Divisions of 1 Critical Care Medicine, 2 Pulmonary Biology, and 3 Pulmonary Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
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ABSTRACT |
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Interleukin (IL)-8 is
an important mediator of acute lung injury. Hyperoxia induces IL-8
production in some cell types, but its effect on IL-8 gene expression
in respiratory epithelium is not well described. In addition, IL-8 gene
expression resulting from the combined effects of hyperoxia and
proinflammatory cytokines has not been well characterized. We treated
cultured respiratory epithelial-like cells (A549 cells) with hyperoxia
alone, tumor necrosis factor (TNF)- alone, or the combination of
TNF-
and hyperoxia and evaluated IL-8 gene expression. Hyperoxia
alone had a minimal effect on IL-8 gene expression, and TNF-
alone increased IL-8 gene expression in a time-dependent manner. In contrast,
the combination of TNF-
and hyperoxia synergistically increased IL-8
gene expression as measured by ELISA (TNF-
alone for 24 h = 769 ± 89 pg/ml vs. hyperoxia + TNF-
for 24 h = 1,189 ± 89 pg/ml) and
Northern blot analyses. Experiments involving IL-8 promoter-reporter
assays, electromobility shift assays, and Western blot analyses
demonstrated that hyperoxia augmented TNF-
-mediated activation of
the IL-8 promoter by a nuclear factor (NF)-
B-dependent mechanism and
increased the duration of NF-
B nuclear translocation after
concomitant treatment with TNF-
. Additional reporter gene assays
demonstrated, however, that increased activation of NF-
B does not
fully account for the synergistic effect of hyperoxia and that the
NF-IL-6 site in the IL-8 promoter is also required for the synergistic
effect of hyperoxia. We conclude that hyperoxia alone has a minimal
effect on IL-8 gene expression but synergistically increases IL-8 gene
expression in the presence of TNF-
by a mechanism involving
cooperative interaction between the transcription factors NF-
B and
NF-IL-6.
respiratory epithelium; inflammation; chemokines; tumor necrosis
factor-; nuclear factor-
B; nuclear factor-interleukin-6
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INTRODUCTION |
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ACUTE LUNG INJURY (ALI) remains a significant cause of morbidity and mortality in critically ill patients. Although oxygen is a key supportive therapy for these patients, high levels of oxygen (hyperoxia) have been shown to directly cause or exacerbate ALI (18, 34). Many factors appear to be involved in hyperoxia-induced ALI. Hyperoxia can directly cause lung injury by generation of reactive oxygen species. These species directly cause cellular damage by mechanisms such as lipid peroxidation, oxidation of cellular proteins, DNA damage, and mitochondrial damage. Other important mechanisms in hyperoxia-induced ALI include the apparent ability of hyperoxia to cause lung inflammation. In rodent models of hyperoxia-induced ALI, neutrophil chemotactic activity of bronchoalveolar lavage (BAL) fluid was significantly increased after exposure to hyperoxia (6, 15). These data suggest that hyperoxia contributes to lung inflammation by modulating chemokine gene expression.
Interleukin (IL)-8 is an 8-kDa protein belonging to the C-X-C family of chemokines (37). The primary function of IL-8 is to promote recruitment and activation of neutrophils to areas of inflammation (19, 22). In various forms of human ALI, neutrophil infiltration is an early and important pathophysiological event, and IL-8 appears to have an important role in mediating this process (5, 31, 32). Clinical studies demonstrated increased IL-8 levels in serum and BAL fluid of patients with ALI (11, 16, 17, 27). Increased BAL fluid levels of IL-8 predicted the development of ALI in at-risk patient populations and were associated with increased mortality in patients with ALI (2, 10, 25). In animal models of ALI, administration of IL-8 antibody conferred protection (4, 14, 30). Collectively, these data demonstrate the importance of IL-8 in the pathophysiology of ALI.
