1 Departments of Laboratory Medicine and Respiratory Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; 2 Fourth Department of Internal Medicine, Nippon Medical School, Tokyo 113-860; 3 Department of Molecular Pathology, Institute of Tuberculosis, Kiyose 204-0022; 4 Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; 5 World Health Organization Collaborating Center, Tokyo Medical College, Tokyo 160-0022; and 6 Faculty of Health Sciences, Aomori University of Health and Welfare, Aomori 030-8505, Japan
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ABSTRACT |
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Fine particles derived from diesel
engines, diesel exhaust particles (DEP), have been shown to augment
gene expression of several inflammatory cytokines in human airway
epithelial cells in vitro. However, it remains unclear whether or not
DEP have any effect on the expression and production of eotaxin, an
important chemokine involved in eosinophil recruitment into the
airways. We studied the effects of DEP by using a conventional
suspended DEP and by a recently established in vitro cell exposure
system to diesel exhaust (Abe S, Takizawa H, Sugawara I, and Kudoh S, Am J Respir Cell Mol Biol 22: 296-303, 2000). DEP
showed a dose-dependent stimulatory effect on eotaxin production by
normal human peripheral airway epithelial cells as well as by bronchial
epithelial cell line BET-1A as assessed by specific ELISA. mRNA levels
increased by DEP were shown by RT-PCR. DEP showed an additive effect on IL-13-stimulated eotaxin expression. DEP induced NF-B activation by
EMSA as previously reported but did not induce signal transducer and
activator of transcription (STAT) 6 activation according to Western
blot analysis. Finally, antioxidant agents (N-acetyl
cysteine and pyrrolidine dithiocarbamate), which inhibited NF-
B
activation but failed to affect STAT6 activation, almost completely
attenuated DEP-induced eotaxin production, whereas these agents failed
to attenuate IL-13-induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-
B-dependent, but
STAT6-independent, pathways.
airway epithelium; signal transduction; interleukin-13
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INTRODUCTION |
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EOTAXIN IS A CC CHEMOKINE
that plays an important role in eosinophil accumulation in a variety of
allergic disorders (14, 17, 23). Animal models of allergic
asthma showed that targeted disruption or antibody against eotaxin
partially blocked eosinophil accumulation in the lung (24,
32). Histopathological studies demonstrated that there is an
intense protein expression of eotaxin in airway epithelium from
patients with bronchial asthma (11, 17, 19). Recently,
genomic structure of human eotaxin has been reported (12),
and subsequent studies demonstrated that its transcriptional regulation
was dependent on promotor regions that contain nuclear factor NF-B
binding sites and signal transducer and activator of transcription
(STAT) 6 binding sites (18).
There is increasing circumstantial data suggesting that exposure to
increased levels of inhalable particulate pollutants (PM10) is related
to the increased prevalence of allergic airway disorders such as asthma
and allergic rhinitis (5, 6, 9, 14, 21). Recent reports
(4, 22) suggest that particles with diameters of <2.5
µm (PM2.5) have an important role in triggering the biological
responses within the lung. In urban areas, fine particulate matter
produced from diesel engines [diesel exhaust particles (DEP)] is one
of the major constituents of PM2.5. Repeated exposure to DEP in mice
induces intense inflammatory reactions that mimic those found in
bronchial asthma (25). Intranasal administration of DEP
extracts induced local Th2-type cytokine production in human atopic
volunteers (8). It is quite likely that these biological
responses to DEP were via cytokines, chemokines, and other inflammatory
mediators locally produced in the airways. To date, several factors,
such as IL-1, TNF-, and Th2-type cytokines (IL-4), have been
reported to upregulate eotaxin gene expression (9, 17, 18,
26). However, it remains unclear whether or not environmental
pollutants such as DEP have any effect on eotaxin expression.
In the present study, we attempted to study the effects of DEP on eotaxin gene expression in human bronchial epithelial cells by using conventional suspended particles and by a newly established in vitro cell exposure system to diesel exhaust (DE) (1).
