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 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.

Various cationic polyelectrolytes (poly-alpha-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-L-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2-3 min with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These "multiple hits" induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL responses in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the "cocktail" of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by superoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, alpha-methyl mannoside, and poly-L-glutamic acid. The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of "cocktails" of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.

The effects of T-2 toxin on bacterial infection and leukocyte function and structure were examined in vivo and in vitro. Rats were innoculated with staphylococci after pretreatment with or without T-2 toxin. The T-2 pretreated rats failed to mount a cellular response to the bacteria. Blood and bone marrow cells were markedly suppressed by the T-2 toxin, the myeloid series being the most affected. In vitro studies with human leukocytes showed that small, nonkilling doses of T-2 toxin inhibited chemotaxis, chemiluminescence stimulated by bacteria, and phagocytosis of bacteria. It was concluded that this inhibition may contribute towards sepsis and rapid onset of death in T-2 toxin poisoning.

Introduction. Although a wealth of knowledge exists today on the biochemical pathways of biosynthesis, turnover and autolysis of bacterial cell wall components in vitro (1, 2), surprisingly very little is actually known about the mechanisms of biodegradation of microbial constituents in_vivo. One should differentiate between bactericidal and bacteriolytic processes induced by leukocytes since killed, but non-degraded, microbial cells may persist within macrophages to trigger chronic inflammation (3, 4). The present communication further supports our contention (5, 6) that the degradation of microbial cell wall components by leukocytes may be due to activation, by leukocytic cationic proteins, of autolytic wall enzymes rather than to the direct cleavage of the cells by lysosomal hydrolases. The modulation of bacteriolysis by anionic polyelectrolytes will be described and discussed in relation to the pathogenesis of chronic inflammation and afequelae.

This year marks the centennial anniversary of Elie Metchnikoff’s discovery of the pivotal role played by "professional" phagocytes in body defenses against invading microorganisms. His cellular theory dealt with the phagocytic events and the postphagocytic killing, and also alluded to the digestion of the internalized bacteria by the "cystases," later shown to be associated with the lysosomal apparatus of leukocytes. Fo date, despite the fact that numerous studies have described in great detail the mechanisms by which serum and leukocytes kill microorganisms (1, 8, 13, 24), surprisingly little is actually known about the biochemical pathways of degradation and mechanisms of disposal of microbial constituents once they have been sequestered within phagolysosomes (3, 9, 10, 13, 24). It is usually taken for granted that the numerous hydrolytic enzymes, including the key bacteriolytic enzyme lysozyme (muramidase), present in lysosomes of "professional" phagocytic cells [granulocytes or polymorphonuclear neutrophils (PMNs), and macrophages] are capable, (at least theoretically) of stripping off bacterial coats, thus exposing the peptidoglycan to cleavage by touramidase. Yet, the majority of pathogenic microorganisms are highly refractory to lysozyme action (9, 10, 17). One should also bear in mind that, while a massive breakdown of microbial cell walls eventually may lead to a bactericidal reaction, the mere killing of a microorganisms, either by leukocyte or by serum factors, may not necessarily be followed by a bacteriolytic reaction. The importance of elucidating the mechanism of microbial biodegradation in tissues stems from the observation that in many infectious diseases there is "storage" of nonbiodegraded microbial cell wall components within macrophages for long periods, which may be responsible for the perpetuation and propagation of chronic inflammatory sequelae and tissue destruction (10, 18). We have recently postulated (11, 14, 15) that bacteriolysis, and the biochemical degradation that ensues after bacteria have been attacked by serum or by leukocytes, may involve close cooperation among heat-stable serum factors, cationic proteins and phospholipase A2 of leukocytes, and heat-labile endogenous bacterial autolyric wall enzymes. This cooperation is affected markedly by anionic polyelectrolytes, likely to accumulate in inflammatory exudates, which may shut down autolysis and, thus, contribute to unfavorable postinfectious sequelae (10, 19). The present communication is a summary of efforts from our laboratory to gain insight into the mechanisms of lysis of Staphylococcus attreus, chosen as a model, by lysosomal enzymes of human blood leukocytes (3, 8-11, 14, 15, 19).

