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Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267
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-
4 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.
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