Eosinophilic Inflammation in Stable Chronic Obstructive Pulmonary ...

Eosinophilic Inflammation in Stable Chronic Obstructive Pulmonary Disease Relationship with Neutrophils and Airway Function GIOVANNI BALZANO, FRANCESCO STEFANELLI, CARMELA IORIO, ALBERTO DE FELICE, ENRICO M. MELILLO, MICHELE MARTUCCI, and GAETANO MELILLO Division of Pneumology, Rehabilitation Institute of Telese Terme, Salvatore Maugeri Foundation, Telese Terme, Benevento, Italy

The number and significance of airway eosinophils in stable COPD is controversial. Aims of this study were to evaluate airway inflammation in patients with stable COPD compared with other groups, and to examine the correlations between inflammatory markers and functional indices of airway obstruction. Cellular analysis and evaluation of eosinophil cationic protein (ECP) levels in induced sputum were made in 46 subjects (10 patients with clinically stable COPD, 15 patients with asthma, 11 asymptomatic smokers, and 10 healthy control subjects). As expected, eosinophils were significantly (p , 0.01) higher in patients with asthma (22.2%) than in other groups (COPD, 0.7%; smokers, 0.2%; control subjects, 0.2%), and neutrophils were significantly (p , 0.01) higher in patients with COPD (77.5%) than in the other groups (asthma, 26.7%; smokers, 33.1%; control subjects, 35.9%). However, eosinophils were also increased in patients with COPD, as compared with healthy controls (p , 0.05). Sputum ECP levels were significantly and similarly higher in both asthma and COPD groups than in the other two groups (p , 0.01). In patients with COPD and asymptomatic smokers, considered as a whole, good correlations were found between eosinophils and ECP, on the one hand, and between FEV1 and the FEV1/FVC ratio, on the other. Our data suggest that eosinophils may be involved in the airway inflammation of COPD. Balzano G, Stefanelli F, Iorio C, De Felice A, Melillo EM, Martucci M, Melillo G. Eosinophilic inflammation in stable chronic obstructive pulmonary disease: relationship with neutrophils and airway function. AM J RESPIR CRIT CARE MED 1999;160:1486–1492.

Asthma and chronic obstructive pulmonary disease (COPD) are both characterized by airway inflammation (1, 2). Studies using bronchoalveolar lavage (BAL), bronchial biopsy or, more recently, induced sputum have shown that the main cellular markers of intralumenal airway inflammation are eosinophils in asthma (3–7), and neutrophils in COPD (2, 8–12). Moreover, good correlations have been found between eosinophils and airway hyperresponsiveness in asthma (6, 13,14), as well as between neutrophils and airway obstruction in COPD (2, 11, 12). However, there is some debate about the number and possible significance of eosinophils in the airways of patients with COPD. In fact, although airway eosinophilia has been found in COPD during exacerbations (15), an increase in airway eosinophils in patients with stable conditions has been found in some studies (2, 10, 16, 17) but not in others (8, 9, 11). We have evaluated airway inflammation, with particular regard to some eosinophilic markers, such as sputum eosinophils and sputum eosinophil cationic protein (ECP), in patients with clinically stable COPD, patients with mild asthma, asymptomatic smokers, and healthy control subjects. We have analyzed (Received in original form October 28, 1998 and in revised form April 28, 1999) Correspondence and requests for reprints should be addressed to Giovanni Balzano, M.D., Division of Pneumology, Rehabilitation Institute of Telese Terme, Salvatore Maugeri Foundation, Telese Terme (BN), 82037 Italy. Am J Respir Crit Care Med Vol 160. pp 1486–1492, 1999 Internet address: www.atsjournals.org

the correlations between markers of airway inflammation and indices of airway obstruction (i.e., FEV1 and the FEV1/FVC ratio) in the two groups (patients with COPD and asymptomatic smokers) considered as a whole. In fact, asymptomatic smokers may be considered to be in a condition intermediate between COPD and healthy status (18).

