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Division of Clinical Immunology and Allergy, Department of Medicine, University of California School of Medicine, Los Angeles, CA 90095
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Abstract |
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Introduction |
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Key scientific issues about the role of DEP that need to be addressed are: 1) what are the cellular targets? and 2) what are the mechanisms of action of DEP on those target cells? These are complicated issues due to the diversity of cell types that participate in airway inflammation as well as the chemical composition of DEP (1, 22). Among the participating cell types in allergic inflammation, DEP or DEP extracts have been shown to affect eosinophil, epithelial, B cell, and macrophage function (1, 23, 24, 25, 26, 27 27A ). However, we do not know whether these are direct or indirect effects. DEP are comprised of an inert carbonaceous core coated with unburnt petrochemicals. DEP contain >450 different organic compounds, including xenobiotics such as polycyclic aromatic hydrocarbons (PAH), halogenated aromatic hydrocarbons (HAH), and redox-active quinones (22, 27 27A, 28, 29). Xenobiotics, including PAH, exert stimulatory or toxic effects via the generation of reactive oxygen radicals (ROR) (24, 30, 31, 32, 33). Macrophages, in particular, are enriched for xenobiotic converting enzymes, which contribute to ROR generation (1, 33, 34, 35, 36, 37, 38). Examples of these enzymes include cytochrome P4501A1, a phase I drug-metabolizing enzyme that is induced by PAH and HAH (37, 39), as well as NADH-cytochrome P450 reductase, which is induced by DEP-derived chemicals (33). ROR generation has also been linked to the mutagenic effects of DEP chemicals (30) as well as to the activation of stress-activated protein kinases (SAPKs), which regulate the expression of proinflammatory genes in macrophages (24, 40). Based on these findings, we have proposed that macrophages are a key cellular target for DEP in the respiratory tract (1). Moreover, the targeting of macrophages may explain the adjuvant effects of DEP, e.g., enhancement of their Ag-presenting function (1). This process may involve ROR generation (1). In addition to their effects on cellular activation, ROR may play a role in the acute respiratory effects of PM10 pollutants.
One manifestation of the acute toxicity of DEP at the cellular level is the induction of apoptosis in macrophages. Xenobiotics can induce cellular apoptosis (41, 42) and may contribute to DEP-induced apoptosis. Macrophage apoptosis may impact tissue responses in two ways. First, the shedding of apoptotic bodies may spread toxic chemicals to neighboring cells, which engulf the apoptotic remnants from dying cells. Second, damage to macrophages may decrease tissue defenses to infection, e.g., increased multiplication of respiratory viruses in the lungs of mice exposed to DEP (43). Apoptosis may also be relevant to chronic airway inflammation (44), including COPD, where macrophages play a role in perpetuating inflammation in small airways (45). We were interested whether the proapoptotic effects of DEP are related to the ability of macrophages to phagocytose this particulate matter, and whether the chemicals contained in DEP play a role in cellular toxicity. Our data show that apoptosis could be induced by phagocytosis of intact DEP or adding the organic extracts made from these particles back to the macrophage cell lines. In contrast, organically extracted DEP or carbon black particles did not induce apoptosis in macrophages. Apoptosis could be reversed by a caspase inhibitor as well as the antioxidant, N-acetyl-cysteine (NAC). This system provides us with a cellular model to study the role of oxygen radicals in DEP-related tissue injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 and FCS were purchased from Irvine Scientific (Santa Ana, CA). DMEM, penicillin-streptomycin, and L-glutamine were purchased from Life Technologies (Gaithersburg, MD). DEP were a gift from Dr. Masaru Sagai (National Institute of Environmental Studies, Tsukuba, Japan) (33). An E-Toxate kit, NAC, EDTA, and propidum iodide (PI) were purchased from Sigma (St. Louis, MO). Z-VAD was obtained from Enzymes Systems Products (Dublin, CA). The annexin-FITC kit was purchased from Trevigen (Gaithersburg, MD). 2',7'-Dichlorofluorescein diacetate (DCF-DA) was purchased from Molecular Probes (Eugene, OR). Anti-caspase 8 and anti-caspase 9 Abs were obtained from PharMingen (San Diego, CA). Anti-phospho-JNK and anti-phospho-p38 MAPK Abs were purchased from New England Biolabs (Beverly, MA). Anti-JNK1/2 and anti-p38 MAPK Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The fluorescent yellow/green latex beads (0.75 µm) were purchased from Polysciences (Warrington, PA). Six-well plastic tissue culture dishes were purchased from Fisher Scientific (Pittsburgh, PA). The diameter of each polystyrene well is 3.5 cm.
Cell culture and stimulations
RAW 264.7 cells (murine) were cultured in a 5% CO2 atmosphere in DMEM containing 10% FCS, 1% penicillin-streptomycin, and 1% glutamine. THP-1 cells (human) were cultured under the same conditions, except that RMPI 1640 medium was used. Pulmonary alveolar macrophages, obtained by saline lavage of the lung of an adult Fisher rat, were provided by Dr. J. Seagrave (Lovelace Respiratory Research Institute, Albuquerque, NM). In phagocytosis assays, aliquots 3 x 106 cells were cultured in six-well plates in 3 ml of medium. These cultures were replenished with sterilized fluorescent latex beads, native DEP, methanol-extracted DEP, or carbon black particles to final concentrations as indicated. To assess phagocytosis of the latex beads, cells were gently scraped from the plates 18 h later, washed three times in PBS, and fixed in 1% paraformaldehyde for 20 min on ice (46). Cells were then viewed under a fluorescence microscope. To assess the phagocytosis of DEP, washed DEP, or carbon black particles, cells were harvested after 18 h, washed, and viewed under a Kodac light optic microscope (Eastman Kodak, Rochester, NY) at a x1000 magnification.
