Delineation of the catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils

Four catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils have been identified. DCIP reductase, cytochrome c reductase and a chromophore 450-455 reductase are present in phorbol myristate acetate stimulated neutrophils and absent in resting cells and phorbol myristate acetate stimulated chronic granulomatous disease cells. Quinol dehydrogenase activity has also been demonstrated in activated and resting cells. Furthermore, a chromophore absorbing in the reduced state at 450-455 nm participates in superoxide production. This chromophore is reduced by NADPH or duroquinol and is missing in cell lysates derived from a patient with chronic granulomatous disease.     

Human polymorphonuclear leukocyte-mediated cytotoxicity against varicella-zoster virus-infected fibroblasts

Polymorphonuclear leukocytes (PMN) were studied for their ability to mediate cytotoxicity against varicella-zoster virus (VZV)-infected and uninfected human fibroblasts in 51Cr release assays. PMN were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) against VZV-infected targets. Maximal ADCC was obtained with effector-to-target ratios of 100:1 and 18 h of incubation. Percent 51Cr release for 26 normal adults was 14.1 +/- 0.6 (mean +/- standard error) in the presence of pooled human seropositive sera (final dilution, 1:100) and 0.5 +/- 0.6 in the presence of pooled human seronegative sera. Addition of phorbol myristate acetate (PMA) enhanced PMN-mediated cytotoxicity against VZV-infected and uninfected targets. PMA-stimulated cytotoxicity was optimal with PMA concentrations of 200 ng/ml and effector-to-target ratios of 10:1, and antibody was not required; killing was detected as early as 3 h after incubation and was maximal after 18 h. Highly purified PMN were capable of mediating both ADCC and PMA-stimulated lysis. Catalase completely inhibited PMA-stimulated PMN cytotoxicity, but had no effect on PMN-mediated ADCC. PMN from patients with chronic granulomatous disease were capable of mediating ADCC, but not PMA-stimulated killing, against VZV-infected targets. Thus, PMN could kill VZV-infected targets by two different mechanisms: ADCC, which required antibody but not hydrogen peroxide (H2O2), and PMA-stimulated cytotoxicity, which required H2O2 but not antibody.     

Mechanism of thyroxine-mediated oxidation of reduced nicotinamide adenine dinucleotide in peroxidase-H2O2 system.

The oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the horseradish peroxidase (HRP)-H2O2 system is greatly increased by the addition of thyroxine or related compounds. On the basis of a study of the rate of NADH oxidation in the presence of various concentrations of thyroxine, it is clear that thyroxine acts as a catalyst for NADH oxidation. Spectral changes of a HRP-H2O2 complex (compound I) indicate that thyroxine acts as an electron donor to both compounds I and II. The rate of electron donation from thyroxine is much faster than that from NADH. The HRP-H2O2 system requires 0.83 mol of O2 for the oxidation of 1 mol of NADH. Ferricytochrome c is reduced to ferrocytochrome c by the system, and causes an inhibition of O2 consumption which can be abolished by superoxide dismutase. JUDGING FROM THE INHIBITION OF O2 uptake by ferricytochrome c, about 54% of the total flux of electrons from NADH to oxygen appears to proceed by way of O2-. These results suggest that the initial step of thyroxine-mediated NADH oxidation by HRP and H2O2 is the formation of oxidized thyroxine, a phenoxy radical, which attacks NADH to produce NAD.     

Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase

Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide.     

The bactericidal mechanisms of polymorphonuclear leukocytes against Bacteroides fragilis: significance of the oxygen-dependent system

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen-radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2- and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen-dependent and oxygen-independent mechanisms.     

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.     

Phagocytosis of immunoglobulin G and C3-bound human sperm by human polymorphonuclear leukocytes is not associated with the release of oxidative radicals.

Antisperm antibody (ASA)- and complement (C)-mediated immune injury to human sperm is thought to be caused in part by phagocytic neutrophils. To investigate this process, we co-cultured purified human polymorphonuclear leukocytes (PMN) with swim-up sperm in the presence of ASA-positive and ASA-negative sera and assayed for PMN respiratory burst activity, monitored by the release of superoxide anion (O2-) and hydrogen peroxide (H2O2). Phorbol myristate acetate (PMA) and opsonized zymosan were used as positive controls. Phagocytosis of ASA-positive and C-bound sperm by PMN did not enhance O2- production when compared to incubation of sperm with ASA-negative sera. Phagocytosis of ASA-positive and C-bound sperm also resulted in minimal release of H2O2 when compared with ASA-positive and C-negative sperm that were not phagocytosed. In contrast, PMN were maximally stimulated to release O2- in response to either opsonized zymosan or PMA. The kinetics of PMA-induced O2- release was unaffected by the presence of ASA-positive and C-bound sperm. Cytocentrifuge preparations of PMN incubated with ASA-positive and C-bound sperm revealed limited O2- release at the site of PMN/sperm contact. These results indicated that 1) phagocytosis of motile sperm by PMN requires the binding of both ASA and C to the sperm surface; 2) phagocytosis of ASA-positive and C-positive sperm by PMN fails to release reactive oxygen species; and 3) metabolic processes associated with PMN respiratory burst activity may not be coupled to the ingestion of ASA-positive and C-bound sperm.     