METHODS Subjects Forty-six subjects were enrolled in the study: 10 patients had moderate to severe COPD and were in clinically stable conditions, 15 patients had mild asthma, 11 were asymptomatic smokers, and 10 were healthy control subjects. The study was approved by the local ethics committee. All patients and subjects gave their informed written consent to the study.

Inclusion Criteria Rigorous inclusion criteria for patients and subjects were applied (Table 1). Our aim was to establish the diagnosis according to wellaccepted criteria (19), and to ensure that baseline airway inflammation was not affected by recent or current exposure to such proinflammatory stimuli as seasonal allergens (20) and respiratory tract infections (21), and to such antiinflammatory agents as oral and inhaled glucocorticosteroids (22).

Study Design This was a consecutive 2- or 3-d study. On Day 1, the history was reviewed, and subjects underwent a clinical examination, allergy skin prick tests, and spirometry with assessment of reversibility of airway

1487

Balzano, Stefanelli, Iorio, et al.: Inflammation and COPD TABLE 1

Skin Tests

INCLUSION CRITERIA Patients with COPD Both sexes, age . 40 yr Smoking history (> 20 cigarettes/d by > 20 yr) Presently smokers or ex-smokers by , 1 yr Negative skin prick tests FEV1 < 60% of the predicted value and FEV1/FVC < 70% FEV1 after inhaled bronchodilator < 112% of the baseline value and DFEV1 (post bronc-pre bronc) < 200 ml No exacerbation and no treatment with oral or inhaled glucocorticoids in the last 8 wk Patients with asthma Both sexes, age 18 to 65 yr Nonsmokers or ex-smokers by > 10 yr Skin prick tests either exclusively positive to perennial allergens or negative to the commonly inhaled allergens FEV1 > 70% of the predicted value FEV1 after inhaled bronchodilator > 115% of the baseline value or, alternatively, PD20-methacholine , 400 mg (which means, in our laboratory, a moderate to severe increase in bronchial responsiveness) No exacerbation and no treatment with oral or inhaled glucocorticoids in the last 8 wk Asymptomatic smokers Both sexes, . 30 yr Smoking history (> 20 cigarettes/d for > 10 yr), presently smokers Negative skin prick tests Normal airway function (FEV1 > 80% pred, FEV1/FVC > 80% and FEV1 change after inhaled bronchodilator < 10% of the baseline value) No chronic bronchitis or asthma in the past PD20-methacholine . 1,600 mg (which means, in our laboratory, a normal bronchial responsiveness) No upper or lower airway infection in the last 8 wk Healthy control subjects Both sexes, age 18 to 65 yr No smoking habit Negative skin prick tests Normal airway function (FEV1 > 80% pred, FEV1/FVC > 80% and FEV1 change after inhaled bronchodilator < 10% of the baseline value) PD20-methacholine . 1,600 mg (which means, in our laboratory, a normal bronchial responsiveness) No chronic bronchitis or asthma in the past No upper or lower airway infection in the last 8 wk

obstruction. On Day 2, patients and subjects presenting with an FEV1 > 70% of the predicted value and an FEV1/FVC ratio > 70% underwent a methacholine inhalation test. On Day 3 (Day 2 for patients not given the methacholine test), the sputum induction procedure was performed. Induced sputum measurements were repeated within 1 wk in 10 of the subjects (2 patients with asthma, 3 with COPD, 3 asymptomatic smokers, and 2 healthy control subjects) to assess reproducibility of the procedure.

Skin prick tests were done with 12 commonly inhaled allergens, including house dust mites, pollens, molds, animal danders, and positive and negative control extracts (Lofarma Allergeni, Milan, Italy).

Spirometry We adhered to ATS recommendations (23) for spirometry, for which we used a dry wedge spirometer (Vitalograph, Buckingham, UK). After baseline evaluation, spirometry was repeated 15 min after the patients had inhaled 400 mg (four puffs) of salbutamol, delivered from a metered dose inhaler attached to a large-volume spacer device (Volumatic; Glaxo, Verona, Italy). Reversibility of airway obstruction was expressed in terms of the percentage of baseline FEV1.

