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Journal of Asthma, 46:470–476, 2009Copyright C© 2009 Informa Healthcare USA, Inc.ISSN: 0277-0903 print / 1532-4303 onlineDOI: 10.1080/02770900902846349
ORIGINAL ARTICLE
Smoking Affects Eotaxin Levels in Asthma Patients
Algirda Krisiukeniene,1 Agne Babusyte,2 Kristina Stravinskaite,1 Jan Lotvall,3 RaimundasSakalauskas,1 and Brigita Sitkauskiene1,2,∗
1Department of Pulmonology and Immunology, Kaunas University of Medicine, Kaunas, Lithuania2Laboratory of Pulmonology, Institute for Biomedical Research, Kaunas University of Medicine, Kaunas, Lithuania
3The Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Internal Medicine, GoteborgUniversity, Goteborg, Sweden
Background. Chronic airway inflammation is most important pathological finding in asthma. Cigarette smoking may modify type of inflammationas well as may influence disease severity and response to the treatment. Objective. Thus the aim of this study was to investigate whether cigarettesmoking may have an influence on the levels of eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 in patients with stable mild/moderate asthma. Methods. 45steroid naive asthmatics (mean age: 55.2 ± 2.2 yrs) and 23 “healthy” smokers and non-smokers control subjects (mean age: 54.4 ± 9.7 yrs) wereinvestigated. Asthmatics were divided into two subgroups according to their smoking histories: asthmatic smokers (n = 19) who currently smoke andhave a history of >10 pack-years and asthmatic never-smokers (n = 26). BAL and induced sputum were performed. Cytospins of induced sputumand BAL were stained with May-Grünwald-Giemsa for differential cell counts. Eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 concentrations in serum,sputum and BAL supernatant was measured using a commercial ELISA kit. Results. In sputum supernatant from asthma smokers was significantlyhigher concentration of eotaxin-1 than in non-smokers asthmatics (203.4 ± 10.0 vs. 140.2 ± 9.5 respectively, p < 0.05). In non-smokers asthmapatients levels of BAL eotaxin-1 strongly related to percent and absolute numbers of BAL eosinophils and neutrophils (Rs = 0.737 and Rs = 0.514respectively, p < 0.05). The number and percent of sputum neutrophils and eosinophils, obtained from smokers asthmatics, significantly correlatedwith eotaxin-2 concentration in sputum supernatant (Rs = 0.58 and Rs = 0.75 respectively, p < 0.05). IL-5 levels in the serum and sputum fromasthmatic never-smokers were significantly higher than they were from asthmatic smokers and “healthy” smokers. Asthmatic never-smokers showeda significantly higher amount of IL-5 in serum and sputum than the asthmatic smokers showed. Conclusions. This study showed the elevated levelsof sputum eotaxin-1 as well as serum, sputum and BAL eotaxin-2 in asthmatic smokers without a significant increase of eosinophils compared toasthmatic never-smokers. The eotaxin concentrations were related not only with number of eosinophils but also with the number of neutrophils inall the studied tissue compartments. The data herein permits a suggestion that smoking may influence change in asthmatic airway inflammation bystimulating the production of eotaxins.
Keywords asthma, smoking, eotaxin-1, eotaxin-2, eotaxin-3, IL-5.
IntroductionAsthma, one of the most prevalent airway diseases, is char-
acterized by the presence of typical symptoms, functionalchanges, and chronic airway inflammation (1). Inflamma-tion, a central feature of asthma, is associated with an airwayinfiltration of inflammatory cells and an increased expressionof cytokines, chemokines, growth factors, enzymes, and ad-hesion molecules (2). It is well established that eosinophilsare some of the main effector cells in asthma pathogenesis (1,3). Since eosinophilc airway inflammation has been proposedas playing an important role in asthma, extensive investiga-tions into the molecular processes that regulate eosinophilrecruitment and activation have been initiated. Among awide range of identified chemokines, only the eotaxin sub-family (eotaxin-1, eotaxin-2, and eotaxin-3) is selective foreosinophil chemotaxis by activating the CC chemokine re-ceptor 3 (CCR3) (4). Eotaxin-1 was first identified as thedominant eosinophil chemoattractant in a guinea pig modelof allergic airway inflammation (5), promoting both localeosinophil recruitment to the lung and, in cooperation withinterleukin 5 (IL-5), rapid mobilization of eosinophils and
∗Corresponding author: Brigita Sitkauskiene, Eiveniu 2, LT-50009,Kaunas, Lithuania; E-mail: [email protected]
their progenitors from the bone marrow (5). Two other eo-taxin homologues (named eotaxin-2 and eotaxin-3) haverecently been identified by applying molecular biologicalmethods (6). Despite a low sequence identity between theeotaxins, they are important chemokines in asthma patho-genesis: eotaxin-1 is involved in the early phase of allergen-induced recruitment of eosinophils; whereas eotaxin-2 andeotaxin-3 are associated with subsequent persistence ofallergen-induced eosinophilic inflammation (6–8). Althoughthe correlation between allergen exposure and eotaxin pro-duction has been confirmed, the influence of other envi-ronmental factors on eotaxin production has not been wellestablished.