Introduction. In previous studies it could be shown that autolytic wall enzymes of bacteria can be activated by some cationic proteins (1). Recently we determination of firming our data lytic enzyme but obtained results from chemical and end group determination of the cleavage products from peptidoglycan confirming our data that even lysozyme acted not only as a muralytic enzyme but also as a cationic protein (2). In order to elucidate the mechanisms of the activation of autolytic wall processes by cationic proteins and of the direct respectively indirect muralytic actions of lysozyme we performed investigations on heated cells.

Although a voluminous literature exists today on the mechanisms by which "professional" leukocytes (granulocytes and maerophages) intercept with, engulf and eventually kill phagocytosed microorganisms (1, 2), surprisingly very little is known about the mechanisms of degradation and elimination of bacteria from tissues. It is well established that phagocytic cells are endowed with numerous hydrolyric enzymes, including the key cell wall splitting enzyme-lysozyme, which can theoretically cleave, surface, eeU wall and cytoplasmic constituents of bacteria. Also, fresh mammalian sera are known to contain a complex mixture of heat-labile complement components (3) heat-stable lysozyme and platelet-derived cationic proteins ( -lysins) (4) which have been shown to kill and partially lyse certain microbial constituents. Surprisingly, however, the majority of virulent microorganisms are highly refractory to both leukocyte and serum lyric agents. Throughout this communication we shall use the general term bacteriolysis to denote the degradation of the cell walls, the outer membranes and the cytoplasmic constituents of bacteria. The term cell wall lysis will be used to describe the specific biochemical degradation of the bacterial peptidoglycan. This may also be accompanied by the rupture of the protoplast membrane and the release of cytoplasmic constituents. The inability of leukocyte and serum factors to induce bacteriolysis is linked to the presence, upon most bacterial surfaces, of’lipopolysaccharides, polysaccharides-teichoic complexes and certain lipids and waxes, which hinder the accessibility of the major cell wall splitting enzyme-lysozyme to the peptidoglycan (1). Once however this obstacle is overcome, the peptidoglycan ist degraded, and the protoplasts burst due to their high osmotic pressure, releasing degradation products of both cell wall and cytoplasmic constituents into the surrounding medium. The very extensive literature on these subjects has been recently summarized and reviewed (5-7). It has als0 been suggested that the process of killing and biochemical degradation of microbial constituents, either following phagocytosis or following treatment with fresh complement and lysozyme-sufficient serum are probably mediated by different mechanisms (6-8). While extensive loss of wall material and cytoplasmic entities is usually accompanied by a bactericidal reaction, the killing of bacteria either by the oxygen-dependent (9) or by the non-oxygen dependent bactericidal systems of leukocytes (5, 10) and serum (3), is not necessarily accompanied by a substantial bacteriolysis. The distinction between a bactericidal and a bacteriolyric process is important, in view of the observations that poorly degraded non-viable microbial constituents may persist for long periods both extracellularly and within phagocytic cells, to trigger and perpetuate chronic inflammatory sequellae (6, 7, 11-13). Furthermore, while degradation products of grampositive and acid-fast bacteria have been shown to be endowed with distinct pharmacological properties (14), to modulate the immune responses (15), to activate the complement cascade (16) and to be cytopathic for mammalian cells (14), the in vivo release of lipopolysaccharides of the outer membrane of gramnegative bacteria may result in severe coagulation defects (Shwartzman phenomenon) and in endotoxic shock (17). Poorly degraded cell wall components of bacteria have also been shown to be translocated within macrophages from one tissue site to another, thus contributing perhaps to the dissemination of granulomatosis (18-20). Although the nature of the biochemical pathways involved in bacterial biodegradation in tissues has not been fully elucidated, it has been recently suggested that a cooperation among leukocyte factors (21, 22), serum components (23), the bacterial own autolytic wall enzymes (21, 22) and certain antibiotics (24), may act in accord to induce a massive breakdown of cell wall and cytoplasmic constituents of bacteria. It is well established that the autolytic systems present in every bacterial cell, control cell division, the deposition of new cell wall material and the regeneration after treatment with certain antibiotics (25, 36). Autolytic enzymes have been isolated from many bacterial species and were found to possess muramidase, Nacetyl glucosaminidase, amidase and peptidase activities (25). It is also known that certain antibiotics, mainly of the penicillin and cephalosporin series, are capable of killing microorganisms, presumably by activating their autolytic wall enzymes (27). These intraceUular enzymes are thought to be controlled by endogenous lipid material (e.g.- phospholipids, lipoteichoic acid) (27). Thus, any agent present in leukocytes or in tissue fluids, which will disrupt the balance between autolytic enzymes and their naturally occurring inhibitors, may lead to the activation of autolysins, and concomitantly to the release of toxic bacterial agents. In view of the complex interrelationship which exist between bacteria and host factors in infectious and imflammatory sites, it was of interest to clarify some of the mechanisms and the factors involved in the biodegradation and persistence of microbial constituents in tissues. The following is an overview of our studies on this subject, employing staphylococci and streptococci as model systems, and using biochemical and electron microscopical techniques (28). The peptidoglycan of Staphylococcus aureus was labelled during the logarithmic phase of growth with 14C-N-acetylglucosamine. When such labelled cells were incubated for several hours at 37 in acetate buffer pH 5.0, with small amounts of crude human leukocyte extracts or with more purified lysosomal extracts, a substantial amount of the radioactivity, associated with the cell walls was solubilized. Electron microscopical analysis of these reaction mixtures revealed the accumulation of cell wall fragments, and both amorphous and intact cytoplasmic constituents still retaining their typical morphologies (6, 7, 21, 22, 28, 29). Since the pH optimum for this reaction process was found to be on the acid side and compatible with the pH optima of many of the acid hydrolases known to be present in the leukocyte preparation (1) we postulated that the breakdown of the bacterial ceils was mediated by acid hydrolases. The findings, however, that heat treatment did not destroy the capacity of the leukocyte extracts to induce cell wall degradation, and that purified radiolabelled staphylococcal cell walls became completely refractory to the lytic effect of the extracts (21, 22), suggested that the wall degradation observed was probably not caused by the leukocyte hydrolases. Since leukocyte lysosomes are known to be rich in heat-stable argininerich bactericidal cationic proteins (LCP) (30) and in myeloperoxidase (MPO) (1) (also a cationic protein) it was reasonable to try and employ them, instead of