ICAM‐1 cytoplasmic sequence and membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) regulate monocyte transendothelial migration (TM).

Abstract

The extravasation of leukocytes to sites of inflammation is mediated through the ligation of leukocytes with intercellular cell adhesion molecule (ICAM-1) on endothelial cells (EC). The role of ICAM-1 cytoplasmic sequence and the protease mediating ICAM-1-dependent leukocyte migration were studied. Human EC were transduced with a viral vector that contained ICAM-1 constructs with the cytoplasmic tail truncated (TR), and tyrosine (Y) to alanine (A) substitutions at residues 474, 476 and 485. EC were seeded on transwells to form a monolayer through which human monocytes were allowed to migrate. 33.2% of monocytes migrated through wild-type ICAM-1 expressing EC, while in non-transduced EC TM was substantially low (7%). Antibodies against ICAM-1 and beta-2 integrins blocked TM. TR and Y485A failed to support TM. Y476A remained unaffected, but Y474A reduced TM down to 46.4%. Synthetic MMP inhibitors blocked TM. When EC were transduced with tissues inhibitors of MMPs (TIMPs), TIMP-2 and -3 blocked TM, whereas TIMP-1 was ineffective, thus, suggesting involvement of MT-MMPs. Using the over expression strategy, we found that MT1-MMP, but not MT2-MMP, is a strong mediator of TM, whereas MT3-MMP also has a moderate role in TM. Antibodies and siRNA directed against MT1-MMP blocked TM. Also, ICAM-1 and MT1-MMP co-localizes in the presence of PMA, in ICAM-1 transfected-293 cells. However, over expression of MT1-MMP in ECs expressing TR failed to rescue whereas Y485A cells were regained TM up to 60%. ICAM-1 independently regulates monocyte TM, in the absence of other adhesion receptors and exogenous cytokine mediators, through MT1-MMP and MT3-MMP. The cytoplasmic sequence and phosphorylation at 485 and 474 regulate ICAM-1 dependent TM. The loss of TM function in TR is not due to MT1-MMP, but is likely due to an intermediate signal required for TM.