Lymphocyte Migration Through Monolayers of Endothelial Cell Lines Involves VCAM-1 Signaling Via Endothelial Cell NADPH Oxidase

Abstract

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.

Superoxide activates very late antigen‐4 on an eosinophil cell line and increases cellular binding to vascular cell adhesion molecule‐1

“…Many investigations have shown that ROS produced by leukocyte NADPH oxidase modulate the expression of adhesion molecules involved in leukocyte trafficking [13,14]. Recently, an intriguing report showed that activation of NADPH oxidase is required for VCAM-1-dependent lymphocyte migration [23]. NO synthesis is catalyzed by isoforms of NO synthase expressed in leukocytes, endothelial cells and various other cells [24].…”

Reactive Oxygen Species in Inflammation and Tissue Injury

“…Furthermore, OS increased mRNA levels of NOX2 and NOX1, and monocyte adhesion was shown to be blocked by siRNA against NOX1. Interestingly, Matheny et al have shown that VCAM-1 ligand binding induced ROS generation via NADPH oxidases in endothelial cells and promoted lymphocyte migration by actin restructuring in endothelial cells (278). Inhibiting NADPH oxidase activity with DPI or Apocynin blocked lymphocytic migration across mouse endothelial cells to greater than 65% (278).…”

An Antibody to the Sixth Ig-like Domain of VCAM-1 Inhibits Leukocyte Transendothelial Migration without Affecting Adhesion

“…In addition to a4b1 integrin, VCAM-1 can bind other integrins, including a4b7, adb2, aMb2, and a9b1 (23)(24)(25)(26)(27)(28). It is known that a4b1 integrin binds Ig-like domains 1 and 4 (29)(30)(31)(32), and that activated integrins are necessary for firm leukocyte adhesion to the endothelium via Ig-like domains 1 and 4 of VCAM-1 (33).…”