Methacholine Inhalation Test The methacholine inhalation test was carried out according to the dosimeter method currently used in our laboratory (24).

Sputum Induction Sputum was induced by inhalation of an ultrasonically nebulized 3% hypertonic saline, using an Orion 2 nebulizer (Nova, Heyer, Germany). Nebulizer output was regulated at 2.5 ml/min. Nebulization time consisted of 5-min sessions at 5-min intervals until a maximum cumulative nebulization time of 20 min. After each inhalation step, the patient, after rinsing his/her mouth with 20–25 ml of water, for 30 s each time, then expectorated. The procedure was continued until either a sufficient amount (. 2 ml) of sputum was obtained, or a cumulative nebulization time of 20 min was reached. The sputum was collected in a sterile plastic container, and the total amount obtained was recorded. Fifteen minutes before starting the hypertonic saline inhalation, we measured the FEV1/FVC of the patient, and administered a bronchodilator (salbutamol, 400 mg by inhalation with a spacer). FEV1 was measured again 10 min after the bronchodilator inhalation and, at the end of each period of hypertonic saline inhalation, to detect any eventual hypertonic saline-induced bronchoconstriction (25). If, indeed, an FEV1 fall > 20% of the postbronchodilator value occurred, the procedure was stopped and the patient treated with a bronchodilator.

Sputum Processing The volume of sputum was measured and an equal volume of freshly prepared dithiothreitol (Sigma, Milan, Italy), 0.1% in phosphate-buffered saline (PBS), was added. The sample was then incubated at 378 C for 20 min, and, during this time, vortexed every 5 min to maximize cell dispersion. At the end of incubation, the sputum sample was filtered on a sterile gauze, and a small volume was used to assess total cell count (TCC) and viability, with a standard hemacytometer and the trypan blue dye method, respectively. The remaining sample was divided in two aliquots. The first one was diluted in PBS to obtain a final cellular concentration of 2 3 105 cells/ml, and amounts of 100 ml (about 20,000 cells) were used for cytospin slides (Cytospin 3; Shandon, Pittsburgh, PA). The slides were stained with Diff-Quik (Dade AG, Düdingen, Switzerland) and 400 nonsquamous cells were counted

TABLE 2 CLINICAL AND FUNCTIONAL CHARACTERISTICS OF PATIENTS AND SUBJECTS*

Sex, M/F Age, yr FEV1, % pred FEV1/FVC, % FEV1 post-bronc, % baseline PD20-methacholine, mg Atopy,† no. of subjects

Patients with COPD

Patients with Asthma

Asymptomatic Smokers

Healthy Control Subjects

10/0 66.3 6 10.0 46.6 6 15.9 53.3 6 12.8 105.8 6 3.6 Not done 0

11/4 33.2 6 16.7 90.4 6 17.2 74.8 6 7.3 112.3 6 11.1 101.8 6 135.6 7

6/5 44.0 6 12.3 98.7 6 11.5 78.0 6 7.9 104.8 6 4.8 . 1,600 0

6/4 43.9 6 13.4 104.4 6 11.2 83.3 6 5.0 104.1 6 4.8 . 1,600 0

Definition of abbreviations: PD20-methacholine 5 provocative dose of methacholine causing a 20% fall in FEV1; post-bronch 5 after administration of bronchodilator. * Data are expressed as means 6 SD. † Atopy means positive results to one or more allergens as demonstrated by skin prick tests.

