Production of H2O2 by bovine blood and milk polymorphonuclear leucocytes

The ability of bovine polymorphonuclear leucocytes (PMN) to release H2O2 was investigated. Resting PMN suspended in buffer released only small amounts of H2O2 which was appreciably increased during phagocytosis of heat killed coliforms. However, in the presence of bovine serum (BS), foetal calf serum (FCS) and milk whey (MW) no increase of H2O2 could be detected unless sodium azide (NaN2) was added. It appears that the enzyme content of these fluids (catalase and lactoperoxidase) consumed the released H2O2 and NaN2, which inactivates these enzymes, abolished this interference. Live organisms required BS or MW both for phagocytosis and for H2O2 production. Bovine IgG2 and, to a lesser extent, IgG1 but not SIgA or IgM stimulated the release of H2O2 independently of phagocytosis; this indicates the presence of receptors specific for IgG2 and IgG1 on the cell surface. Ingestion of casein micelles triggered the greatest burst of H2O2 production by cells suspended in buffer. In general, PMN isolated from blood were more active than cells isolated from milk. Since the extracellular release of H2O2 reflects the intracellular level of H2O2, the lower metabolic activity of milk PMN may contribute to the lesser intracellular bactericidal activity of milk leucocytes. The possibility that the release of H2O2 may activate extracellularly the lactoperoxidase system, known to be bactericidal in milk, is discussed.     

Interrelationship between oxygen consumption,superoxide anion and hydrogen peroxide formation in phagocytosing guinea pig polymorphonuclear leucocytes

The paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2- release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows: (a) The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2- and H2O2. (b) O2- is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2-, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2- detected and the oxygen given back is 1. (c) The amount of O2- released from the cells accounts for only a small part of oxygen actually reduced. (d) H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2- released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources. (e) In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.     

T. cruzi: sensitization to macrophage killing by eosinophil peroxidase

In this study, we report that trypomastigotes of T. cruzi coated with eosinophil peroxidase (EPO) become sensitized to killing by normal macrophages that are unable to kill uncoated organisms. EPO bound to the surface of the organisms without affecting their extracellular viability. The intracellular killing of EPO-coated trypomastigotes could be inhibited by catalase and azide, suggesting that toxicity was mediated through the small amounts of hydrogen peroxide generated by the phagocytic event in normal macrophages and the peroxidatic activity of EPO. EPO-coated organisms could be killed in a cellfree system by the addition of H2O2 and either iodide, bromide, or chloride. Omission of H2O2 decreased but did not prevent the killing of trypanosomes by the cellfree system and this residual toxicity was abolished by catalase. This suggests that H2O2 generated by trypanosomes contributes to the death of EPO-coated organisms. EPO-coated organisms could also be killed extracellularly when exposed to normal macrophages at high parasite to cell ratios or when a high phagocytic load of another particle was given simultaneously. This effect could be inhibited by both azide and catalase, but not by superoxide dismutase. This suggests that enough H202 is released by phagocytosis of a high number of organisms to generate toxic concentrations of H2O2 outside the confines of the vacuolar system.     

Effect of local interaction of reactive oxygen species with prostaglandin F(2alpha) on the release of progesterone in ovine corpora lutea in vivo

