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The Journal of Immunology, 2000, 165: 5472-5479.
Copyright © 2000 by The American Association of Immunologists

IFN-{gamma} and IFN-{alpha} Posttranscriptionally Down-Regulate the IL-4-Induced IL-4 Receptor Gene Expression1

Eui-Young So, Hyun-Hee Park and Choong-Eun Lee2

Department of Biological Science and Institute for Basic Science, SungKyunKwan University, Suwon, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As Th1 and Th2 cytokines, IFN-{gamma}/{alpha} and IL-4 counterregulate diverse immune functions. In particular, IFN-{gamma} and IFN-{alpha} have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (Fc{epsilon}RII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-{gamma}/{alpha} on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R({alpha}) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R({alpha}) is significantly down-regulated by both IFN-{gamma} and IFN-{alpha} to a similar extent. The inhibitory effects of IFN-{gamma}/{alpha} on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-{gamma}/{alpha} is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced Fc{epsilon}RII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytokines are protein mediators that serve as a communication link between cells in the immune system controlling innate as well as adaptive defense mechanisms. The intricate regulation of immune response, therefore, is performed through a complex network interaction among diverse cytokines. Some of the most prominent features of the cytokine network include cascade reaction, synergism, and antagonism. As the Th1 vs Th2 paradigm has emerged as a critical concept governing cellular and humoral immunity, the elucidation of mechanisms underlying the counterregulation by Th1 and Th2 cytokines can provide an important clue to our understanding of the immune regulation network (1). IFN-{gamma} and IL-4 are prototypic Th1 and Th2 cytokines, respectively, whose antagonistic actions against each other have been well known. These include Th1/Th2 cell differentiation, IgG subclass switching, IgE production, and modulated expression of class II MHC, IL-1R, Fc{gamma}RI, and Fc{epsilon}RII (2, 3, 4, 5, 6, 7, 8, 9).

IL-4 is a pluripotent cytokine whose regulatory effects on cell growth and differentiation are exerted in diverse cell types, such as B cells, T cells, and monocytes, as well as cells of nonhemopoietic origin (10). The signal transmission of IL-4 is mediated by the high affinity receptor, of which two types are known: type I IL-4R composed of IL-4R {alpha}-chain (p140) plus common {gamma}-chain, and type II receptor composed of IL-4R{alpha} plus IL-13R{alpha} (11). Although IFN-{gamma} and IFN-{alpha} are primarily produced by different cell types and act on target cells through distinct receptors, IFN-{alpha} has been also recognized as a cytokine promoting Th1 differentiation (12, 13). Thus, its regulatory effects on the IL-4 action have recently become a subject of interest. In fact, IFN-{gamma} and IFN-{alpha} both effectively suppress the IL-4-induced IgE production (4, 14) and the low affinity IgE receptor (Fc{epsilon}RII/CD23) expression (9, 15). It has been reported that while the inhibitory action of IFN-{gamma} on the IgE production is exerted by a direct suppression of the IL-4-induced IgE C region transcript transcription (16), the down-regulation of the IL-4-induced CD23 by IFN-{gamma} mainly involves posttranscriptional inhibition of CD23 mRNA (17). The mechanism of IFN-{alpha} inhibition on the IL-4-evoked responses, however, remains largely unknown.

Because receptor modulation is an important event during cytokine signal transduction, we have been studying the molecular mechanism of the IL-4R regulation as a part of our ongoing investigation on the mechanism of IgE production and responses. Previously, we have reported that IL-4 and anti-CD40 up-regulate IL-4R via tyrosine kinase-dependent pathways, and that the costimulatory effect of anti-CD40 on the IL-4-induced response in B cells is partly due to the increase in IL-4R expression by CD40-mediated signal (18). In the present study, we have examined regulation mechanisms of IFN-{gamma} and IFN-{alpha} on the IL-4R({alpha}) expression, and present strong evidence that in human primary immune cells IFN-{gamma} and IFN-{alpha} both down-regulate the IL-4-induced IL-4R expression as a delayed response requiring an IFN-induced protein factor, and the inhibition occurs not at the transcriptional, but at the posttranscriptional level by decreasing stability of IL-4R mRNA. The accelerated decay of IL-4R mRNA may serve as a means to suppress the IL-4-induced response and thus constitutes one of the mechanisms underlying the counteraction between IL-4 and IFNs. Furthermore, when taken together with our previous report on the mechanism of IFN-{gamma} inhibition of Fc{epsilon}RII/CD23 expression (17), mRNA destabilization of the IL-4-stimulated genes may be a common mechanism by which type I and II IFNs counterregulate the IL-4 response in various immune functions such as allergy, inflammation, and infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