1488

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

VOL 160

1999

TABLE 3 CELLULAR CHARACTERISTICS AND EOSINOPHIL CATIONIC PROTEIN LEVELS IN INDUCED SPUTUM

Total cell count, 3 105/ml Squamous cells, %TCC Viability, %NSC Eosinophils, %NSC Neutrophils, %NSC Macrophages, %NSC Lymphocytes, %NSC Epithelial cells, %NSC Sputum ECP, mg/L

Patients with COPD

Patients with Asthma

Asymptomatic Smokers

Healthy Control Subjects

19.9 6 9.9 9.1 6 7.4 77.2 6 10.3 0.7 6 0.5* 77.5 6 9.7† 19.4 6 10.5† 1.1 6 0.9 1.3 6 1.3 711.5 6 1,179.9†

19.1 6 12.9 9.1 6 7.9 76.6 6 7.9 22.2 6 18.6† 26.7 6 15.4 46.0 6 21.8 1.8 6 1.2 3.2 6 2.2 895.9 6 1,193.2†

38.1 6 50.6 11.4 6 9.4 79.6 6 6.3 0.2 6 0.3 33.1 6 17.0 61.0 6 16.4 2.4 6 2.8 3.5 6 2.6 138.8 6 176.7

13.8 6 7.7 13.5 6 8.7 82.4 6 5.4 0.2 6 0.2 35.9 6 14.4 57.0 6 13.0 2.1 6 1.5 4.8 6 4.7 81.7 6 72.3

Definition of abbreviations: NSC 5 nonsquamous cells; TCC 5 total cell count. * Indicates a significant difference as compared with healthy subjects (p , 0.05). † Indicates a significant difference as compared with the other groups (p , 0.01).

for differential cell count by two separate blinded investigators. The second aliquot was centrifuged at 4,500 rpm for 10 min and supernatants were stored at 2858 C for later analysis.

Eosinophil Cationic Protein Measurement ECP levels in supernatant of induced sputum were assessed by a fluoroimmunoenzymatic assay (CAP-system FEIA; Pharmacia, Uppsala, Sweden). The results were expressed as micrograms per liter, and adjusted for the dilution factor.

Statistical Analysis Differences between groups were first analyzed using the Kruskall– Wallis test. In case of a significant difference between groups, intergroup comparisons were assessed by a nonparametric method using the Mann–Whitney U test. A p value of , 0.05 was considered significant after a Bonferroni correction. The correlations between variables were examined by the Spearman rank correlation coefficient for patients with COPD and asymptomatic smokers, considered as a whole. For the correlations regarding ECP values, the latter were log10 transformed. Only the correlations with both an R value > 0.4 and a p value of < 0.05 were considered relevant. Reproducibility was expressed as an intraclass correlation coefficient [Ri 5 between-subject variance/(within- 1 between-subject variance)] (26) and as a coefficient of repeatability (Cr 5 2 SD of the mean difference of repeated measurements) in percentage for different cell types, as a number 3 105/ml for TCC, and as doubling concentrations for ECP levels (27).

RESULTS The clinical and functional characteristics of patients and subjects are listed in Table 2. All patients were able to produce sputum. A sufficient amount of sputum was obtained after 3 hypertonic saline inhalation steps in 33 of 46 (72%) subjects, whereas 4 steps were necessary in the remaining subjects. No relevant FEV1 fall was recorded during the inhalation procedure; thus sputum induction was completely safe in all subjects. The cellular characteristics and ECP levels in induced sputum are reported in Table 3. There was no difference in TCC between groups, although asymptomatic smokers tended to have higher values. Eosinophils were much higher in the asthma group than in the COPD group (p , 0.01, Figure 1A); moreover, they were also higher in patients with COPD versus asymptomatic smokers and healthy control subjects, although significance (p , 0.05) was reached only for the last comparison (Figure 1B). Neutrophils were higher in patients with COPD than in the other groups (p , 0.01). Sputum ECP levels were significantly (p , 0.01) higher in both asthma and COPD groups than in the other groups. There was no significant difference in sputum ECP levels between asthma and COPD groups, or between asymptomatic smokers and healthy control subjects. Significant inverse correlations were found between FEV1 (% pred) and neutrophils, eosinophils and sputum ECP on the one hand (Figures 2A, 2C, and 2E) and between FEV1/FVC

Figure 1. Eosinophils in induced sputum: (A) COPD versus asthma; (B) COPD versus asymptomatic smokers and healthy controls.

