Immunopathology of Vitreous and Retinocchoroidal Biopsy in Posterior Uveitis

Immunopathology of Vitreous and Retinochoroidal Biopsy in Posterior Uveitis LESLIE

s.

FUJIKAWA, MD, JAN-PETTER HAUGEN, MD

Abstract: The authors used immunopathologic techniques to study vitreous and/or retinochoroidal biopsies from 23 patients with posterior uveitis unresponsive to conventional therapy or who had developed significant complications despite therapy. Results indicated that during active uveitis from many causes, T-helper cells predominated in the vitreous and retinochoroidal biopsies. Monocytes were not prominent constituents except in several cases of granulomatous etiology (e.g., syphilis and acute retinal necrosis). Class II major histocompatibility complex (MHC) antigens were increased on the retinal vascular endothelium, implicating an important role for these cells in the local cellular immune response. These results may be of great importance in our understanding of uveitis, as well as being helpful in categorizing posterior uveitis, permitting appropriate therapy to be given. Ophthalmology 1990; 97:1644-1653

The immunopathogenesis of most intraocular inflammatory disease is unknown and is the subject of intense current research. Diseases such as Behc;et's syndrome, pars planitis, and birdshot retinochoroidopathy are thought to be autoimmune diseases in which immune responses against autologous retinal antigens play an important role. 1•2 Many other uveitides are known to be caused by particular infectious agents (e.g., toxoplasmosis and acute retinal necrosis caused by herpesviruses).3 However, even in the diseases in which infection plays a major role, the associated immune response both against the infectious organism and against autologous retinal tissue plays a crucial role in the outcome of these diseases. Our current knowledge of the mechanisms which induce and perpetuate uveitis is largely indirect (i.e., through

Originally received: October 30, 1989. Revision accepted: July 2, 1990. From the Ocular Immunology Laboratory, Smith-Kettlewell Eye Research Institute, Pacific Presbyterian Medical Center, San Francisco. Presented in part as a poster at the American Academy of Ophthalmology Annual Meeting, Dallas, November 1987. Supported by NIH grant EY-06948, Bethesda, Maryland, Pacific Vision Foundation, San Francisco, Califomia, and Research to Prevent Blindness, Inc, New York, New York. Reprint requests to Leslie S. Fujikawa, MD, Ocular Immunology, PO Box 190922, San Francisco, CA 94119-0922.

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experimental animal models of uveitis, studies of blood and serum components obtained from patients with uveitis, and by the response ofuveitic diseases to certain types of antiinflammatory and immunosuppressive medications). Thus, it is believed that retinal antigens such as the retinal S antigen and interphotoreceptor retinoidbinding protein play an important role in the autoimmune response during uveitis since these are the antigens that cause severe uveoretinitis in experimental animals, and immune responses to these antigens can be demonstrated in patients with uveitis. 2,4,5 In addition, T-helper cells are believed to playa major role in the generation of many types of uveitis responsive to cyclosporine. 2 This medication has its greatest effect on T -helper cell function by blocking production of interleukin-2 and other lymphokines, thereby inhibiting active expression of the immune response. Recently, immunologic advances have allowed direct and precise analysis of biopsy tissues for the type of immunologic response, and immunopathologic studies have gained increasing importance as diagnostic aids in many areas of medicine. Thus, it is possible to define local immunologic responses, as well as to identify possible etiologic agents (e.g., viruses, fungi, etc.). Such studies, therefore, may allow a more accurate assessment of immunopathogenic events in uveitis than those indirect studies described above. In the current article, we report our results on the immunopathology of vitreous and/or retinochoroidal biopsies from 23 patients with posterior uveitis.

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Table 1. Uveitis Cases Case No.

Age (yrs)/ Race

1 2

61/W 32/W

3 4 5 6 7 8 9

13/W 7/W 35/W 43/W 36/B 48/W 72/W

10 11 12 13 14 15 16

32/W 78/W 36/B 77/W 30/B 52/W 60/W

17

18 19

56/0 27/W 42/W

20 21 22 23

44/0 28/W 65/W 45/W

Degree of Inflammation*

Diagnosis Pan uveitis Acute retinal necrosis, chronic phase Vitreitis Lens-induced uveitis Retinitis, ARC Retinitis, ARC Uveal effusion with pan uveitis Panuveitis Retinitis, rheumatoid arthritis on methotrexate Juvenile rheumatoid arthritis Vitreitis Sarcoid uveitis Pan uveitis Pan uveitis Vitreitis Pan uveitis, tuberculosis treated with isoniazid in past Panuveitis Retinal vasculitis Fuchs' heterochromic iridocyclitis Panuveitis Panuveitis Vitritis Retinitis, SIP heart transplant, on cyclosporine, azathioprine, corticosteroids

