Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells
Go Tanaka1,
Sachiko Kanaji1,
Ayumi Hirano2,
Kazuhiko Arima1,
Akira Shinagawa2,
Chiho Goda1,
Shin'ichiro Yasunaga1,
Koichi Ikizawa3,
Yukiyoshi Yanagihara4,
Masato Kubo5,
Yoshiaki Kuriyama-Fujii6,
Yuji Sugita2,
Akira Inokuchi7 and
Kenji Izuhara1,8
1 Division of Medical Biochemistry, Department of Biomolecular Sciences, Saga Medical School, 5-1-1 Nabeshima, Saga, 849-8501, Japan
2 Genox Research Inc., 5-12-8, Koishikawa, Tokyo, 112-8088, Japan
3 Hanno Research Center, Taiho Pharmaceutical Co., Ltd., 1-27, Misugidai, Hanno, 357-8527, Japan
4 Clinical Research Center, National Sagamihara Hospital, 18-1, Sakuradai, Sagamihara, 228-8522, Japan
5 RIKEN Research Center for Allergy and Immunology, (RCAI), RIKEN Yokohama Institute, Yokohama, 1-7-22, Suehiro-cho, Yokohama, 230-0045, Japan
6 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1, Tenoudai, Tsukuba, 305-8577, Japan
7 Department of Otolaryngology-Head and Neck Surgery and 8 Division of Medical Research, Center for Comprehensive Community Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga, 849-8501, Japan
Correspondence to: K. Izuhara; E-mail: kizuhara{at}med.saga-u.ac.jp
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Abstract
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It is widely known that IL-4 and IL-13 act on various kinds of cells, including B cells, resulting in enhancement of proliferation, class switching to IgE and expression of several surface proteins. These functions are important for the recognition of the various antigens in B cells and are known to be involved in the pathogenesis of allergic diseases. However, it has not been known whether IL-4/IL-13 is involved in the metabolism of various kinds of xenobiotics including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), and it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, exaggerating allergic reactions. We identified IL-4- or IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39, using microarray technology, in which the AHR gene was included. The AHR gene product, the aryl hydrocarbon receptor (AhR), was induced by IL-4 in both mouse and human B cells in a STAT6-dependent manner. IL-4 alone had the ability to translocate the induced AhR to the nuclei. TCDD, a ligand for AhR, rapidly degraded the induced AhR by the proteasomal pathway, although IL-4-activated AhR sustained its expression. AhR activated by IL-4 caused expression of a xenobiotic-metabolizing gene, CYP1A1, and TCDD synergistically acted on the induction of this gene by IL-4. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.
Keywords: CYP1A1, dioxin, gene regulation, microarray, xenobiotics
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Introduction
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IL-4 and IL-13, produced by various kinds of cells including Th2 cells, mast cells, basophils, NKT cells and eosinophils, exert similar biological activities by binding to their receptors on the surface of target cells (1). IL-4 and IL-13 act on various kinds of cells including B cells, in which IL-4/IL-13 causes enhancement of proliferation together with the stimuli via the surface IgM receptor and/or CD40, class switching to IgE and IgG4 (IgG1 in case of mice) and expression of surface proteins such as CD23, MHC class II antigen, surface IgM, CD71 and CD72 (25). It is thought that these events are important for the recognition of the various antigens, particularly those derived from parasites, and for the pathogenesis of allergic diseases. However, it has not been known whether IL-4 or IL-13 is involved in the metabolism of various kinds of xenobiotics, including polycyclic aromatic hydrocarbons such as 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene and benzo[a]pyrene. Additionally, it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, although a couple of reports have described that TCDD enhances IgE synthesis in B cells (6, 7).
