The reaction of nitric oxide (NO) and superoxide anions (O2 −) in the airway results in the formation of peroxynitrite, a highly reactive oxidant species. Peroxynitrite reacts with tyrosine residues in proteins to form the stable product nitrotyrosine. We investigated whether nitrotyrosine in exhaled breath condensates may be increased in patients with asthma. Four groups of nonsmoking subjects were studied. We measured exhaled NO, nitrotyrosine, and leukotrienes concentrations in breath condensate in healthy nonatopic subjects (n = 15) and in patients with mild asthma (steroid naive, n = 15), moderate asthma (inhaled steroid treatment, n = 12), and severe asthma (oral steroid treatment, n = 12). Exhaled NO was increased significantly in patients with mild (19.2 ± 2.7 ppb, p < 0.01) and moderate asthma (14.0 ± 1.53 ppb, p < 0.05), as compared with normal control (6.58 ± 0.61 ppb). The levels of LTC4/D4/E4 and LTB4 were increased significantly in patients with moderate and severe asthma treated with steroids. Nitrotyrosine concentrations were detectable (6.3 ± 0.8 ng/ml) in breath condensate of normal subjects, and were increased significantly in patients with mild asthma (15.3 ± 2.0 ng/ml, p < 0.01). However, the levels of nitrotyrosine in exhaled condensate were lower in patients with moderate (5.0 ± 0.6 ng/ml) and severe asthma (3.3 ± 0.6 ng/ml, p < 0.05). There was a significant correlation between nitrotyrosine in breath condensate and exhaled NO in patients with mild asthma (r = 0.65, p < 0.05). We conclude that nitrotyrosine formation in exhaled breath condensates may be a marker of oxidative stress in airways of asthma.
Oxidative stress, defined as an increased exposure to oxidants and/or decreased antioxidant capacities, is implicated in airway inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma (1-3). Inflammatory cells, especially eosinophils, which produce more superoxide anions (O2 −) than neutrophils or macrophages, release several reactive oxygen- and nitrogen-derived species such as nitric oxide (NO). Levels of NO are elevated in the air exhaled by patients with asthma (4), and may contribute to airway edema and inflammation (5). High levels of O2 − have also been detected in bronchoalveolar lavage of patients with asthma, and the concentration was inversely correlated with forced expiratory volume in 1 s (FEV1) (6). A reaction between NO and O2 − results in the formation of peroxynitrite anions, a highly reactive oxidant species (7). Peroxynitrite adds a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine to produce the stable product nitrotyrosine (8). Peroxynitrite induces hyperresponsiveness in airways of guinea pigs (9), inhibits pulmonary surfactant (10), and damages pulmonary epithelial cells (11). The formation of peroxynitrite is now well established in various forms of airway diseases in the lung, including endotoxic shock, adult respiratory distress syndrome (ARDS), hyperoxia, and ischemia-reperfusion injury (7, 12, 13). Recent study also suggests that there was strong immunoreactivity for nitrotyrosine in the airway epithelium, lung parenchyma, and inflammatory cells in the airways of patients with asthma (14, 15), and that inhaled steroid treatment resulted in a significant reduction in nitrotyrosine immunoreactivity (14). Furthermore, a number of in vitro studies have established changes in enzyme activity on nitration of critical tyrosine residues, which has raised suggestions that protein nitration in vivo may be causally linked to inflammation-related forms of lung injury (16, 17).
The cysteinyl-leukotrienes (Cys-LTs: LTC4/D4/E4) are released from chopped human lung from allergic subjects in response to challenge with allergen (18). Calhoun and coworkers reported that zafirlukast, a LTD4 receptor antagonist, significantly blunted the antigen-derived augmentation of superoxide release by alveolar macrophages after challenge in patients with asthma (19). This study provides evidence that Cys-LTs can also modify cell activation in allergic inflammation, and generate reactive oxygen species. By contrast, LTB4 is a potent chemoattractant and activator of neutrophils, without significant effects on airway smooth muscle (20). LTB4 also activates the NADPH oxidase in guinea pig eosinophils, causing hydrogen peroxide (H2O2) generation and enhancing inflammation by oxidative stress (21). These findings suggest that Cys-LTs and LTB4 may be important mediators in the pathogenesis of asthma, including bronchial smooth muscle contraction, mucus production, inflammatory cell recruitment, and enhancement of oxidative stress.
