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Biological effects of atmospheric particles on human bronchial epithelial cells. Comparison with diesel exhaust particles
Toxicology in Vitro 17 (2003) 567–573 www.elsevier.com/locate/toxinvit
Biological effects of atmospheric particles on human bronchial epithelial cells. Comparison with diesel exhaust particles Augustin Bauliga,*, Matthieu Sourdevala, Martine Meyerb, Francelyne Maranoa, Armelle Baeza-Squibana a
Laboratoire de Cytophysiologie et Toxicologie cellulaire, Universite´ Paris 7, 2 place Jussieu, Tour 53-54, 3e e´tage, case courrier 7073, 75251 Paris cedex 05, France b UET qualite´ de l’air, Technocentre, TCR Lab 2 50, 1 avenue du Golf, 78288 Guyancourt, France Accepted 31 May 2003
Abstract Epidemiological studies have associated the increase of respiratory disorders with high levels of ambient particulate matter (PM) levels although the underlying biological mechanisms are unclear. PM are a complex mixture of particles with different origins but in urban areas, they mainly contain soots from transport like Diesel exhaust particles (DEP). In order to determine whether PM biological effects can be explained by the presence of DEP, the effects of urban PM, DEP and carbon black particles (CB) were compared on a human bronchial epithelial cell line (16-HBE14o-). Two types of PM were used : reference material (RPM) and PM with an aerodynamic diameter 42.5 mm collected in Paris with a high volume sampler (VPM). From 10 to 30 mg/cm2, cell viability was never modified whatever the particles. However, DEP and to a lower extent PM inhibited cell proliferation, induced the release of a pro-inflammatory cytokine, GM-CSF, and generated a pro-oxidant state as shown by the increased intracellular peroxides production. By contrast, CB never induced such effects. Nevertheless CB are more endocytosed than DEP whereas PM are the less endocytosed particles. In conclusion, PM induced to a lower extent the same biological effects than DEP in 16-HBE cells suggesting that particle characteristics should be thoroughly considered in order to clearly correlate adverse effects of PM to their composition and to clarify the role of DEP in PM effects. # 2003 Elsevier Ltd. All rights reserved. Keywords: Particulate matter; Diesel exhaust particles; Pro-inflammatory response; GM-CSF; Reactive oxygen species; Phagocytosis
1. Introduction Nowadays, air pollution is a real problem of public health for the general population in urban areas and for road workers since epidemiological studies have shown that fine particulate atmospheric pollution was associated with the increase of respiratory and cardiovascular mortality and morbidity (Kunzli et al., 2000). Particularly, particulate matter with an aerodiameter 42.5 mm (PM2.5) have been recently implicated in the Abbreviations: PM2.5, Particulate matter with an aerodiameter <2.5 mm; DEP, Diesel exhaust particles; RPM, Reference particulate matter; VPM, Vitry particulate matter; CB, Carbon black particles; 16HBE, 16-HBE 14o-cell line; GM-CSF, Granulocyte macrophagecolony stimulating factor; DCF, Dichlorofluorescein; ROS, Reactive oxygen species. * Corresponding author. Tel.: +33-144-276-062; fax: +33-144276-999. E-mail address: [email protected] (B. Augustin). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00115-2
increase of death (Arden Pope III et al., 2002) and in the respiratory adverse effects (Schwartz and Neas, 2000). Particulate matter (PM) are a complex mixture of different types of particles such as soots resulting from combustion processes, terrigenous particles from erosion and scrap-iron. In addition, chemical and biological substances can interact and adsorb on these particles (Soukup and Becker, 2001). PM composition greatly varies according to seasons and locations. In urban areas, Diesel exhaust particles (DEP) are probably a major component of PM, especially in Europe where drive many Diesel vehicles. The DEPmechanism of action is now better understood whereas few informations are available on PM. DEP have been shown to induce an inflammation in the airway of exposed volunteers (Diaz-Sanchez et al., 1997). It may result from the pro-inflammatory responses induced by DEP on the two main targets of inhaled particles: macrophages and airway epithelial cells.
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An important problem still unsolved is to determine whether DEP could contribute to PM-induced biological effects. For these reasons, the aim of our study was to compare on human bronchial epithelial cells the biological effects induced by DEP sampled after a tailpipe to those induced by PM sampled in urban areas. In particular, in vitro studies have shown that bronchial epithelial cells respond to DEP-exposure endocyting particles and releasing pro-inflammatory cytokines like granulocyte macrophage-colony stimulating factor (GM-CSF) (Boland et al., 1999). This release occurs after activation of transduction pathways implicating MAPK, reactive oxygen species (ROS) and the transcription factor (NF-kB) (Boland et al., 2000; Bonvallot et al., 2000; Bonvallot et al., 2001). However, these in vivo and in vitro experiments were performed with DEP directly collected after the tailpipe that are likely very different from that present in PM. Indeed, when spred in the atmosphere, DEP undergo many transformations which could modulate their toxicity. Two types of PM were used: reference urban particulate matter (RPM) purchased from National Institute of Standard and Technology (NIST) in USA and collected in St. Louis, and PM2.5 (VPM) collected with a high volume sampler in a school playground at Vitry-sur-Seine near Paris. After a cellular viability study performed to determine the non cytotoxic concentration, particles were compared according to the extent of their phagocytosis, their effect on cellular proliferation and their ability to induce the release of the pro-inflammatory cytokine GM-CSF and the production of peroxides.
