Increased PMN adherence on endothelial cells after hypoxia: Involvement of PAF, CD18/CD11b, and ICAM-1

Ischemia is a well-known situation occurring in several diseases. There is a large body of evidence for the accumulation of neutrophils in the microvascular injury and the transformation of ischemic tissue into an inflammatory territory. However, the molecular mechanisms underlying this phenomenon are still poorly understood. The effects of hypoxia were investigated on human umbilical vein endothelial cells (HUVEC) in culture, and a very strong activation of these cells was obtained with an induction of the platelet-activating factor (PAF) synthesis which was optimal after 90 min of hypoxia. PAF was chemically identified by gas chromatography-mass spectrometry. Along with incubation under hypoxia, a constant increase in the adherence of unstimulated human polymorphonuclear neutrophils (PMN) to endothelial cells was observed. The role of PAF and of adhesion glycoproteins in this hypoxia-induced neutrophil adherence to HUVEC was then assessed. The adherence was mediated by PAF after 90 min of hypoxia as indicated by the inhibition obtained with PAF receptor antagonists and with PAF synthesis inhibition. When tested on HUVEC incubated for 120 min under hypoxia, PAF antagonists could not inhibit the PMN adherence, whereas inhibition of PAF during hypoxia could block the process, suggesting a role of PAF acting as a second messenger. In addition, the inhibitory effects obtained using monoclonal antibodies indicate that this increased adherence was also mediated by intracellular adhesion molecule 1 on HUVEC and by CD18/CD11b on neutrophils. GMP-140 seems also to be involved after 90 min hypoxia but not after 120 min hypoxia, which correlated well with the presence of PAF. Protein synthesis was not required for this increased adherence, as shown by the lack of effect of cycloheximide. These results indicate that hypoxia by itself can activate endothelial cells and this activation can account for the PMN increased adherence observed in ischemic tissue.