Autoimmunity, 1992, Vol. 13, pp. 303-309 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
IMMUNOPATHOLOGY OF EXPERIMENTAL AUTOIMMUNE UVEORETINITIS IN PRIMATES YUJIRO FUJINO*, QIAN LI, HUM CHUNGT, NAOFUMI HIKITA, ROBERT B. NUSSENBLATT, IGAL GERY and CHI-CHAO CHAN tuhorutory of Immunology, Nutiotzal Eye Institute. Bethesdu, Maryland, USA
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(Received April 3 , 1 9 9 2 ; in final form June I S . 1992)
The eyes and pineal glands from 10 monkeys immunized with S-antigen were studied using routine histopathological and immunohistochemical techniques. Seven out of 10 animals developed uveitis between 19 and 33 days after the initial immuniration. Histopathology of the eyes harvested 70 days after immunization showed moderate to marked uveoretinitis, subretinal fibrosis, retinal necrosis and gliosis. The pineal glands demonstrated chronic pinealitis. The infiltrating cells were both CD3 and CD19/CD22 lymphocytes with a ratio of 1.4 in the eye and 2.2 in the pineal gland. The ratio of CD4 to CD8 lymphocytes was 1.5: I . MHC Class I1 antigens and adhesion molecule (ICAM-I) were observed on resident cells. The influx of B lymphocytes and the formation of subretinal fibrosis differentiate the disease in the monkey from that in the rat and mouse. These findings are similar to Vogt-Koyanagi-Harada syndrome and subretinal fibrosis with uveitis syndrome in human. KEY WORDS: Experimental autoimmune uveoretinitis, pinealitis, primate, subretinal fibrosis, B lymphocyte, ocular inflammation.
INTRODUCTION Experimental autoimmune uveoretinitis (EAU) can be induced in various species by active immunization with ocular antigens, in particular the retinal soluble antigens (S-Ag)',' and interphotoreceptor retinoidbinding protein (1RBP)j. The clinical presentation, histopathological findings and cellular immune responses of EAU resemble certain human uveitides4-'. The detailed clinical and histopathological manifestations may vary from species to species, EAU in primates is characterized clinically by vitritis, retinal vascular sheathing, focal deep chorioretinal yellowish lesions, retinal hemorrhage and edema, localized exudative retinal detachment and papilledema'-'''. The histopathological changes of the eye include loss of the photoreceptor outer segment, retinal periphlebitis, retinitis, diffuse or multi-focal inflammatory cellular infiltration in the choroid and Dalen-Fuchs nodule formation'-"'. The cellular histopathology of the pineal gland shows focal marked lymphocytic infiltration and vasculitisx. These features of the ocular disease mimic certain uveitic syndromes in humans, such as sympathetic ophthalmia, Dr. Fujino is now at the Department of Ophthalmology, University of Tokvo. Tokvo. I Jaoan. . t D r ~Chung is now at the Department of Ophthalmology, Seoul University, Seoul, Korea. Correspondence to: Chi-Chao Chan, M.D., Building 10, Room ION206, NIH/NEI, Bethesda, MD 20892, USA. Telephone: (301) 496-1243; FAX: (301)402-0485.
Vogt-Koyanagi-Harada syndrome (VKH) and birdshot retinochoroidopathy. Immunized monkeys can demonstrate both cellular and humoral responses to the immunizing retinal antigen by the lymphocyte in vitrn proliferative response and the anti-retinal antibodies in the serum, findings which have also been reported in some uveitic patients",". The present study was aimed at examination of the immunopathology of EAU in primates, which has not been described previously.
MATERIALS AND METHODS Animals Ten rhesus monkeys (Macaca mulatta; six male, four female) were utilized in this study. All were obtained from National Institutes of Health, USA approved random sources and housed in environmentally controlled rooms with 12-hr light and dark cycle. All animals were provided with food and water ad libitum. Body weight and foodtwater intake were monitored daily. The study was approved by the institutional animal care and use committee and complies with the Public Health Policy on the Humane Care and Use of Laboratory Animals. Immunization S-Ag was prepared using bovine retinas according to
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the methods of Dorey et ~ 1 . The ' ~ antigen was emulsified (1:l) in complete Freund's adjuvant (CFA; Difco, Detroit, MI) containing Mycohacterium tuherculosis H37Ra at a concentration of 1.25 mglml. The emulsion was injected aseptically into multiple intradermal sites over the dorsal thoracic region of each monkey. The injected volume was limited to 4 0 p I per site and sites were spaced approximately 2.5-3 cm apart. A dosage of S-Ag given to the animal was 2 0 p g per kg body weight. The animals were immunized again by the same procedure 14 days later.
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EAU evaluation Ocular examinations, including slit lamp examination of the anterior segment and dilated funduscopic examination of the posterior segment, were performed twice a week. Each eye was classified as follows, based on the clinical presentation characteristics: -, no inflammation; +, inflammation limited to the posterior segments; ++, inflammation involving both anterior and posterior segments as well as mild lid swelling; +++, ocular inflammation extending into the orbit. Photographs of the eyes, including fluorescein angiography, were taken at the onset of the disease and one or two weeks later. All monkeys were sacrificed on day 70 after the initial immunization and the eyes and pineal glands were dissected out for study.
