Microbicidal mechanisms of human granulocytes: synergistic effects of granulocyte elastase and myeloperoxidase or chymotrypsin-like cationic protein.

The antibacterial activity of a myeloperoxidase (MPO)-glucose oxidase system was found to be greatly increased by granulocyte elastase, present in azurophil granules of human neutrophils. The MPO-H2O3-mediated killing of both Escherichia coli and Staphylococcus aureus was potentiated by granuocyte elastase at an acid pH, whereas at pH 7.4 only killing of E. coli was potentiated. The potentiating effect of elastase was not dependent on the enzymatic properties of the protein since it was not abolished by heating, which destroys the enzymatic activity. A peptide chloromethyl ketone elastase inhibitor abolished both elastolytic activity and the pctentiating effects on MPO-H2-O2-mediated bacterial killing. The antibacterial activity of chymotrypsin-like cationic protein of human neutrophils was also potentiated by elastase. Other degradative enzymes isolated from human granulocytes, e.g., collagenase and lysozyme, did not potentiate MPO-H2O2-mediated or cationic protein-dependent bacterial killing. The present study indicates that a neutrophil constitutent, elastase, which is not microbicidal by itself, can initiate sublethal changes that render some microorganisms more susceptible to the action of microbicidal agents like MPO and chymotrypsin-like cationic protein.     

Role of myeloperoxidase in the killing of Naegleria fowleri by lymphokine-altered human neutrophils.

Previously we have shown that human neutrophils treated with conditioned medium from phytohemagglutinin-stimulated mononuclear leukocytes (sCM) in the presence of antisera have amoebicidal properties for Naegleria fowleri, a pathogenic free-living amoeba. The data now presented show that neutrophils which lack myeloperoxidase (MPO) but have a normal oxygen-dependent respiratory burst could not be altered by sCM to express the amoebicidal activity. Catalase inhibited this amoebicidal activity of sCM-treated neutrophils. Various components and products of the neutrophils were examined for effects on naegleriae. A granule extract was found to have no effect at concentrations up to 100-fold that which killed Salmonella minnesota R595. Hydrogen peroxide appeared to have little effect even at 100 microM. However, in the presence of MPO, H2O2 was amoebicidal at 2.5 microM. The generation of amoebicidal activity required the presence of chloride ions. Azide inhibited the effects of the MPO-H2O2-Cl- system. Arginine, a scavenger of hypochlorite, significantly depressed the ability of sCM-treated neutrophils to kill amoebae and also prevented the amoebicidal properties of the MPO-H2O2-halide system. These results suggest that the MPO-H2O2-halide system is important in the killing of naegleriae by sCM-treated neutrophils and that hypochlorite may be the amoebicidal agent.     

Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans.     

Oxidative Peptide Cleavage and Decarboxylation by the MPO-H2O2-Cl− Antimicrobial System

The antimicrobial activities of the myeloperoxidase-H(2)O(2)-halide system have received considerable attention recently. The precise mechanism by which this system exerts its lethal activity is presently not clear. In an effort to learn more regarding a possible mechanism of action, the susceptibility of protein-bound amino acids to enzymatic attack by myeloperoxidase (MPO) in the presence of chloride ions was investigated. [1, 7-(14)C]diaminopimelic acid (DAP) was incorporated into Escherichia coli W-7 proteins with little randomization of the radioactivity. Under appropriate conditions, it was observed that the MPO-H(2)O(2)-halide system released approximately 94% of the radioactivity from labeled bacteria. This would indicate that, in addition to decarboxylation, peptide bonds are also split during this reaction. The oxidative decarboxylation of DAP-labeled bacteria by MPO (i) is Cl(-) dependent, (ii) has an acid pH optimum, (iii) requires a specific concentration of H(2)O(2) for activity, (iv) reaches a plateau by 25 min, and (v) is markedly inhibited by taurine. These properties are similar to those observed with free amino acids. It appears from these data that MPO can not only decarboxylate free and bound amino acids, yielding aldehydes, but also it can actively participate in oxidative peptide cleavage. Both of those activities may play a critical role in the microbicidal action of the leukocyte.     