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MATERIALS AND METHODS |
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Culture of bronchial epithelial cells. Normal small airway epithelial cells were obtained from healthy volunteers as reported previously (31). Briefly, the subjects underwent a bronchofiberscopic examination with a BF-XT20 fiberscope (Olympus, Tokyo, Japan) in a standard fashion. 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 (30). The cells were immediately collected by vortexing the brush in RPMI 1640 medium supplemented with 10% FCS (GIBCO, 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). The number of the cells was counted by a standard hemocytometer, and the cell viability was assessed by trypan blue dye exclusion. The cells were plated onto collagen-coated, 48-well, flat-bottomed tissue culture plates (Koken, Tokyo, Japan) at a density of 2 × 104 cells/well in duplicates with hormonally defined SAGB medium (Clonetics; SankoJunyaku, Tokyo, Japan). Morphological changes during culture were studied by a phase-contrast microscopy showing polygonal, nonciliated cells with tight connections to each other. Confluent monolayers of epithelial cells were stained with anti-keratin (KL-1; Immunotech, Marseille Cedex, France ) or anti-vimentin (DAKO-Vimentin; DAKOPatts, Glostrup, Denmark ) or with control IgG1 monoclonal antibodies using an avidin-biotin complex method to show that the cells were of epithelial cell origin (30). The three- to four-passaged cells were used for the experiments.
The human bronchial epithelial cell line BET-1A (a kind gift from Drs. J. F. Lechner and C. C. Harris, National Cancer Institute, Bethesda, MD) was cultured as reported (16). Briefly, the cells were plated onto collagen-coated, 24-well, flat-bottomed tissue culture plates (Koken) at a density of 5 × 104 cells/well in hormonally defined Ham's F-12 medium, which contained 1% penicillin-streptomycin, 5 µg/ml insulin (GIBCO), 5 µg/ml transferrin (GIBCO), 25 ng/ml epidermal growth factor (Collaborative Research, Lexington, MA), 15 µg/ml endothelial cell growth supplement (Collaborative Research ), 2 × 10Preparation of DEP and conventional exposure to the cells. The engine used for preparation of DEP was a 2,300-cc diesel engine (Isuzu Automobile, Tokyo, Japan). The engine was connected to an EDYC dynamometer (Meiden-Sya, Tokyo, Japan) and was operated using a standard diesel fuel at 1,050 rpm under a load of 6 torque (kg/m). The exhaust was introduced into a stainless steel dilution tunnel (450 mm diameter × 6,250 mm). The DEPs were collected on glass fiber filters (203 mm × 254 mm) in a constant-volume sampler system equipped at the end of the dilution tunnel. The temperature at the sampling point was <50°C. Different concentrations of DEP suspended in the sterile medium were added to the cells. Preliminary experiments showed that DEP at 0.1-50 µg/ml had no significant cytotoxicity to BET-1A cells and normal human bronchial epithelial cells as assessed by trypan blue dye exclusion, lactate dehydrogenase release assay, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In some experiments, different doses of human recombinant IL-13 with and without DEP were added to the cells.
In vitro cell exposure system to DE. As Bayram and colleagues pointed out (2), in vitro studies have several limitations, because defense mechanisms in vivo are absent and the particles may change once suspended. We have recently developed a system that enabled the cells to be exposed to freshly generated DE (22).
Briefly, a 2,300-ml diesel engine (Isuzu Automobile) was operated at a speed of 1,050 rpm and 80% load with a commercial light oil. The concentration of fine particles and the densities of gaseous materials including CO, NO2, and SO2 were measured as described in detail previously (1) with the averaged levels of 1 mg/m3, 10.6 ppm, 7.3 ppm, and 3.3 ppm, respectively. The concentration of DEP at the tubes just before the inlet of the culture container was averaged to 100 µg/m3. The cells were exposed to DE in a constant-flow system at different time intervals (0, 120, 240, 480, and 840 min). Sham exposure was carried out while the engine was turned off. To evaluate the effects of the gases in DE on cytokine production, a glass fiber filter paper (Toyo Roshi, Tokyo, Japan) was introduced just before the inlet of the culture container to remove >99.99% of the DEP (1).Cytokine assay. Specific immunoreactivity for eotaxin in culture supernatants was measured by ELISA kits (R & D Systems, Minneapolis, MN). Each sample was assayed in duplicate as recommended by the manufacturer.
Northern blot analysis for eotaxin mRNA.
Northern blot analysis was performed to study the effect of DEP on
eotaxin mRNA expression in human bronchial epithelial cells by the
method described previously (4). Briefly, total cellular RNA was extracted by the method of Chomczynski and Sacchi
(3) and was electrophoresed on formaldehyde-denatured
agarose gel (10 µg/lane) followed by capillary transfer onto a
Biodyne nylon membrane. RNA integrity and equivalency of loading were
routinely evaluated by ethidium bromide fluorescence. Blots were baked, prehybridized, and hybridized with 32P 5'-end-labeled
oligonucleotide probes specific for human eotaxin and -actin. A
human complementary DNA for human genomic eotaxin (9) was
a kind gift from Dr. P. D. Ponath (LeukoSite, Cambridge, MA). Blots were stringently washed after hybridization and
exposed to X-ray film.