Tobacco smoke is an environmental factor associated withasthma attacks and their severity (9). Cigarette smoke dam-ages the bronchi in various ways: direct toxicity to thebronchial epithelium, oxidative damage, recruitment of in-flammatory cells, and increased epithelial permeability (10).In asthmatic smokers, an increased imbalance between theproduction of pro-inflammatory and anti-inflammatory cy-tokines and alterations of inflammatory cells has been es-tablished (11, 12). By considering all the published data,it can be presumed that active cigarette smoking may influ-ence chronic airway inflammation by changing the expres-sion of cytokines, chemokines, and receptors and by altering
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EOTAXINS AND SMOKING 471
the recruitment of the inflammatory cells. Considering themolecular distinctions identified between eotaxin-1, eotaxin-2, and eotaxin-3, the hypothesis can be raised that chronictobacco smoke may affect the expression of these threechemokines differently.
Thus, the aim of this study was to investigate whethercigarette smoking may have an influence on the levels ofeotaxin-1, eotaxin-2, and eotaxin-3 in patients with stablemild/moderate asthma.
Materials and MethodsStudy Population
The investigation was of 45 nonatopic patients with sta-ble, mild to moderate asthma (as per Global Initiative forAsthma [GINA]) (1). All asthmatic patients were steroidnaive. Asthmatics were divided into two subgroups accord-ing to their smoking histories: asthmatic smokers (n = 19)who currently smoke and have a history of > 10 pack-yearsand asthmatic never-smokers (n = 26). Pack-years were cal-culated as cigarettes per day divided by 20 and multiplied bythe number of years smoked.
Two control subgroups of healthy individuals were se-lected: healthy never-smokers (n = 13) and “healthy” smok-ers (n = 10) who are current smokers with a normal lung func-tion (forced expiratory volume in 1 second [FEV1] ≥90% ofpredicted) and have a history of >10 pack-years.
Exclusion criteria included evidence of acute respiratoryinfection during the last one month before this investigation,pregnancy, presence of any other acute or chronic respira-tory disorder except asthma, and the use of long-acting β2-agonists or methylxanthines within the preceding 4 weeks.
This study was approved by the Regional Bioethics Com-mittee at Kaunas University of Medicine. All participantsprovided their written, informed consent before entering thestudy.
Lung Function TestingLung function was tested using the CustovitM pneumo-
tachometric spirometer (Custo Med, Germany). All subjectswere asked to avoid the use of short-acting β2-agonists for atleast 8 hours before the testing. Patients were investigated in asitting position and a nose clip was used. The highest value offorced expiratory volume in one second (FEV1) and forcedvital capacity (FVC) from at least three acceptable blowswas recorded. Normal values were characterized accordingto Quanjer et al. (13).
