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
37°C. Apoptosis was analyzed by annexin V-FITC/PI double staining ac-
cording to the manufacturer’s instructions. Time- and dose-dependent cel-
lular cytotoxicity were determined by staining the cells in 1 ?g/ml PI.
DiOC6and annexin V-FITC fluorescence were analyzed in the fluores-
cent-1 channel, while PI and HE fluorescence were analyzed in FL-2 and
-3 channels, respectively.
Determination of GSH/GSSG ratio
Total glutathione (GSH plus 1/2 GSSG) and GSSG were measured in a
recycling assay that uses 5,5?-dithio-bis(2-nitrobenzoic acid) and glutathi-
one reductase (37–39). Briefly, cells were lysed and deproteinized in 3%
5-sulfosalicylic acid. Whole cell lysates were cleared by centrifugation at
4°C at 14,000 rpm in an Eppendorf centrifuge. The supernatant was used
for the measurement of total and oxidized glutathione. The amount of total
glutathione from each sample was calculated from a GSH standard curve
prepared in 5-sulfosalicylic acid. For GSSG assay, 100 ?l supernatant was
incubated with 2 ?l 2-vinylpyridine and 6 ?l triethanolamine for 60 min on
ice. GSSG standards were treated in the same way as samples. The amount
of GSSG in the samples was calculated from the GSSG standard curve. The
amount of reduced GSH was calculated by subtracting the amount of
GSSG from that of total glutathione.
Procedures for routine electron microscopy have been previously described
by Yang et al. in detail (40). Briefly, cells were fixed in glutaraldehyde and
postfixed in osmium tetroxide. The cells were then dehydrated in a series
of ethanol and embedded in Epon-Spurr. Thin sections for electron mi-
croscopy were cut with a Reichert-Jung Ultracut and Ultramicrotome (Vi-
enna, Austria). Copper grids were stained with lead citrate and uranyl ac-
etate and photographed in a Hitachi (Tokyo, Japan) electron microscope.
Measurement of cellular ATP levels
Cellular ATP levels were measured as previously described (12). Briefly,
cells were harvested by scraping and were lysed in H2O. The cell lysates
were boiled for 5 min, and the ATP concentration was determined using a
luciferase assay kit according to the manufacturer’s instruction.
Data were analyzed using SAS statistical software (SAS Institute, Cary,
NC). Scheffe’s method of multiple comparisons with F test was used for
Organic DEP extracts induce ROS production and oxidative
stress effects in bronchial epithelial cells
Using macrophages as a target cell for DEP, we have previously
demonstrated that these particles elicit biological effects that can
be ascribed to their organic carbon content (7–9, 11–14, 20). More-
over, treatment of PAM or THP-1 cells with a methanol DEP
extract mimics the effect of intact particles, including their ability
to generate ROS. This effect can be demonstrated by DCF fluo-
rescence, which reflects mostly H2O2production (17, 18). While
treatment of the bronchial epithelial cell line BEAS-2B with the
same type of extract failed to induce DCF fluorescence (data not
shown), these cells demonstrated increased HE fluorescence (Fig.
1A). HE is oxidized to ethidium bromide by ROS and is mostly
likely a reflection of O2.production (18). In contrast to HE con-
version in BEAS-2B cells, THP-1 cells did not exhibit an appre-
ciable increase in HE fluorescence (Fig. 1A).
Cells use intracellular GSH to neutralize ROS and protect them-
selves against oxidative damage. In case of a vigorous antioxidant
defense, intracellular GSH stores may become depleted, leading to
a drop in GSH/GSSG ratios. When exposed to increasing amounts
of a methanol DEP extract, both THP-1 and BEAS-2B cells ex-
hibited a dose-dependent decrease (p ? 0.005) in GSH/GSSG
ratios (Fig. 1B). Similar changes in intracellular glutathione ratios
the effect of the antioxidant, NAC. A, Time-dependent increase in HE
fluorescence. BEAS-2B and THP-1 cells were treated with 100 ?g/ml DEP
extract for the indicated period. O2.levels were analyzed by flow cytometry
using HE staining as described in Materials and Methods. B, Dose-depen-
dent decrease in the GSH/GSSG ratio induced by DEP extract. BEAS-2B
and THP-1 cells were exposed to the indicated concentrations of DEP
extract for 5 h. Determination of total and oxidized glutathione and GSH/
GSSG ratios was performed as described in Materials and Methods. Values
represent the mean ? SEM. p ? 0.005 at extract doses ?10 ?M. C, The
effects of NAC on cellular GSH/GSSG ratios. Cells were treated with 50
?g/ml DEP extract for 5 h in the absence or the presence of 20 mM NAC.
a, p ? 0.005; b, p ? 0.007 (compared with the controls). c, p ? 0.0002
(compared with 50 ?g/ml DEP).