The aim of this study was to examine whether nitrotyrosine could be detected in breath condensate of patients with asthma, as a marker of oxidative stress in airways of patients with asthma, and to investigate the effect of steroid treatment on nitrotyrosine production in breath condensates of patients with asthma, comparing LT release by inflammatory cells.
Four groups of nonsmoking subjects were studied. Fifteen healthy nonatopic subjects and 15 patients with mild asthma, 12 with moderate asthma, and 12 with severe asthma were recruited (Table 1). There was no significant difference in ages among the subject groups. Atopy was assessed by skin prick tests for common allergens. The diagnosis of bronchial asthma was based on the criteria of the American Thoracic Society (22). Severity of asthma was classified according to the National Institutes of Health/World Health Organization (NIH/ WHO) guidelines (23). Briefly, subjects with mild asthma had symptoms twice a week or less often, with an FEV1 ⩾ 80% predicted, and were taking regular medication, but used inhaled β2-agonists as needed for symptom relief. Subjects with moderate asthma had daily symptoms, used inhaled short-acting β2-agonists daily, had an FEV1 between 60% and 80% predicted, and were taking regular inhaled glucocorticoids (budesonide, 0.4 to 3.2 mg; fluticasone propionate, 0.5 to 2 mg; or beclomethasone, 1 to 2 mg). Subjects with severe asthma were treated with oral prednisolone (4 to 50 mg/d) and inhaled steroids (fluticasone propionate: 1 to 4 mg/d, or budesonide: 0.8 to 4 mg/d). The protocol was approved by the Ethics Committee of the Royal Brompton Hospital, and informed consent from each subject was obtained.
Subjects' details were obtained and then baseline spirometry (Vitalograph Ltd., Buckingham, UK) and exhaled NO were measured, followed by collection of expired breath condensate.
Expired breath condensate was collected by using a condenser, which allowed the noninvasive collection of nongaseous components of the expiratory air (EcoScreen, Jaeger, Würzburg, Germany). Subjects breathed through a mouthpiece and a two-way nonrebreathing valve, which also served as a saliva trap. They were asked to breathe at a normal frequency and tidal volume, wearing a noseclip, for a period of 10 min. The condensate, at least 1 ml, was collected as ice at −20° C and stored at −70° C immediately.
Nitrotyrosine was measured with a specific enzyme immunoassay (EIA) (Cayman Chemical, Ann Arbor, MI). Initially, assays were performed on unconcentrated condensate samples. The lower limit of detection for this assay was 3.9 ng/ml. If nitrotyrosine was not detected in unconcentrated condensate samples, the breath condensates were concentrated threefold, using a freeze dryer (Modulyo; Edwards, Crawley, UK), and reanalyzed. If nitrotyrosine was not detected even after threefold concentration, a value of 1.3 ng/ml (equal to the lower limit of detection, 3.9 ng/ml, divided by 3 to account for the threefold concentration of breath condensates) was arbitrarily assigned to it. LTC4/D4/E4 and LTB4 concentrations were also measured by EIA (LTC4/D4/E4; Amersham Pharmacia Biotech, Amersham, UK; LTB4, Cayman Chemical). The lower limits of detection for these assays were 15.0 pg/ml and 4.4 pg/ml, respectively. If LTs were not detected in the breath condensate samples, a value of 15.0 pg/ml for LTC4/D4/E4 or 4.4 pg/ml for LTB4 was arbitrarily assigned to it.
Exhaled NO was measured by a chemiluminescence analyzer (Model LR2000; Logan Research, Rochester, UK), sensitive to NO from 1 to 500 ppb by volume, and with a resolution of 0.3 ppb. The analyzer was designed for online recording of exhaled NO concentrations. It was calibrated with certified NO mixtures (90 and 436 ppb) in nitrogen (BOC Special Gases, Guilford, UK). Measurements of exhaled NO were made by slow exhalation (5 to 6 L/min) from TLC for 20 to 30 s against a resistance (3 ± 0.4 mm Hg), to prevent nasal contamination.
