Adenoviral E1A primes alveolar epithelial cells to PM10-induced transcription of interleukin-8

Peter S. Gilmour1, Irfan Rahman1, Shizu Hayashi2, James C. Hogg2, Kenneth Donaldson3, and William MacNee1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , 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-kappa 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-kappa B; activator protein-1; lung epithelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa 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)-alpha (46) and amplifies inflammation in response to cigarette smoke in guinea pig lungs (53). IL-8 is induced by cytokines such as TNF-alpha , IL-1beta , 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-kappa 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-kappa 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
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-alpha (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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Fig. 1.   Agarose gel of RT-PCR products of early region 1A (E1A) DNA mRNA extracted from E1A-transfected (E1A+ve) and control-transfected (E1A-ve, vector only) A549 cells. -ve, RT-PCR in the absence of added RNA. Amplification of the E1A target of 486 bp (see ladder), a slower-migrating product representing linear amplifications beyond the second primer site and a faster-migrating product representing the single-strand form, are only observed in RNA extracted from E1A-transfected cells.



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Fig. 2.   Release of interleukin (IL)-8 from E1A+ve and E1A-ve cells without treatment (control) or after 18 h of treatment with lipopolysaccharide (LPS), environmental particulate matter < 10 µm (PM10), and tumor necrosis factor (TNF)-alpha . Each bar represents the mean ± SE value from 4 experiments. **P < 0.01 and ***P < 0.001 compared with E1A-ve samples.



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Fig. 3.   A: IL-8 RT-PCR product (top) from either E1A+ve or E1A-ve cells after 4 h of treatment with LPS (10 µg/ml), H2O2 (200 µM), or PM10 (100 µg/ml) compared with control. B: densitometric quantification of IL-8 RT-PCR products from either E1A+ve or E1A-ve cells after 4 h of treatment with LPS (10 µg/ml), H2O2 (200 µM), or PM10 (100 µg/ml) compared with control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Each bar represents mean ± SE values from 3 experiments. *P < 0.05 compared with E1A-ve samples.



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Fig. 4.   IL-8 RT-PCR (A) and PCR product quantification (B) from E1A+ve cells treated with control (solid bars), PM10 (100 µg/ml; open bars), or LPS (10 µg/ml; hatched bars) for 4, 8, 18, and 24 h.



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Fig. 5.   A: IL-8 RT-PCR from E1A+ve cells without treatment (control) or treated for 4 h with LPS (10 µg/ml), H2O2 (200 µM), or PM10 (100 µg/ml) with or without actinomycin D (Actin-D; 100 ng/ml), an inhibitor of mRNA transcription. GAPDH PCR product (bottom in A) is shown as a housekeeping gene control. B: densitometric quantification of IL-8 RT-PCR products from E1A+ve cells treated for 4 h with LPS (10 µg/ml), H2O2 (200 µM), or PM10 (100 µg/ml) with or without actinomycin D (100 ng/ml). Each bar represents mean ± SE values from 3 experiments. ***P < 0.001 and *P < 0.05 compared with E1A-ve samples.



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Fig. 6.   A: IL-8 RT-PCR from E1A+ve cells without treatment (control) or treated for 4 h with LPS (10 µg/ml) or PM10 (100 µg/ml) with or without glutathione monoethyl ester (GSHMEE; 5 mM), a thiol antioxidant. B: densitometric quantification of IL-8 RT-PCR products from E1A+ve cells treated for 4 h with LPS (10 µg/ml) or PM10 (100 µg/ml) with or without GSHMEE (5 mM). *P < 0.05 and **P < 0.01, PM10 vs. PM10 + GSHMEE and vs. control, respectively.



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Fig. 7.   Release of IL-8 from E1A+ve and E1A-ve cells without treatment (control) or after 4 h of treatment with LPS and H2O2 (200 µg/ml). Bars represent mean ± SE values from 4 experiments. *P < 0.05 and ***P < 0.001 compared with relevant control. #P < 0.05 compared with E1A-ve.



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Fig. 8.   Activator protein-1 (AP-1; A), nuclear factor (NF)-kappa B (B), and CCAAT/enhancer-binding protein (C/EBP; C) nuclear binding in E1A+ve and E1A-ve cells in response to 4 h of treatment with LPS (10 µg/ml), H2O2 (200 µM), and PM10 (100 µg/ml). Treatments are compared with HeLa cell nuclear extract positive (HeLa +ve) and no protein-binding reaction (-ve) controls. Each autoradiograph is representative of 3 separate experiments. Graphs show quantitation by densitometry of the AP-1, NF-kappa B, and C/EBP bands. NS, nonspecific bands. Bands specific to each transcription factor are determined by the inclusion of an unlabeled competitor oligonucleotide (Comp) for each relevant transcription factor and an unlabeled noncompetitor, i.e., an oligonucleotide representing an alternative transcription factor (Non-Comp). *P < 0.05 and **P < 0.01 compared with E1A-ve samples.



