Showing papers on "Myeloperoxidase published in 1985"

Lung myeloperoxidase as a measure of pulmonary leukostasis in rabbits.

Abstract: Pulmonary leukostasis can be associated with acute lung injury. We studied lung peroxidase activity using myeloperoxidase (MPO) as a granulocyte marker to quantitate pulmonary leukostasis in rabbits. Lungs were homogenized in detergent, freeze-thawed, sonified, and centrifuged, and supernatants were assayed for MPO. Seven extractions were performed, and greater than 80% of cumulative MPO was found in the first three extractions. By use of a three-extraction procedure, the mean lung MPO (delta A X min-1 X g tissue-1) was determined in normal [20.9 +/- 5.2 (SE)], granulocyte-depleted (6.5 +/- 2.0), saline-injected (22.2 +/- 5.6), and pneumococcus (PNC)-challenged (69.7 +/- 10.6) animals. Lung MPO was significantly decreased in granulocyte-depleted compared with normal animals (P less than 0.005) and significantly increased in PNC-challenged compared with saline-injected animals (P less than 0.001). MPO extracted from granulocytes and lungs from normal as well as PNC-challenged animals were all biochemically identical. Lung extract did not inhibit MPO, and no MPO was detected in bronchoalveolar lavage fluid obtained from leukostatic lungs. Lung MPO significantly (P less than 0.01) correlated with intravascular intrapulmonary granulocytes. Determination of lung MPO is a relatively simple quantitative method that can be used to detect pulmonary leukostasis.

Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.

Abstract: Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase.

Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

Abstract: A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis.

Reduced neutrophil superoxide anion release after prolonged infusions of lidocaine.

Abstract: Lidocaine is used extensively in coronary care units, yet the effect of lidocaine infusions on neutrophil function has not been known. Lidocaine and other local anesthetics impair leukocyte antibacterial functions when added in vitro. We found that lidocaine added to human neutrophils in vitro markedly impaired the release of superoxide anion (O2-) and the granule enzymes lysozyme and myeloperoxidase after stimulation by phorbol myristate acetate or opsonized zymosan. We then measured production of O2- during stimulation of neutrophils from eight normal subjects, five coronary artery disease patients not receiving lidocaine and 13 coronary artery disease patients receiving lidocaine infusions for at least 12 hr. Release of O2- by cells from lidocaine-treated patients (14.2 +/- 3.8 nmol/2.5 X 10(6) neutrophils per 15 min) was significantly lower than from cells of the normal subjects (78.4 +/- 7.2 nmol; P less than .001) and the coronary patients not receiving lidocaine (70.6 +/- 4.0 nmol; P less than .001). Bactericidal assays at a high concentration (2 mg/ml) of lidocaine demonstrated slight reductions in 2 hr killing rates for Escherichia coli (70% with lidocaine vs. 95% control). Inhibition by lidocaine of the release of toxic oxygen metabolites from neutrophils could potentially reduce infarct size in patients with acute myocardial infarction; but as there is only a slightly reduced ability to kill bacteria, increased susceptibility to infections is unlikely although it cannot be excluded.

Antiarthritic drugs containing thiol groups scavenge hypochlorite and inhibit its formation by myeloperoxidase from human leukocytes. A therapeutic mechanism of these drugs in rheumatoid arthritis

Abstract: We investigated the effect of antiarthritic drugs containing thiol groups, such as D-penicillamine, tiopronin (N-[2-mercaptopropionyl]glycine), sodium aurothiomalate, and aurothioglucose, on the chlorinating activity of myeloperoxidase purified from human leukocytes. Hypochlorite, the reactive product of the reaction catalyzed by myeloperoxidase, was effectively scavenged by these antiarthritic drugs, and in addition, D-penicillamine and tiopronin inhibited myeloperoxidase itself. The above-mentioned effects of these drugs were observed at concentrations that occur in the serum of rheumatoid arthritis patients treated with these agents. We suggest that the therapeutic effect of these antiarthritic drugs may be due to the protection of tissues against the reactive HOCI released by activated granulocytes at inflamed sites.

Myeloperoxidase activity as a quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct--the effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation.

