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

Figure 4: The migration behaviour of individual lymphocytes over a 6min period for HUVEC
treated with TNF+IFN. (A) Video-micrographs of a lymphocyte migrating from above the
endothelial monolayer (phase-bright) to underneath (phase dark), back to the top and then
under again. (B) Schematic representation of typical lymphocyte migratory behaviours.
Individual cells were tracked over a 6min period. Positions above (a) or below (b) the
HUVEC monolayer are plotted for 5 cells: Cell 1 stayed above the monolayer and cell 5
stayed below. The others made one or more transits between the compartments. Similar
behaviours were seen for HUVEC treated with TNF or IFN separately.

Figure 5: Comparison of recruitment of different lymphocyte sub-sets to endothelial cells
cultured in multi-well plates. HUVEC were stimulated with TNF+IFN for 24h. PBL, or
purified CD3⁺ or CD4⁺ T cells were allowed to settle for 5min, non-adherent cells were
washed off and adhesion and transmigration were analysed by phase contrast microscopy.
(A) Effects of cytokines on lymphocyte adhesion measured at 2min and 17min after wash-
off. (B) Lymphocyte transmigration through HUVEC at 2min and 17min after wash-off. Data
are mean ± SEM from 4 independent experiments.

Figure 6: Effects of different cytokine treatments on recruitment of lymphocytes
to endothelial cells in a flow-based assay. A 4 min bolus of lymphocytes was perfused over
HUVEC that had been cultured on chamber slides and treated with TNF alone, IFN alone or
with both for 24h. (A) Effects of cytokines on lymphocyte adhesion measured at 2min after
wash-off. (B) The behaviour of the adherent lymphocytes (rolling, stationary adherent or
migrated through the endothelial monolayer) measured 11min after wash-off. Data are mean
± SEM from 3-4 independent experiments. In (A), ANOVA showed a significant effect of
treatment (p<0.01). * = p<0.05 and ** = p<0.01 compared to untreated by Dunnett test.

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