Short-term modulation of interleukin-1{beta} signaling by hyperoxia: uncoupling of I{kappa}B kinase activation and NF-{kappa}B-dependent gene expression

Kelli Odoms, Thomas P. Shanley, and Hector R. Wong

Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center and Children's Hospital Research Foundation, Cincinnati, Ohio 45229

Submitted 16 June 2003 ; accepted in final form 12 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-{alpha} (TNF-{alpha})-mediated activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by a mechanism involving increased activation of I{kappa}B kinase (IKK). Because the signal transduction mechanisms induced by IL-1{beta} are distinct to that of TNF-{alpha}, herein we sought to determine whether hyperoxia modulates IL-1{beta}-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1{beta} caused increased activation of IKK, prolonged the degradation of I{kappa}B{alpha}, and prolonged the nuclear translocation and DNA binding of NF-{kappa}B compared with cells treated with IL-1{beta} alone in room air. Hyperoxia did not affect IL-1{beta}-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-{beta} alone. In contrast to the effects on the IKK/I{kappa}B{alpha}/NF-{kappa}B pathway, simultaneous treatment with hyperoxia and IL-1{beta} did not augment NF-{kappa}B-dependent gene expression compared with treatment with IL-1{beta} alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1{beta} caused hyperphosphorlyation of the NF-{kappa}B p65 subunit compared with treatment with IL-1{beta} alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1{beta} augments activation of IKK, prolongs degradation of I{kappa}B{alpha}, and prolongs nuclear translocation and DNA binding of NF-{kappa}B. This activation, however, is not coupled to increased expression of NF-{kappa}B-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.

cell signaling; oxidant stress; transcription factors; lung epithelium


WE HAVE BEEN INTERESTED in elucidating how simultaneous stimuli modulate the proinflammatory signal transduction pathways of lung parenchymal cells. The rationale for this stems from the clinical realization that lung parenchymal cells are subjected to multiple, simultaneous stimuli during acute lung injury (ALI). Our previous studies demonstrated that high concentrations of ambient oxygen (hyperoxia) modulate tumor necrosis factor-{alpha} (TNF-{alpha})-mediated signal transduction pathways in cultured human respiratory epithelium (1, 32). We demonstrated that when cells were treated simultaneously with hyperoxia and TNF-{alpha}, the NF-{kappa}B pathway and NF-{kappa}B-dependent gene expression were both activated in a synergistic manner compared with cells treated with TNF-{alpha} alone. These data have led us to further investigate the effects of hyperoxia on modulating proinflammatory signal transduction pathways in lung epithelium.

NF-{kappa}B is a pluripotent transcription factor that regulates the expression of many proinflammatory genes known to be involved in the pathobiology of ALI (12, 15). Increased activation of NF-{kappa}B was demonstrated in patients with ALI (19, 25), and increased activation of NF-{kappa}B was associated with increased mortality in patients with septic shock, a condition that is typically associated with ALI (3). In animal models of ALI, inhibiting activation of NF-{kappa}B conferred protection (2, 17, 24, 34). Thus a better understanding of how multiple, simultaneous stimuli interact to modulate the activity of NF-{kappa}B in lung parenchymal cells could lead to a greater understanding of the signaling pathways involved in ALI.

IL-1{beta}-dependent signaling plays a major effector role in many inflammatory processes including septic shock and ALI (9). IL-1{beta} shares many biological and physiological properties with TNF-{alpha}, but the signaling mechanisms that lead to IL-1{beta}-dependent proinflammatory gene expression are distinct. IL-1{beta}-mediated signaling begins when IL-1{beta} binds to the IL-1 receptor 1 (IL-1R1), which leads to the recruitment of the cytosolic adapter proteins MyD88 and Tollip (4, 5, 22, 27, 30). The IL-1{beta}-IL-1R-MyD88-Tollip complex serves to recruit IL-1 receptor-associated kinase (IRAK), a serine-threonine kinase. The inclusion of IRAK in this signalsome serves to recruit a variety of other kinases and binding proteins, even-tually leading to activation of I{kappa}B kinase (IKK) and subsequent NF-{kappa}B activation.

Given our previous investigations regarding the synergistic effects of hyperoxia on TNF-{alpha}-induced proinflammatory signaling and the distinct and central role played by IL-1{beta} in proinflammatory signaling, herein we tested the hypothesis that hyperoxia modulates IL-1{beta}-dependent signal transduction pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents. A549 human respiratory epithelial cells (American Type Culture Collection, Rockville, MD) were used for all experiments. A549 cells are derived from a human lung adenocarcinoma, retain several features of type II pneumocytes, and have been used as a model for NF-{kappa}B activation in vitro (1, 32, 33). Cells were maintained in a room air/5% CO2 incubator at 37°C in DMEM (GIBCO-BRL) containing 8% FBS and penicillin/streptomycin (GIBCO-BRL).

