- Email: [email protected]
Absence of association of peripheral blood eosinophilia or increased eosinophil cationic protein with bronchial hyperresponsiveness during asthma remission
Absence of association of peripheral blood eosinophilia or increased eosinophil cationic protein with bronchial hyperresponsiveness during asthma remission Young Yull Koh, MD*; Hee Kang, MD*; Kyu Min Nah, MD*; and Chang Keun Kim, MD†
Background: The mechanisms responsible for persistent bronchial hyperresponsiveness (BHR) in adolescents with long-term asthma remission are poorly understood. Objective: To determine whether BHR in adolescents with asthma remission is associated with peripheral blood eosinophilia, increased serum levels of eosinophil cationic protein (ECP), or both findings. Methods: We classified 51 adolescents with long-term asthma remission (neither asthma-related symptoms nor medication during the previous 2 years) into 28 BHR-positive patients (methacholine PC20 [provocative concentration causing a 20% decrease in forced expiratory volume in 1 second] ⬍18 mg/mL) and 23 BHR-negative patients. The peripheral blood eosinophil counts and serum ECP concentrations were compared between these 2 groups. Twenty-eight patients with symptomatic asthma (symptomatic group), matched for methacholine PC20 level with study subjects in the BHR-positive remission group, and 28 healthy adolescents (control group) were also studied. Results: No significant differences in the peripheral blood eosinophil counts (262.1 ⫾ 117.0/L vs 253.9 ⫾ 165.0/L) and the serum ECP levels (15.6 ⫾ 10.0 g/L vs 15.8 ⫾ 11.9 g/L) were found between the BHR-positive and BHR-negative remission groups, respectively. The BHR-positive remission group differed from the symptomatic group (372.9 ⫾ 190.3/L, P ⬍ 0.05; 26.6 ⫾ 11.3 g/L, P ⬍ 0.01) in both blood indices but resembled the control group (214.6 ⫾ 118.6/L and 12.1 ⫾ 4.8 g/L; both, no significant difference). Conclusions: BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. This finding suggests that the mechanism underlying BHR in this clinical setting may differ from that in symptomatic asthma. Ann Allergy Asthma Immunol. 2003;91:297–302.
INTRODUCTION Epidemiologic studies have demonstrated that, in many children with asthma, long-lasting clinical remission ensues at adolescence.1,2 Bronchial hyperresponsiveness (BHR), often called “the hallmark of asthma,” is known to be an important risk factor for the development of asthma3 and to be indicative of the severity of symptomatic asthma.4 Some studies have shown, however, that BHR persists in a considerable proportion of adolescents with asthma in long-term clinical remission.5,6 Little is known about the mechanism underlying BHR in this clinical setting. Airway inflammation is considered a characteristic feature of asthma, and eosinophils are recognized as the most important inflammatory cells.7 Certain studies8,9 have attempted to * Department of Pediatrics and Clinical Research Institute, Seoul National University Hospital, Seoul, Korea. † Department of Pediatrics, Inje University Sanggye Paik Hospital, Seoul, Korea. This study was supported in part by BK 21 Project for Medicine, Dentistry, and Pharmacy and by grant no. 04 –2001-044 from the Seoul National University Hospital Research Fund. Received for publication October 6, 2002. Accepted for publication in revised form January 31, 2003.
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determine the role of eosinophils in asthma by using bronchoalveolar lavage and biopsies in patients with asthma. These studies found not only an eosinophilic cellular infiltrate but also increased levels of eosinophilic granular proteins such as eosinophil cationic protein (ECP). Another characteristic feature of asthma is the presence of peripheral blood eosinophilia.10 Increased levels of ECP have likewise been identified in the serum of patients with asthma.11 Investigators have increasingly suggested that serum levels of ECP may reflect the intensity of airway inflammation in asthma because the serum ECP level is closely related to eosinophilic airway inflammation as determined by evaluation of sputum samples or bronchoalveolar lavage.12,13 BHR in symptomatic asthma is thought to be a consequence of underlying airway inflammation,14 and some studies15,16 have noted correlations between measurements of bronchial responsiveness and various cellular aspects of airway inflammation. Thus far, however, investigators do not know whether the persisting BHR in adolescents with asthma remission is caused by airway inflammation or whether it is linked to another mechanism. In this study, our goal was to ascertain whether BHR in adolescents with asthma remission is associated with peripheral blood eosinophilia, increased
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serum ECP levels, or both. We compared total eosinophil counts and serum levels of ECP between groups of adolescents with long-term asthma remission and persisting BHR or nonpersisting BHR. These variables were also compared against those of patients with symptomatic asthma and a similar degree of BHR as well as those of healthy control subjects. PATIENTS AND METHODS Study Subjects We recruited 51 adolescents with long-term remission of atopic asthma from the allergy clinic at Seoul National University Children’s Hospital. All study subjects had a history of wheezing and dyspnea and had been previously diagnosed as having atopic asthma on the basis of the criteria established by the American Thoracic Society.17 At the time of diagnosis, all patients had a methacholine PC20 (provocative concentration causing a 20% decrease in forced expiratory volume in 1 second [FEV1]) level of less than 18 mg/mL. Atopy was defined as at least 1 positive result of a skin prick test to a panel of 12 common aeroallergens in the presence of positive and negative controls. Most study subjects showed a positive skin reaction to house-dust mites. Long-term clinical remission was assumed if a patient reported the complete absence of wheezing and dyspnea at rest and on exertion and had taken no medication to control symptoms of asthma for at least 24 months before the study. These patients underwent a methacholine inhalation test and were classified into BHRpositive and BHR-negative groups on the basis of the methacholine PC20 criterion (18 mg/mL).18 Another group of 28 adolescents with current atopic asthma (symptomatic group) was recruited. These patients had a history of symptoms of asthma (cough, wheezing, and dyspnea) during the previous year, which had been controlled by an as-needed bronchodilator with or without anti-inflammatory agents, and they had a positive skin test result. Those patients with a history of near-fatal asthma or major exacerbations necessitating the use of systemic corticosteroids were excluded from the study. Candidates were further selected on the basis of the results of a methacholine inhalation test, by matching them with the methacholine PC20 levels of study subjects in the BHR-positive remission group. A separate group of 28 healthy adolescents with no history of asthma or other allergic diseases served as control subjects. These subjects underwent a methacholine inhalation test, and those with a methacholine PC20 of higher than 18 mg/mL were selected and matched for sex with those in the BHRpositive remission group. In all 4 study groups, blood samples were obtained for determination of total eosinophil counts and serum ECP levels at approximately 10 AM. Parents of the subjects enrolled in this study gave informed consent, and the study was approved by the Hospital Ethics Committee.
