GENERAL INTRODUCTION. The invasion of the tissues of a host by pathogenic microorganisms is usually followed by a series of sequential humoral and cellular events which include: the generation of chemotactic agents, the directional migration of leukocytes toward the invader, the opsonization of the agents by immunoglobulins and complement components, the intemalization of the agents within phagolysosomes. and eventually by the killing and biodegradation of the ingested agents. The perturbation of the leukocytes membranes by opsonized micro- organisms as well as by a variety of cytolytic agents generated by bacteria is also accompanied by a series of biochemical events which include an “oxygen burst" which culminates in the generation of oxygen radicals, some of which are directly involved in the killing of the ingested agents. Concomitantly with the activation of the oxygen metabolism, granulocytes (PMN)f and macrophages (MQ)f also generate chemiluminescence (CL) which is believed to be a natural consequence of the redox met mbrane perturbation due to phagocytosis. lt may involve the generation of a species of singlet oxygen and hydroxyl radicals or electronically excited carbonyl groups which relax with light emission. A relationship between CL and superoxide production has also been demonstrated by the reduction of CL which occurs following the addition of superoxide dismutase (SOD) to phagocytizing leukocytes. The CL signals which can be further amplified by luminol are also believed to be dependent upon myeloperoxidase (MPO)-catalyzed reactions and/or upon metabolism of arachidonic acid pathway(s). The luminol-de-pendent CL ( LDCL) reaction is currently employed to assess the opsonophagocytic properties of sera as we as for the evaluation of the membrane perturbation which is initiated, in leukocytes, by cytotoxic drugs, and by microbial toxins. ln addition to opsonized particles, a series of soluble ligands, i.e., chemotactic peptides, lectins, phorbol esters, calcium ionopohres, polyanethole sulfonate, and cationic polypeptides have all been shown to stimulate the oxygen burst and to generate CL in neutrophils, monocytes, and macrophages. Because of the relative ease and rapidity with which CL is measured in leukocytes, this method has also become a powerful tool to investigate host-and-parasite interrelationships and to assess defects in leukocyte functions (e.g. , chronic granulomatous disease of childhood (CGD) MPO deficiency, defects in complement components and in immunoglobulins, etc.). Recent studies from our laboratory have described a unique phenomenon which showed that a variety of microbial species can be very effectively “opsonized” by cationic proteins rich in arginine (e.g., histones. poly-L-arginine - PARG). Such opsonized microorganisms are readily internalized not only by "professional" phagocytes (PMNs and macrophages) but also by epithelial cells and by fibroblasts. We have postulated that perturbation of the mammalian cells by the highly-charged polyelectrolytes, coated upon particles, delivers a signal (through electrostatic interactions) to the cytoskeleton resulting in the invagination of the membrane and the formation of a phagocytic vacuole. This phenomenon mimics the cellular events that take place following the stimulation of the leukocyte membrane by antibody and complement-coated particles which function through the F and Cb receptors. Thus, the polycationic ligands may represent "archaic" antibodies capable of stimulating certain membrane sites probably nonspecifically (see Section A.l). Our studies further postulated that if leukocytes “recognize" cationic charges upon particles and respond to them by phagocytosis, such coated particles should also be able to trigger an “oxygen burst" in a fashion similar to that induced either by antibody-coated particles or by other membrane- active agents. Indeed, we have demonstrated tnat very intense LDCL and superoxide production ls triggered in blood leukocytes following stimulation with bacteria and zymosan particles which had been precoated with arginine rich histon PARG and paradoxically also by the anionic polyelectrolytes,.|iquoid. anddextransulfatc. The intensity of the CL signals obtained exceeded by many magnitudes those induced by antibody-coated particles. These findings further suggested that the stimulation of the leukocyte membrane, either simultaneously or sequentially by mixtures of different ligands, each recognizing a different membrane site, may perhaps culminate in a synergistic metabolic response. Such "multiple hits" may therefore generate large quantities of oxygen radicals and CL, presumably due to a more efficient activation of the membrane oxidase and a better assembly of the electron transport system leading to the generation of superoxide. We have chosen to examine agents like the chemotactic peptide F-Met-Leu-Phe (FMLP), a variety of lectins, calcium ionophore, PMA, liquoid, and poly ot-cationic peptides as probes for the stimulation of LDCL and superoxide production by human PMN. We have shown that whereas each ligand alone induced only a very moderate LDCL response in leukocytes, very intense CL signals were generated if the various ligands were employed as “cocktails, suggesting multiple mechanisms of activation of the oxygen metabolism in leukocytes (see also Reference 27). Since inflammatory exudates which accumulate following microbial proliferation in tissues are known to be rich in both cationic and anionic polyelectrolytes, we also postulated that some of these agents may coat either the surface of the leukocytes or the surface of the microorganisms, or both. and thus modulate mutual recognitions leading either to enhanced or depressed membrane perturbation. These changes may be monitored by CL and by the production of superoxide. The present report further expands our observations on the multiple roles played by polycationic and polyanionic agents and of "cocktails" of soluble ligands in the stimulation of the generation of LDCL and superoxide by human blood luekocytes and by mosue peritoneal macrophages, with an emphasis on the luekocyte-bacteria interactions in inflammation.

