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

through the filter after 2 or 4 hours (e.g. 2.4 ± 1.0% of added cells migrated through TNF-
stimulated HUVEC at 4h; mean SEM, n=3). The proportion increased by 24h (e.g. 11.0 ± 3.2%
of added cells which migrated through TNF-stimulated HUVEC; mean SEM, n=4) and so we
made comparisons between variously-treated HUVEC at this time. Figure 1 shows that
endothelial cells treated with cytokines (TNF or INF alone, or together) tended to support greater
lymphocyte transmigration compared to unstimulated HUVEC, although there was no consistent
difference between the cytokine treatments. This trend was also evident when CD4⁺ or CD8⁺ T
cells were analysed separately (Figure 1), and the two types of T-cell behaved similarly to each
other. The proportion of lymphocytes that were adherent was high after 24 hours (~50%) and not
significantly affected by cytokine treatments (data not shown). Such long contact times are not
physiological, and presumably increase non-specific background adhesion. We thus reduced the
initial contact time by washing off non-adherent lymphocytes after 10min, whilst maintaining the
24h migration endpoint. This decreased lymphocyte adhesion, and there was now a tendency
toward greater adhesion for endothelial cells stimulated with TNF+IFN compared to untreated
cells (21.8 ± 3.7% vs. 14.8 ± 6.0% of added PBL adherent respectively; mean ± SEM from 6
experiments). However, transmigration was also much lower (3.4 ± 0.6% vs. 1.6 ± 0.5% of
added PBL transmigrated respectively).

These findings showed that PBL and the major T-cell subclasses took hours to migrate
through endothelial/filter constructs, a distance of only ~10μm, and that cytokine stimulation of
the endothelial cells only increased recruitment about two-fold. Lymphocyte recruitment across
the wall of inflamed vessels is expected to be more rapid and stimulus-specific. Thus we
observed lymphocyte interactions with HUVEC treated with TNF plus IFN (the treatment
inducing most efficient transmigration) on filters under flow conditions. Few flowing PBL

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