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(The FASEB Journal. 2001;15:2355-2364.)
© 2001 FASEB

Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite

SILVIA PFEIFFER, ACHIM LASS, KURT SCHMIDT and BERND MAYER1

Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria

1Correspondence: Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. E-mail: mayer{at}kfunigraz.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosine nitration is considered a key reaction of peroxynitrite-triggered tissue injury in inflammatory diseases. We investigated the potential involvement of peroxynitrite in protein tyrosine nitration in isolated murine peritoneal macrophages activated either in vitro with interferon-{gamma}/lipopolysaccharide or in vivo by priming mice with Corynebacterium parvum (10 mgxkg-1). Both protocols led to release of NO and accumulation of nitrite accompanied by formation of protein-bound 3-nitrotyrosine. Oxidation of dihydrorhodamine 123, a measure of peroxynitrite release, remained close to basal levels upon in vitro activation of the macrophages but was increased ~twofold in vivo. Tyrosine nitration in macrophages activated in vitro was inhibited by catalase and the time course of nitration correlated with nitrite accumulation, whereas superoxide (O2•-) and H2O2 release occurred at much earlier times. To address the contribution of O2•- and peroxynitrite to in vivo nitration, a O2•- scavenger (MnTBAP; 1 mgxkg-1) was given to C. parvum-primed mice. MnTBAP led to almost complete inhibition of C. parvum-triggered O2•- and peroxynitrite release, whereas nitrite accumulation and formation of 3-nitrotyrosine were less affected (~50% of controls). These results argue against an essential role of peroxynitrite in protein tyrosine nitration in vivo.—Pfeiffer, S., Lass, A., Schmidt, K., Mayer, B. Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite.


Key Words: 3-nitrotyrosine • nitric oxide • superoxide anion • activated macrophages • myeloperoxidase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACTIVATION OF MACROPHAGES and neutrophils is of critical importance in the process of nonspecific cellular immune response, resulting in the killing of pathogens and inflammatory tissue injury. Macrophage activation is associated with the induction of inducible nitric oxide synthase (iNOS) and sustained release of NO (1 , 2) . NO itself is not particularly toxic to cells, but may form secondary oxidants that are responsible for cytotoxicity. An important pathway that enhances NO cytotoxicity is the rapid reaction with superoxide anion radicals (O2•-) to yield peroxynitrite (ONOO–), which has a wide range of biological activities, including oxidation of biomolecules and protein tyrosine nitration (3) . It is widely accepted that peroxynitrite is a key mediator of tissue injury in conditions of oxidative stress. However, this concept is challenged by several observations indicating that peroxynitrite formation and action both require special conditions. Despite the high rate of NO reacting with O2•-, scavengers present at comparably high concentrations in cells, in particular SOD and GSH, may prevent peroxynitrite formation (4) . Moreover, oxidation reactions triggered by free radical decomposition products of peroxynitrite have been shown to require precisely balanced rates of NO and O2•- formation (5) , a condition that may occur rarely in a complex biological system. However, a more recent study suggests that oxidative pathways involving a direct reaction with peroxynitrite are not affected by excess NO or O2•- (6) .

Another potential problem in the concept of peroxynitrite as a cytotoxin in human disease is the apparent failure of human mononuclear phagocytes to express iNOS in response to cytokine mixtures that induce high-level expression of the enzyme in rodent phagocytes. However, though human neutrophils appear to express neuronal NOS rather than iNOS (7 , 8) , there is abundant evidence that numerous human diseases are associated with iNOS expression in monocytes/macrophages and that isolated human monocytes do express iNOS in vitro upon microbial infection and other immunological stimuli (for a detailed review of the literature on iNOS expression in human mononuclear phagocytes, see ref 9 ).

Besides the oxidation of cellular biomolecules, peroxynitrite-triggered nitration of protein tyrosine residues is of particular interest in human pathology. The product 3-nitrotyrosine has been identified in numerous inflammatory and infectious diseases, including atherosclerosis, coronary artery disease, congestive heart failure, host-vs.-graft disease, amyotrophic lateral sclerosis, Parkinson’s disease, and stroke (10) . The functional consequences of protein nitration have not been fully clarified, but in some diseases 3-nitrotyrosine formation may be causally linked to pathogenesis through interference with enzyme function (11 , 12) , structural protein assembly (13 , 14) , or modulation of signaling cascades (15 , 16) .

