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.