1 Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris VII, 75251 Paris; and 2 Institut National de la Santé et de la Recherche Médicale Unité 408, Faculté X. Bichat, 75018 Paris, France
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ABSTRACT |
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We have
previously shown that exposure to diesel exhaust particles (DEPs)
stimulates human airway epithelial cells to secrete the inflammatory
cytokines interleukin-8, interleukin-1, and granulocyte-macrophage
colony-stimulating factor (GM-CSF) involved in allergic diseases. In
the present paper, we studied the mechanisms underlying the increase in
GM-CSF release elicited by DEPs using the human bronchial epithelial
cell line 16HBE14o
. RT-PCR analysis has shown an increase in
GM-CSF mRNA levels after DEP treatments. Comparison of the effects of
DEPs, extracted DEPs, or extracts of DEPs has shown that the increase
in GM-CSF release is mainly due to the adsorbed organic compounds and
not to the metals present on the DEP surface because the metal chelator
desferrioxamine had no inhibitory effect. Furthermore, radical
scavengers inhibited the DEP-induced GM-CSF release, showing
involvement of reactive oxygen species in this response. Moreover
genistein, a tyrosine kinase inhibitor, abrogated the effects of DEPs
on GM-CSF release, whereas protein kinase (PK) C, PKA, cyclooxygenase,
or lipoxygenase inhibitors had no effect. PD-98059, an inhibitor of
mitogen-activated protein kinase, diminished the effects of DEPs,
whereas SB-203580, an inhibitor of p38 mitogen-activated protein
kinase, had a lower effect, and DEPs did actually increase the active,
phosphorylated form of the extracellular signal-regulated kinase as
shown by Western blotting. In addition, cytochalasin D, which inhibits the phagocytosis of DEPs, reduced the increase in GM-CSF release after
DEP treatment. Together, these data suggest that the increase in GM-CSF
release is mainly due to the adsorbed organic compounds and that the
effect of native DEPs requires endocytosis of the particles. Reactive
oxygen species and tyrosine kinase(s) may be involved in the
DEP-triggered signaling of the GM-CSF response.
granulocyte-macrophage colony-stimulating factor; 16HBE14o cells; signal transduction; reactive oxygen
species; cytokines; inflammation
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INTRODUCTION |
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ALTHOUGH CLINICAL ATOPIC DISORDERS such as asthma have long been recognized, they have become steadily more prominent in recent decades so that 30% of the population in some communities may manifest atopic diseases. Indeed, a number of carefully controlled studies point to a real rise in the frequency of asthma in developed communities (40). The recent and rapid rise in asthma emphasizes the importance of environmental factors for the development of atopy and asthma. Among the environmental factors, traffic-related air pollution should be considered, in particular diesel exhaust. Indeed, the number of diesel engine-powered cars has been increasing steadily in recent years, and they are one of the major causes of air pollution in urban areas. Diesel exhaust particles (DEPs) aggregate to form agglomerates of ~0.1-0.5 µm, which are of respirable size. DEPs are composed of a carbon core that adsorbs trace amounts of heavy metals and a great number of organic compounds that could represent up to 40% of the particle (39). Until now, it is unclear which component of DEPs is involved in the biological effects: the carbon core, the adsorbed organic compounds, or, as in asbestos or residual oil fly ash (ROFA), the metals present on the particle surface.