Most of our knowledge regarding tyrosine nitration and other biological effects of peroxynitrite is derived from results of treating biological material such as tissues, cells, or isolated proteins with alkaline solutions of chemically synthesized peroxynitrite. In situ generation of peroxynitrite from continuous fluxes of NO/O2•- would obviously be a better approximation to the in vivo situation. We and others have addressed this issue recently and obtained quite divergent results, indicating distinct chemical properties of peroxynitrite generated in situ from NO/O2•- compared with bolus addition of the authentic compound. Three conceivable scenarios could account for these differences. First, peroxynitrite generated from NO/O2•- in situ might be chemically different from the synthesized authentic compound, but this is rendered unlikely by both calculations and experimental data. Second, the distinct behavior of NO/O2•- could be a result of reduced peroxynitrite formation through scavenging of either reactive species. For example, peroxynitrite fails to trigger S-nitrosation of thiols in the presence of CO2 whereas this reaction occurs at fairly high yields with NO/O2•- (4 , 17) , presumably due to scavenging of NO by thiyl radicals giving the corresponding S-nitrosothiol. Finally, certain reactions may rely on the steady-state concentrations of peroxynitrite, which are orders of magnitude higher upon bolus addition than during continuous fluxes of NO/O2•-. In fact, a pronounced concentration dependence of reaction yields appears to explain the poor nitrating efficiency of peroxynitrite generated from NO/O2•- (18 19 20 21 22) vs. bolus addition of either the authentic compound or rapidly mixed solutions containing NO and O2•- at millimolar concentrations (23) . Thus, despite one report claiming the opposite (24) , studies from at least four independent laboratories (18 19 20 21 22) that used different methodological approaches suggest that tyrosine nitration triggered by authentic peroxynitrite has to be judged as an experimental artifact resulting from extremely high initial concentrations of peroxynitrite added as a bolus to tyrosine-containing samples.

However, the physiological relevance of these results obtained under fairly artificial in vitro conditions remains uncertain. In vivo, the nitrating efficiency of peroxynitrite could be enhanced dramatically by various parameters such as the accessibility and concentration of protein tyrosine residues in cells, the site of NO/O2•- generation, or the presence of co-oxidants in macrophages or adjacent inflammatory cells. Recent results suggest that peroxynitrite does not essentially contribute to protein tyrosine nitration in murine RAW 264.7 macrophages (S. Pfeiffer, A. Lass, K. Schmidt, and B. Mayer, unpublished results), an observation that cannot necessarily be extended to primary macrophages triggering tissue injury in inflammatory processes in vivo.

In the present study, we have addressed this issue and investigated the potential role of peroxynitrite in tyrosine nitration in primary murine peritoneal macrophages. For in vitro activation, macrophages were isolated from thioglycollate-treated mice and activated with interferon {gamma} (IFN-{gamma})/lipopolysaccharide (LPS) for various periods to obtain time-resolved information on the formation of several putative mediators of nitration. To study nitration in vivo, peritoneal macrophages were isolated from mice subjected to systemic inflammation by treatment with heat-inactivated Corynebacterium parvum (25 , 26) . Results indicate that in vitro activation with IFN-{gamma}/LPS and in vivo administration of C. parvum both trigger pronounced protein tyrosine nitration in murine peritoneal macrophages, which apparently occurs in an peroxynitrite-independent manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All solvents used were of HPLC grade. Dihydrorhodamine 123 (DHR) and 3-nitrotyrosine were from Fluka-Sigma (Vienna, Austria). Rabbit anti-human myeloperoxidase (MPO) antibody was from DAKO (Vienna, Austria) and purified human MPO was from Planta Naturstoffe (Vienna, Austria). Recombinant mouse IFN-{gamma} and Pronase from Streptomyces griseus were from Roche Diagnostics GmbH (Vienna, Austria). Manganese (III) tetrakis(4-benzoic acid)porphyrin (MnTBAP) from Alexis Corporation (Lausen, Switzerland) was dissolved in DMSO (100 mg/ml) and diluted with PBS (8 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Penicillin, amphotericin B, and fetal calf serum (FCS) were from PAA Laboratories GmbH (Linz, Austria). Centrifuge tube filters (0.22 µm cellulose acetate) were from Szabo (Vienna, Austria). LPS from Salmonella typhosa, bovine erythrocyte SOD, horse heart ferricytochrome c (type VI), and all other chemicals were from Sigma. Heat-inactivated C. parvum was kindly provided by Dr. M. Blobner (Department of Anesthesiology, Technical University Munich, Germany). Polyethylene glycol-labeled SOD (Peg-SOD) and catalase (Peg-Cat) were prepared according to Beckman et al. (27) .

Animals
C57BL mice (3–4 months of age) of either sex were used in this study. Mice were housed under controlled conditions and fed standard cow and water ad libitum. Animals were maintained at the departmental animal care facility of the University of Graz in accordance with guidelines of the Recommendations from the Declaration of Helsinki adopted by the Austrian Council on Animal Care.