Alteration of cellular redox by an organic DEP extract and
4533 The Journal of Immunology
occurred in NHBE during exposure to the DEP extract (Fig. 1C).
Interestingly, THP-1 cells maintain a higher basal GSH/GSSG ra-
tio than BEAS-2B or NHBE cells (Fig. 1, B and C).
We have previously shown that thiol antioxidants are effective
in preventing the oxidative stress effects of DEP chemicals (16).
While NAC could increase basal GSH/GSSG ratios in THP-1
and MnSOD expression in BEAS-2B and THP-1 cells. Cells were stimulated with different concentrations of DEP extract as indicated for 5 h. Immuno-
blotting and RT-PCR were performed as described in Materials and Methods. ?-Actin was used as an internal control. B, Western blotting showing the
effects of DEP extract on HO-1 and MnSOD expression in NHBE cells. Cells were treated with the indicated concentrations of DEP extract for 5 h before
being collected for SDS-PAGE and immunoblotting. C, The effect of NAC on HO-1 expression was demonstrated by immunoblotting. BEAS-2B and
THP-1 cells were incubated with 20 mM NAC for 1 h before addition of 50 ?g/ml DEP extract for 5 h. D, The effects of aliphatic, aromatic, and polar
chemical fractions on HO-1 expression. An organic DEP extract was fractionated by silica gel chromatography as previously described (15). The presence
of alkenes, PAHs, and quinones in these respective fractions were confirmed by chemical analysis as previously described (15). The indicated amounts of
the crude methanol extract as well as each fraction were incubated with THP-1 and BEAS-2B cells for 5 h before Western blotting. The small band in the
aliphatic (5 ?g/ml) lane probably represents spillage from the 25 ?g/ml DEP sample. E, The effect of CoPP on DEP-induced cell death in BEAS-2B, THP-1,
and RAW264.7 cells. Following preincubation with 5 ?g/ml CoPP for 48 h the cells were incubated with DEP extract (100 ?g/ml) in the continuous
presence of CoPP for 16 h. Induction of HO-1 was determined by Western blotting (inset). Cell viability was analyzed by flow cytometry using PI staining.
Values are the mean ? SEM. a, p ? 0.0001; b, p ? 0.0022 (compared with the controls). c, p ? 0.0001 (compared with 100 ?g/ml DEP).
The effects of DEP extracts on cytoprotective pathways. A, Immunoblotting and RT-PCR demonstrating the effects of a DEP extract on HO-1
4534DEP EFFECTS ON BRONCHIAL EPITHELIAL CELLS AND MACROPHAGES
cells, this thiol antioxidant did not affect basal glutathione levels in
BEAS-2B or NHBE cells (Fig. 1C). In addition, while NAC pre-
vented a decline in GSH/GSSG ratios in THP-1 cells during ex-
posure to a DEP extract, this agent did not prevent a drop in glu-
tathione ratios in BEAS-2B and NHBE cells (Fig. 1C). This
suggests that there is no NAC conversion to glutathione in epithe-
A sustained drop in cellular GSH/GSSG ratios is indicative of a
pro-oxidant state and leads to protective cellular responses. Exam-
ples include the inducible expression of MnSOD and HO-1 (24).
While THP-1 and BEAS-2B cells showed constitutive MnSOD
expression, the DEP extract induced HO-1 protein and mRNA
expression in a dose-dependent fashion (Fig. 2A). In contrast, there
was no change in the expression of a household gene, ?-actin (Fig.
