The assumption that cellular injury induced in infectious and in inflammatory sites might be the result of a well-orchestrated, synergistic "cross-talk" among oxidants, membrane-damaging agents, proteinases, and xenobiotics was further investigated in a tissue culture model employing monkey kidney epithelial cells (BGM) labeled either with 51 chromium or [3H]arachidonate. The cells could be killed in a synergistic manner following exposure to combinations among H2O2 and the following membrane-damaging agents: streptolysins S (SLS) and O (SLO), poly-D-lysine, arachidonic acid, eicosapentanoic acid, arachidic acid, lysophosphatidylcholine, lysophosphatidylinositol, lysophosphatidylglycerol, ethanol, and sodium taurocholate. Peroxyl radical (ROO) generated by azobisdiamidinopropane dihydrochloride (AAPH) further enhanced cell killing induced by SLS, SLO, and nitroprusside when combined with H2O2 and trypsin. BGM cells labeled either with chromium or with tritiated arachidonate, which had been treated with increasing concentrations of sodium nitroprusside (a donor of NO) and with subtoxic amounts of SLS and H2O2, were also killed in a synergistic manner and also lost a substantial amounts of their arachidonate label. Both cell killing and the release of membrane lipids were totally inhibited by hemoglobin (an NO scavenger) but not by methylene blue, an antagonist of NO2-BGM cells that had been treated with increasing concentrations of taurocholic acid were killed in a synergistic manner by a mixture of subtoxic amounts of ethanol, H2O2, and crystalline trypsin (quadruple synergism). Normal human serum possessing IgM complement-dependent cytotoxic antibodies against Ehrlich ascites tumor cells were killed in a dose-dependent fashion. Cell killing was doubled by the addition of H2O2. Cell killing and the release of membrane lipids by all the mixture of agonists tested were both strongly inhibited by the antioxidants catalase, Mn2+, vitamin A, and by fresh carrot juice. It appears that in order to overcome the antioxidant capacities of the epithelial cells, a variety of membrane-damaging agents had to be present in the reaction mixtures. Taken together, it might be speculated that the killing of mammalian cells in infectious and in inflammatory sites is a synergistic phenomenon that might be inhibited by antagonizing the cross-talk among the various proinflammatory agonists generated by microorganisms by activated phagocytes or by combinations among these agents. Our studies might also open up new approaches to the assessment of the toxicity of xenobiotics and of safe drugs to mammalian cells by employing tissue culture techniques.

I read with interest the EHP supplement on oxygen radicals and lung injury (vol. 102, supplement 10). I would like to take this opportunity to comment about this supplement and raise a key issue concerning the major concepts regarding the mechanisms of cellular injury in inflammatory diseases. As an active investigator in this field of research, I cannot fully understand why there was no mention in the supplement about the basic understanding that cellular damage in inflammation is multifactorial. The nonexpert reader of this supplement might receive an erroneous impression that oxygen radicals, per se, are the exclusive toxic agonists that induce cellular injury. Many in this field share the view that cellular damage in inflammatory diseases might be caused by a "coordinated cross-talk" among oxidants, membrane-damaging agents, proteinases, arachidonic acid metabolites, phospholipases, cationic proteins, and cytokines. All these agents are likely to be present in sites of infection and inflammation. But sadly, none of the publications elaborating on this multifactorial view are quoted in modern textbooks or in symposia on inflammation and inflammatory diseases. Instead, the literature is filled with publications that insist on a single agonist, be it an oxidant, a protease, a cytokine, etc., in experimental models. No attempt to integrate the various agonists into the full picture is made. Several of our publications (1-7) deal with synergistic interactions among multiple proinflammatory agonists in cellular injury during inflammation. I believe that this issue is important, timely, and might contribute to an understanding of how drugs, chemicals, and xenobiotics function in vivo. Isaac Ginsburg Hadassah School of Dental Medicine Hebrew University Jerusalem

Comment on Effect of lysophosphatidic acid on motility, polarisation and metabolic burst of human neutrophils. [FEMS Immunol Med Microbiol. 1994]

