Recruitment of leukocytes to acute sites of inflammation is a finely orchestrated process initiated by membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selectins, integrins, and Ig-super family ligands. A set of unifying themes have emerged over the tenure of this R01 which begin to define the molecular and force dynamics that govern the transition from leukocyte rolling to firm arrest and transendothelial migration: 1) Selectins are endowed with mechanical and biochemical properties which allow them to function as both adhesive and signal transduction receptors;2) Shear stress and transmembrane calcium release- activated Ca2+ (CRAC) channels regulate intracellular calcium flux which functions to synchronize integrin mediated arrest and cell migration;3) 22-integrin affinity and avidity provide gatekeeper mechanisms associated with inside-out signaling of transendothelial migration. These rules of neutrophil engagement have lead to the primary hypothesis that under conditions of acute inflammation the adhesive and signaling events constitute a system that by design promotes rapid and irreversible transmigration while maintaining basal endothelial barrier function. In this competitive renewal we apply our innovative vascular mimetic microfluidic channels combined with real time immunofluorescence imaging to pursue the following specific aims: 1) Examine the role of calcium release activated calcium flux in orchestration of the multistep process of neutrophil recruitment. 2) Determine how E-selectin ligands on neutrophils serve as mechano-transducers that trigger and amplify chemokine signaling of integrin actvation on inflamed endothelium 3) determine how endothelial barrier function and neutrophil recruitment are coupled in the transition from acute to chronic inflammation studied in a cutaneous wound model. Our strategy entails the use of freshly isolated human neutrophils and murine models of inflammation with the overarching goal of identifying regulatory pathways and molecular targets for prognosis and treatment of inflammatory diseases.
Neutrophil emigration into inflamed tissue may be likened to a double edged sword because it is critical to innate immune defenses against bacterial infection, but if unchecked contributes to deregulation in blood vessel permeability and morbidity in chronic and autoimmune diseases. These studies will elucidate the adhesive and migratory processes relevant to acute and chronic inflammation.
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