RT-PCR for eotaxin mRNA expression in human bronchial epithelial
cells.
To assess the eotaxin mRNA levels in BET-1A cells exposed to DE by a
new system, a semiquantitative assay utilizing RT-PCR was performed as
previously reported (1). Total RNA was isolated by the
guanidinium thiocyanate-phenol-chloroform extraction method as
described by Chomczynski and Sacchi (3). Briefly, after the cell counting and assessment of cell viability, the cells (5 × 105 viable cells) were lysed in solution D
[4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.5%
sarcosyl, 0.1 M 2-mercaptoethanol] and RNA was extracted from the
solution by chloroform extraction. After that, the
isopropanol-precipitate RNA was washed twice with 70% ethanol, dried,
and resuspended in diethylpyrocarbonate-treated water. Extracted RNA
was reverse transcribed to cDNA with a Takara RNA-PCR kit according to
the manufacturer's recommendation. Briefly, total RNA, random
hexadeoxynucleotides as primer, and avian myeloblastosis virus
reverse transcriptase were used for cDNA synthesis. The specific primer
pairs used for PCR amplification are listed below: eotaxin 5'-primer
5'-GCCCTGGACACCAACTATTGCT-3', 3'-primer 5'-AGGCTCCAAATGTAGGGGCAGG-3'; -actin 5'-primer 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3',
3'-primer 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3' (Clontech, Palo
Alto, CA).
EMSA for the detection of NF-B.
After the cells were washed with PBS, the nuclear proteins were
isolated by the method reported previously (29). In brief, 2-3 × 106 cells were harvested with the addition
of trypsin-EDTA solution (GIBCO), rinsed in Tris-buffered saline,
resuspended in lysis buffer (in mM: 10 HEPES, 10 KCl, 0.1 EGTA, 0.1 EDTA, 1 DTT, 0.5 PMSF) and incubated on ice for 15 min. Nonidet P-40
(10%) was added to lyse the cells, and then the cells were centrifuged
for 6 min at 4°C at 600 g. The nuclear pellet was
resuspended in extraction buffer (in mM: 20 HEPES, 50 KCl, 400 NaCl, 1 EDTA, 1 EGTA, 1 DTT, 1 PMSF) and vortexed for 15 min on ice. The
nuclear extract was centrifuged for 15 min at 12,000 rpm at 4°C. The
supernatant was collected, divided into aliquots, and stored at
70°C. Protein concentration was determined by the Bradford
dye-binding procedure (Bio-Rad protein assay), standardized with bovine
serum albumin.
Western blot analysis for STAT6 activation. For the extraction of the protein, the cells were washed twice with Ca2+- and Mg2+-free Dulbecco's PBS and lysed in 2× SDS sample buffer [125 mmol/l Tris · HCl (pH 6.8), 4.6% wt/vol SDS, 20% glycerol, 10% 2-mercaptoethanol]. The samples were heated in a boiling water bath for 5 min to fully denature the proteins and then centrifuged at 1,200 g for 5 min to remove insoluble debris.
Proteins extracted by the above-described methods were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were probed with phosphospecific rabbit polyclonal anti-STAT6 (p-STAT6) antibody (New England Biolabs) or anti-STAT6 antibody (Upstate Biotechnology). The bands corresponding to the protein of interest were visualized with alkaline phosphatase-conjugated secondary antibodies.Effect of pyrrolidine dithiocarbamate and N-acetyl cysteine on
eotaxin production and gene expression.
To evaluate the role of activation of the transcription factor NF-B,
we treated the cells with different concentrations of pyrrolidine
dithiocarbamate (PDTC) or N-acetyl cysteine (NAC) (pH
adjusted to 7.4) 1 h before the addition of DEP (25 µg/ml) or
IL-13 (10 ng/ml) and studied the levels of eotaxin mRNA as well as its
protein production in the supernatants. The effects of NAC on NF-
B
activation and STAT6 activation were also studied.
Statistical analysis. The results were analyzed by Student's t-test for comparison between two groups and by nonparametric equivalents of analysis of variance (ANOVA) for multiple comparisons as reported previously (29, 30).
