Induction of apoptosis by particulate matter: role
of TNF-
and MAPK
Beek Yoke
Chin1,5,
Mary E.
Choi2,3,
Marie D.
Burdick4,
Robert M.
Strieter4,
Terence H.
Risby5, and
Augustine M. K.
Choi1,3,6
6 Section of Pulmonary and
Critical Care Medicine and
2 Section of Nephrology,
Department of Internal Medicine, Yale University School of Medicine,
New Haven 06250; 3 Connecticut
Veterans Affairs HealthCare System, West Haven, Connecticut 06516;
1 Division of Pulmonary and
Critical Care Medicine and
5 Department of Environmental
Health Sciences, The Johns Hopkins Medical Institutions, Baltimore,
Maryland 21205; and 4 Division of
Pulmonary and Critical Care Medicine, University of Michigan, Ann
Arbor, Michigan 48109
 |
ABSTRACT |
Particulate matter (PM) is a major by-product
from the combustion of fossil fuels. The biological target of inhaled
PM is the pulmonary epithelium and resident macrophages. In this study, we demonstrate that cultured macrophages (RAW 264.7 cells) exposed continously to a well-defined model of PM
[benzo[a]pyrene adsorbed on carbon black
(CB+BaP)] exhibit a time-dependent expression and release of the
cytokine tumor necrosis factor-
(TNF-
). CB+BaP also evoked
programmed cell death or apoptosis in cultured macrophages as assessed
by genomic DNA-laddering assays. The CB+BaP-induced apoptosis was
inhibited when macrophages were treated with CB+BaP in the presence of
a neutralizing antibody to TNF-
, suggesting that TNF-
plays an
important role in mediating CB+BaP-induced apoptosis in macrophages.
Interestingly, neither untreated carbon black nor
benzo[a]pyrene alone induced apoptosis or caused
the release of TNF-
in RAW 264.7 cells. Moreover, we observed that TNF-
activates mitogen-activated protein kinase (MAPK) activity, the
extracellular signal-regulated kinases p42/p44, in a time-dependent manner. RAW 264.7 cells treated with PD-098059, a selective inhibitor of MAPK kinase activity, did not exhibit CB+BaP-induced apoptosis and
TNF-
secretion. Furthermore, cells treated with the MAPK kinase
inhibitor did not undergo TNF-
-induced apoptosis. Taken together,
our data suggest that TNF-
mediates PM-induced apoptosis and that
the MAPK pathway may play an important role in regulating this pathway.
tumor necrosis factor-
; mitogen-activated protein kinase; programmed cell death; cytokines; carbon; signal transduction; macrophages
 |
INTRODUCTION |
EPIDEMIOLOGICAL STUDIES conducted in urban centers have
shown correlations between increases in morbidity and mortality in various respiratory ailments and exposure to environmental air pollution. Much of this increased morbidity and mortality has been
demonstrated in susceptible populations, including people suffering
from diseases such as asthma (14) or pulmonary fibrosis (32) as well as
individuals who are immunocompromised (30). Among the multitude of
agents contributing to environmental air pollution in our society,
particulate matter (PM) generated from the combustion of fossil fuels
represents a major culprit. PM derived from the combustion of fossil
fuels consists of an inert carbonaceous core and multiple layers of
adsorbed pollutant molecules. After inhalation and deposition in the
lung, the majority of the adsorbed pollutants are released into the
pulmonary surfactant and will eventually reach the pulmonary epithelial
cells (3, 25). After this initial release, the particles with their
residual burden of adsorbed pollutants will remain on the surface of
the pulmonary surfactant until cleared by the mucociliary escalator or
phagocytosed by the resident macrophages (18, 23) where the residual
pollutants may be eventually released.
Our current understanding of how PM affects lung function and how it
affects the inflammatory process in the lung is poor. Limited data
exist supporting the notion that PM can modulate the inflammatory
response in the lung. For example, an in vivo study (10) demonstrated
that PM can induce an influx of inflammatory cells such as
polymorphonuclear cells and macrophages into the airways. Cultured
macrophages exhibit increased secretion of cytokines such as
interleukin-1
and tumor necrosis factor-
(TNF-
) when exposed
to other types of particles such as fungus
Micropolyspora faeni, mineral dusts,
crystalline silica, titanium dioxide, and asbestos (8, 11, 12, 17, 20,
29).
