Identification and Functional Validation of Novel Autoantigens in Equine Uveitis

Abstract

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers. The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers. Uveitis is a remitting-relapsing autoimmune disease of the inner eye characterized by breakdown of the blood-retinal barrier, infiltration by inflammatory cells, and tissue destruction ultimately leading to blindness (1Forrester J.V. Uveitis: pathogenesis.Lancet. 1991; 338: 1498-1501Abstract PubMed Scopus (112) Google Scholar). Although a diverse set of initiating events have been proposed to cause uveitis, the pathogenesis is finally perpetuated by CD4+ Th1 effector cells (2Martin T.M. Smith J.R. Rosenbaum J.T. Anterior uveitis: current concepts of pathogenesis and interactions with the spondyloarthropathies.Curr. Opin. Rheumatol. 2002; 14: 337-341Crossref PubMed Scopus (78) Google Scholar). Equine recurrent uveitis (ERU), 1The abbreviations used are: ERU, equine recurrent uveitis; CD, cluster of differentiation; cRALBP, cellular retinaldehyde-binding protein; GFAP, glial acidic fibrillary protein; GS, glutamine synthase; IRBP, interphotoreceptor retinoid-binding protein; S-Ag, S-antigen; VEGF, vascular endothelial growth factor; vWF, van Willebrand factor; IL, interleukin; chIL, chicken IL; 2DE, two-dimensional electrophoresis; PBL, peripheral blood-derived lymphocyte; aa, amino acids; H&E, hematoxylin and eosin. a spontaneous, T cell-mediated disease with high prevalence among horses, serves as a suitable model for human autoimmune uveitis due to the common sharing of at least two different autoantigens, S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), between equine and human uveitis patients (3de Smet M.D. Bitar G. Mainigi S. Nussenblatt R.B. Human S-antigen determinant recognition in uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 3233-3238PubMed Google Scholar, 4Deeg C.A. Kaspers B. Gerhards H. Thurau S.R. Wollanke B. Wildner G. Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 393-398PubMed Google Scholar). Furthermore both autoantigens are capable of inducing experimental uveitis in horses (5Deeg C.A. Thurau S.R. Gerhards H. Ehrenhofer M. Wildner G. Kaspers B. Uveitis in horses induced by interphotoreceptor retinoid-binding protein is similar to the spontaneous disease.Eur. J. Immunol. 2002; 32: 2598-2606Crossref PubMed Scopus (0) Google Scholar, 6Deeg C.A. Reese S. Gerhards H. Wildner G. Kaspers B. The uveitogenic potential of retinal S-antigen in horses.Investig. Ophthalmol. Vis. Sci. 2004; 45: 2286-2292Crossref PubMed Scopus (44) Google Scholar), and in both, peripheral blood-derived, autoaggressive T cells specific for S-Ag and IRBP epitopes have been detected (3de Smet M.D. Bitar G. Mainigi S. Nussenblatt R.B. Human S-antigen determinant recognition in uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 3233-3238PubMed Google Scholar, 4Deeg C.A. Kaspers B. Gerhards H. Thurau S.R. Wollanke B. Wildner G. Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 393-398PubMed Google Scholar). In most autoimmune diseases, several autoantigens participate in the pathogenesis (7Mackay I.R. Rowley M.J. Autoimmune epitopes: autoepitopes.Autoimmun. Rev. 2004; 3: 487-492Crossref PubMed Scopus (32) Google Scholar), and epitope spreading is accountable for disease induction, progression, and inflammatory relapses (8Sawalha A.H. Harley J.B. Antinuclear autoantibodies in systemic lupus erythematosus.Curr. Opin. Rheumatol. 2004; 16: 534-540Crossref PubMed Scopus (81) Google Scholar, 9Seissler J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar, 10Yu M. Johnson J.M. Tuohy V.K. A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease.J. Exp. Med. 1996; 183: 1777-1788Crossref PubMed Scopus (247) Google Scholar, 11Deshmukh U.S. Gaskin F. Lewis J.E. Kannapell C.C. Fu S.M. Mechanisms of autoantibody diversification to SLE-related autoantigens.Ann. N. Y. Acad. Sci. 2003; 987: 91-98Crossref PubMed Scopus (52) Google Scholar, 12Ott P.A. Dittrich M.T. Herzog B.A. Guerkov R. Gottlieb P.A. Putnam A.L. Durinovic-Bello I. Boehm B.O. Tary-Lehmann M. Lehmann P.V. T cells recognize multiple GAD65 and proinsulin epitopes in human type 1 diabetes, suggesting determinant spreading.J. Clin. Immunol. 