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Am J Physiol Lung Cell Mol Physiol 276: L175-L185, 1999;
1040-0605/99 $5.00
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Vol. 276, Issue 1, L175-L185, January 1999

Dexamethasone regulation of lung epithelial cell and fibroblast interleukin-11 production

Jingming Wang1, Zhou Zhu1, Robert Nolfo2, and Jack A. Elias1

Section of Pulmonary and Critical Care Medicine, Departments of 1 Internal Medicine and 2 Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8057

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Studies were undertaken to define the effects of corticosteroids on stromal cell interleukin (IL)-11 production. Unstimulated A549 epithelial-like cells produced modest amounts of IL-11, and transforming growth factor (TGF)-beta 1 was a potent, dose-dependent stimulator of A549 cell IL-11 elaboration. Dexamethasone inhibited the levels of basal and TGF-beta 1-stimulated IL-11 elaboration in a dose-dependent fashion. In the setting of TGF-beta 1 stimulation, dexamethasone caused a >90% decrease in IL-11 production at 10-6 M, a 50% decrease in IL-11 production at ~1 × 10-9 M, and significant inhibition at 10-10 M. This dexamethasone-induced inhibition was reversed by the glucocorticoid-receptor antagonist RU-486. Dexamethasone also inhibited respiratory syncytial virus, rhinovirus, and TGF-beta 1-stimulated IL-11 production by MRC-5 lung fibroblasts. In all cases, dexamethasone caused comparable changes in IL-11 mRNA accumulation. Nuclear run-on studies demonstrated that dexamethasone caused a modest (<= 40%) decrease in TGF-beta 1-stimulated IL-11 gene transcription. Actinomycin D pulse-chase experiments demonstrated that dexamethasone simultaneously destabilized IL-11 mRNA. Dexamethasone also inhibited TGF-beta 1-stimulated IL-11 promoter-driven luciferase activity but did not diminish activator protein-1 binding to IL-11 promoter sequences. Glucocorticoids inhibit lung cell IL-11 production via a complex mechanism that involves the inhibition of IL-11 gene transcription and the destabilization of IL-11 mRNA.

transforming growth factor-beta ; corticosteroid; RU-486; fibroblast; messenger ribonucleic acid degradation; respiratory syncytial virus; rhinovirus

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

INTERLEUKIN (IL)-11 was originally cloned from primate bone marrow stromal cells based on its ability to stimulate the proliferation of a murine plasmacytoma cell line (27). It has since been classified with other members of the IL-6-type cytokine family based on the overlapping biological activities of these cytokines and their common usage of the gp130 molecule in their multimeric receptor complexes (7, 18). Studies of the effector functions of IL-11 have demonstrated that it is a multifunctional molecule. It is a major regulator of hematopoiesis, with prominent effects on platelets and a variety of other circulating cells (7). It also regulates B cell function via a T cell-dependent mechanism (44), induces hepatocyte production of acute phase proteins (3, 7), stimulates the production of tissue inhibitor of metalloproteinase-1 (24), regulates neuronal differentiation (26), influences osteoclast development (17), and has protective effects in a variety of models of mucosal injury of the gastrointestinal tract (20). Studies from our laboratory have investigated the effects of IL-11 in the lung. They have demonstrated that the transgenic overexpression of IL-11 in the murine airway induces peribronchial inflammation; airway remodeling with fibrosis and myofibroblast and myocyte hyperplasia; and altered alveolar development (31, 39). They have also demonstrated that IL-11 has protective effects in the lung in the setting of thoracic radiation and that the protective effects of IL-11 may be mediated by its ability to inhibit macrophage production of tumor necrosis factor (TNF) and other cytokines (32, 42). Our studies of potential cellular sources of IL-11 demonstrated that human lung epithelial cells (9, 12), fibroblasts (14, 45), and smooth muscle cells (11) have the ability to produce large amounts of IL-11 in vitro when appropriately stimulated. Potent stimuli include transforming growth factor (TGF)-beta 1, respiratory tropic viruses, including respiratory syncytial virus (RSV), rhinovirus (RV), and parainfluenza virus type III, and, to a lesser extent, IL-1, histamine, and eosinophil-derived major basic protein (9, 12, 14, 33, 45). In contrast to our knowledge of the processes that stimulate IL-11 production, much less is known about the processes that inhibit IL-11 elaboration by stimulated stromal cells.

Corticosteroids are commonly employed in the treatment of inflammatory and fibrotic pulmonary disorders. In this and other settings, they mediate their anti-inflammatory effects, in great extent, via their ability to inhibit the production of a large variety of inflammatory cytokines. This inhibition is mediated via a variety of mechanisms, including the inhibition of gene transcription and destabilization of cytokine mRNA (5). Recent studies have demonstrated that corticosteroids can also inhibit inflammation by stimulating the production of anti-inflammatory cytokines such as the IL-1-receptor antagonist (23). The effects of corticosteroids on IL-11 production, however, have not been investigated.

