Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration

Chemokines CXCL10 and CXCL12 were added to gels at a concentration (80ng/ml) which
would be expected to increase chemotaxis through filters (unpublished observations). However,
in 2 experiments with each, PBL migration through cytokine-treated endothelium or into the gel
were not increased, and the tendency to migrate back and forth across the endothelial monolayer
remained unaltered (data not shown). The chemokines most likely diffused across the EC and
into the surrounding medium during endothelial culture, washing and the adhesion assay itself,
diluting any gradient. We thus increased the concentration of CXCL10 added to the gel 10-fold,
and observed a small but consistent increase in the migration of lymphocytes into the gel when
compared to untreated gels (Figure 7C). Of note, the total level of transmigration across the
endothelial monolayer was not increased (averaging 51.2 ± 9.8% or 54.3 ± 5.6% for gels with or
without chemokine respectively; mean ± SEM from 3 experiments m,easured at 24h), but the
presence of CXCL10 tended to reduce lymphocyte migration back and forth across the
endothelial monolayer. The proportion of adherent cells undergoing one or more transit over 6
minutes was reduced from 14.2 ± 4.8% to 6.9 ± 2.2%, and the proportion undergoing more than
one transit was reduced from 6.9 ± 2.3% to 3.9 ± 1.8% (mean ± SEM from 3 experiments)
although these trends did not reach statistical significance. Thus the presence of a chemokine,
such as CXCL10, could promote lymphocyte migration away from the endothelium into the
underlying matrix.

Discussion

Using direct microscopic observation of endothelial monolayers treated with different
cytokines, we found that freshly-isolated peripheral blood lymphocytes could migrate across
endothelial monolayers in minutes in the presence or absence of flow. While the lymphocytes
migrated at about 5-10μm∕min underneath the endothelial cells, they did not move quickly

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