IMMUNOLOGY OF TRACHOMATOUS

CONJUNCTIVITIS

ABU EL-ASRAR A.M.L.,* GEBOES K.,**MISSOTTEN L.***

ABSTRACT

Trachoma, a chronic follicular conjunctivitis causedby infection with Chlamydia trachomatis, is a lead-ing cause of preventable blindness. The blindingcomplications are associated with progressive con-junctival scarring. Our immunohistochemical stud-ies of conjunctival biopsies from children with ac-tive trachoma demonstrated the presence of both hu-moral and cell-mediated immune responses. Anti-chlamydial antibodies can neutralize Chlamydiae,block attachment and internalization of the organ-ism, and can produce partial immunity. Our obser-vations suggest a role for T-lymphocytes and cell me-diated immunity in the genesis of conjunctival scar-ring. Conjunctival epithelial cells expressed majorhistocompatibility complex (MHC) class II antigenswhich might allow conjunctival epithelial cells topresent Chlamydial antigens to T-cells enhancing theimmune response. The epithelial cells expressingMHC class II antigens might present autoantigensto T-cells leading to induction of an autoimmune re-action. We have demonstrated that the conjunctivalepithelial cells from patients with trachoma ex-pressed interleukin (IL)-1α and IL-1β. In addition,we have detected cytoplasmic expression of IL-1α,IL-1β, tumor necrosis factor α and platelet-derivedgrowth factor by macrophages. These cytokines havethe potential to influence the remodeling and fibro-sis observed in trachoma. Alterations of extracellu-lar matrix components and collagen metabolism oc-cur in the conjunctival tissue from patients with tra-choma. New collagen type V formation was notedin active trachoma and scarred trachoma. The con-junctival tissue from patients with active trachomacontained increased amounts of collagen types I, III

and IV. Scarred trachoma is characterized by markedincrease in basement membrane - collagen IV andmarked decrease in collagen types I and III. In addi-tion, we demonstrated increased activity of gelati-nase B and numbers of inflammatory cells contain-ing gelatinase B in trachoma patients suggesting thatthis enzyme might be involved in matrix degrada-tion and promotion of conjunctival scarring in tra-choma.

SAMENVATTING:

Trachoma is een van de frequentste oorzaken vanvoorkoombare blindheid. Het is een chronische fol-liculaire conjunctivitis die tot blindheid kan leidendoor progressieve schrompeling van de conjunctiva.Onze immunohistochemische studies van conjunc-tiva biopsies bij kinderen met actief trachoma heb-ben zowel humorale als cel gebonden immuun reac-ties aangetoond. Antichlamydia antilichamen kun-nen Chlamydia neutraliseren, ze kunnen de adhesieen absorptie beletten en zo een gedeeltelijke immu-niteit geven. Onze observaties suggereren dat T-lym-phocyten en cel gebonden immuniteit een rol spe-len in de schrompeling van de conjunctiva. Deepitheelcellen van de conjunctiva tonen HLA type IIantigenen zodat deze epitheelcellen Chlamydia an-tigenen kunnen aanbieden aan de T-cellen en zo deimmuun reactie aanwakkeren. De epitheel cellenkunnen ook met hun HLA type II complexen auto-antigenen aanbieden aan de T-cellen en zo een au-toimmuun proces induceren. Wij toonden aan datconjunctivale epitheel cellen van trachoma lijders in-terleukin (IL)-1α en IL-1β aanmaken. Wij toondenook dat macrophagen in hun cytoplasma IL-1α enIL-1β, tumor necrosis factor α en bloedplaatjes groeifactor bezitten. Deze cytokines kunnen een rol spe-len in de veranderingen en de fibrose van de con-junctiva in trachoma. Bij deze patiënten zien we aan-tasting van de extracellulaire matrix en van het col-lageen metabolisme. Collageen type I, III, IV ziet menbij patiënten met actief trachoma. Collageen V vindtmen in actief zowel als in cicatricieel trachoma. Indit laatste stadium ziet men een sterk verdikte basaal

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* Department of Ophthalmology, College of Medicine,King Saud University, Riyadh, Saudi Arabia,

** Laboratory of Histochemistry and Cytochemistryand

*** Department of Ophthalmology, University Hospital,St. Rafael, University of Leuven, Leuven, Belgium

73Bull. Soc. belge Ophtalmol., 280, 73-96, 2001.

membraan, collageen IV en een sterke verminde-ring van collageen I en III. Ook toonden wij een ver-hoogde activiteit van collagenase B en talrijke ont-stekingscellen die collagenase B bevatten, hetgeensuggereert dat dit enzyme kan bijdragen tot de ont-aarding van de matrix en de bevordering van de fi-brose in trachoma.

