INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: asthma; mice; nitric oxide; 3-nitrotyrosine; eosinophil peroxidase; ovalbumin

Recently, it has been reported that eosinophilic airway inflammation in asthma is associated with protein nitration, detected as immunostaining for 3-nitrotyrosine (3NT) (1, 2). Protein nitration, observed in a wide variety of diseases and inflammatory states, is of interest because of its potential to alter function of affected proteins and also as a marker of oxidative injury (3). Although a variety of pathways may lead to protein nitration in vitro (4), it remains unclear which mechanisms are at work in intact organisms. In particular, the relative importance of nitric oxide (NO) production and the action of granulocyte peroxidases remain to be established. Resolution of this issue is central to understanding the mechanisms that underlie oxidative tissue injury in inflammatory disorders and to the design of novel therapeutic strategies aimed at controlling the inflammatory process.

In asthma, detection of 3NT is closely associated with upregulation of the inducible isoform of nitric oxide synthase (NOS II), suggesting the possibility that protein nitration is driven by increased NO production. In particular, it has been proposed that detection of 3NT is a marker of the action of peroxynitrite (ONOO), a highly reactive NO intermediate formed from superoxide (O₂) and molecular NO (3, 5). In vitro experiments have demonstrated the ability of ONOO to nitrate tyrosine residues on proteins as well as to induce lipid peroxidation and DNA damage and it seems likely that ONOO is produced in high concentration in an oxidative inflammatory milieu such as that in the asthmatic airways (6, 7). The production of ONOO is therefore an attractive hypothesis to explain 3NT staining in allergic inflammation, which is characterized by the influx of a variety of cells, including eosinophils, that have the dual capacity to produce NO and O₂. Evidence of oxidative tissue injury has been found in bronchial biopsies from asthmatics (1, 2) in the form of 3NT, which is closely associated with eosinophils. Indeed, many of the cytotoxic actions of eosinophils depend on their ability to generate reactive oxidizing species (ROS) and the respiratory burst of the eosinophil generates several times as much O₂ and H₂O₂ as the neutrophil (8, 9).

Despite the attractive notion that ONOO is responsible for tyrosine nitration in eosinophilic inflammation, other mechanisms may be at work. There is a very close association between the detection of 3NT and the presence of activated granulocytes. This association, which is found in a variety of inflammatory disorders, is of importance because peroxidases, such as myeloperoxidase and eosinophil peroxidase (EPO), can efficiently nitrate proteins through mechanisms independent of ONOO (10). EPO can use nitrite (NO₂), a major end-product of NO metabolism, as a substrate to efficiently nitrate protein tyrosyl residues (4). In in vitro systems, EPO is an even more potent catalyst of 3NT formation than myeloperoxidase (4), raising the possibility that EPO activity is primarily responsible for 3NT formation in asthma. To date, the extent to which peroxidase activity contributes to protein nitration in vivo remains unknown.

To address this issue, we investigated the relative importance of peroxidase and NO production in the formation of 3NT using models of murine airway eosinophilic inflammation. We first studied ovalbumin (OVA)-sensitized and -challenged New Zealand White (NZW) mice, an inbred murine strain with a spontaneous deficiency of EPO (13). We reasoned that to the extent that protein nitration is dependent on peroxidase activity, EPO-deficient mice would not demonstrate the same level of 3NT formation as other strains despite similar capacity for production of NO and ROS. Additionally, we investigated the role of NOS II expression in protein nitration in OVA-sensitized and -challenged NOS II deficient mutant mice (NOS II KO) and their wild-type controls. We hypothesized that if tyrosine nitration were mainly dependent on EPO, significant levels of tyrosine nitration would be found in these mice, despite the failure of these mice to increase NO production after allergen challenge. Our results indicate that in the mouse, 3NT formation after allergic airway challenge is largely dependent on EPO activity rather than increased NO production, and suggest an important role for EPO in the pathogenesis of allergic airway inflammation.

	METHODS

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Two sets of experiments were performed. Protocol 1 evaluated the role of EPO in protein nitration by comparing NZW mice, deficient in EPO, with two other strains, the A/J and the C57BL/6J. Protocol 2 evaluated the role of NOS II in protein nitration by comparing NOS II deficient mutant mice (14) with their wild-type control, the C57BL/6J strain. The methods for Protocols 1 and 2 were identical except for the number of OVA challenges. O₂ measurements, Western blot for 3NT, and EPO assay were only performed in Protocol 1.

