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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The organic compounds of diesel exhaust particles (DEP-PAHs)
have been shown to favor immunoglobulin production and
bronchial hyperresponsiveness and to affect cytokine and
chemokine productions. To evaluate if diesel exhaust could act
in synergy with a house dust mite allergen (Der p 1), peripheral
blood mononuclear cells from allergic patients were exposed
to DEP-PAHs, with or without purified Der p 1. DEP-PAHs and
Der p 1 separately induced an increase in interleukin (IL)-8,
regulated on activation, normal T cells expressed and secreted
(RANTES), and tumor necrosis factor-
concentrations. Interestingly, a synergy between the two stimuli was also observed. In the case of monocyte chemotactic protein (MCP)-1, DEP-PAHs reduced the release, whereas Der p 1 enhanced it. A simultaneous exposure led to reduced production as compared
with allergen exposure alone, but still represented an increase
as compared with the control exposure. Mitogen-activated protein (MAP) kinase Erk1/2 antagonist mainly inhibited the release of MCP-1, whereas MAP kinase p38 antagonist mainly
suppressed the release of IL-8 and RANTES. Messenger RNA expression correlated with protein measurements. Moreover, supernatants from cells exposed to both DEP-PAHs and Der p 1 had a significant chemotactic activity on neutrophils and eosinophils. These findings suggest that simultaneous exposure of
allergic patients to DEPs and allergens could result in high local
chemokine levels via MAP kinase pathways activation, increasing the likelihood of reaching a critical threshold leading to the initiation of respiratory allergic symptoms.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The relationship between allergic inflammation and environmental air pollution has been increasingly studied over the past few years. Air pollution consists mainly of an industrial production of various gases and particulate matters. Evidence showing that pollutants such as O3, SO2, and NO2 can induce and enhance some parameters of allergic inflammation was collected decades ago (1, 2), whereas studies dealing with the particulate phase of air pollution began more recently. A large part of the particles found in industrialized urban atmospheres are the result of diesel engine exhaust (3). One major concern of public health organizations is the rapidly growing prevalence of allergic diseases such as asthma among the populations of industrialized countries. The inflammation process in asthma is characterized by epithelial destruction and selective cellular infiltration, consisting mainly of eosinophils, specific T helper 2 lymphocyte subpopulations, and mast cells (4). A switch toward allergen-specific immunoglobulin (Ig) E antibody production occurs, leading to activation of effector cells. The evolution toward a type 2 cytokine profile further enhances the allergic reaction.
Both in vitro and in vivo, diesel exhaust particles (DEPs) and their associated polyaromatic hydrocarbons (DEP-PAHs) were shown to enhance the recruitment of inflammatory cells and to trigger the release of numerous mediators of inflammation, such as cytokines and adhesion molecules (5, 6). Among the cells overexpressing these mediators peripheral blood mononuclear cells (PBMCs), alveolar macrophages, and bronchial epithelial cells, which represent the first cell barrier in the airways, are of interest, especially when considering air pollutant impacts. Recent studies demonstrated links between allergic diseases and diesel emissions. DEPs and DEP-PAHs were shown to act in synergy with a given allergen, highly enhancing the production of specific IgE and type 2 cytokines (7). In a mouse model of ovalbumin-induced airway inflammation and airway hyperresponsiveness, a simultaneous diesel exposure and antigen challenge enhanced airway hyperresponsiveness and increased the number of eosinophils and mast cells in lung tissue (8). It also induced a significant increase in interleukin (IL)-2, IL-4, IL-5, and granulocyte macrophage colony- stimulating factor protein levels in the lung tissue and bronchoalveolar lavages (9).
An early and important step in the inflammatory reaction is the recruitment of cells to the site of the reaction.
