Abstract
It has been suggested that patients with refractory eosinophilic airway inflammation represent a separate “eosinophilic” asthma phenotype associated with increased morbidity and a poor prognosis. To investigate whether persistent eosinophilia in these patients is a fixed feature or can still be modified by treatment, we investigated the effect of high-dose intramuscular corticosteroids on eosinophils in induced sputum. Twenty-two patients with stable severe asthma (15 women, aged 21–73 years) participated in this double-blind, placebo-controlled study. All were using inhaled corticosteroids (⩾ 1,600 μg/day) or chronic oral prednisone. They were included if the percentage of eosinophils in induced sputum was above the upper limit of normal (⩾ 2%). Two weeks after treatment with triamcinolone, but not placebo, sputum eosinophils almost completely disappeared from a median of 12.6–0.2% (p < 0.001). In 82% of patients, no eosinophils could be observed at all. In addition, the rescue medication score decreased from 1.4 to 0.8 (p = 0.01), and FEV1 improved from a median of 73.8–88.3% predicted (p = 0.001). We conclude that persistent sputum eosinophilia despite extensive antiasthma treatment is not a refractory phenomenon but is still sensitive to high-dose systemic corticosteroids. This implies that these patients with severe asthma need additional or alternative antiinflammatory treatment to combat the eosinophilia and associated poor prognosis.
There is now accumulating evidence that patients with bronchial asthma are heterogeneous with respect to the type of inflammation in the airways and the response to antiinflammatory therapy (1–4). Most patients with asthma have eosinophilic airway inflammation with good response to treatment with inhaled corticosteroids (5). However, a relatively large proportion of adult asthma cases is characterized by neutrophilic airway inflammation (4, 6), in particular those with severe disease (7–9). These patients are more difficult to treat and often require high doses of inhaled or oral corticosteroids to control their disease. Remarkably, bronchial biopsy and bronchoalveolar lavage studies have shown that in a subgroup of patients with severe asthma such neutrophilic airway inflammation is accompanied by persistent eosinophilic inflammation (10, 11).
Eosinophilic inflammation in the airway mucosa that persists despite the use of high doses of inhaled corticosteroids or oral corticosteroids has been observed in several studies (9, 12–18) and has been implicated by Wenzel and colleagues as a feature of a separate asthma phenotype, associated with poor asthma prognosis (10, 12). Indeed, studies have shown that eosinophilic inflammation despite vigorous antiasthma treatment is associated with remodeling of the airways, impaired lung function, and near-fatal asthma attacks (10, 19, 20).
Why eosinophilia is persisting in these patients is unknown. Moreover, it has not been investigated whether persistent eosinophilia is a permanent characteristic of a specific phenotype of severe asthma or can be modulated by more intensive treatment.
In this study, we investigated the effect of high-dose intramuscular corticosteroids on sputum eosinophilia in a subgroup of patients with severe asthma featuring persistent eosinophilia despite extensive antiinflammatory treatment. In addition, the effect of intramuscular corticosteroids on asthma symptoms, lung function, peripheral blood eosinophils, and nitric oxide in exhaled breath was examined. The results of this study have been published previously in the form of an abstract (21).
The patients in this study were part of a cohort of severe asthma patients participating in studies aimed at identifying risk factors of asthma severity (20). From the original cohort of 152 patients, 22 nonsmoking outpatients meeting the criteria of severe bronchial asthma (22) were selected. The patients were included if they were clinically stable for at least 4 weeks while the percentage of eosinophils in their sputum was above the upper limit of normal (⩾ 2%) (23). All patients used 1,600–6,400 μg/day beclomethasone equivalent and long-acting bronchodilators for more than 1 year and had had at least one (median, 4; range, 1–7) course of oral corticosteroids during the past year or 5 mg or more of oral prednisone daily. Patients with contraindications for systemic corticosteroids were excluded. The study was approved by the hospital medical ethics committee, and all patients gave written informed consent.
