Regulation of Neutrophil Migration and Polarity

描述(申请人提供)：中性粒细胞是人类体内最丰富的白细胞，是人体抵御感染微生物的第一道细胞防线。虽然中性粒细胞通常存在于血液中，但它会迅速迁移到组织中的感染部位。这种迁移受到严格的控制；事实上，中性粒细胞的异常聚集会导致组织损伤。因此，了解S控制中性粒细胞迁移的分子机制具有相当重要的临床意义。这项应用侧重于了解小Rho GTP酶CDc42在中性粒细胞迁移过程中的作用。我们对CDC42缺失的中性粒细胞的分析表明，CDC42是中性粒细胞中极性的关键调节因子。在机制水平上，我们发现了一条新的途径来介导CDC42的功能，并涉及到白细胞整合素CD11b/CD18。CD11b/CD18是一种与致死性人类免疫缺陷疾病白细胞黏附缺陷综合征相关的黏附分子。CD11b是中性粒细胞向组织内迁移的重要介质，其机制尚不明确。我们发现在CDC42缺失的细胞中CD11b聚集是有缺陷的，而恢复CD11b聚集挽救了极性--从而暗示CD11b参与了中性粒细胞的极性。有趣的是，我们发现CD11b缺失的中性粒细胞，类似于CDC42缺失的细胞，在迁移过程中表现出有缺陷的极性。这些数据表明，CDC42通过CD11b信号调节中性粒细胞的极性。我们建议确定CDC42-CD11b轴在体外和体内调节中性粒细胞迁移的分子机制和生物学后果。我们计划使用视频显微镜、免疫荧光成像和生化实验来研究(Aim1)CDC42调节极化中性粒细胞中CD11b聚集的分子机制；(目标2)确定中性粒细胞中性粒细胞CDc42-CD11b轴下游激活的信号通路。(目的3)由于CD11b在中性粒细胞迁移到肺中起重要作用，如果CDC42-CD11b轴在中性粒细胞极性中起关键作用，那么CDC42的缺失应该会导致体内肺部炎症过程中的同等缺陷。我们将使用CD11b依赖的肺损伤模型来测试这一点。我们认为，对原代CDC42缺失的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CDC42通过CD11b信号调节中性粒细胞极性的假设被证实，这将显著改变对CDC42和CD11b在中性粒细胞迁移中的功能的看法，从而在中性粒细胞依赖的炎症中发挥作用。拟议的研究有望导致了解并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.