This step is mainly mediated by a group of small molecules
called chemokines performing chemotactic activities. Apart
from their chemotactic activities on various cell populations, chemokines are of particular interest when considering the development of the allergic inflammatory reaction
(10). IL-8 has a strong chemotactic effect on neutrophils and has been shown to enhance adhesion to endothelial
cells and to allow the release of lysosomal enzymes. Monocyte chemotactic protein (MCP)-1, besides its chemotactic
activity on monocytes, performs a potent chemotactic and
histamine-releasing activity on basophils (11). Regulated
on activation, normal T cells expressed and secreted (RANTES) has a chemotactic activity on memory T lymphocytes and monocytes, as well as on eosinophils, and can induce degranulation of the latter. In addition, RANTES can
enhance IgE production by human B cells (12). Finally, increased levels of IL-8, RANTES, and MCP-1 are detected
in bronchoalveolar lavage fluids and bronchial biopsies of
allergic asthmatic patients (13, 14). The early phase of cell
recruitment is also dependent on the expression of endothelial cell adhesion molecules, themselves under the control of a number of cytokines, including tumor necrosis
factor (TNF)-.
In a previous study, we demonstrated that IL-8, RANTES,
and MCP-1 productions were modulated when exposed to
DEP-PAHs (15). Therefore, the aim of the present study
was to investigate the possible synergistic effects of allergen and DEP-PAHs in the early stages of the inflammatory
reaction. To achieve this goal, the effects of a combined
exposure of PBMCs from allergic patients to DEP-PAHs
and allergen Der p 1 (one of the major allergens of the
house dust mite Dermatophagoides pteronyssinus) were
evaluated on the production of TNF- and chemokines
IL-8, RANTES, and MCP-1. In addition, inhibitors were
used to evaluate the involved transduction pathways.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of Diesel Exhaust and Extraction of DEP-PAHs
DEPs were obtained from a light-duty, diesel-powered passenger car (Renault, Pollution Department, Boulogne-Billancourt, France), and DEP-PAHs were extracted in CH2Cl2 solvent as previously described (15).
The stock solution (25 µg DEP-PAHs/ml CH2Cl2) was stored at 4°C in the dark and was diluted freshly each time in RPMI medium before use.
Donors
Venous blood was collected from donors who were asthmatic patients sensitive to house dust mites. All were allergic patients and
presented positive skin-prick tests toward D. pteronyssinus allergen, positive radioallergosorbent test (class 
4), and elevated serum IgE levels (550 ± 149 IU/ml).
Cell Culture
PBMCs were prepared from blood collected on heparin. Briefly,
after removing the platelets, the blood was diluted and layered on
a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). The RPMI 1640 medium (Sigma, St. Louis, MO) used for culture was
supplemented with 2 mmol/ml glutamine, 100 IU/ml penicillin,
100 µg/ml streptomycin, and 10% fetal calf serum. PBMCs (2 × 106 cells/well) were cultured in 12-well, flat-bottomed microculture plates (Nunc, Roskilde, Denmark) with complete RPMI
alone, Ch2Cl2 alone, or DEP-PAH concentrations of 5 and 50 ng/
ml, with or without 100 ng/ml Der p 1 (kindly provided by G. A.
Stewart, University of Western Australia, Perth, Australia). Culture supernatants were collected after 6 or 24 h, filtered through a
0.2-µm-pore-sized filter (Sartorius AG, Göttingen, Germany), aliquoted, and stored at 
20°C for further quantifications.
Quantification of Chemokine and Cytokine Levels in Cell Culture Supernatants
Concentrations of TNF-, RANTES, MCP-1 (R&D Systems, Abingdon, UK), and IL-8 (CLB, Amsterdam, The Netherlands)
were measured by enzyme-linked immunosorbent assay (ELISA)
according to the manufacturer's recommendations. Results are
expressed in picograms per milliliter or as percentage of variation
from the CH2Cl2 control values ([Chemokine Control/Control] × 100%).