In vitro activation of thioglycollate-elicited murine peritoneal macrophages
Mice were injected intraperitoneally (i.p.) with 1 ml of thioglycollate broth [composition in g/l: casein hydrolysate (17) , soy peptone 110 (3) , L-cystine (0.25), glucose (6) , sodium chloride (2.5), sodium thioglycollate (0.5), sodium sulfite (0.1), agar (0.7)] as described (28) . After 3 days, mice were killed by cervical dislocation and macrophages were washed from the peritoneal cavity with 10 ml of sterile PBS and centrifuged at 170 g for 5 min. The pellet was resuspended in Dulbecco’s modified Eagle medium supplemented with 10% (v/v) heat-inactivated FCS, penicillin (100 units/ml), amphotericin B (1.25 µg/ml), and NaHCO3 (3.7 g/l) (DMEM/FCS). The macrophages were plated in either Petri dishes (for subsequent determination of NO and 3-nitrotyrosine) or 12-well plates (to determine O2•-, H2O2, and DHR oxidation). Approximately 4 x 107 macrophages were obtained per mouse and plated in one Petri dish. Cells from two mice were pooled and plated in 12-well plates (~2x106 cells/well). After 3 h of incubation, cells were washed three times with phenol red-free DMEM to remove nonadherent cells and activated with IFN-{gamma} (100 units/ml) plus LPS (0.5 µg/ml) in phenol red-free DMEM/FCS for up to 48 h. Nitrite accumulation in the culture medium was determined with the Griess assay (29) .

Treatment of mice with C. parvum
To induce chronic systemic inflammation, mice were injected with C. parvum suspended in sterile PBS (0.5 mg/ml; 10 mgxkg-1 i.p., unless otherwise indicated) (26) . At the indicated times (usually after 6 days), the mice were killed by cervical dislocation. Peritoneal macrophages were isolated as described above and plated in either Petri dishes (for subsequent determination of NO and 3-nitrotyrosine) or 12-well plates (for determination of O2•- H2O2, and DHR oxidation). Approximately 1 x 107 macrophages were obtained per mouse. The cells from three or four mice were used for plating in a single Petri dish or 12-well plate, respectively. After 3 h of incubation in DMEM/FCS, the cells were washed three times with phenol red-free DMEM to remove nonadherent cells, followed by immediate determination of the various parameters (NO, 3-nitrotyrosine, O2•-, H2O2, and DHR oxidation; see below). Nitrite accumulation in the medium was determined with the Griess assay after 24 h (29) . To study the effect of the SOD mimetic MnTBAP (30) , mice were injected with 10 mg x kg-1 C. parvum on day 0, followed by injection of MnTBAP (0.1 mg/ml in PBS containing 1% (v/v) DMSO; 1 mgxkg-1, unless otherwise indicated) on days 1 through 5. On day 6, peritoneal macrophages were isolated and processed as described above. Treatment of animals with an equivalent volume of PBS containing 0.1% DMSO had no significant effect on the parameters measured in this study (data not shown).

Determination of NO release
At the indicated times, the cells (one Petri dish with ~4x107 macrophages for each experiment) were washed three times with PBS, harvested, centrifuged, and resuspended in 0.5 ml isotonic phosphate buffer, pH 7.5, containing 100 mM Na2HPO4, 1 mM MgCl2, 5 mM KCl, and 12 mM NaCl. NO release was continuously monitored with a Clark-type NO-sensitive electrode (Iso-NO, World Precision Instruments, Berlin, Germany) at 37°C as described previously (31) . After 1 min, 5 µl of a 0.1 M solution of L-arginine (final concentration 1 mM) was added. NO formation was quantified from the initial release rates obtained after addition of L-arginine using the CHART® software program for Apple Macintosh.

Determination of O2•- release
The rates of O2•- generation from macrophages in 12-well plates were measured photometrically as Peg-SOD-inhibitable reduction of acetylated ferricytochrome c as described (32) . At the indicated times, the cells were washed three times with PBS and equilibrated for 30 min in PBS, followed by incubation for 45 min in PBS containing 10 µM acetylated cytochrome c in the presence or absence of 150 units Peg-SOD/ml. The supernatant was centrifuged at 1300 g for 3 min and absorbance was determined at 550 nm against the Peg-SOD-containing samples. The rates of cytochrome c reduction were calculated using an extinction coefficient of 27,700 M-1 x cm-1 at 550 nm (33) .

Determination of H2O2 formation
The rates of H2O2 formation were measured fluorometrically as horseradish peroxidase-catalyzed oxidation of scopoletin as described (34) . Macrophages were washed three times with PBS and incubated for 45 min with an assay mixture containing 30 µM scopoletin, 1 mM NaN3, and 10 units/ml horseradish peroxidase in Krebs-Ringer phosphate buffer (KRP; 129 mM NaCl, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4, 15.8 mM Na2HPO4, pH 7.4). The supernatant was subsequently centrifuged at 1300 g for 3 min, followed by determination of the fluorescence with a RF-5000 spectrofluorometer from Shimadzu at excitation and emission wavelengths of 305 nm and 470 nm, respectively. The fluorescence of the assay mixture without cells was subtracted as blank. The method was calibrated with standard solutions of H2O2 adjusted photometrically using an extinction coefficient of 40 M-1 x cm-1 at 240 nm.