The study had a randomized, parallel, double-blind, placebo-controlled design. At baseline (Visit 1), patients' characteristics were documented, and Borg score, postbronchodilator FEV1, and level of exhaled nitric oxide were assessed. A blood sample was taken, and sputum was induced. If the sputum eosinophil percentage exceeded 2%, patients were randomized to one of the two treatments, with stratification for daily use of oral steroids (strata yes/no), and level of sputum eosinophil percentage (strata 2–10% or > 10%). After 1 week (Visit 2), one single intramuscular injection of 3 ml (40 mg/ml) long-acting triamcinolone acetonide (Kenacort-A40; Bristol-Myers Squibb, Woerden, The Netherlands) or matched placebo (3 ml NaCl 0.9%) was given. Intramuscular administration was chosen instead of a course of oral corticosteroids to avoid any confounding by noncompliance with therapy. Two weeks thereafter (Visit 3), the measurements performed at Visit 1 were repeated. A diary was completed by the patients during the first and third weeks of the study.
Postbronchodilator FEV1 was assessed (24) 30 minutes after the inhalation of 400 μg salbutamol, and exhaled nitric oxide measurements were performed as previously described (25). Sputum was induced according to a validated protocol (15), and whole sputum samples were processed (26). Peripheral blood eosinophil and neutrophilic percentages were measured by standard automated cell counter. A Borg scale (0–10) was used to rate the patient's dyspnea (27) by asking this: “When thinking about daily activities regularly causing dyspnea, how severe was your dyspnea during the last week as compared with the severest dyspnea you ever experienced?” Additionally, on diary cards, the severity of daytime dyspnea, cough, sputum production and limitation in activities, and nocturnal dyspnea and cough were registered on a scale ranging 0 to 3. The cumulative symptom score per day (range 0–18) was analyzed. Finally, rescue medication score was expressed as the mean number of as-needed puffs of β2-agonist or ipratropium bromide per day.
Eosinophil and neutrophil percentages in blood and sputum were log-transformed before statistical analysis. Two-tailed paired and unpaired t tests, Wilcoxon rank tests, or chi-square analyses were used for between-group analyses at baseline and for the analysis of within-group treatment effects.
Between-group changes were explored using analysis of variance with treatment as between-patient factor and age, age at onset of asthma, asthma duration, and baseline value of the measurement as covariate (28). All analyses were performed using SPSS for Windows, version 10.0 (SPSS, Inc., Chicago, IL).
Eleven of the 22 participating patients were randomized to triamcinolone-treatment, whereas the remaining 11 patients received placebo. There were no significant differences between the groups in age and asthma duration (Table 1)
Triamcinolone | Placebo | ||
|---|---|---|---|
| (n = 11) | (n = 11) | p Value | |
| Age, yr* | 45.6 ± 14.3 | 53.8 ± 12.0 | 0.16 |
| Sex, M/F | 3/8 | 4/7 | 0.65 |
| Age at onset asthma, yr† | 8.5 (0.5–44) | 35 (0.5–60) | 0.07 |
| Asthma duration, yr† | 35.5 (3–60) | 15 (4–53) | 0.25 |
| Pack-years† | 2 (0–10) | 2 (0–9) | 0.79 |
| Dose inhaled steroids, μg/day† | 1,600 (1,600–3,200) | 1,600 (1,600–6,400) | 0.47 |
| Maintenance oral steroids, n (%) | 4 (36.4) | 3 (27.2) | 0.65 |
| Positive RAST, n (%) | 6 (60.0) | 4 (36.4) | 0.28 |
The intramuscular injection of triamcinolone/placebo was well tolerated in all patients, and no serious side effects were reported. At baseline, sputum eosinophil percentages in the triamcinolone-treated group were not different from those in the placebo-treated group (median [range] 12.6% [2–64%] vs. 17.0% [2–30%], respectively; p > 0.5). In addition, high-sputum neutrophil percentages were found in the triamcinolone group as well as in the placebo-treated patients (median [range] 70.8% [28–92%] vs. 58.0% [50–85%], p > 0.5).
