ADHESION OF FLOWING NEUTROPHILS TO CULTURED ENDOTHELIAL-CELLS AFTER HYPOXIA AND REOXYGENATION IN-VITRO

Using a novel in-line deoxygenating system linked to an in vitro flow- based adhesion assay and video microscopy, we have studied neutrophil recruitment and migration after hypoxia and reoxygenation of cultured human umbilical vein endothelial cells (HUVEC). Unstimulated purified neutrophils were perfused over reoxygenating HUVEC immediately after v arious periods of endothelial hypoxia. Adhesion to HUVEC was dependent on the duration of hypoxia, with 30, 60, and 100 min of exposure caus ing graded increments in neutrophil recruitment. The degree of hypoxia also markedly influenced the endothelial response. Severe hypoxia (0( 2) < 2.5%) induced stationary attachment and then migration of neutrop hils, in contrast to rolling adhesion alone under a less intense regim e (O-2 = 2.5-4.0%). Judged from studies with monoclonal antibodies, P- selectin was essential for adhesion after severe hypoxia, and neutroph il immobilization was attributable to the activation of neutrophil bet a 2-integrin. Perfusion of neutrophils with an antibody against interl eukin-8 or a platelet-activating factor antagonist reduced levels of a dhesion. However, IL-8 appeared to be the dominant agent involved in t he immobilization from flow, whereas platelet-activating factor was th e more potent agent involved in initiating subendothelial migration. T hus endothelial cells alone can initiate all stages of adhesion and mi gration of flowing neutrophils after hypoxia and reperfusion.