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* From the Department of Pneumology (Drs. Corhay and Louis), CHU Sart-Tilman B-35; and Centre for Integrative Genoproteomic (Mss. Hemelaers, Henket, and Sele), University of Liege, Liège, Belgium.
Correspondence to: Jean-Louis Corhay, MD, PhD, Department of Pneumology, CHU Sart-Tilman B-35, 4000 Liège Belgium; e-mail: jlcorhay{at}chu.ulg.ac.be
Abstract
Background: Several chemoattractants have been measured in exhaled breath condensate (EBC) from patients with COPD. The aim of this study was to compare the eosinophil and neutrophil chemotactic activity contained in EBC from healthy subjects and patients with COPD.
Methods: EBC collected using a commercially available condenser (EcoScreen; Erich Jaeger Viasys; Hoechberg, Germany) was compared in 45 COPD patients and 65 healthy subjects. EBC chemotactic activity for eosinophils and neutrophils was assessed using microchambers (Boyden; Neuro Probe; Cabin John, MD). Chemotactic index (CI) was used to evaluate cell migration.
Results: EBC from patients with COPD (CI, 2.21 ± 0.16 [mean ± SEM]) and healthy subjects (CI, 1.67 ± 0.11) displayed significant neutrophil chemotactic activity (p < 0.0001 for both), which was however higher in patients with COPD (p < 0.001). Healthy smokers had a significantly raised CI for neutrophils by comparison with healthy nonsmokers (p < 0.01) and ex-smokers (p < 0.05). Likewise, current COPD smokers tended to have greater neutrophil CI than COPD who stopped smoking (p = 0.08). COPD ex-smokers had raised chemotactic activity by comparison with healthy ex-smokers (p < 0.05). Anti-interleukin-8 (10–6 g/mL) antibodies reduced neutrophil chemotactic activity by 35.2% (p < 0.05). EBC also contained significant eosinophil chemotactic activity in healthy subjects (CI, 1.68 ± 0.09; p < 0.0001) and patients with COPD (CI, 1.23 ± 0.07; p < 0.01), with a significantly lower CI in patients with COPD as compared to healthy subjects (p < 0.001). Smoking did not influence eosinophil chemotactic activity in healthy subjects or patients with COPD.
Conclusions: Current smoking favors neutrophil chemotactic activity. As compared to healthy subjects, EBC from patients with COPD displays a skewed chemotactic activity toward neutrophils vs eosinophils.
Key Words: airway inflammation • chemotactic activity • COPD • eosinophils • exhaled breath condensate • neutrophils
Several factors in bronchial secretions of patients with COPD have been identified as chemoattractants for neutrophils.1 Exhaled breath condensate (EBC) provides a noninvasive and easily performed means of looking at the local biochemical pattern in the airways of COPD patients and asthmatics.23 Some of the chemoattractants for neutrophils have been measured in the EBC from healthy subjects and patients with COPD. Leukotriene B4 (LTB4) was found to be raised in patients with stable COPD,456 and was further increased during exacerbations while diminishing following antibiotic treatment.5 The increase in exhaled LTB4 in patients with COPD seems to be relatively resistant to inhaled corticosteroid therapy.4 Although interleukin (IL)-8 was found to contribute to sputum neutrophil chemotactic activity in COPD patients,1 its detection is problematic in EBC because its level is close to the lower limit of quantification of the immunoassays used.78 However, the biological activity of EBC has been poorly investigated. A chemotactic activity for neutrophils was demonstrated by Garey et al9 in EBC from a limited series of healthy subjects, and cigarette smoking was found to enhance this neutrophil chemotactic activity. Granulocytic airway infiltration is thought to play a pivotal role in COPD.10 Until now, the overall neutrophil chemotactic activity contained in EBC has not been assessed in COPD, and to the best of our knowledge EBC eosinophil chemotactic activity has never been investigated in healthy subjects or in any type of obstructive airway disease. The aims of this study were to determine eosinophil and neutrophil chemotactic activity in the EBC from smoking and nonsmoking healthy subjects and to compare it with that found in patients with COPD.
Materials and Methods
Subjects
Overall, 45 patients with stable COPD and 64 nonatopic, healthy control subjects were assessed. Demographic, functional, and treatment characteristics are shown in Table 1
. All the COPD patients fulfilled the criteria proposed by Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.11 There were 28 patients with moderate COPD (GOLD stage 2) and 17 patients with severe COPD (GOLD stage 3). Allergy to common aeroallergens as determined by skin-prick testing was not found in healthy subjects or patients with COPD.