Mononuclear cells were isolated from freshly excised human tonsils or peripheral blood using Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density-gradient centrifugation. Typically, the mononuclear preparation from tonsils contained about 70% B cells, 25~30% T cells, and less than 5% non-B/T cells, including monocytes. B cells were further purified from mononuclear cells by negative selection after rosetting twice with 2-aminoethylisothiouronium bromide-treated SRBC and subsequently removing adherent cells. The purity of B cells was determined by staining with anti-CD20, anti-CD3, and anti-CD14 mAbs, and confirmed to be >=98%. Cells were cultured in RPMI media containing 10% FBS (Life Technologies, Grand Island, NY), 10 mM HEPES, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 50 µg/ml gentamicin, and 50 µg/ml amphotericin B (Sigma, St. Louis, MO). Recombinant human IL-4 (R&D Systems, Minneapolis, MN, and KRIBB, Tajeon, Korea), IFN-{gamma} (R&D Systems, and LG Biotech, Tajeon, Korea), IFN-{alpha} (Schering-Plough, Madison, NJ), cycloheximide (CHX),3 and actinomycin D (Sigma, St. Louis, MO) were added to cells at indicated times and the cells were cultured in humidified 5% CO2 at 37°C.

Flow cytometric analysis of IL-4R

Mononuclear or purified B cells (1 x 106) were cultured in the presence of IL-4, IFN-{gamma}, or IFN-{alpha} for 48 h. The cells were washed with PBS and recultured for 2 h in the fresh media according to Zuber et al. (19). The levels of IL-4R expression were then analyzed by staining cells with mouse anti-IL-4R({alpha}) mAbs (M56, kindly provided by Dr. S. Gillis, Immunex, Seattle, WA) as a primary Ab and goat anti-mouse IgG FITC (Immunotech, Marseille, France) as a secondary Ab in HBSS containing 3% FBS and 1% NaN3 for 30 min and 4°C using fluorescence-activated cell scanner (FACSCalibur; Becton Dickinson, Mountain View, CA). An aliquot of each treated cell sample was stained with the secondary Ab alone as control. The surface IL-4R levels were expressed as the mean fluorescence intensity (MFI) (7). {Delta}MFI was calculated as MFI of cells stained with anti-IL-4R and anti-mouse IgG FITC - MFI of cells stained with anti-mouse IgG FITC alone. Each experiment was repeated several times and the values represent a mean of two independent determinations.

Northern analysis of IL-4R mRNA

Total cytoplasmic RNAs were isolated from mononuclear or purified B cells (1 x 108) after treatment with IL-4, IFN-{gamma}, or IFN-{alpha} with and without actinomycin D or CHX for various durations, as indicated, using guanidium isothiocyanate and cesium chloride through ultracentrifugation. For Northern blots, 10 µg of total RNA from each preparation was separated on a 1% agarose-formaldehyde gel, and transferred to nylon membranes (Genescreen Plus; New England Nuclear, Boston, MA). A cDNA probe of IL-4R({alpha}) (provided by Dr. S. Gillis, Immunex) or STAT6 (provided by Dr. B. Groner, Goethe University, Frankfurt, Germany) was labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) at sp. act. of ~5 x 108 cpm/µg and used for hybridization. RNA concentration was determined by OD measurement, and the amount of loaded RNA on the gel was confirmed by ethidium bromide (EtBr) (8) staining. Blots were reprobed using an adenosyl phosphoribosyl transferase (APRT) (9) probe as internal control (20). Northern analyses were conducted several times for each experiment, and a representative blot is shown.

EMSA (10)

Cells were pretreated with IFN-{gamma} or IFN-{alpha} for 3–24 h and stimulated with IL-4 for indicated durations, after which nuclear extract preparations and EMSA were performed essentially as described (21). IL-4-responsive element (IL-4RE) (11) sequence (Fc{epsilon}RIIb IFN-{gamma}-activated site (GAS) (12): 5'-GGGTGAATTTCTAAGAAAGGG-3') was labeled using [{alpha}-32P]dCTP and Klenow, and the binding reaction with the nuclear extract was performed in the buffer containing 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM NaF, and 2 µg poly(dI)·(dC) for 20 min at room temperature. Mobility shift of the oligomer was then analyzed by a 5% PAGE in 0.5x TBE buffer.