Recent studies have emphasized the role of DEPs in the development of
an inflammatory response. It has been shown that DEPs instilled
intratracheally in mice induce asthmalike symptoms (30) and have an
adjuvant activity for IgE production (21). DEP exposure also induces
hyperresponsiveness to histamine in guinea pigs (19) or to
acetylcholine in combination with ovalbumin in mice (22). Nasal
challenges of human beings with DEPs result in the local increase in
IgE and cytokine production (13, 14). Moreover, there is increasing
evidence that airway epithelial cells play an active role in allergic
inflammation. Besides the function as a physical barrier, the airway
epithelium could release a variety of cytokines such as interleukin
(IL)-1, IL-6, IL-8, granulocyte colony-stimulating factor, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) that are
relevant to allergic airway inflammation (1). Because the epithelium is
one of the first targets of inhaled DEPs, it could therefore be a
plausible initial mediator of allergic inflammation. Indeed, Boland et
al. (6) have previously shown that in vitro DEPs
induce an increase in the release of the proinflammatory cytokines
IL-8, IL-1, and GM-CSF by human airway epithelial cells. Among these
cytokines, GM-CSF is a crucial cytokine for the maturation of
granulocytes and macrophages as well as of eosinophils. It also
increases the eosinophil survival rate and stimulates Langerhans cell
activation (37). The stimulation of cytokine secretion could be due to
the release of preexisting pools of cytokines or to de novo protein
synthesis. Increased cytokine secretion by various agents often
involves mRNA stability or enhanced transcription of cytokine genes.
Transcription factors such as activator protein-1 and nuclear
factor-
B (NF-
B) are known to be involved in the upregulation of
many cytokines including GM-CSF. Their activation is dependent on
signaling cascades involving protein phosphorylation and/or reactive
oxygen species (ROS). In a previous paper, Baeza-Squiban et al. (3)
have demonstrated an increased binding of NF-
B to DNA after DEP but
not after carbon black exposure of bronchial epithelial cells
(16HBE14o
).
The aim of this study was to elucidate the underlying molecular and cellular mechanisms of this enhanced GM-CSF release induced by DEPs. A key question is the relative importance of the nonextractable DEP core versus adsorbed organic compounds, which led us to compare the effects of nonextracted and extracted DEPs (SRM 1650) as well as the effects of extracts of DEPs. We also raised the question whether the native DEPs containing the adsorbed organic compounds have to be endocytosed to induce an increase in GM-CSF release. Because signaling cascades involved in the regulation of cytokine production can imply ROS and protein phosphorylation, we evaluated in the present study the contribution of ROS and protein kinases in the regulation of DEP-mediated GM-CSF release by the use of specific inhibitors.
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MATERIALS AND METHODS |
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Culture conditions. Dr. D. C. Gruenert (Cardiovascular Research
Institute, University of California, San Francisco, CA)
kindly provided the human bronchial epithelial cell subclone
16HBE14o, the properties of which have been previously described
(11). The cell line was maintained routinely at the logarithmic phase of growth through subculturing once a week, and experiments were performed at passages 25-45. Cells
were cultured on collagen (type I, 4 µg/cm2; Sigma, Saint
Quentin Fallavier, France)-coated 25- or 75-cm2 flasks or
6-well plates at 20,000 cells/cm2 in Dulbecco's modified
Eagle's medium-Ham's F-12 medium nutrient mixture (DMEM-F-12; 1:1;
GIBCO BRL, Life Technologies, Cergy Pontoise, France) supplemented with
antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin;
Sigma), Fungizone (1 µg/ml; GIBCO BRL), and 10% fetal bovine serum
(GIBCO BRL). The culture medium was changed every 2 days, and the serum
was removed 24 h before treatments. The cultures were incubated in
humidified 95% air with 5% CO2 at 37°C.
Reagents. The diesel particulate matter SRM 1650 was
purchased from the National Institute of Standards and Technology
(Gaithersburg, MD). Carbon black (FR103; 95-nm diameter) was obtained
from Degussa (Frankfurt, Germany). Cytochalasin D (Cyto D; Sigma) was
used at 10 µg/ml, actinomycin D (Sigma) was used at 0.2 µg/ml, H-89 (ICN, Orsay, France) at 5 µM, GF-109203X (Sigma) was
used at 5 µM, genistein (Sigma) was used at 75 µM,
nordihydroguaiaretic acid (Sigma) was used at 10 µM, PD-98059 (Tebu,
le Perray-en-Yvelines, France) was used at 10 µM, and SB-203580
(Calbiochem, Meudon, France) was used at 1 µM. Stock solutions of
these reagents were prepared in dimethyl sulfoxide (DMSO) and stored at
20°C. Pyrrolidinedithiocarbamate (PDTC; Sigma) was used at 5 µM,
-tocopherol (Sigma) was used at 0.1 mM, and indomethacin
(Sigma) was used at 10 µM. Stock solutions of these reagents were
prepared in ethanol and stored at
20°C. Cycloheximide
(Sigma) at 10 µg/ml, N-acetyl-L-cysteine (NAC;
Sigma) at 10 mM (pH 7.4), 1,3-dimethyl-2-thiourea (DMTU; Sigma) at 10 mM, and desferrioxamine (DEF; Ciba-Geigy, Rueil-Malmaison, France) at
0.1 mM were prepared in culture medium and stored at
20°C. The final concentration of ethanol or DMSO in the culture medium did
not exceed 0.1%. MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays after 4 or 24 h of treatment with these
inhibitors and scavengers showed no cytotoxicity at these concentrations.
