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Published ahead of print on August 5, 2004, doi:10.1165/rcmb.2004-0195OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 573-582, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0195OC

Interferon-{gamma} Inhibits STAT6 Signal Transduction and Gene Expression in Human Airway Epithelial Cells

Nicola M. Heller, Satoshi Matsukura, Steve N. Georas, Mark R. Boothby, Paul B. Rothman, Cristiana Stellato and Robert P. Schleimer

Division of Allergy and Clinical Immunology and Department of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland; First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan; Department of Microbiology and Immunology, Medical Center North, Vanderbilt University Medical School, Nashville, Tennessee; Department of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York; and Division of Allergy and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Address correspondence to: Nicola M. Heller, Division of Allergy and Clinical Immunology, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: nheller{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activating and inhibitory cytokine signals that act upon epithelial cells in the human lung are critically important for controlling the production of inflammatory mediators from those cells in the context of allergic disease. The cytokines interleukin (IL)-4 and IL-13, derived from T helper (Th)-2 cells and other cell types, are potent inducers of epithelial cell expression of a host of inflammatory molecules, including the chemokines eotaxin-1, -2 and -3. Intracellular signal transduction in response to IL-4/IL-13 occurs largely through activation of signal transducer and activator of transcription 6 (STAT6). Interferon (IFN)-{gamma}, a Th1-type cytokine, has opposing effects to IL-4/IL-13 in various cell types, including T cells, B-cells, endothelium, and epithelium. In this study, we demonstrate that IL-4–induced STAT6 activation was inhibited profoundly by 24 h pretreatment with IFN-{gamma} in human primary airway epithelial cell cultures. Using Western blotting, we showed that the levels of both cytoplasmic and nuclear-localized phospho-STAT6 were reduced by IFN-{gamma} pretreatment, and this effect was dependent on the concentration of IFN-{gamma} and time of exposure to IFN-{gamma}. The functional activity of STAT6 was also completely inhibited by IFN-{gamma}: IL-4–induced luciferase activity from a STAT6-driven reporter construct was suppressed, as was IL-4–induced expression of messenger RNA (mRNA) and protein for eotaxin-3, a STAT6-dependent gene implicated in allergic inflammation. We found that mRNA for suppressor of cytokine signaling (SOCS)–1 and (SOCS)–3, known inhibitors of IL-4 signaling, and IL-13 receptor {alpha}2, a potential inhibitor of IL-4 signaling, were both strongly induced by IFN-{gamma} pretreatment. IFN-{gamma} also increased the rate of decay of IL-4–induced eotaxin-3 mRNA. We conclude that there are multiple mechanisms by which IFN-{gamma} regulates IL-4– and STAT6-dependent signaling and gene expression in airway epithelial cells. These observations have important implications for the regulation of epithelial cell activation by the balance of Th1/Th2-type cytokines in the airways in allergic disease.

Abbreviations: bronchial epithelial growth medium, BEGM • CCAAT/enhancer binding protein, C/EBP • enzyme-linked immunosorbent assay, ELISA • concentration producing a half-maximum inhibition of effect, IC50 • interferon, IFN • interleukin, IL • IFN-{gamma}–inducible protein of 10 kD, IP-10 • Janus kinase, JAK • phosphate-buffered saline, PBS • PBS with Tween, PBST • polymerase chain reaction, PCR • receptor {alpha} chain, R{alpha} • sodium dodecyl sulfate, SDS • suppressor of cytokine signaling, SOCS • signal transducer and activator of transcription, STAT • T-helper, Th • untranslated region, UTR • wild-type STAT6 expression vector, WT-STAT6


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin (IL)-4 and IL-13 are central cytokine mediators of allergic airways disease that are produced by activated Th2 cells, eosinophils, basophils, and mast cells in the lung as part of the immune response to allergen. These cytokines are important in eliciting many of the pathophysiologic features of the allergic inflammatory response in structural cells, such as endothelial, epithelial, smooth muscle, and nerve cells within the lung. The airway epithelium undergoes particularly dramatic structural and secretory changes in response to IL-4/IL-13, including a characteristic structural derangement and downregulation of tight-junction proteins, leading to diminished epithelial barrier capacity (1). These cytokines also cause the mucus-producing goblet cells to increase in sensitized and challenged animal models of allergic disease (2, 3). The hyperproliferation of goblet cells and increased expression of the mucins, such as MUC5AC (4), are likely to contribute to the extensive mucus plugging of the airway lumen observed in asthma (5). Microarray studies have shown that epithelial cells also upregulate a variety of intracellular and surface molecules in response to STAT6 activation by IL-4 and IL-13 stimulation, including transforming growth factor-ß, IL-8, granulocyte macrophage–colony stimulating factor (GM-CSF), IL-13 receptor {alpha}2 (IL-13R{alpha}2) (6), and CC-chemokine receptor (CCR) 3 (7). Epithelial cells stimulated with IL-4/IL-13 secrete large quantities of eosinophil and basophil chemoattractants, such as eotaxin-1 (CCL11) (8, 9), eotaxin-2 (CCL24) (10, 11), eotaxin-3 (CCL26) (12), monocyte chemoattractant protein-4 (CCL13) (13, 14), and thymus- and activation-regulated chemokine (CCL17) (15). Activation of epithelial cells by IL-4/IL-13 to release these chemokines may comprise part of a feed-forward inflammatory cycle, as it can induce the influx of IL-4/IL-13–producing cells, such as eosinophils, basophils, and Th2 cells.

Although cellular responses to IL-4 and to IL-13 are not identical, signal transduction in response to both cytokines is mediated through the activation of the transcription factor, signal transducer and activator of transcription (STAT) 6. The type I IL-4R is composed of the IL-4R{alpha} chain, which binds IL-4, and {gamma}c, a receptor chain that is shared among many other cytokine receptors. The type II receptor is made up of the IL-4R{alpha} chain and the IL-13R{alpha}1 chain, which can bind IL-13; therefore, the type II IL-4R is also the cell surface receptor for IL-13. Human airway epithelial cells express all three receptor components (16–18). Upon IL-4/IL-13 binding to the receptor complex, STAT6 becomes phosphorylated within minutes (19–21) by receptor-associated Janus kinases (JAKs). The phosphorylated transcription factor then homodimerizes and translocates to the nucleus, where it initiates gene transcription via specific DNA consensus motifs (22). The central importance of STAT6 in allergic airways disease has been highlighted through numerous studies in humans and genetically-manipulated mice. Sensitized STAT6 knockout mice have a significant reduction or absence of the characteristic markers of airway inflammation, such as eosinophilia, airways hyperresponsiveness, goblet cell hyperplasia, mucus secretion, and chemokine production after antigen challenge when compared with their wild-type littermates (23). The importance of activation of airway epithelium by IL-4 through the STAT6 signal transduction pathway was shown by studies using knockout mice in which selective expression of IL-4R{alpha} or STAT6 was reinstated in epithelial cells alone (24, 25): allergic inflammatory responses, such as airways hyperresponsiveness, mucus hyperplasia, and expression of several genes associated with allergic models of asthma were restored. Interestingly, inflammatory cell infiltration and subepithelial fibrosis were not, which suggested that epithelial STAT6 expression alone was not sufficient to reconstitute IL-13–induced inflammation or fibrosis.