2A). Similarly, NHBE cells showed an increase in HO-1 expres-
sion, while MnSOD was constitutively expressed (Fig. 2B). The
role of oxidative stress in HO-1 expression was confirmed by the
ability of NAC to interfere with this response in BEAS-2B cells
and macrophages (Fig. 2C). This suggests that although NAC is
not converted to GSH in epithelial cells, this agent can function as
a radical scavenger.
We have previously shown that the effect of the crude DEP
extract on HO-1 expression can be mimicked by aromatic and
polar chemical groups fractionated from these particles (11). We
have also demonstrated that the aromatic fraction is enriched for
PAHs, while the polar fraction includes quinones, but no PAHs
(11). Using these materials, we showed dose-dependant HO-1 ex-
pression by polar and aromatic chemical groups in THP-1 cells
(Fig. 2D). The polar was more potent than the aromatic fraction,
while an aliphatic fraction lacked activity (Fig. 2D). The same
trend was seen in BEAS-2B cells, except that the potency of the
polar material was of sufficient magnitude to affect cell viability
and HO-1 expression at doses ?5 ?g/ml (Fig. 2D). These results
are in accordance with the ability of quinones to directly engage in
redox cycling, while PAH require enzymatic conversion before
being able to exert this effect (7).
We have previously demonstrated that CoPP-treated RAW264.7
cells are partially protected against the cytotoxic effects of redox
cycling DEP chemicals (11) (Fig. 2E). CoPP is a non-heme HO-1
inducer (11). Interestingly, an attempt to induce HO-1 expression
with CoPP in THP-1 cells failed, and these cells were not protected
against DEP cytotoxicity (Fig. 2E). While CoPP was an effective
HO-1 inducer in BEAS-2B cells, it did not protect those cells
against the effect of oxidizing DEP chemicals (Fig. 2E). This sug-
gests that despite its cytoprotective and antioxidant function, HO-1
is not sufficient to protect epithelial cells against the injurious ef-
fects of redox cycling DEP chemicals. The higher susceptibility of
epithelial cells to cytotoxic DEP effects is discussed below.
Taken together, the above results demonstrate that organic DEP
chemicals induce oxidative stress in bronchial epithelial cells. This
leads to increased HO-1 expression, which commences at rela-
tively low extract amounts (?10 ?g/ml), and escalates as the level
of oxidative stress increases. While this response mimics HO-1
expression in THP-1 cells, there is a difference in the kinetics and
magnitude of O2.production in these cells as determined by HE
Organic DEP extracts induce JNK activation in bronchial
In addition to initiating antioxidant and cytoprotective responses,
oxidative stress can activate intracellular signaling cascades, in-
cluding the mitogen-activated protein kinase and NF-?B cascades.
Treatment of THP-1 and BEAS-2B cells with an organic DEP
extract led to JNK activation (Fig. 3A). Thus, increased phosphor-
ylation of the 45- and 54-kDa JNK isoforms could be seen at
extract doses ?25 ?g/ml; a high rate of cell death diminished the
BEAS-2B response at 100 ?g/ml (Fig. 3A). NHBE also showed
increased JNK phosphorylation in the dose range of 50–100 ?g/ml
(Fig. 3A). These effects were not due to a decrease in the abun-
dance of JNK protein, as demonstrated by anti-JNK immunoblot-
ting (Fig. 3A, lower panel). The importance of oxidative stress in
JNK activation was demonstrated by interference in p45 and p54
phosphorylation when assays were conducted in the presence of
NAC (Fig. 3B). This again suggests that although not converted to
glutathione, NAC functions as a radical scavenger in epithelial
Taken together, the results in Fig. 3 demonstrate that at extract
doses higher than that required to initiate HO-1 expression, organic
DEP chemicals activate the JNK cascade in epithelial cells and
macrophages. Since higher extract doses are associated with lower
GSH/GSSG ratios (Fig. 1B), this suggests response stratification.
