Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor-kappa B activity

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Article

Reference

Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor-kappa B activity

BARAZZONE, Constance, et al.

Abstract

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-kappaB (NF-kappaB) activity was significantly decreased in lungs of sham mice exposed to hyperoxia but was preserved in ADX mice. There was a correlation between NF-kappaB activity in ADX mice and decreased levels of IkappaBalpha. In contrast, activator protein-1 activity increased similarly in both groups of mice. Levels of interleukin-6 (IL-6), a transcriptional target of NF-kappaB, were higher in bronchoalveolar lavage and serum of ADX than sham mice.

However, the protective effect of ADX was not mediated by [...]

BARAZZONE, Constance, et al . Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor-kappa B activity. American Journal of Physiology. Lung Cellular and Molecular Physiology , 2003, vol. 284, no. 1, p. L197-204

DOI : 10.1152/ajplung.00239.2002 PMID : 12388343

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http://archive-ouverte.unige.ch/unige:55336

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Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor- ␬ B activity

CONSTANCE BARAZZONE-ARGIROFFO,^1,2ALESSANDRA PAGANO,^1,2CRISTIANA JUGE,³ ISABELLE ME´ TRAILLER,^1,2ANNE ROCHAT,²CHRISTIAN VESIN,²AND YVES DONATI^1,2

1Department of Pediatrics,²Department of Pathology, and³Division of Endocrinology,

Department of Internal Medicine, University of Geneva Medical School, 1211 Geneva 4, Switzerland

Submitted 19 July 2002; accepted in final form 14 August 2002

Barazzone-Argiroffo, Constance, Alessandra Pa- gano, Cristiana Juge, Isabelle Me´trailler, Anne Rochat, Christian Vesin, and Yves Donati.Glucocorti- coids aggravate hyperoxia-induced lung injury through decreased nuclear factor-␬B activity. Am J Physiol Lung Cell Mol Physiol284: L197–L204, 2003. First published August 23, 2002; 10.1152/ajplung.00239.2002.—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

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 keratin-

ocyte 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 im- paired 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

Address for reprint requests and other correspondence: C. Barazzone- Argiroffo, Dept. of Pathology, Centre Me´dical Universitaire, 1211 Ge- neva 4, Switzerland (E-mail: constance.barazzone@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.

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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.

METHODS

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. Experi- ments were performed with 2- to 3-mo-old mice. Corticoste- rone 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 injec- tion 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␮g/

mouse; Sigma Chemical, St. Louis, MO) was injected ondays 0, 1, 2, and3(7 mg䡠kg^⫺1䡠day^⫺1ip).

To differentiate between GC and catecholamine absence in ADX mice, we inserted subcutaneously pellets of nadolol (a catecholamine blocker, 21-day release, 0.5 mg/pellet; Innova- tive Research of America, Sarasota, FL) or RU-486 (a GC receptor antagonist, 21-day release, 0.5 mg/pellet) into wild- type (C57BL/6) mice (8, 17). The study protocol was approved by the ethical committee on animal experiments (Office Ve´t- e´rinaire Cantonal of Geneva).

PDTC (Sigma Chemical), which has been shown to block NF-␬B activation in rats challenged with lipopolysaccharide (LPS) and recently in mice with collagen-induced arthritis, was administered at doses similar to those described previously in rats and mice (100 mg䡠kg^⫺1䡠day^⫺1ip at 24, 48, and 60 h of oxygen exposure) (12, 28). ADX and sham mice received PDTC or the solvent.

In some experiments, minipumps (3-day pumps, Alzet, Alza, Palo Alto, CA) delivering recombinant human (rh) IL-6 at 150␮g䡠kg^⫺1䡠day^⫺1(kindly provided by A. Okano, Ajino-

moto Pharmaceutical Research Laboratories, Kawasaki, Ja- pan) were implanted subcutaneously just before hyperoxia exposure (39). rhIL-6 levels were assessed by ELISA, which specifically recognizes human IL-6 (catalog no. MKL6 1, Milenia Biotech, Bad Neuheim, Germany). Mouse IL-6 was determined by DuoSet ELISA (R & D Systems, Minneapolis, MN).

