Anna L.P. Chapman

Chlorination of bacterial and neutrophil proteins during phagocytosis and killing of Staphylococcus aureus.

Myeloperoxidase is proposed to play a central role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes. However, it has yet to be demonstrated that these inflammatory cells target hypochlorous acid against bacteria inside phagosomes. In this investigation, we treatedStaphylococcus aureus with varying concentrations of reagent hypochlorous acid and found that even at sublethal doses, it converted some tyrosine residues in their proteins to 3-chlorotyrosine and 3,5-dichlorotyrosine. To determine whether or not ingested bacteria were exposed to hypochlorous acid in neutrophil phagosomes, we labeled their proteins with [13C6]tyrosine and used gas chromatography with mass spectrometry to identify the corresponding chlorinated isotopes after the bacteria had been phagocytosed. Chlorinated tyrosines were detected in bacterial proteins 5 min after phagocytosis and reached levels of approximately 2.5/1000 mol of tyrosine at 60 min. Inhibitor studies revealed that chlorination was dependent on myeloperoxidase. Chlorinated neutrophil proteins were also detected and accounted for 94% of total chlorinated tyrosine residues formed during phagocytosis. We conclude that hypochlorous acid is a major intracellular product of the respiratory burst. Although some reacts with the bacteria, most reacts with neutrophil components.

Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis

We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1μM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF.

Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Background: Myeloperoxidase promotes oxidative stress during inflammation by producing hypochlorous acid. Results: Ceruloplasmin was a potent inhibitor of myeloperoxidase and slowed its activity in plasma from wild type mice compared with ceruloplasmin knock-out animals. Conclusion: Ceruloplasmin is a physiologically relevant inhibitor of myeloperoxidase. Significance: Ceruloplasmin will provide a protective shield against oxidant production by myeloperoxidase during inflammation. Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the FeV redox intermediate of myeloperoxidase, to Compound II, which has FeIV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.

Measuring chlorine bleach in biology and medicine

BACKGROUND Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. SCOPE OF REVIEW We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. MAJOR CONCLUSIONS Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. GENERAL SIGNIFICANCE These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Hypobromous acid and bromamine production by neutrophils and modulation by superoxide.

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate to their respective hypohalous acids. We have investigated the generation of HOBr by human neutrophils in the presence of physiological concentrations of chloride and bromide. HOBr was trapped with taurine and detected by monitoring the bromination of 4-HPAA (4-hydroxyphenylacetic acid). With 100 microM bromide and 140 mM chloride, neutrophils generated HOBr and it accounted for approx. 13% of the hypohalous acids they produced. Addition of SOD (superoxide dismutase) doubled the amount of HOBr detected. Therefore we investigated the reaction of superoxide radicals with a range of bromamines and bromamides and found that superoxide radicals stimulated the decomposition of these species, with this occurring in a time- and dose-dependent manner. The protection afforded by SOD against such decay demonstrates that these processes are superoxide-radical-dependent. These data are consistent with neutrophils generating HOBr at sites of infection and inflammation. Both HOBr and bromamines/bromamides have the potential to react with superoxide radicals to form additional radicals that may contribute to inflammatory tissue damage.

Oxidation contributes to low glutathione in the airways of children with cystic fibrosis

Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. We measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage. The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis. The antioxidant glutathione is low in the airways of children with CF due to oxidation by hypochlorous acid http://ow.ly/tKfyX

Evidence that C-reactive protein or IL-6 are not surrogates for all inflammatory cardiovascular risk factors in hemodialysis patients

Background/Aims: In otherwise healthy adults, high C-reactive protein (CRP) levels are associated with cardiovascular disease and have been linked to an inflammatory state. The presence of vascular disease is also associated with increased expression of adhesion molecules, including soluble intercellular adhesion molecule (sICAM), vascular endothelial growth factor (VEGF) and leukocyte-derived myeloperoxidase (MPO). These associations suggest potential mechanisms whereby inflammation may injure the vascular endothelium, but the recognition of how these mediators act in concert remain poorly characterized. That the prevalence of atherosclerosis and markers of inflammation are increased in renal failure patients suggests that inflammation causes accelerated vascular disease. Methods: In hemodialysis patients, we examined the relationships between plasma CRP and sICAM, VEGF and MPO longitudinally. We determined whether episodes of a high CRP value were paralleled by simultaneous increases in mediators of inflammatory injury or molecules associated with endothelial cell adhesion or growth and whether CRP levels correlated with those of VEGF and MPO. Results: Episodic increases in CRP were accompanied by higher levels of VEGF, sICAM and MPO. However, there was no correlation between serum CRP levels or other acute phase proteins and either MPO or VEGF, nor was there a constant temporal relationship between MPO and CRP. By contrast, MPO and VEGF levels were closely correlated with one another during episodes of inflammation (p = 0.0001), and CRP and interleukin-6 levels were also correlated. Increases in MPO tended to be restricted to patients with grafts or catheters, and not those with AV fistulas. Conclusions: These results suggest that high plasma levels of CRP or other acute phase proteins in cross-sectional studies should be interpreted cautiously when defining mechanisms underlying cardiovascular disease in the hemodialysis patient population. One, or more than one inflammatory repertoire may be activated, one involving hepatic acute phase proteins and the other neutrophil activation and each may contribute separately to outcomes. Better prognostic information may be obtained by measurement of more markers than CRP alone, such as MPO and VEGF.

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix.

BACKGROUND Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.

Neutrophil granule proteins generate bactericidal ammonia chloramine on reaction with hydrogen peroxide

Abstract The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro‐organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H2O2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H2O2. Myeloperoxidase in the extract converted all the H2O2 to HOCl, which reacted with the granule proteins. 3‐Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H2O2, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H2O2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation. Graphical abstract No caption available. HighlightsMPO‐dependent protein oxidation occurs in neutrophil granule extract treated with H2O2.Chloramines are formed and the extract becomes potently bactericidal.Bactericidal activity is due to NH2Cl, formed by decomposition of protein chloramines.NH2Cl formed by this mechanism could contribute to neutrophil bactericidal activity.