Uveitis is an inflammatory disease of the eye afflicting 2.3 million people in the United States that accounts for 10 percent of severe visual handicaps. The long-term objective is to understand the role of a potent antigen-presenting cell (APC), the dendritic cell (DC), in the immunopathogenesis of uveitis and to use this information for therapeutic purposes. A well-established animal model, experimental autoimmune uveitis (EAU), will be studied. This prototypic T cell-mediated autoimmune disease can be induced with a T cell line, SP35, specific for S-antigen, a normal component of photoreceptor cells that are destroyed in the most severe form of EAU. Monoclonal antibodies (mAbs) against DCS prevent the development of EAU. Thus, DCS may provide the antigen stimulation SP35 cells require within the eye in order to be uveitogenic. This proposal tests the hypothesis that DCS are suppressed by macrophages until activated by cytokines produced upon entry of SP35 cells, whereupon DCS function as the principal APCs to stimulate these cells, thereby resulting in the development of EAU. First, the functional activities and phenotypes of DCs in normal and uveitic eyes will be studied. DCs will be isolated from iris-ciliary body and choroid by immunomagnetic selection that makes use of anti-DC mAb. Functional assays will be performed in vitro for: (1) ability to process and present antigen; (2) stimulatory capability in a mixed leukocyte reaction; and (3) accessory activity required for T cell responses to mitogen. Phenotypic analysis will involve the use of mAb against cell surface molecules required for DC-T cell interactions and upregulated on activated DCs. Levels of these molecules on DCs from normal and uveitic eyes will be quantitated by confocal microscopy on stained wholemounts of iris-ciliary body and choroid. Second, anti-DC mAb will be injected intraocularly to determine their effect on DCs in preventing EAU. Effective doses will be established, and confocal microscopy and staining used to determine if DCs have been eliminated, and if not, DCs will be obtained by immunoselection for studies on potential impairment of their functional activities. Because the kinetics of SP35 cells in the eye differs from that of nonspecific T cell blasts and is driven by an APC, SP35 cells will be labeled with a lipophilic fluorescence dye, PKH26, to study by fluorescence microscopy their kinetics in eyes injected with anti-DC mAb and in contralateral control eyes. And finally, two mutually exclusive populations of macrophages in the uveal tract, one ED1+ and the other ED2+, will be purified from normal and uveitic eyes for determination of the three functional activities listed above and for their effect on these functional activities of DCs.
