Summary. Viable Staphylococcus aureus (strain Oxford beta lactamase negative) which had been cultivated either in the absence or presence of penicillin G (subinhibitory concentrations) were injected into the knee joint of adult rats. Tissue sections taken five days following the injections and analyzed by an electron microscope revealed the persistence of apparently intact cell walls within macrophages at the inflammatory sites. The data suggest that even under penicillin effect the macrophages were capable only of digesting the bacterial cytoplasmic constituents (plasmolysis) but failed to degrade the bacterial peptidoglycan. The possible role played by anionic polyelectrolytes, which accumulate at the inflammatory sites, in the inhibition of cell wall degradation by leukocytes in 1/it/0 and the role played by leukocyte factors in the activation of the bacterial own autolytic wall enzymes will be discussed.

Human blood leucocytes generate large amounts of superoxide following stimulation by polyarginine, polyanetholesulphonate and mixtures of a variety of soluble agents. Generation of O2-. by the various "cocktails" of soluble ligands is markedly enhanced by cytochalasins A, B, C, D, E and F. The efficiency of cytochalasin A is, however, at least 50-fold greater than that of the other cytochalasins. Leucocytes that have been treated for a few minutes with the cytolytic agents saponin, digitonin and lysolecithin undergo lysis and lose their superoxide-producing capacities, when a variety of soluble ligands are employed to stimulate superoxide production. A partial reactivation of the superoxide-producing capacities of the leucocytes can be achieved by adding NADPH. However, as the concentration of the cytolytic agents increases, reactivation of the cytochrome C reduction is less inhibitable by SOD, suggesting that cell lysis releases reductases of cytochrome C not connected with the superoxide-producing system of the leucocytes. Both saponin and digitonin can totally replace polyarginine as ingredients of the "cocktail," suggesting that these agents may also function as "priming agents" for superoxide production which can, however, further be enhanced by the addition of mixtures of soluble agents. Thus, leucocytes which had been lysed by membrane-active agents can nevertheless produce superoxide if adequate amounts of NADPH are added.

The interaction of leucocytes with Staphylococcus aureus results in killing of the bacterial cells, but large portions of the bacterial cell walls persist apparently phagocytic cells for long periods. The mechanisms of biodegradation of staphylococci by leucocyte factors have shown that degradation of cell walls in vitro may be the result of the activation, by leucocyte kationic proteins, of the bacterial autolytic wall enzymes that are responsible for degrading the cell walls from within. This process is markedly inhibited by sulphated polysaccharides like dextran sulphate, by heparin, or by polyanetholesulfonate (liquoid). These anionic polyelectrolytes have also been shown to inhibit the lysis of staphylococci treated with bacteriolytic concentrations of penicillin G. Staphylococci injected intraarticularly into the knee joint of rats underwent massive plasmolysis, but structures compatible with cell walls (peptidoglycan) persisted within macrophages in the inflammatory sites, for long periods. It is postulated that the inability of leucocytes to degrade staphylococcal cell-wall components may be the result of the interference, by anionic polyelectrolytes likely to accumulate in the inflammatory sites, with the activation of the autolytic systems. Alternatively, anionic polyelectrolytes may coat the bacterial cells and interfere with the binding of the autolytic enzymes with their corresponding substrates.

Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.

Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

The invasions of tissues by pathogenic microorganisms is followed by a sequence of events which culminate in phagocytosis and the intracellular killing of the ingested agents, by “professional” phagocytes [19]. It is also expected that the rich arsenal of hydrolases present in neutrophils and macrophages, including the muralytic enzyme lysozyme is adequate to degrade the complex architecture of the bacterial cells. Surprisingly, however, most pathogenic bacteria are extremely resistant to lysozyme action [14,21] and the fate of phagocytosed bacteria in vivo is not fully known [7,8,16,23]. The sequelae of the lack of bacterial degradation by leukocytes may be the “storage” of peptidoglycan-polysaccharide or peptidoglycan-lipopolysaccharide complexes within macrophages leading to the generation of granulomas and to the initiation of prolonged immune responses. This is pivotal to the initiation of immunopathological reactions [7, 8, 16, 23]. We have recently proposed [10, 11, 12, 13, 15, 29] that the biodegradation of certain microorganisms can be mediated through the activation, by cationic agents and phospholipases, of the bacterial own autolytic wall enzymes (suicidal phenomenon) which leads to the breakdown of the rigid cell walls. On the other hand, a variety of sulfonated anionic polyelectrolytes [11–13, 15] likely to be present in inflamed issues, may inhibit the biodegradation of the walls by the autolytic enzymes.