Determination of peroxynitrite formation
Oxidation of DHR was determined and used as an indirect measure of peroxynitrite formation (35) . At the indicated times, macrophages were washed three times with PBS and incubated for 45 min in PBS containing 0.1 mM DHR and 0.1 mM of the metal chelator diethylenetriamine pentaacetic acid. The supernatants were centrifuged at 1300 g for 3 min and absorbance was determined at 500 nm. The absorbance of the assay mixture incubated in the absence of cells was subtracted as blank. DHR oxidation was calculated using an extinction coefficient of 78,800 M-1 x cm-1 at 500 nm (35) .

Determination of protein-bound 3-nitrotyrosine
Protein-bound 3-nitrotyrosine was determined by HPLC and electrochemical detection after derivatization to N-acetyl 3-aminotyrosine (N-AcATyr) as described (36) . Macrophages from six Petri dishes (corresponding to the yields from six thioglycollate- or 18 C. parvum-treated mice) were used for single determinations. The cells were washed three times with PBS, harvested, and centrifuged at 1300 g for 5 min. The cell pellets were resuspended in 0.5 ml 0.1 M potassium phosphate buffer, pH 7.4, giving protein concentrations of 6–9 mg/ml according to the Bradford method with bovine serum albumin as a standard (37) . Protein was precipitated with 0.5 ml acetonitrile; the samples were thoroughly vortexed and centrifuged (1000 g, 5 min), followed by resuspension of the precipitate in 0.1 M phosphate buffer, pH 7.4, and sonication for ~10 s at 50 watt. This procedure was repeated three times to remove nonprotein material. The final suspension (in 250 µl buffer) was incubated for 15–18 h at 50°C with 1–2 mg Pronase and 0.5 mM CaCl2. Samples (~300 µl) were centrifuged (20,000 g) and an equal volume of 3 M potassium phosphate buffer, pH 9.6, was added, followed by the addition of acetic anhydride (20 µl). After 10 min of incubation at ambient temperature, ethyl acetate (1 ml) and formic acid (135 µl) were added. The samples were vortexed for 30 s and then centrifuged at 20,000 g for 1 min. The ethyl acetate phase was concentrated to dryness under a stream of N2 at 45°C. For deacetylation of the phenolic acetate group, the samples were resuspended in 1 N NaOH (60 µl). After 30 min of incubation at 37°C, 1 M potassium phosphate buffer (pH 6.5) (60 µl) was added, followed by addition of 0.1 M sodium dithionite (10 µl) to reduce the nitro substituent to the corresponding amine. The samples were incubated for 10 min at ambient temperature, acidified by the addition of concentrated HCl (20 µl), and centrifuged at 20,000 g for 10 min in centrifuge tube filters. Aliquots (100 µl) were injected onto a 250 x 4 mm C18 reversed phase column (LiChrospher 100 RP-18, 5 µm particle size; Merck, Vienna, Austria) and eluted with 10 mM H3PO4 at 0.7 ml/min. The performance of the column decreased gradually in time. This loss in resolution was overcome by supplementing the solvent with up to 2% (v/v) methanol. N-AcATyr was detected electrochemically with an ESA Coulochem II detector. The potentials of the two electrodes were set to -70 mV and +70 mV (vs. Pd), respectively. Calibration was performed daily with authentic N-AcATyr (5–500 mM) prepared as described (36) . The recovery of authentic 3-nitrotyrosine added to peritoneal macrophage homogenates was 75.6 ± 7.1% (n=3).

Detection of MPO by immunoblotting
Cell homogenates were subjected to SDS-PAGE on 12% slab gels (38) and transferred onto nitrocellulose membranes in 25 mM Tris/HCl, pH 8.3, containing 192 mM glycine, 0.02% (w/v) SDS, and 20% (v/v) methanol at 250 mA for 90 min. Unspecific binding sites were saturated by overnight incubation of the membranes at 4°C in 20 mM Tris/HCl buffer, pH 7.7, containing 137 mM NaCl, and 0.05% (w/v) Tween 20 (TBST) supplemented with 3% (w/v) ovalbumin. The membranes were then washed twice for 5 min, followed by incubation for 2 h with the antimyeloperoxidase antibody diluted 1:500 in TBST containing 0.3% (w/v) ovalbumin. Membranes were washed twice for 15 min with TBST and incubated for 1 h with horseradish peroxidase-labeled anti-rabbit IgG antibody that had been diluted 1:5000 in TBST buffer containing 0.3% (w/v) ovalbumin. Finally, the membranes were washed three times for 20 min with TBST buffer and processed with the ECL Western blotting detection system according to the recommendations of Amersham (Arlington Heights, IL).