National Institute of Standards and Technology (Gaithersburg, MD, USA). Atmospheric particulate matter PM2.5 (VPM) was collected with a high volume sampler machine (DA-80, Megatec, Paris, France), equiped with a PM2.5 selective-inlet head, in a school playground at Vitry-sur-Seine near Paris. The machine operated at the flow of 30 m3/h from 15–20 March 2002. During this period, 3690 m3 of air were filtered and particles were recovered on a nitrocellulose filter. Particles were detached from the filter by sonication, concentrated by centrifugation at 20,000 g and their concentration was determined spectrophotometrically by comparing turbidity to a standard curve of DEP at an optical density of 360 nm. Carbon black particles (CB) of 95 nm diameter (FR103) were obtained from Degussa (Frankfurt, Germany). Stock solutions of DEP, RPM and CB particles were performed by suspension in 0.04% DPL and sonicated two times for 5 min each at maximal power (100 W) (Vibracell, Bioblock Scientific, Illkirch, France). Particles were used at 10, 20 or 30 mg/cm2 (concentrations are expressed in mg/cm2 since particles rapidly sediment onto the culture). Controls were made by using 0.04% DPL. 2.3. Analysis of cell viability
2. Material and methods
Cell viability was studied on subconfluent culture using propidium iodide (PI), a DNA staining fluorescent probe that only enters in dead cells. After a 24 h toxic treatment, cells were washed, dissociated by trypsine-EDTA and stained by 0.1 mg/ml of PI. 10,000 cells were analysed by flow cytometry from each assay. Viability was related to those of control cells (control cells represent 100%).
2.1. Cell culture conditions
2.4. Analysis of cell proliferation
Dr. D. C. Gruenert (Cozens et al., 1994) (Colchester, VT, USA) kindly provided the human bronchial epithelial cell subclone 16HBE14o-. The cell line was cultured in DMEM/F12 culture medium supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), glutamine (1%), fungizone (0.125 mg/ml, Invitrogen) and UltroserG (UG) (2%, Invitrogen). Cells were cultured on collagen (type I, 4 mg/cm2) coated 25 or 75 cm2 flasks, 6- or 96-well plates (Costar, Cambridge, MA, USA) at 20,000 cells/cm2. At the time of treatment, UG was not added to DMEM/F12. Cultures were incubated in humidified 95% air with 5% CO2 at 37 C.
16-HBE cells were seeded at 40,000 cells by well in 12 well plates. 24 h after seeding, cells were treated and cell countings were performed after cell dissociation each 24 h during 3 days in a hemacytometer.
2.2. Particles Different kinds of particles were used: Diesel particulate matter SRM 1650a (DEP) and urban particulate matter SRM 1648 (RPM) were purchased from the
2.5. Analysis of intracellular peroxide levels Intracellular peroxide levels were assessed using H2DCF-DA, an oxidation-sensitive fluorescent probe. Once inside the cell, this probe is deacetylated by intracellular esterases forming H2DCF which in the presence of a variety of intracellular peroxides is oxidized to a highly fluorescent compound 20 ,70 -dichlorofluorescein (DCF). Stock H2DCF-DA solution was made at 20 mM in DMSO and stored at 20 C. Prior to the toxic treatment, cells were loaded for 20 min with 20 mm H2DCF-DA in HBSS and the fluorescence was analyzed by flow cytometry. The fluorescence analysis was
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performed by flow cytometry on 10,000 viable cells. Forward angle light scatter and right angle scatter were used to select cells.
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A 15 mW air cooled argon-ion laser tuned at 488 nm was used for DCF fluorescence. DCF and PI fluorescence were, respectively, collected though a 525 and 620 nm band pass-filter.
2.6. Cytokine assay Subconfluent cultures were exposed for 24 h to particles. Culture supernatant was recovered and frozen at 80 C until used. The concentration of granulocytemacrophage colony-stimulating factor (GM-CSF) released into the culture supernatants was measured with human GM-CSF Duoset ELISA development system (R&D systems Europe, Abingdon, United Kingdom). Color development was measured at 450 nm with a microplate photometer MR 5000 (Dynatech Laboratories).
3. Results 3.1. Effects of the different particles on the viability of 16-HBE cells In order to determine if particles could exhibit different cytotoxicities, their effect on cell viability was evaluated measuring PI fluorescence. No significant cell death was observed in 16-HBE cells exposed for 24 h to either DEP, RPM, VPM or CB from 10 to 30 mg/cm2 (Fig. 1).