Semiquantitative Reverse Transcriptase/Polymerase Chain Reaction
After removal of the supernatants, PBMCs were resuspended in TRIZOL reagent (GIBCO BRL Life Technologies, Gaithersburg, MD), and total cellular RNA was extracted according to the manufacturer's procedure. RNA concentrations were measured using a spectrophotometer. RNA integrity was determined by visualizing the 18S and 28S ribosomal RNA bands with ethidium bromide after electrophoresis on a 2% formaldehyde gel. To synthesize complementary DNA (cDNA), 1 µg of RNA was resuspended in a volume of 25 µl with 0.2 µg oligo (dT)12-18 and 8 IU RNasin. After annealing for 5 min at 70°C, the solution was cooled to 4°C. First strand buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, pH 8.3), 2 mM of each deoxynucleotide triphosphate (Pharmacia Biotech, Uppsala, Sweden), 8 IU RNasin ribonuclease inhibitor (Promega, Lyon, France), 40 µg/ml carrier transfer RNA, 4 mM dithiothreitol (GIBCO BRL), and 400 IU Maloney murine leukemia virus (MMLV) reverse transcriptase (GIBCO BRL) were then added in a final volume of 50 µl. The reaction mixture was heated 1 h at 42°C to synthesize the DNA strand, and the reaction was stopped by denaturing the enzyme at 95°C for 5 min.
The polymerase chain reactions (PCRs) were carried out in a
volume of 25 µl and covered with oil. A total of 10 µl of each cDNA that was diluted five times was supplemented with 100 µmol/liter of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanidine triphosphate, and deoxythymidine triphosphate (Boehringer-Mannheim, Mannheim, Germany), 0.5 µmol/
liter of each primer, 0.6 U platinum Taq DNA polymerase (GIBCO
BRL), and 1 mmol/liter MgCl2. PCR primers for human glyceraldehyde phosphate dehydrogenase (GAPDH), TNF-, MCP-1,
RANTES, and IL-8 were purchased from Eurogentec (Seraing, Belgium). The sequences of the primers were as follows: GAPDH sense: 5'-GTCTTCACCACCATGGAG-3'; antisense: 5'-CCAAAGTTGTCATGGATGACC-3'; TNF- sense: 5'-ACAAGCCTGTAGCCCATGTT-3'; antisense: 5'-AAAGTAGACCTGCCCAGACT-3'; MCP-1 sense: 5'-TCCAGCATGAAAGTCTCTGC-3'; antisense: 5'-TGGAATCCTGAACCCACTTC-3'; RANTES
sense: 5'-TCCCCATATTCCTCGGAC-3'; antisense: 5'-GATCTACTCCCGAAGCCA-3'; IL-8 sense: 5'-TTGGCAGCCTTCCTGATT-3'; antisense: 5'-AACTTCTCCACAACCCTCTG-3'.
The number of amplification cycles for each product was determined to define optimal conditions for linearity and to permit semiquantitative analysis of signal strength. A total of 25 cycles was performed for amplification of GAPDH, TNF-, and RANTES, and 22 cycles for IL-8 and MCP-1. Amplification was initiated by a 1-min denaturation step at 94°C and was then followed
by 22 or 25 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a 7-min extension at 72°C using a DNA thermal
cycler (Mastercycler 5330; Eppendorf, Mississauga, ON, Canada). Amplified PCR products were separated by gel electrophoresis in 1.5% agarose after Gelstar nucleic acid staining (FMC
Bioproducts, Rockland, ME). DNA molecular weight marker VI
(0.15 to 2.1 kb) was purchased from Boehringer-Mannheim. The
intensity of each spot was calculated by densitometry analysis,
and results were expressed as percentage of each corresponding
GAPDH housekeeping gene's optical density (Gel Analyst;
Clara Vision, Paris, France). Specificity of the amplified products
was ascertained using specific oligonucleotide probes as previously described (16).
Chemotaxis Assay
Human neutrophils and human eosinophils were purified from the blood of nonallergic donors using Boyüm's technique and of hypereosinophilic patients using magnetic beads coated with anti- CD16 (17) (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany), respectively.
The purity of neutrophil preparation was over 98%. The purity of eosinophil preparation was 95.4 ± 1.6% with 2.4 ± 1.4% lymphocyte and 2.2 ± 1.8% monocyte contamination. Cells were harvested and resuspended in RPMI at a concentration of 106 cells/ml.
The chemotaxis protocol was performed with a 48-well micro chemotaxis chamber as previously described (15) with all controls regarding the distinction between chemotaxis and chemokinesis, using supernatants from cells exposed to CH2Cl2 solvent, 50 ng/ml DEP-PAHs, 100 ng Der p 1, or both DEP-PAHs and Der p 1.