Light 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 Dick- inson).

Isolation of nuclear proteins and assay of NF-␬B and AP-1 activities.Nuclear proteins were extracted according a mod- ification of a previously described protocol (40). Fresh lung tissue (10 mg) was homogenized with a Dounce homogenizer in 0.5 ml ofbuffer A [10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES, pH 7.9, 1 mM dithiothreitol (DTT), 1 mM NaVO4, 5

␮g/ml leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride]. This procedure was repeated after centrifugation at 2,000gfor 10 min at 4°C. The nuclei-containing pellet was carefully resuspended in 2 vol ofbuffer B[420 mM NaCl, 10 mM KCl, 20 mM HEPES, pH 7.9, 20% glycerol, 1 mM DTT, 1 mM NaVO4, 5␮g/ml leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride] and subjected to vigorous agita- tion for 30 min at 4°C. After centrifugation at 13,000gfor 20 min at 4°C, the supernatant was collected, divided into ali- quots, and stored at ⫺80°C. NF-␬B and AP-1 activity in nuclear proteins was estimated using an electrophoretic mo- bility 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⬘-CGCTTGATGAGT- CAGCCGGAA-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␮g/␮g protein), 1 mM EDTA, 1 mM DTT, 4% Ficoll- 400, 4 mg/ml BSA (Boehringer, fraction V), the nuclear extracts (2␮g), and 30,000 counts/min (ideally 30,000 counts/

min/34 fmol in 1 ␮l) of radiolabeled oligonucleotide probe.

Specificity of the bands in the retardation gels was ascer- tained 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.

L198 GLUCOCORTICOIDS AGGRAVATE ALVEOLAR DAMAGE

AJP-Lung Cell Mol Physiol•VOL 284 • JANUARY 2003 •www.ajplung.org

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Western blots.Lungs were homogenized as described previously (5), and 40␮g 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. Mem- branes were blotted with the polyclonal antibody anti-I␬B␣ (catalog no. 554135, PharMingen, 1:1,000 dilution) in TBST and 5% nonfat milk. A goat anti-rabbit IgG-horseradish per- oxidase (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 sub- saturated 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 Pa- thology, 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’st-test. Where appro- priate, two-way ANOVA with multiple comparisons followed by an unpairedt-test was used. Significance levels were set atP⬍0.05.

RESULTS

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. Al- though 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.

Hyperoxia induces thymocyte apoptosis via endoge- nous 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).

Activated caspases were detected in isolated thymocytes using a specific probe (CaspaTag). The per- centage of positive cells was increased in thymocytes isolated from hyperoxic sham mice compared with air-breathing mice (42% vs. 8%) and was not affected by hyperoxia in ADX mice (7%). Moreover, daily injec- tion of hydroxycorticosterone into ADX mice reversed this effect (29% of positive cells). These results demonstrate that thymocyte apoptosis during hyperoxia de- pends on increased circulating levels of endogenous corticosterone (Fig. 3B).

Modulation of NF-␬B and AP-1 activities by hyper- oxia.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,BandD), 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).

Inasmuch as oxidative stress modulates NF-␬B activity by degrading I␬B␣, a protein that is bound to NF-␬B within the cytosol and, thereby, prevents translocation of NF-␬B into the nucleus, we examined I␬B␣