RÉSUMÉ

Le trachome, une conjonctivite folliculaire due à uneinfection par Chlamydia trachomatis, est une causemajeure de cécité évitable. Les complications quimènent à la cécité sont associées à la cicatrisationprogressive de la conjonctive. Nos recherches im-munohistochimiques faites sur des biopsies conjonc-tivales chez des enfants souffrant de trachome austade actif, montrent la présence de réponses im-munologiques humorales et cellulaires. Les anti-corps antichlamydiens sont capables de neutraliserles Chlamydias, de bloquer leur adhérence et leurabsorption et de donner ainsi une immunité partiel-le. Nos observations suggèrent un rôle pour les lym-phocytes-T et l’immunité cellulaire dans la genèsede la fibrose conjonctivale. Les cellules épithélialesde la conjonctive expriment les antigènes du com-plexe majeur d’histocompatibilité de classe II ce quipermet à ces cellules de présenter aux cellules T lesantigènes chlamydiens ainsi que des auto-antigè-nes ce qui peut induire une réaction auto-immuni-taire. Nous avons pu détecter, chez les trachoma-teux, l’expression de l’interleukine (IL)-1α et IL-β parles cellules épithéliales de la conjonctive ainsi quel’expression cytoplasmique par les macrophages decette même interleukine, du facteur de nécrose tu-morale α et du facteur de croissance associé aux pla-quettes. Ces cytokines peuvent influencer la fibroseet le remodelage observé en cas de trachome. Le tis-su conjonctival des trachomateux montre des alté-rations des composants extra-cellulaires et du mé-tabolisme du collagène. La formation du collagènetype V s’observe dans le trachome actif et cicatri-ciel. La conjonctive de patients avec un trachomeactif montre des quantités accrues de collagène I, IIet IV, tandis que le tissus sous-conjontival dans letrachome cicatriciel montre une diminution mar-quée du collagène I et III et un épaississement netde la membrane basale -collagène IV. En outre nousavons montré une activité accrue de la collagénaseB et du nombre de cellules inflammatoires conte-nant cet enzyme. Ceci suggère que la collagénase Bpourrait être une cause de la dégradation du tissusous-conjonctival et de la fibrose de la conjonctiveatteinte de trachome.

KEY-WORDS:

Conjunctiva, trachoma, Chlamydia,cytokines, collagen, matrixmetalloproteinases, major histocompatibilitycomplex antigens.

MOTS-CLÉS:

Conjonctive, trachome, Chlamydia, cytokine,collagène, métalloprotéinase, CMH.

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INTRODUCTION

Trachoma is a chronic follicular keratoconjunc-tivitis caused by Chlamydia trachomatis sero-types A, B, Ba and C (96). It remains a majorworldwide cause of preventable blindness anda major public health problem, particularly inthe Third World. It accounts for some 25% ofworld blindness, and is the commonest infec-tious cause of blindness (103). An estimated500 million people have had the disease and7 to 9 million of these have lost vision, mainlyas a result of corneal opacification (22). Its se-vere effects are especially prominent in the de-veloping countries of Africa, the Middle East,Southeast Asia and the subcontinent of India,and there are pockets of disease in Latin Amer-ica, the Pacific Islands and the Australian out-back (100). It presents serious obstacles to so-cioeconomic development and human produc-tivity and consumes significant human and ma-terial resources allocated to the care of thosewho are blinded by it.Chlamydiae are obligate intracellular patho-gens that resemble gram-negative bacteria insome respects but differ primarily by the lackof energy-yielding metabolic pathways. Theylack the ability to synthesize high-energy com-pounds such as adenosine triphosphate (ATP)and guanosine triphosphate (GTP). These com-pounds, essential for metabolism and respira-tion must be provided by the infected host cell.Thus Chlamydiae have been called ’’energy par-asites’’ (83). Chlamydiae demonstrate two dis-tinct stages within their developmental cycle(110). The infectious form, the 200-300 nmelementary body, is metabolically inactive, andhas a dense DNA core surrounded by a trilam-inar cytoplasmic membrane, external to whichis a trilaminar outer envelope. The elementarybody is phagocytosed by the host cell after spe-cifically binding to the surface of epithelial cells.Within 6-9 hours after infection, the elemen-tary body becomes metabolically active and en-larges (1 µm) to form an initial or reticulatebody. The reticulate body multiples by binaryfission to form a microcolony of pleomorphicChlamydial forms which lies within a cytoplas-mic vacuole. By 20 hours after infection someof the reticulate bodies undergo reorganizationwithin the expanding intracytoplasmic inclu-sion body of Halberstaedter-Prowazek (Figures

1 and 2) into smaller intermediate forms andthen into elementary bodies. The new elemen-tary bodies are released by cell lysis to infectnew cells.Over the last 10 years, much has been learnedabout the antigenic composition of Chlamydi-ae. Their rigid outer membrane is composed ofa number of structural proteins, the most im-portant being the major outer membrane pro-tein (68). Antibodies against this protein are ca-pable of neutralizing Chlamydiae (94) and canproduce at least partial immunity to Chlamy-dial infection. In addition, T-lymphocyte prolif-erative responses and interferon-gamma (IFN-γ)production were observed after challenge withmajor outer membrane protein (90). Other prom-inent components of the outer membrane in-clude a 60 KD cysteine-rich protein and a spe-

Fig. 1 Conjunctival scraping from a child with active tracho-ma showing multiple epithelial cells containing intracy-toplasmic Halberstaedter-Prowazek inclusion bodies (ar-rows) (Giemsa x 800).

Fig. 2 Corneal epithelial cells from a child with activetrachomatous keratitis obtained by corneal replica tech-nique showing a Halberstaedter-Prowazek inclusion body(arrows) (Giemsa x 1120).

