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Published ahead of print on January 18, 2008, doi:10.1165/rcmb.2007-0337OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 707-714, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0337OC

Differential Regulation of Eotaxin Expression by Dexamethasone in Normal Human Lung Fibroblasts

Tomoko Suzuki1, Hirokazu Arakawa1, Takahisa Mizuno1, Kazuhiro Muramatsu1, Hiromi Tadaki1, Takumi Takizawa1, Hiroyuki Mochizuki1, Kenichi Tokuyama2, Satoshi Matsukura3 and Akihiro Morikawa1

1 Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, Gunma, Japan; 2 Department of Pharmacy, Takasaki University of Health and Welfare, Gunma, Japan; and 3 Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Hirokazu Arakawa, M.D., Ph.D., Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: harakawa{at}showa.gunma-u.ac.jp


   Abstract
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 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lung fibroblasts are a major source of several cytokines including CC chemokine eotaxin. We aimed to study the regulation of eotaxin-1/CCL11 production by dexamethasone and analyze its molecular mechanisms in human lung fibroblasts. Normal human lung fibroblast cells were exposed to IL-4 (40 ng/ml) and/or dexamethasone (10–6–10–9 M), and eotaxin mRNA expression and production was evaluated. Mechanisms of transcriptional regulation were assessed by Western blotting and dual luciferase assay for eotaxin promoter. The effects of dexamethasone on suppressor of cytokine signaling (SOCS)-1 and eotaxin mRNA expression in the cells transfected with expression vector (pAcGFP1-C1) or short interfering RNA (siRNA) for SOCS-1 were also investigated. Within 24 hours, dexamethasone inhibited IL-4–induced eotaxin mRNA expression and protein production, while eotaxin production was markedly increased at 48 and 72 hours after coincubation with IL-4 and dexamethasone. IL-4–induced eotaxin promoter activity was inhibited by dexamethasone at 8 hours, but enhanced at 48 hours after coincubation. Dexamethasone suppressed SOCS-1 mRNA expression but enhanced IL-4–induced STAT6 phosphorylation at 36 to 48 hours after coincubation. Enhanced expression of eotaxin mRNA by dexamethasone 48 hours after coincubation was completely diminished in the cells transfected with either expression vector or siRNA for SOCS-1. These results indicated that dexamethasone, depending on the exposure duration, can either inhibit or enhance IL-4–induced expression and production of eotaxin in the lung fibroblasts. The mechanisms of later enhanced production may depend on the prolonged transcriptional activity of the eotaxin gene, in part due to inhibition of SOCS-1 expression.

Key Words: fibroblast • corticosteroid • eotaxin/CCL11 • SOCS • airway remodeling



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our findings showing a lesser antiinflammatory effect of glucocorticoids in fibroblasts may be relevant to the relatively-insensitive-to-steroid therapy for difficult-to-treat asthma with increased progression of airway remodeling.

 
Asthma is a chronic inflammatory disorder of the airways in which many cells, especially eosinophils, may play important roles through the release of various mediators (1, 2). Chronic inflammation may be associated with bronchial hyperresponsiveness, variable airflow limitation, and respiratory symptoms. A prominent pathophysiologic feature of asthma is airway remodeling, along with airway inflammation. A link between airway inflammation and airway remodeling in asthma has recently been proposed (1–3).

In the airways of subjects with asthma, there is usually extensive infiltration of the airway lumen and wall with eosinophils and lymphocytes accompanied by vasodilatation, microvascular leakage, and epithelial disruption (1, 2). Eosinophil recruitment at the airway tissue is a complex mechanism. Chemokines involved in the migration and activation of blood eosinophils such as eotaxin may be produced by several types of cells, including airway fibroblasts, that have the potential to synthesize and release a variety of proinflammatory and profibrotic cytokines (4–7).

Eotaxin/CCL11, a CC chemokine with potent direct chemoattractant effects on eosinophils, is known to be regulated by Th2 cytokines, such as IL-4 and IL-13 (6, 8, 9). Eotaxin also regulates migration of mast cell progenitors into inflamed tissue and mast cell activation, and is likely to play an indirect role in airway remodeling through recruitment of eosinophils and mast cells, which have profibrogenic activity (2, 3, 7). It has been recently demonstrated that eotaxin has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin could participate in airway remodeling in asthma (7).

