Structure And Function Of Phagocyte Proteins

中性粒细胞和其他循环中的吞噬细胞在各种感染性或炎症性刺激下产生高水平的活性氧(ROS)，这一过程被称为呼吸爆发。这种反应归因于NADPH氧化酶的活性，它产生超氧化物，ROS的前体充当杀菌剂和炎症介质。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，我们正在使用基因转染方法来探索磷脂酶D、ADP-核糖化因子-6(ARF-6)及其核苷酸交换因子的作用。有关影响呼吸爆发的信号中间产物的信息可能提供旨在抑制或增强吞噬细胞氧化反应的治疗策略(药理靶点)。在其他研究中，我们正在确定非髓系组织中活性氧物种(Phox同系物)的来源，特别是结肠、肾脏、甲状腺和唾液腺、粘膜表面、脑和血管组织。在这些部位，氧化剂可能参与宿主防御和炎症反应，或提供影响基因表达模式(对生长因子的增殖反应、分化、细胞衰老、细胞凋亡或程序性细胞死亡、氧气感知)的氧化还原“第二信使”。在对结肠特异氧化酶的研究中，我们检测了Nox1在结肠上皮细胞中的表达模式，并证明了Nox1是由末端分化或干扰素-γ诱导的。用编码Nox1的逆转录病毒转导在功能上取代gp91Phox，在共表达胞浆因子p47Phox和p67Phox的细胞中恢复刺激依赖性的超氧化物释放。此外，我们还鉴定了这些胞浆PHOX共因子的独特的结肠特异性同源物，表明NOX1是一种受调控的PHOX样复合体，在结肠上皮中发挥宿主防御和炎症作用。在旨在探索肾脏氧化酶(Renox或NOX4)功能作用的研究中，我们开发了在饮食多西环素控制下，肾脏特异的NOX4过度表达或抑制(反义)的转基因小鼠系。我们还设计了一种破坏NOX4基因的靶向载体，准备克隆到胚胎干细胞中。我们发现甲状腺双重氧化酶(DUOX1和DUOX2)在唾液腺、呼吸道(气管、支气管)和直肠上皮细胞中有功能表达，这表明这些酶是过氧化氢的来源，支持粘膜表面乳过氧化物酶的抗微生物活性。最后，我们证明了培养的呼吸道上皮细胞以依赖于DOOX1(反义抑制)的方式响应钙信号而产生过氧化氢；该系统正在被开发以证实这种氧化酶在呼吸道宿主防御中的作用。

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 act as 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 cellular mechanisms regulating the respiratory burst in phagocytes and is characterizing oxidative responses of related enzymes expressed in non-immune cells. In work aimed at defining signal transduction pathways that trigger activation of the phagocyte oxidase (phox), we are using gene transfection approaches to explore roles of phospholipase D, ADP-ribosylation factor-6 (ARF-6), and its nucleotide exchange factors. Information on signaling intermediates affecting the respiratory burst may provide therapeutic strategies (pharmacological targets) designed to inhibit or enhance oxidative responses of phagocytes. In other studies, we are characterizing sources of reactive oxygen species (phox homologs) in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces, brain, and vascular tissue. In these sites, the oxidants may serve in host defense and inflammatory reactions or provide redox "second messengers" that affect gene expression patterns (proliferation responses to growth factors, differentiation, cellular senescence, apoptosis or programmed cell death, oxygen sensing). In studies on the colon-specific oxidase, we have examined expression patterns of Nox1 in colon epithelial cells and have demonstrated that Nox1 is induced by terminal differentiation or by interferon-gamma. Transduction with a retrovirus encoding Nox1 functionally replaces gp91phox, restoring stimulus-dependent superoxide release in cells co-expressing the cytosolic factors, p47phox and p67phox. Furthermore, we identified unique, colon-specific homologs of these cytosolic phox co-factors, suggesting that Nox1 is a regulated, phox-like complex acting in host defense and inflammation in the colon epithelium. In studies aimed at exploring the functional role of the renal oxidase (renox or Nox4), we have developed transgenic mouse lines for renal-specific Nox4 over-expression or suppression (antisense), under the control of dietary doxycycline. We have also engineered a targeting vector for disruption of the Nox4 gene, which is ready for cloning into embryonic stem cells. We have documented functional expression of thyroid dual oxidases (duox1 and duox2) in epithelial cells of salivary glands, airways (trachea, bronchium), and rectum, suggesting that these enzymes serve as a source of hydrogen peroxide supporting the anti-microbial activity of lactoperoxidase on mucosal surfaces. Finally, we demonstrated that cultured airway epithelial cells produce hydrogen peroxide in response to calcium signals in a duox1-dependent (antisense-inhibited) manner; this system is being developed to confirm the role of this oxidase in airway host defense.