfer. So far, it has not been possible to demonstrate a role for S-Ag sensitized cells in hu-mann patients. Demonstration in humans would require isolation of T cells from an inflamed eye, andd delineation of their proliferative response to retinal antigens. This approach was successful-lyy used to delineate the specificity of T cells towards herpes simplex virus in the vitreous of pa-tientss with acute retinal necrosis, but is unpractical in patients with Behcet's disease as they rarelyy require an operative procedure during an inflammatory episode. Alternatively, one can at-temptt to demonstrate the presence of an expanded pool of responsive T cells in the peripheral bloodd following an intraocular attack. In patients with multiple sclerosis, activated MBP-reactive TT cells were shown to undergo in vivo clonal expansion in both the blood and the cerebrospinal fluid,, indicating the presence of an active traffic of MBP-reactive T cells across the blood-brain barrier.. Assuming that a similar process is present in patients with ocular inflammation, we de-cidedd to prospectively follow a group of Behcet's disease patients using two assays measuring T celll proliferation to S-Ag.

Standardd proliferative assays have been used by numerous groups to demonstrate an association withh human disease. An often quoted cut off for immunological significance is an S.I. of 2.0. Us-ingg this cut off value, our present group of patients all showed a positive immune proliferative re-sponsee during the follow-up period, while only 3 patients developed intraocular inflammation. Usingg a more restrictive cut off of significance (mean of controls + 2 standard deviations), al-lowedd us to identify 2 of 3 patients with ocular inflammation. The heightened response was mea-suredd either before or at the time of ocular inflammation. This second approach more appropri-atelyy selected patients at risk, but missed one individual. In addition, once inflammation developed,, subsequent measurements were significantly reduced in intensity. Thus, standard pro-liferationn assays can help predict patients at risk of developing uveitis, but cannot be used to fol-loww the immune response during the active phase of inflammation. In another study, a patient withh an unspecified retinal scar, present for more than 30 years, was repeatedly tested over an 18 monthh period against S-Ag. A large variation in lymphocyte responsiveness was attributed to the microenvironmentt present in the culture well. Cytokines produced by one of more cell popula-tionss present in the microwell, prevents the S-Ag phenotype to be expressed.

Limitingg dilution assays have traditionally been used to quantify cells with observable function-all qualities. Appropriate dilution of the seeding cell population, allows the interpretation of data accordingg to "single hit kinetics" (0-term) of the Poisson distribution. Use of unfractionated cell sampless dispersed into limiting dilution cultures yields non-linear titration curves due to oppos-ingg forces generated by suppressor and effector cells. To overcome the suppressor effects re-

130 0


quiress prolonged cultivation, and the supply of needed growth factors such as T cell growth fac-tor.. Studies performed in other labs have shown that this approach generates CD4 single cell lin-eagess in the majority of wells which often are responsive to a single peptide determinant. Given thee number of wells showing a positive response in these studies, single hit kinetics apply. Thus, thee number of positively responding wells is reflective of the circulating pool of responsive T cells.. Of the five patients that were followed, 3 developed a transient ocular inflammatory re-sponse.. In each of these cases, there was a rise in the number of identifiable T cell lines concur-rentt with the ocular inflammation. The expansion was between 9 to 30 fold, and this increase rapidlyy disappeared back to base line within one to three months. Assessingg limiting dilution using this approach makes certain assumptions. The cell populations withinn the culture well behave according to single hit kinetics. Memory T cells, likely responsi-blee for the activity observed in these experiments, require multiple hits for activation. However, inn the presence of IL-2, a major component of the T cell stimulant, it is converted back to single hitt behavior. Thus, addition of the T-cell stimulant will favor cells which are capable of growing inn its presence. The adequacy of the technique can be checked by setting up cultures at different dilutions.. Increases in the number of cells should lead to a linear increase in the number of pos-itivee wells. Deviations from linearity, in particular leveling off in the curve indicates the presence off suppressive elements in the culture well. This phenomenon has been observed in patients suf-feringg from multiple sclerosis. We do not know to what extent, this effect was present in the as-sayss performed with our patients, since the chosen cell dilution was based on previous work with uveitiss patients, all of which were under control at the time of analysis. It is possible that the ob-servedd reduction in the proportion of responding wells is partially due to enhanced suppression providedd by other cells present in the culture well. Performing titration experiments on a broad rangee of concentrations which extend beyond the zone of linearity should help to answer this questionn .

Inn experimental models, both CD8 responses to a viral challenge, and CD4 responses to antigen challengee are characterized by 3 distinct phases. Initial activation and expansion of the lym-phocytee pool last for about 7 days. It is characterized for a novel agent by a 100 to 5000 fold increasee in the number of specific lymphocytes. In the case of mice exposed to LCMV, this expansionn was calculated to represent 15 divisions or 1 division every 13 hours for 8 days. Rapid expansionn is followed by a period of cell death, lasting anywhere from 8 to 30 days and is realizedd primarily through apoptosis. After 30 days, a stable pool of memory T cells is generated whichh represent about 5% of the initial response. Re-exposure to an antigen or virus leads too a rapid expansion from this pool of memory T cells. While this response occurs more rapidlyy than the initial response, the measured lymphocyte expansion in peripheral blood orr lymph nodes amounts only to a 5 to 100 fold expansion over baseline. Our data on Behcet's diseasee patients closely parallels these experimental findings. Patients presented within a few dayss of their recurrence, and were noted to have a 9 to 30 fold increase in responsive lympho-cytes.cytes. As seen in experimental models, this increase rapidly disappeared back to the pre-flare-up level. .

Itt is the first time that a correlation is observed between peripheral antigen responsiveness and inflammatoryy disease in humans. Adjusting the culture conditions to favor effector T cell prolif-erationn has allowed us to unmask this responsiveness. These findings suggest a role for S-Ag in thee autoimmune uveitis associated with Behcet's disease, but it does not imply causality. As pre-viouslyy observed, patients with established disease respond to a number of autoantigens. It is

131 1

ChapterChapter 8

likelyy that some if not all of these antigens will contribute or help to perpetuate the inflammato-ryy response.

Inn conclusion, adjustment in culture conditions promoting clonal expansion of reactive T cells havee allowed us to demonstrate a variation in the number of responsive lymphocytes in the pe-ripheralripheral blood of patients with Behcet's disease with active intraocular inflammation. Though technicallyy more demanding, this approach can yield considerably more information on the im-munee status of uveitis patients than is currently provided by standard lymphocyte culture meth-ods.. It is an invaluable tool for quantifying cells at a functional level.

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35.. Murali-Krishna K, Altman JD, Suresh M, Sourdive DJD, Zajac AJ, Miller JD, et al. Counting antigen-specif-icc CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 1998;8:177-87.

36.. Ahmed R, Gray D. Immunological memory and protective immunity: understanding their relation. Science 1996:272:54-60. .

37.. McHeyzer-Williams MG, Davis MM. Antigen-specific development of primary and memory T cells in vivo. Sciencee 1995;268:106-11.

38.. Butz EA. Bevan MJ. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Im-munityy 1998;8:167-75.

39.. Varga SM, Welsh RM. Stability of virus-specific CD4+ Tcell frequencies from acute infection into long term memory.. J Immunol 1998:161:367-74.

133 3

Chapterr 9

Humann S-Antigen Determinant Recognition inn Uveitis Patients

Marcc D. de Smet, George Bitar, Sumeet Mainigi, Robert B. Nussenblatt


i i

136 6

HS-AGG DETERMINANT RECOGNITION IN PATIENTS

Abstract: :

Background:: Soluble antigen (S-Ag) is a member of the arrestin family of protein with which it sharess a high level of homology. It is an immunologieally privileged retinal antigen that can elic-itt experimental autoimmune uveitis (EAU) and is felt to be a target for ocular inflammatory dis-eases. .

Objective:: To identify in humans, the immunogenic determinants of human S-Ag and to estab-lishh whether a specific response profile occurs in particular ocular inflammatory conditions.

Methods:: Peripheral blood lymphocyte responses were measured against a panel of 40 overlap-pingg synthetic peptides of human S-Ag, in patients with chronic uveitis and compared to con-trols.. Patients with Behcet's disease, Sarcoidosis, Vogt-Koyanagi-Harada, and Sympathetic Oph-thalmiaa were tested.

Results:: A limited number of immunodominant determinants were identified for Behcet's disease andd Sarcoidosis patients. These were all located at sites of limited homology with other known ar-restins.. In addition, several individual patients had prominent proliferative responses to multiple determinantss well above that of controls. This determinant spread was observed in all disease en-titiess except Sympathetic Ophthalmia, which did not show any immunoreactivity to S-Ag. Signif-icantt response shifts were also noted over time in two patients who were repeatedly tested.

Conclusions:: Our results indicate that specific immunodominant determinants exist to human S-Agg in patients with certain forms of uveitis. However, in individual patients, response is not lim-itedd to these determinants. In the chronic stage of disease, response is spread over many deter-minants. .

Introductio n n

Solublee Retinal Antigen (S-Ag) also known as rod arrestin or 48K protein is a major component off rod outer segments whose normal physiological role is to quench the visual transduction cas-cadee induced by light activation of rhodopsin. While closely related to B-Arrestins, which are ubiquitouslyy distributed throughout the body, rod photoreceptor arrestins are confined to the reti-naa in an immune privileged site. In addition to its normal physiologic function, S-Ag is one of severall ocular antigens capable of inducing experimental autoimmune uveitis (EAU) in suscep-tiblee hosts. EAU is mediated by activated, antigen specific CD4+ T cells , and can be induced by immunizationn with several S-Ag derived peptides. Pathologically, the process resembles a num-berr of human conditions felt to be of autoimmune origin. S-Ag is thought to be immunopatho-genicc in humans either causing or prolonging certain forms of uveitis in humans. In accordance withh this notion, a large number of patients with uveitis were found to respond to bovine S-Ag in lymphocytee proliferation assays. In addition lymphocyte responses have been noted to a few pep-tidee determinants derived from both the bovine and human S-Ag sequences. Thee immune response against a whole protein is usually targeted toward a small number of pep-tidee determinants. To initiate the immune response requires binding to MHC molecules on the surfacee of antigen presenting cells (APC), and presentation to receptive T cells. Due to the poly-morphismm of MHC molecules, the majority of immunogenic peptide determinants bind strongly

137 7

ChapterChapter 9

too only a single or a limited set of MHC molecules. However, some peptide determinants are ca-pablee of binding to several heterogeneous MHC molecules and may even cross species. In the casecase of myelin basic protein for example, certain peptides, encephalitogenic in experimental an-imals,, were found to be recognized by lymphocytes from patients with multiple sclerosis. In this study,, we decided to use a panel of overlapping synthetic peptides of human S-Ag to test their immunoreactivityy in patients with various forms of uveitis as well as in controls. It is then possi-blee to define a spectrum of immune reactivity for each disease entity, and identify the limited set off determinants with particularly high immunoproliferative responses. Response to a few of thesee determinants was also followed over time.

Material ss and methods

Antigenn Synthesis and Preparation: Fortyy overlapping oligomeric peptide determinants of human S-Ag, spanning the entire length of thee human S-Ag sequence were synthesized by Applied Biosystems (Foster City, CA, USA). Eachh peptide determinant measured 20 amino acids in length except for the last one measuring 155 amino acids. Each peptide determinant overlapped the previous sequence by 10 amino acids. Thee exact sequence as well as the nomenclature used throughout this article is shown in figure 1. Peptidess were synthesized by solid-phase chemistry using t-butyloxycarbonyl derivatives of the aminoo acids on an automated peptide synthesizer and were purified by HPLC to at least 95% purity.. The amino acid composition of each peptide was verified using amino acid analysis and automatedd gas-phase sequencing. In proliferation assays, a final concentration in each well of 20 jjug/mLL and 100 p*g/mL was utilized.

Patientt Selection Patientss participating in this study were selected from the pool of patients followed in the uveitis clinicc of the National Eye Institute. Bethesda, Maryland. Before participating in the study, all pa-tientss gave informed consent for a protocol approved by the Medical Review Board of the Na-tionall Eye Institute. All patients with active uveitis involving the posterior segment were con-trolledd on cyclosporine {1.0 to 5.0 mg/kg/day) and prednisone (2.5 to 30 mg/day). Any patient withh a reactivation of uveitis in the 3 months prior to this study was considered as clinically ac-tive.. A drop in visual acuity by 2 lines or more on an ETDRS reading card associated with evi-

Tablee 1: Characteristics of Uveitis Patients and Controls:

Diagnosis s

Controls s

Behcet'ss D.

Sarcoidosis s

VK H H

Sympatheticc O.

Numberr of Patients s

15 5

12 2

9 9

4 4

4 4

Sex x M/F F

9/6 6

6/6 6

5/4 4

2/2 2

2/2 2

Age e (years) )

35 (23 -55) )

29 (22 -41) )

35 (18 -45) )

27(211 -33)

411 (31 -47)

Diseasee Duration (months) )

47(10-96) )

42(10-82) )

400 (8-70)

44(11-81) )

Diseasee Activitv Active/Inactive e

6/6 6

4/5 5

3/1 1

0/4 4

138 8

H S - AGG DETERMINANT RECOGNITION IN PATIENTS

Q Q Q. . O O > >

>> S >> Q

Q Q

> >

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>--o o LX X

OO CO

> >

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LU U

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c c 'E E

COO S3

c o Q Q

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UJJ Q

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t --D. . t-t-CO O

< <

< <

> > CL L CL L

> > > > OO O)

£^ ^ CCC co

'E E COO s LUU <D

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> > > >

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> > >--> >

(3 3

> >

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C/3 3 Q Q

LU U

< <

>> -LUU E LUU co

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E E

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o o en n * * o o co o o o o o

• * *

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F F <D D <t) )

a a _J J > > D D CL L LU U

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ta ta

a a

CM M

CO O

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LU U

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ca a

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F/gHWW /.- Sequence of human S-Ag and its overlapping peptide determinants. The nomenclature ofof each determinant as used in this article is indicated under each determinant.

139 9

ChapterChapter 9

Tablee III : Maximum number of immunoproliferative determinants recognized by individual patients per disease categoryy 1

Diseasee Entity

Behcet'ss D.

Sarcoidosis s

Vogtt Koyanagi Harada

Sympatheticc Ophth.

Antigen n Concentration n inn Culture (|ig/mL)

20 0 100 0 20 0

100 0 20 0

100 0 20 0

100 0

1 1

4 4 0 0 1 1 0 0 0 0 0 0 2 2 1 1

Numberr of patients responding to the

2-3 3

2 2 4 4 3 3 2 2 2 2 0 0 1 1 2 2

## of determinants 2 4-5 5

2 2 1 1 0 0 1 1 0 0 1 1 0 0 1 1

6-10 0

4 4 5 5 3 3 2 2 1 1 2 2 0 0 0 0

>> 10

0 0 2 2 I I 3 3 I I 1 1 0 0 0 0

Max x

10 0 13 3 13 3 19 9 14 4 17 7 2 2

4 4

Ann immunoproliferative determinant is defined as a S.I. > mean of control subjects + 2 standard deviations. "Numberr of H-SAg determinants to which a given patient responds. Max corresponds to the highest number of re-sponsee sites registered for a patient within the specified disease category and at the specified antigen concentra-tionn in culture.

Results s

PatientPatient characteristics and assessment of lymphocyte response Twenty-ninee patients and fifteen volunteers were tested for their lymphocyte proliferative re-sponsess to a panel of overlapping human S-Ag peptide determinants at 20p,g/mL and 100 jxg/ mL.. Since the optimal culture conditions for lymphocyte proliferation with these peptide deter-minantss was not known, both concentrations were used. All patients had evidence of retinal in-flammatoryy disease for at least 8 months (8 - 120 months) prior to being tested. All patients had aa chronic remitting course of disease at the time, with the exception of the sympathetic ophthal-miaa patients who were quiescent for over one year prior to testing. A number of patients had a historyy of an acute inflammatory episode in the 6 months prior to testing (Table I).

DeterminantDeterminant mapping using analysis of variance Proliferativee responses to individual peptides were highly variable. In general the response to antigenn was strongest when using the higher antigen concentration (graph 1 and 2). To test for in-ternall consistency, one volunteer was repeatedly tested at different time points over a period of 1 year.. Each proliferative response remained well with the mean + 2 standard deviations of con-trolss except on one of the nine time points, when deviant responses were noted to several pep-tides.. Analysis of variance used to compare stimulation indices from each patient group to con-trolss revealed a statistically significant difference in only a limited number of peptides (Table II). Thesee were found in two disease entities, namely Behcet's disease and Sarcoidosis.

DeterminantDeterminant mapping using a tolerance limit threshold Whilee an analysis of variance showed that there was no statistically significant difference for mostt peptide determinants between controls and patients, several patients had stimulation in-dicess considerably higher than that of controls for particular determinants. Since these could cor-respondd to a subset of patients with an active immune response, a different approach was used to identifyy these determinants. Response in these cases can be better characterized as having over-comee a minimal threshold (or tolerance limit) of response defined as the mean of controls + 2

142 2


1.0 0

.75 5

.25 5

00 -

Sarcoidosis s

:flfcafcot.EEJ J MMnr-fan n

Peptidee Concentration

j ! t aa 20 jjg/mL

~ ~ ]] 10Opg/mL

11 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

1.0 0

.75 5

Behcet'ss Disease

fihi .. ni jyr i faM . I lit 11 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

1.0 0

.75 5

.5 5

.25 5

Vogtt Koyanagi Harada

E l l rTHrteonMfn n 11 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

FigureFigure 4: Proportion of respondersper determinant above the tolerance limit. The tolerance lim-itit was set as the mean + 2 standard deviations of the response in control subjects. In sympathet-icic ophthalmia, one responder was found for determinants 2, 3, 6, 11, 21, 25, 27, and 38.

standardd deviations. This analysis showed in all disease entities, a heterogeneous response to S-Ag.. In the case of Sympathetic Ophthalmia, the following determinants gave significant re-sponses:: 2. 6. 11,21, 22, 25. 27 and 38. In each case, only one patient, but not always the same, producedd a stimulation index above threshold. For the remaining disease entities, the number of significantt responders varied considerably among disease entities and among determinants (Graphh 3). Determinant 21 and 22 in Sarcoidosis, and determinant 35 in VKH were prominent in aa majority of patients. Overall patients with Sarcoidosis were most likely to show a heightened response.. Response heterogeneity was observed in all disease entities but was limited in sympa-theticc ophthalmia (table III) . Twoo patients with Behcet's disease were tested on two separate occasions. In one case, the re-sponsess were measured 3 months later, and in the other after 6 months. A shift in antigenic re-sponsee was noted for certain determinants (table 4). A similar shift was not observed in a normal volunteer. .

Discussion n

Patientss with uveitis show a high degree of heterogeneity in their lymphoproliferative response to determinantss of human S-Ag. Such heterogeneity is not surprising given that patients carried a va-rietyy of diagnosis, and were at different stages of disease activity and duration. However. 2 pat-ternss of response could be observed, one which appeared to be disease specific, and the other spe-cificc to individual patients. The first relates to a generalized increase in lymphocyte proliferation too selected determinants in patients with Behcet's disease or Sarcoidosis, while the second relates

143 3

ChapterChapter 9

Tablee JV: Response to selected determinants of S-Ag over time

Diagnosis s

Control l

Behcet'ss D.

Behcet'ss D.

Timee point (months) )

0 0 2 2 6 6 12 2 0 0 6 6

0 0 2 2

I I

1.4 4 1.1 1 1.2 2 0.8 8 1.0 0 2.9 9

0.9 9 1.1 1

2 2

1.0 0 1.2 2

1.0 0 0.8 8 0.5 5 0.9 9

2.2 2 1.3 3

Responsee to

3 3

3.0 0 4.0 0 5.0 0 5.0 0 0.8 8 1.5 5

1.7 7 1.7 7

S-Agg Determinant (stimulationn index)

4 4

1.5 5 1.2 2 1.0 0 1.0 0 0.8 8 1.0 0

1.0 0 1.2 2

5 5

1.9 9 t.4 4 1.2 2 1.2 2 0.8 8 2.3 3

1.1 1 5.9 9

6 6

1.3 3 2.0 0 1.2 2 0.8 8 0.3 3 2.2 2

2.3 3 4.4 4

7 7

1.7 7 2.4 4 1.1 1 1.0 0 1.0 0 9.1 1

1.7 7 1.0 0

8 8

2.4 4 1.4 4 0.6 6 1.0 0 2.5 5 2.1 1

1.0 0 1.8 8

too the observation that a number of patients have heightened immune responses to specific deter-minants,, significantly above the mean of controls. This heightened response, when present to wholee S-Ag, was previously shown to be a sign of disease activity within weeks of the assay. Thee immunogenic determinants associated with Behcet's disease and Sarcoidosis (table II) arc adjacentt to highly immunogenic or pathogenic determinants in the experimental model EAU. Determinantss 19, 20, and 36 induce uveitis in the Lewis rat. Determinant 18 is immunoprolifer-ativee in the eyanomologus monkey. Since the determinants we studied are 20 amino acids long, thee minimal immunologic trigger for each determinant is nested somewhere within the peptide sequence.. Use of smaller synthetic peptides should make it possible to identify the minimal im-munogenicc sequence and in particular reveal whether or not the same sequence is immunogenic inn all species. The identification of appropriate targets may be facilitated by selecting for sequencess which do not contain homology with known non-retinal arrestins, and which are not expressedd in the thymus.

Thee second pattern of response was observed in a limited number of patients for most disease en-titiess studied except in sympathetic ophthalmia. The heightened response was present to a variable butt significant number of determinants. Each response pattern was patient specific and involved moree determinants in patients with long standing chronic disease (table III) . Recent observations inn experimental autoimmune encephalitis have shown that with disease recurrence, there is a de-creasee in autoreactivity to the primary determimant and the emergence of other, previously cryp-ticc determinants whose appearance is linked with disease progression. This process follows a ratherr predictable course in which the spread to new determinants characterizes particular phases off the disease and is amenable to peptide-based therapy. A similar plasticity of self-recognition hass been observed in patients with multiple sclerosis. Over a 12 to 18 month follow-up of multi-plee sclerosis patients, antigen spread was noted, but also an abrupt shift in responses to an exten-sivee array of self determinants. We infer that a similar process is present in uveitis patients. Certainn patients are capable of showing an immune response, which over lime either significant-lyy increases or decreases back to a baseline level (table IV). However, variations are present amongg disease entities, as the response in sympathetic ophthalmia was limited to a few determi-nants.. The relatively limited determinant spread in this latter disease may indicate that S-Ag does nott play a role in this disease process, as the overall response profile is very similar to that of con-troll subjects. Pathological changes in this disease are located within the choroids, and experi-mentally,, a similar disease pattern is induced by using choroidal antigens. Determinant spread-

144 4


ingg is likely to be an integral part of the immune response over time to antigens, which contribute too the disease process. Inherent to determinant spreading, is a shift in response over time. We havee observed in two patients but not in a volunteer, such a shift in antigenic responses over time (tablee IV). This shift in autoreactivity is unlikely to be an artifact, as repeat testing of a volunteer overr an 12 month period showed only limited variations in stimulation indices which remained withinn 2 standard deviations of the mean of all control subjects. These shifts in responses corre-spondd well to the observations made in multiple sclerosis and EAE, and suggest that antigen spreadingg also occurs in uveitis patients. It deserves to be studied in more detail with a number off patients followed prospectively.

Inn summary, the present study demonstrates the existence of a number of disease specific anti-genicc determinants in two disease entities namely Behcet's disease and Sarcoidosis. These were associatedd with unique sequences within the human S-Ag sequence and were related to patho-genicc sites in the experimental model. It also demonstrates the existence of a number of deter-minantss associated with immune responses in individual patients. These responses were present inn patients with a retinal disease pattern. The observations give credence to the notion that reti-nall autoantigens play a role in certain forms of uveitis. Further study of determinant responses in patientss with uveitis may help to identify the exact site of the immune activation, and may pro-videe the basis for a rationale attempt at peptide-based therapy.

References s

1.. Hirsch JA, Shubert C, Gurevich VV, Sigler PB. The 2.8 A crystal structure of visual arrestin: a model for ar-restingg regulation. Cell 1999;97:257-69.

2.. Granzin J, Wilden U, Choe HW. Labahn J, Krafft B, Biildt G. X-ray crystal structure of arrestin from bovine rodd outer segments. Nature 1998:391:918-21.

3.. Wilden U. Duration and amplitude of the light-induced cGMP hydrolysis in vertebrate photoreceptors are regulatedd by multiple phosphorylaton of rhodopsin and by arrestin binding. Biochemistry 1995:34:1446-54.

4.. Lohse MJ, Benovic JL7 Codina J, Caron MG. Lefkowitz RJ. Beta-arrestin: a protein that regulates beta-adren-ergicc receptor function. Science 1990;248:1547-50.

5.. Attramadal H, Arriza JL, Aoki C. et al, fc>-arrestin2. a novel member of the arrestin/lï-arrestin gene family. J BiolChemm 1992;267:17882-90.

6.. Sterne-Marr R, Gurevich VV, Goldsmith P, et al. Polypeptide variants of 8-arrestin and arrestin 3. J Biol Chemm 1993;268:15640-8.

7.. Craft CM, Whitmore DH, Wiechmann AF. Cone arrestin identified by targeting expression of a functional family.. J Biol Chem 1994;269:4613-9.

8.. Gery I, Mochizuki M. Nussenblatt RB. Retinal specific antigen and immunopathogenic processes they pro-voke.. Prog Ret Res 1986;5:75-109.

9.. Mochizuki M, Kuwabara T, McAllister C, Nussenblatt RB, Gery I, Adoptive transfer of experimental au-toimmunee uveoretinitis in rats. Invest Ophthalmol Vis Sci 1985;26:1-9.

10.. Kotake S. de Smet MD. Wiggert B, Redmond TM. ChaderGJ. Gery I. Analysis of the pivotal residues of the immunodominantt and higly uveitogenic determinant of interphotoreceptor retinoid-binding protein (IRBP). JJ Immunol 1991:146:2995-3001.

11.. Hirose S. Singh VK. Donoso LA, et al. An 18-mer peptide derived from the retinal S antigen induces uveitis andd pinealitis in primates. Clin Exp Immunol 1989:77:106-11.

12.. Gregerson DS. Obritsch WF, Fling SP. Identification of a uveitogenic cyanogen bromide peptide of bovine S-antigenn and preparation of a uveitogenic peptide specific T cell line. Eur J Immunol 1987:17:405-11.

13.. de Smet MD, Yamamoto JH. Mochizuki M, et al. Cellular immune responses of patients with uveitis to reti-nall antigens and their fragments. Am J Ophthalmol 1990:110:135-42.

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14.. Yamamoto JH. Minami M. Inaba G, Masuda K. Mochizuki M. Cellular autoimmunity to retinal specific anti-genss in patients with Behcet's disease. Brit J Ophthalmol 1993;77:584-9.

15.. Doekes G. van der Gaag R. van Kooyk Y, et al. Humora! and cellular immune responsiveness to human S-antigenn in uveitis. Curr Eye Res 1987;6:909-19.

16.. Hirose S. Donoso LA. Shinohara T. Palestine AG, Nussenblatt RB, Gery I. Lymphocyte responses to peptide MM and to retinal S-antigen in uveitis patients. Jpn J Ophthalmol 1990:34<3):298-305.

17.. de Smet MD7 Wiggert B. Chader GJ, Mochizuki M, Gery I, Nussenblatt RB. Cellular immune responses to fragmentss of S-antigen in patients with uveitis. In: Usui M. Ohno S, Aoki K, eds. Ocular Immunology Today. Tokyo.. Japan: Elsevier Science Publ, 1990:285-8.

18.. Unanue ER. Cerottini JC. Antigen presentation. FASEB 1989;3:2496-502. 19.. Watts C. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Im-

munoll 1997:15:821-50. 20.. Buus S. Sette A. Colon SM. Miles C. Grey HM. The relation between major histocompatibility complex

(MHC)) restriction and the capacity of la to bind immunogenic peptides. Science 1987:235:1353-8. 21.. Panina-Bordignon P. Tan A, Termijtelen A. Demotz S, Corradin G, Lanzavecchia A. Universally immuno-

genicc T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Europeann J Immunol 1989:19:2237-42.

22.. Sinigaglia F, Guttinger M, Kilgus J. et al. A malaria T-cell epitope recognized in association with most mouse andd human MHC class II molecules. Nature 1988;336:778-80.

23.. Martin R. Howell MD. Jaraquemada D, et al. A myelin basic protein peptide is recognized by cytotoxic TT cells in the context of four HLA-DR types associated with multiple sclerosis. J Exp Med 1991; 173:19-24.

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25.. Shinohara T, Donoso L, Tsuda M. Yamaki K, Singh VK. S-Antigen: Structure, function and experimental au-toimmunee uveitis (EAU).. Prog Retinal Res. New York. USA.: Pergamon Press, 1989:8:51-65.

26.. Behcet's disease research committee of Japan. Behcet's disease: guide to diagnosis of Behcet's disease. Jpn J Ophthalmoll 1974;18:291-4.

27.. Snyder DA. Tessler HA. Vogt-Koyonagi-Harada syndrome. Am J Ophthalmol 1980:90:69-75. 28.. de Smet MD, Dayan M. Prospective determination of T cell responses to S-antigen in Behcet's Disease pa-

tientss and controls. Invest Ophthalmol Vis Sci 2000;fin press). 29.. de Smet MD. Bitar G. Roberge FG. Gery I, Nussenblatt RB. Human S-antigen: presence of multiple im-

munogenicc and immunopathogenic sites in the lewis rat. J Autoimm 1993:6:587-99. 30.. Fukushima A, Lai JC, Chanaud NP, III , et al. Permissive recognition of immunodominant determinants of the

retinall S-antigen in different rat strains, primates and humans. Int Immunol 1996:9:169-77. 31.. Xu H, Wawrousek EF. Redmond TM, et al. Transgenic expression of an immunologically privileged retinal

antigenn extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis. Eur J Im-munoll 2000:30:272-8.

32.. McPherson SW, Roberts JP. Gregerson DS. Systemic expression of rat soluble retinal antigen induces resis-tancee to experimental autoimmune uveoretinitis. J Immunol 1999:163:4269-76.

33.. Egwuagu CE, Charukamnoetkanok P, Gery I, Thymic expression of autoantigens correlates with resistance to autoimmunee disease. J Immunol 1997:159:3109-12.

34.. Tuohy VK, Yu M, Yin L, Kawczak JA, Kinkel RP. Spontaneous regression of primary autoreactivity during chronicc regression of experimental autoimmune encephalomyelitis and multiple sclerosis. J Immunol 1999;189:1033-42. .

35.. Lehmann PV, ForsthuberT. Miller A, Sercarz EE. Spreading of T-cell autoimmunity to cryptic determinants off an autoantigen. Nature 1992;358:155-7.

36.. Yu M, Johnson JM. Tuohy VK. A predictable sequential determinant spreading cascade invariably accompa-niess progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onsett of clinical disease. J Exp Med 1996:183:1777-88.

