KILLING OF ENDOTHELIAL-CELLS AND RELEASE OF ARACHIDONIC-ACID - SYNERGISTIC EFFECTS AMONG HYDROGEN-PEROXIDE, MEMBRANE-DAMAGING AGENTS, CATIONIC SUBSTANCES, AND PROTEINASES AND THEIR MODULATION BY INHIBITORS

Chromium-51-labeled rat pulmonary artery endothelial cells (EC) cultiv ated in MEM medium were killed, in a synergistic manner, by mixtures o f subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amou nts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclea se, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as we ll as by the membrane-damaging agents phospholipases A2 (PLA2) and C ( PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A strept ococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cat ionized by complexing to poly-L-histidine proved an excellent delivere r of membrane-directed H2O2 capable of enhancing EC killing by other a gonists. EC treated with rabbit anti-streptococcal IgG were also kille d, in a synergistic manner, by H2O2, suggesting the presence in the Ig G preparation of cross-reactive antibodies. Killing of EC by the vario us mixtures of agonists was strongly inhibited by scavengers of hydrog en peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin in hibitor, by polyanions, as well as by putative inhibitors of phospholi pases. 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 GTPgammaS had any modulating eff ects on the synergistic cell killing. EC exposed either to 6-deoxygluc ose, puromycin, or triflupromazin became highly susceptible to killing by n-mixtures of hydrogen peroxide with several of the membrane-damag ing agents. While maximal synergistic EC killing was achieved by mixtu res of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [H-3]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The re lease of AA from EC was markedly inhibited either by scavengers of H2O 2, by proteinase inhibitors, by cationic agents, by HClO, by tannic ac id, and by quinacrin. We suggest that cellular injury induced in infla mmatory and infectious sites might be the result of synergistic effect s among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic ca tionic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These ''cocktails'' not only kill cells, but als o solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocy te-derived agonists on dead cells. It is proposed that treatment by '' cocktails'' of adequate antagonists might be beneficial to protect aga inst cellular injury in vivo.