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Published ahead of print on July 1, 2004, doi:10.1165/rcmb.2004-0128OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 456-462, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0128OC

Upregulation of Interleukin-4 Receptor by Interferon-{gamma}

Enhanced Interleukin-4–Induced Eotaxin-3 Production in Airway Epithelium

Shuichi Yamamoto, Ikuko Kobayashi, Kohsuke Tsuji, Natsuko Nishi, Eriko Muro, Michiko Miyazaki, Masafumi Zaitsu, Shigeyasu Inada, Tomohiro Ichimaru and Yuhei Hamasaki

Department of Pediatrics, School of Medicine, Saga University, Saga, Japan

Address correspondence to: Shuichi Yamamoto, M.D., Department of Pediatrics, School of Medicine, Saga University, 5-1-1 Nabeshima, Saga-City, Saga 849-8501, Japan. E-mail: yamamot6{at}med.saga-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cells produce a number of chemokines, including eotaxins. Among the three known eotaxins, T helper (Th) type 2 cytokines have been observed to induce the expression of eotaxin-3 mRNA. This study investigated the effect of interferon (IFN)-{gamma}, a Th1 cytokine, on Th2 cytokine–induced eotaxin-3 production in a bronchial epithelial cell line, BEAS-2B. BEAS-2B cells produced eotaxin-3 after stimulation with the Th2 cytokines interleukin (IL)-13 and IL-4. When BEAS-2B cells were cultured with varying concentrations of IFN-{gamma} for 24 h, dose-dependent inhibition of Th2 cytokine–induced eotaxin-3 mRNA expression and protein production was observed. This was associated with downregulation of signal transducer and activator of transcription 6 activation. On the other hand, 2-d pretreatment of BEAS-2B cells with IFN-{gamma} dose-dependently enhanced Th2 cytokine–induced eotaxin-3 mRNA expression and production. IFN-{gamma} also increased the mRNA expression and protein production of IL-4 receptor (R) {alpha} in a time- and dose-dependent manner. In addition, IL-2R{gamma}, a component of the type 1 IL-4R, was also upregulated by IFN-{gamma}. These results indicate that IFN-{gamma} has opposite effects on Th2 cytokine–induced eotaxin-3 production in BEAS-2B cells, depending on the length of exposure. Because high levels of IFN-{gamma} are produced during viral infection, airway viral infection may affect allergic airway inflammation in vivo by modulation of eotaxin-3 production.

Abbreviations: actinomycin D, ACD • cycloheximide, CHX • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • common {gamma} chain of interleukin-2 receptor, {gamma}C • interferon, IFN • interleukin, IL • IL-4 receptor, IL-4R • mitogen-activated protein kinase, MAPK • nuclear factor-{kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • phosphorylated signal transducer and activator of transcription, p-STAT • reverse transcription–polymerase chain reaction, RT-PCR • rhinovirus, RV • signal transducer and activator of transcription, STAT • T helper type, Th • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bronchial epithelial cells and cultured bronchial cell lines have been shown to produce a wide range of mediators, including chemokines (1). Because bronchial epithelial cells are exposed to the external environment in vivo, chemokines secreted from these cells are thought to contribute to airway inflammation under a number of circumstances, including viral infections of the airway. Eotaxins are one class of chemokines secreted from bronchial epithelial cells (2). Three eotaxins have been identified: eotaxin-1, -2, and -3, also known as CCL11, CCL24, and CCL26, respectively (3–8). The eotaxins are potent chemoattractants of eosinophils and, thus, are believed to play an important role in the pathogenesis of bronchial asthma (9). Cultured bronchial epithelial cells, including normal bronchial epithelium, had been observed to produce eotaxin-1 after stimulation with tumor necrosis factor (TNF)-{alpha} and T helper (Th) type 2 cytokines (10, 11). Th2 cytokines have been demonstrated to induce eotaxin-3 mRNA expression in a lung epithelial line (12). We have also found that Th2 cytokines strongly induce production of eotaxin-3, but not eotaxin-1 or -2, in cultured bronchial epithelial cells, including normal human bronchial epithelium (13). A study by Berkman and colleagues has shown upregulation of eotaxin-3 mRNA alone in the airways of subjects with atopic asthma 24 h after allergen challenge (14). Thus, induction of eotaxins, specifically eotaxin-3, by Th2 cytokines in bronchial epithelial cells seems to play an important role in the pathogenesis of bronchial asthma, especially the late-phase response.