Preparation of DEP and washed DEP
A stock of DEP was prepared by resuspending 100 mg of DEP in 2 ml of PBS supplemented with 10% FCS. The particles were vortex mixed vigorously, sonicated with a sonic disrupter for 2 min on ice, and then stored at 4°C in the dark. Washed DEP were prepared as follows. DEP (100 mg) was suspended in 50 ml of methanol and vigorously vortex mixed (47). The sample was sonicated for 2 min on ice and centrifuged for 10 min at 2500 rpm. Methanol extracts were saved for later use. The DEP pellet was washed three times in 50 ml of methanol, dried, and reweighed to calculate the relative contribution of the extractable and nonextractable components to the overall particle composition. The sample was then resuspended in 10% FCS in PBS to yield a stock concentration of 50 mg/ml washed DEP. This stock was diluted in complete medium to the indicated concentration at the start of every experiment. DEP extracts were prepared for use in culture by aliquoting 1.5 ml of the methanol extract into weighed Eppendorf tubes. After methanol evaporation by nitrogen gas, the tubes were reweighed to calculate the extract amount. The dried extracts were resuspended in DMSO to the give a stock concentration of 100 µg/µl. DEP, washed DEP, and DEP extracts were tested to determine the absence of endotoxin using the E-Toxate kit.
Flow cytometry
Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single 488-nm argon laser. Dead cell fragments were gated out by forward and side scatter analysis. Annexin V-FITC and DCF were analyzed at the same excitation and emission settings of 488 and 535 nm, respectively (FL-1 channel). One-color PI analysis was performed at excitation and emission settings of 488 and 575 nm, respectively. Where dual color PI/annexin V-FITC analysis was performed, PI fluorescence was displayed in channel FL-2, while the FITC fluorescence was displayed in channel Fl-1.
PI staining
Cells (3 x 106) were plated into 3.5-cm plates in 3 ml of medium and rested for 4 h. Some cultures were preincubated with 20 mM NAC for 2 h. Varying concentrations of DEP were added to these cultures for 18 h. Cells were then collected, washed twice in PBS, and resuspended in 500 µl of PBS. PI, suspended in PBS, was added to these samples to a final concentration of 0.5 µg/ml for 5 min.
Determining apoptosis by hypodiploid DNA detection
Aliquots of 3 x 106 RAW 264.7 cells were incubated with 200 µg/ml DEP for 18 h. Samples were then washed twice with PBS and resuspended in 300 µl of saline GM solution (6.1 mM glucose, 137 nM NaCl, 5.4 mM KCl, 1.1 nM Na2HPO4, 1.1 mM KH2PO4, and 5.1 mM EDTA). Cells were fixed in 700 µl of cold 100% ethanol and incubated at -20°C for 1 h. After fixation and washing, cells were resuspended in 1 ml of hypotonic DNA staining buffer (3.4 mM sodium citrate, 0.15 mM propidium iodide, 0.002% (w/v) RNase A, and 0.003% Triton X-100). The samples were then incubated at 4°C in the dark for 30 min and analyzed by flow cytometry.
Annexin V/PI staining
RAW 264.7 cells (3 x 106) were plated in six-well plates. Varying concentrations of DEP, washed DEP, or carbon black were then added to the cultures for 18 h. Cells, at a concentration of 1 x 106 cells/ml, were washed three times in PBS and resuspended in annexin V/PI staining buffer according to manufacturer’s instructions. After incubating at room temperature for 15 min, samples were analyzed by flow cytometry.
DCF staining
RAW 264.7 cells (3 x 106) were incubated together with 200 µg/ml DEP or 50 µg/ml DEP extract for the indicated time periods. Some cultures received 20 mM NAC for 2 h before the addition of DEP or DEP extracts. Cells were collected and washed three times in PBS. Samples were resuspended in DCF-DA diluted in DMEM to a concentration of 2.5 µM and incubated at 37°C for 30 min in the dark. PI was added to samples, which were immediately subjected to flow cytometric analysis.
Western blotting analysis to assess activation of caspases 8 and 9
THP-1 cells (3 x 106) were incubated together with 300 µg/ml DEP in the presence and the absence of 20 mM NAC for the indicated time periods. Cells were lysed in an SDS buffer, and 100 µg of lysate was separated by 10% SDS-PAGE and transferred to Immobilon-P membranes. Immunoblotting for caspase 8 was performed using a 1/500 dilution of the anti-caspase 8 Ab, followed by a 1/3000 dilution of HRP-coupled sheep anti-mouse Ab. Immunoblotting for caspase 9 was performed using a 1/1000 dilution of the anti-caspase Ab, followed by a 1/3000 dilution of HRP-coupled donkey anti-rabbit Ab.
Phospho-JNK and phospho-p38 immunoblots
Immunoblots were performed by treating 3 x 106 THP-1 cells with 50 and 100 µg/ml DEP extracts or 10 µg/ml LPS. Cells were lysed as previously described, and 100 µg of lysate was separated by 10% SDS-PAGE. Blots were overlaid with a 1/1000 dilution of anti-phospho-JNK or anti-phospho-p38 Abs followed by a 1/3000 dilution of HRP-coupled donkey anti-rabbit Ab (1).
