1 Department of Pediatrics, 2 Department of Pathology, and 3 Division of Endocrinology, Department of Internal Medicine, University of Geneva Medical School, 1211 Geneva 4, Switzerland
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ABSTRACT |
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We previously reported that
exposure of mice to hyperoxia is characterized by extensive lung cell
necrosis and apoptosis, mild inflammatory response, and
elevated circulating levels of corticosterone. Administration of
hydroxycortisone acetate during hyperoxia aggravated lung injury. Using
adrenalectomized (ADX) and sham-operated (sham) mice, we studied the
role of the glucocorticoids in hyperoxia-induced lung injury. Lung
damage was attenuated in ADX mice as measured by lung weight and
protein and cell content in bronchoalveolar lavage and as seen by light
microscopy. Mortality was delayed by 10 h. Nuclear factor-B
(NF-
B) activity was significantly decreased in lungs of sham mice
exposed to hyperoxia but was preserved in ADX mice. There was a
correlation between NF-
B activity in ADX mice and decreased levels
of I
B
. In contrast, activator protein-1 activity increased
similarly in both groups of mice. Levels of interleukin-6 (IL-6), a
transcriptional target of NF-
B, were higher in bronchoalveolar
lavage and serum of ADX than sham mice. However, the protective effect
of ADX was not mediated by IL-6, because administration of recombinant
human IL-6 to sham mice did not prevent lung damage. These results
demonstrate that the adrenal response aggravates alveolar injury and is
likely to be mediated by the decrease of NF-
B function involved in
cell survival.
mice; oxygen; apoptosis; steroid
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INTRODUCTION |
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HIGH-OXYGEN EXPOSURE has been used as a valuable model of idiopathic respiratory distress syndrome or acute respiratory distress syndrome in rodents and is characterized by extensive alveolar cell death, leading to disruption of the alveolocapillary barrier and plasma leakage into the alveoli. Many factors can contribute to alveolar cell death, including the increased presence of reactive oxygen species, the modulation of the cell signaling response, and the inflammatory and stress response of the host. Modulation of the cell response by different strategies, such as administration of keratinocyte growth factor, overexpression of IL-6 in Clara cells, or transfection of Akt into alveolar cells, can prevent lung damage during hyperoxia (5, 24, 43). The lung inflammatory response during hyperoxia varies with species, strain, and age (15, 30). We have shown that adult C57BL/6 mice display only mild inflammatory cell recruitment into the lungs (6) and that endogenous corticosterone increased significantly during hyperoxia in mice. Administration of hydroxycorticosterone acetate at high doses aggravated lung injury (8).
Glucocorticoid (GC) effects have been largely studied with respect to lung development, because they impaired alveolar septation (10). However, their direct effects on lung epithelial cells of adult animals are less evident. GC decreased lung epithelial cell proliferation in rats exposed to ozone (35) and facilitated apoptosis in cultured airway epithelial cells (13). It has been established for 20 years that GC are potent inducers of apoptosis in lymphocytes (45), whereas they protect neuronal cells and hepatoma cell lines from cell death (14, 19).
GC bound to their receptors target the nuclear DNA and are able
to transactivate several genes by binding the GC-responsive element but
are also able to repress the DNA binding of transcription factors such
as nuclear factor-B (NF-
B) and activator protein 1 (AP-1)
(29).
NF-B is a redox-sensitive transcription factor, because antioxidant
equilibrium can modulate its activation in vitro (31) and
it is recognized to act mostly as a cell survival and a proinflammatory factor. NF-
B activity has not been reported during hyperoxia-induced lung injury in vivo, nor have its regulatory effects on lung cell death
been reported. Therefore, modulation of NF-
B activity and its relationship to GC by different approaches are important in the
understanding of lung cell death during oxygen toxicity.