Allergen Sensitization and Challenge of the Mice

A/J and C57BL/6J mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN); NOS II deficient mutant mice (14) and NZW mice were obtained from Jackson Laboratory (Bar Harbor, ME). These male mice were housed in a conventional animal facility at our laboratory. All methods used in these experiments were evaluated and approved by McGill University Animal Care Committee.

Protocol 1. A/J, C57BL/6J, and NZW mice (24 to 26 g body weight), were sensitized subcutaneously with 100 µg OVA (grade V; Sigma Chemical Co., St. Louis, MO) in 0.4 ml of a 4 mg/ml suspension of Al(OH)₃ (Sigma). A second injection was given 7 d later. Seven days after the second injection, mice were challenged once intranasally with 10 µg OVA in 50 µl sterile saline under light anesthesia (45 to 50 s) with halothane (MTC Pharmaceuticals, Cambridge, ON, Canada). The OVA challenge procedure induced a marked but transient decrease in breathing frequency. Tissues were harvested 48 h after the OVA challenge.

Protocol 2. The OVA sensitization protocol for the NOS II KO mice and their wild-type controls, the C57BL/6J strain, was identical to that described in Protocol 1. In preliminary experiments, however, we were unable to satisfactorily replicate the phenotype of A/J or NZW mice using a single OVA challenge. As has been previously reported (15), we found that NOS II KO mice failed to mount as vigorous an eosinophil response to OVA. To ensure a comparable level of eosinophilia to that in Protocol 1, we challenged these mice three times 1 d apart and harvested them 24 h after the last challenge.

Bronchoalveolar Lavage and Tissue Extraction

Mice were deeply anesthetized with 50 mg/kg of sodium pentobarbital (Somnotol; MTC Pharmaceuticals) injected intraperitoneally and exsanguinated by cutting the inferior vena cava. After the chest was opened and while the heart was still beating, 2 to 5 ml of sterile saline was injected in the right ventricle to remove blood from the pulmonary circulation. The upper trachea was then cannulated and the lungs were slowly lavaged (injection and recovery: 45 to 50 s), twice, with 0.6 ml of cold (4° C) Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) containing 0.5 mM ethylenediaminetetraacetic acid (EDTA) (Sigma). The recovered fluid (75 to 80% of the injected volume) was centrifuged at 800 g for 10 min at 4° C and the supernatant stored at 80° C. After resuspension in PBS, some cells were stained with 0.4% trypan blue (Gibco BRL, Grand Island, NY) for total cell number counting. Slides of BAL cells were obtained by centrifuging 2 · 10⁴ cells at 120 g for 2 min (Cytospin 2; Shandon, Pittsburgh, PA). For all animals, one slide was stained with May-Grunwald-Giemsa for differential cell counting. For immunocytochemistry, slides were fixed in acetone-methanol (ratio of 6:4; Fisher Scientific, Pittsburgh, PA) at room temperature (RT) for 7 min and stored at 80° C after 1 h air-drying. After the BAL procedure, the left lung was removed, rinsed with PBS, embedded in OCT Tissue-Tek (Sakura Finetechnical Co., Tokyo, Japan), and then slowly frozen by immersion in isopentane (Fisher Scientific) cooled by liquid nitrogen. Next, 8- to 10-µm sections were cut from the cryostat blocks onto poly-L-lysine (Sigma) coated glass slides (Microm HM500; Microm International, Walldorf, Germany). Slides were then fixed in acetone-methanol (6:4) at RT for 7 min and stored at 80° C after 1 h air-drying. The right lung was homogenized with six volumes of ice-cold tissue lysis buffer consisting of 0.05 M Tris-buffered saline (TBS) pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM sodium fluoride (Sigma). Homogenized samples were incubated for 1 h at 4° C and centrifuged for 10 min at 10,000 × g at 4° C.