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Results |
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We have previously shown that PAH and redox-active quinones exert
stimulatory as well as inhibitory effects in macrophages (1, 24). Because DEP contain similar organic chemicals, these
particles may induce a variety of cellular responses in macrophages.
Moreover, macrophages express xenobiotic converting enzymes such as
cytochrome P4501A1, which plays a role in mediating the biological
effects of PAH (34, 35, 36, 37, 38). To develop a relevant macrophage
model for DEP phagocytosis, we used the murine RAW 264.7 and human
THP-1 cell lines, which have previously been used to demonstrate the
cellular effects of xenobiotics (1, 24). First, we had to
establish that these cell types are phagocytic. For that purpose, we
incubated THP-1 and RAW 264.7 cultures with fluorescent latex beads
(46) and followed the cellular uptake of those beads by
fluorescence microscopy and flow cytometry (Fig. 1
). Fluorescence microscopy demonstrated
that both cell types effectively phagocytosed latex beads (Fig. 1). In
subsequent experiments, we looked at DEP phagocytosis under similar
culture conditions. THP-1 and RAW 264.7 cells were incubated with
different amounts of DEP for 18 h. After washing, the cells were
viewed under a light optic microscope. Both cell types ingested DEP
(Fig. 2). Taken together, the data in
Figs. 1 and 2 show that RAW 264.7 and THP-1 cells can be used to study
the cellular effects of DEP related to their phagocytosis.
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In the course of the DEP studies, we noticed the appearance of
detached cells with blebbing surface membranes and apoptotic bodies in
the culture supernatants. We used PI staining of the nucleus to
determine whether these were dead cells. Fig. 3, A and B,
demonstrates that adding increasing amounts of DEP led to a
dose-dependent increase in the percentage of PI-stained cells in the
RAW 264.7 and THP-1 populations. These toxic effects started at the
lowest particle concentration (50 µg/ml) tested and increased
linearly up to a DEP concentration of 400 µg/ml (Fig. 3). We also
tested the effect of the same batch of DEP on the bronchial epithelial
cell line, BEAS-2B. In these cells, DEP lacked the toxic effects seen
in macrophages (Table I). A similar lack
of toxicity was obtained when coincubating lymphoid cell lines with DEP
(not shown).
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, A and B). This difference was not due to
diminished phagocytosis, because the washed particles were equally well
ingested as the native DEP (not shown). In addition, equal amounts of
unwashed carbon black particles, which were readily phagocytosed,
failed to exert toxic effects on THP-1 or RAW 264.7 cells (not shown).
Taken together, these data show that the chemical composition of DEP is
responsible for their toxicity.
DEP contain a wide range of chemicals, some of which are known inducers
of ROR (30, 33). To determine the possible contribution of
oxidative stress to DEP-induced cell death, both macrophage types were
pretreated with the antioxidant, NAC, before coincubation with
different doses of DEP. Using flow cytometry to measure PI staining of
the nucleus as a measure of cell death, it could be seen that NAC
effectively (>80%) inhibited the induction of cell death in RAW 264.7
cells at all DEP concentrations tested (Table II). While NAC reduced the rate of cell
death in THP-1 cells by >60% at a lower (200 µg/ml) DEP
concentration, this antioxidant had little effect on reversing cell
death at the higher (400 µg/ml) particle concentration (Table II).
This shows that while ROR play a role in the induction of cell death,
additional factors may determine the susceptibility of THP-1 cells
to DEP.
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It has recently been shown that biological responses to PAH and
HAH include the induction of apoptosis (41, 42). Because
these toxic chemicals are present in DEP, we asked whether the cellular
events depicted in Figs. 3 and Table II reflect the induction of
apoptosis. The morphological features of the dying macrophages in our
cell culture were compatible with apoptosis (not shown). These
apoptotic features included changes in the plasma membrane, chromatin
condensation, disruption of the nucleus, and the formation of
membrane-bound apoptotic bodies. The early loss of membrane asymmetry
can be conveniently followed using annexin V staining, which detects
aberrantly expressed phosphatidylserine on the outside of the cell
membrane (48). This analysis can be combined with PI
staining in a two-color flow cytometry protocol, which additionally
reflects cell death. Exposure of RAW 264.7 cells to DEP for increasing
lengths of time demonstrated a time-dependent increase in annexin V
staining (Fig. 4A).
Approximately 20% of the cells stained annexin V positive but remained
PI negative after a 6-h incubation period (Fig. 4A,
right lower quadrant). This fluorescence profile is
compatible with early apoptotic events. However, by 18 h this
profile changes to reflect the additional appearance of PI-positive
cells in the cell population (Fig. 4A, right upper
quadrant). Some annexin V-positive but PI-negative cells could
still be seen at this stage, indicating that new apoptotic events were
occurring (Fig. 4A, right lower quadrant). Additional
evidence for induction of apoptosis by DEP was obtained by assessing
hypodiploid DNA in PI-stained RAW 264.7 cells (Fig. 4B). In
the same analysis, which also reflects cell cycle progression, it could
be seen that DEP induce cell arrest in the G1
phase of the cycle (Fig. 4B). Similar observations were made
in THP-1 cells as well as primary alveolar macrophages (see below).
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A). In a parallel experiment
in which RAW 264.7 cells were incubated with washed DEP and carbon
black particles, only marginally increased rates of apoptosis were
recorded compared with the control values (Fig. 5A).
