Neutrophils are a critical component of both the innate and adaptive immune responses that control microbial invasion. Priming is an intermediate step by which resting neutrophils enhance their production of reactive oxygen intermediates (ROI) upon activation, and priming is necessary for optimal microbial killing. Although priming greatly enhances neutrophil bactericidal capability, primed neutrophils are also more dangerous to normal cells. Primed neutrophils participate in a number of diseases prominent in the aging Veteran population, including ANCA-associated vasculitis, ischemia- reperfusion injury, acute respiratory distress syndrome, rheumatoid arthritis, peridontitis, atherosclerosis, and acute inflammatory liver injury. Thus, manipulation of neutrophil priming is a potential therapeutic strategy for pharmacological intervention to restrain inflammation. Recent studies identified granule exocytosis and enhanced translocation of cytosolic components of the NADPH oxidase to the plasma membrane as possible mechanisms of neutrophil priming. However, many gaps in knowledge remain. The relative contribution of each of those events to priming has not been examined and whether both are necessary, or sufficient, for priming is unknown. The molecular events by which granule exocytosis contributes to priming and the molecular events that control enhanced translocation of cytosolic NADPH oxidase components are incompletely understood. Finally, the ability to manipulate neutrophil priming in vivo has not been established. The McLeish laboratory is in a unique position to address each of these gaps based on our extensive experience examining signal transduction pathways, our application of biochemical, genetic, and proteomic approaches to understand neutrophil biology, and our recent development of novel reagents that inhibit exocytosis in vitro and in vivo. The current proposal will address the central hypothesis that neutrophil priming results from the convergence of signal transduction pathways into two responses, an exocytose-dependent increase in plasma membrane expression of membrane components of NADPH oxidase and prolyl isomerase-dependent conformational changes in cytosolic components of NADPH oxidase. The following four specific objectives will be accomplished. Specific Objective 1 will test the working hypothesis that exocytosis of secretory vesicles and gelatinase granules results in increased plasma membrane expression of p91phox and p22phox, which is necessary, but not sufficient, for priming. Specific Objective 2 will test the working hypothesis that activation of the prolyl isomerase Pin1 contributes to priming of human neutrophils by multiple mechanisms, including enhanced p47phox translocation and increased granule exocytosis. Specific Objective 3 will test the working hypothesis that p38 MAPK phosphorylates multiple proteins that mediate events necessary for priming, including exocytosis and Pin1 activation. Specific Objective 4 will test the working hypothesis that neutrophil priming is a necessary component for neutrophil participation in inflammatory diseases and represents a viable therapeutic strategy, using an animal model of acute lung injury (ALI). The proposed work is expected to establish the molecular basis for neutrophil priming. The impact of the project is that molecular targets identified will permit therapeutic manipulation of neutrophil priming in inflammatory diseases. Thus, new approaches to treating Veteran patients with a number of acute and chronic inflammatory diseases are anticipated.
White blood cells (WBCs) protect against infection by producing toxic chemicals that kill invading bacteria. The production of those toxin chemicals is enhanced by prior exposure to a variety of mediators of inflammation by a process termed priming. Although WBC priming plays a critical role in the control of infection, priming is a two-edged sword that also reduces the ability of WBCs to co-exist with normal cells. Primed WBCs produce tissue injury in a number of diseases that affect our Veteran patients. The events that lead to priming are not known, and inhibition of priming has not been used to treat those diseases. The current proposal will define the mechanisms by which WBCs become primed and will establish priming is a therapeutic target in inflammatory diseases. Accomplishing those goals will provide new treatments for Veteran patients with a number of acute and chronic inflammatory diseases.