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Originally published online as doi:10.1189/jlb.1003488 on June 3, 2004

Published online before print June 3, 2004
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(Journal of Leukocyte Biology. 2004;76:416-422.)
© 2004 by Society for Leukocyte Biology

IFN-{alpha} regulates IL-21 and IL-21R expression in human NK and T cells

Mari Strengell1, Ilkka Julkunen and Sampsa Matikainen

Department of Microbiology, National Public Health Institute, Helsinki, Finland

1Correspondence: Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: mari.strengell{at}ktl.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-21 is a T cell-derived cytokine that regulates innate and adaptive immune responses. IL-21 receptor (IL-21R), which is expressed in natural killer (NK) and T cells, is structurally homologous to IL-2Rß and IL-15R{alpha}. These receptors also share a common cytokine receptor {gamma}-chain with IL-4, IL-7, and IL-9. Macrophage- or dendritic cell-derived interferon (IFN)-{alpha}/ß is a key cytokine in regulation of NK and T cell functions. We demonstrate here that in addition to activating IFN-{gamma} gene expression, IFN-{alpha}/ß and IL-12 enhance the mRNA expression of IL-21 in activated human T cells. In addition, IFN-{alpha}/ß enhanced T cell receptor stimulation-induced IL-21 and IFN-{gamma} gene expression in resting T cells. The promoter analysis of IL-21 gene revealed a putative IFN-{gamma} activation site element, which was found to bind signal transducer and activator of transcription 1 (STAT1), STAT2, STAT3, and STAT4 proteins in IFN-{alpha}/ß-stimulated NK or T cell extracts. In contrast to IL-21 expression, IFN-{alpha}/ß down-regulated IL-21R mRNA expression in NK and T cells. IFN-{alpha}/ß-induced down-regulation of IL-21R expression resulted in reduced STAT3 phosphorylation and DNA binding after IL-21 stimulation. In conclusion, our results suggest a novel role for IFN-{alpha}/ß in the regulation of IL-21 response.

Key Words: interferon • cytokine • lymphocyte • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-21 is a T cell-derived cytokine, which is involved in innate and adaptive immune responses. It is a 15-kDa protein closely related to IL-2 and IL-15. In addition to having a similar structure, these cytokines also have similarities in their receptors. The IL-21 receptor (IL-21R) is structurally related to IL-2R and IL-15R. In addition, these receptors, together with IL-4R, IL-7R, and IL-9R, share a common {gamma}-chain. IL-21R appears in T, natural killer (NK), B, and dendritic cells (DC), and it plays an important role in the regulation and function of these cells [1 2 3 4 5 6 7 ]. Studies with IL-21R knockout (KO) mice show that IL-21 plays a critical role in regulating immunoglobulin (Ig) production in B cells. After immunization, IL-21R-deficient mice have a higher IgE but lower IgG1 production than wild-type animals [6 ]. IL-21R KO mice show no abnormalities in the amount or functionality of their NK cells [1 ]. This suggests that IL-21 is not essential in generation and maturation of NK cells in mice. IL-21 has, however, an effect on NK cells depending on their activation state. It enhances the proliferation and cytotoxic activity of activated NK cells, but in these cells, IL-21 also promotes apoptosis and thus limits NK cell response. In contrast, IL-21 inhibits IL-15-induced proliferation and maturation of NK cell precursors [1 ].

A recent study with human cells shows that IL-21 is crucial in maturation of CD34+ precursors toward NK cells [8 ]. Mature NK cells are generated also without IL-21 in the presence of IL-15, stem cell factor, Flt3-L, and IL-7, but their maturation process is much slower, and only a minor population of cells express cell-surface receptors CD2 and CD8 and the killer inhibitory receptor, which are important markers of fully matured NK cells [8 ]. In addition to NK cells, IL-21 affects human T cells by up-regulating the expression of genes associated with innate immunity and the T helper cell type 1 (Th1) response such as interferon-{gamma} (IFN-{gamma}) [5 ]. IL-15- and IL-18-induced IFN-{gamma} production in human NK and T cells is enhanced by IL-21 [9 ].