75

cific Chlamydial lipopolysaccharide (LPS).Chlamydiae also release two heat-shock pro-teins, of 57 KD and 75 KD. The 57 KD heat-shock protein appears to be the major patho-genic antigen in trachoma. It stimulates the per-sistent deleterious cell-mediated immune res-ponse which, if chronically maintained, leadsto cytokine release, causing ongoing inflamma-tion, fibrosis and conjunctival scarring(21,62,73).In the early (active) stage, which is seen prin-cipally among children in endemic areas, tra-choma is characterized by a chronic follicularconjunctivitis, which affects principally the up-per tarsal conjunctiva, but also the limbus. Con-junctival thickening and a papillary responsemay also be seen with active inflammation, es-pecially in the presence of bacterial secondaryinfection. Corneal changes include punctatekeratitis, indolent ulcers and vascular and cel-lular infiltration (pannus), which is occasion-ally so extensive as to impair vision. Among old-er individuals in endemic areas, conjunctivalscarring replaces the follicles and may lead todry eye syndrome, entropion and trichiasis. Cor-neal scarring and blindness might result fromregressed pannus and/or constant corneal abra-sion due to entropion and trichiasis.The pathologic mechanisms by which tracho-ma causes corneal and conjunctival scarringthat lead to blindness are still unclear. Althoughthe growth of C. trachomatis is restricted to theepithelium (43), the consequences of infec-tion, namely conjunctival and subconjunctivalscarring and blindness can be devastating. Thereis a growing evidence that the blinding com-plications of trachoma may result from chronicstimulation of the immune response. Monnick-endam et al (61), using a guinea pig model ofChlamydial eye disease, demonstrated that re-peated reinfection led to chronic conjunctivitiswith pannus and conjunctival scarring. Ocularinfection with the agent of C. trachomatis in-duces both humoral immunity (74,104) andcell-mediated immunity (51,62,118). The im-mune response seems to confer partial protec-tion against subsequent infection, yet appearsalso to be responsible for much of the observedpathology and tissue destruction seen in tra-choma (99).

PHENOTYPIC

CHARACTERIZATION OF

INFLAMMATORY CELLS

IN TRACHOMATOUS

CONJUNCTIVITIS

Conjunctival biopsy specimens from childrenwith severe active trachoma showed compara-ble changes. The epithelium showed mild tomoderate hyperplasia and was infiltrated by amixed inflammatory infiltrate consisting of mono-nuclear and polymorphonuclear leucocytes. Inthe superficial epithelial layers variable num-bers of Halberstaedter-Prowazek inclusion bod-ies were seen. In the underlying substantia pro-pria, the inflammatory infiltrate was organizedin lymphoid follicles and as a diffuse infiltrate.Variably sized secondary lymphoid follicles con-taining a large, clear follicular centre surround-ed by a lymphocytic mantle were seen through-out the biopsy specimens. The follicular centrewas composed of small and large activatedcleaved and non-cleaved lymphoid cells, andcontained many stainable body macrophages.The surrounding lymphocytic mantle was thinnedand contained small darkly stained lympho-cytes and lymphocytes of variable size andshape, indicating a progressive transformation.In the area between the follicles and the epi-thelium, as well as in areas of diffuse inflam-mation, a mixed population of lymphocytes,polymorphonuclear leucocytes, macrophagesand some mast cells and eosinophils was ob-served. The blood vessels in these areas weredistended and lined by prominent endotheli-um. A band of plasma cells was situated di-rectly underneath the epithelium and a denseinfiltration by plasma cells was observed alsoaround the acini of accessory lacrimal glands.Immunohistochemical staining demonstratedthat all epithelial cells expressed β2 -microglo-bulin (class I major histocompatibility com-plex (MHC] associated); membranous MHC classII antigen expression was noted mainly in thesuperficial epithelial layers (Fig. 3). The inflam-matory infiltrate in the epithelium consisted oflarge numbers of 3MA134+ macrophages, aswell as variable numbers of MT1

+ T-lympho-cytes and polymorphonuclear leucocytes.MHC class II + dendritic cells were observedin the deeper epithelial layers as well as in the

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Fig. 3Immunohistochemical staining showing membranous major histocompatibility complex class II expression in the su-perficial epithelial layer (x 385).

Fig. 4Immunohistochemical staining showing that the lymphoid follicle and surrounding mantle are composed mostly ofB-lymphocytes (x 135). (Reproduced with permission from reference 7)

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Fig. 5Immunohistochemical staining showing large immunoreactive macrophages in the follicular centre. Smaller macrop-hages are present also in the surrounding infiltrate and in the epithelium (x 135). (Reproduced with permission fromreference 7)

Fig. 6Immunohistochemical staining showing few T-lymphocytes in the follicular centre, whereas many T-lymphocytes arepresent in the surrounding infiltrate (x 135.)

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underlying substantia propria, where theywere admixed with MT1

+T- lymphocytes,3MA134+ macrophages and scattered MB2

+

lymphoid cells. Occasional dendritic cells weresituated at the interface between epitheliumand substantia propria. The lymphoid folliclesconsisted of MB2

+ LN2+ MHC class II + B-lym-

phocytes (Fig. 4). In the follicular centre large3MA134+ stainable body macrophages (Fig. 5)and some MT1

+ T-cells were observed (Fig. 6).In the subepithelial band of plasma cells IgA+

cells outnumbered IgG+ cells, whereas IgM+

and IgE+ plasma cells were rare (Fig. 7). A sim-ilar Ig distribution was found in the plasma cellspopulating the accessory lacrimal glands (Fig.8) (1,7).These results indicate the presence of distinctcompartments involved in humoral and cell me-diated immune responses. The humoral im-mune response was represented by large, ac-tive lymphoid follicles formed of B-lympho-cytes, scattered transformed B lymphoid cellsin the lymphocytic mantle and area betweenthe follicle and epithelium, and large numbersof plasma cells below the surface epitheliumand around the acini of accessory lacrimalglands. Previous in vitro studies (49) have shownthat C. trachomatis stimulates B-cells to pro-liferate and differentiate into immunoglobulin-secreting plasma cells. Differentiation to plas-ma cells is enhanced by T-cells, leading to thesecretion of large quantities of polyclonal im-munoglobulins. Our results indicate that the invivo counterpart of this in vitro response con-sists of B-cells that are stimulated by Chlamy-dial antigens exposed on the dendritic reticu-lum cells acting as antigen presenting cells inthe follicular centre. Subsequently the B- cellsundergo transformation, leave the follicular cen-tre, and differentiate into plasma cells, whichmigrate through the region between the lym-phoid follicles and the epithelium. That regionwas found to consist mainly of T-cells and den-dritic cells, and it thus appears to represent asuitable microenvironment for the terminal dif-ferentiation of B-lymphocytes into plasma cells.IgA+ plasma cells outnumbered IgG+ plasmacells, whereas IgM+ and IgE+ plasma cells wereconspicuously rare. These findings are in ac-cordance with the detection of type-specificChlamydial IgA and IgG antibodies in the tearsof patients with trachoma (104). Dense accu-