Chemical treatment. Stock solutions of particles were performed by suspension in a solution of 0.04% dipalmitoyl lecithin (Sigma) in distilled water and sonicated three times for 60 s each at maximal power (8 kilocycles). DEPs were extracted by dichloromethane in a Soxhlet apparatus, and the extract was dried and redissolved in DMSO. The recovered particles were extracted a second time to ensure total extraction. Particles were sonicated three times for 20 s each just before addition to the cultures, and dipalmitoyl lecithin was added to control cultures as well as DMSO in the experiments with extracts of DEPs. Concentrations are expressed in micrograms per square centimeter because the particles rapidly sediment onto the culture. Inhibitors or scavengers were added 30 min before DEP treatment.
Cytokine assay. Four-day-old cultures were treated with
particles at 10 µg/cm2, and after 24 h of treatment, the
culture supernatant was centrifuged and the samples were frozen at
80°C until used. The concentration of GM-CSF released into
the culture supernatant was measured with human GM-CSF ELISA kits
(Amersham, Les Ulis, France). Briefly, the GM-CSF present in the
culture supernatant reacts with mouse monoclonal antibodies against
GM-CSF bound to the wells of a microtiter plate. A monospecific
antibody to GM-CSF conjugated to horseradish peroxidase reacts with the
GM-CSF bound to the monoclonal antibody. Any unbound protein was washed
away, and color development after addition of the enzyme substrate was
measured at 450 nm with a Microplate Photometer MRX 5000 (Dynatech
Laboratories). The concentration of GM-CSF in the culture supernatant
was determined with a standard curve established with the use of a
human recombinant GM-CSF.
Detection of GM-CSF mRNA by RT-PCR. The oligonucleotides used for PCR were chosen to encompass at least one intron to detect amplification of contaminating genomic DNA. For GM-CSF, the primers were sense, 5'-GGC GTC TCC TGA ACC TGA GTA G-3', and antisense, 5'-GTC GGC TCC TGG AGG TCA AA-3'(size of the amplified products was 91 bp). For the ribosomal protein S14, used as the standard, the primers were sense, 5'-ATC AAA CTC CGG GCC ACA GGA-3', and antisense, 5'-GTG CTG TCA GAG GGG ATG GGG-3'(137 bp).
Total cellular RNA was isolated from 16HBE14o cultures with
TRIzol Reagent (GIBCO BRL) according to Chomczynski and Sacchi (10).
Purified RNA was dissolved in RNase-free water and quantified by
measurement of absorbance at 260 nm. All samples were demonstrated to
contain undegraded RNA as assessed by electrophoresis of an aliquot of
each preparation on 1% agarose gels and visualization of the products
after ethidium bromide staining. One microgram of total RNA was first
incubated for 5 min at 70°C with 1 µg of oligo(dT) in a volume of
7.5 µl and quick-chilled on ice before RT reaction buffer was added.