The aryl hydrocarbon receptor (AhR) is a transcriptional factor possessing basic helix-loop-helix/Per-aryl hydrocarbon receptor nuclear translocator (Arnt)-Sim motif, which binds to various kinds of xenobiotics, including TCDD (8, 9). Upon binding to the ligand, AhR translocates to the nuclei, followed by formation of the heterodimeric complex with Arnt. The AhRArnt complex induces expression of a number of monooxygenase genes such as cytochrome P4501A1 (CYP1A1), CYP1A2 and CYP1B1, by binding to the xenobiotic responsive element on the target genes, playing a key role in metabolism of xenobiotics (8, 9). In addition, it has been recently reported that AhR directly affects cell cycle regulation in response to an agonist, although it is controversial whether it can inhibit or promote proliferation (10), and that AhR directly interacts with nuclear factor
B, down-regulating its biological activities (11).
To explore the activity of IL-4 and IL-13 on B cells, we employed a microarray analysis, and it turned out that the AHR gene was included in the identified genes. In this article, we characterize the induction and activation mechanism of AhR by IL-4 in B cells. These results suggest that induction and activation of AhR are novel biological activities of IL-4 and IL-13 on B cells and that consequently IL-4 and IL-13 should be involved in the metabolism of xenobiotics by induced and activated AhR in B cells. In contrast, TCDD did not affect IgE production or CD23 expression by IL-4 in B cells, denying the synergistic effect of TCDD on IgE production by IL-4.
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Methods
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Reagents
Cycloheximide, TCDD and MG132 were purchased from SigmaAldrich (St Louis, MO, USA), Wako (Osaka, Japan) and Calbiochem (San Diego, CA, USA), respectively.
Cells
DND-39 cells and HepG2 cells were maintained in RPMI 1640 medium or Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 µg ml1 streptomycin and 100 U ml1 penicillin. IL-13R
1-transfected DND-39 cells were prepared and maintained as described before (12). PBMCs were separated from healthy human volunteers using LymphoprepTM (Axis-Shield, Oslo, Norway), and then B cells were isolated using DynabeadsTM M-450 CD19 (Dynal, Oslo, Norway), and cultured in the same way as DND-39 cells. DND-39 cells, HepG2 cells and human B cells were stimulated with 10 ng ml1 of IL-4 (Peprotech, Rocky Hill, NJ, USA) and/or 10 µg ml1 of anti-IgM antibody (Cappel, Aurora, OH, USA) and/or 0.5 µg ml1 of anti-CD40 antibody (Immunotech, Marseille, France) for the indicated times. Mouse B cells were isolated from spleen cells using StemSepTM B cell enrichment (StemCell Technologies Inc., Vancouver, Canada) and maintained in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 100 µg ml1 streptomycin and 100 U ml1 penicillin. Mouse B cells were stimulated with 10 ng ml1 of IL-4 (Chemicon International, Temecula, CA, USA) and/or 20 µg ml1 of LPS (SigmaAldrich).
Microarray analysis
Procedures of probe preparation and microarray analysis were performed as described before (13). The microarray analyses of complementary RNA from IL-13R
1-transfected DND-39 cells were performed with human high-density oligonucleotide probe arrays (HG-U95Av2 Array) representing
10 000 full-length, non-redundant genes supplied by Affymetrix. Hybridized probe arrays were read using a Hewlett-Packard GeneArray scanner (HP2500A, Hewlett-Packard, Palo Alto, CA, USA). The data were analyzed using Gene Chip software, Suite ver.4.0 (Affymetrix). Integrity of the RNA was verified before reverse transcription (RT) by visualization of the 28S and 18S ribosomal RNA bands on an agarose gel. Hybridization quality was checked by measuring the ratio of hybridization intensities of the 3'5' regions of control genes.
