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The Journal of Immunology, 1999, 162: 4705-4711.
Copyright © 1999 by The American Association of Immunologists

Diesel Exhaust Particles Induce NF-{kappa}B Activation in Human Bronchial Epithelial Cells In Vitro: Importance in Cytokine Transcription1

Hajime Takizawa2,*, Takayuki Ohtoshi{dagger}, Shin Kawasaki{dagger}, Tadashi Kohyama{dagger}, Masashi Desaki{dagger}, Tsuyoshi Kasama{ddagger}, Kazuo Kobayashi§, Kazuhiko Nakahara*, Kazuhiko Yamamoto{dagger}, Kouji Matsushima and Shoji Kudoh||

Departments of * Laboratory Medicine and {dagger} Medicine and Physical Therapy, University of Tokyo School of Medicine, Tokyo, Japan; {ddagger} First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan; § National Institute of Infectious Disease, Tokyo, Japan; Department of Molecular Preventive Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan; and || Fourth Department of Internal Medicine, Nippon Medical School, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fine particles derived from diesel engines (diesel exhaust particles, DEP) have attracted attention, since their density in industrial countries seems related to the increased prevalence of pulmonary diseases. Previous studies have suggested that DEP have a potential to directly activate airway epithelial cells to produce and release inflammatory cytokines and mediators, and thus facilitate inflammatory responses in the lung. To elucidate the molecular mechanisms of their action, we studied here IL-8 gene expression, one of the important cytokines in inflammatory responses, by Northern blot analysis and run-on transcription assay. Suspended DEP (1–50 µg/ml) increased the steady state levels of IL-8 mRNA, which was suggested to be largely due to increased transcriptional rates. Electrophoretic mobility shift assay demonstrated that DEP induced increased binding to the specific motif of NF-{kappa}B, but not of transcription factor AP-1. The luciferase reporter gene assay using wild-type and mutated NF-{kappa}B-binding sequences showed that DEP-induced NF-{kappa}B activation was involved in IL-8 transcription. Finally, both N-acetylcysteine and pyrrolidine dithiocarbamate attenuated the action of DEP on IL-8 mRNA expression, suggesting that oxidant-mediated pathway might be involved in its processes. These results suggested that DEP activate NF-{kappa}B, which might be an important mechanism of its potential to increase the expression of inflammatory cytokines in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is extensive evidence that exposure to increased levels of inhalable particulate pollutants (PM10) is related to the increased respiratory and cardiac morbidity and mortality (1, 2, 3, 4, 5). Recent reports (6, 7, 8) suggest that those with the diameters less than 2.5 µm (PM2.5) have an important role in triggering the biological responses within the lung. In urban areas, fine particulate matters produced from diesel engines (diesel exhaust particles, DEP3) are one of the major constituents of PM2.5. Animal models of repeated exposure to these matters have shown that DEP had carcinogenic activity in airways (9). DEP have also been postulated to induce intense inflammatory reactions in the airways. Recent reports further showed that inhalation of DEP induced airway inflammatory changes associated with hyperresponsiveness with cell infiltration such as eosinophils, which mimic those found in bronchial asthma (10). It is quite likely that these biological responses to DEP were via the production of cytokines, chemokines, and other inflammatory mediators locally produced in the airways. IL-8, one of the prototype CXC chemokines, plays an important role in the pathogenesis of airway inflammation (11), which has been known to be produced by many kinds of airway cells, including epithelial cells (12).

Airway epithelial cells are the first cells that contact a variety of exogenous agents including DEP, and it is now clear that these cells have a potential to express and release cytokines and chemokines important in the airway inflammatory responses. We (13) and others (14) have recently demonstrated that DEP stimulate these cells to release inflammatory cytokines in vitro.