Involvement of Reactive Oxygen Species and Mitogen-Activated Protein Kinase Pathways
To evaluate the transduction pathways involved in the modulation of chemokine production, cells (2 × 106) exposed to CH2Cl2 control, 50 ng/ml DEP-PAHs, and/or 100 ng/ml Der p 1 were coincubated with three different inhibitors: one inhibitor of the reactive oxygen species (ROS) pathway, glutathione at 1 mM (Sigma); and two inhibitors of the mitogen-activated protein (MAP) kinase pathways, a specific Erk1/2 kinase inhibitor at 2.5 µM (PD-98059; Calbiochem, San Diego, CA) and a specific p38 kinase inhibitor at 1 µM (SB-203580; Calbiochem) during the 24 h incubation. IL-8, RANTES, and MCP-1 concentrations in supernatants were then measured as described previously. Results obtained for each CH2Cl2 control was substracted from the results obtained with 50 ng/ml DEP-PAHs and/or 100 ng/ml Der p 1 before calculating the percentage of inhibition obtained with each specific inhibitor.
Statistical Analysis
Statistical analysis for TNF-, IL-8, MCP-1, and RANTES levels
was performed using the nonparametric Wilcoxon's paired rank test. Values of P < 0.05 were regarded as statistically significant. If not specified otherwise, all results are expressed as median and
interquartile (Q1 and Q3) values. Statistical analysis was performed using the Statview 4.11 software (Abacus Concepts, Berkeley, CA) on Macintosh.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Viability
The toxicity of the dichloromethane solvent and the DEP-PAHs solution added to the cell culture was evaluated by a trypan blue test. The volume of dichloromethane or DEP-PAHs solubilized in dichloromethane added to 1 ml medium was limited to 2 µl. No toxic effect of either product could be detected (viability > 97% for all conditions) after 24 h.
Chemokine and Cytokine Concentrations in Supernatants
Control with medium did not show any significant difference with the CH2Cl2 control. It is of note that using increasing concentrations of DEP-PAHs (ranging from 0.5 to 50 ng/ml), a dose-response effect was observed, the strongest effect corresponding to the highest DEP-PAH concentration (data not shown). In this study, results obtained with the optimal dose of 50 ng DEP-PAHs/ml and with a 24 h exposure time are presented.
DEP-PAHs enhanced TNF-, IL-8, and RANTES release
and decreased MCP-1 release. ELISA performed on supernatants of PBMCs from allergic patients after 24 h of culture demonstrated a significant increase in TNF-, IL-8,
and RANTES concentrations as compared with the CH2Cl2
control (Figure 1). When expressed as percentage of variation from the CH2Cl2 control, the increase in TNF-, IL-8, and RANTES was 114, 153, and 25%, respectively. On the
contrary, exposure to DEP-PAHs decreased MCP-1 levels
as compared with CH2Cl2 control (Figure 1). This decrease in MCP-1 reached 76%.
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Der p 1 enhanced IL-8, RANTES, MCP-1, and TNF- release. PBMCs from patients allergic to D. pteronyssinus
were cultured with CH2Cl2 solvent (without DEP-PAHs)
and 100 ng/ml purified Der p 1. The concentrations of TNF-,
IL-8, RANTES, and MCP-1 in supernatants all increased
as compared with the CH2Cl2 control (Figure 1), corresponding to a 529, 507, 404, and 5,941% increase, respectively.
Potentiating effects of DEP-PAHs and Der p 1. PBMCs
from patients allergic to D. pteronyssinus were cultured in
the presence of both DEP-PAHs and Der p 1. As shown in
Figure 1, the concentrations of all measured mediators
were strongly and significantly increased in these supernatants as compared with the CH2Cl2 control. The increase
for TNF-, IL-8, RANTES, and MCP-1 was 4,393, 1,230, 525, and 1,245%, respectively. When comparing these results with the sum of the effects of DEP-PAHs and Der p 1 alone, it corresponded to a 6.8-fold, 1.86-fold, and 1.22-fold increase in TNF-, IL-8, and RANTES, respectively,
showing a potentiating effect between the two stimuli. In
the case of MCP-1, the resulting balance between the inhibitory effect of DEP-PAHs and the positive effect of
Der p 1 resulted in a final level lower than the sum of the
two separate effects, but still largely increased when compared with the CH2Cl2 control.