There has been increasing interest in the relationship between bronchial asthma and various viral infections, particularly rhinovirus (RV) and respiratory syncytial virus (15). Elevated chemokine responses are maintained in the lungs after clearance of viral infections (16). Clinical exacerbations of bronchial asthma are often associated with airway viral infection (17). A virus-infected airway epithelial cell line and primary bronchial epithelial cells secrete a wide range of cytokines and chemokines (18, 19). In addition, interferon (IFN)-{gamma} produced by activated T cells may modulate the chemokine profile produced by bronchial epithelial cells, which may lead to an exacerbation of asthma (18, 19). Indeed, IFN-{gamma} has been shown to induce a number of chemokines, including the regulated on activation, normal T cell expressed and secreted and fractalkine, in a cultured bronchial epithelial cell line and primary cells (20, 21). On the other hand, in a cultured bronchial epithelial cell line, attenuated IL-4– and TNF-{alpha}–induced eotaxin-1 production has been reported after costimulation with IFN-{gamma} (11). Th2 cytokine–induced eotaxin-3 production has also been reported to be attenuated by addition of IFN-{gamma} in cultured bronchial epithelial cell lines and primary cells (22). It seems, however, that these results do not agree with the observation that clinical exacerbations of asthma are associated with airway viral infection (17). In addition, precise mechanisms of inhibitory effect of IFN-{gamma} remain to be elucidated.

We therefore focused on eotaxin-3 and investigated the effect of IFN-{gamma} on Th2 cytokine–induced eotaxin-3 production and the mechanism by which eotaxin-3 production might be modulated by IFN-{gamma} in a human bronchial epithelial cell line, BEAS-2B. The present study demonstrates two opposing effects of IFN-{gamma} on IL-4–induced eotaxin-3 production. IFN-{gamma} inhibits the effect of IL-4 when the two are added simultaneously. When IFN-{gamma} presents prior to IL-4 stimulation, however, it enhances the effect of IL-4. Modulation of IL-4 receptor (IL-4R) expression is likely the mechanism by which enhanced IL-4–induced eotaxin-3 production by IFN-{gamma} occurs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
A human bronchial epithelial cell line, BEAS-2B, was obtained from the American Tissue Culture Collection (Rockville, MD). The cells were cultured in Dulbecco's modified Eagle medium (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 100 U/ml penicillin G, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B (Gibco).

Stimulation of the Cells
When BEAS-2B cells reached 80% confluence in 6 well plates, the culture medium was replaced with Dulbecco's modified Eagle's medium without FBS. The cells were then cultured with various concentrations of IL-13, IL-4, or IFN-{gamma} (Peprotec, London, UK) for different intervals. In some experiments, the cells were pretreated with IFN-{gamma} before IL-4 stimulation. Cycloheximide (CHX) (30 µg/ml) (Sigma) was added along with each of the Th2 cytokines or IFN-{gamma} to inhibit de novo protein synthesis in some experiments. For the inhibitor studies of various signal transduction pathways, cells were pretreated with 10 µM of MG132 (Sigma), or 100 µM each of PD98059 (Sigma), U0126 (Sigma), SB203580 (Sigma), or SP600125 (Biomol, Plymouth Meeting, PA) for 1 h, then cultured with IFN-{gamma} for 12 h.

Measurement of Protein Release into the Culture Medium
Culture supernatant was used to detect the production of eotaxin-3 by sandwich enzyme-linked immunosorbent assay (ELISA) from R&D Systems (Minneapolis, MN), which was performed according to manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Richmond, CA). The minimum detectable dose of eotaxin-3 was 2.3 pg/ml.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from the cells as previously described (23). First-strand cDNA synthesis was then performed from 1 µg of total RNA using oligo (dT) as a primer and reverse transcriptase (Toyobo, Osaka, Japan). A sample of the reaction mixture (0.5 µl), corresponding to 25 ng of total RNA, was combined with 25 µl of the PCR reaction medium, including 0.5 U Taq DNA polymerase (Promega, Madison, WI). Commercially obtained primers were used (Sigma Genosys, Hokkaido, Japan). The eotaxin-3 primers were as previously described (14). Eotaxin-3: forward, 5'-GGA ACT GCC ACA CGT GGG AGT GAC-3'; reverse, 5'-CTC TGG GAG GAA ACA CCC TCT CC-3' (354 bp); ß-actin: forward, 5'-TCC TGT GGC ATC CAC GAA ACT-3'; reverse, 5'-GAA GCA TTT GCG GTG GAC GAT-3' (314 bp); IL-4R{alpha}: forward, 5'-ACA CCA ATG TCT CCG ACA CTC-3'; reverse, 5'-GGA TGA CAA TGC AGG AAA CGC-3' (348 bp); {gamma}C: forward, 5'-TCC GAA GTG CAG CCA CTA TC-3'; reverse, 5'-GAG CCA ACA GAG ATA ACC ACG-3' (510 bp); IL-13R{alpha}1: forward, 5'-ATA GCT CCG GAA ACT CGT CG-3'; reverse, 5'-AAG TAT CAG GAA GAA CAC CAG G-3' (687 bp).

