In vitro studies of ANCA glomerulonephritis pathogenesis

拟议的研究将调查实验动物模型，阐明自身免疫过程的传入（免疫原性）和传出（致病）机制，导致肾小球肾炎和血管炎患者抗中性粒细胞胞质自身抗体（ANCA）。将通过检验ANCA通过与其他促炎刺激的协同相互作用诱导疾病的假设来研究传出机制。这种可能性得到了临床观察的支持，这些观察表明感染过程促使ANCA疾病的发作和恶化，并且得到了体外实验观察的支持，这些观察表明ANCA对中性粒细胞的激活需要促炎细胞因子的协同中性粒细胞引发。通过MPO主动免疫、单克隆鼠抗MPO抗体被动给药和引入产生抗MPO抗体的免疫球蛋白转基因，产生具有循环抗MPO抗体的小鼠。具有循环抗MPO的小鼠、具有循环抗MPO的MPO敲除小鼠和对照小鼠将用亚致新剂量的抗GBM、在正常小鼠中仅诱导轻度肾小球炎症的低剂量致新异源抗原（马脱铁铁蛋白）和促炎细胞因子的全身注射进行攻击。测量尿蛋白、血清肌酸以及肾和其他组织中的病理变化将通过ANCA与肾和其他组织中的变化的协同相互作用来监测肾病的发作。ANCA诱导（ANCA免疫发生）的传入机制将通过检验某些环境因素诱导ANCA自身免疫反应的假设来研究。这一假设得到了临床观察的支持，即ANCA和ANCA疾病的发展可能与环境暴露有关，例如二氧化硅和某些药物。BALB/c、C57 B6、自身免疫易感小鼠（MRL/lpr、NOD）和表达抗MPO的转基因小鼠将暴露于二氧化硅或氨基胍。将监测对照和暴露小鼠的抗MPO产生和ANCA疾病的发展。当建立ANCA免疫发生和ANCA发病机制的模型时，可以使用开发用于研究小鼠中的免疫系统和炎性介质系统的实验工具（测量炎性介质的mRNA表达和蛋白质水平，评估细胞和体液免疫和炎性效应物消耗的作用，以及评估靶向基因敲除（例如趋化因子和细胞因子及其受体的敲除）的作用。

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.