Technique of immuntihisjopatholt~~y Freshly enucleated eyes and pineal glands were bisected. One half was fixed in 10% formalin and processed for routine paraffin embedded sections. The other half was snap frozen in optimal cutting temperature compound (O.C.T., Miles Scientific, Naperville, IL) and stored at -70°C for study. Serial 4-6pm frozen sections were mounted on gelatinized slides. One frozen tissue section from each specimen was stained with standard hematoxylin and eosin. The adjacent sections were stained by the avidin-biotinperoxidase complex method14. The primary monoclonal antibodies were OKT4A (Ortho Lab, Raritan, NJ) for CD4, OKT8F (Ortho Lab, Raritan, NJ) for CD8, Leu M5 (Becton Dickenson, Mountain View, CA) for CDIlclCD18, Leu 14 (B D) for CD22, ICAM-1 (kindly provided by Dr Alfred Singler) for intracellular adhesion molecule - 1 , and HLA-DR (Dako, Carpinteria, CA) for MHC class I1 antigens. Mouse ascites fluid containing 1-2 p g non-specific IgG per milliliter served as a control. The secondary antibody was biotin conjungated horse anti-mouse IgG (Vector Lab, Burlingame, CA).
Evaluation of immunopathology Positive cells, which were stained bluish-black, were
counted and averaged in the same five anatomical areas in each section in order to minimize sampling error. The staining intensity for each marker was scored on an arbitrary scale from 0 to 4 as foflows: 0, as score of 0; 1-10 cells per 40xhigh power field (HPF), as score of 0.5; 11-50 cells per 40xHPF, as score of I+; 51-150 cells per 40xHPF, as score of 2+; 151-250 cells per 40xHPF, as score of 3+; >250 cells per 40xHPF, as score of 4+. The ratio of cellular counts between different subsets were calculated and recorded. RESULTS
Clinical manifestation Seven out of 10 monkeys developed clinical EAU with the onset date between 19 to 33 days after the initial immunization. The remaining three monkeys (9992B, 505C, 3) had no clinical sign of disease. The intensity of disease correlated well with the time of onset (Table 1). Three of the affected animals with early onset dates of EAU presented with orbital inflammation in addition to severe panuveitis, hyphema and papilledema (Figure 1A). Other clinical signs in these seven affected monkeys were retinal vawular sheathing, retinal hemorrhage, edema and exudative detachment, chorioretinal infiltration and scar, vitritis and iridocyclitis. These findings were confirmed by fluorescein angiography. Anterior segment involvement, characterized by conjunctival chemosis, flare and cells in the anterior chamber, anterior and posterior synechiae and cataract formation, were also observed. The ocular inflammation continued to progress (Figure 1B). By day 70 post immunization, affected animals usually developed severe synechiae and cataracts which obscured fundus details and precluded fundus photography.
Table 1 Clinical presenlsiion of S-Ag immunized monkey. Monkey N o .
Se.r
EAU orrsrt duy
EAU s c ~ r r i t y
F913 88 I9SQ 662D 99938 69N 248H 99928
M M F M M M F M F F
19
+++
S05C
3
21 22 24 26 33 33
+++ +++ ++ ++ + +
-
-
-
-
-
-
UVEORETINITIS IN PRIMATES
Immunopathotogicaffindings Eye
cytic and granulomatous infiltrations. Subretinal fibrosis was identified in three monkeys (9993B, 69N, 248H) and papilledema was present in two (F913 and 88). Immunohistochemical staining disclosed the predominant infiltrating cells to be lymphocytes, with a ratio of 1.4: 1 between T and B cells (Figure 2). B cells were most prominent in those eyes with subretinal fibrosis. The ratio between CD4 and CD8 T lymphocytes was approximately 1 : 1. The infiltration was less severe in the monkeys with marked retinal necrosis
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The histopathological changes in eyes of affected monkeys resembled those described in our previous report" and included retinal edema, hemorrhage, necrosis, gliosis, retinitis and periphlebitis. The retinal pigment epithelium showed marked attentuation alternating with hypertrophy, migration and disruption. The choroid was thickened by marked lympho-
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Figure 1 (A) Funduscopic examination of Monkey 19SQ. 35 days post immunization revealed optic disc swelling, retinal hemorrhages, exudates and vascular sheathing. ( B ) Funduscopic examination of Monkey 993, 42 days post immunization showed retinal vascular sheathing and choroidal lesions; three weeks later (63 days post immunization) severe rubeosis iridis and cataracts had developed.
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Figure 2 Immunohistochemical staining showed both T and B lymphocytic infiltration in one subretinal lesion. ( A ) CD4 positive cells (arrows).
and gliosis (Table 2 ) . A few T lymphocytes were identifed in the choroid of two of the three monkeys without clinical EAU. MHC Class 11 HLA DR antigen was expressed on inflammatory cells and the ocular resident cells in the areas of inflammation and/or necrosis (Figure 3 ) , in particular the vascular endothelia, retinal pigment epithelia and glial cells. In contrast, ICAM-1 was observed only on the vascular endothelia and retinal pigment epithelia where lymphocytic infiltration was present. It is interesting to note that DR antigen, but not ICAM-I. was also exmessed on ocular resident cells of the three monkeis which failed to develop clinical EAU.
