Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production

Sonja Boland1, Armelle Baeza-Squiban1, Thierry Fournier2, Odile Houcine1, Marie-Claude Gendron3, Michèle Chévrier4, Gilles Jouvenot4, André Coste5, Michel Aubier2, and Francelyne Marano1

1 Laboratoire de Cytophysiologie et Toxicologie Cellulaire and 3 Laboratoire de Cytométrie, Institut J. Monod, Université Paris VII Denis Diderot, 75251 Paris; 2 Institut National de la Sante et de la Recherche Médicale Unité 408, Faculté X. Bichat, 75018 Paris; 4 Technocentre Renault, 78288 Guyancourt; and 5 Service d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, Centre Hospitalier Universitaire Henri Mondor, 94000 Créteil, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

16HBE14o- cells; human nasal turbinates; air pollution; carbon black


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PREVALENCE OF ALLERGIC DISEASES, notably asthma, has markedly increased in the last 20 years, particularly in industrialized countries (36). Furthermore, high-particulate air pollution periods have been shown to be associated with an exacerbation of asthma symptoms (28) and an increase in hospital admissions for respiratory diseases. Diesel exhaust emissions, which contribute to fine-particulate air pollution (particulate matter 2.5), are suspected to be involved in the genesis of these two epidemiologic observations. Indeed, recent studies have emphasized the role of diesel exhaust particles (DEPs) in the development of an inflammatory response. It has been shown that DEPs instilled intratracheally in mice induce asthmalike symptoms (27) and that nasal challenges of human beings with DEPs result in the local increase of IgE and cytokine production (14, 15). Moreover, in vitro studies (4, 25, 29) have shown that treatment with DEPs significantly increases the electrical resistance of human bronchial epithelial cells in culture and induces the release of cytokines.

DEPs tend to form aggregates of ~0.1-0.5 µm in diameter after the combustion process, placing >90% of these particles within the respirable size. It has been estimated that up to 33% of the inhaled DEPs are deposited on the respiratory tract and about one-third of these particles are deposited in the pulmonary region (8), with an alveolar clearance half-time of 62 days in rats (9). DEPs are composed of a carbonaceous core with high surface-to-volume ratios. These particles adsorb trace amounts of heavy metals such as iron, copper, chromium, and nickel and a vast number of organic compounds such as polyaromatic hydrocarbons (PAHs), nitroaromatic hydrocarbons, quinones, aldehydes, and aliphatic hydrocarbons, representing up to 40% of the mass of the particles (35). Organic solvents as well as animal tissues (5) can easily remove these organic compounds [the soluble organic fraction (SOF)] from the carbonaceous core. However, experiments with extracts of DEPs may not reflect the actual health risk of the inhaled diesel soot because there is increasing evidence that the carbonaceous core of the particles may take part in the effects resulting from its deposition and accumulation in the conducting airways. Until now, it is unclear which component of DEPs is implicated in the biological effects. This is of great importance because exhaust gas posttreatments, such as an oxidation catalyst, strongly diminish the adsorbed organic compounds (SOF) without an effect on the carbon core and particle number.

The aim of this study was, therefore, to compare the biological effects of DEPs (diesel particulate matter SRM 1650), DEPs collected from diesel vehicles in noncatalyst- and catalyst-equipped states, and carbon black particles, which represent the carbonaceous core of the particles mostly devoid of the SOF. We used two in vitro models of human airway epithelium: a bronchial epithelial cell line (16HBE14o-) and primary cultures of human nasal epithelial (HNE) cells. This study focused on 1) the phagocytosis of the different kinds of particles by human airway epithelial cells and 2) the inflammatory response in terms of cytokine production with regard to the nature of the particles and the amounts of adsorbed organic compounds. Regarding the possible contribution of DEPs to the development of allergic diseases, it is now of greatest concern for public health to investigate whether exhaust gas posttreatments could reduce the biological effects of DEPs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Culture Conditions