2.7. Phagocytosis quantification Subconfluent cultures were treated with particles for 24 h. After rinsing and dissociation by trypsine-EDTA, cells were centrifugated (5 mn, 2200 g) and resuspended in 1 ml of PBS with 0.8 mg/ml of PI. Particulate uptake was measured by flow cytometry on 10,000 viable cells, excluding PI, according to the method of Stringer et al. (1995) adapted by Boland et al. (1999). Because ingested or bound particles induce a more granular morphology of the epithelial cells, the laser light is scattered to a greater extent. Thus, the right angle scatter (RAS) signal is dependent on the granularity of the cells. The mean RAS channel number was used for data analysis.
3.2. Inhibition of cell proliferation by the different particles The different particles used at the non cytotoxic concentration of 10 mg/cm2 were tested on proliferating cells over 72 h of treatment. Whereas CB never disturbed cell growth, DEP, RPM and VPM induced an inhibition of 16-HBE cells proliferation that increases with time exposure for DEP and RPM (Fig. 2). This inhibitory effect at 72 h is significantly different according to the type of particles: DEP (52 0.8) > RPM (31 2) > VPM (19 5). 3.3. Peroxides production
2.8. Flow cytometry assay The fluorescence analysis were performed with an EPICS-Elite-ESP flow cytometer (Coultronics-France).
In order to determine the role of oxidative stress in the biological effects of particles, DCF fluorescent probe was used to quantify the peroxide production in 16-HBE cells
Fig. 1. Effects of the different particles on the viability of 16-HBE cells. Cells were treated with DEP, RPM, VPM, CB at 10, 20 or 30 mg/cm2 for 24 h. The percent of cells excluding PI was analysed by flow cytometry over 10,000 analysed cells. Values are means S.D. (n=3).
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Fig. 2. Effect of different particles on 16-HBE cells proliferation. DEP, RPM, VPM and CB were applied in a single dose at 10 mg/cm2. Inhibition of cell proliferation was evaluated after 24, 48 and 72 h of treatment by cell counting and expressed as a percent of control value. Values are meansS.D. (n=3) except for VPM at 24 and 48 h (n=1).
3.5. Phagocytosis of particles
Fig. 3. Peroxide production in 16-HBE cells treated with different particles. Cells were loaded for 20 min with H2DCF-DA prior to the treatment with DEP, RPM, VPM and CB at 10 mg/cm2 for 4 h. DCF fluorescence (in arbitrary fluorescence units) of 10,000 gated viable cells was analysed by flow cytometry.
Previous experiments have shown that phagocytosis is important to initiate the DEP-biological effects (Boland et al., 2000), the occurrence of such mechanism for the different particles was investigated. All types of particles at 10 mg/cm2 induced after 24 h of exposure, an increase of RAS in 16-HBE cells (Fig. 5). However the rate of phagocytosis is different according to the type of particles. CB induced the most important increase of RAS (140% of control) whereas PM have the lowest effect (116% and 107 for RPM and VPM, respectively) and DEP have an intermediary effect (122%).
4. Discussion treated for 4 h with 10 mg/cm2 of particles. Whereas CB had no effect on DCF fluorescence, DEP, RPM and VPM induced a significant increase of DCF fluorescence similar for DEP and VPM but lower for RPM (Fig. 3). 3.4. Pro-inflammatory response The GM-CSF release was used as a biomarker of the pro-inflammatory response induced in 16-HBE cells exposed for 24 h to the different particles. Whatever the concentration used, CB never induced GM-CSF release (Fig. 4). By contrast, DEP, RPM and VPM induced a dose-dependent increase of GM-CSF release that was always higher in DEP-treated cells than in VPM or RPM-treated cells. The DEP-induced GM-CSF release is 2.9-fold greater at 30 mg/cm2 than that induced by PM.
The aim of our study was to determine the cellular effects of PM2.5 on a bronchial epithelial cell model. PM, and particularly the fine particles (PM2.5), are involved in respiratory diseases but their mechanisms of action and the particle component(s) responsible for the biological effects are still unclear. For these reasons, it was convenient to study the PM-mechanisms of action like it has been done with DEP. In previous studies, we have shown the DEP-involvment in the induction of the pro-inflammatory response and the major role of organic compounds adsorbed on DEP (Boland et al., 2000; Bonvallot et al., 2001). Two kinds of PM were used: (1) Reference PM (RPM) that are total suspended particles (TSP). These particles are interesting because they are certified for their metals content, many experimental data are available in literature and they can be used as internal
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Fig. 4. Pro-inflammatory response of 16-HBE cells. Cells were treated for 24 h with DEP, RPM, VPM and CB at 10, 20 or 30 mg/cm2 for 24 h. Values are means S.D. expressed as a percent of control values (n=3).