Procedure

4+ 3+

Vitrectomy Vitrectomy

4+ 1+ 4+

Vitrectomy Vitrectomy Retinochoroidal Retinochoroidal Retinochoroidal Vitrectomyt Retinochoroidal

3+ 2+ 2+ 4+

1+ 1+

biopsy biopsy biopsy biopsy

3+

Vitrectomyt Vitrectomy Vitrectomyt Vitrectomy Vitrectomyt Vitrectomyt Retinochoroidal biopsy

4+ 4+ 1+

Vitrectomyt Vitrectomy Vitrectomyt

2+

Vitrectomy Vitrectomy Vitrectomy Retinochoroidal biopsy

2+ 2+

1+ 1+

4+ 1+

3+

W = white; ARC = acquired immune deficiency syndrome-related complex; B = black; 0 = oriental. * Vitreous cell and haze. t Associated with pars plana lensectomy.

PATIENTS AND METHODS PATIENTS

Twenty-three patients referred to the Uveitis Service with posterior uveitis unresponsive to conventional therapy or who had developed significant complications despite therapy were included in this study. The spectrum of disease included severe autoimmune uveitis, chronic infectious uveitis (e.g., viral, fungal), and relatively inactive uveitis with complications from past inflammation despite therapy requiring the current surgical procedures to be performed. Cases of suspected bacterial infection were excluded from study. Table 1 summarizes these cases categorized as to the type of operative procedure which was performed. Seventeen vitrectomies with or without pars plana lensectomy were performed. These consisted of (1) nine primarily diagnostic vitrectomies, in which chronic fungal or viral infections (e.g., acute retinal necrosis) or reticulum cell sarcoma were suspected, and (2) eight primarily therapeutic vitrectomies, which included chronic uveitic con-

ditions such as juvenile rheumatoid arthritis, sarcoidosis, and idiopathic panuveitis. Retinochoroidal biopsies were performed in a selected group of six patients in whom retinal and uveal inflammation were particularly severe and resistant to all forms of therapy. Table 1 includes these six cases, and Figure 1 shows the fundus photographs from two cases. It is noteworthy that the majority of the cases of retinochoroidal biopsy demonstrated some degree of systemic immunosuppression, and that this contributed to the difficulty in the prior diagnosis and therapy of these cases. Most cases (20 of 23 cases) in our study were off all systemic steroids or other systemic immunosuppressive medication at the time of ocular biopsy. The medication either had not been working or was thought to be contributing to the patient's ocular problem and therefore was discontinued, often by the referring physician. The exceptions to this included three cases in which systemic therapy was unavoidable (case 9, the effects of methotrexate therapy were most likely still present since therapy was discontinued only several days before surgery; case 20, oral prednisone was necessary at the time of biopsy to prevent acute damage from severe uveitis; and case 23, 1645

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flammatory cell markers were attenuated by the therapy, especially the class II major histocompatibility complex (MHC) antigens, these markers were still highly positive in these cases (see Results section below), consistent with the activity of the uveitis. SPECIMENS

Vitreous biopsies were collected into sterile containers. Portions were sent for cultures, cytologic studies, and serologic studies. The remainder was used for the current immunopathologic studies. The vitrectomy specimen was spun down, and the supernatant was separated from the pelleted cells. Cells were resuspended in Hanks balanced salt solution and were plated onto gelatin-coated slides, using between 200 and 1000 cells per slide. These were allowed to air dry completely and were stored dry at - 20° C until use. Portions of the diluted supernatant fluids were concentrated down to approximate the original volume of the vitreous using an Amicon concentrator and an YMIO membrane (Amicon, Danvers, MA). Both unconcentrated and concentrated vitrectomy fluids were used for serologic or other studies, or were frozen in liquid nitrogen for future studies. Control vitreous from two patients without uveitis showed no inflammatory cells and therefore no staining for T cells, class II antigens, or other inflammatory cell markers. Retinochoroidal biopsies consisted of 3 X 4-mm tissue specimens which were divided into three separate parts. Two portions were sent for culture and electron microscopic studies, respectively. The third and largest portion was frozen immediately in OCT compound (Ames Co, Division of Miles Laboratories, Inc, Elkhart, IN) for the current immunopathologic studies. Frozen tissue sections were cut at 4-1lm thickness and placed onto gelatin-coated slides. Slides were stored dry at -20° C before use. PRIMARY ANTIBODIES

Fig 1. Fundus photographs of cases of retinochoroidal biopsies. Top, Case 9. Case of acute retinal necrosis demonstrates severe peripheral retinitis. Center, case 5. Resolution of severe retinitis after treatment for syphilis. Before therapy, no view of the fundus was obtained. Posterior pole is shown with visual acuity of 20/30. Bottom, case 5. Peripheral retina shows mild pigmentary changes.

