INTRODUCTION

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Chronic obstructive pulmonary disease (COPD) is a major medical problem and there is evidence that it is increasing throughout the world (1, 2). Inflammation of the airways seems to play an important role in the pathogenesis of the disease. However, the inflammatory mechanisms in COPD are less understood than those in bronchial asthma (3).

Oxidative stress and imbalances in host defense mechanisms may be among the causes of COPD (3). The increased production of NO during inflammatory immune processes involving the respiratory tract is thought to constitute a host defense mechanism, although this comes at a price, because a high level of NO can also cause respiratory tract injury and thus contribute to the pathophysiology of inflammatory airway diseases such as COPD and asthma (7). Recently, excessive production of nitric oxide (NO), presumably via inducible NO synthase (iNOS), has been reported in asthmatic airways (8), although its presence is controversial in COPD airways.

The adverse effects of NO are thought to be partly engendered by its reaction with superoxide anion released from inflammatory cells, yielding the potent oxidant peroxynitrite (9, 10). Peroxynitrite adds a nitro group to the 3- position adjacent to the hydroxyl group of tyrosine to produce the stable product nitrotyrosine (11, 12). NO also reacts directly with O₂ to form nitrite, the oxidation of which, by neutrophil-derived myeloperoxidase (MPO) or by other related peroxidases (8, 13), yields nitryl chloride and nitrogen dioxide (NO₂). This mechanism has also been found in inflammatory conditions (8). Although nitration of tyrosine is generally attributed to peroxynitrite, the peroxidase-dependent nitrite oxidation pathway is also involved (8). Therefore, nitrotyrosine is a collective indicator of the involvement of reactive nitrogen species (RNS) in a particular process (8, 13).

In the study reported here we quantified the production of RNS in the airways of subjects with COPD, others with asthma, and healthy subjects (HS) by means of immunostaining for nitrotyrosine and iNOS in induced sputum, as well as by measuring exhaled NO levels. We found that the formation of RNS was increased to a significantly greater extent in subjects with COPD than in those with asthma or in healthy subjects.

	METHODS

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Subjects

Ten patients with COPD, 11 patients with asthma, and 10 HS took part in the study after giving informed consent. The study was approved by the local ethics committee. All patients satisfied the American Thoracic Society criteria for COPD or asthma (2), and all had quit smoking at least 1 yr before the study. Eight of the patients with COPD were diagnosed as having pulmonary emphysema through computed tomography (low-attenuation areas of the lungs) and pulmonary function tests, such as measurement of lung volume (both TLC and FRC exceeded 120% of predicted values) and diffusing capacity of carbon monoxide (DL_CO) (less than 60% of predicted values). The remaining two COPD patients were diagnosed as having chronic bronchitis on the basis of clinical manifestation of a chronic productive cough for 3 mo in each of at least two successive years. Asthmatic subjects had their disease diagnosed on the basis of recurrent episodes of wheezing, airway hyperresponsiveness (provocative concentration of methacholine causing a twofold increase in respiratory resistance < 10 mg/ml), and airway eosinophilia assessed by sputum examination. The clinical characteristics of the study subjects are shown in Table 1. FEV₁ was measured with a dry rolling-seal spirometer (Model OST 80A; Chest Co., Tokyo, Japan). All patients were stable and had been without steroid therapy for at least 6 mo before the study, and all bronchodilator therapies were stopped for at least 24 h before the screening examination for the study. COPD and asthmatic patients had significantly lower values of FEV₁% predicted (p < 0.05 in both cases) than did HS.

Measurement of NO Concentration

The exhaled NO concentration was measured with a chemiluminescence analyzer (Model 280NOA; Sievers, Boulder, CO). Because nasally derived NO and the expiratory maneuver affect the exhaled NO concentration (14), we set up the NO measurement system as follows. Subjects exhaled from TLC at a constant flow of 2.5 L/min. Pressure in the oral cavity was maintained at 20 cm H₂O to close the velum, thus excluding contamination by nasally derived NO. The exhaled air was absorbed at a sample flow rate of 250 ml/min via a mouthpiece side port close to the mouth. At least two successive recordings at 2-min intervals were made, and the mean of the peak values of two reproducible readings was used in analysis of the results. Because the maneuvers used to generate a spirogram may affect exhaled NO levels, we measured NO before spirometry.