Macrophages and especially DC produce IFN-{alpha}/ß, initially identified as an antiviral cytokine, in response to viral infections [10 11 12 13 ], which generate double-stranded RNA. This production is enhanced by an autoamplification mechanism involving transcription factors IFN-regulatory factor (IRF)-3 and IRF-7 [14 15 16 ]. IFN-{alpha}/ß is the key cytokine in the development of innate immune responses against viruses [17 18 19 ]. IFN-{alpha}/ß activates NK cells, DC, and macrophages, which are the principal effector cells of innate immunity, and also serves as an important link between innate and adaptive immunity. IFN-{alpha}/ß enhances NK cell cytotoxicity and in these cells, directly activates IFN-{gamma} gene expression [17 , 20 , 21 ]. Also in human T cells, IFN-{alpha}/ß induces IFN-{gamma} production and elevates T cell responsiveness to IL-18; both of these cytokines promote Th1-favoring milieu. Mice lacking a functional type I IFNR are unable to generate a cytotoxic T lymphocyte response to viruses such as lymphocytic choriomeningitis virus [22 23 24 ]. IFN-{alpha}/ß also controls directly or indirectly the expression of other cytokines such as IL-12, IL-15, IL-18, and IFN-{gamma} [25 26 27 ]. At present, no information is available on the possible regulatory role of IFN-{alpha}/ß in regard to novel T cell-derived cytokine IL-21.

IL-21 production from CD4+ T cells is induced during T cell receptor (TCR) stimulation, but the role of cytokines in the regulation of IL-21 gene expression remains largely unknown [3 ]. In this report, we have studied the effect of IFN-{alpha}/ß on IL-21 and IL-21R expression. We show that IFN-{alpha}/ß enhances IL-21 gene expression in resting and activated T cells (AT). Promoter analysis revealed in the IL-21 promoter a functional IFN-{gamma} activation site (GAS), which bound signal transducer and activator of transcription (STAT)1, STAT2, STAT3, and STAT4 in response to IFN-{alpha}/ß stimulation. In contrast to IL-21 expression, IFN-{alpha}/ß down-regulated IL-21R expression in NK and T cells. Our results suggest an important role for IFN-{alpha}/ß in the regulation of IL-21 production and response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
T cell culture
Leukocyte-rich buffy coats were obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). Monocytes were removed by adherence, and T cells were further purified by nylon wool columns. The purity of T cells was ensured by fluorescein-activated cell sorter (FACS) analysis using anti-CD3, anti-CD14, anti-CD19, and anti-CD56 monoclonal antibodies (mAb; Caltag Laboratories, Burlingame, CA). FACS analysis revealed more than 90% of cells being CD3+. An enriched population of resting T cells was used directly in stimulation experiments or activated with 0.5 µg/ml anti-CD3 and 0.5 µg/ml anti-CD28 mAb (R&D Systems, Abingdon, UK) and was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands), 20 mM HEPES, 2 mM L-glutamine, 0.6 µg/ml penicillin, 60 µg/ml streptomycin, and 100 IU/ml human recombinant (hr)IL-2 (R&D Systems) for 5–6 days. T cells were then expanded for 5–6 days in RPMI supplemented with 100 IU/ml hrIL-2. As determined by flow cytometry, >99% of the cells were CD3+, consisting of CD4+ (30%) and CD8+ (70%) cells [27 ]. In each experiment, T cells from two to four donors were used.

Cytokines
Highly purified human leukocyte IFN-{alpha} (13x106 IU/ml) was provided by Dr. Hannele Tölö (Finnish Red Cross Blood Transfusion Service). hrIFN-{alpha}2b (IntronA, 10x106 IU/ml) and IFN-ß (Betaferon, 8x106 IU/ml) were purchased from Schering-Plough (Kenilworth, NJ). hrIL-12 was purchased from R&D Systems, and hrIL-21 was kindly provided by Dr. Don Foster (Zymogenetics, Seattle, WA). The cytokine concentrations used were as follows unless otherwise indicated: IFN-{alpha}, 100 IU/ml; IL-12, 5 ng/ml; and IL-21, 10 ng/ml.