RTPCR
Total RNA was extracted by ISOGEN (Nippongene, Tokyo, Japan). The RT reaction primed with random hexamer was performed using GeneAmp RNA PCR Kit (Applied Biosystems Japan, Tokyo, Japan). The PCR reaction was performed with cDNA as a template using the indicated primers after an initial 1-min denaturation at 96°C, followed by the indicated cycles of 96°C for 1 min, the indicated annealing temperature for 1 min and 72°C for 1 min. The cycles used were 34, 31, 21, 32, 20 and 25, for human AhR, human Arnt, human GAPDH, the hemagglutinin-tagged truncated form of STAT6 (HA-STAT6DN), human ß-actin and the human germline
transcript, respectively. The annealing temperatures used were 58, 58, 54, 58, 62 and 55°C for AhR, Arnt, GAPDH, HA-STAT6DN, ß-actin and the germline
transcript, respectively. The PCR primers used were 5'-AGTCTGTTATAACCCAGACCAG-3', and 5'-GCATCACAACCAATAGGTGTGA-3' for AhR; 5'-GATGCAGGAATGGACTTGGCT-3', and 5'-CTTTCCTAAGAGTTCCTGTGGCT-3' for Arnt; 5'-GAAGGTGAAGGTCGGAGT-3' and 5'-GAAGATGGTGATGGGATTTC-3' for GAPDH; 5'-CTAGCATGTATCCTTATGATGTTCCTGATTATGCTGGTAC-3' and 5'-CCTCAGCCCCCTTCTGCA-3' for HA-STAT6DN;5'-TCACCCACACTGTGCCCATCTACGA-3' and 5'-CAGCGGAACCGCTCATTGCCAATGG-3' for ß-actin and 5'-CTGGGAGCTGTCCAGGAACC-3' and 5'-GCAGCAGCGGGTCAAGG-3' for the germline
transcript, respectively.
Immunoprecipitation and western blotting
Procedures of immunoprecipitation and western blotting were carried out as previously described (14). The samples were applied to SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK). The antibodies used for immunoprecipitation and western blotting were anti-AhR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Arnt antibody (Santa Cruz Biotechnology), anti-actin antibody (Biomedical Technologies, Stoughton, MA, USA) and anti-histone deacetylase class 1 (HDAC1) antibody (Santa Cruz Biotechnology). The proteins were visualized by enhanced chemiluminescence (Amersham Biosciences). Fractionation of cell lysates was performed using NE-PER (Pierce Chemical Co., Rockford, IL, USA).
Mice
C57/BL6 and Balb/c mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). STAT6-deficient mice and AhR-deficient mice were prepared as described before (15, 16). All experiments were approved by the Saga University Animal Care and Use Committee.
Generation of DND-39 cells expressing the truncated form of STAT6
The plasmid encoding STAT6DN was prepared as previously described (17). This plasmid was transfected into DND-39 cells by electroporation, and the transfectants were maintained with a medium containing 2.5 mg ml1 of Geneticin (SigmaAldrich).
Real-time PCR analysis
Quantitative analysis of mRNA expression was performed using the ABI PRISMTM 7000 sequence detection system (Perkin-Elmer Japan, Urayasu, Japan), as described before (13). To calculate the copy numbers for each gene, standard curves were generated using a plasmid encoding that gene whose copy numbers were known. The PCR primers used were 5'-AACCAGTGGCAGATCAACCAT-3' and 5'-CCCATGCCAAAGATAATCACC-3' for human CYP1A1; 5'-CACTCTCTTTGGTTTGGGCA-3' and 5'-CTCCCCTGGAGACACCTTAAA-3' for mouse CYP1A1; 5'-TCACCCACACTGTGCCCATCTACGA-3' and 5'-CAGCGGAACCGCTCATTGCCAATGG-3' for human ß-actin; 5'-ACTATTGGCAACGAGCGGTTC-3', and 5'-GGATGCCACAGGATTCCATACC-3' for mouse ß-actin; 5'-CAATACTTCCACCTCAGTTGGC-3', and 5'-GCATCACAACCAATAGGTGTGA-3' for human AhR and 5'-GAAGGTGAAGGTCGGAGT-3', and 5'-GAAGATGGTGATGGGATTTC-3' for human GAPDH, respectively. The TaqManTM probes used were 5'-CCCTGATGGTGCTATCGACAAGGTGT-3' for human CYP1A1, 5'-AAAGTGCATCGGAGAGACCATTGGC-3' for mouse CYP1A1, 5'-ATGCCCTCCCCCATGCCATCCTGCGT-3' for human ß-actin, 5'-CCTGAGGCTCTTTTCCAGCCTTCCTTCT-3' for mouse ß-actin, 5'-AGCCACCATCCATACTTGAAATCCGG-3' for human AhR and 5'-CAAGCTTCCCGTTCTCAGCC-3' for human GAPDH, respectively.
