Use of induced sputum to examine airway inflammation in childhood asthma

Airway inflammation is important in the pathogenesis of asthma, during which it may lead to symptomatic exacerbations and increases in asthma severity, as well as contribute to future decline in asthma status. The use of induced sputum has emerged as an important and useful technique to study airway inflammation. It has particular advantages in the study of childhood asthma because it is noninvasive and allows samples to be collected on repeated occasions in children over 7 years of age. The results of cell counts are reliable when the sputum is processed in a standardized manner involving selection from saliva, cell dispersion, and quantitative cytology. Children with asthma have increased eosinophils and mast cells, which may persist even with high doses of inhaled corticosteroid therapy. During a severe exacerbation of asthma, there is an intense and heterogeneous inflammatory response involving eosinophil and neutrophil accumulation and activation. Characterization of the relevance of airway inflammation in children with asthma is important. (J Allergy Clin Immunol 1998;102:S100-1.)     

Safety of inhaled corticosteroid therapy in young children with asthma.

BACKGROUND: Physicians have had some reluctance to use inhaled corticosteroids in very young children with asthma because of the possible risks of adverse systemic effects. OBJECTIVE: The purpose of this study was to evaluate the effects of fluticasone propionate on growth and adrenocortical function in young children with asthma. METHODS: We performed an open, prospective study for 24 weeks of 20 children with asthma, 2.5 to 5.0 years of age, who had received fluticasone by a large volume spacer at dosages ranging from 190.50 to 565.40 microg/m2 daily. Growth was evaluated by height standard deviation scores measured by a stadiometer. Adrenocortical function was evaluated twice in each child, before and after the study, by determining fasting serum cortisol concentrations at 8 AM and also at 30 and 60 minutes after adrenocorticotropic hormone stimulation. Posttreatment values of height standard deviation scores and fasting morning serum cortisol concentrations were compared with those of 18 age-matched children, who constituted the control group. RESULTS: The evaluation of mean +/- SEM (and range) of height standard deviation scores revealed a significant decrease from 0.44 +/- 0.27 (-1.46 to 2.22) to 0.28 +/- 0.26 (-1.51 to 2.07; P = 0.01) at week 18 and to 0.25 +/- 0.24 (-1.90 to 2.13; P = 0.04) at the week 24 in fluticasone-treated children. At the end of the treatment, however, height standard deviation scores of these children did not differ significantly (P = 0.35) from those of the control group. Delayed growth with medium-duration treatment was not associated with alterations in serum cortisol measurements, either at baseline or after stimulation. The mean fasting morning serum cortisol concentrations did not differ significantly between the fluticasone-treated patients and the control group. CONCLUSIONS: Some concern prevails about the safety of medium- or long-term treatment with regularly inhaled corticosteroids in young children with asthma. The prepubertal growth may be delayed, but the effect on ultimate height remains uncertain in such children. Growth should be regularly monitored in children who begin inhaled corticosteroid therapy for mild persistent asthma at an age <5 years old.     

激素治疗对运动诱发性哮喘患者痰嗜酸性粒细胞计数的影响

目的探讨气道嗜酸性粒细胞性炎症和运动诱发的支气管痉挛（EIB）之间的关系,及其对吸入糖皮质激素（ICS）治疗的反应。方法本研究为随机、双盲、二阶段交叉试验,将26例有运动诱发性支气管痉挛发作史且从未接受过激素治疗的哮喘患者随机分为两组,每组分别给予两个剂量水平的布地奈德吸入：①100μg/d与400μg/d对比;②200μg/d与800μg/d对比。每一阶段为3周,洗脱期3～8周。治疗前及开始治疗后每隔1周进行1次运动激发试验并留取痰液标本行嗜酸性粒细胞计数。结果高剂量ICS治疗（400μg/d和800μg/d）可显著减少痰嗜酸性粒细胞比例。痰嗜酸性粒细胞百分比与运动诱发性支气管痉挛严重程度相关,且对EIB的严重程度有预测作用;高剂量ICS治疗时,尚可预测EIB对激素治疗有效,而对低剂量ICS组（100μg/d和200μg/d）则无预测作用。低剂量ICS治疗,不管基线痰嗜酸性粒细胞计数是否增多,EIB在第1周末发作显著减轻,尔后几无改善。而高剂量ICS治疗对EIB的改善作用在痰嗜酸性粒细胞增多的患者中显著优于嗜酸性粒细胞计数小于5%者,这种明显的差异在开始治疗1周后即显现,且随时间的推移而继续加大。结论气道嗜酸性粒细胞性炎症可能在EIB的发生及其对ICS治疗有效的调节机制中起重要的作用。测定痰嗜酸性粒细胞计数在预测EIB的严重程度及其对不同剂量ICS治疗的反应具有一定的临床应用价值。     