Two weeks after the intramuscular injection, sputum eosinophilia in the triamcinolone-treated group was almost abolished to a median (range) percentage of 0.2 (0–5.8), whereas in the placebo group, the eosinophil percentage remained unchanged at 24.8 (5–44) (p = 0.01) (Figure 1)
Figure 1. Effect of treatment with intramuscular triamcinolone (circles) or placebo (squares) on sputum eosinophil percentages in 22 patients with severe asthma. (Open symbols) Patients using oral corticosteroids on a daily basis. (Closed symbols) Patients not using oral corticosteroids on a daily basis. Lines represent median values.
[More] [Minimize]There were no significant differences in baseline values between the two groups (p > 0.2), except for a lower diary score (p = 0.02) and a trend toward a lesser use of rescue medication (p = 0.09) in the triamcinolone group as compared with placebo (Table 2)
Triamcinolone (n = 11) | Placebo (n = 11) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Baseline | 2 Weeks | Within-group p Value | Baseline | 2 Weeks | Within-group p Value | Between-group p Value | |||||
| Borg score | 2.0 (0–5) | 0.5 (0–3) | 0.02 | 3.0 (0–9) | 3.0 (0–9) | NS | 0.01 | ||||
| Diary score | 3.5 (1–6) | 1.4 (0–7) | NS | 5.1 (2–14) | 5.8 (2–12) | NS | NS | ||||
| Rescue med score | 1.4 (0–8) | 0.8 (0–3) | 0.04 | 3.6 (0–11) | 4.9 (0–12) | NS | < 0.01 | ||||
| Pb FEV1, %pred | 73.8 (42–107) | 88.3 (44–119) | 0.001 | 73.0 (39–96) | 67.8 (36–91) | NS | 0.02 | ||||
| NO, ppb | 21.5 (2–55) | 7.9 (2–32) | 0.02 | 22.4 (2–72) | 14.0 (2–62) | NS | NS | ||||
| Blood eo, % | 6.2 (1–18) | 1.3 (0–5) | 0.001 | 5.1 (3–8) | 6.4 (2–10) | NS | < 0.01 | ||||
| Sputum eo, % | 12.6 (2–64) | 0.2 (0–6) | < 0.001 | 17.0 (2–30) | 24.8 (5–44) | NS | 0.01 | ||||
| Blood neutro, % | 54.1 (39–77) | 71.7 (44–80) | 0.01 | 63.0 (46–89) | 61.2 (49–75) | NS | NS | ||||
| Sputum neutro, % | 70.8 (28–92) | 75.1 (35–94) | NS | 58.0 (50–85) | 41.4 (18–86) | NS | < 0.01 | ||||
Figure 2. Effect of treatment with intramuscular triamcinolone (circles) or placebo (squares) on postbronchodilator FEV1 (left panel) and on rescue medication score (right panel) in 22 patients with severe asthma. (Open symbols) Patients using oral corticosteroids on a daily basis. (Closed symbols) Patients not using oral corticosteroids on a daily basis. Lines represent median values.
[More] [Minimize]This study shows that persistent sputum eosinophilia despite extensive antiinflammatory treatment in patients with severe asthma is not a refractory phenomenon but is still sensitive to high-dose systemic corticosteroids. In fact, sputum eosinophilia was almost entirely abolished by the administration of high doses of parenteral corticosteroids, whereas sputum neutrophilia remained unchanged. The normalization of sputum eosinophilia was accompanied by an improvement in FEV1 and a decreased use of rescue medication. These results suggest that patients with persistent eosinophilia are candidates for alternative therapeutic approaches to target the eosinophilic inflammation and to improve long-term outcome.
This study provides us with a number of novel findings that have both scientific and clinical implications. First, it suggests that the term “eosinophilic phenotype” of severe asthma should not be handled as a fixed disease entity but rather as an indication of disease activity and persistence. Apparently, airway eosinophilia in these patients is not static. Therefore, the classification of severe asthma phenotypes in “eosinophilic” or “noneosinophilic” (10, 12) probably needs to be reconsidered. This has potential implications for future genetic studies because it has now become clear that persistent airway eosinophilia per se is probably not associated with a specific genotype in severe asthma (29).
