1 Department of Laboratory Medicine and Respiratory Medicine, School of Medicine, Tokyo University, Bunkyo-ku, Tokyo 113; and 2 World Health Organization Collaborating Centre, Tokyo Medical College, Shinjuku-ku; and 3 Department of Diagnostic Radiology, School of Medicine, Keio University, Tokyo 160, Japan
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ABSTRACT |
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To study the
inflammatory responses of small-airway epithelium in smokers, we
harvested enough living epithelial cells (1.97 × 106 ± 0.74 × 106) with a new ultrathin fiberscope from
the very peripheral airways of 22 current smokers and 17 subjects who
never smoked after informed consent was obtained. The cells were
keratin positive and composed mainly of nonciliated cells. The
expression levels of inflammatory markers [interleukin (IL)-8 and
intercellular adhesion molecule (ICAM)-1] were evaluated with
RT-PCR. The magnitude of the mRNA levels corrected by -actin
transcripts of IL-8 and ICAM-1 was significantly higher in the smokers
than in the nonsmokers (P < 0.001). Furthermore, among
current smokers, IL-8 mRNA levels correlated positively with the extent
of smoking history [in pack · years (packs/day × no. of years of smoking); r = 0.754, P < 0.001]. Spontaneously released IL-8 and soluble ICAM-1 levels (n = 12) from cultured epithelial cells were elevated in
subjects with a smoking history than in those without it (IL-8, 1,580 ± 29.6 vs. 354 ± 39.4 pg · 106
cells
1 · 24 h
1;
P < 0.001; soluble ICAM-1, 356.0 ± 45.9 vs. 112.9 ± 12.9 pg · 106
cells
1 · 24 h
1;
P < 0.01 by Student's t-test ). In contrast, the
epithelial cells from the main bronchi did not show such differences
between smokers and nonsmokers. Our study highlighted a close link
between smoking and the expression of inflammatory mediators such as
IL-8 and ICAM-1 in small airways. Our results also suggested that this new ultrathin bronchofiberscope promised a good approach for the evaluation of cellular changes in the small airways.
smoking; interleukin-8; intercellular adhesion molecule-1; peripheral airways
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INTRODUCTION |
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CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is one of the major causes of respiratory failure and eventual death (27). Both epidemiological and experimental studies suggest that tobacco smoke directly and indirectly drives inflammatory cells such as neutrophils into the airways (5, 8, 16) and that the inflammatory processes in small airways (<2 mm in diameter) are involved as the initial steps for the development of COPD (14). Airway epithelial cells have the potential to express chemokines and adhesion molecules important in cell recruitment, such as of neutrophils in vitro. Mio et al. (9) recently showed that bronchial epithelial cells isolated from major bronchi responded to tobacco smoke to release interleukin (IL)-8, a main chemoattractant for neutrophils. Recruited neutrophils express Mac-1, which binds to its countermolecule, intercellular adhesion molecule (ICAM)-1, on airway epithelium, suggesting a role for neutrophil migration and activation in the local lesions (24). However, the precise functional changes in small airways from tobacco smokers have not yet been clarified because of the lack of the technology. Here, we harvested living epithelial cells by brushing the small-airway mucosa under direct vision with a newly developed ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm, with a biopsy channel of 0.8 mm in diameter) (21, 23). Although the total number of recovered cells was relatively small in a previous report (23), we utilized a new large-size brush for harvesting a larger number of epithelial cells (21). In the present report, we evaluated the mRNA levels of inflammatory mediators important in airway inflammation, including IL-8 and ICAM-1, with the RT-PCR technique. In some samples, we cultured epithelial cells from small airways until confluence and evaluated the secretion of IL-8 and the soluble form of ICAM-1 (sICAM-1) with ELISA.
