1 Respiratory Section, Edinburgh Lung and The Environment Group Initiative/Colt Laboratories, Department of Medical and Radiological Sciences, The University of Edinburgh Medical School, Edinburgh EH8 9AG; 3 School of Life Sciences, Napier University, Edinburgh EH10 5DT, United Kingdom; and 2 Pulmonary Research Laboratory, St. Pauls' Hospital, The University of British Columbia, Vancouver, British Columbia V6Z 146, Canada
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ABSTRACT |
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The
presence of the adenoviral early region 1A (E1A) protein in human lungs
has been associated with an increased risk of chronic obstructive
pulmonary disease (COPD), possibly by a mechanism involving
amplification of proinflammatory responses. We hypothesize that
enhanced inflammation results from increased transcription factor
activation in E1A-carrying cells, which may afford susceptibility to
environmental particulate matter < 10 µm
(PM10)-mediated oxidative stress. We measured interleukin
(IL)-8 mRNA expression and protein release in human alveolar epithelial
cells (A549) transfected with the E1A gene (E1A+ve). Both E1A+ve and
ve cells released IL-8 after incubation with TNF-
, but only E1A+ve
cells were sensitive to LPS stimulation in IL-8 mRNA expression and
protein release. E1A+ve cells showed an enhanced IL-8 mRNA and protein
response after treatment with H2O2 and
PM10. E1A-enhanced induction of IL-8 was accompanied by
increases in activator protein-1 and nuclear factor-
B nuclear
binding in E1A+ve cells, which also showed higher basal nuclear binding
of these transcription factors. These data suggest that the presence of
E1A primes the cell transcriptional machinery for oxidative stress
signaling and therefore facilitates amplification of proinflammatory
responses. By this mechanism, susceptibility to exacerbation of COPD in
response to particulate air pollution may occur in individuals
harboring E1A.
early region 1A; environmental particulate matter less than 10 micrometers; interleukin-8; nuclear factor-B; activator protein-1; lung epithelium
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INTRODUCTION |
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THE EARLY REGION 1A (E1A) gene of the adenovirus genome is the first to be expressed after infection and is crucial in adenovirus transformation of the host cell (39). E1A protein promotes cell entry into the S phase of the cell cycle and therefore facilitates viral replication (41). Expression of the E1A gene results in the production of phosphoproteins (47), in particular the major protein products 289R and 243R (49), which regulate transcription of both viral and host cell genes. The E1A proteins influence cellular transcription, not by direct DNA binding or enzymatic activity but by indirectly affecting cell function by interacting with endogenous nuclear regulatory proteins that affect transcription factor DNA binding (9, 30).
Two important transcriptional coactivators that have recently been
shown to bind targets of E1A proteins are the cAMP response element
binding protein (CREB) or the CREB binding protein (CBP) and related
p300 proteins, which influence many transcription factors and cell
signaling events (1, 31, 41). E1A proteins also bind to,
and interact with, specific members of the activator protein-1 (AP-1)
family of transcription factors (16, 34, 52a) and affect activity of
the nuclear factor-B (NF-
B) transcription factor (12, 22,
46). Thus E1A transformation of host cells alters the regulation
of host gene transcription and, in doing so, changes the functional
state of the cell and its response to the surrounding environment.
Adenoviral infections can remain latent in the lung (37),
tonsils (15), and peripheral lymphocytes
(17). Studies have suggested that previous viral
infection, i.e., during childhood, may be a possible risk factor in the
development of chronic obstructive pulmonary disease (COPD), a disease
characterized by airway inflammation in smokers (37).
