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至关重要的细胞元素，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）在先天免疫功能中不可或缺的功能。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改变，可能伴随着细胞体积的短暂改变。导电性氯离子（CI-）通道已被证明在低压应激后人类中性粒细胞的调节体积减少中起作用，但参与这一过程的特定通道以及阴离子通道在与PMN运动或病原体吞噬所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是一种具有广泛组织分布的电压敏感阴离子通道。我们用Clcn3-/-小鼠来研究该基因在高血压中的作用。在血管内放置血压监测仪后，大多数死于明显的败血症，而Clcn3+/+的幼崽未受影响。与Clcn3+/+ PMN相比，分离出的Clcn3-/- PMN表现出NADPH氧化酶活性、杀微生物活性和吞噬能力的降低。用阴离子通道阻滞剂尼氟替酸处理的人PMN在基本的PMN功能上有类似的损伤。缺乏CI通道功能的PMN的跨内皮迁移也明显减少。目前的提案高度探索了阴离子运动在PMN功能中的特定作用，并将测试新的假设，即阴离子通过CIC-3的运动在PMN细胞体积和形状的调节中起着关键的信号作用，这种传导对于正常的中性粒细胞趋化性、跨内皮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。为了确定细胞体积调节、阴离子电导率和中性粒细胞趋化、转运和吞噬所需的形状变化之间的关系，我们将结合基础生理学技术、PMN亚细胞组分的生化分析和中性粒细胞功能测定来探索这些目标。小鼠Clcn-/-和Clcn3+/+ PMNs和人类PMNs经阴离子通道阻断剂处理后的细胞体积调节将使用膜片钳分析CI-电流，测量细胞体积，并使用荧光探针共聚焦显微镜研究CI-通量。细胞运动将使用先进的2D /3D显微图像分析设备进行分析，体外系统将研究跨内皮迁移，并用显微镜分析吞噬作用。相关性：作为对病原体的先天免疫反应的组成部分，正常PMN功能的要求已得到明确证明。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。提出的研究将推进我们对中性粒细胞在维持正常免疫系统功能中的功能的基本认识。

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.