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* From Airways Disease Section, National Heart and Lung Institute, Imperial College, London, UK.
Currently at the Division of Respiratory Disease and Tuberculosis, Department of Internal Medicine, Siriraj Hospital, Mahidol University, Thailand.
Correspondence to: Peter J. Barnes, DM, DSc, FCCP, Airways Disease Section, Dovehouse St, London, SW3 6LY, UK; e-mail: p.j.barnes{at}imperial.ac.uk
Abstract
Background: The role of neutrophils in exacerbations of asthma is poorly understood. We examined the effect of withdrawal of inhaled corticosteroids on sputum inflammatory indexes in a double-blind study in patients with moderate, stable asthma.
Methods: Following a 2-week run in period, 24 subjects were randomized to receive either budesonide (400 µg bid) or placebo, and the study was continued for another 10 weeks.
Results: Loss of asthma control developed in 8 of 12 patients over the 10-week period of steroid withdrawal, whereas only 1 of 10 patients with budesonide treatment had exacerbations. Those with an exacerbation had increased sputum interleukin (IL)-8 (p < 0.0001) and increased sputum neutrophil numbers (p < 0.0001) compared to those without an exacerbation. The significant elevation in sputum IL-8 and neutrophil counts initially occurred 2 weeks prior to an exacerbation. Sputum neutrophilia correlated positively with changes in IL-8 levels (r2 = 0.76, p = 0.01).
Conclusions: Rapid withdrawal of inhaled corticosteroids results in an exacerbation of asthma that is preceded by an increase in sputum neutrophils and IL-8 concentrations, in contrast to an increase in eosinophils reported in previous studies in which inhaled steroids are slowly tapered.
Key Words: asthma • exacerbation • interleukin-8 • neutrophil
Although eosinophilic airway inflammation is recognized as an important feature of some patients with chronic, stable asthma,12 evidence supports an important role of neutrophils in acute exacerbations.345 In stable asthma, there is typically an infiltrate of eosinophils, which occurs in response to cytokines such as interleukin (IL)-5, secreted by T-helper type 2 lymphocytes.6 In contrast, during acute exacerbations of asthma, the airway inflammatory response is both more intense and heterogeneous.3457 The increased intensity of airway inflammation is reflected by an elevated total cell count in sputum.3458 The heterogeneity of the inflammatory response is reflected by an increase in neutrophils as well as eosinophils, and there are reports of exacerbations without sputum eosinophilia.345 There is also evidence for increased expression of the potent neutrophil chemoattractant IL-8,3 in addition to the presence of IL-5. There is heterogeneity in the concentrations of IL-8 in patients with moderate asthma and the strong correlation between the concentration of IL-8 release by peripheral blood mononuclear cells9 and the frequency of asthma exacerbations.10
Leukocytes, particularly neutrophils and eosinophils, are the first cells recruited to the site of tissue damage after an inflammatory insult.11 These cells are then removed by apoptosis during the resolution of the inflammatory response. Neutrophils are the predominant inflammatory leukocyte characterizing airway inflammation in acute severe asthma, and this may influence the clinical presentation, predisposing to a sudden severe attack.345 IL-8 is a key chemokine involved in the recruitment of neutrophils to the area of inflammation, but few studies have characterized the kinetics of IL-8 production in sputum over a period of time prior to the onset of a clinical asthma exacerbation. Because it is not possible to obtain this information prospectively with a naturally occurring exacerbation, we induced loss of control of asthma by withdrawing inhaled corticosteroids so that we were able to make prospective measurements of inflammatory indexes in induced sputum. This was done in a double-blind controlled manner so that steroid withdrawal could be compared with continued therapy with inhaled corticosteroids.
Materials and Methods
Subjects
Twenty-four nonsmoking patients (10 men) with moderate, persistent asthma participated in the study. The inclusion criteria were age 18 to 60 years of age and a history of stable asthma as defined by the American Thoracic Society. Patient characteristics are summarized in Table 1
. All patients had a baseline FEV1 
70% of predicted, FEV1 reversibility 15%, and required regular treatment with moderate doses of inhaled corticosteroids (beclomethasone dipropionate, 800 µg/d or equivalent) for > 3 months. All patients were hyperresponsive as measured by a provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) < 8 mg/mL. None of the patients had a history of respiratory disease other than asthma, and none required oral glucocorticoid treatment within 3 months before study entry and used any other medication except study medication during the trial. The patients were included during a clinically well-controlled period without symptoms of an upper respiratory tract infection for 4 weeks prior to the study. The study was approved by the Royal Brompton and Harefield NHS Hospital Trust Ethics Committee, and informed consent was given by all patients.
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Run-in Phase
During the 2-week run-in phase, all patients had to have stable disease without lower respiratory tract infection. Throughout the study, only terbutaline via Turbuhaler was permitted on an on-demand basis as a rescue inhaler. A study physician was accessible by telephone 24 h/d.