Balzano, Stefanelli, Iorio, et al.: Inflammation and COPD

1489

Figure 2. Correlations between (A) neutrophils and FEV1 (% pred), (B) neutrophils and FEV1/FVC (%), (C) eosinophils and FEV1 (% pred), (D) eosinophils and FEV1/FVC (%), (E) ECP and FEV1 (% pred), (F) ECP and FEV1/FVC (%). Only the correlations with an R value > 0.4 and a p value < 0.05 were considered relevant and are reported here.

1490


VOL 160

1999

TABLE 4 R AND p VALUES FOR CORRELATIONS IN PATIENTS WITH COPD AND ASYMPTOMATIC SMOKERS* FEV1 (% pred) R

p

FEV1/FVC (%) R

p

Eosinophils

Neutrophils

R

R

p

p

Eosinophils 20.55 0.0103 20.47 0.0261 — — 0.61 0.0072 Neutrophils 20.81 0.0003 20.79 0.0004 0.61 0.0072 — — Sputum ECP 20.47 0.0367 20.56 0.0126 0.53 0.0202 0.54 0.0152 * Only the correlations with an R value > 0.4 and a p value < 0.05 were considered relevant and are reported here.

(%) and neutrophils, eosinophils, and sputum ECP (Figures 2B, 2D, and 2F) on the other hand (Table 4). Furthermore, direct correlations were found between neutrophils and eosinophils (Figure 3), between either neutrophils or eosinophils and sputum ECP (Figures 4 and 5, respectively). The reproducibility was good for sputum eosinophils, neutrophils, macrophages, and ECP, but not for TCC, lymphocytes, and epithelial cells (Table 5).

DISCUSSION All patients and subjects studied produced a sufficient sputum sample, without relevant deterioration in lung function or unpleasant symptomatology. Therefore, our study confirms that induced sputum is a simple and safe technique for the assessment of airway inflammation. As expected, we found an increase of eosinophils in asthma and of neutrophils in COPD. However, a significant increase in sputum eosinophils was also found in patients with COPD as compared with healthy subjects, although the sputum eosinophilia in the COPD group (mean, 0.7%; range, 0.2 to 1.7%) was several orders of magnitude less versus the asthma group (mean, 22.2%; range, 0.6 to 55.5%), thus confirming the value of cellular analysis of induced sputum in the differentiation of inflammatory disorders of the airways. Although airway eosinophilia has been found in patients with COPD during exacerbations (15), conflicting results have

Figure 3. Correlation between sputum neutrophils and eosinophils.

Figure 4. Correlation between sputum neutrophils and ECP.

been obtained in patients who are clinically stable. In fact, an increase in airway eosinophils has been reported by some investigators (2, 10, 16, 17), but not by others (8, 9, 11). This could be due, at least in part, to the different inclusion criteria adopted. We used rigorous selection criteria to establish diagnosis and to exclude all avoidable influences on the underlying airway inflammation, so that both the presence of asthmatic features (atopy, reversibility of obstruction) and the occurrence of disease exacerbations or of antiinflammatory treatments can reliably be excluded in our patients with COPD. One of the reviewers of our study expressed concern that our COPD group could have included patients showing reversibility after a trial with oral steroids. In fact, these patients with COPD with features of asthma present BAL fluid eosinophilia (28). However, because in our unit patients with COPD routinely undergo a reversibility trial with oral steroids [1 mg/ (kg/d) for 2 wk] on admission, we retrieved these data from clinical carts and found that all patients but one had showed no significant reversibility according to the criteria used by

Figure 5. Correlation between sputum eosinophils and ECP.

