Molecular Mechanisms of Neutrophil Migration

Abstract

This work is an investigative look behind the mechanisms of neutrophil migration. Each of three chapters involves exploration into a different signaling pathway important for migration downstream of chemoattractant stimulation through inhibition of a kinase or disruption of the function of an effector and examination of the effects on migration, adhesion, and actin reorganization in primary human or equine neutrophils. Chapter II examines the requirement for the signaling molecule p38 Mitogen Activated Kinase (MAPK) in equine neutrophil chemotaxis through use of the p38 specific inhibitor SB203580. SB203580 reduced LTB4- and PAF-induced migration and disrupted the ability of cells to polarize, but did not affect b2 integrin-dependent adhesion or surface b2 integrin expression. Chapter III is a comprehensive inquiry into the regulation of the phosphorylation of serine 157 of the cytoskeletal protein Vasodilator-stimulated Phosphoprotein (VASP). The rapid and transient phosphorylation of VASP serine 157 corresponded with F-actin levels in chemoattractant-stimulated human neutrophils. fMLF-induced serine 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced serine 157 phosphorylation was unaffected by PKC inhibitors, PKG inhibitors, and the CamKII inhibitor KN-62. Inhibition of adhesion did not alter fMLF-induced VASP phosphorylation or dephosphorylation. This study demonstrated that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent and adhesion-independent VASP serine 157 phosphorylation. Chapter IV probed into the function of the actin binding protein and PKC substrate Myristoylated Alanine-Rich C-kinase Substrate (MARCKS) through utilization of a cell permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with 50 μM MANS, but not a scrambled control peptide, significantly inhibited their migration and adhesion in response to fMLF, IL8, or LTB4. MANS significantly reduced F-actin content in neutrophils 30s after fMLF-induced polymerization, but did not alter the ability of cells to polarize, spread, or upregulate surface b2 integrin expression. These data provided evidence that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.