Comparison of the Pro-Oxidative and Proinflammatory Effects
of Organic Diesel Exhaust Particle Chemicals in Bronchial
Epithelial Cells and Macrophages1
Ning Li,* Meiying Wang,* Terry D. Oberley,†Joan M. Sempf,‡and Andre E. Nel2*
Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle
content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that
organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the
expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of
cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun
N-terminal kinase activation and induction of apoptosis-necrosis require incremental amounts of the organic chemicals and
increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced
more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mito-
chondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial mem-
brane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected
by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings
show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid
transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N-acetylcysteine to cyto-
protective glutathione. The Journal of Immunology, 2002, 169: 4531–4541.
effects include an exacerbation of asthma and allergic inflamma-
tion (1–5). While there has been considerable debate about the
contribution of particles vs chemical components (e.g., nitrates,
sulfates, transition metals, and organic chemicals), our studies, us-
ing diesel exhaust particles (DEP) as a model air pollutant, have
shown that organic chemical compounds play an important role in
the pro-oxidative and proinflammatory effects of these particles in
the respiratory tract (6–9). DEP have a mass medium diameter of
0.05–1 ?m (mean, 0.2 ?m), a size that renders them easily respi-
rable and capable of depositing in the airways and alveoli. DEP
pidemiological studies have demonstrated an association
between exposure to ambient particulate matter (PM)3
and adverse cardiorespiratory effects (1–5). These adverse
consist of a carbonaceous core with a large surface area to which
chemicals are absorbed. These include organic chemicals such as
polycyclic aromatic hydrocarbons (PAH), nitro derivatives of
PAH, oxygenated derivatives of PAH (ketones, quinones, and dio-
nes), heterocyclic compounds, aldehydes, and aliphatic hydrocar-
bons (10–14). Our interest lies with the PAH and their oxygenated
derivatives (e.g., quinones), which are able to redox cycle and
generate reactive oxygen species (ROS) in target cell populations
such as macrophages (8, 14–19). The pro-oxidative effects of in-
tact DEP or crude DEP extracts can be reproduced with fraction-
ated aromatic and polar chemical groups, which are enriched for
PAH and quinones, respectively (11–14, 20). Similarly, intact DEP
or organic DEP extracts induce pro-oxidative and proinflammatory
effects in the respiratory tract, which can be negated by thiol an-
tioxidants (10, 16).
Macrophages constitute an important target for DEP in the lung
(17, 18, 21–23). After phagocytosis of these particles, macro-
phages respond in a hierarchical fashion to increasing particle load
and incremental levels of oxidative stress (24). Thus, at low oxi-
dative stress levels, as defined by no or minimal change in the
cellular reduced glutathione (GSH)/glutathione disulfide (GSSG)
ratios, these cells mount antioxidant and cytoprotective responses,
e.g., heme oxygenase 1 (HO-1) and superoxide dismutase expres-
sion (24). HO-1 expression is dependent on the function of anti-
oxidant response element (ARE) in its promoter (11) and is typi-
cally induced by 1–10 ?g/ml of the DEP extract (11, 24). In
contrast, extract doses of 10–50 ?g/ml are required to activate
intracellular pathways, such as the c-Jun N-terminal kinase (JNK)
and NF-?B cascades, which are responsible for proinflammatory
effects (24). Activation of these cascades may constitute the prin-
cipal mechanism by which DEP exert adjuvant effects in the lung
(7, 8, 24). At even higher oxidative stress levels, which coincide
with extract doses of ?50 ?g/ml, macrophages undergo apoptosis
and necrosis (17, 18, 24).
*Division of Clinical Immunology and Allergy, Department of Medicine, University
of California, Los Angeles, CA 90095;†Department of Pathology and Laboratory
Medicine, University of Wisconsin, Madison, WI 53706; and‡Pathology Service,
Veterans Affairs Medical Center, Madison, WI 53705
Received for publication May 15, 2002. Accepted for publication August 2, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by U.S. Public Health Service Grants AI50495 and
2Address correspondence and reprint requests to Dr. Andre E. Nel, Division of Clin-
ical Immunology and Allergy, Department of Medicine, University of California,
10833 Le Conte Avenue, 52-175 CHS, Los Angeles, CA 90095. E-mail address:
3Abbreviations used in this paper: PM, particulate matter; ARE, antioxidant response
element; BEGM, Bronchial epithelial growth medium; CoPP, cobalt protoporphyrin;
DCF, dihydrochlorofluorescein diacetate; DEP, diesel exhaust particles; DiOC6, 3,3?-
dihexyloxacarbocyanine iodide; ??m, mitochondrial membrane potential; GSH, re-
duced glutathione; GSSG, glutathione disulfide; HE, hydroethidine; HO-1, heme ox-
ygenase-1; JNK, c-Jun N-terminal kinase; LL, lower left; MnSOD, manganese
superoxide dismutase; NAC, N-acetylcysteine; NHBE, normal human bronchial ep-
ithelial cells; O2., superoxide radical; PAM, pulmonary alveolar macrophages; PI,
propidium iodide; phospho-JNK, phosphorylated JNK; PT, permeability transition;
ROS, reactive oxygen species; UL, upper left; UR, upper right.
