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.     

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.     

Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation

Leukocyte chemoattractants were inactivated when exposed to human neutrophils and either ingestible particles or phorbol esters. Loss of biologic activity was time- and temperature-dependent, required physiologic concentrations of viable neutrophils and a halide, and was inhibited by azide or catalase. Neutrophils from patients with either hereditary myeloperoxidase deficiency or chronic granulomatous disease failed to inactivate the chemoattractants unless purified myeloperoxidase or H2O2, respectively, was added. Susceptibility to inactivation by neutrophils correlated with the presence of methionine in the attractant. Loss of chemotactic activity was blocked by low concentrations of methionine and by higher concentrations of other reducing agents, but was unaffected by oxidized methionine. Paper chromatography demonstrated that exposure of a formyl-methionyl peptide chemotactic factor to either the cellfree myeloperoxidase system or stimulated neutrophils resulted in its conversion to a molecular species whose location in the chromatographs was identical to that of the peptide containing oxidized methionine. Thus, stimulated human neutrophils inactivate peptide chemoattractants by secretion of myeloperoxidase and H2O2, which combine with halides to form oxidants that react with a critical methionine residue. We suggest that myeloperoxidase-catalyzed oxidation of thioethers may constitute an inflammatory control mechanism as well as a general means of modifying the functional properties of biologic mediators.     

Oxidation of lysine side-chains of elastin by the myeloperoxidase system and by stimulated human neutrophils

Exposure of [3H]-lysine labeled elastin to either purified myeloperoxidase plus H2O2 and halides or human neutrophils plus phorbol myristate acetate resulted in oxidation of lysine side chains quantitated as 3H2O release. In both the enzyme and cell system oxidation was blocked by azide, cyanide or catalase, but not by beta-aminopropionitrile, an inhibitor of lysyl oxidase. Myeloperoxidase-deficient neutrophils were ineffective unless exogenous myeloperoxidase was added. These data provide a biochemical basis for inflammatory changes in connective tissue proteins mediated by oxidant secretory products of neutrophils.     

Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System

An antibacterial effect of myeloperoxidase, a halide, such as iodide, bromide, or chloride ion, and H(2)O(2) on Escherichia coli or Lactobacillus acidophilus is described. When L. acidophilus was employed, the addition of H(2)O(2) was not required; however, the protective effect of catalase suggested that, in this instance, H(2)O(2) was generated by the organisms. The antibacterial effect was largely prevented by preheating the myeloperoxidase at 80 C or greater for 10 min or by the addition of a number of inhibitors; it was most active at the most acid pH employed (5.0). Lactoperoxidase was considerably less effective than was myeloperoxidase when chloride was the halide employed. Myeloperoxidase, at high concentrations, exerted an antibacterial effect on L. acidophilus in the absence of added halide, which also was temperature- and catalase-sensitive. Peroxidase was extracted from intact guinea pig leukocytes by weak acid, and the extract with peroxidase activity had antibacterial properties which were similar, in many respects, to those of the purified preparation of myeloperoxidase. Under appropriate conditions, the antibacterial effect was increased by halides and by H(2)O(2) and was decreased by catalase, as well as by cyanide, azide, Tapazole, and thiosulfate. This suggests that, under the conditions employed, the antibacterial properties of a weak acid extract of guinea pig leukocytes is due, in part, to its peroxidase content, particularly if a halide is present in the reaction mixture. A heat-stable antibacterial agent or agents also appear to be present in the extract.     

The effect of oxidants on neutrophil-mediated degradation of glomerular basement membrane collagen

The contribution of activated oxygen species to neutrophil-mediated degradation of basement membrane collagen was investigated. In preliminary experiments, pre-exposure of either albumin or glomerular basement membrane to neutrophil myeloperoxidase with H2O2 and chloride increased their susceptibility to proteolysis 2-3-fold. In the basement membrane model, neutrophils are stimulated by trapped immune complexes to adhere, produce oxidants and degranulate. Degradation, measured as the amount of hydroxyproline solubilised, was due to neutral proteinases, particularly elastase, and depended on cell number and the amount of proteinase released. Experiments with oxidant scavengers and inhibitors and with neutrophils from donors with chronic granulomatous disease or myeloperoxidase deficiency showed that oxidants did not affect degradation of the basement membrane when this was measured on a per cell basis. However, oxidative inactivation of the released granule enzymes occurred. Activities of elastase, beta-glucuronidase and lysozyme were 1.5-2-times higher in the presence of catalase, but were unaffected by superoxide dismutase or hydroxyl radical scavengers. Inactivation did not occur with chronic granulomatous disease or myeloperoxidase deficient neutrophils. When related to the activity of released elastase, or to other degranulation markers, collagen degradation was decreased in the presence of catalase, or with chronic granulomatous disease or myeloperoxidase deficient cells. This implies that the basement membrane was made more digestible by myeloperoxidase-derived oxidants, as occurred in the cell-free experiments. Taken together, the results indicate that neutrophil oxidants have two opposing effects. They increase the susceptibility of the collagen to proteolysis and inactivate the proteinases responsible.     