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Methacholine Challenge Test FEV1 was recorded as the best result of 3 attempts with use of a computerized spirometer (Microspiro-HI 298; Chest Inc, Tokyo, Japan); measurements that varied by no more than 5% were considered acceptable. Methacholine inhalation tests were performed with use of a modification of the method described by Chai et al.19 At the time of the study, all subjects had been free of any acute respiratory tract infection for 4 weeks and had not taken any medication for 7 days before the study. Because most study subjects were sensitized to housedust mites, the test was performed during the winter season (December to February), when house-dust mites are least prevalent and least variable across the country.20 Methacholine (Sigma Chemical, St. Louis, MO) at concentrations of 0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10, and 25 mg/mL was prepared by dilution in buffered saline (pH 7.4). A RosenthalFrench dosimeter (Laboratory for Applied Immunology, Baltimore, MD), triggered by a solenoid valve set to remain open for 0.6 second, was used to deliver the aerosol generated from a DeVilbiss 646 nebulizer (DeVilbiss, Somerset, PA) with pressurized air at 20 psi. Each study subject inhaled 5 inspiratory capacity breaths of buffered saline and increasing concentrations of methacholine at 5-minute intervals. This technique yielded an output of 0.009 ⫾ 0.0014 mL (mean ⫾ SD) per inhalation. Each subject was encouraged to continue inhaling slowly (approximately 5 seconds to complete the inhalation) and to hold the breath (at total lung capacity) for another 5 seconds. The study was continued only if the postsaline FEV1 was at least 70% of the predicted value.21 The procedure was terminated when the FEV1 decreased by more than 20% of its postsaline value or when the highest methacholine concentration was reached. The largest value of 3 FEV1 values measured 1.5 minutes after each inhalation was used for the analysis. The percentage decline of FEV1 from the postsaline value was plotted against the log concentration of the inhaled methacholine. Methacholine PC20 was calculated by interpolating between 2 adjacent data points if the FEV1 decreased by more than 20%. Measurement of Total Eosinophil Count and Serum ECP Level Blood samples were withdrawn with use of a 21-gauge butterfly needle with an attached syringe; care was taken to avoid hemolysis. The number of eosinophils was counted by means of an automated hematology analyzer (Coulter Counter, STKS; Beckman Coulter, Fullerton, CA) and expressed as number per microliter. Serum ECP measurements were performed according to the method of Venge.22 Blood samples (4 mL) were collected in Vacutainer SST tubes (BD Biosciences, Franklin Lakes, NJ), and allowed to stand for 60 minutes at room temperature. The blood samples were then centrifuged at 1,300 ⫻ g for 10 minutes, and sera were stored at ⫺70°C until the ECP concentration was determined by using an ECP radioimmunoassay kit (Amersham Biosciences, Piscataway, NJ). All assays were performed in duplicate, and mean values were used for statistical analysis.
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The detection limit of the assay was 2 g/L. The intra-assay coefficient of variation was 6.8%. Statistical Analysis Results are reported as means ⫾ SD unless otherwise indicated. Screening of data for differences in total eosinophil counts or serum ECP levels between groups was performed by using the Kruskal-Wallis test. When significant differences were identified, individual groups were compared using the Mann-Whitney U test. A P value of less than 0.05 was considered statistically significant. RESULTS The clinical characteristics of the 4 study groups are summarized in Table 1. No significant differences were found in mean age, sex ratio, and FEV1 level among the 4 groups. Serum IgE levels were significantly lower in the control group than in the other 3 groups. No significant differences were noted between the BHR-positive and the BHR-negative remission groups with respect to age at onset of asthma, history of use of prophylactic medications, duration of asthma remission, or pattern of positive skin responses (data not shown). The peripheral blood total eosinophil counts in the 4 study groups are shown in Figure 1. The mean eosinophil count in the BHR-positive remission group was similar to that found in the BHR-negative remission group (262.1 ⫾ 117.0/L vs 253.9 ⫾ 165.0/L). These values were not significantly different from that of the control group (214.6 ⫾ 118.6/L) but were significantly lower than that of the symptomatic group (372.9 ⫾ 190.3/L; both, P ⬍ 0.05). The serum concentration of ECP showed similar results (Fig 2). No significant difference was found between the mean ECP level of the BHR-positive and the BHR-negative remission groups (15.6 ⫾ 10.0 g/L vs 15.8 ⫾ 11.9 g/L). These ECP levels were intermediate between those of the symptomatic group (26.6 ⫾ 11.3 g/L) and the control group (12.1 ⫾ 4.8 g/L), but differences were statistically significant only when the BHR-positive or the BHR-negative remission group was compared with the symptomatic group (both, P ⬍ 0.01). No significant difference in the blood indices of the BHRpositive and the BHR-negative remission groups could be
Figure 1. Scatterplot of peripheral blood total eosinophil counts in the 4 study groups. Short horizontal lines represent means ⫾ SD. BHR, bronchial hyperresponsiveness; ⫹, positive; ⫺, negative.
attributed to the values of those patients with PC20 levels around the cutoff point (18 mg/mL). We compared the blood indices between those patients (n ⫽ 20) with PC20 levels of less than 8 mg/mL and those (n ⫽ 18) with PC20 levels of more than 25 mg/mL (Fig 3), discounting those with intermediate PC20. Neither total eosinophil counts (271.0 ⫾ 133.1/L vs 255.0 ⫾ 181.4/L) nor serum ECP levels (15.5 ⫾ 10.5 g/L vs 16.1 ⫾ 12.9 g/L) were significantly different between the 2 selected groups. DISCUSSION No differences were found in the peripheral blood eosinophil counts and serum ECP levels of hyperresponsive and normoresponsive adolescents with long-term asthma remission. In terms of the blood indices, hyperresponsive study subjects differed from patients with symptomatic asthma and a similar degree of BHR, but they resembled normal subjects. In our study, subjects were considered in long-term clinical remission when they reported the complete absence of symptoms of asthma and had received no asthma-related treatment for at least 2 years before the study. This definition was
Table 1. Clinical Characteristics of the 4 Study Groups* Characteristic No. of subjects Age (y) Male:female Total IgE (IU/mL)† FEV1 (% predicted) Methacholine PC20 (mg/mL)†
Symptomatic group
BHR-positive remission group
BHR-negative remission group
Control group
28 14.7 ⫾ 1.4 17:11 325.8 (120.8–885.1) 96.1 ⫾ 9.9 4.55 (2.29–9.04)
28 14.9 ⫾ 1.2 19:9 307.6 (115.9–816.6) 97.0 ⫾ 8.7 4.56 (2.31–8.99)
23 15.0 ⫾ 1.0 16:7 310.5 (112.5–857.0) 96.5 ⫾ 8.8 ...