Primary antibodies for immunopathologic study included mouse monoclonal antibodies directed against the various human inflammatory cell types (Table 2). The monoclonal antibodies listed were obtained from BectonDickinson (Mountainview, CA). Primary antibodies against infectious agents were mostly polyclonal reagents and were directed against cytomegalovirus, herpes simplex virus types 1 and 2 (Biogenex Laboratories, San Ramon, CA), as well as toxoplasmosis, syphilis, and herpes zoster virus (Electronucleonics, Inc, Columbia, MD). Monoclonal antibody against the human immunodeficiency virus (anti-p24) was obtained from Cytotech (San Diego, CA). IMMUNOHISTOCHEMICAL STAINING

immunosuppressive therapy to prevent rejection of a cardiac transplant included prednisone, azathioprine, and cyclosporine). In these cases, the systemic immunosuppressive therapy did not control the uveitis and in fact most likely contributed directly to the retinitis in cases 9 and 23. Although it is theoretically possible that the in1646

Slides were stained for inflammatory cell markers using an avidin-biotin complex immunoperoxidase technique. 6 Briefly, tissue sections were incubated with the primary antibody (monoclonal antibody), followed by biotinylated anti-mouse IgG (heavy- and light-chain specific) (Accurate Chemical Co, Westbury, NY), each for 15 minutes at 37°

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Table 2. Monoclonal Antibodies against Inflammatory Cells MoAb Directed against HLA-DR HLA-DO HLA Leu-1 Leu-3a Leu-2a Leu-14 Leu-M1 Leu-6 Leu-7 K chain X chain IL-2 receptor a PBL

=

Antigen Cluster Designation

Predominant Specificity Class II antigen on B cells, monocytes, macrophages, activated T cells Class II antigen distinct from HLA-DR Class I antigen T cells T-helperlinducer cells, monocytes, macrophages T-suppressor Icytotoxic cells B cells Monocytes, granulocytes Langerhans' cells, thymocytes T-cell and natural killer cell subsets K light chain bearing B cells X light chain bearing B cells Tac antigen

peripheral blood lymphocytes; IL-2

=

Normal Range of Lymphocytes 11% of PBL 10% of PBL 100% of PBL 73% of PBL 45% of PBL

CD5 CD4 CD8 CD22 CD15 CD1 CD57

28% of PBL 10% of PBL 0% of PBL 0% of PBL 10-25% of PBL 65% of B cells 35% of B cells 100% of Iymphoblasts

CD25

interleukin-2.

C. After a IS-minute incubation with avidin reagent and biotinylated horseradish peroxidase (Vector Laboratories, Burlingame, CA), sections were stained with 3,3'-diaminobenzidine (Sigma Chemical Co, St. Louis, MO), nickel

sulfate, and hydrogen peroxide. Slides were counterstained with 1% methyl green. Antisera directed against specific infectious agents were used in either the avidin-biotin complex immunoperox-

Table 3. Immunopathology of Vitreous BiopSies Case No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Diagnosis Pan uveitis, RIO RCS ARN, chronic Vitreitis, RIO chronic infection Lens-induced uveitis Retinitis, syphilis Retinitis, CMV Uveal effusion with pan uveitis Panuveitis Retinitis, ARN Juvenile rheumatoid Vitreitis, lens-induced uveitis Sarcoid uveitis Panuveitis, RIO RCS Panuveitis Vitreitis Pan uveitis Pan uveitis, RIO chronic infection Retinal vasculitis, RIO chronic infection Fuchs' heterochromic iridocyclitis Pan uveitis Pan uveitis, chronic fungal infection Vitreitis, RIO RCS Retinitis, SIP heart transplanttoxoplasmosis

Predominant Cell Type in Biopsy

Approximate TH:Ts Ratio in Vitreous

Class II MHC Antigens HLA-DR

HLA-DO

T cells T cells T cells T = macrophages

2.0 1.0 2.0

2+ 2+ 3+ 1+

1+ 2+ 2+ tr

Macrophages T cells T cells T cells B cells T cells T cells T cells Macrophages T cells T cells T cells