ELISA for IgE
Mouse spleen cells were maintained in RPMI 1640 medium supplemented with 10% FCS (HyClone, South Logan, UT, USA), 1 mM sodium pyruvate (Invitrogen Corp., Carlsbad, CA, USA), 10 mM HEPES (pH 7.4), 0.1 mM non-essential amino acids (GIBCO BRL), 50 µM 2-ME, 100 µg ml1 streptomycin and 100 U ml1 penicillin, and incubated with 1 ng ml1 of IL-4 and 0.5 µg ml1 of anti-CD40 antibody (BD Biosciences, San Jose, CA, USA) in the presence of the indicated concentrations of TCDD for 5 days. ELISA for mouse IgE was performed using a mouse IgE ELISA Quantitation kit (Bethyl, Montgomery, TX, USA).
Flow cytometry for CD23
Flow cytometric analysis of CD23 on spleen cells was performed using FITC-labeled anti-B220 antibody (BD Biosciences) and PE-labeled CD23 antibody (BD Biosciences). Quantitation of the surface staining was performed using FACSCalibur, and data were analyzed using Cell Quest software (BD Biosciences).
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Results
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Identification of IL-4- and IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39
To identify the biological activity of IL-4 and IL-13 on B cells, we used a microarray analysis. The microarray experiments were designed based on the guidelines of Minimum Information About a Microarray Experiment. To exclude the complexity related to the heterogeneity of primary human B cells, we used a human Burkitt lymphoma cell line, DND-39. Then the samples were prepared from DND-39 cells expressing IL-13R
1 stimulated with either IL-4 or IL-13 for either 24 or 48 h. Of the 10 000 annotated genes present on the arrays, IL-4 and IL-13 augmented expression of 22 and 16 genes at 24 h, respectively, and 14 and 45 genes at 48 h, respectively. Among these genes, 11 genes at 24 h and 13 genes at 48 h were commonly up-regulated by both IL-4 and IL-13. The findings that the IGHE gene coding for the Ig
H chain constant region and the FCER2 gene coding for CD23 were included in the overlapped genes proved the appropriateness of this microarray analysis; it is well known that IL-4 or IL-13 induces expression of these genes (18, 19). The AHR gene was consistently induced by both IL-4 and IL-13 at both 24 h (8.9-fold and 7.0-fold, respectively) and 48 h (5.1-fold and 5.0-fold, respectively), raising the possibility that the AHR gene is a novel IL-4- or IL-13-inducible gene in B cells (data not shown).
Induction of AhR by IL-4 in DND-39 cells and human and mouse primary B cells
To validate further the result that induction of AhR was up-regulated by IL-4 or IL-13 in the microarray analysis, we analyzed expression of AhR induced by IL-4 by RTPCR. Upon stimulation of IL-4, AhR started to be induced and reached a peak at 3 h in DND-39 cells, and then declined (Fig. 1A). In contrast, another component of the complex, Arnt, was not affected by IL-4. To address whether the induction of AhR by IL-4 in DND-39 cells required de novo protein synthesis, we next analyzed the effect of cycloheximide on the induction. The induction of AhR by IL-4 was not affected up to 50 µg ml1 of cycloheximide (Fig. 1B). These results suggested that induction of AhR by IL-4 probably did not require de novo protein synthesis.

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Fig. 1. Induction of AhR in B cells. (A) Total RNA was extracted from DND-39 cells stimulated with 10 ng ml1 of IL-4 for the indicated times, and then RTPCR for AhR, Arnt and GAPDH was performed. (B) Total RNA was extracted from DND-39 cells stimulated with 10 ng ml1 of IL-4 for 6 h. The indicated concentrations of cycloheximide were added 30 min before the addition of IL-4. Then RTPCR for AhR and GAPDH was performed. (C) Cell lysates were prepared from DND-39 cells and HepG2 cells stimulated with 10 ng ml1 of IL-4 for 24 h, and then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody. (D) Immunoprecipitates with either anti-AhR antibody or anti-Arnt antibody were prepared from spleen B cells derived from C57/BL6 or Balb/c strains stimulated with 10 ng ml1 of IL-4 and/or 20 µg ml1 of LPS for 24 h, and then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody. (E) Cell lysates were prepared from human primary B cells stimulated with 10 ng ml1 of IL-4 and/or 10 µg ml1 of anti-IgM antibody and/or 0.5 µg ml1 of anti-CD40 antibody for 24 h, and then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody.