28 14.6 ⫾ 1.3 19:9 121.6 (55.5–266.7) 99.1 ⫾ 6.3 ...
* Abbreviations: BHR, bronchial hyperresponsiveness; FEV1, forced expiratory volume in 1 second; PC20, provocative concentration causing a 20% decrease in FEV1. † Geometric mean (range of 1 SD).
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Figure 2. Scatterplot of the serum concentrations of eosinophil cationic protein (ECP) in the 4 study groups. Short horizontal lines represent means ⫾ SD. BHR, bronchial hyperresponsiveness; ⫹, positive; ⫺, negative.
Figure 3. Comparisons of peripheral blood total eosinophil counts (left) and the serum concentrations of eosinophil cationic protein (ECP) (right) in those with PC20 (provocative concentration causing a 20% decrease in forced expiratory volume in 1 second) of less than 8 mg/mL and those with PC20 of more than 25 mg/mL, among subjects with asthma remission. Short horizontal lines represent means ⫾ SD.
applied to avoid the inclusion of patients with asthma who had mild symptoms. The eosinophil count and serum ECP levels are known to fluctuate considerably during the day.23 To reduce the influence of this circadian variation, we obtained blood samples from all subjects at approximately the same time of day. Determined serum ECP concentrations depend on the method of preparation of the serum; therefore, we strictly adhered to preparation guidelines for all samples.22 Previous studies10,11 have reported on the eosinophil count and serum ECP concentration in patients with asthma. Confirming previously reported data, we found increased eosinophil counts and increased concentrations of ECP in the peripheral blood of patients with symptomatic asthma. Although eosinophil counts and ECP levels were higher in
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patients with symptoms of asthma than in the other study groups, considerable overlapping of values was evident; therefore, significant differences are unlikely to be clinically relevant. Investigators have shown that the blood eosinophil count or the serum ECP level may correlate with indices of disease severity such as symptom score, pulmonary function, and bronchial responsiveness.24 –26 We found, however, no significant differences in the blood indices of the adolescent groups with long-term asthma remission and persisting BHR or nonpersisting BHR. This result might be attributable to the “borderline” range of BHR27 in the former group. Nevertheless, the blood indices were not different between the specified groups with more extreme levels of methacholine PC20. In contrast, the persisting BHR group had blood indices similar to those found in normal control subjects, but the indices were significantly lower than those in patients with symptomatic asthma and a similar degree of BHR. Medications used to treat asthma may modify blood eosinophil counts and serum ECP levels. In a study of children with chronic asthma,28 serum ECP levels were significantly reduced after treatment with either inhaled corticosteroid or sodium cromoglycate. In light of the fact that some patients in our symptomatic group were receiving these medications, differences in the blood indices of the persisting BHR group and the symptomatic group may have been underestimated. Our results suggest that persistent BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. The absence of an increase in blood eosinophil count or in serum ECP concentration may suggest that no active inflammation is present in adolescents with BHR and long-term asthma remission. Alternatively, the normal blood eosinophil count or normal ECP concentration in some patients with symptomatic asthma may indicate that the blood indices may be an insensitive measure when abnormalities are milder. In patients with mild asthma, induced sputum may be a more accurate medium than blood for evaluation of airway inflammation,29 inasmuch as the inflammatory process in asthma is primarily localized in the airways and the limited inflammation may be insufficient to be reflected in the blood. The finding of no abnormalities in the blood indices may also reflect a different cause of BHR, such as local release of mediators and cytokines by mast cells or lymphocytes30 or structural changes in the airways such as smooth muscle hypertrophy or subepithelial fibrosis, which results from chronic airway inflammation.31 Further investigation by bronchoalveolar lavage, bronchial biopsies, or both procedures will help to clarify this issue. Whether persistent BHR in adolescents with asthma remission is associated with airway inflammation or whether it is linked to another mechanism is not known. The only published study of the presence of airway inflammation in BHR associated with this clinical setting was reported by van Den Toorn et al,32 who showed that adolescents with atopic asthma in clinical remission had increased levels of exhaled nitric oxide, similar to those of patients with symptomatic
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asthma. This finding, however, does not provide definite evidence for ongoing airway inflammation because atopic status independent of asthma diagnosis may be an important determinant of increased production of nitric oxide in the airways.33 Recently, we showed that inhaled budesonide did not yield a significant decrease in BHR in this clinical setting, in contrast to the BHR associated with symptomatic asthma.34 This finding suggests that airway inflammation does not have an important role in BHR in adolescents with asthma remission because reduction of BHR induced by inhaled corticosteroids is mainly attributable to improvements in various measures of airway inflammation.35 Our results lend support to the hypothesis that factors other than airway inflammation may be responsible for BHR in adolescents with asthma remission. Histamine and methacholine are generally used to measure the presence and severity of BHR. Both agonists act on the relevant receptors on airway smooth muscle, stimulating contraction of airway muscle directly. In contrast, adenosine 5⬘-monophosphate is an indirect stimulus for measuring BHR; it acts mainly through the release of inflammatory mediators from mast cells.36 One study37 has suggested that the bronchial response to adenosine 5⬘-monophosphate is a more relevant marker of airway inflammation than direct bronchoconstrictors such as histamine or methacholine. Our finding of no significant differences in the blood indices of the persisting BHR and the nonpersisting BHR adolescent groups with long-term asthma remission may not be surprising, because we used the direct stimulus methacholine. In this respect, evaluation of the bronchial responsiveness to indirect bronchoconstrictor stimuli in adolescents with long-term asthma remission or exploration of the relationship between this responsiveness and the blood indices measured in the current study would be of considerable interest. CONCLUSION On the basis of the findings in our current study, BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. These results suggest that the mechanism underlying BHR in this clinical setting may differ from that in symptomatic asthma. These data support the relevance of peripheral blood eosinophilia and eosinophil activation to symptomatic asthma as opposed to asthma in remission and emphasize the importance of distinguishing asthma from BHR. REFERENCES 1. Gerritsen J, Koeter GH, Postma DS, Schouten JP, Knol K. Prognosis of asthma from childhood to adulthood. Am Rev Respir Dis. 1989;140:1325–1330. 2. Sears MR. Evolution of asthma through childhood. Clin Exp Allergy. 1998;28(Suppl):82– 89, 90 –91. 3. Hopp RJ, Townley RG, Biven RE, Bewtra AK, Nair NM. The presence of airway reactivity before the development of asthma. Am Rev Respir Dis. 1990;141:2– 8.
