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Published ahead of print on December 30, 2003, doi:10.1165/rcmb.2003-0281RC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 421-427, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0281RC

Rapid Communication

Fine Particulate Matter Induces Amphiregulin Secretion by Bronchial Epithelial Cells

Sophie Blanchet*, Kiran Ramgolam*, Augustin Baulig, Francelyne Marano and Armelle Baeza-Squiban

Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris 7-Denis Diderot, Paris, France

Address correspondence to: Armelle Baeza-Squiban, Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris 7-Denis Diderot, 2 place Jussieu, Tour 53-54, 3e etage, case courrier 7073, 75251 Paris cedex 05, France. E-mail: baeza{at}paris7.jussieu.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Particulate matter (PM) is thought to be responsible for respiratory health problems. Epithelial cells exposed to particles release pro-inflammatory cytokines leading to inflammation of airways. However, the signaling cascades triggered by particles are poorly understood. We demonstrate that PM with an aerodynamic diameter < 2.5 µm (PM2.5) or diesel exhaust particles upregulate the expression of amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), in human bronchial epithelial cells. AR secretion was blocked by an inhibitor of the EGFR tyrosine kinase (AG1478), or a selective mitogen-activated protein (MAP) kinase/extracellular regulated kinase (Erk) inhibitor (PD98059), but not by the p38 MAP kinase inhibitor (SB203580). Thus, AR secretion is mediated through the activation of the EGFR and Erk MAP kinase pathway. In addition, AR secretion was inhibited by the antioxidant N-acetyl cysteine, but not by a neutralizing anti-EGFR, suggesting an EGFR transactivation via oxidative stress. AR may be involved in cytokine secretion, as AR can induce granulocyte macrophage–colony-stimulating factor (GM-CSF) release and a neutralizing anti-EGFR reduces the particle-induced GM-CSF release. This study indicates that PM2.5 induces the expression and secretion of AR, an EGFR ligand contributing to GM-CSF release, which may reflect an important mechanism for sustaining the proinflammatory response.

Abbreviations: amphiregulin, AR • betacellulin, BTC • carbon black, CB • diesel exhaust particles, DEP • dipalmitoyl lecithin, DPL • epidermal growth factor, EGF • EGF receptor, EGFR • extracellular regulated kinase, Erk • human bronchial epithelial cells, HBE cells • heparin-binding EGF, HB-EGF • interleukin-8, IL-8 • mitogen-activated protein, MAP • mitogen-activated protein kinase/Erk kinase, MEK • N-acetyl cysteine, NAC • particulate matter with an aerodynamic diameter < 2.5µm, PM2.5 • reactive oxygen species, ROS • transforming growth factor-{alpha}, TGF-{alpha} • Ultroser G, UG


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Exposure to particulate matter (PM) is associated with increased morbidity and mortality for respiratory and cardiovascular disorders (1, 2). Controlled human exposure has revealed that one of the most striking effects of PM exposure is inflammation of the pulmonary airways (3). Furthermore, recent studies highlight how long-term exposure to PM can promote airway remodeling in small airways (4). A study of people living in Mexico City, notorious for its pollution, revealed that their lungs have more particles typical of combustion processes (5) and thickened airway walls, compared with control subjects living in Vancouver (6). Chronic exposure and persistence of PM could be involved in chronic inflammation leading to airway remodeling (6).

Studies of epithelial cells in vitro have defined the molecular events induced by PM in airway cells. The proinflammatory response induced by PM, and characterized by various cytokines release, results from the activation of transcription factors and signaling pathways such as the mitogen-activated protein (MAP) kinase pathways (7–9). In particular, we previously showed that in human bronchial epithelial cells, diesel exhaust particles (DEP), a major component of urban PM in Europe, increase nuclear factor-{kappa}B DNA binding, activate extracellular regulated kinase (Erk1/2) and p38 MAP kinase pathways (10), and induce the expression and secretion of granulocyte macrophage–colony stimulating factor (GM-CSF) (11), a cytokine involved in allergic diseases. These events are mostly induced by organic components of the DEP (10), and are probably mediated by the generation of reactive oxygen species (ROS) during the metabolism of organic compounds (12). However, the initial molecular event(s) leading to this cascade is still misunderstood.

