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RESEARCH COMMUNICATION |
a Departments of Medicine and Pathology, The Montreal General Hospital, McGill University, Montreal, Quebec, Canada H3G 1A4; GenPath Laboratories, Montreal, Quebec, Canada
b National Heart and Lung Institute, London, England
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: nitrotyrosine • asthma • steroids • human
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1
and Table 2.
Persons with asthma (n=22) were included in the study on the basis of a compatible clinical history and either reversible airflow limitation (increase in FEV1 of 15% or more with bronchodilators) or increased airway responsiveness to methacholine (PC20 of <8 mg/ml) by the method of Juniper et al. (24). All but three asthmatic patients were atopic, as defined by positive skin-prick tests to at least 1 of the 12 common aeroallergens. Of the 22 asthmatic patients, 8 were treated with corticosteroid inhalants (400–1000 mg/day) and ß-agonist (200–400µg/day), 6 were treated with ß-agonist alone, and 8 did not require regular treatment. None of the subjects smoked at the time of the study; four asthmatic subjects were exsmokers but had stopped smoking at least 1 year prior to the study. One subject had an FEV1 persistently below 65% of the predicted value but with significant variability in FEV1, both spontaneously and with bronchodilators. Due to the severity of airflow limitation, methacholine bronchoprovocation was not undertaken. This subject required a high dose of corticosteroid inhalants (Budesonide 2.4 mg/day) as well as intermittent oral corticosteroids. We therefore assumed a severe level of airway responsiveness to methacholine and attributed a PC20 of 0.125 mg/ml for purposes of analysis. All control subjects (n=11) except one were atopic. Pulmonary functions were normal and a PC20 was >8 mg/ml.
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Double-blind randomized study
To determine the effect of inhaled glucocorticoid on the production of peroxynitrite and iNOS, we performed a double-blind, crossover randomized-order, placebo-controlled study on an additional 10 untreated mild asthmatic patients, all atopic (sex: five males; mean age 29.3±1.43 years). None of the patients were taking inhalant or oral corticoids or had a history of upper respiratory tract infection for at least a month prior to the study. Each patient received Budesonide (800 µg twice daily) or matched placebo via a multiple dose, dry powder delivery system (Turbuhaler) in randomized order during each 4 wk period of treatment, separated by a 4 wk washout period. Spirometry and exhaled NO measurement (20) were taken at baseline and at the end of each treatment period. Bronchial biopsies were collected at the end of each treatment period. Bronchial biopsies and exhaled NO measurement were also collected from seven normal control subjects (four females, mean age 25.7±0.87 years, mean FEV1% 99.07±2.0, mean exhaled NO 4.6±0.26 ppm). Clinical characteristics of study subjects in the randomized study are shown in 
Table 2.
Bronchoscopy and biopsies
Fiberoptic bronchoscopy and bronchial biopsies were carried out according to NIH/ATS guidelines (25). To minimize bronchoconstriction, all subjects were pretreated with albuterol (400 µg) and ipratropium bromide (40 µg), administered by a meter dose inhaler attached to a spacer device. Topical anesthesia of the upper airways was obtained using a lidocaine solution (2%). A maximal of six biopsies were taken from the segmental and subsegmental carina in the right lung. Each biopsy at most measured 2 mm in diameter. After the procedure, the individuals were kept under observation until the gag reflex returned; FEV1 was verified prior to discharge to make sure that it had returned to baseline. The tissues were fixed in 2% paraformaldehyde and placed in embedding medium.
Immunohistochemistry
Consecutive frozen sections were immunostained with antiserum against nitrotyrosine (26), human endothelial nitric oxide synthase (eNOS)(27), iNOS (28), and the inflammatory cell markers for macrophages (CD68), neutrophils (elastase), and eosinophils (major basic protein). A modification of the avidin–biotin–peroxidase complex method was used as described previously (29). Sections were immersed in 2% hydrogen peroxide for 1 h to block endogenous peroxidase activity. The sections were permeabilized in 0.3% triton for 15 min, incubated with 10% normal serum to reduce background, and followed by incubation of the sections with the first-layer antiserum overnight at 4°C. The sections were incubated with biotin-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG) for 45 min. Then the avidin–biotin–peroxidase complex was added to the sections for 45 min. Immunostaining was visualized by immersion in diaminobenzidine and hydrogen peroxide and counterstained with the nuclear stain, hematoxylin. To confirm the immunostained inflammatory cell types, we immunostained consecutive sections or the double antigen localization method using diaminobenzidine (brown) and benzidine dihydrochloride (blue) (30). Negative control experiments involved immunoabsorption of the nitrotyrosine and iNOS antisera with the respective antigens before incubation with tissue sections or incubating the sections with normal serum instead of the first-layer antiserum.