Prostaglandin (PG) F(2alpha) is implicated in the process of luteal regression in many species, and has been shown to increase the generation of reactive oxygen species. In this study, the role of reactive oxygen species in the local regulatory mechanisms of functional luteolysis in the ewe was examined. In Experiment 1, we studied local effects of hydrogen peroxide (H(2)O(2)) and its interaction with PGF(2alpha) on P secretion in ovine corpus luteum (CL) in vivo. For this purpose, a microdialysis system (MDS) was used, where only the cells surrounding the capillary membrane in the microenvironment of the CL are exposed to these factors, and the P secretory ability of the CL is maintained as if intact. The study used a multiple CL model to implant the MDS, enabling us to examine in parallel several experimental infusions into the MDS implanted in different CLs (one MDS line per CL) developed after superovulation in one ewe. On Day 8 after GnRH treatment, the MDS were implanted into multiple CL in both ovaries of six ewes. A 4-h infusion with PGF(2alpha) (10(-6)M) at 8-12 h slightly increased P release during infusion, while a 4-h infusion with H(2)O(2) (10(-3)M) at 20-24 h decreased P release at 27-38 h. A pre-infusion with PGF(2alpha) for 4h at 8-12h, followed by infusion of H(2)O(2) at 20-24 h rapidly decreased the P release at 20-40 h (P<0.05); this decrease occurred 7h earlier than in the CL treated with H(2)O(2) alone. In Experiment 2, by utilizing the MDS we also applied free radical scavengers to examine their possible weakening effect on the inhibition of P secretion in the microenvironment within the regressing CL induced by PGF(2alpha) treatment. On Day 8 after estrus, the MDS were implanted into the CL (single CL model, two MDS lines per CL). Infusion of free radical scavengers, superoxide dismutase (SOD;50mg/ml)+catalase (CAT; 10mg/ml), at 0-28 h first increased P release until 12 h (P<0.05), and consequently delayed the decrease in P release until 30 h after administration of PGF(2alpha) i.m. (P<0.05). The present results support the concept that the leading pathway from PGF(2alpha) induces an increase of reactive oxygen species in luteolysis in the ewe.     

Mast cell-mediated toxicity to schistosomula of Schistosoma mansoni: potentiation by exogenous peroxidase

Mast cells, when incubated in vitro with hydrogen peroxide (H2O2) and iodide, are cytotoxic to schistosomula of Schistosoma mansoni, as determined morphologically by dye exclusion, motility, and refractility and by transmission and scanning electron microscopy. When intact mast cells were incubated with schistosomula, mast cell degranulation with extracellular release of mast cell granules (MCG) was only observed in the presence of added H2O2 (10(-4) M). The secreted MCG, which contain small amounts of endogenous peroxidase activity, adhered to the surface of schistosomula. By 15 to 30 min, the mast cell-H2O2 system in the presence of iodide (10(-4) M) produced marked disruption of the tegumental and internal structures of the schistosomula. No helminthic damage was noted if any component of the incubation mixture (mast cells, H2O2 or iodide) was omitted. MCG could substitute for intact mast cells in the H2O2 and iodide-dependent cytotoxic system; MCG-mediated killing of schistosomula was inhibited by the hemeprotein inhibitor azide, suggesting that the cytotoxic reaction required endogenous peroxidase. The cytotoxicity was increased by eosinophil peroxidase bound to the MCG surface. These findings suggest a mechanism by which mast cells may contribute to the host cytotoxic response to helminths. H2O2 formed by nearby inflammatory cells may induce mast cell secretion, and the released MCG, through their endogenous peroxidase content (or bound eosinophil or neutrophil peroxidase), may react with H2O2 and a halide to form a system toxic to the adjacent helminth.     

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Metabolic activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine to nucleic acids and proteins.

Human polymorphonuclear leukocytes (PMNs) have been stimulated with either phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 or a combination of both to induce the respiratory burst and myeloperoxidase (MPO) release. Chlorpromazine (CPZ) but not chlorpromazine sulfoxide (CPZSO) inhibited the respiratory burst as measured with lucigenin chemiluminescence. The inhibition was due to interference with processes in the cell leading to the respiratory burst and not to scavenging of produced oxygen radicals that provoke the luminescence. CPZ was metabolized by stimulated PMNs. HPLC analysis revealed formation of CPZSO and an unidentified product. Both products result from decay of chlorpromazine radical cation (CPZ+.), indicating formation of this radical intermediate in CPZSO oxidation by stimulated PMNs. CPZ conversion correlated with H2O2 production and MPO release. The largest CPZ conversion was observed with phorbol ester plus A23187 stimulation. The conversion was reduced by catalase and sodium azide, an inhibitor of MPO, with 70% and 40%, respectively. This indicates only partial involvement of extracellularly released MPO in CPZ metabolism by PMNs. Considerable covalent binding of [3H]CPZ to nucleic acids and proteins of intact stimulated PMNs was observed. This binding was larger upon co-stimulation with phorbol ester and A23187. Azide did not reduce covalent binding. This indicates that covalent binding is not mediated by extracellularly released MPO and that CPZ is probably activated intracellularly. Activation of PMNs and production of H2O2 is a prerequisite for both CPZ conversion and covalent binding. This study demonstrates that phagocytic cells might contribute to drug metabolism and drug-induced toxicity.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Mechanism of oxygen availability from hydrogen peroxide to aerobic cultures of Xanthomonas campestris