37.. Tuohy VK, Yu M, Weinstock-Guttman B, Kinkel RP. Diversity and plasticity of self recognition during the developmentt of multiple sclerosis. J Clin Invest 1997;99:1682-90,

38.. Chan CC. Roberge FG. Sympathetic ophthalmia. In: Pepose JS. Holland GN. Wilhelmus KR, eds. Ocular in-fectionn and immunity. St Louis: Mosby, 1996:723-33.

146 6

Sectionn III : Novel Therapeutic Strategies in Uveitis

"" Pour embrasser Ie problème medical dans son entier, la médecine experimental doit com-prendreprendre trois parties fondamentales: la physiologie, la pathologie et la thé rape utique. La médecinemédecine scientifique ne peut se constituer ainsi que les autres sciences, que par une voie ex-périmentale,,,,périmentale,,,, le raisonnement est toujours Ie même, mais dans chaque genre de science, les phénomènesphénomènes varient et présentent une complexité et des difficultés d'investigation qui leur sont propres.propres.11' '

Claudee Bernard: Introduction a 1'étude de la médecine experimental 1865

Chapterr 10

FK5066 Treatment of Experimental Autoimmune Uveoretinitiss in Primates

Yujiroo Fujino, Chi C Chan, Marc D. de Smet, Naofumi Hikita, Igal Gery, Manabuu Mochizuki, Robert B. Nussenblatt

Transplantationn Proceedings 23: 3335-3338, 1991 (byy permission © Elsevier Science)

FK5066 IN EAU

UVEITIS S

FKK 506 Treatment of Experimental Autoimmune Uveoretinitis inn Primates

Y.. Fujino, C.-C. Chan, M.D. de Smet, N. Hikita, I. Gery, M. Mochizuki, and R.B. Nussenbtatt

FKK 506 is a neutral macrolide isolated from the fermen-tationn broth of Streptomyces tsukubaensis.^'2 It has a

pp h arm ac o physio logic action similar to that of cyclosporine AA (CyA). It suppresses mixed lymphocyte reactions, the productionn of T-cell-mediated soluble factors, and the expressionn of interleukin 2 (IL-2) receptor.2,3 It has been extensivelyy investigated using transplantation models.""* Cyclosporinee is currently used in the treatment of various immunologicallyy mediated diseases in the eye,10 but its usefulnesss is limited by its adverse side effects."

Experimentall Autoimmune Uveitis (EAU) is an experi-mentall model of ocular autoimmune disease that can be inducedd in different animal species by immunization with a retinal-specificc antigen in the presence of an adjuvant.12"1" Thoughh the exact mechanism of EAU is still controversial, TT cells appear to play a central role.15,16 In the Lewis rat, wee have shown that FK 506 was effective in preventing the expressionn of EAU at a dose less than 0.3 mg/kg/d.17

Withinn the eye. FK 506 was able to inhibit the expression off IL-2 receptors on T cells and prevent the expression of MH CC class II antigens on ocular resident ceils. It also significantlyy delayed the cellular kinetics of EAU, causing aa significant increase in the recruitment time of the T-sup-pressor/cytotoxicc cells."*

Moree recently, we reported on the inhibition of clinical EAUU in the primate model with doses as low as 0.125 mg/kg/d."" The EAU model in the primate more closely resembless certain ocular inflammatory conditions found in mann both by the observed clinical course as well as by the histopathologyy of the ocular lesions.20 We now report on thee histopathologic and immunohistochemical nature of thesee lesions as well as on the systemic toxicity observed inn our treated animals.

MATERIALSS AND METHODS Animalss and Immunization Procedures

Ann extensive description of the study design, immunization schedule,, and follow-up is given in our previous report.19 In summary,, 15 rhesus monkeys (Macaca mulatto. 7 males and 8 females)) were used in this study. All animals were provided with foodd and water ad libitum. All animals were obtained from an NIH-approvedd random source and housed in environmentally controlledd rooms with a [2-hour light and dark cycle. The study wass approved by the institutional animal care and use committee andd complies with the Public Health Service Policy on the

Humanee Care and Use of Laboratory Animals. The animals were immunizedd with bovine S-antigen in phosphate buffer saline (PBS) emulsifiedd in complete Freund's adjuvant (CFA, 1:1) containing MycobacteriumMycobacterium tuberculosis H37Ra at 1.25 mg/mL (Ditto. De-troit,, Mich.). The emulsion was injected as described elsewhere19

intoo multiple intradermal sites over the dorsal thoracic region of eachh animal.

Drugg Dosage and Administration

FKK 506 was provided by Fujisawa Pharmaceutical Co., Osaka. Japan.. The drug was dissolved in PBS and administered intramus-cularlyy on a daily basis starting on day 21 or 23 as previously described."" Control animals were similarly injected with the vehiclee alone. The treatment was given for 15 to 49 days depend-ingg on the experimental group and the animal. Three dose levels weree evaluated, namely 0.125, 0.25, and 0.5 mg/kg/d.

Laboratoryy Determinations

Alll animals were anesthetized with 1M ketamine/rompun on a weeklyy basis and phlebotomized. Laboratory studies included a completee blood count and a chem screen (Mid-Atlantic Regional Laboratory,, Rockville Md.).

Histologyy and Postmortem Examination

Thee monkeys were sacrificed on day 70 after immunization or as soonn as they had lost 15% of their pre-immunization weight. An autopsyy was performed on all animals within 2 hours of death. The eyess were enucleated immediately, bisected with one half of the specimenn imbedded in O.C.T. and snap-frozen. The other half wass fixed in 10% formalin and used to prepare sections for histologicc examination.

Immunohistopathofogy y

Usingg the avidin-biotin-peroxidase complex (ABC) method, 6-/im seriall frozen sections of each frozen eye were prepared for immunohistologicc study. Adjacent frozen tissue sections were

Fromm Laboratory of Immunology, National Eye Institute (Y.F., C.-C.C,, M.D.d.S., N.H.. I.G., R.B.N.), Bethesda, Maryland, and Dept.. of Ophthalmology (MM), Kurume University, Kurume, Japan. .

Addresss reprint requests to Robert B. Nussenbtatt, MD, Bldg. 10,, Rm. 10N202, NIH, Laboratory of Immunology, National Eye Institute,, Bethesda, MD 20892.

BB 1991 by Appleton & Lange 0041-1345/91'$3.00,'+0 0

TransplantationTransplantation Proceedings, Vol 23, No 6 (December), 1991: pp 3335-3338

151 1

ChapterChapter 10

FUJINO,, CHAN, OE SMET ËT AL

Tablee 1. Effect of FK 506 on the Development of EAU byy Hist opat hoi ogy

EAU U MonKey y

No' '

6620 0 9992B B 993B B 69N N

248H H 663D D 9906 6

30 0 S591 1

41J J 16S S

902H H 43K K 792 2

7J J

Onsett Day'

24 4

--26 6 33 3 33 3

--33 3

----53 3

--60 0

--

Seventy y

--•++ ^ +

-I-- t- +

-i-- + t

" " --* * ~ ~

++ + + $

--tt 1-5

----

*Tn©© designation given to the monkey by ïhe NJH animal lacility. •"Tromm the day of the first immunization. 'Histopathologyy grading of EAU: H - . severe: H. moderate *•, mild. - no

intlammation. . 5EAUU developed in monkeys 16S and 43K atter treatment was discontinued

(16SJJ or dosage reduced W K )

stainedd with hematoxylin and eosin (H&Et. The primary mono-clonall antibodies were OKT4A (CD4!. OKT8F (CD* I. Leu-I4 (CD22),, Dako Macrophage (CD68I. HLA-DR. and ICAM. All monoclonall antibodies were purchased from Ortho Diagnostic Systemss Inc.. Raritan, NJ, and Becton Dickinson. Mountain View,, Calif. Mouse ascites fluid containing 1 to 2 jig nonspecific proteinn per mL was used as a control. As a secondary antibody, aa hiotin-conjugated horse anti-mouse lgG was used (Vector Lab-oratories,, Burlingame, Calif. I, The immunopathology was scored ass previously described.18 In brief, positive cells were counted andd recorded for the same anatomic area of each sectiun.

Statistics s

Thee significance of differences between groups was determined by Student'ss / lest.

RESULTS S Ocolarr Histopathologic Findings

Histopathologicc findings are summarized in Table 1 for bothh the control and the FK 506-treated animals, EAU was observedd histologically in five of six control animals. Onset off uveitis was between 24 and 33 days after immunization forr the control group, whereas in the treated group the diseasee was limited to 3 of 10 animals; one monkey developedd disease 10 days after the termination of FK 506 therapy,, while in another the disease developed after a dosee reduction to 0,0625 mg/kg/d. The histopathologic changess in the control group resembled those previously reported.200 These changes included necrosis, gliosis, and lymphocyticc infiltration in the retina, particularly sur-roundingg the venules, hypertrophy of the retinal pigment epithelium,, and thickening of the choroid with granuloma-touss lymphocytic infiltrations. Normal ocular morphology

Tablee 2. Final Measure and Change From Baseline for Total WBC,, Lymphocyte Count, and Hematocrit With

FKK 506 Treatment

FK506 6 (mgkgd) )

Nonee (5) 0-55 (2) 00 25 (4) 0.125(4) )

WBCdO3. .

8.2(A-1.0 0 66 4 (A-2.7 -77 0 (.1-0.7 -8.00 (A-0.4

mL) )

-- 1.5) == 0.9} cc 0.9) -- 2.0)

Lymphocyte e

(%) ) 477 (A-4 2: 10) 133 (.1-13 13) 288 (A-5 z. 6) 311 (.1-8 - 12}

Hematocrit t (Vot%) )

388 { i -1 i 3) 27** (i-10 4) 400 (i-6 - 2) 388 (i-5 = 2)

'P'P - 01 compared to baseline

wass observed in most animals treated with FK 506. In the threee animals that developed uveitis, one had extensive changess comparable to those in the control group. His diseasee started after cessation of therapy. In the other animals,, only focal areas of involvement were found with onlyy mild to moderate lymphocytic infiltration limited mainlyy to the choroid and the subretinal space.

Immunohistochemistry y

Inn the control animals, imrnunohistochemical staining re-vealedd that the predominant infiltrating cell population weree lymphocytes with a ratio of 1:1 between B and T cells.. The ratio between the T helper/inducer (CD4) and theTT suppressor/cytotoxic(CD8) was 1,5,1, MHCclass ll antigenss were expressed on ocular resident cells: retinal pigmentt epithelium (RPE), vascular endothelial cells, and retinall glial cells. Intercellular adhesion molecule-1 (ICAM-l )) was identified on the vascular endothelium of vesselss located in the area of inflammation.

Inn the treated animals, no inflammatory cells were identifiedd in those animals with no evidence of clinical or histopathologicall involvement. However, in the three monkeyss with disease, there were infiltrating lymphocytes withh a relative decrease in the f-helper/inducer (CD4) cell populationn as compared to the T-suppressor/cytotoxic (CDSlcelll population (mean ratio of 0.8:1). B cells showed aa marked decrease with a ratio of 1:4 between B and T cells.. ICAM- l was not visible on either the vascular endotheliumm or the RPE, MHC class II antigen expression wass only partially diminished.

Sidee Effects of FK 506

Animalss treated with 0.5 or 0.25 mg/kg had a \09r to 15C? weightt loss during the course of therapy as was previously reported.1''' In some animals, the weight loss was accom-paniedd by anorexia, lethargy, and diarrhea, which may welll have contributed to the change in weight. Table 2 summarizess both the final measurements and the degree of changee from baseline for peripheral blood counts. Adverse effectss were largely limited to the animals treated with 0.5 mg/kg/d.. A statistically significant decrease in the hemato-critt was seen as well as a decrease in the total white celt count.. The white ceil count was noted to transiently drop, earlyy in the treatment in all groups, followed by a normal-izationn of the count by the third week of therapy. A

152 2

FK5066 IN EAU

FKK 506 TREATMENT OF UVEORETINITIS

Tablee 3. Effects of FK 506 on

FK506 6 (mg/kgd) )

Nonee (5)'

0-55 (2)

0.255 (4)

0.125(4) )

Totall Protein (gdu u

7.00 (A-0.3 0.4)

6.88 (A-0.2 0.6)

77 5 (A-0.4 - 0.7)

8.11 {1-0.6 0.6)

Albumin n

(9ÖD D

44 3 ( i -0 .3 0.6)

4.4(A-0.44 0 1)

4.22 ( i-0.1 0.5)

4.66 [A-0 t 0.7)

'Resultss (or each experimental group are expressed as a mean value at the end inn parentheses

'Numberr of animals in each group *PP - 02 lor the change trom the beginning of therapy when compared to none *PP - 01 ior the change trom the beginning of therapy when compared to none

relativee lymphocytopenia was noted in some animals but wass less predictable in onset and duration.

Resultss of the liver-function tests are summarized in Tablee 3. Statistically significant rises in the serum titers weree noted for LDH and SGPT in animals treated with 0.5 mg/kg/d.. The rise in LDH was first noted about 14 days intoo the treatment phase and continued for about 2 weeks afterr cessation of therapy. No abnormalities of blood glucosee were seen in any of the treated animals. Blood ureaa nitrogen (BUN) was found to be elevated in the animalss treated at the 0.5 mg/kg/d dose. This was not associatedd with a rise in serum creatinine and may have beenn the result of dehydration as a result of anorexia and diarrhea. .

Autopsyy showed diffuse multiple granulomas in the liver andd kidneys as well as enteritis in the two animals treated att the 0.5 mg/kg/d dose. Abnormal liver pathology was notedd in two out of four monkeys treated with ü.25 mg/kg/d,, and one of four monkeys treated with 0.125 mg/kg/d.. Two of five control animals also had multifocal granulomass in the liver. None of the control animals or the animalss treated at the 0.25 or 0.125 mg''kg/d dose had any granulomatouss kidney changes. Enteritis was noted in one outt of four monkeys treated with 0.25 mg/kg/d, in one out off four monkeys treated with 0,125 mg/kg/d, and in two out off five control monkeys,

DISCUSSION N

Histopathologicc examination of the eyes from primates treatedd with FK 506 indicates that it can effectively inhibit S-Agg induced EAU in all animals treated at the 0.5 mg/kg/d dosee and the majority of the animals treated with 0.25 and 0.1255 mg/kg/d. Inhibition of EAU occurred despite starting therapyy 3 weeks after the first immunization, at a time whenn the immunopathogenic response was in an advanced stagee of development as judged by the onset of disease in thee control animals shortly thereafter. Thus, FK 506 is a powerfull inhibitor of the effector limb of the immunopatho-genicc response.21 In eyes from animals with only partial responsee to FK 506, a dynamic shift was observed in the naturee of the infiltrating lymphocytes. The increase in the proportionn of CD8 and the decrease in the proportion of CD222 cells are assumed to be a result of the inhibitory effectss of FK 506.18 A shift toward infiltrating CD8 cells

Liverr Functions in Primates*

nn Alk Phos SGPT LDH HUL)) (JUL) (Ll'L)

2444 (A-3 = 37) 49 (A-24 = 24) 673 (A-514 330)

2188 (A-£2 59) 57 ( i - *7 * ~ 30) 943 (A4095 16)

178(A122 * 47) 48 (.12 :t 18) 434 (A-516 383)

176(A366 24) 31 (A-30 9) 441 (A-295 ^ 223}

3tt treatment. The change trom the mean value at the beginning of therapy is given

wass also observed with suboptimal doses of CyA in the Lewiss rat model.22 The decrease in the infiltrating B-cell populationn (CD22) is noteworthy. It is likely to be second-aryy to a reduction in the recruitment of B cells into the ocularr tissue by the reduced number of T helper cells present.. A decrease in both the cellular and humoral responsess to S-Ag have been observed following adminis-trationn of FK 506 during the etferent phase.,g'21 However, thee effect of FK 506 on B cells appears to be primarily mediatedd through its action on T helper cells.33 A reduc-tionn in the expression of class II antigens on ocular residentt cells is also noted as is a decrease in the expres-sionn of ICAM-1 on the vascular endothelium. Both are importantt in the propagation of the local inflammatory response.. Their decreased expression further limits the recruitmentt of inflammatory cells.

Sidee effects in the monkeys treated with FK 506 were largelyy limited to the animals treated with 0.5 mg/kg/d. Previouss reports have suggested that the adverse effects observedd varied considerably from one animal species to thee next. In rats, little acute toxicity has been observed evenn at higher doses of 1 to 3 mg'kg/d.4'24 In dogs, intussusception99 and vasculitis5*1 have both been ob-served.. Liver involvement was found in some animals with aa significant increase in the liver transaminases and alka-linee phosphatase.5 Few toxic changes have been observed too date in primates. Thiru et al6 reported vasculitis in transplantedd baboons, but this was not found by other investigators.7-255 The major toxic changes reported have beenn limited to the kidneys and to the pancreas.25'2" Side effectss were observed in our animals only at the highest dosee tested. We did not see any hyperglycemia. However, thesee changes were only observed in animals receiving t mg/kg'dd or more of FK 506. The increase observed in the BUNN in our animals treated with 0.5 mg/kg/d is in keeping withh other investigators6'2'' and was reversible. The change inn the LDH has not previously been observed. The LDH mayy have come from several sources. It is known that Freund'ss adjuvant can cause muscle atrophy when used repeatedly277 and might explain the initial high titers seen in alll animals. However, in all groups except 0.5 mg/kg/d, theree was a decrease in the titer during the course of treatment.. In the animals treated with 0.5 mg/kg/d, the rise inn SGPT and LDH during therapy suggests that FK 506

153 3

ChapterChapter JO

mayy have been mildly hepatotoxic though it was not severee enough to cause any significant impairment of liver function. .

Finally,, it appears that FK 506 is an effective means of inhibitingg EAU. Littl e acute toxicity is found at doses necessaryy to inhibit EAU in the majority of animals.

REFERENCES S

1.. Kino T, Hatanaka H. Miyata S, et al: J Antibiol 40:1256. 1987 7

2.. Kino T, Inamura N. Sakai F, et al: Transplant Proc I9<suppl 6):36.. 1987

3.. SawadaS. Suzuki G, Kawase Y, et al: J Immunol 1.19:1797, 1987 7

4.. Todo S, Ueda Y, Demetris AJ, el al: Surgery 104:239. 1988 5.. Thirtj S. Collier D St J, Calne R: Transplant Proc 19:98. 1987 6.. Collier DStJ, Calnc R. Thiru S, et al: Transplant Proc

2(Hsuppll l):226, 1988 7.. Todo S, Demetris AJ, Ueda Y. et al: Surgery 106:444, 1989 8.. Inamura N, Nakahara K. Kino T, et al: Transplantation

45:206,, 1988 9.. Ochiai T, Naata M, Nakajima K, et al: Transplantation

44:729,, 1987 10.. Nussenblatt RB, Palestine AG, Chan CC: Am J Ophthalmol

96:275,, 1983 11.. Nussenblatt RB. Palestine AG: Surv Ophthalmol 31:159,

1986 6

12.. Wacker WB, Donoso LA, Kalsow CM. et al: J Immunol 119:1949.. 1977

13.. Faure JP: Curr Eye Res 2:215. 1980 14.. Gery I, Wiggert B. Redmond TM, et al: Invest Ophthalmol

Viss Sci 27:1296. 1986 15.. Roberge FG. Loberboum-Galski H. Le Hoang P. et al: J

Immunoll 143:3498, 1989 16.. Gerv 1, Mochizuki M. Nussenblatt RB: Prog Retinal Res

5:75.. 1976 17.. Kawashima H, f-ujino Y, Mochizuki M: Invest Ophthalmol

Viss Sci 29:1265, 1988 18.. Ming N, Chan CC. Nussenblatt RB, el al: Autoimmunity

8:43.. 1990 19.. Fujino Y, Mochizuki M, Chan CC, et al: Current Eye Res

10:679,, 1991 20.. Nussenblatt RB, Kuwabara T. DeMonasterio FM, el ai:

Archh Ophthalmol 99:1090. 1981 21.. Kawashima H, Mochizuki M: Hxp Eye Res 51:565, 1990 22.. Chan CC, Mochizuki M, Palestine A, et al: Cell Immunol

96:430.. 1985 23.. Stevens C. Lempert N, Freed BM: Transplantation **] •

1240,, 1991

24.. Stephen M. Woo J, Hasan NU: Transplantation 47:60, 1989 25.. Ohara K, Billington R. James RW: Transplant Proc 22:83

1990 0 26.. Calne R, Collier D St J, Thiru S: Transplant Proc 19:63,

1987 7 27.. Broderson JR: Lab Anim Sci 39:400, 1989

154 4

Chapterr 11

Treatmentt of Autoimmune Uveoretinitis in the Ratt with Rapamycin, an Inhibito r of Lymphocyte

Growthh Factor Signal Transduction

Francoiss G. Roberge, Dasheng Xu, Chi C. Chan, Marcc D. de Smet, Robert B. Nussenblat, Huifang Chen

Currentt Eye Research 12: 197-203, 1993 (byy permission © Swets & Zeitlinger Publisher)

RAPAMYCINN IN EAU

Volun:cc [2 niiiiilvr : i 'W. i4? -<^

Current t E\re e Research h

Treatmentt of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocytee growth factor signal transduction

Francoiss G.Roberge, Hashing Xu1 . Chi-Chao Chan, Marc D.de Smel. Robert N Nussenblatt and Huil'ang Chen' '

Laboratoryy of" Immunology. National Lye Institute, Bethesda. MI) . USA and 'Laboratory of experimental Surgery,, Notre Dame Hospital, Montreal. Que., Canada

ABSTRACT T Rapamycinn (RAPA) is a macrolide antibiotic with

uniquee immunosuppressive properties. RAPA inhibits T-celll function by interlering with IL-2 and IL-4 signal transduction.. It does not prevent IL-2 production or IL-2RR expression. The efficacy of RAPA in the treatment off autoimmune diseases was evaluated using the experimentall autoimmune uveoretinitis {EAU} model, EAUU was actively induced in Lewis rats by immunizationn with S-antigen in Hunter's adjuvant. RAPAA and control vehicle were administered by continuouss intravenous infusion over a 14 day period byy miniosmotic pump. RAPA treatment initiated on the dayy of immunization or 7 days later was found to efficientlyy inhibit EAU induction. The minimal effective dosee was 0.1 mg/kg/d. EAU inhibition was correlated withh reduced number of ceils in the immunization site drainingg lymph nodes, as weli as with a shift and loweringg of the peak of the lymphocyte proliferative responsee curve. The anti-S-antigen antibody response wass delayed by 3 days under RAPA treatment and the serumm levels lowered in a dose dependent manner. An initiall body weight loss was observed during the first weekk of drug administration, but there was a normal weightt gain afterward.

INTRODUCTION N

Rapamycinn (RAPA) is a macrolide antibiotic

originallyy studied for its antifungal properties. It has

moree recently attracted attention as an

immunosuppressivee agent (1). It was purified from

isolatess of Streptomyces hygroscopicus collected on

Easterr Island (2). RAPA was shown to have a strong

anti-rejectionn activity in several animal models of organ

transplantation.. Heart and skin allografts were

prolongedd in the mouse by intraperitoneal (i.p.)

injectionn and oral treatment (3, 4). In rats, early

treatmentt prevented the rejection of heart, kidney, and

pancreaticoduodenall grafts, while delayed treatment

couldd reverse the process of ongoing rejection (5-8).

Overr the past two years the mechanism of action of

RAPAA has begun to be understood. RAPA was found to

inhibitt T cell proliferative signals of both Ca2+

dependentt and Ca2+ independent pathways (9-12).

Thee inhibition could not be overcome by the addition of

IL-22 or IL-4. In fact the production of these lymphokines

ass well as the expression of the IL-2 receptor were not

suppressedd by RAPA as they were by cyclosporin A

(CsA)) and FK 506. On B cells the effect of RAPA varied

withh the stimulus tested. Mouse B cetl proliferation

stimulatedd by S-Mercaptoguanine was inhibited in the

presencee of RAPA but only delayed when stimulated by

anti-IgMM or lipopolysaccharide (13). On the other hand

thee pokeweed mitogen driven proliferation and

antibodyy production of human B cells was profoundly

suppressedd by RAPA (14).

Thesee observations and an early report by Martel

ett al indicated that RAPA could be useful in the

treatmentt of autoimmune diseases (15). One of the

bestt described models of these diseases is

experimentall autoimmune uveoretinitis (EAU). EAU is

inducedd in various species by immunization with

purifiedd retinal antigens (16-20). It is better

characterizedd in the Lewis rat in which retinal lesions

aree caused by a monocytic infiltration occurring 12 to

155 days after immunization with S-antigen (S-Ag) (21).

AA critical role was ascribed to T cells because athymic

nudee rats are resistant to EAU induction unless

adoptivelyy transferred with heterozygous syngeneic T

cellss (22). In the Lewis rat, the T cell responsible for

transferr was shown to be phenotypically contained

withinn the CD4+ subpopulation (23). EAU was also

inhibitedd by treatment with CsA (24), and by selective

killingg of activated T cells with a recombinant IL-2-toxin

fusionn protein (25). In this first study on the activity of

RAPAA in EAU, we report the inhibition of actively

(.Wordd UnivLV-itv Pres

157 7

ChapterChapter J1

Current t Eye e Research h

inducedd disease with low dose intravenous (i.v.)

infusionn and characterize its effect on the immune

response. .

MATERIALSS AND METHODS

Immunizationn and, { p imen t protocol

Malee Lewis rats between 8 to 12 weeks old

(Charles-River,, Montreal, Que.) were immunized in one

hindd footpad with 20 ng S-Ag (26) in PBS, emulsified

1:11 v:v in Hunter's adjuvant (TiterMax™, CytRx,

Norcross,, Ga.) for a total volume of 40 |il per rat (27),

RAPAA (provided by Wyeth-Ayerst Reseach, Princeton,

NJ)) was solubiiized in a vehicle composed of 70%

polyethylenee glycol MW 400, 10% Tween 80, and 20%

N,NN dimethylacetamide (Sigma, St. Louis, MO).

Treatmentt was delivered at the indicated doses by

continuouss i.v. infusion over a period of 14 days by

meanss of a miniosmotic pump (Alzet model 2002, Alza,

Paioo Alto, CA) implanted in the abdominal cavty and

connectedd to a iumbar vein. Surgery was pertcmed

underr anesthesia with sodium pentobarbital 40 mg/kg

inn 0.5 ml PBS injected i.p. EAU was evaluated by

histopathologicc examination of the eyes

andd the severity graded from 0.5 to 4.0 as described

previouslyy (25). The eyes were collected on day 17

whenn the treatment started on the day of immunization

(dayy 0), and on day 28 when the treatment started on

dayy 7. The rats were weighed at seven day intervals

andd the percent variation was calculated in reference to

thee treatment initiation weight and is reported as the

averagee + SD of groups of seven rats. The animals

weree cared for according to the guidelines of the

Associationn for Research in Vision and Ophthalmology.

Lymphocytess proliferation assay

Ratss were immunized and the treatment with

RAPAA 1,0 mg/kg/d or vehicle alone was started on the

samee day. The draining lymph nodes (DLN) of the

immunizationn site were removed on day 6,9,11,13,

andd 15, and prepared to single cell suspension. Celts

weree cultured in flat bottom microtiter plates at 2 x 10$

cells/welll in 200 u.1 of RPMI 1640 supplemented with 2

mMM L-glutamine, 1 mM sodium pyruvate, 0,1 niM non

essentiall amino acids, 5 x 10 5 M 2- mercaptoethanol,

andd 50 u.g/ml gentamycin, and containing 10% FBS

(Hyclone,, Logan, UT). Cultures were done in

quadruplicatee in the presence of S-Ag 5 ug/ml, or Con

AA 2 ug/ml (Boehringer Mannheim, Indianapolis, IN), or

mediumm alone. [3H] thymidine 0.5 u.Ci/well was added

forr the last 16 h of the 64 h culture period and the

incorporatedd radioactivity measured by liquid

scintillationn counting. Results for each time point are for

groupss of four to eight rats and are given as the

averagee SEM of the stimulation indices representing

(cpmm of Ag-stimulated cultures) / (cpm of non-stimulated

cultures). .

Measurementt of antibody production

Serumm antibody levels against S-Ag were

measuredd at the indicated time points by solid phase

ELISAA following Tuyen et al (28). Serum samples were

measuredd at a 1 ;100 dilution. The secondary

antibodiess were peroxidase conjugated sheep anti-rat

IgGG and IgM (Kirkegaard and Perry, Gaithersburg, MD).

Statisticall differences were evaluated using unpaired

Student'ss f-test.

RESULTS S

Inhibitionn of EAU induction bv RAPA. and immune

functionn correlates

Treatmentt was initiated at two different time points.

Thee capacity of RAPA to inhibit EAU induction was first

testedd by implanting the osmotic pumps on the day of

immunization.. Treatment was also started seven days

afterr immunization when the immune response was

alreadyy activated to test conditions more pertinent to

actuall disease. Treatment cannot be defayed until EAU

hass started because of its brief evolution course. The

resultss obtained with treatment on the immunization

dayy showed a complete inhibition of the disease at a

dosee of 1.0 mg/kg/day (table 1). When treatment at the

samee dose was delayed for 7 days, the therapeutic

responsee was also complete (table 1). Graded dose

reductionn showed that EAU could still be effectively

inhibitedd at a dose of 0.1 mg/kg/d, while a majority of

ratss had disease at lower doses. The increase in the

numberr of cells contained in the lymph nodes draining

thee site of immunization was significantly reduced by

RAPAA treatment (figurel). The sensitization of

lymphocytess to S-Ag during treatment was evaluated

byy measuring the in vitro proliferative response. The

resultss are depicted in figure 2. There was a shift of

158 8

R.-\\ PA MYCIN IN E A U

EAU§§ Severity E

8 / 88 1.78 5

TABLEE I

EFFECTT OF RAPAMYCIN ON EXPERIMENTAL AUTOIMMUNE UVEORETINITIS'

TREATMENT?? Day 0 to 14 Day 7 to 21

EAU U

VEHICLE E

RAPAA 1.00 mg/kg/d

RAPAA 0.50 mg/kg/d

RAPAA 0.10 mg/kg/d

RAPAA 0.05 mg/kg/d

RAPAA 0.025 mg/kg/d

** Male Lewis rats were immunized (day 0) in one hind footpad with S-Ag 20 jig/rat emulsifiedd 1:1 v/v in 20 |.il Hunter's adjuvant. Results indicate number of rats positive forr EAU on histopathological examination. The severity is given by the average histopathologicc score (+SE) of positive eyes only. Treatment with Rapamycin or vehicle only was given for 14 days by continuous intravascularr infusion in the lumbar vein with an osmotic pump (Alzet model 2002) implantedd i.p. either at the time of immunization or 7 days later where indicated.

§§ The eyes were removed 17 days after immunization. HH The eyes were removed 28 days after immunization.

