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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1371-1376, (2002)
© 2002 American Thoracic Society

Original Article

Effect of Theophylline on Induced Sputum Inflammatory Indices and Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease

Sarah V. Culpitt, Carmen de Matos, Richard E. Russell, Louise E. Donnelly, Duncan F. Rogers and Peter J. Barnes

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom

Correspondence and requests for reprints should be addressed to Sarah V. Culpitt, Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, Dovehouse Street, SW3 6LY, London, UK. E-mail: duncan.rogers{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic obstructive pulmonary disease (COPD) is characterized by a neutrophilic airway inflammation that can be demonstrated by examination of induced sputum. Theophylline has antiinflammatory effects in asthma, and in the present study we investigated whether a similar effect occurs in COPD patients treated with low doses of theophylline. Twenty-five patients with COPD were treated with theophylline (plasma level of 9–11 mg/L) for 4 weeks in a placebo-controlled, randomized, double-blind crossover study. Theophylline was well tolerated. Induced sputum inflammatory cells, neutrophils, interleukin-8, myeloperoxidase, and lactoferrin were all significantly reduced by about 22% by theophylline. Neutrophils from subjects treated with theophylline showed reduced chemotaxis to N-formyl-met-leu-phe (~ 28%) and interleukin-8 (~ 60%). Neutrophils from a healthy donor showed reduced chemotaxis (~ 30%) to induced sputum samples obtained during theophylline treatment. These results suggest that theophylline has antiinflammatory properties that may be useful in the long-term treatment of COPD.

Key Words: COPD • sputum • theophylline • neutrophil • chemotaxis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic obstructive pulmonary disease (COPD) is a severe clinical condition characterized by progressive airflow limitation, and smoking cessation is the only intervention that slows disease progression (1, 2). The pathophysiology of airway obstruction in COPD is multifactorial, and involves neutrophilic airway inflammation (3), protease–antiprotease imbalance (4, 5), oxidative stress (6, 7), T cell predominant interstitial inflammation (8), and recurrent infection (9). These mechanisms are interrelated, and reducing one factor may reduce the stimulus to others.

Airway inflammation in COPD can be demonstrated by examination of induced sputum (3). Smokers and ex-smokers with COPD have increased sputum neutrophil numbers compared with subjects without COPD (10), and increased neutrophils are associated with rapid decline in forced expiratory volume in 1 second (FEV1) (11). Furthermore, the neutrophil activation markers (constituents of neutrophil granules), myeloperoxidase and lactoferrin, are elevated in the sputum supernatants of COPD subjects (12), indicating that neutrophils are active participants in airway inflammation.

Interleukin (IL)-8, found in greater amounts in sputum from patients with COPD than patients with asthma (3), is chemotactic for neutrophils (13), and is important in recruitment of neutrophils to the airways (14). Furthermore, IL-8 and leukotriene B4 (LTB4) are the major chemoattractants in the sputum of patients with bronchial disease (chronic bronchitis and bronchiectasis) (15), and both correlate with myeloperoxidase levels (16), implicating these chemokines in neutrophil recruitment into the airways. There are several potential sources of sputum IL-8, including bronchial epithelial cells (17), alveolar macrophages (18), monocytes, and neutrophils (19). Inhibition of IL-8 production is one therapeutic option for reducing neutrophil recruitment in COPD, with other strategies including receptor antagonism and therapies with a broad spectrum of cellular activity.

Theophylline has been used for many years in the treatment of obstructive airway diseases, although the molecular mechanisms underlying its therapeutic benefits remain unclear. Phosphodiesterase inhibition is believed to underlie the bronchodilator effect (20). Theophylline improves respiratory dynamics by a positive effect on diaphragmatic strength (21), respiratory muscle performance (22), and trapped gas volume (23). Mucocilliary clearance is also increased by theophylline (24). Numerous antiinflammatory actions of theophylline have been demonstrated that may have a beneficial effect in COPD, including effects on T cells (25, 26), eosinophils (27), and cytokines (28, 29). Theophylline has also been shown to reduce eosinophil (30), T cell (31), neutrophil, and mononuclear cell (32) chemotaxis.