Data evaluation
Release rates are given throughout as the amount of product (pmol or nmol) per minute and milligram of total cell protein. The levels of protein-bound N-AcATyr are expressed as picogram per milligram of total cell protein. Results are mean values ± SE of n experiments as indicated in the text or figure legends. If necessary, the statistical significance of the data was evaluated using Student’s t test. P values of <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time course of NO synthesis by IFN-{gamma}/LPS-activated peritoneal macrophages
Treatment of peritoneal macrophages with IFN-{gamma}/LPS led to induction of NO synthesis, which was apparent from the pronounced release of NO and nitrite accumulation in the medium. Figure 1 shows the time course of NO release (measured with a NO-sensitive electrode in macrophage suspensions; filled symbols) and nitrite accumulation measured photometrically in the culture supernatant (open symbols). Release of NO was not detectable during the first 10 h but then increased rapidly, reaching a maximum of 219 ± 41 pmol x min-1 x mg-1 22 h after addition of IFN-{gamma}/LPS. NO release declined at later times and was undetectable 48 h poststimulation. Release of NO was accompanied by an accumulation of nitrite up to a concentration of 83.2 ± 7.6 µM after 48 h



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  		Figure 1. Formation of NO and nitrite by thioglycollate-elicited mouse peritoneal macrophages activated with IFN-{gamma}/LPS in vitro. Thioglycollate-elicited mouse peritoneal macrophages were incubated in the presence of IFN-{gamma} (100 U/ml) and LPS (0.5 µg/ml) for up to 48 h. At the times indicated, NO release (filled symbols) and nitrite concentrations in the culture supernatants (open symbols) were measured as described in Materials and Methods. Data are mean values ± SE of 4–14 (NO) and 6–21 (nitrite) experiments.

Time course of O2•-, H2O2 and peroxynitrite formation by IFN-{gamma}/LPS-activated peritoneal macrophages
In addition to induction of iNOS, activation of macrophages with IFN-{gamma}/LPS resulted in a burst of O2•- and H2O2 production, albeit at much earlier times (Fig. 2 A). Maximal rates of O2•- release (86±5.2 pmolxmin-1xmg-1) were observed 4 h after addition of IFN-{gamma}/LPS, followed by a rapid decline within the next 4 h (Fig. 2A , filled symbols). No significant release of O2•- was detectable throughout the remaining incubation period (up to 48 h). The time course of H2O2 generation was similar but shifted later (Fig. 2A , open symbols). Maximal rates of 1.8 ± 0.1 nmol H2O2 x min-1 x mg-1 were measured 7 h after IFN-{gamma}/LPS, followed by a decline to basal rates within the next 4 h. H2O2 formation was close to basal levels during the remaining incubation period (from 12 to 48 h).



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  		Figure 2. Formation of O2•-, H2O2, and peroxynitrite by thioglycollate-elicited mouse peritoneal macrophages activated with IFN-{gamma}/LPS in vitro. Thioglycollate-elicited mouse peritoneal macrophages were incubated in the presence of IFN-{gamma} (100 U/ml) and LPS (0.5 µg/ml) for up to 48 h. Release of O2•- (A, filled symbols) and formation of H2O2 (A, open symbols) were measured as described in Materials and Methods. Oxidation of DHR was determined as a measure of peroxynitrite formation (B). Data are mean values ± SE of 6–12 (O2•-) and 3–6 (H2O2, DHR oxidation) experiments. {dagger}Not detectable.

Peroxynitrite formation by the IFN-{gamma}/LPS-activated macrophages was rendered unlikely by the striking mismatch in the time course of iNOS induction and O2•- formation. In accordance with these data, DHR oxidation (determined as a measure of peroxynitrite generation) was barely detectable and remained close to background levels (3.6±1.0 pmolxmin-1xmg-1 at time 0) throughout the incubation period of 48 h (Fig. 2B ).

Time course of protein tyrosine nitration by IFN-{gamma}/LPS-activated peritoneal macrophages: potential involvement of MPO
Protein-bound 3-nitrotyrosine was measured in lysates of activated peritoneal macrophages as the N-AcATyr derivative by HPLC and electrochemical detection. As shown in Fig. 3 , treatment of the cells with IFN-{gamma}/LPS led to detectable nitration after 12 h, followed by a continuous increase in the N-AcATyr levels (120.9±14.5 pgxmg-1 at 48 h). This time course resembled that of nitrite accumulation, suggesting that tyrosine nitration in primary peritoneal macrophages involved a nitrite-dependent mechanism as described previously for peroxidase-catalyzed nitration in neutrophils and eosinophils (39 40 41) . The contribution of peroxidase activity to nitration was tested by measuring 3-nitrotyrosine levels upon removal of cellular H2O2 by incubation of the cells with Peg-Cat. However, as previously reported (42) , we observed that catalase markedly inhibited iNOS activity in RAW 264.7 macrophages by decreasing the availability of the essential cofactor of NO synthase, tetrahydrobiopterin (data not shown). To account for this interference with NO synthesis, Peg-Cat was not added along with IFN-{gamma}/LPS but 10 h later, i.e., at the onset of nitration, followed by incubation for 14 h and determination of protein-bound 3-nitrotyrosine. Under these conditions, nitrite concentrations in the culture medium were 49 ± 1.2 and 34 ± 2.9 µM in the absence and presence of Peg-Cat. The corresponding N-AcATyr levels were 93.6 ± 17.1 and 29.6 ± 6.6 pg x mg-1. Thus, Peg-Cat reduced the levels of 3-nitrotyrosine to 31.6% of control, whereas NO synthesis was much less affected (69.4% of control), further suggesting that a peroxidase pathway is essentially involved in macrophage protein tyrosine nitration.