2.0 2.0 2.0

3.0 3.0

1+ 2+ 4+ tr 1+ 2+ 2+ tr 3+ 1+ 1+ 4+

1+ tr 3+ 0 tr 2+ tr tr 2+ tr tr 3+

Macrophages

1.0

tr

tr

T cells T cells

1.0 2.0

2+ 4+

tr 4+

T cells T cells

1.5

tr 3+

0 2+

3.0 2.0

TH:Ts ratio = T-helper to T-suppressor ratio (normal peripheral blood = 2.0); MHC = major histocompatibility complex; RIO = rule out; RCS = reticulum cell sarcoma; ARN = acute retinal necrosis; CMV = cytomegalovirus. 1647

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Vitreous Cells

Vitreous Cells

III

4+

TeaUs

2+

• 0

BeaUs Maerophagas

Fig 2. Immunologic marken; on vitreous cells from 21 cases of posterior uveitis. Top left. T cells were predominant in most of the biopsies. Top right. B cells and macrophages were not promi· nent constituents in these biopsies. Bottom left. staining for HLA-DR (class II MHC antigen) showed positivity for cells including both lymphocytes and macrophages. Bottom right. staining for HLA-DQ (a separate class II MHC antigen) was slightly less prominent than that for HLA-DR.

1+ tr

o

4+

•

4+

HLA·DR

3+

3+

2+

2+

1+

1+

tr

tr

0

0 0

2

3

4

5

6

7

8

9

10

~

0

# Cases

idase technique or an immunoperoxidase technique involving streptavidin-peroxidase conjugate in similar technique to that described above. The differences in technique involve (I) substitution of streptavidin-peroxidase for the avidin-biotin-peroxidase complex, and (2) the use of 3amino-9-ethylcarbazole in N,N-dimethylformamide as the substrate. This yields a red-brown color as the reaction product. These slides were counterstained with hematoxylin solution. DATA EVALUATION

Slides of vitreous cells were evaluated as to the percent of cells stained positively for each antigen examined. Two hundred cells per slide were counted, and each slide stained for a separate antigen was given a numerical grade on a scale of 0 to 4 as follows: 4+ = more than 80% of the cells showed positive staining; 3+ = 60 to 80% were positive; 2+ = 40 to 60% were positive; 1+ = 20 to 40% were positive; trace = 5 to 20% were positive; and 0 = less than 5% were positive staining or no staining. Approximate T -helper to T -suppressor ratios (TH:Ts) were calculated from the above grading system and reported as less ihan 1.0, 1.0, 1.5, 2.0, 2.5, and 3.0, and more than 3.0. Retinochoroidal biopsies were evaluated as to the predominant inflammatory cell type within the tissue and as to the degree of class II antigen expression: 4+ = widespread positive staining of the resident cells (e.g., retinal 1648

2

3

4

5

6

7

HLA·DQ

8

9

10

# Cases

vascular endothelium, in addition to staining of inflammatory cells); 3+ = focal staining of resident cells; 2+ = very limited staining of resident cells; 1+ = positive staining limited to inflammatory cells; and 0 = no staining.

RESULTS VITREOUS

Table 3 and Figure 2 summarize the results of immunologic markers on vitreous cells. T cells were the predominant lymphocyte in all cases except one (case 11), consistent with a significant cell-mediated immune response. Furthermore, T -helper cells predominated over T -suppressor cells in most cases, consistent with active inflammation, possibly of a delayed-type hypersensitivity response. In the three cases in which the TH:Ts ratio was less than 2:1 (cases 2, 19, and 20), the TH:Ts ratio was approximately equal to one, indicating equal numbers of T -helper and T -suppressor cells. There were no cases in which T -suppressor/cytotoxic cells predominated over Thelper/inducer cells. Clinical severity of uveitis correlated partially with the T H:Ts ratio (i.e., all severe uveitis cases with 4+ vitreous cell and haze [cases 1,3,9, 17, 18, and 21] had T H:Ts ratios of 2: 1 or greater). B cells comprised a minority ofthe lymphocytes in the biopsy specimens (Fig 2, top right; Table 3). There was only one case (case 11) in which B cells were present in

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Table 4. Immunopathology of Retinochoroidal Biopsies Age (yrs)