3NT Immunoreactivity in BAL and Tissue

After 5 min washing in 50 mM TBS, defrosted slides were incubated with Universal Blocking Solution (Dako Corporation, Carpinteria, CA) for 15 min. The primary antibody was an anti-3NT rabbit polyclonal IgG (1:60 dilution; Upstate Biotechnology, Lake Placid, NY). Slides were incubated with this antibody in a humidified chamber overnight at 4° C. After washing, slides were incubated with 60 µl of biotinylated anti-rabbit swine immunoglobulin (1:300 dilution; Dako) at RT for 50 min. Slides were washed again and either incubated with Streptavidin-AP (1:200 dilution; Dako) and developed with Fast Red (Sigma) or incubated with horseradish peroxidase (HRP) and developed with diaminobenzidine (DAB) as substrate chromogen (Dako). When the DAB was used as chromogen, the endogenous peroxidase activity was blocked by incubating the tissue with 3% H₂O₂ (Sigma) for 30 min at RT and then washed in TBS 10 min three times. Counterstaining was done by hematoxylin. Negative controls included: absence of staining in the presence of an excess of 3NT (Sigma), absence of staining with a nonspecific purified rabbit IgG, absence of staining with preincubation with 500 mM sodium hydrosulfite (dithionite) dissolved in 100 mM sodium borate (Sigma) that reduces 3NT to aminotyrosine (16). 3NT-positive cells were counted in the peribronchial region from 2 to 3 airways in each animal. Cell counts were carried out by applying a 0.115 mm × 0.115 mm counting grid around the circumference of each airway. The counts were expressed as the number of positive cells per millimeter of airway basement membrane (16).

NOS II Immunoreactivity in BAL Cells

BAL slides were incubated overnight with an anti-mouse NOS II rabbit polyclonal IgG (1:70 dilution; Transduction Laboratories, Lexington, KY) and then with a biotinylated anti-rabbit swine immunoglobulin (1:300 dilution; Dako) at RT for 50 min. After washing, slides were incubated with Streptavidin-AP (1:200 dilution; Dako) developed with Vector Blue (Vector Laboratories, Inc, Burlingame, CA). Negative controls included use of buffer alone and dilution of nonspecific purified rabbit IgG in the primary layer. Slides were counterstained with eosin Y (Fisher).

NO₂/NO₃ in BAL Fluid (BALF)

NO_x (defined as the sum of NO₂ and NO₃) was measured in BALF by the colorimetric Griess reaction according to the manufacturer's instructions (Cayman Chemical Co., Ann Arbor, MI). Briefly, 80 µl of sample were mixed with 20 µl of NO₃ reductase and 10 µl of its enzyme cofactor for 3 h at RT, and then incubated with 100 µl of Griess reagent for 10 min. NO_x concentrations were determined using different concentrations of NaNO₂ or NaNO₃ as standard curves. Colorimetric absorbance was measured at 540 nm with a plate reader (SLT 400 ATC; SLT Lab Instruments, Salzburg, Austria). No NO_x was detected in saline solutions using this assay.

Western Blot for 3NT in Whole Lung Homogenates

Protein extracts at a concentration of 100 µg/lane were mixed with sample buffer (63 mM Tris base-pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.0025% bromophenol blue, 10% glycerol, and 5% -mercaptoethanol) and separated on 4 to 12% Tris-Glycine gels (Novex, San Diego, CA) at 125 V for 2 h. Proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Inc., Baie d'Urfé, PQ, Canada) with 12 mM of Tris base, 96 mM of glycine (Fisher), and 20% of methanol (Fisher) at 25 V for 2 h. The transferred membranes were blocked with 1% bovine serum albumin (BSA) in TTBS solution (0.02 M Tris Base-pH 7.5, 0.5 M sodium chloride, and 0.1% of Tween 20; Sigma) at RT for 1 h and then probed with the same anti-3NT rabbit polyclonal IgG as previously described, diluted 1:5,000 in the blocking solution. Membranes were subsequently rinsed in 200 ml of TTBS for 30 min and exposed to the secondary HRP-conjugated antibody (anti-rabbit immunoglobulin from donkey; Amersham Pharmacia Biotech, Inc.) for 1 h at RT. A chemiluminescence detection system (ECL Plus; Amersham) and Hyperfilm (Amersham) were used to detect the binding of this antibody. For 3NT, the positive control consisted of a mixture of three nitrated proteins (bovine superoxide dismutase, BSA, and rabbit muscle myosin; Upstate Biotechnology) according to the manufacturer's instructions.

