CHANGES IN THE DISTRIBUTION OF LFA-1, CATENINS, AND F-ACTIN DURING TRANSENDOTHELIAL MIGRATION OF MONOCYTES IN CULTURE

To determine changes in the spatial and temporal distribution of cell- cell adhesion molecules during transendothelial migration of monocytes , we examined an in vitro model system of diapedesis using high resolu tion laser scanning confocal microscopy, Human arterial endothelial ce lls were cultured to confluence on coverslips coated with Matrigel and activated with IL-1 beta before the addition of monocytic THP-1 cells , Seventy per cent of monocytes transmigrated through the endothelium within one hour, Diapedesis, but not adhesion and spreading, was inhib ited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor, D ouble immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regi ons, This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration pas sage, monocytes remained round in shape, while underneath the endothel ium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a majo r role of these molecules during the transmigration process itself, En dothelial adherens junctions, as judged by the presence of VE-cadherin and alpha-catenin adjacent to the passage, remained intact during dia pedesis. The presence of catenins at heterotypic contact regions betwe en monocytes and endothelial cells during diapedesis suggested cadheri n-mediated interactions between the two cell types, These results reve al dynamic changes in the distribution of adhesion molecules and the a ctin cytoskeleton during monocyte transendothelial migration in cultur e.