In this study, we have chosen a well-defined model for PM,
benzo[a]pyrene (BaP) adsorbed onto a carbon
black (CB; CB+BaP) at a defined surface coverage, to examine the
mechanism(s) by which PM modulates the inflammatory response in
cultured macrophages. This particle complex has been shown previously
to be a good model for the carbonaceous particles produced by the
combustion of fossil fuels (28). We show in our in vitro model that PM
(CB+BaP) evoked a time-dependent expression and release of the cytokine
TNF-
. Additionally, we demonstrate that PM-induced TNF-
plays an
important role in mediating PM-induced apoptosis and provide evidence
that the mitogen-activated protein kinase (MAPK) pathway plays an
important role in regulating PM- and TNF-
-induced apoptosis in this
cell culture model.
 |
MATERIALS AND METHODS |
Cell culture. Murine peritoneal
macrophage cell line RAW 264.7 cells (American Type Culture Collection,
Manassas, VA) were maintained in DMEM supplemented with 10% fetal
bovine serum and gentamicin (50 µg/ml) at 37°C in a humidified
atmosphere of 5% CO2-95% air.
All experiments were conducted in subconfluent cells.
Model carbon particles. The CB (N339)
selected for study was manufactured under well-defined conditions
specified by the American Society for Testing Materials. This CB was
selected because it has been shown to have similar surface properties
to the carbonaceous particles produced by the combustion of fossil
fuels (27). Moreover, because it has been well established that
combustion of fossil fuels produces gaseous- and particle-phase
pollutants, a defined amount (three-fourths of a unimolecular surface
coverage) of a typical pollutant, BaP, was adsorbed onto the surface of
the CB. This surface coverage was selected because Risby et al.
(26) determined that particles have similar surface
coverages after the initial release of adsorbed molecules into
pulmonary surfactant.
Exposure of cultured macrophages to model carbon
particles. The model particles (CB+BaP) were
deaggregated to form stable particles, with a mean diameter of 0.1 µm
by homogenization in DMEM at 3,000 g
for 1 h. After this deaggregation, the particles remained suspended for
at least 48 h. The cells were exposed to known concentrations of this
suspension (2 µg/ml) for up to 24 h. Suspensions of deaggregated
untreated CB or free BaP (2 µg/ml) were used as controls for this
study.
Western blot analysis. Total cellular
protein extracts were obtained for the Western analyses as previously
described (29). Briefly, cells were lysed in buffer containing 1%
Nonidet P-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM
Na3VO4,
1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of aprotinin.
Protein concentrations of the cell lysates were determined by Coomassie
blue dye-binding assay (Bio-Rad, Hercules, CA). An equal volume of
2× SDS loading buffer (0.125 mM Tris · HCl, pH
7.4, 4% SDS, and 20% glycerol) was added, and the samples were boiled
for 5 min. Protein samples (100 µg) were resolved by 12% SDS-PAGE,
then electroblotted onto polyvinylidene fluoride membranes (Millipore,
Bedford, MA). The membranes were incubated with TNF-
rabbit
polyclonal antibody (1:500; Biosource International, Camarillo, CA) for
1.5 h, followed by incubation with horseradish peroxidase-conjugated
anti-rabbit antibody for 1.5 h. Signal development was carried out with
an enhanced chemiluminescence detection kit (Amersham).
ELISA assay for TNF-
. Cell culture
medium was collected at various time points after exposure to CB+BaP
and stored at
80°C until assayed. TNF-
protein was
determined by ELISA kits (Biosource International) and quantified with
a plate reader (model EL340, Bio-Tek Instruments, Winooski, VT). Values
are reported as nanograms per milliliter of medium. Neutralizing
antibody to TNF-
was obtained from Biosource International.
