Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages

Maximal rates of superoxide (O) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14–18 nmol O/min/10⁷ cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H₂O₂ by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H₂O₂ staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H₂O₂ staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.     

Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO,H2O2)

Alveolar macrophages secrete numerous mediators, playing an important role in host defence. Among these mediators, nitric oxide (NO) and hydrogen peroxide (H₂O₂) are both involved in bactericidal killing and trigger the release of other cellular metabolites. We have analyzed the effect of an atmosphere polluted with ozone (0.03–0.5 ppm v/v) on the monocytic cell line THP-1, as a model for alveolar macrophages,in vitro. NO and H₂O₂ were chosen to evaluate cell response to ozone. Cell injury was evaluated using lactate dehydrogenase (LDH) liberation into the medium. An exposure to 0.5 ppm ozone proved to be more toxic to the cells, than 0.1 or 0.03 ppm, evidenced by more LDH being liberated and cytotoxicity reaching values up to 64%. For all ozone concentrations, H₂O₂ production reached a peak value after 10–15 min of exposure, after which the concentration of extracellular H₂O₂ production diminished rapidly. The highest NO concentrations were measured with 0.5 ppm ozone, reaching a maximum value of 1460 nmol/L per 5×10⁶ cells, which is 1.55 times higher than for nonexposed cells. Lower concentrations barely induced higher NO concentrations compared to nonexposed cells. The results indicate that ozone effects not only the viability of human monocytes but also the release of antibacterial and defense signaling molecules and suggest that ozone-mediated cytotoxicity may be related to the secretion of NO and H₂O₂. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Abstract

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H₂O₂). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H₂O₂. Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H₂O₂ dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H₂O₂ in the presence of trilostane. H₂O₂ attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H₂O₂ inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Leukocyte-deactivating factor from macrophages: Partial purification and biochemical characterization. A novel cytokine

A deactivating factor (MDF) is released from granuloma-like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion (O₂^?) release when stimulated. This failure to manifest O₂ release is observed whether PMA, all-transretinal, or fMet-Leu-Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 11 kD. When the factor was obtained by extraction of the same cells after washing and sonication. Only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cells that utilize release of reactive oxygen species for cytocidal activity. © 1993 Wiley-Liss, Inc.     

Lipopolysaccharides stimulate Na-dependent transport in alveolar cells and protect against oxidant injury

We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium-coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H₂O₂ (2.5 mM; 20 min). LPS (10 μg/ml, 24 h) induced a significant increase in the Na-dependent component of alanine and phosphate uptake while they decreased Na, K-ATPase activity measured by ouabain-sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS-treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H₂O₂-induced decrease of Na-dependent alanine and phosphate uptake. This protective effect of LPS against H₂O₂ injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H₂O₂ clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium-coupled transport by a process involving protein synthesis and partially prevent H₂O₂-induced decrease of Na-coupled transport without discernible change in antioxidant activities. © 1995 Wiley-Liss, Inc.     

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, -glucosidase and acid phosphatase, secreted by the cells during growth, and also -amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H₂O₂ from the cells (oxidative burst). H₂O₂ released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H₂O₂ is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H₂O₂ is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H₂O₂. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H₂O₂ and, when present together with elicitor, induction was synergistically increased.     

Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid

The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC‐treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC‐induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H⁺ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H⁺‐ATPase; (2) treatment with 10 µM erythrosin B (EB) early and totally inhibits net H⁺ and K⁺ fluxes across the PM, indicative of the H⁺ pump activity; nevertheless, also in these conditions a huge FC‐induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC‐induced PM H⁺‐ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂Cl₂‐soluble fraction extracted from the incubation medium by sequential acid–base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC‐induced catalase inhibition belongs to the organic acid class.     