IL-8 was initially discovered as a secreted product of monocytes (37)
and is now known to be produced by several cell types, including the
respiratory epithelium (31). Previous work (7, 24) characterizing the
effects of hyperoxia on IL-8 gene expression involved alveolar
macrophages and isolated peripheral blood monocytes. Relatively less is
known about the effects of hyperoxia on IL-8 gene expression in the
respiratory epithelium. Because the respiratory epithelium is primarily
exposed to hyperoxia during treatment of ALI, understanding the effect
of hyperoxia on IL-8 gene expression on the respiratory epithelium
could provide further insight into the pathogenesis of
hyperoxia-mediated ALI. Most studies (7, 24) investigating
the effect of hyperoxia on IL-8 gene expression focused on hyperoxia as
an isolated stimulus. This scenario is uncommon in clinical medicine.
Patients who require high levels of oxygen typically have a significant
degree of underlying lung inflammation characterized by increased
alveolar levels of cytokines such as tumor necrosis factor (TNF)-
and IL-1
. Therefore, understanding the complex interaction of
hyperoxia with coexistent proinflammatory stimuli is clinically
relevant to patients with ALI.
In this study, we designed a model that attempts to partially mimic the
alveolar milieu of patients with ALI. Respiratory epithelial cells were
simultaneously exposed to hyperoxia and TNF-. In this context, we
evaluated IL-8 gene expression and investigated the effect of hyperoxia
on IL-8 promoter regulation, specifically evaluating the role of
nuclear factor (NF)-
B and NF-IL-6.
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MATERIALS AND METHODS |
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Cell culture and reagents. A549 respiratory epithelial cells (American Type Culture Collection, Manassas, VA) were used for all experiments. These cells, best described as "epithelium-like," are derived from a human lung adenocarcinoma, retain several features of type II pneumocytes, and have been used successfully as a model to evaluate IL-8 gene regulation in vitro (13, 31). 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).
Experimental conditions. In all experiments, the following four
conditions were used: serum-free medium plus room air (control), TNF- (2 ng/ml) plus room air, TNF-
(2 ng/ml) plus hyperoxia (95%
O2), and hyperoxia alone. Hyperoxia was achieved by placing cells in sealed modular chambers (Billups-Rothenberg, Del Mar, CA) and
flushing the chambers with a gas mixture of 95% O2-5% CO2 at 1 l/min for 30 min. Entry and exit ports were
subsequently clamped, and the cells were returned to a 37°C incubator.
IL-8 ELISA. Cells were exposed to the experimental conditions, and supernatants were harvested 8 and 24 h after treatment. Immunoreactive IL-8 levels were determined with a commercially available human IL-8 ELISA kit (Biosource International, Camarillo, CA). All procedures were performed according to the manufacturer's instructions.
Northern blot analysis. Cells were treated with the
experimental conditions and harvested 2, 4, and 24 h after treatment. Total RNA was isolated with the TRIzol Reagent (GIBCO BRL). RNA concentrations were determined by spectrophotometry (260 nm), and 15 µg of RNA from each sample underwent electrophoresis in gels
containing 1% agarose and 3% formaldehyde. Integrity of RNA was
confirmed visually by ethidium bromide staining and brief ultraviolet
light illumination. RNAs were transferred to nylon membranes (Micron
Separations, Westboro, MA) and ultraviolet auto-cross-linked (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 (13). The cDNA probe was labeled
with [-32P]dCTP (specific activity 3,000 Ci/mM, NEN Research Products, Boston, MA) by random
priming (Pharmacia, Piscataway, NJ). Membranes were subsequently washed
twice with 2× saline-sodium citrate-0.1% SDS at 53°C,
developed with a PhosphorImager screen (Molecular Dynamics, Sunnyvale,
CA), and analyzed with ImageQuant Software. To normalize for loading
differences, membranes were stripped by boiling in 5 mM EDTA and
rehybridized with an end-labeled [
-32P]dATP
oligonucleotide probe for 18S rRNA.