Patients with COPD had three times the level of E1A DNA found in
smokers without airway obstruction. The expression of E1A protein was
localized to the airway epithelium and adjacent parenchyma (10,
37). This observation led to a hypothesis that the presence of
E1A could amplify smoking-mediated inflammatory responses, enhancing
the development of COPD. This is supported by studies showing that the
presence of E1A augments intercellular adhesion molecule-1 and
interleukin (IL)-8 expression in response to lipopolysaccharide (LPS;
see Refs. 20 and 21). E1A protein also sensitizes cells to
tumor necrosis factor (TNF)- (46) and amplifies
inflammation in response to cigarette smoke in guinea pig lungs
(53). IL-8 is induced by cytokines such as TNF-
, IL-1
, IL-6, and LPS (22). IL-8 is regulated by the
oxidant redox-sensitive transcription factors AP-1 (35),
NF-IL-6, which recognizes the same nucleotide sequences as
CCAAT/enhancer-binding protein (C/EBP) elements (36), and
NF-
B (40). The levels of IL-8 increased in the sputum
of patients with COPD (19). Because oxidative stress can
regulate the production of IL-8 (4, 25) and the activity
of transcription factors (45), it has been implicated in
the development of the inflammatory response in COPD (33).
We have shown that environmental particulate matter < 10 µm
(PM10) has free radical activity that acts via a transition
metal-dependent mechanism (14, 27). PM10 can
activate transcription factors AP-1 and NF-B and induce the
expression of IL-8 (18). Increases in the concentrations
of PM10 are also associated with adverse health effects,
including loss of lung function (3), increases in
exacerbations of asthma (54) and COPD (52),
and increased mortality (5). We have previously
investigated ultrafine carbon black as a surrogate for the ultrafine
component of PM10 (27). We have hypothesized
that the ultrafine portion of PM10 may be an arbiter of the
health effects (32).
We propose that the presence of E1A may render individuals with COPD more susceptible to oxidative stress imparted by inhalation of PM10, which may perhaps lead to an exacerbation of COPD. The purpose of the present study was to examine whether cells transfected with E1A enhanced IL-8 production in response to PM10. For convenience and because COPD is a disease characterized by parenchymal and airway inflammation, A549 cells were used in this study. Additionally, the role of transcription factor activation in the E1A response was also studied.
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MATERIALS AND METHODS |
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Reagents.
All chemicals and reagents used in this study were obtained from Sigma
Chemical (Poole, UK). Cell culture media and reagents were obtained
from GIBCO BRL (Paisley, UK). Human recombinant TNF- (R&D Systems,
Abingdon, UK) was stored at
70°C at a concentration of 10 µg/ml
in sterile distilled water and was diluted in culture medium to 10 ng/ml for cell treatment. LPS (Escherichia coli 0111:B4) was
dissolved in sterile distilled water at a concentration of 10 mg/ml and
was diluted in culture medium to 10 µg/ml for cell treatment, as
previously described (20). H2O2
was prepared as a stock solution of 2 mM in PBS, and treatments were
carried out at a concentration of 200 µM unless stated in the legends
for Figs. 1-9. Actinomycin D was stored at a concentration of 100 µg/ml, and treatments were carried out at a concentration of 100 ng/ml unless stated in the legends for Figs. 1-9. The thiol
antioxidant glutathione monoethyl ester (GSHMEE) was stored at
20°C
in PBS at a concentration of 500 mM and was used at 5 mM final
concentration.
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Cell culture.
Human lung alveolar type II-like epithelial cells (A549) transfected
with a plasmid carrying the adenovirus 5 E1A gene or the vector without
the E1A gene (20-22) were maintained and treated in
DMEM supplemented with 10% heat-inactivated FCS,
L-glutamine (2 mM), and 280 µg/ml G418 antibiotic in 5%
CO2 at 37°C. These cells are referred to as E1A+ve and
E1Ave, respectively, from this point forward. With the use of RT-PCR,
the expression of the E1A gene was determined in the transfected cells
used in the study (Fig. 1).
Particle suspensions. Carbon black (Huber 990), which was used in the determination of PM10 concentration, was obtained from Degussa (Frankfurt, Germany).
