Immune Responses to Retinal Autoantigens and Peptides in Equine Recurrent Uveitis

Cornelia A. Deeg,1 Bernd Kaspers,1 Hartmut Gerhards,2 Stephan R. Thurau,3 Bettina Wollanke,2 and Gerhild Wildner3

5 PURPOSE. To test the hypothesis that autoimmune mechanisms unclear. Research has focused on the identification of infec- are involved in horses in which equine recurrent uveitis (ERU) tious agents that may induce uveitis, such as bacteria, viruses, develops spontaneously. or parasites, especially on a possible role for Leptospira inter- 6–8 METHODS. Material obtained from horses treated for spontane- rogans as an initiating agent in this process. However, the ous disease by therapeutic routine vitrectomy was analyzed for concept of an infectious factor that exclusively induces and total IgG content and IgG specific for S-Antigen (S-Ag) and maintains the disease is not sufficient to explain certain aspects interphotoreceptor retinoid-binding protein (IRBP). The cellu- of the clinical course and therapeutic approaches. Because of the recurrence of inflammation,5 the positive effect of cortico- lar infiltrate of the vitreous was analyzed by differential counts 2 of cytospin preparations and flow cytometry using equine steroids, and the insufficient therapeutic success of antibiot- lymphocyte-specific antibodies. Antigen-specific proliferation ics, the concept has emerged that the disease is immune assays were performed comparing peripheral blood lympho- mediated. Therefore, ERU is of high value for studying uveitis, because horses represent the only species besides humans in cytes (PBLs) with vitreal lymphocytes by stimulation with S-Ag 9 and several S-Ag– and IRBP-derived peptides. which recurrent uveitis develops spontaneously. Only a lim- ited number of experiments have been designed to examine RESULTS. The total IgG content of specimens from horses with the immunologic reactions of diseased horses. Immunohisto- ERU was very high with great variability among the investi- chemical studies have demonstrated a predominance of T cells gated samples (11.5 Ϯ 8.0 mg). Autoantibodies to S-Ag or IRBP in the inflammatory cellular infiltrates and an increase of major or both were found in 72% of vitreous specimens from horses 8 histocompatibility complex (MHC) class II expression on reti- with uveitis. The leukocyte infiltrates (up to 2 ϫ 10 cells per nal pigment epithelial cells.10 Recently, we have established sample) were dominated by lymphocytes (Ͼ90%) in most pars plana vitrectomy as a therapeutic approach in ERU,4 and cases (22/32). Flow cytometry showed that more than 50% of ϩ this has enabled us to gain inflammatory cells from eyes with these cells were CD4 T cells. In vitro stimulation of vitreal uveitis. The goal of our study was to further investigate the lymphocytes, but not of PBL, showed a strong proliferative underlying immunopathogenic mechanisms in ERU, providing response to peptides derived from S-Ag or IRBP in 9 of 12 evidence that the disease is autoimmune mediated with useful patients. and unique properties in comparison with all other uveitis CONCLUSIONS. In the eyes of horses with ERU, IgG antibodies models. and autoreactive T cells specific for retinal antigens were detected. These results strongly support the hypothesis that ERU is an autoimmune-mediated disease and is highly similar to METHODS recurrent uveitis in humans in both clinical and immunologic parameters. (Invest Ophthalmol Vis Sci. 2001;42:393–398) Patients Sixty-four horses with clinical signs of ERU underwent therapeutic pars quine recurrent uveitis (ERU) is a spontaneous disease plana vitrectomy according to the method described by Werry and Eaffecting up to 15% of horses.1 The disease is characterized Gerhards.4 All patients had at least two uveitic episodes, and ERU was by repeated episodes of intraocular inflammation, and many diagnosed according to clinical criteria, as described.11 Clinical signs of corresponding clinical and pathologic features are similar to intraocular inflammation including aqueous flare, synechiae, vitreitis, recurrent uveitis in humans.2 Clinical findings include blepha- pigment deposition on the anterior lens surface, retinal detachment, rospasm, photophobia, miosis, and pain in the acute phase.3 and cataract. The horses were treated with topical atropine a few days The recurrences vary in phase and severity, with a tendency to before surgery. Neither immunosuppressive therapy nor corticoste- increase in severity. Vision loss or blindness is the result of the roids were applied, because horses undergo surgery only in the quies- inflammatory process that leads to synechiae, cataract forma- cent stages of the disease.3 Physical examination included intraocular tion, retinal detachment, or phthisis bulbi.4 Blind horses have pressure measurement, slit lamp examination, and direct ophthalmos- to be euthanatized for ethical