STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

中性粒细胞和其他循环中的吞噬细胞在呼吸爆发的过程中，对各种感染性或炎症性刺激做出反应，产生高水平的活性氧(ROS)。这种反应归因于NADPH氧化酶的活性，它产生超氧化物，ROS的前体是重要的杀菌剂和炎症介质。慢性肉芽肿性疾病(CGD)患者存在NADPH氧化酶缺陷，对微生物感染和异常炎症反应的易感性增加。该项目探索了调节吞噬细胞呼吸爆发的细胞机制，并表征了在各种非免疫细胞中表达的相关酶的氧化反应。为了确定触发吞噬细胞氧化酶(Phox)激活的信号转导途径，我们采用了两种基因转染方法：1)在未分化的(K562)细胞中重建受体介导的呼吸爆发激活；2)在分化的髓系(PLB-985)细胞中表达修饰的信号分子。重组趋化肽受体的反应分为钙依赖途径和非依赖途径，而后一种途径为小GTP酶、ADP核糖化因子-6(ARF-6)和磷脂酶D参与呼吸爆发提供了新的证据。有关影响吞噬细胞呼吸爆发的信号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(药理靶点)。在其他研究中，我们正在确定其他组织(结肠、肾脏、脑和血管组织)中活性氧物种的来源。在这些部位，氧化变化可以作为氧化还原的“第二信使”，促进炎症信号、氧气感应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p47Phox缺陷的CGD小鼠模型中的研究表明，p47Phox在中性粒细胞激动剂或β-淀粉样蛋白的反应中，在微小细胞释放反应性氧化剂方面发挥了重要作用，这可能与阿尔茨海默病相关的神经退化过程的发展机制有关。在p47Phox基因缺陷的小鼠中，经PDGF或血管紧张素II刺激后的主动脉平滑肌细胞也没有正常的氧化反应，这表明血管组织中有一种吞噬细胞样的氧化酶参与其中。我们还在肾脏中发现了一种独特的gp91Phox同源物(肾脏氧化酶或Renox)，它在近曲小管中表达，并被认为是调节促红细胞生成素合成的氧传感器。当在转基因的成纤维细胞中表达时，Renox会导致超氧化物释放，并诱导细胞衰老。最后，我们鉴定了一种结肠氧化酶同源物(Mox-1)，它表达在上皮细胞表面，由脂多糖、干扰素-γ或末端分化诱导，可能在肠道的宿主防御或炎症反应中发挥作用。

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.