Selected experiments (IL-8 promoter-luciferase reporter assays and EMSA) were duplicated in BEAS-2B cells (American Type Culture Collection, Bethesda, MD), a human bronchial epithelial cell line transformed by an adenovirus 12-SV40 hybrid virus. These cells also serve as a useful in vitro model of NF-{kappa}B activation and IL-8 gene expression (28). BEAS-2B cells were cultured in the same manner and conditions as described above.

Experimental conditions. Cells were maintained in media containing 8% serum during propagation, and serum-free conditions were used during experiments to decrease basal cell activation. In all experiments the following four conditions were used: serum-free media + room air (control), human IL-1{beta} (1 ng/ml) + room air, IL-1{beta} (1 ng/ml) + hyperoxia (95% O2), or hyperoxia alone. Hyperoxia was achieved by placing cells in sealed modular chambers (Billups-Rothenberg, Del Mar, CA) and flushing the chambers with a humidified gas mixture of 95% O2/5% CO2 at 1 l/min for 30 min. The entry and exit ports were subsequently clamped, and cells were returned to a 37°C incubator. Achievement of 95% O2 was confirmed by placing an oxygen sensor (Mini-Ox II, Catalyst Research MSA) in the modular chambers. To control for the time period of flushing, we also placed cells designated for room air treatment in sealed modular chambers and flushed them with a humidified gas mixture of 21% O2/5% CO2/balance nitrogen (1, 32).

Nuclear protein extraction. All nuclear protein extraction procedures were performed on ice with ice-cold reagents. Cells were washed twice with PBS and harvested by scraping into 1 ml of PBS and pelleted at 6,000 rpm for 5 min. The pellet was washed twice with PBS, resuspended in one packed cell volume of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% vol/vol Nonidet P-40, 1 mM DTT, and 0.1 mM PMSF), and incubated for 5 min with occasional vortexing. After centrifugation at 6,000 rpm, one cell pellet volume of extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 0.1 M EDTA, 1.5 mM MgCl2, 25% vol/vol glycerol, 1 mM DTT, and 0.5 mM PMSF) was added to the nuclear pellet and incubated on ice for 15 min with occasional vortexing. The nuclear proteins were isolated by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA) and stored at -70°C until used for EMSA.

EMSA. The NF-{kappa}B oligonucleotide probe used for EMSA (5'-GTGGAATTTCCTCTGA -3') corresponds to the NF-{kappa}B site in the human IL-8 promoter and was synthesized at the University of Cincinnati DNA Core Facility (1). The probe was labeled with {gamma}-[32P]ATP using T4 polynucleotide kinase (GIBCO-BRL) and purified in Bio-Spin chromatography columns (Bio-Rad). For EMSA, 10 µg of nuclear proteins were preincubated with EMSA buffer [12 mM HEPES pH 7.9, 4 mM Tris·HCl pH 7.9, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly(dI-dC), 12% glycerol vol/vol, and 0.2 mM PMSF] on ice for 10 min before addition of the radiolabeled oligonucleotide probe for an additional 10 min. Supershift assays were performed by incubating the nuclear proteins with radiolabeled oligonucleotide plus an anti-p65 IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Protein-nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide-bisacrylamide) and run in 0.5x TBE (45 mM Tris·HCl, 45 mM boric acid, and 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper, dried under a vacuum at 80°C for 1 h, and exposed to photographic film at -70°C with an intensifying screen.

Western blot analysis. Treated cells were washed once in PBS and lysed in ice-cold buffer containing 50 mM Tris (pH 8.0), 110 mM NaCl, 5 mM EDTA, 1% Triton X-100, and PMSF (100 µg/ml). Protein concentrations were determined by the Bradford assay. Whole cell lysates were boiled in equal volumes of loading buffer (125 mM Tris·HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) and 50 µg of protein loaded per lane on an 8-16% Tris-glycine gradient gel (Novex, San Diego, CA). Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Novex) using the Novex Xcell Mini-Gel system. For immunoblotting, membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies against human I{kappa}B{alpha}, human IRAK, or phospho-p65 (Santa Cruz Biotechnology) were applied at appropriate dilutions for 2 h. After being washed two times in TBS containing 0.05% Tween 20 (TTBS), appropriate peroxidase-conjugated secondary antibodies (Calbiochem, La Jolla, CA) were applied at 1:10,000 dilution for 1 h. Blots were washed in TTBS two times over 30 min, incubated in commercial enhanced chemiluminescence reagents (ECL; Amersham, Buckinghamshire, England), and exposed to photographic film.