Immunoprecipitation and immunoblots

Cells were pretreated with IFNs for 3–24 h and stimulated with IL-4 for indicated periods, after which total cell extracts were prepared using a lysis buffer containing 1% Nonidet P-40, as described (22). The extracts (1–2 mg) were immunoprecipitated by incubating with rabbit polyclonal anti-STAT6 Abs (Upstate Biotechnology, Lake Placid, NY), and then with anti-rabbit IgG agarose, after which precipitated samples were subjected to 10% denaturing SDS-PAGE. Gels were transferred to nitrocellulose membrane, which were then blotted with anti-phosphotyrosine mAbs (4G10; Upstate Biotechnology), and reprobed with anti-STAT6 Abs after stripping. The blots were developed using an enhanced chemiluminescence detection kit (Amersham).

Nuclear run-on transcription

Nuclei were prepared by incubating cells (1 x 108) for 5 min on ice in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, 0.3 M sucrose, and 0.2% Nonidet P-40. In vitro transcription reactions were then performed in 200 µl reaction buffer (10 mM Tris-HCl, pH 7.5, 35% glycerol, 5 mM MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, and 4 mM each of ATP, CTP, GTP, and 200 µCi [{alpha}-32P]UTP (3000 Ci/mmol; Amersham)). The nuclei were digested with RNase-free DNase I and proteinase K. Nuclear RNA was then purified as described (23) and used to hybridize linearized plasmids containing 2 µg each of IL-4R({alpha}) cDNA, GAPDH (13), cDNA, and pBluescript DNA, which were previously blotted on nylon membranes. Conditions for hybridization and washing were as described by Celano et al. (24).

Densitometric analysis

Densitometric analysis was performed to quantify the relative intensity of radioactive bands using Image QuaNT phosphor imager system (Bio-Rad, Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} and IFN-{alpha} both suppress the IL-4-induced IL-4R expression in human primary immune cells

Although it has been widely reported that IFN-{gamma}/{alpha} often antagonize the ability of IL-4 to induce specific gene expression in various cell types, relatively little is known about the mechanism by which IFNs down-regulate such IL-4-induced responses. In fact, results from several studies have indicated that the detailed mechanism used by IFNs to inhibit the IL-4-induced response may differ depending on biological effects and cell types analyzed (9, 16, 25, 26, 27, 28, 29). Since we have been studying the mechanism of counterregulation between IL-4 and IFN-{gamma} in human B cells, we have first analyzed the regulatory effects of IFN-{gamma}/{alpha} on the IL-4-induced IL-4R({alpha}) expression using tonsillar mononuclear cells that represent an enriched human B cell source. As shown in Fig. 1Go, IL-4 induced a noticeable increase (2- to 3-fold) in surface IL-4R({alpha}) expression on these cells as analyzed by flow cytometry, and IFN-{gamma} (type II IFN) significantly inhibited the IL-4-induced IL-4R({alpha}) expression in a dose-dependent manner. IFN-{alpha} (type I IFN) also exerted a similar inhibitory effect. At 100 ng/ml, IFN-{gamma} and IFN-{alpha} produced about 80% and 70% inhibition of IL-4-induced IL-4R({alpha}), respectively. With B cells further purified from the tonsillar mononuclear cells, basically the same response was observed (Fig. 2GoA). In addition, very similar results were obtained with other sources of human primary immune cells, such as purified B cells isolated from peripheral blood of normal donor (Fig. 2GoB). Through multiple experiments, we have confirmed that these results were reproducible with tonsil or blood samples obtained from different donors. The data strongly indicate that antagonistic regulation of IL-4R by IL-4 and IFN-{gamma}/{alpha} occurs in human primary immune cells.



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  		FIGURE 1. Inhibitory effects of IFN-{gamma} or IFN-{alpha} on the IL-4-induced surface IL-4R expression in tonsillar mononuclear cells. Tonsillar mononuclear cells (2 x 106/well) were treated with media alone, IL-4 (5 ng/ml), IL-4 plus IFN-{gamma} (10–500 ng/ml), or IL-4 plus IFN-{alpha} (2–100 ng/ml), as indicated, and cultured for 48 h, after which surface IL-4R expression was measured by FACScalibur analysis, as described in Materials and Methods. FACS histogram shown is a representative of multiple experiments. A, Effect of IFN-{gamma}; B, effect of IFN-{alpha}.

 


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  		FIGURE 2. Inhibitory effects of IFN-{gamma} or IFN-{alpha} on the IL-4-induced IL-4R expression on purified B cells from tonsil or peripheral blood. Purified B cells (2 x 106/well) from tonsils (A) and peripheral blood (B) were treated with media alone, IL-4 (5 ng/ml), and IL-4 plus IFN-{gamma} (200 ng/ml) or IFN-{alpha} (100 ng/ml), as indicated. Cell culture and analysis of surface IL-4R expression were done as in Fig. 1Go. FACS histogram shown is a representative of multiple experiments.

