Abstract
We investigated the relationship between the reversibility of airflow limitation, the concentration of nitric oxide (NO) in exhaled air, and the inflammatory cells in the sputum of patients with stable chronic obstructive pulmonary disease (COPD). We examined nine normal healthy control subjects and 20 nonatopic patients with COPD. Ten patients had no reversibility of airflow limitation (increase in FEV1 of < 12% and < 200 ml after 200 μ g of inhaled salbutamol), and 10 patients had partial reversibility of airflow limitation (increase in FEV1 of < 12% but > 200 ml after 200 μ g of inhaled salbutamol). Exhaled NO levels were higher in COPD patients with partial reversibility of airflow limitation than in those with no reversibility of airflow limitation (median 24 [interquartile range 15.3 to 32] ppb versus 8.9 [4.6 to 14.7] ppb; p < 0.01). Compared with healthy control subjects, only COPD patients with partial reversibility of airflow limitation had increased concentrations of sputum eosinophils. We conclude that, in patients with stable COPD, even a partial bronchodilator response to inhaled salbutamol is associated with increased exhaled NO and sputum eosinophilia, suggesting that these patients may have a different response to treatment than do those without reversible airflow limitation.
Chronic obstructive pulmonary disease (COPD) is a syndrome characterized by airflow limitation that is usually only minimally reversible (1, 2). The degree of reversibility of the airflow limitation in response to bronchodilators, although limited, varies significantly among individuals (3, 4). In patients with a significant, albeit partial, reversibility of airflow limitation after a short course of steroids, COPD shares some pathologic abnormalities with asthma, in particular intraluminal eosinophilia (5, 6). Interestingly, in patients with COPD who have a significant response to bronchodilators, there is a clinical and functional response to inhaled corticosteroids that is similar to that observed in asthmatics (3).
COPD has been increasingly recognized as a chronic inflammatory disease characterized by sputum neutrophilia and, in some cases, eosinophilia (7). Evidence indicates that endogenous nitric oxide (NO) plays a key role in the physiologic regulation of airways and that it is involved in the pathophysiology of airway diseases (8, 9). Although exhaled NO concentrations are markedly increased in asthma and correlate with the degree of sputum eosinophilia (10), the increase in exhaled NO in COPD is lower and less constant and does not correlate with inflammatory indices (11-16). However, a recent study has shown that in patients with pulmonary emphysema, airway eosinophilia is related to reversibility of airflow limitation after a short course of steroids (6).
International guidelines distinguish between nonreversible and reversible airflow limitation by stating that reversible airflow limitation results in an increase in FEV1 of > 12% baseline/predicted and/or > 200 ml after inhalation of salbutamol (1, 2, 17, 18). However, the increase of 12% baseline FEV1, and particularly 12% predicted FEV1, may be quite large (up to 400 to 500 ml), particularly in subjects with mild chronic airflow limitation. COPD patients with partially reversible airflow limitation characterized by an increase in FEV1 of < 12% baseline/predicted but > 200 ml after bronchodilator inhalation might have different pathophysiologic characteristics than COPD patients with nonreversible airflow limitation characterized by an increase in FEV1 of < 12% baseline/predicted and < 200 ml after bronchodilator inhalation.
The aim of the present study was to evaluate whether this differing response to inhaled bronchodilators in stable patients with COPD is related to noninvasive markers of inflammation, such as NO concentration in exhaled air and inflammatory cells in sputum.
We examined nine healthy control subjects and 20 patients with COPD (Table 1). All subjects were recruited from the outpatient clinic of the University of Ferrara, Italy. COPD was diagnosed according to the criteria recommended by the European Respiratory Society (2). All patients were nonatopic, skin prick test negative, and had a history of chronic progressive symptoms such as breathlessness, cough, and sometimes wheezing and sputum production. All patients with COPD were in stable condition at the time of the study and free from acute exacerbations of symptoms and from upper respiratory tract infections in the 2 mo preceding the study. They had no history of asthma or other allergic diseases (e.g., rhinitis). Apart from two nonsmokers, all COPD patients were ex-smokers (24.8 ± 2.1 pack-years) (Table 1), and none of them had smoked in the 6 mo preceding the study.