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MATERIALS AND METHODS |
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Subjects. A total of 39 Japanese subjects, 22 current smokers
and 17 who never smoked (29 men and 10 women, mean age 57.0 yr), were
included in the study. The clinical data are summarized in Table
1. Among smokers, the mean
pack · yr (packs/day × no. of yr of smoking)
was 41.3. All the subjects were free of respiratory diseases, with
normal chest X-ray films and no respiratory symptoms for at least 3 mo
before this study. Spirometry was performed on all subjects, with no
abnormal results in percent forced vital capacity (%FVC) and percent
forced expiratory volume in 1 s (%FEV1). Maximal flow
rates at 50 and 25% lung volume (50
and
25, respectively) were also
evaluated and are expressed as percent of predicted values (3). The
data for
25 were significantly
greater in the nonsmokers than in the smokers (P < 0.001). The age distribution and the data for %FVC,
%FEV1, and
50 were not
statistically different between nonsmoking and smoking subjects
(P > 0.05 by Student's t-test). The study was
planned according to the ethical guidelines following the Declaration
of Helsinki and given institutional approval, and an informed consent
was obtained from each subject.
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Bronchoscopy for isolation of very peripheral airway epithelial cells as well as of bronchial epithelial cells. The subjects underwent a bronchofiberscopic examination with a BF-XT20 fiberscope (Olympus, Tokyo, Japan) in standard fashion (20, 21). Under fluorographic guidance, an ultrathin fiberscope (BF-2.7T) was inserted through a 2.8-mm-diameter biopsy channel. A newly modified BC-0.7T brush was then inserted to collect cells by brushing the airway mucosal surfaces several times. Brushing of the mucosa was routinely performed at three or four 9th to 10th lower lobe bronchioles. The cells were immediately collected by vortexing the brush in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; GIBCO BRL, Life Technologies, Grand Island, NY). The cells were centrifuged for 5 min at 1,000 rpm. The recovered cells were washed twice in Hanks' balanced salt solution without calcium and magnesium (GIBCO BRL). The number of the cells was counted with a standard hemocytometer, and cell viability was assessed with the trypan blue dye exclusion technique (21, 23).
Bronchial epithelial cells from the main bronchi were harvested as described elsewhere (7, 20).
Cell counting, staining, and differential counting of the cells. The cytospin preparations from harvested cells were obtained by a cytocentrifuge and were routinely stained with Diff-Quik stain (Midorijuji, Kobe, Japan). The cytospins were also stained with periodic acid-Schiff (PAS) for the detection of secretory granules. For detection of keratin in the cells, the specimens were stained with anti-keratin (KL-1, Immunotech, Marseille, France), anti-vimentin (DAKO-vimentin, DAKOPatts, Glostrup, Denmark), or control IgG1 monoclonal antibodies with the avidin-biotin complex method (11, 13). In some samples, electron-microscopic examination was performed with transmission microscopy with a method similar to one previously reported (22). Briefly, the cells were cytocentrifuged onto coverslips, and the samples were placed into a fixative consisting of picric acid, paraformaldehyde, and glutaraldehyde. After fixation, the samples were embedded in 100% Epon 812 and trimmed, and ultrathin sections were stained with 2% uranyl acetate and 0.4% lead citrate. The differential counts of the harvested cells basically depended on the Diff-Quik and PAS staining and were divided into ciliated, secretory, and nonciliated cells as well as other inflammatory cells.
RT-PCR for IL-8 and ICAM-1 mRNAs in small-airway epithelial
cells. To assess the IL-8 and ICAM-1 mRNA levels in human
small-airway epithelial cells, a semiquantitative assay with RT-PCR, as
previously reported (2, 21), was performed. We used the epithelial cell samples for RT-PCR only after the samples contained <5%
nonepithelial cells as evaluated by Diff-Quik and keratin staining.
Total RNA was isolated from epithelial cell samples by the guanidium
thiocyanate-phenol-chloroform extraction method as described by
Chomczynski and Sacchi (4). Briefly, after cell counting and assessment
of cell viability, the cells (5.0 × 105 viable cells)
were lysed in solution D (4 M guanidium thiocyanate, 25 mM
sodium citrate, pH 7, 0.5% sarkosyl, and 0.1 M 2-mercaptoethanol), and
RNA was extracted from the solution by chloroform extraction. After
that, the isopropanol-precipitated RNA was washed twice with 70%
ethanol, dried, and resuspended in diethyl pyrocarbonate-treated water.
Extracted RNA was reverse transcribed to cDNA by using the Takara
RNA-PCR kit according to the manufacturer's recommendation (25).