Second, this study shows that the elevated percentages of eosinophils in sputum normalized completely after a high dose of parenteral corticosteroids, suggesting that these patients may be relatively insensitive to steroids but do not represent a distinct subgroup of patients with complete steroid resistance at a cellular level. Relative insensitivity to corticosteroids in patients with severe airway inflammation is conceivable and might result from allergen- or infection-induced cell activation or chronic exposure to medications such as β-agonists or corticosteroids (30). If this is the major cause of persistent eosinophilia, alternative antiinflammatory and immunomodulating approaches should be considered to control the inflammatory process further (31).
Third, the study shows that high-dose parenteral corticosteroids have no effect on airway neutrophilia in this category of patients. Before treatment we observed a prominent neutrophilic inflammation that did not change after steroid therapy. This suggests that neutrophilia in these patients is not induced by corticosteroid therapy but is inherent to the type of inflammation in severe asthma (13). Whether neutrophils contribute to steroid resistance or are associated with underlying causes of steroid resistance remains an important question (4).
Fourth, the study is clinically important. Patients with severe asthma are to be treated according to Step 4 of the international guidelines (32). This implies the use of oral corticosteroids. Most patients and doctors are reluctant to use these drugs continuously because of the serious side effects. This is why some patients prefer to remain symptomatic instead of using oral corticosteroids on a daily basis. This study shows that both the intensity of airway inflammation and the level of symptoms and lung function can (and should) be improved by intensifying treatment and/or administering it via the systemic route. Regular monitoring of sputum eosinophils to adjust antiinflammatory treatment might be necessary in these patients to prevent severe exacerbations and airway fibrosis (33, 34).
The question remains why eosinophilia persists in these patients with severe asthma despite high doses of inhaled corticosteroids. Apart from relative steroid resistance (30), an alternative explanation might be that parenteral corticosteroids were able to reach regions of the airways that are hardly accessible to inhaled corticosteroids, such as the peripheral airways (35) or upper airways including the paranasal sinus (36). These drugs also reach the bone marrow and may have influenced eosinophilopoiesis being upregulated by hemopoietic factors produced by inflamed tissue (37). Also, relative undertreatment caused by impaired perception of dyspnea in this category of patients with severe asthma might have been the case (38). Finally, noncompliance with treatment might be a possible explanation of persistent eosinophilic inflammation in some patients, which is suggested by insufficiently suppressed serum cortisol levels in one patient on oral corticosteroids (data not shown). However, this is not the explanation in the majority of patients.
In conclusion, this study shows that in patients with severe asthma with sputum eosinophilia despite extensive antiasthma treatment, a high dose of intramuscular corticosteroids results in normalization of sputum eosinophilia, associated with a decreased use of rescue medication and an improvement of postbronchodilator FEV1. This implies that persistent eosinophilia despite treatment is a phenomenon that can be overcome by intensifying the antiinflammatory treatment. If persistent eosinophilic airway inflammation is causally related to more severe asthma, remodeling of the airways, and a poor prognosis of the disease, then alternative antiinflammatory therapies are urgently needed. Ideally, this would imply the use of novel potent immunomodulatory agents that are safe and capable of reaching the peripheral airways, the paranasal sinus, as well as the bone marrow.
