INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bronchiectasis is a debilitating disease of heterogeneous etiology and affected patients suffer from distressing regular sputum production and recurrent infective exacerbations. Bronchiectasis is often progressive; gradual destruction of the airways arises from a combination of chronic airway inflammation and infection (1). This destructive process may continue even after the initial cause of bronchiectasis such as measles has subsided. Many patients eventually harbor Pseudomonas aeruginosa in their lower respiratory tract which accounts for significant morbidity. The role of some proinflammatory mediators, such as interleukin-1 (IL-1), IL-8, tumor necrosis factor-alpha (TNF-), and leukotriene B4 (LTB4), in recruiting neutrophils into the tracheobronchial tree has been recently established (1, 2). As there is no gold standard in measuring inflammatory activities in bronchiectasis, concentrations of these proinflammatory mediators might act as surrogate end points in disease monitoring.

The safety and efficacy of inhaled steroid therapy in the treatment of airway inflammation in asthma is well established. Although inflammation plays a significant role in the pathogenesis of progressive bronchiectasis, the anti-inflammatory effects of inhaled steroid therapy on bronchiectasis have not been evaluated systematically. Fluticasone propionate is a new synthetic fluorinated glucocorticoid which has negligible oral bioavailability and a good efficacy to risk ratio (8). We have therefore performed a randomized, double-blind, placebo-controlled study to evaluate the effects of administration of high-dose (1 mg daily) inhaled fluticasone on the inflammatory and infective indices in steady-state bronchiectasis.

	METHODS

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Study Design

Each patient entered a baseline period (three consecutive weekly visits), to ensure that they were in steady-state bronchiectasis, before being randomly assigned into receiving either fluticasone (500 µg twice daily) or otherwise identical placebo administered with an Accuhaler, a dry powder inhaler device. Measurement of clinical and laboratory parameters was performed by a research physician (C.S.H.) and a technician who were unaware of the treatment protocol. Patients were followed 1 and 4 wk after commencement of treatment.

Patient Selection

Patients with proven bronchiectasis, diagnosed by high-resolution computed tomography (HRCT), were recruited with written informed consent. Inclusion criteria included: daily sputum > 10 ml; absence of asthma or other unstable systemic diseases; and "steady-state" bronchiectasis (< 10% alteration of 24 h sputum volume, FEV₁, and FVC, and in the absence of deterioration in respiratory symptoms at baseline visits). Exclusion criteria included: unreliable clinic attendance; known adverse reactions to fluticasone; regular user of inhaled corticosteroids; and known asthma defined according to American Thoracic Society guidelines (9). The study protocol was approved by the institutional ethics committee.

Parameters Measured

At each visit, the patients were directly asked about the presence of respiratory symptoms, including cough, dyspnea, hemoptysis, sputum production, chest pain, and wheezing, and examined physically. The number of lung lobes (including lingula as an individual lobe) affected by bronchiectasis was determined by a thoracic radiologist (C.C.G.O.) who examined the HRCT of each patient using standard criteria (10). The number of exacerbations occurring in the preceding 12 mo and during the study was also determined for each patient by meticulous history taking and review of clinical charts. An exacerbation was defined as subjective and persistent ( 24 h) deterioration in at least three respiratory symptoms including cough, dyspnea, hemoptysis, increased sputum purulence or volume, and chest pain; with or without fever ( 37.5° C), radiographic deterioration, systemic disturbances, or deterioration in physical signs in the chest including crackles and dullness on auscultation and percussion, respectively.

Laboratory assessment included: 24 h sputum volume; sputum leukocyte density (/ml); sputum total bacterial, commensal bacterial, and P. aeruginosa densities (colony-forming units [cfu]/ml); and sputum (sol phase) levels of IL-1, IL-8, TNF-, and LTB4. Lung function indices (Table 1) were measured between 10:00 A.M. to 11:00 A.M., using standard protocols, with a SensorMedics 2200 (SensorMedics, Yorba Linda, CA) package. Peak expiratory flow rate (PEFR) readings, performed on rising in the morning and before retiring to bed, were performed with Wright's peak flow meters (Wright, Harlow, UK), by the patients at home for 3 d before each visit, to ensure that there was no asthmatic trends in the variation of PEFR (9). Compliance was enforced by thrice weekly personal phone calls and diary cards, and monitored by checking the number of remaining doses displayed on the Accuhaler device.