We have explored the effects and the possible roles of stress response
in lung damage. We demonstrate that the adrenal response aggravates
hyperoxia-induced lung injury. The deleterious effect of the stress
response was mainly GC dependent, as demonstrated by administration of
GC and catecholamine antagonists. Lung NF-B activity decreased
significantly during hyperoxia, while, in the absence of the adrenal
gland, NF-
B activity was conserved. The preserved NF-
B activity
in adrenalectomized (ADX) mice correlated with decreased levels of
I
B
. Higher NF-
B activity in ADX mice exposed to oxygen was
also supported by significantly higher levels of IL-6
(transcriptionally regulated by NF-
B) in their serum and
bronchoalveolar lavage (BAL). Administration of pyrrolidine dithiocarbamate (PDTC), which has been shown to inhibit NF-
B activity in several experimental inflammatory diseases (12, 23,
28), blocked I
B
degradation but did not modify lung NF-
B activity. In conclusion, our study shows that the adrenal response aggravates hyperoxia-induced lung injury, possibly by decreasing NF-
B activity and its cell survival property.
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METHODS |
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Mice. ADX and sham C57BL/6 mice were purchased from Iffa Credo (Labresle, France) or adrenalectomized at 6 wk of age by us according the same protocol. Mice were kept in our animal facilities for 2 wk before use. The water of ADX mice was supplemented with 0.9% NaCl and 1% glucose. Experiments were performed with 2- to 3-mo-old mice. Corticosterone plasma levels were checked in all mice before the experimental procedures and determined by RIA using 125I-labeled corticosterone (Diagnostic Systems Laboratories, Webster, TX), as described elsewhere (8, 42).
Hyperoxia exposure and in vivo treatment. Mice were placed in a sealed Plexiglas chamber and exposed to 100% oxygen or room air in the same conditions described previously (6). Food and water were available ad libitum. Mice were killed after 72 h of hyperoxia or, for mortality experiments, when the temperature, measured rectally with a clinical thermometer (type HP5310, Philips), dropped below 32°C, an event followed by death within 2 h. All animals (control or oxygen-exposed) were anesthetized with an injection of pentobarbital sodium (50 mg/kg ip) and then bled through the abdominal aorta. The thorax was opened, and the lungs were removed, weighed, frozen, and prepared for mRNA or DNA extraction. Pulmonary edema was evaluated by measuring the wet weight of the lung, as described previously (4, 5).
In some experiments, hydrocortisone 21-acetate (140 µg/mouse; Sigma Chemical, St. Louis, MO) was injected on days 0, 1, 2, and 3 (7 mg · kgLight microscopy and BAL procedure. In some experiments, lungs were fixed by intratracheal instillation of cold buffered formalin (2%) and a hydrostatic pressure of 20 cmH2O. Transhilar horizontal sections were embedded in paraffin and processed for light microscopy. BAL was performed by intratracheal instillation of 2 ml of saline, as described elsewhere (7). After centrifugation, the cells were counted and the supernatant was collected. BAL protein concentration was determined by Bradford's method.
Thymocyte extraction and fluorescein-activated cell sorter analysis. The thymus was weighed and homogenized with a potter, and cells were counted and suspended at a density of 2 × 106/ml. Caspases were assayed using the CaspaTag fluorescein-labeled caspase activity kit, which detects activated caspase in single cells (VAD probe; Intergen), as described elsewhere (32). Briefly, the carboxyfluorescein-labeled fluoromethyl ketone inhibitor (VAD) enters the cells and irreversibly binds to all caspases. Fluorescence intensity was measured with a FACScan flow cytometer (Becton Dickinson).
Isolation of nuclear proteins and assay of NF-B and AP-1
activities.
