© 2003 American Thoracic Society
Increased Leukotriene B4 and Interleukin-6 in Exhaled Breath Condensate in Cystic FibrosisDepartment of Thoracic Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, United Kingdom; and Institute of Respiratory Diseases, University of Bari, Bari, Italy Correspondence and requests for reprints should be addressed to Peter J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College Faculty of Medicine, Dovehouse Street, London SW3 6LY, UK. E-mail: p.j.barnes{at}ic.ac.uk
Chronic neutrophilic airway inflammation is an important feature of cystic fibrosis (CF). Noninvasive inflammatory markers may be useful in monitoring CF. Leukotriene B4 (LTB4) and interleukin (IL)-6 are inflammatory mediators that are increased in chronic neutrophilic inflammation. The aim of this study was to assess whether LTB4 and IL-6 were increased in exhaled breath condensate of CF patients and whether they could be used to monitor inflammation. Twenty patients with CF (13 males, age of 28 ± 9 years) were recruited together with 15 age-matched healthy subjects (8 males, age 35 ± 7 years). LTB4 and IL-6 levels were markedly elevated in patients with acute exacerbations (28.8 ± 4.3 and 8.7 ± 0.4 pg/ml) compared with control subjects (6.8 ± 0.7 and 2.6 ± 0.1 pg/ml, p < 0.0001). We also observed a decrease of exhaled LTB4 and IL-6 concentrations after antibiotic treatment in six patients who were followed until clinically stable (31.1 ± 4.4 and 9.5 ± 0.4 pg/ml vs. 18.8 ± 0.8 and 6.4 ± 0.2 pg/ml, respectively) and an increase in 15 CF patients infected with Pseudomonas aeruginosa (34.3 ± 5.0 and 9.3 ± 0.3 pg/m) compared with those infected with other bacteria (18.3 ± 0.7 and 6.9 ± 0.5 pg/ml). These findings suggest that LTB4 and IL-6 levels are increased in exhaled breath condensate of patients with CF during exacerbation and could be used to monitor airway inflammation in these patients.
Key Words: leukotriene B4 • interleukin-6, cystic fibrosis • exhaled breath condensate • airway inflammation Cystic fibrosis (CF) is due to genetic abnormalities in CF transmembrane conductance regulator, which results in abnormalities of mucus secretion, airway electrolytes, and impaired mucociliary clearance (1). The presence of copious quantity of viscid mucus in the airways predisposes patients to frequent pulmonary infections, mainly due to Pseudomonas aeruginosa leading to a chronic neutrophil-dominated inflammation (2). Neutrophils are thought to play a key role in the pathogenesis of this disease. They are potential sources of 5-lipoxygenase metabolites of arachidonic acid, and some authors have even postulated that increased arachidonic acid availability is linked to the basic genetic defect in CF (3). Leukotriene B4 (LTB4), formed from arachidonic acid as a result of enzymatic hydrolysis of LTA4, may play an important role in neutrophilic inflammation, as it is a potent chemoattractant of neutrophils and induces oxygen-free radical generation (4). Previous studies have demonstrated elevated concentrations of LTB4 in sputum (5), saliva (6), and epithelial lining fluid (7) of patients with CF.
The persistent inflammation in the airways of patients with CF may also be due to cytokines generated by bacterial infection, oxidative stress, or constitutive abnormalities in the regulation of cytokine production (8). Inflammatory cytokines may play an important role in amplification of inflammation and tissue damage in the lung of patients with CF. Interleukin (IL)-6 is a cytokine that has both inflammatory and anti-inflammatory actions. It regulates production of acute phase protein, which may locally limit the actions of inflammatory cells (9). IL-6 provides a negative feedback signal by downregulating tumor necrosis factor- Monitoring of airways inflammation may be important in patients with CF, as airways disease is the predominant cause of death in CF (11). Noninvasive inflammatory markers may be therefore useful in monitoring disease activity and progression (12). There is a need to find potential new markers of inflammation, which could help in early diagnosis or prediction of disease exacerbations so that early administration of antibiotics is possible. However, direct methods to measure airway inflammation such as bronchoalveolar lavage and bronchial biopsies are too invasive for routine use, particularly when airway function is severely impaired. Recently, exhaled breath condensate, which is completely noninvasive, has been used to measure airway inflammation. This technique is easy to perform, is repeatable, and has previously been used to measure inflammatory and oxidative stress markers in patients with CF (13–15). The purpose of our study was to measure LTB4 and IL-6 in exhaled breath condensate of patients with CF, and to assess whether these markers were related to exhaled nitric oxide (NO), lung function, and clinical status.
