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. ? ? ?
