Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with...

To enable a better understanding of the regulation of neutrophil migration, we investigated the kinetics of adhesion and migration over, through and under endothelial monolayers. Neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) which had been treated with tumour necrosis factor-alpha (TNF; 2-1,000 U/ml) for 4 h. Videomicroscopy showed that transendothelial migration was complete within about 5 min of completion of perfusion of a bolus of neutrophils. Separate populations of adherent cells could then be observed, either rolling, migrating over the surface of the HUVEC or migrating underneath, at different characteristic speeds. Increasing concentration of TNF had little effect on the kinetics of migration, but shifted the balance from rolling adhesion to transendothelial migration. When individual neutrophils were followed from the moment they bound to HUVEC treated with 100 U/ml TNF, we found that approximately 40% immobilised essentially immediately on contact, while approximately 40% immobilised after rolling for varying periods (average 26 s) and approximately 20% rolled continuously. Most of the immobilised cells went on to migrate through the monolayer after spending 20-200 s migrating on top, and took about 60 s to pass through. Overall, the time from first binding to completion of transmigration averaged 152 s (range approximately 60-240 s). Interestingly, neutrophils moved relatively slowly on top of the monolayer (about 8 microm/min) but more rapidly underneath (about 16 microm/min). We suggest that the different stages during neutrophil transmigration have characteristic kinetics with separate control mechanisms, which critically influence the efficiency and rate of clearance from the vasculature.