Monkey kidney epithelial cells, labeled with chromium and grown in culture, were killed in a synergistic manner when subtoxic amounts of ethanol were combined either with subtoxic amounts of glucose oxidase-generated hydrogen peroxide, or with mixtures of peroxide and with 2,2'-Azo-bis (2-amidinopropane)HCl (AAPH)-generated peroxyl radical. A further enhancement of cytotoxicity occurred when subtoxic amounts of trypsin were added to mixtures of all three agents. While ethanol alone caused shrinkage of the monolayers and cell rounding, no visible cytotoxic changes were observed. Hydrogen peroxide at the concentrations used (about 1 mM), caused only some cell rounding. On the other hand, cells exposed simultaneously to ethanol and to H2O2 developed extensive membrane damage characterized by the formation of large polar blebs, which is compatible with altered membrane permeability. The presence of trypsin markedly enhanced cellular cytotoxicity induced by mixtures of peroxide, peroxyl radical, and ethanol. This could markedly be depressed by catalase and by dimethylthiourea. The tissue culture model described might serve to further investigate the role played by synergy among oxidants and a variety of membrane-damaging agents, and by xenobiotics in tissue damage induced by inflammatory processes.

The biochemical and biological properties of many of the pro-inflammatory agonists generated by catalase-negative hemolytic streptococci and by activated human phagocytes, and the mechanisms by which both cell types destroy tissues in infections and in inflammatory sites, are astonishingly similar. In the pre-antibiotic era, group A hemolytic streptococci, also known by the name Streptococcus pyogenes, were responsible for causing serious and life-threatening diseases, mainly in young individuals. These highly virulent agents cause suppurative lesions in virtually any part of the body, due perhaps to their ability to disseminate freely in tissues. They do this by virtue of their ability to elaborate numerous “spreading factors” and tissuedamaging agents. However, the hallmark of the streptococcus injuries is their ability to initiate non-suppurative sequelae (rheumatic fever, arthritis, chorea and glomerulonephritis). Activated phagocytes (neutrophils, eosinophils, macrophages) might also be involved in the pathogenesis of many inflammatory diseases because of their ability to generate and secrete numerous tissue-damaging agonists. It is perhaps paradoxical that both phagocytes and hemolytic streptococci possess adhesion molecules (Patarroyo, 199 1; Ofek et al., 1975; Hasty et al., 1992; Sela et al., 1993; Albelda et al., 1994), receptors for IgG and for IgA (Christensen et al., 1976; Ginsburg et al., 1982; Burova and Schalen, 1993), receptors for complement (Petty and Todd, 1993), receptors for a variety of serum proteins, and for fibronectin (Littenberg et al., 1987; Simpson et al., 1987; Sela et al., 1993). Both phagocytes (Greenwald and Jamison, 1977; Wright, 1982; Gallin et al., 1992) and streptococci (reviewed by Ginsburg, 1972, 1985, 1986), generate numerous spreading factors (hyaluronidase, DNAse, RNAse, proteinases, acid and neutral hydrolases and complement-destroying enzymes (Wexler et al.. 1985). All these agents might facilitate the movement of the cells through the endothelial and epithelial barriers and into the intercellular spaces, and to depolymerize extracellular matrix proteins and inflammatory exudates which, otherwise, might limit cell movement and their spread in the tissues of the host. The non-immunogenic hyaluronic acid capsule, present on the surface of virulent streptococci, mimics similar components also present on mammalian cells. This mimicry allows the streptococci to survive, unrecognized, by the phagocytic cells. Both streptococci (Ginsburg, 1972; Ginsburg, 1979b; Alouf, 1990; Bernheimer and Rudy, 1986) and phagocytes (Victor et al., 198 1; Kennedy and Becker, 1987; Gallin et al., 1992) generate potent membraneperforating agents (hemolysins, phospholipases) which are capable of killing host cells by boring “holes” in