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4. Murray AB, Ferguson AC, Morrison B. Airway responsiveness to histamine as a test for overall severity of asthma in children. J Allergy Clin Immunol. 1981;68:119 –124. 5. Boulet LP, Turcotte H, Brochu A. Persistence of airway obstruction and hyperresponsiveness in subjects with asthma remission. Chest. 1994;105:1024 –1031. 6. Gruber W, Eber E, Steinbrugger B, Modl M, Weinhandl E, Zach MS. Atopy, lung function and bronchial responsiveness in symptom-free paediatric asthma patients. Eur Respir J. 1997; 10:1041–1045. 7. Gleich GJ. The eosinophil and bronchial asthma: current understanding. J Allergy Clin Immunol. 1990;85:422– 436. 8. Bousquet J, Chanez P, Lacoste JY, et al. Eosinophilic inflammation in asthma. N Engl J Med. 1990;323:1033–1039. 9. Djukanovic R, Roche WR, Wilson JW, et al. Mucosal inflammation in asthma. Am Rev Respir Dis. 1990;142:434 – 457. 10. Ulrik CS. Peripheral eosinophil counts as a marker of disease activity in intrinsic and extrinsic asthma. Clin Exp Allergy. 1995;25:820 – 827. 11. Zimmerman B, Lanner A, Enander I, Zimmerman RS, Peterson CG, Ahlstedt S. Total blood eosinophils, serum eosinophil cationic protein and eosinophil protein X in childhood asthma: relation to disease status and therapy. Clin Exp Allergy. 1993; 23:564 –570. 12. Niimi A, Amitani R, Suzuki K, Tanaka E, Murayama T, Kuze F. Serum eosinophil cationic protein as a marker of eosinophilic inflammation in asthma. Clin Exp Allergy. 1998;28:233–240. 13. Claman DM, Boushey HA, Liu J, Wong H, Fahey JV. Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects. J Allergy Clin Immunol. 1994;94:861– 869. 14. Holgate ST, Beasley R, Twentyman OP. The pathogenesis and significance of bronchial hyper-responsiveness in airways disease. Clin Sci. 1987;73:561–572. 15. Kirby JG, Hargreave FE, Gleich GJ, O’Byrne PM. Bronchoalveolar cell profiles of asthmatic and nonasthmatic subjects. Am Rev Respir Dis. 1987;136:379 –383. 16. Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis. 1989;140:1745–1753. 17. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987;136:225–244. 18. Koh YY, Lee MH, Kim CK, et al. A familial predisposition in bronchial hyperresponsiveness among patients with allergic rhinitis. J Allergy Clin Immunol. 1998;102:921–926. 19. Chai H, Farr RS, Froehlich LA, et al. Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol. 1975;56:323–327. 20. Paik YH, Cho YJ, You TH, Bae CW, Ahn CI. The seasonal variation of house dust mite allergen and the incidence of bronchial asthma among children. J Korean Med Assoc. 1991; 34:69 –77. 21. Yoon KA, Lim HS, Koh YY, Kim H. Normal predicted values of the pulmonary function test in Korean school-aged children. J Korean Pediatr Assoc. 1993;36:25–37. 22. Venge P. Serum measurements of eosinophil cationic protein (ECP) in bronchial asthma. Clin Exp Allergy. 1993;23(Suppl): 3–7.
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23. Wempe JB, Tammeling EP, Koeter GH, Hakansson L, Venge P, Postma DS. Blood eosinophil numbers and activity during 24 hours: effects of treatment with budesonide and bambuterol. J Allergy Clin Immunol. 1992;90:757–765. 24. Taylor KJ, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax. 1987;42:452– 456. 25. Griffin E, Hakansson L, Formgren H, Jorgensen K, Peterson C, Venge P. Blood eosinophil number and activity in relation to lung function in patients with asthma and with eosinophilia. J Allergy Clin Immunol. 1991;87:548 –557. 26. Wever AM, Wever-Hess J, Hensgens HE, Hermans J. Serum eosinophil cationic protein (ECP) in chronic asthma: relationship to spirometry, flow-volume curves, PC20, and exacerbations. Respir Med. 1994;88:613– 621. 27. Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med. 2000;161:309 –329. 28. Juntunen-Backman K, Jarvinen P, Sorva R. Serum eosinophil cationic protein during treatment of asthma in children. J Allergy Clin Immunol. 1993;92:34 –38. 29. O’Byrne PM, Inman MD. Induced sputum to assess airway inflammation in asthma. Eur Respir J. 1996;9:2435–2436. 30. Broide DH, Lotz M, Cuomo AJ, Coburn DA, Federman EC, Wasserman SI. Cytokines in symptomatic asthma airways. J Allergy Clin Immunol. 1992;89:958 –967. 31. Fish JE, Peters SP. Airway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol. 1999;104: 509 –516. 32. van den Toorn LM, Prins JB, Overbeek SE, Hoogsteden HC, de Jongste JC. Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyper-
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responsiveness. Am J Respir Crit Care Med. 2000;162: 953–957. Martin U, Bryden K, Devoy M, Howarth P. Increased levels of exhaled nitric oxide during nasal and oral breathing in subjects with seasonal rhinitis. J Allergy Clin Immunol. 1996;97: 768 –772. Koh YY, Sun YH, Lim HS, Kim CK, Hong SJ. Effect of inhaled budesonide on bronchial hyperresponsiveness in adolescents with clinical remission of asthma. Chest. 2001;120:1140 –1146. Djukanovic R, Wilson JW, Britten KM, et al. Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am Rev Respir Dis. 1992;145:669 – 674. van den Berge M, Kerstjens HA, Postma DS. Provocation with adenosine 5⬘-monophosphate as a marker of inflammation in asthma, allergic rhinitis and chronic obstructive pulmonary disease. Clin Exp Allergy. 2002;32:824 – 830. van den Berge M, Meijer RJ, Kerstjens HA, et al. PC20 adenosine 5⬘-monophosphate is more closely associated with airway inflammation in asthma than PC20 methacholine. Am J Respir Crit Care Med. 2001;163:1546 –1550.