Purification of NK cells from peripheral blood mononuclear cells (PBMCs)
Mononuclear cells were isolated by density gradient centrifugation as described above using Ficoll-Paque. NK cells were purified from nonadherent PBMCs by nylon wool columns and two-step density gradient centrifugation by Percoll (Amersham Pharmacia Biotech). Percoll density gradient contained high gradient (46% Percoll in RPMI 1640) and low gradient (43% Percoll in RPMI 1640). The cell suspension in RPMI 1640 was carefully added on top. After centrifugation, the monocyte population remained on top, enriched NK cell population was in the middle phase, and T cells were in the bottom. NK cells from the middle phase were collected and purified by a negative selection using magnetic beads coated with anti-CD3, anti-CD14, and anti-CD19 antibodies (Dynal Biotech, Oslo, Norway). As determined by flow cytometry with anti-CD3 and anti-CD56 antibodies, more than 90% of the cells were CD56+, and none was CD3+.

NK cell line
Human NK-92 cell line (CD56+,CD2+,CD7+,CD3–; CRL-2407, American Type Culture Collection, Manassas, VA) displays characteristics of activated NK cells [28 ]. The cell line was maintained in continuous culture in {alpha}-minimum essential medium (Gibco, Life Technologies, Paisley, UK), supplemented with 12% horse serum (Gibco, Life Technologies), 12% FCS, 0.2 mM i-inositol, 20 mM folic acid, 40 mM 2-mercaptoethanol, 2 mM L-glutamine, antibiotics, and 100 IU/ml rIL-2.

RNA isolation and Northern blot analysis
Human primary NK cells, NK-92 cells, and resting or AT were treated with different cytokines, and cells were harvested at different time-points. Total cellular RNA was isolated by RNeasy midi-kit (Qiagen, Valencia, CA) and quantitated photometrically. The samples containing equal amounts of RNA (10 µg) were size-fractionated on a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane (Hybond, Amersham, Buckinghamshire, UK), and hybridized with IL-21, IL-21R, and IFN-{gamma} [29 ] probes. IL-21 and IL-21R probes were cloned from total cellular RNA obtained from anti-CD3/anti-CD28 antibody-AT by reverse transcriptase-polymerase chain reaction using oligonucleotides 5'-TACTGGGATCCACCATGAGATCCAGTCCTGGCAACATG (IL-21 sense), 5'-CAAGTTGGATCCTCAGGAGTCTTCACTTCCGTG (IL-21 antisense), 5'-GCAGAGGGATCCTGCACCTGGCCCTGCAGCTGTGAGG (IL-21R sense), and 5'-TCCAGGGATCCTCATTAGCTGGCCTGGGGTCCAGGGC (IL-21R antisense). Ethidium bromide (EtBr) staining of rRNA bands was used to ensure equal RNA loading. The probes were labeled with ({alpha}-32P) deoxy-adenosine 5'-triphosphate (dATP; 3000 Ci/mmol, Amersham) using a random-primed DNA-labeling kit (Boehringer Mannheim, Mannheim, Germany). The membranes were hybridized under conditions of high stringency [50% formamide, 5x Denhardt’s solution, 5x sodium chloride sodium phosphate + ethylenediaminetetraacetic acid (SSPE), and 0.5% sodium dodecyl sulfate (SDS)]. The membranes were washed twice at room temperature and once at 60°C in 1x saline sodium citrate/0.1% SDS for 30 min each time. The membranes were exposed to Kodak AR X-Omat films at –70°C using intensifying screen.

Oligonucleotide DNA precipitation
NK-92 cells and T cells were left untreated or stimulated with IFN-{alpha} for 1 h, after which the cells were harvested, washed, and lysed in a buffer containing 10 mM HEPES, 400 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.01% Triton X-100, 0.5 mM dithiothreitol (DTT), 1 mM NaVO4, and protease inhibitors (Complete, Roche, Basel, Switzerland). Cleared cell lysates were incubated with streptavidin-agarose beads coupled to 5'-biotinylated oligonucleotide; IL-21 promoter GAS at position –482–(–464) in the promoter 5'-GGATCCGACATAGTTATTACCATAAGAAAAAGTCCT (DNA Technology, Aarhus, Denmark). The binding reactions were performed for 2 h at +4°C in binding buffer containing 10 mM HEPES, 133 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.01% Triton X-100, 0.5 mM DTT, 1 mM NaVO4, and protease inhibitors. After washing, the oligonucleotide-bound proteins were released in SDS sample buffer, separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Rabbit anti-STAT1, anti-STAT2, anti-STAT3, and anti-STAT4 antibodies (all at 0.8 µg/ml) were allowed to bind for 1 h at room temperature in phosphate-buffered saline (PBS) containing 5% nonfat milk. Peroxidase-conjugated goat anti-rabbit IgG (1/2000 dilution, Dako, Glostrup, Denmark) was allowed to bind for 1 h at room temperature, and the proteins on membranes were visualized by the enhanced chemiluminescence (ECL) system (Amersham).