reasons and cause large eco- copy with dilated pupil. Because the donors of specimens are not nomic losses in the equine industry. Although ERU has been experimental but companion animals and were brought to the Depart- known for centuries and occurs worldwide, the cause remains ment of Equine Surgery in the need of medical help, these studies could not include histologic analysis of the eyes. Enucleation of uveitic eyes is performed rarely, because vitrectomy has been successfully established for horses as a routine treatment for progressive stages From the 1Institute of Animal Physiology, the 2Department for Equine Surgery, and the 3Section of Immunobiology, Department of of ERU. Ophthalmology, Ludwig-Maximilians University, Munich, Germany. Supported by Deutsche Forschungsgemeinschaft Grant DE Sample Collection 719/1-1. All animals were managed in accordance with the ARVO Statement for Submitted for publication July 5, 2000; revised September 5 and October 3, 2000; accepted October 16, 2000. Use of Animals in Ophthalmic and Vision Research. Depending on Commercial relationships policy: P. sample size and numbers of collected cells, specimens were used for Corresponding author: Cornelia A. Deeg, Institute of Animal Phys- one or several of the analytical procedures, to determine the type of iology, Ludwig-Maximilians University, Veterina¨rstrasse 13, 80539 Mu- inflammation and the humoral and cellular immune responses to reti- nich, Germany. [email protected] nal antigens. Heparinized blood for in vitro proliferation assays and

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serum were collected before ocular surgery. Vitreous samples were 118717), PI 536 (IRBP; aa 536-54917), and B27PD (HLA-B; aa 125-13815). obtained during pars plana vitrectomy and assayed immediately. Vitre- Peripheral blood lymphocytes (PBLs) were obtained from heparinized ous was diluted with 200 ml sterile balanced salt solution (BSS) during blood samples by Ficoll density gradient centrifugation (Pharmacia, surgery. BSS is routinely used to replace removed vitreous. Control Freiburg, Germany). Cells were washed three times and resuspended vitreous was immediately collected and processed after death from in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany) sup- eyes of euthanatized horses without ocular disease. Control blood plemented with 2 mM L-glutamine (Biochrom, Berlin, Germany), 100 samples were obtained from horses without ocular disease. U/ml penicillin (Biochrom), 100 ␮g/ml streptomycin (Biochrom), and 10% fetal calf serum (ICN Biochemicals, Meckenheim, Germany). Preparation of Cells from Vitreous Vitreal lymphocyte preparations with more than 90% lymphocytes and Flow Cytometry (as assessed by cytospin preparations) were assayed for their antigen- specific proliferation only when more than 1 ϫ 106 cells were recov- Vitreous humor was filtered through a stainless steel sieve before 5 ered from the vitreous. PBLs (5 ϫ 10 cells per well) and vitreous cells analysis. Cells were pelleted by centrifugation at 4°C for 20 minutes at 4 (1 ϫ 10 cells per well) were cultured as triplicates in flat-bottomed 750g. The supernatant was stored at Ϫ20°C until use, and the cell microtiter plates (Nunc) in the presence of antigens for 5 days in pellet was resuspended in RPMI and subsequently analyzed. Differen- humidified atmosphere at 37°C with 5% CO2. Subsequently, cells were tial counts were obtained from Wright-stained cytospin preparations, labeled with 2 ␮Ci [3H]thymidine per well for 18 hours and harvested and the remaining cells were phenotyped by flow cytometry. In 100 ␮l at day 6 (Amersham, Braunschweig, Germany). [3H]thymidine incor- fluorescence buffer (phosphate-buffered saline [PBS] with 1% bovine poration was measured by scintillation counting. The results are pre- serum albumin [BSA], 0.01% sodium azide; Sigma, Deisenhofen, Ger- 6 sented as stimulation index (SI; mean counts per minute [cpm] of many) 1 ϫ 10 cells were incubated for 30 minutes at 4°C with 10 ␮l triplicates with antigen/mean cpm of triplicates without antigen). of monoclonal mouse anti-equine antibodies directed against the following markers: CD4 (clone CVS4), CD8 (CVS 21), and clone CVS1 to Statistical Methods detect B cells (all from Biozol/Serotec, Eching, Germany). Cells were subsequently washed three times with fluorescence buffer and stained The statistical significance of differences between groups was evalu- with fluorescein-isothiocyanate (FITC)–conjugated goat anti-mouse ated using Student’s t-test in the study of vitreous infiltrating cells. The 2 IgG (Sigma). Cells (5 ϫ 103) were each analyzed in a flow cytometer results of the autoantibody assays were compared using the ␹ and (FACScan; Becton Dickinson, Heidelberg, Germany) using Win MDI Fisher’s exact tests. P Ͻ 0.005 was considered significant. 2.5 software (The Scripps Research Institute, La Jolla, CA).