Table 1. Lung injury score in sham and ADX mice

Air Hyperoxia

Sham ADX Sham ADX ADX⫹HCS

Right lung wt, g 0.10⫾0.01 0.10⫾0.02 0.22⫾0.05^b 0.14⫾0.02^d 0.19⫾0.02^e

Protein, mg/ml 0.09⫾0.05 0.13⫾0.09 1.73⫾0.62^b 0.56⫾0.33^c ND

Cell count,⫻10³ 26.7⫾13.6 27.3⫾12.3 45.6⫾24.7 36.7⫾18.9 ND

IL-6, pg/ml 1.3⫾0.3 1.5⫾0.1 9.8⫾3.9^a 217.2⫾138.0^c ND

Serum IL-6, pg/ml 4.3⫾2.4 5.2⫾0.6 3.1⫾2.2 122⫾82.1^d 6.8⫾6.1^f

Values are means⫾SD of 5–10 animals in each group. Sham-operated (sham) mice, adrenalectomized (ADX) mice, and ADX mice treated with hydrocortisone acetate (HCS, 140␮g/day) were exposed to air or hyperoxia for 72 h, and bronchoalveolar lavage was performed by intratracheal instillation of 2 ml of PBS, with hydrostatic pressure of 20 cmH2O. Cells were counted, and proteins and interleukin-6 (IL-6) were measured in supernatant and serum. Significantly different from air:^aP⬍0.05 and^bP⬍0.001. Significantly different from sham:^cP⬍ 0.01 and^dP⬍0.001. Significantly different from ADX:^eP⬍0.05 and^fP⬍0.001. ND, not determined.

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expression by Western blot (Fig. 5). Hyperoxia did not change I␬B␣ expression in sham mice. However, in ADX mice, where NF-␬B activity was conserved, I␬B␣ expression vanished (Fig. 5). These results suggest that GC could act not only directly by repressing NF-␬B but also by decreasing I␬B␣.

PDTC prevents I␬B␣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 un- treated 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.

To investigate whether the protective effect exerted by the absence of the adrenal gland could be mediated by IL-6 elevation, we administered rhIL-6 to wild-type mice by continuous infusion at a dose previously shown to be efficient (39). rhIL-6 levels reached 0.8–1.7 ng/ml and were even higher than those obtained during hyperoxia in ADX mice. This treatment conferred no protective effect on hyperoxia-induced alveolar edema (Fig. 6). These results indicate that IL-6 does not play a major role in the prevention of lung damage in ADX mice.

DISCUSSION

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

Fig. 1. Lung histology of a representative adrenalectomized (ADX) control (air-breathing) mouse (A), an ADX mouse exposed to hyperoxia for 72 h (B), and a sham-operated (sham) mouse exposed to hyperoxia for 72 h (C). Arrow, alveolar edema and hemorrhages;

arrowhead, interstitial edema. Original magnification⫻400.

Fig. 2. Right lung weight of air-breathing and hyperoxia-exposed C57BL/6 mice treated with slow-release pellets of placebo, nadolol, or RU-486. * Lung weight was significantly lower in RU-486- than in placebo-treated animals (P⬍0.05) but was not different from that in nadolol-treated animals. Values are means⫾SD of 9 mice in each group.

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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).

Fig. 3. A: thymus weight of air-breathing and hyperoxia (Hox)-exposed sham and ADX mice. Thymus weight of sham mice exposed to hyperoxia was significantly reduced compared with air-breathing mice (P ⬍ 0.01).

Adrenalectomy significantly increased thymus weight in air-breathing animals (P ⬍ 0.01) and protected thymus from atrophy during hyperoxia. Values are means⫾SD (n⫽ 8).B: detection of activated caspase on isolated thymocytes. Thymocytes of air-breathing and hyperoxia-exposed mice were isolated and labeled with the FITC-labeled CaspaTag fluorescein (VAD) probe and examined by flow cytometry. Results are from a representative experiment. HCS, hydroxycorticosterone acetate.

Fig. 4. A and C: detection of nuclear factor-␬B (NF-␬B) and activator protein-1 (AP-1) activity in lung nuclear extracts. Two micrograms of lung nuclear protein extract were loaded in each lane, and gels were incubated with³²P-labeled oligonucleotide probes for NF-␬B (A) or AP-1 (C) and subjected to EMSA.A: NF-␬B activity decreased during hyperoxia in sham mice compared with air-breathing mice:

*P ⬍ 0.05. NF-␬B activity was preserved during hyperoxia in ADX mice compared with sham mice: #P⬍0.01.

