Mechanisms of leukocyte transendothelial and interstitial migration during inflammation in vivo

The emigration of leukocytes from the circulation is a critical
step during immune surveillance and inflammatory reactions that is
governed by a coordinated interplay involving a spectrum of
adhesion and signal molecules. While a great deal has been learned
about the early steps of leukocyte recruitment, i.e. rolling and
adhesion, little is known about the subsequent steps,
transendothelial and interstitial migration when leukocytes migrate
across the endothelial layer lining the blood vessel and move to
the sites of inflammation. In particular, it is not fully
understood which endothelial receptors are responsible for
extravasation of leukocytes into the perivascular space. Moreover,
the mechanisms of interstitial migration of leukocytes during
inflammation remain to be clarified in vivo. In the first part of
the study, we analyzed the role of ESAM for leukocyte migration in
vivo. ESAM is a novel adhesion receptor which is specifically
expressed at endothelial tight junctions and on platelets. Using
RLOT intravital microscopy of the murine cremaster muscle, we have
shown that IL-1β-induced leukocyte transmigration was reduced by
about 50% in ESAM-deficient mice without affecting leukocyte
rolling and adhesion. In summary, ESAM at endothelial tight
junctions participates in the migration of neutrophils through the
vessel wall. In the second part of the study, we investigated the
role of two other recently discovered receptors, CD99 and CD99L2,
for leukocyte migration. Similar to ESAM, these receptors are
expressed at endothelial cell contacts but did not belong to any of
the known protein families. We demonstrate that CD99 and CD99L2
mediate transendothelial migration of neutrophils in vivo without
any effect on leukocyte rolling and adhesion. Finally, we show that
the inhibitory effect of anti-CD99 and CD99L2 antibodies on
cytokine-induced leukocyte transmigration in cremasteric venules is
amplified in PECAM-1-/- mice. This fact suggests that a functional
relationship between PECAM-1 and CD99/ CD99L2 might exist in
mediating leukocyte transmigration. Taken together, our study
provides the first evidence for a role of CD99 and CD99L2 in the
process of leukocyte transendothelial migration in vivo. In the
third part of the study, we established a novel approach allowing
the visualization and analysis of directional leukocyte
interstitial migration in vivo. Our technique combines RLOT and
multicolor fluorescence microscopy with microinjection for local
application of chemoattractants. In the mouse cremaster muscle, we
show that microinjection of chemoattractants (MIP-1α and PAF)
induced directional leukocyte polarization and migration.
Combination of RLOT microscopy with fluorescence microscopy allowed
simultaneous visualization and analysis of migratory behavior of
different leukocyte subsets upon chemotactic stimulation. Moreover,
this approach enabled an imaging of subcellular events such as
mitochondria redistribution in single polarized interstitially
migrating leukocytes in vivo. This technique opens new avenues for
investigations of the mechanisms of interstitial migration of
leukocytes as well as the observation of morphological changes and
subcellular events in different leukocyte subsets during their
interstitial migration in vivo.