Single-strand cDNA synthesis was carried out in 12.5 µl containing 50 mM Tris · HCl (pH 8.5), 30 mM KCl, 8 mM
MgCl2, 1 mM dithiothreithol, 1 mM deoxynucleotide triphosphates, 25 U of RNase inhibitor, and 40 U of avian
myeloblastosis virus reverse transcriptase (Boehringer Mannheim,
Meylan, France). The reaction mixture was incubated for 60 min at
42°C and then for 5 min at 99°C. cDNA was amplified in 50 µl
of reaction buffer containing 50 mM KCl, 20 mM Tris (pH 8.4), 2 mM (for
S14) or 1.5 mM (for GM-CSF) MgCl2, 0.2 mM deoxynucleotide
triphosphates, and 0.24 µM each primer. Samples were heated to
80°C before 2.5 U of Taq polymerase (Life Technologies)
were added to reduce formation of nonspecific amplification products.
Cycling parameters were as follows: denaturation, 94°C for 1 min;
annealing, 59 (for S14) or 55°C (for GM-CSF) for 1 min; and
extension, 72°C for 1 min. The final extension was carried out for
5 min. Amplification products were analyzed by electrophoresis on 2%
agarose gels and visualized by ethidium bromide staining.
Western blotting. Levels of phosphorylated and
nonphosphorylated extracellular signal-regulated kinase (ERK) 1/2 in
control and DEP-treated cells were determined by SDS-PAGE as described previously (20). Briefly, proteins were collected from pelleted cells
by resuspension in Laemmli buffer, pH 7.4 (0.06 M Tris, 3% SDS, 5%
-mercaptoethanol, and 10% glycerol) with 1 mM phenylmethylsulfonyl fluoride and run on 12% SDS-PAGE gels. Samples were normalized for
protein content before being loaded. Electrophoresed proteins were
electroblotted onto nitrocellulose membrane, and the blots were blocked
with 5% milk in Tris-buffered saline (0.02 M Tris and 0.135 M NaCl, pH
7.6) containing 0.1% Tween. They were incubated overnight with the
primary antibody and 1% milk in Tris-buffered saline-0.1% Tween.
Horseradish peroxidase-conjugated anti-rabbit IgG was used as secondary
antibody. Bands were detected with chemiluminescence reagents and film
as per the manufacturer's instructions (NEN, Les Ulis, France).
Statistical analysis. Student's t-test was used to compare cytokine secretion from control versus treated samples. Values are means ± SE. P values were corrected for multiple comparisons.
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RESULTS |
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16HBE14o cells cultured in the presence of DEPs produced a time-
and dose-dependent increase in GM-CSF release (Fig.
1), which was already significant from 5 µg/cm2 after 24 h of exposure (108.2. ± 0.8 pg/ml
compared with 57.1 ± 2.4 in control cells).
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To investigate whether the DEP-induced GM-CSF release requires
endocytosis of the particles, which Boland et al. (6) have previously
demonstrated, we examined the influence of Cyto D, an inhibitor of
actin filament assembly. Figure 2 shows
that the secretion of GM-CSF in the control cultures was only slightly affected by Cyto D. However, DEPs without Cyto D increased the release
of GM-CSF to 151%, whereas in the presence of Cyto D, the increase
with DEPs was only 78% compared with cultures treated with Cyto D
alone.
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To determine the respective role of the adsorbed organic compounds and
the carbon core, we compared the effects of native DEPs, extracts of
DEPs, extracted DEPs, and carbon black particles (Fig.
3). Dichloromethane extracts of DEPs at 20 µg/ml, a concentration equivalent to 10 µg/cm2 of
native DEPs, had the same effect on GM-CSF release as unextracted DEPs
(172 and 191%, respectively, over control values). However, the
extracted particles induced a lower but still significant increase in
GM-CSF release, whereas carbon black particles had no effect (73 and
3%, respectively, over control values).
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DEPs contain trace amounts of metals on the particle surface that may,
like for particles such as asbestos or ROFA, mediate an inflammatory
response through catalysis of redox reactions directly on the particle
surface. Figure 4 shows,
however, that the metal chelator DEF did not alter the inflammatory
response to DEPs.
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To investigate the molecular events leading to the increase in GM-CSF
secretion, we first studied the effects of the protein synthesis
inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin D. Both inhibitors blocked DEP-induced GM-CSF release, suggesting that de
novo protein and mRNA syntheses are necessary in mediating the DEP
effect (Table 1). Measurement of mRNA
levels by RT-PCR corroborates these results (Fig.