Western Blot for 3NT in BAL Cells

Enriched eosinophil and macrophage populations from BAL cells were obtained by spontaneous adherence of the macrophages to plastic dishes (17) as follows. Forty-eight hours after OVA challenge, lungs from the three strains were lavaged 10 times with 1 ml of Hanks' balanced salt solution (HBSS). After centrifugation and resuspension in HBSS with 5% fetal bovine serum, cells were plated into a 100-mm Petri dish for 45 min at 37° C. The supernatant consisted mainly of eosinophils with a purity greater than 85%. Cells that adhered to the Petri dish were recovered with a cell scraper (Life Technology, Burlington, ON, Canada) and more than 85% of them were macrophages. After centrifugation, cells were resuspended in lysis buffer, sonicated 5 s (Sonic Dismembrator 60, Fisher). After centrifugation, the supernatant was stored at 80° C until use. The protocol for detection of 3NT by Western blot was similar to that used for lung homogenates except that 30 µg of protein per lane was loaded for the BAL cells.

EPO Assay

To confirm that the NZW mice lack peroxidase activity, we measured EPO activity of BALF from the NZW and the C57BL/6J strains of mice under control and challenged conditions. We used a colorimetric assay based on the oxidation of o-phenylenediamine (OPD, P7288, Sigma) by EPO in the presence of hydrogen peroxide (H₂O₂) (18). A volume of 50 µl of BAL supernatant was transferred into a 96-well microplate in duplicate, and a substrate solution of 0.1 mM OPD, 0.05 M Tris pH 8.0, Triton X-100 (Sigma), and 1 mM H₂O₂ (Sigma) was added to each well. As this reaction is not specific for EPO, samples were measured in the presence of and without 50 mM of 3-amino1,2,4-triazole (AMT, Sigma), a specific inhibitor of EPO. After 60 min incubation at RT, the reaction was stopped by adding 50 µl of 4 M sulfuric acid (Sigma). Colorimetric absorbance was measured at 492 nm with a plate reader (SLT 400 ATC).

O₂ Released by BAL Cells

To confirm that the difference in 3NT staining among the strains was not simply a reflection of different capacities for O₂ production, we measured the amount of superoxide produced by BAL cells from OVA-challenged animals using a chemiluminescence technique. With this method, unstimulated macrophages recovered by the BAL of unchallenged animals had no detectable superoxide release. Spontaneous superoxide release by 10 ⁵ BAL cells from challenged animals was measured as follows (16). BAL cells resuspended in 900 µl of HBSS without calcium chloride or magnesium sulfate and phenol red (Sigma) were placed in a 12 × 75 mm glass tube and incubated for 5 min at 37° C. 100 µl of lucigenin (Sigma) was then added (final concentration 0.23 mM), and the tube was placed in the luminometer (Lumat LB 9507; EG&G Berthold, Wildbad, Germany). The resulting light output was monitored every 30 s for 75 min. Background counts were subtracted. Results were expressed as the highest value obtained (peak count) and the area under the curve (AUC) for the whole period.

Statistical Analysis

To compare different strains of mice, we used one-way analysis of variance; post hoc testing was done with Fisher protected least significant difference (PLSD) correction. Results are expressed as the mean ± SE. A probability level of p < 0.05 was considered statistically significant. Statistics were carried out using the Statview program (ver. 5.0; SAS Institute Inc., Cary, NC).

	RESULTS

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Protocol 1

Eosinophils in BAL after OVA challenge. Total and differential cell counts of BAL cells are shown in Table 1. Under control conditions and in all strains, more than 95% of the cells recovered by the lavage were macrophages. Forty-eight hours after OVA challenge, marked eosinophilia (over 40%) was present in all animals. Both the total cell count and percentage of eosinophils were similar in A/J and NZW mice. The eosinophilic influx in the C57BL/6J strain of mice was significantly lower than in the other strains. Forty-eight h after OVA challenge, neutrophils were also observed in BAL, with similar numbers among the strains. Additional animals were challenged with saline alone to assess the nonspecific effect of saline in the airways. Forty-eight h after saline instillation, the total number of cells, as well as the numbers of eosinophils and neutrophils, were similar to those of unchallenged animals (n = 6 to 8 in all groups, data not shown).