Extrapulmonary effects of maintenance corticosteroid therapy with alternate-day prednisone and inhaled beclomethasone in children with chronic asthma

Extrapulmonary effects of alternate-day prednisone and inhaled beclomethasone dipropionate therapy were examined in 24 and 32 children with asthma, respectively. Early morning serum cortisol values were significantly lower among patients receiving alternate-day prednisone than among patients receiving inhaled beclomethasone dipropionate and control subjects at 24 hours but not at 48 hours after an alternate-day prednisone dose. Urinary-free cortisol output during the second 24 hours of the alternate-day prednisone regimen were similar to values among patients receiving inhaled beclomethasone and were significantly lower than among control subjects for both groups. Mean heights among patients before being placed on maintenance corticosteroids were at the thirty-fifth percentile and were similar for both regimens. This was significantly lower than initial measurements for control subjects who, on average, were near the fiftieth percentile for both children with asthma not requiring maintenance corticosteroids and normal healthy Iowa children. Mean heights for both corticosteroid-treated groups remained at the thirty-fifth percentile after more than a 2-year average duration of follow-up. Heights of children with chronic asthma not requiring maintenance corticosteroids were initially significantly higher (fifty-first percentile) than the patients who subsequently required maintenance corticosteroids and increased significantly to the sixty-first percentile during a mean 2.7-year follow-up. Heights of healthy Iowa children remained near the fiftieth percentile during a mean 7-year follow-up. Disproportionate weight gain, although it was not consistently present, was significantly more likely with the alternate-day prednisone. Other extrapulmonary effects of the corticosteroid regimens appeared not to be of clinical importance during the time period of the study.     

Neutrophilic airway inflammation is a main feature of induced sputum in nonatopic asthmatic children

Background:  Asthma phenotypes are well described among children. However, there are few studies comparing airway inflammation in different clinical presentations of pediatric asthma. We tested the hypothesis that nonatopic asthma is associated with a predominant noneosinophilic inflammation in the airways, as assessed by induced sputum. The objective of this study was to evaluate the cytological characteristics of induced sputum (IS) in atopic (AA), nonatopic asthmatics (NAA) and nonatopic nonasthmatic children (NANA).
Methods:  Of 90 selected children, 77 met eligibility criteria for performing IS and were classified as: AA, n  = 28, NAA, n  = 29 and NANA, n  = 19. Subjects answered to a set of ISAAC-based questions and were skin-tested for common aeroallergens. A defined series of exclusion criteria was applied.
Results:  Induced sputum was obtained from 54 (70.1%) subjects (21 AA, 20 NAA and 13 NANA). Demographic data and mean FEV₁ were similar in the three groups. The proportion of eosinophils [median, inter quartile range (IQR)] was significantly higher in the sputum of AA [(6.0.)12)] compared with NAAs [0 (2)] and NANAs [0 (1)], P  < 0.001. The proportion of children with sputum eosinophilia (eos > 3%) was also significantly higher in AA (71.4%) when compared with NAA (28.6%); none of the NANA had sputum eosinophilia. Nonatopic asthmatic children had significantly higher proportions and absolute number of neutrophils than AA and controls.
Conclusions:  The results suggest that nonatopic children present IS with a cell pattern that is predominantly neutrophilic while eosinophilia is the hallmark of airway inflammation in the majority of atopic wheezing children not treated with inhaled steroids.     

A research method to induce and examine a mild exacerbation of asthma by withdrawal of inhaled corticosteroid