Patients The study population consisted of 20 adult patients with CF and 15 age-matched normal control subjects with no history of lung disease (Table 1) . All patients and normal subjects were white and were recruited from Royal Brompton Hospital. Informed consent was obtained from all subjects, and the Ethics Committee of the Royal Brompton, and Harefield National Health Service Trust approved the study.
Patients with CF were studied during the first day of their hospital admission for treatment of acute exacerbations of CF before commencement of antibiotic treatment. All subjects presented a symptomatic exacerbation of their lung disease characterized by a reduction in FEV1 of more than 10% compared with the best in the previous year, an increase in respiratory symptoms, signs of infection, including increased buccal temperature, leukocytosis, C-reactive protein, and bacterial colonization of sputum (15 patients were colonized by P. aeruginosa, 3 by Staphylococcus aureus, and 2 by Aspergillus fumigatus). Six of these patients colonized by P. aeruginosa were recruited after 2 weeks of antibiotic treatment when they were clinically recovered, with normal body temperature, circulating leukocytes, and C-reactive protein.
Patients infected with Burkholderia cepacia or methicillin-resistant S. aureus were excluded from the study to avoid concerns about cross-infection from shared equipment. CF was confirmed in all subjects by high sweat sodium (Na+
Exhaled Breath Condensate
Assays A specific enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) was also used to measure IL-6. The assay was validated directly by gas chromatography/mass spectrometry. The intra-assay and interassay variability were 10% or less. The detection limit of the assay was 1.5 pg/ml. The reproducibility of repeated LTB4 and IL-6 measurements was assessed by the Bland and Altman method and the coefficient of variation (17).
Exhaled NO
Lung Function
Statistical Analysis
LTB4 LTB4 was detectable in exhaled breath condensate of all subjects. LTB4 concentrations were significantly increased in patients with acute exacerbations of CF (30.3 ± 4.1 pg/ml) compared with healthy subjects (6.7 ± 0.7 pg/ml, p < 0.0001) (Figure 1A) . We found the highest levels of exhaled LTB4 in patients with CF and with P. aeruginosa (n = 15) in sputum culture (34.3 ± 5.0 pg/ml) compared with patients with other infectious agents (n = 5) (18.3 ± 0.7 pg/ml, p < 0.005). LTB4 levels in the six patients who were followed for 2 weeks were significantly reduced by antibiotics after 2 weeks of treatment (31.1 ± 4.4 to 18.8 ± 0.8 pg/ml, p < 0.01) (Figure 1B). The concentrations of exhaled LTB4 were significantly higher in stable state than in normal control subjects (p < 0.005). There was no correlation between the amounts of LTB4 in breath condensate and FEV1 (r = -0.1, p = 0.8) or FVC (r = -0.2, p = 0.4).
IL-6 IL-6 was detectable in exhaled breath condensate of all subjects, and the concentrations were significantly increased in patients with acute exacerbations of CF (8.7 ± 0.4 pg/ml) compared with healthy subjects (2.6 ± 0.1 pg/ml, p < 0.0001) (Figure 1C). We found higher levels of IL-6 in patients with CF and with P. aeruginosa in sputum culture (9.3 ± 0.3 pg/ml) than in patients with CF and with other infectious agents (6.9 ± 0.5 pg/ml, p < 0.005). IL-6 levels in patients after 2 weeks of antibiotic treatment were significantly reduced (9.5 ± 0.4 to 6.4 ± 0.2 pg/ml, p < 0.005). The concentrations of exhaled IL-6 were significantly higher in stable CF than in normal control subjects (p < 0.001) (Figure 1D). There was no correlation between the concentration of IL-6 in breath condensate and LTB4 (r = -0.2, p = 0.5), FEV1 (r = -0.2, p = 0.9), or FVC (r = 0.2, p = 0.4). Reproducibility of exhaled LTB4 measurements was assessed in 10 nonsmoking normal (6 males, 35 ± 7 years) subjects. In the majority of measurements, the differences between the two LTB4 values lie within ± 2 SD (mean difference, -0.04 ± 0.18 pg/ml) (Figure 2A) . The coefficient of variation for LTB4 measured was 2.0%.
Reproducibility of exhaled IL-6 measurements was assessed in 10 nonsmoking normal (6 males, 35 ± 7 years) subjects. In the majority of measurements, the differences between the two IL-6 values lie within ± 2 SD (mean difference, -0.03 ± 0.24 pg/ml) (Figure 2B). The coefficient of variation for IL-6 measured was 5.9%.