The airway epithelium and inflammatory cells were graded semiquantitatively from 0 to 4, with 0 representing no staining; grade 1, focal staining; and grades 2, 3, and 4, diffuse weak, moderate, and strong staining, respectively (27). All analyses, including immunohistochemical grading performed by two investigators, were done without prior knowledge of the treatment of individual patients. In the few cases where there was disagreement between the two observers on the final score (2 vs. 3), the mean of the two values (2.5) was registered. Additional sections were stained with hematoxylin and eosin for histological diagnosis.
In situ hybridization
Frozen sections on RNAse-free precoated slides were permeabilized in proteinase K solution. Background noise was reduced by immersing the slides in acetic anhydride and triethanolamine and a solution of N-ethylmaleimide and iodoacetamide. Complementary RNA and sense probes for human macrophage iNOS (28) and constitutive eNOS (27) were used for hybridization. The probes were labeled with either sulfur-35 or digoxigenin and incubated at 42°C overnight. The unbound probe was removed by immersing the sections in an RNAse solution, followed by high-stringency washes in 0.1x to 2x sodium saline citrate at 22–55°C. The radiolabeled sections were processed for autoradiography and exposed in light-tight boxes for 7 to 14 days at 4°C. The digoxigenin-labeled sections were incubated with anti-digoxigenin antiserum conjugated to alkaline phosphatase. The hybridization signals were visualized with nitroblue tetrazolium and X-phosphatase/5-bromo-4-chloro-3-indolyl-phosphate overnight at room temperature. Negative controls involved hybridization with the radiolabeled sense RNA probe or hybridization buffer.
Statistical analysis
Data are expressed as mean±SE. Analysis of variance was used for multiple comparisons between persons with asthma and controls with a commercial Statview program. A linear correlation test was used to correlate peroxynitrite and iNOS expression with pulmonary functions. P values of less than 0.05 were considered significant. IgE values were log-transformed for statistical analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoreactivity for nitrotyrosine was only weakly and infrequently seen in the bronchial epithelium and in a few inflammatory cells of normal subjects ( Fig. 1A).
No staining was evident on other cell types. In contrast, there was diffuse strong immunoreaction for nitrotyrosine in the airway epithelium and inflammatory cells of asthmatic patients ( Fig. 1B, C). In areas where the epithelium was intact, all but goblet cells were immunostained. In areas where the epithelium was denuded, basal epithelial cells showed strong immunoreaction for nitrotyrosine (Fig. C). The inflammatory cell population immunoreactive for nitrotyrosine was characterized as a combination of macrophages, neutrophils ( Fig. 1E), and eosinophils by colocalization studies with anti-CD68, anti-elastase, and anti-major basic protein, respectively.
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In normal human bronchial tissue, iNOS immunoreactivity and mRNA was seen in airway epithelial cells (except in goblet cells) and only in a few inflammatory cells ( Fig. 1F and Fig. 2A).
In bronchial tissue of asthmatic patients, there was moderate to strong expression of iNOS immunoreactivity ( Fig. 1G–I; Fig. 2F, H) and mRNA ( Fig. 2B, C, E, G) in the airway epithelium and inflammatory cells. Colocalization studies with inflammatory cell markers revealed the presence of iNOS in macrophages, neutrophils, and eosinophils ( Fig. 1I, J). Immunoreactivity and mRNA for iNOS were colocalized to the same cells ( Fig. 2E–H).
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Nitrotyrosine and iNOS-producing macrophages were present mainly within the airway epithelium and subepithelial layer. Neutrophils were seen predominantly in the interstitial space in the submucosa and in intravascular space, whereas eosinophils were mainly seen in the subepithelium interstitial space ( Fig. 1E). The distribution of immunostained cells was comparable for nitrotyrosine and iNOS. Moderate expression of iNOS and nitrotyrosine was infrequently observed in the vascular endothelium and smooth muscle cells of asthmatic patients. Negative control experiments for immunohistochemistry or in situ hybridization showed no signals ( Fig. 1D and Fig. 2D). Semiquantitative analysis of the histological data revealed the presence of significantly more immunostaining for nitrotyrosine in the airway epithelium (P=0.002) and inflammatory cells (P=0.02) of asthmatic patients than in normal controls ( Fig. 3).