Fig. 5. Phagocytosis of 16-HBE cells treated with different particles. Cells were treated or not by DEP, RPM, VPM and CB at 10 mg/cm2 for 24 h. RAS (mean channel number) of 10,000 gated viable cells was analysed by flow cytometry. Values are means S.D. (n=3).
standard in biological assays. (2) PM from Paris (VPM) are PM2.5 collected in suburbs of Paris (Vitry-sur-Seine) which represent a typical urban particulate pollution of European cities. Both RPM and VPM induce similar biological effects on 16HBE cells. However, scanning electron microscopy observations have shown that RPM contain few soots but many fly ash whereas VPM are mainly composed of soots from combustion processes especially from motor vehicle emission (data not shown). Moreover, VPM only contains PM2.5, that are the most involved in respiratory diseases due to their small size (Schwartz and Neas, 2000) whereas RPM are TSP with a wide particle size distribution (Don Porto Carero et al., 2001).
The different particles used have no effect on cell viability even at the concentration of 30 mg/cm2 confirming experiments performed on two other human cell lines (alveolar cells and monocytes) with RPM and DEP (Don Porto Carero et al., 2001) and on a human alveolar cell line (A549) with Mexico PM10 (Alfaro-Moreno et al., 2002). Higher concentrations were not tested as 30 mg/cm2 is already an important concentration in disagreement with PM atmosphere levels. Although the different particles didn’t affect the 16HBE viability, DEP and PM (RPM and VPM) inhibited cell proliferation. But CB, which are used as a surrogate of the carbonaceous core of the soots, has no effect. This result could signify that the inhibition of cell
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proliferation is not due to mechanic effect but is related to particles composition (organic compounds, metals. . .). Especially, organic compounds extracted from DEP have been shown to inhibit 16HBE cell proliferation (unpublished results). In addition, a study performed with PM10 collected in different locations in Mexico city, have shown that their different cytotoxicity towards proliferating cells (mouse monocytes and fibroblats) could be related to their composition (Alfaro-Moreno et al., 2002). In addition, DEP, RPM and VPM but not CB induced an increase of peroxides production suggesting that an oxidative stress is involved in the cellular effects of these particles as already observed with Vermont PM2.5 on a murine alveolar cell line (Shukla et al., 2000). Metals are known to generate reactive oxygen species (ROS) (Pritchard et al., 1996) and could be responsible for the increased DCF fluorescence obtained in particles treated-16HBE cells. It is in agreement with studies on alveolar macrophages exposed to concentrated ambient particulate in which an increased DCFH oxidation occurred and was inhibited by a metal chelator desferrioxamine (Goldsmith et al., 1998). Moreover DEP (SRM 1650a) and RPM (SRM 1648) have been shown to induce the generation of ROS involving transition metals (Ball et al., 2000). However organic compounds could also participate to oxidative stress generation. Indeed, DEP-organic extracts used as at the concentration they are on DEP, induced a DCF fluorescence increase similar to that induced by native DEP (Bonvallot et al., 2002; Baulig et al., 2003). PM10 and DEP have been shown to induce an inflammation in humans (Van Eeden et al., 2000; Salvi et al., 1999). Until now, in vitro studies had mainly used macrophages to investigate the pro-inflammatory response to PM (Soukup and Becker., 2000; Imrich et al., 2000; Alfaro-Moreno et al., 2002). GM-CSF was chosen as a marker of the pro-inflammatory response in 16HBE cells as it has been previously shown to be specifically induced by DEP (Boland et al., 2000). PM (RPM and VPM) also induced GM-CSF release but to a lower extent than DEP whereas CB had no effect. These different levels of GM-CSF release according to the type of particles are likely correlated to particle composition. Indeed, they don’t seem related to the phagocytosis since CB which are the most endocytosed particles, are the less efficient one. Nevertheless, phagocytosis may be necessary as we have demonstrated for DEP (Boland et al., 2000). After endocytosis, DEPorganic compounds become available and are likely metabolized since they induce the cytochrome P450 1A1 gene expression (Bonvallot et al., 2001; Baulig et al., 2003). Concerning PM, they are moderately or few endocytosed but they induce a dose-dependent increase of GM-CSF release suggesting that the endocytosis is not always essential. It is possible that PM can act at the
plasmic membrane level triggering receptors. Such mode of action has already been described for asbestos and residual oil fly ash, metal containing particles (Zanella et al., 1996; Huang et al., 2002). Altogether these results show that PM trigger reduced biological effects in comparison to DEP. It could be explained on the one hand by the aging of DEP present in PM samples. They might have lost some components especially organic compounds which have been shown to mainly contribute to DEP-biological effects (Bonvallot et al., 2001). On the other hand it could be due to a low DEP amount in comparison with other types of particles in PM samples. It is the case for RPM but nevertheless they induce biological effects in the same range order as VPM underlying that other components than DEP have to be considered. To summarize, we have shown that PM induced to a lower extent the same biological effects than DEP in 16HBE cells. Further studies are necessary to determine particle component(s) responsible for these biological effects by PM fractionation (aqueous and organic). The identification of implicated component(s) will allow to thoroughly investigate the signalling pathways triggered by PM in bronchial epithelial cells and to assess the involvement of DEP in PM biological effects.
