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

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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.