the total leukocyte mixture, to lyse the staphylococci. Indeed we found that as little as 0.5-1/zg/ml of nuclear histone, poly-L-lysine, poly-L-arginine or MPO and 10-50/ag/ml of crystalline pancreatic ribonuclease or cytochrome C (all cationic in nature) were sufficient to induce massive loss of cell wall material from 108 log-phase staphylococci. Furthermore, small amounts of the membrane.damaging agents phospholipase A2, and polymyxins B and E were also capable of inducing cell wall lysis, as determined by the release of N-acetyl-glucosamine (8, 31-33). Since purified staphylococcal cell walls (devoid of cytoplasmic structures) were extremely refractory to any of the cationic polyelectrolytes or to the membrane-damaging agents, we postulated that perturbation of the staphylococcal membrane by these agents might have resulted in the activation of endogenous enzymes presumably associated with the autolytic systems (21, 22, 25, 26). As autolytic wall enzymes are known to be heat-labile (25, 26), it was of in,terest to try and reactivate the lytic process by the addition of freshly-harvested viable staphylococci (as donors of autolysins) to the heat killed radiolabeled staphylococci or to the purified labelled cell walls, in the presence of several of the cationic proteins or the membrane-damaging agents. Indeed, such mixtures resulted in a substantial loss of radiolabelled wall material. This process was completely blocked by anionic polyelectrolytes. We suggested, therefore, that the activators of autolysins interacted with the viable bacteria to release the autolytic enzymes, which in turn attacked and degraded the radiolabelled substances. Similar results were recently described with Bacillus subtilis (34). It has also been suggested that pneumococci, gonococci, meningococci, Streptococcus faecalis and perhaps listeriae may also likewise be degraded in vivo following the activation of their autolysins, and not through the direct action of lysosomal enzymes (28). Further experiments showed that Staphylococcus aureus, which had been cultivated in the presence of sub-inhibitory concentrations of penicillin G, became much more susceptible to wall lysis, following treatment with leukocyte extracts (24,35) suggesting a collaboration between/3-1actam antibiotics, leukocyte factors and bacterial autolysins in bacteriolysis (see 27). Other studies from our laboratory (23) have also shown that contrary to the accepted belief, both fresh and heat-treated human serum, when properly diluted, also lysed log-phase grampositive staphylococci and Streptococcus faecalis at pH 5.0. Since anionic polyelectrolytes also inhibited cell lysis induced by serum (23), and since heat-killed bacteria became resistant to lysis by serum, we postulated that, as in the case of leukocyte extracts, lysis was induced by a heat-stable factor, presumably -lysin of platelet origin (4) present in serum, which activated the autolytic systems of the bacteria. To further elucidate the mechanism of bacteriolysis, we have analyzed this process by electron microscopy. In collaborative studies with Prof. P. Giesbrecht and Dr. J. Wecke of the Robert Koch Institute in Berlin, we found (36) that a few hours after the addition of either crystalline lysozyme (500/ag) or pancreatic ribonuclease (50/ag/ml) to log-phase staphylococci, the first signs of cell damage could already be seen. These consisted of the formation of small, periodically-arranged lytic sites between the cell wall and the cytoplasmic membrane of the cross wall. This was followed by the formation of a distinct gap between the cell wall proper and the cytoplasmic membrane. The degradation of the peripheral cell wall continued to the opposite side of the cell, and extended gaps underneath the wall could be detected long before the cell wall itself was peeled off as large ribbons. The cross wall often appeared to be already disintegrated during the early phase of lysozyme or ribonuclease action. The release of the wall left apparently intact protoplasts, which still retained their original shape, and even the invagination of the cross walls were conserved. At this point over 70 % of the toal radioactivity associated with the cell wall was solubilized after three to four hours, and the radioactivity could not be sedimented at 100,000 • g suggesting the formation of solubilized peptidoglycan. Since lysozyme which had been heated to destroy its enzymatic activity still retained its ability to induce cell wall lysis, we postulated that lysozyme in this system did not act as an enzyme but as a cationic protein. Further studies from our laboratory (28) and in collaboration with the Robert Koch Institute (to be published in detail) have revealed that very similar ultrastructural changes in the staphylococci also took place following phagocytosis by mouse non-elicited macrophages in culture. On the other hand, although staphylococci, which had been injected into mouse or rat tissues, and which were phagocytosed by both PMNs and macrophages, underwent rapid loss of their cytoplasmic constituents, presumably by digestion with lysosomal hydrolases (37-39), no apparent damageto the cell walls was evident for many days, suggesting that the autolytic wall enzymes might have been inhibited. Since the degradation of the staphylococcal cell walls in vitro was completely inhibited by a variety of anionic polyelectrolytes like heparin, dextran sulfate, polyanethole sulfonate (a synthetic heparin), as well as by cationic polyelectrolytes like histones, poly-L-lysine, poly-L-ar inine, etc., when used at 10-100 /ag/ml/10 staphylococci (concentrations 10- 100 fold higher than those employed to activate the autolytic wall enzymes (see above), we also postulated that a delicate balance between activators and inhibitors may determine whether or not bacterial wall material may be degraded in tissue lesions in vivo (40,41). Finally, iecent studies (42) have also shown that high-molecular-weight degradation products of staphylococcal cell walls derived following treatment of the bacteria either in buffers (spontaneous wall lysis) or by small amounts of leukocyte extracts, were found to possess very strong chemot ctic activities for PMNs in vitro, and to induce severe inflammatory lesions when injected intraarticularly to rats. Thus, it may be concluded that the fate of bacterial peptidoglycans in leukocytes in inflamed tissues may be dependent on the one hand on the availability of agents capable of activating autolytic wall enzymes in bacteria, and on the other hand on the presence in tissues of inhibitory substances (polyelectrolytes) which are capable of blocking bacteriolysis. It is, however, not aimed t9 rule out the possibility that other still unknown mechanisms may function in the complex milieu of inflammation, which may bring about the biodegradation of bacteria. The employment of certain antibiotics and other pharmacological agents, yet to be discovered, which will be capable of changing the balance between activators and inhibitors of autolytic enzymes, may contribute to a better understanding of the mechanisms involved in the survial and persistence of microbial agents in vivo. It is also obvious that the release of large quantities of microbial constituents following incomplete biodegradation may prove to be deleterious to tissues. Finally, our studies on staphylococci do not shed light on the mechanisms of biodegradation of other microbial species of medical imprtance, and is only a reflection of one possible mechanism, which may or may not be common to all microorganisms.