One-way analysis of variance (ANOVA) with the Newman–Keuls test for multiple comparisons was used to compare groups. Linear regression analysis was used to assess the relationship between nitrotyrosine concentrations in breath condensate and exhaled NO. Data were expressed as means ± SEM, and significance was defined as a value of p < 0.05.
Nitrotyrosine concentrations were detectable (6.3 ± 0.8 ng/ml) in breath condensate of normal subjects, and were increased significantly in patients with mild asthma (15.3 ± 2.0 ng/ml, p < 0.01), who were not treated with steroids, as compared with normal control (Figure 1). However, the levels of nitrotyrosine in exhaled condensate were lower in patients with moderate (5.0 ± 0.6 ng/ml) and severe asthma (3.3 ± 0.6 ng/ml, p < 0.05), who were treated with inhaled or oral corticosteroids, than those of control subjects (Figure 1). There was no correlation between nitrotyrosine level and age in subjects in any group. Exhaled NO was increased significantly in patients with mild (19.2 ± 2.7 ppb, p < 0.01) and moderate asthma (14.0 ± 1.53 ppb, p < 0.05), as compared with normal control (6.58 ± 0.61 ppb) (Figure 1). But there was no significant difference in exhaled NO between normal control subjects and patients with severe asthma with steroid treatment. In patients with mild asthma, the levels of nitrotyrosine in exhaled breath condensate correlated with exhaled NO (r = 0.65, p < 0.05) (Figure 2). No correlation was found between nitrotyrosine concentrations in breath condensate and FEV1 in patients with mild asthma (data not shown). The levels of LTC4/D4/E4 and LTB4 in breath condensate were elevated significantly in patients with moderate (19.5 ± 1.3 and 161.0 ± 31.2 pg/ml, respectively, p < 0.01) and severe asthma (21.2 ± 1.7 and 186.8 ± 31.2 pg/ml, p < 0.01), compared with normal subjects (15.5 ± 0.2 and 63.1 ± 17.3 pg/ml) and patients with mild asthma (15.8 ± 0.3 and 79.1 ± 20.9 pg/ml) (Figure 3). There was no correlation between LTC4/D4/E4 or LTB4 and FEV1 in patients with moderate and severe asthma, however (data not shown).
We demonstrated that exhaled nitrotyrosine was increased in patients with mild asthma who were not treated with corticosteroids. Saleh and coworkers reported strong immunoreactivity for nitrotyrosine in the airway epithelium and inflammatory cells in bronchial biopsies of patients with asthma, which was not seen in normal control subjects, and was reduced by inhaled corticosteroid treatment (14). A recent study also demonstrated intense nitrotyrosine immunoreactivity was in the airways and lung parenchyma of patients with asthma who died of status asthmaticus despite steroid treatment, and suggested that status asthmaticus is characterized by a failure of corticosteroids to control the formation of reactive nitrogen species (15). In addition, nitration of proteins in bronchoalveolar lavage fluid was increased in patients with acute respiratory distress syndrome receiving inhaled nitric oxide (24). This evidence suggests that high levels of NO and O2 − produced in the airways may react to form the potent oxidant, peroxynitrite (12). Peroxynitrite causes hyperresponsiveness in airways of guinea pigs (9) and respiratory epithelial damage (11). Peroxynitrite-induced protein nitration may also alter protein function; peroxynitrite inactivates surfactant (25) and inhibits protein phosphorylation by tyrosine kinases, thus interfering with signal transduction mechanisms (26). Furthermore, it has been suggested that eosinophil peroxidase (EPO) and myeloperoxidase (MPO)-catalyzed nitration in the presence of hydrogen peroxide (H2O2) to form nitrating intermediates from nitrite (NO2 −), a main end product of NO, as an alternative mechanism of protein nitration, independent of peroxynitrite (27, 28). Recent studies suggest that tyrosine nitration by peroxynitrite on eosinophil chemoattractant such as RANTES and interleukin-5 (IL-5) may be a mechanism altering their binding and chemoatactic function (29). These findings have supported the hypothesis that tyrosine nitration might result not only in the formation of inactive “footprints” of reactive nitrogen intermediates, but might also be functionally related to the pathobiology of airway inflammatory diseases. In this study we have provided strong evidence that oxidative stress induced by inflammation produces nitrotyrosine, which presumably reflects increased peroxynitrite formation or direct nitration by granulocyte peroxidases. The increased nitrated proteins are presumably found in the airway lumen and collected in the expired condensate of patients with mild asthma who are not treated with corticosteroids.
