Role of Ion Channel in Mononuclear Phagocyte Activation

离子通道在单核吞噬细胞激活中的作用

• 批准号: 7912041
• 负责人: DEBORAH J. NELSON
• 金额: $ 27.53万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912041
• 关键词: Alveolar; Alveolar Macrophages; Anions; Bacteria; Bacterial Infections; Cell membrane; Cells; Chloride Channels; Chronic; Competence; Complex; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasmic Granules; Data; Defect; Dependence; Dyes; Electric Capacitance; Exhibits; Failure; Functional disorder; GTP-Binding Proteins; Generations; Growth Factor; Host Defense; Human; Immune; Immune system; Infection; Instinct; Ion Channel; Laboratories; Lentivirus Vector; Life; Liquid substance; Lung; Lysosomes; Maintenance; Measures; Membrane Fusion; Membrane Potentials; Methods; Microscopic; Molecular; Mononuclear; Movement; Mucous body substance; Mus; Organelles; Oxidants; Pathway interactions; Peritoneal Macrophages; Phagocytes; Phagocytosis; Phagosomes; Play; Population; Potassium Channel; Process; Production; Proton Pump; Protons; Pump; Recycling; Regulation; Resolution; Rodent; Role; Shunt Device; Signal Transduction; Stimulus; Superoxides; System; Techniques; Testing; Total Internal Reflection Fluorescent; Transgenic Mice; Trees; Vesicle; Video Microscopy; Voltage-Clamp Technics; airway epithelium; bactericide; cell type; chemokine; cystic fibrosis patients; cytokine; killings; macrophage; microbial; microbicide; mutant; neutrophil; particle; pathogen; time use; uptake; vacuolar H+-ATPase

SUMMARY Macrophages and neutrophils play major roles in host defense against microbial infections. In order to perform this function, these cell types must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Alongside the pumps are various channels that shunt the transmembrane potential generated by movement of protons; in different organelles these comprise H+ channels, K+ channels and Cl- channels. Nevertheless, the contribution of these pathways to maintenance of intraorganellar pH is poorly studied. Recently, we demonstrated that murine alveolar macrophages (AMs) but not neutrophils employ the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl? channel as a major shunt mechanism. Lysosomes and phagosomes in murine cftr/- AMs failed to acidify and the cells were deficient in bacterial killing compared to wild-type controls. We have also shown that AMs lacking CFTR are deficient in stimulus-induced secretion. Here we propose to extend these observations by investigating the role of Cl- flux in a common set of phagocyte core functions namely, organellar acidification, granule secretion, and microbicidal activity. We will compare the phagocytic and secretory activities of alveolar and peritoneal macrophages as well as neutrophils and the anion channels that are involved in the regulation of these activities. CFTR and ClC-3 chloride channels are the most reasonable channels with which to begin our studies given our preliminary data on CFTR in murine AMs and that of others in human and mouse neutrophils showing a differential functional dependence on either ClC-3 or CFTR depending upon the species. Utilizing primary cells obtained from normal, Cftr-deficient (or mutant) and ClC3-deficient mice as well as human cells from non-CF and CF patients, we will use a variety of molecular, immunochemical, microscopic and electrophysiological techniques, well-established in our laboratories, to provide a multi-faceted systems approach to the problem. NARRATIVE Patients with CF are highly susceptible to chronic bacterial infection. To date, lung dysfunction in CF has been largely attributed to depletion of the liquid layer covering the upper airway epithelium with a consequent accumulation of mucus that is thought to contribute to the persistence of bacteria in the airway tree. We propose that an additional defect may be attributable to a failure of CFTR-deficient alveolar macrophages to exhibit vigorous bactericidal activity. Defects in the behavior of the innate immune system could have important consequences for microbial defense in CF patients.

概括 巨噬细胞和中性粒细胞在宿主防御微生物中起着重要作用 感染。为了执行此功能，这些细胞类型必须摄入并破坏 病原体，通常在吞噬体中，并分泌许多产品 向其他免疫细胞发出响应。低细胞器pH的产生主要是驱动的 通过V-ATP酶，使用细胞质ATP将H+加载到细胞器中的质子泵。 泵旁边是各种通道，可以分流跨膜电势 由质子的运动产生；在不同的细胞器中，这些包括H+通道， K+通道和Cl-通道。然而，这些途径的贡献 对掌内pH的维护研究很少。最近，我们证明了 鼠肺泡巨噬细胞（AMS），但不使用中性粒细胞使用CFTR（囊性 纤维化跨膜电导调节剂）CL？频道作为主要分流 机制。鼠CFTR/-AM中的溶酶体和吞噬体未能酸化，并且 与野生型对照相比，细胞缺乏细菌杀伤。我们也有 表明缺乏CFTR的AM缺乏刺激诱导的分泌。我们在这里 提议通过研究Clux在A中的作用来扩展这些观察结果 普通的吞噬细胞核心功能，即有细节酸化，颗粒 分泌和微生物活性。我们将比较吞噬细胞和分泌 肺泡和腹膜巨噬细胞以及中性粒细胞的活性以及 与这些活动调节有关的阴离子渠道。 CFTR和 CLC-3氯化物通道是开始我们的研究的最合理的渠道 鉴于我们在鼠和人类和老鼠中有关CFTR的初步数据 中性粒细胞显示出对CLC-3或CFTR的差异功能依赖性 在物种上。利用从正常，CFTR缺陷（或突变体）获得的原代细胞 和CLC3缺乏小鼠以及非CF和CF患者的人类细胞，我们将 使用各种分子，免疫化学，微观和电生理学 在我们的实验室中建立良好的技术，以提供多方面的系统 解决问题的方法。叙述 CF患者非常容易受到慢性细菌感染的影响。迄今为止，肺 CF中功能障碍主要归因于覆盖液体液层的耗竭 上呼吸道上皮，随之而来的粘液积聚 有助于气道树中细菌的持久性。我们建议 其他缺陷可能归因于CFTR缺陷型肺泡巨噬细胞的失败 表现出剧烈的杀菌活性。先天免疫行为的缺陷 系统可能会对CF患者的微生物防御产生重要影响。