Low-dose Theophylline Reduces Eosinophilic Inflammation but Not Exhaled Nitric Oxide in Mild Asthma SAM LIM, KATSUYUKI TOMITA, GAETANO CARRAMORI, ANON JATAKANON, BRIAN OLIVER, ANDREAS KELLER, IAN ADCOCK, KIAN FAN CHUNG, and PETER J. BARNES Department of Thoracic Medicine, Imperial College School of Medicine, National Heart and Lung Institute, London, United Kingdom; and Byk Gulden Pharmaceuticals, Konstanz, Germany
Theophylline is well-established in the management of asthma, and there is some evidence of an antiinflammatory effect in asthma. It is not known whether theophylline affects inflammatory markers such as sputum eosinophils and exhaled nitric oxide (NO) in patients with mild asthma not receiving inhaled steroid therapy. In a double-blind, placebo-controlled, cross-over study of 15 patients with mild asthma, we assessed the effect of low-dose theophylline therapy (250 mg twice per day) on eosinophils in induced sputum, bronchoalveolar lavage (BAL) and airway biopsies at the end of both the treatment and placebo periods. Measurements of exhaled nitric oxide (NO) were made at the end of the active and placebo treatment periods of 5 wk each. Low-dose theophylline (mean serum level, 6.1 mg/L) led to a significant reduction in mean (95% confidence interval [CI]) sputum eosinophils from 11.3% (7.80–14.76%) to 8.0% (5.46–10.44%), BAL eosinophils from 3.4% (2.4–4.4%) to 1.7% (1.1–2.3%) and biopsy eosinophils from 1.83% (0.76–2.89%) to 1.20% (0.27–2.13%) compared with placebo (all p 0.05). There was no significant change in levels of exhaled NO or improvement in lung function and bronchial responsiveness. Low-dose theophylline induced antiinflammatory effects in asthma, reflected by a fall in airway eosinophils with no change in exhaled NO or changes in lung function.
flammatory benefits appear to occur at lower plasma theophylline levels ( 10 mg/L), and the incidence of any adverse effects is minimized. Two studies have demonstrated that lowdose theophylline added to inhaled steroids was equally efficacious when compared with increasing the dose of inhaled steroids, in symptomatic patients established on inhaled steroid therapy (10, 11). Previous studies have demonstrated that low-dose theophylline reduced the increase in eosinophils in airway biopsies after allergen challenge (12) and that withdrawal of theophylline in patients with severe asthma was associated with an increase in airway T lymphocytes (13). We studied the effect of low-dose theophylline on these inflammatory markers in order to determine whether this treatment is associated with antiinflammatory effects in vivo. There have been no studies that have examined the effect of low-dose theophylline on noninvasive parameters of inflammation in patients with asthma. We therefore determined the effect of theophylline on exhaled NO and induced sputum in patients with mild asthma, and also compared these effects with those obtained by using more invasive procedures such as bronchial biopsies and bronchoalveolar lavage.