Quantitative Measurement of Total Vitreal IgG RESULTS IgG concentration was quantified by single radial immunodiffusion, as Phenotypic Characterization of described,12 using a polyclonal antiserum specific for the equine Vitreous-Infiltrating Cells ␥-heavy chain (Dianova, Hamburg, Germany). Purified equine IgG The numbers of infiltrating cells from vitrectomy samples of 64 (Sigma) was used as a standard. horses with ERU were determined. Because of large variations in cell counts, it was not possible to perform all assays (differ- Detection of Autoreactive Antibodies ential analysis of cell type, characterization of cell surface Vitreal and serum samples were tested for the presence of autoanti- antigens, and antigen specific proliferation) with every intraoc- bodies against the retinal antigens S-antigen (S-Ag) and interphotore- ular sample. The average cell number obtained was 6.4 ϫ 106 ceptor retinoid-binding protein (IRBP) by enzyme-linked immunosor- cells Ϯ 2.8 ϫ 106 cells per vitrectomy sample. bent assay (ELISA). These antigens were purified from bovine eyes, as Vitreous samples from unaffected control animals were described previously.13,14 Microtiter plates (Maxisorb; Nunc, Wiesba- devoid of infiltrating cells. Cytologic classification of cells in den, Germany) were coated with 10 ␮g of the respective antigen/ml cytospin preparations from eyes of 32 horses with ERU re- PBS overnight at 4°C. Blocking was performed by 1 hour incubation vealed three different types of inflammation, divided into three with 1% BSA in PBS. Samples were diluted (1:1000) in PBS-0.05% groups (Fig. 1). Twenty-two patients (group 1) had a predom- Tween 20 (Sigma), added to the wells in duplicates, and incubated for inant infiltration of lymphocytes in their vitreous (93% Ϯ 5% 1 hour at 37°C. As a secondary antibody, 50 ␮l of a rabbit anti-horse lymphocytes, significantly higher than in the other groups), a IgG no serum, purified antibody labeled with horseradish peroxidase second group (6/32) had 57% Ϯ 15% vitreal lymphocytes and (dilution 1:10,000; Sigma) was added to each well. Between each 34% Ϯ 21% vitreal granulocytes (group 2), which is signifi- incubation, the plates were washed three times with PBS-0.05% Tween cantly fewer lymphocytes than in group 1. In contrast, horses 20. After 1 hour’s incubation with secondary antibody, the assay was of group 3 (4/32) had 89% Ϯ 9% intraocular granulocytes and developed with 100 ␮l tetramethylbenzidine (Sigma) per well as sub- only a few lymphocytes (10% Ϯ 9%) and had hyalitis rather strate. The reaction was stopped after 10 minutes by adding 50 ␮lof1 than iridocyclitis. The mean cell number of lymphocytes and M sulfuric acid per well. The absorbance was determined at 450 nm granulocytes showed significant differences between every using an ELISA reader (Easy Reader; SLT, Salzburg, Austria). Serial group, using Student’s t-test (P Ͻ 0.005). dilutions of a certain serum with high antibody titer, a serum with Lymphocytes from group 1 horses were further analyzed by intermediate reactivity, and a negative serum were included in each flow cytometry. Cell surface markers were determined from assay to validate the sensitivity and the variation of the ELISA. Horses vitreal lymphocytes of 17 horses. We found predominantly are usually vaccinated with tetanus toxoid (Tt), therefore Tt (kindly CD4ϩ T cells (average of 57% Ϯ 17%) and fewer CD8ϩ T cells provided by Klaus-Dieter Hungerer, Chiron–Behring, Marburg, Ger- (29% Ϯ 14%) and B cells (28% Ϯ 16%; by FACScan, data not many) specific antibodies were used as a control and measured exactly shown). as described earlier. Vitreal Antibodies Specific for Retinal S-Ag Lymphocyte Proliferation Assay or IRBP S-Ag was used in cultures at a concentration of 10 ␮g/ml and peptides Analysis of serum and vitreal antibodies was performed with at 5 ␮g/ml. The following peptides derived from bovine S-Ag or IRBP specimens of all three groups. A high variation was detected were used for antigenic stimulation: PDSAg (S-Ag; amino acid [aa] within the amount of total IgG in vitrectomy samples from 30 342-35515), R14 (IRBP; aa 1169-119116), PDIRBP (IRBP; aa 1174- tested horses with uveitis. IgG concentrations in vitreous were,