Electrophoretic mobility shift assay (EMSA)
NK-92 cells were primed with IFN-{alpha} for 18 h or were left untreated and then stimulated with 1, 3, or 10 ng/ml IL-21. Nuclear extracts were made, as described [30 ], by lysing the cells in a buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM NaVO4, and protease inhibitors (Complete, Roche). The isolated nuclei were then lysed in buffer containing 25% glycerol, 20 mM HEPES, 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaVO4, and protease inhibitors. Nuclear protein/DNA-binding reactions were performed as described previously [30 , 31 ]. Human CISGAS1 5'-CCCCGTTTTCCTGGAAAGTTTTG-GAAATCTGT-3' oligonucleotides were purchased from DNA Technology. The probes were labeled with ({alpha}-32P) dATP (3000 Ci/mmol, Amersham) using Klenow polymerase. The binding reaction was done at room temperature for 30 min. Samples were analyzed by electrophoresis on 6% nondenaturing, low ionic-strength polyacrylamide gels in 0.25x Tris-boric acid-EDTA buffer. The gels were dried and visualized by autoradiography.

Western blotting
NK-92 and T cells were primed with IFN-{alpha} for 18 h or were left untreated and then stimulated with 1, 3, or 10 ng/ml IL-21. The proteins were separated on 10% SDS-PAGE using the Laemmli buffer system and transferred electrophoretically onto Immobilon membranes (Millipore). Antiphospho-STAT1 (0.2 µg/ml) and antiphospho-STAT3 (0.2 µg/ml) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were allowed to bind in PBS containing 5% milk for 1 h at room temperature followed by peroxidase-conjugated goat anti-rabbit IgG. The bands were visualized by the ECL chemiluminescence system (Amersham). The membranes were reprobed with anti-STAT1 (0.8 µg/ml) and anti-STAT3 (0.8 µg/ml) antibodies (Santa Cruz Biotechnology) in PBS containing 5% nonfat milk for 1 h at room temperature followed by secondary antibody binding with peroxidase-conjugated goat anti-rabbit IgG for 1 h at room temperature and visualized by the ECL system (Amersham).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{alpha} up-regulates IL-21 gene expression in human T cells
TCR stimulation is known to activate IL-21 and IFN-{gamma} gene expression [3 , 32 ]. To study the effect of IFN-{alpha} on IL-21 and IFN-{gamma} gene expression, enriched, resting human T cells (~90% pure) were stimulated with IFN-{alpha}, anti-CD3 antibody, or their combination, and total cellular RNA was prepared. Anti-CD3 antibody treatment of resting T cells clearly acti-vated IL-21 mRNA expression at 12 h and IFN-{gamma} mRNA expression, already at 3 h after stimulation (Fig. 1A ). IFN-{alpha} enhanced anti-CD3 antibody-induced IL-21 and IFN-{gamma} expression. Compared with anti-CD3 antibody treatment of cells, IFN-{alpha} alone induced weak expression of IL-21 and IFN-{gamma} mRNAs at 3 h after stimulation (Fig. 1A) . AT were generated by stimulating resting human T cells with anti-CD3/CD28 antibodies. The cells were expanded in the presence of IL-2. To study the effect of IFN-{alpha} and IL-12 on IL-21 and IFN-{gamma} expression, the cells were stimulated with these cytokines for the times indicated, after which total cellular RNA was prepared. IFN-{alpha} induced IL-21 mRNA synthesis at 3 h after stimulation (Fig. 1B) . The kinetics of IFN-{alpha}-induced IFN-{gamma} expression was faster starting at 1 h after cytokine treatment (Fig. 1B) . IL-12 induced IL-21 and IFN-{gamma} mRNA expression at 3 h after stimulation (Fig. 1B) .