Chemokine and Cytokine Messenger RNA Expression
Expression of messenger RNA (mRNA) encoding TNF-,
IL-8, RANTES, and MCP-1 was analyzed after 24 h by reverse transcriptase (RT)-PCR for PBMCs from allergic
patients exposed to CH2Cl2 control, DEP-PAHs alone,
Der p 1 alone, and combined DEP-PAHs and Der p 1.
As shown in Figure 2, mRNA expression for TNF-,
IL-8, and RANTES was enhanced for cells exposed to
DEP-PAHs, whereas expression for MCP-1 was decreased
as compared with CH2Cl2 control. In a similar way to
what was observed at the protein level, cells exposed to
Der p 1 expressed higher amounts of mRNA coding for
TNF-, IL-8, RANTES, and MCP-1 than did control cells.
Simultaneous exposure to both DEP-PAHs and Der p 1 further enhanced the mRNA expression for TNF-, IL-8,
and RANTES, whereas mRNA expression for MCP-1 decreased as compared to exposure to Der p 1 alone but increased when compared with control cells.
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Chemotaxis Assay
To evaluate the chemotactic activity, supernatants from PBMCs from allergic patients cultured with control CH2Cl2, DEP-PAHs, Der p 1, or both DEP-PAHs and Der p 1 were used in a chemotaxis assay with either neutrophils or eosinophils.
As shown in Figure 3, supernatants from cells incubated with DEP-PAHs, Der p 1, or a combination of both exhibited a significantly enhanced capacity to attract neutrophils (10.9 [8.6:11.1], 6.7 [6.2:7.1], 21.6 cells/high-power field [hpf] [20.3:25.3], respectively, versus CH2Cl2 control, 2.1 cells/hpf [1.8:2.5]). Similar data were obtained with eosinophils (10.7 [8.7:11.7], 11.2 [9.3:13.2], 19.8 cells/hpf [17.8:21.3], respectively, versus CH2Cl2 control, 2.5 cells/hpf [2.3:3.3]). The strongest effect on both neutrophils and eosinophils was obtained with supernatants from cells exposed to combined DEP-PAHs and Der p 1 (929 and 692%, respectively, versus CH2Cl2 control). In this case, chemotactic activity of the supernatants was significantly enhanced versus CH2Cl2 control but also versus supernatants from cells exposed to DEP-PAHs or Der p 1 alone.
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When adding neutralizing anti-IL-8 antibodies to the supernatants before the assay, the enhanced chemotactic activity toward neutrophils of supernatants from cells exposed to DEP-PAHs, Der p 1, and both DEP-PAHs and Der p 1 was reduced by 71, 55, and 64%, respectively. When neutralizing anti-RANTES antibodies were added to the supernatants before the assay, the chemotactic effects toward eosinophils were reduced by 47, 36, and 24%, respectively.
A control using anti-IL-3 antibodies showed no inhibitory effect on the ability of supernatants from cells exposed to DEP-PAHs and/or Der p 1 to induce chemotactic movements of neutrophils or eosinophils (data not shown).
Involvement of ROS and MAP Kinase Pathways in Chemokine Release
Cells exposed to CH2Cl2 control, DEP-PAHs, and/or Der p 1 were treated with glutathione, a scavenger of some oxygen species. As stated in Table 1, the effects mediated by diesel extracts on the release of IL-8 and RANTES in supernatants were partly reduced. The effects mediated by Der p 1 were only slightly reduced, except for the production of RANTES that was markedly inhibited. Finally, when glutathione was added to the cells exposed to both DEP-PAHs and Der p 1, a partial inhibition was observed in the production of IL-8 and MCP-1, whereas RANTES release was strongly inhibited.