Explant outgrowth cultures. Dr. A. Coste (Centre Hospitalier Universitaire Henri Mondor, Créteil, France) generously provided nasal epithelium from turbinectomies after informed consent and institutional approval were obtained. After resection, the tissues were placed in Dulbecco's modified Eagle's medium-Ham's F-12 nutrient mixture (DMEM/F12; 1:1; GIBCO BRL, Cergy Pontoise, France) supplemented with antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin; Sigma, Saint Quentin Fallavier, France) and Fungizone (1 µg/ml; GIBCO BRL). The epithelium was separated from the underlying tissue and cut into 2-mm2 explants. The explants were deposited on a thick collagen gel (Institut Jaques Boy, Reims, France) supplemented with MEM and 10% fetal bovine serum (both from GIBCO BRL). The cultures were then covered with 200 µl of MEM containing 10% serum, epidermal growth factor (25 ng/ml; Sigma), glutamine (0.3 mg/ml; Sigma), insulin (5 µg/ml; Sigma), hydrocortisone (0.1 µg/ml; Sigma), Fungizone (0.25 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and neomycin (66 µg/ml).

Cell line cultures. Dr. D. C. Gruenert (Cardiovascular Research Institute, University of California, San Francisco) kindly provided the human bronchial epithelial cell subclone 16HBE14o-. The cell line was maintained routinely at the logarithmic phase of growth through subculturing once a week, and experiments were performed at passages 25-40. Cells were cultured on collagen (type I, 4 µg/cm2; Sigma)-coated 25-cm2 flasks or 24-well plates at 10,000 cells/cm2 in DMEM/F12 supplemented with antibiotics, Fungizone, and 2% Ultroser G (GIBCO BRL).

Cell cultures on porous membranes for transmission electron microscopy. Primary cultures of human nasal turbinates were obtained by enzymatic dissociation for 18 h at 4°C with 2 mg/ml of protease (bacterial type XIV, 10.4 U/ml; Sigma) in DMEM/F12 containing antibiotics and Fungizone. Enzyme action was inhibited by the addition of 10% serum, and the cells were centrifuged at 650 g for 5 min. The pellet was suspended in DMEM/F12 and seeded on 25-cm2 plastic flasks for 2 h, preplating to remove fibroblasts. The HNE cells as well as the 16HBE14o- cells were seeded on collagen (collagen type IV of human placenta, 50 µg/cm2; Sigma)-coated Transwell inserts (12-mm diameter; Costar, Dutscher, Issy les Moulineaux, France) with a cell density of 100,000 cells/cm2 in DMEM/F12 supplemented with antibiotics (50 U/ml of penicillin, 50 µg/ml of streptomycin, and 66 µg/ml of neomycin), Fungizone (0.25 µg/ml), transferrin (7.5 µg/ml; Sigma), glutamine (0.3 µg/ml), insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), triiodotyrosine (3 × 10-8 M; Sigma), retinoic acid (5 × 10 -8 M; Sigma), and endothelial cell growth supplement (1.5 µg/ml; Sigma). The culture medium was changed every 2 days, and at confluence, the physiological air-liquid interface was restored by removing the medium from the upper compartment. During treatment, the cultures were again covered with medium for 24 h.

All cultures were incubated in humidified 95% air-5% CO2 at 37°C.

Chemical Treatment

The diesel particulate matter SRM 1650 was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). Fluorescent latex particles (0.05-µm diameter; fluorescence emission at 505 nm) were obtained from Sigma. Carbon black (FR103; 95-nm diameter) was obtained from Degussa (Frankfurt, Germany). Stock solutions of particles were made by suspension in distilled water or a solution of 0.04% dipalmitoyllecithin (DPL; Sigma) in distilled water and sonicated three times for 60 s each at maximal power (8 kilocycles). DEPs were also collected from a diesel car (1.9 liter with an exhaust gas recirculation system; IDI Renault Express) in a noncatalyst- or catalyst-equipped state (standard oxidation type) filled with fuel of standard composition (500 parts/million sulfur). The legislative driving cycle (MVEG/Euro 96) for European emissions regulations, which consists of an urban phase (820 s) and an extraurban phase (400 s), was applied. Driving cycles were carried out three times to collect enough particulate matter for the toxicological tests. The particles were sampled with a standard dilution tunnel equipped with a constant-volume sampler. A portion of this diluted exhaust gas was collected on polyvinylidene fluoride filters (two in series; Durapore, Millipore, St. Quentin Yvelines, France) under controlled conditions. The weight of the filters before and after a test was determined with a microbalance in a humidity- and temperature-controlled room. Filters were sonicated in a solution of 0.04% DPL two times for 30 s each. Different concentrations of the particles were made in serum-free culture medium before being added to the cultures. Medium with the same concentration of DPL was added to the control cultures. Concentrations are expressed in micrograms per square centimeter because the particles rapidly sediment onto the culture.

