adhered to unstimulated HUVEC cultured on filters, but many more were adherent when the
endothelial cells had been stimulated with TNF plus IFN (0.6 ± 0.3% vs. 5.8 ± 2.0% of PBL
perfused, respectively; mean ± SEM, n=4). The cells adherent to the cytokine-treated EC were
firmly attached with only a small percentage rolling, and within 11 minutes about 30% had
migrated through the endothelial monolayer (Figure 2). At this time, very few lymphocytes had
migrated through the filter itself (Figure 2). The migration velocities of the phase-dark cells
under the HUVEC averaged about 5μm∕min (4.9 ± 0.6 μm∕min; mean ± SEM of means from 3
experiments).
Thus, lymphocytes adhered and migrated quickly through cytokine-stimulated
monolayers in the presence of flow. They then migrated freely under the monolayer but did not
appear below the filter within minutes. Indeed, it took hours to negotiate the filter in the static
assay. Since direct observation of kinetics of migration through the endothelium or the filter was
not possible in the 'standard' static filter assay, it is not possible to conclude at this stage the exact
step at which the hold-up occurred. It is possible that transendothelial migration was slower in
the absence, compared to the presence, of flow [27], or that lymphocytes quickly crossed the
endothelial monolayer but were held up by the filter in either case. To clarify this point, we
compared microscopic observations of migration kinetics through HUVEC under static or flow
conditions, using clear, solid substrates.
Lymphocyte migration through endothelial cells on clear substrates in absence of flow
When PBL were allowed to settle for 5min, few (~5%) adhered to unstimulated HUVEC
cultured in multi-well plates, but cytokine-stimulated endothelial cells supported much higher
levels of attachment (Figure 3A). We were surprised to find that 2 minutes after washing, a