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EBC Collection
EBC was collected by using a commercially available condenser (EcoScreen; Erich Jaeger Viasys; Hoechberg, Germany) that yielded nongaseous components of expiratory air. Subjects breathed through a mouthpiece connected to the condenser, and were asked to breathe at a normal frequency and tidal volume, wearing a nose clip, for a period of 10 min. Approximately 2 mL of condensate was collected and immediately stored at – 80°C in the laboratory within 5 min after collection.
Eosinophil Isolation
Eosinophils were obtained from 70 mL of peripheral venous blood of the same nontreated and nonsmoking atopic donor (blood cytology with 6% of eosinophils) by immunomagnetic cell separation (MACS; Miltenyi Biotec; Bergisch Gladbach, Germany) using anti-CD 16 as described by Hatzelmann and coworkers.12
Neutrophil Isolation
Neutrophils were isolated using a two-step procedure from 40 mL of peripheral venous blood of the same healthy nonatopic and nonsmoking volunteer. The whole blood was treated with modified gelatin (Geloplasma; Fresenius Kabi, the Netherlands), 9/1, for 45 min to sediment the RBC. The leukocytes-rich pellet was diluted three times with phosphate-buffered saline solution and was centrifuged through a separating solution (Percoll; Biochrom; Grundau, Germany) gradient (centrifugation at 400g for 30 min at 22°C) to separate the mononuclear leukocytes from the granulocyte fraction (> 95% neutrophils). Neutrophils were then washed twice in phosphate-buffered saline solution and resuspended in Hank’s balanced salt solution (HBSS) with Ca2+ and Mg2+ (pH 7.4) and 2% bovine serum albumin at 1.106 cells/mL.
Eosinophil and Neutrophil Chemotaxis
Aliquots of 28 µL of EBC were placed in the lower chambers, and 50 µL of cells suspension (106 cells/mL) were placed in the upper chambers. The two chambers were separated by a polycarbonate polyvinylpyrrolidone-free filter, 0.8 µm (Nuclepore; Whatman; Florham Park, NJ). Experiments were performed in triplicate in a 48-well microchemotaxis Boyden chamber incubated in 5% CO2 at 37°C for 60 min. The negative controls consisted of a solution of HBSS with Ca2+ and Mg2+ (pH 7.4). Chambers were dismantled and nonmigrated cells were scraped from the upper surface of the filters. The filters were then fixed in methanol and stained (Diff-Quick; Baxter Scientific; Miami, FL). Migrated cells adherent to the lower surface were counted in 10 fields in each well at a magnification x 600. The results were expressed as the number of eosinophils (neutrophils) having migrated/10 high-power field. Chemotactic index (CI) was calculated by the ratio of cell migration with the EBC/cell migration with HBSS.
The reproducibility of repeat EBC chemotactic activity measurements was assessed. The intraclass correlation coefficients for neutrophil (n = 12) and eosinophil (n = 17) chemotactic activity were 0.67 and 0.62, respectively. Spearman rank correlation coefficients for neutrophil and eosinophil chemotactic activity were 0.74 (p < 0.01) and 0.71 (p < 0.005), respectively.
Determination of the Contribution of IL-8 to EBC Neutrophil Chemotaxis
Serial concentrations (10– 11 to 10– 6 g/mL) of the monoclonal anti-IL-8 antibody neutralizing biological activity of IL-8 (R&D Systems; Minneapolis, MN) were added to EBC for 1 h at room temperature before neutrophil chemotactic activity was assessed.
Statistical Analysis
Demographic and functional data are expressed as mean ± SD, and chemotactic results as expressed as mean ± SEM. CIs for each group of samples were evaluated by one-sample t test vs a hypothetical mean of 1.0 (representing zero net chemotactic activity). The influence of dilution on EBC chemotactic activity was tested by Friedman test followed Dunn test for multiple comparisons. Impact of smoking on chemotaxis in healthy subjects and comparison between healthy subjects and COPD were performed by the Kruskal-Wallis test or Mann-Whitney U test. Correlations between variables were performed using a Spearman rank correlation test; p < 0.05 was considered significant.