Pinral gland The pineal glands of all affected animals and two of the three unaffected monkeys were available for study. The size of the affected pineal glands was much greater than the unaffected ones. Focal moderate to marked lymphocytic and monocytic infiltration was
Figure 2 Immunohistochemical staining showed both T and B lymphocytic infiltration in one subretinal lesion. (B) CD22 positive cells (arrows). (R, retina, C, choroid; ABC staining, x200.)
seen in all of these pineal glands (Table 2 ) . In contrast to the ocular findings, T lymphocytes, in particular CD4 cells rather than B cells,'were by far the predominant infiltrating cell qualitatively. Numerous macrophages (CDI Ic/CD18) were also present in the infiltrate (Figure 4). Both MHC class II HLA DR antigen was expressed on the vascular endothelia of all affected monkeys and one unaffected monkey with trace macrophage infiltration. As in the eye, ICAM-1 was expressed on the vascular endothelia of the affected animals only. DISCUSSION The present study illustrates the immunopathology of S-Ag induced EAU in the monkey. Seven of 10 monkeys developed clinical EAU characterized by chorioretinal involvement as described previously in the I i t e r a t ~ r e ~ - " ~ Similar ~'~. features have also been described in primates immunized with S-Ag derived peptide" and IRBP derived peptidesI7. Our data indi-
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307
Table 2 ~mmunopdtho~ogica~ gradings of S-Ag immunized monkey. Monkey NO.
CD4 OKT4A
CD8
CD I I C/I 8
OKT8F
MS
CD 19,20,22 Leu14
CDS4 ICAM- I
MHC Class I1 HLA-DR
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
2+/2+ 1+/3+ 2+/2+
1+/1+ 1 +/2+
0.5/1+
3+/+ 2+/3+/ I + 4+/2+ 1 +/2t -/-/ND -I-
1+/1+ 1+/0.5
2t/2+ 2+/2+ 2+/2+ 3+/1t 1+/3+ 4+/2+ 3+/2+ I +/o.s I+/ND
+
F913
I +/2+
I +/I
8x
1 +/2+
1+/1+
195Q 662D 9993B 69N 248H 9992B 50SC 3
2t/2+ 2+/2+/2t 2t/2+ 0.5/2+
2+/1+
-1-
1 +/o.s
I +/2t 2+/ I + I t/2+ 0.y0.5/ND
0.5/2+ I +/2+ -/3+ -10.5 -/ND -/-
-I-
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0.5/ND -/-
1+/0.5
Figure 3 Immunohistochemical staining showed expression of HLA-DR on inflammatory cells in the choroid. (ABC staining, x 200. )
care that T lymphocytes are the predominant infiltrating cells in both the eye and pineal gland, findings which are similar to the immunopathology of the eye in sympathetic ophthalmia’x-” and VKH”’.23.Interestingly, unlike what the present report indicates for the primate, infiltration with B lymphocytes is rarely
I +12+
2+/0.5 3+/2+
o.s/o.s -1-/ND -/-
-1-
identified in EAU in the rat and m o ~ s e ’ ~ - ’ and ~ , has not been associated with subretinal fibrosis. In the mouse EAU model, which can present with chronic focal chorioretinal lesions and subretinal neovascularization, a relative mild increase of B lymphocytes at the later stages of the disease has been reported”. In the guinea pig EAU, B cells were predominant in the end stage of the lesion”. The influx of B lymphocytes has also been observed in the late stage of the pulmonary inflammatory response to intratracheal silica in mice, when fibrosis occurs in the lung’*. A relative increase of B lymphocytic infiltration occurs in the multifocal choroiditis2’, subretinal fibrosis and uveitis syndrome in humans”’-”. This syndrome is a type of posterior uveitis characterized by choroidal inflammatory lesions with subsequent development of progressive subretinal fibrotic tissue’’. The immunopathology of this syndrome shows that the subretinal fibrotic tissue consists of a mixture of collagen, retinal pigment epithelia and glial cell proliferation, with a primarily B cell infiltration in the choroid and retina’”,”. This immunohistopathological process is similar to the findings in the present EAU monkey study. Expression of MHC class I1 antigen and adhesion molecule has been observed on the ocular resident cells of other animals with EAU3’.”, as well as some human eyes with uveitis35.3h.In the eyes of the monkeys without clinical EAU in the present study, the resident cells and a few T lymphocytes stained positively for MHC class 11, but there was no staining for adhesion molecules. This observation supports the hypothesis that expression of adhesion molecules on ocular resident cells is critical to the development of clinical EAU. Adhesion molecules are pesumably required to regulate and guide the migration of leukocytes to the target sites, in this case the choroid and retina, and are thus necessary for the infiltration and accumulation of inflammatory cells to O C C U ~ ~ ~ How.~’. ever, correlation between the ICAM- l expression and the severity of the disease remains speculative.
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Figure 4 lmmunohistochemical staining disclosed macrophages (arrows, CDI lc/18) infiltration in the pineal gland. (ABC staining, x400.)