Analysis of PAHs

The SOF was extracted with dichloromethane from particulates with a Soxhlet system. Care was taken to screen the process from light to minimize the loss of PAHs. Fifteen particulate PAHs were separated in the SOF, and analysis was performed with an HPLC system. PAHs were detected by fluorescence. Ten particulate nitro-PAHs were identified in the SOF, and analysis was performed with gas chromatography-mass spectrometry equipment. Components were separated on a chromatograph column after sample purification and were detected by chemical ionization.

Cytotoxicity Assays

Release of lactate dehydrogenase (LDH) and trypan blue uptake were performed as previously described (6). Particles were applied in a single dose and at various concentrations. Cytotoxicity measurements were carried out after 24 or 48 h of treatment.

Ultrastructural Studies

Confluent air-liquid interface cultures were treated with DEPs at 2.5 µg/cm2 and fixed after 24 h of treatment for transmission electron microscopy (TEM) as previously described (17). Briefly, the cultures were fixed in 2% glutaraldehyde and postfixed in 1% OsO4. After dehydration, the samples were embedded in Epon. Ultrathin sections were made with a diamond knife and double stained with uranyl acetate and lead citrate before observation with a JEOL 100 C electron microscope. For TEM observations of HNE cells, the transepithelial potential was measured with a World Precision Instruments DVC 1000 voltage-clamp apparatus to ascertain the confluence of the epithelium before and after the incubation period with DEPs.

Flow Cytometry Assay

After 2 days of culture, 16HBE14o- cells were treated with particles at concentrations between 0 and 20 µg/cm2. After different treatment times, the cells were dissociated by incubation in 0.05% trypsin-EDTA (0.2 mg/ml; GIBCO BRL) for 10 min at 37°C. Trypsin action was inhibited by the addition of 10% serum. The cells were centrifuged and suspended in phosphate-buffered saline (PBS; GIBCO BRL) containing propidium iodide (PI; Sigma) at 20 µg/ml. Cell suspensions were analyzed in an Epics-ELITE-ESP flow cytometer (Coultronics). A 488-nm argon-ion laser excitation (15 mW) was used for measurement of PI fluorescence through a band-pass (BP) 620-nm filter to perform the counting of 10,000 viable cells. Right-angle light scatter (RAS) was collected through a BP 488-nm filter at 90° incident to the cell flow, and fluorescent emission from latex particles was recorded with a BP 520-nm filter. Particulate uptake was measured according to the method of Stringer et al. (30). Because ingested or bound particles induce a more granular morphology of the epithelial cells, the laser light is scattered to a greater extend. Thus the RAS signal is increased proportionally to the amount of particles bound or ingested. The uptake of particles was measured with the increased RAS signal from a univariate histogram of RAS vs. the number of events (after selection of viable cells by measurements of PI uptake and gating from a bivariate histogram of forward-angle light scatter vs. RAS). The mean RAS-channel number was used for data analysis. Similarly, the uptake of fluorescent latex particles was determined by fluorescence and RAS measurements. Unbound particles were substantially smaller than epithelial cells and were removed from the forward-angle light scatter vs. RAS window by adjusting the electronic threshold settings.