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To address whether IL-4 up-regulates AhR production at not only the mRNA level but also the protein level, we next analyzed AhR induction by IL-4 using western blotting. IL-4 induced protein synthesis of AhR in DND-39 cells, whereas HepG2 cells constitutively expressed AhR and IL-4 did not affect the induction (Fig. 1C). Mouse spleen B cells derived from Balb/c or C57/BL6 strains also showed that IL-4 alone could enhance AhR production (Fig. 1D). LPS, which had been reported to induce AhR (20), had little effect on AhR induction. Expression of Arnt was unchanged by the stimulation. In human peripheral B cells, IL-4 alone only slightly induced AhR, whereas co-stimulants of anti-IgM antibody and anti-CD40 antibody significantly augmented the induction (Fig. 1E). Although expression of Arnt was augmented by co-stimulants of anti-IgM antibody and anti-CD40 antibody, IL-4 had no effect on the induction. We confirmed that the same amounts of proteins were loaded in each lane by Coomassie blue staining (data not shown). These results demonstrated that IL-4 had the ability to induce AhR in B cells, probably without de novo protein synthesis, although the co-stimuli to maximize the AhR expression were different in mouse and human B cells.
STAT6 dependency of AhR induction by IL-4
It is well known that a transcription factor, STAT6, plays critical roles for biological activities of IL-4 and IL-13 (21, 22). We next investigated whether AhR induction by IL-4 in B cells is dependent on STAT6. For this purpose, we first generated DND-39 cells expressing a truncated form of STAT6. This form lacked the SH2 domain and the activating domain for transcription; we had already shown that it acted as a dominant negative form of STAT6 (17). We used two independent clones expressing the truncated form. We confirmed that induction of the germline
transcript by IL-4 was diminished in these transfectants (Fig. 2A). When these transfectants were stimulated with IL-4, AhR was not induced, whereas induction of AhR was detected in parental cells and mock-transfected cells (Fig. 2B). Furthermore, induction of AhR by IL-4 was significantly decreased in spleen B cells derived from STAT6-deficient mice (Fig. 2C). These results demonstrated that AhR induction by IL-4 was mostly dependent on STAT6 activation.

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Fig. 2. Dependency of STAT6 of AhR induction in B cells. Total RNA (A) or cell lysates (B) were prepared from parental DND-39 cells, mock-transfected DND-39 cells and STAT6DN (#6 and #13) stimulated with 10 ng ml1 of IL-4 for 24 h. Then RTPCR for the germline transcript or ß-actin (A) or western blotting using either anti-AhR antibody or anti-Arnt antibody (B) was performed. (C) Immunoprecipitates with either anti-AhR antibody or anti-Arnt antibody were prepared from spleen B cells derived from wild mice or STAT6-deficient mice of C57/BL6 strain stimulated with 10 ng ml1 of IL-4 for 24 h, and then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody.