Fundamental studies on the availability of oxygen from the decomposition of H(2)O(2), in vivo, by Xanthomonas campestris, when H(2)O(2) is used as an oxygen source are presented. It was found that the H(2)O(2) added extracellularly (0.1-6 mM) was decomposed intracellularly. Further, when H(2)O(2) was added, the flux of H(2)O(2) into the cell, is regulated by the cell. The steady-state H(2)O(2) flux into the cell was estimated to be 9.7 x 10(-8) mol m(-2) s(-1). In addition, it was proved that the regulation of H(2)O(2) flux was coupled to the protonmotive force (PMF) using experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which disrupts PMF. The coupling constant between the rate of free energy availability from PMF and the rate of reduction of H(2)O(2) flux, was found to be 46.4 mol m(-2) s(-1) J(-1) from simulations using a developed model. Also, the estimated periplasmic catalase concentration was 1.4 x 10(-9) M.     

Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium–plant interaction

Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds. We found that introduction of a wild-type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA-gfp expression over 60-fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells. Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A. tumefaciens cells.     

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity

Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2). The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress. The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity. H2O2 resistance correlates well with catalase activity. An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance. These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity.     

Lucigenin chemiluminescence as a probe for measuring reactive oxygen species production in Escherichia coli

Addition of oxygen to whole cells of Escherichia coli suspended in the presence of the chemiluminescent probe bis-N-methylacridinium nitrate (lucigenin) resulted in a light emission increase of 200% of control. Addition of air to cells showed a chemiluminescent response far less than the response to oxygen. The redox cycling agents paraquat and menadione, which are known to increase intracellular production of O2- and H2O2, were also found to cause a measurable increase in lucigenin chemiluminescence in E. coli cells when added at concentrations of 1 and 0.1 mM, respectively. The oxygen-induced chemiluminescent response was not suppressed by extracellularly added superoxide dismutase or catalase. Further, the lucigenin-dependent chemiluminescent response of aerobically grown E. coli to oxygen was significantly greater than that of cells grown anaerobically. Heat-killed cells showed no increase in chemiluminescence on the addition of either oxygen, paraquat, or menadione. These results show that lucigenin may be used as a chemiluminescent probe to demonstrate continuous intracellular production of reactive oxygen metabolites in E. coli.     

Leukotriene production and inactivation by normal, chronic granulomatous disease and myeloperoxidase-deficient neutrophils

Appropriately stimulated neutrophils release peroxidase and undergo a respiratory burst to form hydrogen peroxide (H2O2) and hydroxyl radicals (OH). We report here that both the myeloperoxidase-H2O2-halide system and OH released in this way can degrade the leukotrienes (LT) formed by neutrophils. More LTB4 and LTC4 were recovered from the supernatants of chronic granulomatous disease neutrophils (which are unable to respond to stimulation with a respiratory burst) than from normal or myeloperoxidase-deficient neutrophils when stimulated with the calcium ionophore A23187. When radiolabeled LTC4 was added, 72% of the LTC4 was recovered from the chronic granulomatous disease cells in contrast to 0% from the myeloperoxidase-deficient and normal cells. Inhibitor studies using catalase, superoxide dismutase, azide, mannitol, or ethanol suggested that LTC4 degradation was mediated primarily by the myeloperoxidase system in normal cells and by OH in myeloperoxidase-deficient cells. LTC4 degradation by the cell-free myeloperoxidase-H2O2-halide system and the OH -generating acetaldehyde-xanthine oxidase-Fe2+ system had inhibitor profiles comparable to normal and myeloperoxidase-deficient neutrophils, respectively. LTC4 degradation products formed by the stimulated neutrophils and model systems included the 5-(S), 12-(R)- and 5-(S), 12-(S)-6-trans-isomers of LTB4. Thus phagocytes may modulate LT activity in inflammatory sites by the inactivation of these potent biologic mediators by at least two oxidative mechanisms.     