EAU^ ^

14// 15

0 /15 5

2 /14 4

2 /14 4

5 / 7 7

6 / 8 8

Severityy E

3.777 5

22 50 0

3.500 0

3.699 1

2.722 5


severall days in the proliferation curve as well as a

loweringg of the peak of the response. Measurement of

thee antibody production when treatment started on day

00 showed that it was delayed by three days but

progressivelyy increased to reach levels close to those

off the controls at the end of the treatment period (figure

3-A).. When treatment was started on day 7 with various

dosess of RAPA. there was a dose dependent decrease

off the antibody levels for the duration of the three-week

observationn period (figure 3-B).

Generall toxicity of RAPA

AA limited loss of weight in the rats treated with

RAPAA was observed in all experiments. Compared to

thee vehicle control group, rats treated with RAPA 10

mg/kg/dd from day 0 had a 9% average decrease in

weight.. The variation in the weight of rats after day 7

treatmentt with various doses of RAPA is shown in figure

4.. During the first week after pump placement, a similar

weightt loss was noticeable at all therapeutic RAPA

doses.. However, during the second week of treatment

REDUCTIONN OF DLN CELL NUMBER BY RAPAMYCIN

1200 -

100 0

800 -

600 -

400 -

20 0

0 0

•• Vehicle

(( Rapamycin 1.0 mg 'kg d

|| }

| | 1 1 1 1 JL L

Figuree 1. Viablee cell counts in the DLN of rats, as the average

DD from groups of 4 to 8 animals immunized with S-Agg 20 u.g in Hunter's adjuvant, and treated from day 00 with RAPA 1.0 mg/kg/d or vehicle alone by continuouss i.v. infusion with a miniosmotic pump.

159 9

ChapterChapter 11


EFFECTT OF RAPAMYCIN TREATMENT ON LYMPHOCYTEE PROLIFERATION TO S-ANTIGEN

Figuree 2. Inn vitro proliferative response of DLN cells (2 x 1fj5 cells/well)) to S-Ag 5 ng/ml. Lewis rats were treated fromm the day of immunization with RAPA 1.0 mg/kg/d or vehiclee alone by continuous i.v. infusion with a miniosmoticc pump. Results are given as the average S.I.. M of groups o! 4 to 8 rats.

thee initial loss was regained, and the rats growth

continuedd normally during the following week.

DISCUSSION N

Thee increased understanding of the mechanism of

uveitiss brought about by the study of EAU in animals

hass allowed for improvement in our therapeutic

approach.. In particular the demonstration of the critical

rolee played by T cells has led to the use of CsA with

greatt success (29,30). FK 506 is also currently being

clinicallyy evaluated in Japan for the treatment of severe

uveitiss (31). RAPA is related to CsA and FK 506 by its

non-cytotoxicc mode of action, as opposed to agents

suchh as cyclophosphamide or chlorambucil . CsA and

FKK 506 prevent the transcription of T cell IL-2 and IL-4

geness (32-34). RAPA appears to differ from all other

immunosuppressivee agents through its capacity to

inhibitt lymphocytes by preventing the signal

transductionn of growth factors (9-12). This effect was

reflectedd by the observed reduction in the number of

cellss present in the lymph nodes of immunized animals.

EFFECTT OF RAPAMYCIN TREATMENT ON ANTIBODY PRODUCTION

11 1

1.0 0

0.9 9

0.8 8

0.7 7

0.6 6

0.5 5

0.4 4

0.3 3

0.2 2

0.1 1

.. B

•• / /

'/// '/// f f

11 / Rapamycin / ^

Vehiclee ^ ^

Figuree 3. Antii S-Ag antibody levels in the serum of rats treated in AA from day 0 and in B from day 7 after immunization, withh RAPA 1.0 mg/kg/d (•) , 0.1 mg/kg/d (A) or vehicle

alonee (O). Results are the average M of groups of 77 rats. The difference between RAPA and vehicle treatedd Ab levels w is statistically significant with p<< 0.005 at all time points except days 14 and 17 in A.

160 0

RAPAMYCINN IN EAU


RATSS WEIGHT VARIATION UNDER RAPAMYCIN TREATMENT

DAYY 14 DAY 21 DAY 28

Figuree 4. Variationn in the weight of rats measured at weekly intervalss and given as percent change D relative to thee day of treatment initiation (day 7 after immunization).. Groups of 7 rats were treated with vehiclee alone (D). or RAPA at 1.0 (•) , 0.5 (0),O.1 ( • ) , 0.055 (E3), or 0.025 (B ) mg/kg/d by continuous i.v. infusionn with a miniosmotic pump.

Thee decrease in the capacity of the remaining cells to

respondd to stimulation by S-Ag in culture further

indicatedd that the expansion of the S-Ag specific cell

populationn had been prevented.

Higherr levels of antibody production are usually

obtainedd when immunizing with Hunter's adjuvant

comparedd to Freund's adjuvant (27). Although using

Hunter'ss adjuvant, we observed a sizable reduction in

thee anti-S-Ag antibody levels during RAPA treatment.

Inn previous reports of EAU induced with S-Ag in

Freund'ss adjuvant, CsA treatment did not affect the

antibodyy levels (35), while FK 506 caused a profound

depressionn of these levels (36). In general the

persistencee of an appreciable antibody production is

advantageouss in the immunosuppressive treatment of T

celll mediated diseases such as uveitis, because it

helpss to prevent intercurrent complicating infections.

Onn that account cytomegalovirus retinitis, a common

opportunisticc viral disease in the immunosuppressed,

wass reported in patients treated with FK 506 (37).

Onee of the most serious problems in the treatment

off autoimmune disease is the need for the

immunosuppressionn to be sustained for prolonged

periodss of time. The likelihood of developing

complicationss from the toxic side effects of the drugs is

increasedd accordingly. For both CsA and FK 506, the

mainn concern lies with the kidney toxicity (38. 39). In

contrast,, reports showing a low renal toxicity of RAPA in

animalss (40-42) suggest that it would be an

advantageouss addition to our therapeutic arsenal. The

resultss reported here certainly indicate that it could be

effectivee in the treatment of uveitis. The minimal

effectivee dose of 0.1 mg/kg/d for EAU inhibition is 100 to

4000 times lower than the intramuscular dose of CsA

reportedreported previously for day 7 treatment (35, 43). Even

whenn taking into account the probably increased

effectivenesss of the i.v. delivery route of RAPA compared

too the i.m. administration of CsA, our results indicate a

veryy high degree of therapeutic efficacy of RAPA. The

therapeuticc effect of RAPA also appears to be prolonged

becausee the rats remained free of disease on day 28,

approximatelyy one week after the treatment course was

stopped.. In regards to the general toxicity, the RAPA

treatedd rats lost weight only during the first week of

treatment.. It is possible that the weight loss is due to the

combinedd toxicity of the anesthetic used for surgery

togetherr with RAPA, since during the second week of

treatmentt there was a net weight gain. Rapamycin thus

appearss to be a promising alternative for the control of

autoimmunee diseases.

ACKNOWLEDGEMENTS S

Wee thank Dr. Suren Sehgal and Dr. Bernard Leduc

fromm Wyeth-Ayerst Research for their help. We are also

gratefull to Mr Serge Boucher and Francois Boisvert,

andd to Mrs Nicole Newman and Mary Alice Crawford for

theirr outstanding technical assistance. This work was

supportedd by grants from Wyeth-Ayerst Research, Ville

St.. Laurent, Que., La Fondation Oculus pour les

Maladiess de I'Oeil, Montreal, Que., and Fondation de la

Banquee d'Yeux du Quebec Inc.

CORRESPONDINGG AUTHOR

Drr Francois G. Roberge, National Institutes of Health,

Bldg.. 10, Room 10N 202, Bethesda, MD 20892, USA.

REFERENCES S 1.. Sehgal, S.R., Baker, H. and Vézina, C. (1975)

Rapamycinn (AY-22,989), a new antifungal antibiotic.. II. Fermentation, isolation and characterization.. J. Antibiot. 28_ 727-732.

I6l l

ChapterChapter 11


2.. Vézina, C , Kudelski, A. and Sehgal, S.N. (1975) Rapamycinn (AY-22,989), a new antilungal antibiotic.. I. Taxonomy of the producing streptomycetee and isolation of the active principle. J.. Antibiot. 2fi,721-726.

3.. Morris, R.E., and Meiser, B.M. (1989) Identification off a new pharmacologic action for an old compound.. Med. Sci. Res. 17, 609-610.

4.. Eng, C.P., Gullo-Brown, J., Chang, J.Y. and Sehgal, S.N.. (1991) Inhibition of skin graft rejection in mice byy Rapamycin: A novel immunosuppressive macrolide.. Transplant. Proc. 22, 868-869.

5.. Stepkowski, S.M., Chen, H,, Daloze, P. and Kahan, B.D,, (1991) Rapamycin, a potent immunosuppressivee drug for vascularized heart, kidney,, and small bowel transplantation in the rat. Transplantation,, 51 , 22-26.

6.. Stepkowski, S.M., Chen, K, Daloze, P. and Kahan, B.D.. (1991) Prolongation by Rapamycin of heart, kidney,, pancreas, and small bowel allograft survivall in rats. Transplant. Proc. 23, 507-508.

7.. Chen, H.F., Wu, J.P.. Luo, H.Y. and Daloze, P.M. (1991)) The immunosuppressive effect of Rapamycinn on pancreaticoduodenal transplants in thee rat. Tranplant. Proc. 22, 2239-2230.

8.. Chen, H.F., Wu, J.P., Luo, H.Y. and Daloze, P.M. (1991)) Reversal of ongoing rejection of allografts byy Rapamycin, Tranplant. Proc. 22, 2241-2242.

9.. Dumont, F.J., Staruch, M.J., Koprak, S.L., Melino, M.R.. and Sigal, N.H. (1990) Distinct mechanisms off suppression of murine T ceil activation by the relatedd macrolides FK-506 and Rapamycin. J. Immunol.. 144. 251-258.

10.. Bierer, BE., Mattila, P.S., Standaert, R.F., Herzanberg,, L.A., Burakoff, S.J., Crabtree, G, and Schreiber,, S.L. (1990) Two distinct signal transmissionn pathways in T lymphocytes are inhibitedd by complexes formed between an immunophilinn and either FK506 or Rapamycin. Proc.. Natl. Acad. Sci. USA, £7, 9231-9235.

11.. Bierer, B.E., Schreiber, S.L. and Burakoff, S.J. (1991)) The effect of the immunosuppressant FK-5066 on alternate pathways of T cell activation. Eur.. J. Immunol. 21 . 439-445.

12.. Henderson, D.J., Naya, I., Bundick, R.V., Smith, G.M.. and Schmidt, J.A. (1991) Comparison of the effectss of FK-506, Cyclosporin A and Rapamycin on IL-22 production. Immunology, 72, 316-321.

13.. Wicker, L.S., Boltz, R.C., Matt, V., Nichols, E.A., Peterson,, L.B. and Sigal, N.H. (1990) Suppressionn of B cell activation by Cyclosporin A, FK5066 and Rapamycin. Eur. J. Immunol. 2Q, 2277-2283. .

14.. Luo, H., Chen, H., Daloze, P., Chang, J. and Wu, J. (1991)) Rapamycin suppresses in vitro immunoglobulinn production by human lymphocytes.. Transplant. Proc. 23, 2236-2238.

15.. Martel, R.R., Klicius, J. and Galet, S. (1977) Inhibitionn of the immune response by Rapamycin, a neww antifungal antibiotic. Can. J. Physiol. Pharmacol.. 55, 48-51.

16.. Wacker, W.B. and Lipton, M.M. (1968) Experimentall allergic uveitis. I Production in the

guinea-pigg and rabbit by immunization with retina inn adjuvant, J. Immunol. 101. 151-156.

177 Nussenblatt, R.B., Kuwabara, T., de Monasterio, F.M.. and Wacker, W.B. (1981) S-Antigen uveitis in primates.. Arch. Ophthalmol. 22,1090-1092.

18.. Broekhuyse, R.M., Winkens, H.J., Kuhlmann, E.D., andd van Vugt, A.H.M. (1984) Opsin-induced experimentall autoimmune retinitis in rats. Curr. Eyee Res. 2. 1405-1412.

19.. Fox, G.M., Kuwabara, T., Wiggert, B., Redmond, T.M... Hess. H.H., Chader, G.J. and Gery, I. (1987) Experimentall autoimmune uveoretinitis (EAU) inducedd by retinal interphotoreceptor retinoid-bindingg protein (1RBP): difference between EAU inducedd by IRBP and by S-Ag. Clin. Immunol. Immunopathol.. 43., 256-264.

20.. Caspi, R.R., Roberge,F.G., Chan, C.-C, Wiggert, B., Chader,, G.J., Rozenszajn, L.A., Lando, Z. and Nussenblatt,, R.B. (1988} A new model of autoimmunee disease. Experimental autoimmune uveoretinitiss induced in mice with two different retinall antigens. J, Immunol, 140^1490-1495.

21.. de Kozak, Y., Sakai, J., Thillaye. B. and Faure, J.P. (1981)) S antigen-induced experimental autoimmunee uveo-retinitis in rats. Curr. Eye Res. 1 ,, 327-337.

22.. Salinas-Carmona, M.C., Nussenblatt, R.B. and Gery,, 1. 1982. Experimental autoimmune uveitis in thee athymic nude rat. Eur. J. Immunol. 12, 480-484. .

23.. Mochizuki, M., Kuwabara, T., McAllister, C , Nussenblatt,, R.B. and Gery, I. (1985) Adoptive transferr of experimental autoimmune uveoretinitis inn rats: Immunopathogenic mechanisms and histologicc features. Invest. Ophthalmol. Vis. Sci, 2£, 1-9. .

24.. Nussenblatt, R.B , Rodriguez, M.M., Wacker, W.B., Cevario,, S.J.. Salinas-Carmona, and I. Gery, I. (1981)) Cyclosporin A. Inhibition of experimental autoimmunee uveitis in Lewis rats J. Clin. Invest. 6.7,, 1228-1231.

25.. Roberge, F.G., Lorberboum-Galski, H., Le Hoang, P.,, de Smet, M., Chan, C.-C, FitzGerald, D. and Pastan,, I. (1989) Selective immunosuppression of activatedd T cells with the chimeric toxin IL-2-PE40. Inhibitionn of experimental autoimmune uveoretinitis.. J. Immunol. 142, 3498-3502,

26.. Dorey.C, Cozette, J. and Faure, J.P. (1982) A simplee and rapid method for isolation of retinal S-antigen.. Ophthalmic Res. 14, 249-255.

27.. Roberge, F.G., Xu, D., and Chan, C.-C. (1992) A neww effective and non-harmful chemical adjuvant forr the induction of experimental autoimmune uveoretinitis.. Curr. Eye Res. 11,371-376,

288 Tuyen, V.V., Faure, J.P., Thillaye, B., de Kozak, Y. andd Fortier, B. (1982) Antibody determination by ELISAA in rats with retinal S antigen-induced uveoretinitis.. Curr. Eye Res, 2, 7-12.

29.. Nussenblatt, R.B., Palestine, A.G. and Chan, C.-C. (1983)) Cyclosporin A therapy in the treatment of intraocularr inflammatory disease resistant to systemicc corticosteroids and cytotoxic agents. Am. J.. Ophthalmol. 2£, 275-282.

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30.. Nussenblatt, R.B., Palestine, A.G. and Chan, C.-C W.B. (1982) Modulation of experimental (1985)) Cyclosporin therapy for uveitis: Long-term autoimmune uveitis with cyclosporin A. Arch, tollowup.. J. Ocular Pharmacol. 1. 369-382. Ophthalmol. 100. 1146-1149.

31.. Mochizuki, M., Masuda, K., Sakane, T., Inaba, G.T Ito,, K,, Kogure, M., Sugino, N., Usui, M., Mizushima, Y.,, Ohno, S., Miyanaga, Y., Hayasaka, S. and Ohizumi,, K. (1991) A murticenter clinical open trial off FK 506 in refractory uveitis, including Behcet's disease.. Transplant. Proc. 22. 3343-3346.

32.. Kronke, M., Leonard, W., Depper, J., Arya, S., Wong-Staal,, F. and Gallo, R. (1984) Cyclosporin A inhibitss T-cell growth factor gene expression at the levell of mRNA transcription. Proc. Natl. Acad. Sci. USA,, S I , 5214-5218.

33.. Granelli-Piperno, A,, Inaba, K. and Steinman, P.M. (1986)) Lymphokine and non-lymphokine mRNA levelss in stimulated human T cells: kinetics, mitogenn requirements, and effect of Cyclosporin A. J.. Exp. Med. 1£3, 922-937.

34.. Tocci, M.J., Matkovich, D.M,, Collier, K.A., Kwok, P., Dumont,, F,, Lin, S,, Degubicibus, S., Siekerka, J.J., Chin,, J. and Hutchinson, N.L (1989) The immunosuppressantt FK 506 selectively inhibits expressionn of early T cell activation genes. J. Immunol.. 143. 718-726.

35.. Nussenblatt, R.B., Rodrigues, M.M., Wacker, W.B., Cevario,, S.J., Salinas-Carmona, M.C. and Gery, I. (1981)) Cyclosporin A inhibition of experimental autoimmunee uveitis in Lewis rats, J. Clin. Invest. £7,, 1228-1231.

36.. Kawashtma, H., Fujtno, Y. and Mochizuki, M. (1988)) Effects of a new immunosuppressive agent, FKK 506, on experimental autoimmune uveoretinitis inn rats. Invest. Ophthalmol. Vis. Sci. 22, 1265-1271. .

37.. Paul, A.A., Leeper, H.F. and Friberg, T.R. (1991) CMVV retinitis and the use of FK 506. Transplant. Proc.. 22, 3042-3043.

38.. Palestine, A.G., Austin, H.A., Balow, J.E., Antonovych,, TT., Sabnis, S.G., Preuss, H.G. and Nussenblatt,, R.B. (1986) Renal histopathologic alterationss in patients treated with cyclosporin for uveitis.. N. Engl. J. Med. 3_L4, 1293-1298.

39.. Japanese FK 506 Study Group. (1991) Clinicopathologicall evaluation of kidney transplantss in patients given a fixed dose of FK 506 Transplant.. Proc. 22, 3111 -3115.

40.. Whiting, PH., Woo, J., Adam, B.J., Hasan, N.U., Davidson,, R.J.L. and Thomson, A.W. (1991) Toxicityy of Rapamycin. A comparative and combinationn study with Cyclosporin at immunotherapeuticc dosage in the rat. Transplantation,, 52, 203-208.

41.. Whiting, P.H., Adam, B.J., Woo. J., Hasan, N.U. and Thomson,, A.W. (1991) The effect of Rapamycin on renall function in the rat: a comparative study with cyclosporin.. Toxicology Lett. 5_g, 169-179.

42.. DiJoseph, J.F., Sharma, R.N. and Chang, J.Y. (1992)) The effect of Rapamycin on kidney function inn the Sprague-Dawley rat. Transplantation, 5.2, 507-513. .

43.. Nussenblatt, R.B., Rodrigues, M.M., Salinas-Carmona,, M., Gery, I., Cevano, S. and Wacker,

163 3

Chapterr 12

Treatmentt of Uveitis with Recombinant Humann Interleukin-13

Francoiss G. Roberge, Marc D. de Smet, Jacques Benichou, Martinn F. Kriete, James Raber, John Hakimi

Britishh Journal of Ophthalmology 82: 1195-1198, 1998 (byy permission BMJ Publishing Group)

IL-133 IN EAU

Treatmentt of uveitis with recombinant human interleukin-13 3

Francoiss G Roberge, Marc D de Smet, Jacques Benichou, Martin F Kriete, James Raber, Johnn Hakimi

Natiooall Institutes of Health,, National Eye Institute,, Bethesda, Maryland,, USA FF Ü Rnberge MM Dde Smei MM F Kriele JJ Raber

Nationall Institutes of Health,, National Cancerr Institute, Bioss tat istics Branch, Bethesda,, Maryland, USA A JJ Benichou

Rochee Research Center,, Inflammation andd Autoimmune Diseases, , HH o Htnann * LaRoc he, Nutley,, New Jersey, USA A JJ Hakimi

Correspondencee lo; F r a n c i ss Cï Roberge, MD, L,aa Piue-Salpetnere Hospica!, Departmentt uf Ophthalmology,, 47-R1 Blvd dee rHópital , Bat Bdbinski, 7500 H Paris, l'rance.

Acceptedd fcsr publication III March l ° " 8

Abst rac t t AimAim—To—To evaluate the an t i - i n f l ammato ry cytokinee inter leukin-13 (IL-13) for the t r e a tmen tt of uveitis. MethodsMethods—Uveitis—Uveitis was induced in m o n keyss by immuni sa t ion with h u m a n re t ina l S-ant igen.. S ta r t ing a t the onset of disease, thee an ima l s were t rea ted with IL-13 at 25 Hg/kg,, o r vehicle cont ro l , injected subcu ta -neouslyy once a day for 28 days. In t r aocu la r in f lammat ionn was scored by indi rec t ophtha lmoscopyy for a per iod of 56 days . Ci rcu la t ingg leucocyte levels were mon i tored . . ResultsResults—Uveitis—Uveitis s t a r t ed unilateral ly in all butt one an ima l . IL-13 inhibi ted i n f l ammationn bo th in the eyes in which the disease wass p resen t when the t r e a tmen t was ini t i atedd (p=0.0001), and in the cont ra la te ra l initiallyy negative eyes (p=0.0001). After cessat ionn of therapy, there was a p rog res sivee increase of in f lammat ion in the IL-13 t rea tedd g roup . However, the beneficial effectt of IL-13 extended into the 4 week followw up per iod . IL-13 p roduced an increasee in c i rcula t ing po lymorpho nuc lea rr neut rophi l s and a decrease in lympp hocytes . ConclusionConclusion—Administration—Administration of IL-13 a p pea r ss to be a p romis ing modal i ty of t r ea t m e n tt for severe uveitis. (Br . ?Ophtha lmoll 19y8 ;82 : ] 1 9 5 - 1198)

Thee treatment of severe non-infectious uveitis iss often based on the broad anti-inflammatory propertiess of corticosteroids. These properties are,, however, counterbalanced by serious metabolicc side effects.1 In order to reduce this toxicity,, the steroids are increasingly used at lowerr doses in combination with other cytostaticc and cytotoxic drugs.2 ' Unfortunately, eachh drug has its own toxic side effects. Clearly, thee goals of treatment would be better achieved withh a therapeutic agent possessing a spectrum off activity comparable with corticosteroids but withh reduced side effects. In the last few years, cytokiness such as interleukin-10 (IL-10) and transformingg growth factor p (TGF-[i) have attractedd attention because of their multifac-etedd downregulating effect on the activity of thee immune system. More recently, the newly discoveredd cytokine IL-13 was found to have evenn more promising anti-inflammatory effects.1 1

IL-133 is a pleiotropic cytokine produced by Th-22 lymphocytes/ IL-1 3 shares with the glucocorticoidss a downregulating effect on cell mediatedd immunity. IL-13 inhibits the synthe

siss of the proinflammatory cytokines IL-1 , IL-6,, IL-8, tumour necrosis factor a (TNF-a) , andd macrophage inflammatory protein l a (MIP-lu).111 ' Conversely, the release of the IL-1 receptorr antagonist is stimulated.' IL-13 also suppressess the production of nitric oxide as welll as the induction of cycio-oxygenase-2 in macrophages."" ** In the bone marrow, IL-13 enhancess the proliferation and differentiation off haemopoietic stem cells, and was reported too induce a marked monocytosis in mice.1" I:

IL-133 does not directly affect T cells. However, byy inhibiting the production of IL-12 and interferonn a, IL-13 may prevent the developmentt of the Th-1 lymphocyte pathway which seemss determinant for most posterior uveitis.'' *'" Thus , the anti-inflammatory propertiess of IL-13 suggested that it could be a powerfull new agent for the treatment of uveitis. Wee evaluated the efficacy of human IL-13 in monkeyss to which uveitis was induced by immunisationn with the retinal S-antigen. In addition,, we monitored the effect of IL-13 on thee blood leucocyte levels.

Mate r i a l ss and me thods INDUCTIONN or UVEITIS

Thee animals participating in the experiment weree female cynomolgus monkeys, Macacafas-cicuiaris,cicuiaris, weighing between 2.5 and 3.0 kg. All weree seronegative for the following viruses: SIV,, SRV-1, SRV-2, SRV-5, and STLV-1. Beforee immunisation, a baseline serum sample wass collected, and the normality of the eyes wass verified by indirect ophthalmoscopy and pressuree tonometry. The animals were immunisedd with 40 (ig/kg of recombinant human S-antigenn solubilised at 1 mg/ml in phosphate bufferedd normal saline solution (PBS), pH 7.3, emulsifiedd 1:1 v:v in TiterMax adjuvant (CytRxx Corporation, Norcross, GA, USA), andd injected subcuianeously at four sites in the napee of the neck. The immunisation, as well as subsequentt examinations, was done with the animalss anaesthetised by intramuscular injectionss of ketamine (Aveco, Forth Dodge, IA, USA)) 10-20 mg/kg, and atropine (Astra Pharmaceutical,, Westboro, MA, USA) 0.05 mg/kg. Thee principles of laboratory animal care of NIHH publication No 86-23, revised in 1985, weree followed.

TREATMENTT PROTOCOL

Fromm 14 days after immunisation, the animals weree examined every 3 and 4 days alternatively byy indirect ophthalmoscopy. The pupils were dilatedd with topical atropine sulphate 1 % solutiontion (Akorn Inc, Abita Spring, LA, USA).

167 7

ChapterChapter 12

Treatmentt was initiated at the first sign of intraocularr inflammation (see below). The animalss were randomly assigned to two treatment groupss of five. The treatment consisted of' recombinantt human 11.-13 at 25 ugkg in 0.2 mll PBS, or PBS alone, injected subcutaneously oncee a day lor 28 days. (IL-1 3 was provided by Hoffmann-LaRochee Ine, Nutley, NJ, USA, underr licence from Sanon-Recherche, Paris, France.)) The dose of IL-1 3 was determined in preliminaryy toxicological and pharmacokinetic assayss at Sanofi and Roche respectively, indicatingg safety at that dose and an effective serumm level as estimated on the TF-1 cell assay inn vitro. After the treatment period, the animals weree followed for an additional 28 days.

DISEASEE EVALUATION Thee intraocular inflammation was e%'aluated by indirectt ophthalmoscopy on an alternate 3 and 44 day schedule and scored as follows, accordingg to the published grading of Guex-Crosier el al.'al.'33 The vitreous haziness was graded on a scalee of 0.0 to 4.0 by reference to a standardisedd panel of ocular fundus photographs as described."" Retinal and choroidal inflammationn were each graded from 0.0 to 4.0, based onn areas of cellular infiltration as follows: the funduss was divided into four quadrants centredd on the optic nerve head; the presence of leucocytee infiltration was scored as 1.0 point perr involved quadrant. Similarly, the presence off intraretinal haemorrhages was scored as 1.0 pointt per quadrant. While retinal vasculitis was alsoo scored by quadrant, the grading was adjustedd for partial segmental vascular sheathingg or continuous sheathing scored 0.5 and 1.0 pointt respectively. The sum from 0 to 20 of thesee scores (0.0 to 4.0 for inflammation in the vitreous,, retina, choroid, vascular sheathing, andd retinal haemorrhages), constituted the gradee for each eye. If the vitreous haziness reachedd levels precluding evaluation of the fundus,, the score of the Hindus for this time pointt was derived as the average of the previous

AA First positive eye

scoree with the first score following the loss of visibilityy of the fundus. The scoring examiner couldd not be blinded to the treatment group; butt the scoring was limited to a simple choice off presence or absence of each sign to avoid estimationn bias. Additional examination consistedd in blood samples collection for the determinationn of leucocyte differential count byy automatic cytometry (Coulter Corp, Miami,, FL, USA) at the beginning and end of thee treatment period.

CYTOKINEE LEVEL MEASUREMENT IN 'I 111-: BLOOD Onee ml of serum was collected on the day of immunisationn and at 1 week intervals from the dayy of disease onset, and stored at -70°C. The levelss of the following cytokines were evaluated withh commercial human cytokine assav kits: IL-1B,, IL-12, and TXF-a (R+D Systems, Minneapolis,, MN, USA);IL-2 (DuPont MEN', Boston,, MA, USA); and IL-6 (Endogen, Cambridge,, MA, USA).

STATISTICALL ANALYSIS OF UVEITIS EXPERIMENT AA linear model with normal error was used, withh inflammation grade (from 0 to 20) as the outcomee variable. The model included the followingg covariates: treatment group (two groups,, PBS and IL-13), time (eight time pointss after initiation of treatment), and initial inflammatoryy grade at time point zero. The latterr term was introduced in order to control for individuall differences among animals. Separate modelss were fitted to the first and second positivee eyes. An average overall treatment effect (thatt is, over the entire time range of the experiment)) was estimated from the model and testedd with an F test, through a least squares analysis.. We used the procedure GLM of SAS.;5 5

Results s Thee disease was initially expressed unilaterally forr at least 7 days in all animals, except one in thee IL-1 3 treated group which presented with

BB Second positive eye 14 4

144 21 28 35

Timee (days)

-- IL-13 treatment —• Follow up

'44 21 28 35 Timee (days)

-- IL-13 treatment —• Follow up

HgureHgure I Inhibition of uveitis with 1L-I3. Uveitis was induced by immunisation with retinal S-antigen. Two groups of five monkeysmonkeys were treated with 1I.-I.I (2> ttg!kg),or vehicle control, by subcutaneous injection once a day for 2H days, and followedfollowed for an additional 2X days. Uveitis was detected in one eye at least 7 days before the fellow eye in nine of 10 animals.animals. The results are given separately fir the eyes in which uveitis had already started (A), and for the eyes that were negativenegative or with the least severe disease at the lime of treatment initiation (II); thev represent the mean inflammation score

168 8

ÏI.-133 IN EAU

TableTable 1 Effect of' 1L~/.? irealmenl on blond'leucocyte kï^h

Wirnia// range Tim* Tim* point point

'/buil '/buil 7-17-1 Sxlff'Ipl

PMN PMN 30-30- 55%

Lymphocytes Lymphocytes .15-70% %

Montxyies Montxyies II 5% '

Animall groups: Controls s

IL-133 treated

DayO O Dayy 28 Dayy 0 Dayy 28

6.96(0.61) ) 8.42(1.25) ) 8.711 (1.71)

11.11 (2.21)

•16.22 (1.2) 45.11 ("J.2) 51.7(12.0) ) 66,2(6.1) )

46.0(1 .9) ) 45 .0(8 .7) ) 41 .7(11,4) ) 20.22 (5.8)

1.01.0 (0.7) 4 .2(1.0) ) 1.7(0.8) ) 1.77 (0.8)

Bloodd samples were collected on the day of treatment initiation (day 0) and uL the end of the neat-mentt period (day 28) Blood leucocytes numbers were determined with an automatic Coulter counter. .

ann asymmetrical bilateral inflammation minimall in one eye (one small spot of sheathing on aa retinal vessel). Since the treatment was institutedd at the onset of disease, the results are reportedd separately for the eyes already inflamedflamed when it was started, and for the eyes thatt were negative or with the least amount of inflammationn for the one animal with bilateral disease.. In both eyes, the treatment with IL-13 significantlyy reduced the severity of uveitis comparedd with controls. The effect was pronouncedd both in the eyes in which the diseasee started (Fig 1A), where the average estimatedd reduction in the inflammatory grade wass 6.0 (p=0.0001), and in the eyes with no or minimall disease at treatment initiation (Fig IB),, where the estimated average reduction wass 3.7 (p=0.0001). In the eyes that were free off disease when the treatment started, uveitis inductionn was almost completely inhibited by IL-13.. Of these four eyes, three were still negativee at the end of the treatment period, while fourr of the five initially negative eyes of the controll group were positive for experimental autoimmunee uveitis (EAU). After cessation of therapy,, there was a progressive increase of inflammationn in the IL-13 treated group. However,, the beneficial effect of IL-13 treatmentt was still observed during the 4 week followw up period. The inflammation score over thatt period was significantly lower in the IL-13 groupp in both eyes (p=0.038 and p=0.0001 in thee first and second positive eyes respectively).

Inn the IL-13 treated animals, there was an increasee in the total number of circulating leucocytess (Table 1). The differential count showedd a proportional increase of the polymorphonuclearr neutrophils (PMN) above the normall range, and a decrease of lymphocytes beloww the normal range. The differences could nott be reliably assessed statistically (by non-parametricc paired data test) because of the low numberr of data points. We observed no effect onn the proportion of monocytes.

Thee serum levels of the cytokines IL-Ip, IL-2,, IL-6, IL-12, and TNF-a were assayed by enzymee linked immunosorbent assay (ELISA) beforee immunisation, and at 1 week interval duringg the treatment period. The levels of thesee cytokines did not rise above the detection limitt of the assays used (data not shown).

Discussion n Treatmentt with IL-13 resulted in a dramatic improvementt of the inflammatory signs of uveitis.. The inflammation was inhibited during thee entire treatment period. After cessation of treatment,, disease progression tended to

resume.. No signs of toxicity were observed sys-temicallyy or at the sites of injection. We paid particularr attention to the induction of monocytosis.. It has been reported that in mice infusedd continually for 7 days by means of an osmoticc pump with high doses of murine IL-13,, a 10-fold increase in the number of circulatingg monocytes was induced." In our animals,, such an effect was not encountered afterr 1 month of subcutaneous injections.

Ann aspect of IL-13 treatment, that is not completelyy reflected in the global scoring, is thee rapid improvement of specific variables of inflammation.. The change was most notable in perivasculitis,, where severe continuous sheathingg of a vascular arcade completely disappearedd within 1 week of treatment in some animals.. The inhibitory effect of IL-13 on the productionn of IL-1, IL-6, and TNF-a , which servee to activate the vascular endothelial cells, mayy explain this observation. The activated vascularr endothelium normally produces chemoltiness such as IL-8 and RANTES to attractt macrophages and polymorphonuclear neutrophilss to inflammatory sites. The inhibitoryy effect of IL-13 on the production of these twoo chemokines is thus probably an important aspectt of its anti-inflammatory action in uveitis,, and may explain the reduction of the retinall vascular sheathing."

Inn vivo, the inflammatory activity of cytokiness results from a complex interaction in whichh they often stimulate the production of eachh other and exert combined synergistic effects.. For example TNF-a induces IL-6 and IL-11 j and conversely both IL-6 and IL-1 can stimulatee TNF-a secretion.17 At the present time,, there are limited specific data on the role playedd by cytokines in human intraocular inflammation.188 |S However a vast body of experimentall observations indicate an importantt involvement of IL-1, IL-6, IL-8, and TNF-aa in uveitis.*°~" Various inflammatory conditionss tend to go through common pathwayss using these cytokines. Thus , the broadd inhibitory activity of IL-13 could be advantageouss in the treatment of several types off uveitis.

Theree are also strong indications that T cells aree central to the mechanism of posterior uveitis."" :1 Indeed, a majority of these uveitis cann be improved by treatment with anti-T cell drugss such as cyclosporin A and FK506. ' ;" T cellss do not have receptors for IL-13, but the modificationn of the cytokine profile induced by IL-133 appears to shift the immune response awayy from the T cell directed delayed type hypersensitivityy reaction.12" In this type of inflammation,, the macrophage is the principal effectorr cell under the direction of Th-I cells. IL-133 by deactivating several macrophage functionss can prevent tissue damage.5 * Such ann effect has been observed in a rat model of experimentall autoimmune encephalitis in whichh the brain damage was considerably reducedd in rats that had been injected with cellss secreting IL-13.1" In the present study, the factt that EAU resumed its active progression soonn after stopping IL-13 injections points in thee direction of an inhibition of the efferent

169 9

phasee of the D T H reaction, without much alterationn in the T cells acquired anii-S-Ag cognitivee reactivity.

Inn the past, the inhibitory effect of drugs in thee animal model of experimental autoimmune uveitiss has been reliably predictive of their therapeuticc effect in humans. Contrary to most biologicall peptides used in therapeutic trials to date,, IL-13 did not have to be given intravenously,, but was effective by a simple subcutaneouss injection once a day. This mode of administration,, commonly used for insulin treatmentt in diabetes, is more readily accepted byy patients. Overall, the results that we obtainedd in subhuman primates certainly supportt further study of IL-13 in view of its use in humann uveitis.

11 Rubin B> Palestine AG. <" *i imp ligations of corticosteroids andd immunosuppressive drugs. Inl Ophthalmul CIm 108";29;15O-7| . .

22 Towler 1ÏM, Whiting PH, Forrester JV Combination low dosee cyclosporin A and steroid therapy in chronic intraocularr inflammation. F.ye 1 « 0 , 4 : 5 14-20.

33 Mochizuld M, Masuda K., Sakanc T, at at. A dinicaJ trial of KK.5066 in refractory uveitis. Am J (Jphihaimol 10Q3;M5: 7633 D,

44 Minty A, Chalon P, Derocc] ]yA,eial. Interleukin- I 3 is a new humann lymphokine regulating inflammatory and immune responses,, Mature l<jy3,J62:24R-W.

55 De Waal Malefyl R, Figdor CG, Huijhens R. a at. Lffects of 11.-133 on phenotype, cytokine production, and cytotoxic functionn of human monocytes. Comparison with 1I.-4 and modulationn by fFN-gamma or \L-UXJ Immunol |(I03;151: 63700 81 .

66 Doherty T M , Kastelein R> Menon S, tf al. Modulation of murinee macrophage function by IL-13. J Imrmmi'l 1993; lSl :715ll 60.

77 Cosenlino G, Soprana Ei, Thienes CP, et at. IL-13 down-regulatess CD14 expression and TNF-alpba secre-tiorii in normal human monocytes. J tmmujtol löü5-155 : 3145-51 . .

88 Kndo T, Ogushi I:, Sone S. I .PS-dependent cyclo-oxygrnase-22 induction in human monocytes is down-regulatedd by 1L-I 3, but not by IKN-gamma J Immunol !096:1S6:22100 6.

oo Once Y, Miyaura C, Kaminaliayashiki 'I', et at. IL-13 and IL-44 inhibit bone resorption by suppressing cyctooxygenase-2-dee pendent pmsraglandin synthesis in osteoblasts.. J Immunol 1Q'>6;1S<:758 64.

100 Jacobsen SF, Okkenhaug C, Vetby OP, et at. Interleukin 13: novell role in direct regulation of proliferation and differentiationn of primitive hematopoieytic progenitor cells. J fixp MedMed 10O4;180:75 82

11 1 Lai YH, Hes lan jM, Poppema S, el at. Continuous admims-traiionn of II 13 to mice induces extramedullar)' hemopoi esiss and monocytosis. 1 imtnunoi 1*106; I $6:3166 " 3 .

122 Skeen MJ, Miller MA, Shinmck FM, et at. Regulation of murinee macrophage JL-12 production. 7 Immunol 14Q6, 156:11%-- 206.

133 Guex-Crosier Y, Raber J, Chan C-C, tt at. Humanuted antibodiess against the alpha-chain of the II.-2 receptor arid againstt the beta-chain shared by the 1L-2 and IL-I 5 recep torss in a monkey uveitis model of autoimmune discases. J ImmunolImmunol l<>°7;15S 452-H

144 Fiorenlino DI', Zlotnik A, MosmannTR . et at. II.-It) inhibitss cytokine production by activated macrophages. J Immu-nolnol l v O | ; H " : 3 a i 5 - 2 2 .

155 SAS/S'IAT uitr'J guule Version o. 4th ed Gary, NC : SAS Institutee Inc. JUHQ:Ho]-Cm6

166 Marfaing-Koka A. Devergne 0 , ( io rgone G, et u/Regulation off the production of the RANTF.S chemokine by endotheliall cells. Synergistic induction by ÏFN-gamma plus T N F -ajphaa and inhibition by IL-1 and IL-13. J fmmitrt^l 1005,154:1870-8 8

177 Iûrum S, Oppenheim J.T. [Viinflammalory cytokines and immunity.. In: Paul WLi, ed. Funtitimental immunologv New York:: Raven Pres*, 1093:801-35

IKK Murray PI, Hoekzema R, van Haren MAC, et a!. Aqueous humorr wierleukin-6 levels in uveius. Imvst Ophthalmol l'u oVii 100031:917 20

1°° Wu WC-S, Mannion B, Stone RM Uveitis associated with imerleukin-33 and inlerleukine-6 therapy. Arch (}phihatmt*l l oq i ; l l ï : 108 - t J . .

200 Roberge FG, Caspi RR, Nussenhlatl RR. (ilia! retinal Mullerr cells produce II.-1 activiiyand have a dual elfect on autoimmunee T helper lymphocytes Antigen presentation manifestedd after removal of suppressive activity. .7 Immunol l<)H8;140:21933 6

211 de Vos AF, Moekzema R, Kijlstra A. Cytokines and uveitis, a revieww CvrrHycRa l W 2 ; I l : i 8 1 07.

222 Gucx-Crt^sicr Y, Witiwer AJ, Robergt YG. Intraocular pn> ductwrnn of a cytokine (GINC) responsible for neutrtiphil infiltrationn in endotoxin-induced uveitis. Br J OphikalmM LQQ6;80:6499 'H.

233 Rosenbaum JT Towards cytokine insight in sight Hr JOpfi-rjWni<7;i<)05,79:070-I. .

244 Deschenes J, Char D H , ftakta S. Activated T lymphoevtes inn uveius, Br J Opliihalmtil li)88;72:83 7.

21)) Chan C-C, Nussenblalt RB, Fujikawa I.S, not. Sympathetic ophthalmiaa Immu no pathological findings. OphhalmnLgy 1986;93:6')0-5. .

266 Chan t >C, Wetzig RP, lâlestine Atl,*t at. lmmunohistt>pa-thologyy of ocular sarcoidosis. Repiirt of a case and discussionn of immunopathogencsis. Arch Ophihatmot I987;105: 1308-402 2

277 Chan C.i\ Palestine AG, Kuwabara 'l\ it at. Itrimunopatho-k>gicall study of Vogl-Koyanagi-Harada syndn^me: rerx^T off a case Am J Ophihalmol 1088;105:u07 11.

288 Vitale AT, Rodriguez A, Foster S C tôw-dose cyclosporin A therapyy in treating chronic, noninfectious uveitis. Ophthal-mologymology IW6;103:365 74.

200 Zurawski G, de Vries JE. Interleukin 13, an interleukin -1-likee cytokine that acts on monocytes and B cells, hut not onn I" cells. Immunol To,tay 1004,IS:lv 26,

300 Cash Et Minty A, F'crrara P, et ul. MacrCNphage-inaclivaling IL-133 suppresses experimental autoimmune encephalomyelitiss in rats. J Immunol | u" t ;153:1258-67

file:///L-UXJ

Sectionn IV: Therapeutic Strategies for the Treatment of Human Uveitis

*'4$0matterof'fact,, everything which in the usual ethical valuation of inter-human relations is feefad.ypQn.^goi$feefad.ypQn.^goi$iicmibefraeed:tw&cmibefraeed:tw& to the material and spiritual maintenance QF enhance* wentwent of human life and to the effort, to raise it to its highest level of value"

Albertt Schweitzer, Civilization and Ethics, 1920

Chapterr 13

Clinicall Use of Cyclosporine in Ocular Disease

Marcc D. de Smet, Robert B. Nussenblatt

Internationall Ophthalmology Clinics 33: 31-45, 1993 (byy permission © Little Brown and Co.)

CYCLOSPORINE E

Clinicall Use of Cyclosporine in Ocularr Disease

Marcc D. de Smet, M.D. Robertt B. Nussenblatt, M.D.

Withh the advent of reliable animal models of uveitis, the factors leading too ocular inflammation have slowly been dissected and studied. Several of thesee models have demonstrated the key role of T cells in the development off autoimmune ocular disease and have led to the prediction that a d rug withh predominantly anti-T-cell activity would be of benefit in treating or preventingg ocular inflammation. Cyclosporin A (CSA), the first T-cel! immunosuppressantt to be discovered, was quickly shown to be effective in suppressingg autoimmune uveitis in both controlled and uncontrolled trials [1—3].. Also known as cyclosporine, CSA was discovered in 1969/1970 by Jeann Borel and coworkers in the microbiology laboratories of Sandoz, Ltd, inn Basel, Switzerland [4]. Isolated from Tolypodadium injlalum (iams, this fungall extract was first tested as an antifungal agent but proved disappointingg as its spectrum of activity was too narrow. The narrow spectrum was duee in part to its potent immunosuppressant effect, an effect directed virtuallyy exclusively against T cells. In addition to this specificity, CSA was shownn to be nonmyelotoxic and to have a reversible effect on the immune system.. However, despite these advantages, CSA therapy requires careful guidelines,, because this drug has a variety of nonimmunoiogical toxic side effects. .

•• Clinical Pharmacology

CSAA is a neutral, lipophilic, cyclic endecapeptide (molecular weight 1,2033 gm) with a unique nine-carbon amino acid in position 1 (Fig 1) [5, 6],, Its activity is very much dependent on its stereochemical configuration and,, in particular, on any modification to the residues in positions 1, 2, 3, 10,, and 11. It is insoluble in water. For clinical use, CSA is stabilized in castorr oil for intravenous injection and in olive oil with 12.5% ethanol for

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175 5

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orall administration, which is usually accomplished by dispersing the drug inn juice or milk before ingestion. The absorption rate from an oral dose iss roughly 30% but is highly variable (4 to 60%), due to slow, incomplete andd variable absorption of CSA from the upper small intestine according too a bile-dependent zero-order process. The peak drug level occurs approximatelyy 2 to 4 hours after ingestion. Absorption is adversely affected byy biliary diversion, slow gastric emptying, or increased gastrointestinal motility.. Coadministration of food and prolonged therapy promote absorptionn of the drug.

Mostt of the CSA in circulation is associated with lipoproteins. Although somee CSA circulates unbound, this fraction does not correlate with the totall blood levels of CSA or with adverse clinical events. The extent of tissuee deposition also varies from patient to patient, with the volume of distributionn ranging from 4 to 8 liters per kilogram of body weight. However,, obesity does not appear to correlate with the volume of distribution. Rather,, distribution volume seems to correlate with the levels of cytoplasmicc binding proteins, which are able to retain CSA for months after therapyy is discontinued. The liver is the major depot of the drug, followed byy the pancreas, fat, blood, heart, lung, kidney, and neural and muscular tissue. .

Ocularr bioavailability depends on the mode of administration and the extentt to which the blood-ocular barrier is broken down. In experimental animals,, systemically administered CSA does not appear to penetrate very welll through intact (uninfiamed) ocular tissues [7], However, in eyes of patientss with chronic flare, the concentration in the aqueous humor is approximatelyy 40% of the plasma concentration, indicating good intraocularr penetration of the d rug [8]. Ocular pigment also appears to influence thee intraocular d rug concentration. In rabbits, a statistically greater amountt of d rug is present in the iris and the retina-choroid of pigmented animalss as opposed to albinos [9]. Uptake from local administration is hamperedd by the hydrophobic nature of CSA. Topically applied CSA in oill does not appear to penetrate beyond the cornea and conjunctiva in concentrationss that are therapeutically efficacious, although when the corneall epithelium is damaged, high levels are detected in the aqueous [7]. Thee use of collagen shields [10] or an alpha cyclodextrin vehicle [11] increasess the corneal penetration five- to tenfold. Periocular injections appearr to generate intraocular levels that are potentially therapeutic, but this approachh has not yet been tried in humans [12].

CSAA metabolism involves oxidative alterations by cytochrome P-450 intoo metabolites with a higher polarity. Coadministration of drugs that interferee with cytochrome P-450 will lead to a lowering of CSA metabolism. Inn the case of ketoconazole, this effect is strong enough to reduce the requiredd oral dose by up to 90% [13]. Other common inhibitors of cytochromee P-450 that can influence CSA levels include erythromycin, oral contraceptives,, androgens, methylprednisolone, and some calcium channel

177 7

ChapterChapter 13

Tablee 1 Cyclosporin* Drug Interactions

Drug g

Aminoglycosidee antibiotic

Amphotericin n Ciprofloxacin n

Melphalan n

Cimetidine e Erythromycin n

Ketoconazole e

Mannito) )

NSAID D BCP P

Phenobarbital l

B B

TT Blood Levels s

+ +

+ +

++ + +

+ +

t t Renal l Dysfunction n

4--

++ +

++ +

++ + + + + + + +

ff +

+ +

+ +

T T

t t t t T T t t 1 1 1 1

1 1

Nephrotoxicity y

Nephrotoxicity y

Nephrotoxicity y

Absorption n Bioavailability y

Metabolism m

Renall dysfunction duee to CSA

Renall blood flow Additivee hepato-

T T toxicity y

Metabolism m

CSAA = cyclosporin A: NSAID = nonsteroidal anti-iuflammatoi v drugs; BCP = birth control pill.

blockers,, particularly diltiazem (Table 1). Nifedipine, on the other hand, hass no effect on CSA metabolism. Agents that induce cytochrome P-450 enzvmess cause a decrease in CSA levels. Phenytoin, for example, will cause thee CSA level to d rop more than 509t within 48 hours of its administration [14].. Other agents with a similar effect include rifampin, phenobarbital, carbama/epine,, and valproate.

Excretionn of CSA occurs primarily through the bile and the intestines (mediann half-life, 6.4 to 8.7 hours), with a small amount (6%) appearing inn the urine. Longer dosing intervals may be required in the presence of elevatedd serum levels of' bilirubin or alanine aminotransferase, but not if thee changes are limited to aspartate aminotransferase, lactate dehydrogenase,, or alkaline phosphatase. The clearance rate in children is 45% higher thann in adults, thus indicating the need for higher doses in children [15]. Inn contrast, d rug clearance is slower in the elderly and in patients with hepaticc impairment.

Mechanismm of Action

Borell [4] showed that CSA reversibly inhibits T cell-mediated alloim-munee and autoimmune responses. T-cell precursors, after being generated withinn the bone marrow, migrate to the thymus where they mature. After maturationn they disseminate throughout the body, where they can bind selectivelyy through specific cell surface receptors to antigens that are presentedd by antigen-presenting cells. This immunorecognition primes I cells

178 8

CYCLOSPORINE E

too express surface receptors for lymphokines, which act as humoral immunee signals that trigger cellular maturation. A second series of T-cell recognitionn reactions—the activation cascade—results in the synthesis of lymphokiness that will promote cell division and the acquisition of cytoag-gressivee potential. CSA does not affect the priming reaction, but it does inhibitt the activation cascade necessary for inducing specific immune functionss such as lymphokine production. The synthesis and secretion of in-terleukinn 2 (IL-2), a potent activator of T and B cells, is reduced, as is the productionn of interferon gamma (IFN-7), the lymphokine that provides ann amplification signal to activate macrophages and monocytes, and tumor necrosiss factor alpha (TNF-a), which has a number of immune targets. In addition,, cyclosporine is able to inhibit the synthesis of the alpha and beta chainss of the IL-2 receptor, further reducing the possibility of T-cell activation. .

Thee precise mechanism of inhibition by CSA is poorly understood, owingg to limited knowledge of the exact activation pathways in T lymphocytes.. CSA probably acts at different cellular levels. At the cell surface, it appearss to act through calcium-related cytoplasmic pathways. Alternate celll surface pathways through the CD2 receptor, which activate protein kinasee C, are resistant to the drug. Within the cytoplasm, CSA reduces the generationn of cytoplasmic activation proteins that mediate signal transductionn between the cytoplasm and the nucleus. It reduces the signal that triggerss DNA synthesis by resting T-lyrnphocyte nuclei. The probable targett is a protein called cyclophiiin, whose affinity for CSA is proportional to itss immunosuppressive potency. Cyclophilins are present in most tissues andd exist in many isoforms, only some of which are active. The pig-kidney cyclophiiinn has sequence homology with peptidyl-propyl-«'.s-£ram isomer-ase,, an enzyme that may initiate folding of specific cytoplasmic proteins thatt can lead to the exposure of DNA-binding domains. The nucleus itself cann be a target for the action of CSA and is the third potential site. The drugg appears to bind to nuclear proteins at sites of transcription regulation.. However, CSA has no effect on events that occur after gene activation.. All these effects appear to occur only in helper-inducer and cytotoxic TT cells. Suppressor T cells are spared the inhibitory effects of CSA, thus settingg the stage for an immunoregulatory disequilibrium favoring unresponsivenesss rather than immunity.

•• Toxicity and Side Effects

Despitee its relatively specific effects on the immune system, CSA is associatedd with a number of side effects and potential toxicities. Because mostt uveitis patients requiring CSA therapy will stay on this medication forr a prolonged period of time, one must carefully weigh the risks and benefitss of CSA at every stage of therapy.

179 9

ChapterChapter 13

Nephrotoxicity Nephrotoxicity

Withh long-term CSA use, a particular concern is the development of nephrotoxicityy and hypertension. The most common effect of this drug onn the kidney is characterized by increased serum creatinine and urea. Thiss reflects a dose-dependent decrease in the glomerular filtration rate (GFR)) secondary to a decrease in renal blood flow, which is a result of vasoconstrictionn of the afferent glomerular arteriole. The fall in GFR initiallyy is purely functional. With a CSA starting dose of 5 mg/kg, one can expectt a rise in serum creatinine of approximately 10%, but even with suchh a rise, the serum creatinine remains within the normal range [16, 17]. Thee serum creatinine level plateaus in 4 to 8 weeks and, with proper monitoring,, will remain in that range provided that potentially nephrotoxicc drugs are avoided. These include nonsteroidal anti-inHammatory drugss (NSAIDs), aminoglycosides, amphotericin B, ciprofloxacin, melpha-lan,, and colchicine. The effect of NSAIDs is of particular concern because severall are now available as nonprescription drugs. NSAIDs reduce the GFRR by inhibiting intrarenal cyclooxygenases and adversely affecting thee balance between vasodilatory and vasoconstrictor prostaglandins in the kidnev.. Concomitant use with CSA can lead to a transient but significant risee in serum creatinine f 18], an effect that can last up to 3 weeks.

Permanentt changes in renal function are rare when the dose of CSA iss maintained below 5 mg/kg and when the dose is adjusted to avoid large risess in serum creatinine [19, 20]. However, the risk of irreversible structurall damage to the kidney remains a major concern when using CSA inn long-term treatment. Chronic CSA nephropathy, when established, is irreversiblee and is characterized by a striped interstitial fibrosis accompaniedd by focal tubular atrophy. Renal arteriolar abnormalities are also noted;; these may take the form of necrosis of smooth-muscle cells, a lumpy nodularr protein deposit in the vascular wall of afferent glomerular arterioles,, or an arteriolar intimal hyalinosis [21]. Such changes were commonly seenn in transplant patients who were on high doses of CSA and in whom thee serum creatinine was permitted to remain elevated for extended periodss of time. Renal arteriolar abnormalities were seen also in patients with uveitiss when the initial treatment dose was 10 mg/kg/day and the serum creatininee was allowed to rise to double the pretreatment level [21]. However,, multivariate logistical regression analysis of renal biopsies from 192 patientss with auto immune or inflammatory diseases revealed that the risk off permanent kidney damage is minimal if the CSA dose is maintained at orr below 5 mg/kg and if increases in serum creatinine of more than 30% abovee baseline are avoided [20], The duration of treatment in patients fromm whom biopsies were obtained ranged from 4 to 39 months. In this study,, CSA nephropathy was more closely related to the maximal dose of thee drug than to the dose at the time of the biopsy or to the length of time thee maximal dose was administered [20]. Age also appeared to be a risk

180 0

CYCLOSPORINS S

factor.. The mean age of patients with nephropathy was 31 13 years as comparedd to 23 12 years in those patients without nephropathy. The incidencee of nephropathy in children was extremely low. These results suggestedd that CSA-induced nephropathy may be related less to long-term, cumulativee toxic effects on the arterioles and tubules than to the consequencee of brief insults brought about by the administration of excessive dosess of CSA.

Hypertension Hypertension

CSAA induces a dose-related rise of arterial blood pressure, resulting inn an average increase of 2 to 3 mmHg (diastolic) on a 2.5-mg/kg/day dosagee and of 5 mmHg on a 5-mg/kg/day dose [19]. Hypertension developss in 15 to 25% of patients. The incidence is higher if the patient has predisposingg factors, particularly alterations in renal function. In most instances,, hypertension occurs early, within the first few weeks of starting CSAA therapy. It is responsive to dose reduction and necessitates cessation off CSA therapy in only 1 to 3% of cases. A sudden elevation of blood pressuree after prolonged therapy is often indicative of impending renal toxicityy and should prompt the treating physician to check serum creatininee and obtain a CSA blood level. This situation is more common in obese patientss who, after months of therapy, suddenly develop hypertension and havee higher circulating levels of CSA. On reducing the drug dose, the bloodd pressure normalizes. The practitioner should also be aware of potentiall drug interactions that could increase circulating levels of CSA.

Hypertensionn alone is not a contraindication to using CSA, provided thatt the hypertension is well controlled on medication [22], Nonetheless, whetherr present before or while on CSA, hypertension must be aggressivelyy treated. The best first-line agents are probably the calcium channel blockers,, especially nifedipine or isradipine, because these agents do not interferee with CSA pharmacokinetics.

Malignancies Malignancies

Itt is known from organ transplantation that all potent immunosuppressivee therapy can lead to an increased risk of some malignancies, particularlyy those that are virus-associated such as B-cell lymphomas and squamouss cell skin carcinomas. With CSA, the highest risk occurs in patients whoo require the highest level of immunosuppression. Among renal transplantt patients, the risk of lymphoma is 0.4%. For patients with autoimmunee disease, the incidence of lymphoma is 0.05% or less (2 of 3,700 patientss studied) [19]. Early diagnosis is important because, at an early stage,, the lymphoproliferative disorder may spontaneously regress after reducingg or stopping the immunosuppression. There is no specific screeningg procedure that is of benefit other than a careful periodic clinical exami-

181 1

ChapterChapter 13

Incidencee in 9r*

500 (33)

399 (50) 37 7

311 (30)

31 1 277 (34)

16 6

16(18) )

UU (6) y y

77 (5) 5 5 44 (7)

Tablee 2 Incidence of Side Effects due to Cyclosporin? in Patient.) with Autoimmune Disease

Sidee Effect

Hyperesthesia,, paresthesia, numbness Hypertrichosis s

Gastrointestinall problems

Renall dysfunction

Fatigue,, weakness, malaise

Gingivall hyperplasia Hypertension n

Headache e Tremor r

Hepaticc dysfunction Weightt gain

Anemiaa (severe)

Musclee cramps, skeletal pain

*Dataa collected on patients treated at an initial dose of 10 mg/kg'dav [3-1]. In parentheses, the percentages correspondd to an initial dosage of 5 mg.kg/day in patients with multiple sclerosis [35j.

nation.. Squamous cell skin carcinomas have not been seen in patients with autoimmunee disease except those with psoriasis. No definite association hass been made between CSA and other malignancies. CSA is known to increasee serum prolactin levels, causing gynecomastia in men and promotingg the development of benign breast adenomas in women. No conclusive associationn has been demonstrated between breast carcinoma and CSA, butt older women should be closely monitored, as prolactin can promote thee growth of such tumors.

OtherOther Adverse Reactions

Thee most common side effects of CSA are listed in Table 2. Mild numbnesss and tingling of the extremities as well as temperature hypersensitivityy are noted by 50 to 90% of patients within several days of starting onn CSA [34, 35], Nausea and gastrointestinal distress beginning with the firstt dose are noted in up to 40%, but only rarely does this alter a patient's abilityy to use the drug. Increased hair growth on the face, arms, shoulders, andd back develops in 50% of patients and is usually noted in the first few-monthss of therapy. Gingival hyperplasia, similar to that seen in phenytoin toxicity,, is noted in 25% of patients also within the first several months of therapy.. Its severity is exacerbated by poor oral hygiene and can largely bee controlled by careful attention to periodontal disease. A fine hand tremorr occurs in the early months of treatment, but this often will improve withh continued therapy. A mild normochromic normocytic anemia may be notedd in 25% of patients. The hematocrit rarely falls below 30%. Hepatic toxicityy has not been a major problem with CSA. There is a mild, dose-

182 2

CYCLOSPORINN E

dependentt elevation of the serum transaminases and serum bilirubin and, withh prolonged use, a possible increase in the incidence of cholelithiasis andd choledocholithiasis [5].

Ann acute overdose of oral CSA does not lead to severe complications. Evenn the ingestion of a whole bottle of CSA (10 gm) produces only a mildd syndrome of hypertension, dysesthesias, flushing, and stomach upset, whichh lasts no more than a few days.

•• Optimal Use of CSA in Autoimmune Uveitis

Fromm the foregoing discussion, it is evident that CSA is a very useful agentt to treat autoimmune ocular disease, but it also has potentially severe sidee effects, particularly when the d rug is used over a long term. Thus onlyy patients with a reasonable chance of responding to CSA should be selected.. Optimal use of this drug implies avoiding unnecessary treatment, stoppingg CSA administration in patients in whom it does not appear to havee the desired effect, and lowering the dose to less than 5 mg/kg/day as soonn as possible in patients who show a response to the medication. Therefore,, it is important to define for each patient, before treatment is initiated, thee parameters that will be used to measure a positive therapeutic response. .

Wheneverr possible, objective criteria should be selected, particularly whenn treating posterior pole inflammation as it is very difficult to judge improvementt in cell and Hare. Measuring the patient's visual acuity, particularlyy in the setting of macular edema, may be all that is necessary, providedd that a cataract is not a major component of the visual impairment. Inn all cases, it is important to rule out the possibility of an infectious cause andd to reconsider this possibility if the condition worsens while on therapy. Theree must also be potential for visual improvement. Ancillary tests such

ass laser interferometry or fluorescein angiography may be useful in determiningg the full extent of this potential [23]. Results of this evaluation shouldd be discussed with the patient, as well as the expected benefits and sidee effects of therapy. The decision to use CSA should be a joint one.

