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Research
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Induction of Proinflammatory Cytokines and C-Reactive Protein in Human Macrophage Cell Line U937 Exposed to Air Pollution Particulates Christoph Franz Adam Vogel,1 Eric Sciullo,1 Pat
Wong,1 Paul Kuzmicky,1 Norman Kado,1,2 and
Fumio Matsumura1,3 1Department of Environmental Toxicology, University of California,
Davis, California, USA; 2California Environmental Protection Agency,
Air Resources Board, Sacramento, California, USA; 3Center for Environmental
Health Sciences, University of California, Davis, California, USA Abstract Exposure to particulate matter air pollution causes inflammatory responses and is associated with the progression of atherosclerosis and increased cardiovascular mortality. Macrophages play a key role in atherogenesis by releasing proinflammatory cytokines and forming foam cells in subendothelial lesions. The present study quantified the inflammatory response in a human macrophage cell line (U937) after exposure to an ambient particulate sample from urban dust (UDP) and a diesel exhaust particulate (DEP) . The effect of native UDP and DEP was compared with their corresponding organic extracts (OE-UDP/OE-DEP) and stripped particles (sUDP/sDEP) to clarify their respective roles. Exposure to OE-UDP, OE-DEP, UDP, DEP, and 2,3,7,8-tetrachlorodibenzo-p-dioxin led to a greater increase of interleukin (IL) -8, tumor necrosis factor-, and cyclooxygenase-2 mRNA expression than did the stripped particles, whereas sUDP, sDEP, UDP, and DEP led to a greater production of C-reactive protein and IL-6 mRNA. The particles and the organic extract-induced expression of cyclooxygenase-2 and cytochrome P450 (CYP) 1a1 was significantly suppressed by co-treatment with an aryl hydrocarbon receptor (AhR) antagonist, indicating that these effects are mainly mediated by the organic components, which can activate the AhR and CYP1a1. In contrast, the induction of C-reactive protein and IL-6 seems to be a particle-related effect that is AhR independent. The inflammatory response induced by particulate matter was associated with a subsequent increase of cholesterol accumulation, a hallmark of foam cells. Together, these data illustrate the interaction between particulate matter and the inflammatory response as well as the formation of cholesterol-accumulating foam cells, which are early markers of cardiovascular disease. Key words: aryl hydrocarbon receptor, cyclooxygenase-2, C-reactive protein, cytokines, foam cells, interleukin-8, inflammation, macrophages, particles, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. Environ Health Perspect 113:1536-1541 (2005) . doi:10.1289/ehp.8094 available via http://dx.doi.org/ [Online 21 July 2005] Address correspondence to C. Vogel, Department of Environmental Toxicology, University of California, Davis, CA 95616,USA. Telephone: (530) 752-1337. Fax: (530) 752-5300. E-mail: cfvogel@ucdavis.edu This study was supported by grants ESO5233 and ESO05707 from the National Institute of Environmental Health Sciences and by grant IRG 95-125-07 from the American Cancer Society. The authors declare they have no competing financial interests. Received 7 March 2005 ; accepted 21 July 2005. |
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Numerous epidemiologic studies have observed
that exposure to particulate matter (PM) air pollution,
which occurs in many urban and industrial environments,
is associated with an increase of cardiovascular
diseases and mortality (Brook et al. 2003; Morris
2001). Although the exact components of PM and
the exact mechanism leading to cardiovascular disease
and cardiopulmonary disease mortality from exposure
to PM are still unknown, several studies have shown
that systemic inflammation may be a key step in
these pathological processes through inflammatory
mediators (Salvi et al. 1999) such as cyclooxygenase-2
(COX-2), interleukin (IL)-1β,
and tumor necrosis factor- (TNF ),
which are among the most important mediators of
the inflammatory response (Ross 1999)
in the development of atherosclerotic vascular
disease (Libby 2002). A positive correlation of
C-reactive protein (CRP) and coronary artery disease,
which could be explained by the atherogenic effects
of chronic inflammation, is well known (Beck et
al. 1999; Mendall et al. 1996). Recently, an association
between minor but chronic elevation of serum CRP
levels and future major cardiovascular events has
been shown (Yeh 2004). Elevated levels of proinflammatory
cytokines and CRP play a significant role in the
genesis of atherosclerosis and in plaque instability
(Libby 2002). CRP activates complement through
binding to the Fc receptor
and enhancing phagocytosis of low-density lipoprotein,
leading to the formation of foam cells (Zwaka et
al. 2001), thus directly contributing to the development
of atherosclerosis. Despite the epidemiologic evidence,
experimental verification of the causal relationship
among air pollution, CRP, and cardiovascular disease
is still limited.