Human neutrophils transform prostaglandins by a myeloperoxidase-dependent mechanism

Intact human neutrophils, incubated with the soluble stimulant phorbol myristate acetate, discharge lysosomal components, generate oxygen metabolites, and transform exogenous 6-keto-prostaglandin F1 alpha, prostaglandin E2, and prostaglandin F2 alpha as assessed by thin layer radiochromatography. Neutrophils alone were incapable of transforming the prostaglandins. The addition of catalase or the myeloperoxidase inhibitor, azide, protected all three prostaglandins from the phorbol-stimulated neutrophils. Neither superoxide dismutase, heat-inactivated catalase, nor albumin had any inhibitory effect in this system. A model system consisting of glucose-glucose oxidase, as a source of H2O2, purified myeloperoxidase, and chloride was also able to transform the prostaglandins in an identical fashion. Neither glucose-glucose oxidase alone nor glucose-glucose oxidase and myeloperoxidase under chloride-free conditions were able to mediate this transformation. Thus, it appears that intact human neutrophils can transform prostaglandins by a mechanism dependent on H2O2, the lysosomal enzyme myeloperoxidase, and chloride. Given the importance of prostaglandins in regulating immune function, neutrophil-dependent prostaglandin transformation could play a novel role in modulating the inflammatory response.     

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

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.     

The effect of catalase and methionine-S-oxide reductase on oxidised alpha 1-proteinase inhibitor

Oxidative damage to alpha 1-proteinase inhibitor (alpha 1-PI) may be important in the pathogenesis of emphysema. We have studied the ability of 2 enzymes (catalase and methionine-S-oxide reductase) to prevent and reverse oxidation of alpha 1-PI by hydrogen peroxide. Pre-incubation of catalase with H2O2 protected alpha 1-PI from oxidation, but the enzyme could not reverse prior oxidation of alpha 1-PI. In contrast, methionine-S-oxide reductase fully restored activity to H2O2-oxidised alpha 1-PI. Sputum sol-phase from smokers and non-smokers contained alpha 1-PI that was only about 30% active. Functional activity increased in both smokers (p less than 0.025) and non-smokers (p less than 0.05) approximately 2-fold following incubation with the reductase. Western blotting of the samples showed that about 20% of the alpha 1-PI was present as an enzyme-inhibitor complex and 20% was proteolytically cleaved. These observations suggest proteolysis, complexing with enzyme and oxidation are mechanisms of inactivation of alpha 1-PI in lung secretions.     

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.     

Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes

Both normal and chronic granulomatous disease (CGD) neutrophils were able to degrade the subendothelial matrix secreted by human endothelial cells via an elastase-dependent process. In the absence of the plasma antiproteinase, alpha-1-proteinase inhibitor (alpha-1-PI), normal neutrophils protect their released elastase from inactivation by using the chlorinated oxidants hypochlorous acid and endogenous N-chloroamines to suppress the antiproteinase's activity. In contrast, CGD neutrophils were unable to generate either class of chlorinated oxidant or to inactivate the porcine pancreatic elastase inhibitory capacity of alpha-1-PI unless the cells were supplemented with exogenous hydrogen peroxide. Despite the reliance of normal neutrophils on chlorinated oxidants to inactivate alpha-1-PI, neutrophils triggered in the presence of agents that block the generation of these reactive species continued to degrade the subendothelial matrix at a suppressed but significant rate in the presence of a 50-fold excess of the antiproteinase. The continued solubilization of the matrix by normal neutrophils was not due to the incomplete inhibition of oxidant generation because triggered CGD neutrophils were also able to degrade the matrix in the presence of excess alpha-1-PI. If CGD neutrophils were stimulated in the presence of an exogenous source of H2O2 and alpha-1-PI, the proteolytic potential of the cells was identical to that observed with normal stimulated neutrophils. We conclude that normal neutrophils can enhance their ability to degrade the subendothelial matrix by oxidatively protecting elastase from inactivation by alpha-1-PI but both normal and CGD neutrophils possess non-oxidatively linked mechanisms for sequestering and using elastase to mediate proteolytic effects in the presence of native antiproteinase.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

Inactivation of alpha-1-proteinase inhibitor in serum by stimulated human polymorphonuclear leucocytes. Evidence for a myeloperoxidase-dependent mechanism

Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha-1-proteinase inhibitor (alpha-1-PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha-1-PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha-1-PI in the presence of exogenously-added PMNL-derived supernatants which contain this enzyme. Cell-free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6.2, the pH optimum for myeloperoxidase-catalysed inactivation of alpha-1-PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha-1-PI by generating hypochlorous acid in the presence of chloride and respiratory burst-derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactivate the most important antielastase, alpha-1-PI.     