To further understand the processes that regulate IL-11 elaboration, studies were undertaken to define the effects of dexamethasone on lung stromal cell IL-11 elaboration. These studies demonstrate that glucocorticoids are potent dose-dependent inhibitors of TGF-beta 1 and virus-stimulated IL-11 elaboration by alveolar epithelial-like cells and lung fibroblasts. They also demonstrate that these inhibitory effects are mediated by a complex mechanism that involves dexamethasone binding to the glucocorticoid receptor, the inhibition of IL-11 gene transcription, and the enhanced degradation of IL-11 mRNA.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Reagents

Human recombinant TGF-beta 1 was purchased from R&D Systems (Minneapolis, MN). Human recombinant IL-11 and monoclonal antibodies 11h3.19.6.1 and 11.h3.16.6.1 against human-IL-11 were obtained from Dr. Paul Schendel (Genetic Institute, Cambridge, MA). Clone pHuIL-11/PMT, a 1,250-bp IL-11 cDNA in the EcoR I site of vector PXM, was a gift of Dr. Paul Schendel. Dexamethasone and actinomycin D were purchased from Sigma Chemical (St. Louis, MO). The glucocorticoid-receptor antagonist RU-486 was a kind gift from Dr. D Martini (Roussel UCLAF, Romainville, France). [alpha -32P]dCTP (3,000 Ci/mmol), [gamma -32P]dATP (3,000 Ci/mmol), and [alpha -32P]dUTP (3,000 Ci/mmol) were purchased from Amersham (Arlington Heights, IL).

Cell Culture and Supernatant Collection

A549 human alveolar epithelial type II-like cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown to confluence in 5% CO2-95% air in 100-mm petri dishes in DMEM supplemented with nonessential amino acids, L-glutamine, penicillin, streptomycin (GIBCO BRL, Grand Island, NY), and 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT). Once confluent, the cells were rinsed two times with serum-free DMEM and incubated in the presence and absence of TGF-beta 1 (10 ng/ml) and/or dexamethasone (10-6 to ~10-10 M) and/or RU-486 (10-6 to 10-7 M). At designated time points (4, 12, 24, and 48 h), the supernatants were removed, clarified by low-speed centrifugation, separated into aliquots, and stored at -70°C until assayed for IL-11. The cell monolayers were then rinsed with cold PBS and used for mRNA analysis or nuclear run-on studies.

IL-11 ELISA

Human IL-11 protein was quantitated by ELISA as previously described by our laboratory (11, 12, 14).

mRNA Isolation and Analysis

Total cellular RNA was extracted from cell monolayers at designated time points using acid guanidinium isothiocyanate-phenol-chloroform extraction as previously described (11-14). Equal amounts (20 µg) of RNA were loaded on 1% agarose gels containing 17% formaldehyde, electrophoresed at 80 volts for 3 h, transferred to nylon membranes, and hybridized with cDNA probe labeled to a high specific activity with [alpha -32P]dCTP (109 counts · min-1 · mg DNA-1) using the random-primer method. After hybridization, the membranes were washed under conditions of high stringency and evaluated using autoradiography. The adequacy of RNA loading was assessed using ethidium bromide staining and ultraviolet illumination. Densitometry was performed using a Hoefer GS 300 densitometer, and densitometry curve integration and analysis were accomplished using the Hoefer GS 370 Data System software package (Hoefer Scientific Instruments, San Francisco, CA).

Analysis of mRNA Half-Life

IL-11 mRNA stability was assessed as previously described by this laboratory (10, 47). A549 cells were incubated with TGF-beta 1 alone or with TGF-beta 1 plus dexamethasone for 24 h. A pair of monolayers was then harvested for baseline mRNA values, and actinomycin D (10 µg/ml) was added to the remaining cultures. The rates of decay of IL-11 mRNA were determined by quantitating the levels of IL-11 mRNA at intervals thereafter. Half-life values were obtained from log-linear plots comparing densitometrically determined absorbance units versus time.

Analysis of Relative Rates of Nuclear Transcription

The relative rates of nuclear transcription were assessed using modifications of protocols previously employed by this laboratory (10, 14, 47). Confluent A549 cell monolayers were incubated in the presence of TGF-beta 1 and/or dexamethasone (10-6 M) for 16 h. The supernatants were then removed, and the cells were washed, mechanically detached, resuspended in lysis buffer (10 mM Tris, pH 7.6, 2 mM MgCl2, 10 mM NaCl, 0.6% Triton X-100, and 3 mM CaCl2), incubated for 5 min, and pelleted again. The nuclei were stored in glycerol at -70°C until used. When needed, 108 nuclei per condition were thawed and incubated in transcription buffer (10 mM Tris, pH 8.0, 0.3 M KCl, 5 mM MgCl2, 5 mM dithiothreitol, and 1 mM ATP, CTP, and GTP) with [alpha -32P]UTP (0.5 mCi, 3,000 Ci/mmol) for 30 min at 30°C. The incubation was terminated by addition of RNase-free DNase (Boehringer Mannheim, Indianapolis, IN), followed by treatment with proteinase K. RNA was extracted with chloroform-phenol-isoamyl alcohol (10:10:1) and precipitated with 2 M sodium acetate in 100% alcohol. The pellet was then washed with 90% alcohol and recovered after centrifugation. A slot-bolt apparatus was used to prepare nitrocellulose membranes containing 20 µg/slot of plaid DNA, with cDNA insert encoding IL-11 and 3 µg/slot of cDNA encoding 28S rRNA. Twenty micrograms of insert-free pUC18 vector were included as a control for nonspecific binding. The membranes were hybridized with equal numbers of counts of precipitated 32P-labeled RNA per condition and were washed in solutions of increasing stringency. DNA-RNA binding was evaluated by autoradiography and densitometry.