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Biological effects of atmospheric particles on human bronchial epithelial cells. Comparison with diesel exhaust particles Augustin Bauliga,*, Matthieu Sourdevala, Martine Meyerb, Francelyne Maranoa, Armelle Baeza-Squibana a
Laboratoire de Cytophysiologie et Toxicologie cellulaire, Universite´ Paris 7, 2 place Jussieu, Tour 53-54, 3e e´tage, case courrier 7073, 75251 Paris cedex 05, France b UET qualite´ de l’air, Technocentre, TCR Lab 2 50, 1 avenue du Golf, 78288 Guyancourt, France Accepted 31 May 2003
Abstract Epidemiological studies have associated the increase of respiratory disorders with high levels of ambient particulate matter (PM) levels although the underlying biological mechanisms are unclear. PM are a complex mixture of particles with different origins but in urban areas, they mainly contain soots from transport like Diesel exhaust particles (DEP). In order to determine whether PM biological effects can be explained by the presence of DEP, the effects of urban PM, DEP and carbon black particles (CB) were compared on a human bronchial epithelial cell line (16-HBE14o-). Two types of PM were used : reference material (RPM) and PM with an aerodynamic diameter 42.5 mm collected in Paris with a high volume sampler (VPM). From 10 to 30 mg/cm2, cell viability was never modified whatever the particles. However, DEP and to a lower extent PM inhibited cell proliferation, induced the release of a pro-inflammatory cytokine, GM-CSF, and generated a pro-oxidant state as shown by the increased intracellular peroxides production. By contrast, CB never induced such effects. Nevertheless CB are more endocytosed than DEP whereas PM are the less endocytosed particles. In conclusion, PM induced to a lower extent the same biological effects than DEP in 16-HBE cells suggesting that particle characteristics should be thoroughly considered in order to clearly correlate adverse effects of PM to their composition and to clarify the role of DEP in PM effects. # 2003 Elsevier Ltd. All rights reserved. Keywords: Particulate matter; Diesel exhaust particles; Pro-inflammatory response; GM-CSF; Reactive oxygen species; Phagocytosis
1. Introduction Nowadays, air pollution is a real problem of public health for the general population in urban areas and for road workers since epidemiological studies have shown that fine particulate atmospheric pollution was associated with the increase of respiratory and cardiovascular mortality and morbidity (Kunzli et al., 2000). Particularly, particulate matter with an aerodiameter 42.5 mm (PM2.5) have been recently implicated in the Abbreviations: PM2.5, Particulate matter with an aerodiameter <2.5 mm; DEP, Diesel exhaust particles; RPM, Reference particulate matter; VPM, Vitry particulate matter; CB, Carbon black particles; 16HBE, 16-HBE 14o-cell line; GM-CSF, Granulocyte macrophagecolony stimulating factor; DCF, Dichlorofluorescein; ROS, Reactive oxygen species. * Corresponding author. Tel.: +33-144-276-062; fax: +33-144276-999. E-mail address: [email protected] (B. Augustin). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00115-2
increase of death (Arden Pope III et al., 2002) and in the respiratory adverse effects (Schwartz and Neas, 2000). Particulate matter (PM) are a complex mixture of different types of particles such as soots resulting from combustion processes, terrigenous particles from erosion and scrap-iron. In addition, chemical and biological substances can interact and adsorb on these particles (Soukup and Becker, 2001). PM composition greatly varies according to seasons and locations. In urban areas, Diesel exhaust particles (DEP) are probably a major component of PM, especially in Europe where drive many Diesel vehicles. The DEPmechanism of action is now better understood whereas few informations are available on PM. DEP have been shown to induce an inflammation in the airway of exposed volunteers (Diaz-Sanchez et al., 1997). It may result from the pro-inflammatory responses induced by DEP on the two main targets of inhaled particles: macrophages and airway epithelial cells.
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An important problem still unsolved is to determine whether DEP could contribute to PM-induced biological effects. For these reasons, the aim of our study was to compare on human bronchial epithelial cells the biological effects induced by DEP sampled after a tailpipe to those induced by PM sampled in urban areas. In particular, in vitro studies have shown that bronchial epithelial cells respond to DEP-exposure endocyting particles and releasing pro-inflammatory cytokines like granulocyte macrophage-colony stimulating factor (GM-CSF) (Boland et al., 1999). This release occurs after activation of transduction pathways implicating MAPK, reactive oxygen species (ROS) and the transcription factor (NF-kB) (Boland et al., 2000; Bonvallot et al., 2000; Bonvallot et al., 2001). However, these in vivo and in vitro experiments were performed with DEP directly collected after the tailpipe that are likely very different from that present in PM. Indeed, when spred in the atmosphere, DEP undergo many transformations which could modulate their toxicity. Two types of PM were used: reference urban particulate matter (RPM) purchased from National Institute of Standard and Technology (NIST) in USA and collected in St. Louis, and PM2.5 (VPM) collected with a high volume sampler in a school playground at Vitry-sur-Seine near Paris. After a cellular viability study performed to determine the non cytotoxic concentration, particles were compared according to the extent of their phagocytosis, their effect on cellular proliferation and their ability to induce the release of the pro-inflammatory cytokine GM-CSF and the production of peroxides.
