Myeloperoxidase produces nitrating oxidants in vivo

Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available.

Nitric oxide (NO^•) produced by the constitutive form of nitric oxide synthase plays a critical role in normal physiology by regulating vasomotor tone (1, 2). The larger amounts of NO^• produced by the inducible isoform of the enzyme contribute to the ability of inflammatory cells to kill microbial organisms and tumor cells. Under pathological conditions, however, reactive nitrogen species derived from NO^• may injure normal tissue (3). The most widely studied mechanism involves the rapid reaction of NO^• with superoxide (4, 5) to produce peroxynitrite (ONOO^–). In vitro, ONOO^– yields potent hydroxylating and nitrating species. Immunohistochemical studies suggest that ONOO^– contributes to a variety of pathological conditions, including atherosclerosis, acute respiratory distress syndrome, and ischemia/reperfusion injury (3, 5).

Evidence that ONOO^– provides a mechanism for oxidative damage rests largely on the detection of 3-nitrotyrosine in inflamed tissue (3, 5). However, immunohistochemical approaches are only semiquantitative. Moreover, cross-reacting but structurally distinct molecules may confound immunodetection. Many investigators have noted that artifact formation complicates analysis of oxidation products, and this has been a particular problem with 3-nitrotyrosine (6–10). It is also unclear whether NO^• and superoxide produced at physiologically plausible rates will nitrate tyrosine efficiently in vitro (11–13), leaving open the possibility that 3-nitrotyrosine has a different origin in vivo.

NO^• can also autoxidize to nitrite (NO₂^–) and nitrate (NO₃^–) (14). Plasma levels of NO₂^–, which range up to 4 μM in healthy humans, increase markedly during acute and chronic inflammation (14). Because NO₂^– is a substrate for myeloperoxidase, a heme enzyme secreted by activated phagocytes, it might also be used for tyrosine nitration in vivo (15). Indeed, myeloperoxidase uses hydrogen peroxide (H₂O₂) and NO₂^– to generate reactive nitrogen species that nitrate tyrosyl residues in vitro (16–18). These reactions might be physiologically relevant because human neutrophils can use the myeloperoxidase-H₂O₂-NO₂^– system to chlorinate and nitrate tyrosine analogues (17).

Despite intense interest in the role of reactive nitrogen species in host defense mechanisms and oxidative tissue injury, the in vivo pathways that promote nitration remain poorly understood (1, 2). The difficulty of quantifying nitrated tyrosine in biological samples has contributed to the lack of progress (6–10). In the current study, we use two clinically relevant models of sepsis and gas chromatography/mass spectrometry (GC/MS) to determine whether myeloperoxidase generates 3-nitrotyrosine in vivo (19). Mass spectrometry has the major advantage of being a quantitative method that permits the use of isotopomers for monitoring any analyte that forms ex vivo during sample work-up and analysis. Our experiments using this method provide strong evidence that myeloperoxidase generates nitrating oxidants in vivo, possibly by a pathway involving NO₂^–.

Myeloperoxidase generates N-acetyl-3-nitrotyrosine in vitro under physiologically relevant conditions. Previous studies have used the tyrosine analogue 4-hydroxyphenylacetic acid to investigate the production of reactive nitrogen species by myeloperoxidase in vitro (17, 18). To confirm that myeloperoxidase can nitrate tyrosine, we incubated the enzyme (5 nM) with NO₂^– (100 μM), N-acetyl-L-tyrosine (0.5 mM), H₂O₂ (50 μM), and plasma concentrations of chloride ion (Cl^–; 100 mM) in phosphate buffer at neutral pH. N-acetyl-L-tyrosine was used instead of L-tyrosine itself to prevent the production of chloramines and p-hydroxyphenylacetaldehyde (26).

Reverse-phase HPLC analysis of the reaction mixture revealed three peaks of new material (peaks I–III, Figure 1a). Peak III contained the only product whose formation required the complete system (Figure 1a). The retention time and absorption spectrum of that product were identical to those of authentic N-acetyl-3-nitrotyrosine (Figure 1a, inset). In contrast, generation of peaks I and II required myeloperoxidase and H₂O₂ but not NO₂^–. Tandem mass spectrometric analysis in the negative ion mode (27) demonstrated spectra that were consistent with N-acetyl-o,o′-dityrosine (peak I, data not shown), N-acetyl-3-chlorotyrosine (peak II, data not shown), and N-acetyl-3-nitrotyrosine (peak III, Figure 1b). The identity of peak III as N-acetyl-3-nitrotyrosine was confirmed with high-resolution proton nuclear magnetic resonance spectroscopy (Figure 1c). Significant features included loss of the C-3 proton resonance, a downfield shift in the C-2 proton, and conversion of the C-2 proton resonance from a doublet to a singlet.

