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* 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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages constitute an important target for DEP in the lung
(17, 18, 21, 22, 23). After phagocytosis of these particles,
macrophages respond in a hierarchical fashion to increasing particle
load and incremental levels of oxidative stress (24).
Thus, at low oxidative 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
expression (24). HO-1 expression is dependent on the
function of antioxidant response element (ARE) in its promoter
(11) and is typically induced by 1–10 µg/ml of the DEP
extract (11, 24). In contrast, extract doses of 10–50
µg/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 principal 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 µg/ml, macrophages undergo apoptosis and necrosis (17, 18, 24).
Bronchial epithelial cells are another primary cell target for PM (25, 26, 27, 28). Not only do these cells play an important role in allergic inflammation, but shedding and dysregulation of bronchial epithelial repair contribute to airway hyper-reactivity in atopic asthmatics (29). Several studies have demonstrated that DEP elicit biological responses in bronchial epithelial cells (25, 26, 27, 28). These effects include the release of proinflammatory mediators as well as the induction of mucoid hyperplasia (30, 31, 32, 33, 34, 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 consequences 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 determine 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 demonstrate 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-acetylcysteine (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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (Carpinteria, CA). HRP-conjugated sheep anti-mouse Ab was
obtained from Amersham (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).
Cell culture
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 epithelial 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/streptomycin, 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 polypropylene tube and dried under nitrogen gas. The tube was reweighed to determine the amount of methanol-extractable DEP components. Dried DEP extract was then dissolved in DMSO at a concentration of 100 µg/µl. 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 exchanging into hexane. The supernatant was concentrated, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (column size, 1 x 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 microbalance by evaporating off a known volume of an aliquot of the sample made up in methylene chloride or methanol. The fractions were dried with N2 gas and redissolved in DMSO.
Western blotting analysis
Western blotting was conducted as previously described (17). One hundred to 150 µg 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 µg/ml and rabbit anti-mouse Ab conjugated to HRP according to the manufacturer’s instructions. Anti-MnSOD Ab was used at 0.3 µg/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).
RT-PCR analysis
Total RNA was extracted using TRIzol RNA extraction reagent
(11). RT was performed at 42°C in a total volume of 20
µl containing 5 µg total RNA; 0.5 µg
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
transcriptase (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'-TAAAACGCAGCTCAGTAACAGTCCG-3'. PCRs for both HO-1 and -actin
were performed in a total reaction volume of 25 µl containing 4 µl
cDNA template, 0.5 µM 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
electrophoresed 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 Laboratories (Baltimore, MD) for measurement of the IL-8 levels by ELISA.
Flow cytometry
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
(O2-·) production and m
were determined by dual staining with HE and
DiOC6. Cells (106/ml) were
incubated with 2 µM HE as well as 20 nM DiOC6
diluted in the serum-free culture medium for 30 min in the dark at
37°C. Apoptosis was analyzed by annexin V-FITC/PI double staining
according to the manufacturer’s instructions. Time- and dose-dependent
cellular cytotoxicity were determined by staining the cells in 1
µg/ml PI. DiOC6 and annexin V-FITC fluorescence
were analyzed in the fluorescent-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 glutathione reductase (37, 38, 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 µl supernatant was incubated with 2 µl 2-vinylpyridine and 6 µl 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.
Electron microscopy
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 microscopy were cut with a Reichert-Jung Ultracut and Ultramicrotome (Vienna, Austria). Copper grids were stained with lead citrate and uranyl acetate 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.
Statistics
Data were analyzed using SAS statistical software (SAS Institute, Cary, NC). Scheffe’s method of multiple comparisons with F test was used for ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 8, 9, 11, 12, 13, 14, 20). Moreover, 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
fluorescence, which reflects mostly
H2O2 production (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. 1
A). HE is oxidized to
ethidium bromide by ROS and is mostly likely a reflection of
O
2 production (18). In contrast to HE conversion
in BEAS-2B cells, THP-1 cells did not exhibit an appreciable increase
in HE fluorescence (Fig. 1A).
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B). Similar changes in intracellular glutathione
ratiosoccurred in NHBE during exposure to the DEP extract (Fig. 1C). Interestingly, THP-1 cells maintain a higher basal
GSH/GSSG ratio 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 cells, this thiol antioxidant did not affect basal glutathione
levels in BEAS-2B or NHBE cells (Fig. 1C). In addition,
while NAC prevented a decline in GSH/GSSG ratios in THP-1 cells during
exposure to a DEP extract, this agent did not prevent a drop in
glutathione ratios in BEAS-2B and NHBE cells (Fig. 1C). This
suggests that there is no NAC conversion to glutathione in epithelial
cells.
A sustained drop in cellular GSH/GSSG ratios is indicative of a
pro-oxidant state and leads to protective cellular responses. Examples
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
expression, 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.
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D). 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 µg/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 suggests that despite its cytoprotective and
antioxidant function, HO-1 is not sufficient to protect epithelial
cells against the injurious effects 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 relatively low
extract amounts (
10 µg/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
fluorescence.
Organic DEP extracts induce JNK activation in bronchial epithelial cells
In addition to initiating antioxidant and cytoprotective
responses, oxidative stress can activate intracellular signaling
cascades, including 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
phosphorylation of the 45- and 54-kDa JNK isoforms could be seen at
extract doses 
25 µg/ml; a high rate of cell death diminished the
BEAS-2B response at 100 µg/ml (Fig. 3A). NHBE also showed
increased JNK phosphorylation in the dose range of 50–100 µg/ml
(Fig. 3A). These effects were not due to a decrease in the
abundance of JNK protein, as demonstrated by anti-JNK
immunoblotting (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 cells.