Functional relationship of the cytochrome b to the superoxide-generating oxidase of human neutrophils

A subcellular particulate fraction containing the NADPH-dependent O2.--generating oxidase from stimulated human neutrophils was prepared. This fraction was depleted of certain enzyme markers of primary and secondary granules and was devoid of measurable myeloperoxidase, both enzymatically and spectrally. When prepared from neutrophils which had been previously stimulated with phorbal myristate acetate, this fraction contained cyanide-insensitive, pyridine nucleotide-dependent O2.--generating activity with a specific activity of 260 nmol min-1 mg-1. O2.--generating activity is completely ablated by p-chloromercuribenzoate exposure. Preparations from normal unstimulated neutrophils or stimulated neutrophils from a male patient with chronic granulomatous disease had negligible amounts of this O2.--generating enzymatic activity. The dominant chromophore in this preparation was a b-type cytochrome, the spectral and functional characteristics of which are further described herein. Pyridine nucleotide-dependent reduction of the intrinsic cytochrome b closely parallels O2.- generation in this preparation. Specifically, reduction occurs in preparations from phorbal myristate acetate-stimulated neutrophils and is absent in unstimulated or stimulated p-chloromercuribenzoate-inactivated preparations.     

Inactivation of creatine kinase during the interaction of indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by indomethacin (IM), an effective drug to treat rheumatoid arthritis and gout, with horseradish peroxidase and hydrogen peroxide (HRP-H2O2). IM inactivated CK during its interaction with HRP-H2O2. Under aerobic conditions, inactivation of CK significantly decreased. CK in rat heart homogenate was also inactivated by IM with HRP-H2O2. When IM was incubated with HRP-H2O2, the maximum absorption of IM at 280 nm rapidly decreased and a new peak at 410 nm occurred with isosbestic points at 260 and 312 nm. In contrast, under anaerobic conditions, the spectral change of IM was almost absent, indicating IM was oxidized to the yellow substance by HRP-H2O2. Adding catalase strongly inhibited the production of yellow substance. Sodium azide also blocked the formation of yellow substance and the inactivation of CK. Electron spin resonance signals of IM carbon-centered radical were detected using 2-methyl-2-nitrosopropane during the interaction of IM with HRP-H2O2 under anaerobic conditions. Oxygen was consumed during the interaction of IM with HRP-H2O2. These results suggest that IM carbon-centered radicals may rapidly react with O2 to generate the peroxyl radicals. Sulfhydryl groups and tryptophane residues of CK decreased during the interaction of IM with HRP-H2O2. Other sulfhydryl enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were also readily inactivated during the interaction with HRP-H2O2. Sulfhydryl enzymes seem to be very sensitive to IM activated by HRP-H2O2.     

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.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram-positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products.     

Oxidant membrane injury by the neutrophil myeloperoxidase system. II. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants

The stimulated human neutrophil can damage a variety of target cells, and in some models, a mechanism involving secretion of myeloperoxidase and H2O2 has been demonstrated. We explored the characteristics of this cell-cell interaction by using neutrophils and our recently described liposome model target cell system. Exposure of 51Cr-labeled liposomes to phorbol myristate acetate-stimulated human neutrophils resulted in release of 25 to 30% of the radioactivity. 51Cr release was abrogated by omission of the neutrophils, the phorbol ester or halide (iodide), replacement of the phorbol by an inactive congener, or addition of azide, cyanide, or catalase. Neutrophils from patients with hereditary absence of myeloperoxidase (MPO) or a failure of H2O2 formation (chronic granulomatous disease) did not cause liposome lysis unless purified MPO or a source of H2O2, respectively, was added. These data indicate that 51Cr release from liposomes is a consequence of the secretion of MPO and H2O2, which combine with extracellular halides to form a membrane lytic system. The influence of liposome composition on injury was then examined, with a focus on physiologically relevant lipid soluble antioxidants. Liposomes containing either alpha-tocopherol (0.33 to 1.67% of molar fraction of lipid) or beta-carotene (1.67% of molar fraction of lipid) were markedly resistant to lysis by the cellfree MPO-H2O2-chloride system. When the major structural lipid phosphatidyl choline was replaced by dipalmitoyl phosphatidyl choline, a synthetic phospholipid with no oxidizable double bonds, the resultant liposomes were totally resistant to lysis by the MPO-H2O2-chloride system. The addition of iodide to this system (i.e., both chloride and iodide present) changed the pattern of protection dramatically in that alpha-tocopherol and beta-carotene were no longer protective and the resistance of dipalmitoyl phosphatidyl choline liposomes was partial rather than complete. In contrast to iodide, the addition of bromide or thiocyanate did not have a major effect on the protection by antioxidants. Finally, we demonstrated protection by alpha-tocopherol or dipalmitoyl phosphatidyl choline against liposome lysis by phorbol-activated neutrophils. These studies illustrate the use of model phospholipid membranes in the characterization of oxygen-dependent cell-mediated cytotoxicity. Activated neutrophils lyse liposome targets through a MPO-dependent mechanism. Target properties, especially the content of lipid-soluble antioxidants, have a marked influence on susceptibility to lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peroxidase-mediated irreversible binding of arylamine carcinogens to DNA in intact polymorphonuclear leukocytes activated by a tumor promoter