Reverse transcription–polymerase chain reaction (RT-PCR) was performed by denaturation at 94°C for 2 min, followed by denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 2 min. This was performed for eotaxin-3, ß-actin, IL-4R{alpha}, {gamma}C, and IL13R{alpha}1. For eotaxin-3, 28 cycles of amplification were performed to allow for semiquantitative comparison because a plateau in the production of PCR products was observed after 32 cycles (data not shown). For ß-actin, IL-4R{alpha}, {gamma}C, and IL-13R{alpha}1, 20 cycles, 35 cycles, 40 cycles, and 32 cycles were performed, respectively. The number of PCR cycles used was based on the results of prior densitometric analyses (23). Electrophoresis of PCR products was performed on 1.5% agarose gel (Cosmo Bio, Tokyo, Japan) and photographed.

Western Blot Analysis
Total cell extracts were prepared using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% Sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1 µg/ml each of aprotinin, pepstatin and leupeptin, 1 mM Na3VO4, and 1 mM NaF. After centrifugation, supernatant was collected as total cell extract. Protein concentration was determined by the Bradford method (Bio-Rad). A 20 µg aliquot of each sample was subjected to 10% SDS-PAGE. Each gel was transferred to a nitrocellulose membrane (Bio-Rad), which was then blotted with anti–signal transducer and activator of transcription (STAT) 6 (#sc-621) (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-phosphorylated STAT (p-STAT) 6 (#sc-11762-R) (Santa Cruz), which recognizes the phosphorylated Tyrosine-641 of STAT6. To investigate nuclear factor (NF)–{kappa}B activation, an antibody that recognizes the nuclear localization signal region of NF-{kappa}B p50 subunits (Santa Cruz) was used. The blots were developed using an ECL-plus detection kit (Amersham, Uppsala, Sweden).

Flow Cytometry
After detachment, the cells were washed twice with phosphate-buffered saline (PBS) and resuspended in staining buffer (PBS supplemented with 1.0% FBS and 0.1% sodium azide, pH 7.4). The cells were then incubated for 20 min on ice with phycoerythrin-conjugated anti-human IL-4R{alpha} (CD124) (Immunotech, Marseille, France) or anti-human IL-2R{gamma} (CD132; Becton Dickinson, Mountain View, CA). After being washed once in PBS, labeled cells were analyzed on a FACScan (Becton Dickinson) and 10,000 events were collected.

Statistical Analysis
Data are presented as means ± SD. Significant differences were assessed using the paired Student's t test. P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Th2 Cytokine–Induced Eotaxin-3 Production Was Inhibited by IFN-{gamma}
BEAS-2B cells were cultured with varying concentrations of IFN-{gamma} and 50 ng/ml of either IL-4 or IL-13 for 24 h. Both IL-4 and IL-13 induced eotaxin-3 mRNA expression as previously demonstrated (12). Eotaxin-3 production was also induced by IL-4 or IL-13. IFN-{gamma} significantly inhibited Th2 cytokine–induced eotaxin-3 production in a dose-dependent manner (Figures 1A and 1B). Decreased production of eotaxin-3 was associated with decreased eotaxin-3 mRNA expression (Figures 1A and 1B).



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  		Figure 1. Inhibitory effect of IFN-{gamma} on Th2 cytokine–induced eotaxin-3 production and mRNA expression. BEAS-2B cells were incubated with varying concentrations of IFN-{gamma} and 50 ng/ml of either IL-13 (A) or IL-4 (B) for 24 h. Eotaxin-3 released into the culture medium was measured by ELISA. Results shown are means ± SD of values from three separate experiments (*P < 0.01; {dagger}P < 0.05). Eotaxin-3 mRNA expression was investigated by RT-PCR. The ethidium bromide gel shown is representative of results obtained from three experiments.

