The goal of this proposal is to foster the career development of Dr. A. Paige Volk while undertaking the investigation of a fundamental component of innate host defense, neutrophil (PMN) chemotaxis, i.e. directed motility. Dr. Volk's long term career goal is become an independent physician-scientist with the education, research skills and experience necessary to make a significant contribution to bio-medical science. Her more short-term goals are: 1) to advance Dr. Volk's expertise in cellular and molecular immunology through both coursework and molecular biological experimentation;and 2) to define mechanisms by which anion transporters and oxidants regulate PMN chemotaxis. Dr. Volk's career development would take place in the nurturing environment of the NIH-supported Inflammation Program, with Dr. Jessica Moreland as her mentor, and Dr. Fred Lamb as co-mentor. This environment provides Dr. Volk full access to the resources and scientific expertise required to make a significant contribution to the study of innate host defense. Dr. Volk began to investigate the role of the anion transporter, ClC-3, in PMN function after mice lacking this protein demonstrated impaired innate immunity and deficits in PMN function. The hypothesis of this proposal is that cell volume regulation via the swelling-induced chloride current, IClswell, is mediated by oxidant signaling and required for chemotaxis to select stimuli. This hypothesis will be addressed with the following aims: 1) To characterize mechanisms of cell volume regulation in PMN chemotaxis;and 2) To characterize the oxidant-sensitive signaling interactions that distinguish chemotaxis to select stimuli and determine the role of ClC-3 in this process. To explore these aims a combination of molecular analyses and functional assays of human and murine PMN will be used. Localized volume changes and chloride movement will be studied using advanced 2D/3D microscopic image analysis, confocal fluorescence microscopy and electrophysiology techniques. Regulation of chemotaxis signaling pathways by ClC-3- modulated oxidants will be assessed by confocal microscopy and flow cytometry using a novel competitive chemotaxis assay. The role of ClC-3 and intracellular oxidants in chemotaxis will be studied in vivo using advanced two-photon intravital microscopy techniques. This proposal is highly innovative in that we will elucidate a previously unappreciated role for intracellular oxidants in chemotaxis signaling. The requirement for normal PMN function in the innate immune response during sepsis has been demonstrated unequivocally and the anion transporters ClC-3 and IClswell are required for normal PMN chemotaxis. Uncontrolled PMN activation and migration into healthy tissues during severe sepsis leads to multiple organ system dysfunction. The current project will significantly advance the understanding of neutrophil migration mechanisms that can ultimately be translated into therapeutic strategies to reduce the burden of illness and disability due to sepsis