Sputum Induction and Examination

Fifteen minutes after pretreatment with fenoterol (400 µg by inhalation), patients inhaled hypertonic saline (4%) from an ultrasonic nebulizer (Model MU-32; Sharp Ltd., Osaka, Japan) (the mean mass median aerodynamic diameter of the aerosol particles and volume output were 5.4 µm and 2.2 ml/min, respectively). Because the collected sputum samples contained saliva, we eliminated contaminated specimens by visual inspection and inverted microscope examination (15). Inhalation of hypertonic saline was continued for 15 to 30 min, until the volume of collected sputum was approximately 1 ml.

The sputum sample was immediately treated with Sputasol (Oxoid Ltd., Bashingstoke, UK) at a volume four times that of the sample, to dissociate the sulfide bonds of the mucus. The mixture was vortexed for 15 s and gently aspirated in and out of a Pasteur pipette to ensure mixing. The sample was then shaken for 15 min and phosphate-buffered saline (PBS) was added to stop the effect of Sputasol. After the sample was centrifuged at 790 × g for 10 min, the cell pellet was resuspended in a half volume of the sputum with 0.1 M PBS, and the total cell count of leukocytes was obtained in a hemocytometer. Cell viability was determined by the trypan blue exclusion method. The total and absolute number of cells per milliliter of processed sputum was calculated. A 100-µl aliquot of the cell suspension, adjusted to contain a volume of 1.0 × 10⁶/ml of the processed sputum, was placed into the cups of a Shandon III cytocentrifuge (Shandon Southern Instruments, Sewickley, PA), and four preparations were obtained from each sample (16). The samples were stained with Hansel's stain (Torii Pharmaceutical, Tokyo, Japan) to make the cell differential count (17), and were stored at 80° C until immunocytochemical analysis.

Immunocytostaining

Two samples were immunostained with antisera to nitrotyrosine and iNOS, respectively. Briefly, each sample was fixed in 4% paraformaldehyde fixative solution for 30 min. Endogenous peroxidase activity was reduced by incubation in 3% hydrogen peroxide in 100% methanol for 5 min at room temperature. After being washed in PBS, the preparation was incubated with primary antibody (rabbit polyclonal antinitrotyrosine IgG, 1:100 dilution; Upstate Biotechnology, Lake Placid, NY [18] or rabbit anti-iNOS antiserum, 1:200 dilution; Wako Pure Chemical Industries, Osaka, Japan) (19) for 12 h at 4° C. In order to reduce nonspecific binding of antibody, we preincubated the preparations with 4% skim milk in PBS containing 0.3% Triton-X for 30 min, and then incubated them with 10% inactivated normal goat serum for 30 min at room temperature. When we incubated the antibody to nitrotyrosine with excess doses of nitrotyrosine in this preparation, we did not observe immunoreactivity, indicating the high specificity of the antibody for nitrotyrosine. Immunoreactivity was visualized through the indirect immunoperoxidase method, using ENVISION polymer reagent, a goat antirabbit IgG conjugated with peroxidase-labeled dextran (DAKO Japan Ltd., Kyoto, Japan), following 1 h of incubation at room temperature. The diaminobenzidine reaction was used for color development, followed by counterstaining with Hansel's stain.

Differential and immunoreactive cell counts were made by two qualified cytopathologists blind to the origin of the samples, who counted 500 cells in each sample. The mean of the two scores was used for analysis.

Statistical Analysis

Data are expressed as mean ± SEM. Comparisons of mean data were made through analysis of variance, and subsequent multiple comparisons were made with Scheffe's test. A linear regression analysis was done using the method of least squares. A value of p < 0.05 was considered significant.

	RESULTS

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As shown in Table 1, percent predicted FEV₁ values were significantly lower in COPD (p < 0.05) and asthma (p < 0.01) patients than in HS. The ranges of FEV₁/FVC values as percents for healthy, asthmatic, and COPD subjects were 73.4 to 87.7%, 46.6 to 94.0%, and 29.2 to 69.1%, respectively. The exhaled NO concentration was significantly higher for asthma patients (p < 0.01) than for COPD patients or HS. There was no significant difference between the exhaled NO levels of COPD patients and those of HS.

