INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bronchiectasis is a chronic inflammatory lung disease characterized by irreversible dilatation of the bronchi and, in most cases, by persistent production of purulent sputum (1). Understanding of the pathogenesis of the disease has increased in recent years, but many aspects remain obscure (2). High levels of proinflammatory cytokines are present in airway secretions, and neutrophils are the predominant cells in the airway lumen (3). In patients with bronchiectasis bronchial damage is thought to be due to neutrophil inflammatory products released in response to bacterial infection (4).

Activation of inflammatory cells such as neutrophils, eosinophils and macrophages induces a respiratory burst resulting in marked production of superoxide anion (O₂), which then undergoes spontaneous or enzyme-catalyzed dismutation to form hydrogen peroxide (H₂O₂) (5). H₂O₂ appears to be an important reactive oxygen species causing cellular injury, perhaps via further reactions leading to more reactive species such as hydroxyl radical and lipid peroxidation products. In humans, increased levels of H₂O₂ detected in breath condensate indicate an elevated production of oxidants in various inflammatory lung disorders such as asthma (6, 7), adult respiratory distress syndrome (ARDS) (8), and chronic obstructive pulmonary disease (COPD) (9).

Because bronchiectasis involves chronic inflammation with relentless traffic of neutrophils in the bronchial wall and H₂O₂ is produced by activated neutrophils, we investigated whether H₂O₂ concentration of breath condensates is elevated in patients with bronchiectasis and whether levels of expired H₂O₂ might be related to the disease severity, as assessed by lung function. There is also some evidence indicating that asthmatic patients treated with inhaled corticosteroids may have values of H₂O₂ lower than those of steroid-naïve subjects (7). Therefore we also compared the values of exhaled H₂O₂ from bronchiectatic patients with and without treatment with inhaled corticosteroids.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Twenty-five normal subjects (18 men; mean age, 42 ± 2 yr) (Table 1) were recruited from volunteers taking part in other studies and from the staff of the National Heart and Lung Institute. All were nonsmokers and were free of respiratory infections for at least 6 wk before starting the study. They had no history of chronic cardiovascular or respiratory disease and were not receiving any regular medication. These subjects had a negative history of allergy (negative skin prick tests to common allergens), normal spirometry (FEV₁, 90 ± 0.6% pred), and normal bronchial reactivity with a provocative concentration of methacholine causing a 20% fall in FEV₁ (PC₂₀) > 32 mg in all subjects.

Thirty-seven patients (13 men; mean age, 45 ± 2.5 yr; FEV₁ 59 ± 3% pred) (Table 1) with diagnosed bronchiectasis were recruited from the Host Defence Unit at Royal Brompton Hospital. All patients had bronchiectasis diagnosed on the basis of clinical and radiological features and confirmed by high resolution computed tomography (CT) of the thorax. Patients were included in the study only if they were clinically stable and had no evidence of acute infective exacerbations (lower or upper airways) for at least 4 wk prior to the study. Bronchiectasis was believed to be secondary to tuberculosis in three patients, primary ciliary dyskinesia (PCD) in nine patients, IgA deficiency in two patients, Young's syndrome in three patients, deficiency of a subclass of IgG in three patients, and with no definite antecedent cause identified in the remaining 17 patients (idiopathic). All patients had a negative sweat test. Patients with cystic fibrosis, allergic bronchopulmonary aspergillosis, asthma, and atopic diseases were excluded. None of our patients was a current smoker. Four patients stopped smoking > 1 yr prior to study (mean smoking history, five pack-yr). Twenty-three were taking regular inhaled ₂-agonists, and 20 were receiving inhaled corticosteroids (fluticasone propionate, 500 to 2,000 µg daily).

The study protocol was approved by the Ethics Committee of the Royal Brompton Hospital, and all subjects gave written informed consent.