Sputum Induction and ProcessingSputum was induced as described previously (14, 15). Be-
fore the sputum induction, volunteers were pretreated with200 µg of salbutamol. Then the subjects each inhaled for 5minutes the mist of sterile 3%, 4%, and 5% hypertonic salinegenerated by a low-output, ultrasonic nebulizer (DeVilbissHealth Care, USA). Sputum was collected into a plastic Petridish, placed on ice, and processed within one hour. Spu-tum was processed by the previously described protocol (15)with some modifications. Keeping in mind that dithioery-thritol (DTE) affects the measurement of sputum eotaxin bymeans of immunoassay (16), the expectorated samples weredivided and processed using two parallel procedures. A small
quantity of sputum was weighed and dispersed with a four-fold volume of 0.1% DTT (Sigma-Aldrich, Germany) thatwas freshly diluted in Dulbecco’s phosphate-buffered saline(D-PBS) (Sigma, Germany). The mixture was wortexed ona bench rocker for 15 minutes at room temperature. Then anequal volume of cooled D-PBS was added, and the samplewas filtered through a 40-m cell strainer (Becton Dickin-son, USA). The filtered sample was centrifuged for 10 min-utes at 4◦C, and the supernatant was aspirated and stored at−70◦C. The total cell counts, cell viability, and percentage ofsquamous cells were investigated using a Neubauer hemocy-tometer (Heinz-Herenz, Germany) by trypan blue staining(Sigma, Germany). Sputum samples were considered ac-ceptable when cell viability was more than 70%, and thepercentage of squamous cells was less than 10%. Cytospinswere prepared on a cytocentrifuge (Shandon Southern Instru-ments, USA). The other part of the sputum was processedwith PBS as described previously (15), and the samples wereused for immunoassay.
Bronchoscopy and Bronchoalveolar Lavavge (BAL)Processing
Bronchoscopy was performed in the morning one weekafter the sputum induction by using a local anesthesia with5 mL of 2% lidocaine (Grindex, Latvia). Before the bron-choscopy, subjects were asked not to smoke for at least 10hours and not to drink or eat for 4 hours. During the proceduresubjects were monitored with pulse oximetry. Seven separate20-mL aliquots of sterile saline solution (0.9% NaCl) wereinstilled into the right middle lobe segment of the lungsthrough a wedged flexible fibreoptic bronchoscope (OlympusBF-B3R, Tokyo, Japan) and then gently suctioned. The fluidwas filtered through a 40-µm cell strainer (Becton Dickinson,USA) and centrifuged for 10 minutes at 4◦C. Supernatantswere aspirated and stored at −70◦C. BAL cytospins wereprepared as described above.
Sputum and BAL Cell AnalysisPrepared cytospins from the sputum and BAL were stained
using the May-Grunwald/Giemsa method for differential cellcounts. Then 400 non-squamous cells were counted per slide.The type of cell was identified using standard morphologicalcriteria. Percentage (percentage of total nonsquamous cells)and absolute values (106/mL) of all cell counts were recorded.
Eotaxins and IL-5 ImmunoassayEotaxin-1, eotaxin-2, eotaxin-3, and IL-5 concentrations
were determined by the enzyme-linked immunosorbent assay(ELISA) using the R&D Systems kits (Minneapolis, USA).The detection limits for eotaxin-1 and eotaxin-2 were 5pg/mLfor IL-5- 2pg/mL; whereas the detection limit for eotaxin-3was 20pg/mL. To prevent the exclusion of low values thatwould distort the group statistics, samples with eotaxin con-centrations below detection limits were assigned a value ofzero for the statistical analysis.
Statistical AnalysisData are expressed as the mean ± standard error of mean
(SEM), and p values of less than 0.05 were considered sta-tistically significant. The statistical analysis was performed
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Table 1.—Characteristics of study subjects.
Variables Asthmatic never-smokers Asthmatic smokers Healthy never-smokers “Healthy” smokers
Subjects (n) 26 19 13 10Male/female 6/20 14/5 4/9 8/2Age (years)† 56.9 ± 1.7 54.7 ± 5.1 53.4 ± 2.3 55.4 ± 3.3Disease duration (years) † 11.3 ± 2.5 9.5 ± 6.1 — —Smoking (pack-years)† — 17.4 ± 7.3 — 22.3 ± 6.1FVC (% pred.) 89.3 ± 3.6 78.8 ± 6.4 108.6 ± 4.7 97.6 ± 9.5FEV1 (% pred.) † 85.3 ± 4.8∗ 80.4 ± 9.7∗ 117.5 ± 4.1 112.6 ± 5.3FEV1/FVC ratio † 69.8 ± 3.4 67.4 ± 7.5 80.5 ± 1.0 79.0 ± 4.6PD20 (mg of methacholine) 0.17 ± 0.07 0.16 ± 0.08 — —Blood eosinophils (×106) 0.4 ± 0.1∗ 0.8 ± 0.6∗ 0.2 ± 0.03 0.1 ± 0.01
†Values are mean ± SEM.∗p <0.05 compared to “healthy” smokers and healthy never-smokers.FEV1 =forced expiratory volume in 1 second; FVC = forced vital capacity; PD20 = provocative dose of methacholine producing a 20% decrease in FEV1.
