Blockade of Focal Clustering and Active Conformation in β2-Integrin-Mediated Adhesion of Eosinophils to Intercellular Adhesion Molecule-1 Caused by Transduction of HIV TAT-Dominant Negative Ras

Abstract

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to β2-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for β1-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for β2-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.