Briefly, total RNA, random hexadeoxynucleotides as primers, and avian
myeloblastosis virus reverse transcriptase were used for cDNA
synthesis. The specific primer pairs used for PCR amplification were
5'-ATGACTTCCAAGCTGGCCGTGCT-3' (5' primer) and
5'-TCTCAGCCCTCTTCAAAAACTTCTC-3' (3'
primer) for IL-8, 5'-TATGGCAACGACTCCTTCT-3' (5'
primer) and 5'-CATTCAGCGTCACCTTGG-3' (3' primer) for
ICAM-1, and 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' (5' primer) and
5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3' (3' primer) for -actin (Clontech, Palo Alto, CA).
The reaction mixture contained 10 mM Tris · HCl (pH
8.3 at 25°C), 50 mM KCl, 1.5 mM MgCl2, 1 mg/ml of
gelatin, 0.4 µM each primer, 0.25 M diethyl p-nitrophenyl
monothiophosphate, 0.3 µg of cDNA, and 1 U of Taq polymerase
(Perkin-Elmer Cetus, Norwalk, CT) in 25 µl. Amplification was
performed for the allotted cycles of denaturation (94°C for 2 min),
annealing (60°C for 30 s), and extension (72°C for 1.5 min)
with a thermal cycler (Progene, Techne, Cambridge, MA).
The PCR cycle was determined by preliminary experiments showing a
linear relationship between PCR cycle and intensity of the signals on
ethidium bromide-stained agarose gels. For semiquantitative evaluation
of IL-8, ICAM-1, and -actin mRNAs, 30, 30, and 25 cycles,
respectively, were chosen. PCR product was run on a 1.0% agarose gel,
and the intensity of ethidium bromide fluorescence was evaluated by
National Institutes of Health Image version 1.61.
Cell culture. In some experiments where enough numbers of epithelial cell samples were obtained, the cells were plated in duplicate onto collagen-coated 48-well flat-bottom tissue culture plates (Koken, Tokyo, Japan) at a density of 5 × 104 cells/well with commercially available hormonally defined small-airway growth medium that consists of bovine pituitary extracts, hydrocortisone, human epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, bovine serum albumin, gentamicin, and amphotericin B (Clonetics, Sanko Junyaku, Tokyo, Japan). Morphological changes during culture were studied by phase-contrast microscopy showing polygonal, nonciliated cells with a tight connection to each other. Confluent monolayers of epithelial cells were stained with anti-keratin (KL-1, Immunotech), anti-vimentin (DAKO-vimentin, DAKOPatts), or control IgG1 monoclonal antibodies with the avidin-biotin complex method (11, 13) to show that the cells were of epithelial cell origin. Although only primary cells described above were used in the experiments, the cells could be passaged three to five times, frozen in liquid nitrogen, and reused for culture.
Evaluation of IL-8 and sICAM-1 release by cultured epithelial cells. On confluency, the epithelial cell-conditioned medium was harvested. Immunoreactive IL-8 and sICAM-1 were measured by specific ELISAs (R&D Systems, Minneapolis, MN) and are expressed in picograms per 106 cells per 24 hours (22).
Statistics. The results were analyzed by Student's t-test for parametric data, by the Mann-Whitney U-test for nonparametric data between the two groups, and by nonparametric equivalents of analysis of variance for multiple comparisons as previously reported (21, 22). Spearman's rank correlation test was used for correlation analysis between the two groups.
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RESULTS |
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Cell harvest, viability, and morphology. The total cell number
and cell differential counts evaluated by Diff-Quik, PAS, and keratin
staining are shown in Table 2. The total
cell number, viability, and cell differential counts were not
statistically different between smokers (cases
1-22) and nonsmokers (cases
23-39). The number of recovered cells ranged from 1.50 × 106 to 3.56 × 106, with a mean of
1.97 × 106. Cell viability ranged from 60.5 to
78.5%, with a mean of 65.8%. Most of the cells were nonciliated round
cells that were positive to keratin staining (Fig.
1, A and B). By
transmission electron microscopy, these cells had tonofilaments with no
apparent basal bodies, suggesting that they were nonciliated epithelial
cells (Fig. 1C).
