Role of Ion Channel in Mononuclear Phagocyte Activation

摘要 巨噬细胞和中性粒细胞在宿主对微生物的防御中起主要作用 感染。为了执行这一功能，这些细胞类型必须摄取和破坏 病原体，通常在吞噬小体中，以及分泌许多产物， 向其他免疫细胞发出信号，让其做出反应。低细胞器pH的产生主要是由 通过V-ATPase，质子泵利用细胞质的ATP将H+加载到细胞器中。 与泵并排的是各种分流跨膜电势的通道。 由质子运动产生；在不同的细胞器中包括H+通道， K+通道和Cl-通道。然而，这些途径对 细胞器内pH的维持研究很少。最近，我们展示了 小鼠肺泡巨噬细胞(AM)而不是中性粒细胞使用CFTR(囊性 肝纤维化跨膜电导调节剂)氯？作为主要分流渠道的航道 机制。小鼠CFTR/-AM中的溶酶体和吞噬体不能酸化 与野生型对照相比，细胞在杀菌方面存在缺陷。我们还有 研究表明，缺乏CFTR的AM在刺激诱导的分泌方面存在缺陷。在这里我们 建议通过研究氯离子通量在 常见的一套吞噬细胞核心功能，即细胞器酸化、颗粒化 分泌物和杀微生物活性。我们将比较吞噬和分泌 肺泡巨噬细胞、腹膜巨噬细胞、中性粒细胞的活性 参与调节这些活动的阴离子通道。Cftr和 ClC-3氯通道是我们开始研究的最合理的通道 鉴于我们在小鼠肺泡巨噬细胞和其他人和小鼠肺泡巨噬细胞中CFTR的初步数据 中性粒细胞对ClC-3或CFTR的功能依赖程度不同 对物种的威胁。利用从正常、CFTR缺陷(或突变体)获得的原代细胞 和ClC3缺乏的小鼠以及来自非CF和CF患者的人类细胞，我们将 使用各种分子、免疫化学、显微和电生理 技术，在我们的实验室中很好地建立，以提供一个多方面的系统 解决这个问题的方法。叙事 CF患者对慢性细菌感染的易感性很高。到目前为止，肺 CF的功能障碍在很大程度上是由于覆盖在皮肤上的液层耗尽 上呼吸道上皮细胞，随后粘液聚集，被认为是 有助于细菌在呼吸道树中的持久性。我们建议一个 其他缺陷可能归因于CFTR缺陷的肺泡巨噬细胞的失败 表现出强烈的杀菌活性。先天免疫的行为缺陷 系统可能对CF患者的微生物防御产生重要影响。

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.