Figure 1

Analysis of N-acetyl-L-tyrosine exposed to the myeloperoxidase-NO₂^–-Cl^–-H₂O₂ system. (a) HPLC analysis. Inset: Absorbance spectra of authentic N-acetyl-L-3-nitrotyrosine (N-Ac-nitrotyrosine [N-Ac-NO₂-Y]) and peak III. (b) Electrospray ionization tandem mass spectrometric analysis of peak III. (c) Aromatic region of a proton nuclear magnetic resonance spectrum of peak III. MPO, myeloperoxidase.

Formation of N-acetyl-3-nitrotyrosine required NO₂^–, N-acetyl-L-tyrosine, H₂O₂, and myeloperoxidase. The reaction depended linearly on NO₂^– concentration over a physiologic range (1–100 μM) and was complete in less than 10 minutes. It was inhibited by about 20% by plasma concentrations of Cl^– and was also inhibited by the peroxide scavenger catalase and the heme poison sodium azide. These results indicate that myeloperoxidase nitrates N-acetyl-L-tyrosine by a reaction that requires active enzyme, NO₂^–, and H₂O₂, and that N-acetyl-3-nitrotyrosine is the major stable product.

Myeloperoxidase generates N-acetyl-3-nitrotyrosine by directly oxidizing NO₂^–. It has been proposed that myeloperoxidase uses two distinct pathways to generate reactive nitrogen species (16–18, 28, 29). In the first pathway, the enzyme generates HOCl, using H₂O₂ and Cl^– (Equation 1 below) (30–32). The HOCl then reacts with NO₂^– to form nitryl chloride, a nitrating species (Equation 2). In the second pathway, myeloperoxidase uses a one-electron reaction to directly oxidize NO₂^– to nitrogen dioxide radical, NO₂^• (Equation 3). NO₂^• then might directly oxidize tyrosine, or it might react with the tyrosyl radical that myeloperoxidase also generates (28, 33).

Equation 11

Equation 22

Equation 33

To distinguish between these two pathways, we first compared the effect of pH on the ability of either a mixture of reagent HOCl and NO₂^– or the myeloperoxidase-H₂O₂-NO₂^–-Cl^– system to generate N-acetyl-3-nitrotyrosine. Interestingly, the HOCl-NO₂^– system operated optimally at neutral pH, whereas the enzymatic system had an acidic pH optimum (Figure 2, a and b). This difference suggests that myeloperoxidase might be able to nitrate N-acetyl-L-tyrosine by a mechanism not involving HOCl. Alternatively, myeloperoxidase might generate HOCl most efficiently under acidic conditions.

Figure 2

Reaction requirements for generation of N-Ac-nitrotyrosine by HOCl and myeloperoxidase. (a and b) Effect of pH. (c) Effect of taurine. Results represent means ± SEM of three independent experiments.

We next determined whether taurine (2-aminoethanesulfonic acid, a potent scavenger of HOCl) (31, 34), inhibited nitration by the HOCl-NO₂^– mixture or the enzymatic system. Increasing concentrations of taurine progressively inhibited the conversion of N-acetyl-L-tyrosine to N-acetyl-3-nitrotyrosine by HOCl-NO₂^– at neutral pH (Figure 2c). In marked contrast, taurine had no effect when myeloperoxidase nitrated N-acetyl-L-tyrosine. This result provides strong evidence that myeloperoxidase nitrates N-acetyl-L-tyrosine by a pathway not involving HOCl. The pathway likely involves direct oxidation of NO₂^– by compound I (Equation 3) and the reaction of NO₂^• with tyrosyl radical. It is noteworthy that myeloperoxidase preferentially oxidizes NO₂^– under these conditions, despite the presence of 2000-fold greater levels of Cl^–.