Northern blot analysis. Total RNA was isolated using the TRIzol reagent (GIBCO-BRL). RNA concentrations were determined by spectrophotometry (260 nm), and 15 µg of RNA for each sample underwent electrophoresis in gels containing 1% agarose and 3% formaldehyde. RNA integrity was confirmed visually by ethidium bromide staining and brief UV light illumination. RNAs were transferred to nylon membranes (Micron Separations, Westboro, MA) and UV auto-crosslinked (UV Stratalinker 1800; Stratagene, La Jolla, CA). Membranes were prehybridized for 4 h at 42°C and subsequently hybridized overnight with a radiolabeled IL-8 cDNA probe (1). The cDNA probe was labeled with {alpha}-[32P]dCTP (specific activity 3,000 Ci/mM; New England Nuclear Research Products, Boston, MA) by random priming (Pharmacia, Piscataway, NJ). Membranes were subsequently washed twice with 2x SSC/0.1% SDS at 53°C, developed using a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA), and analyzed using ImageQuant Software (Molecular Dynamics).

IKK assay. Treated cells were washed in ice-cold PBS containing 1 mM PMSF, 100 µM Na3VO4, 2 mM p-nitrophenyl phosphate (PNPP), and 210 mU/ml aprotinin. Cells were scraped into 0.5 ml of the above modified PBS and centrifuged at 3,000 rpm for 5 min at 4°C, and the resulting pellet was resuspended in 200 µl of lysis buffer containing 50 mM Tris·HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 100 µM Na3VO4, 2 mM PNPP, 1 mM PMSF, and 210 mU/ml aprotinin. The resuspended pellet was rocked for 30 min at 4°C, followed by centrifugation at 10,000 rpm for 10 min at 4°C. The resulting supernatants were assayed for protein concentration, and 600 µg of each sample were rocked for 60 min at 4°C with 4 µl of IKK{gamma} polyclonal antibody (Santa Cruz Biotechnology) in 500 µl of a pull-down buffer containing: 20 mM Tris (pH 8.0), 3 mM EDTA, 3 mM EGTA, 250 mM NaCl, 0.05% Nonidet P-40, 500 µM 4-2-aminoethyl-benzenesulfonyl fluoride, 20 mM {beta}-glycerolphosphate, 100 µM Na3VO4, 2 mM PNPP, and 10 µl/ml of a protease/phosphatase inhibitor cocktail (Sigma Chemical, St. Louis, MO). Fifty microliters of protein A/G agarose beads (Santa Cruz Biotechnology) were then added to the cell extract-antibody mixture and rocked overnight at 4°C. The cell extract-antibody mixture was centrifuged at 3,000 rpm for 5 min at 4°C. The resulting pellets were resuspended in 25 µl of the above kinase assay buffer with the addition of 100 µM ATP, 12 µg glutathione S-transferase (GST)-I{kappa}B{alpha} (1-54, Ref. 2) as substrate, and 1 µl {gamma}-[32P]dATP (10 µCi/µl, New England Nuclear), and incubated for 30 min at 30°C. The reaction was stopped in an ice bath, mixed with 25 µl of Western blot loading buffer, boiled for 3 min, and loaded on 10% Tris-glycine gels (Novex). After electrophoresis, gels were dried, washed, and developed using a PhosphorImager screen and analyzed using ImageQuant Software. The bands corresponding to phosphorylated GST-I{kappa}B{alpha} were interpreted as an indirect measure of IKK activity.

Transient transfection and luciferase assay. To measure the down-stream effects of hyperoxia- and TNF-{alpha}-mediated activation of NF-{kappa}B, we transiently transfected cells with either an IL-8 or an intercellular adhesion molecule-1 (ICAM-1) promoter-luciferase reporter plasmid (1, 32). Cells were transfected with the respective reporter plasmids in triplicate, in six-well plates, at a density of 300,000 cells per well by incubation with FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) and serum-free DMEM for 5 h. After transfection, cells were washed once with PBS and allowed to recover overnight in basal growth media. After treatment with the experimental conditions for 4 h, cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer's instructions (Promega, Madison, WI) using a Berthold AutoLumat LB953 luminometer. Luciferase activity is reported as fold induction over control cells (transfected and treated with basal growth media in room air) and corrected for total cellular protein. The 4-h time point for harvesting cellular proteins for luciferase assay is based on our previous experience indicating that luciferase activity is maximal after this period of incubation (1, 32).