Assessment of Cell Viability

Cell viability was assessed using trypan blue dye exclusion, cell counting, and measurements of cell supernatant lactate dehydrogenase (LDH). LDH was quantitated with a commercial kit (Sigma) according to the manufacturer's instructions.

IL-11 Promoter-Reporter Gene Construction

As previously described (40), a 786-bp Pvu II fragment of the IL-11 promoter containing sequences between -728 and +58 bp relative to its transcription start site was obtained from Dr. Yu-Chung Yang (Indiana University School of Medicine, Indianapolis, IN) and was cloned into the Sma I site of the luciferase gene vector pXP2-luc (ATCC) to generate construct pXP2-IL-11-728.

Cell Transfection and Reporter Gene Assay

Plasmid DNA was introduced in A549 cells using a modification of the DEAE-dextran transfection protocol described by our laboratory (40, 46). Briefly, A549 cells were grown until 60-80% confluent in 60-mm petri dishes in complete DMEM with 10% FBS. They were washed and incubated with the mixture of DNA (4.5 µg) and DEAE-dextran (1 mg/ml) in a volume of 300 ml for 30 min at room temperature. At the end of this incubation period, the cells were washed and incubated in the presence and absence of TGF-beta 1 (10 ng/ml) and/or RU-486 (10-6 M) and/or dexamethasone (10-6 to ~10-11 M) for 24 h at 37°C in 5% CO2 and air. The cells were then washed, mechanically detached, pelleted, and resuspended in 0.25 M Tris · HCl, pH 7.8, in the presence of lysis reagent (Promega, Madison, WI). The lysates were then clarified by centrifugation and stored at -20°C. Luciferase activity was measured using the Luciferase Assay System from Promega. Quantification was obtained in a luminometer (model LB9501, Lumat, Bethold, Germany). Transfection efficiency was simultaneously assessed by cotransfecting (1.5 µg) the construct pCMV-beta -Gal (ATCC), a construct that contains the beta -galactosidase gene driven by the cytomegalovirus immediate-early promoter. beta -Galactosidase activity was measured using the chromogenic technique of Eustice et al. (15). All luciferase measurements were normalized for transfection efficiency using the beta -galactosidase values. The resulting data are expressed as relative light units.

Electrophoretic Mobility Shift Assay

Nuclear extract preparation. Nuclear extracts were prepared using modifications of the techniques of Schreiber et al. (36) as previously described (46). A549 cells were incubated in the presence of TGF-beta 1 and/or dexamethasone as described above. At the desired time points, the cells were mechanically detached, suspended in Tris-buffered saline freshly supplemented with protease inhibitors (1 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), pelleted at 4°C, and resuspended and swelled in 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), and freshly added protease inhibitors as above for 15 min on ice. Membrane lysis was accomplished by adding 25 µl of 10% Nonidet P-40 followed by vigorous agitation. The nuclei were collected by centrifugation, resuspended in 80 µl of 20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and freshly added protease inhibitors as above, and agitated vigorously at 4°C for 30 min. The membrane debris was discarded, and the protein concentration of each nuclear extract was measured using the DC Protein Assay System (Bio-Rad). The extracts were then separated into aliquots and stored at -70°C until used.

Oligonucleotide probes. Classic activator protein-1 (AP-1), classic nuclear factor-kappa B (NF-kappa B) and IL-11 5' AP-1 oligonucleotides were used in these studies. The AP-1 and NF-kappa B oligonucleotides were obtained commercially from Santa Cruz Biotechnology (Santa Cruz, CA) and Bio-Synthesis (Denten, TX), respectively. The IL-11 promoter AP-1 oligonucleotides were prepared at the Yale University Oligonucleotide Synthesis Facility. Their sequences were as follows: classic AP-1, 5'-CGCTTGATGACTCAGCCGGAA-3'; classic NF-kappa B, 5'-TGGACAGAGGGGACTTTCCGAGAGGC-3'; and IL-11 5' AP-1, 5'-GGGAGGGTGAGTCAGGATGTG-3'.

Electrophoresis. Electrophoretic mobility shift assays (EMSA) were performed using modifications of the techniques of Schreiber et al. (36) as previously described (40, 46). Radiolabeled double-stranded oligonucleotide probes were prepared by annealing complementary oligonucleotides and end labeling with [gamma -32P]ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled probes were purified by push-column chromatography, diluted with buffer (1 mM Tris · HCl, pH 8.0, and 1 mM EDTA) to the desired concentration, and incubated with equal aliquots of nuclear extract and poly[dI-dC] at room temperature for 30 min. Resolution was accomplished by electrophoresing 10 µl of the reaction solution on vertical 6% nondenaturing polyacrylamide gels containing 2% glycerol using 22.3 mM Tris · HCl, 22.3 mM boric acid, and 0.25 mM EDTA, pH 8.0. DNA-protein binding activity was assessed via autoradiography.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Dexamethasone Regulation of TGF-beta 1-Stimulated IL-11 Production