National Institute of Standards and Technology (Gaithersburg, MD, USA). Atmospheric particulate matter PM2.5 (VPM) was collected with a high volume sampler machine (DA-80, Megatec, Paris, France), equiped with a PM2.5 selective-inlet head, in a school playground at Vitry-sur-Seine near Paris. The machine operated at the flow of 30 m3/h from 15–20 March 2002. During this period, 3690 m3 of air were filtered and particles were recovered on a nitrocellulose filter. Particles were detached from the filter by sonication, concentrated by centrifugation at 20,000 g and their concentration was determined spectrophotometrically by comparing turbidity to a standard curve of DEP at an optical density of 360 nm. Carbon black particles (CB) of 95 nm diameter (FR103) were obtained from Degussa (Frankfurt, Germany). Stock solutions of DEP, RPM and CB particles were performed by suspension in 0.04% DPL and sonicated two times for 5 min each at maximal power (100 W) (Vibracell, Bioblock Scientific, Illkirch, France). Particles were used at 10, 20 or 30 mg/cm2 (concentrations are expressed in mg/cm2 since particles rapidly sediment onto the culture). Controls were made by using 0.04% DPL. 2.3. Analysis of cell viability
2. Material and methods
Cell viability was studied on subconfluent culture using propidium iodide (PI), a DNA staining fluorescent probe that only enters in dead cells. After a 24 h toxic treatment, cells were washed, dissociated by trypsine-EDTA and stained by 0.1 mg/ml of PI. 10,000 cells were analysed by flow cytometry from each assay. Viability was related to those of control cells (control cells represent 100%).
2.1. Cell culture conditions
2.4. Analysis of cell proliferation
Dr. D. C. Gruenert (Cozens et al., 1994) (Colchester, VT, USA) kindly provided the human bronchial epithelial cell subclone 16HBE14o-. The cell line was cultured in DMEM/F12 culture medium supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), glutamine (1%), fungizone (0.125 mg/ml, Invitrogen) and UltroserG (UG) (2%, Invitrogen). Cells were cultured on collagen (type I, 4 mg/cm2) coated 25 or 75 cm2 flasks, 6- or 96-well plates (Costar, Cambridge, MA, USA) at 20,000 cells/cm2. At the time of treatment, UG was not added to DMEM/F12. Cultures were incubated in humidified 95% air with 5% CO2 at 37 C.
16-HBE cells were seeded at 40,000 cells by well in 12 well plates. 24 h after seeding, cells were treated and cell countings were performed after cell dissociation each 24 h during 3 days in a hemacytometer.
2.2. Particles Different kinds of particles were used: Diesel particulate matter SRM 1650a (DEP) and urban particulate matter SRM 1648 (RPM) were purchased from the
2.5. Analysis of intracellular peroxide levels Intracellular peroxide levels were assessed using H2DCF-DA, an oxidation-sensitive fluorescent probe. Once inside the cell, this probe is deacetylated by intracellular esterases forming H2DCF which in the presence of a variety of intracellular peroxides is oxidized to a highly fluorescent compound 20 ,70 -dichlorofluorescein (DCF). Stock H2DCF-DA solution was made at 20 mM in DMSO and stored at 20 C. Prior to the toxic treatment, cells were loaded for 20 min with 20 mm H2DCF-DA in HBSS and the fluorescence was analyzed by flow cytometry. The fluorescence analysis was
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performed by flow cytometry on 10,000 viable cells. Forward angle light scatter and right angle scatter were used to select cells.
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A 15 mW air cooled argon-ion laser tuned at 488 nm was used for DCF fluorescence. DCF and PI fluorescence were, respectively, collected though a 525 and 620 nm band pass-filter.
2.6. Cytokine assay Subconfluent cultures were exposed for 24 h to particles. Culture supernatant was recovered and frozen at 80 C until used. The concentration of granulocytemacrophage colony-stimulating factor (GM-CSF) released into the culture supernatants was measured with human GM-CSF Duoset ELISA development system (R&D systems Europe, Abingdon, United Kingdom). Color development was measured at 450 nm with a microplate photometer MR 5000 (Dynatech Laboratories).
3. Results 3.1. Effects of the different particles on the viability of 16-HBE cells In order to determine if particles could exhibit different cytotoxicities, their effect on cell viability was evaluated measuring PI fluorescence. No significant cell death was observed in 16-HBE cells exposed for 24 h to either DEP, RPM, VPM or CB from 10 to 30 mg/cm2 (Fig. 1).