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Effects of TCDD and IL-4 on turnover of the AhR protein in B cells
It is known that the binding of a ligand to AhR triggers degradation of AhR by the ubiquitination/proteasome pathway in hepatocytes (2325). To explore the possibility that this would also be the case with B cells, we analyzed the effect of TCDD and also IL-4 on turnover of the AhR protein in DND-39 cells. After AhR was induced by IL-4, incubation with TCDD for 6 h caused a significant decrease of AhR (Fig. 3A). We next tested the effects of MG132, a proteasomal inhibitor, on this TCDD-induced AhR degradation. We confirmed that co-incubation of 5 µM of MG132 with 10 nM of TCDD for 12 h completely recovered AhR degradation by TCDD in HepG2 cells, as previously reported (23) (data not shown). However, the same treatment by MG132 alone caused disappearance of both AhR and Arnt in DND-39 cells, indicating the non-specific inhibitory effect of MG132 at the transcription and/or translation level (data not shown). Therefore, we incubated IL-4-stimulated DND-39 cells with 2 µM of MG132 and 10 nM of TCDD for only 2 h. The treatment of 10 nM of TCDD for 2 h degraded AhR by almost half, and co-incubation of MG132 completely recovered AhR expression (Fig. 3B). In contrast, expression of the AhR protein, which was induced by IL-4, was sustained up to 48 h (data not shown). These results suggested that TCDD rapidly degraded the induced AhR protein through the proteasomal pathway in B cells, although IL-4 alone did not cause AhR degradation.

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Fig. 3. Degradation of AhR proteins by TCDD. (A) Cell lysates were prepared from DND-39 cells stimulated with 10 ng ml1 of IL-4 for 24 h in the presence of 10 nM of TCDD for the indicated times. TCDD was added at the indicated times before cell harvest. Then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody. (B) Cell lysates were prepared from DND-39 cells stimulated with 10 ng ml1 of IL-4 for 24 h. Ten nM of TCDD and 2 µM of MG132 were added 2 h before harvesting cells. Then western blotting was performed using either anti-AhR antibody or anti-Arnt antibody.
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Nuclear location of AhR by IL-4 in B cells
It has been widely believed that AhR exists in the cytoplasm at the steady state and that binding to a ligand such as TCDD leads to translocation of AhR to the nuclei, followed by formation of the ternary complex for transcription together with Arnt (8, 9). We next explored the possibility that IL-4 alone would induce translocation of AhR from the cytoplasm to the nuclei. To address this question, we fractionated the cytoplasm and the nuclei of the cell lysates and then analyzed the amounts of AhR in each fraction. We used actin and HDAC1 as the marker proteins for the cytoplasmic and nuclear fractions, respectively. Based on the amounts of these proteins, no cytoplasmic protein was detected in the nuclear fraction, whereas the cytoplasmic fraction contained some nuclear protein in our system (Fig. 4). Upon stimulation of IL-4, AhR protein was observed in not only the cytoplasmic but also the nuclear fraction in DND-39 cells, demonstrating that IL-4 was able to induce AhR expression and translocate AhR to the nuclei. Co-culture with TCDD decreased the amount of AhR in the cytoplasmic fraction in both DND-39 cells and HepG2 cells by degradation through the ubiquitination/proteasome pathway; however, the amount of AhR in the nuclear fraction was sustained or increased in both DND-39 cells and HepG2 cells. These results indicated that IL-4 not only induced AhR but also translocated it to the nuclei and that although TCDD degraded AhR via the proteasomal pathway, the amounts of AhR in the nuclei were not affected in the presence of TCDD in DND-39 cells.

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Fig. 4. Fractionation of AhR in the cytoplasm and nucleus. Cell lysates prepared from DND-39 cells and HepG2 cells stimulated with 10 ng ml1 of IL-4 for 24 h in the presence of 10 nM of TCDD for the indicated times were separated into cytoplasmic and nuclear fractions. Then western blotting was performed using either anti-AhR antibody or anti-actin antibody or anti-HDAC1 antibody. The asterisk depicts a non-specific band.
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Induction of CYP1A1 by IL-4 and/or TCDD in B cells
It has been shown that several xenobiotic-metabolizing genes such as CYP1A1, CYP1A2, CYP1B1 and CYP2A8 are the main targets for the transcriptional complex of AhR and Arnt (8). Based on the present findings that IL-4 induced expression of AhR and translocated AhR into nuclei, we reasoned that AhR induced and activated by IL-4 in B cells would enhance induction of these CYP enzymes. When DND-39 cells were incubated with TCDD alone, the induction of CYP1A1 was very slight (
2-fold, Fig. 5). In the presence of both IL-4 and TCDD, expression of CYP1A1 was significantly enhanced (
18-fold). It is of note that IL-4 alone had the ability to cause CYP1A1 induction (
5-fold). As the cells were harvested after incubation with TCDD for 24 h, AhR protein was almost completely diminished by TCDD (Fig. 3A).