 
IFN-{gamma} and IFN-{alpha} inhibit IL-4R mRNA expression as a delayed response requiring de novo protein synthesis

To further examine the mechanism of IFN inhibition of the IL-4-induced surface IL-4R, we have analyzed the kinetics of inhibition at IL-4R mRNA level. Cells were first treated with IL-4 for at least 6 h to initiate induction of IL-4R and then cultured in the absence or presence of IFN-{gamma} or IFN-{alpha} for various periods. Fig. 3GoA shows that IL-4-induced IL-4R mRNA gradually accumulates with time. In fact, we have previously reported that IL-4R mRNA starts to increase after 2~4 h of IL-4 stimulation in tonsillar B cells, and becomes saturated at about 12 h posttreatment (18). Fig. 3Go also demonstrates that the counterregulatory effect of IFNs on the IL-4-induced gene expression appears as a rather delayed response. The suppressive effect of IFN-{gamma} was not apparent at 2 h, and the inhibition became prominent by 8 h after IFN-{gamma} treatment (A). In case of IFN-{alpha}, the inhibition was not significant up to 4–6 h and was gradually evident by 8–12 h after IFN-{alpha} treatment (B). These data indicate that the inhibitory action of IFNs requires a lag period of 6–8 h. Thus, we wanted to examine the effect of IFN pretreatment before IL-4 stimulation. As expected, pretreatment of B cells with IFN-{gamma} or IFN-{alpha} for 12 h followed by IL-4 treatment for 12 h yielded an effective inhibition of the IL-4 response (Fig. 4Go). Importantly, CHX (14), a translational inhibitor, abolished the suppressive effect of IFN-{gamma} or IFN-{alpha}; i.e., in the presence of CHX, IFN-{gamma} had no inhibitory effect on the IL-4-inducible level of IL-4R mRNA (A, lanes 5–7). The same effect was observed for the case of IFN-{alpha} (B, lanes 4 and 5). CHX itself caused only slight change (0.9–1.1-fold) in the control or the IL-4-inducible level of IL-4R mRNA. The control STAT6 blot (C) demonstrates that IL-4 or IFNs do not apparently affect the STAT6 mRNA level under our experimental conditions, ruling out a possibility that counterregulation of IL-4R mRNA by IL-4 and IFN-{gamma} occurs via modulation of STAT6 gene expression.



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  		FIGURE 3. Kinetics of IFN-{gamma}/{alpha} inhibition of the IL-4-induced IL-4R mRNA levels. Tonsillar mononuclear cells (1 x 108/well) were treated with IL-4 (5 ng/ml) for 6 h (A) or 12 h (B), after which 100 ng/ml of IFN-{gamma} (A) or IFN-{alpha} (B) was added. After culturing for indicated periods, cells were harvested and processed for IL-4R mRNA analysis. RNA blots were normalized with EtBr staining (A) or with reprobing with APRT (B).

 


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  		FIGURE 4. Down-regulation of the IL-4-induced IL-4R mRNA by IFN-{gamma} or IFN-{alpha} and effect of CHX. Tonsillar B cells were pretreated with IFN-{gamma} (A) or 100 ng/ml IFN-{alpha} (B) for 12 h, after which 5 ng/ml IL-4 was added, and cultured for additional 12 h. Where indicated, cells were treated with CHX (30 µg/ml) for 4 h and washed before IFN treatment. The membranes were stripped and reprobed with APRT and then with STAT6. Top, IL-4R Northern blot; middle, APRT blot; bottom, STAT6 blot. Through densitometric analysis, fold induction of IL-4R mRNA is calculated and shown as bar graphs using APRT level as an internal control.

 
These results suggest that while IL-4-induced IL-4R mRNA is a primary response, the inhibitory action of IFN-{gamma}/{alpha} on the IL-4-stimulated IL-4R gene expression is delayed, requiring a lag period and ongoing protein synthesis. The observed lag period for IFN inhibition seems to be necessary for the production of an IFN-induced protein factor, i.e., a liable and/or newly synthesized protein factor playing a role in mediating the inhibitory action of IFNs on the IL-4R gene expression.