 
Effect of CHX on Th2 Cytokine–Induced Eotaxin-3 mRNA Expression
We next investigated the effect of CHX, an inhibitor of de novo protein synthesis, on eotaxin-3 gene expression. IL-4–induced eotaxin-3 mRNA expression was significantly inhibited by CHX (Figure 2A). CHX could not reverse the inhibition of IL-4–induced eotaxin-3 mRNA expression by IFN-{gamma}. Similar results were obtained in cells treated with IL-13 and CHX (data not shown). These results indicate that IL-4– or IL-13–induced eotaxin-3 mRNA expression involves de novo protein synthesis. IFN-{gamma} may directly affect cellular signaling of IL-4, leading to eotaxin-3 gene expression.



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  		Figure 2. (A) Effect of CHX on eotaxin-3 mRNA expression. BEAS-2B cells were cultured with 30 µg/ml CHX and/or 50 ng/ml IL-4 and/or 100 ng/ml IFN-{gamma} for 24 h. Eotaxin-3 mRNA expression was determined by RT-PCR. Eotaxin-3 mRNA expression is expressed as a ratio of a ß-actin mRNA expression. Results shown are means ± SD of values from triplicate samples (*P < 0.01; {dagger}P < 0.05). The ethidium bromide gel shown is representative of results obtained in triplicate samples. (B) Effect of IFN-{gamma} on IL-4–induced eotaxin-3 mRNA expression. After induction of eotaxin-3 mRNA by 24 h incubation with 50 ng/ml IL-4, cells were washed with PBS, after which they were treated with ACD alone (black bars), or ACD plus 100 ng/ml IFN-{gamma} (white bars), for the indicated time intervals. Eotaxin-3 mRNA expression is expressed as a ratio of ß-actin mRNA expression. Results shown are means ± SD of values from triplicate samples.

 
IFN-{gamma} Did Not Affect IL-4-Induced Eotaxin-3 mRNA Stability
We investigated the possibility that decreased eotaxin-3 protein production might be regulated by IFN-{gamma} via a decrease in eotaxin-3 mRNA stability. After induction of eotaxin-3 mRNA by IL-4 for 12 h, BEAS-2B cells were cultured with 1 µg/ml actinomycin D (ACD) alone, or in combination with 100 ng/ml IFN-{gamma}. The addition of IFN-{gamma} had no effect on eotaxin-3 mRNA expression before 8 h of culture (Figure 2B).

Addition of IFN-{gamma} Inhibited Th2 Cytokine–Induced STAT6 Activation
STAT6 and p-STAT6 levels were investigated by immunoblotting. IL-4 induced phosphorylation of STAT6 (Figure 3A). When cultured with IFN-{gamma}, p-STAT6 generation was attenuated by IFN-{gamma} in a dose-dependent manner. IL-13 also induced phosphorylation of STAT6, although it produced a much weaker signal intensity than was observed with IL-4. IL-13–induced p-STAT6 generation was also inhibited by IFN-{gamma} in a dose-dependent manner (Figure 3B). However, 24 h pretreatment with IFN-{gamma} showed no inhibitory effect on IL-4–induced p-STAT6 generation; rather, it enhanced p-STAT6 generation (Figure 3C). Similar results were obtained in IL-13–stimulated cells (data not shown).



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  		Figure 3. Effect of IFN-{gamma} on Th2 cytokine–induced STAT6 activation. (A) Cells were incubated with 50 ng/ml IL-4 alone, or IL-4 plus varying concentrations of IFN-{gamma}, for 30 min. Cell lysate samples were analyzed by immunoblotting for STAT6 and p-STAT6, as described in MATERIALS AND METHODS. (B) Cells were incubated with or without IL-13 or IFN-{gamma} as indicated. (C) Cells were pretreated with varying concentrations of IFN-{gamma} for 24 h after 30 min of incubation with 50 ng/ml IL-4. In all experiments, the membranes were exposed to the film for 30 sec. The results presented are from one of two experiments that produced similar results.

 
IFN-{gamma} Induced IL-4R{alpha} mRNA Expression and Protein Expression at the Cell Surface
Cell responses may be influenced by the number of receptors on the surface membrane. We therefore analyzed IL-4R{alpha} mRNA expression. Unexpectedly, 24 h incubation with IFN-{gamma} upregulated the expression of IL-4R{alpha} mRNA in a dose-dependent manner (Figure 4A). Thus, we further analyzed IL-4R{alpha} protein expression at the cell surface by flow cytometry. After 48 h incubation with IFN-{gamma}, dose-dependent upregulation of IL-4R{alpha} surface expression was observed (Figure 4B). A sample of 100 ng/ml IFN-{gamma} also showed a time-dependent increase in IL-4R{alpha} expression at the cell surface (Figure 4C). When the cells were cultured with CHX, constitutive expression of IL-4R{alpha} at the cell surface was significantly inhibited. IFN-{gamma}–induced surface IL-4R{alpha} expression was also inhibited by CHX to a similar degree (Figure 4D). Addition of IFN-{gamma} did not change the rate of IL-4R{alpha} mRNA turnover (Figure 4E). These results indicate that induction of IL-4R{alpha} expression at the cell surface by IFN-{gamma} is the result of de novo protein synthesis after increased IL-4R{alpha} gene transcription.