2004; 24: 327-339Crossref PubMed Scopus (43) Google Scholar). Knowledge of the targeted autoantigens is therefore indispensable for understanding the nature of acute autoimmune responses during recurrent attacks. Furthermore identifying persons at risk to develop several autoimmune diseases is based upon the presence of an autoreactive response to several autoantigens prior to disease state (13Scofield R.H. Autoantibodies as predictors of disease.Lancet. 2004; 363: 1544-1546Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14Verge C.F. Gianani R. Kawasaki E. Yu L. Pietropaolo M. Chase H.P. Eisenbarth G.S. Number of autoantibodies (against insulin, GAD or ICA512/IA2) rather than particular autoantibody specificities determines risk of type I diabetes.J. Autoimmun. 1996; 9: 379-383Crossref PubMed Scopus (129) Google Scholar, 15Maclaren N.K. Lan M.S. Schatz D. Malone J. Notkins A.L. Krischer J. Multiple autoantibodies as predictors of Type 1 diabetes in a general population.Diabetologia. 2003; 46: 873-874Crossref PubMed Scopus (17) Google Scholar, 16LaGasse J.M. Brantley M.S. Leech N.J. Rowe R.E. Monks S. Palmer J.P. Nepom G.T. McCulloch D.K. Hagopian W.A. Successful prospective prediction of type 1 diabetes in schoolchildren through multiple defined autoantibodies: an 8-year follow-up of the Washington State Diabetes Prediction Study.Diabetes Care. 2002; 25: 505-511Crossref PubMed Scopus (139) Google Scholar). The accuracy of prediction increases by combining autoantigens (14Verge C.F. Gianani R. Kawasaki E. Yu L. Pietropaolo M. Chase H.P. Eisenbarth G.S. Number of autoantibodies (against insulin, GAD or ICA512/IA2) rather than particular autoantibody specificities determines risk of type I diabetes.J. Autoimmun. 1996; 9: 379-383Crossref PubMed Scopus (129) Google Scholar, 15Maclaren N.K. Lan M.S. Schatz D. Malone J. Notkins A.L. Krischer J. Multiple autoantibodies as predictors of Type 1 diabetes in a general population.Diabetologia. 2003; 46: 873-874Crossref PubMed Scopus (17) Google Scholar). The profiling of autoantibodies serves best in predicting T cell-mediated autoimmune diseases (13Scofield R.H. Autoantibodies as predictors of disease.Lancet. 2004; 363: 1544-1546Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Although autoantibodies play a subordinated role in the pathogenesis of Th1-mediated diseases, they are detectable in both ERU cases and human autoimmune uveitis patients (4Deeg C.A. Kaspers B. Gerhards H. Thurau S.R. Wollanke B. Wildner G. Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 393-398PubMed Google Scholar, 17de Kozak Y. Antibody response in uveitis.Eye. 1997; 11: 194-199Crossref PubMed Scopus (8) Google Scholar). To identify potential novel candidate autoantigens in autoimmune uveitis, we tested the IgG binding profile to the retinal proteome from horses with spontaneous ERU. For the analysis of cellular retinaldehyde-binding protein (cRALBP) as an autoantigen in the spontaneous ERU model, we sampled a total of 344 ERU cases and 137 controls for Western blotting, ELISA, and T cell assays. In detail, sera of 35 ERU cases and 32 healthy control horses were used for the autoantibody profiling on the retinal proteome. Sera of 289 additional ERU cases and 95 control horses were used in the cRALBP autoantibody screening with ELISA. All cRALBP-positive samples were verified using one-dimensional Western blots with 1 μg of cRALBP/lane (84 cRALBP-positive ERU sera, 20 cRALBP-negative ERU sera as negative control, 11 cRALBP-positive healthy control horses, and 20 cRALBP-negative, healthy control horses). We examined the autoreactive T cell response of 20 additional ERU cases and 10 healthy control horses; 10 ERU cases and five control horses were repeatedly tested for their T cell response in a long term study (22 months) on epitope spreading. All cases were selected randomly; we tested the next cases brought to the Equine Clinic in Munich without further selection in a respective experimental condition (autoantibody profiling, large scale autoantibody screening, T cell response, and epitope spreading). We used 20-week-old Lewis rats (Elevage Janvier; 14 rats, Table I) and German standard outbreed horses for the induction of uveitis (six horses, Table I). Initial uveitis induction experiment in rats involved immunization of six Lewis rats subcutaneously with 