Asthma is a chronic inflammatory disorder of the airways characterized by infiltration of the airways with inflammatory cells such as eosinophils, and by the presence of airway hyperresponsiveness (AHR) to a variety of stimuli. The noninvasive markers of inflammation such as levels of nitric oxide in exhaled breath (1), the number of eosinophils in induced sputum (2), and the number of eosinophils in induced sputum or in bronchial biopsies and bronchoalveolar lavage are elevated in asthma (3, 4), and are inhibited by inhaled steroids. Theophylline is used worldwide for the treatment of asthma, and recent studies indicate that theophylline has antiinflammatory effects. Thus, theophylline has been shown to inhibit eosinophil degranulation and release of eosinophil basic proteins (5), prevent the release and expression of tumor necrosis factor (TNF-) and interleukin 1 (IL-1) from blood monocytes (6, 7), and reduce IL-2 production by T cells and IL-2-dependent T cell proliferation (8, 9). The use of theophylline, however, has declined owing to the widespread use of inhaled steroids, which remain the most effective treatment for asthma. One of the limitations of theophylline in the past has been the side effects observed in many patients at the traditional bronchodilator doses associated with plasma levels of theophylline between 10 and 20 mg/L. However, antiin-
METHODS
(Received in original form June 8, 2000 and in revised form January 2, 2001) Correspondence and requests for reprints should be addressed to Prof. K. F. Chung, National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail:
[email protected] Am J Respir Crit Care Med Vol 164. pp 273–276, 2001 Internet address: www.atsjournals.org
Patients Fifteen patients with mild stable asthma, receiving treatment with only the inhaled 2-adrenergic agonist aerosol albuterol for intermittent relief of wheeze, were recruited. All patients demonstrated a 15% improvement in forced expiratory volume in 1 s (FEV1) after administration of 200 g of albuterol and airway hyperresponsiveness to methacholine, with a provocative concentration of methacholine producing a 20% fall in FEV1 (PC20) of 4 mg/ml. All patients were atopic as defined by two or more positive skin prick tests to common allergens. None of the subjects studied had received oral or inhaled corticosteroids for the preceding 12 mo, or any other treatment apart from inhaled 2-agonists. Current smokers or ex-smokers of more than 5 pack-years and patients with FEV1 less than 80% predicted were excluded. Although theophylline is usually used as add-on therapy, we chose to study patients with mild asthma not receiving inhaled steroid treatment.
Study Design The study was a 14-wk double-blind randomized crossover study comparing the effects of low dose theophylline (Euphylong, 250 mg twice daily) with placebo. Each treatment was administered for 5 wk, separated by a 4-wk washout phase. We chose this dose of theophylline with the knowledge that it would give serum levels below the usual accepted level of 10 mg/L (10). Patients were asked to chart their peak expiratory flow (PEF), their symptom scores, and medication use twice daily. All patients were reviewed on Day 28, spirometry and airway responsiveness to methacholine were measured, and sputum induction was performed. On Day 35, venous blood was drawn for the measurement of serum theophylline and fiberoptic bronchoscopy was performed. The study was approved by the Royal Brompton Hospital (London, UK) Ethics Committee, and all patients gave their informed consent.
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Lung Function and Challenge Tests Baseline prebronchodilator spirometric parameters were recorded from the best of three attempts using a dry wedge spirometer (Vitalograph, Buckingham, UK). Spirometric measurements and methacholine provocation tests were performed at the beginning of each treatment period and on Day 26 of the treatment period. PEF measurements were undertaken by patients at home, using a mini Wright peak flow meter (Clement Clarke, Harlow, UK). Patients were instructed to record their peak flow rates and symptom scores on a diary card. PEF variability was calculated from the difference between the highest and lowest daily reading divided by the highest PEF multiplied by 100.
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were made by two experienced observers unaware of the clinical status or origin of the sections. The coefficient of variation between observations was less than 10% between the two different observers.
Data Analysis Data are presented as means (95% confidence intervals), unless otherwise indicated. The data sets were explored for normality of distribution and differences between pre- and post-treatment and between treatment periods were assessed with the appropriate paired sample tests (16). We examined for differences between the two treatment periods and found no significant treatment order and period effect.
Exhaled Nitric Oxide Measurements
RESULTS
Exhaled NO was measured by chemiluminescence analyzer (model LR2000; Logan Research, Rochester, UK), with sensitivity from 1 ppb to 100 ppm of NO, accuracy 0.5 ppb, and response time of 2 s to 90% of full scale. Details have been previously published (14).
Fifteen patients with asthma (mean [SD] age, 30.5 [7.85] yr; 8 males and 7 females) with FEV1%pred of 88.0 (13.5) and PC20 methacholine of 0.85 (1.26) mg/ml completed the study. Mean (SD) serum theophylline levels after the active treatment period was 6.1 (2.9) mg/L compared with less than 1 mg/L with placebo. No patients complained of side effects during the theophylline treatment period.