Nuclear proteins were extracted according a modification of a
previously described protocol (40). Fresh lung tissue (10 mg) was homogenized with a Dounce homogenizer in 0.5 ml of buffer A [10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES, pH 7.9, 1 mM
dithiothreitol (DTT), 1 mM NaVO4, 5 µg/ml leupeptin, and
1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride]. This procedure was
repeated after centrifugation at 2,000 g for 10 min at
4°C. The nuclei-containing pellet was carefully resuspended in 2 vol
of buffer B [420 mM NaCl, 10 mM KCl, 20 mM HEPES, pH 7.9, 20% glycerol, 1 mM DTT, 1 mM NaVO4, 5 µg/ml leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride] and subjected to
vigorous agitation for 30 min at 4°C. After centrifugation at 13,000 g for 20 min at 4°C, the supernatant was collected, divided into aliquots, and stored at
80°C. NF-
B and AP-1
activity in nuclear proteins was estimated using an electrophoretic
mobility shift assay, which measures the abundance of protein species
with the ability to bind a specific nucleotide sequence. The
oligonucleotide probes were the specific consensus-binding site
NF-
Bcons2 (5'-ATGTGAGGGGACTTTCC-3') for NF-
B and the 21-mer
sequence (5'-CGCTTGATGAGTCAGCCGGAA-3') for AP-1. Probes were labeled
with [
-32P]dCTP using the Klenow method. Binding
reactions were performed in DNA binding buffer containing
poly[d(I-C)] (0.5 µg/µg protein), 1 mM EDTA, 1 mM DTT, 4%
Ficoll-400, 4 mg/ml BSA (Boehringer, fraction V), the nuclear extracts
(2 µg), and 30,000 counts/min (ideally 30,000 counts/min/34 fmol in 1 µl) of radiolabeled oligonucleotide probe. Specificity of the bands
in the retardation gels was ascertained by supershift (p65 of NF-
B,
using anti NF-
B p65, TransCruz gel supershift antibody, sc-109 X)
and/or by competition with cold probes. Briefly, competition was
performed by preincubating the samples with the specific 10-nucleotide
sequence 5'-GGGTATTTCC-3' (core sequence for NF-
B) or the
11-nucleotide sequence 5'-GCTGACTCATC-3' (containing the core sequence
for AP-1) before incubation with the respective radiolabeled probes at
molar ratios of 1:1, 1:10, 1:100, and 1:1,000.
Western blots.
Lungs were homogenized as described previously (5), and 40 µg of protein were loaded per lane onto 10% polyacrylamide gels and
electrophoresed. Proteins were transferred to nitrocellulose membranes,
and nonspecific binding was blocked with 5% nonfat milk in
Tris-buffered saline + 0.1% Tween 20 (TBST) overnight at 4°C.
Membranes were blotted with the polyclonal antibody anti-IB
(catalog no. 554135, PharMingen, 1:1,000 dilution) in TBST and 5%
nonfat milk. A goat anti-rabbit IgG-horseradish peroxidase (catalog no.
170-6515, Bio-Rad) diluted 1:3,000 in TBST and 5% nonfat milk was used
as secondary antibody. Membranes were washed again with TBST and
revealed with an enhanced chemiluminescence detection reagent kit
(Amersham International, Amersham, UK) at room temperature before being
exposed to Biomax MR film (Eastman Kodak, Rochester, NY). Results were
quantified using subsaturated emulsions on X-ray film, as described
elsewhere (5), and normalized with the actin signal
detected with a polyclonal antiactin (kind gift of G. Gabbiani, Dept.
of Pathology, University of Geneva).
Statistical analysis. For each parameter measured or calculated, the values for all animals in an experimental group were averaged, and the standard deviation of the mean was calculated. The significance of differences between the values of an experimental group and those of the control group was determined with the unpaired Student's t-test. Where appropriate, two-way ANOVA with multiple comparisons followed by an unpaired t-test was used. Significance levels were set at P < 0.05.
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RESULTS |
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Adrenalectomy protects mice from alveolar edema.
To evaluate lung injury and alveolar damage, we measured lung injury
markers at 72 h of oxygen exposure. ADX mice showed a significant
decrease in lung weight and BAL protein content compared with sham mice
(Table 1). When corticosterone was
reinjected into ADX mice, the susceptibility to hyperoxia was restored.