Absence of peroxidase activity in NZW mice. We confirmed the lack of peroxidase activity in the NZW strain of mice by two different methods, using a similar approach to that of Ohmori and coworkers (13). First, an intracellular brown staining was observed in the majority of the A/J and C57BL/6J cells from BAL slides that were incubated with chromogen substrate DAB, confirming the presence of endogenous peroxidase activity. In contrast, no staining was observed in NZW cells. DAB-positive cells from A/J and C57BL/6J mice were positive for major basic protein (MBP) (data not shown). Second, peroxidase activity in BALF from OVA-challenged C57BL/6J mice was substantially higher than that in NZW mice, in which it was nearly absent (Figure 1). We used the specific EPO inhibitor AMT to confirm that observed peroxidase activity was almost entirely accounted for by EPO (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1.   EPO activity was measured in BALF 48 h after OVA challenge with (gray bars) or without AMT (white bars), a specific EPO inhibitor. No significant activity was found in NZW mice despite marked eosinophilia in BAL (see Table 1). Results are mean ± SE with 4 mice per group.

NO production. In all strains, NO_x levels in OVA-challenged animals were higher than in control animals (Figure 2). No significant difference in the NO_x level was found among strains of mice in either the challenged or unchallenged groups (p > 0.1). In addition, we evaluated the expression of NOS II by immunocytochemistry in BAL cells from all strains after OVA challenge. NOS II was present in eosinophils from all three strains. Representative examples are shown in Figures 3D and 3E. Expression of NOS II was also detected by Western analysis of proteins from the eosinophil-rich subset of BAL cells from A/J, C57BL/6J, and NZW mice (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 2.   NOx concentrations (sum of nitrite and nitrate) measured by the Griess reaction in BALF of A/J, C57BL/6J, and NZW strains of mice, unchallenged (control; white bars) or after a single OVA challenge (OVA; gray bars). In all strains NOx concentrations increased after OVA challenge. Under both conditions, no significant difference was found among the strains. Results are mean ± SE, n = 9 to 17 per group. *p < 0.05 versus control.

View larger version (107K): [in this window] [in a new window]   Figure 3.   Panels A-F : BAL cells from OVA-challenged animals were incubated with an anti-3NT antibody revealed by Fast Red and counterstained with hematoxylin (Panels A-C ) or with an anti-NOS II antibody revealed by Fast Blue and counterstained with eosin (Panels D-F  ). All animals had more than 42% eosinophils and less than 7% neutrophils in the BAL. Despite strong positive staining for NOS II, the NZW mice, which lack peroxidase activity, failed to show any positive staining for 3NT. BAL cells from the NOS II KO mice show strong 3NT formation, despite the absence of NOS II staining. Panels G- J : Immunostaining for 3NT in lung sections from OVA-challenged animals. Blue is counterstaining by hematoxylin. Strong positive staining (appearing in brown when revealed with DAB) is found in A/J (Panel G) whereas no staining is observed in the NZW mice (Panel H ). Strong positive staining was present in the C57BL/6J mice (I ) as well as in NOS II KO mice (3NT appears in red).

O₂ production. The peak of superoxide production by BAL cells from challenged animals was similar among the strains (105 ± 19, 77 ± 37, 186 ± 67 for A/J, C57BL/6J, and NZW mice, respectively, n = 4 to 5, arbitrary units). The AUC was also similar (4,701 ± 1,156, 4,143 ± 2,016, 6,776 ± 1,731 for A/J, C57BL/6J, and NZW mice, respectively).

3NT immunostaining in BAL cells. As illustrated in Figure 3A, BAL eosinophils from A/J mice were strongly positive for 3NT. This was also the case in eosinophils from the C57BL/6J strain (data not shown). In contrast, no 3NT staining was observed in eosinophils from the NZW strain of mice (Figure 3B). In all mice including NZW, the anti-3NT antibody stained the majority of the macrophages, although the density of the staining varied both within and among strains.

3NT immunostaining of lung sections. Occasional faint 3NT staining could be detected in lung sections of control mice from all strains. In OVA-challenged animals, 3NT was observed in the peribronchial region of A/J (Figure 3G) and C57BL/6J mice (not shown) whereas only very faint staining was present around NZW airways after OVA challenge (Figure 3I). Figure 4 shows the number of positive cells around the airways for the three strains studied. Although most of the positive staining was located around the airways (Figure 3, Panels G-J), weak staining could be observed around the vessels in all strains. Faint positive staining was also observed in the parenchyma of A/J and C57BL/6J mice but not in that of the NZW strain. In these experiments, no staining at all was observed in the epithelium. Tissue stained with Fast Red or DAB as chromogen yielded the same results.