100 μg of recombinant human cRALBP emulsified in 50 μg of complete Freund’s adjuvant (Sigma). As a negative control, three Lewis rats were immunized with an unrelated protein (chicken IL-12p40 in 50 μg of complete Freund’s adjuvant (CFA)) expressed and purified using the same bacteria and protocols. In a second experiment, Lewis rats were immunized with 75 μg of cRALBP in 50 μg of CFA (n = 3); separate animals (n = 2) were immunized with the same amount of IL-12p40 in 50 μg of CFA as a negative control. For the induction of uveitis in horses, three horses received 500 μg of cRALBP in 500 μl of CFA subcutaneously at the neck. were in for potential disease control horses received cRALBP by an protein were by the of induced uveitis and cRALBP cell μg of μg of μg of μg of μg of μg of rats μg of μg of μg of μg of μg of μg of μg of μg of μg of μg of μg of μg of μg of μg of in a were with and The protein were in and were in μg of protein additional and on a for 100 at 20 by on at a set of for and the second for Western were with and with sera from ERU cases and binding detected with and To to detected on we the with were from in 100 μl of and to in μl of 1 were on a as described S.M. S. F. van M. of protein in is with in Full Text Full Text PDF PubMed Scopus Google Scholar). samples were with a of in and in in a on were using the and accuracy in in the are as Human cRALBP by expressed in bacteria and purified on a on an and by a The purified by as human cRALBP For large scale autoantibody screening, an used to were with 1 μg of of Sera were tested in a A control tested in a on samples were defined as than the of negative controls with the standard All of the cRALBP were verified with Western blots using 1 μg of cRALBP samples from ERU and healthy control group were by Western cRALBP used as a control. We T cell using the spontaneous ERU (n = and healthy control horses (n = uveitis horses and negative control horses (n = and negative control rats (n = blood-derived or were by and cells were for with or without cRALBP, peptides 14 and peptides S-Ag S-Ag or complete were with of for and Uveitis in horses induced by of 500 μg of cRALBP emulsified in CFA and an of control horses (n = received the same but cRALBP by group of rats = received 100 μg of cRALBP in the second group of rats = received 75 μg in rats = = 2) received the same amount of of We clinical between and disease and based on of the and retinal were on a scale from to based on infiltration by inflammatory cells, retinal and destruction of the retinal were in at for in The a from E. We used specific for glutamine and glial fibrillary acidic protein as cell van Willebrand and vascular endothelial growth were used to were with the to or or with to or of cRALBP autoantibodies in ERU and control using the The remitting-relapsing of several autoimmune diseases be by epitope spreading. of the epitopes to the and a autoaggressive To the of involved autoantigens in the spontaneous disease ERU, we tested the of sera from affected horses on the retinal proteome. protein by to and with sera autoantibodies from 35 ERU cases and 32 protein detected by ERU sera and not by controls were selected for further by As to the characterized autoantigens S-Ag and IRBP were additional were by autoantibodies of ERU cases of we identified as and cRALBP The of had been in the Lewis Smet M.D. C.C. of experimental and human have 2001; PubMed Scopus Google Scholar). Furthermore cRALBP is a novel autoantigen not described we the of in a group of horses with spontaneous We tested the of cRALBP horses with ERU by sera from 289 horses 95 controls and an with human recombinant cRALBP as were verified with Western blots of ERU cases (84 of a to human cRALBP with of control sera 11 of of cRALBP autoantibodies in ERU than in As most autoimmune diseases are T we next examined the cRALBP recognition by autoaggressive T from 20 ERU cases and 10 control horses were in with μg of human cRALBP in For we used an cRALBP to to from the E. expression We from five horses with spontaneous uveitis an to human cRALBP additional to human cRALBP, a to an ERU from healthy control horses not after in with cRALBP, or IRBP not We next to the of cRALBP as autoantigen in a long term study on epitope spreading in 10 samples were and the in cellular response