Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0–24 h. This migration was inhibited by anti-VCAM-1 or anti-α4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.

Lymphocytes bound at endothelial cell junctions extravasate within minutes. Lymphocyte-endothelial cell binding is mediated by receptors such as vascular cell adhesion molecule 1 (VCAM-1). VCAM-1 activates endothelial cell nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in minutes, and this activity is required for VCAM-1-dependent lymphocyte migration. In this report, we examined mechanisms for activation of matrix metalloproteinases (MMPs) during VCAM-1-dependent lymphocyte migration. Lymphocyte binding to VCAM-1 rapidly activated endothelial cell-associ- IntroductionLymphocytes migrate out of the blood between endothelial cells and into tissues where the lymphocytes can interact with antigen. Endothelial cells bind lymphocytes through cell surface adhesion molecules. One of these adhesion molecules is vascular cell adhesion molecule 1 (VCAM-1). It is important to understand VCAM-1 signaling because it is involved in several diseases. For example, VCAM-1 is required for eosinophil infiltration into the lung in experimental ovalbumin-induced asthma, 1 as well as T-cell infiltration across the blood-brain barrier in experimental allergic encephalomyelitis (EAE). 2 In addition, VCAM-1 functions in combination with other adhesion molecules during chronic inflammation and tumor metastasis. Understanding VCAM-1 signaling may have important implications for disease intervention.We have reported that VCAM-1 signaling in endothelial cells is required for VCAM-1-dependent lymphocyte migration. 3 Stimulation of VCAM-1 activates endothelial cell nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which catalyzes the release of low levels of reactive oxygen species (ROS) in cytokinetreated human umbilical vein endothelial cells (HUVECs) and in endothelial cell lines. 4,5 These ROS are required for VCAM-1-stimulated endothelial cell actin restructuring and lymphocyte migration. 3,6,7 Therefore, ROS are involved in modulating endothelial cell function to promote VCAM-1-dependent lymphocyte migration.It has been reported that VCAM-1-dependent adhesion of a T-cell line activates lymphocyte matrix metalloproteinases (MMPs) after 5 hours. 8 However, the mechanism(s) for VCAM-1 activation of lymphocyte MMPs is not known. It is also not known whether VCAM-1 signaling activates endothelial cell MMPs. Activated MMPs degrade extracellular matrix, cell surface receptors in cell-cell junctions, and tight junction proteins. 9-11 MMP activation can be regulated by ROS. In smooth muscle cells, the latent form of MMP-2 (pro-MMP-2) is released after mechanical stretchstimulated production of ROS by NADPH oxidase. 12 In cell-free systems, low concentrations of ROS can activate pro-MMPs by oxidation of the sulfide bond in the prodomain of the MMP followed by release of this prodomain by autocatalytic cleavage. 13 In this report, we demonstrate that VCAM-1 rapidly activates endothelial cell-associated MMPs and that this activation is mediated by endothelial cell-derived ROS. In addition, endothelial cell-derived ROS are i...

Leukocyte migration from the blood into tissues is vital for immune surveillance and inflammation. During this diapedesis of leukocytes, the leukocytes bind to endothelial cell adhesion molecules and then migrate across the vascular endothelium. Endothelial cell adhesion molecules and their counter-receptors on leukocytes generate intracellular signals. This review focuses on the active function of endothelial cells during leukocyte-endothelial cell interactions. We include a discussion of the "outside-in" signals in endothelial cells, which are stimulated by antibody cross-linking or leukocyte binding to platelet-endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Some of these signals in endothelial cells have been demonstrated to actively participate in leukocyte migration. We suggest that some of the adhesion molecule signals, which have not been assigned a function, are consistent with signals that stimulate retraction of lateral junctions, stimulate endothelial cell basal surface adhesion, or induce gene expression.

During lymphocyte migration, engagement of VCAM-1 stimulates the generation of endothelial cell-derived reactive oxygen species (ROS) and activation of matrix metalloproteinases, facilitating endothelial retraction. Because bilirubin is a potent antioxidant, we examined the hypothesis that this bile pigment inhibits VCAM-1-dependent cellular events. The migration of isolated murine splenic lymphocytes across monolayers of murine endothelial cell lines (which constitutively express VCAM-1) is significantly inhibited by physiological concentrations of bilirubin, in the absence of an effect on lymphocyte adhesion. Bilirubin administration also suppresses VCAM-1-stimulated ROS generation and reduces endothelial cell matrix metalloproteinase activity. In a murine asthma model characterized by VCAM-1-dependent airway inflammation, treatment of C57BL6/J mice with i.p. bilirubin decreases the total leukocyte count in the lung parenchyma and lavage fluid, through specific inhibition of eosinophil and lymphocyte infiltration. Blood eosinophil counts were increased in bilirubin-treated animals, while VCAM-1 expression in the capillary endothelium and cytokine levels in both lung lavage and supernatants from cultured lymph node lymphocytes were unchanged, suggesting that bilirubin inhibits leukocyte migration. Conclusion: bilirubin blocks VCAM-1-dependent lymphocyte migration in vitro and ameliorates VCAM-1-mediated airway inflammation in vivo, apparently through the suppression of cellular ROS production. These findings support a potential role for bilirubin as an endogenous immunomodulatory agent.

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase.

Objective Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. Methods and Results We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-κB were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-κB activity and VCAM-1 expression under conditions of atheroprone flow. Conclusion These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-κB–mediated gene expression.

Chemokines, including CXCL1, participate in neutrophil recruitment by triggering the activation of integrins, which leads to arrest from rolling. The downstream signaling pathways which lead to integrin activation and neutophil arrest following G-protein-coupled receptor engagement are incompletely understood. To test whether G␣ i2 is involved, mouse neutrophils in their native whole blood were investigated in mouse cremaster postcapillary venules and in flow chambers coated with P-selectin, ICAM-1, and CXCL1. Gnai2 Ϫ/Ϫ neutrophils showed significantly reduced CXCL1-induced arrest in vitro and in vivo. Similar results were obtained with leukotriene B 4 (LTB 4 ). Lethally irradiated mice reconstituted with Gnai2 Ϫ/Ϫ bone marrow showed a similar defect in chemoattractant-induced arrest as that of Gnai2 Ϫ/Ϫ mice. In thioglycollate-induced peritonitis and lipopolysaccaride (LPS)-induced lung inflammation, chimeric mice lacking G␣ i2 in hematopoietic cells showed about 50% reduced neutrophil recruitment similar to that seen in Gnai2 Ϫ/Ϫ mice. These data show that neutrophil G␣ i2 is necessary for chemokineinduced arrest, which is relevant for neutrophil recruitment to sites of acute inflammation.(Blood.

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)α activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCα in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCα and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.