Assessment of Sputum Physical Characteristics

The volume of 24 h sputum was determined as the mean of a three consecutive day collection (9:00 A.M. to 9:00 A.M.). Twenty-four hour sputum collection was made by the patients at home in clear sterile plastic (60 ml) pots which were prelabeled and stored at 4° C. Patients were instructed and trained to completely empty the contents of their mouth before they expectorated into the sputum pots to ensure that there was minimal contamination by saliva and food debris. The latter were infrequently encountered after the three baseline visits, and were pipetted from the pots by the research physician prior to volume assessment. The volume of a 24 h sputum specimen was determined as the volume of water (to the nearest 0.1 ml) in an adjacent and identical pot containing water at the same level as the sputum in the sputum-containing pot. Patients received chest physiotherapy (at least 15 min of expectoration-aiding maneuvers and until no further sputum was obtained) on arrival at the clinic. Fresh sputum was then collected by the research physician in sterile clear plastic pots between 10:00 A.M. to 11:00 A.M. after thorough mouth emptying, and within 1 h of physiotherapy in the sitting position. Sputum leukocyte density was assessed within 2 h of collection by the same technician. This was assessed on five aliquots chosen randomly from the center of a fresh specimen, which were then serially diluted with phosphate-buffered saline (PBS) and assessed with light microscopy and a hemocytometer.

Determination of Sputum Bacterial Densities

Standard microbiological procedures were employed to identify all the sputum bacteria and classify them into pathogens (P. aeruginosa, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus) or commensal bacteria (Neisseria species, -hemolytic streptococci, diphtheroids, and coagulase-negative staphylococci). The following enriched and selective media were used for determining the microbial density (cfu/ml) of pathogens and commensal bacteria: blood agar (Oxoid CM271; Oxoid, Basingstoke, UK), with 5% defibrinated horse blood, chocolate agar supplemented with 18.9 U/ml bactracin (Sigma, St. Louis, MO), mannitol salt agar (Oxoid CM85) and cetrimide-nalidixic acid agar (Oxoid CM559 and SR102). Fresh sputum was homogenized by using SPUTASOL (Oxoid SR089A), which contained 0.1% dithiothreitol, 0.78% sodium chloride, 0.02% potassium chloride, 0.112% disodium hydrogen phosphate, and 0.02% potassium dihydrogen phosphate, according to the manufacturer's recommendation. The microbial densities of various bacteria were determined by inoculating the media with 10 µl of PBS-diluted sputum (10⁴, 10⁵, and 10⁶) using a standard plastic loop. All plates were inoculated at 37° C in 5% CO₂ and the resulting dilution in 30 to 300 cfu after overnight incubation was measured. Selective plates with negative results were reincubated and reexamined daily for 4 d before disposal.

Measurement of Sputum Proinflammatory Cytokine and LTB4 Concentrations

Fresh sputum was stored at 70° C within 15 min of collection until ultracentrifugation (100,000 g for 30 min at 4° C) to obtain the sol phase used for enzyme-linked immunoabsorbent assay of cytokine and LTB4 levels. Samples were added to a 96-well plate (R&D Systems, Minneapolis, MN) coated with monoclonal antibody against one of the cytokines or LTB4 and incubated for 2 h at room temperature. Following this, the samples were removed and washed three times with buffer and an enzyme-linked antibody specific for a particular cytokine or LTB4 was added to each well and incubated at room temperature for 2 h. After a final wash to remove all unbound antibody, a substrate solution was added to each well and incubated for 20 min before the reaction was terminated by adding a "stop solution" (1 M sulfuric acid). The optical density was determined by using a plate reader at 450 nm to determine the concentration of the cytokines or LTB4 in the sputum, and the mean concentration for each sample was obtained from the triplicate measurements.

