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 various periods of endothelial hypoxia. Adhesion to HUVEC was dependent on the duration of hypoxia, with 30, 60, and 100 min of exposure causing graded increments in neutrophil recruitment. The degree of hypoxia also markedly influenced the endothelial response. Severe hypoxia (O2 < 2.5%) induced stationary attachment and then migration of neutrophils, in contrast to rolling adhesion alone under a less intense regime (O2 = 2.5-4.0%). Judged from studies with monoclonal antibodies, P-selectin was essential for adhesion after severe hypoxia, and neutrophil immobilization was attributable to the activation of neutrophil beta 2-integrin. Perfusion of neutrophils with an antibody against interleukin-8 or a platelet-activating factor antagonist reduced levels of adhesion. However, IL-8 appeared to be the dominant agent involved in the immobilization from flow, whereas platelet-activating factor was the more potent agent involved in initiating subendothelial migration. Thus endothelial cells alone can initiate all stages of adhesion and migration of flowing neutrophils after hypoxia and reperfusion.