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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 epithelial cells
A consequence of the activation of intracellular signaling
cascades is the transcriptional activation of proinflammatory genes,
including genes that encode for cytokines, chemokines, and adhesion
receptors. One example is the IL-8 gene, which is under dual regulation
by NF-B and AP-1 response elements in its proximal promoter
(41, 42). IL-8 is particularly relevant to the
proinflammatory effects of DEP in the lung (22, 26, 27, 28).
To compare IL-8 induction in epithelial and THP-1 cells, cultures were
treated with 10–100 µg/ml of the DEP extract for 14 h before
measuring IL-8 in the culture medium. While THP-1 cells showed a
dose-dependent response over the entire dose range (10–100 µg/ml),
NHBE cells showed an incremental response in the range 10–50 µg/ml,
followed by a rapid decline at 100 µg/ml (Fig. 4). This is probably due to a high rate
of apoptosis in NHBE at doses >25 µg/ml (see below). While BEAS-2B
responded to 10 µg/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.
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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
µg/ml in THP-1 cells, BEAS-2B cells showed a significant rise in the
rate of cell death at doses >10 µg/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).
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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+ macrophages
is more prominent at DEP extract doses <100 µg/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 linking
GSH depletion to the induction of apoptosis via a mitochondrial effect
(17, 18, 43). Indeed, ultramicroscopic visualization of
THP-1 and BEAS-2B cells showed that the appearance of apoptotic bodies
is accompanied by changes in the mitochondrial morphology (Fig. 6, A and B). These changes, which include
mitochondrial 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 shown).
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). The resulting flow diagram shows that
the DEP extract induced a decrease in the mitochondrial membrane
potential (DiOC6 fluorescence, 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
HEbright cells (UR quadrant) in the BEAS-2B
population increased 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 number of
DiOC6low cells increased to
40.2% (LL and UL quadrants) 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 quadrants) in THP-1 cells (Fig. 7A).
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).
Similarly, ATP levels in NHBE cells decreased by >80% within
2 h of stimulation (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.
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indicate that at relatively high
concentrations of DEP extract, there is a transition from
cytoprotective 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. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and were more susceptible to cytotoxic effects than
macrophages. 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
expression 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 µg/ml, a dose leading to a more drastic decline in GSH/GSSG
levels. While induction of cellular toxicity in THP-1 cells required an
extract dose >25 µg/ml, the onset of cell death in BEAS-2B cells was
more linear at doses 10 µg/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 chromatography
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 directly responsible
for the perturbation of cellular function (7, 44, 45, 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 propensity 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 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 established 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
exquisite 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 susceptibility may be due to the lower
basal GSH/GSSG ratios in bronchial epithelial cells (Fig. 1B).
If of sufficient intensity, oxidative stress can initiate
proinflammatory effects in macrophages and bronchial epithelial cells
(21, 22, 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 cascade 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 importance 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 µg/ml in NHBE, there was a precipitous decline in
IL-8 production in BEAS-2B at doses >10 µg/ml. This is probably due
to the higher rate of apoptosis in BEAS-2B cells.
Another consequence of oxidative stress is the induction of cellular
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 mitochondrial PT pore (17, 18). A variety of redox
cycling and oxidizing 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 (Fig. 6).
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
unknown, we know that cellular GSH levels play a role in regulating
mitochondrial permeability transition, possibly by preventing the
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 epithelial 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 antioxidant effectively prevents decline of the
GSH/GSSG ratios in THP-1 cells, NAC did not exert the same effect in
bronchial epithelial 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 related 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 demonstrated by its ability to modulate
biomarker induction by air particulate matter in rat and murine lung
(16, 60). NAC also interferes 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 effects 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 cytoprotective responses. If these protective mechanisms fail, further escalation of oxidative stress may lead to proinflammatory or cytotoxic effects. We propose that activation of intracellular signaling cascades, e.g., the JNK pathway, and perturbation of the mitochondrial 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 responses.
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 relevant 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.,
expression of phase II drug-metabolizing enzymes (62), may
be induced at this low stress level. When exposed to higher PM chemical
doses, screening should include markers for inflammation, including
cytokines and chemokines. This approach has been demonstrated 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 conceal
the proinflammatory effects of the PM. One possibility is that
apoptosis of macrophages and participating immune cells may interfere
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 detoxification pathways that play a role in defending against the adverse biological effects of PM.
In conclusion, we have shown that organic DEP chemicals induce a range of biological responses in epithelial cells and macrophages that depend on the generation of oxidative stress. Epithelial 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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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andre E. Nel, Division of Clinical Immunology and Allergy, Department of Medicine, University of California, 10833 Le Conte Avenue, 52-175 CHS, Los Angeles, CA 90095. E-mail address: anel{at}mednet.ucla.edu
3 Abbreviations 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, reduced glutathione; GSSG, glutathione disulfide; HE, hydroethidine; HO-1, heme oxygenase-1; JNK, c-Jun N-terminal kinase; LL, lower left; MnSOD, manganese superoxide dismutase; NAC, N-acetylcysteine; NHBE, normal human bronchial epithelial 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.
Received for publication May 15, 2002. Accepted for publication August 2, 2002.
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