When human monocytes were added twice at intervals of ≈ 30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈ 1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50 % of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments).
These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.