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Published online before print September 5, 2007, 10.1183/09031936.00085907
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Eur Respir J 2007; 30:1064-1073
Copyright ©ERS Journals Ltd 2007

Expression and role of EGFR ligands induced in airway cells by PM2.5 and its components

M. Rumelhard, K. Ramgolam, R. Hamel, F. Marano and A. Baeza-Squiban

Laboratory of Cytophysiology and Cellular Toxicology, University of Paris Diderot, Paris, France.

CORRESPONDENCE: M. Rumelhard, Laboratory of Cytophysiology and Cellular Toxicology, Tour 53-54, Case Courrier 7073, University of Paris Diderot-Paris 7, 2 place Jussieu, 75 251 Paris Cedex 05, France. Fax: 33 144276999. E-mail: melina.rumelhard{at}univ-diderot-paris.fr

Keywords: Ambient particles, epidermal growth factor receptor ligands, inflammation, organic particulate matter with a diameter <2.5µm, respiratory epithelial cells

Received: July 10, 2007
Accepted August 17, 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The aim of the current study was to establish the epidermal growth factor receptor (EGFR) ligand expression profile in human airway epithelial cells exposed to either particulate matter (PM) with an aerodynamic diameter <2.5 µm (PM2.5) or its components and the involvement of EGFR ligands in PM2.5-provoked airway inflammation.

EGFR ligand mRNA and protein expression were studied in a human bronchial epithelial cell line and normal nasal cells exposed to noncytotoxic concentrations of PM2.5 or its components. The autocrine role of EGFR ligands in airway epithelial cell pro-inflammation was determined by adding conditioned media from PM2.5-treated cells to fresh cells and measuring the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), a pro-inflammatory biomarker.

PM2.5 increased amphiregulin, transforming growth factor-{alpha} and heparin-binding EGF-like growth factor mRNA expression and protein secretion, with a slight contribution of aqueous metallic compounds and a strong participation of organic components putatively attributed to PM polyaromatic hydrocarbon content. PM2.5-induced EGFR ligands were involved in cellular GM-CSF release.

The current study revealed upregulation of several epidermal growth factor receptor ligands by airway epithelial cells exposed to particulate matter with an aerodynamic diameter <2.5 µm and their contribution to bronchial epithelial cell granulocyte-macrophage colony-stimulating factor secretion by an autocrine action, suggesting that these ligands could elicit and sustain the particulate matter-induced airway pro-inflammatory response and contribute to bronchial remodelling.

Considerable attention has been devoted to the health effects of atmospheric pollution, particularly in urban areas. Numerous epidemiological studies have correlated exposure to fine particulate matter (PM) with cardiovascular and respiratory morbidity and mortality, including the development of chronic bronchitis, asthma and lung cancer 1. Urban PM with a diameter <2.5 µm (PM2.5) is a complex mixture, including soot, produced by combustion with which organic, metallic and biological components are associated.

Human and animal studies have shown lung inflammation following brief exposure to diesel exhaust particles or to concentrated ambient particles 2, 3. In addition, the in vitro release of pro-inflammatory cytokines, i.e. granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, etc. by bronchial epithelial cells exposed to PM2.5, has shown the role of epithelium in this airway pro-inflammatory response 4. Long-term exposure to pollution has been associated with histopathological lung damage, such as bronchioli wall thickening, concomitant with the presence of numerous particles in the lungs of exposed individuals 5. It is thus suspected that PM2.5 may provoke or exacerbate airway remodelling, a phenomenon characterised, especially in chronic obstructive pulmonary disease, by epithelial mucous metaplasia associated with airway wall fibrosis, inflammation, enlargement of bronchial smooth muscle mass and modifications of lung growth factor expression and/or functions (such as epidermal growth factor (EGF) and basal fibroblast growth factor, etc.) 6. Furthermore, as recently shown in the current authors’ laboratory, exposure of human bronchial epithelial cells to PM2.5 or diesel exhaust particles leads to the release of amphiregulin, a ligand of the EGF receptor (EGFR) 7.

EGFR is a 173 kD receptor tyrosine kinase predominantly located at the basolateral surface of polarised epithelial cells. Several ligands are able to bind to the mammalian EGFR (c-erbB1, human epidermal receptor 1) with various affinities: EGF, transforming growth factor (TGF)-{alpha}, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, epiregulin and epigen. EGFR ligands have autocrine properties, which may explain the preferential co-localisation of the ligands and their receptor at the basolateral surface. Indeed, TGF-{alpha} and amphiregulin are functionally involved in an EGFR autocrine growth loop in human normal bronchial epithelial cells 8.