Sputum Induction and Processing The method of sputum induction and processing has been previously described 15. Subjects inhaled 3.5% saline nebulized via an ultrasonic nebulizer (DeVilbiss 99; DeVilbiss, Heston, UK) at maximum output. Subjects were encouraged to cough deeply after 5 min and at 3-min intervals thereafter until an adequate amount of sputum had been obtained. At least 2 ml of sputum was collected into a 50-ml polypropylene tube, kept at 4 C, and processed within 2 h. Cytospin slides were prepared and stained with May–Grünwald–Giemsa. Differential cell counts were performed by an observer blind to the clinical characteristics of the subjects. At least two slides were used for counting and 300 inflammatory cells were counted on each slide.
Fiberoptic Bronchoscopy Subjects were pretreated with atropine (0.6 mg, intravenous) and midazolam (5–10 mg, intravenous). Oxygen (3 L/min) was administered via nasal prongs throughout the procedure and oxygen saturation was monitored with a digital oximeter. Under local anesthesia with lidocaine (4%) to the upper airways and larynx, a fiberoptic bronchoscope (BF10 Key-Med; Olympus, Southend, UK) was passed through the nasal passages into the trachea. Bronchoalveolar lavage (BAL) was performed from the right middle lobe using 0.9% NaCl warmed to 37 C, with four successive aliquots of 60 ml. Four mucosal biopsies were taken from segmental and subsegmental bronchi. Cytospins were prepared and stained with May–Grünwald–Giemsa stain for differential cell counts. Cell viability was assessed by trypan blue exclusion.
Bronchial Biopsy and Processing of Tissues Bronchial mucosal biopsies were immediately placed in O.C.T. embedding medium, and then snap frozen in isopentane precooled with liquid nitrogen and stored at 70 C. All biopsies were frozen within 20 min of collection. Six-micron sections were cut on a cryostat and placed on poly-L-lysine-coated microscope slides (Sigma, Poole, UK), air dried for 30 min, and then wrapped in aluminum foil and stored at 70 C before immunostaining. To stain for the presence of inflammatory cells, a mouse monoclonal anti-human major basic protein antibody (Monosam; Bradsure Biological, Loughborough, UK) was used for eosinophils. After the primary antibody had been administered, a biotinylated rabbit anti-mouse immunoglobulin (1:100) followed by peroxidase-conjugated avidin (1:200) was used. Chromogen fast diaminobenzidine was used for 5 min and the slides were counterstained in hematoxylin and mounted on mounting medium (DPX; Electron Microscopy Sciences, Fort Washington, PA).
Cell Counts Counts of positive cells were made on all sections. The number of eosinophil major basic protein-positive (MBP ) cells was expressed as the number per high-power field. At least four fields at 400 magnification were examined on each biopsy for it to be considered of adequate quality. One field was defined as a length of intact epithelium of 175 m together with an area of 175 175 m beneath the epithelium, representing the subepithelium. Counts were expressed as the total number of MBP cells in the epithelium and subepithelium. All counts
Effect on Lung Function, Asthma Symptoms, and Medication Usage
After low-dose theophylline, there was no significant change in lung function, morning and evening PEF rates, or any significant reduction in PEF variability over the two treatment periods (Table 1). There was no significant difference in rescue 2-agonist usage or symptom scores over the recorded periods, although baseline values were low in these subjects with mild asthma. There were no changes in bronchial responsiveness with PC20 methacholine (0.94 mg/ml after placebo, and 1.24 mg/ml after theophylline). Effect on Exhaled NO, Eosinophils in Sputum, BAL, and Bronchial Biopsies
There was no significant difference in exhaled nitric oxide levels between the two treatment periods (Figure 1). Eleven pairs of sputum cytospins, 14 pairs of BAL cytospins, and 13 pairs of biopsies of adequate quality were available for examination. The reasons for the discrepancy in the number of sputum and BAL cytospins and biopsies available for examination included inadequate sputum (3), lost slides (2), and inadequate biopsies (2). The effects of theophylline on sputum, BAL, and mucosal eosinophils are shown in Figure 1. There were significant effects of low-dose theophylline on sputum, BAL, and mucosal eosinophils: 11.3% (7.8–14.8) to 8.0% (5.5–10.4) (p 0.05), 3.4% (2.4–4.4) to 1.7% (1.1–2.3), (p 0.05), and 1.8 per