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  		Figure 4. Effect of IFN-{gamma} on IL-4R{alpha} mRNA expression and protein expression at the cell surface. (A) Cells were incubated with varying concentrations of IFN-{gamma} for 24 h. IL-4R{alpha} mRNA was determined by RT-PCR. IL-4R{alpha} mRNA expression was expressed as a ratio of ß-actin mRNA expression. The results presented are from one of two experiments that produced similar results. (B) IL-4R{alpha} expression at the cell surface was investigated by fluorescence-activated cell sorter using mouse anti-human IL-4R{alpha} conjugated with phycoerythrin. Cells were incubated for 48 h with or without IFN-{gamma}. Dotted line, cells not treated with IFN-{gamma}, labeled with isotype-matched mouse IgG; shaded area, cells not treated with IFN-{gamma}, labeled with mouse anti-human IL-4R{alpha}; thin line, cells treated with 1 ng/ml of IFN-{gamma}, labeled with anti-IL-4R{alpha}; thick line, cells treated with 100 ng/ml of IFN-{gamma}, labeled with anti-IL-4R{alpha}. (C) Kinetics of IL-4R{alpha} expression at the cell surface over time. Cells were incubated with 100 ng/ml IFN-{gamma} for 0 h (shaded area), 24 h (thin line), 48 h (dotted line), and 72 h (thick line). (D) Effect of CHX on IFN-{gamma}–induced IL-4R{alpha} expression at cell surface. Cells were treated with CHX alone (dotted line), CHX plus 100 ng/ml of IFN-{gamma} (thin line), and 100 ng/ml IFN-{gamma} alone (thick line) for 72 h. Shaded area, constitutive expression of IL-4R{alpha}. (E) Cells were treated with ACD alone (black columns), or ACD plus 100 ng/ml IFN-{gamma} (white columns), for the indicated intervals. IL-4R{alpha} mRNA expression is expressed as a ratio of ß-actin mRNA expression. Results shown are means ± SD of values from triplicate samples.

 
IFN-{gamma} Induced IL-2R{gamma} Expression
Bronchial epithelial cells have been observed to express both type 1 and type 2 IL-4Rs (24, 25). We therefore investigated the effect of IFN-{gamma} on expression of the common {gamma} chain of the IL-2R ({gamma}C), which is a component of the type 1 IL-4R. Although no apparent increase in mRNA expression was observed (data not shown), flow cytometry analysis revealed that 48 h incubation with IFN-{gamma} upregulated {gamma}C protein expression at the cell surface (Figure 5A). {gamma}C protein upregulation was not observed in cells incubated with IL-4 or IL-13 (Figures 5B and 5C). Constitutive mRNA expression of IL-13R{alpha}1, a component of the type 2 IL-4R, was also observed, although upregulation by IFN-{gamma} was not observed (data not shown).



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  		Figure 5. Effect of IFN-{gamma} on {gamma}C protein expression at the cell surface. Cells were incubated with either IFN-{gamma} (A), IL-4 (B), or IL-13 (C) for 48 h. The shaded area indicates those cells not incubated with IFN-{gamma} labeled with rat anti-human {gamma}C. (A) Dotted line, cells labeled with isotype-matched rat IgG; Narrow line, cells incubated with 1 ng/ml of IFN-{gamma}; Bold line, cells incubated with 100 ng/ml of IFN-{gamma}; (B) Thin line, cells incubated with 50 ng/ml of IL-4; (C) Thin line, cells incubated with 50 ng/ml IL-13.

 
IFN-{gamma} Pretreatment Increased IL-4–Induced Eotaxin-3 Production
Next we investigated whether increased IL-4R{alpha} surface expression enhanced IL-4–induced eotaxin-3 production. BEAS-2B cells were pretreated with IFN-{gamma} before IL-4 stimulation. Pretreatment with 100 ng/ml of IFN-{gamma} for 2 d resulted in a significant increase in IL-4–induced eotaxin-3 mRNA expression (Figure 6A). Although no significant enhancement of eotaxin-3 production was observed after 24 h pretreatment (data not shown), 48 h pretreatment with IFN-{gamma} significantly enhanced IL-4–induced eotaxin-3 production (Figure 6B).