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc.0022-1767/02/$02.00
Bronchial epithelial cells are another primary cell target for PM
(25–28). Not only do these cells play an important role in allergic
inflammation, but shedding and dysregulation of bronchial epithe-
lial repair contribute to airway hyper-reactivity in atopic asthmat-
ics (29). Several studies have demonstrated that DEP elicit bio-
logical responses in bronchial epithelial cells (25–28). These
effects include the release of proinflammatory mediators as well as
the induction of mucoid hyperplasia (30–35). However, since
these cells are not phagocytic and differ in many other respects
from macrophages, the mechanism of PM action in epithelial cells
is unknown. We do know that bronchial epithelial cells endocytose
DEP and are able to mount biological responses to oxidative stress
(33). However, the extent to which the oxidative stress response
differs in epithelial cells and macrophages is unknown. This is a
key area to explore, since rational therapy for the adverse health
effects of PM should consider effective ways to curb the conse-
quences of oxidative stress in the lung.
The aim of this study was to investigate the sensitivity of human
bronchial epithelial cells to organic DEP chemicals and to deter-
mine whether there is a link between the level of oxidative stress
and the cellular response. To perform these studies we compared
normal human bronchial epithelial cells as well as a bronchial
epithelial cell line, BEAS-2B, to macrophages. Our data demon-
strate that while organic DEP extracts generate oxidative stress in
epithelial cells, these cells differ from macrophages in the types of
ROS being produced and the sensitivity to a programmed cell
death pathway. Similar to THP-1 cells, there was good correlation
between the extract dose, the drop in cellular GSH/GSSG ratios,
and ensuing cellular responses. Unlike macrophages, N-acetylcys-
teine (NAC) was ineffective in protecting bronchial epithelial cells
from cytotoxic death. These results suggest similarities as well as
key differences between macrophages and epithelial cells in their
responses to redox cycling DEP chemicals.
Materials and Methods
RPMI 1640, DMEM, F12K Nutrient Mix (F12K), penicillin-streptomycin,
and L-glutamine were obtained from Life Technologies (Gaithersburg,
MD). Bronchial epithelial growth medium (BEGM) was purchased from
Clonetics (Walkersville, MD). FBS was purchased from Irvine Scientific
(Santa Ana, CA). Type I rat tail collagen was purchased from Collaborative
Research (Bedford, MA). DEP were a gift from Dr. M. Sagai (National
Institute of Environment Studies, Tsukuba, Japan). Anti-HO-1 mAb was
purchased from Stressgen (Victoria, Canada). Anti-manganese superoxide
dismutase (anti-MnSOD) Ab was obtained from Upstate Biotechnology
(Lake Placid, NY). Monoclonal anti-phospho-JNK and polyclonal anti-
JNK Abs were from Cell Signaling (Beverly, MA). Biotinylated rabbit
anti-mouse and swine anti-rabbit Abs were obtained from Dako (Carpin-
teria, CA). HRP-conjugated sheep anti-mouse Ab was obtained from Am-
ersham (Piscataway, NJ). Hydroethidine (HE), dihydrochlorofluorescein
diacetate (DCF), 3,3?-dihexyloxacarbocyanine iodide (DiOC6), and the
ATP assay kit were purchased from Molecular Probes (Eugene, OR). NAC,
propidium iodide (PI), GSH, GSSG, ?-NADPH, and glutathione reductase
were obtained from Sigma (St. Louis, MO). Cobalt protoporphyrin (CoPP)
was purchased from Porphyrin Products (Logan, UT). Annexin-FITC kit
was purchased from Trevigen (Gaithersburg, MD). ECL reagents were
purchased from Pierce (Rockford, IL).
Human bronchial epithelial cells (BEAS-2B) and the human (THP-1) and
murine (RAW 264.7) macrophage cell lines were obtained from American
Type Culture Collection (Manassas, VA). Normal human bronchial epi-
thelial cells (NHBE) were purchased from Clonetics (Walkersville, MD).
Human pulmonary alveolar macrophages (PAM) were provided by Dr. J.
Balmes (University of California, San Francisco, CA). THP-1 and PAM
were cultured in RPMI 1640 supplemented with 10% FBS, penicillin/strep-
tomycin, and glutamine. NHBE were cultured in BEGM. BEAS-2B cells
were cultured in BEGM in type I rat tail collagen-coated flasks or plates.
RAW264.7 were grown in DMEM plus 10% FBS. All cell cultures were
conducted in a 37°C humidified incubator supplied with 5% CO2.