Glucocorticoids are a first-line therapy to control airway inflammation and to improve both bronchial hyperresponsiveness and hyperreactivity in patients with asthma (2). There are, however, conflicting results; showing that in regard to fibroblast function, glucocorticoids may either reduce or increase fibroblast proliferation that may be related to airway remodeling (10). It is unclear whether glucocorticoids either reduce or increase eotaxin production in lung fibroblasts, although they repressed the expression of eotaxin protein and mRNA induced by TNF-{alpha} and IL-4 in airway epithelial cells (11, 12). In the present study, we investigate the regulation of eotaxin expression by dexamethasone and analyze its molecular mechanisms in human lung fibroblasts.


   MATERIALS AND METHODS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture and Stimulation of the Cells
Normal Human Lung Fibroblasts (NHLF) (Clonetics, San Diego, CA) were cultured at 37°C with 5% CO2 in fibroblast cell basal medium (Clonetics) supplemented with fibroblast growth medium-2 (FGM-2 Single Quots; Clonetics), 1.0 µg/L human Fibroblast Growth Factor-Basic (rhFGF-B), 5.0 mg/l insulin, 2% fetal bovine serum (FBS), 30 mg/ml gentamicin, and 15 µg/ml amphotericin. NHLF cells were seeded into 12-well plates for enzyme-linked immunosorbent assay (ELISA) and luciferase assay, and 6-cm dishes for Western blot and mRNA analysis. Cells were allowed to grow to 70% confluence. In one type of experiment, cells were exposed to IL-4 (40 ng/ml) (R&D Systems, Minneapolis, MN) or dexamethasone (DEX, 10–6–10–9 M) (Sigma-Aldrich Co., St. Louis, MO) alone, or a combination of both. In other experiments, cells were treated with tumor necrosis factor (TNF)-{alpha} (40 ng/ml) (R&D Systems) and DEX.

Assay of Eotaxin Protein Release into the Culture Medium
Concentrations of eotaxin in the collected culture medium were determined with a commercial system for ELISA (R&D Systems) in accord with the manufacturer's instructions. The limit of detection in the assay of eotaxin was 5 pg/ml.

Real-Time Quantitative PCR Analysis
Expressions of eotaxin, IL-4R{alpha}, suppressor of cytokine signaling (SOCS)-1, and SOCS-3 mRNA in fibroblasts were determined by reverse transcription (RT), followed by real-time quantitative PCR. Total RNA was extracted from cells after incubation with or without indicated cytokines using Isogen reagent (Nippon Gene, Tokyo, Japan). Reverse transcription was performed using 1 µg of total RNA and oligo (dT) primers in a 20-µl reaction in accord with the manufacturer's protocol (Applied Biosystems, Branchburg, NJ). The sequences of the specific primer sets that were used in the real-time PCR analysis are displayed in Table 1, as previously described (13–15).


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  		TABLE 1. PRIMERS USED FOR QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ANALYSIS OF GENE EXPRESSION

 
Real-time PCR was performed on an ABI Prism 7900HT sequence detection system (PE Applied Biosystems, Foster City, CA) using SYBR green (Applied Biosystems, Warrington, UK) as a dsDNA-specific binding dye. Reactions for eotaxin and β-actin were cycled 40 times after the initial denaturation of 95°C, 10 minutes followed by 95°C, 15 seconds. The annealing and extension temperature was 60°C, 1 minute, except for IL-4R{alpha}, where it was 58°C, 1 minute. The threshold cycle (CT) was recorded for each sample to reflect mRNA expression levels. A validation experiment proved the linear dependence of the CT value for both eotaxin and β-actin concentrations and the consistency of {Delta}CT (eotaxin average CT minus β-actin average CT) in a given sample at different RNA concentrations. {Delta}CT was therefore used to reflect relative eotaxin mRNA expression levels. To determine the effects of different stimuli on eotaxin gene expression as compared with nonstimulated cells, {Delta}{Delta}CT was calculated ({Delta}{Delta}CT = {Delta}CT stimulus – {Delta}CT nonstimulated cells). Eotaxin mRNA was indexed to β-actin using the formula 1/(2{Delta}CT) x 100%. 2{Delta}{Delta}CT was calculated to demonstrate the fold change of eotaxin gene expression in stimulated cells as compared with nonstimulated ones.

