Regulation of phagocyte function by Rac2

Rho家族GTPase Rac在调节多种吞噬细胞功能中发挥关键作用，包括NADPH氧化酶催化的超氧化物生成，Fcgamma受体介导的吞噬作用，以及膜褶皱和细胞运动过程中的细胞骨架变化。为了研究造血特异性Rac2 GTPase的作用，该酶与更广泛表达的Rac1和Rac3亚型大约90%相同，我们产生了靶向破坏Rac2基因的小鼠。缺乏rac2的中性粒细胞（仍然表达Rac1）在fMLP、IgG- opsonized颗粒和phorbol酯的作用下，NADPH氧化酶的激活显著降低，但在补体包被酶酶体的作用下，NADPH氧化酶的激活正常。在最初的研究中观察到的其他中性粒细胞缺陷包括肌动蛋白聚合受损、趋化性受损和l -选择蛋白依赖性粘附不良。此外，小鼠体内渗出物形成减少，对侵袭性曲霉病的易感性增加。Rac在中性粒细胞功能中的重要作用已被进一步证实，在一名出现复发性化脓性感染和与Rc2-/-相似的功能性中性粒细胞缺陷的婴儿中发现了Rac2的显性阴性突变体。这些数据表明，Rac2在特定受体激活途径下游的吞噬细胞中调节多种细胞反应，并且在宿主感染和炎症中与Rac1和Rac3具有不重叠的功能。项目3将侧重于测试这一假设，特别是因为它与NADPH氧化酶的激活有关，并探索潜在的生化机制。有四个特定目标，它们将利用Rac2-/-鼠标。这些建议：(1)进一步定义Rac2-/-吞噬细胞中的缺陷，以检测巨噬细胞超氧化物的产生、吞噬作用和化学引诱剂诱导的运动；(2)检测功能缺陷是否存在于Rac2-/-吞噬细胞中，以及趋化剂诱导的运动；(2)检查Rac2-/-吞噬细胞的功能缺陷是否反映了Rac2的选择性激活、定位或使用，以及是否需要特异性的Rac2效应序列；(3)研究与rac依赖性NADPH氧化酶复合物组装相关的上游和下游信号事件；(4)确定Rac2缺乏对宿主防御和炎症的影响。这项工作将进一步深入了解rho - gtpase如何调节吞噬白细胞功能以及介导特异性激动剂诱导的细胞反应的生化机制。这些研究也可能导致在炎症和宿主防御中调节吞噬细胞功能的新方法。

The Rho family GTPase Rac plays a key role in regulating a variety of phagocyte functions, including NADPH oxidase-catalyzed superoxide generation, Fcgamma receptor-mediated phagocytosis, and cytoskeletal changes during membrane ruffling and cell movement. To examine the role of the hematopoietic specific Rac2 GTPase, which is approximately 90% identical to the more widely expressed Rac1 and Rac3 isoforms, mice with a targeted disruption of the Rac2 gene were generated. NADPH oxidase activation in Rac2-deficient neutrophils )which still express Rac1) was substantially reduced in response to fMLP, IgG- opsonized particles, and phorbol ester, but normal in response to complement-coated zymosan. Other neutrophil defects observed in initial studies include impaired actin polymerization, chemotaxis, and poor L-selectin-dependent adhesion. In addition, mice exhibited decreased exudate formulation in vivo and an increased susceptibility to invasive Aspergillosis. An important role for Rac in neutrophil function has been further established by the identification of a dominant-negative mutant of Rac2 in an infant who presented with recurrent pyogenic infections and functional neutrophil defects similar to those found in Rc2-/-. These data suggest that Rac2 regulates multiple cellular responses in phagocytes downstream of specific receptor-activated pathways, and has non-overlapping functions with Rac1 and Rac3 in the host infection and inflammation. Project 3 will focus on testing this hypothesis, particular as it relates to activation of the NADPH oxidase, and explore underlying biochemical mechanisms. There are four Specific Aims, which will take advantage of the Rac2-/- mouse. These propose to (1) further define defects in Rac2-/- phagocytes to examine macrophage superoxide production, phagocytosis, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes, and chemoattractant-induced movement; (2) examine whether functional defects in Rac2-/- phagocytes reflect selective activation, localization, or usage of Rac2, and if specific Rac2 effector sequences are required; (3) investigate upstream and downstream signaling events involved in Rac-dependent assembly of the NADPH oxidase complex and (4) determine the impact of Rac2 deficiency on host defense and inflammation. This work will provide further insight into how Rho-GTPases regulate phagocytic leukocyte functions and the biochemical mechanisms that mediate specific agonist-induced cellular responses. These studies may also result in new approaches to modulating phagocyte function in inflammation and host defense.