In vivo, leukocyte transendothelial migration (TEM) occurs at endothelial cell junctions (paracellular) and nonjunctional (transcellular) locations, whereas in vitro models report that TEM is mostly paracellular. The mechanisms that control the route of leukocyte TEM remain unknown. IntroductionLeukocyte transendothelial migration (TEM) is a key step in their recruitment to sites of inflammation, injury, and immune reactions. Leukocyte recruitment involves a multistep cascade consisting of leukocyte rolling, firm adhesion, and, ultimately, transmigration. 1 These steps are regulated by shear flow, apical chemokines, and inducible adhesion molecules expressed by endothelium. 2 The actual path of leukocyte egress (paracellular versus transcellular) has been examined in several models (reviewed in Muller, 3 Kvietys and Sandig, 4 and Engelhardt and Wolburg 5 ). Experiments carried out in several animal models of inflammation found that leukocyte TEM can occur at both paracellular and transcellular locations. [6][7][8][9][10][11][12] In contrast, most in vitro studies have reported that leukocyte TEM occurs primarily at paracellular locations. 3,[13][14][15] As a result, in vitro models of polymorphonuclear leukocyte (PMN) transcellular TEM do not exist, and the factors that dictate whether leukocytes use a transcellular or paracellular route and the mechanisms underlying transcellular TEM are unknown.Intercellular adhesion molecule-1 (ICAM-1) interacting with its leukocyte counterreceptors lymphocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1) is key for leukocyte adhesion and TEM. [16][17][18][19] ICAM-1 is a transmembrane glycoprotein with 5 extracellular immunoglobulin G (IgG)-like domains and a short cytoplasmic tail that associates with multiple cytoskeletal linker proteins (reviewed in Springer 1 ). Vascular endothelium expresses low levels of ICAM-1, and inflammatory stimuli can markedly increase ICAM-1 surface expression. 20,21 In acute and chronic inflammatory diseases, endothelial cells become activated and express high levels of ICAM-1, in addition to vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. [22][23][24][25] ICAM-1 has been shown to signal in endothelium during leukocyte adhesion or when cross-linked by anti-ICAM-1 antibodies. ICAM-1 occupancy triggers elevations in intracellular free Ca 2ϩ and myosin contractility, 26 activation of p38 kinase, 27 and the small guanosine triphosphatases (GTPases), in particular, members of the Rho family 28,29 and the tyrosine kinase p60 Src . 30 Activation of these signaling pathways results in extensive cytoskeletal remodeling events that alter endothelial cell contractility and function, 27 possibly facilitating leukocyte diapedesis. Recently, we have shown that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 during PMN transmigration at cell junctions. 31 Others have reported that ICAM-1-enriched projections engulf leukocytes during their firm adhesion and locomot...

The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-α–activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil–endothelial junctional interface through which transmigration occurred. As transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.