Genomic DNA isolation and DNA laddering
analysis. Genomic DNA isolation was performed as
specified by the manufacturer's protocol (Puregene kit, Gentra
Systems, Minneapolis, MN). Briefly, cells were lysed directly on the
plate after medium removal with lysis buffer, followed by a 1-h
incubation with RNase A. The cell lysates were precipitated for
proteins and spun at 2,000 g for 15 min. Supernatant was precipitated with isopropanol for isolation of DNA. After an alcohol wash, DNA was hydrated and quantified, and 20 µg were analyzed in 1.5% agarose gel electrophoresis fractionation.
MAPK activity assays. MAPK activity
assays were performed according to the manuufacturer's instructions
(New England Biolabs, Beverly, MA) with minor modifications. Briefly,
cells were lysed in a buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3VO4,
1 µg/ml of leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and
sheared by passage through a 25-gauge needle. Protein concentrations
were determined as described in Western blot
analysis. Total protein samples (200 µg) were
incubated with phospho-specific p44/42 MAPK rabbit polyclonal antibody
(1:50) overnight on a rocker at 4°C. The p44/42 MAPK antibodies
detect only the 204-tyrosine-phosphorylated forms of extracellular
signal-regulated kinase (ERK) 1 and ERK2 and thus select for the
activated (phosphorylated) MAPK. As a positive control, 20 ng of active
MAPK (ERK2) were incubated with a control cell extract. Protein
A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) were then added
to immunoprecipitate the activated MAPK complex. The immunoprecipitated
pellets were incubated with 1 µg of Elk1 fusion protein in the
presence of 100 µM ATP and a kinase buffer (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4,
and 10 mM MgCl2). The reaction was terminated with SDS loading buffer [62.5 mM
Tris · HCl, pH 6.8, 2% (wt/vol) SDS, 10%
glycerol, 50 mM dithiothreitol, and 0.1% (wt/vol) bromophenol
blue]. The samples were analyzed by 12% SDS-PAGE and
electroblotted as described in Western blot
analysis. ERK activity was assayed by detection of
phosphorylated Elk1 with a phospho-specific Elk1 rabbit polyclonal
antibody (1:1,000). After overnight incubation with the primary
antibody at 4°C, the membrane was incubated for 1 h with a
horseradish peroxidase-conjugated anti-rabbit secondary antibody
(1:2,000) at room temperature with gentle rocking. The proteins were
subsequently detected with LumiGLO (New England Biolabs) and exposed to
X-ray film. Because ERK2 is known to be capable of phosphorylating
Elk1, MAPK activity was determined by a phospho-specific Elk1 antibody
that detects only the 383-serine-phosphorylated Elk1.
Statistical analysis. Data are
expressed as means ± SE. Differences in measured variables between
experimental and control groups were assessed with Student's
t-test. Statistical calculations were
performed on a Macintosh personal computer with the Statview II
statistical package (Abacus Concepts, Berkeley, CA). Significant difference was accepted at P < 0.05.
 |
RESULTS |
CB+BaP-induced TNF-
expression and
secretion. RAW 264.7 cells were treated with CB+BaP (2 µg/ml), and the cells and medium were collected for Western blot and
enzyme-linked immunosorbent assay (ELISA) analyses, respectively.
Figure
1A shows
rapid induction of TNF-
protein expression by Western blot analysis,
with increased TNF-
expression by 1 h and sustained elevated TNF-
expression up to 8 h of CB+BaP treatment. The increase in TNF-
protein expression was associated with increased TNF-
protein
secretion in RAW 264.7 cells in response to CB+BaP as assessed by
ELISA. Figure 1B illustrates a
time-dependent induction of TNF-
secretion in RAW 264.7 cells by
CB+BaP.

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Fig. 1.
Macrophages (RAW 264.7 cells) exposed to carbon black (CB) and
benzo[a]pyrene (BaP; CB+BaP) induce expression
and secretion of tumor necrosis factor (TNF)- .
A: total cellular protein was
extracted after CB+BaP (2 µg/ml) treatment and then analyzed for
TNF- protein expression by Western blot analyses as described in
MATERIALS AND METHODS.
Lane 1, untreated control;
lane 2, 1-h CB+BaP;
lane 3, 2-h CB+BaP;
lane 4, 4-h CB+BaP;
lane 5, 8-h CB+BaP;
lane 6, 24-h CB+BaP. Data are
representative of 3 independent experiments.