Transient transfections and luciferase assays. Four separate
promoter-luciferase reporter plasmids were used for these studies. The
first plasmid (wild type) contained a 200-bp segment (97 to +103
bp) of the IL-8 promoter immediately 5' to the firefly luciferase gene
(13). The second plasmid (mutant NF-
B) had four base substitution
mutations in the NF-
B binding site of the IL-8 promoter, as
indicated by lowercase letters:
82 to
72 bp, GTGGAATTTCC
cTGcAATgTCg (12). The third plasmid (mutant NF-IL-6) had five
base substitution mutations in the NF-IL-6 binding site of the IL-8
promoter, as indicated by lowercase letters:
92 to
81 bp,
GTTGCAAATCGT
GcTaCgcAgCGT (12). The fourth plasmid (NF-
B
dependent) was a synthetic construct in which the luciferase gene was
driven by three tandem NF-
B binding motifs, followed by a minimal
interferon-
promoter (a kind gift from Dr. Roland M. Schmid,
University of Ulm, Ulm, Germany). This plasmid was previously
demonstrated to be a sensitive tool to specifically evaluate NF-
B
activation (33).
Cells were transfected in duplicate by incubation with cationic liposomes (Lipofectin, GIBCO BRL) suspended in Opti-MEM (GIBCO BRL) for 4 h. For all four plasmids, the liposome-to-DNA ratio was 10:2 µg. After transfection, cells were washed with PBS and allowed to recover overnight. 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) with a Berthhold AutoLumat LB953 luminometer. Total protein was measured by the Bradford assay (Bio-Rad, Hercules, CA), and luciferase activity was normalized to the respective total cellular protein. Data are reported as multiples of induction of luciferase activity above that in control cells (transfected and treated with basal growth medium in room air). To account for differences in transfection efficiency, all transfection experiments were performed in duplicate on five separate occasions.
Nuclear protein extraction. Nuclear protein extracts were
prepared from treated cells grown to 80% confluence in 100-mm2
dishes. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were washed twice with PBS, 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 dithiothreitol
(DTT), and 0.1 mM phenylmethylsulfonyl fluoride (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) and stored at 70°C until used for electrophoretic mobility shift assay (EMSA).
EMSA. The NF-B oligonucleotide probe used for EMSA
(5'-GTGGAATTTCCTCTGA-3') corresponds to the NF-
B site in the IL-8
promoter and was synthesized at the University of Cincinnati DNA Core
Facility. The probe was labeled with
[
-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% (vol/vol) glycerol, and 0.2 mM PMSF] on
ice for 10 min before addition of the radiolabeled oligonucleotide
probe for an additional 10 min. Protein-nucleic acid complexes
were resolved with a nondenaturing polyacrylamide gel
consisting of 5% acrylamide (29:1 ratio of acrylamide to
bis-acrylamide) and run in 0.5× Tris-borate-EDTA buffer (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. Nuclear proteins were boiled in equal
volumes of loading buffer (125 mM Tris · HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), and 20 µg of
protein were loaded per lane on 8-16% Tris-glycine gradient gels
(Novex, San Diego, CA). Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Novex) with 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
antibody against the p65 subunit of NF-B (polyclonal, Santa Cruz
Biotechnology, Santa Cruz, CA) was applied at 1:100 for 1 h. After two
washes in TBS-0.05% Tween 20, secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Sigma) was applied at
1:10,000 for 1 h. Blots were washed in TBS-Tween 20 two times over 30 min, incubated in commercial enhanced chemiluminescence reagents (ECL,
Amersham), and exposed to photographic film.
Statistical analysis. Differences in immunoreactive IL-8 levels and luciferase activity between the experimental groups were evaluated by one-way analysis of variance and the Student-Newman-Keuls test. P < 0.05 was considered significant.