C: AP-1 activity increased during hy- peroxia in sham and ADX mice: *P⬍ 0.05. Values are means⫾ SD of 3–5 different samples for each condition. A representative autoradiogram is shown attop. AU, arbitrary units. B and D: competition experiments per- formed by preincubating lung nuclear extracts with cold specific DNA 10-nucleotide or PU.1-specific DNA 11-nucleotide sequence at molar ratios of 1:1, 1:10, 1:100, and 1:1,000.

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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 (un- published 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 corticoste-

rones 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 contradic- tory. 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 contro- versial 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 pleu- risy model. Supporting this concept are the results obtained with intraperitoneal administration of dexamethasone in LPS-challenged mice that express lucif-

Fig. 6. Lung weight of mice treated by continuous infusion of recombinant human interleukin-6 (rhIL-6) or 1% normal mouse serum (NMS). Difference between groups was not significant. Values are means⫾SD (n⫽5).

Fig. 5. Western blot for detection of I␬B␣in total lung extracts. Forty micrograms of protein were loaded in each lane. Sham and ADX mice were treated with solvent (⫺) or pyrrolidine dithiocarbamate (PDTC,

⫹) for 3 days. I␬B␣ was equally detected in air-breathing and hyperoxia-exposed sham mice and decreased during hyperoxia in ADX mice. PDTC slightly increased I␬B␣expression in all samples.

Actin was used to normalize loading. A representative gel is shown.

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erase under the control of NF-␬B promoter (34). Dexa- methasone 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 al- most 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 subcu- taneous 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 en- dothelial cells might be opposed (3, 11, 26, 27). The concomitant ongoing destructive phase, where steroid treatment might accelerate cell death, and prolifera- tive 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.

We thank P.-F. Piguet for scientific advise, D. Belin for critical reading of the manuscript, and A. Scherrer for technical assistance.

This work is supported by Swiss National Foundation Grant 3200-067865.02 (C. Barazzone-Argiroffo) and the Wolfermann- Na¨gele Foundation.

REFERENCES

1. Allen GL, Menendez IY, Ryan MA, Mazor RL, Wispe JR, Fiedler MA, and Wong HR. Hyperoxia synergistically increases TNF-␣-induced interleukin-8 gene expression in A549 cells.Am J Physiol Lung Cell Mol Physiol278: L253–L260, 2000.

2. Auphan N, DiDonato JA, Rosette C, Helmberg A, and Karin M.Immunosuppression by glucocorticoids: inhibition of

NF-␬B activity through induction of I␬B synthesis.Science270:

286–290, 1995.

3. Bancalari E.Changes in the pathogenesis and prevention of chronic lung disease of prematurity. Am J Perinatol18: 1–9, 2001.

4. Barazzone C, Belin D, Piguet PF, Vassalli JD, and Sappino AP.Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.J Clin Invest98: 2666–2673, 1996.

5. Barazzone C, Donati YR, Rochat AF, Vesin C, Laperrousaz CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects both alveolar epithelium and endothelium from oxygen- induced injury in mice.Am J Pathol154: 1479–1487, 1999.

6. Barazzone C, Tacchini-Cottier F, Vesin C, Rochat AF, and Piguet PF.Hyperoxia induces platelet activation and lung se- questration: an event dependent on tumor necrosis factor-␣and CD11a.Am J Respir Cell Mol Biol15: 107–114, 1996.

7. Barazzone Argiroffo C, Donati YR, Boccard J, Rochat AF, Vesin C, Kan CD, and Piguet PF.CD40-CD40 ligand disruption does not prevent hyperoxia-induced injury.Am J Pathol 160: 67–71, 2002.

8. Barazzone-Argiroffo C, Muzzin P, Donati YR, Kan CD, Aubert ML, and Piguet PF.Hyperoxia increases leptin production: a mechanism mediated through endogenous elevation of corticosterone.Am J Physiol Lung Cell Mol Physiol281: L1150–

L1156, 2001.