2.7. Phagocytosis quantification Subconfluent cultures were treated with particles for 24 h. After rinsing and dissociation by trypsine-EDTA, cells were centrifugated (5 mn, 2200 g) and resuspended in 1 ml of PBS with 0.8 mg/ml of PI. Particulate uptake was measured by flow cytometry on 10,000 viable cells, excluding PI, according to the method of Stringer et al. (1995) adapted by Boland et al. (1999). Because ingested or bound particles induce a more granular morphology of the epithelial cells, the laser light is scattered to a greater extent. Thus, the right angle scatter (RAS) signal is dependent on the granularity of the cells. The mean RAS channel number was used for data analysis.
3.2. Inhibition of cell proliferation by the different particles The different particles used at the non cytotoxic concentration of 10 mg/cm2 were tested on proliferating cells over 72 h of treatment. Whereas CB never disturbed cell growth, DEP, RPM and VPM induced an inhibition of 16-HBE cells proliferation that increases with time exposure for DEP and RPM (Fig. 2). This inhibitory effect at 72 h is significantly different according to the type of particles: DEP (52 0.8) > RPM (31 2) > VPM (19 5). 3.3. Peroxides production
2.8. Flow cytometry assay The fluorescence analysis were performed with an EPICS-Elite-ESP flow cytometer (Coultronics-France).
In order to determine the role of oxidative stress in the biological effects of particles, DCF fluorescent probe was used to quantify the peroxide production in 16-HBE cells
Fig. 1. Effects of the different particles on the viability of 16-HBE cells. Cells were treated with DEP, RPM, VPM, CB at 10, 20 or 30 mg/cm2 for 24 h. The percent of cells excluding PI was analysed by flow cytometry over 10,000 analysed cells. Values are means S.D. (n=3).
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Fig. 2. Effect of different particles on 16-HBE cells proliferation. DEP, RPM, VPM and CB were applied in a single dose at 10 mg/cm2. Inhibition of cell proliferation was evaluated after 24, 48 and 72 h of treatment by cell counting and expressed as a percent of control value. Values are meansS.D. (n=3) except for VPM at 24 and 48 h (n=1).
3.5. Phagocytosis of particles
Fig. 3. Peroxide production in 16-HBE cells treated with different particles. Cells were loaded for 20 min with H2DCF-DA prior to the treatment with DEP, RPM, VPM and CB at 10 mg/cm2 for 4 h. DCF fluorescence (in arbitrary fluorescence units) of 10,000 gated viable cells was analysed by flow cytometry.
Previous experiments have shown that phagocytosis is important to initiate the DEP-biological effects (Boland et al., 2000), the occurrence of such mechanism for the different particles was investigated. All types of particles at 10 mg/cm2 induced after 24 h of exposure, an increase of RAS in 16-HBE cells (Fig. 5). However the rate of phagocytosis is different according to the type of particles. CB induced the most important increase of RAS (140% of control) whereas PM have the lowest effect (116% and 107 for RPM and VPM, respectively) and DEP have an intermediary effect (122%).
4. Discussion treated for 4 h with 10 mg/cm2 of particles. Whereas CB had no effect on DCF fluorescence, DEP, RPM and VPM induced a significant increase of DCF fluorescence similar for DEP and VPM but lower for RPM (Fig. 3). 3.4. Pro-inflammatory response The GM-CSF release was used as a biomarker of the pro-inflammatory response induced in 16-HBE cells exposed for 24 h to the different particles. Whatever the concentration used, CB never induced GM-CSF release (Fig. 4). By contrast, DEP, RPM and VPM induced a dose-dependent increase of GM-CSF release that was always higher in DEP-treated cells than in VPM or RPM-treated cells. The DEP-induced GM-CSF release is 2.9-fold greater at 30 mg/cm2 than that induced by PM.
The aim of our study was to determine the cellular effects of PM2.5 on a bronchial epithelial cell model. PM, and particularly the fine particles (PM2.5), are involved in respiratory diseases but their mechanisms of action and the particle component(s) responsible for the biological effects are still unclear. For these reasons, it was convenient to study the PM-mechanisms of action like it has been done with DEP. In previous studies, we have shown the DEP-involvment in the induction of the pro-inflammatory response and the major role of organic compounds adsorbed on DEP (Boland et al., 2000; Bonvallot et al., 2001). Two kinds of PM were used: (1) Reference PM (RPM) that are total suspended particles (TSP). These particles are interesting because they are certified for their metals content, many experimental data are available in literature and they can be used as internal
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Fig. 4. Pro-inflammatory response of 16-HBE cells. Cells were treated for 24 h with DEP, RPM, VPM and CB at 10, 20 or 30 mg/cm2 for 24 h. Values are means S.D. expressed as a percent of control values (n=3).