Study Design

On enrollment into the study all normal subjects were seen in a screening visit during which medical history, physical examination, spirometry, PC₂₀ methacholine, and skin prick tests to common allergens were performed. Before entering the study, patients were seen by the investigator who was the bronchiectatic patient's usual physician (P.J.C). At the time of this examination, this investigator recorded whether the patients were in their usual state of health or whether there was clinical evidence of infective exacerbation of their disease (pyrexia, hemoptysis, increase of sputum volume) for at least 4 wk prior to the study. Patients with infective exacerbation were excluded from the study. Medical history was taken and physical examination was performed. After spirometry, breath condensate was collected. The investigator who performed the H₂O₂ measurements was blinded to the clinical and functional status of the subjects.

Lung Function

FEV₁ was measured using a dry spirometer (Vitalograph, Buckingham, UK). The best value of three maneuvers was expressed as a percentage of the predicted value. Airway responsiveness was measured by methacholine provocation challenge. The solution was nebulized with a hand-held nebulizer (Dosimeter MB₃; MEFAR, Bovezza, Italy) with an output of 10 µl. The PC₂₀ was calculated by interpolation of the logarithmic dose-response curve.

Collection of Expired Breath Condensate and Hydrogen Peroxide Measurement

Expired breath condensate was collected in the morning using a specially designed glass condensing chamber. The condensing chamber contained a double wall glass and the inner side of the glass was cooled by ice. Breath condensate was collected between the two glass surfaces. Exhaled air entered and left the chamber through one-way valves at the inlet and at the outlet keeping the chamber closed. After rinsing their mouths, subjects breathed tidally through a mouthpiece connected to the condenser for 15 min while wearing noseclips. Approximately 1 ml of breath condensate was collected and stored at 70° C in a 2-ml sterile plastic tube. Measurements of H₂O₂ were performed within 2 d of collection.

H₂O₂ was measured as described previously (9, 10). Briefly, 100 µl of 420 µM 3',3,5,5' tetramethylbenzidine (dissolved in 0.42 M citrate buffer at pH 3.8) and 10 µl of 52.5 U/ml of horseradish peroxidase (HRP) (Sigma Chemicals, Poole, UK) were reacted with 100 µl of the condensate for 20 min at room temperature. Subsequently, the mixture was acidified to a pH of 1 with 10 µl of 18 N sulphuric acid. The reaction product was measured spectrophotometrically at the absorbency of 450 nm using an automated microplate reader (Model AR 8003; Labtech Int. Ltd, Uckfield, UK). The detection limit was approximately 0.1 µM H₂O₂.

Statistical Analysis

Data are expressed as means ± SEM. Comparisons between groups were made by Student's unpaired t test. Regression analysis was performed by Pearson's rank correlation coefficients. A p value < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

H₂O₂ was detectable in all subjects. Patients with bronchiectasis had values significantly higher than those of the normal subjects (0.87 ± 0.01 versus 0.26 ± 0.04 µM, p < 0.001) (Figure 1A).

View larger version (13K): [in this window] [in a new window]   Figure 1.   (A) Hydrogen peroxide (H2O2) concentration in expired breath condensate of 37 patients with bronchiectasis (closed circles) and 25 normal subjects (open circles), p < 0.001. (B) Correlation between hydrogen peroxide (H2O2) and FEV1% predicted in patients with bronchiectasis.

There was a significant negative correlation between lung function as assessed by FEV₁ and H₂O₂ (r = 0.76, p < 0.0001) (Figure 1B). The main clinical difference between those patients with high values of H₂O₂ and low FEV₁% pred and those with normal values and normal or nearly normal lung function was the duration of their disease. Most of patients with high values had experienced the disease from their childhood (PCD, immunoglobulin deficiency).

Patients with bronchiectasis who received inhaled corticosteroids had values similar of H₂O₂ similar to those of the steroid-naïve patients (0.8 ± 0.1 versus 0.9 ± 0.1 µM, respectively, p > 0.05). Both groups had similar lung function (FEV₁, 58 ± 4% pred versus 61 ± 4% pred, p > 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study is the first to demonstrate increased concentrations of H₂O₂ in exhaled air condensate from patients with bronchiectasis compared with those from normal subjects, indicating an enhanced oxidative stress in these patients. It also shows a significant negative correlation between H₂O₂ levels and lung function as assessed by FEV₁. In a previous study we demonstrated that patients with bronchiectasis had an increase in exhale nitric oxide (NO), which may reflect inflammation in the airways but is not an index of oxidative stress (11).