The effect of reducing agents on proteolytic enzymes and oxidation of alpha 1-proteinase inhibitor

We have studied the effect of the mucolytic agent N-acetylcysteine and dithiothreitol on the oxidation of alpha 1-PI by hydrogen peroxide, and their effect on porcine pancreatic elastase and leukocyte elastase. In addition, the effect of S-(carboxymethyl)cysteine (= carbocisteine, a mucolytic agent which does not have reducing properties) was studied in vitro and in patients with chronic obstructive bronchitis. Following addition of 59.6mM N-acetylcysteine, the amidolytic activity of leukocyte elastase was decreased by 55.3% and that of porcine pancreatic elastase by 57.0%. Dithiothreitol (5.7 mM) caused the loss of 97.4% and 67.6% of amidolytic activity of leukocyte elastase and porcine pancreatic elastase respectively whereas S-(carboxymethyl)cysteine had no effect. Similar results were found for the effect on elastolytic activity. Oxidation of alpha 1-PI by 8.6mM H2O2 resulted in partial loss of inhibitory function (mean 68.7% activity of native alpha 1-PI). N-Acetylcysteine and dithiothreitol prevented oxidation of alpha 1-PI when pre-incubated with H2O2 or incubated with alpha 1-PI and H2O2 simultaneously (94.5% and 94.4% activity of native alpha 1-PI for N-acetylcysteine; 78.3% and 87.6% activity for dithiothreitol - p less than 0.025). S-(Carboxymethyl)cysteine, when pre-incubated with H2O2 or incubated concurrently with alpha 1-PI and H2O2, caused a further decrease in the porcine pancreatic elastase inhibitory capacity of alpha 1-PI (53.1% and 63.0% respectively - p less than 0.025). None of the agents reversed oxidative inactivation once it had occurred. S-(Carboxymethyl)cysteine had no effect on alpha 1-PI function in sputum at the dose used.     

Myeloperoxidase-dependent oxidative inactivation of neutrophil neutral proteinases and microbicidal enzymes.

The susceptibility of a number of human neutrophil granule enzymes to oxidative inactivation was investigated. Addition of H2O2 to the cell-free medium from stimulated neutrophils resulted in inactivation of all enzymes tested. This was inhibited by azide and methionine, indicating that inactivation was due to myeloperoxidase-derived oxidants. Lysozyme was more than 50% inactivated by one addition of 100 nmol of H2O2/ml, whereas myeloperoxidase, beta-glucuronidase, gelatinase and collagenase were almost completely inactivated by three additions. Cathepsin G was slightly less susceptible, whereas elastase was extremely resistant to oxidative attack. Myeloperoxidase-dependent enzyme inactivation may be a means whereby the neutrophil can terminate the activity of its granule enzymes and control the release of degradative enzymes into the tissues.     

Inactivation of transferrin iron binding capacity by the neutrophil myeloperoxidase system

Human serum apotransferrin was exposed to the isolated myeloperoxidase-H2O2-halide system or to phorbol ester-activated human neutrophils. Such treatment resulted in a marked loss in transferrin iron binding capacity as well as concomitant iodination of transferrin. Each component of the cell-free system (myeloperoxidase, H2O2, iodide) or neutrophil system (neutrophils, phorbol ester, iodide) was required in order to observe these changes. In the cell-free system, the H2O2 requirement was fulfilled by either reagent H2O2 or the peroxide-generating system glucose oxidase plus glucose. Both loss of iron binding capacity and transferrin iodination by either the myeloperoxidase system or activated neutrophils were blocked by azide or catalase. The isolated peroxidase system had an acidic pH optimum, whereas the intact cell system was more efficient at neutral pH. The kinetics of changes in iron binding capacity and iodination closely paralleled one another, exhibiting t1/2 values of less than 1 min for the myeloperoxidase-H2O2 system, 3-4 min for the myeloperoxidase-glucose oxidase system, and 8 min for the neutrophil system. That the occupied binding site is protected from the myeloperoxidase system was suggested by 1) a failure to mobilize iron from iron-loaded transferrin, 2) an inverse correlation between initial iron saturation and myeloperoxidase-mediated loss of iron binding capacity, and 3) decreased myeloperoxidase-mediated iodination of iron-loaded versus apotransferrin. Since as little as 1 atom of iodide bound per molecule of transferrin was associated with substantial losses in iron binding capacity, there appears to be a high specificity of myeloperoxidase-catalyzed iodination for residues at or near the iron binding sites. Amino acid analysis of iodinated transferrin (approximately 2 atoms/molecule) demonstrated that iodotyrosine was the predominant iodinated species. These observations document the ability of neutrophils to inactivate transferrin iron binding capacity via the secretion of myeloperoxidase, formation of H2O2, and subsequent myeloperoxidase-catalyzed iodination. This sequence of events may help to explain the changes in iron metabolism associated with the in vivo inflammatory response.     

Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants

Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.     

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.     

Myeloperoxidase inactivation in the course of catalysis of chlorination of taurine.

Myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated from leukocytes of patients with chronic granulocyte leukemia. In the presence of H2O2 and Cl- at pH 4.0-6.6 the myeloperoxidase catalyses chlorination of taurine to monochloramine taurine and simultaneously undergoes inactivation. The myeloperoxidase inactivation rate depends on the concentration of H2O2 and Cl-: both the initial rate of chlorination and myeloperoxidase inactivation rate increase with increasing concentration of H2O2. However, an increase in concentration of Cl- results in a decrease in enzyme inactivation. At a given H2O2 concentration, myeloperoxidase inactivation is a first order reaction, which implied that the enzyme may react with a substrate a limited number of times.