TABLE 1. EFFECT OF THEOPHYLLINE ON EOSINOPHILS, EXHALED NITRIC OXIDE, LUNG FUNCTION, AND -AGONIST USE*
FEV1, L FVC, L PC20, mg/ml NO, ppb Symptom scores AM PM
PEF AM, L/min PEF variability, % 2-Agonist use, puffs
Placebo
Theophylline
p Value
3.3 (2.9, 3.6) 4.47 (4.03, 4.71) 0.94 ( 0.01, 1.89) 24.1 (16.18, 32.04)
3.4 (3.2, 3.6) 4.50 (4.13, 4.87) 1.24 (0.46, 1.99) 20.6 (14.66, 26.63)
NS NS NS NS
0.76 (0.33, 1.18) 0.47 (0.05, 0.89) 455.5 (425.5, 485.6) 13.56 (7.97, 19.15) 1.03 (0.41, 1.65)
0.44 (0.11, 0.77) 0.12 (0.00, 0.24) 472.8 (433.6, 511.9) 11.14 (5.65, 16.64) 1.02 (0.24, 1.80)
NS NS NS NS NS
Definition of abbreviations: BAL bronchoalveolar lavage; FEV1 forced expiratory volume in the first second; MBP major basic protein; NO nitric oxide; PC20 provocative concentration of methacholine causing a 20% drop in FEV 1; PEF peak expiratory flow; ppb parts per billion. * Data shown as means (95% confidence intervals).
Lim, Tomita, Carramori, et al.: Low-dose Theophylline and Inflammation in Asthma
Figure 1. Effects of theophylline (Th) and placebo (Pl) on eosinophils in mucosal biopsies (A) induced sputum (B), and bronchoalveolar lavage (BAL, C), and on exhaled NO (D) in patients with mild asthma. Measurements were made at the end of each treatment period, but exhaled NO was measured before and after placebo or theophylline administration. Each treatment had no significant effect on exhaled NO. Solid circles represent individual data points. Squares represent means SEM.
175 m epithelium (0.8–2.9) to 1.2 (0.3–2.1) (p 0.05), respectively. Representative effects of theophylline treatment on mucosal eosinophils are shown in Figure 2.
DISCUSSION In this study, we have demonstrated the ability of low-dose theophylline to reduce airway eosinophilic inflammation in
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vivo, in patients with mild asthma, without significant changes in levels of exhaled NO. The effects of theophylline in inhibiting eosinophilia were consistent in that significant reductions were observed in three different compartments of the airways as assessed in the bronchial biopsies, in induced sputum, and bronchoalveolar lavage fluid. These findings support the antiinflammatory potential of low-dose theophylline for the treatment of asthma and are in agreement with other in vivo and in vitro theophylline studies (13, 17, 18). Several studies have demonstrated the clinical utility of theophylline in the management of asthma (10, 11, 19, 20). Previously, the lack of clarity of the antiinflammatory activity of theophylline has limited its acceptance as a disease-modifying agent, particularly in western countries, where inhaled steroids are generally accepted as the first-line antiinflammatory agent of choice. One of the advantages of theophylline is that compliance to therapy may be better than with inhaled glucocorticoids (21). We found no significant effect of theophylline on lung function, which is likely to be due to the mild nature of the asthma and to the absence of airflow limitation in these patients. In addition, plasma levels of theophylline of less than 10 mg/L would not be expected to cause bronchodilatation. Previous studies of low-dose theophylline therapy in patients with more severe asthma suggest that the improvement in physiological parameters observed in these patients may not be due solely to the bronchodilator capacity of theophylline (10). We also studied the effect of low-dose theophylline on exhaled NO. Exhaled NO has been advocated as a noninvasive means to monitor the inflammatory process in asthma. Exhaled levels of NO are elevated in patients with asthma not treated with inhaled corticosteroids, and can be reduced with steroid treatment (3, 22). Because corticosteroids can also decrease inflammatory parameters such as sputum and mucosal eosinophils, NO may be used to monitor the inflammatory process noninvasively. Significant correlations, albeit weak, between exhaled levels of NO with measurement of airway
Figure 2. Representative photomicrographs of sections from bronchoscopic biopsies obtained from patients with mild asthma treated with placebo or with theophylline. The biopsies have been stained for eosinophils, using a mouse monoclonal antihuman major basic protein antibody. The eosinophils stain brown. (A and B) and (C and D) are from the same patients while receiving placebo and theophylline, respectively. Arrowheads indicate the presence of eosinophils in the submucosa and in the epithelium. Scale bars: 50 m.