In the present study, we attempted to study the molecular mechanisms of the effect of DEP on IL-8 gene expression in human bronchial epithelial cells. DEP up-regulated IL-8 gene expression at the transcriptional level in a human bronchial epithelial cell line in vitro. The action seemed to be via, at least in part, molecular processes that involve activation of the transcription factor NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of DEP

The engine used for preparation of DEP was a 4JB1-type, light duty (2740 cc), four-cylinder diesel engine (Isuzu Automobile Company, Tokyo, Japan). The engine was connected to an EDYC dynamometer (Meiden-Sya, Tokyo, Japan) and was operated using a standard diesel fuel with speeds of 2000 rpm under the load of 6 torque (kg/m). The exhaust was introduced into a stainless steel dilution tunnel (300 mm {phi} x 8400 mm). The DEPs were collected on glass fiberfilter (203 mm x 254 mm) in a constant-volume sampler system equipped to the end of the dilution tunnel. The temperature at the sampling point was below 50°C. The diameter of the particles was measured by Anderson Air Sampler (Shibata Scientific Technology, Tokyo, Japan) of low pressure type (15), and the mean diameter was 0.4 µm. Most of the shapes analyzed by a scanning electron microscope were globular. The details of the information of DEP that we used were previously described (10, 13).

Culture of bronchial epithelial cells

A human bronchial epithelial cell line BEAS-2B (16)(a kind gift from Drs. J.F. Lechner and C.C. Harris, National Cancer Institute, Bethesda, MD) was cultured as reported (17). Briefly, the cells were plated onto collagen- coated 24-well flat-bottom tissue culture plates (Koken, Tokyo, Japan) at the density of 5 x 104 cells/well in hormonally defined Ham’s F12 medium (HD-F12) as reported (13, 17, 18, 19). HD-F12 contained 1% penicillin-streptomycin, 5 µg/ml insulin (Life Technologies, Grand Island, NY), 5 µg/ml transferrin (Life Technologies), 25 ng/ml epidermal growth factor (Collaborative Research, Lexington, MA), 15 µg/ml endothelial cell growth supplement (Collaborative Research), 2 x 10-10 M triiodothyronin (Life Technologies), and 10-7 M hydrocortisone (Life Technologies). The cells were incubated in a humidified atmosphere at 37°C and 5% CO2. The medium was changed at day 1 and subsequently every 2 days. 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 BEAS-2B cells as assessed by trypan blue dye exclusion, lactate dehydrogenase release assay, and 3-4,5-(dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium MTT assay.

Northern blot analysis for IL-8 mRNA expression in human bronchial epithelial cells

Northern blot analysis was performed to study the effect of DEP on IL-8 mRNA expression in human bronchial epithelial cells by the method described previously (20). Briefly, total cellular RNA was extracted by the method of Chomczynski and Sacchi (21) and electrophoresed on formaldehyde-denatured agarose gel (10 µg/lane) followed by capillary transfer onto Biodyne nylon membrane (Pall Corporation, East Hills, NY). RNA integrity and equivalency of loading were routinely evaluated by ethidium bromide fluorescence. Blots were baked, prehybridized, and hybridized with a 32P 5' end-labeled oligonucleotide probe specific for human IL-8 and ß-actin. The probes used in this study were reported previously (22). Blots were stringently washed after hybridization and exposed to x-ray film (Kokak, Rochester, NY).

Run-on transcription assay

Nuclear run-on transcription assay was performed by the methods previously reported with modifications (23). Briefly, after the medium was removed, the cells were washed three times with ice-cold PBS, scraped in PBS, and pelleted at 500 x g for 5 min. The cell pellets (5 x 106 cells) were resuspended in 1 ml Nonidet P-40 lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P-40), incubated for 5 min on ice, and centrifuged at 500 x g for 5 min. The nuclear pellet was washed once with 2 ml Nonidet P-40 lysis buffer and centrifuged at 500 x g. The supernatant was discarded, and the nuclei were resuspended in 100 µl 50 mM Tris-HCl (pH 8.3), 40% (v/v) glycerol, 5 mM MgCl2, and 0.1 mM EDTA, and frozen in liquid nitrogen. The nuclei were thawed for the run-on transcription assay and mixed with 100 µl reaction buffer (10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 5 mM DTT, 0.5 mM each of ATP, CTP, and GTP) and 100 µCi of [{alpha}-32P]UTP (760 Ci · mmol-1) and reacted at 37°C for 1 h.

