ocular immunopathology of behçet's disease

SURVEY OF OPHTHALMOLOGY VOLUME 42 l NUMBER 2 l SEPTEMBER-OCTOBER 1997

CLINICAL PATHOLOGICAL REVIEW DAVID APPLE AND MILTON BONIUK, EDITORS

Ocular Immunopathology of Behcet’s Disease MAJ. ROGER IL GEORGE, MD,’ CHI-CHAO CHAN, MD,’ SCOTT M. WHITCUP, MD,2 AND ROBERT B. NUSSENBLATT, MD2

‘Madigwz Army Medical Cet&r, Tli2comn, Washinffton, USA, and “Laboratov of Immunology, National &ye Institute, Bethesda, Maryland, UTA

Abstract. A patient developed progressive, severe, recurrent bilateral iridocyclitis, retinal vasculitis,

and hemorrhagic inf’arction of the retina that led to blindness despite immunosuppressive therapy. His-

topathology of an enucleated blind and painful eye revealed marked nongranulomatous uveitis with a predominantly CD4+ T-lymphocytic infiltration, as well as B-cell and plasma cell aggregation. Extensive expression of adhesion molecules on vascular endotbelial cells were found. This finding suggests that

adhesion molecules play an important role in the vasculitic process, the trademark of Behcet’s disease.

The ocular pathology and the therapeutic approach to Behcet’s disease are briefly reviewed. (Surv Ophthalmol42:157-162, 1997. 0 1997 by Elsevier Science Inc. All rights reserved.)

Key words. adhesion molecule l Behcet’s syndrome l immunopathology l ocular

Be&et’9 disease l plasma cell l T-lymphocyte . vasculitis

Behcet’s disease is a chronic, relapsing, systemic

vasculitis. It is a multisystem, idiopathic disorder with worldwide distribution, but it is much more prevalent in the Mediterranean basin, Middle East, and Far East. The disease accounts for up to 20% of uveitis cases in Japan.‘” It is found less frequently in the USA, accounting for 0.2-0.4% of uveitis pa-

tients.’ Several diagnostic criteria for Behcet’s disease have been developed over the years, but in 1990 the International Study Group for BehCet’s Disease proposed a standardized set of diagnostic criteria.” These include recurrent oral ulceration and two of

the following indications: recurrent genital ulceration, characteristic eye lesions, characteristic skin lesions, or a positive pathergy test.

The ocular manifestations of Behcet’s disease typically include a bilateral nongranulomatous inflammation of the iris and ciliary body, a marked vitritis, and an occlusive retinal vasculitis. Vasculitis, charac-

terized by both a perivascular and intramural infiltrate of lymphomononuclear cells, is the common histopathologic mechanism in all organ systems af- fected by the disease.‘,” This vasculitis can lead to vas-

cular obliteration and fibrinoid necrosis of tissues. We report the clinical and histological findings, in- cluding immunopathology, on the left eye of a 24 year-old man, which was enucleated during an acute

flare-up of his Behcet’s disease. We provide a literature review on the ocular pathology of this disease.

Case Report CLINICAL COURSE

A 24year-old man, who worked as a mechanic, presented to a local ophthalmologist complaining of a red eye after getting rust in his left eye at work. He was diagnosed with a cornea1 abrasion and appropri- ately treated. Several weeks later he again sought

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158 Surv Ophthalmo142 (2) September-October 1997 GEORGE ET AL

medical attention when he noted diminished vision, photophobia, and ocular irritation of the left eye (OS). His visual acuity was 20/20 right eye (OD) and 20/80 OS. Th e d’ g la nosis of uveitis with cystoid mac- ular edema was made, and he was treated with prednisone 60 mg orally daily. Several months later there was a precipitous loss of vision in the left eye to 20,’ 100, while the vision in the right eye remained at 20/20. Examination revealed 1 + anterior-chamber cells OD and 4+ anterior chamber cells with a lay- ered hypopyon OS. On retinal evaluation, perivascular sheathing was noted OS and the diagnosis of Beh- Get’s disease was considered. Serologies for Lyme disease, the Venereal Disease Research Laboratories test, fluorescent treponemal antibody absorption test, angiotensin converting enzyme, serum lysozyme, chest radiograph, and a purified protein derivative were normal.