Similarly, in THP-1 cells, DEP induced dose-dependent apoptosis (Fig. 5B). Although NAC effectively interfered with apoptosis at
the lower (200 µg/ml) DEP concentration (18.4 vs 46.1%), this
antioxidant had only a minor blocking effect (64 vs 47%) on the higher
(400 µg/ml) DEP concentration. However, in RAW 264.7 cells, NAC was
effective in decreasing apoptosis at all DEP concentrations tested (not
shown). These data agree with the PI staining data in Table II.
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A). Moreover, the proteolysis
of this procaspase yielded a 37-kDa fragment that represents the active
caspase (Fig. 6A, lane 4). The time point for caspase 9
activation agrees with the time point for positive annexin V staining
(Fig. 4A). The addition of NAC to THP-1 cells prevented
caspase 9 activation (Fig. 6A, lanes 6–10),
confirming the importance of oxidative stress in DEP-induced
apoptosis.
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B, lanes 1–5). As for caspase 9 activation, NAC
effectively interfered with this proteolytic event (Fig. 6B,
lanes 6–10). Whatever the mechanism of initiation of these caspase cascades, the afferent caspases activate a series of downstream caspases that play a role in the execution of apoptosis (49). A broad spectrum caspase inhibitor, the fluoro-methyl ketone derivative, Z-VAD-fmk, inhibited DEP-induced apoptosis by >50% at a concentration of 30 µM (not shown). This confirms the importance of caspases in DEP-induced apoptosis.
Organic DEP extracts induce apoptosis in macrophage cell lines as well as primary alveolar macrophages
In Fig. 3 we have demonstrated that washed or organically
extracted DEP lack the toxicity of native DEP. Using PI staining, we
could demonstrate a dose-dependent increase in the rate of cell death
when the dried methanol extracts, resuspended in DMSO, were added back
to RAW 264.7 cultures (Table III).
Thirty, 60, and 100 µg of extract represent 80, 160, and 250 µg
DEP, respectively. Two-color flow cytometry confirmed that this is an
apoptotic event (Fig. 7A).
Moreover, the induction of apoptosis could be reversed by the
antioxidant, NAC (Table III and Fig. 7A).
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B).
Methanol extraction decreased the toxicity of the DEP (Fig. 7B). Taken together with the data in Fig. 3, we conclude
that methanol-extractable DEP chemicals are responsible for apoptosis
in primary macrophages as well as cell lines. DEP and DEP extracts generate ROS in an NAC-sensitive manner
Because DEP-induced apoptosis can be reversed by NAC, we studied
ROS generation with the dye, DCF-DA (52). DCF-DA is a
nonfluorescent compound that is freely taken up into cells, where it is
trapped by removing the DA group. Upon interaction with peroxides, DCF
is converted into a fluorescent product. Treatment of RAW 264.7 cells
with 100 µg/ml of a DEP extract generated an almost 2 log-fold
increase in mean DCF fluorescence intensity compared with that in
untreated cells (Fig. 8A). The
addition of 20 mM NAC diminished this response by >90% (Fig. 8A). Although native DEP exerted similar effects,
methanol-extracted DEP did not generate a significant increase in DCF
fluorescence (not shown). Increased DCF fluorescence could be observed
within an hour of adding the DEP extract and peaked after 3 h
(Fig. 8B). This is compatible with the latency of early
apoptotic events (Fig. 5A). NAC effectively suppressed this
response for the entire observation period (Fig. 8B).
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We have previously shown that PAH and redox-active quinones
activate stress-activated protein kinases (SAPKs) in RAW 264.7 and
THP-1 cells by an oxidant-sensitive mechanism (24).
Because these kinases are involved in the cellular response to stress,
including oxidant stress, we were interested in whether DEP extracts
were successful in activating these kinases. Activation of JNK isoforms
and p38MAPK can be assessed by immunoblotting,
which reveals the phosphorylation of these kinases upon activation
(1, 24). Our data show that DEP extracts induced
dose-dependent phosphorylation of two different JNK species, as well as
p38MAPK (Fig. 9).
These increases in phosphorylation were not due to changes in the
abundance of the kinase enzymes, because parallel immunoblotting for
kinase protein showed equal staining intensity in control and treated
samples (see insets at bottom of Fig. 9, A and
B). Ten micrograms of SB 230580, a
p38MAPK inhibitor, decreased the rate of
apoptosis during treatment of THP-1 cells with 100 µg/ml DEP extract
by 22% (not shown). Unfortunately, we could not directly test the
involvement of the JNK cascade in these apoptotic events due to the
lack of a specific inhibitor. The contribution, if any, of these
kinases to DEP-induced apoptosis remains to be determined.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 7), we conclude
that DEP chemicals are responsible for initiating a programmed cell
death pathway. The particulate nature of DEP may be important, however,
for targeting phagocytic cells (Fig. 2). Although the exact mechanism
for the initiation of apoptosis remain to be elucidated, ROR play an
integral role in this event (Fig. 8), as demonstrated by the ability of
an antioxidant, NAC, to suppress the pro-apoptotic effects of DEP or
DEP extracts (Figs. 5 and 7). This idea is further substantiated by the
ability of NAC to inhibit DEP-induced generation of ROS (Fig. 8). In
addition to the generation of oxidative stress, DEP extracts activated
the stress-activated protein kinases, JNK and
p38MAPK, which may contribute to the cellular
response to oxidative stress (Fig. 9). These findings have important
implications for treating the effects of particulate pollutants on
airway inflammation. DEP is a major component of PM10 pollutants. For instance, in the Los Angeles basin, DEP comprise 40% of total PM10 levels, and the daily rate of exposure may be as high as 300 µg of DEP (4). This dose is equivalent to what was used in human nasal challenge studies to demonstrate the adjuvant effects of DEP on allergic inflammation. Considering the limited surface area of the nose, a 300-µg DEP dose may deliver the same number of particles per unit mucosal area as to the lawn of cells in the culture dish. Moreover, even if a single in vivo exposure is not sufficient to achieve these concentrations, the slow clearance of DEP from the respiratory tract may allow particle accumulation during repeated exposure. In addition, the mobility and chemotactic properties of macrophages are likely to induce their accumulation at the DEP deposit sites; this may lead to exposure to high particle concentrations.