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RESULTS |
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Hyperoxia augments TNF--mediated immunoreactive
IL-8 production. We first determined if hyperoxia would modulate
TNF-
-induced production of immunoreactive IL-8. A549 cells were
treated with the experimental conditions, and immunoreactive IL-8
levels were determined by ELISA. Treatment with hyperoxia alone
minimally increased immunoreactive IL-8 levels compared with those in
unstimulated control cells (Fig. 1).
Treatment with TNF-
alone significantly increased immunoreactive
IL-8 levels at 8 and 24 h compared with those in unstimulated control
cells. Concomitant treatment with TNF-
and hyperoxia significantly
increased immunoreactive IL-8 levels compared with those in cells
treated with TNF-
alone. The amount of IL-8 generated after
treatment with the combination of TNF-
and hyperoxia was greater
than would be anticipated if the effects of TNF-
and hyperoxia
were simply additive. These data demonstrate that the combination of
TNF-
and hyperoxia synergistically increases production of
immunoreactive IL-8.
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Hyperoxia augments TNF--mediated expression of IL-8
mRNA. Having demonstrated that hyperoxia augments production of
immunoreactive IL-8 in the presence of TNF-
, we next investigated
the effects of hyperoxia on steady-state IL-8 mRNA levels. Cells were
treated with the experimental conditions, and IL-8 mRNA was measured by Northern blot analyses. Treatment with hyperoxia alone did not detectably affect IL-8 mRNA expression (data not shown). Treatment with
TNF-
alone increased IL-8 mRNA expression in a time-dependent manner
(Fig. 2). Concomitant treatment with
TNF-
and hyperoxia increased IL-8 mRNA expression compared with that
in cells treated with TNF-
alone. As in the ELISA-related
experiments, the amount of IL-8 mRNA expression after treatment with
the combination of TNF-
and hyperoxia was greater than would be
anticipated if the effects of TNF-
and hyperoxia were simply
additive. These data indicate that hyperoxia augments TNF-
-induced
IL-8 production by mechanisms that increase IL-8 mRNA expression.
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Hyperoxia augments TNF--induced IL-8 promoter
activity. Transcriptional control of the IL-8 promoter is known to
have an important role in IL-8 gene expression (20, 23, 26). Therefore, we investigated the effect of hyperoxia on IL-8 promoter activation. Cells were transiently transfected with an IL-8 promoter-luciferase reporter plasmid (13) and treated as indicated in MATERIALS AND
METHODS. Treatment with hyperoxia alone did not significantly increase luciferase activity compared with that in control cells (Fig.
3). Treatment with TNF-
alone caused an
~2.2-fold induction of luciferase activity. The combination of
TNF-
and hyperoxia caused an ~4.2-fold induction of luciferase
activity, a greater induction of luciferase activity than would be
expected if the effects of TNF-
and hyperoxia were simply additive.
These data demonstrate that hyperoxia augments TNF-
-mediated
activation of the IL-8 promoter.
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Mutation in the NF-B binding site of the IL-8
promoter abolishes TNF-
- and hyperoxia-mediated
activation of the IL-8 promoter. The transcription factor NF-
B
is known to be involved in IL-8 gene regulation (20, 23, 26). To
determine the role of NF-
B in our experimental system, we
transfected cells with an IL-8 promoter-luciferase plasmid containing a
mutated NF-
B binding site (12). Treatment with hyperoxia alone,
TNF-
alone, or the combination of TNF-
and hyperoxia did not
induce luciferase activity in cells transfected with the mutant NF-
B
promoter-luciferase reporter plasmid (Fig.
4). From these results, we conclude that induction of the IL-8 gene by TNF-
and the combination of TNF-
and hyperoxia are NF-
B dependent in A549 cells.