| Treatment | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Group | n | Age | Sex | Smoking History | None | Steroids | Bronchodilators | |||||||
| COPD-Rev | 10 | 63.8 ± 2.7 | 9 M, 1 F | 1 nonsmoker | 1 | 8i | 9 | |||||||
| 9 ex-smokers | ||||||||||||||
| (26 ± 4.1 pack-years) | ||||||||||||||
| COPD-nonRev | 10 | 66.7 ± 3.1 | 9 M, 1 F | 1 nonsmoker | 2 | 7i | 8 | |||||||
| 9 ex-smokers | ||||||||||||||
| (23.6 ± 2.7 pack-years) | ||||||||||||||
| Controls | 9 | 61.7 ± 4.5 | 6 M, 3 F | 2 nonsmoker | 9 | |||||||||
| 7 ex-smokers | ||||||||||||||
| (7.3 ± 3.5 pack-years) | ||||||||||||||
| FEV1before 200 μg Salbutamol | FEV1after 200 μg Salbutamol | He-FRC | RV | TLC | Kco | |||||||||
| % pred | % pred | % pred | % pred | % pred | % pred | |||||||||
| Group | (L) | (L) | (L) | (L) | (L) | (mmol · min−1 · L−1 ) | ||||||||
| COPD-Rev | 59.2 ± 4.8% | 67.3 ± 4.9% | 118 ± 8.5% | 135.4 ± 8.9% | 110.7 ± 5.7% | 62.2 ± 5.7% | ||||||||
| 1.75 ± 0.18 L | 1.96 ± 0.15 L | 4.20 ± 0.39 L | 3.34 ± 0.36 L | 6.98 ± 0.46 L | 1.07 ± 0.12 | |||||||||
| COPD-nonRev | 57.3 ± 5.7% | 60.2 ± 6.4% | 120.7 ± 6.8% | 139.2 ± 11% | 112.8 ± 7.1% | 58.6 ± 5.4% | ||||||||
| 1.64 ± 0.15 L | 1.78 ± 0.16 L | 4.31 ± 0.32 L | 3.42 ± 0.33 L | 7.09 ± 0.44 L | 0.98 ± 0.09 | |||||||||
| Controls | > 80% | |||||||||||||
Chronic airflow limitation was defined as FEV1/FVC < 88% predicted and FEV1 < 80% predicted both before and after 200 μg of inhaled salbutamol. These parameters did not change significantly in the 5 mo preceding the study.
The nine control subjects were nonatopic, nonasthmatic, had no history of respiratory diseases, and had FEV1/FVC > 88% predicted and FEV1 > 80% predicted.
The study was approved by the ethics committee of our institution, and all patients gave their informed consent. Patients were characterized by medical history, physical examination, pulmonary function tests, exhaled NO levels, and collection of sputum (spontaneous or induced).
Pulmonary function tests (Biomedin Spirometer, Padova, Italy) were performed as described previously (19). We measured FEV1, FVC, TLC, He-FRC, residual volume (RV), and diffusing capacity (KCO) in all subjects. Predicted normal values were those from the European Steel and Coal Community (CECA) (20). All patients with COPD had an increase in FEV1 < 12% baseline/predicted 30 min after inhalation of 200 μg of salbutamol (Table 1), which is the common definition of nonreversible airflow limitation. However, 10 COPD subjects had partially reversible airflow limitation, arbitrarily defined as an increase in FEV1 of < 12% baseline/predicted but > 200 ml after inhalation of salbutamol, and 10 patients with COPD had nonreversible airflow limitation, arbitrarily defined as an increase in FEV1 of < 12% baseline/predicted and < 200 ml after inhaled salbutamol.
End-exhaled NO was measured with a high-resolution (0.5 ppb) chemiluminescence analyzer (NOA 280; Sievers, Boulder, CO) with sensitivity from 1 to 500 ppb by volume, accuracy within 0.5 ppb, and response time < 2 s. The analyzer was equipped with Teflon mouthpiece tubing, and the method used was that described by Kharitonov and coworkers (21). NO was sampled from a side arm attached to the mouthpiece. Expiratory flow and pressure, exhaled volume in real time along with CO2 (range 0 to 10% CO2, resolution 0.1%, response time 200 ms to 90% full scale) (Medical Gas Analyzer LB2; Beckman Instruments, Schiller Park, IL) were also sampled and measured from the side arm attached to the mouthpiece. A biofeedback display unit was used to provide visual guidance for the subject so that he or she could maintain pressure and flow within limits that would not affect the NO recording. Sampling rate was 250 ml/min for all measurements. In accordance with the European guidelines on exhaled NO measurements (22), subjects exhaled slowly from total lung capacity with an exhalation flow of 4 to 5 L/min. The subjects exhaled against a mild resistance to create the positive pressure of 20 cm H2O that ensures the closure of the soft palate (22, 23). Subjects performed five consecutive maneuvers. Results of the analyses were computed and graphically displayed on a plot of NO and CO2 concentrations, pressure, and flow against time. The mean value of end-exhaled NO was taken from the point corresponding to the plateau of end-exhaled CO2 reading (5 to 6% CO2), representing the lower respiratory tract sample (21).