The aim of the present study was to assess the effect of theophylline on induced sputum inflammatory indices in COPD. In particular, we wished to evaluate the effect of theophylline on neutrophil chemotaxis and neutrophil chemoattractants in induced sputum.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Twenty-five COPD patients with ATS criteria (33), were recruited. Subjects had a smoking history of more than 20 pack years (11 current smokers) and less than 15% reversibility of predicted FEV1 after a steroid trial (oral prednisone 30 mg daily for 2 weeks). Subjects who had inhaled or oral steroids, or an exacerbation of COPD in the previous 6 weeks were excluded. Treatment with albuterol alone (200 µg as needed) or with ipratropium bromide (40 µg four times a day) was continued. No other medications were taken 6 weeks before or during the study. Subjects gave written informed consent, and the study was approved by the ethics committee of the Royal Brompton and Harefield NHS Trust.

Study Design
The study had a randomized, double-blind, placebo-controlled crossover design with a run-in period of 2 weeks, during which a test dose of theophylline was given. The dose (150–300 mg twice a day) used in the study was calculated to give a plasma theophylline level of 8–15 mg/L. Subjects received theophylline (Byk Gulden; Konstanz, Germany) or placebo for 4 weeks, separated by 2 weeks. Subjects recorded peak expiratory flow, and attended the laboratory at the start and end of treatment periods for spirometry (Vitalograph, Buckingham, UK), sputum induction, venous blood aspiration, and plasma theophylline measurement.

Sputum Induction, Processing, and Assays
Sputum was induced and processed by a standard method (3). IL-8 was measured using a paired antibody (Genzyme Diagnostics, Cambridge, MA) (34). Myeloperoxidase and lactoferrin were measured using kits (R&D Systems, Oxon, UK, and Oxis International, Portland, OR, respectively).

Neutrophil Chemotaxis
Neutrophil chemotaxis was measured using an ATP assay (ATP-Lite; Packard Bioscience, Groningen, The Netherlands). In validation studies, the ATP assay gave equivalent data to that generated using a Boyden chamber. For example, maximal chemotaxis occurred at equivalent concentrations of chemoattractant. Neutrophils from healthy volunteers were used to compare the chemotactic activity of sputum (40– 5-fold dilutions). All samples from an individual subject were run on the same day using neutrophils from the same healthy volunteer. Neutrophils from subjects were used to measure chemotaxis to concentration gradients of N-formyl-met-leu-phe (fMLP, 10-10–10-5 M), LTB4 (10-11 –10-6 M), and IL-8 (1.56–100 ng/ml; equivalent to 0.2–12.5 nM). For each subject, chemotaxis to each chemoattractant was assessed in the same chamber, using a buffer solution control.

Chemoattractants were placed in a 96-well microtiter plate (Packard Bioscience) in the lower section of a chemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD). For the sputum chemotaxis assays, each chamber included internal controls of buffer solution (negative control) and IL-8 (positive control). A 3-µm polycarbonate filter (Neuro Probe Inc.) was applied to the plate, and the upper section of the chamber was secured to create a watertight seal. Neutrophils (0.1 million), separated from whole blood by discontinuous percoll gradient, were placed in the upper wells and incubated at 37° C for 90 minutes, after which the microtiter plate was centrifuged to adhere migrated cells. Dilutions of a 1 x 106 neutrophil suspension were added to empty wells to generate a standard curve of luminescence per cell number. Cell lysis solution was added to each well, followed by ATPLite luciferase/luciferin solution. Luminescence was measured (Anthos, Durham, NC), and the number of migrated cells was determined by interpolation from the standard curve. The assay was linear over the range used.

Statistical Analysis
Parametric data are expressed as the mean ± SEM and were compared using Student's t test. Nonparametric data are expressed as medians and ranges with comparisons made using Wilcoxon's signed rank sum test between pre- and post-placebo and pre- and post-theophylline. A p value of < 0.05 was considered significant (two-tailed tests). Where results are summarized in the text as percentage change, the post-minus pre-theophylline value is expressed as a percentage of the pre-theophylline value, and the mean ± SEM of this change is presented.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All 25 subjects completed the study and provided adequate sputum samples for analysis. All subjects tolerated the sputum induction procedure without significant change in FEV1 and symptoms, including the eight subjects who had severe COPD (FEV1 < 40% predicted). Five subjects experienced mild nausea while taking theophylline, which resolved after the first 3 days of treatment. One subject had mild sleep disturbance, which did not necessitate withdrawal of theophylline.

All sputum samples were analyzed for cell counts and IL-8. There was insufficient sputum to perform all assays on all samples, and lactoferrin and myeloperoxidase were measured for 15 subjects, and chemotactic activity was measured for the remaining 10 subjects.

There was no effect of placebo treatment and no carryover effects between treatment periods for any parameter measured.

Clinical Parameters
Subjects were aged 62 ± 2 years, and had moderate to severe airflow limitation (FEV1 20–68% predicted). Mean serum theophylline level during treatment was 9.5 ± 0.3 mg/L (range 9–11 mg/L). Theophylline significantly increased mean % predicted FEV1 by approximately 2% (absolute values: 1.36 ± 0.1 L versus 1.42 ± 0.1 L; p < 0.01 pre- versus post-theophylline). Theophylline treatment increased mean peak expiratory flow % predicted by approximately 2% (absolute values: 206 ± 15 L/minute versus 214 ± 15 L/minute; p < 0.001 pre- versus post-theophylline).

Cell Counts
Data are summarized in Table 1 and Figure 1 . Total inflammatory cell counts (millions/ml) were significantly reduced by approximately 21% after theophylline (Figure 1A). The percentage of neutrophils was not significantly changed, although the absolute number (millions/ml) was reduced after theophylline by approximately 22% (Figure 1B). In contrast, the percentage of macrophages was significantly increased by approximately 36% after theophylline treatment, whereas the actual macrophage number (millions/ml) was not significantly changed. The percentage of lymphocytes (< 3%) and eosinophils (< 3%) did not differ significantly between the treatment periods (data not shown). Cell viability was 85% (range 81–91%) across samples, with no difference between treatment periods.


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  		TABLE 1. Effect of theophylline on inflammatory indices in induced sputum

 


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  		Figure 1. Effect of theophylline on induced sputum inflammatory cells in COPD. Data for 25 patients are presented for pre- and post-placebo and pre- and post-theophylline. (A) Total inflammatory cells millions/ml (neutrophils, macrophages, lymphocytes, and eosinophils). (B) Number of neutrophils/ml (millions/ml).

 
Supernatant Measurements
Data are summarized in Figure 2A–2C . IL-8, myeloperoxidase, and lactoferrin (LTF) were present in all sputum samples. Theophylline significantly reduced IL-8 by 24 ± 7% (Figure 2A), myeloperoxidase by 31 ± 10% (Figure 2B), and LTF by 10 ± 4% (Figure 2C).



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  		Figure 2. The effect of theophylline on induced sputum inflammatory markers in COPD. Data are presented for pre- and post-placebo and pre- and post-theophylline, with median and interquartile range. (A) Interleukin-8 (IL-8, ng/ml). Data are presented for 25 patients. (B) Myeloperoxidase (ng/ml). Data are presented for 15 patients. (C) Lactoferrin (ng/ml). Data are presented for 15 patients.

 
Neutrophil Chemotaxis
Neutrophil chemotaxis to fMLP was maximal at 10-7 M, and was significantly reduced by theophylline (mean percent reduction 28 ± 8%, Figure 3A) . Neutrophil chemotaxis was also significantly decreased during treatment with theophylline at submaximal concentrations of fMLP of 10-10 and 10-8 M.



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  		Figure 3. Effect of theophylline on neutrophil chemotaxis to chemoattractants and induced sputum. Neutrophils were purified from the venous blood of 10 study subjects. Neutrophil numbers are expressed as a percentage of the total number used in each chemotaxis experiment. Data are presented for pre- (open inverted triangles) or post-placebo (open circles) and pre- (filled inverted triangles) or post-theophylline (filled circles). (A) Percentage of neutrophils migrating to a concentration gradient of fMLP. (B) Effect of IL-8. (C) Effect of LTB4. (D) Effect of dilutions of induced sputum. Data are mean % migrating cells (error bars are standard error of the mean of percentage migrated neutrophils) at each concentration of chemoattractant or dilution of sputum. *p < 0.05, **p < 0.01 untreated compared with theophylline treated at matched concentrations or dilutions of chemoattractant.

 
Neutrophil chemotaxis to IL-8 was maximal at 12.5 ng/ml, and was significantly decreased by theophylline treatment (mean percent reduction 59 ± 4%; Figure 3B). After theophylline treatment, chemotaxis to a concentration curve of IL-8 was maximal at 50 ng/ml IL-8, but the number of migrating cells was significantly lower than the pretheophylline maximal.

Neutrophil chemotaxis to LTB4 was maximal at 10-8 M, and was not reduced by theophylline (Figure 3C). However, small statistically significant reductions in chemotaxis to LTB4 were observed at 10-11, 10-10, 10-9, and 10-7 M.

Chemotaxis of healthy donor neutrophils to induced sputum was maximal at 20-fold sputum dilutions, and was decreased after theophylline treatment (mean percent reduction 34 ± 4%; Figure 3D). Chemotaxis to a 40-fold sputum dilution was also reduced by theophylline (mean percent reduction 32 ± 7%), but no effect was seen at other dilutions. Healthy donor neutrophils migrated less than COPD neutrophils in response to 12.5 ng/ml IL-8 (used as a positive control) (29.2% migrating cells, range 26.9–34.4 versus 45.4, range 36.6–57.3, healthy donor versus COPD, respectively, p < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show that treatment of COPD patients with theophylline reduces sputum neutrophils, sputum chemotactic activity, and the chemotaxis of peripheral blood neutrophils to chemotactic stimuli.

Theophylline treatment resulted in a statistically significant increase of approximately 2% in FEV1 (% predicted) that is not explained by a training effect in the performance of spirometry because subjects attended outpatient clinics prior to enrolment. This small increase is of doubtful clinical significance. Larger clinical studies have demonstrated that theophylline increases FEV1 (22, 35, 36), with the greater increases being in longer studies and those where plasma theophylline levels exceeded 12 mg/L. However, there are limitations to the use of FEV1 as a method of assessing clinical improvement with drug therapy. Lung function measurements should include other factors such as mid-expiratory flow rates, forced vital capacity, slow vital capacity, appropriate ratios, and, possibly, inspiratory capacity. It is recognized that flow limitation and changes in inspiratory capacity, or slow vital capacity, can have a major effect on symptomatology without there being a significant change in FEV1 (37). However, FEV1 can be predictive of clinical outcomes during exacerbations of COPD (38).

Induced sputum is a technique that has been used to investigate cellular and cytokine changes in response to oral and inhaled steroids (34, 39). The present study utilized similar methodology, and showed a reduction in the numbers of inflammatory cells in sputum, accounted for by a reduction in neutrophil number, after theophylline treatment. Although this reduction is numerically small, the concurrent reduction in neutrophil granule constituents, myeloperoxidase, and lactoferrin, supports the significance of this observation. Neutrophils are elevated in the sputum of COPD patients (10). They are implicated as active participants in the disease process by an association with an accelerated decline in FEV1 (11), and may contribute by the production of proteases (4, 40), oxidants (41, 42), and inflammatory mediators (43, 44). Therefore, interventions that reduce neutrophil recruitment might be anticipated to modify the progression of COPD. Anticholinergics are effective bronchodilators (45, 46), and there is controversy concerning the effectiveness of corticosteroids in slowing disease progression (47–51).

Neutrophil recruitment to the airways is mediated by IL-8 and LTB4 (14, 15), and the reduction of IL-8 shown in the current study suggests that this may be one mechanism for the observed reduction in neutrophil numbers. This suggestion is supported by the observation that in vitro theophylline inhibits IL-8 release from a human respiratory epithelial cell line (52). However, the source of the IL-8 in the present study is unknown, and may be neutrophils. If neutrophils were the source, the reduction in neutrophil numbers might be expected to lead to a reduction in local IL-8 production in the lung. Neutrophils from COPD patients show enhanced chemotaxis (53), which suggests that a reduction in chemotactic stimulus would not be sufficient to reduce neutrophil accretion. The present study shows that theophylline significantly reduced neutrophil chemotaxis to fMLP, IL-8 and, to a lesser extent, LTB4. The reduction by theophylline in chemotaxis to IL-8 of 59% found herein is greater than that of 27–43% in response to sputum of varying purulence in the presence of an anti–IL-8 antibody (15). The reason for this discrepancy is unclear, but may be related to the complexity of sputum. For example, it is possible that there is activity of sputum IL-8 that may not be blocked by a monoclonal anti–IL-8 antibody. It is also noteworthy that theophylline shifts the chemotactic response to IL-8, whereas it decreases the responses to fMLP and LTB4 (compare Figure 3B with Figure 3A and C). The shift indicates that the sensitivity of the neutrophils to IL-8 has been selectively reduced by theophylline. The mechanism underlying the reduced sensitivity is unclear from the present study, but may be related to a change in kinetics of the IL-8 (chemokine C-X-C) receptor altering the polarization of the neutrophils (54).

The present data suggest that LTB4 is a less potent chemoattractant than fMLP or IL-8. However, neutrophils from patients with COPD may have reduced LTB4 receptors, reduced LTB4 response, or receptors that are blocked by prior LTB4 release in vivo. In addition, the reduced response to LTB4 may be due to the method used to assess chemotaxis, as previous studies have demonstrated that methodology can affect results (55, 56). Several in vitro studies of the effect of theophylline demonstrate a similar reduction in neutrophil chemotaxis (57, 58), although this is dependent on the dose used (59). A clinical study of theophylline treatment in children with asthma (32) demonstrated a reduction in neutrophil chemotaxis after theophylline treatment, which concurs with the present study, but no previous clinical studies have been reported in COPD patients. The reduction in sputum neutrophils observed in the present study could, therefore, be accounted for by reduced chemoattractant in the airways and reduced neutrophil chemotactic response. In addition, although our patients had not had a recent exacerbation, we did not assess pulmonary bacterial colonization. Consequently, in the present study we cannot exclude a direct bactericidal effect of theophylline, with subsequent reduction in inflammatory indices.

Combinations of chemoattractants are more potent than individual agents (60). Induced sputum contains a variety of chemoattractants and, in the present study, induced a greater neutrophil response than fMLP, IL-8, and LTB4 alone. Diluted sputum samples showed greater chemotactic activity, suggesting that the more concentrated samples contained chemokine levels that saturated neutrophil receptors, and therefore, did not induce directional movement. Diluted samples may more accurately reflect the concentration gradient of chemokines from interstitium to airway that result in the movement of neutrophils into the airway. That theophylline treatment reduced the chemotactic activity of sputum suggests that other, unmeasured, chemokines were also reduced by theophylline. However, if theophylline was present in the sputum samples, it might be expected to have had an influence on the normal neutrophils used for this part of the assay, independent of any changes in chemokine levels.

In summary, we have demonstrated that theophylline reduces sputum neutrophils, IL-8 concentrations, and chemotactic activity, and reduces peripheral blood neutrophil chemotaxis to fMLP, IL-8, and LTB4. In combination, the data suggest that theophylline could reduce neutrophil recruitment and, therefore, neutrophil-mediated damage in the airways. These effects may result in theophylline having a beneficial effect on the rate of decline of lung function in COPD.


   Acknowledgments
 
The authors thank Andreas Keller, Byk Gulden, Konstanz, Germany, for his support of this study.

Received in original form May 22, 2001; accepted in final form February 1, 2002


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 RESULTS
 DISCUSSION
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