Pretreatment of IFN-{gamma} or IFN-{alpha} only transiently attenuates STAT6 activation without affecting IL-4R transcription

Although STAT6 has been strongly implicated in the activation of several IL-4-inducible genes, such as IgE C region transcript, Fc{epsilon}RII, IL-4, and IL-1R and murine IL-4R (30, 31), direct role of STAT6 in human IL-4R gene regulation has not been reported. Therefore, we wanted to examine the possibility that the inhibitory effect of IFN-{gamma}/{alpha} on the IL-4R mRNA is exerted through the attenuation of STAT6 activity by IFNs, thereby regulating IL-4-induced IL-4R gene activation. We conducted STAT-DNA-binding assays using an IL-4RE GAS probe as a STAT6-binding sequence. As shown in Fig. 5GoA, IL-4 induced specific activation of a STAT factor, which binds to the IL-4RE sequence. The immunoreactivity of the complex to anti-STAT6 Ab confirmed that the IL-4-activated factor is STAT6 (18). The pretreatment of IFN-{gamma} or IFN-{alpha} for various periods caused a gradual inhibition of the IL-4-mediated STAT6 binding to the IL-4RE at 30 min post-IL-4 stimulation (Fig. 5GoA). The immunoprecipitation and Western blot analysis of total cellular extracts revealed that IL-4-stimulated tyrosine phosphorylation of STAT6, and IFN-{gamma} or IFN-{alpha}, upon pretreatment, suppressed the IL-4-induced tyrosine phosphorylation of STAT6 (Fig. 5GoB). To examine whether the ability of IFNs to modulate STAT6 activity correlates with IFN regulation of IL-4-induced IL-4R mRNA, as observed in Fig. 4Go, cells were pretreated with IFN-{gamma} or IFN-{alpha} for 16 h, a duration sufficient to exert inhibitory effects, and further incubated with IL-4 for 4–10 h to allow substantial induction of IL-4R mRNA by IL-4. Under this condition, however, no inhibitory effects of IFNs on STAT6 activity were observed (Fig. 5GoC). There was also no apparent inhibitory effects of IFNs on the tyrosine phosphorylation of STAT6 (data not shown). To find out whether even transient attenuation of STAT6 activity by IFNs underlies the molecular mechanism of IFN-mediated down-regulation of IL-4R mRNA level via suppression of IL-4-induced IL-4R gene transcription, we have performed nuclear run-on assays. In Fig. 6Go, we have observed an enhancement of IL-4R gene transcription by IL-4. Densitometric analysis revealed that IL-4 induced a significant increase (2- to 3.9-fold) in nuclear IL-4R transcription over untreated samples, while the levels of control GAPDH transcripts were basically not affected by IL-4. The treatment of IFN-{gamma} or IFN-{alpha} at least for 12 h, a duration sufficient for the manifestation of IFN-induced down-regulation of IL-4R mRNA, however, did not affect the IL-4-activated IL-4R gene transcription. These results indicate that while IFN-{gamma} and IFN-{alpha} can cause the transient inhibition of the IL-4-induced STAT6 activation, they do not adversely affect the overall transcriptional activity of IL-4 gene, suggesting that modulation of STAT6 activity is not responsible for the down-regulation of IL-4-induced IL-4R gene expression by IFNs in these cells.



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  		FIGURE 5. Inhibitory effect of IFN-{gamma} or IFN-{alpha} on the IL-4-induced STAT6 activity is only transient and is not sustained throughout IL-4 treatment. A, B cells were pretreated with 100 ng/ml each of IFN-{gamma} or IFN-{alpha} for various times and stimulated with IL-4 (5 ng/ml) for 0.5 h. Cells were then harvested and processed for EMSA using IL-4RE probe as in Materials and Methods. B, Cell lysates from A were also analyzed for tyrosine phosphorylation of STAT6 by immunoprecipitation of STAT6, followed by Western blot with {alpha}-phosphotyrosine using 4G10, and subsequently with {alpha}-STAT6 Ab. C, B cells were pretreated with 100 ng/ml each of IFN-{gamma} or IFN-{alpha} for 16 h, after which IL-4 was added. Cells were then cultured for indicated periods (4–10 h) and subjected to EMSA as in A. Data shown are a representative of multiple experiments.

 


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  		FIGURE 6. IFN-{gamma} or IFN-{alpha} does not adversely affect the IL-4-induced IL-4R nuclear transcription. Cells were pretreated with 100 ng/ml each of IFN-{gamma} for 16 h (A) or IFN-{alpha} for 12 h (B), and further cultured in the presence of 5 ng/ml IL-4 for 10 h. Cells were harvested, and nuclear run-on transcription assays were performed. To normalize the amount of transcripts applied to the membrane, a GAPDH probe was used as an internal control. Densitometric analysis of blots is also shown.

