Ginsburg I, Mitra R S, Gibbs D F, Varani J, Kohen R
Department of Oral Biology, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel.
Inflammation. 1993 Jun;17(3):295-319. doi: 10.1007/BF00918992.
51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.
在MEM培养基中培养的51铬标记的大鼠肺动脉内皮细胞（EC），会被亚毒性剂量的葡萄糖氧化酶产生的H2O2与亚毒性剂量的下列物质的混合物以协同方式杀死：阳离子物质、核组蛋白、防御素、溶菌酶、聚-L-精氨酸、精胺、胰核糖核酸酶、多粘菌素B、洗必泰、十六烷基三甲基溴化铵，以及膜损伤剂磷脂酶A2（PLA2）和C（PLC）、溶血卵磷脂（LL），还有A组链球菌的链球菌溶血素S（SLS）。亚毒性剂量的胰蛋白酶或弹性蛋白酶会进一步增强此类混合物诱导的细胞毒性。通过与聚-L-组氨酸络合而阳离子化的葡萄糖氧化酶被证明是一种出色的膜定向H2O2传递体，能够增强其他激动剂对EC的杀伤作用。用兔抗链球菌IgG处理的EC也会被H2O2以协同方式杀死，这表明IgG制剂中存在交叉反应抗体。过氧化氢清除剂（过氧化氢酶、二甲基硫脲、MnCl2）、大豆胰蛋白酶抑制剂、聚阴离子以及磷脂酶的假定抑制剂能强烈抑制激动剂的各种混合物对EC的杀伤作用。单宁酸和茶提取物也能强烈抑制细胞杀伤，但血清的抑制作用较小。另一方面，去铁胺、HClO、TNF或GTPγS对协同细胞杀伤没有任何调节作用。暴露于6-脱氧葡萄糖、嘌呤霉素或三氟丙嗪的EC对过氧化氢与几种膜损伤剂的混合物的杀伤变得高度敏感。虽然H2O2与PLA2、PLC、LL或SLS的混合物能实现最大程度的协同EC杀伤，但只有在激动剂混合物中也加入蛋白酶时，才会有非常大量的[3H]花生四烯酸（AA）、PGE2和6-酮-PGF释放。H2O2清除剂、蛋白酶抑制剂、阳离子剂、HClO、单宁酸和喹吖因都会显著抑制EC中AA的释放。我们认为，炎症和感染部位诱导的细胞损伤可能是白细胞衍生的氧化剂、溶酶体水解酶、细胞毒性阳离子多肽、蛋白酶和微生物毒素之间协同作用的结果，这些物质可能存在于渗出物中。这些“混合物”不仅杀死细胞，还能溶解AA及其几种代谢产物。然而，白细胞衍生的激动剂攻击死细胞后，各种激动剂也能导致AA释放。有人提出，用适当拮抗剂的“混合物”进行治疗可能有利于在体内预防细胞损伤。