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with ERU. All horses except for one, which was not vaccinated, were positive for anti-Tt antibodies. Whereas only 3 of 10 horses with ERU of the inflammatory group 2 (mixed infiltration, Fig. 1) and group 3 (granulocytic infiltration, Fig. 1) showed intraocular autoantibodies, 20 of 22 group 1 horses’ lymphocytic infiltrations where positive for retinal autoantibodies in the eye. Autoreactive T Cells Preferentially Respond to IRBP Peptides Because autoimmune uveitis is a T-cell–mediated disease, we investigated in vitro responses of T cells to retinal antigens. In preliminary experiments we found that PBL of horses with ERU proliferated only weakly in response to retinal antigens (2/10). Lymphocytes of healthy control horses did not respond to stimulation (data not shown). To further investigate the antigen specificity of lymphocytes from horses with ERU, we took advantage of the availability to gain sufficient numbers of T cells from the target organ eye. Vitreal lymphocyte preparations of group 1 horses (with more than 90% lymphocytes as assessed by cytospin preparations) were used. In 14 of 22 horses from this group we collected sufficient numbers of cells to compare the responses of lymphocytes from PBL and vitreous. Whereas in PBL autoantigen- FIGURE 1. Differentiation of vitreal lymphocytes. The mean percent- age of lymphocytes (lymph.), granulocytes (gran.) and monocytes (mono.) of vitrectomy samples is shown, which were grouped according to the following criteria: of 32 horses, 22 had a mainly lymphocytic infiltrate (group 1, lymphocytes 93% Ϯ 5%), 5 showed a mixed infiltration (group 2, lymphocytes 57% Ϯ 15%; granulocytes 34% Ϯ 21%), and 5 had a predominant infiltration of granulocytes (group 3, 89% Ϯ 9%). Statistical difference was determined using Student’s t-test.

on average, 11.5 Ϯ 8 mg (data not shown). However, no IgG was detectable in any vitreous specimen of 15 healthy control eyes. Using an ELISA to detect antigen-specific IgG-antibodies in vitreal specimens of ERU-affected eyes revealed a mean optical density (OD) of 0.086 Ϯ 0.094 (SD) for IRBP, 0.080 Ϯ 0.095 for S-Ag, and 0.108 Ϯ 0.139 for Tt–specific antibodies (healthy control animals: IRBP: 0.008 Ϯ 0.005; S-Ag: 0.006 Ϯ 0.003; Tt 0.018 Ϯ 0.012; patients versus healthy control animals P Ͻ 0.001, Fig. 2, top). To separate positive and negative samples, a cutoff was set at a fivefold increased OD compared with healthy control animals, thus minimizing false-positive results. Under these conditions, retina-specific antibodies were detected in the vitreous of 73% of horses with uveitis (24/33). Four specimens had only antibodies to IRBP, 7 to S-Ag only, whereas the remaining 13 positive vitreous samples contained autoantibodies against both retinal antigens. Ten of these 24 horses had intraocular antibodies against Tt, indicating leakiness of the blood–retina barrier in these patients. In the vitreous of one horse with ERU only Tt-specific antibodies were detected, but no antibodies against ocular antigens. We further tested the prevalence of retinal autoantibodies in the sera of horses with ERU (Fig. 2, bottom). The cutoff was set at a fivefold increased SD above the average OD of sera from healthy horses. The average ODs in ERU sera were 0.178 Ϯ 0.143 (IRBP), 0.213 Ϯ 0.121 (S-Ag), and 0.386 Ϯ 0.169 (Tt). Sera from five horses with ERU reacted only with IRBP, FIGURE 2. Determination of anti-S-Ag and anti-IRBP autoantibodies in whereas six horses had only S-Ag–specific antibodies (healthy vitreous (top) and serum (bottom) of horses with ERU and healthy control animals: IRBP: 0.060 Ϯ 0.034; S-Ag: 0.054 Ϯ 0.034; Tt: horses by ELISA. The value (OD450) indicates the mean OD corrected 0.312 Ϯ 0.081; anti-IRBP and anti-S-Ag-antibodies in ERU-af- for background binding of rabbit anti-horse IgG. Filled symbols: Re- fected versus healthy control animals: P Ͻ 0.001, anti-Tt-anti- sults in horses with ERU; open symbols: healthy control horses. Black horizontal lines: cutoff. Autoantibody titers were significantly higher bodies in ERU-affected versus healthy control animals, no sta- in horses with ERU than in control horses (eye and serum). Statistical tistically significant difference). difference was determined using ␹2 ϭ P Յ 0.001. There was also a Of control horse sera, 22% (8/35) were positive for one or significant difference between intraocular anti-Tt antibodies in ERU both retinal antigens compared with 81% (27/33) of horses versus control horses (P Յ 0.005).