Although BAL cell count increased during hyperoxia in sham mice, it was not significantly different from that in air-breathing sham mice. Lung
histology of sham mice showed interstitial and alveolar edema, septal
breakdowns, and focal alveolar hemorrhages, which were attenuated in
ADX mice (Fig. 1). Mortality in ADX mice
was delayed by 6-10 h (data not shown). To determine whether the
adrenal medulla or cortex was implicated in lung damage, we
subcutaneously inserted selective inhibitors of GC receptors (RU-486)
or -blockers (8, 17). Only RU-486 treatment
significantly decreased alveolar damage (Fig.
2). These experiments demonstrate that GC
aggravate the alveolar damage induced by hyperoxia.
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Hyperoxia induces thymocyte apoptosis via endogenous
increase of corticosterone.
Steroids are potent inducers of thymocyte apoptosis; therefore,
we explored the role of endogenous GC elevation in thymic atrophy
induced by hyperoxia. Thymus weight was significantly reduced by
hyperoxia (84 h) in sham, but not ADX, mice (Fig.
3A). Adrenalectomy in
air-breathing mice increased thymus weight, which did not change during
hyperoxia (Fig. 3A).
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Modulation of NF-B and AP-1 activities by hyperoxia.
We examined DNA binding of NF-
B and AP-1 in lung extracts during
hyperoxia in sham and ADX mice by gel-shift assay (Fig. 4A). Two bands (p55 and p65)
were detected in lung nuclear extracts (37). These bands
were specific for NF-
B and AP-1: they could be abolished by
competition with the oligonucleotide specific for the core sequence and
not for the mutated sequence (PU.1; Fig. 4, B and
D), and the p65 protein was displaced by supershift (data
not shown). NF-
B activity decreased significantly during hyperoxia
in sham mice (P < 0.01), whereas it was conserved in ADX mice (Fig. 4A). We also analyzed AP-1 activity, which
increased in sham and ADX mice during hyperoxia (Fig. 4C).
Although AP-1 activity was slightly more elevated in ADX than in sham
mice exposed to oxygen, the difference was not significant (Fig.
4C).
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PDTC prevents IB
degradation.
We administered PDTC, a drug that can block NF-
B by preventing I
B
degradation (23), to sham and ADX mice (Fig. 5). Lung NF-
B activity was not affected by daily administration of PDTC in
sham or ADX mice, and lung weight was not modified by PDTC treatment
during hyperoxia (data not shown). I
B expression, analyzed by
Western blot, was higher in PDTC-treated mice than in untreated mice
(Fig. 5). These data suggest that PDTC does not modify total lung
NF-
B activity in hyperoxia-induced injury in sham or ADX mice,
although it prevents I
B degradation.
Effect of IL-6 on hyperoxia-induced lung damage.
IL-6 is a cytokine that is transcriptionally regulated by NF-B
(41). Inasmuch as mice overexpressing IL-6 in Clara cells were clearly protected in hyperoxia (43), we first
examined IL-6 levels in serum and BAL of ADX and sham mice (Table 1). During hyperoxia exposure, the level of IL-6 was 10-fold higher in ADX
than in sham mice (Table 1). Interestingly, the number of cells counted
in BAL was similar in both groups of mice exposed to hyperoxia. These
data correlate with an enhanced NF-
B activity in ADX mice compared
with sham mice during hyperoxia.
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DISCUSSION |
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We have demonstrated that lung damage is aggravated by the GC produced in response to hyperoxia. Lung weight and protein content, reflecting alveolar damage, were significantly reduced in ADX mice, and when hydroxycortisone acetate was administered to ADX mice, the susceptibility to hyperoxia was restored. If at 72 h of oxygen exposure the attenuation of lung injury was evident, ADX mice showed only a delayed mortality of several hours, suggesting that the adrenal response contributes to lung damage. We previously reported that hyperoxia increased circulating levels of endogenous corticosterone in mice and that administration of hydroxycorticosterone at high doses worsened lung injury (8). In humans, cortisol elevation has been reported in acute respiratory distress syndrome and is part of the normal stress response (27). Furthermore, low levels of cortisol have been associated with a poor outcome in septic patients (9).
