Mechanism of Neutrophil Activation

描述(由申请人提供)： 中性粒细胞是控制微生物入侵的先天和获得性免疫反应的关键组成部分。启动是静止的中性粒细胞在激活时增强其产生活性氧中间产物(ROI)的中间步骤，启动是最佳杀灭微生物所必需的。尽管预充大大增强了中性粒细胞的杀菌能力，但预充的中性粒细胞对正常细胞也更危险。中性粒细胞参与了许多在老年退伍军人中突出的疾病，包括ANCA相关性血管炎、缺血再灌注损伤、急性呼吸窘迫综合征、类风湿性关节炎、牙周炎、动脉粥样硬化和急性炎症性肝损伤。因此，操纵中性粒细胞启动是一种潜在的药物干预抑制炎症的治疗策略。最近的研究发现，颗粒胞吐和促进NADPH氧化酶胞液成分向质膜的移位可能是中性粒细胞启动的机制。然而，知识上的许多差距仍然存在。这些事件中的每一个对启动的相对贡献还没有被检验，也不知道这两个事件对于启动是必要的还是充分的。颗粒胞吐作用促进启动的分子事件以及控制胞质NADPH氧化酶组分的增强移位的分子事件还不完全清楚。最后，在体内操纵中性粒细胞启动的能力还没有确定。McLeish实验室拥有丰富的研究信号转导途径的经验，我们应用生化、遗传学和蛋白质组学方法了解中性粒细胞生物学，以及我们在体外和体内抑制胞吐作用的新型试剂的最新发展，因此McLeish实验室处于独特的地位来填补这些差距。目前的建议将解决中性粒细胞启动的中心假设，即中性粒细胞启动的结果是信号转导途径汇聚为两种反应，即胞外依赖的NADPH氧化酶膜组分的表达增加和NADPH氧化酶胞浆组分的Pro-异构酶依赖的构象变化。将实现以下四个具体目标。具体目标1将测试工作假设，即分泌囊泡和明胶酶颗粒的胞吐作用导致质膜p91Phox和p22Phox表达增加，这是启动所必需的，但不是充分的。特定的目标2将检验一个工作假设，即激活Prolyl异构酶Pin1通过多种机制促进人中性粒细胞的启动，包括增强p47Phox易位和增加颗粒胞吐。具体的目标3将测试工作假设，即p38MAPK磷酸化多个蛋白质，这些蛋白质介导启动所需的事件，包括胞吐和Pin1激活。特定的目标4将测试 使用急性肺损伤(ALI)的动物模型，工作假说是中性粒细胞启动是中性粒细胞参与炎症性疾病的必要组成部分，并代表了一种可行的治疗策略。这项拟议的工作有望为中性粒细胞启动奠定分子基础。该项目的影响是，确定的分子靶点将允许在炎症性疾病中对中性粒细胞启动进行治疗操作。因此，治疗患有一些急性和慢性炎症性疾病的退伍军人患者的新方法是可望的。

DESCRIPTION (provided by applicant): Neutrophils are a critical component of both the innate and adaptive immune responses that control microbial invasion. Priming is an intermediate step by which resting neutrophils enhance their production of reactive oxygen intermediates (ROI) upon activation, and priming is necessary for optimal microbial killing. Although priming greatly enhances neutrophil bactericidal capability, primed neutrophils are also more dangerous to normal cells. Primed neutrophils participate in a number of diseases prominent in the aging Veteran population, including ANCA-associated vasculitis, ischemia- reperfusion injury, acute respiratory distress syndrome, rheumatoid arthritis, peridontitis, atherosclerosis, and acute inflammatory liver injury. Thus, manipulation of neutrophil priming is a potential therapeutic strategy for pharmacological intervention to restrain inflammation. Recent studies identified granule exocytosis and enhanced translocation of cytosolic components of the NADPH oxidase to the plasma membrane as possible mechanisms of neutrophil priming. However, many gaps in knowledge remain. The relative contribution of each of those events to priming has not been examined and whether both are necessary, or sufficient, for priming is unknown. The molecular events by which granule exocytosis contributes to priming and the molecular events that control enhanced translocation of cytosolic NADPH oxidase components are incompletely understood. Finally, the ability to manipulate neutrophil priming in vivo has not been established. The McLeish laboratory is in a unique position to address each of these gaps based on our extensive experience examining signal transduction pathways, our application of biochemical, genetic, and proteomic approaches to understand neutrophil biology, and our recent development of novel reagents that inhibit exocytosis in vitro and in vivo. The current proposal will address the central hypothesis that neutrophil priming results from the convergence of signal transduction pathways into two responses, an exocytose-dependent increase in plasma membrane expression of membrane components of NADPH oxidase and prolyl isomerase-dependent conformational changes in cytosolic components of NADPH oxidase. The following four specific objectives will be accomplished. Specific Objective 1 will test the working hypothesis that exocytosis of secretory vesicles and gelatinase granules results in increased plasma membrane expression of p91phox and p22phox, which is necessary, but not sufficient, for priming. Specific Objective 2 will test the working hypothesis that activation of the prolyl isomerase Pin1 contributes to priming of human neutrophils by multiple mechanisms, including enhanced p47phox translocation and increased granule exocytosis. Specific Objective 3 will test the working hypothesis that p38 MAPK phosphorylates multiple proteins that mediate events necessary for priming, including exocytosis and Pin1 activation. Specific Objective 4 will test the working hypothesis that neutrophil priming is a necessary component for neutrophil participation in inflammatory diseases and represents a viable therapeutic strategy, using an animal model of acute lung injury (ALI). The proposed work is expected to establish the molecular basis for neutrophil priming. The impact of the project is that molecular targets identified will permit therapeutic manipulation of neutrophil priming in inflammatory diseases. Thus, new approaches to treating Veteran patients with a number of acute and chronic inflammatory diseases are anticipated.