Additional studies have suggested that intracellular signals induced by particles may also originate from the cell membrane. The epidermal growth factor receptor (EGFR) can be activated by asbestos fibers (13) and PM, leading to activation of the MAP kinase signaling cascade and cell proliferation (14), or cytokine expression and secretion (15). In other respects it has been shown that the interleukin (IL)-8 release observed in bronchial epithelial cells exposed to cigarette smoke was mediated by EGFR ligands having an autocrine action on the EGFR (16). Such autocrine effect of EGFR ligands on cytokines de novo synthesis has also been observed in keratinocytes (17). Both normal and tumorigenic human lung epithelial cells are known to release EGFR ligands such as transforming growth factor-{alpha} (TGF-{alpha}), amphiregulin (AR), betacellulin (BTC), and heparin–binding EGF (HB-EGF) (18, 19). Their secretion is increased in bronchial epithelial cells exposed to a chemical stress such as vanadium (20) or cigarette smoke (16). Secretion of EGFR ligands may contribute to the persistent proinflammatory response, as well as to the repair process and airway remodeling events that are characteristic features of asthma and chronic obstructive pulmonary diseases. In addition, it is well documented that the EGFR is highly expressed in the bronchial mucosa of patients with asthma (21).

These observations led us to investigate, in a model of human bronchial epithelial cells, the effects of atmospheric particles on EGFR ligand expression and secretion and their involvement in GM-CSF release. We used PM with an aerodynamic diameter < 2.5 µm (PM2.5) collected in the Paris suburbs, as they are representative of ambient particulate air pollution in European urban areas. They were compared with reference DEP (SRM 1650a) as a model of PM component and carbon black (CB) particles mimicking the carbonaceous core of DEP and many PM.

We provide the first evidence that PM2.5 and DEP, but not CB, induce the expression and secretion of AR. AR expression is due to activation of EGFR and Erk1/2 pathways via a ligand-independent pathway that probably involves ROS. AR contribute to GM-CSF release by an autocrine effect. Increased secretion of AR could play a pivotal role in the induction of inflammation, repair response, and further airway remodeling by autocrine and paracrine pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Human bronchial epithelial 16HBE cells were obtained from Dr. D. C. Gruenert (University of Vermont, Burlington, VT). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham F12 (containing 365 mg/ml L-glutamine) culture medium (Laboratoires Eurobio, Les Ulis, France) supplemented with UltroserG (UG) (2%; BioSepra, Cergy-St.-Christophe, France) as previously described (10). Four hours before particle exposure, cells were incubated in DMEM/Ham F12 without UG.

Chemicals
EGF, H2O2, and N-acetylcysteine (NAC) were purchased from Sigma (St. Quentin Fallavier, France) and SB203358 from Calbiochem (VWR, Paris, France). PD98059 and AG1478 were purchased from Biomol Research Laboratories (Tebu, Le Perray en Yvelines, France). The mouse monoclonal antibody against the EGFR (neutralizing, clone LA1) and a nonspecific mouse IgG were from Upstate Biotechnologies (Euromedex, Mundolsheim, France) and Chemicon (Euromedex), respectively. Recombinant human AR was from R&D Systems Europe (Abingdon, UK).

Particles
Atmospheric particulate matter PM2.5 was collected with a high-volume sampler machine (DA-80; Megatec, Paris, France) in a school playground at Vitry-sur-Seine near Paris from March 15–20, 2002. During this period, 3690 m3 of air were filtered and particles were recovered on nitrocellulose filters. Particles were detached from the filters by sonication in water and dessicated by lyophylization. Diesel particulate matter SRM 1650a was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). Their metal contents have been described by Huggins and coworkers (22). The PM2.5 have been physico-chemically characterized (A. Baulig, personal communication). Briefly, their soluble organic fraction is 10% instead of 20% for DEP, but they have a higher Fe (10,000-fold) and Cu (17,000-fold) content than DEP. CB particles of 95 nm diameter (FR103) were obtained from Degussa (Frankfurt, Germany), and titanium dioxide (TiO2) of 150 nm diameter from Hunstman Tioxide Europe S.A.S (Calais, France).

Stock solutions of particles were generated by suspension in a solution of 0.04% dipalmitoyl lecithin (DPL; Sigma) in distilled water and sonicated twice for 1 min each at maximal power (100 W) (Vibracell; Bioblock Scientific, Illkirch, France). DPL is a surfactant component used to create the particle suspensions. Particles were used at 0.1, 1, 10, or 30 µg/cm2 (concentrations are expressed in µg/cm2 because particles rapidly sediment in the culture). Control solutions were prepared using 0.04% DPL.