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FIGURE 3. Proliferative responses of PBL and vitreal lymphocytes (VL) from the same horses to peptides PDSAg (S-Ag–derived), S-Ag; PDIRBP, R14, PI536 (IRBP-derived), and B27PD (human HLA-class I peptide). Results are given as stimulation index (SI). The cutoff was set at an SI of 2. PBL from 3 of 14 horses responded weakly, whereas VLs from 10 of 14 horses proliferated in response to the antigens.

specific responses were either weak in incidence and intensity have been performed with eyes and pineal glands of horses (3/14 horses) or completely absent, vitreal lymphocytes with ERU, demonstrating findings that resemble human in- strongly reacted to IRBP-derived peptides and to the HLA- traocular inflammatory disease, the function of immune cells peptide B27PD (10/14 horses; Fig. 3). In two horses no prolif- using vitreal specimens from ERU-affected eyes has not been erative responses to any autoantigen were observed with pe- analyzed. Our research focused on the immunologic mecha- ripheral or with vitreal lymphocytes. Both humoral (Fig. 4) and nisms involved in ERU. This study provides strong evidence cellular (Fig. 3) responses of the same horses were examined that ERU is an autoimmune-mediated disease comparable to but due to technical difficulties, the same antigens could not be human uveitis, in that we found autoantibodies and vitreal used for both procedures, thereby hindering any direct com- lymphocytes reacting with retinal autoantigens. parison. Moreover, besides the spontaneously developing disease, the horse offers the possibility to experimentally induce uve- DISCUSSION itis. This would enable us to compare histopathology and immunopathology of experimentally induced autoimmune dis- ERU is a disease with high prevalence. The disorder shows ease with naturally occurring disease in the same species. clinical and pathologic similarities to recurrent uveitis in hu- First attempts to show an autoimmune reaction associated mans, indicating its potency as a valuable model for this ocular with ERU were made by Maxwell et al., 18 who investigated the inflammatory disease in humans. In contrast to experimentally humoral immune response to S-antigen and rhodopsin in af- induced animal models, ERU develops spontaneously similar to fected horses and healthy control animals and found no signif- human uveitis. Although some immunohistochemical studies icant difference between both groups. Furthermore, S-Ag, which is the best characterized autoantigen in humans and rats, was not a target for the response of PBL in horses with ERU as shown by Hines and Halliwell.19 To extend these early studies we compared the humoral immune response in the periphery to that of the vitreous from horses with ERU. In addition to S-Ag, we also tested bovine IRBP as autoantigen. We used bovine retinal antigens, because highly immunogenic and uveitogenic epitopes are usually highly conserved among species.20 This, however, remains speculative with respect to the still unknown sequences of equine retinal antigens. The latter fact also prevented us from using peptides with the horse- specific sequences. There was a marked difference between ERU-affected and healthy horses regarding autoantibodies in the periphery. Whereas 81% of tested ERU sera were positive for one or both retinal antigens, only 22% of sera from healthy horses reacted positively. These findings are in contrast to studies in human uveitis, in which no differences in antibodies were seen be- FIGURE 4. Anti-S-Ag and anti-IRBP autoantibodies in vitreous and se- tween control animals and horses with uveitis.21 Human pa- rum of horses with ERU shown in Figure 3. The value (OD450) indicates the mean OD corrected for background binding of rabbit anti-horse tients are usually vigorously treated with immunosuppressive IgG. The cellular response in vitro was not correlated with antibody agents, probably resulting in impaired B-cell reactivity. ERU- reaction in antigen specificity and antibody titers. affected horses, however, are usually not treated with immu-