This study evaluated a research method to examine an exacerbation of asthma induced by corticosteroid withdrawal. Ten non-smoking adult asthmatics who were stable on treatment with inhaled steroid underwent a graded reduction of the daily dose by 200 micrograms at weekly intervals until an exacerbation of symptoms occurred. A daily symptom, peak expiratory flow rate (PEF) and medication diary was kept. Weekly clinic visits were used to assess symptoms, spirometry, methacholine airway responsiveness (expressed as the provocative concentration to cause a fall in FEV1 of 20%, PC20), circulating eosinophils, basophils and their progenitors (Eo/B-CFU), and sputum inflammatory cells. The laboratory tests were performed blind to the clinical details. Each subject developed an exacerbation of symptoms, on average at 16 (7-26) days after the onset of steroid reduction. This was accompanied by a deterioration in each of the objective measures. There was a fall in FEV1 by 320 ml (s.e.m. 9.5) and in PC20 from 0.8 to 0.43 mg/ml. Circulating eosinophils rose from 114 (24) x 10(3)/ml to 227 (50) x 10(3)/ml and Eo/B-CFU rose from 31 (5.6) to 44 (11.3)/10(6) cells. Sputum developed in five subjects and contained 36 (5.2)% eosinophils and 1.98 (0.21)% metachromatic cells (mast cells or basophils). The symptom diary and weekly questionnaire were demonstrated to be valid and responsive to change. A deterioration indicated by the daily symptom score preceded changes in PEF. Treatment by an increase in steroid was followed by reversal of each of the changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-invasive assessment of bronchial inflammation in asthma: no correlation between eosinophilia of induced sputum and bronchial responsiveness to inhaled hypertonic saline

Bronchial inflammation in mild asthma has been investigated using bronchoscopical techniques. The safety of bronchoscopy in patients with more severe asthma has been questioned. We have used the non-invasive technique of hypertonic saline (HS) inhalation to induce sputum samples for cellular analysis whilst simultaneously yielding a measure of bronchial responsiveness. Ten normal subjects and a heterogenous group of 24 asthmatic patients (range % predicted FEV₁ 43.3-111.5) underwent HS challenge. Sputum samples induced were analysed. Total and differential cell counts between the two groups were compared. The association between bronchial responsiveness to HS and sputum cell counts was examined in the asthma group. Mean maximum fall in FEV₁ for normal subjects was 4.0 (2.1-5.9, 95%CI)% after saline. Geometric mean PD20HS for asthma patients was 7.7 (range 0.68-40.92) ml. Adequate sputum samples were obtained from 9/10 normals and 23/24 asthmatic patients. Sputum from normal subjects contained a median of 3.8 (2.8-8.1, interquartile range)% eosinophils compared with 17.6 (8.9-34.1)% in sputum from asthma patients (P < 0.001). Sputum from asthma patients contained fewer of all other cell types compared with normals, with the difference in macrophages reaching significance. There was no correlation between PD20HS and cell count for any cell type in asthma subjects. Analysis of induced sputum represents a simple, safe, non-invasive and well-tolerated method of assessment of bronchial inflammation, suitable for use in patients with a range of asthma severity. There was no relationship between inflammation, as assessed by sputum cell counts and a measure of ‘Indirect’ bronchial responsiveness.     

Differential effects of maintenance long-acting beta-agonist and inhaled corticosteroid on asthma control and asthma exacerbations

BACKGROUND: Combination therapy with long-acting beta-agonists (LABAs)/inhaled corticosteroids (ICSs) has become established as effective maintenance treatment for asthma. OBJECTIVE: To compare and contrast the efficacy and safety of LABAs/ICSs against different maintenance ICS strategies in adults with asthma. METHODS: Cochrane systematic reviews of randomized controlled trials (to April 2004) were identified that compared the addition of LABA to ICS against 3 inhaled corticosteroid strategies: (1) a similar dose (n = 4312 subjects), (2) a higher dose (n = 4951), and (3) a similar dose in steroid-naive subjects (n = 968). The outcomes evaluated were asthma exacerbations, asthma control, and adverse effects. Pediatric studies were excluded. RESULTS: The addition of LABA to ICSs significantly reduced the risk of exacerbations compared with a similar ICS dose, number needed to treat = 18. The effects of LABA/ICSs on exacerbations compared with the other maintenance inhaled corticosteroid strategies were not statistically significant. LABA added to inhaled corticosteroids led to significant improvements in asthma control compared with all 3 maintenance ICS strategies. There was an increased risk of tremor with LABA/ICSs that reached significance for initial therapy, number needed to harm = 21, and compared with higher ICS doses, number needed to harm = 74. CONCLUSION: Maintenance asthma therapy with LABA/ICSs has differential effects on asthma control and asthma exacerbations. CLINICAL IMPLICATIONS: The greatest benefit and least harm of LABAs comes when they are added to a similar ICS dose in adults with symptomatic asthma.     

Airway inflammation in sheep with acute airway hypersensitivity to inhaled Ascaris suum.