pattern of were tested to human cRALBP and several peptides various epitopes of the retinal autoantigens S-antigen and same horses were during this for of inner eye In the of an an additional at particular to on the response during The total of from to and analysis of cRALBP during the The response in five healthy horses tested with experimental We a T cell to cRALBP in three ERU horses during the study and two of are here and or by a to retinal and epitope spreading in ERU horses and the of particular ERU a cRALBP response during a in ERU in due to a to the for revealed uveitis of the eye and uveitis with tissue destruction of the eye and Although complete destruction of retinal for the eye infiltration of the eye to the and a complete of acidic fibrillary protein in both and lesions the of We tested the uveitogenic potential of cRALBP on horses and the to spontaneous ERU For this we three healthy horses with 500 μg of cRALBP emulsified in 500 μl of CFA subcutaneously at A cRALBP response detected between and after immunization with T cell in response to cRALBP at I). All three horses uveitis between and 14 after the second immunization and with in the and in the retinal with and uveitis in the to the of the eye by clinical of = and of uveitis in horses of analysis of equine uveitis destruction of the retinal with complete and a and and of the blood-retinal The infiltration by cells were in the In of tissue we observed a in the of retinal as by van Willebrand were with analysis of the cell and glutamine GFAP, GS, revealed by a retinal = and control horses were with at the same as the We could not detect of uveitis in the of animals and = and To complete the characterization of cRALBP as autoantigen, we next tested the uveitogenic potential of human cRALBP in the Lewis rats were immunized subcutaneously with 100 μg of recombinant human cRALBP emulsified with 50 μg of In the control cRALBP with an unrelated protein (chicken expressed and purified using the same bacteria and protocols. The for this experiment 32 and the were examined of six rats of uveitis with lesions typical for uveitis the and complete destruction of the retinal observed in rats by of a and healthy and and the We the of inflammatory cells the inner as as infiltration in the and inner expression retinal The of rats had as by for and expression to in retinal and in expression the The of the rats and with a five of six In a second experiment, the cRALBP immunization we observed in this group of with similar to in the group and an of and of uveitis were detected in animals the control A autoreactive T cell response detectable in of rats using cells for the in I). cells from chicken IL-12p40 rats from both not to in with cRALBP I). Autoantibodies to cRALBP were in rats from the cRALBP immunization I) in to the IL-12p40 negative control rats I). 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Gottlieb P.A. Putnam A.L. Durinovic-Bello I. Boehm B.O. Tary-Lehmann M. Lehmann P.V. T cells recognize multiple GAD65 and proinsulin epitopes in human type 1 diabetes, suggesting determinant spreading.J. Clin. Immunol. 2004; 24: 327-339Crossref PubMed Scopus (43) Google Scholar), multiple V.K. Yu M. L. of autoreactivity during progression of experimental autoimmune and multiple Exp. Med. PubMed Scopus Google Scholar), and autoimmune uveitis (3de Smet M.D. Bitar G. Mainigi S. Nussenblatt R.B. Human S-antigen determinant recognition in uveitis.Investig. Ophthalmol. Vis. Sci. 2001; 42: 3233-3238PubMed Google Scholar). the to identify the complete of autoantigens J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar). a we to additional autoantigens in a spontaneous autoimmune disease in outbreed a a of from the The two-dimensional of in of The of protein from Western blots to protein pattern on for of potential autoantigens by by the blots with R. of with and of and with and PubMed Scopus Google Scholar). The the complete protein pattern to after with sera, the of to and the of the and of the protein We to be an for autoantigen to identify not described autoantigens and but a previously autoantigen in autoimmune uveitis, and protein with several to the cRALBP in the and is expressed in retinal cells and retinal M. J. Cellular retinaldehyde-binding protein with 50 in retinal Ophthalmol. Vis. Sci. 