Requests for reprints should be addressed to: Young Yull Koh, MD Department of Pediatrics Seoul National University Hospital 28 Yongon-dong Chongno-gu Seoul 110 –744 Korea E-mail: [email protected]
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Background: The mechanisms responsible for persistent bronchial hyperresponsiveness (BHR) in adolescents with long-term asthma remission are poorly understood. Objective: To determine whether BHR in adolescents with asthma remission is associated with peripheral blood eosinophilia, increased serum levels of eosinophil cationic protein (ECP), or both findings. Methods: We classified 51 adolescents with long-term asthma remission (neither asthma-related symptoms nor medication during the previous 2 years) into 28 BHR-positive patients (methacholine PC20 [provocative concentration causing a 20% decrease in forced expiratory volume in 1 second] ⬍18 mg/mL) and 23 BHR-negative patients. The peripheral blood eosinophil counts and serum ECP concentrations were compared between these 2 groups. Twenty-eight patients with symptomatic asthma (symptomatic group), matched for methacholine PC20 level with study subjects in the BHR-positive remission group, and 28 healthy adolescents (control group) were also studied. Results: No significant differences in the peripheral blood eosinophil counts (262.1 ⫾ 117.0/L vs 253.9 ⫾ 165.0/L) and the serum ECP levels (15.6 ⫾ 10.0 g/L vs 15.8 ⫾ 11.9 g/L) were found between the BHR-positive and BHR-negative remission groups, respectively. The BHR-positive remission group differed from the symptomatic group (372.9 ⫾ 190.3/L, P ⬍ 0.05; 26.6 ⫾ 11.3 g/L, P ⬍ 0.01) in both blood indices but resembled the control group (214.6 ⫾ 118.6/L and 12.1 ⫾ 4.8 g/L; both, no significant difference). Conclusions: BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. This finding suggests that the mechanism underlying BHR in this clinical setting may differ from that in symptomatic asthma. Ann Allergy Asthma Immunol. 2003;91:297–302.
INTRODUCTION Epidemiologic studies have demonstrated that, in many children with asthma, long-lasting clinical remission ensues at adolescence.1,2 Bronchial hyperresponsiveness (BHR), often called “the hallmark of asthma,” is known to be an important risk factor for the development of asthma3 and to be indicative of the severity of symptomatic asthma.4 Some studies have shown, however, that BHR persists in a considerable proportion of adolescents with asthma in long-term clinical remission.5,6 Little is known about the mechanism underlying BHR in this clinical setting. Airway inflammation is considered a characteristic feature of asthma, and eosinophils are recognized as the most important inflammatory cells.7 Certain studies8,9 have attempted to * Department of Pediatrics and Clinical Research Institute, Seoul National University Hospital, Seoul, Korea. † Department of Pediatrics, Inje University Sanggye Paik Hospital, Seoul, Korea. This study was supported in part by BK 21 Project for Medicine, Dentistry, and Pharmacy and by grant no. 04 –2001-044 from the Seoul National University Hospital Research Fund. Received for publication October 6, 2002. Accepted for publication in revised form January 31, 2003.
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determine the role of eosinophils in asthma by using bronchoalveolar lavage and biopsies in patients with asthma. These studies found not only an eosinophilic cellular infiltrate but also increased levels of eosinophilic granular proteins such as eosinophil cationic protein (ECP). Another characteristic feature of asthma is the presence of peripheral blood eosinophilia.10 Increased levels of ECP have likewise been identified in the serum of patients with asthma.11 Investigators have increasingly suggested that serum levels of ECP may reflect the intensity of airway inflammation in asthma because the serum ECP level is closely related to eosinophilic airway inflammation as determined by evaluation of sputum samples or bronchoalveolar lavage.12,13 BHR in symptomatic asthma is thought to be a consequence of underlying airway inflammation,14 and some studies15,16 have noted correlations between measurements of bronchial responsiveness and various cellular aspects of airway inflammation. Thus far, however, investigators do not know whether the persisting BHR in adolescents with asthma remission is caused by airway inflammation or whether it is linked to another mechanism. In this study, our goal was to ascertain whether BHR in adolescents with asthma remission is associated with peripheral blood eosinophilia, increased
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serum ECP levels, or both. We compared total eosinophil counts and serum levels of ECP between groups of adolescents with long-term asthma remission and persisting BHR or nonpersisting BHR. These variables were also compared against those of patients with symptomatic asthma and a similar degree of BHR as well as those of healthy control subjects. PATIENTS AND METHODS Study Subjects We recruited 51 adolescents with long-term remission of atopic asthma from the allergy clinic at Seoul National University Children’s Hospital. All study subjects had a history of wheezing and dyspnea and had been previously diagnosed as having atopic asthma on the basis of the criteria established by the American Thoracic Society.17 At the time of diagnosis, all patients had a methacholine PC20 (provocative concentration causing a 20% decrease in forced expiratory volume in 1 second [FEV1]) level of less than 18 mg/mL. Atopy was defined as at least 1 positive result of a skin prick test to a panel of 12 common aeroallergens in the presence of positive and negative controls. Most study subjects showed a positive skin reaction to house-dust mites. Long-term clinical remission was assumed if a patient reported the complete absence of wheezing and dyspnea at rest and on exertion and had taken no medication to control symptoms of asthma for at least 24 months before the study. These patients underwent a methacholine inhalation test and were classified into BHRpositive and BHR-negative groups on the basis of the methacholine PC20 criterion (18 mg/mL).18 Another group of 28 adolescents with current atopic asthma (symptomatic group) was recruited. These patients had a history of symptoms of asthma (cough, wheezing, and dyspnea) during the previous year, which had been controlled by an as-needed bronchodilator with or without anti-inflammatory agents, and they had a positive skin test result. Those patients with a history of near-fatal asthma or major exacerbations necessitating the use of systemic corticosteroids were excluded from the study. Candidates were further selected on the basis of the results of a methacholine inhalation test, by matching them with the methacholine PC20 levels of study subjects in the BHR-positive remission group. A separate group of 28 healthy adolescents with no history of asthma or other allergic diseases served as control subjects. These subjects underwent a methacholine inhalation test, and those with a methacholine PC20 of higher than 18 mg/mL were selected and matched for sex with those in the BHRpositive remission group. In all 4 study groups, blood samples were obtained for determination of total eosinophil counts and serum ECP levels at approximately 10 AM. Parents of the subjects enrolled in this study gave informed consent, and the study was approved by the Hospital Ethics Committee.