Oncee a decision has been made to try CSA, it is important to continue thee treatment long enough to notice improvement. In most cases, response willl be evident within 1 month and rarely will occur beyond 3 months. Therefore,, a maximal trial period of 3 months appears reasonable. If there hass been no measurable improvement by the end of the third month, we discontinuee therapy, even if the patient insists that there has been some subjectivee improvement. An abrupt stop serves two purposes: It prevents prolongedd use of a potentially toxic drug, and it can also serve as a measure off CSA's efficacy. In patients who respond to CSA, the disease usually recurss in one to two weeks following treatment interruption. On reintro-

183 3

ChapterChapter 13

Patientt with ocular inflammation that is potentially reversible

Initiatee therapv with CSA 2.5 mg/kg given two times per day (5 mg.•'kg/day) (Addd 0.2 to 0.5 mg/kg/day of prednisone)

Response e Continuee treatment for

aa total of 3 MONTHS or untill completely resolved

Keepp on high-dose CSA for 1 month Thenn tapet to 5 mg/kg/day by 11 mg/kg'dav every 3-4 weeks

Taperr even in the presence of a partiall response provided that as vouu taper there is no significant increasee in inflammation

Noo Response (I Month)

Increasee CSA to 7.5 mg/kg/day

Noo Response (2 Weeks)

Maximizee prednisone dose (upp to 1 mg/kg/day)

Noo Response (4 Weeks)

Stopp CSA. Tryy Cytotoxic Agent

TT If Whenn remission is achieved or after 3 months:

Taperr CSA by 0.5 mg/kg/day every 4 - 6 weeks to the minimumm dose that prevents a recurrence or 2.5 mg/kg/day; thenn taper the steroid to about 5—10 ing/day.

Oncee both drugs are at a low dosage, attempt to take the patient offf both medications.

Iff a recurrence occurs while tapering, reinstate previous CSA dosage andd increase steroids until the patient is under control.

Figuree 2 Treatment schedule for the use of ndüsporin A (CSA) in uveitis patients.

duction,, the disease comes rapidly under control. Consequently, an abrupt cessationn of therapy can serve as a therapeutic trial.

Ass a starting dose, we often use 2.5 mg/kg given twice daily with 0.2 too 0.5 mg/kg/day of prednisone. The therapy is continued for 2 to 3 monthss or until the inflammation has come under control. We then taper thee steroid or the CSA, depending on the needs of the patient (Fig 2). Over thee next 3 months, we taper to the minimal dose of CSA or CSA-steroid combinationn that will control the inflammation [24], In situations where CSA,, 2.5 mg/kg given twice daily, does not seem to be effective, we increase thee dosage up to 7.5 mg/kg/day in divided doses. However, we will maintainn this dosage for only a short period of t ime—no more than 4 to 6 weeks—andd taper it back down to 5 mg/kg/day once the inflammation has beenn brought under control. Recently, we tried a combination of CSA and

184 4

CYCLOSPORINE E

aa microsomal enzyme blocker (ketoconazole) to reduce CSA metabolism. Thiss combination has the advantage of maintaining serum CSA levels at a moree uniform level throughout the day [13], which we found led to improvedd visual acuity in patients who had been under marginal control. Onee added benefit of this approach is that a much smaller volume of CSA iss needed to control the inflammation.

Whenn switching from CSA alone to a combination of CSA and ketoconazole,, the CSA dose should be reduced to one-third of the original dose,, but there are considerable variations between individuals, with some patientss requiring only one-tenth of the original dose. Thus one must takee care in using this approach. Initially, careful monitoring of serum creatininee levels, as well as the whole blood levels of CSA, is needed.

Thee most effective and safe monitoring schedule for CSA is still being debated.. Traditionally, both the CSA blood level and the renal function havee been monitored. The clinical relevance of drug levels has recently beenn reviewed in patients with psoriasis and nephrotic syndrome: Frequent measurementt of CSA serum trough levels did not lead to a significant gain inn safety or efficacy in these patients [25]. This may be particularly true in patientss whose starting dose is 5 mg/kg/day or less, as was the case in this study.. Routine monitoring of the trough level probably is not necessary in mostt circumstances, provided that one carefully monitors renal function. However,, some situations do arise in which blood monitoring is needed, suchh as when there is a potential for drug interaction, when there is liver dysfunction,, or in cases of unexpected inefficacy (potential compliance problems).. If one intends to do a trough measurement, it is important to rememberr that it must be taken 12 hours after the last dose and that the accuracyy is poor with doses below 3 mg/kg/day. Several methods exist forr measuring CSA levels, with different reference ranges for each. Each measurementt must be judged using the specific reference range provided. Inn general, the more .accurate measurements are obtained from whole bloodd rather than serum levels [15, 26].

Whereass it might be unnecessary to measure CSA levels on a regular basis,, it is important to monitor renal function and blood pressure. An acutee rise in the blood pressure may be the first indication of renal toxicity. Inn patients with normal renal function, serum creatinine appears to be a goodd indicator of renal function changes. The lag time between a change inn the CFR (the gold standard for renal function) and a rise in serum creatininee is usually short. As an added safety measure, we also routinely obtainn a urinary creatinine clearance every 3 to 4 months. Although not ass precise as a CFR measurement by inulin clearance or with isotopes, it doess give a somewhat better idea of a patient's renal reserve than the serumm creatinine alone. A 30% rise in the serum creatinine or a 30% drop inn the urinary creatinine clearance should prompt one to reduce the CSA dosee sufficiently to normalize these values. It is further recommended that

185 5

ChapterChapter 13

patientss have their renal function monitored every 2 weeks during the first 22 months of t reatment and every 2 to 3 months thereafter.

•• Clinical Use in Ocular Disease

SystemicSystemic CSA

Basedd on observations in this and other centers, CSA appears to be particularlyy useful in patients with active, bilateral sight-threatening uveitis off a noninfectious nature. It is especially advantageous in patients who are unablee to tolerate moderate doses of systemic corticosteroids (>25 mg prednisone)) and who require steroids to control their intraocular inflammation.. In a recent randomized study, one of the coauthors (RBN) has shownn that CSA, when used as the sole agent, was effective in controlling inflammationn in 46% of patients who were steroid-intolerant [2], When combinedd with steroids, CSA was effective in an additional 35% of patients. Otherss have found that when used as an initial agent, it was effective in 97%% of patients [17].

Nonetheless,, in most cases, it is probably best to use CSA only when otherr more traditional forms of therapy have failed. CSA does not appear too induce a state of immune tolerance, and therefore most patients will be facedd w:ith an extended therapeutic course once CSA is started. An abrupt withdrawall of the d rug before the disease has run its course can cause a ratherr dramatic flare-up, as was discussed earlier.

Behcet'ss disease is an exception to this general recommendation. Ma-sudaa and colleagues [3] have clearly demonstrated in a double-masked studyy that CSA as a sole therapeutic agent was superior to colchicine, the previouss d rug of choice, in preventing recurrences of ocular disease. In anotherr study comparing CSA to a combination of cytotoxics and steroids inn Behcet's disease, CSA was again found to be more effective at preventing recurrencess (p = 0.016) [27]. Thus Behcet's is one disease for which CSA cann be considered as the initial therapeutic agent, but only if specific diagnosticc criteria are met.

Theree have been some indications that CSA may be useful to treat scleritiss and granulomatous optic neuropathy. However, a true assessment off its efficacy in these conditions will require controlled studies.

•• Topical CSA

Ass discussed previously, CSA does not penetrate very well into the eye;; however, it is possible to use it topically to inhibit surface immune-mediatedd processes. Goichot-Bonnat and associates [28] reported that a

186 6

CYCLOSPORINE E

Openn a container of 50 ml cyclosporine oral solution (1000 mg/ml) and leave it for 24 hours under a horizontal

laminarr How hood to evaporate the alcohol

I I Asepticallyy filter olive oil through a silo with a prefitter andd a 0.22-micron fitter, collecting it in a sterile 100-ml

container r

I I Asepticallyy measure 73.5 ml of olive oil

Asepticallyy filter the cyclosporine solution through a 0.22-micronn filter for a total volume of 1.5 ml

Mixx the two solutions and aliquot as needed

! ! Storee the vials at room temperature

Figuree 3 Procedure for Ike preparation of 2% cyclosporine eye dropsdrops using commercially available systemic cyclosporine.

2%2% topical CSA solution in high-risk corneal grafts had an 89% success ratee during a 16-month average follow-up. Similar results were obtained byy Belin and coworkers [29], who reported a success rate of 91% by giving thee drops preoperatively every 2 hours for 2 days followed by four times dailyy after the corneal graft was inserted. In a double-masked trial, 2% topicall CSA was also found to be effective in treating severe vernal keratoconjunctivitis,, with a significant decrease in the conjunctival hyperemia, papillaryy hypertrophy, and the number of Trantas ' dots [30]. Improvementt may be noted within the first 15 days of therapy, but relapses occurredd 2 to 4 months after therapy was discontinued [31].

CSAA eye drops can be prepared in a number of ways. Cenerally, we employy the commercial oral preparation from Sandoz using the protocol outlinedd in Figure 3. It is important to vent the CSA solution for at least 244 hours to remove the alcohol, which otherwise is a strong corneal irritant. Usingg this preparation, we were able to successfully treat a case of recurrentt ligneous conjunctivitis, and we have used it in other situations as well wheree there was a strong suspicion of surface immune activation [32]. Topicall CSA may play a role in the treatment of paracentral corneal rheumatoidd ulceration. Preliminary results using a 2% solution were very encouragingg [33]. However, its usefulness in this and other conditions such ass oculocutaneous syndromes and Sjogren's syndrome needs to be elucidatedd by further studies.

187 7

ChapterChapter 13

•• Conclusion

CSAA is a useful agent in the treatment of serious inflammatory diseases

off the eye. When used carefully and in patients with the appropriate indi

cations,, it is both safe and effective. In the future, we hope agents will appearr that will be less toxic. Agents that might prove to be useful include

FK5066 and rapamycin, two drugs with a potent immunosuppressive potentiall against T cells. Another approach that is being investigated is to com

binee CSA with other immunomodulatory drugs or to cycle the use of CSA withh cytotoxic agents. These newer approaches still need to be carefully

evaluatedd in controlled settings before they can be considered as effective

andd safe. The ultimate goal is to control immune-mediated inflammatory diseasee with the minimal number of adverse side effects.

•• References

1.. Nussenblatt RB, Palestine AG, Chan CC. Cyclosporine A therapy in the treatment off intraocular inflammatory disease resistant to systemic corticosteroids and cytotoxicc agents. Am J Ophthalmol 1983;96:275-282'

2.. Nussenblatt RB, Palestine AG, Chan CC, et al. Randomized, double-masked study off cyclosporine compared to prednisolone in the treatment of endogenous uveitis. Amm J Ophthalmol 1991;112:138-146

3.. Masuda K, Nakajima A. Uravama A. et al. Double masked trial of cyclosporin versus colchicinee and long term open study of' cyclosporin in Behcet's disease. Lancet 1989;1:1093-1096 6

4.. Bore! JF. The history of cyclosporine A and its signihcance. In: White DJG. ed, Cyclosporinee A. Amsterdam: Elsevier Biomedical Press, 1982:5—8

5.. Kahan BD. Cyclosporine. N Engl J Med 1989;321:1725-1738 6.. Nussenblatt RB, Palestine AG. Cyclosporine: immunology, pharmacology and ther

apeuticc uses. Surv Ophthalmol 1986;31:159-169 7.. BenEzra D, Maftzir G. Ocular penetration of cyclosporine A in the rat eye. Arch

Ophthalmoll 1990; 108:584-587 8.. Palestine AG, Nussenblatt RB, Chan CC. Cyclosporine penetration into the anterior

chamberr and cerebrospinal fluid. Am j Ophthalmol 1985;99:210-21 1 9.. Tabbara KF, AI Sayyed Y. Ocular bioavailability of cyclosporin in pigmented and

albinoo rabbits. | Autoimmun 1993 (in press) 10.. Reidy J ] , Gebhardt BM, Kaufman HE. The collagen shield. A new vehicle for

deliveryy of cyclosporin A to the eye. Cornea 1990;9:196-199 11.11. Kanai A, Aiba RM, Takano 'I', et al. The effect on the cornea of alpha cyclodextrm

vehiclee for cyclosporin eye drops. Transplant Proc 1989:21:3150—3152 12.. Nussenblatt RB. The expanding use of immunosuppression in the treatment of

non-infectiouss ocular disease. J Autoimmun 1992:5:247-257 13.. de Smet MD, Rubin Bj, Whitcup SNf, et al. Combined use of cyclosporine and

ketoconazolee in the treatment of endogenous uveitis. Am ] Ophthalmol I992;l 13: 687-690 0

]] 1. Tilney NL, Strom TB, Kupiec-Weglinski JW, Pharmacologic and immunologic agonistss and antagonists of cyclosporine. Transplant Proc 1988;20(suppl 3): 13—22

15.. Vine VV, Bowers L.D. Cyclosporine: structure, pharmacokinetics, and therapeutic drugg monitoring. Crit Rev Clin Lab Sci 1987;25:275-311

188 8

CYCLOSPORINS S

16.. Ellis CN, Fradin MS, Messana JM, et al. Cyclosporine for plaque-type psoriasis. Resultss ofamult idose, double-blind trial. N Engl J Med 1991;324:277-284

17.. Towler HMA, Lightman SL, Forrester JV. Low-dose cyclosporin therapy of ocular inflammation:: preliminary report of a long-term follow-up study. J Autoimmun 1992;5{suppll A):259-264'

18.. Harris KP, Jenkins D, Walls J. Nonsteroidal antiinflammatory drugs and cyclosporine.. A potentially serious adverse interaction. Transplantation 1988;46; 598-599 9

19.. Feutren G. The optimal use of cyclosporin A in autoimmune diseases. J Autoimmun 1992;5:183-195 5

20.. Feutren G, Mihatsch MJ. Risk factors for cyclosporine-induced nephropathy in patientss with autoimmune diseases. N Engl J Med 1992;326:1654-1660

21.. Palestine AG, Austin HA, Balow JE, et al. Renal histopathologic alterations in patientss treated with cyclosporine for uveitis. N Engl j Med 1986;314:1293-1298

22.. Feutren G, Abeywickrama K, Friend D, von Graffenried B. Renal function and bloodd pressure in psoriatic patients treated with cyclosporin A. Br J Dermatol 1990;122(supP136):57-69 9

23.. Palestine AG, Alter GJ, Ghan CC, Nussenblatt R. Laser interferometry and visual prognosiss in uveitis. Ophthalmology 1985;92:1567-1569

24.. BenEzra D, Nussenblatt RB, Timonen P. Optimal use of Sandimmune in endogenouss uveitis. Berlin: Springer-Verlag, 1988:22

25.. Feutren G, Miller C, Low predictive value of cyclosporine level for efficacy or renall dysfunction in psoriasis and idiopathic nephrotic syndrome. Transplant Proc 1990;22:1299-1302 2

26.. Masri MA, Cyclosporine blood level monitoring by three specific methods; R1A H , RIAA II5S, and fluorescence polarization: comparison of accuracy, cost, reproducibilityy and percent recovery. Transplant Proc 1992;24:1716-1717

27.. BenEzra DE, Cohen E, Chajek T, et al. Evaluation of conventional therapy versus cyclosporinee A in Behcet's syndrome. Transplant Proc 1988;20(suppl 4):143-146

28.. Goichot-Bonnat L, De Beauregard C, Saragoussi J ] , Pouliquen Y. Usage de la cyclosporinee A collyre dans la prevention dy rejet de greffe de cornée chez 1'homme: 1.. Evolution préopératoire de 4 yeux atteints de kératite métaherpétique. J Fr Oph-calmoll 1987;10:207-211

29.. Belin MW, Bouchard CS, Frantz S, Chmielinska J. Topical cyclosporine in high-risk corneall transplants. Ophthalmology 1989;96:1144-1150

30.. Bleik PH, Tabbara KF. Topical cyclosporine in vernal keratoconjunctivitis. Ophthalmologyy 1991;98:1679-1684

31.. Secchi AG, Tognon MS, Leonardi A. Topical use of cyclosporine in the treatment off vernal keratoconjunctivitis. AmJ Ophthalmol 1990;110:641-645

32.. Rubin Bl, Holland EJ, de Smet MD, et al. Response of reactivated ligneous conjunctivitiss to topical cyclosporine. AmJ Ophthalmol 1991;112:95-96

33.. Kervick GN, Pflugfelder SC, Haimovici R, et al. Paracentral rheumatoid corneal ulceration:: clinical features and cyclosporine therapy. Ophthalmology 1992;99: 80-88 8

34.. Nussenblatt RB, Palestine AG. L'veitis: fundamentals and clinical practice. Chicago: Yearr Book, 1989

35.. Kappos L, Patzold U, Domatsch S, et al. Cyclosporin versus azathioprine in the longg term treatment of multiple sclerosis. Ann Neurol 1988;23:56-63

189 9

Chapterr 14

Combinedd Use of Cyclosporine and Ketoconazole inn the Treatment of Endogenous Uveitis

Marcc D. de Smet, Benjamin I. Rubin, Scott M. Whitcup, Juan S. Lopez, Howardd A. Austin, Robert B. Nussenblatt

Americann Journal of Ophthalmology 113: 687-690, 1992 (byy permission © Elsevier Science Inc)

CSAA AND KFTOCONAZOLE - 1

Combinedd Use of Cyclosporine and Ketoconazole in the Treatmentt of Endogenous Uveitis

Mar cc D. de Smet, M.D. , Ben jamin I . Rub in , M.D. , Scott M . Wh i t cup , M.D. , Juann S. Lopez, M.D. , Howard A . Aus t in , M.D. , and Robert B. Nussenb la t t, M .D .

Tenn patients wit h endogenous uveit is were inn clinical remission attributabl e to treatment wit hh cyclosporine and prednisone. After the cyclosporinee dose was reduced by two thirds , thesee patients were randomly assigned to treatmentt wit h or without ketoconazole, a potentt inhibito r of cytochrome P-450, in a double-maskedd placebo-controlled study. The dosee was reduced over three days. Durin g a three-monthh follow-up, no patients treated wit hh ketoconazole had a relapse of uveitis, whi l ee four of six (66%) control subjects had a flare-up.. Toxicit y in the ketoconazole-treated groupp was l imite d to a transient decrease in glomerularr filtration rate (20% from baseline) att one month in two of six (33%) patients. Renall function was stabil ized by furthe r re-ductionn of the cyclosporine dose.

OEVERALL STUDIES have shown that many forms off endogenous uveitis can be successfully treatedd with a combination of cycïosporine and corticosteroids.133 One major drawback to the disseminatedd use of cyclosporine has been its considerablee cost. Reducing the metabolism of cyclosporinee would allow a dose reduction and therebyy diminish the cost. Cyclosporine metabolismm is mediated almost exclusively by the liverr cytochrome P-450 microsomal enzymes.4

Thee antifungal ketoconazole inhibits this enzymee system both in vivo and in vitro,1 resultingg in increased blood concentrations of cyclo-

Acceptedd for publication March 10, 1992. Fromm the Laboratory of immunology, National Eye

Institutee (Drs. de Smet, Rubin, Whitcup, Lopez, and Nussenblatt);; and Kidney Disease Section, National Institutee of Arthritis, Diabetes, and Digestive and Kidnev Diseasess (Dr. Austin), National Institutes of Health, Bethesda,, Maryland.

Reprintt requests to Marc D. de Smet, M.D., Laboratory off Immunology, National Lye Institute, BIdg. 10, Rm. 10N202,, Bethesda, MD 20892.

sporinee that may become toxic if the dose is not reduced. s , :: Recently, First and associates7 used 30%% of their standard cyclosporine dose (8 mg /kgg of body weight) in connection with ketoconazolee to prevent graft rejection in patientss who had undergone renal transplantation.. None of their 18 patients developed any markedd toxicity in up to 13 months of follow-up.. This combination has not yet been tried in autoimmunee diseases. We tested this drug combinationn in patients with endogenous uveitis whosee conditions had been stabilized using our standardd dose of cyclosporine and prednisone*1

forr at least three months.

Patientss and Methods

Patientss whose endogenous uveitis was controlledd with cyclosporine (5 mg /kg of body weight /day)) and prednisone (0 to 0.5 mg /kg of bodyy weight /day) signed an informed consent approvedd by the Institutional Review Board, andd had their cyclosporine dose decreased by 70%% in two increments over three days. At the timee of the initial reduction in the cyclosporine dose,, patients were randomly assigned in a maskedd fashion to treatment with or without ketoconazole.. Patients were monitored for threee months or until the development of a flare-upp (defined as a decrease in best-corrected visuall acuity by two lines as measured by the Earlyy Treatment of Diabetic Retinopathy Study eyee chart3 or an increase by two steps in vitreal hazee as defined previously.1" Patients were also monitoredd at regular intervals for development off hepatic or renal toxicity. A doubling of the hepaticc enzymes, a 30% increase in serum creatininee concentration, or a 30% decrease in the 24-hourr urinary creatinine clearance led to a furtherr reduction in the dosage of cyclosporine. Hydratedd clearance studies using technetium Tcc 99m diethylene triaminopenta-acetic acid andd iodohippurate sodium I 131 were per-

193 3

ChapterChapter 14

TABLE E CHARACTERISTICSS OF PATIENTS WITH UVEITIS

DECREASE E

FROMM INITIAL 'NITIAL

CASE E

NO O

1 1

2 2

3 3

4 4

5 5

6 6

7 7

8 8

9 9

10 0

DIAGNOSIS S

Behpets s disease e

Intermediate e uveitis s

Sarcoid d

Behcet's s disease e




Behcet's s disease e

Behcett s disease e

intermediate intermediate uveitis s

TREAtWENt t

Ketoconazole e

Ketoconazole e

Ketoconazole e

Placebo o

Placebo o

Placebo o

Placebo o

Ketoconazole e

Placebo o

Placebo o

CVCLOSPORINE E

DOSEE fc)

85 5

92 2

87 7

72 2

66f f

82 82

70 0

71 1

72' '

50* *

PREDNISONE E

DOSEE (MQi

30--

15 5

None e

15 5

5 5

15 5

5 5

30' '

15 5

None e

FLAHEE UP

No o

No o

No o

Yes.. at 77 days

Yes.. at 188 days

No o

No o

No o

Yes,, at 188 days

Yes,, at 66 days

"Reducedd to 10 mg at two months in the study. Treatmentt with ketoconazole started after a flare-up. 'Reducedd to 5 mg at two months in the study

formedd at days 0, 30, and 90 to obtain an accuratee measurement of the true glomerular filtrationn rate.11

Results s

Tenn patients were entered in the studv; ketoconazolee was administered to four patients and placeboss were administered to six patients (Table).. Use of corticosteroids at the time of entry intoo the study was determined. The dose was nott modified during the study. None of the patientss treated with ketoconazole developed a flare-up,, as compared to four of six patients administeredd placebos who did develop flare-upss (P = .035, chi square). All of the flare-ups developedd within three weeks of random assignmentt to treatment and the decrease of the cvclosporinee dose. All flare-ups were caused by aa decrease in visual acuity by two lines or more. Afterr flare-up of their ocular disease, three patientss elected to be treated with ketoconazole whilee maintaining the same reduced cvclosporinee dose. In these three patients, the ocular

inflammationn resolved with no further ffare-up inn the next three months. Patient 4 had a severe ocularr attack of Behcet's disease after reduction inn the cvclosporine dose To achieve rapid control,, it was in the patient's best interest to be treatedd with standard doses of cvclosporine and noo ketoconazole. None of the patients initially administeredd placebos had a permanent decreasee in visual acuity. Two of the four patients whoo were treated with ketoconazole had an improvementt in visual acuity although their visuall acuities had been considered stable beforee entering the study,

AA 30% increase in serum creatinine concentrationn was noted in two patients while they weree being treated with ketoconazole. This was paralleledd bv a decrease in the glomerular filtrationn rate, which is a more sensitive measure off change in renal function than creatine clearancee or serum creatinine (by 20% in one patient andd 30% in the other patient), in both patients, aa dosage reduction led to a normalization of the serumm creatinine concentration by the next clinicc visit, two months later. The glomerular filtrationn rate returned to the baseline value in onee patient, whereas it remained at the lower

194 4

CSAA AND KETOCONAZOLE - 1

glomerularr nitration r

Dloofll urea nitrogen

Figuree (de Smet and associates). Changes in renal valuess at the beginning and end ot the study in patientss treated with ketoconazole and cyclosporine.

valuee in the other pat ient . At three mon ths in mostt pa t ients , the renal and hepat ic profiles weree unchanged as compared to basel ine valuess (Figure).

Discussion n

achievee a good t rea tment effect. The dose shouldd initially be decreased to 30% of the originall dose . Once the pat ient has been t reated withh this lower dose for several days (min imum, fourr half-lives), a cvclosporine whole b lood levell should be obta ined. If the level r emains high,, the cvclosporine dose can safely be reducedd further. When s tar t ing t rea tment with ketoconazole ,, a pat ient should be carefully moni toredd for clinical signs of acute cvclosporinee toxicity. These would manifest as an increasee in the blood pressure , an increase in the bloodd urea n i t rogen concent ra t ion , serum crea t in inee concent ra t ion , or an increase in liver enzymee concent ra t ion . Manifestation of anv of thesee clinical signs should prompt further reduct ionn of the cvclosporine dose .

Somee pat ients who were t reated with the ketoconazole-cyclospor inee combina t ion had a furtherr improvement in visual acuity even thoughh their clinical remission was cons idered stabilizedd before enter ing the s tudy. This possiblyy indicates that sus ta ined levels of cyclosporinee are better at main ta in ing a remission than is thee usual t rea tment schedule in which d rug concent ra t ionn changes dramat ical ly in a given day.. The addi t ion of ke toconazole to the regimenn of coadminis te red reduced-dose cvclospor inee (5 m g / k g of bodv we igh t / d av or less) andd prednisone seemed to be effective in maintainingg remiss ions in pa t ien ts with uveit is . Clearly,, further s tudies are needed to de te r minee the long-term toxicity of this combina t ion therapyy for newly d iagnosed pa t ien ts as well as forr pat ients with chronic uveitis and severe , bilaterall s igh t - th rea ten ing disease .

Wee demons t ra ted the efficacy of t rea tment withh coadminis tered ke toconazole , cyclosporine,, and p redn isone in pa t ien ts with autoimmunee disease. Ketoconazole proved to be a potentt inhibi tor of cyclospor ine metabol ism. Cvclosporinee doses should be reduced quickly dur ingg a two- to three-day period to prevent deve lopmentt of markedly increased levels of circulat ingg cyclosporine. Cyclosporine-related toxicityy can be avoided if the cvclospor ine wholee blood levels are main ta ined in the lower rangess of the normal values (500 to 1,000 n g / l ) . Thiss was achieved in these pa t ien ts by reduc ing thee orally adminis tered cvclosporine dose to betweenn 10% and 30% of the basel ine dose .

Thee exact extent of the dose reduct ion varies be tweenn pa t ien ts , but by main ta in ing whole bloodd cvclosporine levels in the lower range of thee normal values, one can avoid toxicity and

References s

1.. Nussenblatt, R. B. Palestine. A C . , Chan. C.. C , Mochizuki, M., and Yancey, K.: Lffectiveness off cyclosporin therapy for Behcet's disease. Arthritis Rheum.. 28:671, 1985".

2.. Masuda, K., Nakajima, A.. L'rayama. A., \ a k a e . K.,, Kogene, M., and Inaba. C : Double masked trial of cyclosporinn versus colchicine and long term open studyy of cyclosporin in Behcet's disease, lancet 1:1093,, 1989.

3.. Nussenblatt. R B . and Palestine. A. C : Cyclosporine.. Immunology, pharmacology and therapeu ticc uses. Surv. Ophthalmol. 31:159, 1986.

4.. Tilney, N. I.., Strom. T B., and Kupiec-Weglin-ski.. J. W.: Pharmacologic and immunologic agonists andd antagonists of cvclosporine. Transplant Proc. 20(suppl.. 3): 13, 1988.'

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5.. Gumble ton , M., Brown, J, E., Hawksworth, G., andd Whit ing, P. H : The possible relationship b e tweenn hepat ic drug metabol ism and ketoconazole enhancemen tt of cyclosporine nephrotoxici ty . Transp lan ta t ionn 40:454,' 1985.

6.. Anderson , ]. E., Morris, R, E., and Blaschke, T.. F.: Pharmacodynamics of cyclosporine-ketocona-zolee interact ion in mice. Combined therapy potentiatess cyclosporine immunosuppress ion and toxicity. Transplanta t ionn 43:529, 1987.

7.. First, M. R., Schroeder, T. J., Weiskittel, P., Myre,, S. A., Alexander, J. W., and Pesce, A. J.: Concomitantt adminis t ra t ion of cyclosporin and ketoconazolee in renal t ransplant recipients . Lancet 2:1198, 1989. .

8.. BenEzra, D., Nussenblat t , R. B., and Timonen, P.:: Optimal Use of Sandimmun in Endogenous Uveit is .. Berlin, Springer-Verlag, 1988, p . 18.

9.. The Diabetic Retinopathy Study Research Group:: Report 6. Design, methods , and basel ine results .. Invest. Ophtha lmol . Vis. Sci. 21:149, 1981.

10.. Nussenblatt , R. B., Palestine, A. G., Chan, C.. C , and Roberge, F.: Standardizat ion of vitieal inflammatoryy activity in intermediate and posterior uveitis.. Ophthalmology 92:467, 1985.

11.. Shemesh, O., Golbetz, H„ Kriss, ) . ! ' . , and Myers,, B. D.: Limitations of creatinine as a filtration markerr in g lomeru lopa thy pat ients . Kidney Int. 28:830,, 1985.

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Longg Term Follow-up of Patients with Endogenouss Uveitis Treated with Combination

Cyclosporinee and Ketoconazole

Alii Ramadan, Robert B. Nussenblatt, Marc de Smet

Ophthalmologyy 104: 706-711, 1997 (byy permission © Elsevier Science

! !

I I

! !

CSAA AND KKTOCONAZOLE - 2

Abstract t

Purpose:: Combined treatment with cyclosporine and ketoconazole in autoimmune diseases has receivedd little attention. This report outlines the long term outcome of a cohort of patients that weree placed on combination therapy for endogenous uveitis. Method: Six patients who were initiallyy treated with only cyclosporine (CSA) were followed long term on a combination of cyclosporinee and ketoconazole (KCZ). Data was analyzed for visual acuity, number of flare-ups andd signs of systemic toxicity. Results: Patients were on CSA for a mean of 13 months and CSA/KCZZ for a mean of 33 months. While patients had a number of flare-ups prior to combinationn therapy, only 2 flare-ups in 2 patients were noted during combined therapy P = 0.055. Three patientss showed signs of renal toxicity on CSA, 2 continued to show signs of toxicity on CSA/KCZ.. One patient stabilized and maintained normal renal parameters. Elevation of systolic andd diastolic pressure was present in 3 of 6 patients with CSA. While the systolic pressure remainedd the same with CSA/KCZ, diastolic pressure was within normal parameters in all patients (PP = 0.03). No toxicity related to ketoconazole alone was observed. Conclusion:: Combination of cyclosporine and ketoconazole is safe and effective in the treatmentt of endogenous uveitis. It appears to be more effective in preventing recurrences than cyclosporinee alone, and does not lead to an increased risk of renal toxicity over cyclosporine alone. Itt also represents a significant cost saving over cyclosporine monotherapy.