View larger version (41K): [in this window] [in a new window]   Figure 4.   Number of positive cells for 3NT around the airways per 0.1 mm2, in A/J, C57BL/6J, and NZW mice under control conditions (white bars) or 48 h after a single intranasal OVA challenge (gray bars). Only a small number of positive cells is observed in EPO-deficient NZW mice. *p < 0.05 compared to control.

3NT immunoblotting: whole lung tissue. Whole lungs were homogenized and proteins were immunoblotted after electrophoresis with the same anti-3NT antibody as in the immunochemistry experiments. Several bands were detected in unchallenged animals (n = 5). After OVA challenge (n = 7) and in all strains, some bands increased in density whereas others remained constant. No difference was found among the strains in the pattern of nitration. A representative Western blot obtained after pooling proteins from three animals in each group is shown in Figure 5A.

View larger version (70K): [in this window] [in a new window]   Figure 5.   Panel A : Detection of 3NT by Western blotting in whole lung homogenates from C57BL/6J (lanes 1 and 2), NZW (lanes 3 and 4), and A/J mice (lanes 5 and 6) under control (lanes 1, 3, 5) or OVA-challenged conditions (lanes 2, 4, 6). 100 µg of protein extract were separated on 4-12% Tris-Glycine gels and transferred onto PVDF membranes. The positive control for 3NT (M) is a mixture of nitrated superoxide dismutase (16 kD), dimerized superoxide dismutase (32 kD), and BSA (66 kD). This representative immunoblot of pooled proteins from 3 animals per lane shows an overall increase in protein nitration in all strains. Panel B : 3NT immunoblotting of proteins extracted from BAL cells of C57BL/6J (lanes 1 and 4), NZW (lanes 2 and 5), and A/J mice (lanes 3 and 6). An eosinophil-rich population (lanes 1, 2, and 3) and a macrophage-rich population (lanes 4, 5, and 6) were obtained by spontaneous adherence of the macrophages to plastic dishes (purity > 85%). In contrast to other strains, two large bands were markedly decreased in NZW mice eosinophils, whereas some other bands were enhanced. Molecular markers are similar as in Panel A. No differences among strains were seen for the macrophage-rich population.

BAL cells. When proteins were extracted from an eosinophil-rich subset of BAL cells, we found that unlike C57BL/J and A/J mice, two large bands were markedly decreased in NZW mice, whereas some other bands were enhanced (Figure 5B). In contrast, these large bands were present in the macrophage-rich population of all three strains.

Protocol 2

In this set of experiments, we assessed the contribution of NOS II driven NO production in protein nitration by comparing NOS II KO with their wild-type control, the C57BL/6J strain.

Eosinophils in BAL after OVA challenge. As shown in Table 2, total and differential cell counts in BAL were similar in the two groups under baseline conditions. After OVA challenge, BAL eosinophilia as well as the total cell number were significantly lower in the NOS II KO mice than in the wild-type C57BL/6J mice. Of note, the number of cells and the percentage of eosinophils in the C57BL/6J strain were higher in Protocol 2 compared to Protocol 1, owing to multiple OVA challenge of the airways.

NO_x concentrations and NOS II staining. NO_x concentrations were similar under baseline condition in both groups. After OVA challenge, NO_x levels increased in the wild-type mice, whereas no increase was detected in the mice lacking NOS II activity (Figure 6). Furthermore, BAL eosinophils did not stain for NOS II protein, as shown in Figure 3F.

View larger version (33K): [in this window] [in a new window]   Figure 6.   NOx concentrations (sum of nitrite and nitrate) were measured with the Griess reaction in BALF in C57BL/6J and NOS II KO mice, unchallenged (control; white bars) or 24 h after three daily OVA challenges (OVA; gray bars). NOx concentrations did not increase after OVA challenge in the NOS II KO mice. *p < 0.05 versus unchallenged; n = 5 to 8 per group.

3NT staining in BAL and in tissue sections. Despite the absence of NOS II staining, BAL eosinophils from the knockout mice were strongly positive for 3NT (Figure 3C). The NOS II KO mice also showed strong 3NT-positive staining around the airways (Figure 3J). The number of 3NT-positive cells around the airways was lower than in the wild-type C57BL/6J animals, but this did not reach statistical significance (Figure 7).