EGFR and its ligands play a pivotal role in repair and maintenance of epithelial tissues 9. Indeed, it has been shown that EGF and EGFR are involved in repair of scrape-wounded human bronchial epithelial (16HBE)14o- monolayers in vitro 10. Moreover, EGFR is overexpressed in the airway epithelium of asthmatic subjects 10. In addition to this implication in tissue repair, amphiregulin and HB-EGF have been associated with pro-inflammatory cytokine release (GM-CSF, IL-8) after exposure to PM or cigarette smoke, respectively 7, 11.

The aims of the current study were to establish the expression pattern of EGFR ligands induced in human respiratory epithelial cells by PM2.5 and its different components, and then to determine the resulting consequences, for airway epithelial cells, of increased EGFR ligand production, due to their potential involvement in pro-inflammatory responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Particles
PM2.5 was collected in Vitry-sur-Seine, a suburban city near Paris (France), according to a previously published method 12.

The polyaromatic hydrocarbon (PAH) and metal content of the PM was characterised, respectively, by high-pressure liquid chromatography after extraction with dichloromethane, and by inductively coupled plasma-atomic emission spectrometry or flame photometry (table 1Go) according to previously described methods 12.


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  		Table 1— Polyaromatic hydrocarbon(PAH) and metal content in particulate matter with a diameter <2.5 µm

 
Stock suspensions of native particles (2 mg·mL–1) were prepared in a 0.04% dipalmitoyl phosphatidyl lecithine (DPL; Sigma-Aldrich, Saint-Quentin-Fallavier, France) solution. DPL is a surfactant component used to maintain particles in suspension, and is consequently used as a negative control for particle exposure. For treatment, PM was sonicated six times for 10 s (Vibracell; Bioblock Scientific, Illkrich, France) and suspended in culture medium.

The organic extract was obtained by extraction of PM2.5 with dichloromethane in an accelerated solvent extractor (ASE 200; Dionex, Sunnyvale, CA, USA). The extract was dried and dissolved in dimethyl sulfoxide.

The aqueous extract containing hydrosoluble components was obtained after two 10-min centrifugations of the 2 mg·mL–1 particle suspension, at 10,000xg, followed by filtration of the supernatant through a 0.22 µm filter (Millex® GV, Carrigtohill, Cork, Ireland). Soluble metals present in the particle aqueous extract were depleted by filtration on chelating ion-exchange resins with Chelex® resin according to the manufacturer's instructions (Chelex 100, Bio-Rad, Marnes-la-Coquette, France).

Particles collected after the two centrifugations and suspended in a solution of DPL at 2 mg·mL–1 constituted the washed PM2.5 devoid of hydrosoluble components.

Black carbon particles at 2 mg·mL–1 (95 nm diameter; Degussa, Frankfurt, Germany) were used to mimic the carbonaceous core of PM2.5, as production of PM2.5-devoid of its organic compounds was technically impossible.

Treatment
Cells were exposed to noncytotoxic concentrations of native PM2.5 (1, 5 or 10 µg·cm–2) or to a volume of washed PM2.5, aqueous extract and black carbon particles equivalent to the volume of native PM2.5. Organic extract was used at a concentration equivalent to that found on PM2.5, this value was calculated according to the proportion of soluble organic fraction in the total dry weight of PM2.5 (9%).

For the conditioned media, the following protocol was followed: cells were exposed for 24 h to PM2.5 and the culture medium was removed, centrifuged for 10 min at 10,000 g to eliminate particles, and the GM-CSF content measured. This constituted the conditioned media, which was applied for 24 h to fresh cells to further investigate the secreted GM-CSF, amphiregulin and TGF-{alpha}.

Anti-EGFR monoclonal antibody (clone LA1) was purchased from R&D Systems (Abingdon, UK) and used at 0.5 µg·mL–1. Anti-amphiregulin, anti-HB-EGF and anti-TGF-{alpha} polyclonal antibodies were also purchased from R&D Systems and used at respective concentrations of 20, 10 and 1 µg·mL–1.

