Each Step during Transendothelial Migration of Flowing Neutrophils is Regulated by the Stimulatory Concentration of Tumour Necrosis Factor-α

Abstract

Migration of circulating neutrophils occurs in several steps: capture and rolling adhesion are followed by activation of β2-integrins and immobilisation, and then neutrophils move over and through the endothelium. However, it is not clear how the underlying mechanisms and completion of each step depend on the concentration of stimulatory cytokines such as tumour necrosis factor-α (TNF). We therefore perfused neutrophils over human umbilical vein endothelial cells (HUVEC) which had been cultured with varying concentration of TNF (1–1000 U/ml) for 4 h, and recorded adhesion and migration by videomicroscopy. The number of adherent neutrophils increased with increasing TNF up to 5 U/ml, but changed little at higher concentrations. Interestingly, rolling adhesion at first predominated, but an increasing proportion of adherent cells became immobilised and migrated through the HUVEC monolayer over the complete TNF range. Immobilisation was inhibited by treating neutrophils with antibody against CD18, so that the major change in adhesive behaviour at higher levels of TNF occurred because the surface of the HUVEC presented agent(s) able to activate neutrophil β2-integrins. It was also evident that the selectins initiating capture of flowing neutrophils varied with concentration of TNF. At 100 U/ml TNF, both E-selectin and P-selectin supported capture and rolling adhesion, and antibody blockade of both receptors was required to inhibit adhesion. At lower dose (10 U/ml TNF), stable adhesion was blocked by antibody against E-selectin, although short-lived attachments could still be seen which were inhibited by antibody against P-selectin. Expression of selectins increased with increasing concentration of TNF, judging from surface ELISA and reduction in the velocity of rolling adherent cells. Thus the efficiency of capture, the selectins mediating capture and the proportion of captured cells immobilised and migrating all depend on the concentration of TNF to which endothelial cells are exposed. These results suggest a model in which highly localised and efficient migration of neutrophils is achieved if a concentration gradient of TNF exists around an inflammatory locus.