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Methacholine Challenge Test FEV1 was recorded as the best result of 3 attempts with use of a computerized spirometer (Microspiro-HI 298; Chest Inc, Tokyo, Japan); measurements that varied by no more than 5% were considered acceptable. Methacholine inhalation tests were performed with use of a modification of the method described by Chai et al.19 At the time of the study, all subjects had been free of any acute respiratory tract infection for 4 weeks and had not taken any medication for 7 days before the study. Because most study subjects were sensitized to housedust mites, the test was performed during the winter season (December to February), when house-dust mites are least prevalent and least variable across the country.20 Methacholine (Sigma Chemical, St. Louis, MO) at concentrations of 0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10, and 25 mg/mL was prepared by dilution in buffered saline (pH 7.4). A RosenthalFrench dosimeter (Laboratory for Applied Immunology, Baltimore, MD), triggered by a solenoid valve set to remain open for 0.6 second, was used to deliver the aerosol generated from a DeVilbiss 646 nebulizer (DeVilbiss, Somerset, PA) with pressurized air at 20 psi. Each study subject inhaled 5 inspiratory capacity breaths of buffered saline and increasing concentrations of methacholine at 5-minute intervals. This technique yielded an output of 0.009 ⫾ 0.0014 mL (mean ⫾ SD) per inhalation. Each subject was encouraged to continue inhaling slowly (approximately 5 seconds to complete the inhalation) and to hold the breath (at total lung capacity) for another 5 seconds. The study was continued only if the postsaline FEV1 was at least 70% of the predicted value.21 The procedure was terminated when the FEV1 decreased by more than 20% of its postsaline value or when the highest methacholine concentration was reached. The largest value of 3 FEV1 values measured 1.5 minutes after each inhalation was used for the analysis. The percentage decline of FEV1 from the postsaline value was plotted against the log concentration of the inhaled methacholine. Methacholine PC20 was calculated by interpolating between 2 adjacent data points if the FEV1 decreased by more than 20%. Measurement of Total Eosinophil Count and Serum ECP Level Blood samples were withdrawn with use of a 21-gauge butterfly needle with an attached syringe; care was taken to avoid hemolysis. The number of eosinophils was counted by means of an automated hematology analyzer (Coulter Counter, STKS; Beckman Coulter, Fullerton, CA) and expressed as number per microliter. Serum ECP measurements were performed according to the method of Venge.22 Blood samples (4 mL) were collected in Vacutainer SST tubes (BD Biosciences, Franklin Lakes, NJ), and allowed to stand for 60 minutes at room temperature. The blood samples were then centrifuged at 1,300 ⫻ g for 10 minutes, and sera were stored at ⫺70°C until the ECP concentration was determined by using an ECP radioimmunoassay kit (Amersham Biosciences, Piscataway, NJ). All assays were performed in duplicate, and mean values were used for statistical analysis.
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The detection limit of the assay was 2 g/L. The intra-assay coefficient of variation was 6.8%. Statistical Analysis Results are reported as means ⫾ SD unless otherwise indicated. Screening of data for differences in total eosinophil counts or serum ECP levels between groups was performed by using the Kruskal-Wallis test. When significant differences were identified, individual groups were compared using the Mann-Whitney U test. A P value of less than 0.05 was considered statistically significant. RESULTS The clinical characteristics of the 4 study groups are summarized in Table 1. No significant differences were found in mean age, sex ratio, and FEV1 level among the 4 groups. Serum IgE levels were significantly lower in the control group than in the other 3 groups. No significant differences were noted between the BHR-positive and the BHR-negative remission groups with respect to age at onset of asthma, history of use of prophylactic medications, duration of asthma remission, or pattern of positive skin responses (data not shown). The peripheral blood total eosinophil counts in the 4 study groups are shown in Figure 1. The mean eosinophil count in the BHR-positive remission group was similar to that found in the BHR-negative remission group (262.1 ⫾ 117.0/L vs 253.9 ⫾ 165.0/L). These values were not significantly different from that of the control group (214.6 ⫾ 118.6/L) but were significantly lower than that of the symptomatic group (372.9 ⫾ 190.3/L; both, P ⬍ 0.05). The serum concentration of ECP showed similar results (Fig 2). No significant difference was found between the mean ECP level of the BHR-positive and the BHR-negative remission groups (15.6 ⫾ 10.0 g/L vs 15.8 ⫾ 11.9 g/L). These ECP levels were intermediate between those of the symptomatic group (26.6 ⫾ 11.3 g/L) and the control group (12.1 ⫾ 4.8 g/L), but differences were statistically significant only when the BHR-positive or the BHR-negative remission group was compared with the symptomatic group (both, P ⬍ 0.01). No significant difference in the blood indices of the BHRpositive and the BHR-negative remission groups could be
Figure 1. Scatterplot of peripheral blood total eosinophil counts in the 4 study groups. Short horizontal lines represent means ⫾ SD. BHR, bronchial hyperresponsiveness; ⫹, positive; ⫺, negative.