IL-8 ELISA. Cells were exposed to the experimental conditions, and supernatants were harvested at 24 h after treatment. The 24-h time point for harvesting supernatants is based on our previous experience indicating that supernatant levels of IL-8 peptide are maximal at this time point. Immunoreactive IL-8 levels were determined using a commercially available human IL-8 ELISA kit (Biosource International, Camarillo, CA). All procedures were performed according to the manufacturer's instructions. Interassay variability is 4.3%, and intraassay variability is 4.9% (www.biosource.com). Standard curves for IL-8 were performed using the diluent supplied in the kit as recommended by the manufacturer.

Statistical analysis. Quantitative data were analyzed by ANOVA using SigmaStat software (Jandel, Chicago, IL). A P value < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperoxia synergistically increases IL-1{beta}-mediated activation of IKK. In these experiments we determined whether hyperoxia modulates IL-1{beta}-mediated activation of IKK. Activation of IKK is generally regarded as the rate-limiting step leading to activation of NF-{kappa}B (14), and we previously demonstrated that simultaneous stimulation with TNF-{alpha} and hyperoxia synergistically increased IKK activation compared with stimulation with TNF-{alpha} alone (32). Exposure to hyperoxia alone, for 0.5-2 h, did not increase activation of IKK (Fig. 1, lanes 2, 5, and 8). Exposure to IL-1{beta} alone, for 0.5-2 h, increased activation of IKK in a time-dependent manner (Fig. 1, lanes 3, 6, and 9). Peak activity of IKK occurred at 0.5 h after treatment with IL-1{beta} and decreased thereafter. Concomitant exposure to hyperoxia and IL-1{beta}, for 0.5-2 h, increased IKK activation further compared with cells treated with IL-1{beta} alone (Fig. 1, lanes 4, 7, and 10). This effect was evident at 0.5 and 1 h, but not at 2 h, after concomitant exposure to hyperoxia and IL-1{beta}. These data demonstrate that although hyperoxia alone does not modulate IKK activity, it augments IL-1{beta}-dependent activation of IKK.



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Fig. 1. Representative I{kappa}B kinase (IKK) assay demonstrating that hyperoxia augments IL-1{beta}-dependent activation of IKK. Cells were exposed to the experimental conditions for 0.5-2 h, as indicated. In vitro phosphorylation of a glutathione S-transferase (GST)-I{kappa}B{alpha} substrate is indicative of IKK activity. Gel is representative of 3 separate experiments with similar results. The average band densities (± SE) of the 3 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. IL-1{beta} alone.

 

Hyperoxia prolongs IL-1{beta}-mediated degradation of I{kappa}B{alpha}. A primary functional consequence of IKK activation is phosphorylation and subsequent rapid degradation of I{kappa}B{alpha} (15). Having demonstrated the synergistic effects of hyperoxia on IL-1{beta}-mediated IKK activation, we next examined the effects of hyperoxia on I{kappa}B{alpha} degradation. Exposure to hyperoxia alone, for 0.5-3 h, did not significantly affect I{kappa}B{alpha} levels (Fig. 2, lanes 2, 5, 8, and 11). Treatment with IL-1{beta} alone, for 0.5-3 h, caused degradation of I{kappa}B{alpha} in a time-dependent manner (Fig. 2, lanes 3, 6, 9, and 12). Maximal degradation of I{kappa}B{alpha} occurred 0.5 h after IL-1{beta} treatment and returned to baseline levels by 1 h, in keeping with our previous studies. Concomitant exposure to hyperoxia and IL-1{beta}, for 0.5-3 h, prolonged I{kappa}B{alpha} degradation compared with treatment with IL-1{beta} alone (Fig. 2, lanes 4, 7, 10, and 13). I{kappa}B{alpha} levels remained significantly reduced up to 3 h after concomitant treatment with hyperoxia and IL-1{beta}. These data demonstrate that hyperoxia alone does not cause degradation of I{kappa}B{alpha}, but concomitant hyperoxia and IL-1{beta} cause prolonged degradation of I{kappa}B{alpha}. In addition, this observation correlates temporally with the synergistic effect of hyperoxia on IL-1{beta}-mediated activation of IKK.



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Fig. 2. Representative Western blot analysis demonstrating that hyperoxia prolongs IL-1{beta}-dependent degradation of I{kappa}B{alpha}. Cells were exposed to the experimental conditions for 0.5-3 h, as indicated. Gel is representative of 3 separate experiments with similar results. The average band densities (± SE) of the 3 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. IL-1{beta} alone.