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  		Figure 1. IFN-{alpha} activates IL-21 gene expression in human T cells. (A) Resting human T cells were stimulated with IFN-{alpha} or anti-CD3 antibody or their combination for 3 or 12 h. Total cellular RNA was isolated, and IL-21 and IFN-{gamma} mRNA expression was analyzed by Northern blotting. (B) AT were stimulated with IFN-{alpha} or IL-12 for the times indicated, and total RNA was analyzed by Northern blotting. EtBr staining of rRNA bands was used to ensure equal RNA loading. (C) T cells and NK-92 cells were stimulated with IFN-{alpha} for 1 h or left untreated, and cell lysates were prepared. The proteins were precipitated with IL-21 promoter GAS oligonucleotides and analyzed by Western blotting with anti-STAT1, -STAT2, -STAT3, and -STAT4 antibodies. The results are representative of three separate experiments.

 
IFN-{alpha} activates STAT DNA binding to IL-21 promoter GAS
With computer analysis, we identified a putative GAS element at position –482–(–464) in the IL-21 promoter. GAS elements are known to bind STAT transcription factors in response to cytokine stimulation [33 ]. To study the functionality of IL-21 GAS, we performed oligonucleotide precipitation experiments. AT or NK-92 cells were stimulated with IFN-{alpha}, after which cell lysates were prepared. Specific proteins were precipitated with oligonucleotides containing IL-21 GAS and analyzed by Western blotting with anti-STAT antibodies. IFN-{alpha} induced STAT1, STAT2, STAT3, and STAT4 DNA binding to IL-21 GAS in NK-92 and T cells (Fig. 1C) .

IFN-{alpha} down-regulates IL-21R mRNA expression in human NK and T cells
IFN-{alpha} is known to regulate the expression of several genes encoding cytokine receptors [27 , 34 35 36 ]. To study the effect of IFN-{alpha} on IL-21R gene expression, resting human T cells were stimulated with IFN-{alpha}, anti-CD3 antibody, or their combination, and total cellular RNA was prepared for Northern blot analysis. Anti-CD3 antibody stimulation clearly enhanced IL-21R mRNA expression at 6-h and 24-h time-points (Fig. 2 ). IFN-{alpha} stimulation was found to down-regulate IL-21R mRNA expression, and this down-regulation was seen throughout the experiment. IFN-{alpha} also inhibited anti-CD3 antibody-induced expression of IL-21R mRNA at 6 h (Fig. 2) . This effect, however, appeared to be transient, as at a 24-h time-point, anti-CD3-induced IL-21R expression was at an equal level in the presence or absence of IFN-{alpha} (Fig. 2) .



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  		Figure 2. IFN-{alpha} down-regulates IL-21R expression in resting T cells, which were stimulated with IFN-{alpha} or anti-CD3 antibody or their combination for 6 or 24 h, and total RNA was isolated, followed by analysis by Northern blotting using the IL-21R probe. EtBr staining of rRNA bands was used to ensure equal RNA loading.

 
Next, we studied the effect of IFN-{alpha} and IL-12 on IL-21R expression in NK cells and AT. Human primary NK cells, NK-92 cells, and AT were stimulated with IFN-{alpha} or IL-12, total cellular RNA was isolated, and Northern blot analysis was performed. IFN-{alpha} clearly and rapidly decreased IL-21R mRNA expression in primary NK (Fig. 3A ), NK-92 cells (Fig. 3B) , as well as in AT (Fig. 3C) . Similarly, IL-12 down-regulated IL-21R mRNA expression in primary NK cells (Fig. 3A) and in NK-92 cells (Fig. 3B) . However, in contrast to IFN-{alpha}, IL-12 had little effect on IL-21R gene expression in AT (Fig. 3C) .



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  		Figure 3. IFN-{alpha} down-regulates IL-21R expression in human NK and T cells. Primary human NK cells (A), NK-92 cells (B), and T cells blasts (CD4+ 30% and CD8+ 70%; C) were stimulated with IFN-{alpha} or IL-12 for the times indicated. Total RNA from stimulated and unstimulated control cells was isolated, and IL-21R expression was analyzed by Northern blotting. EtBr staining of rRNA bands was used to ensure equal RNA loading. Comparable data were obtained in three separate experiments.

