OK COBRE: NEUTROPHIL PROTEINASE-3 AUTOANTIGEN EXPRESSION AND REGULATION

促炎细胞因子，如肿瘤坏死因子（TNF），在以组织破坏为特征的疾病的病理生理学中起重要作用。最近，TNF前体被发现是中性粒细胞蛋白酶-3（PR 3）的底物。PR 3是一种在中性粒细胞表面表达的丝氨酸蛋白酶，是韦格纳肉芽肿病（一种严重的自身免疫性血管炎）患者中促炎性PR 3-ANCA抗体的自身抗原。我们发现，可溶性内皮蛋白C受体（sEPCR），蛋白C抗凝剂和抗炎途径的成员，通过PR 3和PR 3-ANCA与活化的中性粒细胞结合，部分但不是全部韦格纳病患者减少sEPCR与中性粒细胞的结合。我们目前的数据包括新的观察结果，即PR 3对TNF前体的活性被sEPCR下调。这些观察结果导致我们的假设，即中性粒细胞PR 3的表达提供了对炎症的立即宿主反应，以及sEPCR结合和随后下调反应的模板。由ANCA识别的共同或独特的PR 3表位可能会改变这种反应途径，并有助于疾病的表现。目的1研究使用诱变技术和功能测定来确定控制sEPCR与PR 3相互作用的结构要求。目的2研究评估这些分子相互作用如何随后受PR 3-ANCA关于表位特异性和疾病表现的影响。目的3研究确定了层流剪切应力对中性粒细胞PR 3表达的重要性，推断体内流变力导致中性粒细胞PR 3暴露并在小型至中型血管中发现的流动条件下接触韦格纳自身抗体。目的4研究确定PR 3 DNA序列和PR 3表达水平之间的关系，以确定导致个体之间观察到的PR 3表达表型稳定变异的机制。独特的多态性或启动子胞嘧啶甲基化水平可能影响PR 3表达 水平或改变对结合sEPCR重要的区域，可能导致韦格纳血管炎或疾病严重程度的风险。结构和功能数据与患者临床状况的相关性将有助于更好地理解导致这些患者内皮损伤的潜在过程。这些研究还将影响我们对PR 3如何促进细胞因子产生以及sEPCR在调节自身免疫介导的血管炎中中性粒细胞炎症反应中的作用的理解。这种监管机制 可能有助于定位对ANCA的反应，并防止细胞因子引起的更系统的有害作用。

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF), play an important role in the pathophysiology of diseases characterized by tissue destruction. Recently, the TNF precursor was found to be a substrate for neutrophil proteinase-3 (PR3). PR3 is a serine proteinase expressed on the surface of neutrophils and is the autoantigen of pro-inflammatory PR3-ANCA antibodies in patients with Wegener's granulomatosis, a severe autoimmune vasculitis. We showed that the soluble endothelial protein C receptor (sEPCR), a member of the protein C anticoagulant and anti-inflammatory pathway, binds to activated neutrophils via PR3 and PR3-ANCA from some, but not all, Wegener's patients reduced sEPCR binding to neutrophils. Our current data include the novel observation that PR3 activity toward the TNF precursor is down-modulated by sEPCR. These observations lead to our hypothesis that expression of neutrophil PR3 provides for both an immediate host response to inflammation, as well as a template for sEPCR binding and subsequent down-modulation of the response. Common or unique PR3 epitopes recognized by ANCA may alter this response pathway and contribute to the disease manifestations. Aim 1 studies use mutagenesis techniques and functional assays to identify the structural requirements governing sEPCR's interaction with PR3. Aim 2 studies evaluate how these molecular interactions are subsequently influenced by PR3-ANCA with respect to epitope specificity and disease manifestations. Aim 3 studies determine the importance of laminar shear stress forces on neutrophil PR3 expression, reasoning that in vivo rheological forces lead to neutrophil PR3 exposure and access to Wegener's autoantibodies under flow conditions found in small-to-medium sized vessels. Aim 4 studies determine relationships between PR3 DNA sequence and PR3 expression levels to identify mechanisms that result in the observed stable variability in PR3 expression phenotype between individuals. Unique polymorphisms or promoter cytosine methylation levels may influence PR3 expression levels or alter regions important for binding sEPCR, potentially contributing to risk of Wegener's vasculitis or disease severity. Correlation of the structural and functional data with the patient clinical condition will contribute to a better understanding of the underlying processes that result in endothelial damage in these patients. These studies will also impact our understanding of how PR3 contributes to cytokine generation and the role of sEPCR in modulating neutrophil inflammatory responses in auto-immune-mediated vasculitis. This regulatory mechanism may contribute to localizing the response to ANCA and preventing more systemic, deleterious effects by the cytokine.