In vitro studies show increased levels
of proinflammatory cytokines including TNF,
IL-1β, IL-6,
and IL-8, which have been described in various
cell types after exposure to PM (Mathiesen et al.
2004; Monn et al. 2003; Monn and Becker 1999; van
Eeden et al. 2001). Acute exposure to diesel exhaust
also increased IL-8 production in human airways
(Salvi et al. 2000). However, elevated serum levels
of CRP, the classic acute-phase protein, has only
been found to be associated with exposure to an
elevated concentration of PM in humans (Kim et
al. 2005; Peters et al. 2001; Pope et al. 2004a;
Seaton et al. 1999). Other harmful effects described
by these authors involved the triggering of acute
vasoconstriction and the development of atherosclerosis.
A few animal models have shown the harmful effects
of inhalation of air pollutants on cardiovascular
functions (Campen et al. 2003; Gordon et al. 1998),
as well as on the etiology of atherosclerosis (Suwa
et al. 2002). Suwa et al. (2002) showed that exposure
of rabbits to PM10 (PM with areodynamic
diameter ≤ 10 µm)
causes progression of atherosclerotic lesions,
and a number of alveolar macrophages phagocytosed
PM10. Direct effects of PM may occur
via components that are able to cross the pulmonary
epithelium into the circulation, such as gases,
ultrafine particles (Nemmar et al. 2002),
and soluble co-pollutants (e.g., polycyclic aromatic
hydrocarbons and transition metals).
To clarify the contribution of each component
of PM in the induction of the inflammatory response,
we systematically compared the effects induced
by PM derived from different sources such as diesel
exhaust particulates (DEP) and urban dust particulates
(UDP) with those induced by their organic extracts
OE-DEP/OE-UDP and the fine particles or coarse
fraction, represented by their stripped particles
sDEP/sUDP and the ultrafine particles carbon black
(CB).
The present study provides evidence that the
organic components of the native particles DEP
and UDP play a major role in mediating the increase
of the inflammatory cytokines TNF,
IL-8, and COX-2. We also demonstrate, for the first
time, an increased expression of CRP in macrophages
induced by the particles that is mediated by the
particulate composition rather than their organic
components.
Reagents. National Institute of
Standards and Technology (NIST) Standard Reference
Material (SRM) 1649, an atmospheric particulate
material collected in an urban area, and a diesel
exhaust particulate sample, NIST SRM 2975, were
purchased from NIST (Gaithersburg, MD). CB 95 nm
in diameter (FR103) were provided by
Degussa (Frankfurt, Germany). We prepared stock
solutions of particles by suspending them in autoclaved
distilled water and by ultrasonication for 2 min
at maximum power (100 W). Particles were used at
2.5, 10, or 40 µg/cm2, equivalent
to 12.5, 50, or 200 µg/mL. Concentrations
are preferentially expressed in micrograms
per square centimeter because particles rapidly
sediment onto the cell layer. UDP and
DEP were extracted by dichloromethane in a soxhlet
apparatus. After sonication the extract was filtered
(0.45 µm Acrodisc) and concentrated to 1
mL by TurboVap and stored in precleaned amber vials.
The extract obtained was dried and then redissolved
in dimethylsulfoxide. We used the OE-DEP and OE-UDP
at concentrations corresponding to the amount of
particles at 10 µg/cm2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD, > 99% purity) was originally obtained
from Dow Chemicals Co. (Midland, MI). Dimethylsulfoxide
and phorbol-12-myristate-13-acetate (TPA) were
obtained from Aldrich Chemical Co. (St. Louis,
MO). Other molecular biological reagents were purchased
from Qiagen (Valencia, CA) and Roche (Indianapolis,
IN).
Cell culture and differentiation. We
obtained human U937 monocytic cells from the American
Tissue Culture Collection (Manassas, VA) and maintained
them in RPMI 1640 medium containing 10% fetal bovine
serum (Gemini, Woodland, CA), 100 U penicillin,
and 100 µg/mL streptomycin supplemented with
4.5 g/L glucose, 1 mM sodium pyruvate, and 10 mM
HEPES. Cell culture was maintained at a cell concentration
between 2 105 and
2 106 cells/mL.
For differentiation into macrophages, U937 cells
were treated with TPA (5 µg/mL) and allowed
to adhere for 48 hr in a 5% CO2 tissue
culture incubator at 37°C, after which they
were fed with TPA-free medium.