The probes contained cDNA inserts for human IL-8 (0.45 kb in pUC19), and human ß-actin (pHFßA-1) (24, 25). Vector plasmid DNA served as control. For binding to nitrocellulose, plasmids were linearized by restriction enzyme digestion, and the DNA was denatured by incubation with 0.2 M NaOH for 30 min at room temperature, followed by neutralization with 10 volumes of 6x SSC. The DNA was spotted onto nitrocellulose using a dot blot apparatus. Ten micrograms of DNA was applied per dot.

The 32P-labeled RNA was isolated as described elsewhere (23) for hybridization to filters. Before ethanol precipitation, 32P-labeled RNA was treated with a final concentration of 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M, and the RNA was precipitated with ethanol. After centrifugation, the 32P-labeled RNA pellet was resuspended in 10 mM N-tris(hydroxymethyl)methyl 2-aminoethanesulfonic acid (TES) (pH 7.4), 0.2% SDS, 10 mM EDTA at 5 x 106 cpm/ml. This RNA solution was mixed with an equal volume of 10 mM TES (pH7.4), 0.2% SDS, 10 mM EDTA, and 600 mM NaCl, and 2 ml of the RNA solution was hybridized at 65°C for 36 h to DNA immobilized on nitrocellulose. After the hybridization, the filters were washed several times with 2x SSC (1x SSC = 0.15 M NaCl, 0.0125 M Na citrate (pH 7.0)) for 2 h at 65°C and incubated at 37°C in 2x SSC with 10 µg/ml RNase A for 30 min. The filters were then washed again in 2x SSC at 37°C for 1 h, air dried, and exposed to Kodak x-ray film.

Electrophoresis mobility shift assay

After the cells were washed with PBS, the nuclear proteins were isolated by the method reported previously (26) with modifications. In brief, 2 to 3 x 106 cells were harvested with the addition of trypsin-EDTA solution (Life Technologies), rinsed in Tris-buffered saline, resuspended in lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT), 0.5 mM 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 x g. The nuclear pellet was resuspended in extraction buffer (20 mM HEPES, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM 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, Richmond, CA), standardized with BSA.

For the detection of NF-{kappa}B DNA binding, a NF-{kappa}B binding protein detection kit (Life Technologies) was used. The sequence of the oligonucleotides containing a tandem repeat of the consensus sequence for NF-{kappa}B DNA binding site (underlined) was as follows: 5'-GATCCAAGGGGACTTTCCATGGATCCAAGGGGACTTTCCATG-3', 3'-GTTCCCCTGAAAGGTACCTAGGTTCCCCTGAAAGGTACCTAG-5'. The specific dsDNA for AP-1, obtained from Promega (Madison, WI), was as follows: 5'-CGCTTGATGAGTCAGCCGGAA-3', 3'-GCGAACTACTCAGTCGGCCTT-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% (v/v) 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% w/v), 2.5% glycerol in 0.5x 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 antisera was added to the reaction mixture containing the {kappa}B oligonucleotide. Binding of the Ab to the appropriate transcriptional factor was indicated by a supershift in the EMSA.

Plasmid construction

The EcoRI-HindIII fragment of the genomic human IL-8 DNA, which spans nucleotides -1480 to +44 bp from the start of the first exon (27), was subcloned into pGL3 (Promega). Site-directed mutagenesis of the IL-8 {kappa}B-like sites was conducted using PCR, which converted the {kappa}B-like site GGAATTTCCT (-80 to -71 bp) to TAACTTTCCT, as reported previously (28, 29). This construct was designated as {kappa}B-like site-mutated plasmid. All plasmids were prepared with Qiagen tip 500 (Diagen, Dusseldolf, Germany) according to the application protocols. The SV-ß-galactosidase (pSV-ß-Gal) vector (Promega) containing the SV40 early promotor and enhancer linked to lacZ was used as an internal positive control to monitor transfection efficiency.

Transient cell transfection and luciferase assays

BEAS-2B cells were plated and cultured as described above to 70–80% confluence. Cells were transfected with 0.2 µg of pSV-ß-Gal plasmid and 0.8 µg of the luciferase vector by using 6 µg of the liposomal transfection reagent DOTAP (Boehringer Mannheim, Mannheim, Germany) for 8 h. After the medium was changed, human recombinant IL-1{alpha} (5 ng/ml) or DEP (25 µg/ml) was added to the cells for 8 h. Then, cell lysates were prepared, and protein concentrations were measured by the method of Bradford (Bio-Rad protein assay kit). Luciferase activity and ß-galactosidase activity were evaluated with a chemiluminescence assay kit (Tropix, Bedford, MA). Preliminary experiments using a plasmid containing the luciferase gene without IL-8 promotor sites showed no increase in luciferase activity after IL-1{alpha} stimulation, suggesting that the reaction was specific. Results were confirmed by three independent transfections.

