Arnould T, Michiels C, Remacle J
Laboratoire de Biochimie Cellulaire, Facultés Universitaires ND de la Paix, Namur, Belgium.
Blood. 1994 Jun 15;83(12):3705-16.
Several 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.
有多项证据表明，活化的中性粒细胞在缺血和再灌注组织中蓄积，导致缺血组织转变为炎症区域，并在再氧合过程中加剧组织损伤。然而，导致这些现象的分子机制以及内皮细胞在此过程中所起的确切作用仍知之甚少。在本研究中，我们使用了一种模拟这种情况的体外模型，来研究缺氧培养的人脐静脉内皮细胞（HUVEC）对多形核白细胞（PMN）功能的影响。当这些细胞与缺氧的HUVEC共同孵育时，观察到PMN强烈活化，其特征为细胞内钙浓度增加、超氧阴离子释放以及白三烯B4生成。另一方面，缺氧培养的HUVEC的条件培养基未能激活PMN，这可通过PMN钙浓度未增加、超氧阴离子生成未增强以及对整合素CD18、CD11a和CD11b表达无影响来确定。这些结果表明，缺氧培养的HUVEC的存在对于获得PMN的活化是必要的，可能是通过黏附过程。一旦通过与缺氧的HUVEC共同孵育而被激活，PMN就会变得具有细胞毒性，预标记的HUVEC释放的51Cr可证明这一点。超氧化物歧化酶和过氧化氢酶的组合可防止活化的PMN对缺氧内皮细胞的这种细胞毒性作用（抑制率为94%），而单独的超氧化物歧化酶则无效。抗蛋白酶（α2-巨球蛋白）和特异性弹性蛋白酶抑制剂（MAAPV-CMK）也无效。这些结果与以下事实非常相关：与缺氧的HUVEC共同孵育的PMN的上清液中未观察到弹性蛋白酶释放增加。此外，当黏附过程被油酸或抗ICAM-1单克隆抗体阻断时，保护率分别为90%和72%。因此，我们证明与缺氧的HUVEC共同孵育的活化PMN释放的自由基而非弹性蛋白酶参与了HUVEC损伤。从这些结果我们得出结论，PMN的活化是由PMN黏附于缺氧的HUVEC引发的。这些观察结果表明，缺氧的HUVEC可能部分导致了在缺血组织中观察到的中性粒细胞活化，这是组织损伤放大过程的一部分。