Monoclonal mouse immunoglobulin (Ig)G antibody used as a control antibody was purchased from DakoCytomation (Dako France, Trappes, France). {alpha}-naphthoflavone (Sigma-Aldrich) was used as antagonist of the aryl hydrocarbon receptor (AhR). Benzo(a)pyrene (Sigma-Aldrich) at 1 µM was used as a positive control for AhR activation.

Cell cultures
16HBE14o- cells were kindly provided by D.C. Gruenert (Research Facility, California Pacific Medical Center, San Francisco, CA, USA). Primary cultures of normal human nasal epithelial (NHNE) cells were obtained from turbinates of patients undergoing turbinectomy (kindly supplied by J. Soudant, Service ORL, CHU La Pitié-Salpétrière, Paris, France).

Cell culture details are provided in the online supplementary material. During treatment and for 4 h before cell exposure, 16HBE and NHNE cells were grown in DMEM/F12 without growth factors.

Gene expression analysis
Total RNA were extracted from 16HBE14o- cells using an SV Total RNA Isolation kit (Promega, Charbonnieres, France) according to the manufacturer's instructions. Reverse transcription (RT) of 1 µg of total RNA was then performed as described by Rumelhard et al. 13, one-tenth of the RT reaction was then amplified using quantitative PCR in the presence of 1X Brilliant® SYBR® Green Master mix (Stratagene, La Jolla, CA, USA) and forward and reverse primers (table 2Go). Amphiregulin, betacellulin, HB-EGF and reduced glyceraldehyde phosphate dehydrogenase primers were purchased from Operon Biotechnologies (Cologne, Germany) and TGF-{alpha} primers from Invitrogen (Paisley, UK). Samples were amplified in a MX3000P® QPCR System (Stratagene) under the following conditions: 10 min at 94°C; 40 cycles of 30 s at 94°C, 1 min at 58°C and 45 s at 72°C; 1 min at 95°C; and a ramp from 55°C to 95°C.


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  		Table 2— Forward and reverse primers of epidermal growth factor ligands and reduced glyceraldehyde phosphate dehydrogenase(GAPDH)

 
The reduced glyceraldehyde phosphate dehydrogenase gene was used as an internal standard. The comparative cycle threshold method (2-{Delta}{Delta}Ct method) was used to analyse data and determine the relative abundance of the target genes. Ct is the threshold cycle at which SYBR® Green emission is determined to be statistically different above background. To obtain the {Delta}{Delta}Ct values, the difference between Ct of a target gene (CtA) and house keeping gene (CtB) was calculated in treated cells ({Delta}CtT = CtA,T–CtB,T) and in untreated cells ({Delta}CtUT = CtA,UT –CtB,UT). Finally, the relative abundance was calculated as 2-{Delta}{Delta}Ct.

Analysis of EGFR ligand and GM-CSF release
Cell culture media was removed from cells and centrifuged for 10 min at 10,000xg (4°C). Enzyme-linked immunosorbent assay kits for amphiregulin, TGF-{alpha}, betacellulin and GM-CSF (R&D Systems) were then used following the manufacturer's instructions.

Statistical analysis
Results are expressed as mean±SD. Data comparisons were carried out using one way ANOVA followed by Dunnett's post-test to compare treatments with their controls, or by Student–Newman–Keuls to compare the different treatment groups. Differences were considered significant for p<0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Effects of PM2.5 on EGFR ligand mRNA and protein expression in respiratory epithelial cells
A time-course study (2, 6, 18, 24 and 48 h) with 16HBE cells treated with PM2.5 at 10 µg·cm–2, a noncytotoxic concentration (data not shown), revealed that among the four EGFR ligands studied, the earliest upregulated genes were amphiregulin and HB-EGF from 6 h (fig. 1aGo). Amphiregulin and HB-EGF were further upregulated by 3.6- and 2.1-fold, respectively, up to 18 h, this then diminished in the following 30 h but remained sustained for amphiregulin (2.5-fold at 48 h). TGF-{alpha} mRNA expression was significantly increased to approximately the same extent (two-fold) as that of HB-EGF mRNA after 18–24 h of exposure, but was more transient as it descended to basal levels at 48 h. Betacellulin mRNA expression in 16HBE cells was virtually unmodulated by PM2.5 exposure.