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In a second step, the effects of specific MAP kinase inhibitors, PD-98059 (Erk1/2 antagonist) and SB-203580 (p38 antagonist), were evaluated on the production of the chemokines IL-8, RANTES, and MCP-1. As shown in Table 2, the effect of diesel exhausts on IL-8 and RANTES production was only partly reduced by addition of the Erk1/2 inhibitor (57 and 48% inhibition, respectively) but was strongly inhibited by addition of the p38 inhibitor (96 and 83%, respectively). In a similar way, the effect mediated by Der p 1 on IL-8 and RANTES release was reduced by only 58 and 54% with the Erk1/2 inhibitor but reached 86 and 90%, respectively, when using the p38 inhibitor. In contrast, the production of MCP-1, which was inhibited by exposure to diesel, was restored by both Erk1/2 and p38 inhibitors (79 and 93%, respectively). Under exposure to allergen Der p 1, only the Erk1/2 inhibitor markedly inhibited the production of MCP-1 (97%), whereas the effect of the p38 inhibitor was very limited (35%).
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When Erk1/2 and p38 antogonists were used at the same time, the release of IL-8, RANTES, and MCP-1 exposed to diesel extracts, allergen, or both stimuli was almost completely inhibited (> 90%, data not shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Diesel organic extracts have been shown recently to interfere with various parameters of the inflammatory process
and more precisely to be able to modulate the cytokine
and chemokine pathways. In this study, we evaluated the
combined effects of DEP-PAHs and a specific allergen on
chemokine release by PBMCs from allergic patients. Diesel organic extracts and a major house dust mite allergen
acted in synergy, strongly affecting the chemokine production of PBMCs from allergic patients. In a fashion similar
to that which had previously been observed with PBMCs
from normal donors, DEP-PAHs enhanced the IL-8 and
RANTES release in supernatants after a 24-h incubation.
In contrast, MCP-1 release was blocked. Corresponding
variations at the mRNA level confirmed that DEP-PAHs affected the IL-8, RANTES, and MCP-1 expression at the
transcriptional level. It is of note that the relative importance of mRNA productions was not matching the corresponding protein releases. This could be due to either intracellular storage or post-transcriptional regulation, the
second hypothesis being the most likely because the storage of chemokines or TNF- has not been reported until now in PBMCs. IL-8, RANTES, and MCP-1 are of particular interest when considering allergic patients, in part because they have been found overexpressed in allergic asthmatic bronchoalveolar lavage fluids and bronchial biopsies
(13, 14). Here, incubation of PBMCs from mite-sensitive
patients with Der p 1 led to a strong increase in the production of all three chemokines, both at the protein and
the mRNA levels. This suggests that a high local release of
chemokines in allergic patients can occur both through
structural cell production and migrating PBMCs, therefore
amplifying the local inflammatory cell recruitment. These
in vitro data are for the main part in agreement with the in
vivo results observed with bronchial biopsies and bronchoalveolar lavage fluids. Yet the case of MCP-1 would require further investigation because contradictory observations concerning the in vivo MCP-1 release in fluids from asthmatic patients have been obtained, depending on the
studies (18). Therefore, the in vitro potentiation by DEP-PAHs of the allergen-induced chemokine production suggests that exposure of allergic patients to diesel may further amplify the local process of inflammation. Another issue that remains to be assessed is the exact cell population
involved in chemokine overexpression. It is likely that
monocytes rather than lymphocytes are mainly responsible
for the massive chemokine production, but this will have to
be further explored using separated PBMC subpopulations.
In parallel to the chemokine evaluation, we investigated whether the production of TNF- was also affected.
