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

Figure Legends

Figure 1: Effects of different cytokine treatments on migration of PBL and T-cell subsets
through endothelial cells and their supporting Transwell filters. HUVEC were stimulated with
100U/ml TNF alone, 10ng/ml IFN alone or with both for 24h. Lymphocytes were allowed to
adhere and migrate for 24h after which the number of lymphocytes transmigrating was
counted and expressed as a percentage of the added cells. Data are mean ± SEM from 2-8
independent experiments.

Figure 2: Behaviour of lymphocytes recruited to cytokine-stimulated endothelial cells in a
flow-based assay. A 4 min bolus of lymphocytes was perfused over HUVEC that had been
cultured in Transwell inserts and treated with TNF+IFN for 24h. The behaviour of the
adherent lymphocytes (rolling, stationary adherent, migrated through the endothelial
monolayer or migrated through the filter) was evaluated 11min after bolus perfusion. Data are
mean ± SEM from 4 independent experiments.

Figure 3: Effects of different cytokine treatments on lymphocyte recruitment to endothelial
cells cultured in multi-well plates. HUVEC were stimulated with 100U/ml TNF alone,
10ng/ml IFN alone or with both for 24h. Lymphocytes were allowed to settle for 5min, non-
adherent cells were washed off and lymphocyte adhesion and transmigration were analysed
by phase contrast microscopy. (A) Effect of cytokines on lymphocyte adhesion measured at
2min after wash-off. (B) Effect of cytokines on lymphocyte transmigration through HUVEC
at 2min and 17min after wash-off. (C) Time courses of lymphocyte transendothelial migration
for different cytokines. Data are mean ± SEM from 4 independent experiments. In A and B,
ANOVA showed a significant effect of cytokine treatment on lymphocyte adhesion and
transmigration; p<0.01. * = p<0.05, ** = p<0.01 compared to untreated by Dunnett test.

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