To characterize the effects of corticosteroids on IL-11 production, A549 cells were grown to confluence and stimulated with TGF-beta 1 in the presence and absence of dexamethasone. Cell supernatants were harvested at intervals thereafter, and the levels of IL-11 were assessed by ELISA. Unstimulated A549 cells produced levels of IL-11 that were near or below the levels of detectability with our assay. In contrast, TGF-beta 1 was an impressive stimulator of IL-11 protein elaboration by these cells. As previously described (12), TGF-beta 1-stimulated IL-11 production was seen readily after 12 h and was more impressive after 24-48 h of cytokine-epithelial cell incubation (Fig. 1). Dexamethasone diminished the baseline levels of IL-11 production by these cells. This effect was hard to quantify, however, because of the low levels of IL-11 production in the unstimulated state. In contrast, the ability of dexamethasone to inhibit TGF-beta 1-stimulated IL-11 elaboration was easily appreciated. This inhibition was noted at all time points between 12 and 48 h (Fig. 1). It was also dose dependent. Maximal inhibition was noted at 10-6 M dexamethasone. A549 cells incubated for 48 h with 10 ng/ml TGF-beta 1 and 10-6 M dexamethasone produced 5.2 ± 0.3% as much IL-11 as cells incubated with TGF-beta 1 alone (P < 0.001, Student's t-test). Significant inhibition was noted with doses of dexamethasone as low as 10-10 M. Overall, dexamethasone manifests an IC50 of ~1 × 10-9 M (Fig. 2).


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  		Fig. 1.   Effect of dexamethasone (Dex) on interleukin (IL)-11 production of unstimulated (Unsti) and transforming growth factor (TGF)-beta 1-stimulated A549 cells. A549 cells were grown to confluence and incubated in the presence (bullet  and ) and absence (open circle  and ) of TGF-beta 1 (10 ng/ml). These incubations were done in the presence ( and ) and absence (open circle  and bullet ) of dexamethasone (10-6 M). At the designated time points, supernatants were removed, and IL-11 content was evaluated by ELISA. Noted values represent the means of a minimum of 3 determinations. In all cases, SE was <= 10% of the noted values.


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  		Fig. 2.   Dose response of dexamethasone regulation of A549 cell IL-11 production. Confluent A549 cells were incubated in the presence (+) and absence (-) of TGF-beta 1 (10 ng/ml) and the noted concentrations of dexamethasone for 24 h. Levels of supernatant IL-11 were evaluated by ELISA. Amount of IL-11 produced by TGF-beta 1-stimulated A549 cells incubated in the presence of dexamethasone is expressed as a percentage of the IL-11 produced in the absence of dexamethasone. Noted values represent the means of at least 3 determinations at each time point. SE was consistently <= 10% of the noted values.

Dexamethasone Regulation of TGF-beta 1-Stimulated IL-11 mRNA Accumulation

To further understand the regulatory effects of dexamethasone, the levels of IL-11 mRNA in unstimulated and TGF-beta 1-stimulated A549 cells incubated in the presence and absence of dexamethasone were evaluated using Northern blot analysis. At baseline, the levels of IL-11 mRNA in A549 cells were near the limits of detection with our assay. In contrast, TGF-beta 1 was an impressive stimulator of IL-11 mRNA accumulation. This induction could be appreciated after as little as 4 h and peaked after 24 h of cytokine-epithelial-like cell incubation (Fig. 3). Dexamethasone alone did not increase the levels of IL-11 mRNA in unstimulated cells. Dexamethasone was, however, an impressive inhibitor of TGF-beta 1-stimulated IL-11 mRNA accumulation. This effect was seen after as little as 4 h and was most prominent after 24 h of TGF-beta 1-A549 cell incubation (Fig. 3). It was also dose dependent, with maximal inhibition being noted with 10-6 M and significant inhibition being noted with 10-10 M dexamethasone (data not shown). These studies demonstrate that the inhibitory effects of dexamethasone are associated with a comparable decrease in IL-11 mRNA accumulation and are, in great extent, pretranslationally mediated.


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  		Fig. 3.   Effect of dexamethasone on TGF-beta 1-stimulated IL-11 mRNA accumulation in A549 cells. Confluent A549 cells were unstimulated or incubated in the presence of TGF-beta 1 (10 ng/ml) and/or dexamethasone (10-6 M) for the noted periods of time. Levels of IL-11 mRNA were evaluated via Northern blot analysis. A: IL-11 mRNA transcripts. B: ethidium bromide loading controls. C: densitometric analysis of the mRNA autoradiogram.