1491

Balzano, Stefanelli, Iorio, et al.: Inflammation and COPD TABLE 5 REPEATABILITY OF SPUTUM MEASUREMENTS*

TCC, 3 105/ml Eosinophils, %NSC Neutrophils, %NSC Macrophages, %NSC Lymphocytes, %NSC Epithelial cells, %NSC ECP, mg/L

Day 1

Day 2

Ri

p Value

Cr

41.4 6 51.2 2.1 6 3.4 50.7 6 29.2 43.3 6 27.7 2.2 6 2.7 1.5 6 1.4 116.1 6 110.2

28.1 6 27.2 2.4 6 3.3 54.1 6 25.9 40.0 6 25.6 1.8 6 1.3 1.5 6 1.3 89.9 6 77.2

0.33 0.98 0.95 0.96 0.19 0.56 0.89

0.26 0.0001 0.0001 0.0001 0.37 0.10 0.0007

104.9 1.7 23.4 18.5 5.9 3.1 1.3

Definition of abbreviations: NSC 5 Nonsquamous cell; TCC 5 total cell count. * Values are presented as means 6 SD. Repeatability is expressed as coefficient of repeatability (Cr, in percentage for different cell types, n 3 105/ml for TCC and doubling concentrations for ECP levels), and as intraclass correlation coefficient (Ri).

In conclusion, this study confirms that induced sputum analysis is a practical, safe, and reproducible method for the assessment of airway inflammation. Like eosinophils in asthma, an increase in sputum neutrophils is the main characteristic of COPD. However, in our patients with stable COPD disease there was also eosinophilic airway inflammation, expressed in terms of sputum eosinophils and ECP levels, which, in addition, was well correlated with both neutrophilic inflammation and indices of airway obstruction. These results may have important implications for our understanding of the pathogenesis of disease. Acknowledgment : The authors are grateful to Prof. Paul O’Byrne for reviewing the manuscript, to Dr. Bruno Balbi for comments and suggestions, and to Jean Gilder for revising the text.

References

Chanez and coworkers (FEV1 change after oral steroids , 12% and , 200 ml). The only patient presenting reversibility had had an FEV1 change of 12.3% and 200 ml in absolute value; however, this patient presented a sputum eosinophilia of 0.9%, which did not differ from the mean value for the group (0.7 6 0.5%). In addition to an increase in airway eosinophils we also found an increase in ECP levels in induced sputum from patients with COPD, as compared with asymptomatic smokers and healthy subjects. Furthermore, the COPD group had a sputum ECP level comparable to that of the asthma group. A possible explanation of this intriguing finding is that eosinophils in patients with COPD could be in a higher state of activation than eosinophils in patients with asthma. This finding is consistent with results obtained in patients with stable COPD by BAL (8) and induced sputum (17), although a biopsy study from France has found that eosinophils in the submucosa of patients with stable COPD are indeed increased to a level comparable to that found in asthma, but are not degranulating (10). When we analyzed the correlations between sputum markers of inflammation and indices of airway obstruction in both patients with COPD and asymptomatic smokers, we found that sputum neutrophils were inversely correlated with functional indices of airway obstruction, such as FEV1 and the FEV1/FVC ratio; and this is in agreement with previous results (2, 11). However, to our knowledge, the present study is the first to demonstrate that sputum eosinophils and sputum ECP levels are also inversely correlated with indices of airway obstruction in these patients. Moreover, the significant correlations we found between sputum eosinophils and neutrophils, as well as between sputum neutrophils and sputum ECP, suggest that eosinophilic and neutrophilic inflammation may be closely related. Thus, it is conceivable that eosinophils may play a role in the pathogenesis of chronic airway inflammation in COPD. In our study reproducibility was good for all sputum measurements, with the exception of TCC, lymphocytes, and epithelial cells. To our knowledge, only one other study has demonstrated such a level of reproducibility for measurements performed on “whole” sputum samples (29), whereas a good reproducibility has indeed been shown in several studies for the selected sputum or “plugs” method (5, 6, 30, 31). Although the last method has undoubtedly been better studied, especially from a technical point of view (32), our data show that the whole sputum method has the potential to produce reliable results. A crucial point in the induced sputum technique is the avoidance of salivary contamination. We have previously shown that careful rinsing of the oral cavity before sputum collection reduces contamination to negligible levels (33).