Statistical Methods

The objective of this trial was to examine the effects of topical fluticasone on airway inflammatory markers. The outcome variables of interest were the sputum concentrations of cytokines and the daily sputum volume. As there were no previous data on sputum cytokine, the study size was estimated using the daily sputum output which varied by as much as 10% between days in our stable bronchiectatic patients. Accepting a type I error of 0.05 and a type II error of 0.20 (power 0.80), a randomized placebo-controlled study with a sample size of 24 subjects (12 in each treatment group) would allow 13% change in sputum output to be detected using two-tail t statistics.

Preliminary inspection of data revealed that the lung function and sputum cytokine data were log-normally distributed and were therefore logarithmically transformed before analysis. These variables were compared between treatment groups by analysis of variance (ANOVA) with Bonferroni correction and reported as geometric mean and 95% confidence intervals. Within-group changes after treatment were examined with paired Student's t tests. Data that were highly skewed (sputum volume, and microbial and leukocyte densities) were compared between and within treatment groups by Wilcoxon's rank sum test and reported as median and interquartile range. All statistical analyses were carried out using a Statistical Analysis System (11) software package. A p value of < 0.05 was taken as indicative of statistical significance.

	RESULTS

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Patient Demography and Clinical Details

Between September 1996 and July 1997, 12 patients were recruited into each treatment group. The patient characteristics are shown in Table 2. There was a significant difference in age (p = 0.01) but not smoking history, the number of bronchiectatic lung segments, or the number of exacerbations in the previous 12 months between the two treatment groups (p > 0.05). No adverse reactions attributable to the use of placebo or fluticasone therapy had been reported in either group. One patient in the fluticasone group experienced an exacerbation in the second week while three patients in the placebo group in the first, second and second weeks during the treatment phase. These episodes were treated at home with oral sparfloxacin (200 mg daily) for 10 d without any alteration of medications otherwise. None of the patients had any alteration in their regular medications throughout the study. All patients achieved 100% drug compliance.

Lung Function Indices

The pre- and post-treatment lung function indices for both groups are shown in Table 1. There was no significant difference between the baseline and the post-treatment spirometry in either treatment group (p > 0.05). There was only a borderline increase in PEFR after 4 wk of fluticasone treatment (p = 0.05) although there was a trend toward an improvement in all other lung function indices in the fluticasone group (p = 0.05). The baseline and post-treatment spirometry values were not significantly different between the fluticasone and placebo groups (p > 0.05).

Sputum Physical, Microbial and Proinflammatory Assessment

P. aeruginosa was the only pathogen identified in all patients. The findings at 1 wk were similar to baseline values (data not shown). In the fluticasone but not the placebo group, the sputum leukocyte density (p < 0.05) improved significantly after 4 wk but the 24 h sputum volume did not change significantly in either group (p > 0.05) (Table 3). Within- and between-group comparisons did not reveal any significant difference between sputum densities of commensal, P. aeruginosa, or total bacteria at all time points in both groups (p > 0.05).

Within-group comparisons showed a significant (p < 0.05) reduction in the levels of IL-1, IL-8, and LTB4 after 4 wk of treatment in the fluticasone but not the placebo group (Table 4). There was also a decrease in the sputum concentration of TNF- in the fluticasone group although the change failed to reach statistical significance (p > 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study show that treatment with high-dose (1 mg daily) inhaled fluticasone therapy improves sputum inflammatory indices including leukocyte density and levels of IL-1, IL-8, and LTB4 in steady-state bronchiectasis. There were, however, no significant changes in the spirometry or sputum bacterial densities. The lack of improvement in lung function indices might reflect the underlying irreversible airway damage in our patients but might also be due to the short treatment duration. Despite the randomized design of this study, we nevertheless recruited a significant younger placebo group (p = 0.01) by chance although the disease severity markers (Table 2) appeared otherwise comparable between these two groups. The theoretical biasing of the "younger and immunosuppressant-treated" placebo group toward "doing better" was not observed (Tables 1, 3, and 4). The 100% compliance also needs to be viewed with some caution although we closely monitored the patients throughout the study. It is also distinctly possible that some patients might have overreported their compliance.