Role of Cytotoxicity and Glucocorticoid Receptor

Studies were undertaken to determine whether cell cytotoxicity played a role in mediating the inhibitory effects of dexamethasone and whether these inhibitory effects were mediated via an interaction of dexamethasone with the glucocorticoid receptor. Cell cytotoxicity was assessed via trypan blue dye exclusion and LDH release. In all experiments, dexamethasone-mediated cell cytotoxicity was not appreciated. A comparable level of trypan blue dye exclusion and LDH release was seen in cultures of A549 cells incubated in the presence and absence of TGF-beta 1, in the presence and absence of varying concentrations of dexamethasone (10-6 to 10-10 M; data not shown). To characterize the role of the glucocorticoid receptor in this inhibition, the effects of dexamethasone were assessed in the presence and absence of RU-486, a well-characterized glucocorticoid-receptor antagonist (47). As noted above, dexamethasone was a potent dose-dependent inhibitor of TGF-beta 1-stimulated IL-11 protein production and mRNA accumulation. The addition of RU-486 to these cultures abrogated this inhibitory effect. This reversal was significant but incomplete at high concentrations of dexamethasone (10-6 and 10-7 M) and was complete at lower concentrations (10-8 and 10-9 M; Fig. 4 and data not shown). These studies demonstrate that the inhibitory effects of glucocorticoids are not mediated via direct cell cytotoxicity and do require dexamethasone-glucocorticoid receptor interaction.


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  		Fig. 4.   Effect of RU-486 on dexamethasone inhibition of TGF-beta 1-stimulated IL-11 production. Confluent A549 cells were incubated in the presence and absence of TGF-beta 1 (10 ng/ml), dexamethasone (10-6 M), and RU-486 (10-6 M). Levels of IL-11 in the resulting cell supernatants were evaluated by ELISA. Noted values represent the means of a minimum of 3 determinations. SE was consistently <= 10% of the noted values.

Specificity of Dexamethasone Effect

Previous studies from our laboratory demonstrated that, in addition to epithelial cells, lung fibroblasts are potent producers of IL-11 (14). In addition, we demonstrated that a variety of respiratory tropic viruses can stimulate stromal cell IL-11 elaboration (9, 12). Thus studies were undertaken to determine whether the inhibitory effects of dexamethasone were specific for TGF-beta 1 or A549 epithelial-like cells. This was done by characterizing the effects of dexamethasone on RSV- and RV-stimulated IL-11 elaboration by A549 cells and TGF-beta 1 and virus-stimulated IL-11 elaboration by MRC-5 human lung fibroblasts. As previously noted (9, 12), RSV and RV were potent stimulators of A549 cell IL-11 protein production and mRNA accumulation. Dexamethasone was a potent dose-dependent inhibitor of these inductive processes (Figs. 5 and 6 and data not shown). Overall, RSV-stimulated A549 cells and RV-stimulated A549 cells incubated for 24 h in the presence of dexamethasone (10-6 M) produced 4.5 ± 0.4 and 6.3 ± 0.5%, respectively, as much IL-11 as cells infected with viruses in the absence of dexamethasone (P < 0.01 for each, Student's t-test).


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  		Fig. 5.   Effect of dexamethasone on respiratory syncytial virus (RSV)-stimulated IL-11 production by A549 cells. Confluent A549 cells were incubated in the presence and absence of RSV [multiplicity of infection (MOI) = 3; bullet  and ]. These incubations were performed in the presence ( and ) and absence (open circle  and bullet ) of dexamethasone (10-6 M). Levels of supernatant IL-11 were evaluated by ELISA. Noted values represent the means of at least 3 determinations. SE was consistently <= 10% of the noted values.


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  		Fig. 6.   Dose response of dexamethasone inhibition of IL-11 mRNA accumulation in virus-infected A549 cells and MRC-5 lung fibroblasts. A549 and MRC-5 cells were grown to confluence and then were infected with RSV or rhinovirus (RV) 14 (MOI = 3). Incubations were performed in the presence and absence of the noted concentrations of dexamethasone. Levels of IL-11 mRNA were evaluated with Northern blot analysis as described in EXPERIMENTAL PROCEDURES.

Unstimulated MRC-5 fetal lung fibroblasts produced detectable levels of IL-11 in the absence of exogenous stimulation. The levels of IL-11 produced by these cells were increased further by TGF-beta 1 and RSV (Fig. 7). Dexamethasone inhibited the basal levels of IL-11 production by unstimulated MRC-5 cells (data not shown). Dexamethasone was also a potent dose-dependent inhibitor of TGF-beta 1- and RSV-stimulated IL-11 mRNA accumulation and protein production by MRC-5 cells (Figs. 6 and 7). This inhibition was comparable in potency to the inhibition noted in A549 cells. Thus the IL-11 inhibitory effects of dexamethasone are not specific for TGF-beta 1 or A549 alveolar epithelial-like cells.


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  		Fig. 7.   Dexamethasone regulation of TGF-beta 1-stimulated and RSV-stimulated IL-11 production by MRC-5 lung fibroblasts. MRC-5 fibroblasts were grown to confluence and then were incubated with TGF-beta 1 (10 ng/ml; bullet  and ; A) or RSV (MOI = 3; bullet  and ; B). These incubations were performed in the presence ( and ) and absence (open circle  and bullet ) of dexamethasone (10-6 M). Noted values represent the means of at least 3 determinations. SE was consistently <= 10% of the noted values.