Myeloperoxidase uses NO₂^– and H₂O₂ to generate bactericidal oxidants. Because myeloperoxidase-deficient mice are more vulnerable to infection with the Gram-negative bacterium K. pneumoniae than are wild-type mice (our unpublished observation), we determined how NO₂^–, Cl^–, and myeloperoxidase affect the killing of K. pneumoniae under neutral and acidic conditions (24). When the myeloperoxidase-H₂O₂ system in phosphate buffer at pH 7 was supplemented with physiologically plausible concentrations of NO₂^– (0.1 mM) or Cl^– (100 mM), it killed about 70% of the bacteria in the reaction mixture. Similar results were observed when both NO₂^– and Cl^– were included. Peroxide alone failed to kill bacteria under these conditions.

Different results were observed at pH 5. The myeloperoxidase-H₂O₂ system supplemented with 100 mM Cl^– killed essentially all of the bacteria. In contrast, only 82% of the bacteria were killed when the reaction mixture included NO₂^– at a level (10 mM) 100-fold higher than that observed in plasma during inflammation. These observations indicate that the bactericidal activity of the myeloperoxidase-H₂O₂-NO₂^– system depends critically on the pH of the reaction mixture. Klebanoff reported that killing of Escherichia coli showed a similar pH dependency (15).

Myeloperoxidase nitrates bacterial proteins in vitro. To explore the potential contribution of protein nitration to the cytotoxic activity of the myeloperoxidase-H₂O₂-NO₂^– system, we used a polyclonal antibody to nitrotyrosine to search for bacterial proteins that become nitrated in vitro. After exposing K. pneumoniae to myeloperoxidase, H₂O₂, and NO₂^– (Figure 3a), we observed multiple bands of immunoreactive material in bacterial proteins subjected to SDS-PAGE and Western blotting. A protein of about 65 kDa was particularly prominent (Figure 3a). Protein nitration required both myeloperoxidase and NO₂^– (Figure 3a).

Figure 3

Western blot analysis and immunoprecipitation of K. pneumoniae proteins exposed to the myeloperoxidase-H₂O₂-NO₂^– system at neutral PH. (a) Western blot analysis of nitrated proteins isolated from a reaction mixture containing K. pneumoniae (KP), myeloperoxidase, H₂O₂, and NO₂^– (100 or 500 μM). Proteins were probed with a rabbit polyclonal antibody to 3-nitrotyrosine. (b) Solubilized proteins from K. pneumoniae exposed to myeloperoxidase, H₂O₂, and NO₂^– were immunoprecipitated with a mouse mAb to 3-nitrotyrosine. Only the protein of about 65 kDa was immunoreactive with a rabbit polyclonal antibody to 3-nitrotyrosine. (c) Tandem mass spectrometric analysis of a trypsin digest of the immunoreactive 65-kDa protein. (M+H)⁺, molecular ion.

To identify the nitrated proteins, bacterial proteins were immunoprecipitated with a mouse monoclonal anti-nitrotyrosine antibody. When the isolated material was separated using SDS-PAGE, protein staining revealed four major bands (Figure 3b). Once again, a protein of about 65 kDa was particularly prominent. When the isolated material was subjected to Western blot analysis with the polyclonal anti-nitrotyrosine antibody, the only immunoreactive band was again the approximately 65-kDa protein (data not shown). Tandem mass spectrometric analysis of trypsin digests of the isolated protein identified the sequence VLVNTKATLAAFR (Figure 3c), a peptidyl aspartic acid metalloendopeptidase of the bacterium Pseudomonas fragi. This sequence is not present in any known K. pneumoniae protein, but the complete genomic sequence of this organism is not available. These observations raise the possibility that reactive nitrogen species generated by myeloperoxidase contribute to bacterial killing by oxidizing specific protein targets such as metalloproteinases.

Depriving neutrophils of myeloperoxidase impairs their ability to nitrate cytoplasmic proteins. To determine whether nitration of tyrosine by myeloperoxidase might be physiologically relevant, we isolated neutrophils from wild-type and myeloperoxidase-deficient mice. We then incubated the cells with K. pneumoniae, a major cause of sepsis in humans, in the presence (100 μM) or absence of NO₂^–. One hour after infection, the neutrophils were immunostained with an anti–3-nitrotyrosine antibody. In the presence of NO₂^–, the wild-type cells showed distinct cytoplasmic immunostaining (Figure 4a). A similar pattern of immunoreactivity was observed in the absence of NO₂^– (Figure 4b), but the immunostaining was less intense. In contrast, no immunostaining was observed in the cells that lacked functional myeloperoxidase in the presence (Figure 4c) or absence of NO₂^– (data not shown). Omitting the primary antibody or preincubating the 3-nitrotyrosine antibody with 10 mM of 3-nitrotyrosine also prevented immunostaining of wild-type cells (data not shown). Because we observed immunoreactivity for 3-nitrotyrosine in the absence of NO₂^–, we hypothesized that mouse neutrophils generate NO^• after they are activated by K. pneumoniae (24, 35). Indeed, both wild-type and myeloperoxidase-deficient cells immunostained intensely for the inducible form of nitric oxide synthase (Figure 4, d–f). These results suggest that mouse neutrophils use myeloperoxidase to nitrate cytoplasmic proteins.

