The tight junction (TJ) is a specialized intercellular structure responsible for the regulation of ionic and macromolecular flux across cell monolayers. Because plasma leakage is believed to occur mainly across the microvasculature, we hypothesized that microvascular endothelial cells (MVEC) may form more intact, regulatable TJ than other endothelial cell (EC) types, allowing further insight into the control of EC permeability. Primary cultures of MVEC monolayers produced transmonolayer electrical resistances (TER) of 120-155 omega.cm2, approximately 10 times that of large-vessel EC. Treatment with tumor necrosis factor and interferon-gamma caused a 50% decrease in the TER and a striking fragmentation of the basal, continuous interendothelial cell zonula occludens-1 protein (ZO-1) distribution determined by immunofluorescence. Fragmentation was inhibited by cytochalasin D, and confocal microscopy demonstrated a colocalization between F actin and ZO-1. These findings suggest that the F actin cytoskeleton plays a central role in endothelial TJ barrier regulation and that dynamic cytoskeletal alterations may primarily control vascular permeability.

We assessed the relative contribution of CD31/PECAM-1 (platelet-endothelial cell adhesion molecule-1) to T lymphocyte transmigration by the use of transfected murine fibroblasts stably expressing either the human CD31/PECAM-1 or the intercellular adhesion molecule-1 (CD54/ICAM-1). Unlike CD54/ICAM-1, CD31/PECAM-1 supported migration of activated T cells in the absence of chemokines: most of the migrating lymphocytes were CD31+ and displayed a phenotype corresponding to the naive subpopulation (LFA-1 dull and CD45RA+). Migration of activated T lymphocytes through CD54/ICAM-1+ transfected monolayers could be induced by creating a chemotactic gradient with the chemokine monocyte chemotactic protein-1, and the migrating cells mainly displayed a memory phenotype (LFA-1 bright CD45RO+) under these conditions. Furthermore, we found that in transfected cells CD54/ICAM-1 is uniformly distributed along the apical surface of the cells, while CD31/PECAM-1 is concentrated at the intercellular junctions, suggesting the existence of a haptotactic gradient (i.e. a gradient of substrate- or cell-bound molecules) responsible for T cell migration. This was also confirmed by the finding that monolayers of murine fibroblasts transfected with a CD31/PECAM-1 mutant lacking the cytoplasmic domain (CD31/PECAM-1-delta cyto), which has a reduced tendency to localize at cell-cell contact areas, supported efficient adhesion but were unable to induce migration of activated T cells unless a chemotactic gradient was created. We propose that in lymphocytes, homophilic CD31/PECAM-1 adhesion may be primarily involved in transmigration of naive T cells and that its role is complementary to that of CD54/ICAM-1.

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