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与中性粒细胞的细胞毒性有关。基底膜的破坏不仅导致了血管炎的发病，还可能暴露了诱导体液免疫的新表位。Mac-I(CD11b/CD18，CR3)是一种白细胞特异性的β2整合素，支持吞噬细胞的黏附和细胞毒作用。它也是补体片段C3bi的主要受体。对Mac-1缺陷小鼠(MAC1-/-)的两项研究表明，Mac-1是炎症诱导的细胞毒作用所必需的，导致基底膜损伤。尽管肾小球中性粒细胞聚集，MAC1-/-在抗肾小球基底膜肾炎的反应中缺乏补体依赖性蛋白尿。此外，对于出血性血管炎模型皮肤中的Shwartzman反应，Mac-1基因缺陷的小鼠没有出现出血，这与血管壁层粘连蛋白降解的缺失有关。这是尽管MAC1-/-中性粒细胞的积累与野生型动物相当。对相关基因敲除小鼠的研究表明，出血需要补体C3，但不需要中性粒细胞聚集，NADPH氧化酶衍生的氧自由基在病理中没有明显作用。这项建议的目的是了解Mac-L在补体依赖的中性粒细胞毒性中的作用的细胞和分子机制。我们将验证我们的假设，即Mac-1与C3bi在血管壁上的黏附产生一个封闭的隔室(“免疫突触”)，用于集中释放蛋白酶，并刺激脱颗粒，这两个步骤可能是中性粒细胞细胞毒所必需的。此外，我们认为这两个步骤需要CD1lb细胞质尾巴和选择下游的整合素信号分子。在目标I中，我们将阐明Mac-1介导的细胞毒性所需的细胞内序列以及选择的信号分子在这一过程中的作用。在AIM II中，将寻找脱颗粒导致蛋白酶释放并在Shwartzman反应中形成依赖于Mac-1的免疫突触的证据。信号分子src、syk和avv在Shwartzman反应中的作用将被评估。在目标III中，将阐明补体结合和Mac-t的胞浆结构域在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-L在中性粒细胞细胞毒性中的作用的理解，并可能导致确定可能干扰这一功能的治疗靶点。

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.