Organic DEP extracts induce IL-8 production in bronchial
A consequence of the activation of intracellular signaling cascades
is the transcriptional activation of proinflammatory genes, includ-
ing genes that encode for cytokines, chemokines, and adhesion
receptors. One example is the IL-8 gene, which is under dual reg-
ulation by NF-?B and AP-1 response elements in its proximal
promoter (41, 42). IL-8 is particularly relevant to the proinflam-
matory effects of DEP in the lung (22, 26–28). To compare IL-8
induction in epithelial and THP-1 cells, cultures were treated with
10–100 ?g/ml of the DEP extract for 14 h before measuring IL-8
in the culture medium. While THP-1 cells showed a dose-depen-
dent response over the entire dose range (10–100 ?g/ml), NHBE
cells showed an incremental response in the range 10–50 ?g/ml,
Dose-dependent activation of JNK by DEP extract. BEAS-2B, NHBE, and
THP-1 cells were stimulated with the indicated concentrations of DEP
extract for 2.5 h. Western blotting for phospho-JNK and JNK was con-
ducted as described in Material and Methods. B, Inhibition of JNK acti-
vation by antioxidant, NAC. Cells were exposed to 50 ?g/ml DEP extract
in the absence and the presence of 20 mM NAC. Immunoblotting was
performed as described in A.
JNK activation by DEP extract and prevention by NAC. A,
4535 The Journal of Immunology
followed by a rapid decline at 100 ?g/ml (Fig. 4). This is probably
due to a high rate of apoptosis in NHBE at doses ?25 ?g/ml (see
below). While BEAS-2B responded to 10 ?g/ml of the extract,
cellular toxicity led to a sharp drop in IL-8 production at higher
doses (Fig. 4). These data strengthen the idea that incremental
levels of oxidative stress lead to a transition from cytoprotective to
injurious cellular responses.
Organic DEP extracts induce cellular apoptosis and necrosis in
epithelial cells by perturbation of mitochondrial function
Previous studies from our laboratory demonstrated that intact DEP
as well as organic extracts made from these particles induce a
cytotoxic response in PAM and macrophage cell lines (17, 18).
Compared with increased cytotoxicity at extract doses ?25 ?g/ml
in THP-1 cells, BEAS-2B cells showed a significant rise in the rate
of cell death at doses ?10 ?g/ml (Fig. 5A). Moreover, cell death
commenced ?2 h in BEAS-2B cells, while increased PI uptake in
THP-1 cells was delayed for at least 8 h or longer (Fig. 5B). NHBE
cells also showed an enhanced rate of cytotoxicity compared with
macrophages (not shown).
We have previously demonstrated that DEP cytotoxic effects in
macrophages involve programmed cell death, dependent on the
ability of these particles and their organic chemicals to generate
oxidative stress (17, 18). This death event is characterized by the
appearance of apoptotic bodies in epithelial cells and macrophages
as well as positive annexin V/PI staining during two-color flow
cytometry (Table I and Fig. 6). The annexin V?/PI?population
was especially prominent in the BEAS-2B and NHBE populations,
while THP-1 and PAM stained mostly PI positively, but annexin V
negatively (Table I). While the exact reason for this difference in
membrane asymmetry is unclear, it is possible that macrophages
more rapidly degrade annexin V on the cell surface. We have
previously shown that the percentage of annexin V?/PI?macro-
phages is more prominent at DEP extract doses ?100 ?g/ml (18).
Treatment of human PAM and THP-1 cells with NAC interfered
significantly in the generation of cytotoxicity (Table I). In contrast,
NAC did not appreciably decrease the number of dead cells in the
BEAS-2B and NHBE populations (Table I). Although this may
suggest that epithelial toxicity is not dependent on oxidative stress,
it should be noted that NAC is ineffective in preventing glutathione
depletion in epithelial cells (Fig. 1C). There is good evidence link-
ing GSH depletion to the induction of apoptosis via a mitochon-
drial effect (17, 18, 43). Indeed, ultramicroscopic visualization of
THP-1 and BEAS-2B cells showed that the appearance of apopto-
tic bodies is accompanied by changes in the mitochondrial mor-
phology (Fig. 6, A and B). These changes, which include mito-
chondrial swelling as well as a loss of cristae, are indicative of
apoptosis-necrosis and may represent a change in mitochondrial
function (Fig. 6). Similar changes were seen in NHBE cells (not
To study changes in mitochondrial function, we used dual-color
DiOC6and HE fluorescence (Fig. 7). The resulting flow diagram
shows that the DEP extract induced a decrease in the mitochon-
drial membrane potential (DiOC6fluorescence, lower left (LL) and
upper left (UL) quadrants) as well as an increase in O2.generation
(HE fluorescence, UL and upper right (UR) quadrants) in
BEAS-2B and THP-1 cells (Fig. 7). More specifically, the number
of HEbrightcells (UR quadrant) in the BEAS-2B population in-
creased from 1.4 to 74.9% within 2 h of introducing the DEP
extract (Fig. 7A). At this point, there was no drop in ??m, but the
NHBE, and THP-1 cells were stimulated with the indicated concentrations
of DEP extract for 14 h before culture media were collected. The IL-8
concentration in the medium was determined by ELISA as described in
Materials and Methods. Values are the mean ? SEM. Values of p ?