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nosuppression, which may explain the enhanced antibody ERU or that processing of the protein in cell culture was not production to retinal autoantigens.22 adequate. IRBP was not tested as a whole antigen because of This new observation was further confirmed by the dem- the content of concanavalin A within the preparations as a onstration of retinal antigen-specific antibodies in vitreous sam- result of the purification procedure. However, the results ob- ples of horses with ERU and the complete absence of autoan- tained with IRBP peptides clearly point to IRBP as the domi- tibodies in vitreous samples obtained from healthy control nant autoantigen in ERU. In 9 of 14 horses, the vitreal lympho- animals. In contrast to previous reports,18,19 our findings indi- cytes reacted to at least one IRBP-derived peptide. R14 (aa cate a specific generation of autoantibodies in affected horses, 1169-1191) is the immunodominant determinant in the Lewis either in the peripheral immune system or in the eye. A break- rat and can induce EAU in these animals.29 In accordance with down of the blood–retinal barrier or infiltration of unspecific B observations in human uveitis,30 there was no positive or cells could be monitored by the presence of Tt-specific anti- negative correlation between detectable autoantibodies and bodies in the vitreous. Because Tt is a common vaccine for the frequency of cellular response. There was also no clear horses in Germany, most animals have specific serum anti- pattern of reactivity to certain antigens in individual horses. In bodies. conclusion, we observed a predominant location of retinal Penetration of anti-tetanus antibodies from blood into the antigen-specific T cells within the eye (10/14 vitreous cell vitreous is probably due to leakiness of choroidal and iris samples proliferating to one or several antigens and peptides), vessels. As in humans, fluorescein angiograms in nonhuman whereas only 4 of 14 horses showed intraocular but no periph- primates with experimental autoimmune uveitis (EAU) have eral autoantibodies as indicators for B-cell reactivity. Although shown that severe uveitis leads to extravasation of dye21 from not supportive of a Th1 immune response, these findings retinal vessels. B cells were recently demonstrated in the eyes demonstrate strong T-cell–mediated autoreactivity and are con- of horses with ERU by immunohistology, indicating a local sistent with the previous demonstration of a Th1 cytokine antibody production. However, autoantigen specificity of pattern in eyes of affected horses.31,32 these B cells remains to be determined.10 In humans, a possible mechanism for the induction of Because anti-retinal antibodies seem to be nonessential for uveitis has been described by antigenic mimicry between ret- the induction of uveitis, as shown in several EAU models,21 and inal autoantigen peptide and a peptide derived from disease- the predominating infiltrates in eyes of horses with ERU are associated HLA-antigens (B27PD).14 This work inspired us to T-helper cells, we were highly interested in the cell-mediated test the reactivity of horse lymphocytes to these peptides, mechanisms involved in ERU. The therapeutic routine proce- although the equine MHC antigens known so far do not con- dure of vitrectomy allowed us to obtain sufficient numbers of tain a sequence corresponding to peptide B27PD. It was inter- vitreous-infiltrating cells from diseased horses for immunologic esting that 6 of 14 horse vitreal lymphocyte preparations tested studies and to compare the reactions of peripheral and intraoc- with this peptide strongly proliferated in response to B27PD. ular lymphocytes. HLA-peptide B27PD was shown to mimic S-Ag peptide PDSAg A hallmark of organ-specific autoimmune diseases is the in the rat model of EAU and also to play a role in human uveitis. response of lymphocytes to certain autoantigens. In human It is also used as an oral tolerogen in rat EAU15 and in human autoimmune uveitis and EAU animal models, S-Ag, IRBP, and patients.28 To explain the tolerogenic properties of B27PD in peptides of these autoantigens have been shown to activate IRBP-induced EAU in the Lewis rat,15 peptide PI536 and CD4ϩ T cells in vivo and in vitro.23–25 Furthermore, uveitis was PDIRBP were selected as possible cross-reactive epitopes ac- induced successfully by adoptive transfer of antigen-specific cording to certain amino acid sequence homologies.16 CD4ϩ T cells and T cell lines.26 Consequently, we set out to In conclusion, our results point toward an autoimmune study the cellular response to a selected panel of retinal anti- pathogenesis