using the SPSS programme (version 15.0, SPSS Inc.,Chicago, IL, USA). The data between independent groupswere compared using the non-parametric Kruskal–Wallis H-test. If differences were found to be significant, the Mann–Whitney U-test was applied to compare differences betweentwo samples. Spearman’s rank correlation was calculated toassess correlations between parameters.
ResultsCharacteristics of Subjects
The characteristics of studied subjects are presented inTable 1. The average age did not differ between any of theinvestigated groups. The number of pack-years did not sig-nificantly differ between asthmatic smokers and “healthy”smokers. FEV1 was significantly lower in both asthma sub-groups compared with the control subjects. Asthma patientshad a significantly higher number of blood eosinophils thanthe healthy volunteers.
Cellular Patterns of Sputum and BALAll subjects tolerated the sputum induction procedure well.
However, successful induction could not be performed on 3asthmatic never-smokers, 2 asthmatic smokers, and 3 healthycontrol subjects. Consequently sputum was obtained from 23asthmatic never-smokers, 17 asthmatic smokers, 10 healthynever-smokers, and 10 “healthy” smokers. Table 2 showsthe cellular pattern of induced sputum from all the investi-gated groups. The total cell counts of induced sputum didnot significantly differ between any of the groups. Cellviability was greater than 85% in all samples. The per-centage of epithelial cells in asthmatic patients was signif-icantly higher (3.5% with a range of 2.5% to 7.0%) thanit was in nonasthmatic subjects (0.95% with a range of0.4% to 1.5%,p < 0.05). Asthmatic never-smokers and asth-
matic smokers had a significantly higher number of sputumeosinophils as compared to both control subgroups. In thesputum from asthmatic smokers, a slightly higher numberof neutrophils was detected as compared to asthmatic never-smokers; however, the difference was not statistically signif-icant (p = 0.074). The composition of sputum inflammatorycell counts did not significantly differ between the patientswho were healthy never-smokers and the “healthy” smokers.A tendency for sputum neutrophils to increase in “healthy”smokers compared to healthy never-smokers was noted, butthis was not statistically confirmed (p = 0.082).
The total cell counts in BAL did not significantly differbetween any of the investigated subgroups (Table 3). Asth-matic never-smokers had a significantly higher number ofBAL eosinophils as compared with both control subgroups.The cellular composition of BAL did not significantly differbetween “healthy” smokers and healthy never-smokers.
Concentrations of Eotaxin-1, Eotaxin-2, Eotaxin-3 and IL-5The mean ranges of eotaxin-1, eotaxin-2, eotaxin-3, and
IL-5 in serum, sputum supernatant, and BAL fluid are rep-resented in Table 4. Eotaxin-1 concentrations in the serumand sputum supernatant from asthmatic smokers and asth-matic never-smokers were significantly higher than they werein healthy never-smokers and “healthy” smokers. The su-pernatant in the sputum from asthmatic smokers was de-tected to contain a significantly higher concentration ofeotaxin-1 than it did from asthmatic never-smokers. A sim-ilar tendency was seen in the serum; however, the differ-ence was not statistically significant (p = 0.062). Asthmaticsmokers had significantly higher levels of serum, BAL,and sputum eotaxin-2 than the asthmatic never-smokers andhealthy never-smokers. BAL levels of eotaxin-2 were signif-icantly higher in both subgroups of asthma patients com-pared with the control subgroups. The levels of sputum
Table 2.—Induced sputum total cell numbers and differential cell counts in the studied groups.
