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. 2001 Oct 9;98(21):11961-6.
doi: 10.1073/pnas.211190298. Epub 2001 Oct 2.

Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis

J P Gaut  1 , G C Yeh, H D Tran, J Byun, J P Henderson, G M Richter, M L Brennan, A J Lusis, A Belaaouaj, R S Hotchkiss, J W Heinecke
Affiliations

Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis

J P Gaut et al. Proc Natl Acad Sci U S A. .

Abstract

The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant in vitro. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis in vivo by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase in vitro. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants in vivo, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.

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Figures

Figure 1
Figure 1
Myeloperoxidase deficiency impairs survival in a CLP model of sepsis. Mortality was monitored in myeloperoxidase-deficient (MPO−/−) mice and littermate wild-type (WT) controls.
Figure 2
Figure 2
Electron capture-negative chemical ionization GC/MS analysis of the ethyl heptafluorobutyrate, MtBSTFA derivatives of 3-bromotyrosine and 3-chlorotyrosine in peritoneal inflammatory fluid of a wild-type mouse subjected to CLP. Note the simultaneous monitoring of endogenous (m/z 489; a), isotope-labeled (m/z 495; b), and artifactual (m/z 499; c) 3-chlorotyrosine (3-Cl-Tyr) and 3-bromotyrosine (3-Br-Tyr).
Figure 3
Figure 3
Isotope dilution GC/MS quantification of 3-chlorotyrosine (3-Cl-Tyr; a) and 3-bromotyrosine (3-Br-Tyr; b) in the peritoneal inflammatory fluid of sham-operated and CLP-subjected mice. Oxidation products were monitored in myeloperoxidase-deficient (MPO−/−) mice and in wild-type (WT) mice in the 129/SvJ and C57BL/6J background.
Figure 4
Figure 4
Reverse-phase HPLC analysis of N-acetyl-L-tyrosine (N-Ac-tyrosine) exposed to the myeloperoxidase-H2O2-Cl−-Br− system. Reactions proceeded for 60 min at 37°C in Chelex-treated buffer A (100 mM NaCl/50 mM sodium phosphate/100 μM diethylenetriaminepentaacetic acid, pH 4.5) supplemented with 3 nM myeloperoxidase, 1 mM N-Ac-tyrosine, 10 μM NaBr, and 50 μM H2O2. The reactions were initiated with H2O2 and terminated with 0.1 mM methionine. MPO, myeloperoxidase; Ac, acetyl.
Figure 5
Figure 5
Reaction requirements for the generation of N-acetyl-L-3-bromotyrosine by phagocyte peroxidases and hypohalous acids at neutral pH. Reactions were carried out in buffer A (Fig. 4) supplemented with 10 μM Br−. (a) Effect of pH on the generation of N-acetyl-bromotyrosine by myeloperoxidase. (b–d) Effect of taurine (200 μM) on the generation of N-acetyl-bromotyrosine by HOCl, HOBr, myeloperoxidase, or eosinophil peroxidase at pH 7. Amino acids were quantified by reverse-phase HPLC. Results are representative of those found in three independent experiments. MPO, myeloperoxidase; EPO, eosinophil peroxidase; Ac, acetyl.
Figure 6
Figure 6
Reaction requirements for the generation of N-acetyl-L-3-chlorotyrosine and N-acetyl-L-3-bromotyrosine by phagocyte peroxidases and hypohalous acids under acidic conditions. Effect of [Br−] on the generation of N-acetyl-chlorotyrosine and N-acetyl-bromotyrosine by: HOCl (a) or myeloperoxidase (b). Effect of taurine on the generation of N-acetyl-chlorotyrosine and N-acetyl-bromotyrosine by hypohalous acid (HOCl, HOBr) (c) or phagocyte peroxidases (MPO, EPO) (d). Reactions for a and b were carried out as described in the legend to Fig. 4. Reactions for c and d were performed as described in the legend to Fig. 4, except the pH was 5.9. Amino acids were quantified by reverse-phase HPLC. Results are representative of those found in three independent experiments. MPO, myeloperoxidase; EPO, eosinophil peroxidase; Ac, acetyl.
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