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Hyperoxia prolongs TNF--mediated nuclear
translocation of NF-
B. In these experiments, we
directly examined the effect of hyperoxia on TNF-
-mediated nuclear
translocation of NF-
B. Hyperoxia alone for 1-4 h did not
detectably increase nuclear translocation of NF-
B compared with that
in control cells as measured by EMSA (Fig.
5, lanes 3-6). Treatment with
TNF-
alone for 1 or 2 h increased nuclear translocation of NF-
B
compared with that in control cells (Fig.
6, lanes 1, 2, and
4). The specificity of the shifted band was demonstrated in a
previous report (36). The amount of NF-
B nuclear translocation was
greatest after 1 h of TNF-
alone (Fig. 6, lane 2) and was
decreased by 2 h (Fig. 6, lane 4). Concomitant treatment with
TNF-
and hyperoxia for 1 h did not significantly alter NF-
B
nuclear translocation compared with that in cells treated with TNF-
alone for 1 h (Fig. 6, lanes 2 and 3). In contrast, cells that were concomitantly treated with TNF-
and hyperoxia for 2 h demonstrated relatively more NF-
B nuclear translocation compared
with that in cells treated with TNF-
alone for 2 h (Fig. 6,
lanes 4 and 5). The amount of NF-
B nuclear
translocation after 2 h of treatment with TNF-
and hyperoxia was
similar to that of cells treated with TNF-
alone for 1 h (Fig. 6,
lanes 2 and 5).
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To confirm our EMSA data, we next performed Western blot analyses for
the NF-B subunit p65 using nuclear proteins of cells treated with
TNF-
with and without hyperoxia. Treatment with TNF-
alone increased nuclear levels of p65 compared with those in control
cells (Fig. 7, lanes 1, 2,
and 4). As with our EMSA data, the amount of nuclear p65 was
greatest after 1 h of TNF-
(Fig. 7, lane 2) and was
decreased by 2 h (Fig. 7, lane 4). Concomitant treatment with
TNF-
and hyperoxia for 1 h did not significantly alter
nuclear p65 levels compared with those in cells treated with
TNF-
alone (Fig. 7, lanes 2 and 3). In
contrast, cells that were concomitantly treated with TNF-
and hyperoxia for 2 h demonstrated increased nuclear p65 levels
compared with cells treated with TNF-
alone for 2 h (Fig. 7,
lanes 4 and 5). Collectively, the EMSA and Western blot
data suggest that hyperoxia increases the duration of
TNF-
-mediated nuclear translocation of NF-
B.
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Hyperoxia does not augment TNF--mediated luciferase
activity in cells transfected with a NF-
B-dependent
promoter-luciferase reporter plasmid. Having demonstrated that
induction of the IL-8 gene by TNF-
is a NF-
B-dependent process
and that hyperoxia increases the duration of TNF-
-mediated
NF-
B nuclear translocation in our model, we hypothesized that
augmentation of TNF-
-mediated IL-8 production by hyperoxia results
from increased NF-
B activity. To test this hypothesis and to measure
NF-
B activity, we performed transient transfections with a synthetic
promoter-luciferase reporter plasmid containing three NF-
B sites in
tandem. This plasmid was previously reported as an effective tool to
specifically evaluate in vitro NF-
B activation (33). Treatment with
hyperoxia alone did not activate NF-
B (Fig.
8). Treatment with TNF-
alone resulted in an ~3-fold induction of luciferase activity, and the addition of
hyperoxia to TNF-
did not increase luciferase activity compared with
those in cells treated with TNF-
alone. From these data, we conclude
that hyperoxia does not augment TNF-
-induced activation of NF-
B
in A549 cells.
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The NF-IL-6 site in the IL-8 promoter is also required for
synergistic activation by hyperoxia and TNF-. Data
derived from transient transfection assays indicate that the NF-
B
site in the IL-8 promoter is required for the synergistic effect of
hyperoxia in the presence of TNF-
. Data derived from EMSA and
Western blot analyses indicate that hyperoxia increases the duration of
TNF-
-mediated nuclear translocation of NF-
B. NF-
B-dependent
reporter gene assays, however, indicate that increased activation of
NF-
B alone does not fully account for the mechanism by which
hyperoxia augments TNF-
-mediated expression of the IL-8 gene.