Variable Asthmatic never-smokers Asthmatic smokers Healthy never-smokers “Healthy” smokers
Total cell count ×106/mL 2.93 ± 0.99 3.45 ± 1.09 2.27 ± 0.44 2.67 ± 1.86Neutrophils (%) 28.9 ± 5.16 31.60 ± 6.09 20.64 ± 12.96 25.25 ± 30.75Eosinophils (%) 6.52 ± 1.64∗,∗ast 3.30 ± 1.03∗ 1.4 ± 0.51 0.88 ± 0.13Lymphocytes (%) 7.68 ± 1.02 6.25 ± 2.27 10.67 ± 2.41 6.37 ± 2.13Macrophages (%) 56.9 ± 4.4 58.85 ± 4.80 67.29 ± 11.03 67.50 ± 28.75
Values are expressed as mean ± SEM.∗p < 0.05 compared to both control groups.∗∗p < 0.05 compared to asthmatic smokers.
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Table 3.—Total and differential cell counts in bronchoalveolar lavage (BAL) samples.
Variables Asthmatic never-smokers Asthmatic smokers Healthy never-smokers “Healthy” smokers
Total cell count × 106/mL 0.22 ± 0.03 0.28 ± 0.04 0.21 ± 0.03 0.27 ± 0.02Neutrophils (%) 1.00 ± 0.72 1.33 ± 0.03 0.95 ± 0.25 0.92 ± 0.43Eosinophils (%) 0.78 ± 0.16∗ 0.46 ± 0.01 0.30 ± 0.11 0.22 ± 0.08Lymphocytes (%) 30.96 ± 3.19 30.33 ± 6.78 21.80 ± 6.20 20.94 ± 5.69Macrophages (%) 67.26 ± 3.27 67.88 ± 6.61 76.95 ± 6.10 77.92 ± 6.29
Data are presented as mean ± SEM.∗p < 0.05 compared to healthy never-smokers and “healthy” smokers.
Table 4.—Eotaxin-1. eotaxin-2, eotaxin-3 levels in serum, induced sputum supernatant, and BALF.
Variable Asthmatic never-smokers Asthmatic smokers Healthy never-smokers “Healthy” smokers
Serum eotaxin-1 pg/mL 190.1 ± 21.5∗ 231.5 ± 39.2∗ 48.6 ± 15.0 82.6 ± 11.3Serum eotaxin-2 pg/mL 524.3 ± 68.4 959.4 ± 76.0∗∗ 490.4 ± 68.5 693.4 ± 230.1Serum eotaxin-3 pg/mL 54.4 ± 18.8 67.2 ± 15.7 28.9 ± 8.9 52.1 ± 29.8Sputum eotaxin-1 pg/mL 140.2 ± 9.5∗ 203.4 ± 10.0∗,∗∗∗ 40.1 ± 11.0 45.4 ± 20.0Sputum eotaxin-2 pg/mL 80.1 ± 24.7 118.0 ± 46.3∗, ∗∗∗ 39.3 ± 12.2 21.2 ± 11.2BAL eotaxin-1 pg/mL 50.1 ± 12.4 51.4 ± 11.7 29.0 ± 13.3 24.3 ± 12.7BAL eotaxin-2 pg/mL 243.9 ± 59.7∗ 347.8 ± 79.6* 118.8 ± 29.4 162.5 ± 67.9BAL eotaxin-3 pg/mL 39.9 ± 6.1 40.5 ± 7.8 33.2 ± 9.2 46.3 ± 20.5
Values are expressed as mean ± SEM.∗p < 0.05 compared to both control groups.∗∗p < 0.05 compared to asthmatic never-smokers and healthy never-smokers.∗∗∗p < 0.05 compared to asthmatic never-smokers.
Figure 1.—IL-5 concentrations in different tissue compartments. Serum, Induced sputum and BAL IL-5 concentrations (pg/ml) in asthmatic never-smokers,asthmatic smokers, healthy never-smokers and “healthy” smokers. Data are presented as mean ± SEM. #p < 0.05 compared to asthmatic smokers, ∗p < 0.05compared to “healthy” smokers.
Figure 2.—Correlation between eotaxin-1 and percent of eosinophils (A), neutrophils (B) in BAL fluid from never-smokers asthma patients.
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Figure 3.—Correlation between levels of eotaxin-2 (pg/ml) and percent of eosinophils (A) and neutrophils (B) in induced sputum from asthmatic smokers.
eotaxin-3 were below the detection limit in most of thesamples.