Intense neutrophil infiltration into the tracheobronchial tree occurs in bronchiectasis (2, 12) which aggravates the underlying tracheobronchial damage. Neutrophil-derived toxic products, such as elastase, cause ultrastructural and functional damage (13) and release of proinflammatory mediators in the tracheobronchial tree (14). There is ample evidence to suggest that this neutrophil influx into the bronchiectatic airways is mediated by proinflammatory cytokines and LTB4 (2, 15, 16). For instance, LTB4 promotes neutrophil migration and degranulation (17); IL-1 mediates airway inflammation and fibrosis (4); TNF- interacts synergistically with IL-1 in prostaglandin induction (18); and IL-8 is one of the most potent chemoattractants which also degranulates neutrophils in bronchiectatic airways (2, 13).

There are only a few longitudinal studies on sputum proinflammatory mediator profiles in bronchiectasis that have failed to show any decline despite clinical improvement with antibiotic and recombinant human deoxyribonuclease (rhDNAse) treatment (2, 5, 19). Our study is the first systematic evaluation of sputum proinflammatory mediator profiles in bronchiectasis after steroid therapy. Fluticasone therapy, by reducing the concentrations of IL-1, IL-8, and LTB4, might lead to a dramatic reduction in the inflammatory activities in the bronchiectatic airways in vivo. Nevertheless, the post-treatment sputum proinflammatory mediators were still high, indicating a persistent intense tracheobronchial inflammation in our patients despite fluticasone treatment.

Administration of low-dose oral prednisolone (0.48 mg/kg/ alternate day) was not associated with any improvement in spirometry but was associated with occurrence of glucosuria and pneumothoraces in cystic fibrosis (CF) patients (20). Higher dosage of prednisolone (1-2 mg/kg/alternate day) improves growth (21), spirometry (21), and serum levels of IgG, IL-2, and IL-1 (22), although similar adverse reactions have been reported in one study (23). Inhaled steroids have also been tried in non-CF bronchiectasis (24). Low-dose inhaled steroid therapy (beclomethasone 0.4 mg/d) had no effects on sputum proteolytic or immune complex activities (24), but higher dosage (beclomethasone 1.5 mg/d or budesonide 1.6 mg/d) improved sputum volume (25), bronchial hyperrreactivity (26), dyspnea (26), cough (26), and spirometry (27).

While the precise mechanism for the reduction in sputum proinflammatory mediators and leukocyte density is unclear, this is unlikely to be due to treatment of underlying occult asthma in our patients. Although our patients had not undergone bronchial challenge, none had any typical asthmatic symptoms, signs, significant reversibility (spirometry) after nebulized salbutamol (9) (data not shown), or diurnal PEFR variation (data not shown).

There is no gold standard for measuring disease activity in bronchiectasis. Sputum leukocyte density and proinflammatory mediator levels were used as inflammatory, and bacterial densities as infective markers although their sensitivity and specificity are unknown. Our experience shows that the reproducibility for measurement of 24 h sputum volume, leukocyte density, and bacterial densities are 1.1- (recruitment criterion), 2.0-, and 3.3-fold, respectively, in our patient population (data not shown). The increase, albeit insignificant statistically, in total bacterial, commensal, and P. aeruginosa densities in the fluticasone group is of some concern. Notwithstanding the small study size, the intrinsic low reproducibility for quantitative sputum bacteriology, and the short study duration, this increase was not associated with any deterioration in clinical parameters. This phenomenon, not described by previous studies (20, 21, 23), deserves further evaluation in the future. In addition, three patients in the placebo group had an exacerbation compared with only one patient in the fluticasone group. The apparent dissociation in inflammatory and infective markers in bronchiectasis suggests that these pathogenic components might have to be dealt with separately and has important therapeutic implications.

Despite the improvement in antibiotics, methods of physiotherapy, and other newer modes of therapy (28), patients with CF and some with non-CF bronchiectasis continue to deteriorate relentlessly. There is, sadly, no effective disease-modifying treatment that would halt or reverse further tracheobronchial damage. The results of our study show that high-dose inhaled fluticasone therapy is effective in reducing the sputum inflammatory indices in severe non-CF bronchiectasis. Further long-term and multicenter studies should follow to evaluate the effects of high-dose steroid therapy on the progressive course in bronchiectasis.