Cell viability assay. To assess
the effect of particles on viability of U937 macrophages,
we used the trypan blue exclusion test (McAteer
and Davis 1994). A 10-µL portion of resuspended
cell pellet was placed in 190 µL PBS with
200 µL trypan blue (0.5% dilution in 0.85%
NaCl) added. After 5 min we loaded 10 µL
of the cell suspension into a hemocytometer and
determined the proportion of nonviable to viable
cells.
Cellular cholesterol and protein determinations. We
extracted free and esterified cholesterol (total
cholesterol) directly from macrophage monolayers in
situ in the cell culture dish. After the indicated
time of treatment, each PBS-washed monolayer was
scraped off in 400 µL RI PA buffer and incubated
for 30 min on ice. Unsoluble material was removed
by centrifugation at 12,000 g for
20 min at 4°C and aliquots were used for protein
determination according to Bradford (1976). We
determined the amount of free and esterified cholesterol
(total cholesterol) using a colorimetric method
(Roche) in the presence of cholesterol oxidase
and cholesterol esterase and then measured the
absorbance at 405 nm.
Table 1 |
Quantitative real-time reverse transcription-PCR. We
isolated total RNA from U937 cells using a high-pure
RNA isolation kit (Roche) and carried out cDNA
synthesis as previously described (Vogel et al.
2004b). Quantitative detection of β-actin
and differentially expressed genes was performed
with a LightCycler Instrument (Roche Diagnostics,
Mannheim, Germany) using the QuantiTect SYBR Green
PCR Kit (Qiagen) according to the manufacturer’s
instructions. DNA-free total RNA (1.0 µg)
was reverse-transcribed using 4 U Omniscript reverse
transcriptase (RT; Qiagen) and 1 µg oligo(dT) 15 in
a final volume of 40 µL. The primers for
each gene (Table 1) were designed on the basis
of the respective cDNA or mRNA sequences using
OLIGO primer analysis software provided by Steve
Rozen and Whitehead Institute/MIT Center
for Genome Research (Rosen and Skaletsky 2000),
so that the targets were 100-200 bp in length.
PCR amplification was carried out in a total volume
of 20 µL, containing 2 µL cDNA, 10 µL
2 QuantiTect
SYBR Green PCR Master Mix, and 0.2 µM of
each primer. The PCR cycling conditions were 95°C
for 15 min followed by 40 cycles of 94°C for
15 sec, 60°C for 20 sec, and 72°C for 10
sec. We performed detection of the fluorescent
product at the end of the 72°C extension period.
We ran negative controls concomitantly to confirm
that the samples were not cross-contaminated. A
sample with DNase- and RNase-free water instead
of RNA was concomitantly examined for each of the
reaction units described above. To confirm the
amplification specificity, we subjected the PCR
products to melting curve analysis. We performed
all PCR assays in triplicate. The intra-assay variability
was < 7%. For quantification we analyzed data
with the LightCycler analysis software. The variables
were examined for one-sided Student’s t test.
The results are given as the mean ± the
SDs of the mean.
Antibodies and Western blotting. Monoclonal
anti-human CRP antibody was purchased from Sigma
Chemical Co. Rabbit polyclonal anti-human actin
and a horseradish peroxidase-conjugated secondary
antibody were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). We separated whole-cell proteins
on a 10% SDS-polyacrylamide gel and blotted
them onto a PVDF (polyvinylidene fluoride) membrane
(Immuno-Blot; BioRad, Hercules, CA). The antigen-antibody
complexes were visualized using the chemoluminescence
substrate SuperSignal West Pico (Pierce, Rockford,
IL) as recommended by the manufacturer. For quantitative
analysis, we quantified respective bands using
a ChemiImager 4400 (Alpha Innotech Corp., San Leandro,
CA).
Statistical analysis. All experiments
were repeated a minimum of 3 times and results
were expressed as mean ± SD. We determined
statistical differences using Student’s t test
and for the analysis of the significance between
pairs of mean values, we used the Bonferroni post-hoc
test.
Table 2 |
Figure 1. Cytotoxicity
of U937 macrophages exposed to DEP or UDP.
Percent
cytotoxicity was assessed by trypan blue
exclusion test. Cells were exposed to DEP
or UDP at various concentrations (2.5, 10,
or 40 µg/cm2). Control cells
(Ctrl) received 100 µL PBS only. Error
bars represent mean ± SD of three
independent experiments.
*Significantly different from control cells
(p < 0.05) |
Figure 2. Effect of DEP (A),
UDP (B), their corresponding stripped
particles, or their organic extracts preparation
on TNF,
COX-2, and IL-8 mRNA expression in U937 macrophages.