0.0001 compared with the controls. Note that for this time duration, there
was a high rate of cell death in BEAS-2B cells at doses ?10 ?g/ml and in
NHBE cells at doses ?25 ?g/ml.
Increased IL-8 production by DEP extract. BEAS-2B,
The percentage of dead cells was determined by flow cytometry, using PI
staining. Values represent the mean ? SEM. A, Dose-dependent increases
in cell death. Cells were treated with different concentrations of DEP ex-
tract for 16 h. B, Time course of DEP toxicity. BEAS-2B and THP-1 cells
were exposed to 100 ?g/ml DEP extract for the indicated time before
staining for flow cytometry.
Cytotoxic effects of DEP extract on BEAS-2B and THP-1.
4536 DEP EFFECTS ON BRONCHIAL EPITHELIAL CELLS AND MACROPHAGES
number of DiOC6
rants) by 7 h (Fig. 7A). Although the extract-induced increase in
HE fluorescence was not as pronounced in NHBE, the drop in the
??m was more pronounced than that in BEAS-2B cells (Fig. 7B,
LL and UL quadrants). In contrast to epithelial cells, the number of
THP-1 cells showing an increase in O2.production after 2 and 7 h
was limited to 20 and 10% (UR quadrant), respectively (Fig. 7A).
These results agree with the data in Fig. 1A, which show higher HE
fluorescence in BEAS-2B compared with THP-1 cells. Moreover,
the drop in ??m cells was limited to ?20% (LL and UL quad-
rants) in THP-1 cells (Fig. 7A).
Apoptosis-necrosis is a process in which the morphological fea-
tures of apoptosis (e.g., the presence of apoptotic bodies) is com-
bined with the features of cellular necrosis (mitochondrial disin-
tegration and cellular fractionation). A key feature of apoptosis-
necrosis is the inability to sustain ATP production due to damage
to the mitochondrial inner membrane. Direct measurement of cel-
lular ATP levels demonstrated a sharp and precipitous (p ?
0.0001) drop in ATP levels in BEAS-2B cells (Fig. 8). Similarly,
ATP levels in NHBE cells decreased by ?80% within 2 h of stimu-
lation (p ? 0.0001) and remained low thereafter (Fig. 8). In contrast,
the rate of ATP decline in THP-1 cells was slower and only reached
the epithelial low point by 8 h (Fig. 8). The significance of apoptosis-
necrosis is that residual cellular fragments are proinflammatory, while
apoptotic bodies are removed in a “silent” manner.
Taken together, the data in Figs. 4–8 indicate that at relatively
high concentrations of DEP extract, there is a transition from cy-
toprotective to cytotoxic effects. This idea is compatible with a
stratified oxidative stress model in which antioxidant responses
transition to proinflammatory and cytotoxic effects as the level of
oxidative stress increases.
