In vitro studies of ANCA glomerulonephritis pathogenesis

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The proposed research will investigate experimental animal models that elucidate afferent (immunogenic) and efferent (pathogenic) mechanisms of the autoimmune process that cause glomerulonephritis and vasculitis in patients with anti-neutrophil cytoplasmic autoantibodies (ANCA). The efferent mechanisms will be investigated by testing the hypothesis that ANCA induce disease by synergistic interactions with other pro- inflammatory stimuli. This possibility is supported by clinical observations that suggest that infectious processes prompt the onset and exacerbation of ANCA- disease, and by in vitro experimental observations indicating that neutrophil activation by ANCA requires synergistic neutrophil priming by pro-inflammatory cytokines. Mice with circulating anti-MPO antibodies will be generated by active immunization with MPO, passive administration of monoclonal murine anti-MPO antibodies, and introduction of immunoglobulin transgenes that produce anti-MPO antibodies. Mice with circulating anti-MPO, MPO-knockout mice with circulating anti-MPO, and control mice will be challenged with sub-neophritogenic doses of anti-GBM, low dose neophritogenic heterologous antigen (horse apoferritin) that induces only mild glomerular inflammation in normal mice, and systemic injection of pro-inflammatory cytokines. Measuring urine protein, serum creatine, and pathologic changes in renal and other tissues will monitor the onset of renal disease by the synergistic interaction of ANCA with changes in renal and other tissues will monitor the onset of renal disease by the synergistic interaction of ANCA with pro-inflammatory stimuli. Afferent mechanisms of ANCA induction (ANCA immunogenesis) will be studied by testing the hypothesis that certain environmental factors induce the ANCA autoimmune response. This hypothesis is supported by the clinical observation that the development of ANCA and of ANCA-disease can be correlated with environmental exposures, for example silica and certain drugs. BALB/c, C57B6, autoimmune prone mice (MRL/lpr, NOD), and transgenic mice expressing anti-MPO will be exposed to silica or aminoguanidine. Control and exposed mice will be monitored for anti- MPO production and development of ANCA-disease. When models of ANCA immunogenesis and ANCA pathogenesis are established, the underlying mechanisms can be evaluated using experimental tools developed to investigate the immune system and inflammatory mediator systems in mice (measuring mRNA expression and protein levels of inflammatory mediators, evaluating effects of cellular and humoral immune and inflammatory effector depletion, and assessing the effects of targeted gene knock outs (e.g. of chemokines and cytokines, and their receptors.