INTRODUCTION. Although much is known today about the mechanisms by which virulent Staphylococcus aureus induce tissue lesions and cause clinical manifestations in mammals, our knowledge of the role played by “professional” phagocytes in host and parasite interrelationships in staphylococcal infections, is not fully understood. The extensive literature on staphylococci and their role in human disease has been the subject of several excellent comprehensive reviews (Whipple, 1965; Cohen, 1972; Jeljaszewicz, 1976). It is accepted that the interception of staphylococci by granulocytes (PMNs) takes place soon after the penetration of the cocci into the tissues. This involves the release of chemotactic factors, by the bacteria themselves (Pusell et al., 1975) or the activation, by staphylococcal factors, of chemotactic agents from complement (Ginsburg and Quie, 1980). Subsequent phagocytosis is markedly enhanced by opsonins (Koenig, 1972; Ekstadt, 1974), and in most cases the engulfed bacteria may be killed intracellularly by a variety of bactericidal agents generated by activated PMNs (Klebanoff, 1972). It is also suggested that certain lysosomal enzymes, which are released into the phagolysosome, may digest the staphylococcal cells (Cohn, 1963a; De Voe et al., 1973; Ginsburg and Sela 1976; Ginsburg, 1979) (see Sections II.B and VI). Several reports have, however, shown that intracellular staphylococci may sometimes survive within PMNs, where they multiply and eventually kill the cell (Koenig, 1972; Cohen, 1972; Pearce et al., 1976). Surprisingly, very little is known about the fate and mechanism of biodegradation of staphylococcal cell constituents, once they have been sequestered within the phagolysosomes of leucocytes. The importance of this field of research stems from the findings that non-biodegradable cell wall components of a variety of microbial species may persist within macrophges for prolonged periods, to trigger chronic infiammatory sequelae (Dannenberg, 1968; Kanai and Kondo, 1974; Ginsburg et al., 1975a; 1975b; 1976a; Ginsburg and Sela, 1976; Adams, 1976; Page et al., 1978; Ginsburg, 1979). The inability of “professional” phagocytes to degrade intracellular bacteria may be due (1) to the presence, on the surface of certain microorganisms, of shielding capsular material (Dossett et al., 1969; Smith, 1977; Wilkinson, et al., 1979; Densen and Mandell, 1980), (2) to the lack of fusion between lysosomes and phagosomes (Goren et al., 1976; Densen and Mandell, 1980), (3) to the production of leucocidins (Gladstone and Van Heyningen, 1957; Woodin, 1960; Ginsburg, 1970; Van Heyningen, 1970; Bernheimer. 1970; Ginsburg, 1972), (4) to the lack of adequate lysosomal enzymes capable of cleaving bacterial peptidoglycans (Dannenberg, 1968; Ginsburg, 1972; Ginsburg and Sela, 1976; Page et al., 1978; Ginsburg, 1979), or (5) to the presence, in serum and in inflammatory exudates, of agents which interfere with the interaction of bactericidal and bacteriolytic age with engulfed bacteria. These fields were comprehensively reviewed EW’ Jeljaszewicz, 1976; Smith, 1977; Ginsburg, 1979; and by Densen and Mandell, 1980. During the last 8 years our laboratory has been studying the host- and-parasite interrelationships in streptococcal and staphylococcal infections using biochemical, electron microscopical and tissue culture techniques. In particular we studied the mechanisms by which leu- cocytes and their isolated lysosomal agents bring about the degrada- tion of staphylococcal cell wall components, and the role which may be played by such degradation products in the initiation of chronic inflammation. The present report is an updated overview of these studies.