Figure 1
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  		Fig. 1— Epidermal growth factor receptor ligand expression in respiratory cells exposed to native particulate matter with a diameter below 2.5 µm (PM2.5). a) Amphiregulin ({circ}), betacellulin ({blacksquare}), heparin-binding epidermal growth factor-like growth factor (•) and transforming growth factor (TGF)-{alpha} ({square}) mRNA expression in human bronchial epithelial (16HBE) cells exposed for 2–48 h to 10 µg·cm–2 of PM2.5, measured by quantitative PCR. Relative abundance was obtained by the 2-{Delta}{Delta}Ct method. Reduced glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control and the mRNA expression of PM2.5-treated cells was related to that of PM2.5 control. b) Amphiregulin ({circ}) and TGF-{alpha} ({square}) release by 16HBE cells exposed for 2–48 h to 10 µg·cm–2 of PM2.5 (– – –) or its control (–––). c) Amphiregulin and d) TGF-{alpha} release by 16HBE cells and normal human nasal epithelial (NHNE) cells exposed to 10 µg·cm–2 of PM2.5 (blk12) or its control (blk14) over a 24-h period and related to 16HBE or NHNE cell number. Data are expressed as mean±SD, n = 4 (mRNA) or n = 6 (protein secretions). *: p<0.05, significantly different from PM2.5 control; #: p<0.05, significantly different from the preceding period of exposure; : 2.4-fold increase; +: 1.4-fold increase; §: 1.3-fold increase.

 
The time-course study (2, 6, 18, 24 and 48 h) shown in figure 1bGo for 10 µg·cm–2 PM2.5 exposure revealed a significant induction of both amphiregulin and TGF-{alpha} secretion in 16HBE cell culture supernatants starting at 18 h of exposure. Amphiregulin secretion increased throughout the time, reaching a concentration of 7.4 ng·mL–1 after 48 h of exposure, whereas TGF-{alpha} secretion did not evolve and even decreased (24 pg·mL–1 at 18 h and 21 pg·mL–1 at 24 h). For constitutive and also inducible release, the amount of amphiregulin (in the ng·mL–1 range) was higher than that of TGF-{alpha} (in the pg·mL–1 range). The dose-dependent study (supplementary fig. 1Go) revealed a significant secretion of both ligands from 1 µg·cm–2 of PM2.5 that seemed to be maximal from 5 µg·cm–2 of PM2.5. No release of betacellulin was detectable in the supernatants of cells either at the basal level or after particle exposure (data not shown).

The comparison of EGFR ligand secretion induced by 10 µg·cm–2 of PM2.5 in 16HBE cells and in primary cultures of epithelial cells (fig. 1cGo) revealed that amphiregulin and TGF-{alpha} secretion was also induced by PM2.5-treated NHNE cells. In addition, when relating secretion data to cell number, figure 1cGo revealed that the amphiregulin concentration was six-fold higher in NHNE than in 16HBE supernatants. TGF-{alpha} was also more strongly secreted by NHNE cells than 16HBE cells, but to a higher extent (16-fold). Again, betacellulin secretion was not detectable (data not shown).

Involvement of PM components in PM2.5-induced EGFR ligand upregulation
Amphiregulin and TGF-{alpha} mRNA expression were measured by quantitative PCR after the treatment of 16HBE cells by noncytotoxic concentrations (data not shown) of PM2.5 organic and aqueous extracts. PM2.5 organic extract induced both amphiregulin and TGF-{alpha} mRNA expression (fig. 2a and bGo), to approximately the same extent as native PM over 24 h of exposure, but with a sustained upregulation up to 48 h of treatment. In contrast, PM2.5 aqueous extract has only a very transient and weak effect on amphiregulin mRNA expression (fig. 2aGo) and no significant effect on TGF-{alpha} mRNA expression (fig. 2bGo).