The relatively small size of DEP also allows their deposition in the lung and may be instrumental in the increased morbidity and mortality in people with asthma and COPD, which follow when there are sudden surges in PM10 levels (20, 21). Until recently, we had little understanding of the pathogenicity of DEP in the respiratory tract, but have now obtained evidence that at least two independent, but possibly interrelated, processes are at work. The first process is the adjuvant effects of DEP on allergic inflammation, as reflected in both human and animal studies (1, 2, 3, 4, 13, 14, 15, 16, 17, 18, 19). Examples of such adjuvant effects include enhanced IgE and Th2 cytokine production during nasal challenge with an allergen in the presence of DEP (13). We have recently shown that macrophages may be involved in this adjuvant effects, based on the ability of DEP to increase CD86 expression in the nose in vivo and in macrophages in vitro (1). CD86 expression may enhance the properties of the macrophage as an APC (1). Longer term, these adjuvant events may play a role in the increased prevalence of asthma and allergic rhinitis, such as has been observed in Japan since the introduction of the diesel engine (11).
The second mechanism of action of DEP appears to be acute injurious events, such as the apoptotic events described in this paper. These events may be relevant to the acute symptomology and mortality that follow a rise in PM10 levels (20, 21). We postulate that ROR generation by macrophages may contribute to these acute symptomatic flares in asthmatics and people with COPD. The occurrence of apoptosis in macrophages may be useful as a marker for the acute injurious effects of DEP. It is also possible that macrophage apoptosis may directly contribute to the pathogenesis of respiratory disease. Although macrophage apoptosis has not been systematically studied in asthma, there are a limited number of studies that indicate that apoptosis pathways are relevant to allergic inflammation (44). We propose a number of ways in which macrophage apoptosis may contribute to chronic inflammation or perturbation of immune defenses in the lung. One consequence of macrophage cell death may be a decreased phagocytic capability, as has been shown in pulmonary alveolar macrophages after DEP ingestion (47). This interference in macrophage function may be responsible for an increased propensity toward respiratory tract infection, e.g., increased multiplication of influenza virus in DEP-exposed mice (43). Another contribution by macrophage apoptosis may be the shedding of apoptotic bodies when these cells die. Because these apoptotic bodies probably contain active chemicals, their uptake by surrounding inflammatory cell types may spread the tissue damage. We are particularly interested in whether this putative Trojan horse effect may induce epithelial cell damage, a prominent cause of increased bronchial hyper-reactivity in people with asthma. We are currently studying that possibility, including the effects of ROR being generated in macrophages on epithelial cells.
How do DEP induce toxic levels of ROR in macrophages? Although
phagocytosis-related enzyme systems, such as membrane NADPH-oxidase,
can contribute to ROR generation (53), macrophages are
generally protected from their own respiratory burst activity. This
explains the lack of an apoptotic effect by carbon black particles or
organically extracted DEP (Fig. 5). Instead, native DEP or organic
extracts made from those particles are effective in inducing apoptosis
(
Figs. 4–7). This suggests that DEP chemicals are responsible for
generating the additional oxidant stress levels that are required to
initiate apoptosis. Although these DEP-associated chemicals need to be
identified, we know that DEP contain polycyclic aromatic compounds,
halogenated aromatic hydrocarbons, and redox-active quinones
(22, 27 27A, 28, 29), all known inducers of ROR (24, 30, 31, 32, 33) and
apoptosis (41, 42). ROR generation by these chemicals
involves drug-metabolizing enzymes such as microsomal p450 reductase
and quinone reductases (33). In this regard, Kumagai et
al. have demonstrated that the interaction of DEP-derived chemicals
with NADPH-cytochrome p450 reductase generates reactive oxygen species
(33). The same group demonstrated that DEP extracts induce
superoxide and hydroxyl radical generation upon coincubation with lung
microsomal preparations (33). Because macrophages express
xenobiotic converting enzymes, it is likely that a major source of ROR
generation in the DEP-exposed lung is macrophages. Our data using DCF
fluorescence confirm that DEP and DEP extracts generate ROR in
macrophages (Fig. 8). Although we lack data about the specific
chemicals in DEP extracts that are responsible for this effect, we do
know that this is rapid event that peaks within 3 h (Fig. 8B). Moreover, this event appears to be specific for
macrophages, because we did not find any evidence for ROR generation in
a bronchial epithelial cell line, BEAS-2B, coincubated with DEP (Table I). The selective targeting of macrophages depends on the ability of
these cells to ingest and concentrate DEP in the cell. At least one
other group has demonstrated generation of oxidant stress by
particulate pollutants in alveolar macrophages (54).
