Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium.
BACKGROUND
During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.


RESULTS
Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.


CONCLUSIONS
Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.
van Buul
Jaap D
JD
Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. j.vanbuul@sanquin.nl
van Rijssel
Jos
J
van Alphen
Floris P J
FP
Hoogenboezem
Mark
M
Tol
Simon
S
Hoeben
Kees A
KA
van Marle
Jan
J
Mul
Erik P J
EP
Hordijk
Peter L
PL
D015153:Blotting, Western; D002448:Cell Adhesion; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1