Regulation of phagocyte function by Rac2

Rho家族GTPase Rac在调节多种吞噬细胞功能中起关键作用，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细胞膜褶皱和细胞运动过程中细胞骨架的变化。为了检测造血特异的rac2 GTPase的作用，该酶与更广泛表达的rac1和rac3亚型大约90%相同，建立了定向破坏rac2基因的小鼠。对fMLP、免疫球蛋白调理颗粒和佛波酯的反应，仍表达rac1的中性粒细胞中的NADPH氧化酶活性显著降低，但对补体包被的酵母多糖的反应正常。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、趋化作用受损，以及L选择素依赖的黏附能力差。此外，小鼠体内渗出液配方减少，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，在一名反复出现化脓性感染和功能性中性粒细胞缺陷的婴儿中，Rac2的显性阴性突变与Rc2-/-相似。这些数据表明，在特定受体激活途径下游的吞噬细胞中，rac2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将重点检验这一假说，特别是当它与NADPH氧化酶的激活有关时，并探索潜在的生化机制。有四个具体的目标，这将利用rac2-/-鼠标。这些建议建议：(1)进一步定义rac2-/-吞噬细胞中的缺陷，以检查巨噬细胞超氧化物的产生、吞噬作用和趋化物质诱导的运动；(2)检查rac2-/-吞噬细胞中的功能缺陷是否反映了rac2的选择性激活、定位或使用，以及是否需要特定的rac2效应序列；(3)调查RAC依赖的NADPH氧化酶复合体组装中涉及的上下游信号事件；以及(4)确定rac2缺陷对宿主防御和炎症的影响。这项工作将进一步深入了解Rho-GTP酶如何调节吞噬白细胞的功能，以及介导特定激动剂诱导的细胞反应的生化机制。这些研究还可能导致调节吞噬细胞在炎症和宿主防御中的功能的新方法。

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.