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  		Figure 6. Effect of IFN-{gamma} pretreatment on IL-4–induced eotaxin-3 mRNA expression and protein production. (A) Cells were incubated with varying concentrations of IFN-{gamma} for 48 h. Cells were then washed with PBS and incubated with 100 ng/ml IL-4 for 24 h. Eotaxin-3 mRNA expression is expressed as a ratio of ß-actin mRNA expression. Results shown are means ± SD of values from triplicate samples (*P < 0.01; {dagger}P < 0.05). (B) Cells treated with no IFN-{gamma} (black columns), 1 ng/ml IFN-{gamma} (hatched columns) or 100 ng/ml IFN-{gamma} (white columns), for 48 h were washed with PBS, then stimulated with varying concentrations of IL-4, as indicated, for 24 h. Eotaxin-3 release into the culture medium was determined by ELISA. Results shown are means ± SD of values from triplicate samples (*P > 0.05; {dagger}P < 0.01).

 
NF-{kappa}B Inhibitor Inhibited IFN-{gamma}–Induced IL-4R{alpha} Expression
To study which cellular signals result in IL-4R{alpha} gene expression, we investigated the effect of MG132, an inhibitor of NF-{kappa}B, on IFN-{gamma}–induced IL-4R{alpha} mRNA expression and expression at the cell surface. IFN-{gamma} induced NF-{kappa}B activation in a dose-dependent manner (Figure 7A). A 1 h pretreatment with MG132 significantly inhibited IFN-{gamma}–induced IL-4R{alpha} mRNA expression, but had no apparent effect on constitutive expression (Figure 7B). MG132 also inhibited IFN-{gamma}–induced IL-4R{alpha} expression at the cell surface (Figure 7C). PD98059 and U0126, inhibitors of mitogen-activated protein (MAP)/extracellular signal-regulated kinase kinase 1/2 in the p44/p42 MAP kinase (MAPK) pathway, and SP600125, an inhibitor of the c-JUN N-terminal kinase MAPK pathway, also inhibited IFN-{gamma}–induced IL-4R{alpha} mRNA expression. However, SB203580, an inhibitor of p38 in the MAPK pathway, failed to inhibit IFN-{gamma}–induced IL-4R{alpha} mRNA expression (Figure 7B).



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  		Figure 7. Effect of selective inhibitors of transduction pathways on IFN-{gamma}–induced IL-4R{alpha} expression. (A) Immunoblotting of NF-{kappa}B p50 subunit. Cells were incubated with varying concentration of IFN-{gamma} for 30 min. (B), Cells were pretreated with vehicle (dimethyl sulfoxide), MG132 (MG), PD98059 (PD), U0126 (U), SB203580 (SB) or SP600125 (SP), for 1 h. Cells were then incubated with (black bars) or without (white bars) 100 ng/ml IFN-{gamma} for 12 h. Gray bar indicates constitutive expression of IL-4R{alpha} mRNA (cons.). IL-4R{alpha} mRNA expression is expressed as a ratio of ß-actin mRNA expression. Results shown are means ± SD of values from triplicate samples. The ethidium bromide gel shown is a result of the cells treated with IFN-{gamma}. A representative of RT-PCR from three separate experiments is shown. (C), Cells were pretreated with MG132 or vehicle as described above. Cells were then incubated with 100 ng/ml IFN-{gamma} for 72 h. Shaded area, cells pretreated with vehicle and incubated without IFN-{gamma}; thick line, cells pretreated with vehicle and incubated with IFN-{gamma}; thin line, cells pretreated with MG132 and incubated with IFN-{gamma}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed inhibition of Th2 cytokine–induced eotaxin-3 production from bronchial epithelial cells by costimulation with IFN-{gamma}. This inhibited eotaxin-3 production was linked with a reduction in eotaxin-3 mRNA expression. Because IFN-{gamma} did not affect the turnover of eotaxin-3 mRNA, the inhibited eotaxin-3 production must be due to decreased eotaxin-3 gene transcription. We showed attenuation of IL-4– and IL-13–induced STAT6 activation by IFN-{gamma}. These results suggest that, although the precise mechanism is unknown, IFN-{gamma} directly inhibits IL-4–mediated signal transduction pathways. However, incomplete inhibition of IL-4–induced STAT6 activation by IFN-{gamma} was observed; thus, there seems to be less attenuation of STAT6 activation than eotaxin-3 protein production. 100 ng/ml IFN-{gamma} resulted in an almost 80% decrease in protein production, whereas densitometric analysis showed only a 50% decrease in p-STAT6 intensity. This suggests that other mechanisms of inhibition of Th2 cytokine–induced eotaxin-3 production by IFN-{gamma} might exist (26).