Diagnosis/Infectious Agent

35

Retinitis, ARC/syphilis

Penicillin

43

Retinitis, ARC/ cytomegalovirus

36

Treatment

Predominant Cell Type in Biopsy

Class II MHC Antigens (R/C/V) HLA-DR

HLA-DQ

Macrophage

2+/2+/-

2+/2+/-

DHPG

T cells

H/H/-

H/H/-

Uveal effusion with panuveitis/no organism identified

Systemic steroids

T=B=macrophage

4+/4+/H

tr/tr/H

72

Retinitis, rheumatoid arthritis on methotrexate/herpes simplex virus

Acyclovir

T-helper cells

4+/3+/4+

4+/3+/3+

60

Pan uveitis, tuberculosis in past/no organism identified

Systemic steroids

T cells

H/2+/H

tr/H/tr

45

Retinitis, SIP heart transplant on cyclosporine, azathioprine, steroids/ toxoplasmosis

Pyrimethamine/ sulfa

T-helper cells

4+/4+/3+

2+/H/2+

R/C/V = retina/choroid/vitreous; ARC = acquired immune deficiency syndrome-related complex; DHPG = 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

slightly greater numbers than T cells. In this case, the overall vitreal cellular inflammation was graded clinically as being only 1+ at the time of surgery (1 + vitreous cell and haze), which was one of the least acutely active cases of uveitis included in this study. Natural killer cells were not seen by staining for the Leu-7 antigen. Although one may assume that natural killer cells comprised a portion of the non-T, non-B lymphocytes (null cells), this component formed only a small minority of the total cellular population. Macrophages were less prominent than T cells in the vast majority of cases (Fig 2, top right; Table 3). There was one case (case 4, lens-induced uveitis) in which the numbers ofT cells was equal to that of macro phages, and three cases (case 7, uveal effusion with panuveitis; 15, idiopathic vitreitis; and 19, Fuchs' heterochromic iridocyclitis) in which macrophages were slightly more numerous than T cells. Most of the inflammatory cells stained positively for the class II MHC antigen HLA-DR, including both macrophages and lymphocytes (Fig 2, bottom left). Slightly fewer cells stained positively for the separate class II antigen HLA-DQ (Fig 2, bottom right). Of the diagnostic vitrectomies, there were two cases of acute retinal necrosis and one chronic fungal infection which were diagnosed by biopsy (cases 2, 9, and 21, respectively). Three possible reticulum cell sarcoma cases yielded negative vitreous biopsies (cases 1, 13, and 22). Two cases of suspected lens-induced uveitis were con-

firmed by the demonstration of significant levels of antilens antibodies by indirect immunofluorescence studies (cases 4 and 11). In patients in whom no infectious organism was found, immunosuppressive therapy was given. Significant improvement in inflammation and visual acuity was noted in the great majority of these patients postoperatively. RETINOCHOROIDAL BIOPSIES

Table 4 summarizes the immunopathology of the cases ofretinochoroidal biopsies. T cells (mostly T-helper cells) were the predominant inflammatory cell type in most of the retinochoroidal biopsies. Many of these cells stained positively for the interleukin-2 receptor, indicating that they were in an activated state. B cells were present in small numbers. Natural killer cells were not present to any significant degree in any of the retinochoroidal biopsies. Macrophages were not generally present in great numbers, but were found to be increased in two cases of granulomatous etiology, one case of syphilis and one of acute retinal necrosis (Fig 3; Table 4). Class II MHC antigens were greatly increased on endothelial cells of the retina and choroid (Fig 3; Table 4), as well as being present on inflammatory cells within the infiltrates. Staining for HLA-DR was more widespread than for HLA-DQ, although staining for both components was quite strong. Staining of the retina was greater than that of the choroid in five of six cases (Table 4), indicating

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Fig 3. Immunopathology of retinochoroidal biopsies. Top left. case 5. Class II antigens are expressed on the retinal vascular endothelium in the retinochoroidal biopsy from a patient with retinitis and retinal vasculitis (stained for HLA-DQ). The brown-black color identifies positive staining. The nuclei are counterstained green. Bottom left. case 9. Vascular endothelium of the choroid stained positively for class II antigens (stained for HLADR). Top right. case 9. Granulomatous response is seen in the choroid from a case of acute retinal necrosis. T cells are seen in the choroid (top right), which surround a focus of macrophages, shown in the serial section in the bottom right figure (Leu I and Leu MI, respectively) (original magnification, X513).