Th1-type cytokines, such as interferon (IFN)-{gamma}, IL-12, and IL-10 are known to antagonize the activating effects of IL-4 and IL-13 in a variety of cell types (reviewed in Ref. 26) and Th1-type cytokines can inhibit or even reverse allergic inflammation (27–29). IFN-{gamma} is known to inhibit many respiratory epithelial cell responses to IL-4 important in allergic disease, such as the production of transforming growth factor-ß (30) and 15-lipoxygenase (31). Thus, the balance between IL-4 and IFN-{gamma} appears to determine whether various mediators of allergic disease are induced or inhibited in airway epithelium. There is no information on the mechanism by which IFN-{gamma} inhibits these IL-4–induced responses in airway epithelial cells. Based upon studies in other cell types (32), it is possible that IFN-{gamma} inhibits the IL-4–initiated STAT6 signal transduction pathway in epithelial cells. Because the airway epithelium is a major source of IL-4–induced chemokines and other mediators of allergic disease, and STAT6 is a central transcriptional activator of allergic inflammation, we sought to determine whether IL-4–induced STAT6 activation and function were inhibited by IFN-{gamma} in airway epithelial cells. Such an effect could provide a possible mechanism by which many IL-4–elicited epithelial responses are inhibited by IFN-{gamma}.

Our results show that IFN-{gamma} decreased dramatically the amount of IL-4–induced phospho-STAT6 in the nucleus of airway epithelial cells in a concentration and time-dependent fashion. Analysis of STAT6 and phospho-STAT6 levels in all cellular compartments revealed that this was not due to an IFN-{gamma}–mediated decrease in overall cellular STAT6 levels. This IFN-{gamma}–mediated reduction in nuclear phospho-STAT6 corresponded to a striking decrease in STAT6-dependent luciferase activity from a STAT6-driven reporter gene construct, as well as a profound inhibition of the expression of eotaxin-3, a STAT6-dependent chemokine gene. Inhibition of nuclear phospho-STAT6 by IFN-{gamma} was associated with the induction of messenger RNA (mRNA) for both suppressor of cytokine signaling-1 (SOCS-1) and IL-13R{alpha}2, potential inhibitors of IL-4–induced STAT6 activity. Reduction of eotaxin-3 expression by IFN-{gamma} was due, in part, to the increased rate of decay of eotaxin-3 mRNA. We present data suggesting that IFN-{gamma} employs multiple mechanisms to act as an important negative regulator of IL-4–induced STAT6 signaling and gene expression in human airway epithelium. These findings are relevant to airway inflammation, insofar as the balance between IL-4 and IFN-{gamma} in the airways can dictate the activation state of the respiratory epithelium and the nature of the inflammatory mediators produced.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Primary human bronchial epithelial cells were harvested from human lungs that had been rejected for transplantation purposes (obtained from NDRI, Philadelphia, PA) and were seeded in bronchial epithelial growth medium (BEGM; Clonetics, Walkersville, MD) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.2% fungizone (GIBCO BRL, Grand Island, NY) and 1x nystatin (Sigma, St. Louis, MO) in collagen-coated tissue culture flasks. After three feedings, the medium was changed to BEGM without additional antibiotics and 24 h before IFN-{gamma} treatment, the medium was changed to BEGM lacking hydrocortisone. Normal human bronchial epithelial cells were purchased from Clonetics and cultured according to the supplier's instructions. Primary cells from both sources were grown at 37°C in 5% CO2 humidified air.

Cytokine Stimulation of Cultured Cells
Cells were used for experiments upon reaching 70% confluency. Cells were treated for 24 h with recombinant human IFN-{gamma} (R&D Systems, Minneapolis, MN) diluted from a 100 mg/ml stock solution stored in phosphate-buffered saline (PBS)/0.1% bovine serum albumin at –70°C. For analysis of phospho-STAT6 protein levels, cells were stimulated with 0.2 ng/ml recombinant human IL-4 (R&D Systems) for 30 min, then harvested by trypsinization, washed with Hanks buffered saline solution, and collected by centrifugation at 1,200 rpm. For all the transfection studies, cells were stimulated for 6 h with IL-4 and then harvested for measurement of luciferase activity. For analysis of eotaxin-3 mRNA and protein levels, cells were stimulated for 24 h (or more as noted) with 100 ng/ml IL-4, supernatants were removed for eotaxin-3 enzyme-linked immunosorbent assay (ELISA), and the cells were then washed as described for the phospho-STAT6 protein analysis prior to isolation of RNA.

Transfection with the STAT6-Driven Luciferase Reporter Construct and STAT6 Expression Vector
The STAT6-driven luciferase reporter construct designated mut-(CCAAT/enhancer binding protein [C/EBP])-N4-luc ("—N4" [TPU475], generously provided by Tularik, Inc., South San Francisco, CA) was derived from a composite C/EBP-STAT6 site in the human germ line immunoglobulin {epsilon} promoter (33), in which the C/EBP sites have been mutated. We therefore refer to this plasmid as mut-N4-luc. The wild-type STAT6 (WT-STAT6) expression vector (described in Ref. 33), also from Tularik, Inc, and the empty expression vector, pcDNA3 (Invitrogen, Carlsbad, CA), were used for cotransfection and as a control, respectively.

Normal human bronchial epithelial or primary cells were plated at 50% confluency (400,000 cells/well) into 6-well plates in BEGM and allowed to adhere. Twenty four hours later, cells were transfected in triplicate wells for each treatment condition, as previously described (34), using 0.95 µg luciferase reporter plasmid with or without 0.05 µg WT-STAT6 expression vector or empty pcDNA3 vector. A total of 150 µl luciferase cell lysis buffer (Pharmingen, San Diego, CA) was used to solubilize the cells, and the protein concentration of the lysates was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) according to the manufacturer's instructions. The luciferase activity was measured using the luciferase assay system (Promega, Madison, WI) and a luminometer (Analytic Luminescence Laboratories, Sparks, MD). The luciferase activity was normalized to the protein concentration of the lysates. The normalized luciferase activity was expressed as the fold induction of luciferase activity above the unstimulated control condition, which was given a value of one.