Figure 2
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  		Fig. 2— Contribution of particulate matter (PM) with a diameter below 2.5 µm (PM2.5) components to epidermal growth factor receptor (EGFR) ligand upregulation. a) Amphiregulin and b) transforming growth factor (TGF)-{alpha} relative abundances compared with mRNA expression of reduced glyceraldehyde phosphate dehydrogenase (GAPDH) in human bronchial epithelial (16HBE) cells exposed to 10 µg·cm–2 of PM2.5 (•), or to an equivalent concentration of PM organic extract (OEx; 4.27 µg·mL–1, {blacksquare}) or to an equivalent volume of PM aqueous extract (AEx; 19 µL, {blacktriangleup}) from 2–48 h of exposure. c) Amphiregulin and d) TGF-{alpha} release by 16HBE cells exposed from 2–48 h. {triangleup}: particle control; •: PM2.5; {blacksquare}: OEx; {circ}: OEx control; {blacktriangleup}: AEx. e) Amphiregulin and f) TGF-{alpha} release induction by 16HBE cells exposed for 18 h to 10 µg·cm–2 of PM with a diameter <2.5µm (PM2.5), or to the equivalent volume of PM AEx (19 µL) alone or with pre-treatment by a Chelex resin (R), washed PM2.5 (WPM; 10 µg·cm–2), black carbon (BC; 10 µg·cm–2) and their relative control (C) or to the equivalent concentration of PM OEx (4.27 µg·mL–1) and its control. Data are expressed as mean±SD, n = 3. *: p<0.05, significantly different from treatment control; #: p<0.05, significantly different from PM2.5 treatment; : p<0.05, significantly different from AEx treatment.

 
A kinetic study (fig. 2cGo and d) revealed that amphiregulin and TGF-{alpha} release was significantly induced by the organic PM extract from 6 h of exposure, with a level of induction almost similar to the one of native PM. The aqueous extract also induced amphiregulin and TGF-{alpha} release with a significant induction from 18–24 h of exposure (fig. 2c and dGo). The level of induction was far weaker than that of native PM (fig. 2c–fGo) and was almost completely abrogated when metals were removed from the aqueous extract using a Chelex resin (fig. 2eGo and f).

Washed particles, devoid of their hydrosoluble compounds, induced amphiregulin and TGF-{alpha} to approximately the same level as native PM2.5 (1.8- and 2.5-fold for amphiregulin and TGF-{alpha}, respectively; fig. 2eGo and f), while there was virtually no effect of black carbon on amphiregulin and TGF-{alpha} release (fig. 2eGo and f). NHNE cells responded in a similar fashion to 16HBE cells (fig. 2Go, supplementary online material) except for a slight increase or absence of response in TGF-{alpha} release by NHNE cells treated by PM organic and aqueous extracts (1.8- and 1.7-fold, respectively).

Since PM organic extract had the greatest effect on EGFR ligand expression and PAH represent constituents of this extract, the effect of PM2.5-induced EGFR ligand secretion as mediated by the AhR pathway, was investigated. Figure 3Go demonstrates that both amphiregulin and TGF-{alpha} release provoked by 10 µg·cm–2 of PM2.5 diminished with the addition of {alpha}-naphthoflavone, an AhR antagonist, in a concentration-dependent manner. Furthermore, {alpha}-naphthoflavone decreased the amphiregulin and TGF-{alpha} secretion induced by 1 µM of benzo(a)pyrene (used as positive control) more than the secretion induced by PM2.5.


Figure 3
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  		Fig. 3— Involvement of the aryl hydrocarbon receptor (AhR) pathway in particulate matter (PM)-induced epidermal growth factor receptor ligand secretion. a) Amphiregulin and b) transforming growth factor (TGF)-{alpha} release induction by human bronchial epithelial cells exposed for 24 h to 10 µg·cm–2 of PM with a diameter below 2.5 µm (PM2.5: {square}) or 1 µM of benzo(a)pyrene (blk12) alone or with 30 min pre-treatment by 0.1–2.5 µg·mL–1 of {alpha}-naphthoflavone, an AhR antagonist. Data are expressed as mean±SD, n = 3. *: p<0.05, significantly different from PM2.5 control; #: p<0.05, significantly different from benzo(a)pyrene control; : p<0.05, significantly different from release without pre-treatment by {alpha}-naphthoflavone.

 
EGFR ligands mediate a PM2.5-induced epithelial cell autocrine pro-inflammatory response
When 16HBE cells were exposed for 24 h to human recombinant amphiregulin or TGF-{alpha} (fig. 4Go), the GM-CSF pro-inflammatory cytokine secretion was induced by an exposure to 5 ng·mL–1 of amphiregulin or 20 pg·mL–1 of TGF-{alpha}. Co-treatment with both amphiregulin and TGF-{alpha} had no additive effect on GM-CSF release (fig. 4Go).