In order to investigate the relationship between airway inflammation and the development of naturally occurring allergic airway hypersensitivity, the lower respiratory tract was studied in eighteen sheep 14 days after challenge with inhaled Ascaris suum antigen. Sheep could be classed into three groups on the basis of their response: group A (n = 6) had significant changes in both airway resistance and dynamic lung compliance; group B (n = 6) had changes in only dynamic lung compliance, and group C (n = 6) were nonresponders. The results showed that the volume density of secretory granules in lung mast cells was greater in hypersensitive sheep than in nonreacting sheep (p less than 0.01). This high volume density was due to a high numerical density of granules in the mast cells. There was, however, no significant difference between groups in the numerical density of mast cells and eosinophils, or the observed degree of degranulation of mast cells. Although lymphocytes were commonly seen within the airway epithelium and subepithelial regions, infiltration of neutrophils or aggregation of dense and nodular lymphoid tissues were not common in the airways of any sheep. There was no significant difference in the infiltration of neutrophils or the aggregation of lymphocytes and lymphoid tissue in airways between the three groups. These findings indicate that there is an inherent difference in the volume density of lung mast cell granules between hypersensitive and nonreacting sheep but that other inflammatory cells are probably not directly involved in the initial development of allergic airway hypersensitivity in this species.     

miR-221在哮喘患儿痰液的表达及对哮喘小鼠气道炎症的影响

目的探讨miR-221在哮喘患儿及哮喘小鼠中的表达及其对气道炎症的作用。方法应用realtime PCR方法检测哮喘患儿和健康儿童痰液,以及卵清蛋白诱导的哮喘小鼠和正常小鼠肺组织中miR-221的表达。化学合成miR-221抑制剂与阴性对照,并分别鼻滴至哮喘小鼠,HE染色验证哮喘小鼠模型的成功建立,免疫荧光方法和western blot方法检测各组小鼠肺组织IL-1β与IL-4的蛋白表达。结果 HE染色显示哮喘模型组小鼠炎症细胞浸润明显高于正常对照组,哮喘小鼠模型成功建立。与对照组相比,哮喘患儿痰液及哮喘小鼠肺组织中miR-221表达水平显著升高(P0.01)。哮喘小鼠肺组织IL-1β与IL-4的蛋白表达水平显著高于正常对照组,而低于miR-221抑制剂组(P0.01)。结论 miR-221在支气管哮喘患儿及支气管哮喘小鼠模型中高表达,miR-221抑制剂能够抑制哮喘小鼠的气道炎症。     

Effect of an inhaled corticosteroid on methacholine airway reactivity

Methacholine airway reactivity was studied in seven asthmatic patients before and at the end of 4 mo of beclomethasone dipropionate therapy, as well as in a control group. There was no statistically significant change in reactivity in either group, suggesting that a change in airway cholinergic receptor activity is not part of the mechanism of action of corticosteroids in asthma.     

Noninvasive monitoring of airway inflammation and steroid reduction in children with asthma

PURPOSE OF REVIEW: Management of pediatric asthma is currently based on symptoms (often a second-hand report from parents) and lung function. Inhaled steroids are the mainstay of asthma management targeted at controlling airway inflammation. They should be used in the lowest possible doses. A number of noninvasive methods to assess inflammation have been developed in an effort to optimize anti-inflammatory treatment. RECENT FINDINGS: The first longitudinal studies have been published demonstrating an improvement in asthma control in children by adding noninvasive monitoring of inflammation into the clinical management. New methods include exhaled nitric oxide measurements, induced sputum and markers in exhaled breath condensate. SUMMARY: Further studies will show the practicability of including these measurement methods into everyday clinical practice. Their addition to the conventional assessment of asthma control appears promising. Using these methods to evaluate the current inflammatory state seems obligatory in research into new asthma therapeutics and management strategies. Managing asthma in children in specialist practice relying only on symptoms and lung function is no longer state of the art.     

Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma

Background Hypertonic saline-indueed sputum has recently been used for the evaluation of airway inflammation in asthma. Objective To assess the effect of hypertonicity on airway inflammation. Methods We compared the inflammatory cell composition of hypertonic saline-induced sputum with that of isotonic saline-induced sputum in 21 asthmatic subjects and, at baseline and 30min after each sputum induction, we measured bronchial hyper-responsiveness to methacholine as an indirect marker to detect increased airway inflammation. On two different days, the patients inhaled hypertonic saline (3–5% NaCl) or isotonic saline (0.9% NaCl) for 30 min via an ultrasonic nebulizer, while monitoring FEV₁. Sputum was collected for inflammatory cell analysis. Results There was no difference in inflammatory cell percentages obtained with the two methods. Eosinophils were >1% in 20 subjects after hypertonic saline and in 16 subjects after isotonic saline, but this difference was not statistically significant. Intraclass correlation coefficients for sputum inflammatory cells obtained with the two methods were +0.642 for eosinophils, +0.644 for neutrophils. +0.544 for lymphocytes and +0.505 for macrophages. Hypertonic saline induced bronchoconstriction in a significantly greater number of subjects than isotonic saline. Also, hypertonic saline increased bronchial responsiveness to methacholine. while isotonic saline did not. Conclusion We conclude that hypertonicity does not affect sputum cell composition, suggesting that inflammatory cells in hypertonic saline-induced sputum are probably preexisting and not acutely recruited in the airways by the hypertonic stimulus. However, the bronchoconstriction and the increase in bronchial hyper-responsiveness after hypertonic saline inhalation may imply the release of inflammatory mediators. This fact must be considered in the evaluation of soluble markers of inflammation in hypertonic salineinduced sputum.     