Human blood leukocytes and platelets and mouse peritoneal macrophages emit very rapid and very intense Luminol-dependent chemiluminescence (CL) signals when treated with streptococci, staphylococci, or with zymosan, which have been preopsonized with arginine-rich histone, dextran sulfate or polyanetholesulfonate (liquoid). Liquoid alone at 10-30 micrograms/2 X 10(5) leukocytes also triggers intense CL responses in the absence of a carrier. Strong CL can also be triggered, and at the same levels, when the various polyelectrolytes are simply mixed with the bacteria or zymosan and added to the leukocyte suspensions. The CL responses induced by the polyelectrolyte-bacteria complexes greatly exceed those triggered in leukocytes by antibody-complement-coated particles. Liquoid also shows a unique property of markedly augmenting CL signals which have already been induced by other ligand-coated bacteria or zymosan particles. Streptococci and staphylococci were found to be much superior to zymosan, Gram-positive bacilli, or E. coli as carriers for the various polyelectrolytes in the CL reaction. Neither protamine sulfate, lysozyme, myeloperoxidase, crystalline ribonuclease (all cationic in nature), chondroitin sulfate, heparin, nor alginate sulfate acted as ligands for triggering CL, when used to opsonize bacteria or zymosan. The induction of CL in blood leukocytes by the various ligand-coated bacteria is markedly inhibited by azide, KCN catalase, aminotriazole, and EDTA, agents known to inhibit the production of oxygen radicals following stimulation of leukocytes by opsonized bacteria. Two children diagnosed for chronic granulomatous diseases (CGD) of childhood and an apparently healthy sister of one of the male patients completely failed to respond with CL either to the polyelectrolyte-bacteria complexes, liquoid or antibody-coated bacteria and zymosan. It is proposed that liquoid be employed for the rapid screening of defects in certain oxygen-dependent metabolic processes in both PMNs and macrophages. It is also suggested that polyelectrolytes like the ones described in this study may markedly enhance the bactericidal properties of leukocytes and macrophages towards both extracellular and intracellular microorganisms and may perhaps also augment the tumoricidal effects of activated macrophages.