Cytokine Assay

Four-day-old cultures were grown for 24 h in culture medium without serum and then treated with particles at 10 µg/cm2. After 6, 12, or 24 h of treatment, the culture supernatant was centrifuged, and samples were frozen at -80°C until used. The concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, and IL-1beta released into the culture supernatants was measured with human GM-CSF, IL-8, and IL-1beta ELISA kits (Amersham, Les Ulis, France). Color development was measured at 450 nm with a microplate photometer MR 5000 (Dynatech Laboratories).

DNA Assay

DNA assays were adapted from the method of Labarca and Paigen (22) and were based on the enhancement of fluorescence seen on bisbenzimide (Hoechst 33258; Sigma) binding. Standards of calf thymus DNA (Sigma) or samples in saline-phosphate buffer (0.05 M NaH2PO4 · H2O-Na2HPO4 1:2 vol/vol and 2 M NaCl, pH 7.4) were added to Hoechst 33258 (0.1 µg/ml in saline-phosphate buffer). Fluorescence measurements (excitation at 356 nm and emission at 458 nm) were made with a spectrofluorimeter.

Statistical Analysis

Student's t-test was used to compare LDH, trypan blue uptake, RAS, and cytokine secretion from control samples with those from treated samples. Values are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemical Analysis of Particles

HPLC analysis of DEPs collected from diesel engines with and without a catalytic converter and of carbon black particles are shown in Table 1. The use of this oxidation catalyst reduces the PAH and nitro-PAH content by 50-60%. On the other hand, carbon black particles contain only 1.5% of the PAH content of DEPs.

                              
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Table 1.   PAH and nitro-PAH particulate composition

Cell Cultures

Primary cultures of epithelial cells obtained by the explant method have already been described for rabbit tracheal epithelial cells (2). The cell sheet was obtained by the migration of cells out of the explant and by proliferation of the cells. As already shown for tracheal epithelial cells (2), the use of a thick collagen gel allows reconstitution of a stratified epithelium composed of basal, secretory, and functional ciliated cells. On the other hand, confluent primary cultures of HNE cells on porous membranes were stratified (Fig. 1), contained ciliated cells (Fig. 1C), and presented a transepithelial potential (data not shown), revealing the presence of functional tight junctions. At this stage of culture, the cells have a very low proliferation rate.


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Fig. 1.   Transmission electron microscopy (TEM) observations of confluent primary cultures of nasal epithelial cells on porous membranes treated for 24 h with diesel exhaust particles (DEPs) at 2.5 µg/cm2. A: general view of stratified epithelium showing presence of DEPs in intercellular spaces. AP, apical pole; BP, basal pole. B: detail of DEPs in an intercellular space. Note that DEPs are in contact with plasma membrane. C: ciliated cell containing DEPs. Arrows, DEPs. Bars, 1 µm.

The properties of 16HBE14o- cells have been previously described (13). Cultures were devoid of ciliated cells but expressed cytokeratins, intermediate filaments characteristic of epithelial cells (13). At confluence, cultures tend to stratify (Fig. 2A) and continue to proliferate.


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Fig. 2.   TEM observations of confluent cultures of bronchial epithelial cells (16HBE14o-) on porous membranes treated for 24 h with DEPs at 2.5 µg/cm2. A: general view of culture showing stratification of epithelium and a large DEP-filled vacuole. B: note presence of DEPs in a pit of plasma membrane. DEPs that underwent endocytosis were only partially surrounded by a membrane. Clathrin-coated pits did not contain DEPs. C: necrotic cell (N) beside a DEP-filled cell. D: DEPs in cytoplasm and nucleus. Thin arrows, DEPs; thick arrow, clathrin-coated pit. Bars, 1 µm.

Toxic Effects of DEPs

The release of cellular LDH and trypan blue uptake by 16HBE14o- cells as a measure of the cytotoxicity of DEP exposure are shown in Table 2. After a 24-h treatment, no significant difference between control and treated cultures was seen by the two tests. In contrast, a significant increase in LDH release was obtained at 48 h with a single dose of 10 µg/cm2 (193% of control value), indicating membrane damage of the treated cells, whereas a 5 µg/cm2 DEP treatment had no effect. Thus the LDH release observed is a time- and dose-dependent process. However, with trypan blue viability assays, no toxicity could be observed even after a treatment of 48 h with 10 µg/cm2 of DEPs, a dose for which an increase in LDH release was observed.