Tables 2 and 3 show the cell differential and immunopositive cell counts in induced sputum. Total cell numbers were significantly higher in asthmatic (p < 0.05) and COPD subjects (p < 0.05) than in HS. The total cell counts were not significantly different in COPD and asthma patients.

Immunoreactivity for iNOS was more abundant in other cell populations than lymphocytes in both COPD and asthma patients as compared with HS (Tables 2 and 3; Figures 1A, 1C, and 1E), and was positively correlated with the exhaled NO concentration in asthma (p < 0.01) but not in COPD patients (Figure 2). The exhaled NO concentration was negatively correlated with % predicted FEV₁ values in asthma but not in COPD patients (Figure 3).

View larger version (164K): [in this window] [in a new window]   Figure 1.   Immunohistochemical staining of iNOS (A, C, and E ) and nitrotyrosine (B, D, and F ) in the induced sputum. Data from healthy (A and B), asthmatic (C and D), and COPD subjects (E and F ) are shown.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Relation of exhaled NO concentration to iNOS-positive cell counts in induced sputum of asthma and COPD patients. r is correlation coefficient; the line and p value correspond to the fitted regression equation. N.S. = not significant.

View larger version (10K): [in this window] [in a new window]   Figure 3.   Relation between FEV1% predicted (%FEV1) and exhaled NO concentration. r is correlation coefficient; the line and p value correspond to the fitted regression equation. N.S. = not siginficant.

Abundant nitrotyrosine immunoreactivity was also observed in macrophages and neutrophils of COPD patients and in eosinophils of asthma patients, but not in these cells from HS (Tables 2 and 3; Figures 1B, 1D, and 1F). The nitrotyrosine-immunopositive cell counts were positively related to the iNOS-positive cell counts in both asthma and COPD patients (Figure 4). Both the actual numbers and the percentages of nitrotyrosine-immunopositive cells were significantly higher in COPD than in asthma patients (Figure 5). In contrast, the same level of iNOS immunoreactivity was observed in both COPD and asthma patients (Figure 5). The nitrotyrosine-positive cell counts were negatively correlated with the values of % predicted FEV₁ in COPD (p < 0.01) but not in asthma patients (Figure 6).

View larger version (12K): [in this window] [in a new window]   Figure 4.   Correlation between nitrotyrosine (NT)-positive and iNOS-positive cell counts in induced sputum of asthma and COPD patients. r is correlation coefficient; the line and p value correspond to the fitted regression equation.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Actual cell counts and iNOS- and nitrotyrosine (NT)-positive cells as percentages of total cell counts. **p < 0.01 compared with healthy subjects,  p < 0.05 compared with asthma patients.

View larger version (11K): [in this window] [in a new window]   Figure 6.   Relation between %predicted FEV1 and nitrotyrosine (NT)- positive cell counts in induced sputum of asthma and COPD patients. r is correlation coefficient; the line and p value correspond to the fitted regression equation. N.S. = not significant.

	DISCUSSION

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In the present study, abundant cells showing staining for nitrotyrosine, as well as abundant iNOS-positive cells, were observed in induced sputum from both COPD and asthmatic patients as compared with HS. The nitrotyrosine positive cells were significantly more obvious in sputum from COPD than from asthma patients, suggesting that the oxidative stress imposed by RNS may be exaggerated in the airways of patients with these diseases, and especially COPD. Further, because the counts of nitrotyrosine-positive cells were significantly correlated with the airway obstructive changes in COPD, hyperproduction of RNS may be an important factor in the pathogenesis of COPD.

iNOS, which is responsible for hyperproduction of NO, has been reported to be expressed abundantly in epithelial cells (18, 19) and inflammatory cells including macrophages and eosinophils (18) in asthmatic airways. In the present study, we also observed abundant iNOS expression in eosinophils in induced sputum of asthmatic subjects, although we did not examine epithelial cells for the presence of iNOS. In COPD patients we also observed a significant increase in iNOS expression in neutrophils and macrophages. The total counts of iNOS- expressing cells assessed by immunoreactivity were almost the same in COPD and asthma patients, indicating that NO hyperproduction occurs in COPD as well as in asthmatic airways.