The mechanisms of biodegradation of microbial cell wall components is discussed. Employing Staphylococcus aureus as a model it is proposed that bacteriolysis following phagocytosis is mediated by the activation by leukocyte cationic proteins of the bacterial own autolytic wall enzymes. The role of lysozyme in bacteriolysis might not be due to its muramidase activity but to its cationic nature. A variety of sulfated polysaccharides, proteinases and oxygen radicals which might be present in inflamed tissues might inactivate the bacterial autolytic wall enzymes leading to the persistence of highly-phlogistic peptidoglycan-polysaccharide complexes within macrophages and to tissue damage.

Heparinoids and related negatively-charged substances caused suppression of the penicillin-induced bacteriolysis of staphylococci and a higher viability rate. Furthermore, the penicillin-induced release of cell wall material was reduced by these substances. The main reason for this suppression of bacteriolysis was an inhibition of the activity of cell wall autolytic enzymes while the penicillin-specific perturbations of wall morphogenesis were not affected.

Murine tumor cells were induced to phagocytize either Candida albicans or group A streptococcal cells. The presence of microbial particles within the tumor cell cytoplasm had no effect on in vitro tumor cell growth. However, when Candida albicans-infected tumor cells were injected into syngeneic mice, they formed tumors that grew faster, invaded the surrounding normal tissue more rapidly and metastasized more rapidly than control tumor cells. Tumor cells infected with group A streptococcal particles did not grow faster or show increased malignant behavior. These data indicate that the in vivo behavior of malignant tumor cells can be modulated by microbial particles, which are often present in the microenvironment of the growing tumor.

Poly-L-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of superoxide (O2-) and hydrogen peroxide (H2O2) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O2- took place with 4-5 X 10(-6) M of PHSTD, starting after a lag of about 25 sec and proceeding for 15-17 min at a rate of 150 nmol/10(7) PMNs/min, suggesting that this polycation is one of the most potent stimulators of O2- generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O2- by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O2- and H2O2 by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O2- and H2O2 following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O2- which were inhibited by CYB. Generation of O2- and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-L-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H2O2 by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)

Considerable attention has been focused on the role of electrostatic charge in the pathogenesis of immune complex-mediated tissue injury. The authors have examined the ability of cationic (histone, polyhistidine, polyarginine) and anionic (polyanetholsulfonate) polyelectrolytes to modulate acute immune complex-mediated tissue injury. Tissue injury elicited in rats by the reversed dermal Arthus reaction was increased 26-43% by addition of polyelectrolytes to antibody prior to its intradermal injection. Kinetic studies using 111In-labeled neutrophils indicated that the enhanced tissue injury was not the result of increased influx of neutrophils. Infusion of polyethylene glycol-conjugated superoxide dismutase prior to induction of the Arthus reaction resulted in 40-68% suppression of tissue injury. Concomitant in vitro functional studies (enzyme secretion, O-2 and H2O2 generation, and chemiluminescence) of rat neutrophils demonstrated that addition of polyelectrolytes to preformed immune complexes (IgG-bovine serum albumin) resulted in marked increases in O-2, H2O2, and chemiluminescence, but no increases in enzyme secretion, compared with neutrophils stimulated with immune complexes alone. The cationic polyelectrolytes did not alter the capacity of preformed immune complexes to activate complement in vitro. These studies suggest that both cationic and anionic polyelectrolytes can increase the pathogenic potential of immune complexes and that this modulation is, at least in part, mediated by enhanced generation of toxic oxygen-derived metabolites by neutrophils.

Voluminous literature exists today on the involvement of cationic polyelectrolytes (CPs) in host and parasite interrelationships. It has been shown that CPs of neutrophil (1-14), eosinophil (15, 16), macrophage (17), and platelet (18) origins function as distinct microbicidal agents. These probably constitute a "secondary" defense line supplementary to the main oxygen-dependent microbicidal systems of "professional" phagocytes. CPs have, however, also been implicated as modulators of blood clotting (19) and fibrinolysis (20), as a permeability-enhancing factors (21-23), in mast cell degranulation and in histamine release (24), as pyrogenic agents (25), as enhancers of complementmediated lysis (26), as modulators of PMN adherence (27-29) and chemotaxis (30-34), and as modulators of endocytosis (35-45) to list only several of the properties ascribed to these agents. Since the effects of CPs probably involve the interaction, through electrostatic forces, with negatively charged sites on target cells (36), it is plausible that the complex polyelectrolytic milieu found in infectious and inflammatory sites might function to modulate and regulate several important interactions of the host with invaders (46-48). Although CPs are primarily recognized for their distinct killing properties (10-13), recent studies have suggested that CPs might also be involved in a variety of additional biological, biochemical, and immunopathological phenomena which are seldom discussed in the general context of host and parasite interrelationships. The present review deals primarily with the possible involvement of CPs (1) in endocytosis and in cell adherence, (2) as activators of the respiratory burst in "professional" phagocytes, (3) as activators of the autolytic wall enzymes in certain microbial species and its relation to bacteriolysis and to the pathogenesis of chronic inflammation induced by bacterial cell walls, and (4) as agents capable of modulating the pathogenicity of immune complexes. It was felt that a discussion of these "other" properties of CPs is timely as it may shed a new light on the role of surface charge in cell-to-cell interactions as seen in inflammatory and infectious sites.