The authors thank M. C. Timmers and H. van der Veen for technical assistance and the chest physicians of the participating hospitals for their cooperation (P. I. van Spiegel, G. Visschers, Slotervaart Hospital, Amsterdam; A. H. M. van der Heijden, Rode Kruis Hospital, Beverwijk; B. J. M. Pannekoek, Reinier de Graaf Gasthuis, Delft; H. H. Berendsen, K. W. van Kralingen, Bronovo Hospital, Den Haag; H. G. M. Heijerman, A. C. Roldaan, Leyenburg Hospital, Den Haag; A. H. M. van der Heijden, Spaarne Hospital, Heemstede; H. C. J. van Klink, Diaconessenhuis, Leiden; C. R. Apap, St. Antoniushove, Leidschendam; and A. Rudolphus, K. Y. Tan, St. Franciscus Gasthuis, Rotterdam).
| 1. | Peters SP. Heterogeneity in the pathology and treatment of asthma. Am J Med 2003;115(Suppl 3A):49S–54S. |
| 2. | Wardlaw AJ, Brightling CE, Green R, Woltmann G, Bradding P, Pavord ID. New insights into the relationship between airway inflammation and asthma. Clin Sci (Lond) 2002;103:201–211. |
| 3. | Douwes J, Gibson P, Pekkanen J, Pearce N. Non-eosinophilic asthma: importance and possible mechanisms. Thorax 2002;57:643–648. |
| 4. | Green RH, Brightling CE, Woltmann G, Parker D, Wardlaw AJ, Pavord ID. Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids. Thorax 2002;57:875–879. |
| 5. | Tattersfield AE, Knox AJ, Britton JR, Hall IP. Asthma. Lancet 2002;360:1313–1322. |
| 6. | Gibson PG, Simpson JL, Saltos N. Heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin-8. Chest 2001;119:1329–1336. |
| 7. | European Network for Understanding Mechanisms of Severe Asthma. The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma. Eur Respir J 2003;22:470–477. |
| 8. | Jatakanon A, Uasuf C, Maziak W, Lim S, Chung KF, Barnes PJ. Neutrophilic inflammation in severe persistent asthma. Am J Respir Crit Care Med 1999;160:1532–1539. |
| 9. | Wenzel SE, Szefler SJ, Leung DY, Sloan SI, Rex MD, Martin RJ. Bronchoscopic evaluation of severe asthma: persistent inflammation associated with high dose glucocorticoids. Am J Respir Crit Care Med 1997;156:737–743. |
| 10. | Wenzel SE, Schwartz LB, Langmack EL, Halliday JL, Trudeau JB, Gibbs RL, Chu HW. Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. Am J Respir Crit Care Med 1999;160:1001–1008. |
| 11. | Booth H, Richmond I, Ward C, Gardiner PV, Harkawat R, Walters EH. Effect of high dose inhaled fluticasone propionate on airway inflammation in asthma. Am J Respir Crit Care Med 1995;152:45–52. |
| 12. | Miranda C, Busacker A, Balzar S, Trudeau J, Wenzel SE. Distinguishing severe asthma phenotypes: role of age at onset and eosinophilic inflammation. J Allergy Clin Immunol 2004;113:101–108. |
| 13. | Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M, Djukanovic R. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med 2000;161:9–16. |
| 14. | Sont JK, Han J, van Krieken JM, Evertse CE, Hooijer R, Willems LN, Sterk PJ. Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax 1996;51:496–502. |
| 15. | ten Brinke A, de Lange C, Zwinderman AH, Rabe KF, Sterk PJ, Bel EH. Sputum induction in severe asthma by a standardized protocol: predictors of excessive bronchoconstriction. Am J Respir Crit Care Med 2001;164:749–753. |
| 16. | Chakir J, Hamid Q, Bosse M, Boulet LP, Laviolette M. Bronchial inflammation in corticosteroid-sensitive and corticosteroid-resistant asthma at baseline and on oral corticosteroid treatment. Clin Exp Allergy 2002;32:578–582. |
| 17. | Synek M, Beasley R, Frew AJ, Goulding D, Holloway L, Lampe FC, Roche WR, Holgate ST. Cellular infiltration of the airways in asthma of varying severity. Am J Respir Crit Care Med 1996;154:224–230. |
| 18. | Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003;167:1360–1368. |