In the majority of lung illness, there is a component of alveolar damage and a component of inflammatory response. GC effects are diverse and often opposite, depending on the type of cell. Several studies have demonstrated negative effects of steroids on lung development when given to pregnant rats or newborn pups (see Ref. 18 for review). Luyet et al. (25) reported recently that early treatment of newborn rats with dexamethasone decreased cell proliferation and increased the number of TdT-mediated dUTP nick end label-positive cells. In adult rats, absence of the adrenal gland resulted in a higher compensatory growth response of the contralateral lung after pneumonectomy (18); however, this increased growth phenomenon was not detected in ADX rats without pneumonectomy. We were unable to detect any DNA ladder in lungs of control mice treated with dexamethasone in vivo (unpublished observations), suggesting that GC alone do not induce significant alveolar cell apoptosis in adult mice but are likely to synergize with the effects of oxygen on alveolar damage.
Corticosterone elevation is part of the normal stress response to injuries, and in this context it is still debated whether this normal response to stress can overwhelm the normal defense mechanisms and be deleterious for the organism (27). Among its effects, the stress response has been known for a very long time to result in thymus atrophy (38). Thymus atrophy was later shown to be due to an apoptosis-dependent mechanism, which was induced by GC (45). Indeed, thymocyte apoptosis, evaluated by CaspaTag labeling, was completely abrogated in ADX mice exposed to hyperoxia and reversed by hydrocortisone administration. These data demonstrate that the elevation of GC is clinically relevant since it induces thymus atrophy and that GC are directly responsible for thymus apoptosis during hyperoxia-induced lung injury.
Taking into account that among the major effects of GC is repression of
the activity of NF-B and, to a lesser extent, AP-1 (29), we examined NF-
B and AP-1 activities in vivo
during hyperoxia. Our results showed that lung NF-
B activity
decreased significantly during hyperoxia in sham mice, and, in
contrast, AP-1 activity increased. Although the pattern of AP-1
activity was similar in sham and ADX mice, this was not the case for
NF-
B. Indeed, NF-
B activity did not decrease but was conserved in
ADX mice during hyperoxia. These data suggest that endogenous
corticosterones mediate extinction of the NF-
B signal during
hyperoxia and that higher levels of NF-
B protect from cell death.
The direct effects of oxidative stress on NF-
B activity in vitro
have been somehow contradictory. Li et al. (21) showed
that oxidative stress increased NF-
B activity as a result of I
B
degradation in epithelial cell lines. Conversely, Allen and Wong and
their co-workers (1, 44) did not observe an increase in
NF-
B activity or I
B degradation in human epithelial cells exposed
to 95% oxygen. Interestingly, in the study of Li et al.
(21), despite increased NF-
B activity, epithelial cells
were not protected from cell death. When cells were preexposed to
H2O2 or hyperoxia or were transfected with I
B-dominant negative, they were protected from subsequent exposure to hyperoxia, suggesting that a higher basal level of NF-
B was able
to prevent cell death (16, 33). I
B
expression could easily be detected by Western blot in our total lung extracts and did
not change in any group of mice, except ADX mice exposed to oxygen,
where the conserved NF-
B activity correlated with the extinction of
I
B
. Indeed, GC also diminish NF-
B activity by increasing the
transcription of I
B
, which, when bound to NF-
B, prevents
NF-
B translocation into the nucleus (2, 36). However, it is possible that, in the absence of GC, NF-
B activity could be preserved also by a mechanism decreasing I
B
synthesis.