                              
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Table 2.   Effects of DEP on LDH release and trypan blue uptake by HBE cells

TEM

To investigate the reasons for this cellular damage, we performed ultrastructural studies with TEM. The 16HBE14o- cells and the HNE cells in primary culture were treated for 24 h with 2.5 µg/cm2 of DEPs. As shown in Fig. 2, the 16HBE14o- cells clearly caused endocytosis of DEPs that were accumulated in large phagosomes, probably resulting from the fusion of several smaller vesicles. Figure 2B shows the existence of cellular expansions at the site of contact of the plasma membrane with DEPs. Thus DEPs may enter the cell by phagocytosis rather than by clathrin-coated pits in which no DEPs could be observed (Fig. 2B). DEPs were mostly surrounded by a membrane but could also be in direct contact with the cytoplasm (Figs. 1, B and C, and 2B) or occasionally be observed in the nucleus (Fig. 2D). Nevertheless, the structure of cells containing large amounts of DEPs was not damaged, whereas necrotic cells that were sometimes present in the culture did not seem to contain DEPs (Fig. 2C). Nasal epithelial cells in primary culture could also phagocytose DEPs, which entered the intercellular spaces (Fig. 1, A and B). Ciliated cells also showed endocytosis of DEPs (Fig. 1C). Despite the presence of functional tight junctions (ascertained by the measurement of a transepithelial electric potential difference; data not shown), DEPs were observed in the intercellular spaces and in apical as well as basal cells. Thus the particles may pass through the epithelial cell sheet by transcytosis.

Quantification of Phagocytosis by Flow Cytometry

The uptake of particles induced an increase in the granularity of the cells, which led to a greater scattering of the laser light in the flow cytometer (30). The 16HBE14o- cell line was used for these investigations because these cells present a more homogeneous granularity than HNE cells. Moreover, Boland et al. (7) previously showed that on a ciliated epithelium the particles were swept to the border of the culture due to the ciliary beat. Thus the absence of ciliated cells in this cell line allowed a homogeneous deposition of the particles. The cultures were first treated with fluorescent latex beads, allowing the evaluation of phagocytosis by both RAS and fluorescence measurements. As expected, the fluorescence of the cells due to the phagocytosis of latex beads increased in the same manner as the mean RAS of the cells (Table 3).

                              
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Table 3.   Measurements of particle uptake after 24 h of exposure

DEPs also induced a shift in the RAS, with 29.2% of the cells having a greater granularity than the control cells after a treatment for 24 h at 5 µg/cm2 (Fig. 3). The uptake of DEPs was a dose-dependent process (Fig. 4A). A significant increase in cell granularity was seen after 24 h of exposure to 0.5 µg/cm2 of DEPs, and the mean RAS increased with the concentration of DEPs. Figure 4B shows the time dependency of the uptake of DEPs. The mean RAS increased significantly after 4 h of treatment with 10 µg/cm2 of DEPs (239 ± 2 compared with 199 ± 4 in control cells) and was still raised after 18 h (284 ± 1).


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Fig. 3.   Flow cytometric histograms of control and DEP-treated cultures (5 µg/cm2 for 24 h) of 16HBE14o- cells from a representative experiment. RAS, right-angle light scatter. After treatment, 29.2% of cells had a greater granularity than control cells.


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Fig. 4.   Effects of DEPs on RAS of 16HBE14o- cells. A: treatment for 24 h with DEPs at indicated concentrations. B: treatment with DEPs at 10 µg/cm2 for indicated times. Values are means ± SE from 4 cultures. * P < 0.05 compared with control value.