Effect of pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on IL-8 gene expression

To evaluate the role of activation of the transcription factor NF-{kappa}B, we treated the cells with different concentrations of PDTC (30) and NAC (31) (pH adjusted to 7.4) 1 h before the addition of 25 µg/ml DEP and studied the levels of IL-8 mRNA as well as its protein in the supernatants.

Cytokine assay

Specific immunoreactivity for IL-8 in culture supernatants was measured by ELISA kits (R&D Systems, Minneapolis, MN) (20). Each sample was assayed in duplicates as recommended by the manufacturer.

Statistical analysis

The results were analyzed by Student’s t test for comparison between the two groups and by nonparametric equivalents of ANOVA for multiple comparison as reported (13, 20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DEP increased the steady state levels of IL-8 mRNA in human bronchial epithelial cells

Total cellular RNA was extracted after different time periods with and without 25 µg/ml DEP, and the steady state levels of IL-8 mRNA were studied by Northern blot analysis. As shown in Fig. 1Go, A and B, DEP at 25 µg/ml showed a time-dependent stimulatory effect on IL-8 mRNA levels up to 12 h. As shown in Fig. 1Go, C and D, DEP at the ranges of 1–50 µg/ml showed a dose-dependent stimulatory effect on IL-8 mRNA levels when evaluated 12 h after the addition to the cells.



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  		FIGURE 1. Effect of DEP on the IL-8 mRNA levels in human bronchial epithelial cells BEAS-2B in vitro. BEAS-2B cells were cultured in HD-F12 until confluence. A, Then, the cells were treated with 25 µg/ml of DEP. The cellular RNA was extracted, and Northern blot analysis was performed to evaluate the changes in IL-8 mRNA levels. There was a time-dependent increase in IL-8 mRNA levels with DEP (25 µg/ml) up to 12 h. B, Results from three representative experiments were shown (*, p < 0.01, ANOVA). C, Different doses of suspended DEP were addded to the cells, and the levels of IL-8 mRNA was evaluated after 12 h. DEP at the doses of 1–50 µg/ml showed a dose-dependent stimulatory effect on IL-8 mRNA levels. D, Results from three representative experiments were shown (*, p < 0.01, ANOVA).

 
Increased production of immunoreactive IL-8 by DEP

IL-8 protein levels in culture supernatants were measured by the specific ELISA after different time periods. As shown in Table IGo, DEP increased IL-8 protein levels in a dose-dependent fashion.


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  		Table I. Effect of DEP on IL-8 production by human bronchial epithelial cells

 
DEP up-regulated the transcriptional rate of IL-8 mRNA but had no effect on the RNA degradation

To further investigate the mechanism of DEP-induced increase of IL-8 mRNA levels, we evaluated the transcriptional rate of IL-8 mRNA by nuclear run-on assay. The bronchial epithelial cells were treated with 25 µg/ml DEP for 8 h, and the nuclei were isolated and frozen until use. The transcription reaction proceeded in vitro with radiolabeled UTP as described in Materials and Methods. As shown in Fig. 2GoA, DEP increased the transcriptional rate of IL-8 gene. DEP showed a dose-dependent increasing effect on transcriptional rates as shown in Fig. 2GoB.



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  		FIGURE 2. Nuclear run-on assay for the evaluation of the transcriptional rate of IL-8 mRNA. To investigate the mechanism of DEP-induced increase of IL-8 mRNA levels, the transcriptional rate of IL-8 mRNA was studied by nuclear run-on assay. The bronchial epithelial cells were treated with DEP for 8 h, the nuclei were isolated, and the transcription reaction proceeded in vitro. A, DEP (25 µg/ml) increased the transcriptional rate of IL-8 gene, while that of ß-actin showed no change. B, DEP showed a dose-dependent increasing effect on the transcriptional rates (n = 3; *, p < 0.01 as compared with the baseline) ANOVA.