their plasma membranes. Both streptococci (Suzuki and Vogt, 1966; Vogt et al., 1983) and phagocytes (Elsbach and Weiss, 1992; Spitznagel, 1990; Lehrer, 1993) also generate a large variety of highly cationic arginine- and cysteine-rich bactericidal and cytocidal proteins. These agents are also capable of activating the respiratory burst in neutrophils (Ginsburg, 1987, 1989) and also of functioning as opsonins (Ginsburg, 1987, 1989). Polycations might also enhance the adherence of neutrophils to targets (Oseas et al., 1981) and thus facilitate delivery of the toxic agonist directly upon the targets. This property is also shared by streptococci possessing cell-bound streptolysin S (Ginsburg and Harris, 1965; Ginsburg and Varani, 1993). Phagocytes and hemolytic streptococci produce either cytokines (West, 1990; Badwey et al., 1991) or a pyrogenic super-antigen (erythrogenic toxin; see Hensler et al., 1993), respectively, which prime phagocytes to generate excessive amounts of reactive oxygen species (ROS) and of lipid mediators of inflammation. Streptococci also generate a surface amphiphile (lipoteichoic acid-LTA) (Ginsburg et al., 1988) which, like lipopolysaccharides (LPS) of Gramnegative rods (Forehand et al., 1989, 1991) also primes neutrophils to generate excessive amounts of ROS. A possible “genetical” linkage between the highly anti-phagocytic surface component, the M-protein of streptococci and human proteins, has been found (Fischetti et al., 1988). Seventy percent of the Mprotein molecule has a tertiary structure of coiled-coil, which is also a characteristic either of tropomyosin, myosin or fibrinogen. Group A hemolytic streptococci also possess two sets of antigens which crossreact with human heart, kidney, brain, skin, myosin and perhaps also with leukocytes (Ayoub and Kaplan, 1991: Stollerman, 1975, 1991; Trentin, 1967; Kaplan, 1967; Ginsburg, 1972; Krisher and Cunningham, 1985; Swerlick and Cunningham. 1986). This led to the hypothesis that the development of crossreactive immunity, in susceptible hosts (Stollerman, 1975, 1991) might be responsible for the pathogenesis of rheumatic fever. arthritis, chorea and nephritis, that are the hallmarks of the post-streptococcal sequelae. Since the crossreactive antibodies isolated from rheumatic fever patients were not cytotoxic to cardiac tissue, their role, if any, in the pathogenesis of tissue damage remains to be established. Most importantly, however, both activated phagocytes and the catalase-negative hemolytic streptococci generate large amounts of H2 O2 (Avery and Morgan, 1924; Ginsburg, 1972; Halliwell and Gutteridge, 1989; Klebanoff and Clark, 1978; Klebanoff, 1992). It therefore stands to reason that both phagocytes and streptococci might cause cellular damage by a tight and wellorchestrated and synergistic collaboration among their secreted agonists (see below). Furthermore, extracellular products elaborated by both phagocytes and streptococci during their encounter in infectious sites, might also interact to amplify cellular damage. Such interactions might take place when H20Z generated by streptococci might be effectively utilized by neutrophils of patients suffering of chronic granulomatous disease of childhood (CGD), which possess a defective NADPH oxidase (Smith and Curnutte, 1991). Such an interaction might not only restore the ability of the CGD phagocytes to kill bacteria, but might also, paradoxically, lead to enhanced cellular damage provided that additional agonists are also present (see below). It is thus tempting to speculate that, mainly from functional and perhaps also from evolutionary points of view, hemolytic streptococci and other toxigenic bacteria (Clostridiae) might perhaps be considered “forefathers of modern phagocytes”. However, it should also be emphasized that evolution displays many examples where basic and parallel biological phenomena might appear in phyla far removed from each other, with no apparent common genetical basis. This emphasizes the successfulness of the strategy, since two totally separate evolutionary pathways have led to it.