PM10 particles were removed directly from the collection filters of the tapered element oscillating microbalance in the Marylebone and Bloomsbury London monitoring sites of the United Kingdom enhanced urban network. Filters were cut in half, and each half was sonicated in 1 ml of PBS for 1 min and vortexed vigorously to remove the particles, after which the spent filters were removed. The concentration of particles was estimated by spectrophotometric comparison of turbidity at 340 nm with a standard curve of serial dilutions of carbon black. The use of a carbon black standard curve allows the dose of PM10 to be both estimated and standardized relative to a toxicologically important component of PM10. For all PM10 treatments, cells were exposed to 100 µg/ml PM10 in culture medium.RT-PCR.
After treatment, RNA was isolated from PBS-washed cells using the
TRIzol reagent (GIBCO BRL) according to the manufacturer's instructions and dissolved in 50 µl diethyl pyrocarbonate
(DEPC)-treated water. SuperScript II (GIBCO BRL) was used to
transcribe cDNA from 1 µg of RNA according to the manufacturer's
instructions. The genes tested were the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primers (Table
1) from Stratagene (Cambridge, UK) and
IL-8 with primers according to Lindley et al. (Ref. 29 and
Table 1) from MWG-Biotech (Milton Keynes, UK).
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ELISA for IL-8. The ELISA method of IL-8 detection was performed as previously described (8). Typically, standard curves were generated with 25-2,500 pg/ml IL-8.
Nuclear extract preparation.
After treatment, 18 h in the case of AP-1 analysis and 4 h
for NF-B and C/EBP, cells were washed two times with PBS, scraped from the culture plate, and harvested by centrifugation at 1,000 g for 10 min at 4°C. The nuclear proteins were extracted
by a previously described method (13). The combined
cytoplasmic and nuclear proteins, including NF-
B and C/EBP, were
extracted in a one-step process from a cell pellet after a 20-min
incubation in 10% glycerol, 10% Nonidet P-40, 50 mM HEPES, 50 mM KCl,
300 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 M sodium orthovanadate, 0.2 mM NaF, 0.4 mM phenylmethylsulfonyl fluoride, 0.3 µg/ml leupeptin, and 1 µg/ml aprotinin followed by centrifugation for 10 min at 1,000 g at 4°C. The resulting supernatant
containing the nuclear proteins was decanted.
Electrophoretic mobility shift assays.
Electrophoretic mobility shift assay (EMSA) of specific nuclear
proteins was carried out as previously described (13).
Briefly, 7 µg of nuclear protein extract from sample cells (HeLa for
+ve control; Promega) or no protein (ve) was incubated with 0.25 mg/ml poly(dI-dC) · poly(dI-dC), 1 mM DTT, and binding buffer (Promega) for 5 min at room temperature followed by the addition of
[
-32P]ATP end-labeled double-strand consensus
oligonucleotides for AP-1, NF-
B (Promega), or C/EBP (Santa Cruz
Biotechnology, Santa Cruz, CA) transcription factors and further
incubation for 20 min. The sequences of the oligonucleotides carrying
the transcription factor binding sites are listed in Table 1.
Oligonucleotides bound by transcription factors were separated from
unbound oligonucleotides on a 6% nondenaturing polyacrylamide gel that
was dried on Whatman filter paper for phosphorimager analysis.
Statistical analysis. The data are expressed as means ± SE. Treatment-related differences were evaluated using one-way ANOVA followed by Tukey's post hoc test for multigroup comparisons (48). Statistical significance is reported at P < 0.05, P < 0.01, and P < 0.001 and expressed in Figs. 1-9 as one, two, or three asterisks, respectively.
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RESULTS |
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E1A mRNA expression.
After the RT-PCR amplification of cDNA prepared from E1A+ve and E1Ave
A549 cells, the E1A gene was shown to be expressed only in the cells
transfected with the E1A gene (Fig. 1).
Effect of PM10, LPS, and TNF- on IL-8 release.
