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

significant proportion of the adherent cells had transmigrated, and that after a further 15 minutes,
this proportion remained essentially the same (Figure 3B). Transmigration was higher for
cytokine-treated monolayers, particularly in the presence of IFN. Although some adherent cells
did transmigrate through unstimulated HUVEC in minutes, the absolute number observed was
small due to the low level of adhesion. Examining migration through cytokine-treated EC in
more detail, we recorded individual fields and repeatedly assessed transmigration. While there
were minor fluctuations in the proportion of PBL transmigrated, there were no significant upward
trends (Figure 3C). We also noted the velocity of migrated cells, which averaged about 8μm∕min
and tended to be faster in the presence of IFN (Table 1A). Thus, in a static assay with short
initial contact times, we detected cytokine-specific induction of lymphocyte adhesion and could
observe transendothelial migration within minutes.

The detailed analysis of individual fields revealed another unexpected phenomenon. PBL
were seen to continue transmigrating (going from phase-bright to phase-dark) throughout the
entire 15min period, but other cells were migrating in the opposite (basal to apical) direction.
The continual forward and reverse migration explained the nearly constant level of
transmigration observed at any time. Some cells made several transits back and forth within the
observation period, and some stayed in the same compartment throughout. To quantify this
behaviour, we followed individual cells second-by-second over a period of 6 minutes, and
recorded if and when they moved between the basal and apical surfaces of the HUVEC. A
sequence of pictures of a multiply-migrating cell is shown in Figure 4A, and some typical
behaviours are illustrated schematically in Figure 4B. For cytokine-treated HUVEC, about 20%
of PBL made at least one transit during the observation period and about a third of these made
multiple transits (Table 1A). By measuring the number of transits during the 6-minute period, we

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