Both antibodies and complement components are essential for successful phagocytosis of many virulent microorganisms (1,2). Although the mechanisms by which opsonins promote particle uptake are not fully understood, it has been suggested that both electrostatic and hydrophobic forces act in concert with specific receptors for Fc and C3b to facilitate interiorization of particles (2,3). In the case of group A streptococci, opsonization by immunoglobulins abolishes the anti-phagocytic properties of the M-antigen (4,5). Since one mechanism by which opsonins may act is to decrease repulsion forces between negative charges present on the surface of the particle and phagocyte, cationic ligands may function as effective opsonins (6–11). In addition, cationic substances may participate in bacteriolysis. We recently suggested (11) that the breakdown of bacterial cells following phagocytosis is mediated indirectly by leukocyte cationic proteins and phospholipases which activate autolytic enzymes and not by lysosomal enzymes directly.

Various polyelectrolytes were investigated regarding their capacity to inhibit the binding of human IgG to Fc-receptors on group A streptococci, type M1. Of cationic substances, protamine and arginine-rich histone inhibited significantly, while lysine-rich histone, concanavalin A, lysozyme, polymyxin B, ribonuclease and tuftsin did not. Of anionic materials, liquoid was inhibitory, in contrast to chondroitin sulphate, dextran sulphate, DNA and heparin. Washing experiments showed that the inhibition was caused by binding of the polyelectrolytes to the streptococci. The finding that heated IgG inhibited the binding of histone to the streptococci also indicated a close relation between the binding sites for these compounds. Diffusion-in-gel experiments with alkaline extract of M1 demonstrated that the substances blocking the IgG Fc-receptor were bound to polyglycerophosphate, suggesting that the inhibition of the IgG uptake was due to interaction with lipoteichoic acid. Leukocyte and platelet extracts could modify the binding of IgG, probably by an enzymatic digestion of the receptors. The arginine-rich histone was also capable of inhibiting the binding of IgG to type M15 group A streptococci and to one group G strain. However, the polyelectrolytes had no effect on the binding of IgG to Staphylococcus aureus or of IgA to type 4 group A streptococci.

In contrast to former findings lysozyme was able to attack the cell walls of Staphylococcus aureus under acid conditions. However, experiments with 14C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. "attack from the inside"). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but "stabilized protoplasts" were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.

Although much is known today about the mode of action of antibiotics on microorganisms, relatively little has been done to evaluate the possible collaboration between antibiotics and the host defenses in the containment and elimination of pathogens from host tissues. Since certain antibiotics are known to interfere with the biosynthesis of bacterial cellular and extracellular components, it is conceivable that such modified bacterial cells may be more readily intercepted, killed, and eventually digested by professional phagocytes. On the other hand, certain antibiotics may have adverse effects on mammalian cells by interfering with their normal metabolism and subsequently with their antimicrobial functions. Although the role of bacteriolysis in host and parasite interrelationships has been recognized for over a decade, this field of research has surprisingly been almost totally neglected. The importance of understanding the mechanisms of biodegradation of microbial cells in vivo stems from the recognition that the inability of the enzymes of the host to degrade the rigid cell wall of microorganisms is a contributory factor to the formation of chronic granulomatous responses, and to the destruction of tissues [1, 6, 16, 17, 22, 30]. Thus, any antibiotics which will collaborate with leukocytes or with serum factors in the elimination of bacterial constitutents from infected tissues may greatly contribute to the well-being of the individual.