A focal mononuclear ceilular infiltration consisting mainly of CD4 cells was present in the affected pineal glands although a relatively larger proportion of macrophages was present than that in the eye. Unlike in the eye, no fibrosis developed in these pineal glands in spite of the presence of moderate numbers of B lymphocytes in three monkeys (88, 69N, 2488). This observation suggests that the fibrogenic process, in particular subretinal fibrosis in the eye, is regulated not only by the inflammatory mediators secreted by both T and B lymphocytes, but also by the organ resident cells, such as RPE and glial (Muller) cell inside the eye. It is likely that the development of fibrosis in the eye is a time dependent process. In the early stages of disease T and B cell infiltration predominates, whereas later in the disease fibrosis occurs. A similar process is proposed to occur in multifocal choroiditis, which is felt to progress to subretinal fibrosis and uveitis syndrome only later in its course3'. The effects on the primate EAU model of several cytokines with stimulatory effects to induce fibrosis, such as transforming growth factor beta, interleukin 1, and tumor necrosis factor alpha39,as well as the kinetics of the disease, are currently being investigated in our laboratory.
Acknowledgements The authors are grateful for the editing help provided by Louis Kasner. the technical help provided by Ms Deborah Luyo and the secretarial assistance provided by Ms Dee NaQuin.
References I . Wacker WB, Lipton MM. Experimental allergic uveitis. I. Homologous retina as uveitogenic antigen. Narure 1965; 206: 253-254 2. de Kozak' Y, Yuan WS, Bogossian M, Faure J-P. Humoral and cellular immunity to retinal antigens in guinea pigs. Mod Prohl Ophthalmol 1976; 16: 51-58 3. Gery I, Wiggert B, Redmond TM, Kuwabara T, Crawford MA, Vistica BP, Chader GJ. Uveoretinitis and pinealitis induced by immunization with interphotoreceptor retinoid-binding protein. lnvesr Ophthalmol Vis Sci 1986; 27: 1296-1300 4. Faure J-P. Autoimmunity and the retina. Curr Top Eye Res 1980; 2: 215-302 5. Gery I, Mochizuki M, Nussenblatt RB. Retinal specific antigens and immunoapthogenic processes they provoke. In: Osborne N, Chader J. eds. Progress in Retinal Resear-ch. Vol. 5 . New York: Pergamon Press, 1986; 75-109 6. Caspi R.R. Basic mechanisms in immune-mediated uveitic disease. In: Lightman S. ed. lniniunology of Eye Diseases. Chapt. 5 . Lancaster: Kluever Academy Publisher, 1989; 61-86 7. Schalken JJ, Winkens HJ, Van Vugt AHM, De Geip WJ. Broekhuyse RM. Rhodopsin-induced experimental autoimune uveoretinitis in monkeys. B r J Ophthulrnol 1989; 73: 168-172 8. Hirose S, Kuwabara T, Nussenblatt RB, Wiggert B, Redmond TM, Gery I. Uveitis induced in primates by interphotoreceptor retinoid-binding protein. Arch Ophthulmol 1986; 104: 1698-1 702 9. Nussenblatt RB, Kuwabara T, de Monasterior FM, Wacker WB. S-antigen uveitis in primates. A new model for human disease. A w h Ophthalmol 1981; 99: 1090-1092 10. Faure J-P, Phuc LH, Takano S, Sterkers M , Thillage B, de Kozak Y . Experimental uveoretinitis induced in monkeys by retinal S-antigen. Induction, histopathology. J F r Ophthalrnol 1981; 4: 465-472 1 1 . Forrester JV, Stott DI, Herus KM. Naturally occurring antibodies to bovine and human retinal S-antigen: A comparison between uveitis patients and healthy volunteers. E r J Ophthalmol 1989; 73: 155-159 12. Opremcak EM, Cowans AB, Orosz C G , Adams PW, Whisler
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UVEORETINITIS IN PRIMATES RL. Enumeration of autoreactive helper T lymphocytes in uveitis. Invest Ophthalmol Vis Sci 1991; 32: 2561-2567 13. Dorey C, Cozette J, Faure J-P. A simple and rapid method for isolation of retinal S-antigen. Ophthalmic Res 1982; 14: 249-255 14. Hsu SM, Raine L, Franglor H. The use of aviden-biotin-peroxidase complex (ABC) in immunoperoxidase technique. J Histochem Cytochem I98 I ; 29: 577-580 15. Fujino Y. Mochizuki M, Chan C-C, Raber J, Kotake S , Gery I, Nussenblatt RB. FK506 treatment of S-antigen induced uveitis in primates. Curr Eve Res 1991; 10: 679-690 16. Hirose S, Singh VK, Donoso LA, Shinohara T, Kotake S, Tanaka T, Kuwabara T, Yamaki K, Gery I, Nussenblatt RB. An 18-mer peptide derived from the retinal S-antigen induces uveitis and pinealitis in primates. Clin Exp Immunol 1989; 77: 106-111. 