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Effects of inhaled Budesonide
In the double-placebo controlled study, bronchial biopsies of asthmatic patients collected after placebo inhalation showed abundant expression of nitrotyrosine ( Fig. 4A)
and iNOS ( Fig. 4C) in the airway epithelium and inflammatory cells. In contrast, the expression of both molecules was either considerably reduced or completely diminished after Budesonide inhalation ( Fig. 4B, D). The level of NO in the exhaled air of control subjects (6.6±0.5, n=7) was significantly lower compared with those of asthmatic patients measured before administration of placebo (34.5±8.83) or Budesonide (42.5±7.69; P=0.01) ( Table 2). Levels of exhaled NO were significantly reduced after Budesonide inhalation compared with baseline ( Table 2; P=0.02), but not after placebo inhalation. Semiquantitative analysis of the data showed a significant reduction in nitrotyrosine immunoreactivity in the airway epithelium (2.9±0.4 vs. 1.4±0.3, P=0.0025) and inflammatory cells (2.0±0.6 vs. 0.7±0.2, P=0.022) after Budesonide inhalation compared with placebo. Expression of iNOS was also reduced in the airway epithelium (2.6±0.3 vs. 1.6±0.3, P=0.018) and inflammatory cells (2.4±0.5 vs. 1.1±0.5, P=0.007).
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Correlation data
The presence of nitrotyrosine in airway epithelium correlated inversely with methacholine PC20 (r=-0.841, P<0.0001), FEV1 (r=-0.771, P=0.0004), and FVC (r=-0.612, P=0.014). Immunoreactivity for nitrotyrosine in inflammatory cells also correlated inversely with methacholine PC20 (r=-0.727, P=0014), FEV1 (r=-0.681, P=0.004), and FVC (r=-0.573, P=0.024). There was a significant correlation between expression of nitrotyrosine and iNOS in airway epithelium (r=0.829, P<0.0001) and inflammatory cells (r=0.576, P=0.018). Levels of exhaled NO correlated significantly with the expression of nitrotyrosine (r=0.60, P=0.0093; r=0.631, P=0.0073) and iNOS (r=0.589, P=0.0088; r=0.523, P=0.0299) in airway epithelium and inflammatory cells, respectively. There was no significant correlation between nitrotyrosine or iNOS and age, sex, or IgE levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oxidant formation is thought to play an important role in cellular injury/damage seen in a number of inflammatory diseases (4). An example of these oxidants is peroxynitrite, which has been implicated in the pathology of adult respiratory distress syndrome and acute lung injury (3, 31). Inflammatory cells produce a great many cytokines, chemokines, and oxidants that can activate iNOS expression through endocrine or paracrine pathways (32, 33). Several reports have shown that inflammatory cells produce high levels of superoxide anions in asthma with a significant inverse correlation between superoxide production in neutrophils and FEV1, suggesting that the worsening of airway obstruction in asthma is associated with increased superoxide production by leukocytes (19, 22, 23). High levels of NO and superoxide produced in the same or neighboring cells would react rapidly to form the potent oxidant, peroxynitrite (8, 9). We demonstrated a significant increase in the expression of nitrotyrosine (a marker of protein nitration by peroxynitrite) and iNOS in neutrophils, eosinophils, and macrophages in the airways of asthmatic patients. We also demonstrated a significant inverse correlation between the presence of nitrotyrosine in inflammatory and epithelial cells with PC20, FEV1, and FVC. Our data suggest that formation of the potent oxidant peroxynitrite in airway inflammatory cells of asthmatic patients may serve as an important marker and/or mediator of cellular oxidative stress in airway disease.
In a fashion similar to inflammatory cells, airway epithelial cells produce various substances that regulate airway tone and homeostasis. In addition, the airway epithelium represents an important physical barrier against noxious substances. Cytokine treatment induces nitric oxide synthases expression and increases NO production by human bronchial epithelial cells (34). Strong expression of iNOS has previously been found in airways of asthmatic patients compared to the low levels in airway epithelial cells of normal control subjects (34). We demonstrate the expression of iNOS in airways of both control subjects and asthmatic patients, with the latter showing greater expression. Our data in the control group are supported by the recent report of constitutive expression of iNOS in the airway epithelium of normal control subjects (35). More important, we demonstrated abundant expression of peroxynitrite in the airways of asthmatic patients, which correlated inversely with airway responsiveness to methacholine and pulmonary function. Asthmatic patients often exhibit shedding of the airway epithelium and impaired airway relaxation after methacholine or histamine challenge (18). Peroxynitrite formation has been shown to cause epithelial cell damage (14, 16). A previous report has shown that the average rate of peroxynitrite formation could be 0.8 micromolar per minute within the whole lung and 1 millimolar per minute in the epithelial lining fluid (9). Sadeghi-Hashjin et al. (14) have shown that administration of 10 micromolar of peroxynitrite increases airway hyperresponsiveness, epithelial damage, and eosinophil activation in guinea pigs. Therefore, we propose that peroxynitrite formation in the airway epithelium, as shown in this study, causes epithelial damage that may result in increased airway hyperresponsiveness.