9. Bollaert PE, Charpentier C, Levy B, Debouverie M, Audi- bert G, and Larcan A. Reversal of late septic shock with supraphysiologic doses of hydrocortisone. Crit Care Med 26:

645–650, 1998.

10.Burri P.Structural aspects of prenatal and postnatal development and growth of the lung. In:Lung Growth and Development, edited by McDonald JA.New York: Dekker, 1997, p. 1–35.

11.Collard HR and King TE.Treatment of idiopathic pulmonary fibrosis: the rise and fall of corticosteroids. Am J Med 110:

326–328, 2001.

12.Cuzzocrea S, Chatterjee PK, Mazzon E, Dugo L, Serraino I, Britti D, Mazzullo G, Caputi AP, and Thiemermann C.

Pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation.Br J Pharmacol135: 496–510, 2002.

13.Dorscheid DR, Wojcik KR, Sun S, Marroquin B, and White SR. Apoptosis of airway epithelial cells induced by corticosteroids.Am J Respir Crit Care Med164: 1939–1947, 2001.

14.Evans-Storms RB and Cidlowski JA.Delineation of an anti- apoptotic action of glucocorticoids in hepatoma cells: the role of nuclear factor-␬B.Endocrinology141: 1854–1862, 2000.

15.Fox RB, Hiodal JR, Brown DM, and Repine JE.Pulmonary inflammation due to oxygen toxicity: involvement of chemotactic factors and polymorphonuclear cells. Am Rev Respir Dis 123:

521–523, 1981.

16.Franek WR, Horowitz S, Stansberry L, Kazzaz J, Koo HC, Li Y, Davis JM, Mantell AS, Scott W, and Mantell L.Hy- peroxia inhibits oxidant-induced apoptosis in lung epithelial cells.J Biol Chem276: 569–575, 2001.

17.Ghoshal K, Wang Y, Sheridan JF, and Jacob ST.Metallo- thionein induction in response to restraint stress.J Biol Chem 273: 27904–27910, 1998.

18.Gilbert KA, Petrovic-Dovat L, and Rannels E. Hormonal control of compensatory lung growth. In: Lung Growth and Development,edited by McDonald JA.New York: Dekker, 1997, p. 627–660.

19.Gorman AM, Hirt UA, Orrenius S, and Ceccatelli S.Dexa- methasone pre-treatment interferes with apoptotic death in gli- oma cells.Neuroscience96: 417–425, 2000.

20.Lawrence T, Gilroy DW, Colville-Nash PR, and Wil- loughby DA. Possible new role for NF-␬B in the resolution of inflammation.Nat Med7: 1291–1297, 2001.

21.Li Y, Zhang W, Mantell LL, Kazzaz JA, Fein AF, and Horowitz S. Nuclear factor-␬B is activated by hyperoxia but does not protect from cell death.J Biol Chem272: 20646–20649, 1997.

22.Liu SF, Ye X, and Malik AB.Inhibition of NF-␬B activation by pyrrolidine dithiocarbamate prevents in vivo expression of proinflammatory genes.Circulation100: 1330–1337, 1999.

(9)

23.Liu SF, Ye X, and Malik AB. Pyrrolidine dithiocarbamate prevents I␬B degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs.Mol Pharmacol 55: 658–667, 1999.

24.Lu Y, Parkyn L, Otterbein LE, Kureishi Y, Ray A, and Ray P.Activated Akt protects the lung from oxidant-induced injury and delays death of mice.J Exp Med193: 545–549, 2001.

25.Luyet C, Burri PH, and Schittny JC. Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development. Am J Physiol Lung Cell Mol Physiol282: L477–L483, 2002.

26.Meduri GU and Kanangat S. Glucocorticoid treatment of sepsis and acute respiratory distress syndrome. Time for a critical reappraisal. Crit Care Med26: 630–633, 1998.

27.Munford RS and Pugin J.Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med163: 316–321, 2001.