Whole Cell, Cytoplasmic, and Nuclear Extraction
Whole cell extracts from the primary cells were made as previously described (35), with the following modifications: protease and phosphatase inhibitors added to the boiling lysis buffer included complete, Mini protease inhibitor cocktail tablets (Roche Diagnostics, Basel, Switzerland) per the manufacturer's instructions, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 2 µg/ml pepstatin A. The protein concentration of cell extracts was determined by bicinchoninic acid assay as described for the luciferase lysates above. The samples were boiled and then stored in sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis loading buffer (25 mM Tris-HCl [pH 6.8], 2% SDS, 5% ß-mercaptoethanol, 25% glycerol, 0.01% bromophenol blue) at –70°C before use in SDS–polyacrylamide gel electrophoresis. Nuclear and cytoplasmic extracts were prepared using the NE-PER kit (Pierce); complete, Mini protease inhibitor cocktail tablets, 50 mM NaF, 1 mM Na3VO4 and 2 µg/ml Pepstatin A were added to the cytoplasmic extraction reagent (CER) I and nuclear extraction reagent (NER) solutions of the NE-PER kit. The protein concentration determination and storage of nuclear and cytoplasmic extracts were performed in the same manner described for the luciferase lysates.

Western Blot Analysis
Whole cell, cytoplasmic, and nuclear extracts were subjected to 7.5% Tris-glycine gel electrophoresis (PAGEr Gold Precast gels; BioWhittaker Molecular Applications, Rockland, ME) and then transferred to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked overnight with 4% bovine serum albumin (Fisher Scientific, Fernwood, NJ) in PBS with 0.1% Tween 20 (PBST) before incubation with either mouse anti-human STAT6 (BD Pharmingen Transduction Labs, Franklin Lakes, NJ) or mouse anti-human phospho-STAT6 (BD Pharmingen Transduction Labs) for 2 h at room temperature. Membranes were then washed with PBST and incubated with anti-mouse immunoglobulin antibody conjugated to horseradish peroxidase (Amersham, Piscataway, NJ) for 1 h. Enhanced chemiluminescence (ECL Western blot detection system; Amersham) substrate was added after further washing with PBST, and the membrane was then exposed to film.

ELISA and Real-Time Polymerase Chain Reaction Analysis
Supernatants were removed from the treated cells, spun for one minute at 14,000 rpm to remove cellular debris, aliquotted and stored at –70°C. Eotaxin-3 (R&D Systems), IFN-{gamma}–inducible protein of 10 kD (IP-10; R&D Systems) and IL-13R{alpha}2 ELISA (Antigenix America, Huntington Station, NY) were performed according to the manufacturer's instructions.

For quantitative real-time polymerase chain reaction (PCR) analysis of eotaxin-3 mRNA levels, 500 ng total RNA extracted from washed primary cells per the manufacturer's instructions (using the RNeasy RNA isolation kit; Qiagen, Valencia, CA) was reverse transcribed in 20 µl and 5 µl of a 1:10 dilution of the resulting complementary DNA (cDNA) was analyzed by LUX (Light Upon eXtension) primer quantitative real-time PCR using a pair of eotaxin-3–specific primers (a kind gift of Brian Lowe, Invitrogen) at a final concentration of 200 nM. The sequence of the 6-carboxy fluorescein (FAM)-labeled LUX forward primer was: 5' CAC AGC CTC TCC TTG GCC TCT GCT G FAM G 3', and the sequence of the reverse primer was: 5' GCT TGT GGC TGT ATT GGA AGC A 3' (synthesized by Invitrogen). The same cDNA samples were also analyzed for the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase, to allow for normalization of the target eotaxin-3 sequence for the amount of input cDNA. The sequences of the specific glyceraldehyde-3-phosphate dehydrogenase primers were as follows: for the forward primer, 5' GGC ATC CTG GGC TAC ACT GA 3'; and the sequence of the 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE)-labeled LUX reverse primer was: 5' GAC CAC GGA AAT GAG CTT GAC AAA GTG G JOE C 3'. The master reaction mixture was prepared using platinum quantitative PCR SuperMix-UDG according to the manufacturer's instructions (Invitrogen). The thermal cycling protocol for PCR tubes/plates and the analyses were performed using an ABI 7700 for real-time PCR (Applied Biosystems, Foster City, CA). The amount of eotaxin-3 mRNA in each sample was calculated according to the 2–{Delta}{Delta}CT method (36). Briefly, the threshold cycle (or CT, defined as the fractional cycle number at which the fluorescence passes the fixed threshold) for the target sequence in stimulated cells was normalized to the value obtained for the housekeeping gene and then that value was expressed as a relative change (compared with a control or unstimulated sample) in the level of target gene expression.

The sequences of the specific primer sets and minor groove-binding probes that were used in the real-time PCR analysis of other genes of interest are displayed in Table 1. All minor groove-binding probes and primers were designed using the published gene sequences available from GenBank (National Center for Biotechnology Information) and the Primer Express software from Applied Biosystems, Inc.


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  		TABLE 1. Minor groove-binding probes and primers used for quantitative TaqMan real-time polymerase chain reaction analysis of gene expression

 
Software and Statistical Analysis
Bands detected by Western blotting on the autoradiographic film were captured digitally (Kodak 1D Image Analysis Software v. 3.5; Eastman Kodak, New Haven, CT) and band density was quantitated using Scion Image 1.62c (Scion Corporation, Frederick, MD). Primers and probes used for TaqMan real-time PCR analysis were designed using Primer Express, v. 1.5 (Applied Biosystems Inc.). Statistical analysis was performed using a paired t test or analysis of variance with Statview v. 5.0 (Cary, NC) and Kaleidagraph 3.6 (Reading, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4 and IL-13 mediate allergic inflammatory responses in large part via phosphorylation and activation of STAT6. We have reported previously, as have others, that IFN-{gamma} can inhibit activation of epithelial cells to express STAT6-dependent genes, such as eotaxin (37, 38). Therefore, we tested whether IFN-{gamma} pretreatment would inhibit the appearance of phospho-STAT6 in the nuclei of primary airway epithelial cells in response to IL-4. Primary bronchial epithelial cell cultures were incubated for 24 h with 100 ng/ml IFN-{gamma} and then stimulated for 30 min with IL-4, after which lysates from each cellular compartment were separated and analyzed by Western blotting using specific monoclonal antibodies for STAT6 and for phospho-STAT6. The nuclear lysates from cells stimulated with IL-4 contained substantial amounts of phospho-STAT6, as shown by the appearance of an intense band detected at ~ 105 kD (Figure 1A, panel i, lane 2) that was not present in the nuclear lysates from unstimulated cells (Figure 1A, panel i, lane 1). Pretreatment with IFN-{gamma} before stimulation with IL-4 dramatically reduced the amount of nuclear phospho-STAT6 (Figure 1A, panel i, lane 3). Nuclear lysates from cells that were pretreated with IFN-{gamma} but not stimulated with IL-4 did not contain any phospho-STAT6 (Figure 1A, panel i, lane 4), as expected.