 

Effect of hyperoxia on IRAK degradation. IL-1{beta}-mediated degradation of IRAK precedes IKK activation (4, 5, 22, 27, 30). Having demonstrated the effects of hyperoxia on IL-1{beta}-mediated activation of IKK and degradation of I{kappa}B{alpha}, we next determined the effects of hyperoxia on IL-1{beta}-mediated degradation of IRAK. Exposure to hyperoxia alone did not induce degradation of IRAK (Fig. 3, lanes 2, 5, 8, and 11). Treatment with IL-1{beta} alone, for 0.5-3 h, caused degradation of IRAK (Fig. 3, lanes 3, 6, 9, and 12). Concomitant treatment with hyperoxia and IL-1{beta}, for 0.5-3 h, did not affect IRAK degradation differently compared with treatment with IL-1{beta} alone. These data demonstrate that hyperoxia does not alter IL-1{beta}-mediated degradation of IRAK.



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Fig. 3. Representative Western blot analysis demonstrating that hyperoxia does not modulate IL-1{beta}-dependent degradation of IL-1 receptor-associated kinase (IRAK). Cells were exposed to the experimental conditions for 0.5-3 h, as indicated. Gel is representative of 3 separate experiments with similar results. The average band densities (± SE) of the 3 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. hyperoxia alone.

 

Effects of hyperoxia on IL-1{beta}-mediated activation of NF-{kappa}B. Having demonstrated the effects of hyperoxia on IL-1{beta}-mediated activation of IKK and degradation of I{kappa}B{alpha}, we next determined the effects of hyperoxia on IL-1{beta}-mediated nuclear translocation and DNA binding of NF-{kappa}B. Exposure to hyperoxia alone, for 0.5-3 h, did not affect activation of NF-{kappa}B (Fig. 4A, lanes 2, 5, 8, and 11). Treatment with IL-1{beta} alone, for 0.5-3 h, increased activation of NF-{kappa}B in a time-dependent manner (Fig. 4A, lanes 3, 6, 9, and 12). Maximal NF-{kappa}B activation occurred within 0.5 h of IL-1{beta} treatment and decreased thereafter. Exposure to concomitant hyperoxia and IL-1{beta}, for 0.5-3 h, modified the kinetics of NF-{kappa}B activation compared with treatment with IL-1{beta} alone (Fig. 4A, lanes 4, 7, 10, and 13). Hyperoxia prolonged IL-1{beta}-mediated activation of NF-{kappa}B up to 3 h after treatment. The specificity of NF-{kappa}B binding was confirmed by supershift assays using a p65 subunit IgG. Treatment with IL-1{beta} alone or concomitant treatment with IL-1{beta} and hyperoxia for 1 h caused a similar shift of the putative NF-{kappa}B band (Fig. 4B, lanes 2 and 4). These data demonstrate that the effects of hyperoxia on IL-1{beta}-mediated activation of IKK and I{kappa}B{alpha} degradation lead to prolonged nuclear translocation and DNA binding of NF-{kappa}B in A549 cells.



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Fig. 4. A: representative EMSA demonstrating that hyperoxia prolongs IL-1{beta}-dependent nuclear translocation and DNA binding of NF-{kappa}B. Cells were exposed to the experimental conditions for 0.5-3 h, as indicated. Gel is representative of 4 separate experiments with similar results. The average band densities (± SE) of the 4 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. IL-1{beta} alone. B: representative supershift assay with an anti-p65 IgG demonstrating the specificity of DNA-nuclear protein binding. Cells were exposed to the indicated conditions for 1 h.

 

Effects of hyperoxia on IL-1{beta}-mediated phosphorylation of p65. Phosphorylation of the NF-{kappa}B p65 subunit provides another regulatory mechanism for NF-{kappa}B-dependent gene expression (26, 29). Having demonstrated that hyperoxia augments IL-1{beta}-mediated activation of the IKK/I{kappa}B{alpha}/NF-{kappa}B pathway, we next sought to determine the effects of hyperoxia on IL-1{beta}-mediated phosphorylation of p65 using a phospho-p65 specific antibody and Western blot analysis. Exposure to hyperoxia alone, for 0.5-3 h, did not cause phosphorylation of p65 (Fig. 5, lanes 2, 5, 8, and 11). Treatment with IL-1{beta} alone, for 0.5-3 h, increased phosphorylation of p65 in a time-dependent manner (Fig. 5, lanes 3, 6, 9, and 12). Maximal phosphorylation of p65 occurred between 0.5 and 1 h of IL-1{beta} treatment and decreased thereafter. Exposure to concomitant hyperoxia and IL-1{beta}, for 0.5-3 h, augmented p65 phosphorylation at all time points tested except for the 3-h time point (Fig. 5, lanes 4, 7, 10, and 13). These data demonstrate that hyperoxia by itself does not affect phosphorylation of p65 but augments IL-1{beta}-dependent phosphorylation of p65 in A549 cells.