 
We also examined the effect of DEP on the decay of IL-8 mRNA. After 8 h incubation with and without DEP (25 µg/ml), actinomycin D (5 µg/ml) was added to the cell culture, and total RNA was isolated at specific time points (Fig. 3Go). The t1/2 of IL-8 mRNA from 8-h DEP-stimulated BEAS-2B cells were not significantly different between the groups with and without the treatment of DEP. The magnitudes of ß-actin transcripts remained stable as long as 12 h after the actinomycin D treatment (101 ± 11.6% as compared with the baseline = 100%, p > 0.05), suggesting that the changes in IL-8 mRNA levels were not due to the cytotoxicity by actinomycin D. These data suggest that a significant portion of DEP-induced increase in IL-8 mRNA was at the transcriptional level.



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  		FIGURE 3. Effect of DEP on the decay of IL-8 mRNA. After 8 h incubation with and without DEP (25 µg/ml), actinomycin D (5 µg/ml) was added to the cell culture, and total RNA was isolated at specific time points. The t1/2 of IL-8 mRNA from 8-h DEP-stimulated BEAS-2B cells (6.28 ± 0.33 h) were not significantly different from the groups without the treatment of DEP (6.98 ± 0.85 h). p > 0.05, Student’s t test, n = 3. The data were shown in mean values.

 
EMSA for the detection of NF-{kappa}B activation by DEP

Since it has been suggested that a nuclear transcription factor NF-{kappa}B plays an important role in the transcriptional regulation of IL-8 gene expression (32), 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 DEP for 6 h, and the nuclear extracts were isolated for EMSA as described in Materials and Methods. DEP at 1 to 50 µg/ml increased the nuclear bonding to the labeled oligonucleotide dsDNA (Fig. 4GoA). The specificity of the binding was ascertained by the supershift of the bands with Abs to p65 and p50 as well as the reduced intensity of the signals with excess amount (x100) of cold DNA NF-{kappa}B probes (Fig. 4GoB). In contrast, AP-1 binding to the specific oligonucleotides was not affected by DEP as shown in Fig. 5Go.



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  		FIGURE 4. Effect of DEP on NF-{kappa}B activation in human bronchial epithelial cells as assessed by EMSA. A, The 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 to 50 µg/ml increased the nuclear binding to the labeled oligonucleotide dsDNA. B, The specificity of the binding was ascertained by the supershift of the bands with Abs to p65 and p50, as well as the reduced intensity of the signals with excess amount of cold DNA probes (x100). Addition of control antiserum showed no effect.

 


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  		FIGURE 5. Effect of DEP on AP-1 binding activity in human bronchial epithelial cells. The BEAS-2B cells were treated with DEP for 6 h, and the nuclear extracts were isolated. EMSA demonstrated AP-1 binding to its specific oligonucleotide DNA, but DEP showed no significant effect. Human recombinant IL-1{alpha} (5 ng/ml) increased AP-1 binding.

 
DEP-induced increased NF-{kappa}B binding to its motif was involved in increased IL-8 gene expression in BEAS-2B cells.

To determine whether DEP-induced NF-{kappa}B binding to its motif was really involved in increased IL-8 gene expression, transient transfections of a plasmid containing the 5' promotor region from human genomic IL-8 gene linked to a luciferase reporter gene were performed. The promotor region had either the wild-type or a {kappa}B-like site-mutated sequence. As shown in Fig. 6Go, treatment with DEP (25 µg/ml) as well as IL-1{alpha} (5ng/ml) of BEAS-2B cells transfected with the normal {kappa}B-like site resulted in a significant increase in luciferase activity, but the cells with mutated {kappa}B-like site showed no response to IL-1{alpha} and DEP. These results demonstrated that DEP-induced increased NF-{kappa}B binding to its motif was actually important in increased IL-8 gene expression in BEAS-2B cells.