Oxygen-derived species are implicated in the pathogenesis of tissue damage in experimental models such as ethanol-induced gastric injury as well as in certain clinical conditions. The aim of this study was to examine the protective effect of manganese and glycine, previously shown to act as H202 scavengers, on ethanol-induced gastric lesions in the rat: MnCl2 and glycine (12.5-50mg/rat) were injected s.c up to 6 hours prior to oral administration of 96% ethanol and the extent of mucosal damage was evaluated 1 hour later by gross and microscopic score. Mn and glycine pre-treatment induced a dose-dependent inhibition of lesion formation. Maximal protection was observed when agents were applied 4 hours prior to the insult. Gross damage was also markedly prevented by pre-treatment with dimethyl-thiourea (DHTU,75mg/Kg), but not by allopurinol. Mixtures of subtoxic concentrations of ethanol and H202 were highly lethal for monkey kidney epithelial cells in culture. Cell killing in this model was markedly attenuated by catalase and DMTU and to a lesser degree by Mn+2 . These results imply that ethanol-induced gastric damage may in part, involve generation of oxygen derived species, independent of the xanthine oxidase system. Mn+2 and glycine provide marked gastro- protection, acting possibly as oxygen radical scavengers.

Cimetidine, a known H2 blocker, markedly inhibited the generation of luminol-dependent chemiluminescence (LDCL) and the generation of Superoxide by human neutrophils (PMNs) stimulated by polycation-opsonized streptococci. Cimetidine also inhibited LDCL generation in peritoneal PMNs derived from mice pre-injected with this drug. The elucidation of the mechanisms of LDCL inhibition involved the employment of a variety of cimetidine analogues. The most effective inhibitory activity, besides cimetidine, was displayed by histamine, histidine, imidazole acetate, anserine and ergothionine. Imidazole, carnosine and homocarnosine had no inhibitory effect on oxygen radical generation. The possible mechanisms by which cimetidine and certain of its analogues affect the respiratory burst in leucocytes is discussed.

Previous studies have shown that the streptococcal hemolysin, streptolysin S, is capable of interacting with hydrogen peroxide (H2O2) to injure vascular endothelial cells (Free Radic. Biol. Med. 7:369-376; 1989). To extend these observations, intact group A streptococci (strain 203S) were examined for ability to injure endothelial cells alone and for ability to injure the same cells in the presence of sublethal concentrations of H2O2 (generated from glucose/glucose oxidase). While neither control bacteria nor bacteria that had been pretreated with poly-L-histidine to render them cationic were cytotoxic to endothelial cells by themselves under the conditions of the experiment, endothelial cells were injured by combinations of streptococcal cells and sublytic amounts of H2O2. Taken together, these data suggest that the sequelae which often occur following primary infection with group A streptococci may be the result of a combined assault of host inflammatory cells and the invading bacteria on the vascular lining cells of the host.

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.

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the result of the interaction among H2O2, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H2O2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and Co2+ very markedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by superoxide dismutase (SOD) and by deferoxamine LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.

A striking similarity exists between the pathogenetic properties of group A streptococci and those of activated mammalian professional phagocytes (neutrophils, macrophages). Both types of cells are endowed by the ability to adhere to target cells; to elaborate oxidants, hydrolases, and membrane-active agents (hemolysins, phospholipases); and to freely invade tissues and destroy cells. From the evolutionary point of view, streptococci might justifiably be considered the forefathers of “modern” leukocytes. Our earlier findings that synergy between a streptococcal hemolysin (streptolysin S, SLS) and a streptococcal thiol-dependent proteinase and between cytotoxic antibodies + complement and streptokinase-activated plasmin readily killed tumor cells, led us to hypothesize that by analogy to the pathogenetic mechanisms of streptococci, the mechanisms of tissue destruction initiated by activated leukocytes in inflammatory sites, as well as in tissues undergoing episodes of ischemia and reperfusion, might also be the result of the synergistic effects among leukocyte-derived oxidants, phospholipases, proteinases, cytokines, and cationic proteins. The current report extends our previous synergy studies with endothelial cells to two additional cell types-monkey kidney epithelial cells and rat beating heart cells. Monolayers of51Cr-labeled cells that had been treated by combinations of sublytic amounts of hydrogen peroxide (generated either by glucose oxidase, xanthine-xanthine oxidase, or by paraquat) and with sublytic amounts of a variety of membrane-active agents (streptolysin S, phospholipases A2 and C, lysophosphatides, histone, chlorhexidine) were killed in a synergistic manner (double synergy). Crystalline trypsin markedly enhanced cell killing by combinations of oxidant and the membrane-active agents (triple synergy). Injury to the cells was characterized by the appearance of large membrane blebs that detached from the cells and floated freely in the media, looking like lipid droplets. Cytotoxicity induced by the various combinations of agonists was depressed, to a large extent, by scavengers of hydrogen peroxide (catalase, dimethyl thiourea, and by Mn2+) but not by SOD or by deferoxamine. When cationic agents were employed together with hydrogen peroxide, polyanions (heparin, polyanethole sulfonate) were also found to inhibit cell killing. It is proposed that in order to effectively combat the deleterious toxic effects of leukocyte-derived agonists on cells and tissues, antagonistic “cocktails” comprised of cationized catalase, cationized SOD, dimethylthiourea, Mn2+ + glycine, proteinase inhibitors, putative inhibitors of phospholipases, and polyanions might be concocted. The current literature on synergistic phenomena pertaining to mechanisms of cell and tissue injury in inflammation is selectively reviewed.