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Fig. 5. Representative Western blot analysis (using a phospho-p65 antibody) demonstrating that hyperoxia does not decrease IL-1{beta}-dependent phosphorylation of p65. Cells were exposed to the experimental conditions for 0.5-3 h, as indicated. Gel is representative of 3 separate experiments with similar results. The average band densities (± SE) of the 4 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. IL-1{beta} alone.

 

Effects of hyperoxia on IL-1{beta}-mediated NF-{kappa}B-dependent gene expression. Having demonstrated the effects of hyperoxia on IL-1{beta}-mediated activation of the IKK/I{kappa}B{alpha}/NF-{kappa}B pathway, we next determined the effects of concomitant hyperoxia and IL-1{beta} on NF-{kappa}B-dependent gene expression. We focused on IL-8 gene expression because we have previously demonstrated that IL-8 is an NF-{kappa}B-dependent gene in A549 cells. Exposure to hyperoxia alone did not affect expression of IL-8 mRNA (Fig. 6, lanes 3 and 4) or IL-8 peptide (Fig. 7). IL-8 peptide was not detectable in the media of control cells or cells treated with hyperoxia alone (data not shown). Treatment with IL-1{beta} alone increased both expression IL-8 mRNA (Fig. 6, lanes 5 and 6) and IL-8 peptide (Fig. 7). Unexpectedly, concomitant exposure to hyperoxia and IL-1{beta} decreased expression of IL-8 mRNA (Fig. 6, lanes 7 and 8) and IL-8 peptide (Fig. 7) compared with treatment with IL-1{beta} alone.



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Fig. 6. Representative Northern blot analysis demonstrating that hyperoxia does not augment IL-1{beta}-dependent expression of IL-8 mRNA. Cells were exposed to the experimental conditions for 2 h, as indicated. Conditions are represented in duplicate, and the gel is representative of 4 separate experiments with similar results. The average band densities (± SE) of the 4 experiments are represented in the graph and plotted as fold induction over control cells and corrected for the respective 18S rRNA band densities. *P < 0.05 vs. IL-1{beta} alone.

 


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Fig. 7. ELISA demonstrating that hyperoxia does not augment IL-1{beta}-dependent expression of IL-8 peptide. Cells were exposed to the experimental conditions for 24 h, as indicated. Data represent 4 separate experiments with each condition carried out in triplicate. IL-8 peptide levels were not detectable in the media of control cells or cells exposed to hyperoxia alone (data not shown). *P < 0.05 vs. IL-1{beta} alone.

 

To confirm these unexpected observations, we transiently transfected cells with either an IL-8 promoter-luciferase reporter plasmid or an ICAM-1 promoter-luciferase plasmid and exposed cells to the experimental conditions. Exposure to hyperoxia alone did not increase the promoter of activity of either IL-8 (Fig. 8) or ICAM-1 (Fig. 9). Treatment with IL-1{beta} alone increased the promoter activity of both IL-8 (Fig. 8) and ICAM-1 (Fig. 9). Concomitant treatment with hyperoxia and IL-1{beta} decreased the promoter activity of both IL-8 (Fig. 8) and ICAM-1 (Fig. 9) compared with treatment with IL-1{beta} alone.



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Fig. 8. Luciferase assay demonstrating that hyperoxia does not augment IL-1{beta}-dependent activation of the IL-8 promoter. Cells were transiently transfected with an IL-8 promoter-luciferase promoter plasmid and exposed to the experimental conditions for 4 h, as indicated. Data represent 5 separate transfections with each condition carried out in triplicate. *P < 0.05 vs. IL-1{beta} alone.

 


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Fig. 9. Luciferase assay demonstrating that hyperoxia does not augment IL-1{beta}-dependent activation of the ICAM-1 promoter. Cells were transiently transfected with an ICAM-1 promoterluciferase promoter plasmid and exposed to the experimental conditions for 4 h, as indicated. Data represent 5 separate transfections with each condition carried out in triplicate.

 

Collectively, these data demonstrate that hyperoxia by itself does not affect NF-{kappa}B-dependent gene expression in A549 cells, in keeping with the above data involving hyperoxia alone and activation of the NF-{kappa}B pathway. In contrast, hyperoxia attenuates IL-1{beta}-mediated activation of NF-{kappa}B-dependent genes despite the observation that hyperoxia augments IL-1{beta}-mediated activation of the IKK/I{kappa}B{alpha}/NF-{kappa}B pathway.

