MRL-lpr/lpr and C3H-gld/gld mice develop lymphadenopathy, autoantibody production, a T cell tolerance defect, and autoimmune disease. The lpr mutation has recently been identified as a mutation of the fas apoptosis gene. Despite this molecular defect, an apoptosis defect has not been clearly defined in MRL -lpr/lpr mice, or in autoimmune disease in other strains of mice or in humans. However, we have demonstrated that in CD2- fas transgenic MRL-lpr/lpr mice, correction of fas in T cells eliminates all aspects of autoimmune disease including autoantibody production and renal disease. Therefore, understanding the apoptosis defect and development of strategies to correct the apoptosis defect might lead to elimination of the tissue damage resultant from SLE. The overall goals of the project are to determine the role of defective apoptosis in lpr/lpr and gld/gld mice and determine the mechanism by which the lymphoproliferative autoimmune disease develops from the apoptosis defect. The first goal of this project will be to identify the Fas ligand using a purified Fas fusion protein. This is important, as the Fas ligand has been proposed to be the normal counterpart of the gld mutation, a second autoimmune gene. Analysis of tissue sections stained with the fas fusion protein has shown expression of the fas ligand in the thymic medulla of +/+ and lpr/lpr but not gld/gld mice. The second goal will be to determine if the fas ligand is defective in C3H-gld/gld mice. This will be carried out using high molecular weight DNA and RNA from C3H-gld and C3H-+/+ mice and analysis of restriction fragment lengths, differences in expression of fas ligand, difference in the sequence of the fas ligand cDNA obtained by PCR cloning. If a defect in the cDNA is found, the functional significance will be verified by transfection of the mutated fas ligand cDNA into Cos cells and crosslinking with fas ligand fusion protein. The third goal will be to characterize the apoptosis defect in lpr and gld mice. This will be carried out by first examining apoptosis using the terminal deoxynucleoside transferase assay in cells that express normal or abnormal levels of both fas and the fas ligand. Defective apoptosis will also be studied in the Db/HY TCR transgenic C57BL/6-lpr/lpr mice and CD2-fas transgenic lpr/lpr mice to determine if defective fas expression by T cells results in a B cell hyperactivity due to defective interaction with the fas ligand. These experiments should also help determine if defective apoptosis gives rise to abnormal clonal deletion in the thymus and production of non-tolerant peripheral T cells in the Db/HY TCR transgenic lpr/lpr male mice. The fourth goal will be to produce a fas-ligand (Fas-L) transgenic C3H- gld/gld mouse and determine if the lymphproliferative autoimmune disease is reduced or eliminated. These experiments should allow an detailed characterization of the apoptosis defects in lpr and gld mice.
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