Sputum was collected after bronchodilator inhalation and was analyzed as described previously (24). When necessary, sputum was induced by inhalation of hypertonic saline nebulized with an ultrasonic nebulizer (Mistogen EN 145 electronic nebulizer; Mistogen Equipment Co., Oakland, CA) for 5-min periods up to 20 min. The concentration of saline was increased from 3 to 4% at intervals of 10 min. Spirometric tests were repeated at 5-min intervals, and subjects were then asked to rinse their mouths and throats and to try to expectorate sputum into a petri dish.
Sputum plugs arising from the lower respiratory tract were selected and incubated with 0.1% dithiothreitol until complete homogenization. The cell suspension was filtered through 52-μm nylon gauze and then centrifuged at 800 × g for 10 min. The cell pellet was resuspended in phosphate-buffered saline, and total cell counts with evaluation of cell viability (trypan blue exclusion method) were performed. The cell suspension was adjusted to a final concentration of 400,000 cells/ml and spun in a cytocentrifuge (Shandon Cytospin 2; Shandon, Oakland, CA). Two slides were stained with Diff-Quick (Merz-Dade, Duningen, CH) for differential cell counts of leukocytes and squamous epithelial cells. The slides were coded, and 400 cells were counted, masked for differential leukocyte count. A sample was considered adequate when the percentage of squamous cells was lower than 20%. To correct for variable salivary contamination, the results of differential leukocyte counts were expressed as a percentage of nucleated cells, excluding squamous cells. On average, two slides were prepared for each patient.
Group data were expressed as mean ± SEM or median and interquartile range when appropriate. Exhaled NO concentrations and FEV1 changes after salbutamol inhalation showed a non-normal distribution (chi-square test for normal distribution). Groups were compared by means of the Kruskal-Wallis test and the Mann-Whitney U test when appropriate. Correlations were determined by using Spearman rank correlation. Probability values of < 0.05 were considered significant.
The characteristics of the three groups of subjects examined are reported in Table 1. All three groups were similar in age, and the two groups of COPD patients were similar in smoking history, pharmacologic treatment (particularly the use of inhaled steroids), and baseline pulmonary function, especially FEV1, lung volumes, and Kco.
Exhaled NO levels were significantly increased in COPD patients with partially reversible airflow limitation (24 ppb), as compared with both COPD patients with nonreversible airflow limitation (8.9 ppb) and control subjects (9.9 ppb) (p < 0.01) (Figure 1). Eight of the 10 subjects with partially reversible airflow limitation had an exhaled NO of > 15 ppb.
Fig. 1. Exhaled NO levels in healthy control subjects (Control) and in COPD patients with (COPD-REV) and without (COPD-nonREV) an increase in FEV1 > 200 ml after 200 μg of salbutamol. Horizontal bars represent median values. **p < 0.01 compared with control.
[More] [Minimize]Conversely, when we grouped the patients with COPD on the basis of NO concentrations (> 15 ppb or < 15 ppb), we observed a significantly larger increase in FEV1 after inhalation of salbutamol in the group with exhaled NO levels > 15 ppb (220 [200 to 230] ml) than in the group with exhaled NO levels < 15 ppb (65 [10 to 180] ml) (p < 0.01).
In COPD patients, the concentration of exhaled NO correlated with the response to bronchodilators, expressed as increased milliliters of FEV1 after 200 μg of inhaled salbutamol (r = 0.7, p = 0.003) (Figure 2).
Fig. 2. Relationship between reversibility of airway obstruction after 200 μg of salbutamol and exhaled NO levels in patients with COPD (r = 0.7, p = 0.003). The vertical line represents 15-ppb cutoff value.
[More] [Minimize]Results of the counts of inflammatory cells in sputum of patients with COPD and control subjects are shown in Figure 3. The percentage of sputum neutrophils was significantly higher in both groups of COPD patients (75.5 [70 to 79]% and 83 [68.5 to 87.2]% for patients with partially reversible and nonreversible airflow limitation, respectively) than in control subjects (40.6 [28.7 to 43.9]%) (p < 0.01). The percentage of sputum macrophages was significantly lower in both groups of COPD patients (12 [10 to 14]% and 12.5 [11 to 20]%, respectively) than in control subjects (55 [52.2 to 68.7]%) (p < 0.01). In contrast, the percentage of sputum eosinophils in control subjects (0 [0 to 1.2]%) was significantly lower than that in COPD patients with partially reversible airflow limitation (6 [2.7 to 10.5]%) (p < 0.01) but was not significantly different from that in patients with nonreversible airflow limitation (1 [0 to 2]%) (p > 0.05). The reversibility of airflow limitation, expressed as percent predicted or absolute increase in FEV1 after inhaled salbutamol, did not correlate with any sputum inflammatory cell type.