attributed to the values of those patients with PC20 levels around the cutoff point (18 mg/mL). We compared the blood indices between those patients (n ⫽ 20) with PC20 levels of less than 8 mg/mL and those (n ⫽ 18) with PC20 levels of more than 25 mg/mL (Fig 3), discounting those with intermediate PC20. Neither total eosinophil counts (271.0 ⫾ 133.1/L vs 255.0 ⫾ 181.4/L) nor serum ECP levels (15.5 ⫾ 10.5 g/L vs 16.1 ⫾ 12.9 g/L) were significantly different between the 2 selected groups. DISCUSSION No differences were found in the peripheral blood eosinophil counts and serum ECP levels of hyperresponsive and normoresponsive adolescents with long-term asthma remission. In terms of the blood indices, hyperresponsive study subjects differed from patients with symptomatic asthma and a similar degree of BHR, but they resembled normal subjects. In our study, subjects were considered in long-term clinical remission when they reported the complete absence of symptoms of asthma and had received no asthma-related treatment for at least 2 years before the study. This definition was
Table 1. Clinical Characteristics of the 4 Study Groups* Characteristic No. of subjects Age (y) Male:female Total IgE (IU/mL)† FEV1 (% predicted) Methacholine PC20 (mg/mL)†
Symptomatic group
BHR-positive remission group
BHR-negative remission group
Control group
28 14.7 ⫾ 1.4 17:11 325.8 (120.8–885.1) 96.1 ⫾ 9.9 4.55 (2.29–9.04)
28 14.9 ⫾ 1.2 19:9 307.6 (115.9–816.6) 97.0 ⫾ 8.7 4.56 (2.31–8.99)
23 15.0 ⫾ 1.0 16:7 310.5 (112.5–857.0) 96.5 ⫾ 8.8 ...
28 14.6 ⫾ 1.3 19:9 121.6 (55.5–266.7) 99.1 ⫾ 6.3 ...
* Abbreviations: BHR, bronchial hyperresponsiveness; FEV1, forced expiratory volume in 1 second; PC20, provocative concentration causing a 20% decrease in FEV1. † Geometric mean (range of 1 SD).
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Figure 2. Scatterplot of the serum concentrations of eosinophil cationic protein (ECP) in the 4 study groups. Short horizontal lines represent means ⫾ SD. BHR, bronchial hyperresponsiveness; ⫹, positive; ⫺, negative.
Figure 3. Comparisons of peripheral blood total eosinophil counts (left) and the serum concentrations of eosinophil cationic protein (ECP) (right) in those with PC20 (provocative concentration causing a 20% decrease in forced expiratory volume in 1 second) of less than 8 mg/mL and those with PC20 of more than 25 mg/mL, among subjects with asthma remission. Short horizontal lines represent means ⫾ SD.
applied to avoid the inclusion of patients with asthma who had mild symptoms. The eosinophil count and serum ECP levels are known to fluctuate considerably during the day.23 To reduce the influence of this circadian variation, we obtained blood samples from all subjects at approximately the same time of day. Determined serum ECP concentrations depend on the method of preparation of the serum; therefore, we strictly adhered to preparation guidelines for all samples.22 Previous studies10,11 have reported on the eosinophil count and serum ECP concentration in patients with asthma. Confirming previously reported data, we found increased eosinophil counts and increased concentrations of ECP in the peripheral blood of patients with symptomatic asthma. Although eosinophil counts and ECP levels were higher in
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patients with symptoms of asthma than in the other study groups, considerable overlapping of values was evident; therefore, significant differences are unlikely to be clinically relevant. Investigators have shown that the blood eosinophil count or the serum ECP level may correlate with indices of disease severity such as symptom score, pulmonary function, and bronchial responsiveness.24 –26 We found, however, no significant differences in the blood indices of the adolescent groups with long-term asthma remission and persisting BHR or nonpersisting BHR. This result might be attributable to the “borderline” range of BHR27 in the former group. Nevertheless, the blood indices were not different between the specified groups with more extreme levels of methacholine PC20. In contrast, the persisting BHR group had blood indices similar to those found in normal control subjects, but the indices were significantly lower than those in patients with symptomatic asthma and a similar degree of BHR. Medications used to treat asthma may modify blood eosinophil counts and serum ECP levels. In a study of children with chronic asthma,28 serum ECP levels were significantly reduced after treatment with either inhaled corticosteroid or sodium cromoglycate. In light of the fact that some patients in our symptomatic group were receiving these medications, differences in the blood indices of the persisting BHR group and the symptomatic group may have been underestimated. Our results suggest that persistent BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. The absence of an increase in blood eosinophil count or in serum ECP concentration may suggest that no active inflammation is present in adolescents with BHR and long-term asthma remission. Alternatively, the normal blood eosinophil count or normal ECP concentration in some patients with symptomatic asthma may indicate that the blood indices may be an insensitive measure when abnormalities are milder. In patients with mild asthma, induced sputum may be a more accurate medium than blood for evaluation of airway inflammation,29 inasmuch as the inflammatory process in asthma is primarily localized in the airways and the limited inflammation may be insufficient to be reflected in the blood. The finding of no abnormalities in the blood indices may also reflect a different cause of BHR, such as local release of mediators and cytokines by mast cells or lymphocytes30 or structural changes in the airways such as smooth muscle hypertrophy or subepithelial fibrosis, which results from chronic airway inflammation.31 Further investigation by bronchoalveolar lavage, bronchial biopsies, or both procedures will help to clarify this issue. Whether persistent BHR in adolescents with asthma remission is associated with airway inflammation or whether it is linked to another mechanism is not known. The only published study of the presence of airway inflammation in BHR associated with this clinical setting was reported by van Den Toorn et al,32 who showed that adolescents with atopic asthma in clinical remission had increased levels of exhaled nitric oxide, similar to those of patients with symptomatic
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asthma. This finding, however, does not provide definite evidence for ongoing airway inflammation because atopic status independent of asthma diagnosis may be an important determinant of increased production of nitric oxide in the airways.33 Recently, we showed that inhaled budesonide did not yield a significant decrease in BHR in this clinical setting, in contrast to the BHR associated with symptomatic asthma.34 This finding suggests that airway inflammation does not have an important role in BHR in adolescents with asthma remission because reduction of BHR induced by inhaled corticosteroids is mainly attributable to improvements in various measures of airway inflammation.35 Our results lend support to the hypothesis that factors other than airway inflammation may be responsible for BHR in adolescents with asthma remission. Histamine and methacholine are generally used to measure the presence and severity of BHR. Both agonists act on the relevant receptors on airway smooth muscle, stimulating contraction of airway muscle directly. In contrast, adenosine 5⬘-monophosphate is an indirect stimulus for measuring BHR; it acts mainly through the release of inflammatory mediators from mast cells.36 One study37 has suggested that the bronchial response to adenosine 5⬘-monophosphate is a more relevant marker of airway inflammation than direct bronchoconstrictors such as histamine or methacholine. Our finding of no significant differences in the blood indices of the persisting BHR and the nonpersisting BHR adolescent groups with long-term asthma remission may not be surprising, because we used the direct stimulus methacholine. In this respect, evaluation of the bronchial responsiveness to indirect bronchoconstrictor stimuli in adolescents with long-term asthma remission or exploration of the relationship between this responsiveness and the blood indices measured in the current study would be of considerable interest. CONCLUSION On the basis of the findings in our current study, BHR in adolescents with long-term asthma remission is not associated with peripheral blood eosinophilia or an increase in serum ECP concentration. These results suggest that the mechanism underlying BHR in this clinical setting may differ from that in symptomatic asthma. These data support the relevance of peripheral blood eosinophilia and eosinophil activation to symptomatic asthma as opposed to asthma in remission and emphasize the importance of distinguishing asthma from BHR. REFERENCES 1. Gerritsen J, Koeter GH, Postma DS, Schouten JP, Knol K. Prognosis of asthma from childhood to adulthood. Am Rev Respir Dis. 1989;140:1325–1330. 2. Sears MR. Evolution of asthma through childhood. Clin Exp Allergy. 1998;28(Suppl):82– 89, 90 –91. 3. Hopp RJ, Townley RG, Biven RE, Bewtra AK, Nair NM. The presence of airway reactivity before the development of asthma. Am Rev Respir Dis. 1990;141:2– 8.
