The long term objective of this investigation is to elucidate the mechanisms for autoantibody induction related to certain therapeutics such as procainamide (PA) and the associated immunopathology of drug-induced lupus (DIL). It appears that drug metabolites, not the parent molecules,are involved in the initiation and maintenance of autoimmunity. PA is oxidized to the highly reactive hydroxylamine (PAHA) by activated neutrophils, and PAHA is cytotoxic to lymphocytes at micromolar concentrations. One goal of this project is to determine the basis for the capacity of medium from PAHA-treated lymphocytes to activate quiescent lymphocytes in a murine splenocyte In vitro immune system. Optimum conditions for generating mitogenic factor and its composition will be determined by purifying and isolating the active material. Production of mitogenic activity is associated with appearance of partially degraded histones in nucleosomes, and the substrate requirements and the nature of the putative endogenous protease will be determined. A well-defined xenogeneic system involving size-fractionated calf thymus oligonucleosomes will be characterized and tested for mitogenic function. The phenotype and activation status of responding lymphocytes will be determined by measuring immunoglobulin and autoantibody production, constitutive and activation antigen expression, biochemical events associated with T cell activation and cytokine gene expression. Evidence for similar events occurring in vivo in patients treated with procainamide will be sought. In these longitudinal studies changes in appearance and composition of circulating nucleohistone will be determined by immunoassay with a set of monoclonal anti-chromatin antibodies on plasma from patients with DIL and control groups. Special emphasis will be placed on nucleosome particles containing the histone-DNA complex (H2A-H2B)-DNA, since a component epitope in this but not in other subnucleosome particles is recognized by most patients with DIL. Peptides derived from histones H2A and H2B that are predicted to bind autoantibody based on analysis of the 3-dimensional structure of the nucleosome octamer will be used to identify this ubiquitous epitope. Together with other work on the role of activated neutrophils in drug metabolism,the proposed studies offer an opportunity to determine how chronic exposure to a medication can lead to a systemic autoimmune disease.
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