Collectively, these data suggest that other IL-8 promoter elements may
be involved. Accordingly, we investigated the role of NF-IL-6 because
this transcription factor was previously reported to play a cooperative role with NF-
B in the regulation of IL-8 (20, 23, 26).
The role of NF-IL-6 was investigated by transiently transfecting A549
cells with an IL-8 promoter-luciferase reporter plasmid containing base
substitution mutations in the NF-IL-6 site. Treatment with hyperoxia
alone did not increase luciferase activity in cells transiently
transfected with the mutant NF-IL-6 promoter-luciferase plasmid (Fig.
9). Treatment with TNF- alone
significantly increased luciferase activity, but the addition of
hyperoxia did not augment TNF-
-mediated luciferase activity. These
data indicate that an intact NF-IL-6 site is required for the mechanism
by which hyperoxia augments TNF-
-mediated activation of the IL-8
promoter.
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DISCUSSION |
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Previous studies involving alveolar macrophages, U937 cells, isolated peripheral blood monocytes, and human whole blood demonstrated that hyperoxia modulates IL-8 gene expression (7, 8, 24). Oxidant stress other than hyperoxia was previously described to induce IL-8 expression in respiratory epithelial cells. DeForge et al. (9) and Lakshminarayanan et al. (21) demonstrated that A549 cells exposed to H2O2 produced IL-8. In our current study, we used hyperoxia as an oxidant stress model because hyperoxia is a clinically relevant form of oxidant stress. In addition, we used a respiratory epithelial cell model because the respiratory epithelium is primarily exposed to hyperoxia in patients with ALI.
We evaluated how the interaction between hyperoxia and TNF- affects
IL-8 gene expression in A549 cells. In these experiments, hyperoxia
alone had a minimal effect on IL-8 gene expression. In contrast, the
combination of hyperoxia and TNF-
synergistically increased IL-8
gene expression. Synergy was evident by increased IL-8 peptide
expression (ELISA) and increased IL-8 mRNA expression (Northern blot
analysis) in cells treated with the combination of hyperoxia and
TNF-
compared with cells treated with TNF-
alone. To provide more
mechanistic insight into these findings, we performed transient
transfections using an IL-8 promoter-luciferase reporter plasmid. These
experiments demonstrated that the augmentation of TNF-
-mediated IL-8
gene expression by hyperoxia is secondary to increased activation of
the IL-8 promoter.
The amount of induced luciferase activity measured in transiently transfected cells was proportionally less compared with induced IL-8 levels measured by ELISA. This discrepancy most likely represents differences in assay sensitivities. Alternatively, this discrepancy may indicate that the observed effects on IL-8 gene expression are not solely transcriptional but may also involve posttranscriptional regulation of the IL-8 gene. Future studies are needed to establish whether changes in mRNA stability are relevant in our experimental model.
Previous studies demonstrated the importance of NF-B in the
regulation of IL-8 gene expression (12, 13, 20, 23, 26). Our current
results are partially consistent with these data. Site-directed
mutagenesis of the NF-
B binding site in the IL-8 promoter abolished
the ability of TNF-
to induce luciferase activity. Furthermore,
hyperoxia had no intrinsic ability to induce luciferase activity in
cells transfected with this mutant NF-
B promoter and did not
interact with TNF-
to increase luciferase activity.