Figure 4

Immunohistochemical staining of wild-type and myeloperoxidase-deficient neutrophils for 3-nitrotyrosine (red immunostaining; a–c) or inducible nitric oxide synthase (iNOS) (brown immunostaining; d–f). Cells were incubated with K. pneumoniae in the presence (a, c, d, and f) or absence (b and e) of NO₂^– and then subjected to immunostaining.

Free 3-nitrotyrosine is detectable in peritoneal fluid isolated from septic mice. To determine whether tyrosine is nitrated in vivo during inflammation, peritoneal fluid from septic mice was analyzed by isotope dilution GC/MS. We were aware that ex vivo formation of 3-nitrotyrosine can be a major problem during analysis of biological materials (6, 7, 9, 10). Indeed, some studies have suggested that virtually all of the 3-nitrotyrosine detected in plasma and tissue is generated artifactually (6, 7, 9). To circumvent this problem, we used a method that avoids strongly acidic conditions during sample preparation and amino acid derivatization (7, 10, 23). To confirm that any 3-nitrotyrosine detected in inflammatory fluid was endogenous rather than artifactual, an isotope-labeled tyrosine (L-[¹³C₉, ¹⁵N]tyrosine) was routinely added to each sample before analysis. We reasoned that any procedure that converted endogenous tyrosine to 3-nitrotyrosine would also convert L-[¹³C₉, ¹⁵N]tyrosine to 3-nitro[¹³C₉, ¹⁵N]tyrosine. The latter would be detectable by GC/MS because its m/z differs from those of 3-nitrotyrosine and the internal standard. Thus, its appearance would indicate that artifactual oxidation had taken place.

To determine whether L-tyrosine is nitrated during sepsis, the peritonea of wild-type mice were infected with K. pneumoniae. Sixteen hours later, the peritoneum was lavaged to obtain fluid containing white blood cells, bacteria, and inflammatory exudate. This lavage fluid was immediately frozen and stored at –80°C. For analysis, the fluid was thawed and centrifuged, and the supernatant was harvested. Less than 3% of the free tyrosine in the lavage fluid remained in the cellular pellet, demonstrating that free tyrosine from both the cellular and extracellular components of peritoneal lavage fluid was recovered quantitatively by this procedure.

Isotope dilution GC/MS was used to quantify free 3-nitrotyrosine in the supernatant (7, 10, 23). The derivatized material contained a compound that exhibited the major ion and retention time identical to those of authentic 3-nitrotyrosine. Selected ion monitoring showed that the ion derived from the amino acid (Figure 5a) co-eluted with the ion derived from ¹³C-labeled internal standard (3-nitro[¹³C₆]tyrosine; Figure 5b). In contrast, there was little evidence for 3-nitrotyrosine formation during sample work-up and analysis (3-nitro[¹³C₉, ¹⁵N]tyrosine; Figure 5c). These observations indicate that free 3-nitrotyrosine is detectable in peritoneal lavage fluid from mice infected with K. pneumoniae and that the compound is not generated ex vivo during analysis.

Figure 5

Detection of free 3-nitrotyrosine in peritoneal inflammatory exudate of a wild-type mouse infected with K. pneumoniae. Amino acids were isolated from peritoneal fluid by solid-phase chromatography, converted to their heptafluobutyryl t-butyl-dimethylsilyl derivatives, and subjected to GC/MS analysis. (a) Endogenous (m/z 518), (b) isotope-labeled (m/z 524), and (c) artifactual (m/z 528) 3-nitrotyrosine was monitored simultaneously using selected ion monitoring in the negative ion electron capture mode.