17. Sanu H, Redmond TM, Kotake S, Wiggert B, Tanaka T, Chader GJ, Gery 1. Uveitis and immune responses in primates immunized with IRBP-derived synthetic peptides. Curr Eyz Res 1990; 9: 193-199 18. Jakobiec FA, Marboe CC, Knowles DMII, Iwamoto T, Harrison W. Chang S , Coleman DJ. Human sympathetic ophthalmia: An analysis of the inflammatory infiltrate by hybridoma-monoclonal antibodies, immunochemistry, and correlative electron microscopy. Ophthalmology 1983; 90: 76-95 19. Chan CC, BenEzra D, Rodrigues MM, Palestine AG, Hsu SM, Murphree AL, Nussenhlatt RB. lmmunohistochernistry and electron microscopy of choroidal infiltrates and Dalen-Fuchs nodules in sympathetic ophthalmia. Ophthalmology 1985; 92: 580-590 20. Chan CC, Nussenblatt RB, Fujikawa LS, Palestine AG, Stevens G Jr, Parver LM, Luckenbach MW, Kuwabara T. Sympathetic ophthalmia: Immunopathological findings. Ophfhu/mology 1986; 93: 690-695 21. Muller-Hermelink HK, Kraus-Mackin E, Dans W. Early stage of human sympathetic ophthalmia: Histologic and immunopathologic findings. Arch Opthalmol 1984; 102: 13.53-1357 22. Chan CC, Palestine AG, Kuwabara T, Nussenblatt RB. lmmunopathological study of Vogt-Koyanagi-Harada syndrome. Report of a case. Am J Opthalmol 1988; 105: 607-61 1 23. Sakamoto T, Murata T, lwamoto H. lmmunohistochemical studies of Vogt-Koyanagi-Harada disease with sunset sky fundus. Curr Eye Res 1990; 9 (supp): 35-40 24. Chan CC, Mochizuki M, Nussenblatt RB, Palestine AG, McAllister C, Gery I , BenEzra D. T-lymphocyte subsets in cxperimental autoimmune uveitis. Clin Immunol /mmunopathol 1985; 35: 103-1 10
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25. Chan CC, Caspi RR, Ni M, Leake WC, Wiggert B, Chader GJ, Nussenblatt RB. Pathology of experimental autoimmune uveoretinitis in mice. J Autoimmunity 1990; 3: 247-255 26. Brown EC, Kasp E, Dumonde DC. Morphometric analysis of T lymphocyte compartmentation in experimental autoimmune uveoretinitis. Clin Exp Immunol 1989; 77: 422427 27. Liversidge J, Forester JV. Experimental autoimmune uveitis (EAU): immunophenotopic analysis of inflammatory cells in chorio retinal lesions. CurrEve Res 1988; 7: 1231-1241 28. Kumar RK. Quantitative immunohistologic assessment of lymphocyte populations in the pulmonary inflammatory response to intratracheal silica. Am J Ophthalmol 1989; 135: 605-614 29. Chan CC, Palestine AG, Denis JL, de Smet MD, McLean IW, Burnier M, Drouilhet JH, Nussenhlatt RB. Role of chorioretinal biopsy in inflammatory eye disease. Ophthalmology 1991; 98: 128 1-1286 30. Palestine AG, Nussenblatt RB, Chan CC, Hooks JJ, Friedman L, Kuwabara T. Histpathology of the subretinal fibrosis and uveitis syndrome. Ophthalmology 1985; 92: 838-844 31. Kim M, Chan CC, Belfort R Jr, Farah M, Burnier MP, Nussenblatt RB, Kuwabara T, Palestine AG. Histopathologic and irnmunohistochemical features of subretinal fibrosis and uveitis syndrome. Am J Ophthalmol 1987; 104: 15-23 32. Palestine AG, Nussenblatt RB, Parver L, Knox D. Progressive subretinal fibrosis and uveitis. Br J Ophthalmol 1984; 68: 667-6? 33. Chan CC, Hooks J J , Nussenhlatt RB, Detrick B. Expression of la antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis. Curr Eye Res 1986; 5 : 325-330 34. DeBarge LR, Chan CC, Caspi RR, Harming R, Nussenblatt RB, Whitcup SM. Expression of cell adhesion molecules in mice with experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 1992; 33 (suppl): 796. 35. Chan CC, Detrick B, Nussenblatt RB, Palestine AG, Fujikawa LS, Hooks JJ. HLA-DR antigens on retinal pigment epithelial cells from patients with uveitis. Arch Ophrhaimol 1986; 104: 725-729 36. Whitcup SM, Chan CC, Li Q, Nussenblatt RB. Expression of cell adhesion molecules in posterior uveitis. Arch Ophthulmol 1992; 110: 662-666. 37. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 4 2 5 4 3 4 38. Cantrill HL, Folk JC. Multifocal chorioditis associated with progressive subretinal fibrosis. A m J Ophthalmol 1986; 101: 170-1 80 39. Kunico GS, Neilson EG, Haverty T. Mechanisms of tubulointerstitial fibrosis. Kidney /,If 1991; 39: 550-556
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
IMMUNOPATHOLOGY OF EXPERIMENTAL AUTOIMMUNE UVEORETINITIS IN PRIMATES YUJIRO FUJINO*, QIAN LI, HUM CHUNGT, NAOFUMI HIKITA, ROBERT B. NUSSENBLATT, IGAL GERY and CHI-CHAO CHAN tuhorutory of Immunology, Nutiotzal Eye Institute. Bethesdu, Maryland, USA
Autoimmunity Downloaded from informahealthcare.com by York University Libraries on 11/06/14 For personal use only.