lowcells increased to 40.2% (LL and UL quad-
We demonstrate that organic DEP extracts, including polar and
aromatic fractions, induce oxidative stress in epithelial cells, in
response to which these cells exhibit HO-1 expression, JNK acti-
vation, IL-8 production and induction of apoptosis-necrosis. While
THP-1 cells responded in similar fashion, epithelial cells produced
more O2.and were more susceptible to cytotoxic effects than mac-
rophages. Cytotoxicity is the result of mitochondrial damage,
which manifests as a decrease in the ??m, ROS production, and
ATP depletion. Another key difference between epithelial cells and
macrophages is the ability of NAC to elevate GSH/GSSG ratios
and prevent cytotoxicity in macrophages, while failing to do so in
epithelial cells. Since treatment with NAC interfered with JNK
activation and HO-1 expression in epithelial cells, this suggests
that despite acting as a radical scavenger, this thiol agent is not
converted to glutathione in epithelial cells. Induction of HO-1 ex-
pression at a low extract dose coincides with a minimal change in
the GSH/GSSG ratio, while IL-8 production and JNK activity
commences at 10–50 ?g/ml, a dose leading to a more drastic de-
cline in GSH/GSSG levels. While induction of cellular toxicity in
THP-1 cells required an extract dose ?25 ?g/ml, the onset of cell
death in BEAS-2B cells was more linear at doses ?10 ?g/ml.
The data in this study are of considerable importance to the
priorities for airborne particulate matter as formulated by an expert
committee of the National Academy of Sciences (6). Among the
committee’s top 10 priorities, particular emphasis is given to the
elucidation of molecular mechanisms by which ambient airborne
PM cause adverse health effects (6). We are particularly interested
in the role of organic chemical compounds and have selected DEP
as a model air pollutant to clarify some of these mechanistic issues
(8). The idea that PAH and their oxygenated derivatives on DEP
(e.g., quinones) participate in redox cycling and ROS generation
was confirmed by the data in Fig. 2D showing that aromatic and
polar compounds fractionated from DEP by silica gel chromatog-
raphy induce HO-1 expression (11). These fractions are enriched
for PAH and quinones, respectively. It is important to clarify that
our data do not exclude the contribution of transition metals and
other PM components in the biological effects of PM.
GSH and GSSG are the major redox pair involved in cellular
redox homeostasis. A decline in the cellular GSH/GSSG ratio is
regarded as a representative marker for oxidative stress and is di-
rectly responsible for the perturbation of cellular function (7, 44–
Table I. The effects of NAC (20 mM) on DEP-induced apoptosis in BEAS-2B, NHBE, PAM, and THP-1a
Cell Type Treatment
% of Cells
? ? NAC
DEP 100 ?g/ml
DEP ? NAC
? ? NAC
DEP 100 ?g/ml
DEP ? NAC
? ? NAC
DEP 100 ?g/ml
DEP ? NAC
? ? NAC
DEP 100 ?g/ml
DEP ? NAC
aCells were treated with DEP extract (100 ?g g/ml) with or without 20 mM NAC for 16 h. Apoptosis was determined by
flow cytometry using annexin V and PI double staining as described in Materials and Methods.
4537The Journal of Immunology
46). This includes activation of antioxidant defense pathways, as
well as induction of proinflammatory and cytotoxic responses (24).
An interesting difference between epithelial cells and macrophages
is the lower basal GSH/GSSG ratios in the former compared with
the latter cell type (Fig. 1). This may explain the increased pro-
pensity toward cytotoxicity in epithelial cells.
An example of a cellular antioxidant defense mechanism is
HO-1 expression (11, 47). Not only does HO-1 constitute a very
sensitive marker of oxidative stress, but its catalytic action on
heme generates a potent antioxidant, bilirubin, as well as a gaseous
substance, CO, that exert anti-inflammatory effects in the lung
(48). Not surprising, therefore, the CO level in exhaled air is a
electron microscopic study. Electron microscopy was performed as described in Materials and Methods. A, BEAS-2B. a and b, Control; c and d,
DEP-treated cells. Magnification: a, ?3500; b, ?15,100; c, ?2400; d, ?12,400. B, THP-1. Magnification: a, ?2800; b, ?15,100; c, ?2,400; d, ?15,100.
AP, apoptotic body; M, mitochondrion.
Electron microscopy showing structural features of cell death. Cells were treated with 100 ?g/ml DEP extract for 6 h before being fixed for
4538 DEP EFFECTS ON BRONCHIAL EPITHELIAL CELLS AND MACROPHAGES
sensitive in vivo marker for the proinflammatory effects of DEP in
the lung (49). The molecular basis for initiating this antioxidant
defense mechanism is the transcriptional activation of the HO-1 gene
by a series of ARE in its promoter (11, 47). It has now been estab-
lished that the transcription factor Nrf-2 is involved in ARE activation
in vivo and in vitro (50, 51). Not only have we confirmed that ARE
is involved in activation of the HO-1 promoter by aromatic and polar
DEP chemicals in macrophages (11), but we also demonstrate ex-
quisite sensitivity of HO-1 to oxidative stress in epithelial cells (Fig.