B: supernatants were collected from
cells at indicated times of CB+BaP (2 µg/ml) exposure, and ELISAs
were performed as described in MATERIALS AND
METHODS. CTL, untreated control cells. Data are means ± SE of measurements from 3 independent experiments.
P < 0.03 for each CB+BaP-treated
sample compared with CTL.
|
|
TNF-
mediates CB+BaP-induced
apoptosis. In view of our observations that CB+BaP
induces induction of TNF-
expression and secretion (Fig. 1) and that
TNF-
has been strongly implicated in the induction of apoptosis in
various in vitro models (1, 9, 15), we investigated whether TNF-
induction by CB+BaP may mediate CB+BaP-induced apoptosis in RAW 264.7 cells. Initially, we established whether CB+BaP alone can induce cell
death via programmed cell death or apoptosis. Genomic DNA was isolated
from RAW 264.7 cells after CB+BaP treatment and analyzed for
nucleosomal fragmentation or DNA laddering. Figure
2A shows
DNA laddering at 24 h, with pronounced DNA fragmentation at 48 h of
CB+BaP treatment. We then demonstrated that TNF-
can also induce
apoptosis in RAW 264.7 cells. Genomic DNA was isolated from RAW 264.7 cells after treatment with TNF-
(1 ng/ml) and then analyzed for DNA
fragmentation by gel electrophoresis. Figure
2B shows marked DNA laddering in cells
after treatment with TNF-
(1 ng/ml). To determine whether TNF-
played a role in the CB+BaP-induced apoptosis in RAW 264.7 cells, we
exposed cells to CB+BaP in the absence and presence of a neutralizing
antibody against TNF-
. As shown in Fig.
3A, DNA
laddering is evident in RAW 264.7 cells exposed to CB+BaP (lane 2). However, when cells were
exposed to CB+BaP in the presence of neutralizing antibody to TNF-
(lane 4), no evidence for DNA laddering was observed. These observations suggest that CB+BaP-induced apoptosis in RAW 264.7 cells may be mediated by TNF-
. Interestingly, RAW 264.7 cells exposed to either untreated CB or free BaP alone had a
negligible effect on TNF-
secretion as assessed by ELISA, whereas
the combination of CB and BaP induced a marked secretion of TNF-
(Fig. 4). Based on the observations (Figs.
1-3) that TNF-
may play an important role in mediating
CB+BaP-induced apoptosis in RAW 264.7 cells, we hypothesized that CB or
BAP alone would not induce apoptosis in RAW 264.7 cells due to their
inability to induce TNF-
. This was confirmed by exposing RAW 264.7 cells to CB and BAP alone: no apoptosis as assessed by DNA-laddering assays was exhibited (Fig. 5).

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Fig. 2.
A: CB+BaP-induced apoptosis in RAW 264.7 cells. Genomic DNA
was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 and 48 h
and fractionated in a 1.5% agarose gel electrophoresis as described in
MATERIALS AND METHODS. MW, molecular-weight marker.
B: TNF- -induced apoptosis in RAW 264.7 cells. Genomic DNA
was isolated from cells exposed to TNF- (1 ng/ml) for 24 h and
fractionated in a 1.5% agarose gel electrophoresis as described in
MATERIALS AND METHODS. Data are representative of 4 independent experiments.
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Fig. 3.
Effect of neutralizing antibody to TNF- on CB+BaP-induced apoptosis.
A: genomic DNA was isolated from cells
exposed to CB+BaP (2 µg/ml) for 24 h in absence and presence of
neutralizing antibody to TNF- (100 U) and then fractionated in a
1.5% agarose gel electrophoresis as described in
MATERIALS AND METHODS.
Lane 1, CTL; lane
2, CB+BaP alone; lane
3, TNF- antibody alone; lane
4, CB+BaP + neutralizing antibody to TNF- . Data are
representative of 3 independent experiments.
B: genomic DNA was isolated from cells
exposed to CB+BaP (2 µg/ml) for 24 h in absence and presence of
control isotype IgG antibody and then fractionated in a 1.5% agarose
gel electrophoresis as described in MATERIALS AND
METHODS. Lane 1, CTL;
lane 2, CB+BaP alone;
lane 3, IgG antibody alone;
lane 4, CB+BaP + IgG antibody.