There are at least two major caspase cascades that play a role in
apoptosis, one commencing with the activation of caspase 8 and the
other with caspase 9 (49, 50, 51). Procaspase 8 is activated
by membrane receptors that recruit proteins with death effector domains
(49), while procaspase 9 is activated by a mitochondrial
released protein, cytochrome c (50). The
ability of DEP to induce both caspases may reflect distinct initiation
pathways (Fig. 6). It is known, however, that some membrane receptors
that activate caspase 8 may induce cytochrome c release, and
it is possible, therefore, that caspase 8 activation may lead to
caspase 9 activation. Although we still have to obtain evidence for
mitochondrial involvement in our in vitro culture system, ROR
generation has been shown to induce lipid peroxidation and protein
oxidation in mitochondrial membranes (50). This may lead
to cytochrome c release, which in the presence of the
cofactor, Apaf-1, will induce procaspase 9 activation
(49). This idea fits with our observations that ROR
generation precedes the onset of apoptosis in RAW 274.7 and THP-1 cells
(Figs. 4A and 8). Moreover, the inhibitory effect of NAC in
our cells agrees with observations that NAC disrupts stress-induced
apoptosis in other cell types (55). We are in the process
of investigating the role of mitochondria in DEP-induced apoptosis.
Regarding the role of receptor-mediated apoptosis pathways, it is
interesting that the stress-activated protein kinase (JNK) plays a role
in Fas ligand and TNF-
expression (56, 57). However, we
did not observe any reduction in DEP-induced apoptosis using the Fas-Fc
recombinant protein, which interferes with Fas ligand binding to Fas
(not shown). There was also no significant effect of the DEP extract on
TNF- production, arguing against a role of the p55 TNF receptor
(57). We did, however, observe a small decrease in the
rate of apoptosis in the presence of a p38MAPK
inhibitor, but it is not clear whether this inhibitory effect involves
a receptor-mediated death event. There is no direct way to explore the
involvement of JNK cascade in DEP-induced apoptosis at present.
Elucidation of target cells and the mechanism of action of DEP in the respiratory tract are of considerable clinical importance. The demonstration, for instance, that NAC effectively inhibited DEP-induced ROR generation and apoptosis induction predicts that antioxidants may be helpful in alleviating the effects of particulate pollutants in diseases such as asthma and COPD. Although a number of oral antioxidants are available, it may be useful to consider specific intrabronchial drug formulations that will target macrophages. For instance, it has been demonstrated that polyethylene glycol-aggregated superoxide dismutase, instilled intratracheally, reverses DEP-induced airway hyper-reactivity in mice (58).
In summary, we have elucidated a toxic cellular response that may explain the adverse effects of DEP in the respiratory tract. We predict that this type of cellular response may also be applicable to understanding the mechanisms of action of other toxic environmental pollutants in the lung.
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Acknowledgments |
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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 School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; DEP, diesel exhaust particles; DCF-DA, dichlorofluorescein diacetate; HAH, halogenated aromatic hydrocarbons; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAC, N-acetyl-cysteine; PM10, particulate matter (10 µm); PI, propidium iodide; PAH, polycyclic aromatic hydrocarbon; ROR, reactive oxygen radicals; SAPK, stress-activated protein kinase; z-VAD-fmk, z-Val-Ala-Asp(OMe)-fluoromethyl ketone.
Received for publication May 7, 1999. Accepted for publication August 25, 1999.
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References |
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  F. M. Arantes-Costa, F. D.T.Q.S. Lopes, A. C. Toledo, P. A. Magliarelli-Filho, H. T. Moriya, R. Carvalho-Oliveira, T. Mauad, P. H. N. Saldiva, and M. A. Martins Effects of Residual Oil Fly Ash (ROFA) in Mice with Chronic Allergic Pulmonary Inflammation Toxicol Pathol, July 1, 2008; 36(5): 680 - 686. [Abstract] [Full Text] [PDF]
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 T. Stevens, Q. T. Krantz, W. P. Linak, S. Hester, and M. I. Gilmour Increased Transcription of Immune and Metabolic Pathways in Naive and Allergic Mice Exposed to Diesel Exhaust Toxicol. Sci., April 1, 2008; 102(2): 359 - 370. [Abstract] [Full Text] [PDF]
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 J. A. Araujo, B. Barajas, M. Kleinman, X. Wang, B. J. Bennett, K. W. Gong, M. Navab, J. Harkema, C. Sioutas, A. J. Lusis, et al. Ambient Particulate Pollutants in the Ultrafine Range Promote Early Atherosclerosis and Systemic Oxidative Stress Circ. Res., March 14, 2008; 102(5): 589 - 596. [Abstract] [Full Text] [PDF]
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 N. Amara, R. Bachoual, M. Desmard, S. Golda, C. Guichard, S. Lanone, M. Aubier, E. Ogier-Denis, and J. Boczkowski Diesel exhaust particles induce matrix metalloprotease-1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox-dependent mechanism Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L170 - L181. [Abstract] [Full Text] [PDF]