Expressions of IL-4R{alpha}, SOCS-1, and SOCS-3 mRNA in fibroblasts were determined in the same manner as eotaxin mRNA expression.

Western Blot Analysis
Cells stimulated with IL-4 were solubilized with NP-40 lysis buffer (0.5% NP-40, 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 3 mM p-amidinophenylmethanesulfonyl fluoride [Sigma, St. Louis, MO], 5 mg/ml aprotinin [Sigma], 2mM sodium orthovanadate [Sigma], 5 mM EDTA). Whole cell extracts were subjected to 7.5 to 12% Tris-glycine gel electrophoresis (XV Pantera Gel; DRC, Tokyo, Japan) and then transferred to Sequi-Blot polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked for 30 min with 5% skimmed milk in TBS-T (Tris-buffered saline with 0.05% Tween 20) before incubation with either rabbit anti-human STAT6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit anti-human phospho-STAT6 (Santa Cruz Biotechnology, Inc.) for 1 hour at room temperature. Membranes were then washed by TBS-T and incubated with anti-rabbit immunoglobulin antibody conjugated to horseradish peroxidase (Amersham, Buckinghamshire, UK) for 30 minutes. Enhanced chemiluminescence (ECL plus Western blot detection system; Amersham) substrate was added after further washing with TBS-T, and the membrane was then exposed to film.

Transient Transfection and Luciferase Assay
Eotaxin promoter-luciferase reporter plasmid, generously supplied by Prof. R. P. Schleimer (Division of Allergy-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL), is a 1,363-bp fragment of the promoter region of the eotaxin gene (site 1,363 to 1) (16). NHLF cells were seeded into 12-well plates and allowed to grow to 50 to 70% confluence. Cells were transfected with 0.75 µg of reporter plasmids and 10 ng of a control Renilla luciferase vector pRL-TK (Promega Corporation, Madison, WI) using 1.5 µl of Fugene 6 transfection reagent (Roche Diagnostics Co., Indianapolis, IN) and incubated for 12 hour in 1 ml medium. Eight or 48 hours after coincubation with or without dexamethasone (10–6–10–8 M) and IL-4 (40 ng/ml), cells were washed with Ca2+ and Mg2+-free phosphate-buffered saline (PBS), solubilized by incubation in 250 µl of lysis buffer for 20 minutes, transferred to microtubes, and then centrifuged to pellet cellular debris. The supernatants were measured for luciferase activity using a Dual-Luciferase Assay System (Promega Corporation). The firefly luciferase activity of the eotaxin promoter-reporter plasmid was normalized using Renilla luciferase activity.

Eotaxin and IL-4R{alpha} mRNA Stability
NHLF cells were treated for 36 hours with IL-4 (40 ng/ml) and DEX (10–6 M). Cells were subsequently harvested at time 0 (as control) or further treated with actinomycin D (ACD, 1 µg/ml) (Sigma) for each specified time to block further transcription of mRNA. Eotaxin mRNA expression was analyzed 12, 24, and 36 hours after ACD was added, and IL-4R{alpha} mRNA expression at 2, 4, 8, and 12 hours after ACD was added, as mentioned above.

Cloning of SOCS-1 Expression Vector and Transfection into NHLF Cells
A DNA fragment of the coding sequence of SOCS-1 was amplified by PCR using cDNA from IL-4–treated NHLF cells. The purified PCR product was digested with BglII and EcoRI and cloned into the pAcGFP1-C1 vector (Clontech Laboratories Inc., Shiga, Japan). The plasmid was analyzed by digestion with restriction enzymes and DNA sequencing. Plasmids for transfection were purified with HiSpeed Plasmid Maxi Kit (QIAGEN Sciences, Germantown, MD).

