Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

Edwin Kanters,Jos Van Rijssel,Paul J Hensbergen,David Hondius,Frederik P.J Mul,André M Deelder,Arnoud Sonnenberg,Jaap D Van Buul,Peter L Hordijk

doi:10.1074/jbc.m804888200

Abstract

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migration.

Highlights

Endothelial cells are highly specialized to create a relatively impermeable barrier between the circulating blood and underlying tissue
Identification of Proteins Associating to the Intracellular Domain of intercellular cell adhesion molecule-1 (ICAM-1)— the intracellular domain of ICAM-1 is short (28 amino acids) and contains no identified signaling domains, it has been shown that the lack of the intracellular tail of ICAM-1 results in a decrease of leukocyte transmigration and a loss of formation of apical cups around adhered leukocytes [4, 10, 16, 29]
To identify proteins that bind to the intracellular domain of ICAM-1 we performed pull-down assays with lysates of primary human umbilical vein endothelial cells (HUVEC) using biotinylated peptides encoding the intracellular tail of ICAM-1

Summary

Introduction

Endothelial cells are highly specialized to create a relatively impermeable barrier between the circulating blood and underlying tissue. Reducing filamin expression in primary human endothelial cells results in impaired ICAM-1 clustering and membrane dynamics and reduced adhesion and transendothelial migration of leukocytes. Addition of the ␣ICAM-1 antibody-coated beads to an endothelial cell lysate did not result in the co-precipitation of detectable amounts of filamin B (Fig. 2D), indicating that the interaction with endogenous filamin B is promoted upon clustering of ICAM-1.

Results

Conclusion