Myeloperoxidase generates 3-nitrotyrosine during acute inflammation. To determine whether myeloperoxidase could have generated the 3-nitrotyrosine observed in peritoneal fluid, we investigated 3-nitrotyrosine formation in two models of acute inflammation characterized by an intense neutrophil response (23, 24). The first model (described above) employed the intraperitoneal injection of K. pneumoniae. Neutrophils were the predominant cells in lavage fluid from both the wild-type animals (n = 3; 97% ± 1% neutrophils, 1% ± 0% macrophages, 2% ± 1% eosinophils) and the myeloperoxidase-deficient animals (n = 3; 96% ± 1% neutrophils, 2% ± 1% macrophages, 3% ± 2% eosinophils). Occasional red blood cells and abundant intracellular and extracellular microorganisms were also apparent. Importantly, the cellular response to sepsis of the myeloperoxidase-deficient animals was comparable to that of the wild-type animals.

The second model was a clinically relevant mouse model that creates sepsis through cecal ligation and puncture (CLP), which allows intestinal bacteria to leak into the peritoneum. Neutrophils predominate in the lavage fluid, and myeloperoxidase deficiency does not affect their abundance (23). However, the peritoneal inflammatory fluid contains more red blood cells and cellular debris than does lavage fluid from mice infected with K. pneumoniae.

Peritoneal lavage fluid from wild-type mice subjected to either CLP or infection with K. pneumonia had markedly higher levels of immunoreactive myeloperoxidase than did peritoneal lavage fluid from control mice. In contrast, peritoneal fluid isolated from the septic myeloperoxidase-deficient mice contained no immunoreactive protein. We have previously shown that myeloperoxidase-deficient mice produce active eosinophil peroxidase (23). These results indicate that the number of myeloperoxidase-containing cells increases markedly in the normal mouse peritoneum during sepsis, that myeloperoxidase-deficient mice lack immunoreactive myeloperoxidase, and that both the wild-type mice and the genetically altered mice make eosinophil peroxidase.

Previous studies have demonstrated that neutrophils employ myeloperoxidase to generate chlorinating intermediates in vivo (23). Consistent with these observations, we detected a marked increase in levels of 3-chlorotyrosine in both models of inflammation (Figure 6a). This increase was almost completely abrogated in myeloperoxidase-deficient mice. To determine whether myeloperoxidase similarly generates nitrating species in vivo, 3-nitrotyrosine levels were quantified by isotope dilution GC/MS (Figure 6b). In this analysis, we observed a striking difference between the two models. Peritoneal lavage fluid from wild-type mice and myeloperoxidase-deficient mice subjected to CLP contained similar levels of 3-nitrotyrosine (Figure 6b). However, the level of 3-nitrotyrosine in lavage fluid from wild-type mice infected with K. pneumoniae was 2.5-fold higher than the level in fluid from myeloperoxidase-deficient animals (Figure 6b) (P < 0.01). The latter was similar to the levels observed after wild-type mice and myeloperoxidase-deficient mice were subjected to CLP. Collectively, these results indicate that a myeloperoxidase-dependent pathway nitrates tyrosine during the acute inflammation that follows K. pneumoniae infection but not during the tyrosine nitration that occurs after CLP.

Figure 6

Quantification of (a) free 3-chlorotyrosine and (b) free 3-nitrotyrosine in peritoneal fluid isolated from wild-type and myeloperoxidase-deficient mice subjected to either CLP or infection with K. pneumoniae. 3-Chlorotyrosine (3-Cl-Tyr) and 3-nitrotyrosine (3-NO₂-Tyr) were quantified by isotope dilution GC/MS with selected ion monitoring in negative ion electron capture mode. KP, infection with K. pneumoniae.

K. pneumoniae infection markedly increases NO₂– and NO₃– levels. Myeloperoxidase generates reactive nitrogen species in vitro by a reaction pathway that requires NO₂^–. To determine whether NO₂^– is available during inflammation, levels of NO₂^– and NO₃^– were quantified in peritoneal lavage fluid from mice subjected to the two different models of inflammation. Levels of total NO₂^– and NO₃^– were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to CLP (Figure 7). Levels of total NO₂^– and NO₃^– were similar in sham-operated mice and mice subjected to CLP (data not shown). There was no significant difference in the levels of total NO₂^– and NO₃^– between the wild-type mice and myeloperoxidase-deficient mice in either model. These observations strongly suggest that myeloperoxidase requires elevated levels of NO^• or products derived from NO^• to produce reactive nitrogen species in vivo.

