Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-alpha

To enable a better understanding of the regulation of neutrophil migration, we investigated the kinetics of adhesion and migration over, through and u nder 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 tran sendothelial migration was complete within about 5 min of completion of per fusion of a bolus of neutrophils. Separate populations of adherent cells co uld then be observed, either rolling, migrating over the surface of the HUV EC or migrating underneath, at different characteristic speeds. Increasing concentration of TNF had little effect on the kinetics of migration, but sh ifted the balance from rolling adhesion to transendothelial migration. When individual neutrophils were followed from the moment they bound to HUVEC t reated with 100 U/ml TNF, we found that similar to 40% immobilised essentia lly immediately on contact, while similar to 40% immobilised after rolling for varying periods (average 26 s) and similar to 20% rolled continuously. Most of the immobilised cells went on to migrate th rough the monolayer aft er 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 avera ged 152 s (range similar to 60-240 s). Interestingly, neutrophils moved rel atively slowly on top of the monolayer (about 8 mu m/min) but more rapidly underneath (about 16 mu m/min). We suggest that the different stages during neutrophil transmigration have characteristic kinetics with separate contr ol mechanisms, which critically influence the efficiency and rate of cleara nce from the vasculature. Copyright(C) 1999 S. Karger AG, Basel.