NHLF cells were seeded into 6-well plates and allowed to grow to 50% confluence. Cells were transfected with 4 µg of expression vector with 10 µl Lipofectamine 2000 (Promega) and grown in fibroblast cell basal medium containing FGM-2 Single Quots without antibiotics. After 24 hours, the medium of the cells was changed to fibroblast cell basal medium with antibiotics; 30 mg/ml gentamicin, and 15 µg/ml amphotericin. Then, cells were exposed to IL-4 (40 ng/ml) or DEX (10–6 M) alone, or a combination of both. Forty-eight hours after coincubation, eotaxin or SOCS-1 mRNA expression was evaluated.

Knockdown of Gene Expression with short interfering RNA
Pre-designed short interfering RNA (siRNA) for SOCS-1 (catalog #45060) was purchased from Ambion (Tokyo, Japan). NHLF cells were seeded into 6-well plates and allowed to grow to 50% confluence. Cells were transfected with 16.5 nM of siRNA with 5 µl Lipofectamine 2000. Then, the same procedures described for the knockdown of gene expression were performed. Scrambled siRNA was used as non-specific negative control of siRNA (Ambion).

Statistical Analysis
Data are expressed as means ± SD. Statistical differences were determined by ANOVA first before confirming significance with a paired Student's t test. Data were analyzed with Dr. SPSS II (SPSS Japan Inc., Tokyo, Japan). P values less than 0.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Eotaxin Protein Production and mRNA Expression
The minimum level of eotaxin protein was detected in the medium of unstimulated NHLF (Figure 1A). Coincubation with DEX (10–6 M) alone until 48 hours did not interfere with eotaxin production or mRNA expression in NHLF. DEX incubation for 72 hours slightly increased eotaxin mRNA expression and protein, although it did not reach significance for eotaxin mRNA expression (P = 0.056) (Figures 1A and 1B). At all of the time points monitored, stimulation with IL-4 (40 ng/ml) alone increased eotaxin production and mRNA expression. The combination of IL-4 (40 ng/ml) and DEX (10–6 M) partially inhibited eotaxin production and mRNA expression at 12 and 24 hours after stimulation, while at 48 and 72 hours it increased eotaxin production and mRNA expression compared with incubation with IL-4 alone. Coincubation of IL-4 (40 ng/ml) and DEX (10–6–10–9 M) for 24 hours slightly, but concentration-dependently, suppressed the expression of eotaxin mRNA. Coincubation of IL-4 and DEX for 72 hours markedly increased eotaxin mRNA in a concentration-dependent manner compared with IL-4 alone (Figure 1C).


Figure 1
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  		Figure 1. Effect of dexamethasone (DEX) and IL-4 on the production of eotaxin protein and mRNA in normal human lung fibroblast (NHLF) cells. (A) Cells were incubated with or without IL-4 (40 ng/ml) and/or DEX (10–6 M) for 24, 48, and 72 hours, and the concentration of eotaxin protein in the medium analyzed by enzyme-linked immunosorbent assay. Data are presented as the mean ± SD of two independent experiments. (B) Quantitative real-time PCR assessment of the fold changes in eotaxin mRNA at 12, 24, 48, and 72 hours after coincubation with IL-4 (40 ng/ml) and/or DEX (10–6 M) or the unstimulated values (control). Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). Data are presented as the mean ± SD of four to six independent experiments (*P < 0.05). (C) Concentration-dependent effect of dexamethasone on expression of eotaxin mRNA. Cells were coincubated with IL-4 (40 ng/ml) and DEX (10–6–10–9 M) for 24 and 72 hours. Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). Data are presented as the mean ± SD of four independent experiments (*P < 0.05, **P < 0.01).

 
Effect of DEX on IL-4R{alpha} mRNA Expression
We next investigated IL-4R{alpha} mRNA expression stimulated with IL-4 (40 ng/ml) and DEX (10–6 M) in NHLF. IL-4R{alpha} mRNA expression was not enhanced after 48 hours of coincubation with IL-4 and DEX (Figure 2A).