Dexamethasone Regulation of IL-11 Gene Transcription

Nuclear run-on assays were used next to characterize the effects of glucocorticoids on IL-11 gene transcription. IL-11 gene transcription was barely detectable or undetectable in nuclei from unstimulated A549 cells (data not shown). In contrast, IL-11 gene transcription was readily appreciated in nuclei from TGF-beta 1 (10 ng/ml)-stimulated cells (Fig. 8). Dexamethasone (10-6 M) alone did not augment IL-11 gene transcription. Dexamethasone did, however, inhibit TGF-beta 1-induced IL-11 gene transcription. Comparable alterations in the rate of transcription of the genes encoding 28S rRNA and glyceraldehyde-3-phosphate dehydrogenase were not noted (Fig. 8 and data not shown). Interestingly, high concentrations of dexamethasone (10-6 M) caused only a partial (<= 40%) decrease in TGF-beta 1-stimulated IL-11 gene transcription. These same concentrations of dexamethasone caused a 90-95% decrease in IL-11 protein production and mRNA accumulation. Thus dexamethasone inhibition of TGF-beta 1-stimulated IL-11 can only be partially accounted for by effects of dexamethasone on IL-11 gene transcription.


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  		Fig. 8.   Nuclear run-on assay characterizing the effect of dexamethasone on TGF-beta 1-stimulated IL-11 gene transcription. A549 cells were grown to confluence and stimulated for 16 h with TGF-beta 1 (10 ng/ml) in the presence and absence of dexamethasone (10-6 M). Nuclei were then harvested, and nuclear run-on analysis was performed as described in EXPERIMENTAL PROCEDURES. Rates of IL-11 and 28S gene transcription are illustrated. Levels of nonspecific binding are illustrated using the pUC18 construct that does not have a cDNA insert.

Dexamethasone Regulation of IL-11 mRNA Stability

To further define the mechanism(s) of dexamethasone inhibition, we compared the rates of degradation of IL-11 mRNA transcripts in cells stimulated with TGF-beta 1 (10 ng/ml) and TGF-beta 1 plus dexamethasone (10-7 M). The half-life of the IL-11 mRNA in cells stimulated for 24 h with TGF-beta 1 was ~3 h (Fig. 9). Dexamethasone destabilized this mRNA, decreasing the half-life of IL-11 mRNA to <2 h (Fig. 9). When viewed in conjunction with the nuclear run-on findings, the data demonstrate that dexamethasone inhibits IL-11 production by simultaneously decreasing IL-11 gene transcription and destabilizing IL-11 mRNA transcripts.


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  		Fig. 9.   Effect of dexamethasone on the half-life of IL-11 mRNA. Confluent A549 cells were incubated for 24 h with TGF-beta 1 (10 ng/ml) in the presence and absence of dexamethasone (10-7 M). At the end of this incubation (time = 0), the levels of IL-11 mRNA in cells stimulated with TGF-beta 1 in the presence and absence of dexamethasone were assessed, and actinomycin D was added to the remaining cultures. Levels of mRNA in the remaining cultures were evaluated 1, 2, and 3 h after actinomycin D addition. A: levels of IL-11 mRNA. B: ethidium bromide loading controls. C: densitometric evaluations of 3 experiments. Levels of IL-11 mRNA after actinomycin D addition are expressed as a percentage of those at time 0. Values represent the means of 3 evaluations (n). SE values were <= 10% of the noted values.

Dexamethasone Regulation of IL-11 Promoter Activity

Previous studies from our laboratory demonstrated that TGF-beta 1 stimulates IL-11 gene transcription in A549 cells via a mechanism that is dependent on cis elements between -100 and -82 in the IL-11 promoter (40). To further understand the mechanism by which dexamethasone regulates IL-11 transcription, studies were undertaken to determine if dexamethasone could regulate the activity of IL-11 promoter fragments containing this important response region. In these experiments, IL-11 promoter-reporter gene constructs were transfected into A549 cells and incubated in the presence and absence of TGF-beta 1 (10 ng/ml) and/or dexamethasone (10-6 M) for 24 h. The luciferase activity in unstimulated A549 cells was near or below the lower limits of detection in our assay. As previously described (40), TGF-beta 1 was a potent stimulator of this IL-11 promoter-reporter gene construct (Fig. 10). Dexamethasone did not stimulate IL-11 promoter activity in unstimulated cells. It did, however, inhibit, in a dose-dependent fashion, the IL-11 promoter-driven luciferase activity in TGF-beta 1-stimulated cells (Fig. 10). This inhibition was glucocorticoid receptor dependent because it was reversed by RU-486 (Fig. 10). These studies demonstrate that dexamethasone is a potent, dose-dependent inhibitor of IL-11 promoter activity in A549 cells.


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  		Fig. 10.   Dexamethasone regulation of IL-11 promoter-driven luciferase activity. A549 cells were grown to confluence, transfected with IL-11 promoter-luciferase constructs, and incubated for 24 h in the presence and absence of TGF-beta 1 (10 ng/ml), dexamethasone, and/or RU-486 as noted. At the end of the incubation period, luciferase activity was assessed as described in EXPERIMENTAL PROCEDURES. RLU, relative light units.