Induction of Exacerbation
Steroid withdrawal was performed by inhalation of placebo, and the study was discontinued when an exacerbation developed or when no exacerbation of asthma occurred within the 10-week study period. Patients undergoing exacerbation during treatment period were withdrawn from the study. Thereafter, inhaled budesonide was increased to 800 µg bid, and systemic corticosteroids (prednisolone 30 mg/d) were administered.
An exacerbation was defined as at least one of three criteria1213: (1) a drop in morning peak flow 20% below baseline (mean of the last 7 days run-in period) on 2 consecutive days; (2) wakening due to asthma on 2 consecutive nights, and requiring rescue medication; (3) 50% increase in 24-h rescue medication use on at least 2 consecutive days compared to mean use during the last 7 days of the run-in period, which also exceeded the equivalent of four puffs of terbutaline. FEV1 was then measured within 24 h of the exacerbation.
Lung Function Measurements and Methacholine Challenge Tests
FEV1 and FVC were measured using a dry wedge spirometer (Vitalograph; Buckingham, UK). Values are expressed as percentage of predicted. Baseline values were measured after 15 min of rest and taken as the highest of three readings. Single readings only were taken at other times. Airway hyperresponsiveness to methacholine was assessed using the method of Sterk and colleagues.13 Methacholine was inhaled by tidal breathing in doubling concentrations (0.015 to 32 mg/mL) for 2 min at 5-min intervals. Measurements of FEV1 were made at baseline and after each dose. The challenge test was discontinued if FEV1 dropped 20% from baseline. PC20 was calculated by linear interpolation of the log-dose response curves.13
Sputum Induction and Processing
Sputum was induced by inhalation for 15 min of 3.5% saline solution via an ultrasonic nebulizer (model 2000; DeVilbis; Heston, UK), as previously described.14 After each inhalation period, patients were asked to rinse their mouth and were encouraged to expectorate sputum. Briefly, the whole sputum sample was processed with dithiothreitol (Sigma Chemicals; Poole, UK). The homogenized sputum was centrifuged at 1,500g for 10 min. The supernatant was separated and frozen at – 70°C until further analysis. Total cell counts were made on a hemocytometer slide, using Kimura stain, and slides were prepared (Cytospin; Shandon; Runcorn, UK) and stained with May-Grunwald-Giemsa stain. Differential cell counts were made by a blinded observer. Three hundred nonsquamous cells were counted on two slides for each sample in a blind way. Differential cell counts are expressed as percentages of nonsquamous cells. Two of the sputum samples from the same patient in budesonide treatment group at different visits were withdrawn from analysis because of containing > 80% squamous cells (Table 2
).
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Statistical Analysis
Results are reported as mean ± SEM. We compared patients who lost control of asthma with those who did not after corticosteroid withdrawal. All statistical tests were two sided, and significance was accepted at the level of 95% and p < 0.05 using statistical software (GraphPad Prism; GraphPad Software; San Diego, CA). To determine whether changes in sputum neutrophil counts and sputum IL-8 levels accompanied asthma exacerbation, the differences of the last-visit measurements from baseline were used for analysis of correlation with Pearson product-moment technique. The data from subjects with and without exacerbation at each time point were categorized as a group before analysis. Sputum neutrophil counts, percentage of neutrophils, and IL-8 levels 2 weeks prior to an exacerbation were compared to those of the week-8 inflammatory markers in nonexacerbation group using the unpaired t test. The last-visit measurements of the exacerbation and nonexacerbation groups were also evaluated by the unpaired t test.
Results
Patient Characteristics
Patient characteristics are shown in Table 1. Two patients in the budesonide treatment group were excluded from the analysis: one was unable to attend for all of the study visits, and the other provided induced-sputum specimens containing squamous cell counts > 80%.
Exacerbations
Eight of 12 patients had an exacerbation over the 10-week period of steroid withdrawal (Table 3
). The remaining four subjects did not have an exacerbation during the 10 weeks of follow-up. Only 1 of 10 patients in the budesonide treatment group had an asthma exacerbation at week 2 (Table 2).
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Sputum Eosinophils
No significant differences in the percentages of eosinophils and absolute eosinophil number in sputum were observed either at the onset of exacerbation or 2 weeks before an exacerbation, compared with nonexacerbation data (Table 4, Fig 2, top, left, A, and top, right, B).