more severe retinal inflammation than choroidal inflammation. The degree of vitreous cellular staining for class II MHC antigens most closely approximated that of the retina (Table 4). Direct immunopathologic evidence of infectious organisms was demonstrated in the ocular tissues of three of six infectious retinitis patients and confirmed serologically in the remaining patients (Fig 4; Table 4). These patients were treated with specific anti-microbial therapy (Table 4). It is of importance that cultures, including viral cultures, were invariably negative for infectious organisms, despite the fact that antigens of the infectious organisms

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were clearly demonstrated in the tissues. In patients in whom no infectious organism was found, immunosuppressive therapy was given. Significant improvement in inflammation and visual acuity was noted in most of these patients. OTHER OCULAR BIOPSY

Specimens of iris and conjunctiva were obtained in selected patients and were stained for the presence of inflammatory cell markers. In general, the profile of inflammatory cells was similar to that described for the vitreous

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and retinochoroidal biopsies with a predominance of Thelper cells and few B cells and macrophages. Langerhans' cells were seen in the basal epithelium of the conjunctiva. Natural killer cells were not demonstrated in these tissues. There was a significant vasculitic component in the conjunctiva overlying scleritis associated with panuveitis (e.g., case 17) (Fig 5).

DISCUSSION The current study demonstrates the immunopathology of vitreous and retinochoroidal biopsies from patients with a wide variety of active uveitic conditions. Results showed that T cells (mostly T-helper cells) formed the predominant inflammatory cell type in the vitreous and retinochoroidal biopsies. B cells were relatively scarce in most of the specimens. Monocytes and macro phages were not the predominant constituent of the inflammatory infiltrate in most cases, but were present in increased numbers in the retinochoroidal biopsies from two cases of granulomatous etiology (i.e., syphilis and acute retinal necrosis). Class II MHC antigens were increased on endothelial cells of the retina, suggesting an important role for these cells in the immunopathogenesis of uveitis. These results may contribute significantly to our understanding of mechanisms of uveitis. The fact that Thelper cells were the predominant inflammatory cell type in most of the cases of uveitis is consistent with the central role for T cells as regulators of in vivo immune responses, and in many cases also may indicate a delayed-type hypersensitivity response. 2 Exudation of T-helper cells and other inflammatory cells into these tissues occurs both from breakdown of blood-ocular barriers and from the preferential accumulation of these cells in response to chemotactic inflammatory stimuli, such as gamma interferon and interleukin-2, important mediators of cell-mediated immune responses. 7,8 These mediators serve to amplify the local immune response by activating and recruiting nonantigen-specific inflammatory cells to the site of inflammation. 7,8 Thus, T H:Ts ratios in the vitreous specimens did not necessarily correlate with those in the peripheral blood and in several cases were increased above levels normally found in the blood (Table 3). Furthermore, since T -helper cells are the greatest producers of gamma iriterferon and interleukin-2, one would expect that the patients in whom the T H:Ts ratio in the vitreous was the highest (cases 12, sarcoidosis; 17, panuveitis; and 18, retinal vasculitis) contain large increases in gamma interferon and interleukin-2 in the vitreous. This is an area of current investigation in the laboratory. Class II MHC antigens are known to be induced on the surfaces of cells by lymphokines secreted by activated T cells, specifically gamma interferon. 7 The fact that Thelper cells were the predominant cell type in the biopsy specimens of vitreous, retina, and choroid is consistent with the observed large increase in class II antigens seen on the retinal vascular endothelium in this study. Class