Abstract: The accumulation of polymorphonuclear leukocytes (PMN) into a canine occlusion/reperfusion model of myocardial infarction (MI) has been quantitated using a spectrophotometric assay for the PMN-specific enzyme myeloperoxidase (MPO). In anaesthetized beagle dogs subjected to 1 h of occlusion of the left anterior descending coronary artery followed by coronary reperfusion for 1 h, MPO activity was found in fourfold greater amounts in infarcted myocardium as opposed to normal myocardium. MPO activity in infarcted myocardium increased with coronary reperfusion in a time-related manner, the greatest accumulation occurring at 5 h of coronary reperfusion. The time-related increase in MPO activity in ischemic myocardium was closely correlated with the observed accumulation of PMN as assessed by histological sectioning, thus supporting the specificity of the method. Pretreatment of dogs with ibuprofen (12.5 mg/kg i.v.) did not alter the MPO content and therefore PMN invasion of infarcted myocardium, a finding confirmed by light microscopy. However, pretreatment with ibuprofen increased myocardial infarct size (from 11.5 to 37.4%, p less than 0.01) and increased the incidence of haemorrhagic infarction and ventricular fibrillation. In conclusion, our studies demonstrate that MPO can be used as a sensitive and quantitative marker of PMN accumulation into an evolving MI. Furthermore, we have demonstrated that PMNs rapidly infiltrated injured myocardium in a time dependent manner and this accumulation was unaffected by ibuprofen pretreatment.

Hydrogen peroxide as a tumoricidal mediator of murine polymorphonuclear leukocytes induced by a linear β-1,3-D-glucan and some other immunomodulators

Abstract: Polymorphonuclear leukocytes (PMN) of mice can destroy tumor cells effectively in vitro in the presence of antitumor polysaccharide, linear β-1,3-d-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 (TAK), and some other immunomodulators. In the present study, we investigated the mechanism of the tumoricidal activity of PMN induced by these immunomodulators and especially TAK. The TAK-induced PMN cytotoxicity was concluded to involve hydrogen peroxide from the following results: ( a ) the cytotoxicity depended on glucose consumption; ( b ) it was almost completely inhibited by catalase but not affected by superoxide dismutase; ( c ) it was not reduced by cyanide or azide, which are inhibitors of myeloperoxidase; ( d ) it was not affected by scavengers of singlet oxygen or hydroxyl radical; ( e ) release of hydrogen peroxide from PMN was observed by the addition of TAK; ( f ) MM46 target cells were lysed directly by hydrogen peroxide in the absence of myeloperoxidase; ( g ) the supernatant of PMN in the presence of TAK, tested as a stable cytotoxic factor, did not have cytotoxic activity, and protease inhibitors had no effect on this cytotoxicity. These results suggest that hydrogen peroxide is a direct cytotoxic mediator in TAK-induced PMN cytotoxicity. Next, the mechanism of PMN cytotoxicities induced by other immunomodulators was also examined and was compared with that induced by TAK. The results suggest that hydrogen peroxide is also important for these cytotoxicities whereas, unlike the results with TAK, the H2O2:halide:myeloperoxidase system may partly participate in the cytotoxicities with some immunomodulators.

Differential effects of oxidizing agents on human plasma alpha 1-proteinase inhibitor and human neutrophil myeloperoxidase.

Abstract: Human alpha 1-proteinase inhibitor is easily susceptible to inactivation because of the presence of a methionyl residue at its reactive site. Thus, oxidizing species derived from the myeloperoxidase system (enzyme, H2O2, and C1-), as well as hypochlorous acid, can inactivate this inhibitor, although H2O2 alone has no effect. Butylated hydroxytoluene, a radical scavenger, partially protects alpha 1-proteinase inhibitor from the myeloperoxidase system and completely protects it from hypochlorous acid. Each oxidant also reacts differently with the inhibitor, in that the myeloperoxidase system and hypochlorous acid can each oxidize as many as six methionyl residues, but hypochlorous acid can also oxidize a single tyrosine residue. Myeloperoxidase can be inactivated by hypochlorous acid, by autoxidation in the presence of H2O2 and C1-, as well as by H2O2 alone. Butylated hydroxytoluene completely protects this enzyme from hypochlorous acid inactivation, does not affect the action of H2O2, and enhances autoinactivation. As many as six methionyl residues and two tyrosine residues could be oxidized during autoxidation and six methionine residues by H2O2 alone. Eight methionine residues and one tyrosine residue could be oxidized by hypochlorous acid. The tyrosine residue in myeloperoxidase was oxidized only at a relatively high concentration (600 microM) of hypochlorous acid at which point the enzyme simultaneously and completely lost its enzymatic activity. Loss of activity of myeloperoxidase could also be correlated with the loss of the heme groups present in the enzyme when a relatively high concentration of hypochlorous acid (600 microM) was used and also during autoxidation. It appears that once there is sufficient oxidant to modify one of the tyrosine residues, the heme group itself becomes susceptible.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Abstract: Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

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

Abstract: 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)

Increased neutrophil myeloperoxidase activity associated with cigarette smoking.