Steroid treatment has previously been shown to diminish iNOS expression in cultured cells and to cause a significant reduction in the level of NO in exhaled air of asthmatic patients (36–38). We have performed a double-blind, randomized, placebo-controlled study of the effect of the inhaled glucocorticoid Budesonide on expression of nitrotyrosine and iNOS in airways of patients with mild asthma. We found a significant reduction in the expression of nitrotyrosine and iNOS in the airways of asthmatic patients after Budesonide inhalation. Indeed, we found no significant difference between the expression of both molecules in airways of persons with asthma treated with Budesonide and normal control subjects. Furthermore, inhalation of Budesonide reduced the levels of exhaled NO, which correlated significantly with tissue nitrotyrosine and iNOS in the airways of asthmatic patients. This is consistent with the finding that inhalation of glucocorticosteroids decreases exhaled NO in asthmatic patients to normal levels but does not affect normal individuals, suggesting the ongoing induction of nitric oxide synthase in asthma (37, 38). In the same group of asthmatic patients, we also observed a significant correlation between the expression of nitrotyrosine and iNOS. The latter findings suggest that formation of nitrotyrosine in the airways of asthmatic patients is associated with increased production of NO through the inducible enzyme and that corticosteroid inhalation blocks induction of the enzyme and inhibits the formation of the potent oxidant peroxynitrite. The therapeutic mode of Budesonide in asthma may involve direct inhibition of nitrotyrosine and NO or indirect inhibition of inflammatory cytokines known to induce the formation of both substances.
Vascular endothelial and smooth muscle cells produce superoxide, iNOS, and peroxynitrite after activation with cytokines (39–42). In the present study, we observed moderate expression of iNOS and nitrotyrosine in vascular smooth muscle and endothelial cells in persons suffering from asthma. Our data are consistent with previous reports of increased peroxynitrite formation in pulmonary vessels of patients with acute inflammatory diseases and in atherosclerotic coronary arteries (3, 26, 31). Expression of these molecules by vascular endothelial and smooth muscle cells and by inflammatory cells may contribute to increased vascular permeability, mucus secretion, inflammatory cell recruitment, and reduction of eNOS in asthma (14, 31, 32, 43).
It has recently been suggested that the formation of nitrotyrosine may not be exclusive to the actions of peroxynitrite, and may be the result of other reactive nitrogen species such as nitrogen dioxide, nitrous acid, or nitryl chloride (44). Unlike peroxynitrite, however, there is no in vivo evidence showing that other NO derivatives are formed in significant concentrations and have the ability to induce tyrosine nitration. It is highly probable that tyrosine nitration, particularly during periods of inflammation, is a result of peroxynitrite. Nevertheless, in vivo expression of nitrotyrosine is undoubtedly a marker for oxidant injury whether it is due to the effects of peroxynitrite or other NO-derived oxidants.
In summary, we have provided strong evidence for increased production of peroxynitrite and increased expression of iNOS in the airways of persons with asthma compared to control subjects, and an inverse correlation between tissue nitration and pulmonary function. In a controlled study, we have shown for the first time that inhaled glucocorticoid reduces the formation of nitrotyrosine and NO as well as expression of the inducible enzyme in the airways of asthmatic patients. Increased formation of peroxynitrite as shown in this study may contribute to the pathophysiology of asthma. This is supported by the wide range of damaging effects for peroxynitrite in the respiratory system including destruction of glutathione (11) and surfactant (15), oxidation of lipids (11), inhibition of type II pneumocyte metabolic activity (16), eosinophil activation, and enhanced airway responsiveness (14). Finally, use of specific iNOS inhibitors and/or antioxidants may provide new tools in the management of asthma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; PC20, provocation concentration for methacholine; Ig, immunoglobulin.
Received for publication December 10, 1997. Accepted for publication March 5, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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