To determine whether maintained NF-B activity could be responsible
for protecting the lung in ADX mice, we administered PDTC, a drug that
can prevent I
B degradation (20), to ADX and sham mice.
We found that PDTC was able to raise I
B in both groups of mice;
however, it did not affect NF-
B activity. We are not surprised by
the lack of PDTC effect on lung damage, because NF-
B activity was
not changed by this treatment. Several studies using PDTC have shown
controversial effects on NF-
B activation (20, 22, 23,
28). For instance, in the rat model of LPS-induced inflammation,
the time and route of PDTC administration strongly influenced the
inhibition of NF-
B activity (23, 28). Recently, Cuzzocrea et al. (12) showed that, in mice, PDTC inhibited
NF-
B activity in pleural macrophages and decreased the amount of
fluid in a pleurisy model. Supporting this concept are the results
obtained with intraperitoneal administration of dexamethasone in
LPS-challenged mice that express luciferase under the control of
NF-
B promoter (34). Dexamethasone did not decrease
luciferase expression after LPS administration in the lungs, whereas it
decreased NF-
B activity in peritoneal macrophages (34).
It is likely that different cell types did not respond similarly in
terms of NF-
B activation and might be more or less sensitive to PDTC treatment.
Recently, Ward and co-workers (43) reported that mice
overexpressing IL-6 in Clara cells removed were protected from
hyperoxia, and they explained lung cell resistance through increased
basal levels of Bcl-2 in their transgenic mice. IL-6 is a well-known proinflammatory cytokine synthesized within the lungs mainly by alveolar macrophages. Inasmuch as NF-B is almost the exclusive transcription factor for IL-6 (41), we measured IL-6
levels in response to hyperoxia. ADX mice display much higher levels of
IL-6 in serum and BAL than sham mice in response to hyperoxia. IL-6
levels were completely dependent on the presence of GC, since administration of hydroxycorticosterone to ADX mice restored the sham
phenotype. These results confirm the belief that adrenal hormones and,
most likely, GC act on IL-6 production via increased NF-
B activity.
To further test the hypothesis that higher IL-6 levels in ADX mice
might prevent oxygen-induced lung damage, we performed continuous
infusion of rhIL-6. Although we reached IL-6 levels in serum that were
similar to those measured in ADX mice exposed to oxygen reported by
Ward et al. (0.5-1.5 ng/ml; Ref. 43), we did not
observe any protective effect with rhIL-6 administration, suggesting
that the protective mechanism of ADX mice might not be related to the
IL-6 response. However, it cannot be excluded that subcutaneous
administration did not reach the epithelial side of the alveoli in
sufficient quantity.
Taken together, our data show that the adrenal response and, in
particular, GC aggravates alveolar damage, which is correlated with the
extinction of NF-B activity. This work provides an explanation for
the failure of the steroid to markedly improve several acute or chronic
inflammatory lung disorders, since its effects on inflammatory cells
and/or epithelial and endothelial cells might be opposed (3, 11,
26, 27). The concomitant ongoing destructive phase, where
steroid treatment might accelerate cell death, and proliferative
inflammatory phase, where steroids might stop cell proliferation and
prevent proinflammatory cytokine transcription, could explain the
conflicting results in assessing the efficacy of steroid treatment.
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ACKNOWLEDGEMENTS |
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We thank P.-F. Piguet for scientific advise, D. Belin for critical reading of the manuscript, and A. Scherrer for technical assistance.
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FOOTNOTES |
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This work is supported by Swiss National Foundation Grant 3200-067865.02 (C. Barazzone-Argiroffo) and the Wolfermann-Nägele Foundation.
Address for reprint requests and other correspondence: C. Barazzone-Argiroffo, Dept. of Pathology, Centre Médical Universitaire, 1211 Geneva 4, Switzerland (E-mail: constance.barazzone{at}medecine.unige.ch).
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.
August 23, 2002;10.1152/ajplung.00239.2002
Received 19 July 2002; accepted in final form 14 August 2002.
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