STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

中性粒细胞和其他循环吞噬细胞产生高水平的活性氧（ROS），以响应各种感染或炎症刺激，这一过程称为呼吸爆发。 这种反应归因于NADPH氧化酶的活性，NADPH氧化酶产生超氧化物，超氧化物是ROS的前体，ROS是重要的杀微生物剂和炎症介质。 患有慢性肉芽肿病（CGD）的患者具有NADPH氧化酶缺陷，并且遭受对微生物感染和异常炎症反应的增强的易感性。 该项目探索调节吞噬细胞呼吸爆发的细胞机制，并表征各种非免疫细胞中表达的相关酶的氧化反应。 在旨在确定触发吞噬细胞氧化酶（phox）激活的信号转导途径的工作中，我们采用了两种基因转染方法：1）在未分化（K562）细胞中重建受体介导的呼吸爆发激活，以及2）在分化的髓样（PLB-985）细胞中表达修饰的信号分子。重建的趋化肽受体反应已被描绘成钙依赖性和独立的途径，而后者的方法提供了新的证据参与的小GTdR，ADP-核糖基化因子-6（ARF-6），和磷脂酶D的呼吸爆发。 影响吞噬细胞呼吸爆发的信号传导中间体的信息将为旨在抑制或增强吞噬细胞氧化反应的治疗策略提供基础（药理学靶点）。 在其他研究中，我们正在描述其他组织（结肠、肾脏、脑和血管组织）中活性氧的来源。在这些部位，氧化变化可作为氧化还原“第二信使”，促进炎症信号、氧传感和基因表达模式的变化（对生长因子的增殖反应、分化、细胞衰老、凋亡或程序性细胞死亡）。 在CGD的p47 phox缺陷小鼠模型中的研究表明，p47 phox在响应于中性粒细胞激动剂或β-淀粉样蛋白的小胶质细胞释放反应性氧化剂中起重要作用，这可能与阿尔茨海默病相关的神经退行性过程的发展机制有关。 在p47 phox缺陷小鼠中，PDGF或血管紧张素II刺激后主动脉平滑肌细胞的正常氧化反应也不存在，表明血管组织中涉及吞噬细胞样氧化酶。 我们还确定了一个独特的gp 91 phox同系物在肾脏（肾氧化酶或Renox），在近曲小管中表达，并提出作为氧传感器调节促红细胞生成素的合成。 当在转染的成纤维细胞中表达时，Renox引起超氧化物释放并诱导细胞衰老。 最后，结肠氧化酶同源物已被表征（Mox-1），其在上皮表面上表达并由脂多糖、干扰素-γ或终末分化诱导，并且可以在肠道中的宿主防御或炎症反应中起作用。

Neutrophils and other circulating phagocytes generate high levels of reactive oxygen species (ROS) in response to a variety of infectious or inflammatory stimuli in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies and suffer from enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst in phagocytes and is characterizing oxidative responses of related enzymes expressed in a variety of non-immune cells. In work aimed at defining signal transduction pathways triggering activation of the phagocyte oxidase (phox), we have engaged two gene transfection approaches: 1) reconstitution of receptor-mediated activation of the respiratory burst in undifferentiated (K562) cells and 2) expression of modified signaling molecules in differentiated myeloid (PLB-985) cells. The reconstituted chemotactic peptide receptor responses have been delineated into both calcium-dependent and independent pathways, while the latter approach has provided new evidence for involvement of the small GTPase, ADP-ribosylation factor-6 (ARF-6), and phospholipase D in the respiratory burst. Information on signaling intermediates affecting the respiratory burst in phagocytes will provide a basis (pharmacological targets) for therapeutic strategies designed to inhibit or enhance oxidative responses of phagocytes. In other studies we are characterizing sources of reactive oxygen species in other tissues (colon, kidney, brain, and vascular tissue). In these sites, the oxidative changes can serve as redox "second messengers" promoting inflammatory signals, oxygen sensing, and changes in gene expression patterns (proliferation responses to growth factors, differentiation, cellular senescence, apoptosis or programmed cell death). Studies in the p47phox-deficient mouse model of CGD indicate an essential role for p47phox in the release of reactive oxidants by microgial cells in response to neutrophil agonists or beta-amyloid, which may relate to mechanisms of development of neurodegenerative processes associated with Alzheimer's disease. The normal oxidative responses of aortic smooth muscle cells following stimulation by PDGF or angiotensis II are also absent in p47phox-deficient mice, indicating involvement of a phagocyte-like oxidase in vascular tissue. We have also identified a distinct gp91phox homologue in the kidney (renal oxidase or Renox) that is expressed in proximal convoluted tubules and proposed to serve as an oxygen sensor regulating erythropoietin synthesis. When expressed in transfected fibroblasts, Renox causes superoxide release and induces cellular senescence. Finally, a colon oxidase homologue has been characterized (Mox-1), which is expressed on epithelial surfaces and induced by lipopolysaccharide, interferon-gamma or terminal differentiation, and may function in host defense or inflammatory responses in the gut.