Results
Effect of EBC Dilution on Chemotactic Activity
The effect of dilution on neutrophil chemotactic activity was measured in the EBC from 10 subjects (6 COPD patients and 4 control subjects). Mean CI without dilution was 1.63 ± 0.25 and decreased significantly to 1.48 ± 0.15 and 1.01 ± 0.14 when diluted 1/10 and 1/100 by HBSS, respectively (Fig 1
). EBC without dilution and with a dilution 1/10 exhibited a significant chemotactic activity (p < 0.05), whereas dilution 1/100 suppressed any measurable activity. In our model, serum activated by casein was used as positive control and caused a CI of 8.13 ± 1.16 (n = 16).
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Effect of Heating on Chemotactic Activity
The effect of heating (during 5 min at 95°C) on neutrophil chemotactic activity was measured in the EBC from eight COPD patients. Mean neutrophil CI without heating was 3.64 ± 0.88 and decreased significantly to 1.60 ± 0.18 after heating (Fig 2
).
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Neutrophil and Eosinophil Chemotactic Activity in Healthy Control Subjects
The EBC from control subjects exhibited a significant neutrophil chemotactic activity (CI, 1.67 ± 0.11; p < 0.0001). In those subjects, the CI was significantly increased in the EBC from current smokers (Fig 3
) in comparison with long-life nonsmokers and ex-smokers (CI, 2.03 ± 0.15 vs 1.41 ± 0.12 and 1.54 ± 0.26, respectively; p < 0.05), but chemotactic activity remained significant in each group (CI significantly different from 1 in each group; p < 0.01). Neutrophil CI was not correlated with the age irrespective of the smoking habit (whole group, r = – 0.036, p = 0.78; nonsmokers, r = 0.09, p = 0.67; ex-smokers, r = 0.03, p = 0.93; smokers, r = – 0.11, p = 0.67). In addition, there was no significant difference between female and male subjects (CI, 1.6 ± 0.19 vs 1.37 ± 0.14, respectively; p = 0.17).
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Neutrophil and Eosinophil Chemotactic Activity in the EBC From COPD Patients
A significant neutrophil chemotactic activity was demonstrated in the EBC from COPD (CI, 2.21 ± 0.16; p < 0.0001). There was a trend to a significant difference between smokers and ex-smokers (CI, 2.51 ± 0.24 and 1.89 ± 0.17, respectively; p = 0.08) [Fig 3], and mean CI for neutrophils in both groups significantly differed from 1 (p < 0.0001). There was no significant difference in the neutrophil chemotactic activity according to the stage of COPD (CI, 2.07 ± 0.16 in stage 2, and 2.44 ± 0.32 in stage 3; p = 0.43). Likewise, there was no difference for neutrophil chemotactic activity between patients with COPD treated with inhaled steroids and untreated COPD patients (CI, 2.32 ± 0.22 and CI, 1.99 ± 0.23, respectively; p = 0.31).
Significant eosinophil chemotactic activity was also demonstrated in the EBC from COPD (CI, 1.23 ± 0.08; p < 0.01). There was no difference for eosinophil chemotactic index between smokers and ex-smokers (CI, 1.26 ± 0.10 and 1.21 ± 0.12, respectively; p = 0.84) nor significant difference according to the GOLD stage of COPD (CI, 1.28 ± 0.10 in stage 2 and 1.15 ± 0.12 in stage 3; p = 0.40). Finally no significant difference was found between inhaled steroid treated and untreated COPD (CI, 1.22 ± 0.12 and CI: 1.25 ± 0.11, respectively; p = 0.86).
Comparison Between COPD and Control Subjects
When the two groups were taken as a whole, there was a significant difference in the neutrophil CI between COPD and control subjects (p < 0.001). We also found a significant increase of CI for neutrophils in the EBC from COPD ex-smokers by comparison with controls ex-smokers (CI, 1.89 ± 0.17; and CI, 1.54 ± 0.26, respectively) [p < 0.05; Fig 3], but we did not find a significant difference between COPD smokers and control smokers (CI, 2.51 ± 0.24 and 2.03 ± 0.15, respectively; p = 0.25). In contrast with CI for neutrophils, CI for eosinophils appeared to be lower in COPD when compared to healthy subjects (p < 0.001; Fig 4
).
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We confirm by this study that collection of EBC is a noninvasive and successful method for obtaining samples from the lungs in patients with COPD. EBC from both healthy subjects and COPD patients display significant neutrophil and, as shown for the first time, eosinophil chemotactic activity. Our data show that current smoking and COPD independently favor chemotactic activity toward neutrophils.