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Fig. 5. Induction of CYP1A1 in DND-39 cells. Total RNA was extracted from DND-39 cells stimulated with 10 ng ml1 of IL-4 and/or 10 nM of TCDD for 24 h. Then real-time PCR analysis for CYP1A1 and ß-actin was performed.
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We next analyzed induction of CYP1A1 by IL-4 in mouse spleen B cells. When mouse spleen B cells were stimulated by TCDD alone, a slight induction of CYP1A1 occurred (
2-fold, Fig. 6A). In the presence of IL-4 and TCDD, significant induction of CYP1A1 as well as DND-39 cells was detected (
9-fold). Again, IL-4 alone induced several fold of CYP1A1 (
4-fold). Induction of CYP1A1 by IL-4 tended not to be observed, but rather decreased, in STAT6-deficient mice and AhR-deficient mice. Effects of TCDD remained in STAT6-deficient mice, but disappeared in AhR-deficient mice, as expected. CYP1B1 was induced in a way similar to CYP1A1 by IL-4 and/or TCDD, but the copy numbers of CYP1B1 were extremely low compared with those of CYP1A1 (data not shown). No induction of CYP1A2 was observed in mouse B cells (data not shown). ß-Actin was slightly induced by IL-4 (
2-fold, Fig. 6B), and expression of ARNT was not affected by either IL-4 or TCDD (Fig. 6C). These results clearly demonstrated that AhR induced and translocated to nuclei by IL-4 in B cells was functional, inducing expression of a xenobiotic-metabolizing gene, CYP1A1, predominantly in B cells, and that TCDD exerted a synergistic effect on this induction.

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Fig. 6. Induction of CYP1A1 in mouse B cells. Total RNA was extracted from spleen B cells derived from wild mice, STAT6-deficient mice or AhR-deficient mice of C57/BL6 strain stimulated with 10 ng ml1 of IL-4 and/or 5 pM of TCDD for 24 h. Then real-time PCR analysis for CYP1A1 (A), ß-actin (B) and Arnt (C) was performed.
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Effect of AhR on IgE synthesis and CD23 expression
IgE synthesis and CD23 expression are major well-known functions of IL-4 on B cells. It has been already reported that TCDD, an environmental pollutant, enhances IgE synthesis in B cells (6, 7). To explore the possibility that AhR induced and activated by IL-4 was involved in expression of IgE and CD23, we compared the abilities to synthesize IgE and express CD23 in wild mice and AhR-deficient mice. Although when mouse B cells were stimulated with IL-4 and anti-CD40 antibody, significant IgE synthesis was observed, 5 pM of TCDD did not affect IgE synthesis and it was not impaired in AhR-deficient mice (Fig. 7A). CD23 expression was not affected by the presence of TCDD or in AhR-deficient mice (Fig. 7B). Furthermore, neither was human IgE synthesis induced by IL-4, anti-IgM antibody and anti-CD40 antibody affected by the co-culture with up to 1 nM of TCDD, nor was CD23 expression by IL-4 influenced by TCDD (data not shown). These results indicated that expression of AhR had no effect on IgE synthesis or CD23 expression induced by IL-4 in B cells.

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Fig. 7. Effect of AhR on IgE synthesis and CD23 expression in B cells. Spleen cells derived from wild mice or AhR-deficient mice of C57/BL6 strain were cultured with 1 ng ml1 of IL-4 and 0.5 µg ml1 of anti-CD40 antibody (only A) in the presence of 5 pM of TCDD for 5 days (A) or 24 h (B). Then the amounts of IgE in the supernatant (A) or the expression of CD23 on the surface (B) was assayed.