To investigate whether this phagocytosis is specific to DEPs and to ascertain whether DEPs without PAHs could also be phagocytosed by epithelial cells, carbon black particles were tested. Indeed, a 24-h exposure to either DEPs or carbon black at 5 µg/cm2 induced an increase in the mean RAS, which was even significantly more important for carbon black (124 and 142%, respectively, compared with that in control cells; Fig. 5). The presence of DPL in the culture medium, used to suspend the particles, did not influence the uptake of DEPs (129% compared with control value; Fig. 5).


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Fig. 5.   Comparison of RAS of 16HBE14o- cells treated for 24 h with DEPs and carbon black at 5 µg/cm2. DPL, dipalmitoyllecithin. Values are means ± SE from 4 cultures. * P < 0.05 compared with control value.

Secretion of Cytokines

To assess whether DEP exposure could lead to an inflammatory response of the epithelial cells, the release of cytokines involved in allergic inflammation (GM-CSF, IL-8, and I-1beta ) was measured.

16HBE14o- cells (Fig. 6) and HNE cells (Fig. 7) were able to secrete GM-CSF and IL-8, which were not affected by the presence of DPL in the culture medium (data not shown). In addition, primary cultures of HNE cells secreted IL-1beta , whereas the cell line did not. On the other hand, we also studied the release of Th2-type cytokines: IL-4, IL-5, and IL-13 were undetectable by ELISA in the culture supernatant of 16HBE14o- cells as well as in that of HNE cells (data not shown).


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Fig. 6.   Effect of DEPs and carbon black at 10 µg/cm2 on granulocyte-macrophage colony-stimulating factor (GM-CSF; A) and interleukin (IL)-8 (B) release by 16HBE14o- cells after indicated treatment times. Values are means ± SE from 3 cultures. * P < 0.05 compared with control value.


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Fig. 7.   Effect of DEPs at 10 µg/cm2 on GM-CSF, IL-8, and IL-1beta release by nasal epithelial cells in primary culture after different treatment times. Values are means ± SE from 3 cultures where 100% corresponds to control value. P values compare control with DEP-treated cultures.

Treatment of 16HBE14o- cells with 10 µg/cm2 of DEPs induced a significant increase in GM-CSF secretion after 24 h of treatment (1,642% compared with control cultures), which still increased after 48 h (Fig. 6A). An increase in GM-CSF secretion was also observed after treatment of HNE cells in primary culture (307% compared with control value after 6 h and 474% after 24 h; Fig. 7). In the 16HBE14o- cell line, the increase in IL-8 release became significant after a 48-h exposure with 10 µg/cm2 of DEPs (361% compared with control culture; Fig. 6B). In HNE cells in primary cultures, this increase was also observed (150% compared with control cultures at 6 h and 197% at 24 h). The production of IL-1beta was also stimulated by DEPs after 24 h of treatment (723%). The secretion of cytokines by HNE cells in primary cultures exhibited a greater heterogeneity compared with the cell line because we used tissues from different individuals. Despite the great variability in response between individuals, we always observed an increase in secretion after DEP treatment, and the P values (Student's t-test) were close to statistical relevance.

When the effects of DEPs and carbon black particles on cytokine release by the 16HBE14o- cell line were compared, we found that carbon black induced a significant but lower increase in GM-CSF than DEPs after 24 h and a slight but nonsignificant increase in GM-CSF release after 48 h as well as a nonsignificant increase in IL-8 release at 24 and 48 h (Fig. 6, A and B). Furthermore, the comparison of DEPs generated by diesel engines with and without an oxidation catalyst that reduced their PAHs up to 60% revealed that the release of GM-CSF is strongly reduced by this exhaust gas posttreatment (403% compared with control value for a noncatalyst-equipped vehicle versus 40% for a catalyst-equipped vehicle, with the control value being 100%; Fig. 8).


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Fig. 8.   GM-CSF release by 16HBE14o- cells treated for 24 h with DEPs collected from a diesel engine with and without an oxidation catalyst. Values are means ± SE from 6 cultures obtained from 2 different filters. * P < 0.05 compared with control value.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In an attempt to understand the toxicity of DEPs on human airway epithelial cells, cell culture systems of nasal and bronchial epithelial cells are of great interest because they allow the elimination of interference with the inflammatory response produced by surrounding nonepithelial cells. To simulate real exposure conditions, native DEPs were suspended in a solution of DPL, a phospholipid that had no extraction effect on the PAHs adsorbed on the surface of the particles (data not shown).