Increased mRNA levels of TNF,
COX-2, and IL-8 are shown. (A) U937
macrophages were treated for 24 hr with 10 µg/cm2 DEP.
To examine the effect of the stripped particles
and the organic components of these particle
samples, cells were treated with equivalent
amounts of the corresponding sDEP or OE-DEP.
As a control, cells were treated with 10 µg/cm2 CB.
(B) U937 macrophages were treated
for 24 hr with 10 µg/cm2 UDP.
To examine the effect of the stripped particles
and the organic components of these particle
samples, cells were treated with equivalent
amounts of the corresponding sUDP or OE-UDP.
Values are given as mean ± SD of triplicates
of three independent experiments.
*Significantly increased compared to control
cells (p < 0.05). **Significantly
lower than in native particle-treated cells
(p < 0.05). #Significantly
increased compared to native particles-treated
cells (p < 0.05) |
Figure 3. Effect of DEP (A),
UDP (B), their corresponding stripped
particles, or their organic extracts preparation
on IL-6 and CRP mRNA expression inflammatory
mediators in U937 macrophages. Increased
mRNA levels of IL-6 and CRP are shown. (A)
U937 macrophages were treated for 24 hr with
10 µg/cm2 DEP. To examine
the effect of the stripped particles and
the organic components of these particle
samples, we treated cells with equivalent
amounts of the corresponding sDEP or OE-DEP.
As a control, cells were treated with 10 µg/cm2 CB.
(B) U937 macrophages were treated
for 24 hr with 10 µg/cm2 UDP.
To examine the effect of the stripped particles
and the organic components of these particle
samples, cells were treated with equivalent
amounts of the corresponding sUDP or OE-UDP.
Values are given as mean ± SD of triplicates
of three independent experiments.
*Significantly increased compared to control
cells (p < 0.05). **Significantly
lower than in native particle-treated cells
(p < 0.05). #Significantly
increased compared to native particle-treated
cells (p < 0.05) |
Figure
4. Increased intracellular protein
level of CCRP. (A) The levels of
CRP in whole-cell lysates from U937 macrophages
48 hr after treatment with 10 µg/cm2 DEP,
UDP, sDEP, sUDP, OE-DEP, OE-UDP, or 1%
PBS as vehicle control (C) were determined
by Western blot analysis. Equivalent amounts
of whole-cell lysates (100 µg protein)
were loaded in each lane on 10% SDS-polyacrylamide
gels and analyzed by immunoblotting using
a CRP- or actin- (housekeeping protein
as control) specific antibody. (B)
Densitometric evaluation of CRP protein
band intensities normalized to actin protein
band intensities. Mean values of two independent
experiments are shown.
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Figure 5. Effect of various inhibitors
on DEP- or UDP-induced mRNA expression of
CRP. U937 macrophages were pretreated for
15 min with 100 µg/mL aggregated human
IgG, 10 µM Lut, or 0.1 µM wortmannin
(Wort). Cells were then treated with 10 µg/cm2 DEP,
UDP, or sDEP, sUDP for 24 hr. The mRNA expression
was analyzed by real-time reverse transcription-PCR
and results were normalized to β-actin
and given as fold increase compared to the
mRNA level in control cell (= 1). Values
are given as mean ± SD of triplicates
of three independent experiments.
*Significantly increased compared to control
cells (p < 0.05). **Significantly
lower than in cells treated with native particles,
stripped particles, or their organic extracts
(p < 0.05) |
Figure 6. Effect of various inhibitors
on DEP- or UDP-induced mRNA expression
of COX-2. U937 macrophages were pretreated
for 15 min with 100 µg/mL aggregated
human IgG, 10 µM luteolin (Lut),
or 0.1 µM wortmannin (Wort). Cells
were then treated with 10 µg/cm2 DEP,
UDP or OE-DEP, OE-UDP for 24 hr. The mRNA
expression was analyzed by real-time RT-PCR
and results were normalized to β-actin
and given as fold increase compared to
the mRNA level in control cell (= 1). Values
are given as mean ± SD of triplicates
of three independent experiments.
*Significantly increased compared to
control cells (p < 0.05). **Significantly
lower than in cells treated with native
particles, stripped particles, or their
organic extracts (p < 0.05)
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Figure 7. Effect of the AhR-antagonist
luteolin on DEP-, UDP-, or TCDD-induced CYP1a1 expression.