In November 1990 the patient was referred to the National Eye Institute for further evaluation. Medi- cations at the time included prednisone 40 mg orally daily and prednisolone acetate 1 “/o, one drop every 2 hours in each eye. Visual acuity was 5/200 OD and sufficient vision to count fingers OS. Examination revealed 3+ anterior chamber cells and cystoid mac- ular edema in each eye. In addition, a branch retinal vein occlusion was present inferotemporally OD, and there was marked attenuation of the retinal vasculature OS. Significant disk pallor OS was noted as well (Fig. 1). Systemic signs included acnelike skin lesions, oral ulceration, and arthritis. A diagnosis of Behcet’s disease was made and cyclosporine A was added to the treatment regime.

In January 1991 visual acuity was 20/63 OD and light perception OS. Intraocular tension was 11 mm

Fig 1. Left eye 5 months prior to enucleation. Note the areas of retinal edema and the hazy view from vitreous inflammation.

Fig. 2. Iris neovascularization of the left eye that extends from the pupillary margin into the iris stroma. Note also the extensive posterior synechiae.

Hg OD and 34 mm Hg OS. Gross neovascularization of the iris OS and a retinal detachment OS were noted (Fig. 2). No surgical intervention was under- taken at the time. Three months later the patient’s left eye became phthysical and painful, and was sub- sequently enucleated. Medications at the time of sur- gery included prednisone 20 mg and cyclosporine A 400 mg daily and acetazolamide 500 mg and timolol maleate 0.5% OS twice-daily. During the hospitalization the patient developed a large uvular ulcer, which was initially treated as a bacterial infection (Fig. 3). A subsequent biopsy of the ulcerated uvula showed an ongoing vasculitic process.

Despite multiple operations and immunosuppres- sion with corticosteroids and cyclophosphamide, the right eye had recurrent attacks of retinal vasculitis, a

Fig. ?. Uvular ulcer that developed during hospitalization for enucleation of the left eye.

OCULAR IMMUNOPATHOLOGY OF BEHCET’S DISEASE 159

rhegmatogenous retinal detachment, and a loss of vision to hand motions (Fig. 4).

Pathology

The left eye was obtained immediately after enu-

cleation and was examined before fixation. The globe, which measured 25 X 23 X 24 mm, was opened horizontally by removal of the superior cap. Intraocular examination revealed that the angle was

obliterated by a neovascular membrane. The lens was cataractous, the vitreous was filled with blood, the choroid was thickened, and the retina was totally de-

tached (Fig. 5). The fresh superior cap was embedded in an opti-

mal cutting temperature-freezing medium (O.C.T., Miles Scientific, Naperville, IL) and snap-frozen

for immunohistologic study. Serial frozen sections were stained by the avidin-biotin-peroxidase complex method. The primary monoclonal antibodies included

leukocyte markers of CD4 (T-helper/inducers), CD8 (T-suppressor/cytotoxic cells), CD3 (T-lymphocytes), CD16 (NK cells), CD19 (B-cells), CDllc/l8 (mac-

rophages), CD25 (interleukin-2 receptor), CD54 (ICAM-1, intercellular adhesion molecule-l), CD1 la/ 18 (LFA-1 a and b, lymphocyte function-associated antigen-l), CD62E (E-selectin), CD106 (VCAM, vascular cell adhesion molecule), and major histocompatibility classes 1 and 11 of HLA-DR and HLA-DQ.

Mouse ascites fluid containing 1 kg of nonspecific IgG per mL served as a control. The secondary antibody was biotin conjugated horse antimouse IgG

(Vector Laboratories, Burlingame, CA). The central portion of the globe with the inferior

cap was fixed in 10% formalin for 48 hours and then embedded in paraffin for routine serial sections.

Microscopic examination showed mild nonspe-

cific infiltration and congestion of the conjunctiva.

The cornea1 endothelium was attenuated and ab-

sent. There were 2 mm of anterior synechiae. The iris was adherent to the lens by a membrane of dense

connective tissue with partial destruction of the pig- ment epithelium. This newly formed membrane ap- peared to arise from the surface of the iris to cover

the anterior lens. The iris vessels were engorged and surrounded by inflammatory cells, particularly many lymphocytes and plasma cells. The lens showed cata-

ractous changes in the posterior cortex and subcap- sular region. The ciliary body was diffusely infil- trated by lymphocytes with focal aggregations of

plasma cells. Scattered polymorphous neutrophils were also observed, and a cyclitic membrane was

tightly adherent to the detached retina. The retina was totally detached and had extensive

gliosis (Fig. 6). It was separated from the lens and

ciliary body by newly formed vessels and fibroglial tissue with a marked infiltrate of lymphocytes, macrophages, plasma cells, and a few polymorphous

neutrophils. The retina was connected to the optic nerve by a stalk of gliotic retinal tissue that con- tained occasional remnants of both patent and oc-

cluded venous vessels. Fibrinoid necrosis was present in a few vascular walls, and clusters of focal hemorrhagic necrosis and monocytic inflammatory cells were present. The choroid demonstrated mild cellu-

lar infiltration, mainly lymphocytes. The optic nerve revealed atrophy with mildly thickened meninges.