| 19. | Fabbri LM, Romagnoli M, Corbetta L, Casoni G, Busljetic K, Turato G, Ligabue G, Ciaccia A, Saetta M, Papi A. Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:418–424. |
| 20. | ten Brinke A, Zwinderman AH, Sterk PJ, Rabe KF, Bel EH. Factors associated with persistent airflow limitation in severe asthma. Am J Respir Crit Care Med 2001;164:744–748. |
| 21. | ten Brinke A, Goos C, Timmers M, van der Veen H, Sterk PJ, Rabe KF, Bel EH. Persistent sputum eosinophilia in severe asthma despite treatment: the triamcinolone experience [abstract]. Am J Respir Crit Care Med 2001;163:A871. |
| 22. | Chung KF, Godard P, Adelroth E, Ayres J, Barnes N, Barnes P, Bel E, Burney P, Chanez P, Connett G, et al. Difficult/therapy-resistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies: ERS Task Force on Difficult/Therapy-Resistant Asthma: European Respiratory Society. Eur Respir J 1999;13:1198–1208. |
| 23. | Belda J, Leigh R, Parameswaran K, O'Byrne PM, Sears MR, Hargreave FE. Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000;161:475–478. |
| 24. | Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced ventilatory flows: Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal: Official Statement of the European Respiratory Society. Eur Respir J Suppl 1993;16:5–40. |
| 25. | de Gouw HW, Grunberg K, Schot R, Kroes AC, Dick EC, Sterk PJ. Relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects. Eur Respir J 1998;11:126–132. |
| 26. | in 't Veen JC, de Gouw HW, Smits HH, Sont JK, Hiemstra PS, Sterk PJ, Bel EH. Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma. Eur Respir J 1996;9:2441–2447. |
| 27. | Wilson RC, Jones PW. A comparison of the visual analogue scale and modified Borg scale for the measurement of dyspnoea during exercise. Clin Sci 1989;76:277–282. |
| 28. | Armitage P, Berry G. Statistical methods in medical research, 2nd ed. Oxford: Blackwell Scientific; 1987. |
| 29. | Hall IP. Genetic factors in asthma severity. Clin Exp Allergy 1998;28:16–20. |
| 30. | Leung DY, Bloom JW. Update on glucocorticoid action and resistance. J Allergy Clin Immunol 2003;111:3–22. |
| 31. | Barnes PJ, Adcock IM. How do corticosteroids work in asthma? Ann Intern Med 2003;139:359–370. |
| 32. | National Heart, Lung, and Blood Institute, National Institutes of Health. Global initiative for asthma. Global Strategy for asthma management and prevention: NHLBI/WHO Workshop report. Bethesda, MD; 2002. |
| 33. | Sont JK, Willems LN, Bel EH, van Krieken JH, Vandenbroucke JP, Sterk PJ. Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment: the AMPUL Study Group. Am J Respir Crit Care Med 1999;159:1043–1051. |
| 34. | Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw AJ, Pavord ID. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002;360:1715–1721. |
| 35. | Hauber HP, Gotfried M, Newman K, Danda R, Servi RJ, Christodoulopoulos P, Hamid Q. Effect of HFA-flunisolide on peripheral lung inflammation in asthma. J Allergy Clin Immunol 2003;112:58–63. |
| 36. | ten Brinke A, Grootendorst DC, Schmidt JT, De Bruine FT, van Buchem MA, Sterk PJ, Rabe KF, Bel EH. Chronic sinusitis in severe asthma is related to sputum eosinophilia. J Allergy Clin Immunol 2002;109:621–626. |
| 37. | Dorman SC, Sehmi R, Gauvreau GM, Watson RM, Foley R, Jones GL, Denburg JA, Inman MD, O'Byrne PM. Kinetics of bone marrow eosinophilopoiesis and associated cytokines after allergen inhalation. Am J Respir Crit Care Med 2004;169:565–572. |
| 38. | in 't Veen JC, Smits HH, Ravensberg AJ, Hiemstra PS, Sterk PJ, Bel EH. Impaired perception of dyspnea in patients with severe asthma: relation to sputum eosinophils. Am J Respir Crit Care Med 1998;158:1134–1141. |
