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



calculated the average interval between transits for those cells that moved between compartments
(mean ~4 min) (Table 1A). The values varied little between the cytokine treatments (TNF, IFN
or TNF+IFN). Transits were seen with unstimulated HUVEC (data not shown), but again, the
number of cells observed was small. On average, half of the transits were forward and half in the
reverse direction, which was consistent with the observation noted above, that overall levels of
transmigration were constant over the total observation period.

We wondered whether specific lymphocyte sub-populations might be more efficient in
migration or prone to reverse migration, and so carried out a series of experiments comparing
PBL to purified CD3+ and CD4+ T cells from the same donors. We observed no significant
difference in the proportion of these lymphocyte populations adhering or transmigrating through
endothelial cells stimulated with TNF+IFN (Figure 5). In addition, the multiple-transit behaviour
of purified T cell subpopulations, and the velocities of migrated cells were not significantly
different from PBL (Table 1B).

Lymphocyte migration through endothelial cells on clear substrates under flow

Unstimulated endothelial cells cultured in chamber slides consistently failed to recruit
flowing lymphocytes (Figure 6A). However, after cytokine stimulation, the endothelial cells
efficiently captured much greater numbers of flowing lymphocytes, with greater adhesion
observed on endothelial cells stimulated with TNF or TNF+IFN compared to IFN alone (Figure
6A). As with HUVEC cultured on filters, few captured lymphocytes rolled, and most adhered
firmly. Nearly a half of the adherent PBL transmigrated through the cytokine-stimulated
monolayers within 11 minutes, with IFN being the most effective inducer of migration (Figure
4B). Velocity of migrated cells averaged about 8μm∕min, which is similar to that observed in



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