2, A and B). However, while HO-1 exerts a cytoprotective effect in
RAW264.7 cells, it failed to do so in BEAS-2B cells despite the fact
that HO-1 expression could be induced by CoPP (Fig. 2B). This sus-
ceptibility may be due to the lower basal GSH/GSSG ratios in bron-
chial epithelial cells (Fig. 1B).
If of sufficient intensity, oxidative stress can initiate proinflamma-
tory effects in macrophages and bronchial epithelial cells (21–23, 27,
32). These effects are mediated by phosphorylation-dependent cell
signaling pathways, including activation of the mitogen-activated
protein kinase and NF-?B kinase cascades. In this communication
we demonstrate that organic DEP extracts activate the JNK cas-
cade in BEAS-2B and NHBE cells in a dose-dependent fashion
(Fig. 3B). Interestingly, JNK activation required higher extract
doses than that needed to initiate HO-1 expression, suggesting that
this may constitute a hierarchical oxidative stress effect (Figs. 2A
and 3A). This idea is in agreement with the progressive decrease in
GSH/GSSG ratios at higher extract doses (Fig. 1B). The impor-
tance of JNK activation is that this may lead to transcriptional
activation of proinflammatory cytokines and chemokines (8, 28,
52, 53). An example is IL-8 production, which could be induced by
DEP extracts in BEAS-2B and NHBE cells (Fig. 4). While this
response achieved a plateau at 25–50 ?g/ml in NHBE, there was
a precipitous decline in IL-8 production in BEAS-2B at doses ?10
?g/ml. This is probably due to the higher rate of apoptosis in
Another consequence of oxidative stress is the induction of cel-
lular apoptosis and necrosis (Fig. 5, A and B, and Table I). In this
regard we have demonstrated that organic DEP chemicals induce
cellular apoptosis and necrosis through perturbation of the mito-
chondrial PT pore (17, 18). A variety of redox cycling and oxi-
dizing chemicals has been shown to perturb the PT pore (18, 54,
55). This leads to a cascade of events that includes a decrease in
??m, cytochrome c release, and activation of cellular caspases
(Fig. 7, A and B, and Table I). Damage to the mitochondrial inner
membrane also disrupts four-electron reductions of O2, switching
this process instead to one-electron reductions (56, 57). This leads
to O2.generation and could be responsible for the increased HE
fluorescence shown in Fig. 7. Ultimately, damage to the inner
membrane and interference in electron transfers lead to decreased
ATP production and energy failure (Fig. 8). This leads to cellular
necrosis, reflected by mitochondrial swelling and appearance of
PI?/annexin V?cells, in addition to other features of apoptosis
Epithelial cells appear to be more susceptible to the cytotoxic
effects of DEP extracts than macrophages (Table I and Fig. 5, A
and B). While the reason for this increased susceptibility is un-
known, we know that cellular GSH levels play a role in regulating
mitochondrial permeability transition, possibly by preventing the
drial ?? were measured by flow cytometry using dual-color HE/DiOC6
staining as described in Materials and Methods. Definitions of quadrants:
induced changes in O2.and mitochondrial ?? in BEAS-2B and THP-1.
Cells were exposed to 100 ?g/ml DEP extract for 2 or 7 h. B, Dual-color
analysis in NHBE cells. Cells were treated with 100 ?g/ml DEP extract for
2 or 7 h before staining for flow cytometry.
Mitochondrial functional perturbation. O2.and mitochon-
bright/HEbright. A, DEP-
bright/HElow; and upper right (UR), DiOC6
treated with DEP extract (100 ?g/ml) for the indicated time period. The
ATP concentration was determined by luciferase assay following the man-
ufacturer’s instructions. Values are the mean ? SEM. For BEAS-2B, p ?
0.0009; for THP-1, p ? 0.0001.