Oxidant stress is known to induce NF-B activity in certain
experimental models (1, 3, 29). Therefore, we evaluated the effect of
hyperoxia on NF-
B nuclear translocation (EMSA and Western blot
analyses) and NF-
B activation (reporter gene assays). Hyperoxia
alone did not detectably affect NF-
B nuclear translocation as
measured by EMSA and Western blot analyses. In contrast, hyperoxia modestly prolonged the time during which NF-
B was present in the
nucleus after concomitant treatment with TNF-
. Thus increased activation of NF-
B could account for the mechanism by which
hyperoxia augments TNF-
-mediated IL-8 gene expression.
To test this hypothesis, we performed transient transfections with a
synthetic NF-B-dependent promoter-luciferase reporter plasmid, which
allowed us to specifically evaluate NF-
B activity. These experiments
demonstrated that hyperoxia did not augment TNF-
-mediated NF-
B
activity. Also, treatment with hyperoxia alone did not increase NF-
B
activity. These results provide functional evidence suggesting that
hyperoxia does not augment TNF-
-mediated IL-8 gene expression by
simply augmenting NF-
B activation in A549 cells. These data further
indicate that other promoter elements may be involved in the mechanism
by which hyperoxia augments TNF-
-mediated expression of the IL-8
gene. Indeed, transient transfections with an IL-8 promoter-luciferase
plasmid containing a mutant NF-IL-6 site indicated that NF-IL-6 is also
required for the synergistic effect of hyperoxia. In combination, our
data demonstrate that NF-
B is required for induction of IL-8 gene
expression by TNF-
and by the combination of TNF-
and hyperoxia.
Increased activation of NF-
B alone, however, is not sufficient to
fully account for the synergistic effect of hyperoxia in the presence
of TNF-
. The NF-IL-6 site is also required for the synergistic
effect of hyperoxia. Thus the mechanism by which hyperoxia augments
IL-8 gene expression appears to involve interactions between NF-
B and NF-IL-6. This assertion is well supported by previous studies demonstrating cooperative interactions between NF-
B and NF-IL-6 in
other experimental models (20, 23, 26).
The pathophysiology of ALI is a complex process involving multiple stimuli. Hyperoxia can directly cause ALI by generating cytotoxic reactive oxygen species. Hyperoxia is also known to cause lung inflammation, but the mechanisms of this effect are not fully understood. Rats exposed to 95% O2 alone demonstrated pulmonary infiltration with neutrophils within 48 h of exposure (28). BAL fluid from rats exposed to 95% O2 had increased chemotactic properties, and the degree of chemotactic activity was highly correlated with the degree of lung injury (6, 15). In combination with our current data, these data indicate that hyperoxia may also cause ALI, indirectly, by modulating chemokine gene expression.
The scenario of exposing patients without lung disease to hyperoxia
alone is clinically uncommon. Virtually all patients with ALI severe
enough to require high levels of O2 have a significant degree of underlying inflammation. Levels of cytokines such as TNF-
are increased in ALI, and the interaction of these mediators with
hyperoxia may influence gene expression in parenchymal lung cells. It
is becoming increasingly apparent that interactions between
simultaneous or sequential stimuli significantly impact the course of
ALI and other forms of organ injury during proinflammatory states. The
term "multiple hits" has been used to describe this concept in
which there are important additive, synergistic, or unexpected effects
of exposing cells or whole organs to multiple stimuli (35). The results
of our current study suggest that cytokines, such as TNF-
, may have
important interactions with hyperoxia that influence IL-8 gene
expression. Our data also suggest that the respiratory epithelium,
previously thought to be a passive target in hyperoxia-induced ALI, is
actually an active participant in this process. Further elucidation of
the mechanisms by which hyperoxia modulates IL-8 gene expression in the
respiratory epithelium may allow for the development of more rational
therapeutic interventions for hyperoxia-induced ALI.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the Children's Hospital Research Foundation and National Heart, Lung, and Blood Institute Grant K08-HL-03725 (to H. R. Wong).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. R. Wong, Division of Critical Care Medicine-OSB5, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: wonghr{at}chmcc.org).
Received 11 August 1999; accepted in final form 15 September 1999.
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