Dexamethasone Regulation of AP-1 in A549 Cells

Our previous studies demonstrated that TGF-beta 1 stimulates IL-11 gene transcription in A549 cells via an AP-1-dependent mechanism characterized by enhanced AP-1 transcription factor binding to response elements between -100 and -82 bp in the IL-11 promoter (40). Thus studies were undertaken to determine whether dexamethasone altered this TGF-beta 1-induced AP-1 response. This was done by performing EMSA using nuclear extracts from A549 cells that were unstimulated or stimulated with TGF-beta 1 in the presence and absence of dexamethasone. As previously described (40), nuclei from unstimulated A549 cells contained protein moieties that bound to the major AP-1 site in the IL-11 promoter, and TGF-beta 1 caused in a further increase in this AP-1-DNA binding. This inductive response was appreciated after 12-24 h of TGF-beta 1-A549 cell incubation (Fig. 11). EMSA also demonstrated that it was AP-1-specific because unlabeled AP-1, but not NF-kappa B oligonucleotides, competed for trans factor binding (data not shown). Interestingly, dexamethasone did not alter the levels of baseline or TGF-beta 1-stimulated AP-1-DNA binding in a significant fashion (Fig. 11).


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  		Fig. 11.   Effect of dexamethasone on TGF-beta 1-stimulated activator protein (AP)-1-DNA binding. Confluent A549 cells were incubated with TGF-beta 1 (10 ng/ml) in the presence and absence of dexamethasone (10-6 M) for the noted periods of time, and nuclear extracts were prepared. Electrophoretic mobility shift assays were then performed as described in EXPERIMENTAL PROCEDURES using an oligonucleotide encoding the major IL-11 AP-1 response sequences.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Glucocorticoids are extremely important therapeutic agents that are extensively utilized to treat inflammatory and fibrotic pulmonary and nonpulmonary disorders. In accord with this widespread clinical use, the mechanisms of glucocorticoid action have been analyzed extensively. Studies in the 1950s and 1970s suggested that steroids mediated their effects via their ability to stabilize lysosomal membranes and inhibit arachidonic acid metabolism, respectively (reviewed in Ref. 5). Later studies focused on the ability of steroids to inhibit the production of proinflammatory cytokines and suggested that this inhibition is the major anti-inflammatory mechanism of steroid action (5). Most recently, it has become clear that the effects of steroids are cytokine specific. Although a wide variety of inflammatory cytokines are inhibited by glucocorticoids, a variety of others are either not inhibited or augmented in a modest fashion (5, 23). To further define the biological profile of corticosteroids, we characterized the effects of glucocorticoids on human lung stromal cell IL-11 production. Our studies demonstrate that glucocorticoids inhibit TGF-beta 1- and virus-stimulated IL-11 production by lung fibroblasts and epithelial cells. Importantly, they also demonstrate that the doses of dexamethasone that mediate these effects are in the range of the concentrations used clinically and approximate the concentrations that are present under physiological circumstances.

Studies of the cellular and molecular mechanisms of glucocorticoid action have demonstrated that glucocorticoids can mediate their effects via their ability to alter the transcription of target genes and/or their ability to alter the posttranscriptional processing and degradation of target gene mRNA (5, 47). Our studies demonstrate that dexamethasone inhibits IL-11 protein production and that this inhibition is associated with a comparable decrease in IL-11 mRNA accumulation. This indicates that this inhibition is mediated, to a great extent, via a pretranslational mechanism. Nuclear run-on assays demonstrated that this decrease in IL-11 mRNA accumulation was due, in part, to glucocorticoid inhibition of IL-11 gene transcription. However, the magnitude of transcriptional inhibition could not fully explain the observed decrease in steady-state mRNA. This apparent discrepancy was explained, at least in part, by our finding that glucocorticoids also destabilize IL-11 mRNA. Thus steroids inhibit IL-11 production via a complex mechanism(s) that involves the combined action of transcriptional and posttranscriptional inhibitory processes. These findings are in accord with previous studies from our laboratory demonstrating that transcriptional and posttranscriptional regulatory events mediate the inhibitory effects of glucocorticoids on fibroblast IL-6 production (47) and studies from other laboratories showing similar patterns of corticosteroid regulation of interferon-gamma , IL-2, and granulocyte-macrophage colony-stimulating factor (5, 28, 41).