Uncoupling of IL-8 promoter activation and NF-{kappa}B activation in BEAS-2B cells. The data above demonstrate that concomitant exposure of A549 cells to hyperoxia and IL-1{beta} leads to prolonged NF-{kappa}B activation, but this effect does not lead to enhanced expression of the IL-8 gene. To determine whether this observation is limited to A549 cells or if it is operative in other types of respiratory epithelial cells, we treated BEAS-2B cells with the same experimental conditions and measured IL-8 promoter activation and NF-{kappa}B activation. BEAS-2B cells were transiently transfected with an IL-8 promoter-luciferase reporter plasmid and exposed to the above experimental conditions. In BEAS-2B cells, exposure to hyperoxia alone did not significantly increase IL-8 promoter activity, whereas exposure to IL-1{beta} alone significantly increased IL-8 promoter activity (Fig. 10). Concomitant exposure to hyperoxia and IL-1{beta} did not increase IL-8 promoter activity beyond that seen with IL-1{beta} alone.



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Fig. 10. Luciferase assay demonstrating that hyperoxia does not augment IL-1{beta}-dependent activation of the IL-8 promoter in BEAS-2B cells. Cells were transiently transfected with an IL-8 promoter-luciferase promoter plasmid and exposed to the experimental conditions for 4 h, as indicated. Data represent 4 separate transfections with each condition carried out in triplicate.

 

We next measured the effects of hyperoxia on IL-1{beta}-mediated nuclear translocation and DNA binding of NF-{kappa}B in BEAS-2B. Exposure to hyperoxia alone, for 0.5-3 h, had a minimal effect on NF-{kappa}B activation (Fig. 11, lanes 2, 5, 8, and 11). Treatment with IL-1{beta} alone, for 0.5-3 h, increased activation of NF-{kappa}B in a time-dependent manner (Fig. 11, lanes 3, 6, 9, and 12). Maximal NF-{kappa}B activation occurred within 0.5-1 h of IL-1{beta} treatment and returned to baseline by 3 h. Exposure to concomitant hyperoxia and IL-1{beta}, for 0.5-3 h, prolonged activation of NF-{kappa}B up to 3 h compared with cells exposed to IL-1{beta} alone (Fig. 11, lanes 4, 7, 10, and 13). Collectively, these data demonstrate that in BEAS-2B cells concomitant exposure to hyperoxia and IL-1{beta} prolongs NF-{kappa}B activation compared with cells exposed to IL-1{beta} alone, but this effect does not lead to enhanced activation of the IL-8 promoter.



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Fig. 11. Representative EMSA demonstrating that hyperoxia prolongs IL-1{beta}-dependent nuclear translocation and DNA binding of NF-{kappa}B in BEAS-2B cells. Cells were exposed to the experimental conditions for 0.5-3 h, as indicated. Gel is representative of 3 separate experiments with similar results. The average band densities (± SE) of the 4 experiments are represented in the graph and plotted as fold induction over control cells: hyperoxia alone (light gray bars), IL-1{beta} alone (black bars), and hyperoxia + IL-1{beta} (dark gray bars). Control cells are assigned a value of 1 and are not shown on the graph. *P < 0.05 vs. IL-1{beta} alone.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-1{beta}, TNF-{alpha}, and hyperoxia are canonical cellular signals centrally involved in the pathophysiology of ALI (6-8, 10, 13, 16, 20, 23, 31). The effects of each signal, in isolation, are well characterized in the lung and other tissues. During ALI, however, it is unlikely that either of these two signals act in an isolated manner. Rather, lung parenchymal cells are often exposed to these signals simultaneously. Thus a better understanding of how these signals interact may be relevant to the signal transduction pathways involved in ALI.

Using an in vitro model that employs simultaneous stimulation with hyperoxia and TNF-{alpha}, our laboratory recently demonstrated an unexpected interaction between these two stimuli in cultured human lung epithelium (1, 32). In combination, hyperoxia and TNF-{alpha} synergistically induced expression of the IL-8 gene, which is generally regarded as an important proinflammatory mediator during ALI. The mechanism of this effect involves increased activation of IKK and subsequent prolonged activation of NF-{kappa}B. Using the same in vitro model, we have now demonstrated another unexpected interaction between hyperoxia and IL-1{beta}.

