Analysis of tight junctions during neutrophil transendothelial migration

Intercellular junctions have long been considered the main sites through wh ich adherent neutrophils (PMNs) penetrate the endothelium, Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact b etween adjacent endothelial cells. Whether PMN transmigration involves disr uption of the TJ complex is unknown. We report here that endothelial TJs ap pear to remain intact during PMN adhesion and transmigration, Human umbilic al vein endothelial cell (HUVEC) monolayers, a commonly used model for stud ying leukocyte trafficking, were cultured in astrocyte-conditioned medium t o enhance TJ expression, Immunofluorescence microscopy and immunoblot analy sis showed that activated PMN adhesion to resting monolayers or PMN migrati on across interleukin-l-treated monolayers does not result in widespread pr oteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial b orders. Ultrastructurally, TJs appear intact during and immediately followi ng PMN transendothelial migration. Similarly, transendothelial electrical r esistance is unaffected by PMN adhesion and migration. Previously, we showe d that TJs are inherently discontinuous at tricellular corners where the bo rders of three endothelial cells meet and PMNs migrate preferentially at tr icellular corners. Collectively, these results suggest that PMN migration a t tricellular corners preserves the barrier properties of the endothelium a nd does not involve widespread disruption of endothelial TJs.