Monocyte activation and endothelial small G‐proteins control the route of transendothelial migration

Abstract

Monocyte extravasation can occur across endothelial junctions (paracellular migration, PM) or through endothelial cells (transcellular migration, TCM). Both routes employ similar molecules, but factors controlling the choice of transmigration route are not known. We studied the interaction of freshly isolated human monocytes on primary human umbilical vein endothelial cells (HUVEC) as well on an established cell line (iHUVEC) using live video microscopy and 3D imaging of fixed immunostained cells. Although most endothelial cells supported PM only, some cells, especially those with morphology suggestive of increased Rho activity, allowed both TCM and PM. The monocytes that chose the TCM pathway were defective in establishing polarity and directionality of migration towards the junctions, but not in movement per se. Such monocytes were better spread, had more podosomes, and deeply invaginated the endothelial surface. We treated the endothelial cells and/or monocytes with small molecule Rac/Cdc42 and Rho activators or inhibitors. TCM can be increased by activating Rac and Cdc42 in monocytes, or by activating monocytes with chemokines. Rho activation in endothelial cells significantly increased the proportion of monocytes migrating transcellularly (~3 times). Rho inhibition had the opposite effect, and prevented the effect of Rho activation. The monocytes undergoing TCM were surrounded by a zone of high RhoA activity in the underlying endothelial cells, as revealed by a FRET biosensor. We conclude that the route of monocyte transmigration is controlled by Rho activity in the endothelial cells or by the state of activation of the monocytes. Funded by NIH R37 HL064774 to WAM