(Received April 3 , 1 9 9 2 ; in final form June I S . 1992)
The eyes and pineal glands from 10 monkeys immunized with S-antigen were studied using routine histopathological and immunohistochemical techniques. Seven out of 10 animals developed uveitis between 19 and 33 days after the initial immuniration. Histopathology of the eyes harvested 70 days after immunization showed moderate to marked uveoretinitis, subretinal fibrosis, retinal necrosis and gliosis. The pineal glands demonstrated chronic pinealitis. The infiltrating cells were both CD3 and CD19/CD22 lymphocytes with a ratio of 1.4 in the eye and 2.2 in the pineal gland. The ratio of CD4 to CD8 lymphocytes was 1.5: I . MHC Class I1 antigens and adhesion molecule (ICAM-I) were observed on resident cells. The influx of B lymphocytes and the formation of subretinal fibrosis differentiate the disease in the monkey from that in the rat and mouse. These findings are similar to Vogt-Koyanagi-Harada syndrome and subretinal fibrosis with uveitis syndrome in human. KEY WORDS: Experimental autoimmune uveoretinitis, pinealitis, primate, subretinal fibrosis, B lymphocyte, ocular inflammation.
INTRODUCTION Experimental autoimmune uveoretinitis (EAU) can be induced in various species by active immunization with ocular antigens, in particular the retinal soluble antigens (S-Ag)',' and interphotoreceptor retinoidbinding protein (1RBP)j. The clinical presentation, histopathological findings and cellular immune responses of EAU resemble certain human uveitides4-'. The detailed clinical and histopathological manifestations may vary from species to species, EAU in primates is characterized clinically by vitritis, retinal vascular sheathing, focal deep chorioretinal yellowish lesions, retinal hemorrhage and edema, localized exudative retinal detachment and papilledema'-'''. The histopathological changes of the eye include loss of the photoreceptor outer segment, retinal periphlebitis, retinitis, diffuse or multi-focal inflammatory cellular infiltration in the choroid and Dalen-Fuchs nodule formation'-"'. The cellular histopathology of the pineal gland shows focal marked lymphocytic infiltration and vasculitisx. These features of the ocular disease mimic certain uveitic syndromes in humans, such as sympathetic ophthalmia, Dr. Fujino is now at the Department of Ophthalmology, University of Tokvo. Tokvo. I Jaoan. . t D r ~Chung is now at the Department of Ophthalmology, Seoul University, Seoul, Korea. Correspondence to: Chi-Chao Chan, M.D., Building 10, Room ION206, NIH/NEI, Bethesda, MD 20892, USA. Telephone: (301) 496-1243; FAX: (301)402-0485.
Vogt-Koyanagi-Harada syndrome (VKH) and birdshot retinochoroidopathy. Immunized monkeys can demonstrate both cellular and humoral responses to the immunizing retinal antigen by the lymphocyte in vitrn proliferative response and the anti-retinal antibodies in the serum, findings which have also been reported in some uveitic patients",". The present study was aimed at examination of the immunopathology of EAU in primates, which has not been described previously.
MATERIALS AND METHODS Animals Ten rhesus monkeys (Macaca mulatta; six male, four female) were utilized in this study. All were obtained from National Institutes of Health, USA approved random sources and housed in environmentally controlled rooms with 12-hr light and dark cycle. All animals were provided with food and water ad libitum. Body weight and foodtwater intake were monitored daily. The study was approved by the institutional animal care and use committee and complies with the Public Health Policy on the Humane Care and Use of Laboratory Animals. Immunization S-Ag was prepared using bovine retinas according to
303
304
Y. FUJINO ET AL.
the methods of Dorey et ~ 1 . The ' ~ antigen was emulsified (1:l) in complete Freund's adjuvant (CFA; Difco, Detroit, MI) containing Mycohacterium tuherculosis H37Ra at a concentration of 1.25 mglml. The emulsion was injected aseptically into multiple intradermal sites over the dorsal thoracic region of each monkey. The injected volume was limited to 4 0 p I per site and sites were spaced approximately 2.5-3 cm apart. A dosage of S-Ag given to the animal was 2 0 p g per kg body weight. The animals were immunized again by the same procedure 14 days later.
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EAU evaluation Ocular examinations, including slit lamp examination of the anterior segment and dilated funduscopic examination of the posterior segment, were performed twice a week. Each eye was classified as follows, based on the clinical presentation characteristics: -, no inflammation; +, inflammation limited to the posterior segments; ++, inflammation involving both anterior and posterior segments as well as mild lid swelling; +++, ocular inflammation extending into the orbit. Photographs of the eyes, including fluorescein angiography, were taken at the onset of the disease and one or two weeks later. All monkeys were sacrificed on day 70 after the initial immunization and the eyes and pineal glands were dissected out for study.
Technique of immuntihisjopatholt~~y Freshly enucleated eyes and pineal glands were bisected. One half was fixed in 10% formalin and processed for routine paraffin embedded sections. The other half was snap frozen in optimal cutting temperature compound (O.C.T., Miles Scientific, Naperville, IL) and stored at -70°C for study. Serial 4-6pm frozen sections were mounted on gelatinized slides. One frozen tissue section from each specimen was stained with standard hematoxylin and eosin. The adjacent sections were stained by the avidin-biotinperoxidase complex method14. The primary monoclonal antibodies were OKT4A (Ortho Lab, Raritan, NJ) for CD4, OKT8F (Ortho Lab, Raritan, NJ) for CD8, Leu M5 (Becton Dickenson, Mountain View, CA) for CDIlclCD18, Leu 14 (B D) for CD22, ICAM-1 (kindly provided by Dr Alfred Singler) for intracellular adhesion molecule - 1 , and HLA-DR (Dako, Carpinteria, CA) for MHC class I1 antigens. Mouse ascites fluid containing 1-2 p g non-specific IgG per milliliter served as a control. The secondary antibody was biotin conjungated horse anti-mouse IgG (Vector Lab, Burlingame, CA).