H2O2- or l-DOPA-injured dopaminergic neurons trigger the release of soluble mediators that up-regulate striatal GDNF through different signalling pathways

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective molecule for dopaminergic neurons of the nigrostriatal pathway that degenerate in Parkinson's disease. We have previously shown that H₂O₂- or l-3,4-dihydroxyphenylalanine (l-DOPA)-challenged dopaminergic neurons trigger the release of soluble factors that signal ventral midbrain astrocytes to increase GDNF expression. In the present work, we evaluated whether the factors released by ventral midbrain-challenged cells were able to alter GDNF expression in striatal cells, the targets of dopaminergic neurons projecting from the substantia nigra, and investigated the signalling pathways involved. Our data showed that soluble mediators released upon H₂O₂- or l-DOPA-induced dopaminergic injury up-regulated GDNF in striatal cells, with different temporal patterns depending on the oxidative agent used. Conditioned media from H₂O₂- or l-DOPA-challenged midbrain astrocyte cultures failed to up-regulate GDNF in striatal cultures. Likewise, there was no direct effect of H₂O₂ or l-DOPA on striatal GDNF levels suggesting that GDNF up-regulation was mediated by soluble factors released in the presence of failing dopaminergic neurons. Both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways were involved in striatal GDNF up-regulation triggered by H₂O₂-induced dopaminergic injury, while diffusible factors released in the presence of l-DOPA-challenged dopaminergic neurons induced GDNF expression in striatal cells through the activation of the MAPK pathway. These soluble mediators may constitute, in the future, important targets for the control of endogenous GDNF expression enabling the development of new and, hopefully, more efficient neuroprotective/neurorestorative strategies for the treatment of Parkinson's disease.     

Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells

Endothelial cells are critical targets in both hypoxia-and reoxygenation-mediated lung injury. Reactive O₂ species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H₂O₂ generation and extracellular H₂O₂ and O/₂ release were measured after exposure to normoxia (room air-5% CO₂), hypoxia (0% O₂ -95% N-5% CO₂), or hypoxia followed by normoxia or hyperoxia (95% O₂-5% CO₂). Exposure to hypoxia results in significant reductions in intracellular H₂ O₂ formation and extracellular release of H₂ O₂ and O₂ by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley-Liss, Inc.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

Abstract: A cDNA encoding a guinea pig histamine H₁ receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H₁), the H₁ receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a G_i/G_o protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca²⁺ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca²⁺ level of which only the second sustained phase was dependent on the extracellular Ca²⁺ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca²⁺. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca²⁺- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A₂, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H₁ receptor, the histamine-induced arachidonic acid release was also Ca²⁺- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H₁) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca²⁺, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H₁ receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca²⁺ and protein kinase C activation.     

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H₂O₂ plays an essential role in the activation of these channels and that H₂O₂ per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl⁻ current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H₂O₂ VSOR Cl⁻ current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H₂O₂, exogenous addition of ATP in the presence of extracellular Ca²⁺ resulted in a decrease in the half-time for VSOR Cl⁻ current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl⁻ current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl⁻ current onset in a extracellular Ca²⁺-dependent manner.     

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H₂O₂) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H₂O₂ was 34.8±2.3% at day 9, and was 70% larger than that without H₂O₂ (21.5±0.8%). Further, H₂O₂ addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H₂O₂, the BrdU assay clearly indicated that H₂O₂ suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H₂O₂ is due to synergistic inhibition of cytokinesis with PMA. Although H₂O₂ did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H₂O₂) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H₂O₂ evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H₂O₂ resulted in a increase in [Ca²⁺] _i , indicating the release of calcium from intracellular stores. H₂O₂ abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H₂O₂-induced calcium release. We conclude that H₂O₂ induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Role of LytF and AtlS in eDNA Release by Streptococcus gordonii

Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general, the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii, eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H₂O₂). Addition of H₂O₂ to cells grown under conditions non-permissive for H₂O₂ production causes eDNA release. Furthermore, eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H₂O₂ production. Obvious cell lysis, however, does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report, we investigated the role of both proteins in the H₂O₂ dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions, but the AtlS mutant was still inducible for eDNA release by external H₂O₂. Moreover, we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H₂O₂. Our data support a role of LytF in the H₂O₂ eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress.