Abstract
Inhaled corticosteroids are widely prescribed for the treatment of stable chronic obstructive pulmonary disease (COPD), despite lack of proven efficacy. Because COPD involves airway inflammation and probable protease-antiprotease imbalance, we examined the effect of high dose fluticasone propionate on markers of activity of both pathogenetic mechanisms. Thirteen patients with COPD were treated with fluticasone propionate (500 μ g twice a day) for 4 wk, delivered via MDI and spacer, in a double-blind crossover study. There was no clinical benefit in terms of lung function or symptom scores, and induced sputum inflammatory cells, percentage neutrophils, and IL-8 levels were unchanged. Sputum supernatant elastase activity, matrix metalloproteinase (MMP)-1, MMP-9, and the antiproteases secretory leukoprotease inhibitor (SLPI) and tissue inhibitor of metalloproteinase (TIMP)-1 were similarly unaffected by treatment. These results add to previous evidence that inhaled steroids have no anti-inflammatory action in stable COPD. Furthermore, inhaled steroids do not appear to redress the protease-antiprotease imbalance that is thought to be important in the pathogenesis of airway obstruction. Culpitt SV, Maziak W, Loukidis S, Nightingale JA, Matthews JL, Barnes PJ. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease.
Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, and to date cessation of smoking is the only intervention that has been shown to slow disease progression (1). The pathogenesis of airway obstruction in COPD is multifactorial, involving neutrophilic airway inflammation (2), protease-antiprotease imbalance (3, 4), oxidative stress (5), and recurrent infection. These mechanisms are interrelated such that reducing one factor may also reduce the stimulus to others.
Airway inflammation in COPD can be demonstrated by examination of induced sputum, and it clearly differs from that seen in asthma (6). Smokers and ex-smokers with COPD have increased sputum neutrophil numbers compared with subjects without COPD (6, 7), and increased neutrophils are associated with rapid decline in FEV1 (2). Furthermore, neutrophil activation markers are elevated in the sputum supernatants of subjects with COPD (8), suggesting that neutrophils are active participants in airway inflammation. Such evidence would predict that sputum is an effective tool for assessing airway inflammation and that a reduction in neutrophilic inflammation would be a marker of therapeutic success.
The protease-antiprotease imbalance hypothesis of emphysema has seen many advances since the initial reports of Laurell and Eriksson (9) and Gross and colleagues (10). Much attention has focused on the proteolytic potential of neutrophils (11), which includes neutrophil elastase and matrix metalloproteinases (MMPs) with collagenase and gelatinase activity. MMPs contribute as much as 50% of the elastolytic activity of bronchoalveolar lavage fluid in smokers (12). More recently, the macrophage has been identified as a source of a family of MMPs with the ability to degrade all extracellular matrix components (13, 14). The elastinolytic activity of macrophages from subjects with emphysema is greater than that from control subjects (15), and macrophage metalloelastase is essential in the development of cigarette-smoke-induced emphysema in mice (16), observations that have contributed to growing evidence of the importance of macrophages and MMPs in the pathogenesis of emphysema (17). MMP-1 and MMP-9 are elevated in bronchoalveolar lavage fluid of subjects with emphysema, suggesting that these two MMPs may be important in pathogenesis (18). A similar body of literature has arisen around the inhibitors of proteolysis. The major antineutrophil elastase shield in the lung is provided by α1-proteinase inhibitor (α1-PI) in peripheral lung and secretory leukoprotease inhibitor (SLPI) in airways (19). The MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs) of which there are at least three types (13). Thus, the balance of protease- antiprotease activity remains an active area of investigation.
There is evidence that steroids increase lung defenses against neutrophil elastase (20, 21). Steroids may also modify MMP and TIMP activity (22, 23). Systemic corticosteroids reduce neutrophil recruitment and activation (24), and in vitro studies have demonstrated a reduction in neutrophil chemotaxis by fluticasone (25). Treatment with fluticasone also reduces sputum chemotactic activity and increases neutrophil elastase inhibitory capacity (26). Therefore, steroids could be expected to have an anti-inflammatory action in COPD. The effect of steroids on inflammation in COPD has been studied using the method of induced sputum (27), and no benefit was demonstrated in this 2-wk trial. We aimed to extend the latter study, using a more potent steroid and for a longer duration of 4 wk. In addition we looked at the effect of fluticasone on MMP-1, MMP-9, TIMP-1, elastase activity, and SLPI levels in the induced sputum of subjects with COPD.
Twenty-five patients (13 male, eight female) 43 to 73 yr of age, with stable COPD were recruited from the outpatient department of the Royal Brompton Hospital. All subjects had a smoking history of at least 20 pack-years, 11 were current smokers, and the remainder had ceased smoking for a minimum of 8 mo. Inclusion criteria for entry were FEV1/FVC ratio < 0.7, postbronchodilator FEV1 < 85% predicted value, reversibility with inhaled β2-agonist of < 15% of predicted FEV1: all three criteria were required. Subjects who had taken inhaled or oral steroids or who had suffered an exacerbation of their airway disease in the previous 6 wk were excluded. Three subjects had concomitant treatment with albuterol (200 μg twice a day) and ipratropium bromide (40 μg twice a day), one subject with albuterol (200 μg as needed) alone. No other medications were taken in the 6 wk before or during the study. Subjects had no history of asthma or variability of symptoms and were nonatopic. All subjects gave written informed consent, and the study was approved by the ethics committee of the Royal Brompton Hospital.
The study was randomized, double-blind, placebo-controlled crossover design with a run-in period of 2 wk. Subjects received treatment with either fluticasone propionate (FP) 500 μg (Glaxo-Wellcome, Stamford, UK) twice a day or placebo, delivered by metered-dose inhaler via a spacer device, for 4-wk. Treatment periods were separated by 2 wk of washout during which no study medications were taken. Each subject kept a diary card to record diurnal peak expiratory flow rate (PEF), use of reliever inhaler (albuterol), use of study medication, and symptoms. Dyspnea was assessed daily as none, mild (one or two episodes of breathlessness), moderate (three or more episodes but not breathless most of the time), or severe (breathless most of the time). Cough was assessed daily as none, mild (one or two episodes), moderate (three or more episodes) or severe (troubled by cough most of the time). Daily sputum production was ranked as none, only on rising, less than one eggcup full, or more than one eggcup full, and the color was recorded.
Patients attended the laboratory at the end of the run-in and treatment periods. At each visit spirometry (Vitalograph, Buckingham, UK) and sputum induction were performed. Diary cards were collected, and investigators checked treatment compliance as recorded by the patients. The run-in period of 2 wk enabled assessment of PEF variability, ability to comply with filling a diary card, and assessment of the stability of spirometry on two occasions.
Subjects inhaled 3.5% saline for 15 min via an ultrasonic nebulizer (DeVilbiss 2000; DeVilbiss Co., Heston, UK) with a calibrated mass median diameter of 4.5 μm and the output set at 4.5 ml/min. Prior to inhalation and before each expectoration, subjects discarded saliva into a separate bowl and mouthwashed thoroughly. Any secretions collected during the first 5 min were discarded in order to minimize squamous epithelial cell contamination (6). Secretions expectorated over the subsequent 10 min hereafter referred to as “sputum,” were analyzed. Fresh samples were kept at 4° C for no more than 2 h before further processing.
The whole sputum sample was diluted with Hank's balanced salt solution (HBSS) containing dithiothreitol (DTT) (Sigma Chemicals, Poole, UK), with a final DTT concentration of 0.2%, and was gently vortexed at room temperature. When homogeneous, the volume was recorded and the sample was further diluted with HBSS and centrifuged at 300 × g for 10 min. The supernatant was separated and stored at −70° C and the cell pellet resuspended in 1 ml HBSS. Cell viability was determined by exclusion of Trypan Blue. Total cell counts were determined on a hemocytometer slide using Kimura stain, and slides were prepared using a cytospin (Shandon, Runcorn, UK) and stained with May-Grunwald-Giemsa. Differential cell counts were performed by a blinded observer with 300 nonsquamous cells counted on each of two slides for each sample. Differential cell counts were expressed as a percentage of lower airways cells (i.e., excluding squamous epithelial cells). Squamous cell counts were expressed as a percentage of total cells. Samples were considered adequate for analysis if there was < 50% squamous cell contamination.
IL-8 assay. IL-8 was measured with an amplified sandwich-type enzyme-linked immunosorbent assay (ELISA) using a paired antibody system (Genzyme Duoset; Genzyme Diagnostics, Cambridge, MA). Plates were washed between incubations with phosphate-buffered saline (PBS) containing 0.05% vol/vol Tween. Ninety-six-well microtiter plates (Greiner Labortecnik Ltd., Dursley, UK) were coated with 100 μl of mouse antihuman IL-8 at a concentration of 2.5 μg/ml and left overnight at 4° C. Blocking buffer of 0.1 M PBS containing 1% bovine serum albumin (BSA) was applied for 2 h at 37° C. Plates were decanted, and 100 μl of samples and standards were added and incubated at 37° C for 1 h. Then 100 μl of secondary antibody, rabbit anti-human IL-8 biotinylated were added at a concentration of 0.5 μg/ml and plates were incubated at 37° C for 1 h. The plates were incubated with Streptavidin-horseradish peroxidase for 15 min and 3,3′,5,5′-tetramethylbenzidine (TMB) (Pharminagen, San Diego, CA) for 20 min. The reaction was stopped with 2N sulfuric acid, and the optical density of the wells was read using a plate photometer at 450 nm within 30 min. The lower limit of sensitivity of the assay was 15.6 pg/ml. The assay was linearly affected by DTT, and all standards were prepared using an equivalent concentration of DTT to that in the samples. The upper limit of detection of the assay was 1 ng/ml, and samples containing higher levels were diluted prior to assay.
SLPI assay. SLPI was measured by ELISA using a commercially available kit (R&D Systems, Minneapolis, MN). The assay measured in the range 62.5 to 4,000 pg/ml. Similar to that of IL-8, the effect of DTT was a linear reduction in the gradient of standard curves, which was compensated for by adding an equivalent concentration of DTT to standards.
Elastase assay. Elastolytic activity was measured by a method that uses N-methoxysuccinyl-ala-ala-pro-val p-nitroanilide (Sigma Chemicals) as a substrate. Samples, substrate, and TRIS buffer (pH, 8.6) were shaken at 1,000 rpm for 2 min and incubated for a further 60 min at 37° C. The plates were read using a photometer at 405 nm. Samples were compared with standards of human sputum elastase (HSE). The lower limit of detection of the assay was 0.05 μg/ml. Elastolytic activity was not altered by the presence of DTT in standards or samples.
MMP-1, MMP-9, and TIMP-1 assays. ELISA assays were performed using commercially available kits (Amersham Life Science; Amersham, Buckinghamshire, UK) and according to manufacturers instructions. The MMP-1 assay recognizes total MMP-1, i.e., free MMP-1, pro-MMP-1, and that complexed with TIMP-1. There is no cross-reactivity with MMP-2, 3, or 9, and the measurement range is 6.25 to 100 ng/ml. The MMP-9 assay recognizes pro-MMP-9 and pro-MMP-9 complexed with TIMP-1. MMP-9 can be measured in the range 1 to 32 ng/ml. The TIMP-1 assay recognizes free TIMP-1 and that complexed with MMPs. It does not cross-react with TIMP-2, and the lower limit of detection is 3.13 ng/ml. DTT was added to standard curves at an equivalent concentration to that in the sputum samples as the assays were affected similarly to the IL-8 assay.
Parametric data are expressed as the mean ± SEM; nonparametric data are expressed as medians and ranges. Data are presented as baseline values followed by post-FP values. Parametric data were compared using Student's t test. For nonparametric data comparisons were made using Wilcoxon's signed rank sum test between two groups—baseline (start of FP treatment period) and end of FP treatment period. Two-tailed tests were performed, and a p value of < 0.05 was considered significant.
A total of 12 subjects were withdrawn from the study. Five subjects failed to meet the inclusion/exclusion criteria after the run-in period: two of five had postbronchodilator FEV1 of 85% predicted on retesting, and the remainder were unable to complete diary cards or were unwilling to continue with the study. Of the seven withdrawn during the treatment phase, two suffered exacerbation of their airway disease (increased dyspnea, purulent sputum, and positive sputum culture requiring antibiotic therapy), one failed to take the study medication, and four completed treatment but failed to provide adequate sputum samples so were not evaluable. All subjects tolerated the medication and sputum induction procedure. The results presented are an analysis of the 13 subjects (8/13 male, 4/13 ex-smokers) who completed the study and had provided adequate samples from each study period. The subject characteristics remain the same as those outlined in Methods. For all results there was no effect of placebo, baseline results for the placebo and treatment periods were equivalent, and there were no carryover effects of treatment. All 13 subjects were compliant with study medication, as assessed by diary card records: none reported missing more than two doses during the study.
Subjects were 62 ± 2 yr of age and had moderate to severe airflow limitation, with a mean FEV1 49.5 ± 4.6% predicted. There was no significant difference in daily PEF between the placebo and FP treatment periods (278 ± 22 L/min versus 295 ± 22 L/min, p > 0.05). Similarly, there were no differences in median dyspnea score, cough, sputum production, sputum color, or percentage of relief medication-free days (Table 1).
| Clinical Parameter | Baseline | FP | ||
|---|---|---|---|---|
| FEV1, % pred | 49.5 ± 4.6 | 50.4 ± 4.1 | ||
| PEF, L/min | 278 ± 22 | 295 ± 22 | ||
| Dyspnea score | ||||
| None | 3 (23%) | 2 (16%) | ||
| Mild | 4 (31%) | 4 (31%) | ||
| Moderate | 5 (38%) | 6 (46%) | ||
| Severe | 1 (8%) | 1 (8%) | ||
| Cough | ||||
| None | 4 (31%) | 4 (31%) | ||
| Mild | 7 (54%) | 6 (46%) | ||
| Moderate | 2 (16%) | 3 (23%) | ||
| Severe | 0 (0%) | 0 (0%) | ||
| Sputum production | ||||
| None | 3 (23%) | 4 (31%) | ||
| On rising | 6 (46%) | 4 (31%) | ||
| All day < 1 eggcup full | 3 (23%) | 4 (31%) | ||
| All day > 1 eggcup full | 1 (8%) | 1 (8%) | ||
| Sputum color | ||||
| None | 5 (38%) | 4 (31%) | ||
| Grey/white | 8 (62%) | 9 (69%) | ||
| Yellow/green | 0 (0%) | 0 (0%) |
Total inflammatory cell counts (millions/ml) were unchanged after FP (1.9, range, 0.6 to 4.3 versus 1.4; range, 0.3 to 3.3). The percentage of inflammatory cells identified as neutrophils was also unchanged (85%; range, 39 to 95% versus 76%; range, 28 to 95%) (Figure 1A) as was the absolute neutrophil number (millions/ml) (1.6; range, 0.3 to 6.9 versus 1.3; range, 0.3 to 2.9) (Table 2). The percentage of macrophages (Table 2), lymphocytes (< 3%), and eosinophils (< 3%) did not differ between the treatment periods (data not shown). Cell viability was 85% (range, 82 to 91%) in all samples, with no difference between treatment periods.
Fig. 1. Effect of fluticasone propionate (FP) on inflammatory indices in induced sputum of patients with COPD. (Panel A) Percentage of neutrophils in induced sputum at baseline and after 4 wk of treatment. (Panel B) Interleukin-8 levels in induced sputum at baseline and after 4 wk of treatment.
[More] [Minimize]| Sputum Parameter | Baseline | FP | ||
|---|---|---|---|---|
| Total cell counts | 1.9 (0.6–4.3) | 1.4 (0.3–3.3) | ||
| Cell viability, % | 85 (82–89) | 86 (82–91) | ||
| Neutrophils, % | 85% (39–95%) | 76% (28–95%) | ||
| Absolute neutrophils | 1.6 (0.3–6.9) | 1.3 (0.3–2.9) | ||
| Macrophages, % | 15% (4–60%) | 22% (4–69%) | ||
| Absolute macrophages | 0.27 (0.11–1.71) | 0.23 (0.02–2.49) | ||
| IL-8, ng/ml | 10.9 ± 3.4 | 6.9 ± 1.8 | ||
| SLPI, μg/ml | 5.7 ± 0.8 | 5.3 ± 1.0 | ||
| Total elastase activity, μg/ml | 4.2 ± 0.3 | 5.2 ± 0.5 | ||
| MMP-1, ng/ml | 19.1 ± 2.0 | 20.5 ± 2.8 | ||
| MMP-9, ng/ml | 13.7 ± 3.5 | 13.2 ± 3.8 | ||
| TIMP-1, ng/ml | 14.7 ± 5.1 | 15.1 ± 4.0 |
All samples tested contained measurable amounts of all parameters. There were no changes in sputum IL-8 (Figure 1B), MMP-1, MMP-9 (Figure 2), or TIMP-1 (Figure 3) after FP treatment. Similar results were obtained for SLPI (Figure 3A) and elastase activity (Figure 2A). Results are summarized in Table 2. There was no relationship between the non-significant changes in cell counts, cytokines, or proteases.
Fig. 2. Effect of fluticasone propionate (FP) on proteases in induced sputum of patients with COPD. (Panel A) Total elastase activity of induced sputum at baseline and after 4 wk of treatment. (Panel B) Matrix metalloproteinase-1 (MMP-1) levels in induced sputum at baseline and after 4 wk of treatment. (Panel C ) Matrix metalloproteinase-9 (MMP-9) levels in induced sputum at baseline and after 4 wk of treatment.
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Fig. 3. Effect of fluticasone propionate on antiproteases in induced sputum of patients with COPD. (Panel A) Secretory leukoprotease inhibitor (SLPI) levels in induced sputum at baseline and after 4 wk of treatment. (Panel B) Tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in induced sputum at baseline and after 4 wk of treatment.
[More] [Minimize]The results of this study show that FP, at a dose of 500 μg twice a day, has no effect on lung function, inflammatory indices, proteases, or antiproteases that were measured in the induced sputum of patients with COPD.
Studies on induced sputum in asthmatic subjects suggest that the technique is sensitive enough to demonstrate changes that reflect reduced inflammation (27). Induced sputum analysis can also distinguish between asthmatic and healthy subjects and subjects with COPD (6) and has been validated in these groups (28). Therefore, the lack of a demonstrable treatment effect is unlikely to be solely attributable to the methods used. FP is a potent corticosteroid and the dose of 500 μg once a day should be therapeutic, especially when delivered via a spacer device. Therefore, treatment failure is unlikely to be due to the dose used or the method of administration. A 4-wk treatment period is short in clinical terms, and this could account for the lack of clinical improvement. Many studies have used longer treatment courses and also failed to show clinical benefit (29). However, a number of published studies have shown improvement in airway inflammation (30, 31) and lung function (32). Four weeks of treatment may not be sufficient to observe reduction in cell counts, but the time course of steroid action on gene expression suggests that an effect on cytokines and MMPs would be observed in this time period. The current study included smokers and ex-smokers, but numbers were too small to identify a difference in response between them. There was no change in the smoking habit of the subjects during the study and no differences in the sputum characteristics of smokers and ex-smokers at baseline or after FP treatment. However, we cannot exclude a differential response to steroids between patients with COPD who smoke and those who abstain. This was a small study and no formal power calculation was carried out. Underpowering of the study may be responsible for the negative results.
The lack of anti-inflammatory effect may account for the failure of FP to modify elastase activity. IL-8 is a potent chemoattractant for neutrophils (33), which in turn provide elastase activity, and we have demonstrated that IL-8 levels are not reduced after treatment. MMPs are also upregulated by mediators of inflammation (34). It is possible that whereas inflammation is present it outweighs the downregulation of MMP mRNA and protein synthesis achieved by steroids (35). Oxidative stress, thought to be important in COPD (5), may also contribute to an ongoing stimulus for MMP activation. Neutrophil collagenases are activated by chlorinated oxidants, thiol-driven activation of MMPs has been demonstrated, and oxidants can inactivate the antiprotease shield (11), all of which contribute to ongoing protease-antiprotease imbalance.
In summary we have demonstrated the presence of measurable quantities of MMP-1, MMP-9, and TIMP-1 in the induced sputum of subjects with COPD. FP has no effect on the levels of these proteases, nor does it alter elastase activity or SLPI. In addition we have confirmed the results of previous studies, which demonstrated no effect of inhaled steroids on induced sputum inflammatory indices.
Supported by Glaxo-Wellcome UK.
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