Evaluation of immunopathology Positive cells, which were stained bluish-black, were
counted and averaged in the same five anatomical areas in each section in order to minimize sampling error. The staining intensity for each marker was scored on an arbitrary scale from 0 to 4 as foflows: 0, as score of 0; 1-10 cells per 40xhigh power field (HPF), as score of 0.5; 11-50 cells per 40xHPF, as score of I+; 51-150 cells per 40xHPF, as score of 2+; 151-250 cells per 40xHPF, as score of 3+; >250 cells per 40xHPF, as score of 4+. The ratio of cellular counts between different subsets were calculated and recorded. RESULTS
Clinical manifestation Seven out of 10 monkeys developed clinical EAU with the onset date between 19 to 33 days after the initial immunization. The remaining three monkeys (9992B, 505C, 3) had no clinical sign of disease. The intensity of disease correlated well with the time of onset (Table 1). Three of the affected animals with early onset dates of EAU presented with orbital inflammation in addition to severe panuveitis, hyphema and papilledema (Figure 1A). Other clinical signs in these seven affected monkeys were retinal vawular sheathing, retinal hemorrhage, edema and exudative detachment, chorioretinal infiltration and scar, vitritis and iridocyclitis. These findings were confirmed by fluorescein angiography. Anterior segment involvement, characterized by conjunctival chemosis, flare and cells in the anterior chamber, anterior and posterior synechiae and cataract formation, were also observed. The ocular inflammation continued to progress (Figure 1B). By day 70 post immunization, affected animals usually developed severe synechiae and cataracts which obscured fundus details and precluded fundus photography.
Table 1 Clinical presenlsiion of S-Ag immunized monkey. Monkey N o .
Se.r
EAU orrsrt duy
EAU s c ~ r r i t y
F913 88 I9SQ 662D 99938 69N 248H 99928
M M F M M M F M F F
19
+++
S05C
3
21 22 24 26 33 33
+++ +++ ++ ++ + +
-
-
-
-
-
-
UVEORETINITIS IN PRIMATES
Immunopathotogicaffindings Eye
cytic and granulomatous infiltrations. Subretinal fibrosis was identified in three monkeys (9993B, 69N, 248H) and papilledema was present in two (F913 and 88). Immunohistochemical staining disclosed the predominant infiltrating cells to be lymphocytes, with a ratio of 1.4: 1 between T and B cells (Figure 2). B cells were most prominent in those eyes with subretinal fibrosis. The ratio between CD4 and CD8 T lymphocytes was approximately 1 : 1. The infiltration was less severe in the monkeys with marked retinal necrosis
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The histopathological changes in eyes of affected monkeys resembled those described in our previous report" and included retinal edema, hemorrhage, necrosis, gliosis, retinitis and periphlebitis. The retinal pigment epithelium showed marked attentuation alternating with hypertrophy, migration and disruption. The choroid was thickened by marked lympho-
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Figure 1 (A) Funduscopic examination of Monkey 19SQ. 35 days post immunization revealed optic disc swelling, retinal hemorrhages, exudates and vascular sheathing. ( B ) Funduscopic examination of Monkey 993, 42 days post immunization showed retinal vascular sheathing and choroidal lesions; three weeks later (63 days post immunization) severe rubeosis iridis and cataracts had developed.
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Y. FUJINO ET AL.
Figure 2 Immunohistochemical staining showed both T and B lymphocytic infiltration in one subretinal lesion. ( A ) CD4 positive cells (arrows).
and gliosis (Table 2 ) . A few T lymphocytes were identifed in the choroid of two of the three monkeys without clinical EAU. MHC Class 11 HLA DR antigen was expressed on inflammatory cells and the ocular resident cells in the areas of inflammation and/or necrosis (Figure 3 ) , in particular the vascular endothelia, retinal pigment epithelia and glial cells. In contrast, ICAM-1 was observed only on the vascular endothelia and retinal pigment epithelia where lymphocytic infiltration was present. It is interesting to note that DR antigen, but not ICAM-I. was also exmessed on ocular resident cells of the three monkeis which failed to develop clinical EAU.
Pinral gland The pineal glands of all affected animals and two of the three unaffected monkeys were available for study. The size of the affected pineal glands was much greater than the unaffected ones. Focal moderate to marked lymphocytic and monocytic infiltration was
Figure 2 Immunohistochemical staining showed both T and B lymphocytic infiltration in one subretinal lesion. (B) CD22 positive cells (arrows). (R, retina, C, choroid; ABC staining, x200.)
seen in all of these pineal glands (Table 2 ) . In contrast to the ocular findings, T lymphocytes, in particular CD4 cells rather than B cells,'were by far the predominant infiltrating cell qualitatively. Numerous macrophages (CDI Ic/CD18) were also present in the infiltrate (Figure 4). Both MHC class II HLA DR antigen was expressed on the vascular endothelia of all affected monkeys and one unaffected monkey with trace macrophage infiltration. As in the eye, ICAM-1 was expressed on the vascular endothelia of the affected animals only. DISCUSSION The present study illustrates the immunopathology of S-Ag induced EAU in the monkey. Seven of 10 monkeys developed clinical EAU characterized by chorioretinal involvement as described previously in the I i t e r a t ~ r e ~ - " ~ Similar ~'~. features have also been described in primates immunized with S-Ag derived peptide" and IRBP derived peptidesI7. Our data indi-
UVEORETINITIS IN PRIMATES
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Table 2 ~mmunopdtho~ogica~ gradings of S-Ag immunized monkey. Monkey NO.