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  		Figure 3. Protein tyrosine nitration by thioglycollate-elicited mouse peritoneal macrophages activated with IFN-{gamma}/LPS in vitro. Thioglycollate-elicited mouse peritoneal macrophages were incubated in the presence of IFN-{gamma} (100 U/ml) and LPS (0.5 µg/ml) for up to 48 h. Protein-bound 3-nitrotyrosine was measured as N-AcATyr derivative by HPLC and electrochemical detection as described in Materials and Methods. Data are mean values ± SE of 3–5 experiments.

As MPO and other heme peroxidases have been shown to catalyze nitration in the presence of nitrite and H2O2, we probed lysates of thioglycollate-elicited peritoneal macrophages for MPO by immunoblotting with a specific antibody. As shown in Fig. 4 , the antiserum recognized a protein with an apparent molecular mass of ~57 kDa, which comigrated with the 57 kDa band of purified human myeloperoxidase (lane 1). The staining intensity was not significantly changed on in vitro activation of the cells with IFN-{gamma}/LPS for 18 h (lanes 2 and 3 vs. lanes 4 and 5). These results suggest that murine peritoneal macrophages contain small amounts of MPO that might contribute to tyrosine nitration on induction of NO/nitrite synthesis in these cells.



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  		Figure 4. Detection of myeloperoxidase in lysates of thioglycollate-elicited murine peritoneal macrophages by immunoblotting. Homogenates of thioglycollate-elicited murine peritoneal macrophages (0.1 mg of protein) were probed for MPO by SDS-PAGE and immunoblotting using an antibody raised in rabbits against human MPO. Lane 1: 4 ng of purified human MPO, lanes 2 and 3: nonactivated cells, lanes 4 and 5: cells activated in vitro with IFN-{gamma}/LPS(18 h). The blot shown is representative of three.

In vivo activation of peritoneal macrophages in the C. parvum model of systemic inflammation
Activation of primary macrophages with IFN-{gamma}/LPS in vitro does not necessarily reflect the in vivo response of the cells to inflammatory stimuli. Therefore, we studied a mouse model of systemic inflammation and measured the formation of 3-nitrotyrosine and putative mediators of nitration in peritoneal macrophages isolated form mice treated with heat-inactivated C. parvum. As shown in Fig. 5 A, i.p. injection of C. parvum at a dose of 100 mg x kg-1 led to expression of iNOS in peritoneal macrophages that appeared as a pronounced accumulation of nitrite when the isolated cells were kept in culture for 24 h. As reported previously (26) , induction of macrophage iNOS activity was delayed by several days, reaching a maximum of ~95 µM nitrite 6 days after C. parvum application. Figure 5B shows that the effect of C. parvum was dose dependent. All subsequent measurements were performed with peritoneal macrophages isolated 6 days after i.p. application of 10 mg C. parvum/kg body weight. Following this protocol, the isolated cells produced 64 ± 7.6 µM nitrite within 24 h of incubation. It would have been helpful to obtain time-resolved information on in vivo nitration, as shown for IFN-{gamma}/LPS-activated macrophages. However, treatment with C. parvum yielded ~threefold less macrophages than thioglycollate, resulting in an unacceptable expense of animals for such experiments. Thus, we used a different strategy to obtain additional information. The O2•- scavenger MnTBAP (43) was reported to protect against NO toxicity in vivo, presumably due to interference with peroxynitrite formation and associated tissue injury and tyrosine nitration. MnTBAP was considered a potentially useful tool to test for peroxynitrite-triggered nitration in vivo. Based on preliminary evaluation of the dose-response relationship (not shown), 1 mg x kg-1 was injected daily to C. parvum-treated mice, followed by isolation of peritoneal macrophages on day 6.



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  		Figure 5. Time course (A) and dose-response (B) of nitrite formation by mouse peritoneal macrophages activated with C. parvum in vivo. A) Mice were injected with 100 mg C. parvum/kg body weight. On the days indicated, macrophages were isolated and incubated in phenol red-free DMEM/FCS for 24 h. Nitrite accumulation was determined in the culture supernatants photometrically with the Griess assay. Data are mean values ± SE of 6–9 determinations performed with macrophages isolated from different mice. B) Mice were injected with 1, 10, and 100 mg C. parvum/kg body weight. After 6 days, macrophages were isolated and incubated in phenol red-free DMEM/FCS for 24 h. Nitrite accumulation was determined in the culture supernatants photometrically with the Griess assay. Data are mean values ± SE of 9 determinations performed with macrophages isolated from different mice.