Very recently, Sato and colleagues (27) demonstrated that IFN-{gamma} inhibits IL-4– and TNF-{alpha}–induced eotaxin-1 production in mouse embryonic fibroblasts. The study demonstrated that IFN-{gamma} inhibited phosphorylation of STAT6 at 6 h after stimulation but not immediately after stimulation. The study also showed that inhibition of STAT6 phosphorylation is mediated by suppressor of cytokine signaling–1, which was induced by IFN-{gamma}–induced STAT1 activation. However, immediate inhibition of phosphorylation of STAT6 after addition of IFN-{gamma} was observed in the present study. Although this discordance between the present study and that of Sato and colleagues (27) may depend on the difference of cell type used in the experiments, immediate inhibition of phosphorylation of STAT6 in this study may suggest that IFN-{gamma} inhibits eotaxin-3 mRNA expression by suppressor of cytokine signaling–1–independent pathway in BEAS-2B cells. Interestingly, 24 h pretreatment with IFN-{gamma} had no inhibitory effect on IL-4–induced STAT6 phosphorylation and subsequent eotaxin-3 protein production in the present study. These observations indicate that the inhibitory effect of IFN-{gamma} on STAT6 phosphorylation is transient. Thus, effects on other signal transduction pathways may play a larger role in the response to IFN-{gamma} (26). We suggest that induction of IL-4R{alpha} gene expression may be one such pathway.

Receptor regulation is an important mechanism by which several mediators, including IL-4 and IL-13, work. IL-4 and IL-13 signal through IL-4R complexes (24). Two types of IL-4Rs have been demonstrated: the type 1 IL-4R is composed of IL-4R{alpha} and {gamma}C subunits and is primarily expressed in hematopoietic cells (24, 25); the type 2 IL-4R is composed of IL-4R{alpha} and IL-13R{alpha}1 subunits and is primarily expressed in nonhematopoietic cells (24, 28). In bronchial epithelial cells, both types of IL-4R are present; thus, IL-4 and IL-13 are thought to signal through both types of IL-4R in bronchial epithelial cells (29, 30). In bronchial epithelial cells, a combination of phorbol acetate and calcium ionophore have been shown to upregulate IL-4R{alpha} expression, whereas cytokines, such as IL-1ß, IL-4, and IL-6, have not been observed to upregulate IL-4R{alpha} expression (29). Among other cell types, such as human mononuclear cells and B lymphocytes, IL-4R{alpha} upregulation by IL-4 has been shown; specifically, increased IL-4R expression has been observed to play a role in IL-4–induced responses in B cells (31). Inhibition of IL-4–induced upregulation of IL-4R{alpha} expression has been observed with IFN-{gamma} and IFN-{alpha} (32). The mechanism for this has been identified as accelerated decay of IL-4–induced IL-4R mRNA (32). Thus, we first investigated the possibility that the inhibitory effect of IFN-{gamma} on IL-4–induced eotaxin-3 production in bronchial epithelial cells might be due to decreased expression of IL-4R{alpha}. However, upregulation of IL-4R{alpha} mRNA expression by IFN-{gamma} was observed in cells after 24 h of incubation. Although minimal upregulation of surface IL-4R{alpha} protein was observed after 24 h, a significant increase in IL-4R{alpha} protein expression was observed after 48 h of stimulation with IFN-{gamma}. In addition, upregulation of {gamma}C protein, a component of the type 1 IL-4R, was also observed. However, increased mRNA expression of {gamma}C was not observed by RT-PCR, even though the primer pair clearly amplified {gamma}C mRNA when peripheral blood mononuclear cells were used as a positive control (13). These results may suggest post-transcriptional or post-translational upregulation of {gamma}C by IFN-{gamma} in bronchial epithelial cells. We also checked the effect of IL-4 on the mRNA and protein expression of IL-4R{alpha}, during which no changes in IL-4R{alpha} expression were observed. This is in keeping with previous results (29, 30). On the other hand, IL-13 might inhibit IL-4R{alpha} and {gamma}C expression at the cell surface (Figure 5C and unpublished observations).