IL-5 levels in the serum and sputum from asthmatic never-smokers were significantly higher than they were from asth-matic smokers and “healthy” smokers (Figure 1). Analogoustrends were in the BAL fluid, but the inequality was notstatistically significant. Asthmatic never-smokers showed asignificantly higher amount of IL-5 in serum and sputum thanthe asthmatic smokers showed.
Relationship between Eotaxin Levels, Lung Function, andSmoking History
In asthmatic never-smokers, the level of BAL eotaxin-1was strongly related to the percentage and absolute numbersof BAL eosinophils and neutrophils (Figure 2). In this sub-group of patients, an inverse correlation between the BALeotaxin-2 and FEV1/FVC ratio (%) (Rs = −0.45, p < 0.05)was observed.
The number and percentage of sputum neutrophils andeosinophils obtained from asthmatic smokers correlated sig-nificantly with the eotaxin-2 concentration in the sputumsupernatant (Figure 3). In this subgroup of patients, negativecorrelations between the FEV1/FVC ratio (%) and sputumeosinophils as well as sputum eotaxin-1 (Rs = −0.75, Rs= −0.45 respectively, p < 0.05) were observed.
No significant correlations were observed in either of thetwo subgroups of healthy volunteers.
DiscussionThe key finding of this study is that increased levels of spu-
tum eotaxin-1, as well as serum, sputum, and BAL eotaxin-2were detected in asthmatic smokers as compared to asthmaticnever-smokers.
The hypothesis at the start of this study was that activecigarette smoking may influence asthmatic airway inflam-mation by changing the pattern of inflammatory cells andchemokine production. To prove this, asthma patients weregrouped according to their smoking histories, examined, andthen compared with the volunteer healthy smokers and never-smokers. During this study the focus was on the cellular partof chronic inflammation and the features of chemokine (i.e.,
eotaxin-1, eotaxin-2, and eotaxin-3) concentrations. To as-sess the profile of local and systemic inflammatory biomark-ers, different tissue compartments were used: blood serum,induced sputum supernatant, and BAL fluid. Moreover, totaland differential cell counts were obtained in the induced spu-tum and BAL to evaluate the inflammatory cells in proximaland distal airways. The use of induced sputum has previouslybeen shown to be an appropriate and feasible method to as-sess airway inflammation in chronic lung diseases (17). Evenso, induced sputum mainly represents larger airways (18) andmay be a combination of resident mucus (19). Nevertheless,although the use of bronchoscopy and BAL in research islimited owing to invasiveness, this method is still importantto evaluate the inflammatory patterns in distal airways.
In this study it was detected that, in non-atopic asthmat-ics, eosinophils are the predominant inflammatory cells ininduced sputum independent of the smoking status. Inducedsputum inflammatory cell counts and the total number ofcells did not differ between asthmatic smokers and never-smokers. As previously described (1,2), asthma is charac-terized by the presence of chronic airway inflammation inwhich eosinophils play an important role. However, a num-ber of various environmental factors (e.g., tobacco smoke andair pollution) may influence the pathogenesis of this disease.It was shown that tobacco smoke is associated with moresevere asthma symptoms and a poorer response to inhaledcorticosteroids mainly because of the increased number ofneutrophils in asthmatic airways (20, 21). In light of the re-sults of this study, it can be proposed that the attraction ofneutrophils to the asthmatic airways is a complex of patho-logical processes that depends on more than merely tobaccosmoke. However, the impact of smoking on airway inflam-mation can not be diminished. Our investigated asthmaticsmokers had higher numbers of sputum neutrophils than theasthmatic never-smokers, and this difference was close tothe formal significance level. Similar results were obtainedin BAL; however, the predominance of eosinophils in asth-matic smokers was not established as compared to those in thehealthy control subgroups. These data suggest that tobaccosmoke may modify allergic inflammation via suppression ofimportant proinflammatory cells and chemokines (22).