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  		Figure 1. Twenty-four hour pretreatment with IFN-{gamma} decreased IL-4–induced nuclear- and cytoplasmically-localized phospho-STAT6 in primary human airway epithelial cells. Cells were pretreated with 100 ng/ml IFN-{gamma} and then stimulated with 0.2 ng/ml IL-4 for 30 min as indicated; nuclear, cytoplasmic, and whole cell lysates were extracted and subjected to Western blot analysis with either anti-STAT6 or anti–phospho-STAT6 monoclonal antibodies. (A) Representative Western blot films from each cellular fraction probed as indicated (panels i – vi). The number of replicate experiments represented was: (i) n = 10; (ii) n = 3; (iii) n = 3; (iv) n = 3; (v) n = 4; and (vi) n = 1. (B) The detected phospho-STAT6 band densities in the nuclear fraction were measured and the densitometric values were normalized to the IL-4–stimulated value. Data are shown as the mean normalized values ± SEM of ten independent experiments (*P < 0.0001).

 
The Western blots were reprobed for histone deacetylase 1 to control for the amount of nuclear protein, and the densitometric value for each phospho-STAT6 band was normalized to histone deacetylase 1. In ten independent experiments, the resulting normalized value was then expressed as a percentage of the value obtained for the IL-4–stimulated condition, which was set at 100%. Data in Figure 1B show that pretreatment of primary airway epithelial cells with IFN-{gamma} reduced the amount of IL-4–induced phospho-STAT6 in the nucleus to 35% of the maximal value observed in IL-4–stimulated cells. Levels of total nuclear STAT6 (Figure 1A, panel ii) showed a similar pattern as that observed with nuclear phospho-STAT6 (36 ± 11% of the maximal amount, n = 3, P < 0.05). It is unlikely that reduced phospho-STAT6 was a result of a decrease in the total pool of cellular STAT6 because there was no change in the level of total cellular STAT6 after 24 h IFN-{gamma} treatment of the airway epithelial cells (Figure 1A, panel iv). In addition, total cellular phospho-STAT6 levels (Figure 1A, panel iii) under the four different experimental conditions showed a pattern similar to that observed in the nuclear fraction. The amount of total cellular phospho-STAT6 after IL-4 stimulation was reduced (47 ± 2%, n = 3, P < 0.001) by the 24 h pretreatment with IFN-{gamma}. A similar reduction of IL-4–induced cytoplasmic phospho-STAT6 was observed (Figure 1A, panel v) after IFN-{gamma} pretreatment (27 ± 12% of the maximal amount, n = 4, P < 0.01), whereas the total cytoplasmic STAT6 levels were not affected by 24 h treatment (Figure 1A, panel vi).

Because type I IFNs can also inhibit allergic inflammation (39–41), we tested whether IFN-{alpha} would reduce the amount of phospho-STAT6 in the nucleus after IL-4 stimulation, as was observed with IFN-{gamma}. Pretreatment of the cells for 24 h with 100 ng/ml IFN-{alpha} reduced the amount of IL-4–induced nuclear-localized phospho-STAT6 (63 ± 14% of the maximal amount, n = 7, P < 0.05). Thus, inhibition of IL-4–induced STAT6 activation is a common property of different classes of IFNs.

The reduction in the amount of phospho-STAT6 in the nucleus after IL-4 stimulation was dependent upon the concentration of IFN-{gamma} used in the pretreatment, as shown in Figure 2A. Statistically significant reductions of IL-4–induced nuclear phospho-STAT6 were observed with IFN-{gamma} at 10 ng/ml and 100 ng/ml. From the kinetic analysis (Figure 2B), it was observed that reduction of the amount of phospho-STAT6 in the nucleus after IL-4 stimulation was dependent upon the duration of the IFN-{gamma} pretreatment before IL-4 stimulation. Incubation with IFN-{gamma} for a minimum of 3 h was necessary to achieve a statistically significant reduction in the amount of IL-4–induced phospho-STAT6 in the nucleus.



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  		Figure 2. Inhibition by IFN-{gamma} of IL-4–induced nuclear phospho-STAT6 levels in human airway epithelial cells was dependent on both the concentration of the IFN-{gamma} pretreatment and the duration of IFN-{gamma} pretreatment before IL-4 stimulation. The methods were the same as in Figure 1, except that the IFN-{gamma} concentration (A) and duration (B) of pretreatment were varied as indicated. Data are shown as the mean normalized values ± SEM of four to six independent experiments (*P < 0.05; **P < 0.01).

 
Next we sought to determine whether the transcriptional activity of STAT6 was also reduced by pretreatment with IFN-{gamma} using two different approaches. The first method used transient transfection assays with a STAT6-dependent luciferase reporter construct to measure IL-4–induced STAT6 transcriptional function after IFN-{gamma} pretreatment. Cells were cotransfected overnight with a STAT6 reporter construct plus the WT-STAT6 expression vector (to enhance the luciferase signal as previously described [42]). After pretreatment for 24 h with varying concentrations of IFN-{gamma} (0.1–100.0 ng/ml), IL-4–induced luciferase activity was measured after stimulation with 0.2 ng/ml IL-4. The data in Figure 3 show that the IL-4–induced luciferase activity was strikingly diminished by IFN-{gamma} pretreatment in a concentration-dependent manner. Inhibition was statistically significant across the entire range of IFN-{gamma} concentrations tested.



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  		Figure 3. Twenty-four hour pretreatment with IFN-{gamma} inhibited IL-4–induced luciferase activity from a STAT6-driven luciferase reporter construct in primary human airway epithelial cells, and inhibition by IFN-{gamma} was dependent on the concentration of IFN-{gamma} used in the pretreatment. Cells were transiently transfected in duplicate with 0.95 µg mut-(C/EBP)-N4-luc plus 0.05 µg wild-type STAT6 expression vector for 18 h. Cells were pretreated with the indicated concentrations of IFN-{gamma} for 24 h, stimulated with 0.2 ng/ml IL-4, and then lysed 6 h later. Luciferase activity for each well was determined, averaged, and the data are shown as the mean ± SEM fold induction of luciferase activity above the unstimulated condition, as a percentage of the value obtained for the IL-4–stimulated condition, set at 100%, for five independent experiments (*P < 0.005).

 
To monitor the impact of IFN-{gamma} pretreatment on the ability of STAT6 to enhance the expression of an endogenous gene as opposed to a transfected reporter plasmid, we measured the expression of eotaxin-3, a STAT6-dependent chemokine gene, at both the mRNA and protein level in IFN-{gamma}–pretreated cells. mRNA and secreted protein were quantitated by real-time PCR and ELISA, respectively, in IL-4–stimulated cells after 24 h pretreatment with IFN-{gamma}. Pretreatment with IFN-{gamma} resulted in dose-dependent inhibition of both eotaxin-3 mRNA and protein (Figure 4). Interestingly, IFN-{gamma} was more potent at inhibiting eotaxin-3 protein production than was eotaxin-3 mRNA expression: the concentration producing a half-maximum inhibition of effect (IC50) values were 2 ng/ml versus 9 ng/ml, respectively (n = 6), suggesting an additional effect at the translational level. Thus, IFN-{gamma} blocks expression of both a STAT6-dependent reporter construct as well as expression of an endogenous STAT6-dependent chemokine gene in airway epithelium.