Fig. 5. Phagocytosis of 16-HBE cells treated with different particles. Cells were treated or not by DEP, RPM, VPM and CB at 10 mg/cm2 for 24 h. RAS (mean channel number) of 10,000 gated viable cells was analysed by flow cytometry. Values are means S.D. (n=3).
standard in biological assays. (2) PM from Paris (VPM) are PM2.5 collected in suburbs of Paris (Vitry-sur-Seine) which represent a typical urban particulate pollution of European cities. Both RPM and VPM induce similar biological effects on 16HBE cells. However, scanning electron microscopy observations have shown that RPM contain few soots but many fly ash whereas VPM are mainly composed of soots from combustion processes especially from motor vehicle emission (data not shown). Moreover, VPM only contains PM2.5, that are the most involved in respiratory diseases due to their small size (Schwartz and Neas, 2000) whereas RPM are TSP with a wide particle size distribution (Don Porto Carero et al., 2001).
The different particles used have no effect on cell viability even at the concentration of 30 mg/cm2 confirming experiments performed on two other human cell lines (alveolar cells and monocytes) with RPM and DEP (Don Porto Carero et al., 2001) and on a human alveolar cell line (A549) with Mexico PM10 (Alfaro-Moreno et al., 2002). Higher concentrations were not tested as 30 mg/cm2 is already an important concentration in disagreement with PM atmosphere levels. Although the different particles didn’t affect the 16HBE viability, DEP and PM (RPM and VPM) inhibited cell proliferation. But CB, which are used as a surrogate of the carbonaceous core of the soots, has no effect. This result could signify that the inhibition of cell
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proliferation is not due to mechanic effect but is related to particles composition (organic compounds, metals. . .). Especially, organic compounds extracted from DEP have been shown to inhibit 16HBE cell proliferation (unpublished results). In addition, a study performed with PM10 collected in different locations in Mexico city, have shown that their different cytotoxicity towards proliferating cells (mouse monocytes and fibroblats) could be related to their composition (Alfaro-Moreno et al., 2002). In addition, DEP, RPM and VPM but not CB induced an increase of peroxides production suggesting that an oxidative stress is involved in the cellular effects of these particles as already observed with Vermont PM2.5 on a murine alveolar cell line (Shukla et al., 2000). Metals are known to generate reactive oxygen species (ROS) (Pritchard et al., 1996) and could be responsible for the increased DCF fluorescence obtained in particles treated-16HBE cells. It is in agreement with studies on alveolar macrophages exposed to concentrated ambient particulate in which an increased DCFH oxidation occurred and was inhibited by a metal chelator desferrioxamine (Goldsmith et al., 1998). Moreover DEP (SRM 1650a) and RPM (SRM 1648) have been shown to induce the generation of ROS involving transition metals (Ball et al., 2000). However organic compounds could also participate to oxidative stress generation. Indeed, DEP-organic extracts used as at the concentration they are on DEP, induced a DCF fluorescence increase similar to that induced by native DEP (Bonvallot et al., 2002; Baulig et al., 2003). PM10 and DEP have been shown to induce an inflammation in humans (Van Eeden et al., 2000; Salvi et al., 1999). Until now, in vitro studies had mainly used macrophages to investigate the pro-inflammatory response to PM (Soukup and Becker., 2000; Imrich et al., 2000; Alfaro-Moreno et al., 2002). GM-CSF was chosen as a marker of the pro-inflammatory response in 16HBE cells as it has been previously shown to be specifically induced by DEP (Boland et al., 2000). PM (RPM and VPM) also induced GM-CSF release but to a lower extent than DEP whereas CB had no effect. These different levels of GM-CSF release according to the type of particles are likely correlated to particle composition. Indeed, they don’t seem related to the phagocytosis since CB which are the most endocytosed particles, are the less efficient one. Nevertheless, phagocytosis may be necessary as we have demonstrated for DEP (Boland et al., 2000). After endocytosis, DEPorganic compounds become available and are likely metabolized since they induce the cytochrome P450 1A1 gene expression (Bonvallot et al., 2001; Baulig et al., 2003). Concerning PM, they are moderately or few endocytosed but they induce a dose-dependent increase of GM-CSF release suggesting that the endocytosis is not always essential. It is possible that PM can act at the
plasmic membrane level triggering receptors. Such mode of action has already been described for asbestos and residual oil fly ash, metal containing particles (Zanella et al., 1996; Huang et al., 2002). Altogether these results show that PM trigger reduced biological effects in comparison to DEP. It could be explained on the one hand by the aging of DEP present in PM samples. They might have lost some components especially organic compounds which have been shown to mainly contribute to DEP-biological effects (Bonvallot et al., 2001). On the other hand it could be due to a low DEP amount in comparison with other types of particles in PM samples. It is the case for RPM but nevertheless they induce biological effects in the same range order as VPM underlying that other components than DEP have to be considered. To summarize, we have shown that PM induced to a lower extent the same biological effects than DEP in 16HBE cells. Further studies are necessary to determine particle component(s) responsible for these biological effects by PM fractionation (aqueous and organic). The identification of implicated component(s) will allow to thoroughly investigate the signalling pathways triggered by PM in bronchial epithelial cells and to assess the involvement of DEP in PM biological effects.
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