 
Both IFN-{gamma} and IFN-{alpha} posttranscriptionally down-regulate IL-4R through mRNA destabilization

Having observed that IFNs down-regulate the IL-4-induced steady state IL-4R mRNA level without affecting transcription of IL-4R gene, we conducted mRNA stability studies to examine a possible posttranscriptional control by IFNs. Mononuclear cells were first treated with IL-4 for 12–15 h to induce the IL-4 activation of IL-4R gene expression in the presence or absence of IFNs. Actinomycin D (15) was then added to block further synthesis of IL-4R mRNA, and time-dependent changes in mRNA levels by spontaneous degradation were analyzed. As shown in Fig. 7Go, A and B, an accelerated decay of IL-4R mRNA in cells treated with IL-4 plus IFN-{gamma} or IFN-{alpha} was noted compared with cells treated with IL-4 alone. The t1/2 of IL-4R mRNA was found to be significantly reduced in both IFN-{gamma} (80 vs 180 min) and IFN-{alpha} (50 vs 95 min)-treated cells. With purified B cells, very similar results were obtained (C), in that the stability of IL-4R mRNA was drastically decreased by IFN-{gamma} or IFN-{alpha} treatment. These data strongly suggest that IFN-{gamma}/{alpha} posttranscriptionally rather than transcriptionally modulate IL-4R through accelerating mRNA decay.



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  		FIGURE 7. IFN-{gamma} and IFN-{alpha} accelerate the IL-4R mRNA decay. Tonsillar mononuclear cells (A and B) or purified B cells (C) were treated with 5 ng/ml IL-4, IL-4 plus 100 ng/ml IFN-{gamma}, or IL-4 plus 100 ng/ml IFN-{alpha}, as indicated, and cultured for 12 h. Actinomycin D was then added to cells, and cells were further incubated for indicated periods before IL-4R mRNA analysis. Autoradiograms were subjected to densitometric analysis of IL-4R mRNA to determine relative intensity of radioactive bands. Top, Northern blot autoradiogram; middle, EtBr-stained RNA gel or APRT blot; bottom, decay curve for IL-4R mRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Specific responses to cytokines regulate the development, differentiation, and activation of cells in the immune systems. IL-4 mediates diverse immunoregulatory effects on cells by targeting specific gene expression, and one of the genes regulated by IL-4 is IL-4R({alpha}) (32, 33, 34). Among the known two types of IL-4R complex, IL-4R({alpha}) is a common and essential subunit (35, 36). In fact, modulation of IL-4R({alpha}) has been recognized as an important event during the IL-4-mediated signal transduction, which leads to the regulation of IL-4-induced responses. It has been reported that in human lymphoblastoid cell line Jiyoye, immediately after IL-4 treatment, the surface IL-4R undergoes a transient down-regulation via internalization and degradation. Then, most likely through IL-4-induced de novo synthesis, the surface IL-4R is up-regulated by 24 h, which is thought be required for the IL-4 induction of Fc{epsilon}RII/CD23 expression (33). According to the well-established Th1/Th2 modality, diverse antagonistic actions have been recognized between IL-4 and IFN-{gamma}. Therefore, as a part of our study on the regulation of IgE response, we investigated the molecular mechanism of IL-4R({alpha}) modulation by these cytokines.

Although IFN-{alpha} has been primarily recognized for antiviral effects rather than immunoregulatory effects, recent studies suggest a potential role of IFN-{alpha} on diverse immunomodulatory functions, such as activation of macrophages and NK cells (37, 38), differentiation of Th1 cells (12), as well as regulation of cytokine secretion and Ig production. Many of these biological effects of IFN-{alpha} are shared by IFN-{gamma} in the antagonistic regulation of IL-4 action, and IFN-{gamma} and IFN-{alpha} both use partially overlapping signaling mechanisms (30, 31). Hence, we explored the possibility that IFN-{gamma} and IFN-{alpha} both employ a common mechanism to down-regulate the IL-4-evoked response by modulating the IL-4R expression. Indeed, IFN-{gamma} and IFN-{alpha} both significantly reduced the IL-4-induced IL-4R expression at the surface protein as well as steady state mRNA level in tonsillar mononuclear cells. Basically, the same pattern of induction and antagonistic regulation of IL-4R gene expression by IL-4 and IFNs was also observed when purified B cells were used (Figs. 1Go and 2Go). With purified T cells from tonsils or peripheral blood, a low magnitude of IL-4R induction by IL-4 and down-regulation by IFNs were observed (data not shown). It should be noted that our tonsillar mononuclear population contained about 70% B cells, 25~30% T cells, and less than 5% of non-B/T cells, and we have been obtaining basically the same effects of IL-4/IFN-{gamma} on the IL-4R expression using mononuclear cells or purified B cells from tonsil throughout the experiments conducted in this study.

