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J.C. Cruz, P.S. Bora, J.–H. Sohn, H. Nishihori, Y. Wang, H.J. Kaplan, N.S. Bora; Type I Collagen is the Auto–antigen in Experimental Autoimmune Anterior Uveitis. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):539.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Experimental autoimmune anterior uveitis (EAAU) is an organ specific autoimmune disease of the eye, which is an animal model of idiopathic human anterior uveitis. This study was undertaken to identify and characterize the antigen responsible for the induction of EAAU. Methods: Melanin–associated antigen isolated from bovine iris and ciliary body was digested with the proteolytic enzyme V8 protease to solubilize the proteins and the pathogenic protein was purified to homogeneity by a combination of preparative SDS–PAGE and preparative Isoelectric Focussing (IEF). Lewis rats were sensitized to various fractions and investigated for the development of anterior uveitis and an immune response to the purified antigen. The humoral response to the purified antigen was determined by enzyme–linked immunoassay (ELISA) while the cellular response to the antigen was investigated using an in vitro lymphocyte proliferation assay. Adoptive transfer of EAAU was performed using the purified antigen. The role of glycosylation in defining the tissue specificity of the uveitogenic antigen was investigated by using a chemical (TFMSA) method. Results: The uveitogenic antigen had a mass of 22 kDa (SDS–PAGE) and an isoelectric point of 6.75. The amino–terminal amino acid sequence of this protein demonstrated 100% homology with the bovine type I collagen α–2 chain starting from amino acid 385 and will be referred to as BCI–α2 (22 kDa). Animals immunized with BCI–α2 (22 kDa) developed both cellular and humoral immunity to the antigen. They developed anterior uveitis only if BCI–α2 chain underwent proteolysis and if the bound carbohydrates were intact. EAAU induced by BCI–α2 (22 kDa) can be adoptively transferred to naïve syngenic rats by primed CD4+ T cells. The α–1 and α–2 chains (intact or proteolytically cleaved) of type I collagen from calfskin were not pathogenic suggesting that BCI–α2 (22 kDa) was organ–specific. Conclusions: Our results suggest that BCI–α2 (22kDa), a 22 kDa fragment of Type I collagen, contains the antigenic determinant(s) necessary to induce anterior uveitis in Lewis rats. Furthermore, Type I collagen must undergo tissue specific post–translational modification involving both proteolytic degradation and glycosylation to induce ocular autoimmunity.
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