• T Arnould, C Michiels, and J Remacle.
• Laboratoire de Biochimie Cellulaire, Facultés Universitaires ND de la Paix, Namur, Belgium.
• Blood. 1994 Jun 15;83(12):3705-16.

AbstractSeveral pieces of evidence are reported for the accumulation of activated neutrophils in ischemic and reperfused tissues leading to the transformation of the ischemic tissue into an inflammatory territory and to an enhancement of tissue damages during reoxygenation. However, the molecular mechanisms responsible for these observations and the precise role played by endothelial cells in this process are still poorly understood. In this study, an in vitro model that mimics this situation was used to investigate the effects of hypoxia-incubated human umbilical vein endothelial cells (HUVEC) on polymorphonuclear leukocyte (PMN) functions. A strong PMN activation characterized by an increase in intracellular calcium concentration as well as by superoxide anion release and leukotriene B4 production was observed when these cells were coincubated with hypoxic HUVEC. On the other hand, conditioned medium from hypoxia-incubated HUVEC failed to activate PMN, as determined by the lack of PMN calcium concentration increase, the failure of superoxide anion production enhancement, as well as the absence of effects on the integrin CD18, CD11a, and CD11b expression. These results indicate that the presence of hypoxia-incubated HUVEC is necessary to obtain an activation of the PMN, probably via the adherence process. Once activated by coincubation with hypoxic HUVEC, PMN became cytotoxic, as evidenced by 51Cr released from prelabeled HUVEC. This cytotoxic effect of activated PMN for hypoxic endothelial cells could be prevented by a combination of superoxide dismutase and catalase (94% inhibition), whereas superoxide dismutase alone was inefficient. Antiprotease (alpha 2-macroglobulin) and a specific elastase inhibitor (MAAPV-CMK) were also inefficient. These results correlate very well with the fact that no increase in elastase release could be observed in supernatants from PMN coincubated with hypoxic HUVEC. Furthermore, when adherence process was blocked by oleic acid or by anti-ICAM-1 monoclonal antibodies, protection was, respectively, 90% and 72%. We thus evidenced that free radicals but not elastase released from activated PMN coincubated with hypoxic HUVEC are involved in HUVEC injury. We conclude from these results that PMN activation is initiated by PMN adherence to hypoxic HUVEC. These observations indicate that hypoxic HUVEC may be partly responsible for neutrophil activation observed in ischemic tissues, which is part of the amplification process of tissue damage.

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