Figure 2
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  		Figure 2. IL-4R{alpha} mRNA analysis and representative Western blots. (A) Forty-eight hours after stimulation with or without IL-4 (40 ng/ml) and DEX (10–6 M), IL-4R{alpha} mRNA expression was determined by quantitative real-time PCR. Data are presented as the mean ± SD of four independent experiments. Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). (B) Representative STAT6 and p-STAT6 Western blots from a single culture stimulated with medium alone, DEX (10–5–10–7 M), IL-4 (40 ng/ml), or the combination for 36 hours. In the experiments, membranes were exposed to the film for 30 seconds. The results presented are from one of three experiments that produced similar results. (C) Phospho-STAT6 was expressed as a fold increase in relative intensity. Data are shown as the mean values ± SD of three independent experiments.

 
DEX Enhanced IL-4-Induced STAT6 Activation
STAT6 and phospho-STAT6 levels were investigated by Western blot analysis using specific monoclonal antibodies for them. The cell lysates 36 hours after stimulation with IL-4 contained substantial amounts of phospho-STAT6, as shown by the appearance of an intense band detected at 105 kD (Figure 2B, lane 3) that was not present in the cell lysates from unstimulated and DEX-treated cells (lanes 1 and 2). When cultured with IL-4, phospho-STAT6 generation was enhanced by DEX in a concentration-dependent manner (Figure 2C, lanes 4–6).

Transient Transfection and Luciferase Assay
After 8 hours, IL-4 alone enhanced induction of the eotaxin promoter, pEotx.1363 (Figure 3A). Coincubation with DEX and IL-4 inhibited induction of the eotaxin promoter, and this effect was significant and concentration-dependent. By contrast, after 48 hours, coincubation with DEX and IL-4 significantly enhanced the activities of the eotaxin promoter, pEotx.1363, compared with either stimulus alone (Figure 3B).


Figure 3
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  		Figure 3. Activation of an eotaxin promoter-luciferase reporter plasmid (pEotax.1363) by IL-4 and/or DEX in NHLF cells. IL-4 (40 ng/ml) and/or DEX (10–6–10–8 M) was added to the media, and after 8 hours (A) luciferase assay was performed. Forty-eight hours after coincubation with IL-4 (40 ng/ml) and/or DEX (10–6 M), luciferase assay was done (B). Data are presented as the mean ± SD of a total of four (A) and six (B) independent experiments (*P < 0.05). {dagger}P < 0.05 and {ddagger}P < 0.01 are compared with the value of control.

 
Effect of DEX on IL-4-Induced SOCS-1 and SOCS-3 mRNA Expression
At all of the time points monitored, the expression of SOCS-1 mRNA was significantly inhibited by DEX (10–6 M) alone. Stimulation with IL-4 (40 ng/ml) alone increased SOCS-1 mRNA expression. The combination of IL-4 and DEX significantly inhibited SOCS-1 mRNA expression compared with stimulation with IL-4 alone at all time points (Figure 4A). By contrast, IL-4 (40 ng/ml) alone did not increase SOCS-3 mRNA expression, and DEX (10–6 M) had no effect on IL-4–induced SOCS-3 mRNA expression at any of the time points (Figure 4B). Coincubation with IL-4 (40 ng/ml) and DEX (10–6–10–8 M) for 48 hours concentration-dependently suppressed the expression of SOCS-1 mRNA, with significant suppression found at a concentration of 10–6 M of DEX (Figure 4C).


Figure 4
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  		Figure 4. SOCS-1 and SOCS-3 mRNA analysis. (A) Quantitative real-time PCR assessment of the fold changes in SOCS-1 mRNA at 24, 48, and 72 hours after coincubation with IL-4 (40 ng/ml) and/or DEX (10–6 M) or the unstimulated values (control). Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). Data are presented as the mean ± SD of six independent experiments (*P < 0.05, **P < 0.01). (B) Quantitative real-time PCR assessment of the fold changes in SOCS-3 mRNA at 24, 48, and 72 hours after coincubation with IL-4 (40 ng/ml) and/or DEX (10–6 M) or the unstimulated values (control). Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). Data are presented as the mean ± SD of six independent experiments. (C) Concentration-dependent effects of DEX on the expression of SOCS-1 mRNA. Quantitative real-time PCR assessment of the fold changes in SOCS-1 mRNA 48 hours after coincubation with IL-4 (40 ng/ml) and/or DEX (10–6–10–8 M). Results are expressed as the relative quantity of SOCS-1 mRNA (= fold over control). Data are presented as the mean ± SD of four independent experiments (**P < 0.01).

