The mortality from Gram-negative sepsis arises from development of the sepsis syndrome with uncontrolled systemic inflammation. Polymorphonuclear leukocytes (PMNs) are critical cellular elements of the innate immune response, but are also responsible for host tissue damage when fully activated. Diverse regulatory mechanisms exist to control the level of PMN activation, including priming. A priming stimulus modulates the phenotype of the PMN so that the response to subsequent stimuli is greatly amplified, including enhancement of NADPH oxidase activity. Endotoxin, a pro-inflammatory component of Gram (-) bacteria induces PMN priming in vitro and in vivo with primed PMNs identified in the circulation of patients with sepsis. The anion transporter ClC-3 is required for normal NADPH oxidase activity and mice deficient in ClC-3 appear to have a defect in innate immunity. In addition, redox signaling elicited by inflammatory cytokines is markedly impaired in smooth muscle cells from ClC-3 deficient mice. The overall hypothesis of this proposal is that the anion transporter ClC-3 modulates NADPH oxidase-dependent responses during endotoxin priming. This hypothesis is supported by strong preliminary data demonstrating that PMNs lacking ClC-3 have markedly impaired priming responses after stimulation with endotoxin. The long-term research goal of this project is to better understand PMN priming during Gram (-) sepsis in order to enhance therapeutic options to modulate host defense. The approach to the hypothesis will include first an assessment of the individual contributions of ClC-3 and the NADPH oxidase to PMN priming by endotoxin, followed by a focused investigation of the mechanism of their interaction during the priming process with the following specific aims: 1) To explore the roles of ClC-3 and NADPH oxidase-derived ROS in the generation of the primed phenotype 2) To characterize the interaction between ClC-3 and the NADPH oxidase during LOS priming. To explore these aims biochemical analyses of PMN subcellular fractions in combination with neutrophil functional assays including measurement of NADPH oxidase activity, degranulation, and cell surface receptor expression will be utilized. Confocal and electron microscopy studies will define the localization and kinetics of oxidant generation by priming stimuli, and explore the relevant ions that might participate in this process. Whole cell patch clamp analysis of human and murine PMNs along with differentiated PLB cells will be employed to directly observe the relevant conductances, and will allow the use of molecular tools to focus directly on the role of ClC-3. Human PMNs treated with inhibitors of ClC-3 and the NADPH oxidase will be used in addition to PMNs from patients with chronic granulomatous disease, and murine ClC-3 deficient PMNs.
Sepsis continues to be a major challenge in medicine and a profound health care burden with unacceptably high mortality. The requirement for normal neutrophil function as a component of the innate immune response to bacterial pathogens has been unequivocally demonstrated. The anion transporter ClC-3 appears to have a role in several aspects of basic neutrophil function including modulation of the NADPH oxidase. The studies of neutrophil priming by bacterial products that are proposed will advance our fundamental knowledge of the function of neutrophils in the maintenance of normal immune system function and in the pathogenesis of Gram-negative sepsis.
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