Fig. 3. Characteristics of sputum inflammatory cells in healthy control subjects (Control) and in COPD patients with (COPD-Rev) and without (COPD-nonRev) an increase of FEV1 > 200 ml after 200 μg of salbutamol. Horizontal bars represent median values. **p < 0.01 compared with control.
[More] [Minimize]This study shows that, in stable patients with COPD, partial reversibility of airflow limitation after inhalation of salbutamol is associated with increased exhaled NO levels and sputum eosinophilia and that the concentration of exhaled NO, but not the percentage of sputum eosinophils, positively correlates with the degree of reversibility. To our knowledge, this is the first study that shows that in patients with COPD even a small response to a bronchodilator is associated with an increase in exhaled NO and sputum eosinophilia, two characteristic features of asthma but not of COPD.
Complete reversibility of airflow limitation is typical of asthma (25), but partially reversible airflow limitation may be present in patients with COPD who have no evidence of asthma or atopy (1). Indeed, a significant reversibility of airflow limitation after the use of bronchodilators or steroids, or both, may be present in up to 30% of stable patients with COPD (26, 27).
The precise mechanisms underlying the presence or absence of partially reversible airflow limitation are still debated (28, 29). Our data suggest a possible involvement of inflammatory mechanisms in the pathogenesis of reversible airflow limitation. Previous studies documented increased exhaled NO concentrations in unstable COPD (11) but not in stable COPD (13, 16, 30). Two recent studies reported either a significant increase (12) or no difference (15) in exhaled NO levels in stable patients with COPD compared with matched control subjects. Our findings may partially explain the conflicting data in the literature on exhaled NO levels in COPD (11-16, 30), because reversibility of airway obstruction was not taken into account in most of the previous studies.
Endogenous NO is generated from the terminal guanidine nitrogen of the amino acid l-arginine by a family of nitric oxide synthases (NOS). Three major isoforms of NOS have so far been identified. Two are constitutively expressed in epithelium; one, termed endothelial NOS (eNOS), is found in the endothelium, cardiac myocytes, platelets, and elsewhere, whereas the second, termed neuronal NOS (nNOS), is found in neural tissue and other tissues, including skeletal muscle. Conversely the third isoform, inducible NOS (iNOS), is considered not normally expressed in most tissue, but inducible in many different types of cells in response to infections, inflammation, or tissue injury. Recent studies in humans have demonstrated that iNOS is normally expressed in airway epithelial cells, that it is overexpressed in asthma, and in this condition it is also expressed in inflammatory cells, including eosinophils (31). Because eosinophils represent in our study the inflammatory cell type specifically increased in the sputum of patients with partially reversible airflow limitation, we speculate that eosinophils are probably the main source of the increased exhaled NO concentrations we observed in these subjects. However, we cannot exclude that between the two groups of patients there is a different degree of iNOS expression in airway epithelium, secondary to the different type of inflammation. Indeed, NO may per se stimulate eosinophil migration and infiltration.
Interestingly, exhaled NO concentrations were increased only in COPD patients with partially reversible airflow limitation, despite the fact that most of the patients were taking low doses of inhaled corticosteroids. A recent study reported that exhaled NO levels were increased in unstable patients with COPD treated with inhaled corticosteroids as compared with stable patients not treated with inhaled steroids (11), suggesting that exhaled NO is a marker of COPD instability and that inhaled steroids may not influence exhaled NO in COPD as they do in asthma (32). In agreement with previous reports (24, 33, 34), we found sputum neutrophilia in patients with COPD, whereas macrophages were the most common cell type in the sputum of healthy control subjects. In addition, a significant increase in eosinophils was observed in patients with COPD, but only in the group with partially reversible airflow limitation. Thus, we identified a relationship between airway eosinophilic inflammation and response to bronchodilators in COPD, but, unlike with asthma (10), we found no correlation between exhaled NO concentrations and the percentage of sputum eosinophils. This may be partially explained by the wide variability of eosinophil counts in sputum.
In conclusion, we have identified a subgroup of COPD patients with partial reversibility of airflow limitation that is associated with increased exhaled NO and sputum eosinophilia. Recent studies have shown that patients with COPD who have some pathologic features of asthma, in particular intraluminal eosinophilia, exhibit partially reversible airflow limitation after a short course of oral steroids (5, 6); therefore, they benefit from corticosteroid treatment (35). Our findings provide more evidence for this conclusion by demonstrating that the presence of a nonfixed amount of airway obstruction in COPD is associated with a significant increase in airway inflammatory markers typical of asthma. As a consequence, exhaled NO concentrations in COPD might predict partially reversible airflow limitation associated with eosinophilic inflammation of the airways and may help to identify COPD patients who could respond to pharmacologic treatment. We conclude that in patients with a significant response to bronchodilator inhalation, COPD may share some functional and pathologic features with asthma, such as increased exhaled NO and sputum eosinophilia.
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