A New Assay to Monitor the Degranulation Process in Phagocytizing Human Neutrophils

The microbicidal activity of phagocytes depends on intraphagosomal secretion (i.e., the degranulation process), during which the content of the secretory granules is discharged into the phagosome to kill ingested microorganisms. The availability of a reliable assay to quantify the extent of degranulation can be an important tool to gain a deeper insight into the mechanism of bacterial processing in phagocytes and into the transmembrane signaling that leads to granule–phagosome fusion. In an early study, Baehner et al. (25) showed by using the peroxidase chromogenic substrate DAB that the occurence of intraphagosomal MPO release in human neutrophils. Starting from that finding and from that of Herzog and Fahimi (26), who reported the quantitative evaluation of peroxidase-mediated DAB oxidation, we set up a method for measuring MPO intraphagosomal release in human neutrophils. The method is based on the passive engulfment of DAB together with the phagocytosable particle. Inside the vacuole, this substrate is oxidized by MPO released from the azurophilic granules. The colorimetrical evaluation of the amount of DAB oxidized allows for cheap, rapid quantification of MPO intraphagosomal secretion in whole cells. Using this method, we show that the degranulation process, involving azurophilic granules, can be monitored carefully during phagocytosis. It takes place after the ingestion of zymosan particles opsonized with normal human serum, as well as during IgG-mediated phagocytosis and under conditions where 2 integrins are blocked. However our findings also show that the extent of intraphagosomal secretion depends on either the extent of opsonization or the type of receptor engaged during the phagocytic event.     

Phagosome dynamics during phagocytosis by neutrophils

The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil-pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.     

Oxygen radical-induced natural killer cell dysfunction: role of myeloperoxidase and regulation by serotonin

Natural killer (NK) cells are functionally suppressed and induced to apoptosis by reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs). These inhibitory events are reversed by the biogenic amine serotonin. MPs generate hydrogen peroxide (H(2)O(2)), which is processed further by myeloperoxidase (MPO) to even more toxic compounds. Earlier studies suggest that serotonin scavenges MP-derived oxygen radicals generated by the MPO-H(2)O(2) system. These findings led us to explore the capability of MPO-deficient MPs to induce NK cell dysfunction. We show that MPs recovered from subjects with MPO deficiency trigger inhibition of NK cells. In addition, MPs recovered from healthy subjects conveyed suppression of NK cells in the presence of the MPO inhibitor ceruloplasmin. We conclude that ROS-dependent inhibition of NK cell function is unrestricted by the availability of MPO-derived oxygen radicals and that the protecting properties of serotonin may operate in the absence of functional MPO. Our data suggest a complex mechanism of MP-induced NK cell inhibition, which comprises the generation of interchangeable oxygen radicals.     

Oxygen metabolism of the HL-60 cell line: comparison of the effects of monocytoid and neutrophilic differentiation

HL-60 cells are promyelocytic leukemia cells that respond to culture conditions with differentiation into granulocytelike or macrophagelike phagocytes. O2 metabolism is critical to the microbicidal function of phagocytic cells. O2 metabolism was studied in HL-60 cells differentiated with dimethylsulfoxide (Me2SO) and 1,25(OH)2D3, with the objective of 1) determining the validity of these cells as models for human neutrophils and monocytes, respectively, and 2) determining whether these cells are capable of forming hydroxyl radical. Me2SO-treated cells had morphology consistent with human neutrophils. O2 consumption by these cells in response to phorbol myristate acetate (PMA; 100 ng/ml) or opsonized zymosan (3 mg/ml) was less than that by neutrophils, as was superoxide formation. O2 metabolism was not inhibited by KCN or antimycin A. Myeloperoxidase (MPO) activity decreased during differentiation but remained greater than that of human neutrophils. Cytochalasin B enhanced recovery of superoxide secreted in response to zymosan, implying its release from the phagosome. 1,25(OH)2D3-treated cells had morphology consistent with monocytes. O2 consumption and superoxide release were less than with Me2SO-treated cells. Unlike the case with human monocytes, O2 consumption was not inhibited by KCN or antimycin A. MPO activity was minimally reduced by differentiation. Cytochalasin B inhibited recovery of superoxide. Luminol-dependent luminescence was greater among 1,25(OH)2D3-treated cells than among Me2SO-treated cells. Free radicals were also measured with a spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Spin trapping allows direct, simultaneous detection of superoxide and hydroxyl radicals. Regardless of the mechanism of differentiation, only superoxide was formed by HL-60 cells. These results show that Me2SO-treated HL-60 cells represent an excellent model for the study of human neutrophil oxidative function. However, 1,25(OH)2D3-treated cells are quite different in their O2 metabolism from peripheral blood monocytes.     

