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B Activation in Human Bronchial Epithelial Cells In Vitro: Importance in Cytokine Transcription1
Departments of
*
Laboratory Medicine and
Medicine and Physical Therapy, University of Tokyo School of Medicine, Tokyo, Japan;
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
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Fourth Department of Internal Medicine, Nippon Medical School, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, but not of transcription factor AP-1.
The luciferase reporter gene assay using wild-type and mutated
NF-B-binding sequences showed that DEP-induced NF-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-B, which might be an
important mechanism of its potential to increase the expression of
inflammatory cytokines in vitro. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-B.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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 [
-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-B DNA binding, a NF-B binding protein
detection kit (Life Technologies) was used. The sequence of the
oligonucleotides containing a tandem repeat of the consensus sequence
for NF-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
[
-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 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 B-like sites was conducted
using PCR, which converted the B-like site GGAATTTCCT (-80 to -71
bp) to TAACTTTCCT, as reported previously (28, 29). This construct was
designated as 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 (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 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-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
, 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. 1, 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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IL-8 protein levels in culture supernatants were measured by the
specific ELISA after different time periods. As shown in Table I, DEP increased IL-8 protein levels in a
dose-dependent fashion.
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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. 2A, DEP increased the
transcriptional rate of IL-8 gene. DEP showed a dose-dependent
increasing effect on transcriptional rates as shown in Fig. 2B.
|
). 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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B activation by DEP
Since it has been suggested that a nuclear transcription
factor NF-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-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. 4A). 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-B probes (Fig. 4B). In contrast, AP-1 binding to the specific
oligonucleotides was not affected by DEP as shown in Fig. 5.
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B binding to its motif was involved in
increased IL-8 gene expression in BEAS-2B cells.
To determine whether DEP-induced NF-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 B-like
site-mutated sequence. As shown in Fig. 6, treatment with DEP (25 µg/ml) as
well as IL-1 (5ng/ml) of BEAS-2B cells transfected with the normal
B-like site resulted in a significant increase in luciferase
activity, but the cells with mutated B-like site showed no response
to IL-1 and DEP. These results demonstrated that DEP-induced
increased NF-B binding to its motif was actually important in
increased IL-8 gene expression in BEAS-2B cells.
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PDTC, an antioxidant reagent with an inhibitory potential of
NF-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. 7). It was
also shown that NAC partially blocked DEP-induced IL-8 expression (Fig. 6
). Both of the drugs also inhibited DEP (25 µg/ml)-induced
production of IL-8 protein in the culture supernatants (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, which is considered to play an
important role in the gene regulation of IL-8. To determine whether
DEP-induced NF-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 (5ng/ml) of BEAS-2B cells transfected with the normal B-like
site resulted in a significant increase in luciferase activity, whereas
the cells with mutated B-like site showed no response to IL-1 and
DEP, showing that DEP-induced increased NF-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-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-B, but not of AP-1. To determine whether DEP-induced increased
NF-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
B-like site-mutated sequence. Treatment of BEAS-2B cells transfected
with the normal 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-B binding
to its motif was actually important in increased IL-8 gene expression
in BEAS-2B cells. NF-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-B,
and, therefore, it was suggested that DEP exerted their effect through
processes involving NF-B activation. Recent studies showed that
oxidants play an important role as intracellular signals to induce
NF-B activation (45). Oxidants directly and indirectly activate this
transcription factor by phosphorylation of IB and induce
translocation of NF-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.
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Acknowledgments |
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Footnotes |
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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:
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.
Received for publication September 28, 1998. Accepted for publication January 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and -actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol. 3:787.B activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent. Am. J. Respir. Cell Mol. Biol. 18:384. and interferon synergistically induce interleukin-8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-B-like binding sites of the interleukin-8 gene. J. Biol. Chem. 267:22506.B-like sites are genetic end targets of FK-506-sensitive pathway accompanied by calcium mobilization. J. Biol. Chem. 269:8582.B activation in intact cells. J. Exp. Med. 175:1181.B activation and neutrophilic lung inflammation. J. Immunol. 157:1630.[Abstract]
B: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.B. Methods Enzymol. 234:151.[Medline]
B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Rad. Res. Commun. 17:221.[Medline]
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O. Fahy, H. Hammad, S. Sénéchal, J. Pestel, A.-B. Tonnel, B. Wallaert, and A. Tsicopoulos Synergistic Effect of Diesel Organic Extracts and Allergen Der p 1 on the Release of Chemokines by Peripheral Blood Mononuclear Cells from Allergic Subjects . Involvement of the MAP Kinase Pathway Am. J. Respir. Cell Mol. Biol., August 1, 2000; 23(2): 247 - 254. [Abstract] [Full Text]
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 J. W. Christman, R. T. Sadikot, and T. S. Blackwell The Role of Nuclear Factor-{kappa} B in Pulmonary Diseases Chest, May 1, 2000; 117(5): 1482 - 1487. [Abstract] [Full Text] [PDF]
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 S. HASHIMOTO, Y. GON, I. TAKESHITA, K. MATSUMOTO, I. JIBIKI, H. TAKIZAWA, S. KUDOH, and T. HORIE Diesel Exhaust Particles Activate p38 MAP Kinase to Produce Interleukin 8 and RANTES by Human Bronchial Epithelial Cells and N-Acetylcysteine Attenuates p38 MAP Kinase Activation Am. J. Respir. Crit. Care Med., January 1, 2000; 161(1): 280 - 285. [Abstract] [Full Text]
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S. Boland, V. Bonvallot, T. Fournier, A. Baeza-Squiban, M. Aubier, and F. Marano Mechanisms of GM-CSF increase by diesel exhaust particles in human airway epithelial cells Am J Physiol Lung Cell Mol Physiol, January 1, 2000; 278(1): L25 - L32. [Abstract] [Full Text] [PDF]
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