Introductio n n

Cyclosporinee A (CSA) was introduced for the treatment of endogenous uveitis in late 1983, and hass been used successfully by a number of groups' '. In one report, over 50% of patients with endogenouss uveitis unresponsive to steroids alone were controlled on CSA or a combination of CSAA and steroids"1. Cyclosporine is non cytotoxic. It inhibits the activation of CD4+ lymphocytess by preventing the synthesis and secretion of IL-2 and its receptor1. This inhibition is dose dependent. .

Toxicityy related to cyclosporine is also dose dependent1 N " i : . When the dose of oral CSA is kept beloww 5 mg/kg/day and large swings in serum creatinine levels are avoided, the incidence of irreversiblee changes in renal function are quite low. However, CSA blood levels tend to vary considerablyy between individuals due largely to variations in the rate of absorption from the gas-troinstestinall tract, and the rate of CSA metabolism through cytochrome P-450 microsomal enzymess '' r . Thus, medications that affect cytochrome P450 can have a dramatic effect on blood CSAA levels. Phenytoin, a potent inducer of hepatic cytochrome P450, can cause a significant dropp in serum CSA levels within 2 hours of its administration '* ''. Other agents such as phenobarbetal,, rifampin, isoniazid have similar effects :" : | . Drugs that inhibit the hepatic microsomal enzymee P-450 will increase CSA plasma levels. This inhibition results in increased blood concentrationn of CSA, necessitating a reduction in the oral dose to maintain serum levels within the therapeuticc range -• :\ Drugs like cimetidine, erythromycin ^ : \ oral contraceptives, androgens, andd methylprednisolone are all reported to cause elevations in serum CSA levels. Some calcium channell blockers, especially verapamil have also been observed to increase CSA plasma concentrationn ;'':7.

Ann imidazole derivative, ketoconazole has a broad antifungal spectrum of activity, and is known

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too inhibit cytochrome P-450 : ::"'". A number of studies have shown that it can be combined successfullyy with cyclosporine to prevent graft rejection ! ' " . Ketoconazole has no known direct renall effect which is a major concern of cyclosporine therapy, and it does not alter the systemic bloodd pressure. Its main toxicity is hepatocellular, in a dose dependent manner, whereas cyclosporinee is not commonly reported to severely alter hepatic function \ Thus the association of thee two drugs is not contraindicated at least in terms of additive toxicities. The addition of a cytochromee inhibitor such as ketoconazole can drastically reduce cyclosporine requirements, whichh represents a significant cost saving over the duration of treatment4 !\ On this basis, we havee tried the combination of both agents, i.e. CSA and ketoconaz,ole in the treatment of endogenouss uveitis4. Initial results were extremely encouraging. However, we also wanted to determinee the long-term outcome of combined therapy. In this study, we report on these initial patientss treated with cyclosporine and ketoconazole, now followed for up to five years on combinationn therapy.

Patientt Selection and Method

Thiss study reports on the long term follow-up of patients that were entered in a double masked randomizedd study comparing cyclosporine/ketoconazole to cyclosporine/placebo. The study had beenn approved by the Institutional Review Board of the National Eye Institute. All study participantss gave informed consent prior to enrollment. Patients with endogenous uveitis of non infectiouss origin, treated at the NIH with cyclosporine A were considered elligible for entry provided thatt the uveitis had been stable on medication for at least 1 month. In the initial phase of the study.. 10 patients were randomized. Four patients received ketoconazole (200 mg/day) and 6 patientss were given a placebo. These patients were carefully followed for signs of renal toxicity or recurrencee of uveitis for a period of 3 months. As previously reported in the Journal \ patients on ketoconazolee were able to reduce their dose of cyclosporine by over 70% without any recurrence off uveitis. Four of 6 patients receiving the placebo had a recurrence. Recurrence was defined as aa decrease in visual acuity of 2 or more lines as measured on the Early Treatment of Diabetic Retinopathyy Study eye chart (ETDRS)'\ or a two step increase in vitreal haze •''.

Afterr completing the double masked portion of the study, all patients were given the option of enteringg an open arm, designed to evaluate the long term effect of the combination of cyclosporine AA and ketoconazole. Six patients (3 males and 3 females) entered this arm of the study. Three patientss had Behcet's disease, two intermediate uveitis and one patient had Sarcoidosis. The diseasess were confirmed according to established criteria as previously described 4 '•'. Patients were treatedd for as long as was clinically indicated or until they showed signs of persistent renal or hepaticc toxicity. Renal toxicity was defined as any increase in serum creatinine concentration by 30%% or more, a decrease in the 24-hour creatinine clearance by more than 30%. Hepatic toxicityy was defined as a doubling of the hepatic enzymes. Patients were also given the option of switchingg to an alternate form of therapy if such therapy became available. Cessation of combinedd therapy for any reason was considered the end point of the protocol. At the time of data analysis.. 2 patients were still being treated on protocol. Two patients were taken off the study becausee of depressed creatinine clearances, 3 months after starting therapy. Both of these patients hadd shown signs of renal toxicity while on cyclosporine monotherapy. One patient was taken off alll medication, and one patient elected to enter a different therapeutic protocol. At each clinic visit,, best corrected visual acuity was measured on ETDRS eye charts. Patients had a complete

200 0


Tablee 1: Baseline Characteristics of Patients at Initiation of Cyclosporine or Cyciosporine/Ketoconazole (MeanS.E.M.)) n = 6

Agee (years) Cyclosporinee Dose (mg/kg/day) Prednisonee dose (mg/day) Visuall Acuitv (letters) Serumm Creatinine (mg/dL) Creatininee Clearance (mL/min) Systolicc Blood Pressure (mm Hg) Diastolicc Blood Pressure (mm Hg)

Cyclosporine e

35.77 11.2 6.88 9 (Range

19.22 2 (Range 566 8

0.922 0.08 105.33 2

1222 4 777 5

4 -- 10) 155 to 40)

Cyclosporinee + Ketoconazole e

1.44 2 (Range 14.22 7 (Range

566 7 1.188 8 90.22 7.8 1277 6 844 5

0.8 8

5 --

-2) ) 25) )

PP value

0.002 2 0.111 1 0.932 2 0.043 3 0.008 8 0.415 5 0.206 6

** Mean standard error of the mean.

eyee exam including funduscopy. Blood was taken to measure hepatic and renal functions. Every 33 to 6 months, a 24 hour creatinine clearance was obtained. Prior to final discharge, all measurementss were repeated.

111 patients had been treated at the NIH with cyclosporine for a variable period of time prior to beingg placed on cyclosporine and ketoconazole. For the purposes of this analysis, this initial treatmentt period was compared to the treatment with the drug combination. Visual acuity, recorded ass the number of letters read on an ETDRS chart, serum creatinine levels, creatinine clearance values,, and systolic and diastolic blood pressure measurements were compared at baseline for bothh periods using a two-tailed paired Student's t-test. Similarly, changes in values between baselinee and the end of treatment were compared with the same statistical test. The null hypothesiss was rejected at a significance level of P < 0.05.

Results s

Forr the group as a whole, the mean age was 35.7 years when first treated with cyclosporine. Baselinee characteristics such as visual acuity, prednisone dose, systolic and diastolic blood pressuress were similar for both study periods (table 1). Renal function was somewhat poorer when thee patients were started on combination therapy. Serum creatinine was higher and creatinine clearancee lower at the time than when the patients were first treated with cyclosporine. Both differencess were statistically significant p < 0.05. The cyclosporine dose was also significantly differentt in both periods, being 80% lower (p=0.002) after starting ketoconazole. Thiss baseline dose

Tablee 2: Comparison of the Therapeutic Efficacy of Cyclosporine versus Cyclosporine and Ketoconazole (Mean :: n = 6.

Cyclosporinn Cyclosporine + Ketoconazole P value

Follow-upp Interval (months) 13.3 6.6 32.7 8.3 0.155 (Range:: 3 - - 45) (Range: 3 - - 58)

AA Visual Acuity (number of letters) -0.50 6.3 -1,33+ 1.5 0.875 Numberr of Flare-ups 1.8 + 0.8 0.3 0.2

Flare-upp by months of treatment 0.2 0.1 0.008 0.005 0.055

** Mean standard error of the mean.

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Tablee 3 : Comparison of the Side Effects and Dosage Modifications between Cyclosporine and Cyclosporine/ Ketoconazolee at Baseline and at End of Treatment (Mean S.E.M.):

Cyclosporinee Cyclosporine + Ketoconazole N=66 N=6

PP value

AA Cyclosporine (mg/kg/day) -2.1 0.7 Ketoconazolee (mg/day) AA Prednisone (mg/day) -5.8 4.2 AA Serum Creatinine (mg/mL) 0.27 0.09 AA Creatinine Clearance (mL/min) -15,8 3.5 AA Systolic Blood Pressure (mm Hg) 8.0 4.6 AA Diastolic Blood Pressure (mm Hg) 11.0 2.4

-0.88 0.2

200 0 2.11 0

0.133 5 3.677 0 0.22 4.7

-9.33 5.4

0.125 5

0.098 8 0.137 7 0.247 7 0.376 6 0.033 3

** Mean standard error of the mean,

wass determined in both study periods 3 weeks to 1 month after entry. Thiss time was selected as itt allowed for a readjustment in the cyclosporine dose after starting combination treatment.

Thee follow-up interval is somewhat longer in the second study period with a mean of 32.7 monthss (range 3 to 58 months) as compared to 13.3 months (range 3 to 45 months) for cyclosporinee alone. However, this difference was not statistically significant. On average, the changee in visual acuity was similar for both periods (table 2). A notable difference was seen in thee observed number of flare-ups of uveitis. A total of 11 flare-ups were observed over a cum-mulativee 80 month period with cyclosporine alone, while 2 flare-ups were noted over a cummu-lativee 196 months of combined treatment. When the data is analysed per patient (table 2) a significantt trend is observed particularly when comparing the risk of a flare-up per month of treatment. .

Dosagee adjustments were required in both study periods. The dose was adjusted according to ocularr response and adverse side effects. In both periods, the cyclosporine dose could be lowered further.. Adjustments were also made to the prednisone dose. However, the overall need for steroidss remained about the same during both study periods. At the time of the last evaluation whilee on cyclosporine alone, patients were receiving a mean cyclosporine dose of 4.7 mg/kg/day andd a mean dose of prednisone of 13 mg/day. At the time of the last evaluation on cyclosporine/ ketoconazolee , the mean cyclosporine dose was 0.6 mg/kg/day and the mean prednisone dose wass 16 mg/day. The final cyclosporine concentration in the latter study period represents less thann \0% of the initial cyclosporine dose or about 13% of the final cyclosporine concentration priorr to entering combination therapy.

Duringg the initial study period, 3 patients showed signs of renal toxicity as defined in the methodss section. Two patients had a transient rise in hepatic transaminases and bilirubin that respondedd to a cyclosporine dose reduction. Three patients had an increase in systolic and diastolic bloodd pressures. While on combination treatment, 2 of the 3 patients with signs of renal toxicity progressed.. Renal function did not significantly recover following a cyclosporine dose reduction andd they were taken off cyclosporine therapy. The other patient maintained his serum creatinine andd creatinine clearance within a normal range. No hepatic toxicity was observed. Patients showedd no further change in systolic blood pressure, and the diastolic pressure decreased in all affectedd patients (table 3).

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CSAA AND KETOCONAZOI.E - 2

Discussion n

Thee systematic coadministration of ketoconazole and cyclosporine appears to provide several advantagess to patients suffering from chronic uveitis. In our initial study, we were able to show thatt the combination was both safe and effective over a 3 month period. In several cases, the cyclosporinee dose could be lowered by more than 80%, while still providing adequate if not superiorr immunosuppression \ However, we did not know the long term effects of combining both drugs.. In this study, we followed the same cohort of patients for up to 5 years on combination therapy.. The combination in the majority of patients was well tolerated. Only two patients showedd renal toxicity which required a switch to an alternate form of treatment. In both cases, renall toxicity had been evident before combination therapy was instituted. In another two patients, earlyy signs of renal, hepatic and hemodynamic changes were normalized on cyclosporine/keto-conazolee without any subsequent recurrence of the problem. Such results were possible because off careful and frequent measurements of both hepatic and renal functions throughout the follow-upp period. Frequent cyclosporine levels were obtained in the early stages, until a stable level withinn the therapeutic range was achieved. This is particularly indicated in cases where ketoconazolee is added some time after cyclosporine therapy is started. In such cases, cyclosporine shouldd initially be reduced by 70%, with further adjustments being made over the next 2 to 3 monthss based on the serum cyclosporine levels 41! 1: '\ The lack of renal toxicity observed in the presentt and other similar studies, cannot be explained on the basis of a cyclosporine dose reductionn alone "• •"•*'. Several factors are probably responsible including a reduction in the number of circulatingg metabolites "••", a lessening of the maximum cyclosporine concentration 4:, and possiblyy a direct inhibition of cyclosporine induced toxicity mediated through cytochrome P4500 :"43. At higher in vitro concentrations above 100_M, cyclosporine can block its own hy-droxylationn through cytochrome P450 and can lead to the generation of superoxide free radicals 4\\ These free radicals in turn cause an increase in lipid peroxidation, an important mediator of bothh renal and hepatic CSA toxicity :". Since ketoconazole prevents O, fixation and activation by P450,, it can effectively inhibit both the metabolism of cyclosporine and the generation of superoxidee free radicals which mediate CSA related toxicity11 l\

Ketoconazolee was well tolerated, with no adverse effects related to its long term use being noted inn our patients. Hepatic injury associated with ketoconazole therapy has been reported but appearss to be due to an idiosynchratic drug reaction 4h. It is hepatocellular in nature, leading to an increasee in transaminases. Combination of CSA and ketoconazole can occasionally lead to an increasee in bilirubin and alkaline phosphatase levels , but these usually normalize upon reducing thee CSA dose47. Ketoconazole requires an acid medium for effective absorption, and therefore shouldd not be given at the same time as antacids or drugs that increase the gastric pH. High dose ketoconazolee can lower serum testosterone and lead to a reduction in sexual function • • -4\ In a groupp of heart transplant patients, it was observed that the mean testosterone level decreased fromm 4.1 _ 1.2 ng/mL before ketoconazole to 3.2 _1.3 ng/mL at the end of follow-up (mean of 10.77 months). Nothing is known about the effect of this combination on children, and its use in thiss age group should be carefully monitored, if used at all.

Thee addition of ketoconazole did not have a deleterious effect on cyclosporine's immunosuppressivee activity. Patients appeared to be in tighter control when on combination therapy as comparedd to cyclosporine alone (table 2). The difference nearing statistical significance between the twoo groups. The number of patients in our study is limited, and a larger number would be neces-

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ChapterChapter 15

saryy to fully assess the immunosuppressive effect of combined therapy. While all patients were stablee at the time ketoconazole was added, all patients were still considered to have active disease.. All patients had experienced a flare-up of uveitis in the 4 months preceeding the initiation off ketoconazole. A similar beneficial effect has also been noted in a randomized heart transplant study.. Patients on combined therapy suffered fewer episodes of rejection requiring OKT3 treatmentt ". The exact mechanism responsible for this observation is not known. Ketoconazole is knownn to have some immunomodulatory effect of its own. It is able to block the synthesis of leukotrieness and thromboxanes 4"•5", and it can potentiate the effect of steroids by inhibiting microsomall degradation through 6b-hydroxylation ?1 •". All of our patients were on low to moderate dosess of steroids at the time of the trial Uable3).

Ass a consequence of the reduced oral requirements for cyclosporine following the coadministrationn of ketoconazole, the mean daily cost of cyclosporine is sharply decreased. In our case, patientss were able to reduce their daily cyclosporine requirements by 90%. In heart transplant patients,, this can represent a 73% reduction in cost, even when taking the additional cost of ketoconazolee into account " ". Such savings can be of considerable importance in the face of increasingg pressures for cost containment''. However, these savings must be partially offset by the increasedd need for cyclosporine blood monitoring. This is particularly important shortly after institutingg combination therapy, as we and others have found that the degree of inhibition varies somewhatt between patients \ Inn the future, the coadministration of ketoconazole and cyclosporine may become an important practicall consideration in patients with endogenous uveitis. It appears to offer both significant costt savings and a favorable pharmacodynamic profile.

References: :

1.. Nussenblatt RB. Palestine AG. Chan CC. Cyclosporine A therapy in the treatment of intraocular inflammatoryy disease resistant lo systemic corticosteroids and cytotoxic agents. Am J Ophthalmol 1983; 96:275-82.

2.. Nussenblatt RB, Palestine AG. C CC, Mochizuki M. Yancey K. Effectiveness of cyclopsorin therapy for Behcet'ss disease. Arthritis Rheum 1985: 20:671-9.

3.. de Smet MD. Nussenblatt RB. Clinical use of cyclosporine in ocular disease. Int Ophthalmol Clin 1993: 33:31-45. .

4.. de Smet MD, Rubin BJ. Whitcup SM. Lopez JS, Austin HA. Nussenblatt RB. Combined use of cyclosporine andd ketoconazole in the treatment of endogenous uveitis. Am J Ophthalmol 1992; 113:687-90.

5.. Masuda K. Nakajima A. Urayama A, Nakae K, Kogene M. Inaba G. Double masked trial of cyclosporin versuss colchicine and long term open study of cyclosporin in Behcet's disease. Lancet 1989: 1093-6.

6.. Mochiz.uki M, de Smet MD. Use of immunosuppressive agents in ocular diseases. Prog Ret Eye Res 1994; 13:479-506. .

7.. Secchi AG. Tognon MS. Leonardi A. Topical use of cyclosporine in the treatment of vernal keratoconjunctivitis.. Am J Ophthalmol 1990: 1 10:641-5.

8.. Towler HMA, Lightman SL, Forrester JV. Low-dose cyclosporin therapy of ocular inflammation: preliminary reportt of a long-term follow-up study. J Autoimmun 1992; 5 (Suppl A):259-64.

9.. Wakefield D. McCluskey P. Cyclosporin therapy for severe scleritis. Brit J Ophthalmol 1989; 73:743-6. 10.. Nussenblatt RB, Palestine AG. Chan CC. Stevens G. Mellow SD, Green SB. Randomized, doubled-masked

studyy of cyclosporine compared to prednisolone in the treatment of endogenous uveitis. Am J Ophthalmol 1991;; 112:138-46.

II 1. Feutren G. The optimal use of cyclosporin A in autoimmune diseases. J Autoimmun 1992; 5:183-95. 12.. Feutren G. Mihatsch ML International Kidney Biospy Registry of Cyclosporine in Autoimmune Diseases.

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Riskk factors for cyclosporine-induced nephropathy in patients with autoimmune diseases. New Engl J Med 1992;326:1654-60. .

13.. Grevel J. Optimisation of immunosuppressive therapy using pharmacokinetic principles. Clin Pharmacokinet 1992:23:380-90. .

14.. Grevel J, Post BK, Kahan BD. Michaelis-menten kinetics determine cyclosporine steady-state concentrations:: a population analysis in kidney transplant patients. Clin Pharmacol Ther 1993; 53:651 -60.

15.. Mallet A, Mentre F, Steimer JL, Lokiec F. Nonparametric maximum likelihood estimation for population pharmacokineticss with application to cyclosporine. J Pharm Biopharm 1988; 16:311-317.

16.. Lee GC, Stanley DL, Pessa LJ. Costa TD, Beltz SE, Ruiz J, et al. Effect of grapefruit juice on blood cyclosporinn concentration. Lancet 1995; 345:955-956.

17.. Tilney NL, Strom TB, Kupiec-Weglinski JW. Pharmacologic and immunologic agonists and antagonists of cyclosporine.. Transplant Proc 1988; 20 (suppl 3): 13-22. 18.. Keown PA, Stiller CR, Laupacis AL. The effects and side effects of cyclosporine; relationship to drug phar

macokinetics.. Transplant Proc 1982; 14:659-61. 19.. Keown PA, Laupacis A, Carruthers G, Stawecki M, Koegler J. McKenzie FN, et al. Interaction between

phenytoinn and cyclosporine following organ transplantation. Transplantation 1984; 38:304-6. 20.. Daniels NJ, Dover JS. Schachter RK. Interaction between cyclosporin and rifampicin. Lancet 1984: 2:639. 21.. Herman RJ. Nakamura K, Wilkinson GR, Wood AJ. Induction of propranolol metabolism by rifampicin. Br J

Clinn Pharmacol 1983; 16:565-9. 22.. Gumbleton M, Brown JE, Hawksworth G, Whiting PH. The possible relationship between hepatic drug me

tabolismm and ketoconazole enhancement of cyclosporine nephrotoxicity. Transplantation 1985; 40:454-5. 23.. Anderson JE, Morris RE, Blaschke TF. Pharmacodynamics of cyclosporine-ketoconazole interaction in mice:

combinedd therapy potentiates cyclosporine immunosuppression and toxicity. Transplantation 1987; 43:529-33. 24.. Puurunen J, Sotaniemi E, Pelkonen O. Effect of cimetidine on microsomal drug metabolism in man. Eur J

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tionn with oral anticoagulants in man. Lancet 1979; 2:317-9. 26.. Serino F, Grevel J, Napoli KL, Kahan BD, Strobel HW. Oxygen radical formation by the cytochrome P450

systemm as a cellular mechanism for cyclosporine toxicity. Transplant Proc 1994; 26:2916-7. 27.. Grino JM, Sabate I, Castelao AM, Alsina J. Influence of diltiazem on cyclosporin clearance. Lancet 1986;

1:1387. . 28.. Shepard JH, Canafax DM, Simmons RL, Najarian JS. Cyclosporine-ketoconazole: a potentially dangerous

drug-drugg interaction. Clin Pharm 1986; 5:468. 29.. Cunningham C, Burke MD, Whiting PH, Simpson JG. Wheatley DN. Ketoconazole, cyclosporin, and the kid

ney.. Lancet 1982:2:1464. 30.. Morgenstern GR, Powles R, Robinson B. McElwain TJ. Cyclosporin interaction with ketoconazole and mel-

phalan.. Lancet 1982:2:1342. 31.. First MR. Schroeder TJ, Michael A, Hariharan S, Weiskittel P, Alexander JW. Cyclosporine-ketoconazole in

teraction.. Long-term follow-up and preliminary results of a randomized trial. Transplantation 1993; 55:1000-4. .

32.. First MR, Schroeder TJ, Weiskittel P. Myre S A, Alexander JW, Pesce AJ. Concomitant administration of cyclosporinee and ketoconazole in renal transplant patients. Lancet 1989: 2:1198-201.

33.. Butman SM, Wild JC, Nolan PE, Fogan TC, Finley PR. Hicks MJ, et al. Prospective study of the safety and financiall benefit of ketoconazole as adjunctive therapy to cyclosporine after heart transplantation. J Heart Lungg Transplant 1991; 10:351-8.

34.. Girardet RE, Melo JC, Fox MS, Whalen C. Lusk R, Masri ZH. et al. Concomittant administration of cyclosporinee and ketoconazole for three and a half years in one heart transplant recipient. Transplantation 1989; 48:887-90. .

35.. Albengres E, Tillement JP. Cyclosporin and ketoconazole, drug interaction or therapeutic association? Int J Chnn Pharm Ther Toxicol 1992; 30:555-70.

36.. de Smet MD, Yamamoto JH. Mochizuki M, Gery 1, Singh VK. Shinohara T, et at. Cellular immune responsess of patients with uveitis to retinal antigens and their fragments. Am J Ophthalmol 1990; 110:135-42. .

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37.. Shemesh O. Golbetz H. Kriss JP. Myers BD. Limitations of creatinine as a filtration marker in glomerulopathyy patients. Kidney Int 1985: 28:830-38.

38.. The Diabetic Retinopathy Study Research Group. Report 6. Design, methods, and baseline results. Invest Ophthalmoll Vis Sci 1981: 21:149-209.

39.. Nussenblatt RB. Palestine AG, C CC. Roberge F. Standardization of vitreal inflammatory activity in intermediatee and posterior uveitis. Ophthalmology 1985: 92:467-71.

40.. Ernst W. Hauser J. Balducci M, Fussbinder W. Akute verschlechterung der nierentransplantation dnrch ein pilzbezoar.. Nieren Hochdruckkr 1988; 17:127-32.

41.. Buss WC. Stepanek J. Bennett WB. A new proposal for the mechanism of cyclosporin A nephrotoxicity. Biochemm Pharmacol 1989; 38:4085-93.

42.. Schroeder TJ, Meivin DB. Clardy CW. AL E. Use of cyclosporine and ketocona/ole without nephrotoxicity inn two heart transplant recipients. J Heart Transplant 1987; 6:84-9.

43.. Serino F. Grevel J. Napoli KL, Kahan BD. Strobel HW, Generation of oxygen free radicals during the metabolismm of cyclosporine A: a cause-effect relationship with metabolism inhibition. Mol Cel! Biochem 1993; 122:101-21. .

44.. Sheets JJ. Mason JI. A potent inhibitor of cytochrome P450-dependent drug metabolism in rat liver. Drug Metabb Dispos 1984: 12:603-6.

45.. Meredith CG. Maldonado AL. Speeg KV. The effect of ketoconazole on hepatic oxidative drug metabolism inn the rat in vivo and in vitro. Drug Metab Dispos 1985; 13:156-62.

46.. Lewis JH. Zimmerman HJ. Benson GD. hhak KG. Hepatic injury associated with ketoconazole therapy. Gastroenterologyy 1984: 86:503.

47.. Krupp P. Monka C. Side-effect profile of cyclosporin A in patients treated for psoriasis. Br J Dermatol 1990: 122(Suppl36):47-56. .

48.. Heyns W, Drochmans A, van der ShuerenE. Verhoeven G. Endocrine effects of high-dose ketoconazole therapyy in advanced prostatic cancer. Acta Endocrinol 1985: 110:276-83.

49.. Williams JG. Maier RV Ketoconazole inhibits alveolar macrophage production of inflammatory mediators involvedd in acute lung injury (adult respiratory distress syndrome). Surgery 1992: 1 12:270-7.

50.. Beetens JR. Loots W. Somer Y, Coene MC, DeClerk F, Ketoconazole inhibits the biosynthesis of leukotrienes inn vitro and in vivo. Biochem Pharmacol 1986: 35:883-91.

51.. Ulrich B. Frey FJ. Speck RF. Frey BM. Pharmacokinetics/pharmacodynamics of ketoconazole-prednisolone interaction.. J Pharmacol ExpTher 1992; 260:487-90.

52.. Zürcher RM. Frey BM. Frey FJ. Impact of ketoconazole on the metabolism of prednisolone. Clin Pharmacol Therr 1989:45:366-72.

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Chapterr 16

Intraocularr Inflammatory Disease (Uveitis) and thee Use of Oral Tolerance: A Status Report

Robertt B. Nussenblatt, Scott Whitcup, Marc D. de Smet, Rachell Caspi, Alexander Kozhich, Howard Weiner,

Barbaraa Wistica, Igal Gery

Annalss of the New York Academy of Science 778: 325-337, 1996 (byy permission © The New York Academy of Science)

ORALL ANTIGEN THERAPY

Summary y

Intraocularr inflammatory disease, or uveitis, is a disorder that affects mostly children and young adults.. It is the cause of about 10% of the severe visual handicap in the United States. Many of thee severe, sight threatening uveitic conditions are thought to be driven by putative autoimmune mechanisms,, often with high dose oral prednisone used as treatment, along with cytotoxic agents,, antimetabolites, and cyclosporine adjunctively. The feeding of the uveitogenic retinal S-antigenn to rats immunized with the same antigen resulted in clinical protection. A pilot study in whichh two patients, one with pars planitis and the other with Behcet's disease, were fed with the retinall S-antigen resulted in these patients' immunosuppressive medication being decreased and/orr stopped. The trial also provided us with information concerning dosage and expected immunee responses. A randomized, masked study looking at the effect of feeding retinal antigens to uveitiss patients is ongoing.

Introductio n n

Intraocularr inflammatory disease or uveitis is a common disorder that ophthalmologists must deall with in their everyday practice. The term uveitis is an old one, harking back to the last century.. Though at one time indicating where the nidus of the inflammatory response was thought to reside,, i.e. the uvea, the term today has taken on a generic characteristic, and indicates only that theree is an inflammatory response inside the eye. Therefore disorders such as sympathetic ophthalmia,, which primarily affects the choroid of the eye, and Behcet's disease, which primarily affectss the retina, are both termed an uveitis. Further, an infectious process, such as toxoplasmosis andd cytomegalovirus, both of which have a propensity for the retina, and candidiasis, which beginss in the choroid, are also considered one of the many uveitides. It is the cause of about 10% off the severe visual handicap in the United States. Thee mechanism of action of intraocular inflammatory disease has been debated over the years. Initially,, most physicians caring for patients with this problem considered that the uveitis was a reflectionn of systemic infections. In one study performed at the Wilmer Eye Institute in the 1940's,, essentially all the patients examined with granulomatous uveitis were diagnosed as havingg either tuberculosis or syphilis (1). Over the years, these diagnoses simply could not explain whatt was observed in thee eye. Additionally, immunologic theories and laboratory techniques permittedd a more in depth evaluation of pathologic processes. The presence of circulating immune complexes,, as well as in the eye were described in patients with uveitis (2,3). Because of these observations,, it was suggested that the mechanism of uveitis was a type 111 hypersensitivity reaction.. Therapeutic strategies were initially based on these concepts. Subsequent studies have suggestedd that uveitis patients with immune complexes have better visual outcomes than those withoutt immune complexes (4). An explanation offered has been that immune complexes are present ass a way to more rapidly clear debris and potentially toxic materials from the eye or circulation, andd therefore helping as well to limit the inflammatory process.

Thee development of an animal model for intraocular inflammatory disease altered immeasurably ourr ability to study the underlying mechanisms of this disorder. Several uveitogenic antigens havee been isolated and purified (5). The first and certainly one of the best characterized is the retinall S-antigen (S-Ag), which is found in the photoreceptor region of the retina. Isolated and purifiedd by Wacker et al (6) and Faure et al (7), immunization of genetically susceptible lower

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mammalss with this 55kD protein will result in a bilateral uveitis several weeks later. S-Ag is alsoo found in the pineal and a pinealitis accompanies the ocular inflammatory response. A second uveitogenicc antigen that has been extensively used as a model for human uveitis is IRBP, the in-terphoreceptorr retinoid-binding protein. This antigen, about thrice as large as the S-Ag (140kb), producess an inflammatory response that is similar to that of the S-antigen. While both the S-anti-genn and IRBP are capable of inducing an uveitis in genetically prone rats, mice appear to be muchh more susceptible to IRBP induced EAU (8).