In our study there was a positive correlation between exhaled NO and nitrotyrosine level in breath condensate. There is now persuasive evidence that levels of NO are increased in association with airway inflammation and are decreased by antiinflammatory treatments (30). Exhaled NO has a positive correlation with eosinophil counts in induced sputum in patients with asthma (31). We therefore investigated whether nitrotyrosine concentrations were related with any other markers of airway inflammation, which was detectable in breath condensates and contributed to oxidative stress in airways of asthma, and found significant increases in Cys-LTs and LTB4 in breath condensates of patients with moderate and severe asthma, compared with normal subjects and subjects with mild asthma. The Cys-LTs, generated predominantly by mast cells and eosinophils, possess various bronchoactive properties in vitro, inducing airway smooth muscle contraction (32, 33), microvascular leakage, and mucus hypersecretion (32). A recent study has provided evidence that zafirlukast, the potent Cys-LT receptor antagonist, reduced the number of eosinophils and basophils recovered in bronchoalveolar lavage fluid in patients with asthma after allergen challenge, and blunted the antigen-derived augmentation of O2 − release by alveolar macrophages after challenge in patients with asthma (19). In addition, LTB4 is a potent proinflammatory mediator and an attractant for neutrophils. Although LTB4 has not been closely linked to asthma in contrast to the Cys-LTs, there is increasing evidence that neutrophils may play a role in more severe asthma. Neutrophil number and activation are increased in the airways of subjects with severe persistent asthma and during exacerbations of asthma (34, 35). In our study the levels of LTB4 in breath condensate were significantly higher in patients with moderate and severe asthma than those of normal subjects and patients with mild asthma. This suggests that LTB4 may be involved in exacerbations of asthma, probably in neutrophil recruitment. LTB4 also directly promotes a receptor-mediated activation of the NADPH oxidase in guinea pig eosinophils, to cause the formation of H2O2 (21). LTB4 presumably induces neutrophil recruitment and activation of MPO, resulting in nitrotyrosine production in airways of patients with asthma. However, there was no significant increase in either exhaled NO or nitrotyrosine in breath condensates of patients with moderate and severe asthma whose levels of Cys-LTs and LTB4 in condensates were increased significantly. This finding suggests that an increase in NO production plays a critical role in nitrotyrosine production in airways of patients with asthma.
We found a significant reduction in nitrotyrosine production in breath condensates of patients with severe asthma who required systemic treatment with corticosteroids, compared with healthy control subjects. In contrast, there was no significant difference in exhaled NO between normal control subjects and subjects with severe asthma. This finding suggests that systemic steroid treatment may inhibit oxidative stress induced by inflammation and also inhibits the inflammatory response in the airways. The finding that levels of exhaled nitrotyrosine were lower than in normal subjects suggests that there may be some inflammation and oxidative stress in normal airways as a result of inhalation of oxidants in the urban environment. The recognition of asthma as a chronic inflammatory disease (3), along with the recommendation by asthma management guidelines (23) for early intervention with antiinflammatory agents, has resulted in inhaled corticosteroid therapy becoming the mainstay of steroid treatment for patients with asthma. Despite the availability and use of inhaled corticosteroids for the treatment of asthma, a proportion of patients require long-term adjunct therapy with systemic corticosteroids to achieve control of symptoms. Because of the recognized risk of side effects associated with the use of systemic corticosteroids, the development of therapeutic options that can eliminate the dependence of these patients on oral corticosteroids is important. Our data suggest that expired nitrotyrosine may be a useful marker to control the dose of systemic corticosteroids, or to combine new antiasthma drugs with inhaled steroids, keeping nitrotyrosine in breath condensate at normal levels.
In summary, our study has demonstrated that nitrotyrosine in exhaled breath condensate was increased in patients with mild asthma who were not treated with corticosteroids, compared with normal control subjects, and was reduced in patients with severe asthma receiving steroid therapy. We have also provided evidence that nitrotyrosine formation in exhaled breath condensate may be a more sensitive marker to evaluate the contribution of oxidative stress to airway inflammation of asthma than exhaled NO.
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