 
To study in more detail the effect of type I IFNs on IL-21, IL-21R, and IFN-{gamma} gene expression, AT were stimulated for 3 h with different doses of natural IFN-{alpha}, rIFN-{alpha}2b, and rIFN-ß, after which the cells were isolated, total cellular RNA was prepared, and Northern blot analysis was performed. IFN-{alpha}, IFN-{alpha}2b, and IFN-ß enhanced IL-21 and IFN-{gamma} expression in a dose-dependent manner (Fig. 4 ). IFN-{alpha} and IFN-ß down-regulated IL-21R expression, even at the lowest concentration used (10 IU/ml), whereas IFN-{alpha}2b down-regulated IL-21R mRNA expression at higher concentrations (Fig. 4) .



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  		Figure 4. Regulation of IL-21 and IL-21R gene expression by type I IFNs in AT. Human T cells were stimulated with natural IFN-{alpha}, rIFN-{alpha}2b, or rIFN-ß with three different concentrations (10–1000 IU/ml) for 3 h. Total RNA was isolated, and IL-21, IL-21R, and IFN-{gamma} mRNA expression was analyzed by Northern blotting. EtBr staining of rRNA bands was used to ensure equal RNA loading. A representative of three independent experiments is shown.

 
IFN-{alpha} treatment of NK-92 cells inhibits IL-21-induced STAT3 activation
It has been shown earlier that IL-21 activates STAT3 in NK and T cells [5 , 37 ]. Therefore, we studied whether IFN-{alpha}-induced down-regulation of IL-21R expression would lead to reduce IL-21-induced STAT3 activation. NK-92 cells were treated with IFN-{alpha} for 20 h, after which the cells were stimulated with three different concentrations of IL-21 for 1 h, and cellular lysates were prepared for Western blot analysis. Pretreatment of NK-92 cells with IFN-{alpha} clearly reduced IL-21-induced STAT3 phosphorylation (Fig. 5A ), whereas the effect on STAT1 phosphorylation was marginal. The same effect was seen in T cells (data not shown). We also studied the effect of IFN-{alpha} on IL-21-induced STAT3 DNA binding. NK-92 cells were stimulated with IFN-{alpha} for 20 h, after which the cells were stimulated with three different concentrations of IL-21 followed by preparation of nuclear extracts. The extracts were incubated with oligonucleotides containing CIS GAS1 and analyzed in EMSA. IFN-{alpha} treatment of NK-92 clearly reduced IL-21-induced DNA binding of STAT3 to CIS GAS1 oligonucleotides (Fig. 5B) .



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  		Figure 5. IFN-{alpha} inhibits IL-21-induced STAT3 phosphorylation and DNA binding in NK-92 cells. (A) NK-92 cells were treated with IFN-{alpha} (100 IU/ml) for 20 h and were stimulated with 1, 3, or 10 ng/ml IL-21 for 1 h, and the cell lysates were prepared. Lysates were analyzed by Western blotting with antiphospho-STAT3 and anti-STAT3 antibodies. (B) NK-92 cells were treated with IFN-{alpha} (100 IU/ml) for 20 h and were stimulated with 1, 3, or 10 ng/ml IL-21 for 1 h, and nuclear extracts were prepared. The extracts were incubated with oligonucleotides containing CIS GAS1 element and analyzed in EMSA. The experiments were repeated twice with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Antigen presenting cell (APC)-derived IFN-{alpha}/ß is one of the key cytokines regulating innate and adaptive, Th1-dominated, immune responses against viral infections [38 , 39 ]. In addition to its antiviral effects, IFN-{alpha}/ß activates NK cells, enhances DC maturation, and drives Th1 differentiation [20 , 21 , 26 , 40 41 42 ]. IFN-{alpha}/ß also promotes Th1 response by regulating Th1-associated cytokines. We have previously shown that IFN-{alpha}/ß enhances the expression of IL-2R{alpha}, IL-12Rß2, and IL-18R genes in human NK and T cells [27 , 43 ]. To date, there is no information on the possible role of IFN-{alpha}/ß in the regulation of a novel T cell-derived cytokine IL-21. In this report, we show that IFN-{alpha}/ß enhances IL-21 gene expression in human T cells. Up-regulation of IL-21 expression is, however, accompanied with IFN-{alpha}/ß-induced down-regulation of IL-21R expression in T and NK cells. Our present results suggest a significant role for IFN-{alpha}/ß in the regulation of IL-21 response.