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4. Murray AB, Ferguson AC, Morrison B. Airway responsiveness to histamine as a test for overall severity of asthma in children. J Allergy Clin Immunol. 1981;68:119 –124. 5. Boulet LP, Turcotte H, Brochu A. Persistence of airway obstruction and hyperresponsiveness in subjects with asthma remission. Chest. 1994;105:1024 –1031. 6. Gruber W, Eber E, Steinbrugger B, Modl M, Weinhandl E, Zach MS. Atopy, lung function and bronchial responsiveness in symptom-free paediatric asthma patients. Eur Respir J. 1997; 10:1041–1045. 7. Gleich GJ. The eosinophil and bronchial asthma: current understanding. J Allergy Clin Immunol. 1990;85:422– 436. 8. Bousquet J, Chanez P, Lacoste JY, et al. Eosinophilic inflammation in asthma. N Engl J Med. 1990;323:1033–1039. 9. Djukanovic R, Roche WR, Wilson JW, et al. Mucosal inflammation in asthma. Am Rev Respir Dis. 1990;142:434 – 457. 10. Ulrik CS. Peripheral eosinophil counts as a marker of disease activity in intrinsic and extrinsic asthma. Clin Exp Allergy. 1995;25:820 – 827. 11. Zimmerman B, Lanner A, Enander I, Zimmerman RS, Peterson CG, Ahlstedt S. Total blood eosinophils, serum eosinophil cationic protein and eosinophil protein X in childhood asthma: relation to disease status and therapy. Clin Exp Allergy. 1993; 23:564 –570. 12. Niimi A, Amitani R, Suzuki K, Tanaka E, Murayama T, Kuze F. Serum eosinophil cationic protein as a marker of eosinophilic inflammation in asthma. Clin Exp Allergy. 1998;28:233–240. 13. Claman DM, Boushey HA, Liu J, Wong H, Fahey JV. Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects. J Allergy Clin Immunol. 1994;94:861– 869. 14. Holgate ST, Beasley R, Twentyman OP. The pathogenesis and significance of bronchial hyper-responsiveness in airways disease. Clin Sci. 1987;73:561–572. 15. Kirby JG, Hargreave FE, Gleich GJ, O’Byrne PM. Bronchoalveolar cell profiles of asthmatic and nonasthmatic subjects. Am Rev Respir Dis. 1987;136:379 –383. 16. Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis. 1989;140:1745–1753. 17. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987;136:225–244. 18. Koh YY, Lee MH, Kim CK, et al. A familial predisposition in bronchial hyperresponsiveness among patients with allergic rhinitis. J Allergy Clin Immunol. 1998;102:921–926. 19. Chai H, Farr RS, Froehlich LA, et al. Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol. 1975;56:323–327. 20. Paik YH, Cho YJ, You TH, Bae CW, Ahn CI. The seasonal variation of house dust mite allergen and the incidence of bronchial asthma among children. J Korean Med Assoc. 1991; 34:69 –77. 21. Yoon KA, Lim HS, Koh YY, Kim H. Normal predicted values of the pulmonary function test in Korean school-aged children. J Korean Pediatr Assoc. 1993;36:25–37. 22. Venge P. Serum measurements of eosinophil cationic protein (ECP) in bronchial asthma. Clin Exp Allergy. 1993;23(Suppl): 3–7.
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23. Wempe JB, Tammeling EP, Koeter GH, Hakansson L, Venge P, Postma DS. Blood eosinophil numbers and activity during 24 hours: effects of treatment with budesonide and bambuterol. J Allergy Clin Immunol. 1992;90:757–765. 24. Taylor KJ, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax. 1987;42:452– 456. 25. Griffin E, Hakansson L, Formgren H, Jorgensen K, Peterson C, Venge P. Blood eosinophil number and activity in relation to lung function in patients with asthma and with eosinophilia. J Allergy Clin Immunol. 1991;87:548 –557. 26. Wever AM, Wever-Hess J, Hensgens HE, Hermans J. Serum eosinophil cationic protein (ECP) in chronic asthma: relationship to spirometry, flow-volume curves, PC20, and exacerbations. Respir Med. 1994;88:613– 621. 27. Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med. 2000;161:309 –329. 28. Juntunen-Backman K, Jarvinen P, Sorva R. Serum eosinophil cationic protein during treatment of asthma in children. J Allergy Clin Immunol. 1993;92:34 –38. 29. O’Byrne PM, Inman MD. Induced sputum to assess airway inflammation in asthma. Eur Respir J. 1996;9:2435–2436. 30. Broide DH, Lotz M, Cuomo AJ, Coburn DA, Federman EC, Wasserman SI. Cytokines in symptomatic asthma airways. J Allergy Clin Immunol. 1992;89:958 –967. 31. Fish JE, Peters SP. Airway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol. 1999;104: 509 –516. 32. van den Toorn LM, Prins JB, Overbeek SE, Hoogsteden HC, de Jongste JC. Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyper-
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Requests for reprints should be addressed to: Young Yull Koh, MD Department of Pediatrics Seoul National University Hospital 28 Yongon-dong Chongno-gu Seoul 110 –744 Korea E-mail: [email protected]
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