Redox-sensing release of human thioredoxin from T lymphocytes with negative feedback loops

Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H2O2 and extracellular TRX regulates its own H2O2-induced release. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. The level of TRX release is augmented upon the addition of H2O2, but suppressed upon the addition of N-acetylcysteine. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H2O2. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S; CS) fails to release the protein. H2O2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. Preincubation of the transfectant with rTRX-WT for 1 h at 37 degrees C, but not 0 degrees C, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H2O2, respectively. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions.     

Hypochlorous acid potentiates hydrogen peroxide-mediated DNA-strand breaks in human mononuclear leucocytes.

In this study the formation of DNA single-strand breaks in MNL in close proximity to activated phagocytes, or in contact with added H2O2 and/or HOCl, were evaluated. Neutrophils activated by phorbol myristate acetate (PMA), induced DNA-strand breaks in neighboring lymphocytes which increased after 1-2 h incubation in a repair medium. These DNA-strand breaks could be prevented by the addition of catalase or substitution of the neutrophils with cells from a patient with chronic granulomatous disease. Inclusion of the myeloperoxidase (MPO) inhibitor, sodium azide (NaN3), to the system was associated with less damage after 1-2 h incubation and a faster repair rate. Exposure of MNL to added reagent H2O2 (12-100 microM) was also accompanied by DNA damage. Addition of reagent HOCl (3-25 microM) did not induce any DNA-strand breaks. However, when combined with H2O2 (12.5 microM), HOCl increased H2O2-mediated DNA damage and compromised the repair process. Interactions between the phagocyte-derived reactive oxidants H2O2 and HOCl are probably involved in the etiology of inflammation-related cancer.     

Mechanisms of systemic vasodilation by lysozyme-c in septic shock

In septic shock (SS), cardiovascular collapse is caused by the release of inflammatory mediators. We previously found that lysozyme-c (Lzm-S), released from leukocytes, contributed to systemic vasodilation in a canine model of SS. We then delineated the pathway by which this occurs in a canine carotid artery organ bath preparation (CAP). We showed that Lzm-S could intrinsically generate hydrogen peroxide (H(2)O(2)) and that H(2)O(2) subsequently reacted with endogenous catalase to form compound I, an oxidized form of catalase. In turn, compound I led to an increase in cyclic guanosine 3',5'-monophosphate to produce vasodilation. However, it was not clear from previous studies whether it is necessary for Lzm-S to bind to the vasculature to cause vasodilation or, alternatively, whether the generation of H(2)O(2) by Lzm-S in the surrounding medium is all that is required. We examined this question in the present study in which we used multiple preparations. In a partitioned CAP, we found that when we added Lzm-S to a partitioned space in which a semipermeable membrane prevented diffusion of Lzm-S to the carotid artery tissue, vasodilation still occurred because of diffusion of H(2)O(2). On the other hand, we found that Lzm-S could accumulate within the vascular smooth muscle layer (VSML) after 7 h of SS in a canine model. We also determined that when Lzm-S was located in close proximity to vascular smooth muscle cells, it could generate H(2)O(2) to produce lengthening in a human cell culture preparation. We conclude that there are two mechanisms by which Lzm-S can cause vasodilation in SS. In one instance, H(2)O(2) generated by Lzm-S in plasma diffuses to the VSML to cause vasodilation. In a second mechanism, Lzm-S directly binds to the VSML, where it generates H(2)O(2) to produce vasodilation.