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Fig. 4.
TNF- secretion is not affected in RAW 264.7 cells exposed to
untreated CB or BaP. Supernatants were collected from cells after
exposure to 24 h of untreated CB (2 µg/ml), BaP (2 µg/ml), or
CB+BaP (2 µg/ml), and ELISAs were performed as described in
MATERIALS AND METHODS. Data are means ± SE of measurements from 3 independent experiments.
* P < 0.05 compared with
CTL.
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Fig. 5.
Effect of CB or BaP on apoptosis in RAW 264.7 cells. Genomic DNA was
isolated from cells exposed to 24 h of untreated CB (2 µg/ml), BaP (2 µg/ml), or CB+BaP (2 µg/ml) alone and fractionated in a 1.5%
agarose gel electrophoresis as described in MATERIALS
AND METHODS. Lane 1,
CTL; lane 2, CB; lane
3, BaP; lane 4,
CB+BaP. Data are representative of 3 independent experiments.
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TNF-
activates MAPK (ERK1/ERK2) in RAW 264.7 cells. RAW 264.7 cells were treated with TNF-
(1 ng/ml), and p44/p42erk1/2
immunoprecipitation assays were performed with the transcriptional factor Elk1 as our substrate for measuring MAPK (ERK1/ERK2) activities. Figure 6A
demonstrates that ERK1 and -2 are both activated after 1 h of TNF-
treatment, and their activities are maintained throughout the duration
of the 24-h exposure. Furthermore, we investigated whether CB+BaP alone
can also activate the MAPK (ERK1/ERK2) pathway. Figure
6B illustrates the activation of MAPK
(ERK1/ERK2) in RAW 264.7 cells in response to CB+BaP.

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Fig. 6.
A: effect of TNF- on
mitogen-activated protein kinase (MAPK) activity (p44/p42) in RAW 264.7 cells. Cellular lysates were isolated at indicated times after
treatment with TNF- (1 ng/ml) and were analyzed for MAPK activity as
described in MATERIALS AND METHODS.
+CTL, positive control [extracellular signal-regulated kinase
(ERK) 2] provided by manufacturer as described in
MATERIALS AND METHODS. Data are
representative of 4 independent experiments.
B: effect of CB+BaP on MAPK activity
(p44/p42) in RAW 264.7 cells. Cellular lysates were isolated after
treatment with CB+BaP (2 µg/ml) and were analyzed for MAPK activity
as described in MATERIALS AND METHODS.
Lane 1, CTL; lane
2, 2-h CB+BaP.
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|
Role of MAPK pathway in mediating CB+BaP-induced
apoptosis in RAW 264.7 cells. Based on our observations
that TNF-
or CB+BaP alone can stimulate MAPK activity (Fig. 6) and
may mediate CB+BaP-induced apoptosis in RAW 264.7 cells, we
investigated whether inhibiting the MAPK pathway would result in the
attenuation of both CB+BAP-induced TNF-
release and CB+BAP-induced
apoptosis in RAW 264.7 cells. Cells were treated with CB+BaP in the
presence and absence of the MAPK kinase (MEK) inhibitor PD-098059. MEK
is an upstream activator of ERK1/ERK2 and has been shown to regulate
the activity of ERK1/ERK2 in response to TNF-
(24, 34, 35). We did
not observe any differences in the amounts of TNF-
secreted between the control cells (untreated) and the cells treated with the MEK inhibitor alone (Fig. 7). Marked induction
of TNF-
secretion was observed in cells treated with CP+BaP alone;
however, RAW 264.7 cells exposed to CB+BaP in the presence of the MEK
inhibitor exhibited a marked attenution of TNF-
release, levels
comparable to control levels (Fig. 7). We then performed DNA-laddering
assays from genomic DNA isolated from RAW 264.7 cells that were
pretreated with the MEK inhibitor PD-098059 before exposure to CB+BaP
(Fig. 8). There was no evidence of DNA
laddering in control untreated cells (lane 1)
or MEK inhibitor-treated cells (lane
2). DNA laddering is prominent in cells treated with
CB+BaP alone (lane 3); however, we
did not observe evidence of DNA laddering in cells exposed to CB+BaP in
the presence of the MEK inhibitor (lane
4). Furthermore, we also examined the effect of the
MEK inhibitor on TNF-
-induced apoptosis by DNA-laddering assays. As
illustrated in Fig. 9, the MEK inhibitor
attenuated TNF-
-induced apoptosis in RAW 264.7 cells.