View larger version (42K): [in this window] [in a new window]   Figure 7.   Number of positive cells for 3NT around the airways in NOS II KO and C57BL/ 6J mice, unchallenged (control; white bars) or 24 h after three OVA challenges (OVA; gray bars). No significant difference was found among the strains. Five to nine airways from three animals are analyzed in each control group. Five animals represent 10 to 12 airways in each OVA-challenged group. *p < 0.05 compared to unchallenged controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In OVA-sensitized and -challenged mice, formation of 3NT in the lung was highly dependent on EPO activity rather than on expression of NOS II or the level of production of NO metabolites. 3NT formation was decreased in EPO-deficient NZW mice after specific OVA airway challenge, despite a similar degree of eosinophilic inflammation compared with that in other strains of mice. In contrast, in mutant NOS II deficient mice, which as expected (19) failed to increase NO production after OVA challenge, 3NT formation was unaffected, suggesting that in this model system protein nitration is not driven by substrate availability for ONOO formation. These findings underscore the importance of granulocytes, particularly eosinophils, in mediating oxidative tissue injury and provide strong evidence that peroxidase activity is a major source of 3NT formation during allergic inflammation. Although there is definitive evidence in vitro that EPO can mediate tyrosine nitration by using nitrite to form a tyrosyl radical intermediate (12), the present findings are the first definitive in vivo evidence that EPO promotes 3NT formation.

Recent reports of an association between asthmatic airway inflammation and the detection of 3NT (1, 2) have led to interest in the potential importance of oxidative NO derivatives as mediators of tissue damage or as modulators of the inflammatory response. The mechanism by which production of NO in an oxidative milieu leads to oxidative nitration of tissue proteins remains unresolved however. Although there is support for the widely held notion that ONOO is the principal agent responsible for 3NT formation, this has been hard to confirm in vivo. In particular, it has been observed that it is difficult to induce tyrosine nitration in systems lacking granulocytes (3). Given that granulocyte peroxidases are capable of promoting 3NT formation through mechanisms that are independent of ONOO, it has been suggested that peroxidases rather than ONOO may be responsible for tyrosine nitration in vivo. If so, then therapeutic strategies aimed at preventing NO-mediated oxidative injury should be focused on modifying granulocyte numbers or altering their function, rather than simply blocking NO production. Further progress in understanding the mechanisms of oxidative injury depends on resolution of this question.

We used EPO-deficient NZW mice to directly address the possibility that in allergic inflammation EPO is an important mediator of 3NT formation in vivo. NZW mice responded to specific allergen challenge of the airways in a similar manner to other strains. Indeed, in terms of eosinophilia, the NZW mouse more closely resembles the A/J mouse, a strain known for brisk eosinophilic responses, than the relatively unresponsive C57BL/6J strain (Table 1). NZW mice also resembled other strains in terms of production of NO metabolites (Figure 2) and spontaneous superoxide generation (see RESULTS), excluding the possibility that decreased 3NT formation might simply reflect insufficiency of substrate for ONOO production. Decreased 3NT formation in NZW mice therefore is attributable to the absence of EPO activity in this strain. This is the first demonstration of an altered physiologic response caused by EPO deficiency. Although EPO deficiency, which was originally detected serendipitously on routine blood smears, has been observed in several species including humans (6), it is not associated with disease. While two mutations of the human gene have been already identified (20) and the murine EPO gene has been cloned (21), the molecular basis of this deficiency in NZW mice remains unknown. EPO deficiency appears to be independent of other phenotypic characteristics of NZW mice, which have traditionally been used in lupus research. The F1 generation of a cross between NZW and New Zealand Black mice develops a disease closely resembling systemic lupus erythematosus (22). Although NZW mice are known to have a deletion of the T-cell receptor beta-chain, NZW T lymphocytes are functionally normal (23, 24). NZW mice have a benign phenotype with a normal life span and although elderly NZW mice are at increased risk to develop a mild form of autoimmune renal disease (25), our results indicate that these characteristics do not interfere with the development of eosinophilic inflammation in this model.