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RESULTS |
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Increased production of immunoreactive eotaxin by suspended DEP.
When the cells were incubated with DEP for 24 h, DEP increased
eotaxin release from cultured normal human peripheral airway epithelial
cells (Fig. 1A) and BET-1A
cells (Fig. 1B) in a dose-dependent manner. Human
recombinant IL-13 showed a dose-dependent stimulatory effect on eotaxin
production (Fig. 1C). Simultaneous treatment with a
near-maximal dose of IL-13 (25 ng/ml) and different doses of DEP showed
an additive effect on eotaxin production in BET-1A cells (Fig.
1D). Such was also the case for normal human peripheral airway epithelial cells (data not shown).
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Suspended DEP increased the levels of eotaxin mRNA in human
bronchial epithelial cells.
The total cellular RNA was extracted after different time periods with
and without 25 µg/ml DEP, and the steady-state levels of eotaxin mRNA
were studied by Northern blot analysis. DEP at 25 µg/ml showed a
time-dependent stimulatory effect on eotaxin mRNA levels up to 12 h in both the normal airway epithelial cells and BET-1A cells (data not
shown). As shown in Fig. 2, A and
B, DEP in the range of
1-50 µg/ml showed a dose-dependent stimulatory effect on eotaxin
mRNA levels when evaluated 12 h after the addition to the cells.
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DE exposure in vitro increased eotaxin mRNA levels and production
in BET-1A cells.
We also studied the protein production and mRNA levels of eotaxin in
DE-exposed BET-1A cells by the new exposure system. As shown in Fig.
3A, DE exposure showed a
time-dependent stimulatory effect on eotaxin production, whereas DE
with a glass fiber filter to remove particles had no significant
effect. The mRNA levels for eotaxin as evaluated by RT-PCR were
increased by DE exposure, as shown in Fig. 3B.
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EMSA for the detection of NF-B activation by suspended DEP.
Since it has been suggested that the nuclear transcription factor
NF-
B plays an important role in the transcriptional regulation of
eotaxin gene expression (18), we attempted to evaluate the effect of DEP on NF-
B activation in human bronchial epithelial cells
by EMSA. The cells were treated with different concentrations of
suspended DEP for 6 h, and the nuclear extracts were isolated for
EMSA as described in MATERIALS AND METHODS. DEP at
1-25 µg/ml increased the nuclear protein binding to the labeled
oligonucleotide double-stranded DNA (Fig.
4A). The specificity of the
binding was ascertained by the supershift of the bands with antibodies to p65 and p50. In contrast to the effect of DEP, human recombinant IL-13 failed to activate NF-
B.
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Suspended DEP failed to activate STAT6.
Human recombinant IL-13 induced phosphorylation of STAT6, but it
suspended DEP failed to activate STAT6 by Western blot analysis, as
shown in Fig. 5.
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Effect of NAC and PDTC on production and mRNA levels of eotaxin
induced by suspended DEP.
NAC and PDTC, both being antioxidant reagents with an inhibitory
potential of NF-B activation, showed a dose-dependent inhibitory effect on DEP-induced eotaxin production when studied 24 h after DEP treatment (25 µg/ml; Fig.
6A). It was also shown that
NAC and PDTC (10 mM) blocked DEP-induced eotaxin mRNA levels (Fig. 6B). In contrast, NAC and PDTC failed to suppress eotaxin
production and mRNA levels in IL-13-stimulated BET-1A cells (Fig.
6B).
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NAC attenuated NF-B activation but did not affect STAT6
activation in BET-1A cells.
Pretreatment with NAC 1 h before stimulation with DEP attenuated
NF-
B activation in a dose-dependent fashion, as shown in Fig.
7A. In contrast, NAC failed to
affect the activation of STAT6 induced by the treatment with IL-13
(Fig. 7B). These results suggested that the attenuating
effect of NAC on eotaxin expression was largely via suppression of
NF-
B activation.
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DISCUSSION |
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In the present study, we demonstrated that DEP, one of the important air pollutants, stimulated eotaxin gene expression and protein production in human normal and transformed bronchial epithelial cells. This effect was also ascertained by a new in vitro cell exposure system that we recently established (1). DEP with near-maximal concentration of IL-13 showed an additive effect on eotaxin gene expression, suggesting that DEP might affect eotaxin gene expression via different pathways from those of IL-13.