Human neutrophils (PMNs) stimulated by sub-toxic concentrations of cetyltrimethyl ammonium bromide (CETAB) (37 μmol/L) generated intense luminol-dependent chemiluminescence (LDCL) and moderate non-amplified chemiluminescence (CL), but, paradoxically, generation of superoxide (as assayed by cytochrome c reduction, lucigenin-dependent chemiluminescence, nitroblue tetrazolium reduction test (NBT), spin trapping or hydrogen peroxide (Thurman reaction) and also oxygen uptake, were not observed. LDCL generation, however, was dependent on the viability of the PMNs. On the other hand, CETAB failed to induce CL in PMNs obtained from two children with an X-linked chronic granulomatous disease of childhood. CETAB inhibited superoxide generation by PMNs stimulated by phorbol-12-myristate-13-acetate (PMA), histone or polyhistidine-opsonized streptococci. It also inhibited NBT reduction in PMNs stimulated by PMA or by cationized streptococci. Generation of LDCL by CETAB-stimulated PMNs was inhibited by azide, cyanide, thiourea, dimethylthiourea, histidine, cimetidine, chloroquine, nordihydroguaiaretic acid and bromophenacyl bromide and partially so, about 50%, by superoxide dismutase (SOD), by TEMPOL (a SOD mimetic) and H-7, a protein kinase c inhibitor, but not by catalase, desferrioxamine, taurine or methionine. PMNs stimulated by CETAB in the presence of azide generated a large peak of LDCL when treated with horseradish peroxidase (HRP), suggesting that hydrogen peroxide, perhaps of intracellular origin, was involved. Such enhanced HRP-stimulated light emission was inhibited by catalase and by desferrioxamine, suggesting that the HRP-catalysed reaction also depended on some source of trace metals. CETAB also markedly enhanced CL generated by a cell-free mixture of hydrogen peroxide and HRP, which was quenched to a large extent by catalase, dimethylthiourea or desferrioxamine, again suggesting that light emission might be linked with trace metals present in the salt solutions employed. It is postulated that CETAB-induced CL in human PMNs is the result of the interaction of hydrogen peroxide, presumably of intracellular source, a trace metal, and a peroxidase (myeloperoxidase). This phenomenon might be unrelated to the classical respiratory burst, which is always accompanied by oxygen consumption, and to the generation of a variety of oxygen-derived species linked with the activation of the NADPH oxidase present in the cell membrane.

Carnosine, anserine and homocarnosine are natural compounds which are present in high concentrations (2-20 mM) in skeletal muscles and brain of many vertebrates. We have demonstrated in a previous work that these compounds can act as antioxidants, a result of their ability to scavenge peroxyl radicals, singlet oxygen and hydroxyl radicals. Carnosine and its analogues have been shown to be efficient chelating agents for copper and other transition metals. Since human skeletal muscle contains one-third of the total copper in the body (20-47 mmol/kg) and the concentration of carnosine in this tissue is relatively high, the complex of carnosine:copper may be of biological importance. We have studied the ability of the copper:carnosine (and other carnosine derivatives) complexes to act as superoxide dismutase. The results indicate that the complex of copper:carnosine can dismute superoxide radicals released by neutrophils treated with PMA in an analogous mechanism to other amino acids and copper complexes. Copper:anserine failed to dismute superoxide radicals and copper:homocarnosine complex was efficient when the cells were treated with PMA or with histone-opsonized streptococci and cytochalasine B. The possible role of these compounds to act as physiological antioxidants that possess superoxide dismutase activity is discussed.

Recent evidence indicates that under in vitro conditions, superoxide anion and hydrogen peroxide (H2O2) are unstable in the presence of manganese ion (Mn2+). The current studies show that in the presence of Mn2+, H2O2-mediated injury of endothelial cells is greatly attenuated. A source of bicarbonate ion and amino acid is required for Mn2+ to exert its protective effects. Injury by phorbol ester-activated neutrophils is also attenuated under the same conditions. EDTA reverses the protective effects. Acute lung injury produced in vivo in rats by intratracheal instillation of glucose-glucose oxidase is almost completely blocked in rats treated with Mn2+ and glycine. Conversely, treatment of rats with EDTA, a chelator of Mn2+, markedly accentuates lung injury caused by glucose-glucose oxidase. These data are consistent with the findings of others that Mn2+ can facilitate direct oxidation of amino acids with concomitant H2O2 disproportionation. This could form the basis of a new therapeutic approach against oxygen radical-mediated tissue injury.