in ERU. Witebsky postulated regarding the auto- gens and their peptides. Our initial attempts to activate PBL immune origin of human diseases that the autoimmune re- with these antigens failed in most horses with ERU. Only two sponse must be mediated by an autoantibody or an autoreac- of 14 patients responded to S-Ag- or IRBP-derived peptides; tive T cell, the corresponding antigen must be identified, and however, no response to the whole antigens could be ob- the analogous autoimmune response must be induced in an served. This is in accordance with the findings of Hines and experimental animal, which should develop a similar disease.33 Halliwell,19 who also could not observe an activation of PBL These criteria are now fulfilled with the demonstration of the from horses with ERU to whole S-antigen. presence of autoantibodies and autoreactive lymphocytes spe- In several autoimmune diseases, responses to autoantigens cific for S-Ag and IRBP in horses with ERU. Moreover, we found are rarely seen with PBL. Only in some patients27 or at certain significant proliferation of retinal autoantigen-specific T lym- time points28 do PBL respond to autoantigens. The low fre- phocytes within the target organ eye compared with periph- quency of antigen-specific PBL, even in advanced cases of eral blood, indicating that autoimmunity plays an important uveitis, has been discussed as one reason for poor results in role in this disease. However, the initiating events of the proliferation assays.29 Because equine patients usually have immunopathology remain obscure. In many autoimmune dis- severe advanced uveitis, proliferative responses cannot be orders, infections have been discussed as triggering events, studied during the early phase of disease or even before a either by antigenic mimicry with a pathogen’s antigen or as a relapse, when higher numbers of antigen-specific T cells bystander effect due to the general systemic or local immune should be present in the circulation. To circumvent this prob- stimulation by the pathogen.15 The induction of an autoim- lem, we investigated the proliferative capacity of target organ mune disease through molecular mimicry between a linear infiltrating lymphocytes in vitro. In contrast to PBL, cells from epitope of an infectious agent and host protein was demon- the eye strongly responded to several of the tested peptides. In strated only sporadically.34 most cases, a clear response to one or several IRBP-derived The role of infection with L. interrogans in ERU is currently peptides was observed. The S-Ag–derived peptide PDSAg acti- under investigation. Recently, immunohistopathologic exami- vated vitreal lymphocytes in 5 of 14 cases. PDSAg was chosen, nations of equine ocular tissues with leptospiral antisera because it is a major pathogenic epitope of S-Ag in the rat showed cross-reactivity with iris pigment epithelium and retina model and has also been shown to be highly immunogenic in from horses with ERU. The investigators underscored the rel- humans.15 evance of these studies as a possible model of postinfectious In general, the response to whole S-Ag was weak, suggest- immunopathogenesis.35 It is unclear at present, whether infec- ing S-Ag may not be a major autoantigen for T-lymphocytes in tion with L. interrogans leads to an autoimmune reaction

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through molecular mimicry between the pathogen and retinal 16. Sanui H, Redmond TM, Hu LH, et al. Synthetic peptides derived tissue. The immune reactions to ocular autoantigens demon- from IRBP induce EAU and EAP in Lewis rats. Curr Eye Res. strated in the present study may be the clue for the mainte- 1988;7:727–735. nance of ocular inflammatory disease. Because lymphocytes in 17. Wildner G, Thurau SR. Database screening for molecular mimicry. horses with ERU proliferate strongly in response to peptide Immunol Today. 1997;18:252. B27PD, which is derived from HLA-B antigens that are associ- 18. Maxwell SA, Hurt D, Brightman AH, Takemoto D. Humoral re- ated with uveitis in humans, a similar mechanism of molecular sponses to retinal proteins in horses with recurrent uveitis. Vet mimicry between a self-MHC peptide and a peptide from a Comp Ophthalmol. 1991;1:155–162. retinal autoantigen could also be speculated for horses; how- 19. Hines MT, Halliwell REW. 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