 
Effects of DEX on IL-4-Induced Eotaxin and IL-4R{alpha} mRNA Stability
There was no difference in the stabilities of eotaxin mRNA and IL-4R{alpha} mRNA expressions between coincubation with IL-4 alone and combination with IL-4 and DEX (Figures 5A and 5B).


Figure 5
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  		Figure 5. Eotaxin and IL-4R{alpha} mRNA stability. After induction of eotaxin mRNA (A) and IL-4R{alpha} mRNA (B) by 36 hours of incubation with 40 ng/ml IL-4 and/or DEX (10–6 M), cells were treated with actinomycin D (1 µg/ml). After treatment with actinomycin D, eotaxin or IL-4R{alpha} mRNA expression was analyzed at the indicated time intervals by real-time PCR. Results are expressed as 100% of maximum (eotaxin or IL-4R{alpha} mRNA expression at time 0). Data are presented as the mean ± SD of four to six independent experiments.

 
TNF-{alpha}–Induced Eotaxin and SOCS-1 mRNA
TNF-{alpha} (40 ng/ml) increased the expression of eotaxin mRNA at 24 hours after stimulation, while the combination of TNF-{alpha} (40 ng/ml) and DEX (10–6 M) significantly inhibited eotaxin mRNA. Unlike IL-4, an enhancing effect was not observed at 48 hours with the combination of TNF-{alpha} and DEX (Figure 6A). The expression of SOCS-1 mRNA was significantly inhibited by DEX alone and the combination of TNF-{alpha} and DEX at 48 hours after stimulation, as was the case with IL-4 (Figure 6B).


Figure 6
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  		Figure 6. (A) Effect of DEX on TNF-{alpha}–induced eotaxin mRNA expression. Real-time PCR assessment of the fold changes in eotaxin mRNA at 24 and 48 hours after coincubation with TNF-{alpha} (40 ng/ml) and/or DEX (10–6 M) or the unstimulated values (control). Results are expressed as the relative quantity of eotaxin mRNA (= fold over control). Data are presented as the mean ± SD of four independent experiments (**P < 0.01). (B) Effect of DEX on suppressor of cytokine signaling (SOCS)-1 mRNA expression. Real-time PCR assessment of the fold changes in SOCS-1 mRNA 48 hours after coincubation with TNF-{alpha} (40 ng/ml) and/or DEX (10–6 M) or the unstimulated values (control). Results are expressed in relative quantity of SOCS-1 mRNA (= fold over control). Data are presented as the mean ± SD of eight independent experiments (**P < 0.01).

 
Effect of Expression Vector and siRNA for SOCS-1
Levels of SOCS-1 mRNA were significantly enhanced in the cells transfected with SOCS-1 expression plasmids cloned into pAcGFP1-C1 vector compared with wild-type cells, while reduced in the cells with siRNA for SOCS-1 (Figures 7A and 7C). Enhanced expression of eotaxin mRNA by dexamethasone (DEX, 10–6 M) at 48 hours after coincubation with IL-4 (40 ng/ml) in wild-type cells was completely diminished in the cells transfected with either expression vector or siRNA for SOCS-1 (Figures 7B and 7D).