Comparative effects of azapropazone on cellular events at inflamed sites. Influence on joint pathology in arthritic rats, leucocyte superoxide and eicosanoid production, platelet aggregation, synthesis of cartilage proteoglycans, synovial production and actions of interleukin-1 in cartilage resorption correlated with drug uptake into cartilage in-vitro. Azapropazone (APZ) has been compared with standard NSAIDs in title systems to establish aspects of its mode of action on cellular events at inflamed sites. APZ (150 mg kg-1 day-1) given for 10-13 days exhibited a reduction in joint pathology in established adjuvant arthritis in rats comparable with that of indomethacin (2 mg kg-1 day-1) and clobuzarit (20 mg kg-1 day-1). APZ was shown to be a potent inhibitor of the production of leucocyte superoxide and synovial interleukin-1 (IL-1)-like activity and stimulated articular cartilage proteoglycan synthesis, but was ineffective as an inhibitor of platelet aggregation or IL-1 induced cartilage degradation in-vitro. These in-vitro effects may have relevance to the mode of action of this weak inhibitor of prostaglandin synthesis.

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.

Insoluble complexes of poly-L-histidine (polyhistidine) and catalase were prepared by mixing the two reactants together in solution at pH 5.5 and subsequently elevating the pH to approximately 7.0, at which point they precipitated. Complexes formed at optimal ratios of polyhistidine to catalase contained essentially all of the catalase present in the original solution. The catalase present in such complexes contained greater than 50% of the H2O2-inhibiting activity of the native catalase used to prepare the complexes. The insoluble complexes rapidly bound to viable endothelial cells and were resistant to removal by extensive washing. The presence of polyhistidine-catalase complexes on the cell surface protected the cells against injury mediated by H2O2 or activated polymorphonuclear leukocytes. These data show that polyhistidine-catalase complexes can be prepared that have a high affinity for cells and that retain catalase activity. These complexes may be useful in treating inflammatory conditions in which it is necessary to maintain a high local concentration of inhibitor.

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidylcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma-O-hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly-L-histidine, phytohemagglutinin, and N-formyl-methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O2-). None of the lyso compounds by themselves caused generation of O2-. The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O2- generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O2-. However, in the presence of cytochalasin B, both lysophosphatidylethanolamine and lysophosphatidylserine also markedly enhanced O2- generation induced by immune complexes. O2- generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A2, phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O2-. The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O2- in stimulated PMNs.

Monolayers of murine fibrosarcoma cells that had been treated either with histone-opsonized streptococci, histone-opsonized Candida globerata, or lipoteichoic acid-anti-lipoteichoic acid complexes underwent disruption when incubated with human polymorphonuclear leukocytes (PMNs). Although the architecture of the monolayers was destroyed, the target cells were not killed. The destruction of the monolayers was totally inhibited by proteinase inhibitors, suggesting that the detachment of the cells from the monolayers and aggregation in suspension were induced by proteinases releases from the activated PMNs. Monolayers of normal endothelial cells and fibroblasts were much resistant to the monolayer-disrupting effects of the PMNs than were the fibrosarcoma cells. Although the fibrosarcoma cells were resistant to killing by PMNs, killing was promoted by the addition of sodium azide (a catalase inhibitor). This suggests that the failure of the PMNs to kill the target cells was due to catalase inhibition of the hydrogen peroxide produced by the activated PMNs. Target cell killing that occurred in the presence of sodium azide was reduced by the addition of a "cocktail" containing methionine, histidine, and deferoxamine mesylate, suggesting that hydroxyl radicals but not myeloperoxidase-catalyzed products were responsible for cell killing. The relative ease with which the murine fibrosarcoma cells can be released from their substratum by the action of PMNs, coupled with their insensitivity to PMN-mediated killing, may explain why the presence of large numbers of PMNs at the site of tumors produced in experimental animals by the fibrosarcoma cells is associated with an unfavorable outcome.