Figure 4
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  		Fig. 4— Pro-inflammatory response triggered in bronchial epithelial cells exposed to epidermal growth factor receptor (EGFR) ligands. Granulocyte-macrophage colony-stimulating factor (GM-CSF) release by human bronchial epithelial cells exposed for 24 h to 1–50 ng·mL–1 of amphiregulin (A), 1–100 pg·mL–1 of transforming growth factor (TGF)-{alpha} or to the combination of these two EGFR ligands. Data are expressed as mean±SD, n = 3. C: control. *: p<0.05, significantly different from control.

 
In order to determine whether GM-CSF release, known to be induced by PM2.5 12, was regulated in an autocrine manner by EGFR ligands, conditioned media of cells previously exposed to PM2.5 for 24 h was used (fig. 5Go). Figure 5aGo demonstrates that a 24 h exposure to 10 µg·cm–2 of PM2.5 induced GM-CSF secretion (increase of 35 pg·mL–1 in treated cells versus control cells, fig. 5aGo). The transfer of the conditioned media onto fresh cells further increased GM-CSF secretion by cells exposed to either control conditioned media or to PM2.5 conditioned media, but the increase in GM-CSF secretion was greater in cells exposed to PM2.5 conditioned media (increase of 46 pg·mL–1 in PM2.5-conditioned media versus control conditioned media; fig. 5aGo). Interestingly, when conditioned media was applied to fresh cells in addition to 0.5 µg·mL–1 of an anti-EGFR antibody (fig. 5aGo), the conditioned media-induced GM-CSF release was strongly reduced both for control and PM2.5-conditioned media, in contrast to the addition of 0.5 µg·mL–1 of control (IgG) antibody, which had almost no effect on GM-CSF release (fig. 5aGo). Furthermore, the GM-CSF secretion induced by conditioned media was concentration dependent, occurring from conditioned media of cells exposed to 1 µg·cm–2 of PM2.5 (fig. 3Go in the online supplementary material). The measurements of amphiregulin and TGF-{alpha} release in parallel to GM-CSF release (fig. 5b and cGo) revealed that: 1) secretion levels of these two ligands were increased only slightly or not at all by fresh cells exposed for 24 h to conditioned media; and 2) the addition of an anti-EGFR antibody only slightly modulated secretion levels of amphiregulin, whereas a strong increase in TGF-{alpha} release was observed, especially in PM-treated cultures.


Figure 5
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  		Fig. 5— Pro-inflammatory response of bronchial epithelial cells exposed to particulate matter (PM) or conditioned media (CM). a and d) granulocyte-macrophage colony-stimulating factor (GM-CSF) release induction, b) amphiregulin or c) transforming growth factor (TGF)-{alpha} release by human bronchial epithelial (16HBE) cells exposed directly to 10 µg·cm–2 of PM with a diameter below 2.5 µm (PM2.5) or PM2.5 control for 24 h ({square}). Fresh cells were then exposed to either control- or PM2.5-CM alone (blk14) or CM following: 0.5 µg·mL–1 anti-epidermal growth factor (EGF) receptor antibody treatment (blk34); treatment with 0.5 µg·mL–1 of immunoglobulin G antibody ({blacksquare}); pre-treatment with 20 µg·mL–1 of anti-amphiregulin (AR: blk12) antibody; 1 µg·mL–1 of anti-TGF-{alpha} antibody (Figure 5); 10 µg·mL–1 of anti-heparin-binding EGF-like growth factor antibody (blk14); or the combination of anti-heparin-binding EGF-like growth factor, anti-AR and anti-TGF-{alpha} antibodies (Figure 5). GM-CSF induction is normalised by the cells directly exposed to control. Data are expressed as mean±SD, n = 6 or n = 3. *: p<0.05, significantly different from treatment control; #: p<0.05, significantly different from release by cells directly exposed to PM2.5; : p<0.05, significantly different from release by cells exposed to CM.