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  		Figure 4. Inhibition of IL-4–induced expression of eotaxin-3 mRNA (circles) and protein (squares) in primary human airway epithelial cells was dependent on the concentration of IFN-{gamma} used in the pretreatment. Cells were pretreated with the indicated concentration of IFN-{gamma} for 24 h and stimulated for a further 24 h with 100 ng/ml IL-4. Eotaxin-3 ELISA was performed on the cell supernatants as described in MATERIALS AND METHODS and the amount of eotaxin-3 produced (pg) per million cells was calculated. Data are shown as the mean ± SEM amount of eotaxin-3 produced as a percentage of the IL-4–stimulated condition (squares), from six independent experiments. Mean ± SEM eotaxin-3 production was 407 ± 67 pg/106 cells from IL-4–stimulated cells and 8 ± 8 pg/106 cells from unstimulated cells. Eotaxin-3 mRNA was quantitated from the same treated primary cells by quantitative real-time PCR using specific LUX primers. The amount of eotaxin-3 mRNA relative to the unstimulated value was calculated. Data are shown as the mean ± SEM amount of eotaxin-3 mRNA, as a percentage of the IL-4–stimulated condition (circles), from six independent experiments. The mean maximal eotaxin-3 mRNA levels were 501-fold ± 159-fold from IL-4–stimulated cells, relative to the unstimulated value of 1 (*P < 0.05).

 
To ensure that the considerable reduction of eotaxin-3 protein levels by IFN-{gamma} was not a result of global inhibition of protein synthesis, we measured the production of IP-10, another chemokine known to be secreted from airway epithelial cells in response to IFN-{gamma}. High concentrations of IP-10 were measured by ELISA in cell-free supernatants from the IFN-{gamma}–treated cells (> 500 ng/ml, n = 5), as compared with untreated (23 ± 7 ng/ml, n = 5) or IL-4–stimulated (34 ± 10 ng/ml, n = 5) cells. By performing erythrosin B dye exclusion viability staining of the treated cells in each experiment of this study (cell viability was >= 90%), we also verified that the reduction in eotaxin-3 protein expression was not due to IFN-{gamma}–induced cell death.

Previous studies of the effect of IFN-{gamma} on IL-4/STAT6 signal transduction in human monocytes (43–45) suggested that there are several possible mechanisms by which IFN-{gamma} could inhibit the IL-4–initiated STAT6 signal transduction pathway, including downregulation of IL-4 receptors on the cell surface, inhibition of JAK kinase, induction of phosphatases, and degradation of STAT proteins. To test whether IFN-{gamma} pretreatment reduced expression of IL-4R{alpha} components, total RNA was harvested from IFN-{gamma}–pretreated and IL-4–stimulated cells, and the amount of mRNA for the two different chains of the IL-4 receptor complex (IL-4R{alpha} and IL-13R{alpha}1), as well as IL-13R{alpha}2, was measured by quantitative real-time PCR. As shown in Figure 5A, IFN-{gamma} pretreatment did not reduce, but actually enhanced, the expression of mRNA for all three receptor chains. Based on this finding, it is unlikely that the inhibitory effect of IFN-{gamma} is mediated by downregulation of transcription of the IL-4R{alpha} or IL-13R{alpha}1 genes.



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  		Figure 5. (A) Twenty-four hour pretreatment with IFN-{gamma} induced expression of mRNA for IL-13R{alpha}2 and did not decrease mRNA for IL-4R{alpha} or IL-13R{alpha}1 in primary human airway epithelial cells (light gray bars, IL-4R{alpha} mRNA; white bars, IL-4R{alpha}1 mRNA; dark gray bars, IL-13R{alpha}2 mRNA. (B) Induction of IL-13R{alpha}2 mRNA was dependent on the concentration of IFN-{gamma} used in the pretreatment. The cells were treated and the mRNA was analyzed in the same fashion as described for Figure 4 (circles, without 24-h IL-4 stimulation; squares, with 24-h IL-4 stimulation). Data are shown as (A) the mean ± SEM amount of mRNA for IL-4R{alpha} (mean unstimulated CT = 24.3 ± 0.4), IL-13R{alpha}1 (mean unstimulated CT = 23.6 ± 0.4), or IL-13R{alpha}2 (mean unstimulated CT = 26.9 ± 0.4), relative to the unstimulated value from five independent experiments (*P < 0.05; **P < 0.01), and (B) the mean ± SEM amount of IL-13R{alpha}2 mRNA minus control and as a percentage of the maximal response (the mean maximal IL-13R{alpha}2 mRNA levels were 33-fold ± 12-fold, relative to the unstimulated value of 1) rom five independent experiments (*P < 0.05).

 
In these experiments, we also found that IFN-{gamma} pretreatment led to a striking induction of the expression of mRNA for IL-13R{alpha}2 (~ 64-fold). Interestingly, IL-4 stimulation alone also induced expression of mRNA for IL-13R{alpha}2 (~ 8-fold), which agrees with previous reports in other cell types (6, 46). Although IL-13R{alpha}2 has been shown to act as a decoy receptor for IL-13, it has not been shown to bind IL-4. However, IL-13R{alpha}2 has been shown to downregulate IL-4 signaling in IL-13R{alpha}2-transfected 293T cells and NIH 3T3 cells (47), and IFN-{gamma} has been shown to mobilize intracellular stores of IL-13R{alpha}2 to the surface of human monocytes, leading to downregulation of STAT6 signaling in response to IL-13 (17). Given the striking induction of IL-13R{alpha}2 mRNA by IFN-{gamma}, we used three different approaches to test the possibility that IL-13R{alpha}2 was involved in the mechanism by which IFN-{gamma} inhibits STAT6 signal transduction. First, we examined the expression of IL-13R{alpha}2 after IFN-{gamma} treatment in more detail. There was a clear concentration-dependent induction of IL-13R{alpha}2 mRNA both in the presence and absence of IL-4 stimulation (Figure 5B). Interestingly, IFN-{gamma} was ~ 10-fold more potent in combination with IL-4 (the concentration at which IFN-{gamma} elicited a response halfway between baseline and maximal [EC50] was ~ 1.3 ng/ml and 13 ng/ml in the presence and absence of IL-4, respectively) and the maximal response was similar regardless of whether IL-4 stimulation had occurred.