Nitrotyrosine immunoreactivity, which is a marker of production of RNS, was also significantly increased in eosinophils of asthma patients and in macrophages and neutrophils of COPD patients. In contrast to the results for iNOS immunoreactivity, counts of nitrotyrosine-positive cells were significantly higher in COPD than in bronchial asthma patients, as described earlier, suggesting that production of RNS was greater in COPD airways than asthmatic airways.

Formation of nitrotyrosine depends on oxidation of NO (8). NO reacts with superoxide anion to yield the powerful oxidant peroxynitrite, which is presumed to be largely responsible for the adverse effects of excessive NO generation (20). Peroxynitrite causes tyrosine nitration and has highly proinflammatory actions via oxidative stress and lipid peroxidation (9, 10, 21, 22). Peroxynitrite also activates matrix metalloproteinases released from neutrophils and macrophages, which are capable of degrading all of the components of the extracellular matrix of lung parenchyma (23), and may cause emphysematous changes. An alternative pathway of nitrotyrosine formation via NO involves mechanisms dependent on MPO or related peroxidases (8, 13). NO reacts with O₂ to form nitrite. Oxidation of nitrite by MPO or by other, related peroxidases results in the formation of nitryl chloride and NO₂. These reactive nitrogen intermediates are also involved in the nitration of tyrosine (8). Because neutrophil-derived MPO and eosinophil peroxidase are abundantly present in COPD and asthmatic airways, nitrotyrosine formation via these pathways may also have an important role in the pathophysiology of these diseases. The importance of these mechanisms during inflammation has been reported (8).

Increased nitrotyrosine formation and its relation to the pathobiology of inflammatory changes have been reported in acute respiratory distress syndrome (24), idiopathic pulmonary fibrosis (25), and asthma (18). In asthma especially, the amount of nitrotyrosine staining in airway epithelial and inflammatory cells is significantly correlated with the degree of airway obstruction (18). In the present study, however, we observed a significant correlation between the amount of nitrotyrosine formation in airway inflammatory cells and the degree of airway obstruction only in COPD, and not in asthma. The precise reason for the discrepancy in these results is unknown. Our use of induced sputum to examine the nitrotyrosine immunoreactivity of airway inflammatory cells, and lack of examination of epithelial cells, may explain the differences in the findings.

Increased levels of exhaled NO (7), possibly resulting from iNOS induction (19), and their correlation with the degree of airway obstruction have been reported in asthma (26). In contrast to the case with asthma, conflicting results have been reported for airway NO production levels in COPD (27). In the present study, the exhaled NO levels of COPD patients were not significantly different from those of HS. Whatever its significance, the exhaled NO concentration in COPD was lower than in asthma in previous studies as well as the present one. However, in the present study we found almost the same degree of immunoreactivity for iNOS in inflammatory cells in induced sputum from COPD and asthma patients. Further, nitrotyrosine formation was increased to a significantly greater extent in COPD than in asthma patients. Taken together, these findings suggest that in COPD airways, peroxynitrite formation via the reaction of NO with superoxide anion (30, 31) and/or peroxidase-dependent nitrite oxidation mechanisms (8, 13) may act to consume NO. This might at least partly explain the lower exhaled NO levels in COPD than in asthma.

It remains to be established whether nitrotyrosine is merely a biomarker of RNS or whether it actively contributes to cellular dysfunction and development of the airway inflammatory processes in COPD. In this respect, the recent discovery of apparent enzymatic "nitrotyrosine dinitrase" activity in rat spleen and lung homogenates (32) may further hint at the potential significance of nitration (and dinitration) as a signaling of inflammatory mechanism (8).

In summary, we found that nitrotyrosine immunoreactivity in inflammatory cells obtained by the induced sputum technique was more abundant in COPD patients than in healthy or asthmatic subjects. Further, the degree of airway obstruction and the amounts of nitrotyrosine identified were significantly correlated with one another. Exhaled NO levels were significantly increased in asthma but not in COPD patients as compared with HS, although iNOS-immunopositive cells were observed to almost the same degree in asthma and COPD patients. These results suggest that: (1) RNS may be involved in the pathophysiology of the inflammatory and airway obstructive process in COPD; and (2) NO produced in the airways, presumably via iNOS, appears to be consumed by its reaction with superoxide anion and/or by peroxidase-dependent nitrite oxidation.