Kinetics of DEP-induced decline in ATP levels. Cells were
4539 The Journal of Immunology
cross-linking of vicinal thiol groups in the PT pore (58). Although
GSH predominates in the cytoplasm, a small portion is sequestered
in mitochondria (59). Moreover, it has been suggested that GSH is
the only antioxidant that protects mitochondria against the harmful
effects of H2O2(59). Lower GSH/GSSG ratios in bronchial epi-
thelial cells may limit their ability to protect the mitochondrial PT
pore and may render these cells more susceptible to DEP-induced
oxidative stress. The same reasoning may apply to the failure of
NAC to protect bronchial epithelial cells (Table I). While this an-
tioxidant effectively prevents decline of the GSH/GSSG ratios in
THP-1 cells, NAC did not exert the same effect in bronchial epi-
thelial cells (Fig. 1C). A possible explanation for this finding is
that the drug is not deacetylated to the glutathione precursor in
epithelial cells. The fact that NAC can prevent JNK activation and
HO-1 expression in epithelial cells (Figs. 2C and 3B) may be re-
lated to its activity as a radical scavenger. Taking all these data into
consideration, NAC may be a valuable therapeutic agent that can
be used to modify macrophage and epithelial activation, as dem-
onstrated by its ability to modulate biomarker induction by air
particulate matter in rat and murine lung (16, 60). NAC also in-
terferes in TNF-? production in alveolar macrophages exposed to
air PM (61).
The above studies indicate that organic DEP extracts that are
enriched for PAH and oxy-PAHs induce a range of biological ef-
fects related to the generation of oxidative stress. We propose that
this constitutes a stratified cellular response to oxidative stress. At
the lower end of the oxidative stress scale, cells or tissues are
stimulated to induce ARE-dependent antioxidant and cytoprotec-
tive responses. If these protective mechanisms fail, further esca-
lation of oxidative stress may lead to proinflammatory or cytotoxic
effects. We propose that activation of intracellular signaling cas-
cades, e.g., the JNK pathway, and perturbation of the mitochon-
drial PT pore play a role in these injurious cellular responses. This
implies that the activation threshold for cellular injury requires
higher oxidative stress levels than those required for cytoprotective
A stratified oxidative stress model may prove useful in study of
the adverse health effects of PM. Although some adverse effects
may occur independently of oxidative stress, a stratified stress
model implies that biological end points should be selected rele-
vant to the level of oxidative stress and PM exposure. For instance,
under the experimental conditions chosen by Nightingale et al.
(49), an increased CO level in the expired air was a more sensitive
end point than the bronchoalveolar neutrophil content. This agrees
with the idea that HO-1 is a more sensitive oxidative stress marker
than IL-8 (Fig. 2, A and B). Other ARE-driven events, e.g., ex-
pression of phase II drug-metabolizing enzymes (62), may be in-
duced at this low stress level. When exposed to higher PM chem-
ical doses, screening should include markers for inflammation,
including cytokines and chemokines. This approach has been dem-
onstrated in DEP nasal challenge studies in atopic individuals (9).
Finally, it is important to consider that high levels of oxidative
stress may induce cytotoxic effects, which could override and con-
ceal the proinflammatory effects of the PM. One possibility is that
apoptosis of macrophages and participating immune cells may in-
terfere with allergic inflammation, but could still exacerbate
asthma due to bronchial epithelial shedding.
Another value of the oxidative stress theory is that it may assist
in the identification of human subsets that are more susceptible to
the adverse health effects of PM. An example is HO-1 expression.
This enzyme has potent antioxidant and cytoprotective effects in
the lung (63). Noteworthy, a polymorphism of the HO-1 promoter
has been described that reflects gene expression in the presence of
ROS (64). Moreover, it has been demonstrated that male smokers
with a poorly responsive HO-1 promoter have a higher rate of
emphysema than male smokers with a more inducible HO-1 gene
(64). The same paradigm may apply to antioxidant and detoxifi-
cation pathways that play a role in defending against the adverse
biological effects of PM.
In conclusion, we have shown that organic DEP chemicals in-
duce a range of biological responses in epithelial cells and mac-
rophages that depend on the generation of oxidative stress. Epi-
thelial cells appear to be more sensitive than macrophages,
possibly due to a limited ability to defend them against oxidative
stress. This is true even in the presence of a thiol antioxidant.
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