CIC-3 channels in regulation of cell volume/shape in PMN

描述(由申请人提供)：完整的中性粒细胞功能是免疫系统中的一个关键细胞元素，参与识别微生物颗粒以维持健康。趋化、吞噬和杀菌活性是多形核白细胞(PMN)对天然免疫功能的贡献所不可或缺的功能。这些复杂的过程中的每一个都需要中性粒细胞的形状和大小发生深刻的变化，并可能伴随着细胞体积的瞬时变化。尽管导电氯(CI-)通道已被证明在低渗应激后调节中性粒细胞体积减少中起作用，但参与这一过程的特定通道以及阴离子通道在与PMN运动或吞噬病原体所必需的形状变化相关的细胞体积调节中的作用尚未被研究。CIC-3是一种广泛分布于组织中的电压敏感阴离子通道，在中性粒细胞中表达。为了研究该基因在高血压中的作用，研究了Clcn3-/-小鼠。在血管内放置血压监测仪后，大多数人死于明显的脓毒症，而Clcn3+/+未受影响。与Clcn3+/+PMN相比，Clcn3-/-PMN的NADPH氧化酶活性、杀菌活性和吞噬能力均降低。用阴离子通道阻滞剂尼氟米酸处理的人PMN在基本的PMN功能上也有类似的损害。缺乏CI通道功能的PMN的内皮迁移也明显减少。目前的提议是对阴离子运动在PMN功能中的特定作用的高度探索，并将检验新的假设，即通过CIC-3的阴离子运动在调节PMN的细胞体积和形状中发挥关键的信号作用，而这种电导对于正常的中性粒细胞趋化、跨内皮细胞迁移和吞噬是必不可少的。这一假设将通过以下两个目标进行检验。1)研究CIC-3在中性粒细胞体积调节中的作用；2)结合基本生理学技术、中性粒细胞亚群生化分析和中性粒细胞功能分析，探讨细胞体积调节、阴离子电导与中性粒细胞趋化、迁移和吞噬所必需的形态变化之间的关系。用膜片钳分析CI-电流，测量细胞体积，用荧光探针共聚焦显微镜研究CI-电流，研究用阴离子通道阻滞剂处理的小鼠CLCN-/-和Clcn3+/+PMN和人PMN的细胞体积调节。细胞运动性将使用先进的2D/3D显微图像分析设备进行分析，跨内皮细胞迁移将在体外系统中进行研究，吞噬功能将在显微镜下进行分析。相关性：作为对病原体的先天免疫反应的一部分，正常的PMN功能的要求已被明确证明。阴离子通道CIC-3似乎是中性粒细胞迁移到感染和炎症部位以及吞噬和杀死病原体所必需的。提出的研究将促进我们对中性粒细胞在维持正常免疫系统功能中的作用的基础知识。

DESCRIPTION (provided by applicant): Intact neutrophil function is a crucial cellular element of the immune system involved in recognition of microbial particles for the maintenance of health. Chemotaxis, phagocytosis, and bactericidal activity are functions integral to the contribution of polymorphonuclear leukocytes (PMN) to innate immune function. Each of these complex processes requires profound alterations in the shape and size of the neutrophil, presumably accompanied by transient alterations in cell volume. Although conductive chloride (CI-) channels have been demonstrated to function in the regulatory volume decrease of human neutrophils after hypotonic stress, the specific channels involved in this process and the role of anion channels in cell volume regulation associated with shape change necessary for PMN motility or phagocytosis of pathogens has not been studied. CIC-3, a voltage sensitive anion channel with widespread tissue distribution is expressed in PMN. Clcn3-/- mice were generated to study the role of this gene in hypertension. Following intravascular placement of blood pressure monitors, a majority died from apparent sepsis with Clcn3+/+ littermates unaffected. Clcn3-/- PMNs were isolated and demonstrated diminished NADPH oxidase activity, microbicidal activity, and phagocytic capabilities in comparison to Clcn3+/+ PMN. Human PMN treated with the anion channel blocker niflumic acid had similar impairments in essential PMN functions. Transendothelial migration of PMN lacking CI- channel function was also markedly reduced. The current proposal is highly exploratory of a specific role for anion movement in PMN function and will test the novel hypothesis that anion movement through CIC-3 serves a critical signaling role in the regulation of cell volume and shape in PMNs, and this conductance is essential for normal neutrophil chemotaxis, transendothelial migration, and phagocytosis. This hypothesis will be tested with the following two aims. 1) To characterize the role of CIC-3 in cell volume regulation in polymorphonuclear leukocytes 2) To determine the relationship between cell volume regulation, anion conductance, and shape changes necessary for neutrophil chemotaxis, transmigration, and phagocytosis We will explore these aims with a combination of basic physiology techniques, biochemical analyses of PMN subcellular fractions, and neutrophil functional assays. Cell volume regulation in murine Clcn-/- and Clcn3+/+ PMNs and human PMNs treated with anion channel blockers will be studied using patch clamp analysis of CI- currents, measurements of cell volume, and confocal microscopy study of CI- flux using fluorescent probes. Cell motility will be assayed using an advanced 2D /3D microscopic image analysis facility, transendothelial migration will be studied in an in vitro system, and phagocytosis will be assayed with microscopy. Relevance: The requirement for normal PMN function as a component of the innate immune response to pathogens has been unequivocally demonstrated. The anion channel CIC-3 appears to be required for neutrophil migration to sites of infection and inflammation and for phagocytosis and killing of pathogens. The studies proposed will advance our fundamental knowledge of the function of neutrophils in the maintenance of normal immune system function.