mulations of plasma cells, showing a similar Igdistribution, were seen around the acini of ac-cessory lacrimal glands, suggesting that theseglands constitute part of the conjunctival pro-tective immune system. According to Franklinand Remus (29) these IgA+ plasma cells orig-inate from B-cells present in the conjunctiva-associated lymphoid tissue (CALT) and are in-volved in the formation of secretory IgA, whichhas been found to play a part in the defenceagainst Chlamydial infection by preventing itsadherence to epithelial cells (23). It has beendemonstrated that antichlamydial antibodiesare capable of neutralizing chlamydiae, block-ing attachment and internalization of the or-ganism, and can produce partial immunity(94,112).Scattered MHC class II + dendritic cells wereobserved in the epithelium, in the underlyingsubstantia propria and at the border of both re-gions. By analogy with the skin (87) these den-dritic cells might be antigen-loaded Langer-hans cells, previously shown to occur in theconjunctival epithelium (30,31) which havemigrated towards the underlying substantia pro-pria, where they present their antigens in anMHC class II-restricted manner to T- lympho-cytes. In vitro studies have shown that antigenpresenting cells are able to process and presentChlamydial antigens to T-lymphocytes induc-ing T-cells proliferation. These T- cell prolifer-ative responses were major histocompatibilitycomplex restricted (95). In addition, Stagg etal (90) have demonstrated T-cell proliferativeresponse and interferon-gamma production tomajor outer membrane protein of C. trachom-atis. This response displayed an absolute re-quirement for dendritic cells in the antigen-presenting cell population. In addition to help-er T-cells involved in B-cell differentiation, theantigen-specific proliferation of T-cells inducesthe development of cytotoxic effector T-cells(92). The majority of T-lymphocytes present inthe epithelium are likely to belong to the cyto-toxic T-cell subset engaged in cytotoxicity of in-fected cells. For the cytotoxic T-cells to medi-ate cytotoxicity they must recognize class I MHCantigen displayed by diseased cells.We have demonstrated that CD4

+ T-lympho-cytes were greater in number than CD8

+ T-lym-phocytes (4) (Fig. 9) which is similar to thatdescribed in previous reports of inflamed and

79

Fig. 7IgA staining showing many IgA+ plasma cells in a subepithelial band (x 135). (Reproduced with permission from refe-rence 7)

Fig. 8IgA staining showing dense accumulation of IgA+ plasma cells around the acini of accessory lacrimal glands (x 135).

80

Fig. 9Serial sections immunohistochemical stainings for CD4 (top) and CD8 (bottom). Note that CD4+ helper / inducer T-lymphocytes predominated over CD8+ suppressor / cytotoxic T-lymphocytes (300).

81

active trachoma (16,77). Two different typesof CD4

+ T-cell clones have been identified onthe basis of their differing cytokine profile (63).T helper (TH) 1 cells produce IL-2 and IFN-γand mediate several functions associated withcell-mediated immunity, while TH2 cells pro-duce IL-4, IL-5, IL-6 and IL-10 and are moreeffective in stimulating B- cells to produce an-tibody. Previous studies have demonstrated thatcellular immune responses in the local tissuesassociated with Chlamydial infections are dom-inated by TH1-like responses (18,89) and in-travenous transfer of C. trachomatis-specificTH1 clone induced resolution of murine Chlamy-dial genital infection (41). In our immunohis-tochemical studies of conjunctival specimensfrom patients with active trachoma, we havedemonstrated that the epithelial cells expressedMHC class II antigens (1,7). It is well knownthat the expression of MHC class II antigens re-quires stimulation with IFN-γ (6) produced byactivated TH1 cells. Based on these findingswe may assume that the large preponderanceof CD4

+ T-cells observed in the conjunctival bi-opsies comprised TH1 cells. Igietseme et al(40) have demonstrated that CD8

+ T-cells maycontribute to antichlamydial T-cell immunity invivo. In addition, Starnbach et al (91) charac-terized a cytotoxic T-lymphocyte line derivedfrom mice infected with C. trachomatis. Thiscytotoxic T-lymphocyte line is specific for, andable to lyse, Chlamydia-infected cells, and re-cognizes a peptide epitope present on infectedcells in the context of MHC class I molecule.Based on these observations cytotoxic T-cellscould comprise the major component of CD8

+

T-cells detected in conjunctival specimens.In an immunohistochemical study of conjunc-tival biopsies from children with mild to mod-erate active trachoma (4), lymphoid follicleswere present in six out of nine conjunctival spec-imens. In these specimens the number of B-lymphocytes was greater than T-cells and thefibrosis was confined to the deep substantiapropria. In three specimens lymphoid follicleswere not detected and T-lymphocytes outnum-bered B-cells. In these specimens fibrosis wasmore pronounced and involved the whole sub-stantia propria, starting immediately under theepithelium. Our findings are supported by pre-vious studies (16,77) of lymphocyte subsets inadults with cicatricial trachoma that demon-

