MAC-1(CR3) IN IMMUNE-MEDIATED NEUTROPHIL CYTOTOXICITY

描述（由申请人提供）： 在许多自身免疫性疾病中观察到的微血管损伤涉及神经细胞的细胞毒性。基底膜破坏不仅有助于血管炎的发病机制，而且还可能暴露诱导体液免疫的新表位。Mac-I（CD 11b/CD 18，CR 3）是一种白细胞特异性β-2整合素，支持吞噬细胞的粘附和细胞毒性功能。它也是补体片段C3 bi的主要受体。对Mac-1缺陷小鼠（Mac 1-/-）的两项研究表明，Mac-1是导致基底膜损伤的炎症诱导的细胞毒性所必需的。Mac 1-/-在抗肾小球基底膜肾炎中缺乏补体依赖性蛋白尿，尽管肾小球中性粒细胞聚集。此外，响应于皮肤中的Shwartzman反应（出血性血管炎的模型），Mac-1缺陷的小鼠没有表现出出血，这与血管壁中没有层粘连蛋白降解相关。尽管Mac 1-/-中的中性粒细胞蓄积与野生型动物相当。在相关基因敲除小鼠中的研究表明，补体C3是出血所需的，但不是中性粒细胞蓄积所需的，NADPH氧化酶衍生的氧自由基在病理学中不起重要作用。本提案的目标是了解Mac-I在补体依赖性中性粒细胞细胞毒性中作用的细胞和分子机制。我们将测试我们的假设，即Mac-1粘附到血管壁中的C3 bi产生一个封闭的隔室（“免疫突触”），用于集中的蛋白酶释放，并刺激脱粒，这是嗜中性粒细胞细胞毒性可能需要的两个步骤。此外，我们提出，这两个步骤需要的CD 11b细胞质尾和选择下游整合素信号分子。在目的我，我们将阐明Mac-1介导的细胞毒性和选择信号分子在这一过程中的作用所需的细胞内序列。在目的II中，将寻找脱粒导致蛋白酶释放和在Shwartzman反应中形成免疫突触的证据，该反应是Mac-1依赖的。将评估信号分子src、syk和vav在Shwartzman反应中的作用。在目的III中，将阐明补体结合和Mac-t胞质结构域在Shwartzman发病机制中的体内作用。这些研究的结果将极大地扩展我们对Mac-I在中性粒细胞毒性中的作用的理解，并且可以导致鉴定可以干扰该功能的治疗靶标。

DESCRIPTION (provided by applicant): Neutrophil cytotoxicity is implicated in the microvascular damage observed in a number of autoimmune diseases. Basement membrane disruption contributes not only to the pathogenesis of vasculitis but may also expose neoepitopes that induce humoral immunity. Mac-I(CD11b/CD18,CR3), a leukocyte specific beta-2 integrin, supports adhesion and cytotoxic functions in phagocytes. It is also the primary receptor for complement fragment C3bi. Two studies on Mac-1 deficient mice (Mac1-/-) revealed that Mac-1 is required for inflammation-induced cytotoxicity leading to basement membrane damage. Mac1-/- lacked complementdependent proteinuria in response to anti-glomerular basement membrane nephritis despite glomerular neutrophil accumulation. Furthermore, in response to the Shwartzman reaction in the skin, a model of hemorrhagic vasculitis, mice deficient in Mac-1 exhibited no hemorrhage which correlated with an absence of laminin degradation in the vessel wall. This was despite neutrophil accumulation in Mac1-/- that was comparable to wild-type animals. Studies in relevant knock-out mice revealed that complement C3 was required for hemorrhage but not neutrophil accumulation and that NADPH oxidase derived oxygen radicals did not play a significant role in the pathology. The goal of this proposal is to understand cellular and molecular mechanisms that underly Mac-l's role in complement-dependent, neutrophil cytotoxicity. We will test our hypothesis that Mac-1 adhesion to C3bi in the vessel wall generates a sealed compartment ("immunological synapse") for focalized protease release, and stimulates degranulation, two steps likely required for neutrophil cytotoxicity. Furthermore we propose that these two steps require the CD1 lb cytoplasmic tail and select downstream integrin signaling molecules. In Aim I we will elucidate the intracellular sequences required for Mac-1 mediated cytotoxicity and the role of select signaling molecules in this process. In Aim II, evidence for degranulation leading to protease release and formation of an immunological synapse in the Shwartzman reaction that is Mac-1 dependent will be sought. The role of signaling molecules src, syk and vav in the Shwartzman reaction will be evaluated. In Aim III, the in vivo role of the complement binding and the cytoplasmic domain of Mac-t in the pathogenesis of Shwartzman will be elucidated. The results of these studies will greatly extend our understanding of Mac-l's role in neutrophil cytotoxicity and could lead to the identification of therapeutic targets that can interfere with this function.