1. Djukanovic, R., W. R. Roche, J. W. Wilson, C. R. Beasley, O. P. Twentyman, P. H. Howarth, and S. T. Holgate. 1990. Mucosal inflammation in asthma. Am. Rev. Respir. Dis. 142:434–457. 2. Thompson, A. B., D. M. Daughton, R. A. Robbins, M. A. Ghafouri, M. Oehlerking, and S. I. Rennard. 1989. Intraluminal airway inflammation in chronic bronchitis: characterization and correlation with clinical parameters. Am. Rev. Respir. Dis. 140:1527–1537. 3. Kirby, J. G., F. E. Hargreave, G. J. Gleich, and P. M. O’Byrne. 1987. Bronchoalveolar cell profiles of asthmatic and non-asthmatic subjects. Am. Rev. Respir. Dis. 136:379–383. 4. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barneon, N. Ghavanian, I. Enander, P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F. B. Michel. 1990. Eosinophilic inflammation in asthma. N. Engl. J. Med. 323:1033–1039. 5. Gibson, P. G., A. Girgis-Gabardo, M. M. Morris, S. Mattoli, J. M. Kay, J. Dolovich, J. Denburg, and F. E. Hargreave. 1989. Cellular characteristics of sputum from patients with asthma and chronic bronchitis. Thorax 44:693–699. 6. Pin, I., P. G. Gibson, R. Kolendowicz, A. Girgis-Gabardo, J. A. Denburg, F. E. Hargreave, and J. Dolowich. 1992. Use of induced sputum cell counts to investigate airway inflammation in asthma. Thorax 47:25–29. 7. Fahy, J. V., J. Liu, H. Wong, and H. A. Boushey. 1993. Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am. Rev. Respir. Dis. 147:1126–1131. 8. Linden, M., J. B. Rasmussen, E. Piitulainen, A. Tunek, M. Larson, H. Tegner, P. Venge, L. A. Laitinen, and R. Brattsand. 1993. Airway inflammation in smokers with nonobstructive and obstructive chronic bronchitis. Am. J. Respir. Crit. Care Med. 148:1226–1232. 9. Lusuardi, M., M. Capelli, C. G. Cerutti, E. L. Spada, and C. F. Donner. 1994. Airways inflammation in subjects with chronic bronchitis who have never smoked. Thorax 49:1211–1216. 10. Lacoste, J. Y., J. Bousquet, P. Chanez, T. van Vyve, J. Simony-Lafontaine, N. Lequeu, P. Vic, I. Enander, P. Godard, and F. B. Michel. 1993. Eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis and chronic obstructive pulmonary disease. J. Allergy Clin. Immunol. 92:537–548. 11. Keatings, V. M., P. D. Collins, D. M. Scott, and P. J. Barnes. 1996. Differences in interleukin-8 and tumor necrosis factor-alfa in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am. J. Respir. Crit. Care Med. 153:530–534. 12. Ronchi, M. C., C. Piragino, E. Rosi, M. Amendola, R. Duranti, and G. Scano. 1996. Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax 51:1000–1004. 13. Jeffery, P. K., A. J. Wardlaw, F. C. Nelson, J. V. Collins, and A. B. Kay. 1989. Bronchial biopsies in asthma: an ultrastructural, quantitative study and correlation with hyperreactivity. Am. Rev. Respir. Dis. 140: 1745–1753. 14. Foresi, A., C. Leone, A. Pelucchi, B. Mastropasqua, A. Chetta, R. D’Ippolito, L. Marazzini, and D. Olivieri. 1997. Eosinophils, mast cells, and basophils in induced sputum from patients with seasonal allergic rhinitis and perennial asthma: relationship to methacoline responsiveness. J. Allergy Clin. Immunol. 100:58–64. 15. Saetta, M., A. Di Stefano, P. Maestrelli, G. Turato, M. P. Ruggieri, A. Roggeri, P. Calcagni, C. E. Mapp, A. Ciaccia, and L. M. Fabbri. 1994. Airway eosinophilia in chronic bronchitis during exacerbations. Am. J. Respir. Crit. Care Med. 150:1646–1652.