Thus, the current study demonstrated increased IL-4–induced eotaxin-3 production after 2-d pretreatment with IFN-{gamma}. Although direct evidence is lacking, our results strongly suggest that upregulation of IL-4R protein expression at the cell surface resulted in increased IL-4–induced eotaxin-3 production. Eotaxin-3 release into the culture medium was observed to plateau, despite increasing doses of IL-4, in cells not pretreated with IFN-{gamma}. However, cells pretreated with 100 ng/ml of IFN-{gamma} showed continued significant dose-dependent increases in eotaxin-3 production. Again, this may have been due to an IFN-{gamma}–induced increase in IL-4R expression at the cell surface. Furthermore, the observation that IL-4–induced p-STAT6 generation was enhanced by 24 h pretreatment with IFN-{gamma} also suggests upregulation of IL-4R, although fluorescence-activated cell sorter analysis showed only a slight increase in IL-4R expression at the cell surface.

Acute viral infections, such as respiratory syncytial virus and RV, have been observed in association with acute exacerbations of bronchial asthma in both children and adults (33, 34). Airway epithelial cells are important initiators of the immune response to viruses through the secretion of inflammatory cytokines and chemokines (35). Induction of adhesion molecules, including intercellular adhesion molecule–1, by RV infection has been demonstrated (36). IFN-{gamma}, which is secreted by lymphocytes in response to RV, is a potent inducer of regulated on activation, normal T cell expressed and secreted and fractalkine, in addition to intercellular adhesion molecule–1 (19, 20). Our study also suggests the possibility that virally induced IFN-{gamma} may modulate allergic airway inflammation through the production of eotaxin-3 by bronchial epithelial cells. IFN-{gamma} may suppress allergic inflammation during the early stages of viral infection; however, it may result in exacerbations during the convalescent stage. Our study has revealed a novel mechanism of modulation of allergic inflammation by IFN-{gamma} through upregulation of IL-4R. IFN-{gamma} exerts its effects on cells by interacting with a specific receptor that is expressed on nearly all cell surfaces. In addition to the established notion that IFN-{gamma} signals via the Janus kinase–STAT pathway, STAT-1–independent pathways have been implicated in IFN-{gamma}–dependent signaling (26). Our study also suggests a role for NF-{kappa}B, p44/42 MAPK, and c-JUN N-terminal kinase MAPK pathways in IFN-{gamma}–induced IL-4R{alpha} gene expression.

In summary, these in vitro experiments demonstrate that IFN-{gamma} modulates Th2 cytokine–induced eotaxin-3 production in bronchial epithelial cells. This finding suggests that secretion of IFN-{gamma} by T cells and NK cells within the airway during the early stages of a viral infection might suppress Th2 cytokine–induced eotaxin-3 gene expression through inhibition of p-STAT6 generation. There also may be enhancement of Th2 cytokine–induced eotaxin-3 production through upregulation of IL-4R at the cell surface in bronchial epithelial cells during the convalescence stage of viral infection. The enhanced eotaxin-3 production might result in additional recruitment of eosinophils into the airway. We propose a novel mechanism of exacerbations of bronchial asthma during viral infection, in which modulation of IL-4R by IFN-{gamma} is involved (Figure 8). Further understanding of effects of IFN-{gamma} on eotaxins and IL-4Rs may lead to new insights regarding the relationship between viral infections and allergic airway inflammation.



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  		Figure 8. Proposed mechanism of exacerbations of asthma during viral infection. Virus infection induces secretion of several chemokines from airway epithelium. These chemokines recruit many kinds of inflammatory cells to the airway mucosa, which results in airway inflammation. In addition, IFN-{gamma} secreted from activated T cells enhances IL-4R expression at the cell surface of airway epithelium. Increased expression of IL-4R allows enhanced IL-4R–mediated signal followed by increased eotaxin-3 production. Eotaxin-3 further recruits eosinophils to the airway. Thus, in subjects with asthma, exacerbation of asthma may occur.

 

   Acknowledgments
 
This study was supported, in part, by a grant from the Ministry of Education, Science, Sports, and Culture of Japan (C2–14570753).


   Footnotes
 
Conflict of Interest Statement: S.Y. has no declared conflicts of interest; I.K. has no declared conflicts of interest; K.T. has no declared conflicts of interest; N.N. has no declared conflicts of interest; E.M. has no declared conflicts of interest; M.M. has no declared conflicts of interest; M.Z. has no declared conflicts of interest; S.I. has no declared conflicts of interest; T.I. has no declared conflicts of interest; and Y.H. has no declared conflicts of interest.

Received in original form April 22, 2004

Received in final form June 8, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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