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



through TNF-treated HUVEC cultured in glass capillaries [31]. We described reverse migration
of neutrophils over hours in a similar flow model [32]. Reverse migration of neutrophils has
since been observed directly in a inflamed microvessels of the zebra fish [33]. Here, some PBL
and T-cells shuttled back and forth between these compartments over minutes. Repeat migration
of lymphocytes between the lumen and sub-endothelial spaces has not been described previously.
Nearly 10% of all adherent cells made more than one transit in a 6-minute period, and of cells
that started underneath the endothelial monolayer, over 40% underwent reverse migration in the
same time. Whether comparable behaviour occurs in vivo is unknown, and we are not aware of
any published real-time observations of migration of lymphocyte (as opposed to neutrophils) in
inflamed vessels.

In conclusion, lymphocytes (and specifically T-cells) migrated across endothelial cells in
minutes, were highly motile thereafter, and some at least migrated back and forth across the
endothelium repeatedly. The presence of flow improved the specificity of the assays (in the
sense that binding to unstimulated EC was negligible and effects of cytokines more clear-cut than
in static assays), but did not influence strongly the initial migration, or the back and forth
movement in the models used here. It appears that lymphocytes may be actively retained by
endothelial cells and require a signal from another source to induce their migration onward from
the sub-endothelial space. Consequently, the process of lymphocyte recruitment appears to
involve an additional regulated stage, in all probability to minimise non-specific sub-set
recruitment.



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