U937 macrophages were treated with 10 µg/cm2 DEP,
UDP, sDEP, sUDP, OE-DEP, OE-UDP, or 10
nM TCDD. To antagonize AhR binding and
activation, cells were co-treated with
10 µM luteolin (Lut) and OE-DEP,
OE-UDP, or TCDD. After 24 hr of treatment,
CYP1a1 mRNA expression was analyzed by
real-time RT-PCR and results were normalized
to β-actin
and given as fold increase compared to
the mRNA level in control cell (= 1).
Values are given as mean ± SD
of triplicates of three independent experiments.
*Significantly
increased compared to control cells (p < 0.05). **Significantly
lower than in cells treated with native
particles, their organic extracts, or
TCDD (p < 0.05).
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Figure 8. Cholesterol accumulated
in U937 macrophages. Cells were treated
for 5 days with 10 µg/cm2 DEP,
UDP, sDEP, sUDP, or corresponding amounts
OE-DEP, OE-UDP. Vehicle control cells
received 1% PBS. Total cholesterol
was determined using a colorimetric
method in the presence of cholesterol
oxidase and cholesterol esterase. Values
are given as mean ± SD of triplicates
of three independent experiments.
*Significantly different from vehicle
control (p < 0.05)
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Dose-dependent effect of DEP and UDP on
inflammatory factors and CYP1a1 expression. To
address the dose-dependent effect of the particles,
we studied the mRNA expression of inflammatory
factors and CYP1a1 24 hr after treatment with
various concentrations of DEP and UDP. As shown
in Table 2, treatment of U937 macrophages with
the native particles of DEP or UDP in the range
of 2.5, 10, or 40 µg/cm 2 cell
culture area led to dose-dependent mRNA induction
of COX-2, TNF ,
IL-6, IL-8, CRP, C/EBPβ (CCAAT/
enhancer binding protein), and CYP1a1.
Except for COX-2, the UDP-induced expression
on these genes was more prominent than the effect
of DEP. All parameters were significantly induced
by UDP at the low concentration of 2.5 µg/cm2.
In contrast, COX-2, IL-6, C/EBPβ,
and CRP were significantly increased only at 10
or 40 µg/cm2 DEP. The most conspicuous
effect of UDP was found in the case of IL-8 (31.6-fold),
whereas DEP showed the strongest effect on CRP
expression (19.7-fold). To estimate the toxic potency,
we compared the effects of DEP and UDP with various
concentrations of TCDD, which has been shown to
be an efficient inducer of inflammatory factors
and foam-cell formation in U937 macrophages. Except
for IL-6, which was downregulated by TCDD, we observed
concentration-dependent increases of COX-2, TNF,
and IL-8, showing a 5.1-, 2.8-, and 2.5-fold increase,
respectively, at the lowest concentration of TCDD
tested (0.1 nM). CRP was also significantly increased
at higher concentrations of TCDD (1 and 10 nM),
which correlated with the induction of C/EBPβ.
Evaluation of cytotoxicity of DEP and UDP.Cytotoxic
effects on U937 macrophages were measured for DEP,
UDP, sDEP, sUDP, as well as for their corresponding
extracts OE-DEP and OE-UDP. The viability of cells
cultured in medium alone with 100 µL PBS added
served as control. After incubating the cells with
particles or extracts for 24 hr at 37°C, we determined
cytotoxicity by trypan blue exclusion test. Cell
death in the unexposed U937 macrophages was 8% (Figure
1). In U937 macrophages treated with 2.5 or 10 µg/cm 2 DEP,
UDP, sDEP, sUDP, OE-DEP, or OE-UDP, no significant
effect on cell viability compared to control cells
was found (data not shown). However, at the highest
dose of 40 µg/cm 2, treatment with
DEP as well as with UDP led to a significant increase
of the number of dead cells by 7 and 10%, respectively
(Figure 1).
Effect of organic compounds in DEP- and
UDP-induced expression of proinflammatory marker
genes COX-2, TNF,
and IL-8. Macrophages
involved in atherosclerotic lesions are a primary
source of inflammatory cytokines. Cytokines
can contribute to initiation and progression
of atherosclerotic lesions by triggering multiple
cellular functions such as leukocyte recruitment
and synthesis/degradation of extracellular
matrix. Therefore, we tested the expression
of selected biomarkers of the proinflammatory
response after treatment with DEP and UDP.
To determine which component of DEP and UDP
is the most critical one for this response,
the DEP- and UDP-induced mRNA expressions were
compared with those induced by sDEP, sUDP,
and their organic components OE-DEP and OE-UDP.