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system

An in vitro model system was used to define the mechanism of interaction between human neutrophils and lymphocytes. Blood mononuclear leukocytes were exposed to purified neutrophils in the presence of a neutrophil-activating agent (phorbol ester, lectin, or opsonized particle). The treated mononuclear cells displayed a marked decrease in both natural killer activity and mitogen-dependent DNA synthesis, but no change in viability. This functional suppression was dependent on neutrophil number, stimulus concentration, and duration of exposure. Lymphocytes were protected by addition of catalase, but not superoxide dismutase. Neutrophils defective in oxidative metabolism (chronic granulomatous disease) failed to suppress lymphocyte function unless an H2O2-generating system, glucose oxidase plus glucose, was added. The patients' neutrophils provided a factor, possibly myeloperoxidase, which interacted with the glucose oxidase system. The immunosuppressive effect of normal neutrophils was diminished when chloride was omitted from the cultures and was enhanced when chloride was replaced by iodide. Myeloperoxidase-deficient neutrophils were partially defective in suppressing lymphocytes and this was corrected by addition of purified myeloperoxidase. Paradoxically, azide caused enhancement of suppression that depended on the neutrophil oxidative burst, but not on myeloperoxidase and was mediated at least in part by an effect of azide on the target mononuclear leukocytes. These data indicate that suppression of lymphocyte function by activated neutrophils is mediated by the secretion of myeloperoxidase and H2O2 that react with halides to form immunosuppressive products. Moreover, the mononuclear leukocytes contain an azide-sensitive factor, probably catalase, which provides partial protection against injury by neutrophil products. These dynamic interactions may be important local determinants of the immune response.     

Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.     

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.     

Suppression of phorbol myristate acetate-triggering of macrophage H2O2 release by sarcoma 180 originating factor

A high molecular weight proteinaceous factor in the cell extract of sarcoma 180 (S-180) was found to inhibit phorbol myristate acetate (PMA)-triggering of macrophage H2O2 release. This factor (S-180 factor) was stable at 56 C for 1 hr and resistant to ultraviolet-irradiation. The S-180 factor inhibited the specific binding of PMA to macrophages and this was accompanied by a parallel reduction of PMA-triggered H2O2 release. S-180 factor preferentially depressed macrophage H2O2 release in response to phorbol diesters including PMA, 4 beta-phorbol 12 13 beta,13 alpha-diacetate, 4 beta-phorbol 12 beta,13 alpha-didecanoate, 4 beta-phorbol 12 beta,13 alpha-dibenzoate, and 4-omicron-methyl-PMA rather than the H2O2 release triggered by wheat germ agglutinin or by phagocytosis of latex particles. The S-180 factor failed to affect the PMA-elicited macrophage cell spreading and macrophage phagocytic activity against latex beads with or without PMA-mediated stimulation. A similar inhibitory factor was found in the extracts of some other murine tumor cells (Ehrlich carcinoma and thymic leukemia) and normal cells (liver, spleen, and peritoneal exudate cells).     

Reaction of human myeloperoxidase with hydrogen peroxide and its true catalase activity

The instability of human myeloperoxidase [EC 1.11.1.7] compound I, which was spontaneously reduced to compound II, and the abnormal stoichiometry of the reaction of myeloperoxidase with H2O2 were investigated. As to the former, a pretreatment of myeloperoxidase with H2O2 did not stabilize compound I, and no difference in its stability was observed between native (alpha 2 beta 2) and hemi (alpha beta) myeloperoxidase. From these results, it was thought that the instability of compound I was caused by neither the presence of endogenous donors nor the intramolecular reduction of compound I to compound II by the other heme in the native enzyme molecule. As for the latter, true catalase activity of myeloperoxidase was demonstrated by monitoring O2 evolution after the injection of H2O2 into the enzyme solution. Myeloperoxidase compound I reacted with H2O2 and returned to the ferric state with concomitant evolution of an O2 molecule. Accordingly, the abnormal stoichiometry of the reaction with H2O2 and a part of the instability of compound I can probably be ascribed to this true catalase activity.