Abstract: Neutrophils from 49 young male smokers contained significantly higher myeloperoxidase (MPO) activity than those from 49 age-matched, nonsmoking controls, while the elastase-like activity was not different in the two populations. MPO activity was increased in some smokers, but did not correlate significantly with the increased number of peripheral blood neutrophils, cigarette usage (present or cumulative), or the mild pulmonary dysfunction detected by forced expiratory spirometry and the single breath nitrogen test. This increased MPO activity in smokers' neutrophils may contribute to the greater risk of obstructive pulmonary disease in some smokers by an exacerbation of the protease-antiprotease imbalance in the lung. This hypothesis is supported by the prior observations that neutrophils are recruited in greater numbers into the lungs of smokers and that MPO (in the presence of H2O2 and chloride ion) oxidatively inactivates antiproteases of both the alveoli and the mucus-lined airways.

Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: Function and granule protein content

Abstract: Polymorphonuclear leukocytes (PMN) demonstrate altered function during acute infections and after administration of corticosteroids. We questioned whether or not such changes are due to population shifts from functionally different compartments of the granulocyte pool. Volunteers were given epinephrine to induce demargination or hydrocortisone (HC) to promote egress of PMN from the bone marrow. PMN obtained before and after drug administration were compared for adherence, chemotaxis, luminol-enhanced chemiluminescence, and total content and release of lactoferrin (LF), myeloperoxidase (MPO), and beta-glucuronidase (beta-glu). Epinephrine induced a significant neutrophilia of mature PMN (segmented neutrophils), but there were no changes in function or granule protein content. HC induced a significant neutrophilia with segmented neutrophils and immature PMN (bands). Circulating PMN obtained 4 hr after HC administration demonstrated less adherence, increased chemiluminescence, increased MPO release, and decreased MPO content. Band neutrophils, however, were more adherent than segmented PMN and showed a similar decrease in adherence following HC in vivo. Thus alteration of PMN adherence following intravenous corticosteroids is not due to an influx of immature neutrophils. On the other hand, it is possible that MPO content and release and capacity for oxidative metabolism change as PMN mature.

Oxidative degradation of leukotriene C4 by human monocytes and monocyte-derived macrophages.

Abstract: Freshly isolated 2-h adherent normal human monocytes, when stimulated, degrade added leukotriene C4 (LTC4) by a myeloperoxidase (MPO) and H2O2-dependent mechanism. Among the stimuli effective in this regard are phorbol myristate acetate (PMA), the calcium ionophore A23187, opsonized zymosan, and N-formyl-methionine-leucine-phenylalanine (FMLP) when combined with cytochalasin B. The predominant products formed are the all-trans isomers of LTB4, 5-(S), 12-(R)-6-trans-LTB4 and 5-(S),12-(S)-6-trans-LTB4. Degradation is inhibited by azide and catalase, but not by superoxide dismutase. LTC4 degradation does not occur when MPO-deficient monocytes are used, unless MPO is added. Stimulated monocytes from patients with chronic granulomatous disease also are unable to degrade LTC4 under these conditions. Normal monocytes maintained in culture lose their ability to degrade LTC4. The addition of MPO to monocyte-derived macrophages increases degradation, particularly when the monolayers are pretreated with gamma-interferon. The oxidative degradation of LTC4 is a capacity shared by neutrophils, eosinophils, and mononuclear phagocytes, and may be an important mechanism for the modulation of leukotriene activity in inflammatory lesions.

Erythrocyte lysis by PMA-triggered neutrophil polymorphonuclears: evidence for an hypochlorous acid-dependent process.

