Regulation of Neutrophil Migration and Polarity

描述（由申请人提供）：中性粒细胞是人体内最丰富的白细胞，是人体抵御微生物感染的第一道细胞防线。虽然中性粒细胞通常存在于血流中，但它们会迅速迁移到组织中的感染部位。这种迁移受到严格监管；事实上，中性粒细胞的异常积累会导致组织损伤。因此，了解控制中性粒细胞迁移的分子机制具有重要的临床意义。这个应用程序的重点是了解小Rho GTPase Cdc42在中性粒细胞极性迁移过程中的作用。我们对Cdc42-null中性粒细胞的分析表明，Cdc42是中性粒细胞极性的关键调节因子。在机制水平上，我们已经确定了介导Cdc42功能并涉及白细胞整合素CD11b/CD18的新途径。CD11b/CD18是一种与致命性人类免疫缺陷疾病白细胞粘附缺陷综合征相关的粘附分子。CD11b是中性粒细胞向组织迁移的重要介质，其机制尚不明确。我们发现CD11b聚集在cdc42缺失的细胞中是有缺陷的，恢复CD11b聚集可以恢复极性，从而表明CD11b参与中性粒细胞极性。有趣的是，我们发现CD11b-null中性粒细胞与Cdc42-null细胞相似，在迁移过程中表现出极性缺陷。这些数据提出了Cdc42通过CD11b信号调节中性粒细胞极性的假设。我们建议确定Cdc42-CD11b轴在体外和体内调节中性粒细胞迁移中的分子机制和生物学后果。我们计划利用视频显微复制、免疫荧光成像和生物化学实验来研究Cdc42调控极化中性粒细胞中CD11b聚集的分子机制；（目的2）确定在中性粒细胞极性中Cdc42-CD11b轴下游被激活的信号通路。（目的3）由于已知CD11b在中性粒细胞向肺迁移中起重要作用，如果Cdc42-CD11b轴在中性粒细胞极性中起关键作用，那么Cdc42的缺乏应该会导致体内肺部炎症过程中的等效缺陷。我们将使用cd11b依赖性肺损伤模型来验证这一点。我们认为，对初级Cdc42-null中性粒细胞的分析为研究Cdc42在中性粒细胞极性中的特定功能提供了难得的机会。如果我们关于Cdc42通过CD11b信号调节中性粒细胞极性的假设得到验证，这将显著改变Cdc42和CD11b在中性粒细胞迁移中的功能，从而改变中性粒细胞依赖性炎症的观点。拟议的研究有望导致了解和最终治疗中性粒细胞相关疾病的策略。

DESCRIPTION (provided by applicant): Neutrophils are the most abundant white blood cell in humans, and form the body's first line of cellular defense against infecting microorganisms. While normally found in the blood stream, neutrophils migrate rapidly to sites of infection in tissue. This migration is tightly regulated; indeed, aberrant accumulation of neutrophils causes tissue injury. The understanding of the molecular mechanism(s) controlling neutrophil migration is thus of considerable clinical importance. This application focuses on understanding the role of the small Rho GTPase Cdc42 in neutrophil polarity during migration. Our analysis of Cdc42-null neutrophils shows that Cdc42 is a critical regulator of polarity in neutrophils. At a mechanistic level, we have identified a new pathway mediating Cdc42 functions and involving the leukocyte integrin CD11b/CD18. CD11b/CD18 is an adhesion molecule associated with a lethal human immunodeficiency disorder Leukocyte Adhesion Deficiency syndrome. CD11b is an important mediator of neutrophil migration into tissue, of ill-defined mechanism. We show CD11b clustering is defective in Cdc42-null cells, and restoring CD11b clustering rescues polarity - thereby suggesting the involvement of CD11b in neutrophil polarity. Interestingly, we show that CD11b-null neutrophils, similarly to Cdc42-null cells, exhibited defective polarity during migration. These data suggest the hypothesis that Cdc42 regulates neutrophil polarity through CD11b signaling. We propose to determine the molecular mechanism and biological consequences of the Cdc42-CD11b axis in modulating neutrophil migration both in vitro and in vivo. We plan to use video microcopy, immunofluorescence imaging and biochemistry experiments to examine (Aim1) the molecular mechanism by which Cdc42 regulates CD11b clustering in polarized neutrophils; (Aim 2) to determine the signaling pathway that is activated downstream of the Cdc42-CD11b axis in neutrophil polarity. (Aim 3) Since CD11b is known to play significant roles in neutrophil migration into lungs, if the Cdc42-CD11b axis is critical in neutrophil polarity, then deficiency of Cdc42 should result in equivalent defects in lung inflammatory processes in vivo. We will test this using a model of CD11b-dependent lung injury. We believe that analysis of primary Cdc42-null neutrophils offers a rare opportunity to study the specific function of Cdc42 in neutrophil polarity. If our hypothesis that Cdc42 regulates neutrophil polarity though CD11b signaling is validated, this will significantly change the view of Cdc42 and CD11b functions in neutrophil migration and thus in neutrophil-dependent inflammation. The proposed studies are expected to lead to strategies to understand and ultimately treat neutrophil-related disorders. PUBLIC HEALTH RELEVANCE: Rapid and directed migration of neutrophils to sites of infection is the first line of defense employed by the body. Failure to regulate neutrophil migration leads to an inability to control infection. However, aberrant accumulation of neutrophils in tissues is also a known cause of tissue injury. The lungs are particularly vulnerable to neutrophil mediated inflammation and resulting disorders are amongst the leading causes of morbidity and mortality in the United States of America. These patients respond poorly to current therapeutics modalities. Despite its clinical importance, the molecular mechanisms regulating neutrophil migration remains incompletely understood both in physiological and pathological settings. The studies proposed in this application will provide important information of the physiologic role of Cdc42 in the regulation of neutrophil migration. The inhibition of Cdc42 activity in vivo has just become possible with the development of small pharmacologic inhibitor. Therefore, our study will guide designing strategies to modulate neutrophil functions via inhibition of Cdc42 that could be used in the future for novel therapeutics of inflammatory diseases.