Because IFN-{gamma} was found to be a potent inducer of IL-13R{alpha}2 mRNA in epithelial cells, we tested whether IFN-{gamma}–induced IL-13R{alpha}2 protein on the cell surface (experiments) by flow cytometry or (by ELISA and washing experiments) was soluble IL-13R{alpha}2 acting as a decoy receptor. The presence of IL-13R{alpha}2 has been demonstrated on the cell surface and in the intracellular compartment of human monocytes and airway epithelial cells, as shown by flow cytometry and immunocytochemistry with a fluorescein isothiocyanate–conjugated anti–IL-13R{alpha}2 antibody (17). We used the same antibody but conjugated to phycoerythrin (see MATERIALS AND METHODS). Unstimulated primary airway epithelial cells expressed very low levels of the IL-13R{alpha}2 protein on their surface (8 ± 4% of live cells positive for IL-13R{alpha}2, mean fluorescence intensity = 7 ± 1, n = 3), and these low levels did not appear to increase after 24 h treatment with IFN-{gamma} (6% of live cells positive for IL-13R{alpha}2, mean fluorescence intensity = 6, n = 2). Soluble IL-13R{alpha}2 in the supernatants from cells treated with IFN-{gamma} in four independent experiments was determined using a commercially available ELISA. In these experiments, we were unable to detect any soluble IL-13R{alpha}2 in the supernatants from IFN-{gamma}–treated cells, suggesting that levels of soluble IL-13R{alpha}2 were below 15.6 pg/ml, which is the detection limit of the assay. To further eliminate the possibility that the inhibitory effect of IFN-{gamma} might result from induction of other soluble IL-4–binding decoy receptors or other soluble IFN-{gamma}–inducible inhibitors, we removed epithelial cell supernatants and washed the cells after the 24 h pretreatment with IFN-{gamma} and immediately before stimulation with IL-4 to eliminate any soluble inhibitors. Data from the eotaxin-3 ELISA showed that there was no restoration of IL-4–induced (0% of the maximal response, n = 3) or IL-13–induced (0% of the maximal response, n = 1) eotaxin-3 protein production after IL-4 stimulation of the washed, IFN-{gamma}–pretreated cells. Taken together, these results indicate that IL-13R{alpha}2 is unlikely to be part of the mechanism by which IFN-{gamma} inhibits STAT6 signaling in airway epithelial cells.

We tested next whether IFN-{gamma} might regulate early receptor-mediated events, including JAK activation and expression of the SOCS regulatory proteins. In these experiments, we detected only low levels of JAK1 phosphorylation in response to 0.2 ng/ml IL-4 using immunoprecipitation and Western blot and the anti-phosphotyrosine antibody, 4G10 (n = 3, data not shown). Similar amounts of phospho-JAK1 were detected in the immunoprecipitate from cells pretreated for 24 h with 100 ng/ml IFN-{gamma}, both with and without IL-4 stimulation. The SOCS proteins, SOCS-1 (48), SOCS-3 (48, 49), and SOCS-5 (50), have been reported to inhibit the IL-4/STAT6 signal transduction pathway, and the mRNA for SOCS-1 and SOCS-3 is induced by IFN-{gamma} in a variety of cell types (51, 52). Therefore, we sought to determine whether expression of SOCS-1 and SOCS-3 was induced by 24 h pretreatment with IFN-{gamma} in the airway epithelial cell cultures using TaqMan real-time PCR. Treatment with IFN-{gamma} alone significantly upregulated the mRNA for SOCS-1 (16-fold) and SOCS-3 (~9-fold) (Figure 6A), whereas stimulation with IL-4 alone induced SOCS-1 mRNA 13-fold, and SOCS-3 mRNA to a much lesser degree (1.4-fold). Although there was a trend toward additive induction of SOCS-1 mRNA when both IFN-{gamma} and IL-4 were used (24-fold), no additivity was observed in the case of SOCS-3 mRNA (Figure 6B). Induction of SOCS-1 mRNA was concentration-dependent both in the presence and absence of IL-4 stimulation (Figure 6B). As was observed at maximal concentrations of IFN-{gamma} in Figure 6A, there was a trend toward additivity between IL-4 and IFN-{gamma} throughout the concentration-response curve.



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  		Figure 6. (A) Twenty-four hour pretreatment with IFN-{gamma} induced expression of mRNA for the STAT6 inhibitors, SOCS-1 and SOCS-3, in primary human airway epithelial cells. (B) Induction of SOCS-1 mRNA was dependent on the concentration of IFN-{gamma} used in the pretreatment. The cells were treated and the mRNA was analyzed in the same fashion described in Figure 4. Data are shown as (A) the mean ± SEM amount of SOCS-1 or SOCS-3 mRNA , relative to the unstimulated value from seven independent experiments for SOCS-1 analysis and three independent experiments for SOCS-3 analysis, and (B) the mean ± SEM amount of SOCS-1 mRNA minus control and as a percentage of the maximal value (mean maximal SOCS-1 mRNA levels were 15-fold ± 6-fold relative to the unstimulated value of 1) from five independent experiments (*P < 0.05).

 
Due to the lack of commercial availability of an antibody to the endogenous human SOCS-1 protein, Western blots to correlate the upregulation of SOCS-1 mRNA with SOCS-1 protein expression were not possible. Therefore, the involvement of SOCS-1 in the inhibition of IL-4–induced STAT6 activation by IFN-{gamma} was tested by analyzing STAT6 phosphorylation in a known "SOCS-1–deficient" hepatic epithelial cell line, Hep3B (53). In this cell type, the expression of the SOCS-1 gene is completely silenced by DNA methylation, shown by methylation-sensitive PCR and bisulfite sequencing, as well as by Northern blotting (53). These cells are responsive to IFN-{gamma}, upregulate a variety of cell surface molecules (54, 55), and have significant levels of IFN-{gamma}R{alpha} on their surfaces (56). Interestingly, IFN-{gamma} did not significantly affect the appearance of nuclear phospho-STAT6 in these cells (91 ± 11% of control, n = 3, P = 0.49). This suggests that induction of SOCS-1 is involved in inhibition of nuclear phospho-STAT6 by high concentrations of IFN-{gamma}.

Because IFN-{gamma} was significantly more potent as an inhibitor of STAT6-driven luciferase expression (Figure 3; IC50 ~0.6 ng/ml) and expression of eotaxin-3 mRNA (Figure 4; IC50 ~2 ng/ml) than as an inhibitor of the phosphorylation of STAT6 (Figure 1; IC50 ~33 ng/ml), it is likely that inhibition of eotaxin-3 expression at low concentrations of IFN-{gamma} results from additional mechanisms beyond inhibition of STAT6 activation. Because IFN-{gamma} has been shown to have posttranscriptional effects on mRNA turnover (for example, Fc{epsilon}RII [57], c-fos [58], transferrin receptor [59], and IL-4R{alpha} in monocytes and B-cells [44]), we determined whether IFN-{gamma} influenced the decay of the mRNA for eotaxin-3. Primary airway epithelial cell cultures were stimulated with IL-4 for 24 h to generate significant amounts of eotaxin-3 mRNA, the cells were then incubated with or without the transcriptional inhibitor, actinomycin D, and with or without IFN-{gamma}. Actinomycin D was used both to block further transcription of eotaxin-3 and to assess whether the effect of IFN-{gamma} on mRNA decay required gene transcription. Total RNA was harvested from the treated cells over a range of time points (0, 4 and 8 h) after addition of IFN-{gamma}/actinomycin D, and the amount of eotaxin-3 mRNA was quantitated using real-time PCR (see Figure 7A for the experimental protocol). The amount of eotaxin-3 mRNA that remained at each time point after the addition of IFN-{gamma} +/– actinomycin D was calculated as a percentage of the initial amount of eotaxin-3 mRNA present at time 0. Curve fits were performed to calculate the rate of decay of the eotaxin-3 mRNA.