Abstract: Human red blood cells (HRBC) were efficiently lysed when incubated with neutrophil polymorphonuclears (PMN) in the presence of phorbol-myristate-acetate (PMA), as detected by a 4-hr 51Cr release assay. The lysis was virtually absent in the presence of catalase, azide or cyanide and in the absence of chloride ions. These findings indicate the involvement of the myeloperoxidase (MPO)-hydrogen peroxide (H2O2)-chloride (Cl-) system in the cytolytic process. As the MPO-H2O2-Cl- system is capable of generating the powerful oxidant hypochlorous acid (HOCl), cytotoxicity assays were performed in the presence of taurine, glycine, serine and valine to scavenge this potentially lytic agent. Each of these compounds efficiently inhibited the HRBC lysis by PMA-triggered PMN, as well as the lysis caused by HOCl in a cell-free system. Thus, the results suggest that HOCl, or an agent with similar reactivity, plays a key role in the PMA-dependent PMN-mediated cytotoxicity against HRBC targets.

Erythrocyte lysis byPMA-triggered neutrophil polymorphonuclears: evidence foran hypochlorous acid-dependent process

Abstract: Summary. Humanredbloodcells (HRBC)were efficiently lysed whenincubated withneutrophil polymorphonuclears (PMN)inthepresence ofphorbolmyristate-acetate (PMA),asdetected bya 4-hr 51Cr release assay. Thelysis wasvirtually absent inthe presence ofcatalase, azide orcyanide andinthe absence ofchloride ions. Thesefindings indicate theinvolvement ofthemyeloperoxidase (MPO)-hydrogenperoxide (H202)-chloride (Cl-) system inthe cytolytic process. AstheMPO-H202-Cl- system is capable ofgenerating thepowerful oxidant hypochlorous acid(HOCl), cytotoxicity assays wereperformed inthepresence oftaurine, glycine, serine and valine toscavenge this potentially lytic agent. Eachof these compounds efficiently inhibited theHRBClysis byPMA-triggered PMN,aswell asthelysis caused by HOClinacell-free system. Thus, theresults suggest that HOC1,oranagent withsimilar reactivity, plays a keyrole inthePMA-dependent PMN-mediated cytotoxicity against HRBC targets. Abbreviations: Cl-, chloride ion; GO,glucose oxidase, H202,hydrogen peroxide; HOCl,hypochlorous acid; HRBC,humanredbloodcells; HRP,horseradish peroxidase; HVA,homovanillic acid; MPO,myeloperoxidase; Of-, superoxide anion; PMA,phorbol myristate acetate; PMN, neutrophil polymorphonuclears;

Stimulation of human neutrophilic granulocyte chemotaxis by monoclonal antibodies.

Abstract: Two mouse monoclonal antibodies, L12.2 and S5.22, were developed that are specific for human neutrophilic granulocytes and produce a twofold to threefold stimulation of n-formyl-methionine-leucyl-phenylalanine (FMLP)-induced chemotaxis. Stimulation of chemotaxis by the antibodies is specific for FMLP and is concentration dependent. L12.2 appears to be more potent in stimulating chemotaxis and is isotypically distinct from S5.22. In addition, although L12.2 reacts only with mature peripheral blood granulocytes, S5.22 reacts with leukemic cells of both myeloid and monocytic origin and with immature granulocyte precursor cells. This suggests that L12.2 interacts with an antigen that appears late in the differentiation pathway, whereas S5.22 binds to an antigen that is present throughout the myeloid lineage. By means of the under-agarose and Boyden chamber techniques, L12.2, but not S5.22, by itself was also found to be a potent granulocyte chemoattractant. Cells in a gradient of L12.2 display polarized and oriented morphology. L12.2 alone, but not S5.22, also stimulates granulocyte phagocytosis and induces superoxide anion production. Neither L12.2 nor S5.22 affected the release of myeloperoxidase or lysozyme from granulocytes either alone or in combination with FMLP, C5a, or the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). These results suggest that L12.2 interacts with a single antigenic determinant on granulocytes that is involved in chemotaxis, phagocytosis, and superoxide anion release.

Neutrophil myeloperoxidase concentration: changes with development and during bacterial infection.

Abstract: In experimental animals, the quantity of myeloperoxidase (MPO) in a volume of whole blood, and the neutrophil concentration in that same volume, were determiner and the results expressed as units of MPO (10-7)/neutrophil. Two situations are repored in which the concentration of MPO/neutrophil was found to change; during the growth and development of animals and during bacterial infection. Premature and newborn rats had only 25% or the MPO/neutrophil found in adults. One to three wk olds had 50% of the adult enzyme concentration. During fatal bacterial infection, MPO/neutrophil fell rapidly, often to undetectable levels, but during sublethal infections, following a 24–h lag period in adults and a 48–h lag in neonates, the concentration increased to twice normal.