2004; 45: PubMed Scopus Google Scholar). in the human cRALBP cause a condition characterized by and H. in the cellular retinaldehyde-binding protein in a of Ophthalmol. Vis. Sci. Google Scholar). identifying autoantibodies in ERU cases by blotting, further for the of cRALBP in ERU by a large scale screening for cRALBP autoantibodies in horses uveitis, = healthy = We a in between ERU cases and healthy controls and T cells were in six ERU cases but not in healthy control the of the autoantibody profiling in identifying autoantigens in T cell-mediated diseases as we could a link between the to cRALBP and epitope spreading in two horses and from a long term T cell spreading Although to response profile to cRALBP we could a to cRALBP at the of the were this previously the response pattern to cRALBP with the observed recurrence of uveitis, this could have been by the A further long term study several ERU cases is to the role of cRALBP in the uveitis spreading In complete characterization of cRALBP as autoantigen, we could uveitogenic potential in the and Lewis In both the cells were and for at between and and and GS, and and cell S.M. S. M. profiling of retinal cells a in expression upon to in 2003; PubMed Scopus Google Scholar). The of VEGF, a vascular and could be in retinal and cells in the equine model In a role in the of and in breakdown of the blood-retinal characterized by of retinal D. M. S. I. R. of the with but not in 2004; PubMed Scopus Google Scholar). In the ERU we to the uveitis model in and of a long term study on epitope spreading and a with a to cRALBP as autoantigen The of a role of cRALBP in ERU as is a of autoantigens is for at risk of autoimmune is best autoantibodies to different autoantigens are in the J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar). Type I diabetes is and best in this The presence of autoantibodies three different autoantigens, the of and insulin, a predictive for diabetes between and in the general increases to in J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar). the of a diabetes screening J.M. Brantley M.S. Leech N.J. Rowe R.E. Monks S. Palmer J.P. Nepom G.T. McCulloch D.K. Hagopian W.A. Successful prospective prediction of type 1 diabetes in schoolchildren through multiple defined autoantibodies: an 8-year follow-up of the Washington State Diabetes Prediction Study.Diabetes Care. 2002; 25: 505-511Crossref PubMed Scopus (139) Google Scholar, S. M.S. M. epitopes and during the in of with type 1 diabetes.J. Autoimmun. 2004; PubMed Scopus Google Scholar, J.M. Yu L. D. J.M. Eisenbarth G.S. M. Prediction of autoantibody and progression to type 1 Diabetes in the Clin. 2004; PubMed Scopus Google Scholar), and a study is induction to in cell H. S. M. J. M. of and prediction of type I diabetes in a 2001; PubMed Scopus Google Scholar). prior to clinical been between and onset of systemic lupus R. M. Antinuclear the onset of systemic lupus Rheumatol. Google and between and in van D. van van J.P. B.A. of protein in from the onset of 2004; PubMed Scopus Google Scholar). and increases both the predictive and to of in persons van D. van van J.P. B.A. of protein in from the onset of 2004; PubMed Scopus Google Scholar). The predictive of cRALBP autoantibodies for autoimmune uveitis to be tested in additional control of a of autoantigens in type I diabetes the three autoantigens are most for and prediction J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar). cRALBP autoantibodies have been in control horses, we to for several multiple have the of autoantibodies for disease in T cell-mediated autoimmune diseases J. Scherbaum W.A. Are we ready to predict and prevent endocrine/organ specific autoimmune diseases?.Springer Semin. Immunopathol. 2002; 24: 273-295Crossref PubMed Scopus (12) Google Scholar, R.H. Autoantibodies as predictors of disease.Lancet. 2004; 363: 1544-1546Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, van D. van van J.P. B.A. of protein in from the onset of 2004; PubMed Scopus Google Scholar). In been the autoimmune clinical for or As cRALBP healthy horses could be at risk for uveitis in the the for autoantibody profiling of various autoimmune diseases is a and for autoantigens involved in the dynamic spreading cascade and be as a for predictive We for the of human with