Both types of particles were used at a concentration
of 10 µg/cm2, and the final
concentration of the organic extracts was equivalent
to 10 µg/cm2 of the particles.
Treatment for 24 hr with DEP and UDP significantly
induced a 7.5- and 10-fold increase of COX-2,
a 4- and 7-fold increase of TNF,
and 8- and 25-fold increase of IL-8,
respectively (Figure 2). The increases of COX-2,
TNF, and
IL-8 mRNA levels by the organic compounds from
the diesel particulates (OE-DEP) were more
than 2-fold higher than the effect of the native
particle DEP (Figure 2A), whereas the organic
compounds of the urban dust particulates (OE-UDP)
led to increases of these cytokines comparable
to the native particle UDP (Figure 2B). For
the stripped particles, sDEP, the effects on
COX-2, TNF,
and IL-8mRNA expression remained 3-fold
lower than that induced by DEP, and about 8-fold
lower than that induced by their organic compounds,
suggesting the role of organic compounds in
these responses (Figure 2A). The increase of
COX-2, IL-8, and TNF mRNA
after treatment with sUDP was about 2-fold
less pronounced than the effects of UDP or
the organic extract OE-UDP (Figure 2B). Treatment
with CB (10 µg/cm2) had no
significant effect on the mRNA expression of
COX-2, TNF,
or IL-8 (Figure 2A).
Role of the particles on DEP- and UDP-induced
expression of CRP and IL-6. CRP
participates in the systemic response to inflammation
and is an important cardiovascular risk factor.
Elevated concentrations of PM have been shown
to be associated with increases of serum CRP
levels in men (Peters et al. 2001; Pope et
al. 2004b). In the present study we observed
a 6- and 13-fold increase of CRP mRNA expression
after exposure to 10 µg/cm2 DEP
or 10 µg/cm2 UDP for 24 hr,
respectively (Figure 3). Treatment with DEP
or sDEP resulted in similar induction rates
of CRP mRNA (6-fold), but OE-DEP had a significantly
lesser effect (2.5-fold) on CRP mRNA expression
than DEP or sDEP (Figure 3A). sUDP led to significantly
higher induction (22-fold) of CRP mRNA than
did UDP or the organic compounds OE-UDP (3-fold)
(Figure 3B).
To test whether elevated mRNA levels correspond
with increased accumulation of the CRP protein,
we performed Western blots. In whole-cell protein
of U937 macrophages, we observed 3- to 4-fold higher
protein levels of CRP in DEP-, UDP-, sDEP-, and
sUDP-treated cells compared to control, OE-DEP-,
or OE-UDP-treated cells (Figure 4).
IL-6 has been shown to be elevated after exposure
to particles in macrophages in numerous studies
(e.g., Monn and Becker 1999) and is the most potent
inflammatory cytokine for the induction of human
CRP. We found that after 24 hr, DEP, UDP, and their
corresponding stripped particles significantly
increased the mRNA level of IL-6 by 5- and 7-fold,
respectively (Figure 3). However, similar to the
results found for CRP, the organic compounds OE-DEP
or OE-UDP did not significantly induce mRNA expression
of IL-6 (Figure 3). Treatment with CB (10 µg/cm2)
had no significant effect on the mRNA expression
of IL-6. Only the CRP mRNA level was slightly elevated
after exposure to CB; however, the effect was not
statistically significant (Figure 3A).
The fact that treatment with TCDD, OE-DEP, or
OE-UDP did not increase IL-6 and led to only a
moderate increase of CRP mRNA expression compared
to sDEP, sUDP, DEP, or UDP suggests that particles
rather than the co-pollutants mediate the increase
of CRP and IL-6.
Effect of various inhibitors on DEP- and
UDP-mediated CRP and COX-2 induction.CRP induction
by DEP, UDP, sDEP, or sUDP was blocked by about
75% after pretreatment for 15 min with 100 µg/mL
aggregated IgG, which blocks binding to the
Fc receptor.
Preincubation for 15 min with 100 nM wortmannin,
which inhibits Fc receptor-dependent
ingestion and activation, blocked the induction
of CRP mediated by DEP, UDP, sDEP, and
sUDP by about 50% (Figure 5). Neither aggregated
IgG nor wortmannin led to a significant inhibition
of DEP- or UDP-mediated COX-2 induction
(Figure 6). Conversely, the aryl hydrocarbon
hydroxylase (AhR) antagonist luteolin had no
significant effect on the particle-induced
expression of CRP (Figure 5). However, the
induction of COX-2 mediated by DEP/UDP,
as well as their organic extracts, was significantly
suppressed (50%) by luteolin (Figure 6).