Myeloperoxidase-dependent generation of hypochlorite-modified proteins in human placental tissues during normal pregnancy

Myeloperoxidase (MPO), which is released from cytoplasmic granules of activated phagocytes by a degranulation process, reacts with H(2)O(2) (generated during the oxidative burst) and chloride ions to generate hypochlorous acid/hypochlorite (HOCl/OCl(-)). HOCl, a strong oxidant, in turn reacts with proteins to form HOCl-modified proteins. The presence of these cytotoxic chloramines during inflammatory conditions, eg, atherosclerosis and glomerular and tubulointerstitial injury, suggested that chloramines are powerful oxidants that can have profound biologic effects. In the present study, immunoreactive MPO was identified in fetal membranes and the basal plate and in maternal and fetal blood cells of human placental tissues. Monocytes/macrophages represent the major cell source for MPO in human placental tissues. Immunohistochemical findings revealed that HOCl-modified proteins are present in normal human term placenta but not during the first trimester of pregnancy (Weeks 7 to 12). HOCl-modified proteins were localized in areas formed by fetally derived cells as well as maternal decidual tissues, ie, areas where fetal extravillous trophoblast cells invade the maternal tissue and stimulate the maternal immune system. HOCl-modified proteins, products of the MPO-H(2)O(2)-chloride system in vivo, were not present intracellularly, but immunoreactivity for HOCl-modified proteins was cell-associated and/or present in the extracellular matrix. Extravillous trophoblast cells, which may also exert phagocytic activities, showed no intracellular immunoreactivity for MPO or HOCl-modified proteins. The present findings indicate that the generation of HOCl-modified proteins during normal pregnancy is a physiologic rather than a pathophysiologic process.     

Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils.

Neutrophils use a variety of microbicidal mechanisms in their role as one of the primary arms of the human host defense system. We have previously observed that a cell-free system containing myeloperoxidase (MPO), one of the major components of the neutrophil's oxidative antimicrobial systems, inactivated microbial penicillin-binding proteins (PBPs), which mediate the formation of the peptidoglycan layer of eubacterial cell walls. This is a potentially important mechanism of MPO-mediated bacterial toxicity. Since numerous other microbicidal systems, both oxidative and nonoxidative, are used by whole neutrophils, we investigated the effect of intact neutrophils on Escherichia coli PBPs. Penicillin binding activity was progressively reduced by neutrophil exposure for all PBPs. Loss of penicillin binding activity correlated well with loss of microbial viability for almost all PBPs. Azide-treated neutrophils, MPO-deficient neutrophils, and chronic granulomatous disease neutrophils inactivated E. coli PBPs in a manner similar to that of normal neutrophils, suggesting that MPO-independent, and even oxygen-independent, microbicidal systems are also involved in inactivation of PBPs. PBP inactivation, an antimicrobial strategy used by beta-lactam-producing molds (and now by physicians), may be an important microbicidal mechanism used by human neutrophils.     

Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus.

The microbicidal activities of freeze-thaw and high-salt extracts of human and bovine polymorphonuclear leukocyte (PMN) granules were tested against a smooth intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus which differ in virulence and survival within PMNs. Freeze-thaw extracts of human PMN granules were more brucellacidal than high-salt extracts when supplemented with hydrogen peroxide (H2O2) and potassium iodide (KI), whereas the opposite was found with freeze-thaw and high-salt extracts of bovine PMN granules. There was no oxygen-independent killing of either the smooth or rough strain of B. abortus by amounts of granule extracts which caused 100% killing of a deep rough mutant (Re) of Salmonella typhimurium. The oxygen-dependent brucellacidal activity of granule extracts was dependent on concentrations of myeloperoxidase (MPO) units, H2O2, and KI. Maximal brucellacidal activity was observed at pH 5.5 to 6.0. The smooth strain, 45/0, was more resistant to oxygen-dependent killing by granule extracts than was the rough strain, 45/20. Granule extracts were more brucellacidal than purified MPO at equivalent levels of MPO enzyme units, suggesting that at least one other reaction enhances killing by the MPO-H2O2-I- system.     

Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide

A variety of inflammatory stimuli induces NETs. These structures consist of a network of chromatin strands associated with predominately granule proteins, including MPO. NETs exhibit antimicrobial activity, which is proposed to augment the more-established mechanism of phagosomal killing. They may also be detrimental to the host in situations such as chronic inflammation or severe sepsis. The objective of this study was to establish whether MPO associated with NETs is active and able to kill bacteria. Neutrophils were stimulated with PMA to release NETs. Peroxidase activity measurements were performed and showed that enzymatically active MPO was released from the neutrophils, 2-4 h after stimulation, concomitant with NET formation. Approximately 30% of the total cellular MPO was released, with the majority bound to the NETs. The bound enzyme retained its activity. Staphylococcus aureus were not killed when added to preformed NETs under our assay conditions. However, addition of H(2)O(2) to the bacteria in the presence of NETs resulted in MPO-dependent killing, which was observed with NETs in situ and with NETs when they were removed from the neutrophils by limited DNase digestion. Our results show that the enzymatic activity of MPO on NETs could contribute to antimicrobial activity or tissue injury when NETs are released from neutrophils at sites of infection or inflammation.     

Redundant contribution of myeloperoxidase-dependent systems to neutrophil-mediated killing of Escherichia coli.

Neutrophil microbicidal activity is a consequence of overlapping antimicrobial systems that vary in prominence according to the conditions of the neutrophil-microbe interaction, the nature of the microbe, and its metabolic state. In this study, normal, myeloperoxidase-deficient, and respiratory burst-deficient (chronic granulomatous disease [CGD]) neutrophils killed Escherichia coli with equivalent, high efficiencies. Killing by CGD and myeloperoxidase-deficient neutrophils was not augmented by supplements, such as exogenous H2O2 and myeloperoxidase, directed at ameliorating their metabolic defects, suggesting that nonoxidative microbicidal systems were sufficient for a full microbicidal effect. Neutrophils with an intact myeloperoxidase antimicrobial system (normal or appropriately supplemented deficient cells) were capable of rapidly suppressing E. coli DNA synthesis, while unsupplemented CGD or myeloperoxidase-deficient cells were far less effective, indicating that the myeloperoxidase system was active in normal neutrophils. The degree of DNA synthesis inhibition by myeloperoxidase-sufficient neutrophils could account, in a cell-free system, for most of the observed microbicidal activity. While the myeloperoxidase system was active and probably bactericidal, it was not rate limiting for microbicidal activity and appears to have been redundant with other microbicidal systems in the cell. Rapid and extensive inhibition of bacterial DNA synthesis appears to be an indicator of myeloperoxidase activity in neutrophils.     

Oxidative metabolic products released from polymorphonuclear leukocytes in middle ear fluid during experimental pneumococcal otitis media.

To determine whether oxidative metabolic products of phagocytic cells are present in the middle ear during experimental pneumococcal otitis media, we measured the concentration of myeloperoxidase (MPO) in middle ear fluid (MEF) and the capacity of neutrophils isolated from MEF and peripheral blood to produce MPO and superoxide anion (O2-) after in vitro stimulation. Free MPO in MEF was significantly increased 24 and 48 h after either viable or nonviable pneumococci were inoculated into the middle ear. In vitro-stimulated production of MPO and O2- from middle ear neutrophils was significantly less than that from peripheral blood neutrophils 24 h after nonviable pneumococci were inoculated but similar to it after 48 h. Twenty-four hours after viable pneumococci were inoculated, middle ear neutrophils stimulated in vitro produced less MPO but the same amount of O2- as did blood neutrophils. Oxidative metabolic products, therefore, are released from phagocytic cells into the MEF during pneumococcal otitis media, and future studies will need to define the contribution of these products to acute and chronic middle ear tissue injury.     

Active oxygen generating system in immune cells]

Oxygen metabolites, such as O2-, H2O2 and HClO formed during the respiratory burst in phagocytes are known to be essential for killing certain microorganisms. In the initial reaction of the respiratory burst, an electron is transferred from NADPH to molecular oxygen to generate O2-. Other active oxygen species are formed secondarily from O2-. Patients with chronic granulomatous disease, whose phagocytes can not generate O2-, often suffer from recurrent, life-threatening infections. We have demonstrated recently that B lymphocytes but not T lymphocytes (nor NK cells) have an O2- generating system identical or very similar to that in phagocytes. This article deals with the present status of the research on O2- generating system in phagocytes and B lymphocytes.     

Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes.

Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.     