After treatment with LPS and PM10 for 18 h, E1A+ve
cells released a significantly greater amount of IL-8 than untreated
control or E1A
ve cells (Fig. 2). This
increase in IL-8 protein release was associated with an increase in
IL-8 mRNA, detected by RT-PCR, after 4 h of incubation (Fig.
3). After TNF-
treatment for 18 h, both E1A+ve and E1A
ve cells released IL-8, with no significant difference between the two (Fig. 2). IL-8 release from E1A+ve cells
treated for 18 h with PM10 increased with doses of 0 µg/ml (0.141 ± 0.02 ng/ml), 50 µg/ml (0.304 ± 0.03 ng/ml), 100 µg/ml (0.471 ± 0.08 ng/ml), and 150 µg/ml
(0.529 ± 0.06 ng/ml) PM10. Similarly, IL-8 release
from E1A+ve cells treated for 18 h with H2O2 increased with doses of 0 µM (0.116 ± 0.007 ng/ml), 50 µM (0.179 ± 0.007 ng/ml), 100 µM
(0.279 ± 0.011 ng/ml), 150 µM (0.477 ± 0.04 ng/ml), 200 µM (0.522 ± 0.025 ng/ml), and 300 µM (0.575 ± 0.034 ng/ml) H2O2. E1A
ve cells displayed a similar
yet lower IL-8 dose response to these agents (data not shown).
Effect of PM10 and LPS on IL-8 gene expression. PM10- and LPS-mediated IL-8 gene expression was greater than that of untreated controls at 4-, 8-, 18-, and 24-h time points (Fig. 4, A and B). There was a trend to greater IL-8 gene expression with LPS treatment than with PM10 treatment.
Effect of actinomycin D on IL-8 RT-PCR. E1A+ve cells were left untreated or were treated with LPS (10 µg/ml), H2O2 (200 µM), and PM10 (100 µg/ml) for 4 h with or without actinomycin D (100 ng/ml). The presence of actinomycin D inhibited the expression of IL-8 mRNA at least partially in all treatments (Fig. 5, A and B).
Effect of the antioxidant GSHMEE on IL-8 RT-PCR. E1A+ve cells were left untreated or treated with LPS (10 µg/ml) or PM10 (100 µg/ml) for 4 h with or without GSHMEE (5 mM). The presence of GSHMEE inhibited the expression of IL-8 mRNA at least partially from PM10 treatment (Fig. 6, A and B).
Effect of oxidative stress on IL-8 release.
After treatment with H2O2 (200 µM) for only
4 h, both E1A+ve and E1Ave cells released significantly more
IL-8 than untreated control cells (Fig.
7), although the magnitude of the
increase was considerably lower compared with the 18-h response to
TNF-
. After this short stimulation time, E1A+ve cells released
significantly more IL-8 in response to both
H2O2 and LPS compared with E1A
ve cells (Fig.
7). IL-8 mRNA was also increased in E1A+ve and in E1A
ve cells in
response to 4 h of H2O2 treatment (Fig.
3).
Effect of LPS and oxidative stress on AP-1, NF-B, and C/EBP
nuclear binding.
Untreated E1A+ve cells had significantly higher basal levels of AP-1,
NF-
B, and C/EBP nuclear binding than untreated E1A
ve cells (Fig.
8, A-C,
bottom). AP-1 binding was significantly increased after
incubation with LPS only in E1A+ve cells (Fig. 8A). NF-
B binding was significantly enhanced in the E1A+ve cells treated with LPS
compared with untreated controls or LPS-stimulated E1A
ve cells (Fig.
8B). Both E1A+ve and E1A
ve cells showed significantly more
AP-1 and NF-
B binding after 18 and 4 h of treatment,
respectively, with H2O2 compared with untreated
controls (Fig. 8, A and B). There was
significantly more AP-1 and NF-
B activity in the E1A+ve cells
treated with H2O2 than in the E1A
ve cells
(Fig. 8, A and B). The level of C/EBP nuclear
binding did not change upon treatment with LPS or
H2O2 in either cell type (Fig. 8C).