Analysis of AR Gene Expression
16HBE cells were seeded into 25 cm2 flasks at 45,000 cells/cm2 2 d before treatment. The cells were exposed to particles (PM or DEP) at 10 µg/cm2 for different times between 6 and 30 h (18 h for macroarrays). Total cellular RNA was isolated using Tri Reagent (Sigma) according to the manufacturer's instructions. The RNA concentration was determined by measuring absorbance at 260 nm.

Macroarray
A human cytokine array (R&D Systems Europe) consisting of 375 different clones cDNAs was used. Radiolabeled cDNA were generated by 500 ng of poly(A)+ RNA retrotranscription in the presence of [33P]-dCTP, purified and hybridized to the membrane according to the manufacturer's instructions. The membranes were exposed to a phosphor screen and the signal detected with a Storm860 machine (Amersham Biosciences, Orsay, France) and quantified using ArrayVision software (Imaging Research Inc., St. Catherine, ON, Canada). Relative intensity of signals was normalized and compared between the different cell culture conditions with Excel software (Microsoft, Redmond, WA). Genes were considered as overexpressed when relative intensities ratio (particles versus DPL control) was > 1.7.

Northern Blots
Equal amounts (20 µg) of total cellular RNA were separated by 2.2 M formaldehyde/1.2% agarose denaturing gel electrophoresis, and capillary-transferred onto Hybond-N+ membranes (Amersham Biosciences) using 10x SSC buffer (1x: 150 mM NaCl, 15 mM sodium citrate). The blot was then baked for 2 h at 80°C and hybridized with specific sequences obtained by polymerase chain reaction with DyNAzyme II DNA Polymerase (Finnzyme; Ozyme, St. Quentin en Yvelines, France) according to the manufacturer's instructions and with the following primers: AR: (forward: 5'-Tgg TgC TgT CgC TCT TgA TA-3', reverse: 5'-ACAgCA ACA gCT gTg Agg AT-3'); glyceraldehyde phosphate dehydrogenase (GAPDH): (forward: 5'-ACC ACA GTC CAT gCCATC AC-3', reverse: 5'-TCC ACC ACC CTg TTg CTg TA-3').

Probes were synthesized from polymerase chain reaction products with the Redyprime II DNA Labeling system (Amersham Biosciences) according to the manufacturer's instructions. The blots were prehybridized and hybridized in Rapid-Hyb solution (Amersham Biosciences) and washed according to the manufacturer's instructions. The membranes were exposed to a phosphor screen and the signal detected with a Storm860 machine (Amersham Biosciences) and quantified using Bio1D software (Vilber Lourmat Biotechnology, Marne-la-Vallee, France).

Analysis of AR and GM-CSF Secretion
Cells were seeded into 12-well culture plates and exposed to particles at 10 µg/cm2 in DMEM/F12 medium, without UG in the presence or absence of different inhibitors, for 24 h. The inhibitors were added 30 min before particle exposure. The conditioned medium was then removed from the cells, centrifuged, and stored frozen until the assay. Enzyme-linked immunosorbent assay kits for AR or GM-CSF (R&D Systems Europe) were used according to the manufacturer's instructions. Color development was measured at 450 nm with a microplate photometer MR 5000 (Dynex Technologies, Issy les Moulineaux, France).

Statistical Analysis
All data are expressed as the mean ± SEM of three cultures from a representative experiment. Means were compared by ANOVA. The equal variance test is significant with {alpha} = 0.05 (P < 0.001). All pairwise multiple comparisons were made with the Student-Newman-Keuls method (t test, P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Effect of Particle Exposure on EGFR Ligand Expression: AR Overexpression
Gene expression in 16HBE cells treated by PM2.5 or DEP at 10 µg/cm2 for 18 h was studied using a cytokine expression array. Among the 375 genes present on the macroarray, AR mRNA is the most expressed and induced in response to DEP as well as PM2.5 (Table 1). This brought our focus on the expression of the different EGFR ligands genes. Other EGFR ligands (HB-EGF, BTC, TGF-{alpha}) are weakly expressed, especially HB-EGF (Table 1), and are not clearly induced by particles excepting BTC. However, BTC mRNA induction occurs only in PM2.5-treated cells and is moderated in comparison with AR mRNA induction. To confirm AR mRNA induction and define its time course, Northern blot analysis was performed on total RNA from cells exposed to DEP or PM2.5 (10 µg/cm2) for 6–30 h (Figure 1). AR transcripts were constitutively expressed in cells treated with DPL. PM2.5 and DEP exposure induced significant early (within 6 h) and sustained (to 30 h) effects on AR gene transcription. As already observed with macroarray experiments, the AR mRNA induction is more important in PM2.5- than in DEP-treated cells (Figure 1).