CD4 OKT4A
CD8
CD I I C/I 8
OKT8F
MS
CD 19,20,22 Leu14
CDS4 ICAM- I
MHC Class I1 HLA-DR
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
Eye1P.G.
2+/2+ 1+/3+ 2+/2+
1+/1+ 1 +/2+
0.5/1+
3+/+ 2+/3+/ I + 4+/2+ 1 +/2t -/-/ND -I-
1+/1+ 1+/0.5
2t/2+ 2+/2+ 2+/2+ 3+/1t 1+/3+ 4+/2+ 3+/2+ I +/o.s I+/ND
+
F913
I +/2+
I +/I
8x
1 +/2+
1+/1+
195Q 662D 9993B 69N 248H 9992B 50SC 3
2t/2+ 2+/2+/2t 2t/2+ 0.5/2+
2+/1+
-1-
1 +/o.s
I +/2t 2+/ I + I t/2+ 0.y0.5/ND
0.5/2+ I +/2+ -/3+ -10.5 -/ND -/-
-I-
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0.5/ND -/-
1+/0.5
Figure 3 Immunohistochemical staining showed expression of HLA-DR on inflammatory cells in the choroid. (ABC staining, x 200. )
care that T lymphocytes are the predominant infiltrating cells in both the eye and pineal gland, findings which are similar to the immunopathology of the eye in sympathetic ophthalmia’x-” and VKH”’.23.Interestingly, unlike what the present report indicates for the primate, infiltration with B lymphocytes is rarely
I +12+
2+/0.5 3+/2+
o.s/o.s -1-/ND -/-
-1-
identified in EAU in the rat and m o ~ s e ’ ~ - ’ and ~ , has not been associated with subretinal fibrosis. In the mouse EAU model, which can present with chronic focal chorioretinal lesions and subretinal neovascularization, a relative mild increase of B lymphocytes at the later stages of the disease has been reported”. In the guinea pig EAU, B cells were predominant in the end stage of the lesion”. The influx of B lymphocytes has also been observed in the late stage of the pulmonary inflammatory response to intratracheal silica in mice, when fibrosis occurs in the lung’*. A relative increase of B lymphocytic infiltration occurs in the multifocal choroiditis2’, subretinal fibrosis and uveitis syndrome in humans”’-”. This syndrome is a type of posterior uveitis characterized by choroidal inflammatory lesions with subsequent development of progressive subretinal fibrotic tissue’’. The immunopathology of this syndrome shows that the subretinal fibrotic tissue consists of a mixture of collagen, retinal pigment epithelia and glial cell proliferation, with a primarily B cell infiltration in the choroid and retina’”,”. This immunohistopathological process is similar to the findings in the present EAU monkey study. Expression of MHC class I1 antigen and adhesion molecule has been observed on the ocular resident cells of other animals with EAU3’.”, as well as some human eyes with uveitis35.3h.In the eyes of the monkeys without clinical EAU in the present study, the resident cells and a few T lymphocytes stained positively for MHC class 11, but there was no staining for adhesion molecules. This observation supports the hypothesis that expression of adhesion molecules on ocular resident cells is critical to the development of clinical EAU. Adhesion molecules are pesumably required to regulate and guide the migration of leukocytes to the target sites, in this case the choroid and retina, and are thus necessary for the infiltration and accumulation of inflammatory cells to O C C U ~ ~ ~ How.~’. ever, correlation between the ICAM- l expression and the severity of the disease remains speculative.
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Y. FUJINO ET AL.
Figure 4 lmmunohistochemical staining disclosed macrophages (arrows, CDI lc/18) infiltration in the pineal gland. (ABC staining, x400.)
A focal mononuclear ceilular infiltration consisting mainly of CD4 cells was present in the affected pineal glands although a relatively larger proportion of macrophages was present than that in the eye. Unlike in the eye, no fibrosis developed in these pineal glands in spite of the presence of moderate numbers of B lymphocytes in three monkeys (88, 69N, 2488). This observation suggests that the fibrogenic process, in particular subretinal fibrosis in the eye, is regulated not only by the inflammatory mediators secreted by both T and B lymphocytes, but also by the organ resident cells, such as RPE and glial (Muller) cell inside the eye. It is likely that the development of fibrosis in the eye is a time dependent process. In the early stages of disease T and B cell infiltration predominates, whereas later in the disease fibrosis occurs. A similar process is proposed to occur in multifocal choroiditis, which is felt to progress to subretinal fibrosis and uveitis syndrome only later in its course3'. The effects on the primate EAU model of several cytokines with stimulatory effects to induce fibrosis, such as transforming growth factor beta, interleukin 1, and tumor necrosis factor alpha39,as well as the kinetics of the disease, are currently being investigated in our laboratory.
Acknowledgements The authors are grateful for the editing help provided by Louis Kasner. the technical help provided by Ms Deborah Luyo and the secretarial assistance provided by Ms Dee NaQuin.
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