Neutrophil Adhesion to Endothelial Cells Exposed to Disrupted Flow

During ischaemia and reperfusion (I/R), a complex pattern of haemodynamic changes and soluble agonists may act on EC, and influence subsequent leukocyte recruitment. To investigate such responses, human umbilical vein endothelial cells were cultured in glass capillaries and exposed to a wall shear stress of 2.0Pa for 24h. Shear was then reduced to 0.01Pa (creeping flow) or stasis (with 30s perfusion every hour to exchange medium). At chosen times isolated human neutrophils were perfused over the EC at 0.1Pa (effective reperfusion) and viewed by videomicroscopy. After 1h flow reduction of either type, perfused neutrophils made transient adhesions to the EC, but capture returned to baseline by 3h. Adhesion was inhibited by antibody against P‐selectin or pre‐treatment of EC with dipheyleneiodinium chloride (inhibitor of the NADPH‐oxidase). The response of EC to tumor necrosis factor (TNF, 100U/ml), judged by neutrophil recruitment, was greatly suppressed after 24h at high shear, compared to conventional static cultures. The response remained suppressed immediately after cessation of flow and took 48h of stasis to approach the level in conventional cultures. Interestingly, the suppression of TNF response remained in cultures switched to creeping flow. Thus, leukocyte recruitment after I/R may contain a component directly arising from a response of EC to reduction in flow. The increase in sensitivity of EC to soluble inflammatory stimulus, which might be expected to occur with reduction in shear stress, may develop quite slowly. Overall outcome may depend on duration of flow interruption and release of agonists in a complex manner. This work was supported by the British Heart Foundation.