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  		TABLE 1 EGF receptor ligands gene expression in DEP- and PM25-treated 16 HBE cells

 


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  		Figure 1. Time course of induction of AR mRNA in 16HBE cells following exposure to 10 µg/cm2 DEP (DEP) or PM2.5 (PM) compared with controls (C). (A) Northern blots; (B) densitometric analysis.

 
The Effect of Particle Matter on AR Secretion
We next investigated whether the PM-induced increases in AR mRNA expression were accompanied by enhanced AR secretion into the culture medium. Control cultures constitutively release AR, and this level of release is the same in cells treated with DPL (Figure 2A). PM2.5 and DEP (10 µg/cm2) significantly increased AR secretion (7.8- and 3.6–fold, respectively), whereas similar concentrations of CB or TiO2 had no effect on AR release (Figure 2A). PM2.5-induced AR release is dose-dependent (Figure 2B). It occurs from 1 µg/cm2.



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  		Figure 2. Induction of AR protein secretion in 16HBE cells after 24 h of treatment with (A) DEP, PM2.5 (PM), CB, or TiO2 (Ti) at 10µg/cm2. (B) PM2.5 at 0.1, 1, 10, and 30 µg/cm2. Values represent mean ± SD of triplicate cultures. The equal variance test is significant with {alpha} = 0.05 (P < 0.001) and all pairwise multiple comparisons were tested with the Student-Newman-Keuls method (P < 0.05). Asterisks, different from control; open circles, different from other treatments.

 
Signaling Pathways Involved in AR Secretion
To elucidate the signaling pathways necessary for AR secretion, we studied the effects of a series of selective inhibitory drugs specific for the EGFR tyrosine kinase (AG1478) (Figure 3A), the MAP kinase/Erk kinase (MEK) pathway (PD98059) (Figure 3B) or the selective p38 MAP kinase pathway (SB203580) (Figure 3B). Both the AG1478 and PD98059 compounds reduced the basal and induced AR levels, whereas SB203580 had no effect. These results suggest that AR secretion induced by PM2.5 or DEP is mediated by activation of both the EGFR and the Erk MAP kinase pathway.



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  		Figure 3. Effects of signaling pathway inhibitors on DEP- or PM2.5-induced AR secretion in 16HBE cells. Cultures of 16HBE were pretreated for 30 min with inhibitors and then stimulated with 10 µg/cm2 DEP or PM2.5 (PM) for 24 h before collecting culture medium for enzyme-linked immunosorbent assay. (A) Effects of the EGFR tyrosine kinase inhibitor: 1 µM AG1478. (B) Effects of the MAP kinase inhibitors: 10 µM PD98059 or 1 µM SB203580. Values represent mean ± SD of triplicate cultures. The equal variance test is significant with {alpha} = 0.05 (P < 0.001) and all pairwise multiple comparisons were tested with the Student-Newman-Keuls method (P < 0.05). Asterisks, different from respective control; open circles, different from treatments without pathway inhibitors.

 
To determine if EGFR activation is ligand-dependent or ligand-independent via oxidative stress, we studied the effects of antioxidants (N-acetyl cysteine and catalase) (Figures 4A and 4B, respectively) or neutralizing anti-EGFR antibodies (which prevent EGF ligand binding) (Figure 5) on AR secretion. EGF and H2O2 were used as positive control for ligand-dependent or ligand-independent EGFR activation, respectively. NAC inhibited basal and toxin-induced AR secretion (Figure 4A), whereas catalase only inhibited toxin-induced AR secretion (Figure 4B) and the EGFR-neutralizing antibody only inhibited EGF-induced AR release (Figure 5).



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  		Figure 4. The role of oxidative stress in PM-induced AR secretion in 16HBE cells. AR secretion induced by PM2.5 (10 µg/cm2) or H2O2 (0.2 mM) in presence of (A) the antioxidant N-acetyl cysteine (NAC) at 10 mM or (B) the antioxidant enzyme catalase (CAT) at 1,400 U/ml. Values represent mean ± SD of triplicate cultures. The equal variance test is significant with {alpha} = 0.05 (P < 0.001) and all pairwise multiple comparisons were tested with the Student-Newman-Keuls method (P < 0.05). Asterisks, different from respective control; open circles, different from treatments without antioxidants.