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 K. Inoue, H. Takano, K. Hiyoshi, T. Ichinose, K. Sadakane, R. Yanagisawa, S. Tomura, and Y. Kumagai Naphthoquinone enhances antigen-related airway inflammation in mice Eur. Respir. J., February 1, 2007; 29(2): 259 - 267. [Abstract] [Full Text] [PDF]
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J. C. Bonner Lung Fibrotic Responses to Particle Exposure Toxicol Pathol, January 1, 2007; 35(1): 148 - 153. [Abstract] [Full Text] [PDF]
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 F. D. Gilliland, Y.-F. Li, H. Gong Jr., and D. Diaz-Sanchez Glutathione S-Transferases M1 and P1 Prevent Aggravation of Allergic Responses by Secondhand Smoke Am. J. Respir. Crit. Care Med., December 15, 2006; 174(12): 1335 - 1341. [Abstract] [Full Text] [PDF]
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 P E Schwarze, J Ovrevik, M Lag, M Refsnes, P Nafstad, R B Hetland, and E Dybing Particulate matter properties and health effects: consistency of epidemiological and toxicological studies Human and Experimental Toxicology, October 1, 2006; 25(10): 559 - 579. [Abstract] [PDF]
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 Z. Li, X. Hyseni, J. D. Carter, J. M. Soukup, L. A. Dailey, and Y.-C. T. Huang Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells Am J Physiol Cell Physiol, August 1, 2006; 291(2): C357 - C365. [Abstract] [Full Text] [PDF]
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H. Shima, E. Koike, R. Shinohara, and T. Kobayashi Oxidative Ability and Toxicity of n-Hexane Insoluble Fraction of Diesel Exhaust Particles Toxicol. Sci., May 1, 2006; 91(1): 218 - 226. [Abstract] [Full Text] [PDF]
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H. Bayram, K. Ito, R. Issa, M. Ito, M. Sukkar, and K. F. Chung Regulation of human lung epithelial cell numbers by diesel exhaust particles Eur. Respir. J., April 1, 2006; 27(4): 705 - 713. [Abstract] [Full Text] [PDF]
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 M. Alkio, T. M. Tabuchi, X. Wang, and A. Colon-Carmona Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms J. Exp. Bot., November 1, 2005; 56(421): 2983 - 2994. [Abstract] [Full Text] [PDF]
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I. Jaspers, J. M. Ciencewicki, W. Zhang, L. E. Brighton, J. L. Carson, M. A. Beck, and M. C. Madden Diesel Exhaust Enhances Influenza Virus Infections in Respiratory Epithelial Cells Toxicol. Sci., June 1, 2005; 85(2): 990 - 1002. [Abstract] [Full Text] [PDF]
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 T. Ohtani, S. Nakagawa, M. Kurosawa, M. Mizuashi, M. Ozawa, and S. Aiba Cellular Basis of the Role of Diesel Exhaust Particles in Inducing Th2-Dominant Response J. Immunol., February 15, 2005; 174(4): 2412 - 2419. [Abstract] [Full Text] [PDF]
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X. J. Yin, J. Y. C. Ma, J. M. Antonini, V. Castranova, and J. K. H. Ma Roles of Reactive Oxygen Species and Heme Oxygenase-1 in Modulation of Alveolar Macrophage-Mediated Pulmonary Immune Responses to Listeria monocytogenes by Diesel Exhaust Particles Toxicol. Sci., November 1, 2004; 82(1): 143 - 153. [Abstract] [Full Text] [PDF]
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F. Gao, A. Barchowsky, A. A. Nemec, and J. P. Fabisiak Microbial Stimulation by Mycoplasma fermentans Synergistically Amplifies IL-6 Release by Human Lung Fibroblasts in Response to Residual Oil Fly Ash (ROFA) and Nickel Toxicol. Sci., October 1, 2004; 81(2): 467 - 479. [Abstract] [Full Text] [PDF]
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C.-J. G. Pan, D. A. Schmitz, A. K. Cho, J. Froines, and J. M. Fukuto Inherent Redox Properties of Diesel Exhaust Particles: Catalysis of the Generation of Reactive Oxygen Species by Biological Reductants Toxicol. Sci., September 1, 2004; 81(1): 225 - 232. [Abstract] [Full Text] [PDF]
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N. Li, J. Alam, M. I. Venkatesan, A. Eiguren-Fernandez, D. Schmitz, E. Di Stefano, N. Slaughter, E. Killeen, X. Wang, A. Huang, et al. Nrf2 Is a Key Transcription Factor That Regulates Antioxidant Defense in Macrophages and Epithelial Cells: Protecting against the Proinflammatory and Oxidizing Effects of Diesel Exhaust Chemicals J. Immunol., September 1, 2004; 173(5): 3467 - 3481. [Abstract] [Full Text] [PDF]
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C.-C. Lee, J.-W. Liao, and J.-J. Kang Motorcycle Exhaust Particles Induce Airway Inflammation and Airway Hyperresponsiveness in BALB/C Mice Toxicol. Sci., June 1, 2004; 79(2): 326 - 334. [Abstract] [Full Text] [PDF]
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Q. Zhang, S. R. Kleeberger, and S. P. Reddy DEP-induced fra-1 expression correlates with a distinct activation of AP-1-dependent gene transcription in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L427 - L436. [Abstract] [Full Text] [PDF]
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 G. G. Xiao, M. Wang, N. Li, J. A. Loo, and A. E. Nel Use of Proteomics to Demonstrate a Hierarchical Oxidative Stress Response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line J. Biol. Chem., December 12, 2003; 278(50): 50781 - 50790. [Abstract] [Full Text] [PDF]