Effect of PM10 on AP-1, NF-B, and C/EBP binding.
AP-1 binding was significantly greater in response to PM10
in E1A+ve than E1A
ve cells (Fig. 8A). Similarly, the
increase in the binding of NF-
B in response to PM10 was
greater in the E1A+ve cells than the E1A
ve cells (Fig.
8B). PM10 did not increase binding of C/EBP in
either cell type (Fig. 8C).
Role of p50 and p65 subunits in H2O2- and
PM10-induced NF-B binding in E1A+ve cells.
Increased NF-
B binding after PM10 and
H2O2 treatment involved both p50 and p65
subunits, with a predominance for p50 (Fig. 9).
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DISCUSSION |
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Environmental particles have been associated with hospital admissions (3, 54) and mortality (5) from airway diseases. The underlying airway inflammation in patients with COPD and asthma may render them susceptible to the effects of air pollution. Adenoviral infection, in particular the presence of adenoviral E1A protein, is considered to be a factor in susceptibility to COPD (37) as a result of its ability to enhance inflammatory responses (22, 41). IL-8 is a potent neutrophil chemoattractant and activator with an important role in COPD (19). In previous studies, we demonstrated that intratracheal instillation of PM10 in the rat lung led to inflammation and oxidative stress (18, 23, 27). In the present study, we investigated the influence of E1A in the release of IL-8 in response to oxidative stress and PM10.
Here we show that lung epithelial cells that harbor the E1A gene exhibit enhanced IL-8 release after exposure to LPS, PM10 particles, and oxidative stress (H2O2). Our data are therefore supportive of an inflammatory mechanism mediated through IL-8 for the development of the adverse health effects of PM10 (3, 5, 14). The proposed mechanism for IL-8 release in response to PM10 is the generation of free radicals by the Fenton chemical reaction mediated by the presence of transition metals (including iron; see Ref. 14). This is supported by our previous observation that PM10 causes IL-8 release by transition metal-mediated oxidative stress, which may also be influenced by the ultrafine component (18, 28, 32). Ultrafine particles have been thought to play a role in the adverse effects of PM10-mediated oxidative stress (50) and inflammation in vivo (32). We show here that the proinflammatory effect of PM10 is enhanced in E1A+ve cells. Studies on the underlying molecular mechanism for enhanced IL-8 expression that demonstrated a role for oxidative stress-responsive transcription factors (see below) support our hypothesis that ultrafine particles operate via an oxidative stress mechanism (26). The role of oxidative stress in the release of IL-8 from lung epithelial cells has been demonstrated previously (25). Moreover, the E1A-mediated sensitization of cells to oxidative stress is in agreement with a previous study which identified that the presence of the E1A gene enhanced oxidative stress by inhibiting ferritin induction, which has the role of metabolizing reactive free iron (42). Our study, albeit an in vitro study, suggests a mechanism whereby individuals with airway diseases and E1A+ve cells in their lungs after adenoviral infection may be susceptible to agents that cause inflammation or to oxidative stress, causing an enhancement of an ongoing inflammation, resulting in exacerbations of the disease.
