Recruitment of leukocytes to sites of acute 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 provide molecular scale insight into how PMNs integrate force as a spatio-mechanical cue in the transition from 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 bond mechanics in binding ICAM-1 provide a gatekeeper mechanism via regulation of outside-in signaling of transendothelial migration. 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 activation on inflamed endothelium 3) define the molecular and mechanical mechanisms underlying LFA-1 outside-in signaling of neutrophil shape polarization. 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.
White blood cell emigration into inflamed tissue is likened to a double edged sword, it is critical to innate immune defense against bacterial infection, but if unchecked contributes to autoimmune disease and tissue destruction. We develop technologies to measure how neutrophils, the most common white cell, adhere to blood vessels and migrate to sites of inflammation in order to design more effective anti-inflammatory therapeutics.
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