Natural and disease associated anti-myeloperoxidase (MPO) autoantibodies

Myeloperoxidase (MPO) is a cationic protein present in primary azurophilic granules of neutrophils and monocytes. MPO produces a highly deleterious reactive oxygen species, the hypochlorous acid (HOCl), using hydrogen peroxide (H(2)O(2)) and chloride ions as substrate. Anti-MPO antibodies (Abs) are present in 70% of the cases in patients with microscopic polyangiitis (MPA), a small-sized vessel vasculitis. Anti-MPO Abs from patients with MPA can trigger the release of MPO by neutrophils and monocytes. Anti-MPO Abs can activate MPO to generate an oxidative stress deleterious for the endothelium. Thus, we recently demonstrated that MPA sera with anti-MPO Abs activated MPO in vitro, and generated hypochlorous acid, whereas sera from MPA patients with no anti-MPO Abs or healthy individuals did not. Both hypochlorous acid production and endothelial lysis were abrogated by N-acetylcysteine (NAC), an antioxidant molecule. Thus, anti-MPO Abs could play a pathogenic role in vivo by triggering an oxidative burst leading to severe endothelial damages.     

Detection of the production of oxygen-centered free radicals by human neutrophils using spin trapping techniques: a critical perspective

Human neutrophils generate oxygen reduction products as a consequence of membrane interactions with a number of stimuli. One oxygen-centered free radical (superoxide) has been unequivocally shown to result from this "respiratory burst," and some experimental evidence for another (hydroxyl radical) has been published, although debate remains as to its significance. The role of phagocyte-derived free radicals in microbicidal and tumoricidal activity as well as tissue damage at sites of inflammation has been the focus of extensive investigation in recent years. Of the techniques available to study free radical generation in biological systems, spin trapping has emerged as a powerful tool for detection and identification of these reactive species, owing in part to its ability to measure production of free radicals inside the phagosome. However, interpretation of resulting spectra is extremely complex and filled with pitfalls and limitations. In this communication we review spin-trapping techniques and discuss the application of this system to the identification of free radicals resulting from stimulation of human neutrophils. Criteria are established which are necessary for definitive identification of superoxide and hydroxyl radical when employing this technology. In this context a critical perspective of previous studies of neutrophil-derived free radicals is offered.     

The Role of Protegrins and Other Elastase-Activated Polypeptides in the Bactericidal Properties of Porcine Inflammatory Fluids

The mammalian host response to infection includes the production and secretion of antimicrobial peptides from phagocytes and epithelial cells. Protegrins, a group of broadly microbicidal peptides isolated originally from porcine neutrophil lysates, were found to be stored as inactive proforms in porcine neutrophil granules but could be activated extracellularly by neutrophil elastase. We assessed the biological role of protegrins and other elastase-activated polypeptides in the microbicidal activity of neutrophil secretions and inflammatory fluids. When stimulated with phorbol myristate acetate (PMA), neutrophils generated stable microbicidal activity against both Escherichia coli and Listeria monocytogenes under normal-salt conditions and in the presence of 0 to 10% serum. The generation of these antimicrobial substances was dependent on neutrophil elastase, since it was inhibited by 1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone when it was present during activation, but not when this inhibitor was added afterwards. However, elastase-dependent activation of proprotegrins to protegrins in PMA-stimulated neutrophils was not inhibited by the presence of 1 to 2% serum. Porcine neutrophils also released antibacterial activity during phagocytosis of latex beads, and this too was dependent in large part on elastase-activated polypeptides, including protegrins. Moreover, protegrins were found at bactericidal concentrations in cell-free abscess fluid from naturally infected pigs. Taken together, these studies show that protegrins and other elastase-activated polypeptides are important stable antibacterial factors in porcine neutrophil secretions. The potential host defense role of elastase as an activating enzyme for the precursors of microbicidal peptides must be taken into account when therapeutic inhibitors of neutrophil elastase are evaluated for clinical use as anti-inflammatory agents.     

Mycoplasmacidal Activity of Peroxidase-H2O2-Halide Systems

A mycoplasmacidal system consisting of myeloperoxidase (MPO)-containing granules, H(2)O(2), and a halide is described. In all parameters measured, it appears to be identical to the MPO-H(2)O(2)-halide bactericidal system previously reported. It has a pH optimum of approximately 5.5 and an optimal MPO:H(2)O(2) ratio of 1:25. The halide requirement can be satisfied by either chloride or iodide. Through the use of taurine or horseradish peroxidase substitution, chloride-mediated killing can be distinguished from iodide-mediated killing. The relationship of this mycoplasmacidal system to other mycoplasmacidal systems and to host surveillance of mycoplasma is discussed.