Diesel exhaust particles upregulate eotaxin gene expression in human bronchial epithelial cells via nuclear factor-kappa B-dependent pathway

Hajime Takizawa1, Shinji Abe2,3, Hitoshi Okazaki1, Tadashi Kohyama1, Isamu Sugawara3, Yoshinobu Saito2,3, Takayuki Ohtoshi1, Shin Kawasaki1, Masashi Desaki1, Kazuhiko Nakahara1, Kazuhiko Yamamoto1, Kouji Matsushima4, Mitsuru Tanaka5, Masaru Sagai6, and Shoji Kudoh2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa 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-kappa B-dependent, but STAT6-independent, pathways.

airway epithelium; signal transduction; interleukin-13


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-alpha , 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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-10 M triiodothyronin (GIBCO), and 10-7 M hydrocortisone (GIBCO). The cells were incubated in a humidified atmosphere at 37°C and 5% CO2. The cells at 12-15 passages were used for the experiments.

Preparation 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 beta -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'; beta -actin 5'-primer 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3', 3'-primer 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3' (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 gelatin, 0.4 µM of each primer, 0.25 M dNTP, 1.0 µg cDNA, and 1 unit of Taq polymerase (Perkin-Elmer-Cetus, Norwalk, CT) in total volume of 25 µl. Amplification was performed for allotted cycles of denaturing (94°C, 2 min), annealing (60°C, 30 s), and extension (72°C, 1.5 min) using a thermal cycler (Progene; Techne, Cambridge, MA). The PCR cycle was determined by preliminary experiments showing a linear relationship between PCR cycles and intensity of signals on ethidium bromide-stained agarose gels as reported previously (34). For semiquantitative evaluation of eotaxin and beta -actin mRNAs, 30 cycles were chosen for beta -actin and eotaxin. PCR product was run on a 1.0% agarose gel, and the intensity of ethidium bromide fluorescence was evaluated by NIH Image version 1.61.

EMSA for the detection of NF-kappa 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.

For the detection of the NF-kappa B DNA binding, a NF-kappa B binding protein detection kit (GIBCO) was used. The sequence of the oligonucleotides containing a tandem repeat of the consensus sequence for the NF-kappa B DNA binding site (underlined) was 5'-GATCCAAGGGGACTTTCCATGGATCCAAGGGGACTTTCCATG-3', 3'-GTTCCCCTGAAAGGTACCTAGGTTCCCCTGAAAGGTACCTAG-5'.

Synthetic double-stranded oligonucleotides were labeled with [gamma -32P]ATP using T4 polynucleotide kinase as recommended by the manufacturer. The DNA binding reaction was conducted at room temperature for 20 min in a volume of 25 µl. The reaction mixture contained 10 µg nuclear extract, 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (vol/vol) glycerol, 0.08 mg/ml sonicated salmon sperm DNA, and 32P-labeled double-stranded oligonucleotides at 0.7 fmol/µg nuclear extract. After incubation, the samples were loaded onto a 4% polyacrylamide gel [polyacrylamide:bis (30:0.8% wt/vol), 2.5% glycerol in 0.5× Tris-borate-EDTA] and run at 120 V for 2 h. Each gel was then dried and subjected to autoradiography.

For supershift studies, 2 µl of anti-p65, anti-p50, or control antiserum was added to the reaction mixture containing the kappa B oligonucleotide. Binding of the antibody to the appropriate transcriptional factor was indicated by a supershift in the EMSA.

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-kappa 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-kappa 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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effect of suspended diesel exhaust particles (DEP) on eotaxin production by normal and transformed human bronchial epithelial cells. Normal human bronchial epithelial cells and BET-1A cells were cultured in hormonally defined Ham's F-12 medium until confluence. Then the cells were treated with suspended DEP (0-50 µg/ml) for 24 h. The supernatants were harvested, and the amount of eotaxin was measured by specific ELISA. DEP showed a dose-dependent stimulatory effect on eotaxin production by normal human bronchial epithelial cells (A) and BET-1A cells (B). C: human recombinant IL-13 also showed a dose-dependent stimulatory effect in both cells. D: simultaneous treatment with 25 ng/ml IL-13 and DEP showed an additive effect. Results from 3 representative experiments are shown. *P < 0.01, ANOVA.

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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Fig. 2.   Effect of suspended DEP on eotaxin mRNA levels in normal and BET-1A cells in vitro. Normal human bronchial epithelial cells and BET-1A cells were cultured in hormonally defined Ham's F-12 medium until confluence. A: cells were treated with suspended DEP (0-50 µg/ml). The cellular RNA was extracted 12 h after stimulation, and Northern blot analysis was performed to evaluate the changes in eotaxin mRNA levels as normalized by beta -actin transcripts. There was a dose-dependent increase in eotaxin mRNA levels with DEP up to 25 µg/ml in normal bronchial epithelial cells. B: results from 3 representative experiments in BET-1A cells are shown. *P < 0.01, ANOVA. C: different doses of IL-13 were added to the cells, and the levels of eotaxin mRNA as normalized by beta -actin transcripts were evaluated after 12 h. IL-13 at 1-50 ng/ml showed a dose-dependent stimulatory effect on eotaxin mRNA levels. * P < 0.01, ANOVA. D: different concentrations of DEP were added simultaneously with near-maximal dose of human recombinant IL-13 (25 ng/ml). DEP showed an additive effect on eotaxin mRNA levels. Results from 3 representative experiments are shown. *P < 0.01, ANOVA.