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As shown in Table 2, the major contaminating cells were neutrophils, and in two cases (cases 21 and 22), the percentage of neutrophils was >5%. Therefore, the data of these cases were excluded from the subsequent analysis.
Small-airway epithelial cells in smokers showed increased IL-8 mRNA
compared with that in nonsmokers by RT-PCR. Among 20 current smokers (2 cases were excluded as described in Cell harvest,
viability, and morphology) and 17 subjects who never smoked, the
relative intensity of IL-8 mRNA to -actin was statistically
increased in smokers than in nonsmokers as shown in Fig.
2, A and B. To exclude the possibility that this result was because of the
contamination of a few nonepithelial cells such as neutrophils, we
incubated the cells on collagen-coated tissue culture plates for 90 min to allow the cells to attach; then the plates were rinsed twice and
total cellular RNA was extracted [n = 7 in the smoking
group (cases 1-7) and 7 in the nonsmoking group
(cases 23-29)]. With this technique, the
keratin-positive cells were always >98% in all the samples as
assessed by immunostain on collagen-coated LabTec chamber slides. The
results again elucidated that the relative signals for IL-8 mRNA were
significantly increased in smokers (Fig. 2C).
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Small-airway epithelial cells in smokers showed increased ICAM-1
mRNA compared with that in nonsmokers by RT-PCR. As shown in Fig.
3, A and B, the magnitude
of ICAM-1 mRNA corrected by -actin transcripts was significantly
higher in the smokers compared with the nonsmokers. When the attached
cell samples were utilized for RT-PCR, it was again significant for
ICAM-1 mRNA levels (Fig. 3C).
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Correlation between the levels of RT-PCR gene expression of
inflammatory markers and smoking history. Among current smokers, IL-8 mRNA levels correlated positively with the extent of smoking history when the signals were normalized by -actin transcripts (r = 0.754, P < 0.001; Fig.
4A). There was also a significant correlation between IL-8 mRNA levels and current amount of smoking habit (r = 0.717, P < 0.001; Fig. 4B).
However, the levels for ICAM-1 did not show significant correlation
with the magnitude of smoking (Fig. 5,
A and B).
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Correlation between mRNA levels of inflammatory mediators and lung
function tests. None of the mRNA levels of inflammatory mediators
significantly correlated with %FVC, %FEV1,
%50, or %
25 in smokers and nonsmokers (data not shown).
IL-8 and sICAM-1 release versus smoking. The cells from smokers
(n = 12, cases 1-12) and nonsmokers
(n = 7, cases 23-29) were cultured until confluence in small-airway growth medium.
Immunocytochemical studies demonstrated that the cells were keratin
positive (Fig. 1D) but vimentin negative, showing that the
cells were epithelial cells. Spontaneous release of IL-8 protein was
elevated in epithelial cells from smokers compared with that from
nonsmokers (1,580 ± 29.6 vs. 354 ± 39.4 pg · 106
cells1 · 24 h
1;
P < 0.001; Fig. 6A). That
was also the case for release of sICAM-1 into the medium (356.0 ± 45.9 vs. 112.9 ± 12.9 pg · 106
cells
1 · 24 h
1;
P < 0.01 by Student's t-test; Fig. 6B).
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Comparison of IL-8 and ICAM-1 expression between proximal and
peripheral airway epithelial cells. Bronchial epithelial cells from
the main bronchi were harvested from smokers and nonsmokers. The
magnitude of IL-8 and ICAM-1 mRNA levels studied by a similar method
did not show any difference between the two groups (Fig. 7). There was no significant correlation
between the magnitude of IL-8 mRNA levels of proximal and peripheral
airway epithelial cells (r = 0.120, P > 0.05).
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DISCUSSION |
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Our present studies highlighted a close link between smoking and IL-8
gene expression in small-airway epithelial cells. We could safely
harvest bronchial epithelial cells from small airways with high purity
(21, 23). By employing a larger brush than that used in the original
report (23), we obtained enough cells for cell and molecular biological
analyses. The expression levels of IL-8, which is a potent neutrophil
chemotactic factor, were significantly increased in smokers.