strated predominance of T-lymphocytes over B-cells. These observations suggest a role for T-lymphocytes in the genesis of conjunctival scar-ring in cicatricial trachoma. Several lines of ev-idence suggest that T-lymphocytes play a directrole in the pathogenesis of fibrosis(11,108,109). T-lymphocytes produce media-tors including IL-2 and interferon-γ (IFN-γ) whichtrigger macrophage production of fibrogenic cy-tokines. Furthermore, T-lymphocytes producetransforming growth factor-β (TGF-β) whichstimulates collagen synthesis (46). On the oth-er hand, T-lymphocytes and cell-mediated im-munity are essential for the eradication ofChlamydial infections (40,41,93,109). T- lym-phocytes stimulation by C. trachomatis ele-mentary bodies elicit IFN-γ production (28).IFN-γ produced by activated T-cells has beenshown to inhibit the intracellular growth ofChlamydia (97) and participate in the defenseagainst early infection (59). In addition, IFN-γexhibits cytotoxic activity against cells infect-ed with C. trachomatis (17). Therefore, acti-vation of T cells could bring about both protec-tive and deleterious effects.Increased numbers of macrophages were not-ed in the conjunctival biopsy specimens frompatients with active trachoma (4,7). Macro-phages are important components of the im-mune response to foreign agents. It has beendemonstrated, using an in vitro system, thatmacrophages inhibit intracellular C. trachom-atis replication (56). In addition, macrophag-es play a central role in normal wound healingand pathologic fibrosis by virtue of their abilityto release a variety of fibrogenic cytokines (46).

CONJUNCTIVAL

EPITHELIAL CELLS

INFECTED WITH

CHLAMYDIA

TRACHOMATIS EXPRESS

MHC CLASS II

ANTIGENS

The epithelial cells not only expressed the classI MHC antigens associated β2 -microglobulin,but were also found to express MHC class II an-tigens at their surface (Fig. 3) (1,7). MHC classII antigens are present on cells involved in im-

82

mune responses, including B cells, activated Tcells and antigen-presenting cells. The pres-ence of MHC class II antigens on these cells isassociated with antigen recognition and pre-sentation to T cells and the initiation of spe-cific B- and T-cell responses (102,105). Wehave shown that the normal conjunctival epi-thelium does not express MHC class II anti-gens (30). Itsexpressionontheconjunctivalepi-thelial cells in trachoma patients is probablydue to the release of interferon-gamma by ac-tivated T-cells present in the epithelium. Inter-feron-gamma has been shown to induce MHCclass II antigens synthesis and expression by avariety of epithelial cells in vitro (6,12,98) andis produced by lymphocytes stimulated withC. trachomatis (28). MHC class II antigens ex-pression might allow conjunctival epithelial cellsto present Chlamydial antigens to T-cells andthus to enhance the immune response in tra-choma. Moreover, this MHC class II antigensexpression may result in unopposed T-cell pro-liferation and lead to perpetuation of the im-

mune response. In addition, induction of MHCclass II antigens expression on epithelial cellshas been associated with autoimmune reac-tions--for example, autoimmune thyroiditis(14,35) and diabetes mellitus (13). The epi-thelial cells expressing MHC class II antigensmight present autoantigens to T-lymphocyteswhich activate effector B-and T-lymphocytes,leading to induction of an autoimmune reac-tion (50). In this context the appearance of au-toantibodies in patients with Chlamydial sal-pingitis (9) may be relevant. Alternatively, ex-pression of MHC class II antigens may renderthe infected epithelial cells susceptible to im-munological attack by MHC class II-restrictedcytotoxic T-cells (60). The expression of MHCclass II antigens by the conjunctival epithelialcells may therefore contribute to the progres-sion of conjunctival scarring in trachoma.

Fig. 10Anti-IL-1α staining shows cytoplasmic IL-1α expression in the superficial epithelial layers (x 300). (Reproduced withpermission from reference 4)

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Fig. 11Collagen V immunohistochemical staining of scarred trachoma showing thick, continuous immunoreactivity in uppersubstantia propria, around stromal vessels (top) and around acini of accessory lacrimal glands (bottom) (x 300). (Re-produced with permission from reference 5)

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Fig. 12Collagen IV immunohistochemical staining of scarred trachoma showing very thick, intensely stained epithelial base-ment membrane, thick vascular endothelial basement membrane (top) and very thick, intensely stained accessorylacrimal glands basement membrane (bottom) (x 300). (Reproduced with permission from reference 5)

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EXPRESSION OF

FIBROGENIC CYTOKINES

IN TRACHOMATOUS

CONJUNCTIVITIS

Cytokine production during infection may playan important role in modulating host defensesto C. trachomatis. This may also be a factor inthe perpetuation of the inflammation and sub-sequent fibrosis. We have demonstrated thatthe conjunctival epithelial cells from patientswith trachoma showed cytoplasmic expressionof interleukin (IL)-1α and IL-1β (Fig. 10) (4).Normally, conjunctival epithelial cells do not ex-press IL-1. These findings demonstrate the invivo expression and the production of IL-1 bythe conjunctival epithelial cells in trachoma.This production of IL-1 may constitute an im-portant contributing factor in the process ofscarring in trachoma. IL-1 is a pleiotropic cy-tokine produced chiefly by monocytes and mac-rophages but also by cells of epidermal, epi-thelial, lymphoid and vascular origin (25). Ourresults are consistent with the findings of Ro-thermel et al (79) that C. trachomatis inducedIL-1 production by human blood monocytes.They showed that human blood monocytes pro-duced detectable IL-1 when cultured with aslittle as 1 µg of Chlamydial protein per ml, whichcorresponded to 4 to 40 Chlamydiae per mono-cyte. This suggests that during low-grade orsubclinical infections, which are characteristicof Chlamydial disease, very few organisms maybe sufficient to stimulate IL-1 production. Fur-thermore, Magee and associates (52) have dem-onstrated increased m RNA and bioactivity forIL-1 in murine lungs after Chlamydial infec-tion. The relevance of IL-1 to trachoma was alsosuggested by the detection of increased levelsof IL-1 in the tears from children with activetrachoma (79).As an inflammatory mediator, IL-1 affects tis-sue remodeling by inducing the production ofcollagenase (24,36) and collagen (26). Fur-thermore, IL-1 can stimulate fibroblast prolif-eration (26,75,119). Excessive IL-1 produc-tion is thought to contribute to tissue damageand fibrosis in chronic inflammatory conditions,such as pulmonary fibrosis (45,119) and bonemarrow fibrosis (76). The local constitutive pro-duction of IL-1 by the conjunctival epithelial