Figure 7

Quantification of NO₂^– and NO₃^– levels in peritoneal fluid isolated from wild-type and myeloperoxidase-deficient mice subjected to either CLP or infection with K. pneumoniae. Total NO₂^– and NO₃^– in peritoneal lavage fluid was quantified using the Griess reagent.

More than 40 years ago, Chance demonstrated that NO₂^– can serve as a substrate for peroxidases (36). Moreover, nitrating oxidants are implicated in host defense mechanisms and the pathogenesis of many diseases (1, 3, 5). Recent studies show that myeloperoxidase and other peroxidases use H₂O₂ and NO₂^– to nitrate tyrosine in vitro (15–18, 37), leading to the proposal that peroxidases contribute to the generation of reactive nitrogen species and inflammation in vivo. However, it is not yet established whether peroxidases are physiologically relevant sources of nitrating oxidants.

To determine whether myeloperoxidase nitrates tyrosine in vivo, we used isotope dilution GC/MS to quantify 3-nitrotyrosine levels in lavage fluid from septic mice, using two models of acute peritoneal inflammation. We found that, after infection with K. pneumoniae, wild-type mice had markedly higher levels of free 3-nitrotyrosine than did myeloperoxidase-deficient mice. After CLP, however, wild-type and myeloperoxidase-deficient mice had comparable levels of the nitrated amino acid. Although both models of intraabdominal inflammation were characterized by an intense neutrophil response and a marked increase in levels of 3-chlorotyrosine, a marker of myeloperoxidase action, they differed in one important respect. Levels of total NO₂^– and NO₃^– were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to CLP. These results indicate that myeloperoxidase generates oxidants that can nitrate tyrosine after mice are infected with K. pneumoniae. They also suggest that the enzyme generates these oxidants only under conditions associated with marked increases in NO₂^– and NO₃^–. Our observations strongly support the proposal that myeloperoxidase uses NO₂^– or other NO^•-derived species to produce inflammatory nitrating oxidants in vivo.

3-Nitrotyrosine was detectable in myeloperoxidase-deficient mice, indicating that pathways independent of the enzyme contribute to 3-nitrotyrosine formation in vivo. Potential mechanisms include uptake of 3-nitrotyrosine from the gastrointestinal track (perhaps generated by the interaction of NO₂^– and tyrosine in the acidic milieu of the stomach) (38), reactive nitrogen species generated by other peroxidases such as eosinophil peroxidase (37), and ONOO^– derived from the interaction of superoxide and NO^• (4, 5). It is interesting to note that myeloperoxidase-deficient mice injected with K. pneumoniae increased their levels of total NO₂^– and NO₃^– 20-fold but failed to demonstrate any increase in 3-nitrotyrosine. These observations strongly suggest that all of the excess 3-nitrotyrosine generated in the K. pneumoniae model of inflammation is myeloperoxidase-dependent, even though infection with this bacterium markedly increases the production of NO^•, which can be converted into ONOO^–.

Myeloperoxidase is secreted both extracellularly and into the phagolysosome, suggesting that it might generate reactive nitrogen species in either environment. The immunochemical detection of 3-nitrotyrosine in bacteria ingested by cytokine-treated human neutrophils suggests that the enzyme might nitrate in the phagolysosome (35). However, studies using a fluorescent probe failed to demonstrate that reactive nitrogen species are generated in the phagolysosome of human neutrophils (39). Our immunohistochemical results suggest that mouse neutrophils can nitrate cytoplasmic proteins. The reaction appears optimal when physiologically relevant concentrations of NO₂^– are provided, but we observed a readily detectable level of nitration by wild-type mouse neutrophils even in the absence of NO₂^–. In contrast, myeloperoxidase-deficient mouse neutrophils were apparently unable to nitrate cytoplasmic proteins in the presence or absence of NO₂^–, indicating that protein nitration in this system was completely dependent upon myeloperoxidase.