The uvula was edematous and moderately infil-

trated by a combination of polymorphous neutrophils, macrophages, and lymphocytes. Foci of necrosis were also observed.

lmmunohistologic staining showed that most infiltrating cells in the retina and uvea were CD3-t (T-cells) with a CD4:CD8 ratio of 1.5:l.O (Fig. 7A).

Focal clusters of CD19 (B-cells) were also identified (Fig. 7B). In the choroid of the superior cap (frozen

Fig. 4. Posterior view of the right eye 2 years after initial presentation. Note the attenuation of the vasculature, fi- brovascular bands, and the panretinal photocoagulation.

Fig. 5. Macroscopic examination showing a total detachment of the retina (arrow).


Fig. 6. Microphotograph showing numerous inflammatory cells (arrow) and focal hemorrhage (open arrow) in the gliotic retina (hema- toxylin and eosin, 50 X )

section), the predominant infiltrating cells were CD4+ (T-helper/inducer cells). Major histocompatibility classes I and II (HLA-DR and HLA-D(L) and

adhesion molecules (ICAM-1, E-selectin, VCAM-1, LFA-la, LFA-lb) were abundant, particularly on the vascular endothelial cells (Fig. 8).

Discussion

The clinical appearance and the histologic findings in this patient are most consistent with Behcet’s syndrome, which is characterized by marked (active)

intraocular vasculitis, particularly in the early stage of the disease. Even though Behcet’s disease can cause blindness, few reports on the ocular immuno-

histopathology of Behcet’s disease have been pub- lished.?.3,‘” In contrast, many histopathologic studies have been performed on other tissues involved with

Fig. 7. Microphotograph. A: dif- fLlSl e T-lymphocytic infiltration. B: Fo- cal aggregation of B-lymphocytes in the eye (immunoperoxidase, 200 X ) .

Behcet’s disease. They have shown the predominant inflammatory cell to be of T-cell lineage, suggesting that cell-mediated immunity plays a central role in Behcet’s disease. ‘“.‘2,s3 In our patient, the uvular biopsy demonstrated a characteristic inflammatory in-

filtrate of polymorphous neutrophils, macrophages, and T-lymphocytes.

Early reports of the ocular histopathology of Beh- Get’s disease were defined by light microscopy. Find-

ings typically included a panuveitis with associated perivasculitis. Inflammatory cells noted to be present included lymphocytes, plasma cells, scattered macrophages, monocytes, and neutrophils.“,‘“,” More re-

cently, immunohistochemical techniques have been used to characterize lymphocyte subsets present in Behcet’s disease. In 1992, Charteris et al reported on

the histopathology of six cases.? One of these reports

OCULAR IMMUNOPATHOLOGY OF BEHCET’S DISEASE 161

Fig. 8. Microphotograph showing positive ICAM- staining on choroidal vascular endothelial cells (arrows, [immunoperoxidase, 400X]).

involved a %&year-old man who had had Behcet’s disease for 10 years. The histopathology showed intramural and perivascular infiltrate of CD4+ T-lymphocytes. No CDS+ T-lymphocytes were identified. In a second study of five patients, which investigated the immunohistochemical features of Behcet’s disease, Charteris et al found that the cellular infiltrate in the choroid consisted mostly of CD4+ lymphocytes and macrophages, which were both HLA-DR positive.” B-lymphocytes were an infrequent finding. In addition, no complement or immunoglobulin deposition were identified. This last finding con-

trasts Mullaney et al’s finding of mural IgG, IgA, and C, (complement) in both episcleral and choroidal veins in one enucleated eye.16 Our study resembles prior ones in that nongranulomatous uveitis, retinal vasculitis, hemorrhagic infarction, and retinal detachment are the ocular histopathologic features of eyes with a severe course of Behcet’s disease, leading to enucleation (Table 1). T-lymphocytes were the predominant, inflammatory cell type found. How- ever, in contrast to prior reports, we found focal ag- gregates of B-lymphocytes and many plasma cells present. In addition, the vascular endothelium showed extensive expression of adhesion molecules and major histocompatibility class II antigens.