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eosinophilia (2) have been reported, and the inducible isoenzyme of nitric oxide synthase (iNOS) is inhibited by corticosteroid treatment (23). In contrast, theophylline has no known inhibitory activity on iNOS and our data are consistent with this. Exhaled levels of NO reflect iNOS activity and this does not correlate with disease activity (24); furthermore, theophylline decreased eosinophilic inflammation, without decreasing levels of exhaled NO. The apparent dissociation between eosinophils and exhaled NO, due to the lack of effect of theophylline on iNOS, suggests that measurement of exhaled NO may not be suitable for the assessment of the effect of theophylline on the airway inflammation of asthma. Our study raises the issue of the use of exhaled NO levels as a surrogate marker of airway inflammation. When using mucosal eosinophil numbers as a true marker of airway inflammation, we found no significant correlation with exhaled NO in a group of patients with mild asthma (3). However, after treatment with inhaled steroids, both exhaled NO and mucosal eosinophils fell, but the change in exhaled NO did not correlate with the change in mucosal eosinophils (3). Taken together with the results of the current study, measurement of exhaled NO are not a reliable marker of airway mucosal eosinophilia. It is likely that exhaled NO is indicative of an inflammatory pathway different from that of mucosal eosinophilia. These observations also indicate that exhaled NO may not be an appropriate marker of airway inflammation in asthma. In summary, low-dose theophylline has antiinflammatory activity in patients with asthma, as indicated by a reduction in eosinophils in reduced sputum, bronchoalveolar lavage fluid, and the bronchial mucosa, but this was not associated with a reduction in levels of exhaled NO. References 1. Barnes PJ, Kharitonov SA. Exhaled nitric oxide: a new lung function test. Thorax 1996;51:233–237. 2. Jatakanon A, Lim S, Kharitonov S, Chung KF, Barnes PJ. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998;53:91–95. 3. Lim S, Jatakanon A, John M, Gilbey T, O’Connor BJ, Chung KF, Barnes PJ. Effect of inhaled budesonide on lung function and airway inflammation: assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 1999;159:22–30. 4. Oddera S, Silvestri M, Balbo A, Jovovich BO, Penna R, Crimi E, Rossi GA. Airway eosinophilic inflammation, epithelial damage, and bronchial hyperresponsiveness in patients with mild-moderate, stable asthma. Allergy 1996;51:100–107. 5. Kita H, Abu-Ghazaleh RI, Gleich GJ, Abraham RT. Regulation of Iginduced eosinophil degranulation by adenosine 3 ,5 -cyclic monophosphate. J Immunol 1991;146:2712–2718. 6. Ghezzi P, Dinarello CA. IL-1 induces IL-1. III. Specific inhibition of IL-1 production by IFN-gamma. J Immunol 1988;140:4238–4244. 7. Spatafora M, Chiappara G, Merendino AM, D’Amico P, Bellia V, Bon-
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