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EOTAXINS AND SMOKING 475
There are a few significant findings in this study. Thefirst one is that in steroid naıve asthmatic smokers theconcentrations of sputum eotaxin-1, as well as eotaxin-2 inserum, sputum, and BAL fluid are elevated in comparisonwith asthmatic never-smokers and healthy never-smokers.These results partly differ from previously published data.Oltmanns et al. performed an experiment with human air-way smooth muscle cells and suggested that cigarette smokeinhibits eotaxin release from airway smooth muscle (23). Si-multaneously Kubo et al. described the decreased eotaxinlevels in lungs from guinea pigs after repeated exposureto cigarette smoke (24). In contrast to the results of thosestudies, Yamamoto et al. described the elevated sputum andserum eotaxin levels in volunteer smokers as well as inasthma patients compared with healthy non-smokers (25).In concurrence, this study showed elevated concentrationsof sputum eotaxin-1 in asthmatic smokers. The relation be-tween tobacco smoke and elevation of eotaxin levels maybe explained by an increased expression of IL-4 in smokersthat stimulates production of eotaxin-2 and eotaxin-3 by thebronchial epithelial cells (26). Moreover, chronic tobaccosmoke alters the differentiation of the bronchial epithelialcells and may influence production of two chemokines –eotaxin-2 and eotaxin-3 (27). Additionally, to date, severalexperimental studies have been published that demonstratedthat concurrent exposure to allergen and mainstream tobaccosmoke increases the levels of allergic airway inflammationand remodeling in previously sensitized mice (28, 29). Miceexposed to tobacco smoke had an increased expression ofeotaxin-1 in airway epithelium, which was associated withincreased numbers of peribronchial eosinophils (29).
IL-5 is an important cytokine for eosinophil differentiation,chemotaxis, activation, and prolonged survival (1, 3). IL-5acts synergistically with eotaxins to promote eosinophilicairway inflammation (30). An experimental study with oval-bumin sensitized BALB/c mice demonstrated an increasedproduction of IL-5 after chronic exposure to cigarette smoke(31). Conversely, this study reveals decreased levels of serumand sputum IL-5 in asthmatic smokers and “healthy” smok-ers. These results suggested that chronic cigarette smoke de-creases IL-5 levels in the subgroups of smokers involved inthis study. Analogous trends were noted in the experimentalstudy, which demonstrated a suppressed expression of IL-5 inguinea pigs following repeated exposure to cigarette smoke(24). Considering these data together, it can be explainedwhy this study did not find a significant increase of eosinophilcounts in asthmatic smokers with elevated levels of eotaxin-1and eotaxin-2 compared with asthmatic never-smokers.
Another interesting finding of this study was that not onlywere the expected correlations between eosinophils and eo-taxins observed in both subgroups of asthma patients, butthere was also a significant correlation between BAL eotaxin-2 and number of BAL neutrophils detected in asthmaticnever-smokers. In asthmatic smokers, the levels of sputumeotaxin-1 were closely related to the number of sputum neu-trophils. The previously performed studies also detected asignificant neutrophil influx following a cutaneous injec-tion of eotaxin-1 and eotaxin-2 in human volunteers (32)as well as an increased sputum neutrophil count in asthmat-ics after eotaxin inhalation (33). These results highlightedthe important role of eotaxins into the recruitment not only
of eosinophils but also of other inflammatory cells (e.g., neu-trophils) during airway inflammation. Nonetheless, the exactmechanism of neutrophil recruitment is not yet understood.This could be a result of a direct chemotactic signal or re-sponse to eotaxin-induced mast cell degranulation and re-lease of neutrophil-specific chemokines.
In summary, this study showed the elevated levels of spu-tum eotaxin-1, as well as serum, sputum, and BAL eotaxin-2 in asthmatic smokers without a significant increase ofeosinophils compared to asthmatic never-smokers. The eo-taxin concentrations were related not only with number ofeosinophils but also with the number of neutrophils in all thestudied tissue compartments. These data permit a sugges-tion that smoking may influence change in asthmatic airwayinflammation by stimulating the production of eotaxins.
AcknowledgmentThe authors are grateful to Kestutis Malakauskas, Ph.D.,
for constructive discussions, to Elvyra Draugeliene, M.D.,and Vytis Dudzevicius, Ph.D., for their help in perform-ing bronchoscopies, to Sandra Ragaisiene, M.D., and InesaJermalaviciene for their technical support, and to Viole Arbasfor her assistance in editing this manuscript.
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