Effect of leukocyte hydrolases on bacteria XVI. Activation by leukocyte factors and cationic substances of autolytic enzymes in Staphylococcus aureus: modulation by anionic polyelectrolytes in relation to survival of bacteria in inflammatory exudates. The mechanisms involved in the activation of autolytic enzymes in Staphylococcus aureus, by leukocyte extracts, cationic proteins, phospholipase A2, amines, and membrane-damaging agents was studied in a resting cell system as well as by growing staphylococci. The bacteria were labeled with [14C]N-acetylglucosamine and were subjected to a variety of agents either in 0.1 M acetate buffer, pH 5.0, or in phosphate buffer, pH 7.4. While intact log-phase cultures were found to undergo partial autolysis at pH 5.0 and almost complete lysis at pH 7.4, both heat-killed bacteria and bacterial cell walls were completely resistant to autolysis in buffers. Autolysis at pH 5.0 can be further activated by leukocyte extracts, nuclear histone, crystalline ribonuclease, egg-white and human lysozyme, phospholipase A2, as well as by spermine, spermidine, and polymyxins B and E. The addition of viable log-phase bacteria to radiolabeled heat-killed staphylococci or to radiolabeled cell walls which had been cleaned off autolytic enzymes resulted in degradation of the radiolabeled targets. The data suggest that the various inducers of autolysin activation caused leakage of autolytic enzymes from the intact bacteria which attacked the depolymerized the bacterial cell walls. Anionic polyelectrolytes like heparin, dextran sulfate, suramine, polyglutamic acid, and liquid (polyanethole sulfonic acid) markedly inhibited both spontaneous and induced lysis. Staphylococci which had grown in the presence of anionic polyelectrolytes became highly resistant to lysis triggered by any of the inducers of autolysis. Since inflammatory exudates are known to be rich in anionic polyelectrolytes, it is suggested that the prolonged survival of intact bacterial cells in such a milieu may be due to the inactivation of autolytic enzymes. It is also postulated that the degradation of certain bacterial species following phagocytosis or extracellular degradation may not be the result of the action of hydrolytic enzymes but rather the result of activation by leukocyte factors of autolytic enzymes which lead to bacteriolysis.

Group A streptococci strains were grown in broth containing subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin, and tested for possible changes in colonial morphology, activity and amount of cellular and extracellular components. The following components were tested: T protein, M protein, opacity factor, lipoteichoic acid, hyaluronic acid, streptolysin S, streptolysin O, DNase, hyaluronidase and NADase. Sub-MICs of these drugs produced variable changes in the bacteria. They increased the amount of hyaluronic acid and hyaluronidase, decreased the amount of M protein, and enhanced phagocytosis and the release of lipoteichoic acid. The results indicate that sub-MICs of chloramphenicol, erythromycin and penicillin may affect the pathogenicity and toxinogenicity of group A streptococci.

Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5’ end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.

Blast transformation of human peripheral blood lymphocytes by PHA is shown to be modulated by lipoteichoic acid (LTA) of Streptococcus mutans, by a cell-sensitizing factor of Actinomyces viscosus, as well as by a frozen and thawed extract of human leukocytes (LE). While small amounts of LE (5-50 micrograms/10(6) cells) significantly enhanced PHA-induced transformation, higher amounts showed a lesser effect on the blastogenic response. Both LTA and the A. viscosus extract did not cause any lymphocyte blastogenic effect when used alone. On the other hand LTA had an inhibitory effect and the A. viscosus extract had an enhancing effect when lymphocytes were pretreated by these agents and then exposed to PHA.

Isolated human peripheral blood neutrophils were exposed to sonic extracts of Actinobacillus actinomycetemcomitans. Such bacterial preparations contain a potent leukotoxin which rapidly kills the leukocytes as reflected by cellular uptake of trypan blue, extracellular release of lactate dehydrogenase, or discharge of 51Cr from pre-labeled cells. Exogenous phospholipids with a glycerol skeleton esterified by fatty acids or positively charged liposomes inhibited cytotoxic phenomena. The data suggest that cell damage may involve the interaction of leukotoxin with phospholipids in the neutrophil cell membrane and that exogenous lipids either compete for or sterically block binding of the leukotoxin to these moieties in the membrane.