Glucocorticoids are believed to inhibit gene transcription via a variety of mechanisms. At least four mechanisms have been proposed. They include 1) the direct interaction of the ligand-bound glucocorticoid receptor with a cis-acting "negative glucocorticoid response element" in the regulatory region of the gene. This mechanism, although unusual, may mediate the effects of glucocorticoids on prolactin and pro-opiomelanocortin (6, 34); 2) the binding of the ligand-bound glucocorticoid receptor to positive-acting cis elements in the basal promoter and enhancer sequences, thereby blocking stimulatory trans-acting factors. This may be the mechanism by which glucocorticoids inhibit osteocalcin transcription and IL-6 promoter reporter gene activation in HeLa epithelial cell lines (29, 38); 3) the binding to and direct inactivation of transcription factors such as AP-1 and NF-kappa B (19, 30); and 4) the induction of inhibitory proteins such as the inhibitor of NF-kappa B, which sequesters NF-kappa B transcription factors in an inactive form in cellular cytoplasm (2, 35). Previous studies from our laboratory demonstrated that TGF-beta 1 stimulation of stromal cell IL-11 production is mediated, to a great extent, at the level of gene transcription (14, 40). More recent studies have demonstrated that this stimulation is AP-1 dependent and is associated with enhanced AP-1-DNA binding activity in these cells (40). To further understand the mechanism by which glucocorticoids regulate TGF-beta 1-stimulated IL-11 gene transcription, studies were undertaken to determine if glucocorticoids could regulate the expression of an IL-11 promoter-reporter gene construct and alter TGF-beta 1-stimulated AP-1-DNA binding. These studies demonstrated that a promoter construct that contains the important AP-1 sites in the IL-11 promoter was inhibited in a potent dose-dependent fashion by dexamethasone. In accord with our previous findings, TGF-beta 1 was an effective inducer of AP-1-DNA binding in A549 cell nuclei. Glucocorticoids did not, however, inhibit this induction. These observations suggest that glucocorticoid inhibition of TGF-beta 1-stimulated IL-11 gene transcription is not mediated via the ability of the ligand-bound glucocorticoid receptor to quantitatively alter AP-1-DNA binding. All in all, they raise the possibility that these inhibitory effects are mediated via an AP-1-independent mechanism. The recent demonstration of a functional NF-kappa B site in the IL-11 promoter (4) supports this contention and raises the possibility that glucocorticoid regulation of NF-kappa B activity may play a role in this response. Our conclusions regarding AP-1 must be viewed with caution, however. It is well known that very complex alterations in AP-1 transcription factor subunit composition can be seen in the setting of TGF-beta 1 stimulation (40). Thus the present studies do not rule out the possibility that corticosteroids inhibit IL-11 transcription by altering AP-1 subunit composition. Further studies will be required to determine with certainty whether AP-1 is involved in the inhibition of IL-11 induced by dexamethasone.

Gene expression can be regulated, both positively and negatively, by alterations in the stability of mRNA transcripts. Previous studies from our laboratory demonstrated that IL-1 and TNF interact to selectively stabilize IL-6 mRNA transcripts (10). Conversely, IL-4 has been shown to destabilize cytokine mRNAs (16). In the present study, we show that glucocorticoids inhibit IL-11 production, in part, by destabilizing its mRNA. This finding is in accord with previous studies by Yang and Yang (43) demonstrating that heparin also inhibits IL-11 protein production and gene expression via a similar posttranscriptional mechanism. The mechanism by which the mRNA transcripts are destabilized is poorly understood. It has been proposed, however, that cytokine mRNA degradation and destabilization are mediated, in part, by AUUUA motifs (37), which have recently been redefined as UUAUUUAUU motifs (21, 48). These AU-rich sequences are present in the 3'- untranslated region of IL-11. Proteins that interact with these sequences in labile mRNAs, such as the AUUA-binding factors (8, 25) or factors that bind to other sequences in the 3'-untranslated region (1), are felt to mediate this destabilization. Additional investigation will be required to define the cis elements in the 3'-untranslated region of IL-11 that mediate the effects of corticosteroids and the trans-activating factors that bind to these locations.

Our studies of the biological effects of steroids were prompted by a desire to understand the ramifications of steroids when they are administered in the setting of tissue inflammation and ongoing repair. The anti-inflammatory effects that result from steroid inhibition of cytokines such as IL-1, IL-2, and TNF can be understood easily. The consequences of steroid inhibition of IL-11 production are, however, more complex. Studies from our laboratory and others have demonstrated that IL-11 can activate B and T cells (44) and can cause mononuclear cells to accumulate around small bronchioles in the murine lung (39). In contrast, IL-11 also appears to have immunosuppressant and protective effects. These can be easily appreciated in studies from our laboratory and others that demonstrated that IL-11 can inhibit macrophage production of IL-1, TNF, and IL-12 (22, 32, 42) and exert protective effects in the setting of thoracic irradiation (32) and colonic mucosal injury (20). Thus the effect that steroid inhibition of IL-11 would have at the tissue level would depend on whether the pro- or anti-inflammatory effects of the cytokine predominated at that time and at that location. Should steroids inhibit the production of IL-11 that is playing a protective role, adverse consequences of this inhibition might be noted.

In summary, these studies demonstrate that dexamethasone is a potent dose-dependent inhibitor of TGF-beta 1-stimulated IL-11 production by a variety of stromal cells. They also demonstrate that this inhibition is mediated via a complex mechanism that involves the inhibition of gene transcription and destabilization of IL-11 mRNA. Last, these studies demonstrate that dexamethasone inhibition of IL-11 gene transcription is not associated with a decrease in TGF-beta 1-induced AP-1-DNA binding, raising the possibility that this inhibition is mediated via an AP-1-independent mechanism.

    ACKNOWLEDGEMENTS

We thank the investigators and institutions that provided the reagents that were employed and Kathleen Bertier for excellent secretarial and administrative assistance.

    FOOTNOTES

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: J. A. Elias, Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St., 105 LCI, New Haven, CT 06520-8057.

Received 5 June 1998; accepted in final form 14 October 1998.

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Discussion
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Am J Physiol Lung Cell Mol Physiol 276(1):L175-L185
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