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  		Figure 7. IFN-{gamma} treatment of IL-4–induced eotaxin-3 mRNA resulted in an accelerated loss of eotaxin-3 mRNA in primary human airway epithelial cells. (A) Experimental protocol: after 24 h stimulation with IL-4, primary human airway epithelial cells were treated with or without 100 ng/ml IFN-{gamma} and with or without 3 µg/ml actinomycin D. Total RNA was harvested at 0, 4, and 8 h after the IFN-{gamma}/actinomycin D treatment. The amount of eotaxin-3 mRNA remaining at each time point was quantitated by TaqMan real-time PCR and was expressed as the mean ± SEM percentage of the initial amount of eotaxin-3 mRNA present at time zero from four independent experiments, in (B) the absence (line A, IL-4–treated cells and line B, IL-4– and IFN-{gamma}–treated cells) or (C) the presence of actinomycin D (line C, IL-4–treated cells and line D, IL-4– and IFN-{gamma}–treated cells). The mean maximal eotaxin-3 mRNA level at t = 0 was 694-fold ± 181-fold relative to the unstimulated value of 1. (*P < 0.01, compared with IL-4 treatment alone).

 
The level of eotaxin-3 mRNA declined very slowly over the course of 8 h, such that 87% of the initial amount of eotaxin-3 mRNA was still present at the end of the experiment (Figure 7B, line A). A striking and significant acceleration in the rate of decay of the eotaxin-3 mRNA was observed after addition of IFN-{gamma}: the amount of eotaxin-3 mRNA remaining after 8 h was approximately half of the initial amount of eotaxin-3 mRNA (Figure 7B, line B). Addition of actinomycin D to block transcription slightly increased the rate of decay of the IL-4–induced eotaxin-3 message (Figure 7C, line C), leaving ~ 70% of the initial amount of eotaxin-3 mRNA after 8 h. The accelerated rate of decay in the presence of IFN-{gamma} was attenuated by the addition of actinomycin D (Figure 7C, line D), which was significant compared with the rate observed in the presence of IL-4 alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of IL-4 signal transduction and gene expression in human airway epithelial cells is of critical importance in allergic diseases, such as asthma, because epithelial cells stimulated with IL-4 express a multitude of inflammatory mediators that trigger and then maintain airway inflammation. In this study, we investigated whether the classical Th1-type cytokine, IFN-{gamma}, was capable of inhibiting IL-4–initiated STAT6 signal transduction and subsequent gene expression in cultured primary airway epithelial cells. The effect of IFN-{gamma} on STAT6 phosphorylation and nuclear translocation by IL-4 was examined by cellular fractionation and Western blotting for the latent and activated forms of the STAT6 protein. A comprehensive study of STAT6 phosphorylation/translocation in each cellular compartment revealed that the levels of nuclear-localized phospho-STAT6 and total nuclear STAT6 were significantly diminished in cells treated with IFN-{gamma} for 24 h before stimulation with IL-4. This was not due to an IFN-{gamma}–mediated decline in the total level of STAT6 in the cell available for phosphorylation/nuclear translocation, because IFN-{gamma} had no effect on total cellular or total cytoplasmic STAT6 available for phosphorylation. Possible explanations for these findings include decreased formation of phospho-STAT6 in response to IL-4, increased dephosphorylation of phospho-STAT6 in the cytoplasm or increased dephosphorylation and rapid export/degradation of phospho-STAT6 in the nucleus. IFN-{gamma} pretreatment decreased the amount of both nuclear and cytoplasmic phospho-STAT6 observed in response to IL-4, without altering the total level of STAT6 protein in the cell. Therefore, the most likely explanation for the observed decrease in phospho-STAT6 in the nucleus is a reduction in the formation of phospho-STAT6 in the cytoplasm.

The IFN-{gamma}–mediated reduction of nuclear phospho-STAT6 was associated with a striking effect on STAT6 transcriptional function and STAT6-dependent gene expression. In studies using STAT6-dependent luciferase reporter assays, there was a clear concentration-dependent inhibition by IFN-{gamma} of IL-4–induced luciferase activity. Interestingly, activation of the STAT6-dependent luciferase reporter was 10 to 100 times more sensitive to inhibition by IFN-{gamma} than the formation of nuclear phospho-STAT6, suggesting that reduction of phospho-STAT6 is not the only mechanism by which IFN-{gamma} inhibited reporter gene expression. However, because the phosphorylation analysis was performed 30 min after IL-4 stimulation and the reporter analysis was performed after 6 h of IL-4 stimulation, it is possible that IFN-{gamma} accelerated the export of phospho-STAT6/STAT6 from the nucleus after the 30 min point of assay for phospho-STAT6. More likely, this result suggests that low concentrations of IFN-{gamma} may separately inhibit the nuclear function of STAT6, without necessarily diminishing the level of phospho-STAT6 in the nucleus. It is possible that IFN-{gamma} induces inhibitory proteins, which prevent DNA binding by STAT6 or prevent posttranslational modifications of STAT6 important in function, such as acetylation (60) or methylation (61). An additional possibility is that low concentrations of IFN-{gamma} may decrease the function or amount of another factor necessary for transcriptional activation in the reporter system. Further studies will be needed to investigate these possibilities.

STAT6-dependent gene expression was inhibited by IFN-{gamma} in a concentration-dependent manner, as was shown by the reduction in the level of eotaxin-3 mRNA and secreted protein from IFN-{gamma}–treated cells. Interestingly, the production of eotaxin-3 protein was almost 5-fold more sensitive to inhibition by IFN-{gamma} than was eotaxin-3 mRNA production (IC50 of ~ 2 ng/ml versus 9 ng/ml, respectively). These results suggest that IFN-{gamma} may exert posttranscriptional effects on the translation or stability of eotaxin-3 protein. IFN-{gamma} is known to inhibit the production of many proteins, either at the translational level, such as major histocompatibility complex class II (62), collagen and fibronectin (63), ceruloplasmin (64), or at the posttranslational level, such as apolipoprotein E (65). Another possibility is that IFN-{gamma} leads to the production of "sterile" eotaxin-3 transcripts, which are detected by real-time PCR analysis but for some reason cannot be translated efficiently.