cells, could serve for the paracrine stimulationof target cells such as fibroblasts and induceexaggerated production and accumulation ofsubconjunctival fibrous tissue in patients withtrachoma.In addition, we have detected cytoplasmic ex-pression of the fibrogenic cytokines IL-1α, IL-1β (discussed above), tumor necrosis factor(TNF)-α and platelet-derived growth factor(PDGF) by macrophages in the substantia pro-pria. Our results are consistent with previousstudies that murine C. trachomatis infection in-duces TNF-α production (115). TNF-α stimu-lates fibroblast proliferation and collagen syn-thesis (26,119). On the other hand, TNF-α isknown to restrict intracellular Chlamydial rep-lication (97). PDGF is a potent chemoattrac-tant and mitogen for fibroblasts and smoothmuscle cells and a stimulator of collagen syn-thesis by fibroblasts (34,78,86,114). Thesecytokines have the potential to influence the re-modeling and fibrosis observed in the conjunc-tiva of patients with trachoma.

ALTERATIONS IN

CONJUNCTIVAL

COLLAGEN IN THE

CONJUNCTIVA OF

PATIENTS WITH

TRACHOMA

The collagens which are the major proteins ofconnective tissue, provide the extracellular frame-work for all multicellular organisms. A subclassof this family comprises collagens I, II, III, Vand XI which form banded fibrils. They havethus been called the fibrillar collagens, to dis-tinguish them from other collagens unable toaggregate into these highly-ordered fibrils. Col-lagens I, II and III are the most abundant andhave therefore been far more studied than thequantitatively minor collagens V and XI. Thefunction of type I collagen is to give tissue ten-sile strength. Type II collagen is principallypresent in cartilage and type III collagen is as-sociated with tissues and organs that require amotile structural scaffolding such as uterus, ar-teries, skin, intestines and lung. Type IV col-lagen is a non-fibrillar collagen which is onlyfound within basement membranes (27,57).

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Histochemically, we observed that the conjunc-tival substantia propria from patients with ac-tive trachoma and scarred trachoma containedcollagen type V. The extent of deposition of col-lagen V was markedly increased in scarred tra-choma when compared with active trachoma.On the other hand, collagen V was not detect-ed in the normal conjunctiva. New collagen Vdeposition was noted close to the basementmembranes of the conjunctival epithelium, stro-mal vascular endothelium and accessory lac-rimal glands (Fig. 11) (3,5). This is consistentwith previous immunolocalization data that dem-onstrated collagen V localization in the imme-diate vicinity of basement membranes suggest-ing an anchoring function for collagen V be-tween basement membranes and stromal ma-trix (27). Our results are consistent with stud-ies describing accumulation of collagen V inconditions associated with tissue remodelingand new collagen synthesis. Increased type Vcollagen content has been detected in other dis-eased tissues such as cardiac hypertrophy (39),neoplasia (10), Crohn’s disease (33), athero-sclerotic lesions (64), pseudointima of vascu-lar grafts (113) and chronically inflamed gin-gival tissue (67) suggesting that type V col-lagen may play an important role in the pathol-ogy of these diseases. A number of factors havebeen shown to induce type V collagen expres-sion including transforming growth factor-beta1 (69), epidermal growth factor (88), hepaticfibrogenic factor (20), angiotensin II (44) andplatelet derived growth factor (69).We have observed that the conjunctival tissuefrom patients with active trachoma containedincreased amounts of collagen types I, III andIV. Scarred trachoma is characterized by markedincrease in basement membrane-collagen IV(Fig. 12). In contrast, collagen types I and IIIimmunoreactivity was markedly decreased whencompared with active trachoma (3,5). Theseobservations are well in agreement with thefindings reported in wound healing. In the ear-ly phase of wound healing the expression of α1(I) and α1 (III) collagen chains is coordinatelyincreased. In later stages the gene expressionis rapidly down regulated (70,84). In addition,our findings agree well with immunohistochem-ical studies of kidney biopsies from patientswith chronic renal disease. The severity of in-terstitial extracellular matrix accumulation was

accompanied by significant increase in the dep-osition of collagen types IV and V. On the otherhand, the increase in interstitial abnormalitieswas not accompanied by significant increase inthe deposition of the interstitial collagen typesI and III (107). Our results in active trachomaand scarred trachoma are also in agreementwith experimental autoimmune uveoretinitisstudies. Increased immunoreactivity of collagentypes I, III and V was noted at the peak of in-flammation. A decrease in immunoreactivity forcollagen types I and III but persistant immu-noreactivity for collagen type V was observedin later stages (54). Previous histopathologicstudies of conjunctival biopsies from patientswith cicatricial entropion complicating tracho-ma showed a thick and compact subepithelialfibrous membrane (8). Our data indicate thatthe increased deposition of collagen type IV inthe epithelial basement membrane and newtype V collagen formation in the immediate vi-cinity of the epithelial basement membranemight contribute to the formation of this sub-epithelial scar tissue. The additional increaseof immunoreactivity for types IV and V collagenaround the acini of accessory lacrimal glandsmight contribute to dysfunction of these glandsleading to dry eye disease.