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Discussion
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We report here our use of microarray analysis to identify IL-4- or IL-13-inducible genes in B cells, finding that the AHR gene was included in the list (data not shown). Because it is known that IL-4 and IL-13 play central roles in the pathogenesis of bronchial asthma (1, 4, 26), microarray technology has been applied to identify the downstream of IL-4 or IL-13 in several cells including bronchial epithelial cells, smooth muscle cells, lung fibroblasts and macrophages (13, 2729) or in lung tissues derived from asthmatic monkeys induced by IL-4 (30). However, thus far, there is no application of microarray to identify IL-4- or IL-13-inducible genes in B cells. Our present microarray analysis has provided us a novel biological function of IL-4/IL-13 on B cells.
It has been already known that AhR is expressed in human tonsils (31); however, in this article, we demonstrated that AhR was induced and activated by IL-4 in B cells (Figs 1 and 4). In contrast, it has been reported that TCDD, a ligand for AhR, augments IgE synthesis in B cells (6, 7). Considering these results, it was assumed that IL-4/IL-13 and TCDD synergistically act on IgE synthesis in B cells, exaggerating allergic reactions. However, our present results denied this possibility because TCDD had no effect on IgE synthesis and CD23 expression by IL-4 in B cells (Fig. 7). In contrast, IL-4 could induce a xenobiotic-metabolizing gene, CYP1A1, among a number of gene products known to be regulated by AhR (Figs 5 and 6). These data suggested that IL-4 and IL-13 would have a role in metabolizing xenobiotics, including polycyclic aromatic hydrocarbons in B cells, which is a novel biological function of these cytokines.
In this article, we demonstrated that IL-4 had an ability not only to induce AhR, but also to activate AhR (Figs 1 and 46
). Induction of AhR was dependent on STAT6, which is known to be critical for most of the biological activities of IL-4 or IL-13 (21, 22, 32) (Fig. 2). It would be possible that STAT6 binds to the promoter region of the AHR gene, enhancing its induction because the 5'-flanking region of the AHR gene has a consensus STAT6-binding motif (TTCCTGTGAA: 1721 to 1730 nucleotides from the translation start site). The present finding that AhR induction did not require de novo protein synthesis (Fig. 1B) would suggest that STAT6 might bind to this site and be involved in the induction of AhR.
It has already been shown that TCDD degrades AhR via the proteasomal pathway, and ubiquitination of AhR triggers this event (2325, 33). We demonstrated that TCDD rapidly degraded IL-4-induced AhR through the proteasomal pathway in DND-39 cells as well as HepG2 cells (Fig. 3). In contrast, IL-4 alone could cause translocation of AhR into the nuclei, sustaining expression of AhR and inducing expression of CYP1A1 (Figs 46
). These findings suggested that the nuclear translocation of AhR and the formation of the ternary transcriptional complex are not enough to trigger degradation of AhR but that binding of a ligand, such as TCDD, to AhR would be needed for this event. It is assumed that binding of a ligand to AhR may change its conformation, exposing the recognition site for the ubiquitination machinery. Thus, the activation mechanism of AhR is thought to be different between its ligands and IL-4 in that its ligands, but not IL-4 causes degradation of AhR, although both induce translocation of AhR into the nuclei.
In conclusion, we found that IL-4 could induce and activate AhR, inducing a xenobiotic-metabolizing gene, CYP1A1, in B cells. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics by inducing and activating AhR would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.
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Acknowledgements
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We thank Dovie R. Wylie for a critical review of the manuscript. This work was supported in part by a Research Grant for Immunology, Allergy and Organ Transplant from the Ministry of Health, Welfare, and Labor of Japan, a grant-in-aid for Scientific Research from Japan Society for the Promotion of Science and AstraZeneca Research Grant 2002.
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Abbreviations
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AhR | aryl hydrocarbon receptor |
Arnt | aryl hydrocarbon receptor nuclear translocator |
CYP1A1 | cytochrome P4501A1 |
HA-STAT6DN | hemagglutinin-tagged truncated form of STAT6 |
HDAC1 | histone deacetylase class 1 |
RT | reverse transcription |
TCDD | 2,3,7,8-tetra-chlorodibenzo-p-dioxin |
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Notes
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Transmitting editor: A. Falus
Received 9 December 2004,
accepted 17 March 2005.
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