5). Indeed, a treatment for 24 h with DEPs
at 10 µg/cm2 increased the mRNA of GM-CSF compared with
that of the control cultures. Carbon black particles also
induced an increase in this mRNA, which is, however, less important
than after DEP treatment. Transcription factors such as NF-B, which
has been shown to be activated by DEPs (3, 36), are sensitive to
oxidative stress. To investigate whether the DEP-induced increase in
GM-CSF involves ROS, various radical scavengers were added to the cell
cultures. Figure 6 shows the decrease in
the response to DEPs after the addition of NAC (43% over control value
compared with 151% without drug), DMTU (40% over control value
compared with 151% without drug), and PDTC (96% over control value
compared with 213% without drug). However, the lipid radical scavenger
-tocopherol had no effect on GM-CSF release after DEP treatment
(174% over control value compared with 133% without drug). No
decrease in GM-CSF release was observed in control cultures by these
radical scavengers. Furthermore, Fig. 7
shows that in the presence of NAC, the increase in GM-CSF release was
not only significantly reduced in cultures treated with native DEPs but
also in cultures treated with DEP extracts.
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To assess the signal transduction pathways involved in DEP-induced
GM-CSF release, we used specific inhibitors. H-89, an inhibitor of
protein kinase (PK) A, as well as the PKC inhibitor GF-109203X had no
effect on the secretion of GM-CSF in response to DEP treatment. Furthermore, inhibitors of the cyclooxygenase and lipoxygenase pathways
(indomethacin and nordihydroguaiaretic acid, respectively) did not
reduce the secretion of GM-CSF (Table 2). In contrast, the tyrosine kinase inhibitor genistein diminished the release of
GM-CSF in the control cultures and blocked the increase in cytokine secretion after DEP treatment (Fig. 8). In
addition, we studied the effect of specific inhibitors of the
mitogen-activated protein (MAP) kinase pathways. PD-98059, an inhibitor
of MAP or ERK kinase, diminished the release of GM-CSF in control
cultures and strongly reduced the DEP-induced increase in GM-CSF
secretion (58% over control value compared with 213% without drug),
whereas the MAP kinase p38 inhibitor SB-203580 had a lower effect
(102% over control value; Fig. 8). Western blot analysis actually
showed an increase in phosporylated ERK, which is the active form of MAP kinase, after only 30 min of exposure to DEPs (Fig.
9).
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DISCUSSION |
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Exposure of animals or humans to DEPs results in an inflammatory
reaction characterized by enhanced IgE and cytokine production (24).
Airway epithelial cells exposed to DEPs in vitro have been shown to
secrete increased amounts of proinflammatory mediators such as soluble
intercellular adhesion molecule-1 (sICAM-1), IL-6, IL-8,
and GM-CSF (5, 6, 25, 33). However, the underlying cellular mechanisms of this inflammatory response are still unclear. In
the present study, we studied the mechanisms that induce GM-CSF secretion after DEP exposure in the human bronchial epithelial cell
line 16HBE14o. GM-CSF is a key cytokine in the development of
respiratory allergic disease because besides its role in the maturation
of granulocytes and macrophages, it also has several biological
activities on eosinophils, the predominant cells in bronchial asthma.
There is an increase in GM-CSF-positive cells in bronchial asthma (18),
and the nasal epithelial cells of atopic individuals release increased
amounts of GM-CSF that were still enhanced by allergen exposure (8).
Experimental studies have indicated an increased release in GM-CSF
by human bronchial epithelial cells from asthmatic
patients (16), who also present an increased sensitivity to DEPs in
respect to GM-CSF release (4). The increase in inflammatory mediators
could be due to the release of preexisting pools of cytokines or to de
novo protein synthesis involving enhanced translation of preexisting
mRNA or increased transcription of cytokine genes. These mechanisms may involve signal transduction pathways using PKs and/or ROS. In addition,
it is unclear which part of the particle, the adsorbed organic
compounds, the metals, and/or the carbon core, is responsible for the
inflammatory response and if it requires phagocytosis of the particles.