The results obtained with the C. parvum model are summarized in Table 1 . Suspensions of the isolated macrophages released NO at remarkably high rates of ~0.5 nmol x min-1 x mg-1 when supplemented with 1 mM exogenous L-arginine. The rates of O2•- release (44±6.6 pmolxmin-1xmg-1) were in the same range as the maximal rates measured with IFN-{gamma}/LPS-activated macrophages (see Fig. 2A ), but H2O2 formation was ~10-fold lower. The rate of DHR oxidation (12.5±1.7 pmolxmin-1xmg-1) was significantly above the background levels measured with IFN-{gamma}/LPS-activated primary peritoneal macrophages (see Fig. 2B ) or RAW 264.7 cells (S. Pfeiffer, A. Lass, K. Schmidt, and B. Mayer, unpublished results), suggesting that in vivo activation of macrophages does indeed trigger the formation of peroxynitrite. Nevertheless, 3-nitrotyrosine levels (60.1±9.2 pgxmg-1) were in the same range as found in the IFN-{gamma}/LPS-activated cells (see Fig. 3 ), suggesting that peroxynitrite formation did not have a considerable effect on tyrosine nitration in activated macrophages.


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  		Table 1. Effect of a daily dose of 1 mg MnTBAP/kg body weight on in vivo activation of peritoneal macrophages in mice treated for 6 days with heat-inactivated C. parvum (10 mg x kg-1)a

This conclusion was confirmed by results obtained from C. parvum-primed mice treated with MnTBAP. As shown in Table 1 , MnTBAP significantly inhibited C. parvum-triggered O2•- release to 24.6% of controls and reduced the rates of DHR oxidation to basal levels, indicating substantial inhibition of peroxynitrite formation. Unexpectedly, MnTBAP also affected the rates of NO release (70.6% of controls), albeit this effect was not statistically significant due to relatively large variability in the data. Nitrite accumulation, however, was significantly inhibited from 63.8 ± 7.6 µM (n=9) to 29.5 ± 5.8 µM (n=17), corresponding to 46.3% of controls (P<0.05). As expected from the inhibition of NO synthesis, treatment of the mice with MnTBAP led to a reduction of 3-nitrotyrosine levels to 37.1 ± 8.1 pgxmg-1, corresponding to 62.9% of controls (not significant).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that activation of the L-arginine/NO pathway results in pronounced protein tyrosine nitration in primary murine peritoneal macrophages via a mechanism that does not appear to involve peroxynitrite. This was demonstrated in vitro with IFN-{gamma}/LPS-activated peritoneal macrophages isolated from thioglycollate-treated mice as well as in vivo using macrophages from C. parvum-primed animals. The data obtained with the in vitro model were qualitatively similar to those reported previously for cultured RAW 264.7 macrophages activated with IFN-{gamma}/LPS (S. Pfeiffer, A. Lass, K. Schmidt, and B. Mayer, unpublished results). However, in agreement with previous observations (44 45 46) , a clear difference was apparent in the time course of iNOS induction, which occurred with lag phases of ~4 and 12 h after IFN-{gamma}/LPS in RAW cells and primary peritoneal macrophages, respectively. Albeit similar in magnitude, the burst of O2•- occurred later in the primary cells than in RAW cells (4–8 h vs. 1–5 h after IFN-{gamma}/LPS, respectively). The fall in the rates of NO release between 20 and 48 h, accompanied by decreasing rates of nitrite accumulation, was also observed in RAW 264.7 macrophages, though earlier (between 6 and 15 h). It is unlikely that this apparent reduction in iNOS activity was a consequence of substrate depletion, since NO release rates were measured upon addition of 1 mM exogenous L-arginine to the cell suspensions. Alternative explanations would include limited availability of cofactors, particularly tetrahydrobiopterin (42 , 47) , or inactivation of macrophage iNOS by LPS (48) .

The induction of iNOS in thioglycollate-elicited peritoneal macrophages was not associated with detectable generation of peroxynitrite. Although rapid scavenging of peroxynitrite in the cells might have out-competed DHR oxidation, the mismatch in the time course of NO and O2•- formation argues against peroxynitrite as a major reactive nitrogen species formed by IFN-{gamma}/LPS-activated macrophages. Despite this apparent lack of detectable peroxynitrite formation, the activated cells showed pronounced protein tyrosine nitration. The time course of 3-nitrotyrosine formation correlated with nitrite accumulation and was inhibited by catalase, characteristic features of tyrosine nitration catalyzed by heme peroxidases in granulocytes. Several heme peroxidases, including MPO from neutrophils, lactoperoxidase, and eosinophil peroxidase, were shown to use H2O2 for the oxidation of nitrite to ·NO2 radical, a potent nitrating species (39 40 41) . Although macrophages are generally believed to contain little or no heme peroxidases, peroxidase activity has been detected in several types of resident tissue macrophages, including peritoneal macrophages (49 50 51 52) , and a recent study has shown that granulocyte macrophage colony-stimulating factor up-regulates expression of active MPO in macrophages residing in atherosclerotic plaques (53) . Together with the present study demonstrating the presence of small amounts of MPO in thioglycollate-elicited murine peritoneal macrophages, the available experimental evidence renders it likely that MPO or another unidentified heme peroxidase mediates tyrosine nitration in cytokine-activated macrophages under conditions of inflammatory tissue injury.