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Fig. 7.
Effect of MAPK kinase (MEK) inhibitor (I) on CB+BaP-induced TNF-
secretion in RAW 264.7 cells. Cells were exposed to CB+BaP with and
without pretreatment with MEK I PD-098059 (10 µM) for 24 h.
Supernatants were collected from cells after exposure, and ELISAs were
performed as described in MATERIALS AND
METHODS. Data are means ± SE of measurements from 3 independent experiments. * P < 0.05 compared with CB + BAP. ** P < 0.05 compared with
CTL.
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Fig. 8.
Effect of MEK I on CB+BaP-induced apoptosis in RAW 264.7 cells. Genomic
DNA was isolated from cells exposed to CB+BaP (2 µg/ml) for 24 h in
absence and presence of MEK I PD-098059 (10 µM) and then fractionated
in a 1.5% agarose gel electrophoresis as described in
MATERIALS AND METHODS.
Lane 1, CTL; lane
2, MEK I PD-098059; lane
3, CB+BaP; lane 4,
CB+BaP + MEK I PD-098059. Data are representative of 3 independent
experiments.
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Fig. 9.
Effect of MEK I on TNF- -induced apoptosis in RAW 264.7 cells.
Genomic DNA was isolated from cells exposed to TNF- (1 ng/ml) for 24 h in absence and presence of MEK I PD-098059 (10 µM) and then
fractionated in a 1.5% agarose gel electrophoresis as described in
MATERIALS AND METHODS.
Lane 1, CTL; lane
2, MEK I PD-098059; lane
3, TNF- ; lane 4,
TNF- + MEK I PD-098059. Data are representative of 3 independent
experiments.
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DISCUSSION |
Combustion of fossil fuels can be inefficient and results in the
generation of airborne environmental PM consisting of a carbonaceous particle with layers of pollutant-adsorbed molecules (36). This carbonaceous PM is increasingly recognized as a major environmental air
pollutant, contributing to the increasing morbidity and mortality of
various respiratory diseases such as asthma (5, 6). The CB used in this
study has defined surface properties that are similar to those of
carbonaceous particles found in PM. If this CB is coated with a defined
surface coverage of BaP, it represents a well-characterized and defined
model for airborne PM.
We demonstrated that carbon particles such as CB+BaP induce the
expression of an inflammatory cytokine, TNF-
, whereas macrophages exposed to untreated carbon particles alone or to a saturated free
solution of BaP show no induction of TNF-
secretion. Interestingly, noncarbonaceous particles such as asbestos, quartz, and silica (17, 29)
induce TNF-
expression and secretion without surface-adsorbed molecules. Moreover, it has been demonstrated that saturated solutions of BaP can be internalized without the presence of carbon particles (4,
13). These results show that the internalization of CBs or BaP
separately do not trigger the secretion of TNF-
. Triggering the
secretion of TNF-
appears to require that the macrophages be exposed
to BaP adsorbed onto the surface of a particle. Because phagocytosis of
CB+BaP is critical to elicit biological effects in macrophages (21,
23), including the induction of stress gene products (data not shown),
and phagocytosis of PM is coupled to the production of reactive oxygen
species, it is reasonable to propose that the surface-adsorbed BaP on
the phagocytosed CB+BaP is activated by intracellular reactive oxygen
species to trigger the induction of TNF-
. The precise biochemical
mechanism(s) by which CB+BaP triggers TNF-
production will require
further study. Interestingly, our observations of this differential
effect of TNF-
secretion between carbon particles with and without
adsorbed molecules on their surfaces have been reported in other
models. For example, Dasenbrock et al. (7) observed differences in the
rate of tumor formation between diesel PM and extracted diesel PM.