Distinct from the IL-4-induced IL-4R up-regulation that is thought to be manifested as a primary response involving tyrosine kinase-dependent STAT6 action in a CHX-independent manner (18) (Fig. 4Go), IFN-{gamma}/{alpha}-induced inhibitory effects represent a secondary response. The effects of IFN-{gamma} and IFN-{alpha} were both sensitive to CHX, and required a lag period of 6–8 h, a duration probably necessary for the de novo synthesis of IFN-{gamma}/{alpha}-induced factors (Figs. 3Go and 4Go). Yet, the signal transmission of IFN-{gamma}/{alpha} to leading to such secondary response occurred within minutes, in that pretreatment of IFN-{gamma}/{alpha} for less than 1 h, subsequent washing, and continued incubation of cells in the presence of IL-4 for 16 h produced the same inhibitory effects (data not shown). It is likely that the IFN-{gamma}/{alpha}-induced factors are products of IFN-inducible genes whose transcription is mediated by STAT1 activation rapidly occurring upon IFN treatment. Although it has been suggested that STAT1 and STAT6 can recognize the same GAS site, and competitive DNA binding by the two STATs may influence the transcriptional activities of target genes counterregulated by IFN and IL-4 (39), no direct roles of STAT1 and STAT6 in the repression of IL-4-inducible or IFN-inducible genes, respectively, have yet been demonstrated. Aside from STAT1 activation, pretreatment of IFN-{gamma} or IFN-{alpha} caused a substantial attenuation of the IL-4-induced STAT6 activity, as demonstrated by IL-4RE binding and tyrosine phosphorylation of STAT6 (Fig. 5Go, A and B). However, such negative modulation of STAT6 by IFNs was found to be only transient, and was not reflected in the regulation of IL-4R gene transcription, in that under the condition in which IFN-inhibitory action on IL-4R mRNA level is exerted, IFNs do not affect the IL-4-induced STAT6 activity (Figs. 5GoC and 6). This suggests that down-regulation of STAT6 activity by IFNs does not constitute the mechanism of IFN inhibition of the IL-4-induced IL-4R gene expression observed in this study. In fact, although IL-4-stimulated STAT6 activation is correlated with the increased rate of IL-4R transcription by IL-4, the essential role of STAT6 in human IL-4R gene activation has not been demonstrated, possibly due to the unavailability of the exact promoter structure of human IL-4R gene. In case of murine IL-4R gene, a recent article reported that STAT6 binds the GAS site of murine IL-4R promoter and activates the transcription (34). Whatever the authentic role of STAT6 in IL-4R gene regulation in human cells, our data still imply that the inhibitory action of IFNs on the IL-4-induced IL-4R gene expression involves not direct transcriptional repression of IL-4R, but posttranscriptional down-regulation via decreasing IL-4R mRNA stability (Figs. 6Go and 7Go).

There is a recent article describing that IFNs inhibit the IL-4-induced STAT6 activation in monocytes that may be associated with the repression of IL-4-induced IL-1R by IFNs (40). In this study, the authors obtained the inhibitory effect of IFN-{gamma} upon pretreatment of monocytes with IFN-{gamma} for 1 h and subsequent stimulation of cells with IL-4 for 30 min. Considering that their observation of the suppressive effect of IFN-{gamma} on the IL-4-induced IL-1R gene expression in monocytes was made at 6-h incubation of cells cotreated with IL-4 and IFN-{gamma}, it remains to be further explored whether the attenuation of STAT6 activity observed at 30 min of IL-4 treatment is the major mode of IFN action to down-regulate the IL-1R gene, or other delayed responses involving IFN-induced factors also play a role in this process. It is quite possible that IL-4 or IFN-{gamma} can transiently down-regulate STAT1 or STAT6 activation, respectively, in the early phase of cytokine signal transduction, especially when one cytokine is treated to cells before the other. Because IL-4 and IFN-{gamma} both require Janus kinase activity 1 for STAT6 and STAT1 activation (30, 31), either prior receptor occupation or depletion of Janus kinase 1 that is caused by one cytokine may cause a transient suppression of the other’s activity. However, as much as observed biological effect of both IFN-{gamma} and IFN-{alpha} on IL-4R was manifested as a delayed secondary response in the present study (Figs. 3Go and 4Go), there seems to be dissociation between the ability of IFNs to modulate STAT6 activity and to down-regulate the IL-4-induced IL-4R gene expression.