miR-21在哮喘患儿痰液的表达及对哮喘小鼠气道炎症的影响

目的探讨miR-21在哮喘患儿及哮喘小鼠中的表达及其对气道炎症的作用。方法应用实时PCR方法检测哮喘患儿和健康儿童痰液,以及卵清蛋白诱导的哮喘小鼠和正常小鼠肺组织中miR-21的表达;化学合成miR-21模拟物与阴性对照,并分别鼻滴至哮喘小鼠,HE染色验证哮喘小鼠模型的成功建立,免疫荧光方法和western blot方法检测各组小鼠肺组织肿瘤坏死因子-α(TNF-α)与核转录因子-κB(NF-κB)的蛋白表达。结果与对照组相比,哮喘患儿痰液及哮喘小鼠肺组织中miR-21表达显著升高(P<0.01);HE染色显示哮喘模型组小鼠炎症细胞浸润明显高于正常对照组,哮喘小鼠模型成功建立。miR-21模拟物滴入后,哮喘小鼠肺组织TNF-α与NF-κB的蛋白表达显著降低(P<0.01)。结论 miR-21在支气管哮喘患儿及支气管哮喘小鼠模型中高表达,miR-21模拟物能够抑制哮喘小鼠的气道炎症。     

miR-33a在哮喘患儿痰液的表达及对哮喘小鼠气道炎症的影响

目的探讨miR-33a在哮喘患儿及哮喘小鼠中的表达及其对气道炎症的作用。方法应用real-time PCR方法检测哮喘患儿和健康儿童痰液,以及卵清蛋白诱导的哮喘小鼠和正常小鼠肺组织中miR-33a的表达;化学合成miR-33a模拟物与阴性对照,并分别鼻滴至哮喘小鼠,HE染色验证哮喘小鼠模型的成功建立,免疫荧光方法和Western blot方法检测各组小鼠肺组织肿瘤坏死因子-α(TNF-α)与白介素-6(IL-6)的蛋白表达。结果与对照组相比,哮喘患儿痰液及哮喘小鼠肺组织中miR-33a表达显著降低(P0.01);HE染色显示哮喘模型组小鼠炎症细胞浸润明显高于正常对照组,哮喘小鼠模型成功建立。miR-33a模拟物滴入后,哮喘小鼠肺组织TNF-α与IL-6的蛋白表达显著降低(P0.01)。结论 miR-33a在支气管哮喘患儿及支气管哮喘小鼠模型中低表达,miR-33a模拟物能够抑制哮喘小鼠的气道炎症。     

Difficult asthma in children: an analysis of airway inflammation

BACKGROUND: Difficult asthma in children displays distinct clinical patterns, and its physiopathology remains poorly understood. OBJECTIVE: To determine the characteristics of the bronchial inflammatory profile in children with difficult asthma. METHODS: We performed endobronchial biopsy and bronchoalveolar lavage in 28 children with persistent bronchial obstruction despite high doses of inhaled corticosteroids and regular treatment with long-acting beta(2)-agonists: 13 had persistent symptoms and 15 had few or no symptoms. RESULTS: The number of eosinophils (P =.03) and neutrophils (P =.04) in the epithelium was significantly higher in symptomatic children than in children with few symptoms. Reticular basement membrane thicknening was similar in both groups. IFNgamma levels (P =.03) and IFNgamma/IL-4 ratio (P =.01) were significantly higher in children with few symptoms. CONCLUSIONS: In symptomatic children, T(H)2-type inflammation was associated with the presence of activated eosinophils in the epithelium, whereas asthma in children with few symptoms was associated with an increase in T(H)1 cytokine levels. The high levels of IFNgamma suggest that this T(H)1 cytokine may modulate the local inflammatory response.