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Fig. 9.   Electrophoretic mobility shift assay (EMSA) of NF-kappa B binding after nuclear extracts of E1A+ve stimulated with PM10 (100 µg/ml) or H2O2 (200 µM) were incubated with antibodies to the p50 and p65 subunits of this transcription factor before addition of the labeled oligonucleotide. Arrows indicate the position of the bound oligonucleotide and the bound oligonucleotide supershifted by the binding of the monoclonal antibodies to the respective NF-kappa B subunits. NS, nonspecific binding; control, untreated E1A+ve cells.

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 E1A-ve, 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).

For cell treatments, monolayers were seeded and grown to 90% confluence in six-well plates, washed with PBS, and then placed in fresh medium either with or without the added treatment. In assays involving H2O2 where the antioxidant effects of serum were not desirable, the cell monolayers were washed with PBS, and fresh medium without serum was added for 24 h before treatment. The H2O2 treatment was also carried out in serum-free DMEM. Treatment incubation times are as stated in RESULTS and in the legends for Figs. 1-9. All treatments were for 18 h except for H2O2 treatments and where NF-kappa B and AP-1 binding activities were measured, which were 4 h because of the rapid nature of the treatment effect. RT-PCR experiments were performed on 4-h treatments and also on a time-course experiment; the treatment times are indicated in the legends for Figs. 1-9.

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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Table 1.   DNA sequences of RT-PCR primers and double-strand EMSA oligonucleotides used in this study

The primers were diluted to 100 pmol/µl with DEPC water. For each PCR, 5 µl of reverse-transcribed RNA (cDNA) was added directly to a PCR mixture set to a final volume of 50 µl and containing Taq DNA polymerase reaction buffer (Promega), 2.5 mM MgCl2, 0.2 mM dNTP mixture, 1 unit Taq DNA polymerase (Promega), and 1 µM appropriate primer pair. The E1A PCR conditions were 35 thermal cycles at 93°C for 1 min, 63°C for 1 min, and 72°C 90 s, followed by a final extension stage at 72°C for 10 min. The IL-8 PCR conditions were 35 thermal cycles at 94°C for 30 s, 60°C for 1 min, and 68°C for 2 min followed by a final extension step at 68°C for 7 min. The conditions for GAPDH were 35 thermal cycles at 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s followed by a final extension step at 68°C for 7 min. The resulting amplified DNA fragments were separated by electrophoresis through a 1.5% agarose gel, and the resulting bands were visualized and scanned by a white/ultraviolet transilluminator (Ultra Violet Products, Cambridge, UK) and quantified by densitometry.

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-kappa 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-kappa 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 [gamma -32P]ATP end-labeled double-strand consensus oligonucleotides for AP-1, NF-kappa 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.

To determine which subunits of the NF-kappa B bound to the oligonucleotide specifying this transcription factor, nuclear extracts were incubated with 4 µl (1 mg/ml) of anti-p50 or anti-p65 antibody (Santa Cruz Biotechnology) for 30 min on ice before addition of labeled oligonucleotides and analyzed by EMSA as described above. These sera do not interfere with nuclear factor binding. Rabbit preimmune serum (SAPU, Edinburgh, UK) was incubated with nuclear extracts as described above and used as a control.

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.


    RESULTS
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INTRODUCTION
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E1A mRNA expression. After the RT-PCR amplification of cDNA prepared from E1A+ve and E1A-ve 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-alpha 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-alpha 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 E1A-ve 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-alpha . 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-kappa B, and C/EBP nuclear binding. Untreated E1A+ve cells had significantly higher basal levels of AP-1, NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa B binding in E1A+ve cells. Increased NF-kappa B binding after PM10 and H2O2 treatment involved both p50 and p65 subunits, with a predominance for p50 (Fig. 9).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 E1A-ve 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa B and not AP-1 in LPS-treated E1A+ve cells (22), and indeed, NF-kappa 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-kappa 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-kappa 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-kappa 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.


    ACKNOWLEDGEMENTS

We thank Ellen Drost for advice in the preparation of this manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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