Regulation of Human Phagocyte Function by Rac Proteins

描述(申请人提供)：由吞噬细胞的NADPH氧化酶形成的活性氧物种(ROS)在宿主防御和炎症中起主要作用。当遇到微生物产品时，白细胞激活的一个中心特征是两个阶段的反应，首先导致预激活的准备状态，这会在第二个激活的刺激下产生极大的增强反应。与微生物病原体相关的分子模式由Toll样受体(TLRs)识别。这种识别导致TLR激活和信号事件，为化学诱导剂随后的刺激中性粒细胞做准备。为了了解这种与ROS产生相关的中性粒细胞协调反应的分子基础，我们将研究从涉及的受体到NADPH氧化酶激活的最终序列的信号通路。 我们已经证明，氧化酶的活性受GTP酶rac2和效应器p21激活的激酶(PAK)的调节。到目前为止，我们的研究表明，Pak在fMLP诱导的呼吸爆发的多个方面发挥作用，包括多种磷酸化事件和与细胞色素b558的联系。Pak在膜上NADPH氧化酶组装中的作用将被详细研究。对Pak缺失或含有Pak激酶抑制片段的转基因小鼠的研究将确定Pak在体内的功能。 从激活的TLR发出的信号序列似乎在单核细胞和中性粒细胞之间存在分歧，导致ROS的产生而不是启动。我们假设RAC调节的通路是理解不同的先天性免疫细胞如何调节细菌杀灭的关键因素，我们将研究其中涉及的分子机制。连接TLR2激活、启动和化学诱导剂诱导的ROS产生的一个共同特征是依赖于酪氨酸磷酸化。我们将阐明TLRs或甲酰肽受体与酪氨酸激酶受体的潜在联系，并评估这些复合体如何在中性粒细胞和小胶质细胞中传递它们的信号。这些研究将为先天免疫细胞全面控制氧化剂的形成提供新的见解。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) formed by the NADPH oxidase of phagocytic cells play a primary role in host defense and inflammation. A central feature of leukocyte activation upon encountering microbial products is a two-phased response, leading at first to a pre-activated, primed state, which generates a vastly enhanced response upon a secondary, activating stimulus. The molecular patterns associated with microbial pathogens are recognized by Toll-like receptors (TLRs). This recognition leads to TLR activation and signaling events that prime neutrophils for subsequent stimulation by chemoattractants. To understand the molecular basis for this coordinated neutrophil response in respect to ROS generation, we will investigate the signaling pathways from the receptors involved to the final sequence of NADPH oxidase activation. We have shown that oxidase activity is regulated by the GTPase Rac2 and the effector p21-activated kinase (Pak). Our studies so far point to a role of Pak in several aspects of the fMLP-induced respiratory burst including multiple phosphorylation events and association with cytochrome b558. The involvement of Pak in NADPH oxidase assembly at the membrane will be investigated in detail. Studies with Pak-null or transgenic mice harboring a Pak kinase inhibitory fragment will define Pak function in vivo. The signaling sequences emanating from activated TLRs seem to diverge between monocytes and neutrophils, leading to ROS generation versus priming. We hypothesize that Rac-regulated pathways are key elements for understanding how different innate immune cells regulate bacterial killing and we will investigate the molecular mechanisms involved. A common trait connecting TLR2 activation, priming and chemoattractant-induced ROS generation is the dependence on tyrosine phosphorylations. We will elucidate potential associations of TLRs or formyl peptide receptors with tyrosine kinase receptors and evaluate how these complexes relay their signals in neutrophils and microglia. These studies will yield novel insights into the overall control of oxidant formation by innate immune cells.