At least two factors might contribute to the discrepancy between the immunohistochemical and chemical studies. First, immunohistochemistry might be more sensitive than HPLC for detecting 3-nitrotyrosine. It is also possible that the antibodies used for the immunohistochemical studies react with a molecule that is structurally distinct from 3-nitrotyrosine. Second, mouse and human neutrophils might differ in their ability to execute nitration reactions intracellularly or in the phagolysosome (1). One particularly important factor might be the much higher levels of inducible nitric oxide synthase in mouse neutrophils than in human neutrophils (40–42). These observations suggest that the myeloperoxidase system of mouse neutrophils uses at least two pathways to generate reactive nitrogen species. One involves exogenous NO₂^– and/or other species derived from NO^•, and the other involves products derived from inducible nitric oxide synthase in the neutrophil itself. Because human neutrophils contain very low levels of inducible nitric oxide synthase (40–42), they are unlikely to generate reactive nitrogen species intracellularly or in the phagolysosome (39). Instead, human neutrophils are likely to rely on NO₂^– from other sources to generate nitrating oxidants in the extracellular environment. The inflammatory milieu provides all of the factors — numerous phagocytes and a high local concentration of NO^• — necessary for the generation of nitrating oxidants by myeloperoxidase (1).

A key question is whether reactive nitrogen species generated by myeloperoxidase promote tissue damage. For example, enzymatically active myeloperoxidase is present in human atherosclerotic lesions, and 3-nitrotyrosine has been detected in LDL isolated from this tissue (43, 44). Reactive nitrogen species produced by myeloperoxidase peroxidize the lipid moieties of LDL in vitro, converting the lipoprotein to a form that is recognized by the macrophage scavenger receptor (29, 45). Unregulated uptake of such modified lipoprotein may play a role in cholesterol accumulation by macrophages, a critical early step in atherogenesis (45). The detection of elevated levels of 3-nitrotyrosine and 3-chlorotyrosine in LDL isolated from atherosclerotic lesions suggest that reactive intermediates derived from myeloperoxidase may indeed promote atherosclerotic vascular disease (43, 44, 46).

Reactive nitrogen species produced by phagocytic white blood cells might also play a key role in host defense mechanisms by interacting with bacterial proteins, disabling them, and causing dysfunction of the bacterium (1). In a mouse model of Gram-negative pneumonia, products derived from nitric oxide synthase play a critical role in killing K. pneumoniae (47). We found that NO₂^– enables myeloperoxidase to kill K. pneumoniae efficiently at neutral pH, and we have recently shown that neutrophils use myeloperoxidase to generate antimicrobial chlorinating and brominating intermediates in vivo (23), raising the possibility that reactive nitrogen species also promote bacterial killing. An important unresolved issue is the identity of the cellular machinery targeted for damage during oxidative bacterial killing. To approach this issue, we used immunoprecipitation coupled with mass spectrometry to identify bacterial proteins targeted for nitration by the myeloperoxidase-H₂O₂-NO₂^– system in vitro. Our results revealed one susceptible protein; its sequence showed similarities to a bacterial metalloproteinase. Interestingly, metalloproteinases produced by pathogenic microorganisms might be toxic to hosts (48). In future studies, it will be important to identify other bacterial targets for nitration, to investigate the physiologic function of the metalloproteinase-like protein that we identified, and to determine whether nitration of bacterial proteins by myeloperoxidase might impair the pathogenicity of K. pneumoniae.

In summary, our results show that myeloperoxidase secreted by neutrophils generates 3-nitrotyrosine during acute inflammation under conditions where extracellular levels of NO₂^– and NO₃^– increase markedly. One mechanism might involve direct oxidation of NO₂^– to NO₂^•. Because NO₂^– enables myeloperoxidase to kill K. pneumoniae efficiently at neutral pH, this system might represent a novel host defense mechanism. Moreover, elevated levels of 3-nitrotyrosine have been detected in many acute and chronic human inflammatory diseases. We have recently shown that neutrophils employ the myeloperoxidase system to chlorinate tyrosine residues in vivo (23). Levels of myeloperoxidase, 3-nitrotyrosine, and 3-chlorotyrosine are elevated in human atherosclerotic tissue (43, 44, 46). Therefore, reactive nitrogen species produced by myeloperoxidase might also play a role in inflammatory tissue injury and the pathogenesis of human disease.

We thank William Parks (Washington University) for assistance with immunohistochemical studies. Mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource. This work was supported by grants from the NIH (AG-121191, AG-19309, HL-64344, DK-56341, and RR-00954) and the Washington University–Pharmacia Research Program. J.P. Gaut was supported by a Cardiovascular Training Grant at Washington University School of Medicine and a grant from the Glenn/American Federation of Aging Research.

See the related Commentary beginning on page 1287.

Joseph P. Gaut and Jaeman Byun contributed equally to this work.

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