Although the EBC consists of > 99.99% of water vapor,13 the EBC from control subjects and patients with COPD contain chemotactic factors, as demonstrated by a significant chemotactic activity for neutrophils and eosinophils. These chemotactic activities rapidly disappear with dilution of the EBC by HBSS and with heating the EBC at 95°C. These results suggest that thermolabile biological factors are responsible for EBC chemotactic activity. Previous studies have shown a neutrophil chemotactic activity in the EBC of healthy subjects9 as well as in a mixed group of subjects including two asthmatics,14 but the number of subjects studied was limited in these two studies (n = 8 in the first study, and n = 5 in the second study).
In a large number of subjects, we showed that chemotactic activity for neutrophils was significantly increased in the EBC from healthy smokers by comparison with healthy nonsmokers and ex-smokers. This is in keeping with the fact that neutrophils are well known to be increased in the airways of smokers,15 and Garey et al9 showed that cigarette smoking caused an increase in neutrophil chemotactic activity of EBC from healthy subjects. In healthy subjects, no correlation was found between age and the eosinophil CI or neutrophil CI. This might be surprising because Thomas et al16 have shown that the induced-sputum neutrophil, but not eosinophil, count increased significantly with age. It is, however, likely that other factors than chemotactic activity contribute to the determination of neutrophil number in the airways.
In this study, we have clearly shown that EBC from COPD patients contains active chemoattractants for neutrophils as demonstrated by a significant chemotactic activity. Furthermore, EBC from COPD patients display greater neutrophil chemotactic activity than that healthy control subjects. Our observation is linked with the fact that neutrophilic inflammation is indeed a major feature of COPD.17181920 Higher levels of LTB4 found in the EBC of COPD456 may explain the increased neutrophil chemotactic activity reported in the present study. In addition, IL-8, which is not easily measurable in EBC, contributes at least partially to the neutrophil chemotactic activity as shown in our study by the effect of anti-IL-8 antibodies. Active smoking in our COPD patients can account partially for the raised neutrophil chemotactic activity because current COPD smokers tended to have increased neutrophil chemotactic activity by comparison with COPD ex-smokers. As the latter display a higher neutrophil chemotactic activity by comparison with healthy ex-smokers, our results point to an enhanced EBC neutrophil chemotactic activity that is inherent in COPD itself. The influence of treatment by inhaled steroids on the bronchial inflammation of COPD patients is controversial in the literature. In our study, we did not find differences between COPD with or without inhaled steroids on chemotactic activity for neutrophils. Indeed, some studies have shown that inhaled steroids reduce neutrophil cell counts2122 and chemotactic activity23 in induced sputum of COPD patients. An in vitro study24 also demonstrated a reduction in neutrophil chemotaxis by fluticasone. These effects of the inhaled steroids on the neutrophilic inflammation in COPD have not been found by others.2526 Controlled study would be required to establish the effect of steroids on EBC chemotactic activity from COPD patients. Although we did not demonstrate a difference in the EBC neutrophil chemotactic activity according to the stage of COPD, we would like to underline that COPD patients were all stable and that most of them were GOLD stage 2. Thus, we cannot rule out a possible relationship between disease severity and EBC neutrophil chemotactic activity.
An intriguing finding is that COPD patients have a lower chemotactic activity for eosinophils by comparison with control subjects. This observation together with the raised neutrophil chemotactic activity indicates that patients with stable COPD display a markedly skewed chemotactic activity toward neutrophils. This holds true in COPD smokers as well as in COPD ex-smokers by comparison with their controls.
We conclude that chemotactic activity for neutrophils and eosinophils is measurable in EBC from healthy control subjects and patients with stable COPD. Both current smoking and COPD amplified neutrophil chemotactic activity, while COPD alone seems to be associated with reduced chemotactic activity toward eosinophils. The biochemical compounds underlying the EBC chemotactic activity remain to be determined in further studies.
Footnotes
Abbreviations: CI = chemotactic index; EBC = exhaled breath condensate; GOLD = Global Initiative for Chronic Obstructive Lung Disease; HBSS = Hank’s balanced salt solution; IL = interleukin; LTB4 = leukotriene B4
This work was performed at CHU Sart-Tilman B-35, and Centre for Integrative Genoproteomic, University of Liege.
The authors have no financial or other potential conflicts of interest to disclose.
Received for publication September 8, 2006. Accepted for publication January 22, 2007.
References
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