This study shows that, at the 4-h time point, there was no difference
in the IL-8 mRNA expression of E1A+ve and E1Ave cells, indicating
that preformed vesicles of IL-8 may have been, at least in part,
responsible for the increase in IL-8 in response to
H2O2. In the present study, we also show that
E1A-dependent IL-8 secretion in response to LPS and PM10
was the result of increased IL-8 mRNA expression. The transcription of
IL-8 is mediated primarily through the transcription factors AP-1,
NF-
B, and NF-IL-6 (C/EBP; see Refs. 24,
35, 38). To understand the molecular
mechanism involved in PM10-mediated excess IL-8 release, we
studied the role of the redox-sensitive transcription factors such as
AP-1 and NF-
B, which have been shown to be activated in epithelial cells in response to PM10 (18, 23). We
demonstrate that the increased expression of IL-8 in E1A+ve cells was
associated with an increase in the nuclear binding of the transcription
factors AP-1 and NF-
B. Moreover, the basal levels of nuclear binding of these transcription factors and of C/EBP were increased in E1A+ve
cells, suggesting that these epithelial cells are primed for
proinflammatory responses. E1A proteins exert their major effects by
interaction with or altering the function of transcription factors,
including AP-1, NF-
B, and C/EBP, and coactivators such as CBP and
p300 (31, 34, 35, 46, 52a). This direct or indirect interaction may be
involved in the formation of complexes that control the expression of
genes regulated by these factors (30, 31, 52a). The complexes that are
formed may be responsible for the activation of NF-
B and AP-1 in the
nucleus, which was reported in E1A+ve cells in this and a previous
study (22). The presence of E1A protein has been shown to
be related to an activation of NF-
B and not AP-1 in LPS-treated
E1A+ve cells (22), and indeed, NF-
B is presumed to be
the primary regulatory factor in the expression of IL-8
(24). However, there is also evidence that E1A acts through AP-1 (2, 34). Our study also shows the activation of AP-1 in E1A+ve cells in response to PM10 compared with
the basal levels in E1A+ve cells. This study shows that E1A acts
through both AP-1 and NF-
B and that these are both involved in IL-8
gene regulation in E1A+ve cells. Further work is required to clarify the role of E1A in the transcriptional regulation of proinflammatory genes and to quantify the relative importance of the AP-1 and NF-
B pathways.
As previously reported (37), A549 cells harboring the
adenovirus E1A gene are sensitive to LPS exposure as shown by enhanced release of IL-8 and increased NF-B binding, a response that is not
present with cells not expressing the E1A gene. Although LPS is present
in some PM10 samples and may modulate its biological effects (7), we have previously shown that it is present
only in trace amounts in our PM10 samples
(27). LPS stimulation of cells normally requires the
LPS-binding protein and either membrane-bound or soluble CD14
(11). A deficiency of the membrane-bound receptor CD14 is
generally thought to be the principal mechanism of cellular nonresponsiveness to LPS in A549 cells (44). However,
there is no evidence to suggest that E1A-mediated LPS sensitization is
the result of increased CD14 abundance in this cell phenotype (21), and because E1A is a nuclear protein, it is unlikely
to interfere with membrane-related signal transduction
(22). The mechanism of LPS sensitivity in this system may
be the result of a serum-associated LPS-binding protein and requires
further study. This may involve the presence of soluble LPS-binding
proteins or changes in the expression of genes responsible for LPS
recognition, which are as yet largely unknown (55).
These in vitro data provide a plausible mechanism by which the presence of the E1A adenoviral protein may render cells primed and therefore susceptible to an amplification of inflammatory responses resulting from oxidative stress provided by PM10. Acute effects of PM10 on patients with airway disease (3) and chronic effects on airway disease (43) have been reported. An enhanced inflammatory response in E1A+ve lungs, if repetitive, may advance the development of chronic airway disease. Also, this mechanism of E1A susceptibility may be relevant to acute adenoviral infections, where with associated E1A presence, inflammation may be amplified to the level of causing exacerbations of airway diseases.
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ACKNOWLEDGEMENTS |
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We thank Ellen Drost for advice in the preparation of this manuscript.
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FOOTNOTES |
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This study was supported by the Medical Research Council, UK, and the British Lung Foundation. K. Donaldson is the Transco British Lung Foundation Fellow in Air Pollution and Respiratory Health.
Address for reprint requests and other correspondence: W. MacNee, ELEGI/Colt Laboratory, The Univ. of Edinburgh, Dept. of Medical & Radiological Sciences, Respiratory Section, Wilkie Bldg., Teviot Place, Edinburgh EH8 9AG, UK (E-mail: w.macnee{at}ed.ac.uk).
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. Section 1734 solely to indicate this fact.
Received 21 September 2000; accepted in final form 9 April 2001.
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