 


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  		Figure 5. The role of EGFR ligands in PM-induced AR secretion in 16HBE cells. Effect of a neutralizing anti-EGFR antibody (0.50 µg/ml) on AR secretion induced by DEP (10 µg/cm2), PM2.5 (10 µg/cm2), EGF (50 ng/ml), or H2O2 (0.2 mM). Values represent mean ± SD of triplicate cultures. The equal variance test is significant with {alpha} = 0.05 (P < 0.001) and all pairwise multiple comparisons were tested with the Student-Newman-Keuls method (P < 0.05). Asterisks, different from respective control; open circles, different from treatments without EGFR antibody.

 
Involvement of AR in GM-CSF Secretion
To support the hypothesis of an involvement of AR in cytokine release, we first investigated the effect of recombinant human AR on GM-CSF release, a cytokine involved in the proinflammatory response induced by DEP and PM2.5 in 16HBE cells (23, 24). As 16HBE cells treated by 10 µg/cm2 DEP or PM2.5 secrete 10–40 ng AR/ml of culture medium, recombinant human amphiregulin was tested from 5–50 ng/ml. AR dose-dependently induces GM-CSF release from 10 ng/ml (Figure 6A). Second, we investigated the effect of a neutralizing anti-EGFR that prevents EGFR ligand binding on particles-induced GM-CSF release. DEP induce a higher GM-CSF release than PM2.5 (8- and 3.2-fold induction relative to control, respectively) (Figure 6B) as we have previously shown (24). The particles-induced GM-CSF release is reduced in presence of 0.25 µg/ml neutralizing anti-EGFR, whereas it does not occur in the presence of a nonspecific IgG (Figure 6B). The GM-CSF release is now 4.2- and 2.7-fold for DEP and PM2.5, respectively.



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  		Figure 6. Relationship between AR secretion and GM-CSF release. (A) Effect of human recombinant AR from 5–50 ng/ml on GM-CSF release. (B) Effect of a neutralizing anti-EGFR antibody (0.25 µg/ml) on GM-CSF secretion induced by DEP (10 µg/cm2) and PM2.5 (10 µg/cm2). Values represent mean ± SD of triplicate cultures. The equal variance test is significant with {alpha} = 0.05 (P < 0.001) and all pairwise multiple comparisons were tested with the Student-Newman-Keuls method (P < 0.05). Asterisks, different from respective control; open circles, different from treatments without EGFR antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report for the first time that PM2.5 and DEP stimulate human bronchial epithelial cells to express and secrete AR, an EGFR ligand. In addition, we provide evidence of a causal relationship between autocrine AR release and GM-CSF production in response to particles.

Northern blot analysis clearly confirmed preliminary data obtained from macroarray experiments demonstrating that exposure to PM2.5 or DEP at 10 µg/cm2, a noncytotoxic concentration (24), results in upregulation of the AR gene. By contrast, the other EGFR ligands (TGF-{alpha} and HB-EGF) are not overexpressed, except betacellulin, but only in PM2.5-treated cells. This is the reason we focused our study on AR. The increase in AR mRNA expression was accompanied by elevated levels of secreted AR protein. In addition, AR secretion is dose-dependent.

AR secretion is triggered by activation of the EGFR-linked extracellular signal–regulated kinase p44/42 (Erk1/2) pathway, as previously shown in other cell types (25). Indeed, the MEK inhibitor, PD98059, significantly reduced basal- and PM-induced AR secretion, whereas the p38 kinase inhibitor, SB203580, had no effect. In addition, an inhibitor of EGFR tyrosine kinase activity, AG1478, reduced basal- and PM-induced AR secretion in 16HBE cells. PD98059 and AG1478 having no cytotoxic effects at the concentrations used, the reduction of basal AR secretion they induced may reflect the constitutive autocrine stimulation of 16HBE cells. Thus, PM2.5 and DEP may act on the EGFR to initiate the Erk1/2 MAP kinase signaling cascade leading to AR expression and secretion. It provides a new explanation to the clear Erk1/2 phosphorylation we previously observed in DEP-treated 16HBE cells (10). Similarly, vanadium, a metal present in trace amounts in PM, has been shown to induce the release of HB-EGF after EGFR and MAP kinases activation in HBE cells (20).