28.Nathens AB, Bitar R, Davreux C, Bujard M, Marshall JC, Dackiw AP, Watson R, and Rotstein OD.Pyrrolidine dithiocarbamate attenuates endotoxin-induced acute lung injury.

Am J Respir Cell Mol Biol17: 608–616, 1997.

29.Newton R.Molecular mechanisms of glucocorticoid action: what is important?Thorax55: 603–613, 2000.

30.Parrish D, Mitchell BC, Henson PM, and Larsen GL.Pul- monary response of the fifth component of complement-sufficient and -deficient mice to hyperoxia. J Clin Invest 74: 956–965, 1984.

31.Piette J, Piret B, Bonizzi G, Schoonbrodt S, Merville MP, Legrand-Poels S, and Bours V.Multiple redox regulation in NF-␬B transcription factor activation. Biol Chem 378: 1237–

1245, 1997.

32.Piguet PF, Vesin C, and Rochat AF. ␤²-Integrin modulates platelet caspase activation and life span in mice.Eur J Cell Biol 80: 1–7, 2001.

33.Rahman I, Mulier B, Gilmour PS, Watchorn T, Donaldson K, Jeffery PS, and MacNee W.Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-␬B.Biochem Pharmacol62: 787–794, 2001.

34.Sadikot RT, Jansen ED, Blackwell TR, Zoia O, Yull F, Christman JW, and Blackwell TS.High-dose dexamethasone

accentuates nuclear factor-␬B activation in endotoxin-treated mice.Am J Respir Crit Care Med164: 873–878, 2001.

35.Salmon M, Koto H, Lynch OT, Haddad EB, Lamb NJ, Quinlan GJ, Barnes PJ, and Chung KF. Proliferation of airway epithelium after ozone exposure: effect of apocynin and dexamethasone.Am J Respir Crit Care Med157: 970–977, 1998.

36.Scheinman RI, Cogswell PC, Lofquist AK, and Baldwin AS Jr. Role of transcriptional activation of I␬B␣ in mediation of immunosuppression by glucocorticoids. Science 270: 283–286, 1995.

37.Schneider A, Martin-Villalba A, Weih F, Vogel J, With T, and Schwaninger M. NF-␬B is activated and promotes cell death in focal cerebral ischemia.Nat Med5: 554–559, 1999.

38.Seyle H.Thymus and adrenals in response of the organism to injuries and intoxication.Br J Exp Pathol17: 234–248, 1936.

39.Suzuki C, Okano A, Takatsuki F, Myasaka Y, Hirano T, Kishimoto T, Ejima D, and Akiyama Y.Continuous perfusion with IL-6 enhances production of hematopoietic stem cells (CFU- S).Biochem Biophys Res Commun159: 933–938, 1989.

40.Vaisse C, Halaas JL, Horvath CM, Stoffel M, and Fried- man JM. Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 14:

95–97, 1996.

41.Van den Berghe W, Vermeulen L, De Wilde G, De Bosscher K, Boone E, and Haegeman G.Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6.Biochem Pharmacol8: 1185–1195, 2000.

42.Walker CD, Sizonenko PC, and Aubert ML.Modulation of the neonatal pituitary and adrenocortical responses to stress by thyroid hormones in the rat: effects of hypothyroidism and hy- perthyroidism.Neuroendocrinology50: 265–273, 1989.

43.Ward N, Waxman A, Homer R, Mantell L, Einarsson O, Du Y, and Elias JA. IL-6-induced protection in hyperoxic acute lung injury.Am J Respir Cell Mol Biol22: 535–542, 2000.

44.Wong HR, Odoms KK, Denenberg AG, Allen GL, and Shan- ley TP. Hyperoxia prolongs tumor necrosis factor-␣-mediated activation of NF-␬B: role of I␬B kinase.Shock17: 274–279, 2002.

45.Wyllie AH and Morris RG.Hormone-induced cell death. Pu- rification and properties of thymocytes undergoing apoptosis after glucocorticoid treatment.Am J Pathol109: 78–87, 1982.