We have demonstrated that DEPs induce a time- and dose-dependent
increase in the secretion of GM-CSF by 16HBE14o cells, which corroborates results from primary cultures of human nasal or bronchial epithelial cells (5, 25).
Phagocytosis of DEPs observed in airway epithelial cells (7), which is also a time- and dose-dependent process (6), seems to be a prerequisite for the induction of cytokine release because Cyto D, an inhibitor of actin filament polymerization involved in the endocytosis of particles, reduced DEP-induced GM-CSF secretion. An inhibitory effect of particle-induced cytokine release by Cyto D was also seen for asbestos (29). The concentration of Cyto D used in this study only partially inhibits the phagocytosis of DEPs as seen by flow cytometry and transmission electron microscopy (data not shown), which may explain that DEPs still significantly increase the release of GM-CSF despite the presence of Cyto D. Exocytosis, which also depends, in part, on actin filament assembly, was not affected by Cyto D treatment because the release of GM-CSF was only slightly reduced in control cultures in the presence of Cyto D.
A crucial question for understanding the inflammatory response generated by DEPs is the relative importance of the carbon core and the adsorbed organic or metallic compounds in this event. The DEP-induced GM-CSF increase is not inhibited by the metal chelator DEF, suggesting that the trace amounts of metals on the DEP surface are not responsible for the inflammatory effect, in contrast to other air pollution particles such as ROFA (27). Furthermore, we have shown that the increased release of GM-CSF is mainly due to the adsorbed organic compounds because organic extracts of DEPs induce the same response as DEPs. In this connection, Ohtoshi et al. (25) have shown that benzo[a]pyrene, an aromatic hydrocarbon contained in DEPs, increases the release of GM-CSF by the human bronchial cell line BEAS-2B. This corroborates previous results by Boland et al. (6) on the effect of catalysts that reduce the adsorbed organic compounds, thus decreasing the DEP-induced GM-CSF secretion. A predominant role of the adsorbed organic compounds on DEP-induced cytokine release has also been shown for rat alveolar macrophages (41). However, the carbon core may also play a role in the inflammatory response because extracted DEPs have a lower but still significant effect. The differences in response between the carbon core of DEPs and carbon black, which had no effect on GM-CSF release, may be due to different particle number, size, or surface area. Indeed, different types of carbon black particles at the same concentration could have adverse biological effects depending on their physical properties (12). Recently, it has been shown that ultrafine carbon black exhibits greater free radical activity than fine carbon black or quartz (34). This points out the difficulty in predicting the role of the carbon core by the use of other kinds of particles devoid of organic compounds like carbon black, TiO2, or silica. However, because the inflammatory response elicited is a time- and dose-dependent process, carbon black particles may induce an increase in GM-CSF at another time point or at higher concentrations.
Our hypothesis is that phagocytosis may be necessary to allow the
desorption and further metabolism of the adsorbed organic compounds.
DEPs and their extracts both seem to trigger signal transduction
pathways involving ROS because the secretion of GM-CSF is strongly
decreased after treatment with NAC. Indeed, polycyclic aromatic
hydrocarbons such as benzo[a]pyrene and
1-nitropyrene (23, 35) as well as the activation of cytochrome
P-450 have been shown to induce the production of ROS. We have
shown that the increase in GM-CSF after DEP treatment is inhibited by
the radical scavengers NAC, DMTU, and PDTC. However, no increase in the
intracellular levels of peroxides or depletion of thiols could be
observed by flow cytometry with fluorescent probes (data not shown).
This suggests that DEPs did induce signaling pathways involving a
moderate production of ROS, which act as a second messenger leading to
the production of inflammatory mediators, whereas oxidative stress due
to high levels of ROS has been shown to induce cytotoxicity. However,
in acellular systems, DEPs or DEP extracts could produce ROS (31, 38).
Furthermore, the lipid radical scavenger -tocopherol had no effect,
suggesting that the inflammatory effect of DEPs is not due to lipid
peroxidation, which has been shown to be involved in asbestos-induced
transcription factor activation (15).