It is tempting to speculate that EGFR activation could occur by transactivation via intracellularly produced ROS (26). This hypothesis is supported by three observations: (i) the PM-induced AR secretion is abolished in the presence of the antioxidant NAC—a similar result was observed using H2O2 as a positive control for ROS; (ii) catalase, an antioxidant enzyme that cannot enter cells, inhibits H2O2- but not PM-induced AR secretion; and (iii) an anti-EGFR neutralizing antibody that prevents EGF ligand binding, failed to modify the PM2.5- and H2O2-induced AR secretion. The inefficiency of catalase in inhibiting PM-induced AR release revealed that H2O2 are not extracellularly produced by PM. Moreover, the possibility of EGFR transactivation via oxidative stress is further supported by previous reports that ligand-independent EGFR activation could be achieved through ROS inhibition of the phosphatase activity associated with the EGFR kinase domain (27). Ligand-independent EGFR activation was proposed as a mechanism for the mucin synthesis in airway epithelial cells induced by H2O2, neutrophils, and cigarette smoke (28, 29).

The possible involvement of ROS in AR secretion and EGFR activation induced by PM is supported by the chemical composition of particles. DEP contain numerous organic compounds, and our previous studies have shown that they contribute to the production of ROS in airway epithelial cells (12). PM2.5 contain metals (Fe, Cu, and V), in addition to organic compounds, among which are transition metals known for their ability to generate ROS. It has been shown that fibers such as asbestos (14), and particles characterized by a rich metal content (15), trigger the EGFR and the downstream signaling cascades leading to IL-8 cytokine release. Furthermore, we have shown that CB particles, devoid of organic compounds and metals, as well as the biologically inert fine TiO2, do not induce ROS production in airway epithelial cells (12) and are also inefficient in enhancing AR secretion.

We propose that particles could directly and indirectly activate the EGFR to stimulate the expression and secretion of cytokines, autocrine ligands of EGFR being the mediators of this proinflammatory response. Indeed AR can induce GM-CSF release by 16HBE cells from 10 ng/ml. In addition, in the presence of a neutralizing anti-EGFR, the GM-CSF release induced by DEP and PM2.5 is reduced, suggesting that AR induction constitutes one pathway contributing to GM-CSF release. The relay action of EGFR ligands in the induction of cytokine release has already been demonstrated in HBE cells in response to cigarette smoke (16). Such a mechanism could contribute to the sustained proinflammatory response. However, due to their ability to induce the expression and secretion of AR, particles could have other direct effects on epithelial functions linked to cell survival and differentiation. In particular, AR can stimulate the proliferation of HBE cells (30–31). Finally, the secretion of EGFR ligands could also have a paracrine effect on mesenchymal cells underlying the respiratory epithelium, leading to airway remodeling. For example, HB-EGF released by vanadium-exposed HBE cells is a mitogen for lung fibroblasts (20) and vanadium provokes airway fibrosis in rats, characterized by thickening of the smooth muscle cells, mucous cell metaplasia, and peribronchiolar fibrosis (32).

These results were obtained with 16HBE cells, a transformed cell line exhibiting a high basal AR expression as many tumor cell lines (18–19). However, they were confirmed in primary cultures of normal human nasal cells that exhibit a basolateral AR secretion enhanced in the presence of organic extracts of DEP (Auger, personal communication).

In conclusion, we have shown that PM2.5 and DEP enhance the expression and secretion of AR by bronchial epithelial cells mediated by EGFR and Erk1/2 activation and that AR contributes to GM-CSF release. The key role of EGFR and its ligands in various aspects of epithelial growth, repair, and differentiation, as well as respiratory pathologies such as asthma and chronic obstructive pulmonary disease, suggests that any modulation of their expression or activity by atmospheric particles is likely to have important and multiple consequences. Further studies are required to address the consequences of AR secretion and EGFR signaling, with respect to the proinflammatory response but also repair processes following exposure to atmospheric particles.


   Acknowledgments
 
This work was supported by grants from Renault (DIMAT; no. 235), Ademe (no. BOU 9808 and no. BOU 0138), and Primequal (97034). The authors thank Dr. D. C. Gruenert for the human bronchial cell line and Arulraj Nadaradjane for his excellent technical help in cell culture. This work was also supported by CAMPLP (Caisse d'Assurance Maladie des Professions Liberales de Province, Paris, France).


   Footnotes
 
* These authors contributed equally to this work. Back

Received in original form July 28, 2003

Received in final form December 1, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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