Adhesion and signaling events in selectin and beta(2)-integrin mediated neutrophil recruitment.

摘要

The underlying objective of this dissertation is to examine the continuum of cellular events that links initial capture and rolling of neutrophils to subsequent cellular activation and adhesion. In pursuit of this objective, the overall hypothesis tested was that selectin recognition and clustering signals shifts in beta2-integrin avidity and affinity leading to rapid neutrophil arrest on ICAM-I in shear flow.; In Chapter 2, the hypothesis that L-selectin functions as a transmembrane signaling receptor, amplifying the extent of neutrophil activation as a function of clustering by multivalent ligands was tested. We examined how alterations in the topography of L-selectin correlate with the dynamics of CD18 activation and phosphorylation of MAPK. The data demonstrates that L-selectin transduces signals effecting rapid (∼1 sec) neutrophil adhesion that is regulated by the size and frequency of clustering in a p38 and p42144 MAPK dependent mechanism.; In Chapter 3, the hypothesis tested was that multivalent recognition of E-selectin by L-selectin and PSGL-1 on rolling neutrophils results in colocalization of these receptors in self assembled molecular domains, thereby signaling activation of CD18 dependent adhesion. Utilizing chimeric E-selectin constructs and substrate cell monolayers expressing E-selectin, we show that recognition of sLex on L-selectin and PSGL-1 drives their colocalization at the trailing edge of neutrophils. This event signaled the beta2 -integrin activation and elicited neutrophil capture of beta 2-integrin ligands in shear flow.; In Chapter 4, the final objective of this dissertation was to test the hypothesis that optimal neutrophil recruitment involves both a conformational activation of CD18 and macromolecular assembly of LFA-1 and Mac-1 into dynamic clusters on the plasma membrane. We examined the dynamics of CD18 activation and correlated this with the membrane topography of Mac-1, LFA-1 and mAb 327C. E-selectin binding signaled arrest while chemokine ligation signaled polarization and migration that was dependent primarily on clustering of LFA-1 at the neutrophil-endothelial contact region in a calpain and PI(3)K sensitive mechanism. We propose that distinct and sequential roles for E-selectin and IL-8 exists enabling optimal neutrophil arrest and transmigration at sites of inflammation via assembly of an adhesion and signaling complex between neutrophils and endothelium.