It is intriguing that peroxidase-catalyzed nitration occurs under essentially the same conditions that favor peroxynitrite formation, i.e., pronounced synthesis of NO to accumulate nitrite and generation of O2•- to provide H2O2 as an oxidant. Together with the lack of peroxidase inhibitors with sufficient selectivity, these similarities make it difficult to pinpoint the mechanism of tyrosine nitration in vivo. In our in vivo model of systemic inflammation, priming mice with heat-inactivated C. parvum led to a pronounced induction of iNOS activity in peritoneal macrophages that was maximal after 6 days, in agreement with a previous report on C. parvum-triggered systemic iNOS expression in mice (26) . Although neither RAW 264.7 cells nor thioglycollate-elicited primary macrophages activated with IFN-{gamma}/LPS in vitro generated detectable amounts of peroxynitrite, treating mice with C. parvum led to an ~threefold increase in DHR oxidation by the isolated peritoneal macrophages. It was beyond the scope of this study to investigate the molecular basis underlying this difference between in vitro and in vivo activation, but it was of interest to clarify whether the observed peroxynitrite formation was involved in macrophage tyrosine nitration.

To address this issue, we used the manganese-based porphyrin MnTBAP, which has been shown to scavenge peroxynitrite and O2•- both in vitro and in vivo (for review, see ref 54 ). Scavenging of peroxynitrite results in inhibition of DHR oxidation (55) , whereas tyrosine nitration becomes enhanced (56) . As a cell-permeable SOD mimetic (43) , MnTBAP blocks the formation of peroxynitrite from NO and O2•- and associated tissue injury in vivo (30 , 57 , 58) . Thus, MnTBAP is expected to decrease rather than increase peroxynitrite-triggered tyrosine nitration in cells and tissues exposed to oxidative stress or immunological stimuli. Indeed, administration of MnTBAP at a dose of 15 mg x kg-1 led to a pronounced reduction of 3-nitrotyrosine staining in rat models of inflammation (58) and septic shock (30) . Notably, nitrite levels were not affected by MnTBAP in these studies. Our results on the effects of MnTBAP in the C. parvum model of systemic inflammation do not fully agree with these previous reports. Although our data confirm that MnTBAP scavenges O2•- and completely inhibits peroxynitrite formation in vivo, we observed only a moderate decrease in 3-nitrotyrosine levels similar in magnitude to inhibition of nitrite accumulation. Preliminary experiments on the dose-response relationship (data not shown) indicated that superoxide scavenging and inhibition of NO synthesis were closely related, i.e., we were unable to find a dose of MnTBAP that effectively scavenged O2•- without affecting nitrite accumulation. MnTBAP had no effect on nitrite formation by IFN-{gamma}/LPS-activated RAW 264.7 macrophages (A. Lass and B. Mayer, unpublished observation), but Szabo and colleagues reported that the porphyrin reduced nitrite accumulation in IFN-{gamma}/LPS-activated aortic smooth muscle cells by ~30% (57) . Thus, it is conceivable that O2•- modulates iNOS induction in a tissue-specific manner, but effects of MnTBAP unrelated to O2•- scavenging are also feasible. In any case, the inhibition of macrophage NO synthesis by MnTBAP appears to fully account for the slight inhibitory effect of the porphyrin on tyrosine nitration in vivo.

Even if produced in detectable amounts, peroxynitrite does not contribute considerably to overall protein tyrosine nitration in activated macrophages, suggesting that the conclusions we and others reached based on studies with noncellular systems and free tyrosine (18 19 20 21 22) can be extended to in vivo nitration. Our data do not exclude that peroxynitrite is involved in the nitration of reactive and readily accessible specific tyrosine residues of certain cellular proteins representing a minor fraction of total cell protein. However, in light of the overwhelming experimental evidence against peroxynitrite as a potent nitrating agent, each case of allegedly peroxynitrite-mediated protein nitration reported in the literature needs to be reinvestigated with respect to the underlying nitrating pathway. This may help to sort out potential targets of coordinated and specifically regulated nitration by peroxynitrite and perhaps reveal novel mechanisms of nitration in mammalian tissues. A detailed knowledge of the underlying molecular mechanisms appears to be crucial to address the pathophysiological relevance of protein tyrosine nitration and to find new drugs that specifically interfere with this pathway in vivo.


   ACKNOWLEDGMENTS
 
We thank Margit Rehn for excellent technical assistance. This work was supported by grant 13784-MED from the ‘Fonds zur Förderung der Wissenschaftlichen Forschung’ in Austria. S.P. is a recipient of an Austrian Academy of Sciences APART fellowship (APART 7/98).

Received for publication April 26, 2001. Revision received July 18, 2001.
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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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