Several lines of evidence indicate that the Ro/SS-A (Ro) autoantibodies and their target autoantigens play major roles in the pathogenesis of subacute cutaneous lupus erythematosus (SCLE) and neonatal LE (NLE). Recently cDNA clones have been characterized that encode immunologically distinctive 52 kD and 60 kD Ro autoantigens which share no significant amino acid sequence homology. These cDNA clones will be utilized in the proposed studies aimed at further elucidating the role that these autoantigens and autoantibodies play in the pathogenesis of SCLE and NLE skin disease. The specific short term goals of this project are to: 1. Develop a panel of antibodies that are specific for the 52 and 60 kD Ro molecules. To accomplish this, Ro antibodies will be affinity purified from anti-Ro rabbit antisera and Ro autoimmune sera by using recombinant human 52 or 60 kD Ro protein. Murine monoclonal antibodies will also be raised against each recombinant Ro protein. The development of 52 and 60 kD Ro specific antibodies is of paramount importance as these antibodies will allow investigators to more precisely study each Ro autoantigen. These antibodies will serve as valuable tools not only for the proposed studies but for future studies as well. 2. Determine the effects of clinically-relevant perturbations on human keratinocyte expression and intracellular distribution of the Ro autoantigens. These studies will be done on cultured human keratinocytes and in both normal human skin and SCLE patient skin. Ro specific antibodies will detect their respective antigens by microscopy (conventional immunofluorescence, scanning confocal, and immunoelectron). Ro protein expression will be quantified by ELISA inhibition and whole cell ELISA methods. Knowledge gained from these studies should further elucidate the roles that the Ro autoantigens and autoantibodies play in the pathogenesis of SCLE and NLE, and provide a new foundation on which future investigative studies can be devised. It is hoped that these efforts will provide information that serves to expedite the development of more effective therapies for these LE-related skin diseases.