There still is a possibility that IFNs may influence the IL-4R transcription machinery via other constitutive or IFN-inducible transcriptional factors, including proteins of SOCS/SSI/CIS (suppressor of cytokine signaling/STAT-induced STAT inhibitors/cytokine-inducible inhibitors of signaling) (16, 17, 18) family, which have been suggested to play a role in the feedback inhibition of cytokine response (41, 42, 43). Such possibility, however, is clearly ruled out by nuclear run-on transcription assays, the results of which demonstrate that IFN-{gamma} or IFN-{alpha} does not directly or indirectly suppress the IL-4-induced IL-4R transcription rate (Fig. 6Go). A conclusive evidence that IFN-{gamma} and IFN-{alpha} both down-regulate the IL-4-induced IL-4R by accelerating the mRNA decay was then provided by the mRNA stability studies conducted using both mononuclear and purified B cells (Fig. 7Go). Although the absolute t1/2 of IL-4R mRNA turned out to be somewhat different in experiments using primary cells derived from tonsils provided by different donors, ~50% of reduction in the t1/2 of IL-4R mRNA was obtained for IFN-{gamma} or IFN-{alpha}. Such a rapid turnover rate is characteristic for mRNAs of primary response genes induced by mitogens or other growth stimuli, which include mRNAs of a number of protooncogenes, cytokines, and cytokine receptors (44, 45, 46). These mRNAs usually possess AU-rich sequences in their 3' untranslated region (UTR), and the decay mechanism of the mRNAs has been suggested to involve the action of mRNA-destabilizing factor binding to the consensus UUAUUUA motifs. Considering that IL-4R is induced by IL-4 as a primary response gene and that there is an AU-rich motif in the 3'UTR of human IL-4R (47), it is likely that IL-4R mRNA is a target of rapid turnover upon specific extracellular stimuli. The observation that the effects of IFNs for the down-regulation of IL-4-induced IL-4R mRNA required a lag time and ongoing protein synthesis strongly argues for the role of a liable and/or IFN-induced protein factor mediating destabilization of IL-4R mRNA. Although potential candidates of such IFN-{gamma}/{alpha}-inducible factor include a (2'-5') oligoadenylate-dependent endonuclease (RNase L) that is shown to preferentially recognize U-rich sequence of 3'UTR of many mRNAs via (2'-5') adenylate oligomer, an allosteric activator of RNase L (48, 49), more specific IFN-induced factors responsible for mediating the decay of IL-4R as well as other IL-4-inducible genes are to be identified in future investigations. A recent paper by Mozo et al. (50) reported that while the modulation of IL-4R mRNA stability is an important mechanism for IL-4R regulation by PMA and glucocorticoids, IL-4-induced down-regulation of IL-4R by glucocorticoids may involve translational or posttranslational mechanisms in human PBMCs. In this regard, any possible regulations of IL-4-induced IL-4R by IFNs at translational or posttranslational levels are also worthy of investigation.

The results of the present study strongly support our previous report on IFN-{gamma} regulation mechanism for the IL-4-induced Fc{epsilon}RII/CD23 gene expression, in which we observed the same pattern of posttranscriptional regulation by IFN-{gamma} (17). In fact, we have found that IL-4R and Fc{epsilon}RII, two major allergy-associated immune cell receptors, are regulated by cytokines and other costimulatory signals in a highly coordinated manner (18, 51, 52), which may be important for a fine-tuned control of IgE response in normal and disease conditions. In conclusion, our study collectively suggests that mRNA destabilization of IL-4-stimulated genes by IFN-{alpha}/{gamma}-induced factors may provide a common mechanism by which allergy-associated immune cell receptors are regulated via posttranscriptional modulation by type I and II IFNs, and constitute in part, a molecular basis of counterregulation by Th1 and Th2 cytokines.


   Acknowledgments
 
We acknowledge the kind provision of IL-4R{alpha} cDNA and anti-IL-4R mAb (M56) by Dr. S. Gillis at Immunex, and STAT6 cDNA by Dr. B. Groner at Institute for Biomedical Research, Goethe University. We are also grateful to Drs. B. S. Cho and J. S. Cho at Kyung Hee University for the valuable help provided in our experiments with tonsils, and to Mr. J. H. Kim for excellent technical assistance.


   Footnotes
 
1 This study was supported in part by grants provided through the Basic Science Research (KOSEF 971-0507-038-2 and 1999-1-212-001-5) and the 21 Century Frontier Human Genome Research programs. E.-Y.S. was supported by the Brain Korea 21 program. Back

2 Address correspondence and reprint requests to Dr. Choong-Eun Lee, Laboratory of Immunology, Department of Biological Science, SungKyunKwan University, 300 Cheon-Cheon Dong, Jang-An Ku, Suwon, 440-746, Korea. Back

3 Abbreviations used in this paper: CHX, cycloheximide; APRT, adenosyl phosphoribosyl transferase; EtBr, ethidium bromide; GAS, IFN-{gamma}-activated site; IL-4RE, IL-4-responsive element; MFI, mean fluorescence intensity; UTR, untranslated region. Back

Received for publication July 6, 2000. Accepted for publication August 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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