EXPRESSION OF

GELATINASE B IN

TRACHOMATOUS

CONJUNCTIVITIS

Metabolic alterations of extracellular matrix com-ponents and collagen metabolism occur in theconjunctival tissue from patients with tracho-ma (3,5). The matrix metalloproteinases arerecognized as key enzymes both for normal ex-tracellular matrix turnover and for the exagger-ated extracellular matrix breakdown associat-ed with pathologic conditions, including tumorinvasion and metastasis, angiogenesis, inflam-matory reactions, wound healing and scar for-mation (58,72,116). The major members ofthis family include the following: collagenas-es, which degrade and denature fibrillar col-lagen types I, II and III; gelatinases A and B (re-spectively, the 65-Kd to 75 -Kd matrix metal-loproteinase-2 and the 85-Kd to 96-Kd matrixmetalloproteinase-9) which cleave denatured

87

collagens (gelatins), collagen types IV, V, VII andX, elastin and fibronectin; and stromelysins,which degrade proteoglycans, laminin, fibronec-tin, type IV collagen and the globular domainsof other extracellular matrix macromeclules(116). More recently, membrane type matrixmetalloproteinase expressed on cell mem-branes is identified as a fourth category (82).Because of its unique and broad substrate spec-ificity, its involvement in other chronic inflam-matory and autoimmune diseases (71) and itsdistal position in the matrix proteolytic cas-cade we hypothesized that excessive expres-sion of gelatinase B may play a role in matrixdegradation in trachomatous conjunctivitis. Totest this hypothesis, we examined conjuncti-val specimens obtained from patients with ac-tive trachoma using immunohistochemistry andgelatin zymography. The findings in trachomawere compared with the findings in the con-junctiva from normal individuals.Zymography indicated an increasing activity ofgelatinase B in trachoma specimens (Fig. 13).Using immunohistochemistry, we have dem-onstrated that gelatinase B was specifically lo-calized in macrophages and neutrophils present

in the inflammatory infiltrate in trachoma spec-imens (Fig. 14) (2). Macrophages, monocytesand neutrophils are the main producers of ge-latinase B (37,66). Most of the stromal neu-trophils were in the intravascular spaces indi-cating that macrophages were primarily respon-sible for the production of gelatinase B. Mac-rophages synthesize and secrete matrix metal-loproteinases which are capable of degradingall macromolecular constituents of the extra-cellular matrix (111). These enzymes appearto function as a proteolytic cascade of whichgelatinase B is a downstream element and whoseactivation is initiated after that of stromelysin(32). The intracellular localization of gelati-nase B in macrophages can be interpreted asevidence of active synthesis of this enzyme be-cause macrophages do not store this metallo-proteinase (37).The mechanism by which C. trachomatis in-duces gelatinase B expression by macrophag-es has not been determined. The effects of C.trachomatis on the synthesis and release of thisenzyme may be related to the production of cy-tokines. C. trachomatis is an effective inducerof IL-1 and TNF-α production by monocytes(42,79). Whole spleen cells produced TNF-αin vitro in response to murine C. trachomatisin a mouse model of pneumonia caused by mu-rine C. trachomatis (115). IL-1α and IL-1βmRNA and bioactivity are induced in murinelungs in response to Chlamydial infection (52).In agreement with these studies we have dem-onstrated upregulated production of IL-1α, IL-1β and TNF-α in the conjunctiva from patientswith active trachoma (4). TNF-α and IL-1β se-lectively up-regulate gelatinase B by macroph-ages (81,106). As in all gram-negative bacte-ria, the outermost monolayer of C. trachoma-tis is composed of LPS, a complex glycolipidessential for bacterial survival (15,19). LPS isa potent inducer of the biosynthesis and secre-tion of gelatinase B in macrophages(80,101,106,111,117). Ingalls and associ-ates (42) demonstrated that the inflammatorycytokine response to C. trachomatis infectionis mediated primarily through LPS and can becompletely blocked by a specific LPS antago-nist. Chlamydial LPS induces mononuclearphagocytes to produce TNF-α and IL-1β (42,79).Thus it is possible that gelatinase B produc-tion by mononuclear cells may be influenced

Fig. 13 Gelatin zymography of conjunctival biopsy speci-mens from normal subjects (A) and from patients with ac-tive trachoma (B). The zymographies of these samplesshow the presence of both gelatinase a (72 KDa) and ge-latinase B (92 KDa). (Reproduced with permission fromreference 2)

88

A

92KDa

72KDa

B

92KDa

72KDa

Fig. 14Serial sections immunohistochemical stainings for gelatinase B (top) and macrophage marker (CD68) (bottom). Near-ly all macrophages are positive for gelatinase B (x 500). (Reproduced with permission from reference 2)