Preparation of DEP methanol extracts and cell stimulation
DEP methanol extracts were prepared as previously described (17). Briefly,
100 mg DEP were suspended in 25 ml methanol and sonicated for 2 min.
The DEP methanol suspension was centrifuged at 2000 rpm for 10 min at
4°C. The methanol supernatant was transferred to a preweighed polypro-
pylene tube and dried under nitrogen gas. The tube was reweighed to de-
termine the amount of methanol-extractable DEP components. Dried DEP
extract was then dissolved in DMSO at a concentration of 100 ?g/?l. The
aliquots were stored at ?80°C in the dark until use.
Preparation of DEP fractions
Preparation of DEP fractions was conducted as previously described (11).
Briefly, 1 g DEP was extracted with 60 ml methylene chloride five times
using a VirTis homogenizer (Gardiner, NY). The combined extracts were
concentrated by rotoevaporation, and asphaltenes were precipitated by ex-
changing into hexane. The supernatant was concentrated, dried over anhy-
drous sodium sulfate, and subjected to silica gel column chromatography
(column size, 1 ? 30 cm) following the method of Venkatessan et al. (11).
Aliphatic, aromatic, and polar fractions were collected by elution with 20
ml hexane, 40 ml hexane/methylene chloride (3/2), and 30 ml methylene
chloride/methanol (1/1), respectively. The fractions were weighed in a mi-
crobalance by evaporating off a known volume of an aliquot of the sample
made up in methylene chloride or methanol. The fractions were dried with
N2gas and redissolved in DMSO.
Western blotting analysis
Western blotting was conducted as previously described (17). One hundred
to 150 ?g total protein was separated by SDS-PAGE before transfer to
polyvinylidene difluoride membranes. HO-1 protein was detected by anti-
HO-1 mAb at 0.3 ?g/ml and rabbit anti-mouse Ab conjugated to HRP
according to the manufacturer’s instructions. Anti-MnSOD Ab was used at
0.3 ?g/ml. Biotinylated swine anti-rabbit Ab (1/1,000) was used as the
secondary Ab, followed by HRP-conjugated avidin-biotin complex
(1/10,000). Blots were developed with the ECL reagents according to the
manufacturer’s instruction. Phospho-JNK and JNK proteins were detected
using monoclonal anti-phospho-JNK (1/1,000) and polyclonal anti-JNK
(1:1,000) Abs. Biotinylated rabbit anti-mouse (1/1,000) and swine anti-
rabbit (1/1,000) Abs were used as secondary Abs before HRP-conjugated
avidin-biotin complex (1/10,000).
Total RNA was extracted using TRIzol RNA extraction reagent (11). RT
was performed at 42°C in a total volume of 20 ?l containing 5 ?g total
RNA; 0.5 ?g oligo(dT)12–18; 10 mM DTT; 0.5 mM each of dATP, dGTP,
dCTP, and dTTP; and 10 U Moloney murine leukemia virus reverse tran-
scriptase (15). HO-1 primers for PCR amplification of a 350-bp human
HO-1 fragment (36) were obtained from Life Technologies. The primer
sequences of human HO-1 are 5?-CAGGCAGAGAATGCTGAGTT-3?
and 5?-GCTTCACATAGCGCTGCA-3?. The sequences of human ?-actin
primers are 5?-TGGAATCCTGTGGCATCCATGAAAC-3? and 5?-TAA
AACGCAGCTCAGTAACAGTCCG-3?. PCRs for both HO-1 and ?-actin
were performed in a total reaction volume of 25 ?l containing 4 ?l cDNA
template, 0.5 ?M sense and antisense primers, 1.5 mM MgCl2, 0.2 mM
dNTP, and 2.5 U Taq DNA polymerase in a PerkinElmer thermal cycler
(Norwalk, CT). Samples were heated to 95°C for 2 min and subjected to 35
cycles of amplification (30 s at 94°C, 60 s at 58°C, and 60 s at 72°C),
followed by 7 min at 72°C for final extension. PCR products were elec-
trophoresed in 2% agarose gels and viewed by ethidium bromide.
Analysis of IL-8 production
After DEP stimulation, the culture media were collected and centrifuged to
remove the debris. The media were frozen and sent to Cytokine Core Lab-
oratories (Baltimore, MD) for measurement of the IL-8 levels by ELISA.
ROS generation, mitochondrial membrane potential (??m), and apoptosis
were analyzed by flow cytometry using a FACScan equipped with an argon
laser (BD Biosciences, Franklin Lakes, NJ) (17, 18). Superoxide radical
DiOC6. Cells (106/ml) were incubated with 2 ?M HE as well as 20 nM
DiOC6diluted in the serum-free culture medium for 30 min in the dark at
??) production and ??m were determined by dual staining with HE and
4532 DEP EFFECTS ON BRONCHIAL EPITHELIAL CELLS AND MACROPHAGES
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4541 The Journal of Immunology