Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1

Abstract

We tested the hypothesis that TNF-alpha induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the beta2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-alpha resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-alpha challenge. We observed that TNF-alpha induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1-dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-alpha-induced endothelial adhesivity was protein synthesis-independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKCzeta, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-alpha-induced ICAM-1 gene transcription, we determined the possibility that PKCzeta was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-alpha stimulation of endothelial cells induced PKCzeta activation and its association with ICAM-1. Inhibition of PKCzeta by pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-alpha induces early-onset, protein synthesis-independent expression of endothelial adhesivity by PKCzeta-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.