IFN-{alpha}/ß enhances IFN-{gamma} gene expression in NK and T cells and promotes NK cell cytolytic activity [17 , 20 , 21 ]. IFN-{alpha}/ß regulates the immune system also by enhancing or reducing the production of other cytokines, such as IL-12, IL-15, and IL-18 [25 26 27 ]. IL-15 and IL-18 are DC/macrophage-derived cytokines, which synergistically with IL-21 enhance IFN-{gamma} production in human NK and T cells [9 ]. In this report, we show that IFN-{alpha}/ß and IL-12 enhance not only IFN-{gamma} mRNA expression but also IL-21 production in AT. IFN-{alpha}/ß also enhanced TCR stimulation-induced IL-21 gene expression in resting T cells. In vivo, when cytokines usually are present simultaneously in the infection site, the elevated IL-21 production may also contribute to IFN-{gamma} production by itself or in synergy with other cytokines such as IL-15 or IL-18. In contrast to T cells, IFN-{alpha}/ß or IL-12 stimulation of primary NK cells or NK-92 cells was not able to induce IL-21 gene expression (data not shown), suggesting that the T cell is the major cell type responsible for IL-21 production.

Our results show that although IFN-{alpha}/ß induces IL-21 expression in T cells, it simultaneously decreases IL-21R gene expression in NK and T cells. IL-21 promotes NK cell proliferation and maturation and enhances IFN-{gamma} production in these cells. IFN-{alpha}/ß is the first cytokine secreted by virus-infected cells. We have preliminary evidence showing that IFN-{alpha}/ß inhibits NK cell IL-21R expression during influenza A virus infection (data not shown). Decreased expression of IL-21R in NK cells results in decreased NK cell responsiveness to IL-21 and thus may prevent IL-21-induced maturation and activation of NK cells. During viral infections, IL-21 is produced in T cells that have been activated by APCs. At the same time, virus-infected APCs, including DC, secrete IFN-{alpha}/ß. In addition to NK and T cells, IL-21R is expressed on DC, and IL-21 is involved in the regulation of DC responses as well. IL-21 inhibits DC differentiation and maturation [44 ], and therefore, it may be that IFN-{alpha}/ß indirectly enhances DC differentiation and maturation by down-regulating IL-21R in DCs.

A computer analysis of the IL-21 promoter revealed a putative GAS-binding site for STAT transcription factors, which are involved in mediating the IFN response. We clearly show that IFN-{alpha} activates the binding of multiple STATs to this IL-21 GAS site. Similarly, we have previously shown that IFN-{alpha}/ß also directly activates IFN-{gamma} gene expression by inducing STAT4 binding to IFN-{gamma} promoter GAS [21 ]. It seems that IL-21 and IFN-{gamma} genes are turned on by TCR stimulation, likely through activation of nuclear factor (NF)-{kappa}B and NF-AT, after which IFN-{gamma} and IL-21 gene expression is further enhanced through the activation of STATs by IFN-{alpha}/ß.

In summary, our study provides evidence for a unique role of IFN-{alpha}/ß in the regulation of the IL-21 response. IFN-{alpha} enhances IL-21 expression in T cells, but at the same time, it negatively regulates the IL-21 response by down-regulating IL-21R expression in NK and T cells. Further studies are needed to characterize the regulatory elements of the IL-21R promoter that are involved in mediating IFN-{alpha}-induced down-regulation of IL-21R gene expression.


   ACKNOWLEDGEMENTS
 
The Medical Research Council of the Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Cancer Organizations supported this work. We thank Dr. Leena Kinnunen for sequence analysis. Teija Westerlund, Hanna Valtonen, and Mari Aaltonen are acknowledged for their excellent technical assistance.

Received October 20, 2003; revised March 8, 2004; accepted March 20, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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