IL-13 showed a dose-dependent stimulatory effect on eotaxin mRNA levels, as shown in Fig. 2C, and combination of IL-13 (25 ng/ml) with DEP showed an additive effect on eotaxin mRNA levels in BET-1A cells (Fig. 2D).

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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Fig. 3.   Effect of in vitro exposure to diesel exhaust (DE) by a new system. BET-1A cells were exposed to DE as described in MATERIALS AND METHODS. A: supernatants were harvested after different time periods, and eotaxin levels were evaluated by ELISA (n = 3). Data are means ± SE. *P < 0.01, ANOVA. B: total cellular RNA was extracted, and the levels of eotaxin mRNA were evaluated by semiquantitative RT-PCR. The eotaxin mRNA levels as normalized to beta -actin transcripts were increased at all time points up to 24 h.

EMSA for the detection of NF-kappa B activation by suspended DEP. Since it has been suggested that the nuclear transcription factor NF-kappa B plays an important role in the transcriptional regulation of eotaxin gene expression (18), we attempted to evaluate the effect of DEP on NF-kappa 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-kappa B.


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Fig. 4.   Effect of DEP on NF-kappa B activation in BET-1A cells as assessed by EMSA. A: cells were treated with different concentrations of DEP for 6 h, and the nuclear extracts were isolated for EMSA assay as described in MATERIALS AND METHODS. DEP at 1-50 µg/ml increased the nuclear binding to the labeled oligonucleotide double-stranded DNA. Human recombinant IL-13 showed no effect. B: specificity of binding was ascertained by supershift of the bands with antibodies to p65 and p50 as well as the reduced intensity of the signals with excess amount of cold DNA probes (×100).

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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Fig. 5.   Western blot analysis for the phosphorylation of STAT6. BET-1A cells were treated with indicated concentrations of DEP or 10 ng/ml IL-13 for 30 min, and the cellular proteins were extracted. Western blot analysis demonstrated that DEP showed no significant effect on the phosphorylation processes of STAT6 (p-STAT6), whereas human recombinant IL-13 (10 ng/ml) showed a clear activation of STAT6.

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-kappa 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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Fig. 6.   Effect of N-acetyl cysteine (NAC) on eotaxin production and mRNA levels in BET-1A cells. A: antioxidants NAC and pyrrolidine dithiocarbamate (PDTC) showed an attenuating effect on DEP (25 µg/ml)-induced eotaxin production in BET-1A cells in a dose-dependent fashion when studied 24 h after treatment with DEP in vitro. *P < 0.01 vs. DEP alone (25 µg/ml). B: NAC and PDTC showed an attenuating effect on DEP (25 µg/ml)-induced eotaxin mRNA levels in bronchial epithelial cells in a dose-dependent fashion when studied 12 h after treatment with DEP in vitro, whereas the 2 drugs showed no effect on IL-13-induced eotaxin mRNA levels. *P < 0.01 vs. DEP alone (25 µg/ml); **P < 0.01 vs. baseline; n = 3 (ANOVA).

NAC attenuated NF-kappa B activation but did not affect STAT6 activation in BET-1A cells. Pretreatment with NAC 1 h before stimulation with DEP attenuated NF-kappa 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-kappa B activation.


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Fig. 7.   Effect of NAC on the activation processes of NF-kappa B (A) and STAT6 (B). A: BET-1A cells were pretreated with NAC for 1 h and then stimulated with DEP (25 µg/ml). Nuclear extracts were obtained after 6 h, and activation of NF-kappa B was evaluated by EMSA. NAC clearly inhibited the activation of NF-kappa B. B: BET-1A cells were treated with NAC for 1 h and then stimulated with DEP (25 µg/ml) or IL-13 (10 ng/ml) for 30 min. As described in Fig. 5, IL-13 induced activation of STAT6, but NAC failed to affect this process.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B and STAT6. Matsukura and associates (18) studied the transcriptional regulation of the human eotaxin gene and clearly demonstrated that TNF-alpha and IL-4 independently activate NF-kappa 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-alpha and IL-4 in the cells with normal plasmids. When the plasmids that were mutated at the NF-kappa B binding site were used, the response to TNF-alpha , but not to IL-4, was lost. These findings clearly demonstrated that activation of NF-kappa B induced eotaxin gene transcription in vitro. Our studies with EMSA showed that DEP induced the activation of the transcription factor NF-kappa 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-kappa B activation in human bronchial epithelial cell line BET-1A. Finally, antioxidants NAC and PDTC, which had NF-kappa B inhibitory activity but no effect on STAT6 activity, suppressed the mRNA levels of eotaxin. These results suggested that oxidant-dependent, NF-kappa 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-kappa 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.


    ACKNOWLEDGEMENTS

We thank Takako Kobayashi and Makiko Baba for their excellent technical support.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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