Exhaled NO
Our data show that both LTB4 and IL-6 are detectable in exhaled breath condensate and that their concentrations are significantly increased in patients with CF with acute exacerbation of their disease compared with patients with CF with stable disease and to healthy subjects. We also show higher concentrations of exhaled LTB4 and IL-6 in subjects infected with P. aeruginosa compared with other infectious agents and a lack of correlation between exhaled LTB4, exhaled IL-6, exhaled NO, and lung function.
A noninvasive measurement of airways inflammation in patients with CF could be useful in monitoring the disease. However, at the moment, noninvasive inflammatory markers are not available for clinical use. Exhaled and nasal NO levels are significantly lower in CF than in normal subjects, despite the intense neutrophilic inflammation in the airways (19–22). This may be due to release of superoxide anions, which may result in the formation of peroxynitrite (20), or it may be due to a defect in inducible NO synthase expression (19, 21). Increased oxidative stress in CF is likely to be a consequence of this neutrophilic inflammation, malnutrition, and a decrease in IL-10 (22, 23). Although there is a trend toward both exhaled and nasal NO being higher in patients who were not homozygous for the In this study, we analyzed two new inflammatory markers, LTB4 and IL-6, in exhaled breath condensate, which is a completely noninvasive method for obtaining samples that reflects airway lining fluid composition. This technique presents numerous advantages compared with bronchoalveolar lavage and induced sputum because it may be repeated frequently, is simple to perform, and is less expensive in terms of equipment and personnel costs (13, 26). We observed an increase of LTB4 in breath condensate of patients with CF, in agreement with previous studies showing higher concentrations of LTB4 in sputum (5–7) and saliva (6). We also found a marked increase in IL-6 concentrations in breath condensate of patients with CF compared with healthy subjects. The very narrow range of normal value of these exhaled markers that we observed shows the clinical value of these measurements to separate patients with ongoing inflammation from healthy control subjects. Other authors have previously investigated this cytokine with inconsistent results. Decreased concentrations of IL-6 have been found in the sputum (8, 25) and in blood of patients with CF (8, 27, 28). However, an increase in IL-6 has been found in bronchoalveolar lavage fluid of children with CF (29). The very high concentrations of exhaled IL-6 that we found in patients with CF during exacerbations suggest a key role for this cytokine in the inflammatory processes. In our study, we found greater increased concentrations of exhaled LTB4 and IL-6 in the subgroup of patients with CF infected with Pseudomonas. These findings are in line with results of other studies showing that Pseudomonas products such as porins, pyocanins, and lipopolysaccharide are potent stimulants of neutrophils and increase IL-6 production (30–32). Further studies are needed to confirm our findings that P. aeruginosa may stimulate the release of cytokines and of neutrophil-derived mediators. In future studies, it would be of interest to correlate the concentrations of inflammatory markers in exhaled breath condensate with bacterial colony counts in sputum. The reduction of the concentrations of exhaled LTB4 and IL-6 observed in six patients that we analyzed before and after 2 weeks of antibiotic treatment when they were clinically stable (expressed by an improvement in respiratory symptoms and by absence of signs of infection) demonstrates that these markers are related to severity of inflammation and are modified by antibiotic treatment. We did not find any correlation between inflammatory markers and lung function, suggesting that the measurement of lung function, exhaled IL-6, exhaled LTB4, and exhaled NO provide different information about the inflammatory process of the airways. Furthermore, the intensity of ongoing inflammation, as reflected by exhaled LTB4 and exhaled IL-6, may not be directly related to the long-standing lung damage, as reflected by lung function tests, so that a close relationship with spirometric measurements is not expected. In conclusion, our results provide evidence that LTB4 and IL-6 are increased in the respiratory tract of patients with CF during acute infective exacerbation and that their measurement in breath condensate could be useful for the noninvasive monitoring airway inflammation in these patients.
Supported by the National Heart and Lung Institute (London, UK) and the Institute of Respiratory Diseases, University of Bari (Bari, Italy). Received in original form March 6, 2002; accepted in final form May 26, 2002
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ABSTRACT
METHODS
, which is a potent mediator of inflammation and is the first cytokine to be released after interaction of host cells with a variety of microbial products (
50 mM) and genotyping (all showed the 
F508 genotype) (
) and control (
) subjects. (B) LTB4 concentrations in CF patients measured before and after 2 weeks of antibiotic treatment. (C) IL-6 concentrations in breath condensate of CF patients with exacerbations (