We report here that PM such as CB+BaP can induce apoptosis in cultured
macrophages. Our findings are consistent with a recent report that
alveolar macrophages undergo apoptosis when exposed to the
urban particle residual oil fly ash (19). This study, however, did not
examine the signaling pathways or mechanism(s) by which residual oil
fly ash induces apoptosis. We believe that the TNF-
secretion
induced by CB+BaP plays a major role in mediating CB+BaP-induced
apoptosis based on the following observations: 1) TNF-
directly induces
apoptosis in macrophages, and these cells treated with CB+BaP in the
presence of a neutralizing antibody against TNF-
do not exhibit
apoptosis; 2) inhibition of
CB+BaP-induced TNF-
secretion by the MEK inhibitor also inhibits
CB+BaP-induced apoptosis; and 3)
cells treated with BaP or untreated carbon alone do not induce TNF-
release and do not induce apoptosis in macrophages.
The MAPK signaling pathway has been shown to play a major role in
regulating a variety of cellular functions such as cell growth during
proliferation and the stress response to DNA damage; cellular stimuli
including inflammatory cytokines, hyperosmolality, and oxidative
stress; and activation of transcription factors mediating downstream
gene transcription and expression (31, 33). This study suggests that
the ERK1/ERK2 MAPK pathway plays an important role in regulating both
CB+BaP-induced TNF-
secretion and CB+BaP- or TNF-
-induced
apoptosis based on our observations that inhibition of the ERK1/ERK2
pathway resulted in attenuation of both CB+BaP-induced TNF-
secretion and CB+BaP- or TNF-
-induced apoptosis. A schematic diagram
illustrating the possible signaling pathways of CB+BaP-induced
apoptosis in RAW 264.7 macrophage cells is provided to summarize our
proposed model (Fig. 10). Moreover, recent data (2, 22) have also implicated the MAPK pathway in the
regulation of programmed cell death or apoptosis. For example, in a
nerve growth factor withdrawal model of apoptosis, both ERK1/ERK2 and
c-Jun-NH2-terminal kinase MAPKs
play an important role in modulating apoptosis in neuronal tissue. We
have not examined rigorously the role of
c-Jun-NH2-terminal kinase or p38,
the other two MAPK signaling pathways in our CB+BaP model of apoptosis. These studies are currently being pursued.
Is there a functional significance of TNF-
-induced apoptosis in
macrophages exposed to our model of environmental pollutant? Others
(16) have speculated on the role of TNF-
in mediating apoptosis in
parasitic worms, helminths, as "a protective immune response."
These authors have argued that TNF-
induction of an intrinsic
programmed cell death signal may serve to protect the host from
potentially hazardous oxidants and/or radicals generated from
parasitic material. The function of TNF-
in CB+BaP-induced apoptosis
in macrophages may serve as a signal to minimize the inflammatory
process during PM exposure. On the other hand, TNF-
mediates the
recruitment of inflammatory cells such as polymorphonuclear leukocytes
and macrophages, and additional autocrine/paracrine secretion of
TNF-
induced by CB+BaP can further aid in the recruitment of even
more inflammatory cells to the affected areas. This complex regulation
of apoptosis by CB+BaP poses a challenge to a straightforward assessment of the physiological function of apoptosis in lung injury.
Further investigations leading to the identification of cell types
undergoing apoptosis in these models of PM exposure will be helpful in
delineating the functional significance of apoptosis.
 |
ACKNOWLEDGEMENTS |
A. M. K. Choi was supported by National Heart, Lung, and Blood
Institute Grant HL-55330, National Institute of Allergy and Infectious
Diseases Grant AI-42365; and American Heart Association (AHA)
Established Investigator Award. T. H. Risby was supported by National
Institute of Environmental Health Sciences Grant ES-03156. R. M. Strieter was supported by National Cancer Institute Grant CA-66180. M. E. Choi was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant 5-K12-DK-0129809, AHA Grant GIA96015510, and
Department of Veterans Affairs Career Development Award.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. M. K. Choi, Section of Pulmonary and
Critical Care Medicine, Yale Univ. School of Medicine, 333 Cedar St.,
LCI 105, New Haven, CT 06520.
Received 21 May 1998; accepted in final form 30 July 1998.
 |
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