Cytotoxicity of DEPs

In the present paper, we demonstrated that DEP treatment of 16HBE14o- cells induced a dose- and time-dependent increase in LDH release, whereas no increase in trypan blue uptake was observed. These results are in agreement with those obtained by Steerenberg et al. (29) on another bronchial epithelial cell line. Moreover, these results corroborate those obtained in a previous work on rabbit tracheal epithelial cells in primary culture where Boland et al. (7) showed that DEP treatment induced a dose- and time-dependent membrane damage and that the presence of ciliated cells delayed this cytotoxicity.

Phagocytosis of DEPs

The cellular toxicity may be due to the phagocytosis of DEPs that is already observed after 4 h of treatment, an event before LDH release. Indeed, ultrastructural studies have shown that DEPs at a noncytotoxic concentration undergo endocytosis by nasal as well as by bronchial epithelial cells, confirming previous observations by Boland et al. (7) on rabbit tracheal epithelial cells. The increased release of LDH after prolonged DEP treatment may be due to particle-induced membrane disruption. It has been reported that DEPs accumulate in the perinuclear region (10), and thus the presence of DEPs without a surrounding membrane in the cytoplasm during cell division may explain the occasional presence of DEPs in some nuclei.

This phagocytosis is not an artifact of in vitro exposure because endocytosis of DEPs also occurs in vivo as seen for alveolar macrophages (3) as well as for nonprofessional phagocytes like eosinophils, type I pneumocytes, and bronchial epithelial cells (3, 20). We have also shown by the use of confluent cultures on porous membranes that DEPs are able to translocate the epithelium by transcytosis. This could also explain the presence of DEPs in ciliated cells that we reported and that was also observed in vivo (20). Indeed, the ciliary beat prevents the uptake of particles at the apical membrane of ciliated cells. This transcytosis suggests that, in vivo, DEPs may enter the interstitium where the particles could be taken up by macrophages or may get in contact with endothelial cells, which could explain the endothelial cell injury observed after intratracheal instillation of DEPs (21). We adapted flow cytometric evaluation of particle uptake by RAS measurements to 16HBE14o- cells. This method allowed us to quantify this phenomenon and to affirm its time and dose dependency, which is difficult to establish by microscopic observations. The observation that bound particles are largely surrounded by extensions of cytoplasmic processes, leading to spacious phagosomes that are not closely adherent to DEPs, indicates that the phagocytosis of DEPs may be mediated by the recently proposed trigger mechanism rather than by the classic zipper model (for a review, see Ref. 31). Unlike receptor-mediated endocytosis, phagocytosis of particles occurs by extension of pseudopods, which involves actin assembly (for a review see Ref. 1). This phagocytosis of DEPs seems to be nonspecific because carbon black and latex particles were phagocytosed to the same extent as DEPs. Carbon black particles were even more phagocytosed than DEPs, which is in agreement with the findings of Gu et al. (18) showing that DMSO-extracted particles are phagocytosed to a greater extent. This could be due to a different size of the agglomerates, resulting in a different number of particles. It has been proposed that small particles are more readily phagocytosed by epithelial cells and transported across the epithelium than larger particles (11, 12).

Induction of an Inflammatory Response

The uptake of DEPs in human airway epithelial cells may lead to the release of proinflammatory cytokines. We have shown that exposure of primary cultures of HNE cells or 16HBE14o- cells to DEPs induced a time-dependent increase in IL-8 and GM-CSF release, cytokines involved in allergic inflammatory disorders such as asthma. These cytokines are known to have biological activities on eosinophils, the predominant cells in bronchial asthma, and neutrophils. Phagocytosis might be a prerequisite step for a subsequent cytokine secretion because phagocytosis was already observed after 4 h of treatment, whereas the increase in cytokine release by 16HBE14o- cells was significant after 24 h. This is in agreement with the findings by Rosenthal et al. (26) showing that asbestos-induced IL-8 release is dependent on phagocytosis. However, our results further suggest that cytokine production is not linked to phagocytosis per se because carbon black particles, phagocytosed to the same extent as DEPs at the same concentration, did not significantly increase cytokine levels.