Figure 7
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  		Figure 7. Effect of SOCS-1 expression plasmids cloned into pAcGFP1-C1 vector (A and B) and siRNA for SOCS-1 (C and D) on SOCS-1 mRNA and IL-4 (40 ng/ml) induced eotaxin mRNA enhanced by DEX (10–6 M) 48 hours after coincubation. Results are expressed in relative quantity of SOCS-1 mRNA (A and C). Eotaxin mRNA levels were expressed as 100% of value stimulated with IL-4 (B and D). Data are presented as the mean ± SD of four to six independent experiments (*P < 0.05, **P < 0.01). Levels of SOCS-1 mRNA were enhanced significantly in the cells transfected with SOCS-1 expression plasmids cloned into pAcGFP1-C1 vector (A), while reduced in the cells with siRNA for SOCS-1 (C). Enhanced expression of eotaxin mRNA by dexamethasone at 48 hours after coincubation in each wild-type cells was completely diminished in the cells transfected with either expression vector (SOCS-1) (B) or siRNA for SOCS-1 (D).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
A variety of cell types are responsible for eotaxin production, including macrophages, T lymphocytes, bronchial epithelial cells, and endothelial cells (1, 2, 6, 16). We found that eotaxin was produced by stimulation with IL-4 in normal human lung fibroblast (NHLF) cells, indicating that Th2 cytokines may regulate eotaxin production in lung fibroblasts. We also demonstrated here that glucocorticoid inhibited the eotaxin production induced by IL-4 at 24 hours after coincubation, which is in agreement with previous reports (8, 11). Interestingly, coincubation with IL-4 and DEX significantly enhanced eotaxin production at 48 and 72 hours after stimulation. Thus, DEX exhibited dual effects on IL-4–induced eotaxin production in NHLF. Such dual effects were also observed in other glucocorticoids, including hydrocortisone and prednisolone (data not shown).

Glucocorticoids suppress inflammatory genes by many different molecular mechanisms. Matsukura and coworkers (11) demonstrated that fluticasone inhibited eotaxin expression in airway epithelial cells (BEAS-2B) in part through repression of eotaxin transcription, and that this mechanism may not depend on the direct inhibition of transcription factors, such as NF-{kappa}B or STAT6. Lilly and colleagues (12) reported that TNF-{alpha} and IL-1β induced the accumulation of eotaxin mRNA with a maximum at 4 hours in the pulmonary epithelial cell lines A549 and BEAS-2B. In addition, DEX diminished TNF-{alpha}– and IL-1β–induced increases in eotaxin mRNA in a concentration-dependent manner. In the present study, we found that DEX repressed eotaxin production and mRNA expression 24 hours after stimulation with DEX in NHLF, as was the case with epithelial cells. In addition, DEX inhibited activation of the eotaxin promoter-luciferase reporter plasmid pEotx.1363 induced by IL-4, indicating that the transcriptional regulation may be related to the repression of eotaxin gene expression by glucocorticoids in lung fibroblasts.

Studies were performed to investigate the mechanisms of the observed enhancement in IL-4–induced eotaxin production and mRNA expression at 48 and 72 hours after incubation with DEX. One possible mechanism of synergy between IL-4 and DEX might be the up-regulation of IL-4 receptors or the activation of STAT-6 by DEX. Yamamoto and coworkers found that enhanced IL-4–induced eotaxin-3 production by IFN-{gamma} may be due to up-regulation of IL-4R{alpha} in airway epithelium (14). In the present study, we could not find mRNA expression of IL-4R{alpha} enhanced by coincubation of IL-4 and DEX. The effect of DEX on STAT6 phosphorylation by IL-4 was examined by Western blotting for the latent and activated forms of the STAT6 protein at 36 hours after coincubation with DEX and IL-4. IL-4–induced phosphorylation of STAT6 and p-STAT6 generation was enhanced by DEX in a concentration-dependent manner.

In the present study, eotaxin promoter activity was enhanced 48 hours after coincubation with DEX and IL-4, whereas it was inhibited at 8 hours, suggesting that the transcription of eotaxin gene exhibits dual effects similar to the response of eotaxin production and mRNA expression. DEX alone induced an increase in eotaxin promoter activity at 48 hours after stimulation, although it did not reach significance. This may explain the slight increase in eotaxin protein production and mRNA expression at 72 hours after stimulation with DEX alone. Thus, it implies that transcriptional regulation is involved in the enhanced expression of eotaxin by DEX, which was further confirmed by the effect of actinomycin D.

