Tumor Necrosis Factor-α Induces Early-Onset Endothelial Adhesivity by Protein Kinase Cζ–Dependent Activation of Intercellular Adhesion Molecule-1

Abstract

We tested the hypothesis that TNF-α induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the β₂-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-α resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-α challenge. We observed that TNF-α 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-α–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 PKCζ, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-α–induced ICAM-1 gene transcription, we determined the possibility that PKCζ was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-α stimulation of endothelial cells induced PKCζ activation and its association with ICAM-1. Inhibition of PKCζ by pharmacological and genetic approaches prevented the TNF-α–induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-α induces early-onset, protein synthesis–independent expression of endothelial adhesivity by PKCζ-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.