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 M. Dybdahl, L. Risom, P. Moller, H. Autrup, H. Wallin, U. Vogel, J. Bornholdt, B. Daneshvar, L. O. Dragsted, A. Weimann, et al. DNA adduct formation and oxidative stress in colon and liver of Big Blue(R) rats after dietary exposure to diesel particles Carcinogenesis, November 1, 2003; 24(11): 1759 - 1766. [Abstract] [Full Text] [PDF]
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C. L. Galindo, J. Sha, D. A. Ribardo, A. A. Fadl, L. Pillai, and A. K. Chopra Identification of Aeromonas hydrophila Cytotoxic Enterotoxin-induced Genes in Macrophages Using Microarrays J. Biol. Chem., October 10, 2003; 278(41): 40198 - 40212. [Abstract] [Full Text] [PDF]
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R. McConnell, K. Berhane, F. Gilliland, J. Molitor, D. Thomas, F. Lurmann, E. Avol, W. J. Gauderman, and J. M. Peters Prospective Study of Air Pollution and Bronchitic Symptoms in Children with Asthma Am. J. Respir. Crit. Care Med., October 1, 2003; 168(7): 790 - 797. [Abstract] [Full Text] [PDF]
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H.-P. Tzeng, R.-S. Yang, T.-H. Ueng, S.-Y. Lin-Shiau, and S.-H. Liu Motorcycle Exhaust Particulates Enhance Vasoconstriction in Organ Culture of Rat Aortas and Involve Reactive Oxygen Species Toxicol. Sci., September 1, 2003; 75(1): 66 - 73. [Abstract] [Full Text] [PDF]
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A. Baulig, M. Garlatti, V. Bonvallot, A. Marchand, R. Barouki, F. Marano, and A. Baeza-Squiban Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L671 - L679. [Abstract] [Full Text] [PDF]
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 R. A. Canuto, G. Muzio, M. Maggiora, A. Trombetta, G. Martinasso, R. Autelli, P. Costelli, G. Bonelli, and F. M. Baccino Apoptosis induced by clofibrate in Yoshida AH-130 hepatoma cells: role of HMG-CoA reductase J. Lipid Res., January 1, 2003; 44(1): 56 - 64. [Abstract] [Full Text] [PDF]
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N. Li, M. Wang, T. D. Oberley, J. M. Sempf, and A. E. Nel Comparison of the Pro-Oxidative and Proinflammatory Effects of Organic Diesel Exhaust Particle Chemicals in Bronchial Epithelial Cells and Macrophages J. Immunol., October 15, 2002; 169(8): 4531 - 4541. [Abstract] [Full Text] [PDF]
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N. Rudra-Ganguly, S. T. Reddy, P. Korge, and H. R. Herschman Diesel Exhaust Particle Extracts and Associated Polycyclic Aromatic Hydrocarbons Inhibit Cox-2-dependent Prostaglandin Synthesis in Murine Macrophages and Fibroblasts J. Biol. Chem., October 11, 2002; 277(42): 39259 - 39265. [Abstract] [Full Text] [PDF]
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 S B Gordon and R C Read Macrophage defences against respiratory tract infections: The immunology of childhood respiratory infections Br. Med. Bull., March 1, 2002; 61(1): 45 - 61. [Abstract] [Full Text] [PDF]
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M. J. Whitekus, N. Li, M. Zhang, M. Wang, M. A. Horwitz, S. K. Nelson, L. D. Horwitz, N. Brechun, D. Diaz-Sanchez, and A. E. Nel Thiol Antioxidants Inhibit the Adjuvant Effects of Aerosolized Diesel Exhaust Particles in a Murine Model for Ovalbumin Sensitization J. Immunol., March 1, 2002; 168(5): 2560 - 2567. [Abstract] [Full Text] [PDF]
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 V. Bonvallot, A. Baeza-Squiban, A. Baulig, S. Brulant, S. Boland, F. Muzeau, R. Barouki, and F. Marano Organic Compounds from Diesel Exhaust Particles Elicit a Proinflammatory Response in Human Airway Epithelial Cells and Induce Cytochrome p450 1A1 Expression Am. J. Respir. Cell Mol. Biol., October 1, 2001; 25(4): 515 - 521. [Abstract] [Full Text] [PDF]
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 L. J. Pagliari, H. Perlman, H. Liu, and R. M. Pope Macrophages Require Constitutive NF-kappa B Activation To Maintain A1 Expression and Mitochondrial Homeostasis Mol. Cell. Biol., December 1, 2000; 20(23): 8855 - 8865. [Abstract] [Full Text]
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N. Li, M. I. Venkatesan, A. Miguel, R. Kaplan, C. Gujuluva, J. Alam, and A. Nel Induction of Heme Oxygenase-1 Expression in Macrophages by Diesel Exhaust Particle Chemicals and Quinones via the Antioxidant-Responsive Element J. Immunol., September 15, 2000; 165(6): 3393 - 3401. [Abstract] [Full Text] [PDF]
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T. S. Hiura, N. Li, R. Kaplan, M. Horwitz, J.-C. Seagrave, and A. E. Nel The Role of a Mitochondrial Pathway in the Induction of Apoptosis by Chemicals Extracted from Diesel Exhaust Particles J. Immunol., September 1, 2000; 165(5): 2703 - 2711. [Abstract] [Full Text] [PDF]
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O. Fahy, H. Hammad, S. Sénéchal, J. Pestel, A.-B. Tonnel, B. Wallaert, and A. Tsicopoulos Synergistic Effect of Diesel Organic Extracts and Allergen Der p 1 on the Release of Chemokines by Peripheral Blood Mononuclear Cells from Allergic Subjects . Involvement of the MAP Kinase Pathway Am. J. Respir. Cell Mol. Biol., August 1, 2000; 23(2): 247 - 254. [Abstract] [Full Text]
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