Anti-myeloperoxidase autoantibody response

针对髓过氧化物酶(MPO)和蛋白酶3(PR3)的抗中性粒细胞胞浆抗体(ANCA)在大多数低免疫性坏死性肾小球肾炎和小血管炎患者中检测到。这些自身抗体的产生所涉及的因素尚不清楚。我们推测，ANCA及其相关疾病的低发病率部分是由于对特定V区基因重组、特定体细胞突变或两者兼而有之的需求，而小鼠中抗MP抗体的表达是耐受性下降的结果。对来自未免疫的SCG/KJ小鼠的抗MPO抗体的研究揭示了VK基因的使用受到限制，并提供了MPO是抗MPO反应的驱动抗原的证据。本项目的两个具体目的是(1)评估小鼠抗MPO自身抗体的亲和力，(2)研究小鼠抗MPO反应的调节。目标1将通过将抗MPO衍生的H链或L链与多种非抗MPO衍生的链共表达，并引入定点突变来消除特定的体细胞突变。所产生的抗体的特异性和亲和力将通过酶联免疫吸附试验和基于生物传感器的技术进行测量。目标2将通过产生仅表达抗MPO轻链、仅表达重链以及两者都表达的转基因小鼠来实现。这种方法将允许研究他们的成熟水平的抗MPO B细胞受到调控。这两个目标将有助于填补我们目前对这些机制的一个重要空白，这些机制涉及到抗MPO自身免疫反应的产生和调节。

An anti-neutrophil cytoplasmic antibodies (ANCA), directed toward myeloperoxidase (MPO) and proteinase 3 (PR3), are detected in the majority of patients with pauci-immune necrotizing glomerulonephritis and small vessel vasculitis. The factors involved in the generation of these autoantibodies remain unknown. We hypothesize that the low incidence of ANCA and their associated diseases is in part due to requirements for particular V region gene recombinations, specific somatic mutations, or both and that the expression of anti-MP antibodies in mice is the result of a breakdown in tolerance. The study of anti-MPO antibodies derived from unimmunized SCG/KJ mice revealed a restriction in Vk gene use, and provides evidence that MPO is a driving antigen in the anti-MPO response. The two specific aims of this project are to (1) assess the affinity of murine anti-MPO autoantibodies, and (2) study the regulation of the anti-MPO response in mice. Aim 1 will be pursued by co-expressing anti-MPO derived H or L chains with a variety of non-anti-MPO-derived chains, and by introducing of removing specific somatic mutations using site-directed mutagenesis. The specificity and avidity of the resulting antibodies will be measured by ELISA and Biosensor based techniques. Aim 2 will be approached by the generation of mice transgenic for anti- MPO-light chain only, heavy chain only, and both. This approach will allow to study at which level of their maturation anti-MPO B cells are regulated. These two aims will help fill an important gap in our current knowledge of these mechanisms involved in the generation and regulation of the anti- MPO autoimmune responses.