In keeping with our previous findings, hyperoxia by itself did not induce activation of IKK, degradation of I{kappa}B{alpha}, or activation of NF-{kappa}B in A549 cells. When combined with IL-1{beta}, however, the results were different from expected based on the effects of either signal in isolation. Concomitant exposure to hyperoxia and IL-1{beta} increased both the amount and duration of IKK activation compared with treatment with IL-1{beta} alone, and this effect did not appear to be related to differences in IRAK degradation. Functionally, this effect of concomitant hyperoxia and IL-1{beta} led to prolonged degradation of I{kappa}B{alpha} and prolonged nuclear translocation and DNA binding of NF-{kappa}B. Thus similar to that seen with TNF-{alpha} signaling, hyperoxia synergistically increases nuclear translocation and DNA binding of NF-{kappa}B by a mechanism involving increased activation of IKK.

To define the functional consequences of increased NF-{kappa}B activation in response to combined hyperoxia and IL-1{beta}, we measured expression of the NF-{kappa}B-dependent gene IL-8 using Northern blot analyses, ELISA, and an IL-8 promoter luciferase reporter plasmid. We were surprised to find that hyperoxia did not augment IL-8 gene expression when combined with IL-1{beta}, despite the augmentation noted when we measured IKK activity, I{kappa}B{alpha} degradation, and NF-{kappa}B nuclear translocation and DNA binding. In fact, the combination of hyperoxia and IL-1{beta} seemed to decrease IL-8 gene expression compared with IL-1{beta} alone.

Given that three distinct assays were used to measure IL-8 gene expression, we are confident of these data. In addition, these data were confirmed using an ICAM-1 promoter luciferase plasmid. We previously demonstrated that the ICAM-1 promoter, another NF-{kappa}B-dependent gene, is synergistically activated by the combination of hyperoxia and TNF-{alpha} (32). In the current experiments the combination of hyperoxia and IL-1{beta} did not augment activation of the ICAM-1 promoter compared with IL-1{beta} alone. Thus unlike the combination of hyperoxia and TNF-{alpha}, the combination of hyperoxia and IL-1{beta} does not augment activation of the NF-{kappa}B-dependent genes IL-8 and ICAM-1.

IKK activation is generally regarded as the rate-limiting step in the activation of NF-{kappa}B and of NF-{kappa}B gene expression (15). In direct contrast to this concept, our current data demonstrate an uncoupling between IKK activation, NF-{kappa}B activation, and the expression of NF-{kappa}B-dependent genes. Hyperoxia clearly augments IL-1{beta}-mediated activation of IKK in this model. This leads to the expected effects on I{kappa}B{alpha} and NF-{kappa}B but does not lead to the expected effect on NF-{kappa}B-dependent gene expression.

This finding appears to be applicable to at least one other respiratory epithelial cell line since similar observations were made in BEAS-2B cells. Whether or not this finding is applicable to other respiratory epithelial cells and other mammalian systems is an important point to consider, since previous studies involving murine systems demonstrated increased lung epithelial expression of ICAM-1 in response to hyperoxia (14, 21). In addition, previous in vivo studies demonstrated increased neutrophil chemotactic activity and increased TNF-{alpha} expression in the bronchoalveolar lavage fluids of neonatal and adult rats exposed to 95% oxygen (11, 18). At best we can conclude that in two distinct types of human lung epithelial cells, in vitro, there is an uncoupling between NF-{kappa}B activation and NF-{kappa}B-dependent gene expression when the cells are exposed to concomitant hyperoxia and IL-1{beta}. In addition, these observations are limited to 95% oxygen and need to be extended with future dose-response experiments involving lower levels of hyperoxia.

The mechanism of this uncoupling does not appear to involve decreased phosphorylation of the NF-{kappa}B p65 subunit. In fact, the combination of hyperoxia and IL-1{beta} caused hyperphosphorylation of p65 compared with treatment with IL-1{beta} alone. From the current data, it could be suggested that hyperphosphorylation of the p65 subunit by the combination of hyperoxia and IL-1{beta} leads to nuclear translocation of an NF-{kappa}B dimer having decreased transcriptional activity, but this has not been directly demonstrated. In combination with our previous data involving simultaneous hyperoxia and TNF-{alpha}, these data further illustrate that IL-1{beta}-dependent signal transduction mechanisms are distinct from those of TNF-{alpha}. Furthermore, these data provide another example of how simultaneous stimuli can modulate proinflammatory signal transduction pathways in lung cells differently from what would be predicted by the effects of isolated signals.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Institute of General Medical Sciences Grant RO1GM-61723 (H. R. Wong).


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. R. Wong, Div. of Critical Care Medicine, Cincinnati Children's Hosp. Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: wonghr{at}chmcc.org).

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.


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