Experimentall autoimmune uveitis (EAU) can be induced not only in lower mammals but in non-humann primates as well (5). The disorder induced has many characteristics of severe human uveitis.. Studies investigating the underlying mechanism of this model have repeatedly shown the importancee of T cells. The disease cannot be induced in athymic rats, nor will a transfer of antiS-antigenn antibody to naive immunocompetent hosts result in disease. However, the transfer of T cellss to naive hosts will result in disease. T cell lines are efficient in transferring disease (9). These liness bear large numbers of IL-2 receptors on their cell surface and have a cytokine profile that wouldd define them as TH-1 cells. Recent work would suggest that as the disorder progresses, TH-22 cells appear in the eye, apparently in an attempt to down regulate the inflammatory response.

Becausee the animal model for uveitis effectively mimics the human situation, we used this modell to evaluate various immunomodulatory approaches. This practice began with the use of cy-closporinee (CsA) (10). The results demonstrated that CsA very effectively prevented the expressionn of EAU in Lewis rats. The response was dose dependent. Therapy could be initiated even 7 dayss after immunization, a point at which uveitogenic cells can be found in the draining lymph nodess of S-Ag immunized rats. The inhibition of disease was obtained also when lines of uveitogenicc T-cells were transferred to naive hosts. CsA treatment did not, in our hands, induce a longstandingg tolerance, since the disease would appear if therapy was stopped while S-Ag was still presentt at the immunization site. On the basis of these observations, CsA has been used in the treatmentt of uveitis in patients (see below).

EAUU has become a template for the evaluation of various immunomodulatory approaches. To date,, almost all new immunomodulatory agents destined for the treatment of severe intraocular inflammatoryy disease have been tested in this model. FK-506, with a mechanism of action thoughtt to be virtually identical to CsA, was shown to be effective in inhibiting EAU both in rodentss as well as non-human primates (11.12). Other agents, such as rapamycin and mycopheno-latee mofetil, have been used and found to inhibit disease (13,14). Other immunomodulatory approaches,, such as T-cell vaccination (15) and the recombinantly produced material ÏL-2PE40, havee been tried, and found useful in treating the disorder (16).

Withh new approaches to therapy constantly being evaluated in the animal model for human uveitis,, it would be helpful to put into perspective what is presently used to treat patients. The initiall drug of choice is corticosteroid. This drug can be used systemically, in periocular injections orr topically. For most serious sight-threatening uveitides, most patients have posterior pole involvement,, invariably necessitating the use of systemic corticosteroid. Long term use is problematic,, particuarly so because the initial dosages needed to effect a positive therapeutic response aree relatively high, 1 to 1.5 mg/kg, as are the doses needed to maintain this effect, usually 20-40 mgg of prednisone/day. Alternative approaches would include cyclosporine, as well as cytotoxic andd anti-metabolic agents. Cyclosporine has begun to be used widely as a second line agent or as

210 0

O R A LL ANTIGEN THERAPY

aa steroid sparing agent. In cases of Behcet's disease, it may be used as an initial treatment. The usee of cytotoxic agents has become less popular because of their long-term potential side effects. They,, at least anecdotally, appear to be helpful in the treatment of uveitis, and if used for fairly shortt periods of time at low doses, in certain cases may have an acceptable risk of secondary effects.. Anti-metabolites, particularly imuran and methotrexate, have undergone a resurgence of use,, at lower dosages than previously used, and often as a steroid or CsA sparing agent.

Clearlyy there is a practical need for the development of less toxic and hopefully more specific therapiess for uveitis. Oral tolerance, with its effect shown in experimental autoimmune encephalomyelitiss and reports of positive therapeutic effects in 2 clinical studies would be an obviouss choice. We report here in summary the results of some of the animal work using EAU as a modell to evaluate various aspects of oral tolerance and the status of studies in humans.

Material ss and Methods

Studiess with Rats Sixx to 10 week old Lewis rats were gavage fed with S-Ag or a non-specific protein [Keyhole limpett hemocyanin (KLH) or bovine serum albumin (BSA)] at a dose of 1 mg/feeding on specificc intermittant days relative to immunization. In other experiments, rats were fed bovine IRBP peptidee 1179-1191, LPS, GMDP (an analog of muramyl dipeptide) or combinations of the peptidee and either LPS or GMDP. Rats were immunized with either S-Ag (20 p.g) or IRPB peptide 1179-11911 (1 nmol), depending on the experiment. Animals were examined regularly for evidencee of ocular inflammatory disease, then sacrificed at various points after immunization and thee histological changes in their eyes were examined and their immune responses evaluated.

Inn one set of experiments, some Lewis rats were splenectomized prior to feeding while control ratss received sham operations.

Inn another set of experiments, female Lewis rats were treated intraperitoneally with the anti-CD8 antibody,, 0X8 (9 mg total), while control animals received PBS or an isotype-matched irrelevant antibody.. The rats were fed either S-Ag or BSA, as above, at specific points during the injection schedule,, then subsequently immunized with S-Ag (20_g). Evidencee of disease was evaluated for three weeks then the rats were sacrified and their immune responsess measured.

Studiess with Mice: Sixx - 12 week old mice deficient in CD8 lymphocytes [82m (-/-)J and immunologically intact 129/JJ control mice were fed intermittently with either ovalbumin (OVA) or myosin prior to immunizationn with OVA (20_g). Cellular immune responses were measured by the lymphocyte proliferationn assay two weeks after immunization.

Inn vitr o Human Studies 22 x 105 cells isolated from the blood of the patient were placed into microtiter wells. At the initiationn of the cultures, each well was pulsed with 20„g/well of purified bovine retinal S-antigen. Culturess were performed in sextuplicate and harvested on day 5. Sixteen hours before the terminationn of the culture, 'thymidine was added to the wells. The results are expressed as stimulation

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index,, which is obtained by dividing the counts/min obtained in the wells pulsed with retinal S-antigenn by those counts obtained in the controls wells which just received culture medium. A stimulationn index of 2 or more is considered as evidence of an anamnestic response to the retinal S-antigcnn (indicated by the dashed line).

Results s

Feedingg S-Ag Before the Immunization with S-Ag (17) Feedingg S-Antigen 3 times (-7, -5, -3) before immunization with S-Ag resulted in abrogation of thee disease. The observation appeared to be dose dependent, with animals receiving 1 mg at each feedingg being essentially totally protected. Lowerr doses resulted in an attenuated form of the disease, when measured by a masked observerr approximately 2 weeks after immunization. Proliferative responses of T-cells taken from the drainingg lymph nodes to the immunizing antigen were decreased as compared to those animals fedd as a control antigen.

Feedingg antigen after immunization Thee effect of oral tolerance as a means of inhibiting disease after immunization has been evaluatedd as well. Animals fed S-Ag 3. 5, and 7 days after immunization were noted to have either no diseasee or a disease process which was far less severe than control animals.

Rolee of the spleen in oral tolerance Thee effect of splenectomy on oral tolerance was also evaluated (18). It was noted that animals thatt were splenectomized soon after birth developed severe uveitis even if fed with S-Ag at a dosee which was capable of protecting animals who had received sham operations. This would suggestt that an intact gut-spleen-ocular axis is important for the development of oral tolerance.

Effectss of bacterial products on oral tolerance Interestt has been raised as to whether oral tolerance could be enhanced if bacterial products are fedd at the same time as the uveitogenic antigens. Lewis rats immunized with an immuno-domi-nantt fragment of IRBP, peptide 1179-1191, developed disease in all cases. The development of EAUU could be exacerbated by feeding the rats with LP or GMDP prior to immunization. In contrast,, the disease was markedly diminished when these bacterial products were fed along with the 1RBPP peptide used later for immunization (19),

Rolee of CD8 ceils in oral tolerance Ratss which received injections of the anli-CD8 antibody 0X8 and were fed S-Ag prior to immunizationn with S-Ag responded similarly to their controls. As can be seen in Figures 1 A and 1 B. thee use of the antibody did not affect the induction of oral tolerance, as compared to treatment withh PBS. As well. Figure 2 shows that the proliferative responses from the draining lymph nodess of S-antigen immunization gave identical proliferative responses..

Mice: : Cellularr immune responses from CD8 deficient mice [B2m (-/-)] and their 129/J controls were veryy similar; feeding with OVA markedly reduced the cellular response against this antigen (Figuree 3).

212 2

0X8 8 Treatment t


PBS S

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Fig.. 1A S-Ag g BSA A Fedd Antigens

4 4

33 -

22 -

11 -

o o

o o

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FiguresFigures I A and I B. Effect of anti-CD8 monoclonal antibody therapy on the induction of oral tolerancetolerance in experimental autoimmune uveitis (EAU). In panel A, animals received 0X8 (anti-CD8)CD8) therapy and then were fed either S-Ag or BSA. In panel B, the results after PBS therapy are shown.shown. The monoclonal antibody therapy did not abrogate the effect of oral S-Ag therapy.

Animall Studies: Discussion Orall tolerance appears to be a mode of immunosuppression which protects against EAU. The effectt is dose dependent and can be induced when feeding begins before or even after immunization.. While fragments have been reported to be effective in preventing the expressionn of EAU, we havee not seen a particularly remarkable effect with fragments of the retinal S-Ag. The immuno dominantt fragment of the retinal S-antigen in human disease is not clear since patients* cells will respondd to several fragments when tested in vitro. Additionally, usually these fragments are not thosee to which rat lymphocytes respond (21). Experiments not shown here have not been overwhelminglyy convincing in demonstrating a bystander suppression effect such as has been reportedd in other animal models. It may be that doses of the antigen used in our experiments induced anergyy and therefore no bystander suppression was induced. As well, experiments would suggestt that CD8+ cells are not necessary for the induction of oral tolerance. This finding needs to bee discussed in the context of an earlier finding that the addition of anti-CD8 antibody to cultures off splenically derived lymphocytes from S-antigen fed rats reversed the downregulation noted in proliferationn assays (17). We have seen as well the importance of the ocular splenic axis in our system.. This mimics other immunosuppressive mechanisms involving the eye. most notably ACC AID (22). Of interest was the finding that CDS positive cells were not needed for the inductionn of oral tolerance.

Basedd on the observations that the EAU model would be a good '"predictor" of an effect in humann disease, a pilot study involving two patients was initiated.

Humann Studies Twoo patients were included in a pilot study initiated about 2 and one half years ago. The first patientt is a 28 year old man with pars planitis (a common form of intermediate uveitis), who hadd been taking prednisone orally. This medication controlled his ocular inflammatory activity withh maintenance of good visual acuity. However, he would suffer a recurrence of his uveitis

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FigureFigure 2. Panel showing the proliferative re-sponsessponses of cells from draining lymph nodes of S-AgS-Ag immunization site. Some animals were treatedtreated with the anti-CDH antibody syslemi-callycally and others received only PBS. Those fed S-AgS-Ag had significantly lower stimulation in-dicesdices than those fed BSA, whether they were treatedtreated beforehand with antibody or not.

oo l

S-Agg (log ng/ml)

withh any significant decrease in his prednisone dosage. The second patient is a 42 year old white womann with over a 12 year history of Behcet's disease (23). She was begun on cyclosporine in 19833 and has required this medication as well as a low dosage of prednisone in order to maintain remissionn of her ocular disease. In 1986 she received a trial of leukeran but this resulted in multiplee ocular attacks necessitating a return to cyclosporine therapy. These patients were chosen becausee of the documented recurrences of their ocular disease that occurred with dosage reduction, andd because they manifested in vitro cell mediated proliferative responses to the retinal S-antigen.

Bothh patients in this pilot phase of the study initially received 30 mg of bovine derived retinal S-antigen.. given orally, three times a week with a subsequent decrease in S-antigen dosing to once aa week and their subsequent course can be seen on figures 4 and 5. The figures also show the resultss of in vitro proliferative assays to the retinal S-antigen performed over time.

129/JJ Mice B2mm (-/-) Mice

OVAA Cone, (ug/ml)

FigureFigure 3. Lymphocyte proliferation response oJj32m(-/-) mice and their 129/J controls after three feedingsfeedings with either 0.3mg OVA or a control antigen (myosin).

214 4


UVEITIS S

P.O. . PREDNISONE E

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399 THERAPY

20 /200 OD 20 /166 OS

S-ANTIGENN SSNSSSSSBSS 300 MG 3 TIMES

AA WEEK 300 MG ONCE

AA WEEK 300 MG

Q 2 2 WEEKS S

300 MG 0 0

MONTH H

FigureFigure 4. Flow sheet showing clinical course and in vitro proliferative responses to the retinal S-antigenS-antigen in Behcet's disease patient participating in the pilot study evaluating the induction of oraloral tolerance with S-antigen.

20 0

STIMULATION N INDEX X

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prednisone e

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20/25 5 20/32 2

Imuran n 50mg g

t.i.w w qq week qq 2 weeks

FigureFigure 5. Flow sheet showing clinical course and in vitro proliferative responses to the retinal S-antigenantigen in a pars planitis patient participating in the pilot study evaluating the induction of oral tolerancetolerance with S-antigen.

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Modulationn of Human Uveitis with Ocular Antigens

UVEITIS S

CANDIDATE E

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GOODD CONTROL OF OCULAR DISEASE

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GETSS RECURRENCES WITH TAPER

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RANDOMIZEDD STUDY

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BUTT MAINTAIN STANDARD THERAPY

BEGINN TO TAPER STANDARD THERAFY OVER THE NEXT 8 WEEKS

CONTINUEE TO "FEED" ANTIGEN BUT DECREASED TO ONCE A WEEK

ATTEMPTT TO TAKE PATIENT COMPLETELY OFF STANDARD THERAPY

CONTINUEE MAINTAINANCE FEEDING THERAPY ONCE A WEEK FOR AT LEAST 6 MONTHS

FigureFigure 6. Outline of double masked study protocol evaluating the use of orally administered reti-nalnal antigens and the induction of oral tolerance.

Thee patient with Behcet's disease (Figure 4) had a minor inflammatory episode as the decrease off her cyclosporine therapy began, but this problem stabilized, and she remained free of activity forr over two years. She suffered a recurrence at this point, necessitating a short term course of cyclosporinee and prednisone which then was stopped. She was placed on a maintenance of 50 mg off imuran and continued to be fed with S-antigen. Of interest in both cases was the fact that the stimulationn indices for the S-antigen in the main appeared to elevate with decreases in immunosuppressivee or dosing before an attack.

Figuree 5 summarizes the clinical course of the pars planitis patient. As one can see, there was an initiallyy high stimulation index of about 9. After three weeks of S-antigen therapy, the stimulationn index fell to below two. With the discontinuation of prednisone therapy and as well a decreasee in the frequency of S-antigen administration, an increase of the stimulation index back to thee original level was noted, but no change in the clinical status of the. patient was seen. With time,, a decrease in the stimulation index was noted , but again this increased as the S-antigen feedingg was stopped, which was done in an attempt to see if long term immunologic tolerance

216 6

O R A LL ANTIGEN THERAPY

hadd been permanently induced. The patient remained off all medication from week 24 till week 622 when he returned because of an ocular inflammatory attack. He was treated with prednisone andd feeding of the retinal S-antigen (three times a week) was reinstituted. Tested two weeks afterr the initiation of therapy, the stimulation index had once again fallen to well below two. He had aa mild drop in vision in one eye with his local physician giving him a periocular injection, but has nott required systemic immunosuppression; he has continued to take S-antigen and has excellent visionn in both eyes.

Thee results of these two pilot study patients were most helpful. While of course not proving the potentiall universal therapeutic efficacy of this approach, it did give us an indication that such an approachh could be useful since neither clinical course would have been expected, based on the naturall history of either patient's disease. Both have retained good vision for the past 2 and one halff years. The stimulation indices appear to be a sensitive indicator of either a change in immunosuppressionn or of an impending attack. Because of these positive therapeutic responses in thesee two patients, a randomized double masked study has begun where the efficacy not only of thee retinal S-antigen but also a retinal mixture made up of soluble retinal antigens at low concentrations,, is being tested (Figure 6). Patients with intermediate or posterior uveitis of a noninfectiouss cause necessitating systemic immunosuppressive therapy are potential candidates. Their circulatingg lymphocytes are tested for evidence of responsiveness to the retinal S-antigen. If they aree positive, patients are randomized either to placebo, a retinal mixture, S-antigen (bovine source),, or a combination of the retinal mixture and 5-antigen. Once fed one of these combinationss for 3 weeks while maintaining their immunosuppressive therapy necessary to maintain goodd vision, they are tapered off from their therapeutic regimen over 10 weeks while continuing thee feeding of the retinal materials or placebo. The intent is to induce a toleragenic state with the feeding,, therefore obviating the need for further immunosuppressive therapy (or at a lower dose). Thee end point of the study is time to recurrence of disease and the dosage of immunosuppressive agentss used at the time of recurrence.

Inn summary, we have presented here our experience with oral tolerance, first in an animal modell for human disease and then in a pilot study with 2 patients. The randomized masked study we hopee will give more information about the potential utility of this approach.

References s

1.. SchlaegelTFJr: Essentials of Uveitis. Boston. Little Brown & Co. 1969. p. 27. 2.. CharDH, Stein P, Masi R, et al.: Immune complexes in uveitis. Am J Ophthalmol 1979; 87:678-681. 3.. Dernouchamps JP, Vaerman JP. Michiels J. et al.: Immune complexes in the aqueous humor and serum. Am J

Ophthalmoll 1977;84:24-31. 4.. Kasp E. Graham E, Stanford MR, et al.: A point prevalence study of 150 patients with idiopathic retinal vas

culitis;; 2. Clinical relevance of antiretinal autoimmunity and circulating immune complexes. Brit J Ophthalmoll 1989;73:722-730.

5.. Caspi RR: Experimental Autoimmune Uveoretinitis - Rat and Mouse. In: Autoimmune Disease Models: A Guidebook.. (I.Cohen and A. Miller Eds).Chapter 5. pp. 57-81. Academic Press. 1994.

6.. Wacker WB, Donoso LA. Kalsow CM. et al.: Experimental allergic uveitis. Isolation, characterization, and localizationn of a soluble uveitopathogenic antigen from bovine retina. J Immunol 1977; 119:1949-1958.

7.. Faure JP: Autoimmunity and the retina. CurrTop Eye Res 1980.2:215-302. 8.. Caspi, R.R.. Roberge. F.G., Chan, C.C., Wiggert, B., Chader, G.J., Rozenszajn, L.A.. Lando. Z., and Nussen-

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blatt,, R.B.: A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice withh two different retina] antigens. J Immunol 140:1490-1495, 1988.

9.. Caspi. R.R.. Roberge. EG.. McAllister. C.G.. EI-Saied. M.. Kuwabara, T.. Geiy I- Hanna, E. and Nussen-blatt,, R.B.: T-cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J Immunol 1986; 136:928-933. .

10.. Nussenblatt, R.B.. Rodrigues. M.M.. Wacker. W.B., Cevario. S.J., Salinas-Carmona. M.C, and Gery, I.: Cyclosporinn A: Inhibition of experimental autoimmune uveitis in Lewis rats. J Clin Invest 1981: 67:1228-1231.

11.. Kawashima H: Fujino Y; Mochizuki M: Effects of a new immunosuppressive agent. FK506, on experimentall autoimmune uveoretinitis in rats. Invest Ophthalmol Vis Sci 1988: 29:1 265-271.

12.. Fujino, Y„ Chan. C C , de Smet, M.D., Hikita. N.. Gery. I.. Mochizuki, M.. Nussenblatt. R.B. FK506 treatmentt of experimental autoimmune uveoretinitis in primates. Transplantation Proc 1991; 23 :3335-3338.

13.. Roberge FG, Xu D, Char CC,de Smet MD, Nussenblatt RB, and Chen H: Treatment of autimmune uveoretinitiss in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction. Curr Eye Res 1993;; 12:191-196.

14.. Chanaud NP III. Vistica BP. Eugui E, Nussenblatt RB. Alhsion AC. and Gery I; Inhibition of experimental autoimmunee uveoretinitis by mycoplienolate mofetil, an inhibitor of purine metabolism, (submitted)

15.. BeraudE. Kotake 5. Caspi RR. Oddo SM. Char CC. Gery I. and Nussenblatt RB: Control of experimental autoimmunee uveoretinitis by low dose T cell vaccination!. Cell Immunol 1992; 140:112-122.

16.. Roberge FG: Lorberboum-Galski H; Le Hoang P; de Smet M; CharCC: Fitzgerald D: Pastan : Selective immunosuppressionn of activated T cells with the chimeric toxin 1L-2-PE40. Inhibition of experimental autoimmunee uveoretinitis. J Immunol 1989; 143: 3498-3502.

17.. Nussenblatt R.B., Caspi R., Mahdi R., Cha rCC . Roberge F. LiderO.. and Weiner H.L.: Inhibition of S-anti-genn induced experimental autoimmune uveoretinitis by oral induction of tolerance with S-antigen. J Immunol 1990:: 144: 1689-1695.

18.. SuhEDW. Vistica B. CharCC. Gery I. and Nussenblatt RB: Splenectomy abrogates the induction of oral tolerancee in experimental autoimmune uveoretinitis. Curr Eye Res 1993; 12:833-839.

19.. Kozhich AT. Nussenblatt RB. and Gery 1: Enhancement of oral tolerance by bacterial products. In: Nussenblattt RB. Whitcup SM. Caspi RR. and Gery I (eds). Advances in Ocular Immunology. Proceedings of the 6"' Internationall Symposium on the Immunology and Immunopathology of the Eye. Bethesda. USA. Elsevier. Amsterdam.. 1994; pp. 217-220.

20.. Chanaud NP 111, Felix NJ. Silver PB. Rizzo LV. Caspi RR. Nussenblatt RB. and Gery I: Induction of oral tolerancee in CD8 cell deficient mice. In: Nussenblatt RB. Whitcup SM. Caspi RR, and Gery I (eds). Advances inn Ocular Immunology, Proceedings of the 6th Internationa] Symposium on the Immunology and Immunopathologyy of the Eye, Bethesda. USA. Elsevier, Amsterdam. 1994; pp. 213-216.

21.. de Smet MD, Yamamoto JH. Mochizuki M, Gery I. Singh VK. Shinohara T. Wiggert B, Chader GJ. and Nussenblattt RB: Cellular immune responses of patients with uveitis to retinal antigens and their fragments. Amm J Ophthalmol 1990 110:135-142.

22.. Gery 1 and Streilein JW: Autoimmunity in the eye and its regulation. Curr Opinion Ophthalmol 1994; 6:938-945. .

23.. Nussenblatt R. B. ,de Smet M. D. . Weiner H. L. . and Gery 1 :The treatment of the ocular complications of Behcet'ss disease with oral tolerization. The Internaltional Congress on Behcet's disease. Paris , France. June 1993. .

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Conclusions s

"Qui,"Qui, lerêyedotit se nourrii obwurêment la Recherche humaine, c'est aufand deparvenir $ mattmer,mattmer, p&rdeB toutes affinités atomiques ou molécuküres, l*Emrgie defend dont toutes les mttresmttres energies ne sant que les servantes: saisir, réunis tous ensemble, la barre du Monde, en mettantlamettantla main sürle ResH&rt même de Ï/Èv&hdkm?*

Pieftee Tëilh^fd de Chardin: Le Phénomène humain, 1940

Summaryy and Conclusions

Inn the foregoing chapters, a series of observations are presented on both experimental and human uveitis.. The observations in the experimental model are used as a basis to better understand the humann situation. However, human studies have also generated new insights on the pathophysiologicc process, and these will lead to further studies in the animal model. Findings regarding fluctuationss in circulating lymphocytes responsive to ocular autoantigens, their enumeration, or the presencee of determinant spread as seen in humans have not yet been documented in experimentall uveitis models. Indeed, it has rarely been studied in other autoimmune diseases. By contrast, whilee we have considerable knowledge regarding the pivotal uveitogenic determinants of S-Ag andd interphotoreceptor retinoid binding protein (IRBP) in the Lewis rat (section I), we know very littlee of the role of peptide determinants in humans. The first plausible determinants to fulfill this rolee have been identified and presented in section II.

Inn addition to observations relating to the mechanism of immune stimulation, this thesis has alsoo addressed the use of experimental models in testing new therapeutic modalities. For clinicians,, experimental models are particularly useful in testing these novel approaches prior to initiatingg clinical studies. In section III, attempts at modulating experimental uveitis using several differentt approaches are presented. When this thesis was submitted, two of the three tested approachess have made it to human clinical trials or to clinical practice. Whether rapamycin will makee it to the clinic will depend on the company's determination to fund the appropriate trials. Rapamycinn has several characteristics that make it particularly appealing, including its ability to inhibitt both T and B cells proliferation, and to reduce neovascularization. Section IV presents resultss in human trials based on experimental data using antigen feeding, an approach which is felt too hold great promise, but which is still poorly understood. Other articles refer to cyclosporine andd its clinical use. Also introduced to the treatment of uveitis on the basis of experimental results,, cyclosporine has required the development of specific treatment protocols to avoid systemicc toxicity. Since uveitis patients are generally healthy except for their eye disease, avoidance off systemic side effects is of crucial importance. Strategies in this direction based on the use of cyclosporinee on its own, or in combination with an enzyme inhibitor are presented.

Beforee discussing each section in more detail, it is appropriate to reflect on the differences betweenn the experimental model as it is currently used, and human disease. Experimental Autoimmunee Uveitis (EAU) is a T cell mediated process characterized by an acute onset, a self limiting coursee and recurrence only under special circumstances (and in a limited number of species). In contrast,, human disease in most instances is rather indolent, characterized by exacerbations and remissionss over several months to years. Furthermore, S-Ag and IRBP, which are used extensivelyy in this thesis as model ocular autoantigens, generate an inflammation centered in the retinaa (particularly in the rat and mice). Many human uveitic conditions affect the retina and choroid equally,, or appear to cause more damage in the RPE and choriocapillaris. Other antigens such as melanocytee derived antigen may more appropriately portray human disease, and its recurrence pattern.. This model deserves further investigation. At present the antigen causing EMIU is poorlyy characterized, and the immunopathogenic epitopes are not known. Despite its limitations, EAUU has given us a template for the study of autoimmunity in general, and uveitis in particular.

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Thiss thesis has drawn on the elements of this template and hopefully may serve as a model for futuree studies.

Sectionn 1: Immunologic Determinants of Ocular Autoantigens in Experimentall Uveitis

Antigenicc proteins are processed prior to inducing an immune response. In general, the initial immunee response is directed to a limited number of determinants [ see chapter 1]. In IRBP, this immunodominantt determinant is located at 1182-1190 of bovine IRBP. By using amino acid substitutionss along the length of the decapeptide, it was possible to study the role of individual residuess with regards to various immunologic activities that characterize a given determinant, namelyy immunogenicity. immunodominance, and uveitogenicity (pathogenicity). Each of these termss was specifically defined in the context of these studies. Immunogenicity referred to the abilityy to generate an immune response. Uveitogenicity referred to the ability to cause uveitis. Immunodominancee referred to the ability of a determinant to cause disease at the lowest immunizingg dose (causing disease comparable to the whole molecule). Since the peptide determinant servess as a bridge between the antigen presenting cell (APC) and the T cell receptor (TCR), it wass also possible to study the likely role in the TCR-MHC interaction of each residue. Analysis off activities of pivotal residues made it possible to learn about their role in the interaction betweenn the T cell receptor and the MHC molecule on the surface of the antigen presenting cell (APC).. Thus, valine at position 1188 and proline at position 1189 are critical for interaction with thee TCR. while tryptophan at 1 182. and aspartic acid at 1190 are essential for peptide binding to thee MHC. These findings raise another issue: other experiments have shown that certain determinantss were recognized by several different MHCs . and across species. It would be interesting too know if the same binding elements are crucial for TCR and MHC interactions in each of these species. .

Inn the course of certain studies regarding factors affecting the intracellular trafficking of IRBP, wee found that it was strongly bound, through an ATP dependent process, to a cytosolic protein of molecularr weight 70 kD. Immunoprecipitation and partial sequencing identified this protein as beingg part of the chaperone family of proteins, a highly conserved protein family involved in preventingg protein denaturation, and degradation. Its role in the immune process is not clear. There iss considerable evidence in the literature to suggest that chaperones influence antigen presentation.. Their presence may also promote an immune response as has been clearly demonstrated in certainn forms of cancer and suggested n Behcet's disease. Certainly modifications in the peptide sequencee will modify the affinity for the IRBP chaperone (personal communication - K. Ren-garajan).. Expression of hsp in the inflamed retina follows a defined pattern, starting in the ganglionn cell layer and progressing to the RPE which expresses high level of hsps only in late stages off the disease. However, the exact role of this particular chaperone in the immunologic process remainss to be defined.

Nextt human S-Ag (hS-Ag) was studied in the Lewis rat to identify its immunologic and im-munopathologicc determinants. Using overlapping peptides of the whole sequence, several sites weree identified capable of generating an immunopathogenic response. The most active site was identifiedd at position 340-360, similar to findings by other authors who had used the bovine sequence.. Of interest, this most pathogenic sequence was not the most proliferative suggesting a

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separationn between pathogenicity and immunogenicity. This observation, also made by other investigatorss is of critical importance, since the identification of a highly proliferative site in humanss does not necessarily imply a role in pathogenesis.

Finally,, the more pathogenic determinants of S-Ag in the Lewis rat were tested in other rat strains,, some of which shared the same MHC background while others did not. This study attemptedd to identify determinants which had the ability to cross histocompatibility boundaries. In thee case of determinant 340-360, this was certainly the case. However, even in strains that shared thee same MHC background, there was considerable variation in the degree of immunogenicity andd pathogenicity of each tested determinant. While the causes of this variation were not investigatedd in this study, factors such neuro-endocrine modulation, the T cell priming to a Th 1 or Th2 profilee play a critical role. Thus, factors extrinsic to the ability to appropriately process and recognizee an antigen play a role in the phenotypic expression of disease. In outbred species, these externall influences are even more likely to play a critical role. In the light of these results and others,, interpretation of proliferation results in humans must be done with care.

Sectionn II : Immunologic Reponses to Ocular Autoantigens in Humans

Too obtain a profile of the immune response to retinal autoantigens in humans, two patient populationss were tested in the US and in Japan. The profile in both countries was similar. Patients with retinall pathology were more likely to have a positive immune response to S-Ag or IRBP. Furthermore,, several patients responded to both antigens or fragments thereof. This pattern wass seen in 26 of 82 patients tested. The result is even more significant if one considers that aboutt half of the patients did not show any significant proliferative response to any antigen (definedd in this case as a proliferation above the mean of controls plus 2 standard deviations). Sincee most of these patients had a well established disease pattern for several years, it is assumed thatt sensitization to the second retinal autoantigen (and likely more) took place some time after thee initial inflammatory episode began. A proliferative response in control subjects was noted in upp to one third of individuals. Current thoughts regarding autoimmunity support the concept of selff recognition in both B and T cell networks both in normal individuals and in pathological states. .

