Neutrophil recruitment from the circulation to vascular sites of inflammation is a finely orchestrated multistep process initiated by upregulation of membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selections, integrins, and ICAMs. Transgenic mouse models in which a single or multiple genes encoding selectins or beta2-integrins have revealed both unique and overlapping functions of these molecules in supporting cell-cell and cell-matrix adhesion, as well as intracellular signaling of a diverse set of functions. Despite the progress in deciphering the molecular anatomy of leukocyte recruitment via selectins and integrins the sequence of binding and signaling events that direct leukocytes and that may be targeted for therapeutic action is lacking. Over the tenure of our preceding FIRST and R01 awards three important rules of engagement have been discovered and these guide the specific aims of this proposal: l) Selectins function as both adhesive and signal transduction receptors in neutrophil recruitment; 2) membrane clustering of selectins and [32-integrins is critical to their function and occurs via active transport involving MAP kinases; 3) a shift in [32-integfin conformation determines affinity and membrane clustering both of which regulate neutrophil arrest on endothelium at an interface we denote the inflammatory synapse. The primary hypothesis is that neutrophils navigate their journey from the bloodstream to inflamed tissue by precise regulation of the lifetime and strength of adhesive bonds.
Aim 1 of this project is to determine how selectin engagement signals stable adhesion and endothelial transmigration.
In Aim 2, we will examine how leukocytes regulate adhesion strength and lifetime through beta2-integrins.
Aim 3 will validate and target mechanisms of neutrophil signaling and adhesion via CD18 and selectins in recruitment to a cutaneous skin wound model in mouse. Our strategy entails the use of freshly isolated human neutrophils and intravital microscopy of routine microcirculation with the objective of identifying regulatory pathways and molecular targets for more effective prognosis and treatment of inflammatory diseases.
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