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性和适应性免疫反应的关键组成部分。致敏是一个中间步骤，通过该步骤，静息中性粒细胞在活化时增强其活性氧中间体（ROI）的产生，并且致敏对于最佳的微生物杀灭是必要的。虽然致敏能大大增强中性粒细胞的杀菌能力，但致敏的中性粒细胞对正常细胞也更危险。致敏的中性粒细胞参与许多在老年退伍军人群体中突出的疾病，包括ANCA相关的血管炎、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性肝损伤。因此，操纵中性粒细胞引发是抑制炎症的药物干预的潜在治疗策略。最近的研究确定了颗粒胞吐作用和NADPH氧化酶的胞质组分向质膜的增强易位是中性粒细胞引发的可能机制。然而，在知识方面仍然存在许多差距。这些事件中的每一个对启动的相对贡献还没有被检查，并且对于启动来说，两者是否都是必要的或足够的还不清楚。颗粒胞吐促进引发的分子事件和控制胞质NADPH氧化酶组分增强易位的分子事件还不完全清楚。最后，在体内操纵中性粒细胞引发的能力尚未建立。McLeish实验室是在一个独特的位置，以解决每一个这些差距的基础上，我们广泛的经验检查信号转导途径，我们的应用生物化学，遗传学和蛋白质组学的方法来了解中性粒细胞生物学，以及我们最近开发的新试剂，抑制胞吐作用在体外和体内。目前的建议将解决的中心假设，即中性粒细胞引发的结果从收敛的信号转导途径到两个反应，外切依赖性增加质膜表达的膜组件的NADPH氧化酶和脯氨酰异构酶依赖性构象变化的NADPH氧化酶的胞质组分。将实现以下四个具体目标。 具体目标1将检验工作假设，即分泌囊泡和明胶酶颗粒的胞吐作用导致p91 phox和p22 phox的质膜表达增加，这是启动所必需的，但不是充分的。具体目标2将检验工作假设，即脯氨酰异构酶Pin 1的激活通过多种机制（包括增强的p47 phox易位和增加的颗粒胞吐）促进人中性粒细胞的启动。具体目标3将检验p38 MAPK磷酸化介导引发所需事件（包括胞吐和Pin 1激活）的多种蛋白质的工作假设。具体目标4将测试 中性粒细胞启动是中性粒细胞参与炎性疾病的必要组成部分，并代表了一种可行的治疗策略，使用急性肺损伤（ALI）的动物模型的工作假设。这项工作有望为中性粒细胞致敏奠定分子基础。该项目的影响是，确定的分子靶点将允许炎症性疾病中中性粒细胞引发的治疗操作。因此，预期治疗患有许多急性和慢性炎症性疾病的退伍军人患者的新方法。

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.