1 Laboratoire de
Cytophysiologie et Toxicologie Cellulaire and
3 Laboratoire de Cytométrie, 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
16HBE14o 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 Culture Conditions
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) 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-1
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.
cells; human nasal turbinates; air pollution; carbon black
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) 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
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, 16HBE14oCytokine 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 atDNA 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 |
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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.
|
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.
|
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.
|
Toxic Effects of DEPs
The release of cellular LDH and trypan blue uptake by 16HBE14o
|
TEM
To investigate the reasons for this cellular damage, we performed ultrastructural studies with TEM. The 16HBE14oQuantification 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
|
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).
|
|
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).
|
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-116HBE14o 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-1
, 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).
|
|
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-1
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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DISCUSSION |
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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 16HBE14oPhagocytosis 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 16HBE14oWe 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-1 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-1
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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