II MHC antigens playa crucial role in the triggering of T-helper cells and therefore in the triggering and perpetuation of the immune response. Normally, class II antigens expressed on the surface of the macrophage, the traditional antigen-presenting cell, bind to antigen and trigger the T cell. 9 In uveitis, the "aberrant" expression of class II molecules on the retinal vascular endothelium may allow these cells to function as antigen-presenting cells, leading to the local immune response in the retina. Results of the current study are consistent with our previous results in which class II MHC antigens were expressed on the retinal vascular endothelium during experimental autoimmune uveoretinitis, in vitro upon exposure of the retinal vascular endothelium to gamma interferon, and in vivo upon systemic or intravitreal gamma interferon administration.tO,11 These results suggest a critical reciprocal relationship between the T -helper cells in the inflammatory infiltrate and the class II-positive retinal vascular endothelium: class II-positive retinal vascular endothelial cells present autologous retinal antigen to antigen-specific T -helper cells, leading to increased production of lymphokines, including gamma interferon. This then causes more class II MHC antigen expression on resident ocular cells, causing continued expansion of the immune response. These immune mechanisms involving T-helper cells, gamma interferon, and class II antigens may be essential in causing the local immune response during uveitis. We are currently attempting to control the expression of class II MHC antigens on the retinal vascular endothelium to control sight-threatening retinal inflammation. In this regard, we have recently studied the effects of systemic cyclosporine treatment on class II antigen expression in ocular tissues? We have found a coordinate decrease in class II antigen expression and local inflammation with cyclosporine treatment. These results indicate potent ocular actions of cyclosporine and suggest that prevention of increased class II antigen expression may be one of the mechanisms by which cyclosporine exerts its immunosuppressive effect. In addition to T -helper cells and class II-positive retinal vascular endothelium, macrophages play an important role in the generation of uveal and retinal inflammation. Macrophages are the traditional antigen-presenting cell and are a prominent cell type in granulomatous inflammations, in which they form the core of the granulomas. 12 Macrophages were found to be significantly increased in the vitreous in selected cases oflens-induced uveitis, uveal effusion with pan uveitis, idiopathic vitreitis, and Fuchs' heterochromic iridocyclitis. Furthermore, macrophages also were increased in retinochoroidal biopsies from cases of granulomatous etiology induding syphilis and acute retinal necrosis. In these cases, the macrophages formed the core of the granulomas which were surrounded by activated T cells (mostly T-helper cells). The results of our study suggest a central role for the macrophage in the generation of these uveitides. B cells and T-suppressor cells comprised a minority of the inflammatory cells in the vitreous biopsies of the cur1651

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Fig 4. Antigens of infectious organisms in the retina. Left. case 6. Cytomegalovirus is found in the retina of a patient infected with the human immunodeficiency virus. The red-brown color identifies positive staining. Top right. case 5. Human immunodeficiency virus antigens are found on the retinal vascular endothelium of the retinochoroidal biopsy specimen (black stain). Bottom right. serial section of the retina stained positively for factor VIII antigen (black stain), confirming that these cells are retinal vascular endothelial cells (original magnification, X690). Fig 5. Immunopathology of conjunctiva. Left. case 20. T -helper cells surround inflamed vessels of conjunctiva in a patient with panuveitis and scleritis (stained for Leu 3a, black stain). Right. serial section shows class II-positive vascular endothelium (HLA-DR) (original magnification, X367).

rent study, suggesting that they did not playa major role in the generation of local inflammation in most of these cases of uveitis. The single case from our study in which B cells did predominate in the vitreous was obtained from one of the least active cases of uveitis in our study group. Furthermore, T suppressor/cytotoxic cells were most prominent only in cases of relatively chronic disease (chronic acute retinal necrosis, Fuchs' heterochromic iridocyclitis, and idiopathic panuveitis). In these chronic, less-active uveitis cases, T-suppressor cells serve to keep the inflammation under control, whereas T -helper cells may promote inflammation in more active uveitis cases, as has been demonstrated for other types of ocular inflammation. 13 Thus, B cells and possibly T -suppressor cells are increased in ocular tissues during relatively inactive cases of uveitis, in which the principal immune and inflammatory response due to T -helper cells has subsided. Natural killer cells also were not a significant part of the inflammatory cell population, and in fact, were often difficult to demonstrate in the tissues. These cells are thought to be important in natural immunity to tumors (immune surveillance) and to certain infections (e.g., viral 1652

infection), and they are recruited by T-helper cells, secreting interleukin-2, into the site of inflammation. 14 Once recruited, these natural killer cells produce gamma interferon, adding to the gamma interferon being made by the T cells, and thus possibly contributing to amplification of the immune response in vivo. The results of the current study do not necessarily support this hypothesis, since we were unable to demonstrate significant numbers of natural killer cells using the anti-Leu-7 monoclonal antibody in either vitreous, retina, or choroidal tissues. This is despite the fact that there were large numbers of T -helper cells in all of these tissues, presumably secreting significant amounts of interleukin-2. The interleukin-2 receptor (Tac antigen) was found to be increased on cells in the biopsy tissues, presumably primarily on T cells. This indicates that these cells were in an activated state, compatible with the degree of uveitis. The interleukin-2 receptor is comprised of at least two different polypeptide chains, including interleukin-2 receptor a (Tac antigen) and a separate protein known as interleukin-2 receptor fl. Resting T cells and natural killer cells may normally express interleukin-2 receptor fl, which