 
In order to determine the respective roles of the different EGFR ligands in GM-CSF release, antibodies against amphiregulin, TGF-{alpha} or HB-EGF were used either alone or in combination in conditioned media (fig. 5dGo). Only the anti-amphiregulin antibody alone or in combination with the other antibodies significantly reduced the GM-CSF secretion, to the same extent as the anti-EGFR antibody (fig. 5dGo).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
PM2.5 upregulates several EGFR ligands in respiratory epithelial cells
The current study demonstrates for the first time that urban PM2.5 differentially regulates by respiratory epithelial cell mRNA expression of EGFR ligands, with upregulation of TGF-{alpha}, amphiregulin and HB-EGF but unchanged betacellulin expression. The three induced growth factors have similar gene expression kinetics, but different levels of induction. These differences also occur with protein release, since amphiregulin exhibits higher expression both at the mRNA and protein level (3.6- and 2–3.5-fold, respectively) than TGF-{alpha} (2.1- and 1.4–1.7-fold for mRNA and protein, respectively). These data suggest that increased EGFR ligand secretion results from increased gene expression. To the current authors’ knowledge, this effect of PM on EGFR ligand secretion by respiratory epithelial cells has not been published previously. However, TGF-{alpha}, amphiregulin and HB-EGF gene upregulation has already been shown in the lung epithelial cell line (NCI-H292) exposed to cigarette smoke, where amphiregulin secretion was stronger than that of TGF-{alpha} 11. Similarly, the present protein release kinetic study revealed that TGF-{alpha} induction is weaker than that of amphiregulin and does not accumulate in the cell medium. This weaker induction may be misleading, as underlined by the experiment performed in the presence of an anti-EGFR antibody, which blocks binding of TGF-{alpha} to EGFR, leading to a dramatic increase in TGF-{alpha} in the culture medium (fig. 5cGo). This suggests that TGF-{alpha} is quickly consumed by cells due to its strong affinity for EGFR. Moreover, this would explain the picogram amounts of TGF-{alpha} that were measured in vitro as well as those obtained in airway epithelial cells exposed to cigarette smoke 14.

EGFR ligand secretion may be a common response of respiratory epithelial cells to stress, as underlined by miscellaneous data obtained with human bronchial epithelial cells exposed to cigarette smoke 11 or after inhalation of asbestos fibres by rats 15. These data, combined with the present study, performed with a bronchial cell line and nasal primary cells, underline the fact that induced release of EGFR ligands is not limited to cell lines and occurs throughout the respiratory tract: nasal and bronchial cells responding in a similar fashion. Furthermore, amphiregulin 16 and TGF-{alpha} 17 have been described as being preferentially released towards the basolateral compartment, while EGFR is localised in the basolateral membrane 13, which suggests that PM2.5-induced EGFR ligands could mediate responses of bronchial epithelial cells via an autocrine effect.

PM2.5-induced EGFR ligands mediate a pro-inflammatory response of bronchial epithelial cells but not their proliferation
Airway inflammation is the most frequently described and most well-known effect of short-term exposure of healthy volunteers to PM and diesel exhaust particulate (DEP) 18, 19, and can be explained by the capacity of these particles to induce the release of pro-inflammatory cytokines such as IL-8, TNF-{alpha} and GM-CSF 4, 16. Mechanistic studies performed to elucidate signalling pathways triggered by PM in order to induce cytokine release have underlined the involvement of EGFR activation 7, 20. Such EGFR activation could be a direct effect of particles, as shown for asbestos 21, but it is tempting to hypothesise that the PM2.5-induced epithelial cell pro-inflammatory response could be mediated and/or amplified via EGFR activation by PM2.5-induced EGFR ligands. This assumption was assessed using conditioned media in the presence or absence of an antibody to block the binding of EGFR ligands to their receptor. These data highlight the maintenance of GM-CSF release by cells, particularly when exposed to PM, and the strong participation of EGFR ligands in constitutive and PM-enhanced GM-CSF sustained release. Amphiregulin and TGF-{alpha} may be specifically involved in this response, as both ligands appeared to induce GM-CSF secretion by cells when used at concentrations found in PM2.5-treated cells (fig. 4Go), and to be entirely consumed by 16HBE cells exposed to conditioned media, as shown in figure 5b and cGo. Nevertheless, the use of antibodies directed towards the different EGFR ligands suggest that GM-CSF secretion is mainly driven by amphiregulin. These results suggest that PM2.5 could exacerbate and sustain bronchial epithelial cell inflammation. The autocrine effects of EGFR ligands may thus be a parameter explaining exacerbation of asthma by PM 22, since asthmatic subjects exhibit overexpression of EGFR in bronchial epithelium 10.