Bronchoalveolar lavage (BAL) fluid obtained from patients with bronchiectasis contains a significantly higher number of neutrophils compared with BAL from healthy subjects (12). Neutrophils release several molecules that may contribute to inflammatory damage, particularly proteases and oxidants. An increased number of neutrophils or increased production of H₂O₂ by these cells might result in the increased production of H₂O₂ observed in bronchiectatic patients.

Bacterial infections may contribute to oxidative stress by facilitating the recruitment and activation of phagocytic cells in the lung (13). Phagocytic cells produce potentially toxic metabolites of oxygen, including H₂O₂. Even clinically stable bronchiectatic patients are persistently colonized by bacteria on the airway epithelium (2). This chronic exposure of phagocytic cells to bacteria might be the particular stimulus for production of H₂O₂.

Tumor necrosis factor- (TNF-), a proinflammatory cytokine, has been detected in the airway secretions of patients with bronchiectasis (3). It has recently come to light that TNF- induces neutrophil release of H₂O₂ in vitro (14). This means that high concentrations of this proinflammatory cytokine may trigger a prolonged release of H₂O₂.

In the present study a strong negative correlation was observed between H₂O₂ concentration and FEV₁% pred. It is possible the correlation between H₂O₂ and FEV₁% pred could be a coincidence. On the other hand, there is some evidence that the two parameters (lung function and inflammation) change in the course of inflammation and the reduced pulmonary reserve is a consequence of ongoing inflammation (12). In another study, longer duration of disease significantly related to worse spirometry and state of inflammation (15). These earlier observations support the hypothesis that the higher values of exhaled H₂O₂ detected in patients with more severe disease in the present study might be related to the inflammatory pattern as assessed by the number of total cells and neutrophils in BAL. Further studies using BAL in relation to H₂O₂ are needed in order to confirm this hypothesis.

There is some evidence that patients with bronchiectasis treated with inhaled corticosteroids may benefit from a reduction in sputum production and improved lung function (16). However, no inflammatory markers were used in that study in order to confirm the anti-inflammatory action of steroids. The present study shows that bronchiectatic patients treated with inhaled corticosteroids had values of H₂O₂ similar to those not receiving steroid treatment. A previous study has shown that intravenous administration of methylprednisolone failed to alter hydrogen peroxide production (17). Another explanation is that corticosteroids do not alter the predominant neutrophilic inflammation seen in bronchiectasis. This has already been confirmed in a study of induced sputum in patients with COPD (18). Our findings with respect to use of inhaled corticosteroids contrast with results in asthmatic patients where inhaled corticosteroids were associated with lower exhaled H₂O₂ compared with that in steroid-naïve subjects (7). Oral and inhaled corticosteroids have been demonstrated to reduce the number of eosinophils from the airways of patients with asthma (18). It is also established that activation of eosinophils results in the production of H₂O₂ in asthmatic patients (7). This lead to the theoretically plausible explanation that inhaled corticosteroids inhibit H₂O₂ production by decreasing eosinophil numbers in asthmatic patients; however, they cannot decrease exhaled H₂O₂ levels in bronchiectatic patients because of their relative ineffectiveness on neutrophils. Both studies were designed as cross-sectional and cannot clearly demonstrate a causal relationship between steroids and H₂O₂. Controlled studies are needed for that purpose.

In conclusion, this study has shown an increased concentration of H₂O₂ in exhaled air condensate in patients with bronchiectasis. We also found a strong inverse correlation with severity of the disease as assessed by lung function. These observations may have practical implications since the measurement of exhaled H₂O₂ by a simple noninvasive method may provide a simple means of monitoring disease activity and may also give an indication of the extent of the inflammatory process. Further studies are needed to confirm whether H₂O₂ levels and lung function are correlated with direct markers of inflammation.