The suppressors of cytokine signaling (SOCS) represent a recently discovered family of proteins engaged in the negative regulation of cytokine signaling, primarily signaling associated with the Jak-STAT pathway (17). Sato and colleagues provided evidence that SOCS-1 can negatively regulate IL-4– and IL-13–induced eotaxin-1 expression (18). These findings are in agreement with a recent study demonstrating the regulation of eotaxin-3 by SOCS-1 and SOCS-3 but not SOCS-2 (17). To test whether SOCS proteins play an important role in the regulation of eotaxin expressions by DEX, we analyzed SOCS-1 and SOCS-3 mRNA expressions. We detected the induction of mRNA for SOCS-1, but not SOCS-3, from 24 to 72 hours of treatment of NHLF cells with IL-4. We found that SOCS-1, which serves to down-regulate cytokine signaling, was suppressed by DEX alone and in combination with IL-4 stimulation, suggesting that up-regulation of eotaxin was likely to be due to down-regulation of SOCS-1 by DEX. To confirm that the effects seen with increasing eotaxin are in fact due to changes in SOCS-1, we have performed some studies using overexpressing SOCS-1 or siRNA for SOCS-1 in NHLF cells then treating with DEX. We found that reversal or diminished SOCS-1 levels prevented up-regulation of eotaxin in cells transfected with expression vector or siRNA for SOCS-1 after DEX treatment. Our results may be partly supported by the findings of Paul and coworkers showing that glucocorticoids strongly inhibit both basal and IL-6–induced rat SOCS-3 mRNA synthesis in hepatocytes (19). They also found the negative regulation of SOCS-3 promoter by glucocorticoids caused by a glucocorticoid response element–independent pathway. Thus, taken together with these previous studies, our results suggest that SOCS-1 suppression by DEX may possibly be because of the negative regulation of SOCS-1 promoter activity. Furthermore, SOCS-1 suppression could enhance the level of phospho-STAT6 and up-regulate transcription, subsequently enhancing eotaxin production in response to IL-4.

To confirm that the enhancing effect of DEX was not caused by a generalized enhancement of cellular responses, we analyzed the effect of DEX on TNF-{alpha}–induced eotaxin production in NHLF cells. TNF-{alpha} induced a small but significant increase in eotaxin mRNA expression, and this response was not enhanced 48 hours after coincubation with TNF-{alpha} and DEX. Although DEX also suppressed SOCS-1 mRNA expression in this study, it could not induce the enhancing of eotaxin mRNA expression. Thus, taken together with the IL-4 stimulation study, suppression of SOCS-1 by DEX may have an important role for the enhancement of IL-4–induced eotaxin production.

Atasoy and colleagues, using an actinomycin D–based assessment, demonstrated that TNF-{alpha} and IL-4 significantly increase eotaxin mRNA stability (20). While in unstimulated cells eotaxin mRNA is short-lived (with a half-life of ~ 2 h), they showed treatment with either TNF-{alpha} or IL-4 induced up to a 3-fold extension of the eotaxin mRNA half-life. In the present study, DEX had no effect on IL-4–induced eotaxin mRNA expression. This suggested that eotaxin mRNA stability was unrelated; implying that post-transcriptional regulation such as eotaxin mRNA stability may be not involved in the enhanced expression of eotaxin by DEX.

Finally, since Kraft and coworkers (10) found that IL-4 and DEX significantly increased fibroblast proliferation in the biopsy specimens from subjects with mild asthma, we hypothesized that the numbers of NHLF coincubated with IL-4 and/or DEX increased compared with the numbers of untreated cells. However, we did not find any differences in the numbers of viable cells with or without DEX at 24 to 72 hours after coincubation (data not shown).

In conclusion, DEX induced dual effects on the expression and production of eotaxin in lung fibroblasts. The mechanisms of the later enhanced production may depend on the prolonged transcriptional activity of the eotaxin gene, in part due to inhibition of SOCS-1 expression. Our findings showing a lesser antiinflammatory effect of glucocorticoids in fibroblasts may be relevant to the relatively-insensitive-to-steroid therapy for difficult-to-treat asthma with increased progression of airway remodeling (21). Because glucocorticoids are recommended as a first-line therapy for asthma, further study regarding the molecular mechanisms of glucocorticoids and cytokine modulation of fibroblast function is required to determine whether the currently available therapies for asthma provide long-term benefits for patients.


   Acknowledgments
 
The authors thank Tomoko Endo and Chinori Iijima for their excellent technical assistance.


   Footnotes
 
This work was supported by a grant from Research on Eye and Ear Sciences, Immunology, Allergy and Organ Transplantation, Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0337OC on January 18, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 16, 2007

Accepted in final form December 29, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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