DEP- and UDP-induced CYP1a1 mRNA level. The
CYP1a1 mRNA level increased 40-fold in DEP-treated
(10 µg/cm2) cells, whereas the
organic extract OE-DEP led to a markedly higher
increase of 170-fold in comparison to control cells.
Exposure to 10 µg/cm2 UDP- or
OE-UDP led to about 100-fold elevated levels of
CYP1a1 mRNA after 24 hr of treatment. The stripped
particles sUDP had still significant effects and
increased CYP1a1 mRNA levels by 20-fold (Figure
7).
To test the role of the AhR in OE-DEP- and
OE-UDP-mediated increase of CYP1a1,
we co-treated cells with the AhR antagonist luteolin
(10 µM) and OE-DEP or OE-UDP for 24 hr. Co-treatment
with luteolin (10 µM) inhibited OE-DEP- and
OE-UDP-mediated CYP1a1 induction by about
50%, which indicates the involvement of the AhR
in this process. As a positive control, cells were
treated with 10 nM TCDD for 24 hr and co-treated
with luteolin. TCDD led to a 110-fold increase
of CYP1a1 mRNA level, which was inhibited by 95%
after co-treatment with luteolin (Figure 7).
Stimulation of cholesterol accumulation
in U937 macrophages by UDP and DEP. Foam
cells are primarily macrophages laden with
cholesterol ester-rich cytoplasmic lipid inclusions.
To quantify total amount of cholesterol in
U937 macrophages, we used a colorimetric method
in the presence of cholesterol oxidase and
cholesterol esterase. Exposure for 5 days to
10 µg/cm2 DEP as well as to
UDP stimulated the accumulation of cholesterol
by 2- and 2.5-fold, respectively (Figure 8).
Both organic extracts OE-DEP or OE-UDP increased
the amount of cholesterol by about 2.3-fold
above control. The stripped particles sDEP
and sUDP did not significantly increase the
amount of cholesterol in U937 macrophages compared
to control cells (Figure 8). Results from cholesterol
assay were consistent with findings from Oil
Red O staining (data not shown).
There is strong evidence from epidemiologic and
animal studies that exposure to air pollution particulates
play a role in the development of cardiovascular
diseases such as atherosclerosis and heart diseases
(Pope et al. 2004; Suwa et al. 2002). DEP and UDP,
which are the most important components of PM2.5 (PM
with aerodynamic diameter ≤ 2.5 µm)
and PM10, respectively, in many urban
areas, have been suspected. The results presented
in this study show that diesel particles as well
as urban dust cause the induction of several proinflammatory
factors such as COX-2, TNF,
C/EBPβ, IL-6,
and IL-8 in human macrophages. Exposure to these
particles also results in a significant elevation
of CRP mRNA and protein levels in U937 macrophages.
We used U937-derived macrophages because these
cells are frequently used to develop foam cells
after treatment with modified low density protein
(Martens et al. 1998) and as described earlier
by exposure to environmental toxicants like TCDD
(Vogel et al. 2004a). Concomitant with induction
of inflammatory factors, the accumulation of total
cholesterol was significantly increased in DEP-
or UDP-treated macrophages. Cholesterol accumulation
in macrophages is a hallmark of foam-cell formation
indicating early lesion of atherosclerosis (Linton
and Fazio 2003). Thus, the particle-mediated inflammatory
response and subsequent formation of foam cells
may contribute directly to the progression of atherosclerosis
and other cardiovascular events.
Several studies have shown that the toxicity
of PM might be linked to the generation of reactive
oxygen species (ROS) in the lungs (Tao et al. 2003),
which can be detected by their electron spin resonance
signals (Kadiiska et al. 1997). ROS might also
play a role in promoting a state of systemic inflammation.
In the current study we performed experiments to
estimate the signaling mechanisms of the proinflammatory
response induced by DEP or UDP using human monocyte-derived
macrophages. Organic compounds like polyaromatic
hydrocarbons (PAH) adsorbed on UDP, and especially
DEP, which induce CYP1a1 gene expression
(Figure 7) seemed to be mainly involved in the
response of inflammatory factors such as COX-2,
IL-8, and TNF.
Besides COX-2 and TNF,
we observed, for the first time, a dose-dependent
increase of IL-8 mRNA level in cells treated with
the AhR ligand TCDD. Results of this study show
that the organic extract of OE-DEP is significantly
more effective at inducing COX-2, IL-8, and TNF than
its native particle DEP, whereas OE-UDP led to
a similar increase compared to its native particle
UDP. These results suggest that the organic co-pollutants
are highly adsorbed by DEP and thus less bioavailable
compared to UDP. The stripped particles of both
diesel and urban dust had significantly less effect
on the induction of COX-2, IL-8, and TNF.