Importantly, the magnitude of IL-8 signals corrected by -actin
transcripts showed a positive correlation with the amount of cigarette
smoking. ICAM-1 mRNA levels were also increased in smokers, and
spontaneously released IL-8 and sICAM-1 were again statistically
increased in smokers than in nonsmokers. ICAM-1 is believed to be an
important adhesion molecule for neutrophil accumulation, adhesion to
the epithelium, activation, and transepithelial migration (12, 24).
Therefore, these findings suggested a potential mechanism by which
tobacco smoke activates small-airway epithelium to express IL-8 and
ICAM-1, thereby resulting in a local accumulation of neutrophils. We
also studied the expression levels of IL-8 and ICAM-1 in bronchial epithelial cells obtained from the main bronchi, but there was no
statistical difference between smokers and nonsmokers.
It is well known that cigarette smoking causes neutrophil migration and
accumulation in the lungs (5, 8, 16), which impose a major risk for
COPD, especially pulmonary emphysema. Neutrophil-derived products such
as elastases destroy the bronchiolar and alveolar structures, followed
by remodeling of the airway and parenchymal tissues (17, 19). Such
dynamic sequestration of neutrophils into the lung may be induced by
the direct effect of tobacco contents (25). However, recent data
suggest that cigarette smoke stimulates airway epithelial cells to
release chemotactic activities such as IL-8 for neutrophils (9, 18). IL-8 is believed to play an important role in the pathogenesis of
various airway inflammatory diseases (6, 21). Airway epithelial cells,
the first cells to contact the exogenous agents including tobacco
smoke, are capable of expressing and releasing this chemokine (10, 15)
as well as ICAM-1. Mio et al. (9) demonstrated that human bronchial
epithelial cells obtained from proximal airways released IL-8 in
response to cigarette smoke extracts in vitro. They also found that
IL-8 levels in bronchoalveolar lavage fluid (BALF) showed a significant
correlation with neutrophil counts in BALF, supporting the hypothesis
that cigarette smoke induces bronchial epithelial cells to release IL-8
and that this may contribute to airway inflammation in smokers. Because
there is only a thin barrier between airway lumens and submucosal
layers in small airways, the increased levels of IL-8 and ICAM-1
expression in small-airway epithelium could be crucial for cell
migration and activation. It was also interesting to note that such
increased levels of IL-8 and ICAM-1 did not show any correlation to
small-airway obstruction as assessed by
25. Other factors acting on airway
fibroblasts, such as transforming growth factor-
, might be involved
in the airway obstruction frequently found among heavy smokers.
It remains controversial whether functional changes in proximal and small airways are similar in disease states such as COPD and asthma. For example, a number of inflammatory changes such as a thickened lamina reticularis have been linked to chronic obstructive lung functions in asthma (26). However, the vast majority of studies have been performed in large airways, with little regard to the small airways (1). In the present study, there was increased expression of IL-8 and ICAM-1 in small-airway epithelium but not in proximal bronchial epithelium from smokers. The reason for this difference between proximal and small airways is unclear. It is probable that a variety of compounds other than tobacco smoke (i.e., viral particles, bacteria, and air pollutants) may contribute to the functional changes in proximal airways.
In summary, this report demonstrated a possible role of epithelium in sequestration of neutrophils to the small airways in tobacco smokers. Obtaining bronchial epithelial cells from living human donors (7) has greatly facilitated the research on respiratory cell and molecular biology. Our new ultrathin bronchofiberscope in association with a new brush technique promised a good approach for evaluation of cellular and molecular changes in the small airways.
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ACKNOWLEDGEMENTS |
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We thank C. Sakamaki, A. Hashimoto, and T. Kobayashi for excellent technical support.
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FOOTNOTES |
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This work was supported in part by grants from The Japan Ministry of Education, Science and Culture; the Adult Diseases Memorial Foundation (Tokyo, Japan); and the Manabe Medical Foundation (Tokyo, Japan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Takizawa, Dept. of Laboratory Medicine, Univ. of Tokyo, School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan (E-mail: takizawa-phy{at}h.u-tokyo.ac.jp).
Received 2 August 1999; accepted in final form 7 December 1999.
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