Discussion
We evaluated the use of sputum neutrophils and IL-8 as predictors for the development of asthma exacerbations. Our study indicates that neutrophils, rather than eosinophils, are associated with asthma exacerbations following steroid withdrawal. This may result from the increase in sputum IL-8 observed prior to the onset of an exacerbation. Taken together, our findings demonstrated that sequential monitoring of sputum IL-8 and neutrophils might be useful for predicting an episode of severe exacerbation in moderate persistent asthma after corticosteroid withdrawal. This study also confirms that the mechanisms underlying asthma exacerbation are highly variable.212151617
There is growing evidence that reducing asthma exacerbations is achieved by targeting therapy to eosinophilic inflammation. An eosinophilic exacerbation is triggered by allergen exposure. However, several studies34518 have demonstrated increased neutrophil numbers in airway secretions and airway tissue from patients with acute severe asthma. Sputum neutrophilia may be mediated by IL-8, a chemokine that selectively attracts neutrophils. IL-8 is increased in sputum of asthmatic subjects during exacerbations in association with sputum neutrophilia.31516 However, there is no clear relationship between the type of airway inflammation and asthmatic exacerbations,19 possibly due to differences in the methodology, specifically the use of steroid tapering.12 Furthermore, the pathophysiologic mechanisms underlying asthma exacerbations following abrupt steroid withdrawal in moderate persistent asthmatics may be distinct from those seen with gradual steroid tapering. The neutrophilic airway inflammation in the present study may reflect the sudden withdrawal of inhaled corticosteroids and reflect the kinetics of inflammatory cells in an acute exacerbation when neutrophils are the first cells to infiltrate the airways.
There are several studies12171920 suggesting that eosinophilic inflammation is associated with sputum asthma exacerbations in response to controlled withdrawal of steroids. In addition, targeting management based on normalizing sputum eosinophils is able to reduce asthma exacerbations.212223 Unexpectedly, the present study showed that a small number of patients with steroid withdrawal did not have asthma exacerbations, although they had eosinophilic inflammation. The patients in whom the dose of inhaled budesonide was maintained showed a reduction in sputum eosinophils, which may indicate greater compliance with inhaled corticosteroids during the trial period. The difference in the relationship between sputum eosinophils and asthma exacerbation with the majority of current evidence is not easily understood. We suggest that this may be due to the fact that a 10-week period in our study is inadequate to monitor the development of eosinophilic exacerbation. This is supported by the study of Pizzichini and colleagues,24 who showed that breakthrough eosinophilic inflammation occurred at 4 weeks following steroid reduction and subsequent asthma exacerbations developed within 12 weeks thereafter. Clinical exacerbation depends on the perception of breathlessness that is impaired by eosinophilic inflammation.25 Patients with eosinophilic inflammation who had no clinical worsening were moderately severe in terms of FEV1 and might have impaired perception of dyspnea and therefore were not aware of worsening asthma. Although the strategy of titrating the inhaled corticosteroid dose against sputum eosinophil counts reduced exacerbations, it by no means eliminated them. It is possible that the residual exacerbations were neutrophilic in nature.
Our results differ from those of Jatakanon et al20 and in’t Veen and colleagues,12 who induced loss of control in asthma by gradual tapering of the steroid dose, and both studies showed eosinophil predominance during exacerbations. As suggested above, these results suggest that the pathophysiologic mechanism(s) of asthma exacerbation induced by complete withdrawal may be different from that induced by steroid tapering. This may have important clinical consequences in that patients with a loss of asthma control as a result of stepping down inhaled corticosteroid dosage should be able to attenuate eosinophil actions by increasing the inhaled steroid dose.2326 In contrast, exacerbations induced after abrupt steroid cessation may need antineutrophilic agents such as long-acting ß2-agonists to attenuate the neutrophilic inflammation.27
Increases in sputum IL-8 concentrations have been reported to precede an acute asthma attack,28 but the time course of this increase is unknown. In another study15 there was a significant elevation of IL-8 levels in sputum samples from asthmatics who had exacerbated compared to IL-8 concentrations after the resolution of the exacerbation. However, the onset of the elevation of IL-8 levels prior to the development of asthma exacerbation has not been previously investigated. The present study demonstrates that a marked increase in IL-8 level occurred 2 weeks before the symptomatic exacerbation (Fig 1, top left, A). Clinically, this may have important implications because there appears to be a window during which it may be possible to adjust asthma treatment, such as the addition of long-acting ß2-agonist, in an attempt to suppress IL-8 production and subsequent neutrophilia leading to exacerbation. Although corticosteroids promote neutrophil survival as a result of delay in neutrophil apoptosis in vitro,29 we did not detect sputum neutrophilia in patients with budesonide treatment despite a failure to suppress sputum IL-8 levels below that seen in the run-in period.
In summary, the findings in this study in contrast to earlier reports suggest that asthma exacerbations following inhaled corticosteroid withdrawal are associated with increased sputum IL-8 and neutrophils. Significant increase in sputum IL-8 and neutrophil influx occurs 2 week prior to an exacerbation. Our results may be used to encourage asthmatic patients who have poor compliance with inhaled corticosteroid therapy are at risk for neutrophilic exacerbation if they abruptly stop their corticosteroid therapy.
Footnotes
Abbreviations: IL = interleukin; PC20 = provocative concentration of methacholine causing a 20% fall in FEV1
Funding for research was provided by the Royal Government of Thailand for sponsoring Dr. Maneechotesuwan, and AstraZeneca (Lund, Sweden).
Drs. Barnes, Kharitonov, and Adcock obtained research funding from several pharmaceutical companies interested in the treatment of asthma, including AstraZeneca, GlaxoSmithKline, and Novartis.
Received for publication December 12, 2006. Accepted for publication March 23, 2007.
References
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