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IMMUNOPATHOLOGY OF POSTERIOR UVEITIS

binds to interleukin-2 with intermediate affinity. Interleukin-2 binding to this receptor leads to interleukin-2 a gene expression and the generation of a high affinity interleukin-2 receptor comprised of both a and (3 chains. 15 Therefore, interleukin-2 is partially responsible for the increase in expression of the Tac antigen and thus of the high affinity interleukin-2 receptor. This renders the T cell more susceptible to the actions of interleukin-2, thereby leading to amplification of the immune response. In addition to yielding information on immunopathogenic mechanisms, the results of the current studies further demonstrate the importance and utility of ocular biopsy as a diagnostic procedure in uveitis. In particular, retinal or retinochoroidal biopsy is a highly useful diagnostic procedure for a selected group of patients with severe uveitis, including (1) cases of immunosuppression, such as acquired immune deficiency syndrome, in which diagnostic clinical patterns of ocular inflammation may be obscured due to lack of an effective immune response, and in which serologic studies are inaccurate and vitreous biopsy alone may be inconclusive, and (2) other uveitides in which severe inflammation persists despite all attempts at diagnosis and therapy. 16 As shown in the current study, the results of vitreous and retinochoroidal biopsies allow definitive diagnosis to be made in selected cases of sightthreatening uveitis and rule out infectious causes or malignancy in others. Retinochoroidal biopsy as well as other ocular biopsy allows study of disease processes during active disease, as opposed to specimens obtained postmortem or upon enucleation after significant long-term damage has occurred to ocular structures. Therefore, such biopsies are more highly indicative of pathogenic events leading to ocular immune disease. Furthermore, it is clear that immune cell profiles were not restricted to specific diagnostic entities in the current study and therefore there was a significant component of nonspecificity to the local ocular immune response. Profiles of inflammation are still being described in uveitis, and as our experience grows, it will be possible to group larger numbers of cases within diagnostic categories, thus distinguishing various immune responses in different types of uveitis. The current results

may serve as an important basis for such future studies on the immunopathogenesis of specific uveitis entities.

REFERENCES 1. Kraus-Mackiw E. O'Connor GR. Uveitis: Pathophysiology and Therapy. 2nd ed. New York: Thieme Medical Publishers. Inc. 1986; 78-101. 2. Fujikawa LS. Advances in immunology and uveitis. Ophthalmology 1989; 96: 1115-20. 3. Culbertson WW. Blumenkranz MS. Haines H. et al. The acute retinal necrosis syndrome. Part 2: Histopathology and etiology. Ophthalmology 1982; 89:1317-25. 4. Donoso LA. Merryman CF. Sery TW. et al. Human IRBP: characterization of uveitopathogenic sites. Curr Eye Res 1988; 7:1087-95. 5. Faure JP. Autoimmunity and the retina. Curr Top Eye Res 1980; 2: 215-302. 6. Hsu S-M. Raine L. Fanger H. The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. 7. Vilcek J. Gray PW. Rinderknecht E. et al. Interferon gamma. A Iymphokine for all seasons. Lymphokines 1985; 11: 1-32. 8. Mertelsmann R. Welte K. Human interleukin 2: molecular biology. physiology and clinical possibilities. Immunobiology 1986; 172:40019. 9. Unanue ER. Allen PM. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 1987; 236:551-7. 10. Fujikawa LS. Reay CR. Morin ME. Class II antigens on retinal vascular endothelium. pericytes. macrophages and lymphocytes of the rat. Invest Ophthalmol Vis Sci 1989; 30:66-73. 11. Fujikawa LS. Class II histocompatibility complex antigens on the retinal vascular endothelium in uveitis. Proc Int Soc Eye Res 1988; 5:103. 12. Hunninghake GW. Role of alveolar macrophage- and lung T cell-derived mediators in pulmonary sarcoidosis. Ann NY Acad Sci 1986; 465:8290. 13. Bhan AK. Fujikawa LS. Foster CS. T-cell subsets and Langerhans cells in normal and diseased conjunctiva. Am J Ophthalmol1982; 94: 205-12. 14. Ortaldo JR. Herberman RB. Heterogeneity of natural killer cells. Ann Rev Immunol1984; 2:359-94. 15. Lowenthal JW. Greene WC. Contrasting interleukin 2 binding properties of the a (p55) and {3 (p70) protein subunits of the human high-affinity interleukin 2 receptor. J Exp Med 1987; 166:1156-61. 16. Fujikawa LS. AIDS and the Eye. Philadelphia: WB Saunders. 1988; 81-90.

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