Beside the regulation of a pro-inflammatory response in epithelial cells, the current authors speculated whether EGFR ligands secreted after PM2.5 treatment could also mediate a mitogen effect on bronchial epithelial cells, since EGFR ligands are known to exhibit growth factor properties. Nevertheless, the culture of bronchial epithelial cells with conditioned media of PM-treated cells did not reveal any modification of cell proliferation (data not shown).

PM2.5 organic components mainly contribute to amphiregulin and TGF-{alpha} upregulation
Fractionation of PM2.5 reveals that PM aqueous extract induces weak but significant amphiregulin and TGF-{alpha} release by 16HBE cells that is abolished when metals are trapped by a Chelex resin, suggesting a main contribution of metals to growth factor induction by PM aqueous components. These results, already reported for amphiregulin 23, corroborate findings linking pro-inflammatory cytokine production by epithelial cells to the metal content of PM 24. The present study sheds new light on the contribution of organic particle compounds to PM2.5-induced amphiregulin and TGF-{alpha} gene expression and protein secretion. Indeed, extracted organic PM components, as well as water-washed PM2.5 still bearing its organic content, both appear to induce amphiregulin and TGF-{alpha} secretion, whereas inorganic carbonaceous compounds have no effect on such growth factor release (fig. 2eGo and f and supplementary fig. 2Go). This strong involvement of organic components in PM2.5-induced EGFR ligand expression has already been shown for amphiregulin 13, 23.

The organic component of PM2.5 contains PAH, known to have biological activities, especially through specific binding to the AhR. {alpha}-naphthoflavone, an AhR antagonist, diminished PM-induced secretion of amphiregulin and TGF-{alpha} (fig. 3Go), suggesting AhR-dependent transcriptional regulation of amphiregulin and TGF-{alpha}, which has already been described for another EGFR ligand, epiregulin, in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated mouse cells 25. This hypothesis is reinforced by the identification of consensus xenobiotic responsive element sequences upstream of the transcriptional start site in human amphiregulin and TGF-{alpha} promoters, to which a transcriptional complex including PAH and AhR binds to transactivate genes. But the presence of cyclic adenosine monophosphate (cAMP) responsive element (CRE) sequences in amphiregulin and TGF-{alpha} promoters could also explain this AhR-dependent signalling pathway; indeed, Du et al. 26 recently described induced amphiregulin expression triggered by cigarette smoke in bronchial cells that involves an AhR -> cAMP -> PKA -> CRE pathway. An additional regulation pathway could be linked to the ability of PM components to induce oxidative stress. This pathway could activate the oxidative stress-sensitive transcription factor, nuclear factor-{kappa}B, previously shown to be activated in DEP- and PM2.5-exposed epithelial cells 27, 28, and present consensus binding sites in amphiregulin and TGF-{alpha} promoters.

In conclusion, the current study reports upregulation of several epidermal growth factor receptor ligands by human airway epithelial cells exposed to urban particulate matter with a diameter <2.5 µm, and in particular, the strong participation of organic components of particulate matter with a diameter <2.5 µm in amphiregulin and transforming growth factor-{alpha} upregulation and the weaker contribution of aqueous components through mechanisms that require further investigation. The current study demonstrates for the first time that particulate matter with a diameter <2.5 µm induced amphiregulin and transforming growth factor-{alpha} release contribute to the bronchial pro-inflammatory response via an autocrine action. Amplification of bronchial inflammation is highly suspected to be responsible for bronchial remodelling, notably found in asthma or chronic obstructive pulmonary disease. The present findings may thus have clinical implications, especially since amphiregulin and transforming growth factor-{alpha} appear significantly released after exposure to only 1 µg·cm–2 of native particulate matter with a diameter <2.5 µm, a concentration easily reached in the tracheobronchial region of individuals with respiratory diseases living in polluted urban areas 22. In addition to the role of particulate matter-induced epidermal growth factor receptor ligands in sustained bronchial inflammation, they may also have paracrine effects on other cellular actors involved in airway remodelling, such as fibroblasts or vascular smooth muscle cells previously described to be sensitive to such ligands 29, 30.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors would like to acknowledge the technical assistance of A. Jaeger (Laboratory of Cytophysiology and Cellular Toxicology, University of Paris Diderot, Paris, France), J. Bram for English editing and S. Middendorp (Laboratory of Regulation of Immune Responses, University of Paris 7, Paris, France) for precious help in gene promoter analysis.


   FOOTNOTES
 
This article has supplementary material accessible from www.erj.ersjournals.com Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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