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  		FIGURE 6. Reporter gene assay with transient transfection of normal and mutated {kappa}B-like sequence into BEAS-2B cells. The BEAS-2B cells were transiently tansfected with a plasmid containing the IL-8 promotor bound to the luciferase gene. Constructs contained either a normal or mutated {kappa}B-like sequence. The cells were exposed to IL-1{alpha} (5 ng/ml) or DEP (25 µg/ml) for 8 h. The cells were lysed, and luciferase activity was measured, corrected for transfection efficiency relative to ß-galactosidase activity, and expressed as a percentage of unstimulated controls. DEP (25 µg/ml) as well as IL-1{alpha} (5 ng/ml) showed a significant enhancing effect on luciferase activity of the cells transfected with patent NF-{kappa}B binding sequences as compared with baselines. In contrast, neither stimulus showed any augmenting effect on the luciferase activity of the cells transfected with mutant sequences. The data represent the mean ± SEM of three experiments. *, p < 0.01 (ANOVA).

 
Effect of PDTC and NAC on IL-8 mRNA levels as evaluated by Northern blot analysis

PDTC, an antioxidant reagent with an inhibitory potential of NF-{kappa}B activation, showed a dose-dependent inhibitory effect on DEP-induced IL-8 mRNA gene expression when studied 8 h after DEP treatment (25 µg/ml) (Fig. 7Go). It was also shown that NAC partially blocked DEP-induced IL-8 expression (Fig. 6Go). Both of the drugs also inhibited DEP (25 µg/ml)-induced production of IL-8 protein in the culture supernatants (Table IGo).



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  		FIGURE 7. Effect of PDTC and NAC on IL-8 mRNA levels in BEAS-2B cells. Antioxidants PDTC and NAC showed an attenuating effect on DEP (25 µg/ml)-induced IL-8 mRNA in bronchial epithelial cells in a dose-dependent fashion when studied 8 h after the treatment of DEP in vitro. *, p < 0.01 compared with DEP group (25 µg/ml) (second column from the left); **, p < 0.01 compared with baseline, n = 3 (ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that DEP, one of the important air pollutants, stimulated IL-8 gene expression in human bronchial epithelial cells. This effect was largely due to increased transcriptional rates, with no apparent change in the decay process of the mRNA. Studies with EMSA suggested 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 IL-8. To determine whether DEP-induced NF-{kappa}B binding to its motif was really involved in increased IL-8 gene expression, transient transfection of either a wild or mutant IL-8 promotor sequence was done and a luciferase reporter gene assay was performed. Treatment with DEP (25 µg/ml) as well as IL-1{alpha} (5ng/ml) of BEAS-2B cells transfected with the normal {kappa}B-like site resulted in a significant increase in luciferase activity, whereas the cells with mutated {kappa}B-like site showed no response to IL-1{alpha} and DEP, showing that DEP-induced increased NF-{kappa}B binding to its motif was actually important in increased IL-8 gene expression in BEAS-2B cells. Finally, NAC and PDTC, both being potent anti-oxidants with NF-{kappa}B inhibitory activity, suppressed the mRNA levels of IL-8, suggesting an involvement of oxidants-dependent pathways in these events.

Epidemiological studies have suggested that there may be a link between the incidence of respiratory diseases and particulate matters, especially small particles such as PM2.5 (6, 7, 8). DEP have been considered to be the major part of PM2.5, and in vivo and in vitro experiments have suggested their potent activity in the respiratory tracts. DEP have been demonstrated to have an adjuvant activity for IgE synthesis in vivo (33, 34). Inhalation of DEP in mice resulted in increased airway responsiveness (10). Recently, Diaz-Sanzes et al. showed that transnasal challenges of DEP-derived extract in humans enhanced local IgE and cytokine production (35, 36). The same group (37) demonstrated that aromatic hydrocarbons contained in DEP had a direct biochemical effect on B cells to enhance IgE production in vitro. As for the effect of DEP on airway epithelial cells, we (13) and others (13, 14, 38) recently showed that DEP had a direct effect on airway epithelial cells to release inflammatory mediators such as IL-6, IL-8, granulocyte-macrophage-CSF, and soluble ICAM-1. IL-8 is a potent chemotactic factor for eosinophils (39), basophils (40), and T lymphocytes (41), as well as neutrophils. Recent studies have shown that this C-X-C type chemokine plays a role in eosinophil as well as neutrophil transmigration through endothelium and epithelium (42), and induced airway hyperresponsiveness in guinea pigs (43). Bronchial epithelial cell-derived IL-8, as well as human recombinant IL-8 (up to 10 ng/ml), were not cytotoxic to the epithelial cells as assessed by typan blue dye test, but the epithelial cell-derived IL-8 was actually chemotactic for neutrophils in vitro (unpublished data), and therefore seemed important for DEP-induced airway inflammation found in vivo. Therefore, DEP might be involved in the activation of airway epithelial cells to release cytokines and other mediators and, thereby, play an important role in the initiation and prolongation of allergic inflammation.