Adhesion molecules are normally expressed on leukocytes and on vascular endothelium. Their up- regulation facilitates the margination and extravasa- tion of inflammatory cells during an immune response. Enhanced adhesion molecule expression has been shown to be associated with experimental autoimmune uveitis,‘“,2” posterior uveitis,” and idiopathic retinal vasculitis.” In addition, unpublished data from the National Eye Institute have shown ele- vated levels of circulating intracellular adhesion molecules in patients with BehGet’s disease. Thus, excessive expression by multiple adhesion molecules on the vascular endothelium in our patient with Be- hcet’s disease is not surprising and is important in the development of both the extensive vasculitis and ocular inflammation seen in patients with active Behcet’s disease.

Different mechanisms for the pathophysiology of BehGet’s disease have been proposed. Discussions

TABLE 1

Histopathology of Ocular BehCet ‘s Syndrome

Author (reference) Year No. of Eyes Site of Involvement Vasculitis Infarction Inflammatory Cells

Fenton and Easom” 1964 4 Uvea, retina, vitreous + + Winter and Yukins”

Lymph, PMN, plasma 1966 2 Uvea, retina t t

Green and Koo’ 1967 Lymph, MO

1 Uvea, retina - + Kaneko et al” 1976

PMN, lymph, MO 1 Retina, vitreous + - Round cells

Okabe et al” 1982 2 Choroid, retina + - Mullaney et al”’

Lymph, M0 1985 1 Retina, urea + + Lymph, plasma, PMN, IgC,

Charteris et al’ 1992 IgA, C3

2 Retina, uvea + - Charteris et al”

CD4. lymph, HLA-DR 1992 5 Retina, choroid + - T-lymph. M0, PMN,

HLA-DR lnomata et al!’ 1993 25 Uvea, retina + - George et al 1996

Lymph, MO, PMN 1 Uvea. retina, vitreous + +

(this article) T & B-lymph, plasma, MO,

PMN, HLA-DR, HLA-DQ ICAM-1, LFA-1, VCAM, E-selectin

+ = yes; - = no; Lymph = lymphocyte; PMN = polymorphonuclear lymphocyte; MO = macrophage; Ig = immunoglobulin (type A or G); C3 = complement type 3; HLA = human leukocyte antigen (DR or DQ = major histocompatabil- ity complex type II antigens); ICAM = intercellular adhesion molecule; LFA = lymphocyte function-associated antigen; VCAM = vascular cell adhesion molecule.


have centered around whether the process involves immune-complex deposition versus cell-mediated immunity. The predominant ocular infiltrating cells in this study were immunocompetent T-helper lymphocytes (CD4+ cells) and plasma cells. The abundant expression of major histocompatibility class II antigens suggests that there is active presentation of antigen to the CD4+ T-cells, which appear to play a major role in Behcet’s disease via cell-mediated immunity. The abundance of these CD4+ T-cells and the relative lack of T-suppresser cells (CD8-t T-cells) may lead to B-cell stimulation and polyclonal B-cell activation, explaining the numerous fully differenti- ated Ig-secreting cells (plasma cells) which we observed. This local production of antibodies may lead to immune-complex deposition. The pathophysiology of Behcet’s disease may thus be the result of combined immunologic mechanisms (humoral and cellular).

The two agents most frequently used in the treatment of the ocular manifestations of Behcet’s disease are oral corticosteroids and cyclosporine A. Both of these medications have inhibitory effects on T-lymphocytes, but have little, if any, effect on B-lymphocytes. Patients who do not respond adequately to treatment may have a component of humoral immunity not adequately treated by oral corticosteroids and cyclosporine A. In these patients one may con- sider the use of pulse intravenous corticosteroids or cyclophosphamide, which have inhibitory effects on both T- and B-lymphocytes.‘3 Future therapeutic ap- proaches for the ocular inflammation of Behcet’s disease may include antiadhesion molecule therapy, as this case has demonstrated the extensive adhesion molecule expression that occurs. In addition, the serum of patients with Behcet’s disease has been shown to respond to the retinal Santigen.” The re- lease of Santigen may be the result of retinal infarction or necrosis. This immunologic response to Santi- gen may be modulated by the induction of oral tolerance through the oral administration of Santi- gen. Both of these therapeutic modalities are being studied.

In conclusion, more immunopathologic studies will be needed to confirm the finding of prominent amounts of B-cells and adhesion molecules in eyes with Behcet’s disease. Immunotherapy of the ocular disease may need to be directed toward downregu- lating the effects of adhesion molecules as well as both CD4+ T-cells and B-cells.

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Reprint address: Roger K George, MD, Madigan Army Medi- cal Center, Department of Ophthalmology, Tacoma, WA, 98431. 5000, USA.

ocular immunopathology of behçet's disease

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