We detected 3NT formation in the airways of A/J and C57BL/6J mice after OVA challenge (Figure 3) using an immunohistochemical approach similar to that previously described in studies of asthmatic airways (1, 2). In contrast to the airways of asthmatics, in which 3NT was detected in both inflammatory cells and in the bronchial epithelium (1, 2), in the mouse 3NT staining was largely confined to peribronchial inflammatory cells (mainly eosinophils) and there was little or no epithelial staining (Figure 3). Anatomic and physiologic differences between mice and humans likely explain this discrepancy. The submucosal layer of the murine airway wall is nearly absent so that the number of inflammatory cells found between the smooth muscle and the epithelium layer is very low (26). This reduces the likelihood that eosinophils find themselves close enough to the airway epithelium to promote 3NT formation. This effect is compounded by an additional physiologic difference between mice and humans. Unlike human eosinophils, murine eosinophils do not degranulate easily (27, 28), limiting the capacity of granular proteins such as EPO to exert their effects extracellularly. If epithelial 3NT formation in human asthma depends on the action of extracellular EPO, it is perhaps not surprising that we failed to detect evidence of 3NT immunostaining in murine epithelium.

The importance of 3NT formation within eosinophils is underscored by Western blot analysis of protein extracted from lungs and BAL cells after OVA challenge (Figure 5). When we focused attention on an eosinophil-rich subset of BAL cells, we found differences in both the type and amount of nitrated protein between NZW and other strains (Figure 5B). At least two protein bands, present after OVA challenge in A/J and C57Bl/6 mice were almost completely absent from the NZW mice on these gels. In contrast, protein bands from the macrophage-rich subset of BAL cells were similar across strains, indicating that, as would be expected, 3NT formation is independent of EPO in macrophages. It is of interest to compare the results in BAL macrophages with those in lung homogenates, where we detected multiple protein bands positive for 3NT even under control conditions, similar to findings in other model systems (29). As in the immunohistochemical studies in A/J and C57BL/J mice, there was a general increase in the amount of 3NT-positive protein after OVA challenge with a similar pattern of bands to that seen in lungs from unchallenged animals (Figure 5A). In contrast to the immunocytochemistry and proteins from eosinophil-rich BAL, however, the relative increase in nitrated proteins was similar in all strains of mice, including NZW. Although differences in protein preparation and in sensitivity of the techniques make direct comparison between immunocytochemistry and Western blots difficult, taken together these findings indicate that in the mouse, EPO is largely responsible for 3NT formation. Furthermore, in this model the promotion of nitration by EPO is largely restricted to eosinophils themselves.

To determine whether 3NT formation is dependent on NO production by NOS II in this model, we carried out specific OVA challenge in NOS II deficient mice and their wild-type controls, achieving similar degrees of eosinophilic inflammation in both groups (Table 2). As previously reported (15), absence of NOS II prevented any increase in NO production after OVA challenge, confirming the role of NOS II as the major source of induced NO production in this model (Figure 6). Despite the inhibition of NO production, 3NT formation after OVA challenge in NOS II deficient mice was similar to that in wild-type controls (Figure 7), arguing strongly against the hypothesis that protein nitration is primarily driven by increased NO production in this model in which the detection of 3NT after specific OVA challenge was closely associated with the presence of eosinophilia.

What about 3NT formation outside the eosinophil? As might be expected, deficiency of EPO had no effect on 3NT formation in the macrophage-rich subset of BAL cells (Figure 5B). It is thus possible that 3NT formation in macrophages is mediated by ONOO produced as a consequence of increased NO production in an oxidative milieu. In this regard, it is important to note that our findings in NOS II mice contrast with those in models of endotoxin-induced lung injury. After endotoxin exposure, NOS II deficient mice demonstrated decreased pulmonary 3NT immunostaining compared with wild-type control mice (30, 31). Given the marked differences in the inflammatory response to endotoxin and to allergen, this is perhaps not surprising. Macrophages and neutrophils are the principal inflammatory cells in endotoxin-induced lung injury. It is possible that after endotoxin exposure, 3NT formation is driven by the production of ONOO by macrophages and other cells in which peroxidase function is not as prominent as in granulocytes.

Although 3NT formation is closely associated with a variety of inflammatory states, the physiologic importance of protein nitration remains an open question. There is definitive evidence in vitro that tyrosine nitration alters the function of proteins, including regulatory proteins such as tyrosine kinases (32). Nitrated chemokines or cytokines have decreased chemotactic activity for granulocytes (33) and it has been suggested that tyrosine nitration is a reversible phenomenon (34), raising the possibility that nitration acts as a physiologic regulatory mechanism. However, information concerning the importance of protein nitration in vivo remains scanty, and further work is required to better understand how protein nitration helps to shape the allergic inflammatory response.