Inn lymphocyte cultures from patients, fluctuations in triplicate wells were frequently observed. Oftenn one of the three triplicate wells would deviate in its response level. The reason for this deviationn was generally felt to be due to an inhibitory microenvironment preventing growth of responsivee cells. However, the cause could equally have been a low circulating number of responsivee T cells. To test this hypothesis, cultures were set up in a way to minimize both effects. A statisticallyy significant number of wells were placed in culture (490 wells), and T cell cytokine stimulantss were given in each well. This assay was shown by others to act as a modified limiting dilutionn assay (LDA). Thus, it was possible to estimate the number of circulating responsive T cellss in the peripheral blood of patients and controls. Patients have a 100 to 1000 fold higher numberr of responsive circulating cells to S-Ag as compared to controls. Results were in keeping withh those previously published using an 1L-2 release assay .

Havingg shown that this method allowed enumeration of responsive T cells in the peripheral blood off patients and controls, we wanted next to determine the profile of these cells over time. We de-

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cidedd to limit our study to a well defined population of patients, namely those with Behcet's disease.. A group of five patients were followed prospectively for several months using the standard proliferationn assay {as was used in the first study presented in this section), as well as by the modifiedd LDA assay described above. Despite the limited nature of the study, several observations weree possible. (1) The standard assay is poor at predicting the timing of an inflammatory recurrence,, but a stimulation index higher than the mean + 2x standard deviation correlated with clinicall exacerbation some time in the future. (2) Following an ocular attack, the standard assay gave stimulationn indices often below their baseline, making it an unsuitable test to follow patients prospectively.. (3) The LDA appeared to correlate fairly well with ocular activity - demonstrating ann increase in peripheral responsive cells shortly after an episode of ocular activity (4) The in creasee was maintained for a few months before decreasing back to baseline. The curve was similarr to the one observed in experimental models of inflammation or infection. Thus, the standard assayy is able to provide information on the relevance of a given antigen to a disease process (withoutt saying anything on its role in the disease), while the LDA assay might be useful in prognostication.. Future studies may help to delineate the predictive value of LDA assays, but a simplerr methodology than the one used in the current study would be needed.

Finally,, using the same overlapping peptides of human S-Ag that were used in section I. we testedd a number of patients and controls. To insure relevance to the disease process, a substantial numberr of controls were tested. The data were analyzed using 2 approaches. The first aimed at identifyingg determinants for which the proliferative response in patients was statistically significantlyy higher than in controls- in essence these determinants would provide an immunodominant (proliferative)) response in all patients with a given disease. Such determinants were identified in Behcet'ss disease and in Sarcoidosis. They were located adjacent to immunopathogenic determinantss in the rat with which they shared part of the sequence. Next the data was analyzed for determinantss of clinical relevance, as indicated in the previous study by stimulation indices above thee mean of controls plus 2 standard deviations. Several patients had responses to multiple determinantss not limited to the immunodominant determinants identified above. A similar observationn was made by others in patients with multiple sclerosis where it was proposed to be caused byy determinant spreading. With each disease recurrence, the immune response appears to shift to aa new determinant. In experimental autoimmune encephalitis, this determinant spread follows a predictablee course, and modulation of the response to these determinants can limit disease recurrence.. This is the first observationn of such a phenomenon in uveitis. Its relevance to ocular autoimmunityy needs further study.

Sectionn III : Novel Therapeutic Strategies in Uveitis

Overr the years, the EAU model of uveitis has been useful to test new therapeutic modalities. Favorablee observations with FK506 (tacrolimus) in the rat model were extended to the primate model,, and are the subject of the first paper in this section. Side effects were limited to a dose of 0.55 mg/kg/d and were largely limited to liver toxicity. At the lower doses of 0.25 mg/kg/d or 0.1255 mg/kg/d, no toxicity was observed. Protection from uveitis was induced in a majority of animalss though not all were protected, particularly when the drug was given a few days prior to thee onset of disease (in the afferent phase of the immune response). Immunohistochemical study off the ocular infiltrating cells revealed a shift in response to a more inhibitory profile (CD8), with aa reduction in the number of infiltrating B cells.

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AA similar study was carried out in rats using rapamycin, an inhibitor of lymphocyte growth factorr signal transduction. A dose of 0.1 mg/kg/d was found effective at inhibiting EAU induction evenn when treatment was initiated 7 days after immunization.

Wee also looked at the ability of using IL-13 to block further development of uveitis once it had appearedd in one eye. IL-13 is a pleomorphic cytokine produced by Th-2 lymphocytes. As IL-10 andd glucocorticoids, it has a downregulating effect on cell mediated immunity. IL-13 inhibited inflammationn both in the affected eye as well as in the contralateral eye. The effect extended beyondd the treatment period, and lasts at least for the 4 week follow-up period. Modulation of diseasee by promoting a Th2 environment as suggested here appears to be a promising line of investigation. .

Sectionn IV: Therapeutic Strategies for the Treatment of Human Uveitis

Cyclosporinee was introduced about 20 years ago as a potent mediator of T cell activity. It has beenn used extensively in uveitis with beneficial results, particularly in Behcet's disease. However,, a few years after its introduction, it became clear that Cyclosporine was responsible for significantt side effects, particularly in the kidney. Appropriate treatment schemes were required to monitorr patient response to treatment, and to monitor for side effects. A summary of the current knowledgee regarding cyclosporine and its use in Ophthalmology is provided in the first chapter inn this section. Since then, trials have been made to start at lower initial doses , and it has been usedd to treat dry eye syndrome using a topical preparation.

Thee next two articles present a novel treatment combination in an attempt to reduce the required orall dose of cyclosporine. Since cyclosporine is metabolized primarily by hepatic cytochrome P450.. the use of an inhibitor of this enzyme (ketoconazole) leads to a reduced need for the medication,, and more stable systemic drug levels. Additional benefits include an increased efficacy duee to more stable blood levels throughout the day, and a reduction in renal toxicity.

Thee final article presents a novel therapeutic approach. The use of antigen feeding to prevent ocularr inflammation was introduced a few years ago as a highly promising approach to modulate autoimmunee inflammation. The mechanism is felt to involve antigen presentation in intestinal Peyerr patches where a Th2 profile is induced in exposed lymphocytes. These cells are then free too circulate to other target organs such as the eye where their action may lead to a down regulationn of inflammation through secretion of Th2 type cytokines. The pilot results of an S-Ag feedingg study in 2 patients are presented in the last article. Two patients with posterior uveitis were ablee to be tapered off medication. Stopping S-Ag feeding caused an increase in lymphocyte responsivenesss which was followed by disease recurrence. Based on these promising results, a triall of bovine S-Ag versus crude bovine retinal extract was initiated. Results from this study were ratherr surprising. S-Ag fed patients had prolonged remissions compared to controls and lower dosess of anti-inflammatory medications were needed. Patients receiving retinal extract did worse thann controls. While the reason for these results is not known, the literature does support paradoxicall immune responses. Thus, while a promising approach, additional studies are required to identifyy the exact determinants responsible for the ocular inflammatory response. Given our observationss with S-Ag fragments, it would seem that additional information would also be neededd regarding the influence of antigen spreading on the feeding response.

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Manyy unanswered questions remain. While our understanding of human disease still lags behind öürr understanding of the experimental model, approaches similar to those proposed here, supplementedd by obtaining intraocular material (through ocular punctures or biopsies) should help u§§ to fill in part of thisIgapiii years^coaie.

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Samenvattingg en conclusies

Inn de voorafgaande hoofdstukken is een reeks observaties aan de orde gesteld over zowel dierexperimentelee modellen als uveitis bij de mens. De waarnemingen in het experimentele model wordenn gebruikt als basis om de gang van zaken bij de mens beter te begrijpen. De onderzoekingenn bij de mens echter hebben ook nieuwe inzichten betreffende de pathofysiologie opgeleverdd en deze op hun beurt zullen leiden tot voortgezette studies in het diermodel. Bevindingen betreffendee schommelingen van het aantal circulerende lymfocyten gericht tegen oculaire antigenen,, of de spreiding van antigene derterminanten, zoals die gezien wordt bij de mens, zijn nog niett beschreven voor de experimentele uveitis modellen. Dit is echter ook nog nauwelijks bestudeerdd bij andere auto-immuun ziekten. Terwijl wij aanzienlijk veel kennis hebben betreffende de crucialee uveitis veroorzakende epitopen van het S-antigeen en het "interphotoreceptor retinoid bindingg protein" (IRBP) bij de Lewis rat (sectie I), weten we slechts weinig van de rol van antigenee determinanten bij de mens. Mogelijke epitopen zijn geïdentificeerd en worden in sectie II aann de orde gesteld.

Naastt de waarnemingen welke betrekking hebben op het mechanisme van immuun stimulatie steltt dit proefschrift ook aan de orde het gebruik van experimentele modellen om nieuwe therapeutischee mogelijkheden te toetsen. Voor clinici zijn experimentele modellen buitengewoon nuttigg om deze nieuwe benaderingswijze te toetsen alvorens klinische studies in gang te zetten. In sectiee III worden pogingen gerapporteerd om experimentele uveitis te moduleren met gebruikmakingg van verschillende benaderingswijzen. Tijdens het tot stand komen van dit proefschrift zijnn twee van de drie beproefde benaderingswijzen uitgemond in clinical trails bij de mens of opgenomenn in de klinische praktijk (Mochizuki en de Smet 1994). Of rapamycine door zal dringen tott de klinische praktijk zal afhangen van het besluit van de firma om de benodigde onderzoeken tee ondersteunen. Rapamycine heeft diverse eigenschappen die het buitengewoon aantrekkelijk makenn met inbegrip van het vermogen om zowel T als B cel proliferatie te remmen en vaat-nieuwvormingg te verminderen. Sectie IV biedt resultaten van onderzoek bij mensen gebaseerd opp experimentele gegevens waarbij oraal antigeen wordt toegediend, een benadering waar men veell van verwacht maar waarvan het werkingsmechanisme slecht wordt begrepen. Andere artikelenn hebben betrekking op cyclosporine en het klinisch gebruik ervan. Eveneens geïntroduceerd voorr de behandeling van uveitis op grond van experimentele resultaten, heeft cyclosporine de ontwikkelingg van specifieke behandelingsprotocollen noodzakelijk gemaakt teneinde systemi-schee toxiciteit te vermijden. Daar uveitis patiënten in het algemeen gezond zijn behalve hun oogafwijkingg is vermijding van systemische neveneffecten van cruciaal belang. Benaderingswijzen diee dit beogen, gebaseerd op het gebruik van cyclosporine alleen óf in combinatie met een enzymremmer,, worden aan de orde gesteld.

Alvorenss iedere sectie meer gedetailleerd te bespreken is het gepast ons te bezinnen op de verschillenn tussen het experimentele model zoals dat tegenwoordig wordt gebruikt en de ziekte bij dee mens. Experimentele auto-immuun uveitis (EAU) is een door de T-cel gereguleerd proces, gekarakteriseerdd door een acuut begin, een beperkte duur en waarbij recidieven slechts onder bepaaldee omstandigheden voorkomen (en bij beperkt aantal soorten). Hiermee contrasteert het feit datt bij de mens de ziekte meestal indolent verloopt met exacerbaties en remissies gedurende

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maandenn tot jaren. Voorts veroorzaken S-antigeen en IRBP, welke uitgebreid in dit onderzoek gebruiktt worden als model oculaire auto-antigenen, voornamelijk een ontsteking in de retina (voorall bij de rat en de muis). Vele uveitiden bij de mens treffen zowel retina en choroïdea in gelijke mate,, of lijken meer het retinale pigmentepitheel en de choriocapillaris te beschadigen. Andere antigenenn zoals het "melanocyte derived antigen" lijken meer de menselijke ziekte na te bootsen, alss ook zijn recidief patroon (Chan et al. 1994). Dit model behoeft verder onderzoek. Van het an-tigeenn dat "melanine geinduceerde uveitis'" (EMIU) veroorzaakt is nog weinig bekend, ondermeerr zijn de immunopathogene epitopen nog onbekend. Ondanks zijn beperkingen heeft de experimentelee auto-immuun uveitis ons een uitganspunt gegeven voor de bestudering van auto-immuniteitt in het algemeen en uveitis in het bijzonder. Dit proefschrift biedt een schets van dee elementen van dit uitgangspunt en kan hopelijk dienen als een model voor verder onderzoek.

Sectiee I: Immunologische Epitopen van Oculaire Auto-antigenen bij Experimentelee Uveitis

Antigenee eiwitten worden door afweer cellen verwerkt (in stukken geknipt) alvorens een immuunn respons te induceren. In het algemeen is het begin van de immuun respons gericht op een beperktt aantal epitopen [zie hoofdstuk 1|. Bij IRBP, is het dominante epitoop gelokaliseerd op de aminozuurr posities 1182-1190 van het bovine IRBP (Kotake et al. 1991). Door substitutie van aminozurenn over de lengte van het decapeptide, werd het mogelijk de rol van diverse individuele positiess met betrekking tot verschillende immunologische activiteiten welke een gegeven epitoopp karakteriseren n.1. immunogeniciteit, immunodominantie and uveitogeniciteit (pathogenici-teit)) te bestuderen. De definitie van deze begrippen is in de betreffende hoofdstukken uitvoerig besprokenn en kan in het kort als volgt worden samengevat: Immunogeniciteit betreft de mogelijkheidd van een antigeen om in een bepaalde persoon of proefdier een afweer reactie op te wekken.. Uveitogeniciteit wil zeggen de mogelijkheid om een uveitis te veroorzaken. Het epitoop (peptide)) dat in vergelijking met andere epitopen bij lagere doseringen reeds ziekte kan veroorzakenn (vergelijkbaar met het intacte eiwit) wordt het immunodominante epitoop genoemd. Daar hett peptide ais brug dient tussen de antigeen presenterende cel (APC) en deT cel receptor (TCR) wass het ook mogelijk de waarschijnlijke rol te bestuderen van ieder aminozuur bij de TCR-APC interactie.. Analyse van de activiteiten van kardinale aminozuren maakt het mogelijk een rol te lerenn kennen bij de interactie tussen de T cel receptor en het MHC (Major Histocompatibility Complex)) molecuul op het oppervlak van de antigeen presenterende cel (APC). Zo was valine op plaatss 1188 en proline op plaats 1189 essentieel voor de interactie met de TCR, terwijl tryptop-haann op plaats 1182 and asparajinezuur op 1 190 essentieel voor de peptide binding aan het MHC.. Deze bevindingen werpen een nieuw uitgangspunt op: andere experimenten hebben aangetoondd dat dezelfde epitopen herkend kunnen worden door verschillende MHC moleculen, en zelfss door verschillende diersoorten worden herkend (Fukushima et al. 1996). Het zou interessantt zijn om te weten of dezelfde bindende elementen van wezenlijk belang zijn voor T cell receptorr en MHC interactie bij elk van deze diersoorten.

Tijdenss ons onderzoek naar factoren die het intracellulair transport van IRBP beïnvloeden hebbenn wij gevonden een ATP afhankelijk binding tussen IRBP een cytosol eiwit met een molecuul gewichtt van 70 kD. Immunoprecipitatie en gedeeltelijke sequentie analyse toonden aan dat dit eiwitt behoorde tot de chaparone familie van eiwitten (heat shock eiwitten: HSP). Deze eiwitten zijnn sterk betrokken bij het voorkomen van denaturatie van eiwitten en hun opsplitsing. Hun rol

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inn het immuun proces is niet duidelijk. Er is aanzienlijk bewijs in de literatuur dat suggereert dat chaparoness een antigeen presentatie beïnvloeden (Winfield and Jarjour 1991; Pierce 1994). Hun aanwezigheidd kan ook de immuun respons bevorderen zoals duidelijk is aangetoond bij bepaaldee vormen van kanker zoals ook bij de ziekte van Behcet wordt gesuggereerd (Standord et al. 1994;; Janetzki and Srivastava 1995). Wijzigingen in de peptide volgorde zullen zeker de affiniteitt voor de IRBP chaparone wijzigen (persoonlijke mededeling - K. Rengarajan). Expressie van HSPP in het ontstoken netvlies volgt een vast patroon, beginnend in de ganglion cel laag en voortschrijdendd naar het retinale pigmentepitheel. Sterke expressie van HSP's in het retina pigment epitheell werd in de latere stadia van de ziekte gevonden (de Smet et al. 1996). De exacte rol van ditt specifieke chaparone bij het immunologisch proces dient echter nog te worden vastgesteld.

Vervolgenss werd het humane S-antigeen (hS-AG) bij de Lewis rat onderzocht om zijn immuno-logischee en immunopathologische epitopen vast te stellen. Gebruik makend van overlappende peptidess van het gehele eiwitmolecuul werden diverse plaatsen geïdentificeerd die een ontstekingg van het netvlies konden veroorzaken. De meest actieve plaats werd vastgesteld op positie 340-3600 in overeenstemming met de bevindingen van andere auteurs die gebruik hadden gemaaktt van het runder eiwit (Gregerson et al. 1989; Merryman et al. 1991). Van belang is dat het peptidee met de meest pathogene aminozuur volgorde niet het meest proliferatieve effect had hetgeenn een scheiding suggereert tussen pathogeniciteit en immunogeniciteit. Deze bevinding werd ookk gedaan door andere onderzoekers (Gregerson et al. 1989) en is van groot belang, daar de identificatiee van een sterke lymfocyten proliferatie inducerende plek bij mensen niet noodzakelijkerr wijze een rol bij de pathogenese impliceert.

Tenn slotte werden de meer pathogene epitopen van hel S-antigeen bij de Lewis rat onderzocht bij anderee ratten stammen, sommige met dezelfde MHC samenstelling en andere zonder. Deze studiee beoogde epitopen te identificeren welke de histocompatibiliteit grenzen konden overschrijden.. Dit was zeker het geval bij het epitoop 340-360. Zelfs bij stammen met eenzelfde MHC karakteristiekk was er een aanzienlijke variatie in de mate van immunogeniciteit en pathogeniciteit vann elk onderzocht epitoop. Terwijl de oorzaak van deze variatie niet nader werd bestudeerd in dezee studie, spelen neuro-endocrine modulatie, de T cel ontwikkeling volgens Th 1 of Th2 profiell een centrale rol. Zodoende spelen factoren welke buiten de mogelijkheid liggen om antigeen tee herkennen een rol bij de genotypische expressie van de ziekte. Deze externe invloeden spelen waarschijnlijkk bij gemengde stammen een nog kritischer rol. In het licht van deze andere resultatenn moet de interpretatie van in vitro lymfocyten proliferatie door retinale autoantigenen bij de menss kritisch worden bekeken.

Sectiee II : Immunologische Respons van Oculaire Auto-antigenen bij mensen n

Omm een beeld te krijgen van de immuun respons op retinale auto-antigeen bij mensen werden tweee patiënten populaties in de Verenigde Staten en Japan onderzocht. Het profiel van beide groepenn was gelijk. Patiënten met retina pathologie hadden vertoond een positieve immuun responss op S-antigeen of IRBP. Verder hadden verschillende patiënten een respons op beide antigenenn of fragmenten daarvan. Dit patroon werd gezien bij 26 van de 82 onderzochte patiënten. Hett resultaat is zelfs meer significant indien men in aanmerking neemt dat ongeveer de helft van dee patiënten geen in vitro lymfocyten proliferatie toonde op enig antigeen (dit is gedefinieerd als

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eenn proliferatie die boven het gemiddelde van de controle is plus 2 maal de standaarddeviatie). Daarr de meeste patiënten reeds meerdere jaren aan hun ziekte leden, wordt aangenomen dat sen-sibilisatiee voor het tweede retinale autoantigeen (en misschien meer) plaats vond enige tijd na het beginn van de eerste ontstekingsperiode. Proliferatie bij controles werd waargenomen bij een derdee van de onderzochte personen (de Smet et al. 1990). Huidige inzichten over auto-immuniteit ondersteunenn het concept van zelfherkenning bij zowel B en T cel netwerken bij normale individuenn en bij pathologische situaties.

Inn lymfocyten kweken van patiënten werden soms sterk fluctuerende resultaten gevonden bij de inn drievoud uitgevoerde experimenten. Vaak week een van de drie kweek bakjes (wells) afin zijn responss niveau. Als reden hiervoor werd aangenomen dat het microklimaat groei van de cellen remde.. Een andere oorzaak echter zou het aantal circulerende autoreactive T cellen kunnen zijn. Omm deze hypothese te kunnen onderzoeken werden kweken ingezet waarbij deze effecten tot een minimumm werden teruggebracht. Bij deze proefopzet werden 490 wells ingezet in aanwezigheid vann T cel stimulerende cytokines. Door anderen was aangetoond dat deze bepaling fungeerde als eenn gewijzigde limiting dilution assay (LDA). Op deze manier was het mogelijk het aantal circulerendee autoreactive T cellen in het perifere bloed van patiënten en controle individuen nauwkeurigg te bepalen. Patiënten hebben een 100 tot 1000 maal groter aantal circulerende autoreactivee cellen voor S-antigeen in vergelijking met controle individuen. De resultaten waren in overeenstemmingg met gegevens die eerder door anderen waren gerapporteerd, die gebruik maaktenn van een interleukine-2 release assay (Opremcak et al. 1991).

Naa aangetoond te hebben dat deze methode ons in staat stelde het aantal antigeen specifieke autoreactivee T cellen in bloed van patiënten en controle individuen te bepalen wilden wij vervolgenss het profiel van deze cellen in de tijd meten. Besloten werd ons onderzoek te beperken tot eenn duidelijk omschreven patiënten populatie n.1. die met de ziekte van Behcet. Gedurende een reekss van maanden werd een groep van 5 patiënten prospectief gevolgd gebruik makend van de standaardd lymfocyten proliferative methode (zoals in de eerste studie in deze sectie) als ook door dee gemodificeerde LDA assay hierboven beschreven. Ondanks de beperkte aard van het onderzoekk waren diverse conclusies mogelijk. (1) De standaard test voorspelt slecht het moment van eenn ontstekingsrecidief. Een positieve ging echter vaak vooraf een klinische exacerbatie. (2) Na eenn opvlamming van het ziektebeeld, gaf de standaard bepaling stimulatie uitkomsten die dikwijlss lager waren dan hun uitgangswaarden, waardoor dit een onbruikbare test is om patiënten prospectieff te volgen. (3) De LDA leek vrij goed te correleren met ziekte activiteit daar een toenamee van autoreactive cellen in het perifere bloed kon worden aangetoond kort na een episode vann ziekte activiteit. (4) Een toename bleef bestaan gedurende een aantal maanden alvorens af te nemenn naar de uitgangswaarden. Een zelfde kinetiek werd eerder waargenomen bij experimentelee ontsteking- of infectiemodellen. De standaardbepaling is zodoende in staat om informatie te verschaffenn of een bepaald antigeen betrokken is bij een ziekteproces (zonder iets te zeggen over zijnn rol daarin), terwijl de LDA nuttig kan zijn voor de prognose. Studies in de toekomst kunnen behulpzaamm zijn om de voorspellende waarde van LDA's duidelijk te maken, maar een eenvoudigerr methodiek dan degene welke in de huidige studie is gebruikt, zou nodig zijn.

Tenslotte,, gebruik makend van dezelfde overlappende eiwitten van het menselijk S-antigeen zoalss in sectie I werden beschreven, onderzochten wij een aantal patiënten en controle individuen. Omm zeker te zijn dat dit betrekking had op het ziekte proces werd een aanzienlijk aantal controlee individuen in het onderzoek meegenomen. De gegevens werden volgens twee benaderings-

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wijzenn geanalyseerd. De eerste had als doel om epitopen vast te stellen waarvoor de proliferatie-vee respons bij patiënten statistisch significant hoger was dan bij controle individuen - in wezen zoudenn deze epitopen een immunodominante (proliferatieve) respons bij alle patiënten met een bepaaldee ziekte leveren. Zulke epitopen werden geïdentificeerd bij de ziekte van Behcet en bij sarcoïdose.. Zij werden gelokaliseerd in de onmiddelijke nabijheid van de immunopathogene epitopenn bij de rat waarmee zij een deel van de aminozuur volgorde deelden. Vervolgens werden de gegevenss geanalyseerd voor epitopen met klinische betekenis zoals aangeduid in de vorige studiee door middel van stimulatie uitkomsten boven de gemiddelde van de controles plus 2x de standaarddeviatie.. Verschillende patiënten hadden responsen op meervoudige epitopen, die niet beperktt bleven tot de immunodominante epitopen zoals boven vastgesteld. Dezelfde waarneming werdd gedaan bij patiënten met multiple sclerose waarbij men dacht dat het veroorzaakt werd door verspreidingg van de epitopen (Tuohy et al. 1999). Bij ieder ziekte recidief lijkt de immuun responss te verschuiven naar een nieuwe epitoop op hetzelfde eiwit. Bij experimentele auto-immuunn encefalitis volgt deze epitoop verspreiding een voorspelbaar beloop en modulatie van de responss op deze epitopen kan het ziekte recidief beperken. Dit is de eerste waarneming van dit fenomeenn bij uveitis. De toepassing hiervan bij oculaire auto-immuniteit behoeft verder onderzoek. .

Sectiee III : Nieuwe Therapeutische Benaderingswijze bij Uveitis

Gedurendee een reeks vanjaren is het EAU model van nut geweest om nieuwe behandelingswijzenn te introduceren. Vanuit het ratten model werden gunstige waarnemingen met FK506 (tacrolimus)) uitgebreid naar het primaten model en deze zijn onderwerp van het eerste artikel in deze sectie.. Nadelige effecten waren beperkt tot een dosis van 0.5 mg/kg/d en beperkten zich grotendeelss tot leverbeschadiging. Bij een lagere dosering van 0.25 mg/kg/d of 0.125 mg/kg/d werd geenn toxiciteit waargenomen. Bescherming tegen uveitis werd teweeg gebracht bij de meeste dieren,, met name wanneer het middel werd gegeven enkele dagen voor het begin van de ziekte (inn de afferente fase van de immuun respons). Immuunhistochemisch onderzoek van het oog binnendringendee cellen liet een verschuiving naar een meer remmend profiel (CD8) zien met een afnamee van het aantal infiltrerende B cellen. Een zelfde onderzoek werd met rapamycine uitgevoerd,, een remmer van signaal transductie in lymfocyten. Bij een dosis van 0.1 mg/kg/d kon EAUU voorkomen worden, zelfs wanneer behandeling werd begonnen 7 dagen na immunisatie. Ookk werd nagegaan of IL-13 de verdere ontwikkeling van uveitis kon blokkeren wanneer deze zichh in één oog had gemanifesteerd. IL-13 is een pleomorph cytokine dat doorTh-2 lymfocyten geproduceerdd wordt. Evenals IL-10 en glucocorticoïden heeft het een remmend effect op de cellulairee immuniteit. IL-13 remde ontsteking zowel in het aangedane oog als in het contralaterale oog.. Het effect bleef bestaan buiten de behandelingsperiode, en wel minstens gedurende een fol-low-upp periode van 4 weken. Wijziging van het ziektebeloop doorTh2 lymfocyten te stimuleren lijkt,, een veelbelovende onderzoekslijn.

Sectiee IV: Therapeutische Benaderingswijzen voor de Behandeling van Uveitiss bij de Mens

Twintigg jaar geleden werd cyclosporine als een krachtige T cel remmer ingevoerd. Het is uitgebreidd gebruikt met gunstig resultaat bij uveitis, in het bijzonder bij de ziekte van Behcet. Enkele jarenn na zijn introductie echter werd het duidelijk dat cyclosporine verantwoordelijk was voor ernstigee bijwerkingen, vooral in de nier (Palestine et al. 1986). Gepaste behandelingsschemata warenn vereist om het effect van behandeling bij de patiënt te controleren, als om de bijwerkingen tee vervolgen. Een samenvatting van de huidige kennis over cyclosporine en zijn gebruik in de oogheelkundee wordt geleverd in het eerste hoofdstuk van deze sectie. Sindsdien is onderzoek verrichtt naar behandeling met lagere begindoses (Vitale et al. 1996). In tussen wordt cyclosporinee ook gebruikt om het sicca syndroom via lokale toediening te behandelen.

Dee volgende twee artikelen vermelden een nieuwe behandelingscombinatie in een poging om de benodigdee orale dosis cyclosporine te verminderen. Daar cyclosporine vooral door het leverenzymm cytochroom P450 wordt gemetaboliseerd leidt het gebruik van een remmer van dit enzym (ketaconazol)) tot een verminderde behoefte aan dit middel en een meer stabiele geneesmiddelti-ter.. Hen bijkomend voordeel is toegenomen effectiviteit als gevolg van stabielere bloed gehalten gedurendee de dag en afname van niertoxiciteit. Het laatste artikel biedt een nieuwe therapeutischee benadering. Het gebruik van orale antigeen toediening om oogontstekingen te voorkomen werdd enkele jaren geleden als een veelbelovende benadering om auto-immuun ontstekingen te modulerenn geïntroduceerd. Men denkt dat het mechanisme te maken heeft met het aanbieden van antigeenn aan de plaques van Peyer in de darm. waar een Th2 profiel wordt geïnduceerd in blootgesteldee lymfocyten. Deze cellen kunnen dan vrij circuleren naar andere organen, zoals het oog, waarr hun werking kan leiden tot vermindering van ontsteking door afscheiding van Th2 type cytokines.. De resultaten van een piloot studie waarbij twee patiënten oraal S-antigeen kregen toegediendd worden in het laatste artikel gepresenteerd. Bij deze patiënten met uveitis posterior kon dee medicatie geleidelijk worden stopgezet. Beëindiging van het S-antigeen toediening veroorzaaktee een toename van de in vitro tymfocyte proliferatie op retina antigenen en ging gepaard mett een recidief. Op grond van deze veelbelovende resultaten werd een onderzoek gestart met runderr S-antigeen versus ruw runder retina extract. De resultaten van dit onderzoek waren tamelijkk verrassend. Patiënten aan wie het S-antigeen oraal was toegediend hadden langdurige remissiee in vergelijking met patiënten in de controle groep en hadden lagere doses anti-inflammatoire geneesmiddelenn nodig. Patiënten die het ruw retina extract kregen toegediend reageerden slechterr dan personen uit de controle groep. Hoewel de reden voor deze resultaten onbekend is ondersteuntt de literatuur het idee van een paradoxale immuun response (Blanas et al. 1996). Hoewel ditt een veelbelovende benadering lijkt, zijn aanvullende studies nodig om de precieze epitopen tee identificeren welke verantwoordelijk zijn voor de oogontsteking. Op grond van onze waarnemingenn met S-antigeen fragmenten is aanvullende informatie nodig ter bestudering van de verspreidingg van immuun reactieve determinanten in relatie tot het effect van orale antigeen toediening. .

Velee vragen blijven onbeantwoord. Terwijl ons begrip over ziekte bij de mens achterloopt bij onzee kennis ontrent het experimentele model zou de benadering zoals die in dit proefschrift is voorgesteld,, aangevuld met gegevens verkregen van intra-oculair weefsel (via puncties of biopsieën) onss van dienst kunnen zijn om dit hiaat in kennis in de komende jaren op te vullen.

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observations in clinical and experimental ocular autoimmunity

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