Epidemiological studies have suggested that there may be a link between the incidence of respiratory diseases and concentrations of particulate matter in atmosphere, especially small particles such as PM2.5 (11, 12, 19). DEP have been considered to comprise the major part of PM2.5 in urban areas, and in vivo and in vitro experiments have suggested their potent activity in the respiratory tracts. Inhalation of DEP in mice resulted in eosinophilia, Th2-type cytokine production, and increased airway responsiveness (25, 27). Transnasal challenges of DEP-derived extract in humans enhanced local IgE and Th2-type cytokine production (7, 8). DEP have been shown to have a stimulatory effect on airway epithelial cells to express cytokines such as IL-6, IL-8, and granulocyte macrophage-colony stimulating factor and adhesion molecules such as ICAM-1 (2, 20, 28). Eotaxin is a potent chemotactic and activating factor for eosinophils (15, 17, 23) and has been shown to play an important role in eosinophil accumulation in the airways in asthma and allergic rhinitis (11, 19). To the best of our knowledge, this is the first report showing that DEP augments eotaxin gene expression in human bronchial epithelial cells in vitro. Our results suggested that DEP directly induces production of this potent eosinophil chemoattractant, and this chemokine in concert with other potent chemokines such as regulated on activation, normal T cell expressed, and presumably secreted exaggerate local eosinophil infiltration in asthma.
Recently, the gene structure of human eotaxin was reported, and its
promotor regions contain binding sites to both NF-B and STAT6.
Matsukura and associates (18) studied the transcriptional regulation of the human eotaxin gene and clearly demonstrated that
TNF-
and IL-4 independently activate NF-
B and STAT6 to upregulate
eotaxin gene expression. They transfected the human bronchial
epithelial cell line BEAS-2B with luciferase reporter plasmids that
contained normal or mutated eotaxin promoters. Eotaxin promoter
activity was increased by TNF-
and IL-4 in the cells with normal
plasmids. When the plasmids that were mutated at the NF-
B binding
site were used, the response to TNF-
, but not to IL-4, was lost.
These findings clearly demonstrated that activation of NF-
B induced
eotaxin gene transcription in vitro. Our studies with EMSA showed that
DEP induced the activation of the transcription factor NF-
B, which
is considered to play an important role in the gene regulation of
eotaxin as reported previously (18).
We also studied the effect of DEP on the phosphorylation processes of
STAT6 by Western blot analysis, but DEP failed to show any effect. This
was in sharp contrast to the activity of IL-13. This Th2-type cytokine,
partially sharing its receptor with IL-4, showed a significant
stimulatory effect on eotaxin gene expression and STAT6 activation but
did not stimulate NF-B activation in human bronchial epithelial cell
line BET-1A. Finally, antioxidants NAC and PDTC, which had NF-
B
inhibitory activity but no effect on STAT6 activity, suppressed the
mRNA levels of eotaxin. These results suggested that oxidant-dependent,
NF-
B-mediated, but STAT6-independent pathways are involved in
DEP-induced eotaxin expression, although admittedly these antioxidants
can also act via other mechanisms.
IL-13 has been reported to be increased in asthmatic airways and play important roles in its pathogenesis (33). Our data showed that IL-13 and DEP had an additive effect on eotaxin gene expression in human airway epithelial cells via different intracellular pathways. Therefore, it is probable that DEP exposure may exaggerate eosinophil accumulation and activation in asthmatic patients at lower concentrations than in nonasthmatic populations.
In conclusion, DEP stimulates eotaxin gene expression largely via
NF-B-mediated processes in human bronchial epithelial cells. These
findings may give new insight into the molecular mechanisms of DEP
action in the human respiratory tract, especially in cases of allergic
inflammation such as in asthma.
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ACKNOWLEDGEMENTS |
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We thank Takako Kobayashi and Makiko Baba for their excellent technical support.
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FOOTNOTES |
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This work was supported by a grant from Japan Ministry of Education, Science and Culture, the Pollution-Related Health Damage Compensation and Prevention Association of Japan, and The Manabe Medical Foundation.
Address for reprint requests and other correspondence: H. Takizawa, Dept. of Respiratory Medicine, Univ. of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (E-mail: takizawa-phy{at}h.u-tokyo.ac.jp).
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. Section 1734 solely to indicate this fact.
First published February 7, 2003;10.1152/ajplung.00358.2002
Received 25 October 2002; accepted in final form 27 January 2003.
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