Activation of STAT6 by IL-4 is a multistep process involving binding of IL-4 to either type I or type II IL-4 receptors on the cell surface, activation of JAK kinases, and recruitment of cytosolic STAT6 monomers for phosphorylation. The mechanism of inhibition of STAT6 activation and function by IFN-{gamma} may involve one or more of the steps of the signal transduction pathway. Previous studies indicated that IFN-{gamma} can reduce the amount of IL-4 receptors on the surface of a variety of different cell types (44), which would lead to a decrease in the amount of IL-4R–triggered signal transduction. Our results indicate that there was no downregulation of the mRNA for either IL-4R{alpha} or IL-13R{alpha}1 by IFN-{gamma}. Indeed, we found that the mRNA for IL-13R{alpha}2 was induced strongly by IFN-{gamma} treatment of the airway epithelial cells. This receptor has been shown to exert inhibitory effects on IL-13 signaling (17, 18, 66, 67) and may act as an IL-13 decoy receptor (reviewed in Ref. 68). Induction of soluble cytokine decoy receptors is a well-described mechanism by which cytokine signaling, as well as signaling in response to other types of soluble ligands, can be downregulated (e.g., type II IL-1R [69], IL-17R [70], lymphotoxin ß receptor [71], Fas ligand decoy receptor [DcR3, {72}], and osteoprotegerin [73]). The increase in IL-13R{alpha}2 mRNA was dependent upon the concentration of IFN-{gamma} used, and the extent of induction of IL-13R{alpha}2 mRNA corresponded with the decline in phospho-STAT6 levels found in the nucleus in response to IL-4. However, our results do not suggest that IL-13R{alpha}2 is responsible for the inhibitory effects of IFN-{gamma} on phospho-STAT6 signaling. Washout experiments and ELISA assays indicate that a soluble form of the IL-13R{alpha}2 receptor was not released from epithelial cells by IFN-{gamma}, and flow cytometry studies indicated that IL-13R{alpha}2 expression was very low in resting cells. In addition, IFN-{gamma} treatment of epithelial cells did not change the amount of membrane-bound IL-13R{alpha}2 protein on the cell surface. The low level of IL-13R{alpha}2 that we measured on the cell surface after excluding dead and autofluorescent cells agrees with one recent study in which IL-13R{alpha}2 was reported to be unmeasurable by flow cytometry on the surface of primary human airway epithelial cells (18). In addition, IL-13R{alpha}2 does not bind to IL-4 (17, 74), and IL-4–induced STAT6 phosphorylation was not inhibited in IL-13R{alpha}2–transfected BEAS-2B airway epithelial cells (18). Based on our results and these reports, the overall contribution of IL-13R{alpha}2 as an inhibitor of IL-4–induced STAT6-dependent gene expression in our systems at lower concentrations of IFN-{gamma} is likely to be minimal.

IFN-{gamma} can induce expression of several SOCS proteins that serve to downregulate cytokine signaling. We detected induction of the mRNA for two SOCS proteins, SOCS-1 and SOCS-3, after 24 h treatment of epithelial cells with IFN-{gamma}. SOCS-1 was induced by IFN-{gamma} alone and in combination with IL-4 stimulation, which concurs with recent reports in T cells (75) and airway epithelial cells (76). The induction of SOCS-3 mRNA was generally lower than that of SOCS-1. The induction of SOCS-1 mRNA by the higher concentrations of IFN-{gamma} appeared to correlate with the decline in nuclear-localized phospho-STAT6 induced by IL-4, suggesting a possible link between the two processes. This connection is strengthened by the fact that IL-4–induced phospho-STAT6 was not reduced by treatment with 100 ng/ml IFN-{gamma} in Hep3B cells, which do not express SOCS-1. Furthermore, recent studies in SOCS-1–/– mouse embryonic fibroblasts suggest a role for SOCS-1 in the inhibitory effect of IFN-{gamma} on IL-4–induced eotaxin-1 expression (77). Our present studies indicate that SOCS-1 induction is likely to play a role in the reduction in the level of cytoplasmic and nuclear phospho-STAT6 in response to IL-4.

Based on the greater potency of IFN-{gamma} as an inhibitor of eotaxin-3 protein expression than of STAT6 activation, we anticipated the existence of another mechanism by which IFN-{gamma} may downregulate IL-4–/STAT6-dependent gene expression. A simple mRNA decay experiment demonstrated the acceleration of the rate of decay of IL-4–induced eotaxin-3 mRNA in cells treated with IFN-{gamma}. The acceleration of the decay rate appears to require the transcription of an IFN-{gamma}–induced gene because the effect was completely lost in the presence of actinomycin D. Although we did not explore the mechanism of this effect, it could include induction of mRNA binding and destabilizing proteins, such as tristetraprolin, a protein known to destabilize mRNA transcripts, or A + U-rich element RNA binding factor 1 (AUF1) (reviewed in Ref. 78). Because the decay of mRNA is often mediated by AU-rich sequences in the 3' untranslated region (UTR) of many genes, we examined the sequence of the 3'-UTR of the eotaxin-3 gene for the presence of the consensus sequence AUUUA, as well as for abundant A/T-stretches. Indeed, one such AUUUA motif was found, suggesting that further experiments using a reporter mRNA containing the 3'-UTR of eotaxin-3 would be warranted.

We have provided evidence for multiple mechanisms by which IFN-{gamma} inhibits IL-4 signal transduction and gene activation, including inhibition of phospho-STAT6 formation (possibly through induction of SOCS-1), inhibition of STAT6 function (shown by inhibition of STAT6-dependent transcription), posttranscriptional effects (accelerated decay of eotaxin-3 mRNA), and possibly inhibition of eotaxin-3 translation (inferred from data in Figure 4). A combination of these mechanisms is likely to explain the potent inhibitory effect of IFN-{gamma} on the output of eotaxin-3 protein from airway epithelial cells. Whether these observations can be generalized to the effect of IFN-{gamma} on the epithelial expression of all IL-4–/STAT6-dependent genes will need to be evaluated in further experiments. Together, these findings indicate that the prototypical Th1-type cytokine, IFN-{gamma}, is a potent negative regulator of the IL-4–initiated STAT6 signal transduction pathway in human airway epithelial cells. Th1 cells and their secreted cytokine, IFN-{gamma}, are well established inhibitors of allergic inflammation in the airways, and the mechanism by which IFN-{gamma} inhibits the response of airway epithelium is therefore of direct relevance to the regulation of allergic inflammation. Our results highlight a number of different mechanisms by which the balance between IFN-{gamma} and IL-4 can determine whether airway epithelial cells are activated to produce STAT6-dependent inflammatory mediators and thereby maintain the allergic inflammatory state within the airways. These regulatory pathways may prove useful in the search for novel therapies to halt allergic inflammation.


   Footnotes
 
Conflict of Interest Statement: N.M.H. has no declared conflicts of interest; S.M. has no declared conflicts of interest; S.N.G. has no declared conflicts of interest; M.R.B. has no declared conflicts of interest; P.B.R. has no declared conflicts of interest; C.S. has no declared conflicts of interest; and R.P.S. has no declared conflicts of interest.

Received in original form June 17, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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