Targeted at learning about inflammatory eye diseases, this project focused in FY 2002 on three topics: (1) surface markers that characterize T-cells capable of inducing ocular inflammation; (2) analysis of immunity and tolerance in transgenic mice expressing a foreign antigen in their retina; (3) the capacity of the retinal protein RPE65 to induce experimental autoimmune uveitis (EAU) in mice. Topic 1. To identify surface markers that characterize inflammation inducing lymphocytes we have used a system in which T-helper (Th) lymphocytes sensitized against hen egg lysozyme (HEL) induce ocular inflammation in mouse eyes expressing transgenically HEL. Previous studies revealed that Th cells induce inflammation only following activation in culture and that Th type 1 (Th1) are superior to Th type 2 (Th2) in their inflammation-inducing capacity. Th1 and Th2 cells were purified and activated by stimulation with HEL as detailed in Kim et al., Invest. Ophthal. Vis. Sci., 2002, 43:758, and expression of surface markers on the different cell populations was determined by flow cytometry. Major observations: (i) activation of both Th1 and Th2 cells profoundly increased expression of surface molecules CD25 and CD69, moderately elevated levels of CD54 and CD49d (adhesion molecules ICAM-1 and integrin alpha-4, respectively), but reduced the expression of CD62L (L-selectin); (ii) activation also increased expression of chemokine receptor CXCR3 on Th1, but not on Th2 cells. These observations suggest that the changes in surface molecules determine the pathogenic capacity of Th cells and, thus, highly expressed molecules could serve as target for immunosuppressive modalities. Topic 2. Transgenic (Tg) animals expressing foreign antigens are very useful to investigate immunity and tolerance against tissue-specific self antigens. In a previous study (Lai et al., Invest. Ophthal. Vis. Sci., 1998, 39:2049) we have shown that Tg mice expressing HEL under control of the lens alpha-A crystallin develop tolerance against HEL, due to expression of HEL in their thymus and the consequent deletion of HEL-specific T-lymphocytes. On the other hand, Gregerson et al. (J. Imm., 1999, 163:1073) reported that no tolerance could be detected in Tg mice expressing another foreign antigen, beta galactosidase, under control of the rhodopsin promoter. Since both alpha-A crystallin and rhodopsin are similarly expressed in mouse thymi (Charukamnoetkanok et al, Curr. Eye Res., 1998, 17:788), we examined the immune status of Tg mice expressing HEL under control of the rhodopsin promoter. Unlike the observation of Gregerson et al., these mice did develop tolerance to HEL, apparently by thymic elimination of HEL-specific T-cells. These results suggest, therefore, that thymic expression and tolerance against neo-self antigens in Tg mice depends on the type of antigen in use and the level of its thymic expression. Adoptive transfer of HEL-specific T-cells into the Tg mice expressing HEL in the retina produced inflammation that resembles that in mice developing EAU, thus providing a novel model for pathogenic autoimmune process targeted at a retinal antigen. Topic 3. RPE65, a protein that plays a pivotal role in the vision process, is expressed specifically in the retinal pigment epithelium (RPE) cell layer. In a previous study (Annual Report, 2001 and Ham et al., Invest. Ophthal. Vis. Sci., 2002, 43:2258) we found that RPE65 is highly immunopathogenic and induces severe EAU in several strains of rats. This study was extended in FY 2002 to examine the capacity of RPE65 to induce EAU in mice. Unlike the high susceptibility observed among rat strains, only one mouse strain (C57Bl/6) developed mild EAU when immunized with RPE65, whereas no disease was detected in other six tested mouse strains (BALB/c, AKR/J, B10.A, B10.RIII, B10.BR, C57Bl/10). Analysis of thymic expression of RPE65 revealed that this antigen is expressed in all tested mouse strains, but in none of the tested rat strains. This finding thus provides an explanation to the difference between the susceptibility of these two related species and underscores the importance of thymic expression of organ-specific antigens in determining the susceptibility of animals to pathogenic autoimmunity.
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