We found that both DEP-PAHs and Der p 1 separately
enhanced the release of TNF- in supernatants, as well as
its mRNA expression. Once again, when combining DEP-PAHs and Der p 1, the two stimuli had a synergistic effect
and led to a strong TNF- production. It is known that inflammatory blood cells must perform diapedesis through
the vessel wall to enter the actual site of inflammation in
the tissue. This migration involves the transient expression
of successive families of membrane adhesion molecules
such as selectins and integrins (19). TNF- is produced by
monocytes, macrophages, neutrophils, natural killer cells,
and T cells, and is in part responsible of the induction of
expression of intercellular adhesion molecule-1, vascular
cellular adhesion molecule-1, and E-selectin (20). In addition, it is of note that TNF- is involved in bronchial hyperresponsiveness and can activate eosinophils (21, 22). Teran and coworkers (23) recently demonstrated that TNF-
could directly induce the release of RANTES by lung fibroblasts. Similar suggestions also issued from studies
dealing with human peripheral blood monocytes (24) or
rat skin (25), the latter correlating with a recruitment of
eosinophils. In a human bronchial epithelial cell line, TNF-
was also described to induce IL-8 production in a concentration-dependent manner (26). It is therefore tempting to
speculate that the present increase in RANTES or IL-8
production could be partly explained by a direct synergistic effect between DEP-PAHs and the allergen, but also
by the strong parallel increase in TNF- release. It can
also be suggested that combined diesel and allergen exposure, through TNF- overexpression, might affect the inflammatory process in its early stages, indirectly facilitating the diapedesis of circulating inflammatory cells.
In an attempt to evaluate the resulting biologic chemotactic activity of the supernatants, we used them in a chemotaxis assay on neutrophils and eosinophils. The recruitment of both populations was enhanced when using supernatants from cells exposed either to DEP-PAHs or Der p 1, and this effect was clearly further enhanced with supernatants from cells exposed to both. This tends to confirm that chemokine overexpression observed after combined exposure to DEP-PAHs and Der p 1 is responsible for an effective increased chemotactic activity for effector cells potentially harmful in a context of allergic inflammation. The chemotactic activity on neutrophils was mainly mediated by IL-8 because a blocking antibody against IL-8 reduced the activity by 64%. Eosinophils responded partly to the attraction by RANTES because a blocking antibody against RANTES could reduce the supernatant activity by only 24%. This suggests that additional mediators are likely to be involved in eosinophil recruitment.
DEP-PAHs can theoretically trigger cell metabolism
via two main distinct pathways: a direct interaction with
PAH cytosolic receptors such as AhR (receptor for dioxin), which can translocate to the nucleus and act as transcription factor, and the broad ROS pathway, leading to
the induction of promoters of metabolizing enzymes or activation of pluripotent transcription factors such as activator protein (AP)-1 or nuclear factor 
B (NF-B) (27). Interestingly, this latter effect on ROS production seems
specific to the organic phase of DEPs because diesel particles after organic extraction of their adsorbed PAHs no
longer display ROS induction in alveolar macrophages
(28). When exposed to a range of dioxin concentrations
(the main AhR agonist and a component of DEP-PAHs) during 7 or 24 h, PBMCs from allergic patients did not exhibit altered chemokine production as compared with the
diluent control (data not shown). This implies that triggering the AhR in our culture conditions did not mimic the
chemokine dysregulation, suggesting that this pathway is
not involved in the effect of diesel extracts on chemokines.
On the contrary, experiments using glutathione, a scavenger of oxygen species, revealed that the production of IL-8,
RANTES, and to a lesser extent MCP-1 was sensitive to
glutathione, pointing out the involvement of the ROS
pathway. In contrast, except for RANTES, little inhibitory
effect was observed on the effects mediated by Der p 1, showing that the allergen can trigger cytokine and chemokine productions by affecting other pathways such as those
related to its proteolytic activity (29). In the case of
RANTES, it is known that this chemokine is regulated in
part by ROS such as H2O2 (30) and is produced in large
amounts by platelets. It has also been demonstrated that
allergen stimulation of platelets leads to oxygen metabolite production (31). This cell type is a common contaminant of PBMC preparations and accounts for a large part
of RANTES production, especially at early time points.
Therefore in this study, it cannot be excluded that the inhibition of RANTES production by glutathione might be
partly related to platelet inactivation.