We could still observe an increase in cytokine release after 48 h of treatment despite a slight cytotoxic effect at this treatment time. Nevertheless, a noncytotoxic treatment of 24 h did induce a significant increase in cytokine release. Membrane damage due to DEP exposure has been shown to diminish the release of IL-8 by epithelial cells in response to DEPs (29).

Our observation of increased cytokine release after DEP exposure corroborates that obtained in primary bronchial epithelial cells (4, 25), primary cultures of nasal polyps (25), and BEAS 2B cells (25, 29). The observation of increased GM-CSF release is also in accordance with the findings that DEPs enhance eosinophil adhesion to human nasal epithelial cells (34) and increase antigen-induced GM-CSF expression in mice (32). GM-CSF is a key cytokine in acute inflammation, but it is also involved in the development of respiratory allergic disease because it is a chemotactic factor for eosinophils and also stimulates Langerhans cell activation (33), which, in turn, could stimulate T lymphocytes (Th2). On the other hand, IL-8 is a chemotactic factor for neutrophils, which are the predominant cells in chronic bronchitis, but it is also expressed by epithelial cells in bronchial asthma (19).

This inflammatory response seems mainly due to the adsorbed organic compounds because we have shown that carbon black only induced a slight increase in GM-CSF and IL-8 release from 16HBE14o- cells compared with that of DEPs at the same concentration. Moreover, the use of an oxidation catalyst that reduced the PAH content by ~60% abolished the effects of the generated DEPs on GM-CSF secretion. To our knowledge, this is the first report of the efficiency of exhaust posttreatments on the biological response to DEPs. This observation strengthens our hypothesis that the carbonaceous core plays a minor role in this enhanced cytokine secretion. This is in agreement with effects observed after an in vivo exposure of mice showed that carbon black is a less potent adjuvant than DEPs for IgE production in response to ovalbumin (23). Our observations also confirm other in vitro studies showing that incubation of bronchial epithelial cells with DEPs but not with activated charcoal (4, 25), graphite (25), or TiO2 (29) increases the release of cytokines.

To our knowledge, this study also shows for the first time enhanced IL-1beta secretion by HNE cells after DEP exposure, whereas no secretion was observed for the 16HBE14o- cell line. In this regard, it is interesting to note that IL-1beta upregulates GM-CSF synthesis and release (24) and IL-8 production in epithelial cells (4) as well as proliferation of Th2 cells (16), which are involved in allergic reactivity.

In conclusion, the present study shows that human respiratory epithelial cells phagocytose different types of particles and develop a specific inflammatory response in terms of cytokine release after an in vitro exposure to DEPs. Regarding these results, it is of particular interest to note that exhaust posttreatments could diminish or even abolish the increase in GM-CSF release observed after DEP treatments.


    ACKNOWLEDGEMENTS

We acknowledge Dr. D. C. Gruenert for the human bronchial cell line. We gratefully thank Christiane Guennou for excellent technical help in cell culture, Mireille Legrand for cell culture and photographic work, Anne-Catherine Dazy for measurements of the transepithelial potential, and Thomas Boland for reviewing manuscript.


    FOOTNOTES

This work was supported by DIMAT Renault Grant 235, Ademe Grant BOU 9536, a grant from Caisse d'Assurance Maladie des Professions Libérales Provinces, and European Commercial Community Grant BIO4-CT960052.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Boland, Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris VII Denis Diderot, Tour 53/54 E3, case 70-73, 2 place Jussieu, 75251 Paris cédex 05, France (E-mail: marano{at}paris7.jussieu.fr).

Received 28 October 1998; accepted in final form 20 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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