However, it remains unclear how DEP exerts its effect on human respiratory cells. Takenaka et al. (37) studied the activity of DEP-derived aromatic hydrocarbons that were extracted in dichloromethane. We had studied the effects of benzo(a)pyrene, one of the prototypes of aromatic hydrocarbons contained in DEP, and showed that it had a potential to stimulate bronchial epithelial cells to release cytokines (13). It has been shown that these aromatic hydrocarbons activate their receptor (Ah receptor) complexes in cytoplasm, which consists of at least a polyaromatic hydrocarbon ligand-binding protein and a nuclear transporter/DNA-binding protein. The Ah receptor complexes are widespread in human tissues (44). Once aromatic hydrocarbons bind to the Ah receptor, this complex translocates to the nucleus where it induces a variety of biochemical reactions.

Our present studies first demonstrated that DEP induced the activation of NF-{kappa}B, but not of AP-1. To determine whether DEP-induced increased NF-{kappa}B binding to its motif was really involved in increased IL-8 gene expression, transient transfections of a plasmid containing the 5' promotor region from human genomic IL-8 gene linked to a luciferase reporter gene. The promotor region had either the wild-type or a {kappa}B-like site-mutated sequence. Treatment of BEAS-2B cells transfected with the normal {kappa}B-like site showed a significant increase in luciferase activity after exposure to DEP as well as IL-1, but the cells with mutated B-like site showed no response to IL-1 and DEP. These results demonstrated that DEP-induced increased NF-{kappa}B binding to its motif was actually important in increased IL-8 gene expression in BEAS-2B cells. NF-{kappa}B has been considered to play a key role in the regulation of a variety of cytokines and adhesion molecules, being important mediators in airway inflammatory processes. The cis elements of IL-8 had specific binding motifs to NF-{kappa}B, and, therefore, it was suggested that DEP exerted their effect through processes involving NF-{kappa}B activation. Recent studies showed that oxidants play an important role as intracellular signals to induce NF-{kappa}B activation (45). Oxidants directly and indirectly activate this transcription factor by phosphorylation of I{kappa}B and induce translocation of NF-{kappa}B into the nuclei (46). In the present studies, antioxidants NAC and PDTC attenuated DEP-induced IL-8 gene expression, suggesting that oxidants might be involved in these molecular events. These findings may give new insight into the molecular mechanisms of DEP action in human bronchial epithelial cells.


   Acknowledgments
 
We thank Dr. M. Sagai for his kind supply of DEP. We also thank Takako Kobayashi and Asako Hashimoto for their excellent technical support.


   Footnotes
 
1 This work was supported in part by a grant from the Diffuse Lung Disease Research Committee, Japan Ministry of Health and Welfare, by the Pollution-Related Health Compensation and Prevention Association of Japan, and by Manabe Medical Foundation. Back

2 Address correspondence and reprint requests to Dr. Hajime Takizawa, Department of Laboratory Medicine, University of Tokyo School of Medicine, 7–3-1 Hongo, Bunkyo-ku, 113 Japan. E-mail address: Back

3 Abbreviations used in this paper: DEP, diesel exhaust particle; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetylcysteine; EMSA, electrophoretic mobility shift assay; AP-1, activator protein-1; Ah receptor, aromatic hydrocarbon; HD-F12, hormonally defined Ham’s F12 medium; TES, N-tris(hydroxymethyl)methyl 2-aminoethanesulfonic acid; pSV-ß-Gal, SV-ß-galactosidase vector containing the SV40 early promotor and enhancer linked to lacZ. Back

Received for publication September 28, 1998. Accepted for publication January 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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