89

by these cytokines acting through autocrinepathways. However, Saarialho-Kere and asso-ciates (80) have shown that the LPS-mediatedeffects on gelatinase B production are proba-bly transduced primarily by signaling pathwaysthrough the LPS receptor. Although Chlamydi-al LPS has been shown to elicit secretion ofproinflammatory cytokines, Ingalls and associ-ates (42) showed that the potency of Chlamy-dial LPS was 100-fold less than that elicitedby LPS from Neisseria gonorrhoeae or from Sal-monella minnesota. According to these inves-tigators, this relatively weak induction of anacute immune response may partially explainthe asymptomatic nature of Chlamydial infec-tion.Gelatinase B degrades denatured collagens (gel-atin), collagen types IV, V, VII and X, elastin andfibronectin (65,85). Therefore, expression ofgelatinase B in trachoma may contribute to pro-gressive breakdown of conjunctiva by degrad-ing minor constituents of the extracellular ma-trix. The degradation of fibrillar collagen typesI, II and III is initiated by collagenases and com-pleted by gelatinase B. The degradation of col-lagen types IV and V associated with basementmembranes permits the extravasation of ma-crophages into conjunctival tissue. It has beendemonstrated that in vitro migration of T lym-phocytes across a basement membrane equi-valent is mediated by gelatinase B (48). Macro-phage gelatinase B might enhance fibroblastmigration into conjunctival epithelium by cre-ating breaks in epithelial basement membrane.Since collagen fragments may be chemotacticformonocytes,degradationofcollagenbymacro-phages could further promote monocyte recruit-ment (55). In addition, chronic release of spe-cific collagen fragments in the conjunctiva bygelatinase B might contribute to selection of au-toreactive T cells and generation of auto-im-munity (71).

CONCLUSIONS

On the basis of our data and of other in vitroand in vivo animal studies, the following im-munopathogenesis of trachomatous keratocon-junctivitis can be proposed. Chlamydia tra-chomatis replication in the conjunctival andcorneal epithelial cells releases a number ofpathogenic antigenic proteins including the ma-

jor outer membrane protein, 60 KD cysteine-rich protein and 57 KD and 75 KD heat-shockproteins. In addition, Chlamydial lipopolysac-charide is released (21,62,68,73,90,94). C.trachomatis stimulates B-lymphocyte prolifer-ation and differentiation into plasma cells se-creting large quantities of immunoglobulins.This response is enhanced by T-lymphocytes(49). These antibodies are capable of neutral-izing Chlamydiae, block attachment and inter-nalization and can thereby produce partial im-munity (94,112). Stimulation with C. trachom-atis elementary bodies elicits IL-6 formation byB-lymphocytes (28) which induces the final dif-ferentiation of B cells to antibody-producingcells (38). Antigen-presenting cells process andpresent Chlamydial antigens to CD4

+ T-lym-phocytes in an MHC class II molecule-restrict-ed manner, resulting in helper T-lymphocyteproliferation and activation, and in secretion ofseveral cytokines (90,95) that promote differ-entiation and clonal expansion of several dif-ferent subsets of Chlamydia-committed effec-tor CD8

+ cytotoxic T-lymphocytes. The major-ity of CD4 T-lymphocytes are belonging to TH1cells producing IL-2 and IFN-γ (18,89). T-lym-phocytes and cell-mediated immunity are es-sential for the eradication of Chlamydial infec-tions (91,93). T-lymphocyte stimulation by C.trachomatis elementary bodies elicits IFN-γproduction (28) which has been shown to in-hibit the intracellular growth of Chlamydia (97)and to exert cytotoxic activity against cells in-fected with C. trachomatis (17). TNF-α is pro-duced in response to C. trachomatis infection(115) and exhibits a synergistic effect with IFN-γto restrict intracellular growth of C. trachoma-tis (97). CD8

+ cytotoxic T-lymphocytes con-tribute to antichlamydial T-cell immunity.CD8

+ T cells lyse Chlamydia-infected cells andrecognize a peptide epitope on infected cells inthe context of MHC class I molecules. Theirability to resolve Chlamydial infection is corre-lated with the capacity to elaborate IFN-γ andTNF-α (40,91). On the other hand, stimula-tion by IFN-γ induces aberrant MHC class II an-tigens expression by the infected conjunctivaland corneal epithelial cells which may lead toperpetuation of the inflammatory immune re-sponse, induction of an autoimmune reactionand progression of conjunctival and cornealscarring. T-lymphocytes produce mediators in-

90

cluding IL-2 and IFN-γ which trigger macro-phages to produce fibrogenic cytokines. Fur-thermore, T-lymphocytes produce transform-ing growth factor-β which stimulates collagensynthesis (46). Colony-stimulating factors (CSFs)are produced by CD4

+ cells in response to C.trachomatis infection (53). CSFs are cytokinesinvolved in the production, differentiation andactivation of phagocytic cells. Macrophages areeffector cells in cell-mediated immunity to C.trachomatis and inhibit intracellular replica-tion of C. trachomatis (56). On the other hand,macrophages play a central role in normal woundhealing and pathologic fibrosis by virtue of theirability to release a variety of fibrogenic cyto-kines (46). Conjunctival macrophage activa-tion in trachoma is suggested by our findingsof cytoplasmic expression of IL-1α, IL-1β, TNF-αand PDGF that may be important in the patho-genesis of scarring (4). In addition, active syn-thesis of gelatinase B by macrophages mightbe involved in matrix degradation and promo-tion of conjunctival scarring in trachoma (2).Cell-mediated immunity to ocular infection withC. trachomatis may thus be a two-edged sword.Furthermore, the local constitutive productionof IL-1 by the conjunctival epithelial cells asshown by our data (4) could stimulate fibro-blast proliferation and induce exaggerated pro-duction and accumulation of subconjunctival fi-brous tissue in patients with trachoma.

ACKNOWLEDGEMENTS

The authors thank Ms C. Van den Broeck, MsB. Smets and Ms E. Van Dessel for technicalassistance, and Ms C. B. Unisa-Marfil for sec-retarial work.

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Correspondence to:Professor Dr. Ahmed M. Abu El-Asrar,Department of Ophthalmology, King AbdulazizUniversity Hospital,Airport Road,P.O. Box 245, Riyadh 11411, Saudi ArabiaFax: 966 - 1 - 477 5741 / 4775724E-mail: abuasrar@KSU.edu.sa

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