1492


16. Balbi, B., A. Aufiero, A. Pesci, S. Oddera, P. Zanon, G. A. Rossi, and D. Olivieri. 1994. Lower respiratory tract inflammation in chronic bronchitis: evaluation by bronchoalveolar lavage and changes associated with treatment with immucytal, a biological response modifier. Chest 106:819–826. 17. Keatings, V. M., and P. J. Barnes. 1997. Granulocyte activation markers in induced sputum: comparison between chronic obstructive pulmonary disease, asthma and normal subjects. Am. J. Respir. Crit. Care Med. 155:449–453. 18. Burrows, B., R. J. Knudson, M. G. Cline, and M. D. Levowitz. 1979. Quantitative relationship between cigarette smoking and ventilatory function. Am. Rev. Respir. Dis. 115:195–205. 19. American Thoracic Society. 1987. Standards for the diagnosis and care of patients with COPD and asthma. Am. Rev. Respir. Dis. 136(1): 225–244. 20. Balzano, G., C. Gallo, C. Masi, G. Cocco, P. Ferranti, E. Melillo, and G. Seccia. 1992. Effect of azelastine on the seasonal increase in nonspecific bronchial responsiveness to methacholine in pollen allergic patients: a randomized, double-blind placebo-controlled, crossover study. Clin. Exp. Allergy 22:371–377. 21. Fraenkel, D. J., P. G. Bardin, G. Sanderson, F. Lampe, S. L. Johnston, and S. T. Holgate. 1995. Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects. Am. J. Respir. Crit. Care Med. 151:879–886. 22. Djukanovic, R., J. W. Wilson, K. M. Britten, S. J. Wilson, A. F. Walls, W. R. Roche, P. H. Howarth, and S. T. Holgate. 1992. Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am. Rev. Respir. Dis. 145:669–674. 23. American Thoracic Society. 1987. Standardization of spirometry: 1987— update. Am. Rev. Respir. Dis. 136:1285–1298. 24. Balzano, G., I. Delli Carri, C. Gallo, G. Cocco, and G. Melillo. 1989. In-

25.

26.

27. 28.

29.

30.

31.

32.

33.

VOL 160

1999

trasubject between-day variability of PD20 methacholine assessed by the dosimeter inhalation test. Chest 95:1238–1243. Shoeffel, R. E., S. D. Anderson, and R. E. C. Altounyan. 1981. Bronchial hyperreactivity in response to inhalation of ultrasonically nebulized solutions of distilled water and saline. Br. Med. J. 283:1285–1287. Bland, J. M., and D. G. Altman. 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 8: 307–310. Chinn, S. 1991. Repeatability and method comparison. Thorax 36:454–456. Chanez, P., A. M. Vignola, T. O’Shaugnessy, I. Enander, D. Li, P. K. Jeffery, and J. Bousquet. 1997. Corticosteroid reversibility in COPD is related to features of asthma. Am. J. Respir. Crit. Care Med. 155:1529– 1534. in’t Veen, J. C. C. M., H. W. F. M. de Gouw, H. H. Smits, J. K. Sont, P. S. Hiemstra, P. J. Sterk, and E. H. Bel. 1996. Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma. Eur. Respir. J. 9:2441–2447. Pizzichini, E., M. M. M. Pizzichini, A. Efthimiadis, S. Evans, M. M. Morris, D. Squillace, G. J. Gleich, J. Dolovich, and F. E. Hargreave. 1996. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurement. Am. J. Respir. Crit. Care Med. 154:1308–1317. Spanevello, A., G. B. Migliori, A. Sharara, R. Ballardini, P. Bridge, P. Pisati, M. Neri, and P. W. Ind. 1997. Induced sputum to assess airway inflammation: a study of reproducibility. Clin. Exp. Allergy 27:1138–1144. Pavord, I. D., M. M. M. Pizzichini, E. Pizzichini, and F. E. Hargreave. 1997. The use of induced sputum to investigate airway inflammation. Thorax 52:498–501. Stefanelli, F., C. Iorio, E. Melillo, C. Gaudiosi, and G. Balzano. 1997. Variability of salivary squamous cell contamination in induced sputum. Eur. Respir. J. 10(25):182s.