To further define the transduction mechanisms involved in the mediation of the effects on chemokine modulation, we used specific inhibitors to block two MAP kinases downstream of the ROS: Erk1/2 and p38. Both
Erk1/2 and p38 can induce the activation of AP-1, which in
turn can regulate the transcription of several genes coding
for various mediators, including chemokines (30). Here we
show that the effect of DEP-PAHs on the production of
IL-8 and RANTES by PBMCs mostly relied on p38 activity, whereas both Erk1/2 and p38 kinases were important
in diesel-mediated reduced production of MCP-1. Regarding IL-8 and RANTES, this result is in agreement with a
recent study on human bronchial epithelial cells (32). The
effects mediated by Der p 1 alone were similarly mainly
dependent on p38 for the production of IL-8 and RANTES but in contrast were dependent almost solely on Erk1/2 for
enhanced MCP-1 production. When cells were coincubated with diesel exhaust and allergen, RANTES and IL-8
productions were mainly linked to p38 activation, whereas
MCP-1-enhanced production showed a dependency on
Erk1/2, suggesting a predominance of the effects of Der p
1 over DEP-PAHs. When both Erk1/2 and p38 inhibitors
were added, the observed chemokine dysregulations were
almost completely abolished in all cases, suggesting that
whatever the complexity of the primary steps involved in
the fixation and/or processing of DEP-PAHs and allergen
Der p 1, Erk1/2 and p38 kinases are likely to be the two
main intermediates of the transduction signal leading to
IL-8, RANTES, and MCP-1 dysregulation. Promoters of
IL-8, RANTES, and MCP-1 genes have been extensively
studied. All of them can be transcribed after NF-B and
AP-1 activation. Takizawa and colleagues (33) have shown
that DEPs can induce IL-8 expression via NF-B activation (and not AP-1) in bronchial epithelial cells, whereas Ng and coworkers (34) showed that p38 and AP-1 were involved in IL-8 and RANTES expression in macrophages
after tBHQ (a major quinone found in DEP-PAHs) stimulation. In parallel, Hiura and associates (28) demonstrated
the involvement of NF-B in RANTES production by
macrophages. Given the wide variety of chemicals composing the DEP-PAHs and the multiple cell types likely to
encounter them, it is not surprising that many activation
pathways are involved. In this study, we point out the
highly preferential involvement of ROS and the downstream MAP kinase pathway rather than NF-B in the final overexpression of IL-8, RANTES, and MCP-1 after
exposure to a combination of diesel extracts and allergen.
In conclusion, we show that PBMCs from patients allergic to D. pteronyssinus respond to a simultaneous exposure to diesel organic extracts and major specific allergen
Der p 1 by producing high amounts of IL-8, RANTES,
and TNF-, due in part to a synergy between the two stimuli. The inhibition of MCP-1 release by the diesel extracts
is counterbalanced by the effect of the allergen, nevertheless leading to a consistent production. The high chemokine release correlates with a strong chemotactic activity
of the supernatants toward eosinophils and neutrophils.
Therefore, it is likely that allergic patients may present increased symptom severity resulting from exposure to both
diesel exhausts and allergen because of a synergistic effect
of the two stimuli, leading to overexpression of chemotactic factors potentially responsible for the extravasation of
granulocytes and for the recruitment of a broad set of inflammatory cells. Moreover, these data provide us with activation pathway targets to modulate the deleterious effects of diesel on allergic inflammation.
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Footnotes |
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Abbreviations: aryl hydrocarbon receptor, AhR; activator protein-1, AP-1;
complementary DNA, cDNA; dichloromethane, CH2Cl2; diesel exhaust
particle, DEP; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; immunoglobulin, Ig; interleukin, IL; mitogen-activated protein, MAP; monocyte
chemotactic protein, MCP; messenger RNA, mRNA; nuclear factor-B,
NF-B; polycyclic aromatic hydrocarbon, PAH; peripheral blood mononuclear cell, PBMC; polymerase chain reaction, PCR; regulated on activation, normal T cells expressed and secreted, RANTES; reactive oxygen
species, ROS; tumor necrosis factor, TNF.
(Received in original form February 2, 2000 and in revised form April 10, 2000).
Acknowledgments: The authors wish to thank the team of the Pneumology Department of the Calmette Hospital of Lille for allergic patient recruitment, the staff of Renault Lardy for providing the diesel extracts and for the HPLC analysis, and Dr G.A. Stewart for providing purified Der p 1. They are grateful to Philippe Gosset and Philippe Lasalle for critical review of this work. This study was supported by a grant from ADEME and Institut Pasteur de Lille and by PRIMEQUAL-PREDIT grant 97034 from the Ministère de l'Environnement.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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