We confirm the results of Ohtoshi et al. (25) showing that the release
of GM-CSF seems to be due to de novo protein synthesis. This increase
in synthesis seems rather to be due to new transcription of cytokine
genes than to translation of preexisting mRNA because we showed an
increase in GM-CSF mRNA by DEPs. Exposure of bronchial epithelial cells
to DEPs has also been shown to increase transcription of IL-8 mRNA
(36). This is in agreement with previous results (3) of increased
activation of the transcription factor NF-B by DEPs in
16HBE14o
cells. The production of ROS by DEPs may explain the
activation of this oxidative stress-sensitive transcription factor.
To further investigate the signaling pathway elicited by DEPs, we used different specific inhibitors of several PKs. Only genistein, an inhibitor of tyrosine kinases, but not inhibitors of PKC, PKA, or the arachidonic acid pathway prevents the effect of DEPs, whereas they are involved in GM-CSF release induced by other mediators (2, 17, 26). Genistein also reduces the release of GM-CSF in control cultures, suggesting that the same signaling pathways are involved in constitutive and DEP-induced GM-CSF release.
Tyrosine kinases are known to elicit MAP kinase pathways that lead to
phosphorylation-dependent activation of a variety of transcription
factors that modulate cytokine gene expression. ERK, c-Jun
NH2-terminal kinase, and the p38 pathway are the three major MAP kinase pathways. Indeed PD-98059, an inhibitor of the ERK
pathway, also significantly reduces the release of GM-CSF in response
to DEPs, whereas the inhibitor of p38 MAP kinase (SB-203580) has a
lower effect. However, PD-98059 is also an antagonist of the aryl
hydrocarbon receptor involved in the expression of phase I and II
biotransforming enzymes (28), and thus we cannot exclude that its
inhibitory action could also be due to the inhibition of DEP
metabolism. However, we have shown that DEP treatment actually increases the active, phosphorylated form of ERK. Recently, MAP kinases
have been shown to be activated by metals that also induce the
expression of cytokines in the human bronchial epithelial cell line
BEAS-2B (32). Cytokine induction in alveolar macrophages has been shown
to be dependent on both the ERK and p38 kinase pathways for optimal
gene expression (9). MAP kinase pathways are involved in the activation
of the transcription factor NF-B, which is also induced by DEPs (3).
Other cytokines such as IL-1 could also activate these signaling
pathways and increase the secretion of GM-CSF. Thus the DEP-induced
GM-CSF release, which is observed after 24 h, may be secondary to an
autocrine effect. Indeed, Boland et al. (6) have shown an increase in IL-1
release by human airway epithelial cells after DEP treatment.
In conclusion, the present study shows that the increase in GM-CSF release by DEPs is mainly due to the adsorbed organic compounds that may be metabolized after phagocytosis of the particles and induce the production of ROS. Our results further suggest that tyrosine kinase(s) may be involved in the DEP-triggered signaling of the GM-CSF response. These results are of particular interest regarding the development of efficient exhaust gas posttreatments that may reduce the inflammatory response elicited by DEP exposure, which is especially important for asthmatic patients. Indeed oxidation catalysts that reduce the soluble organic fraction are under development, and it has been shown that the use of these catalysts reduces the release of GM-CSF by human epithelial cells after DEP exposure (6).
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. D. C. Gruenert for the human bronchial cell line. We gratefully thank Christiane Guennou for excellent technical help and Mireille Legrand for the cell culture.
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FOOTNOTES |
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This work was supported by DIMAT Renault Grant 235, Ademe Grant BOU 9536, a grant from Caisse d'Assurance Maladie des Professions Liberales Provinces, and European Commercial Community Grant B104-CT960052.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Boland, Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris VII Denis Diderot, Tour 53/54 E3, case 70-73, 2 place Jussieu, 75251 Paris cedex 05, France (E-mail: marano{at}paris7.jussieu.fr).
Received 11 May 1999; accepted in final form 20 August 1999.
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