To analyze the contribution of AhR-activating
compounds, we co-treated cells with the AhR antagonist
luteolin and the particles or their organic extracts.
Our results (Figures 5-7) clearly show that
luteolin is more effective in suppressing COX-2 or CYP1a1 than CRP in
all samples. In turn, both IgG and wortmannin were
much better inhibitors of CRP than COX-2.
These results indicate that the solvent extraction
procedure could effectively separate the AhR agonists
(i.e., luteolin inhibited components) from the
particles. Furthermore, the stripped particles
showed properties different from the solvent extracts
in that affected cells showed high mRNA expression
of CRP by treatment with stripped or native
particles but not with organic extracts (Figure
3).
We have previously shown that dioxin-type chemicals
are powerful inducers of inflammation in U937 macrophages
(Vogel et al. 2004a), and therefore it is logical
to explain the action of solvent extracts to activate COX-2.
However, the finding that stripped particles from
air pollutants selectively activate CRP is
new. It is known that insoluble, fine particles
have the property to affect Fc receptor
activity on the macrophage membrane and thereby
trigger the process of phagocytosis and uptake
of low-density lipoprotein (Kleinman et al. 2003;
Luo et al. 2005; Ohtsuka et al.
2000; Swanson and Hoppe 2004; Zwaka et al.
2001). The notion that the stripped particles are
also acting through the Fc receptor
is supported by our observation that aggregated
IgG, the specific ligand for Fc receptor,
is effective in suppressing the same cell response
indicates that the phagocytosis of those particles
is the key event accompanying the Fc receptor
stimulating action of sDEP and sDEP, since phosphatidylinositol
3-kinase, which is sensitive to wortmannin, is
a crucial factor mediating phagocytosis of macrophages
(Song et al. 2004). Activation of Fc receptor
by aggregated IgG might also trigger inhibitory
signaling pathways that suppress the effects of
particles on the expression of CRP. The
effect of the ultrafine CB on CRP expression was
rather small, which indicates that not only the
chemical components of the PM but also other factors
such as surface properties and shape affect toxicity.
One interesting aspect is that the timing of
particle-mediated induction of CRP was correlated
with elevated levels of IL-6 mRNA, which can mediate
transcriptional activation of CRP. Both CRP and IL-6 induction
by PM were blocked by aggregated IgG or wortmannin,
which inhibits Fc receptor-dependent
ingestion and activation. Neither aggregated IgG
nor wortmannin led to a significant inhibition
of DEP- or UDP-induced COX-2, TNF,
or IL-8. The close relationship between
IL-6 and CRP has been pointed out by many scientists
(e.g., Monton and Torres 1998), but in most cases
CRP production is carried out in liver as a result
of stimulation by circulating IL-6. The fact that
IL-6 acts as an autocrine factor to stimulate CRP
production in macrophages is not well known; however,
the increased synthesis and secretion of CRP, IL-6,
and soluble IL-6 receptor by macrophage-derived
foam cells in the arterial intima has been demonstrated
(Ballou and Lozanski 1992; Jones et al. 1999; Libby
2002; Lusis 2000; Ross 1999). Recent studies also
show that binding of C/EBPβ is
critical for induction of CRP expression
(Agrawal et al. 2003), which could explain the
increase of CRP by TCDD, as TCDD induces the expression
of C/EBPb (Vogel et al. 2004a; Vogel and
Matsumura 2003) but not IL-6 (Table 2).
According to Du Clos and Mold (2004), CRP acts
through Fc receptor
to play important roles in infection, inflammation,
and autoimmune diseases. This phenomenon of action
of stripped particles deserves close attention
in the future.
In conclusion, we have shown that air pollution
particles have two major classes of toxic components.
One is the dioxin-type AhR agonist, which is extractable
by solvents, and the other type is the stripped
particle, which elicits a different pattern of
mRNA activation from that induced by dioxin-type
chemicals. These findings may contribute to a better
understanding of the differential toxicity of various
constituents and sources of PM, including their
chemical/biological components alone or in combination
with PM. Further research is necessary to fully
elucidate this mechanism of differential toxicity.
Other relevant sources of PM should be collected
and tested, such as those from the combustion of
alternative fuels, and evaluated for their potential
contribution as risk factors for cardiovascular
disease in both urban and rural environments. |
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Last Updated: October 17, 2005 |
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