1,25-Dihydroxyvitamin D3 Induces CCR10 Expression in Terminally Differentiating Human B Cells

This information is current as Aiko-Konno Shirakawa, Daisuke Nagakubo, Kunio of September 28, 2021. Hieshima, Takashi Nakayama, Zhe Jin and Osamu Yoshie J Immunol 2008; 180:2786-2795; ; doi: 10.4049/jimmunol.180.5.2786 http://www.jimmunol.org/content/180/5/2786 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

1,25-Dihydroxyvitamin D3 Induces CCR10 Expression in Terminally Differentiating Human B Cells1

Aiko-Konno Shirakawa,2 Daisuke Nagakubo,2 Kunio Hieshima, Takashi Nakayama, Zhe Jin, and Osamu Yoshie3

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19؉ B cells into IgD؊CD38؉ plasma cells in vitro. A minor proportion of the ؉ ؊ ؉ resulting CD19 IgD CD38 cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite ؉ ؊ ؉ of vitamine D3, dramatically increased the proportion of CD19 IgD CD38 cells expressing high levels of CCR10. The 1,25- ؉ ؉ (OH)2D3 also increased the number of CCR10 cells expressing surface IgA, although the majority of CCR10 cells remained Downloaded from negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3.We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion,

1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human http://www.jimmunol.org/ CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3. The Journal of Immunology, 2008, 180: 2786–2795.

lasma cells represent the terminal stage of B cell differ- gration and tissue localization of lymphocytic cells (2, 4). As for entiation and secrete large amounts of Ab. The transcrip- plasma cells, CXCR4 has been demonstrated to guide the move- P tion factors Blimp-1 and XBP-1 play essential roles in the ment of plasma cells to splenic red pulp, lymph node medullary plasma cell differentiation and function (1). Plasma cells exhibit cords, and bone marrow, where its ligand CXCL12 is abundantly preferential tissue localization in accordance with the route of im- produced (5). CXCR3 mobilizes IgG-secreting plasma cells to in- munization and the isotypes of Ig secreted. IgG-secreting plasma flammatory sites, where its ligands CXCL9, CXCL10, and/or by guest on September 28, 2021 cells are mainly induced by a systemic route of immunization and CXCL11 are strongly up-regulated (6–8). CCR9 contributes to the preferentially localize in the bone marrow. In contrast, IgA-secret- localization of IgA-secreting plasma cells to the small intestine ing plasma cells are predominantly induced by a mucosal route of (9–11), where its ligand CCL25 is selectively produced by the immunization and preferentially localize in the mucosal tissues (2). intestinal epithelial cells (11–13). Differential expression of tissue-specific adhesion molecules has CCR10 was originally identified as the receptor for CCL27 (14), 4 been demonstrated in Ab-secreting cells (ASCs) depending on the which is selectively expressed by epidermal keratinocytes (15, 16). route of immunization (3). Recently, and their recep- Accordingly, CCR10 has been shown to be expressed by skin- tors have been attracting much attention as the regulators of mi- homing effector/memory T cells expressing cutaneous lymphocyte Ag (14, 17, 18). Subsequently, another ligand of CCR10 was iden- tified and termed CCL28, which is widely expressed by the Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, epithelial cells of various mucosal tissues (11, 19–21). Thus, cer- Osaka, Japan tain types of CCR10-expressing cells must be present in the mu- Received for publication November 29, 2006. Accepted for publication December 13, 2007. cosal tissues. Previously, we have demonstrated that EBV-immor- talized human B cells express CCR10, although CCR10 is not The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance inducible by EBV-encoded latent genes (22). This was rather un- with 18 U.S.C. Section 1734 solely to indicate this fact. expected because the expression of CCR10 had not been described 1 This work was supported by a grant-in-aid from the Ministry of Education, Culture, at any of the developmental or differentiation stages of B cells Sports and Technology, Japan; by Solution-Oriented Research for Science and Tech- nology from the Japan Science and Technology Corporation; and by the High-Tech examined to date (23). Because EBV-immortalized B cells resem- Research Center Project for Private Universities: matching fund subsidy from the ble plasma cells in terms of the continuous production of Igs (24), Ministry of Education, Culture, Sports, Science and Technology of Japan, 2002–2009. we hypothesized that CCR10 might be selectively expressed at the 2 A.-K.S. and D.N. contributed equally to this work. terminal differentiation stage of B cells. We have indeed demon- 3 Address correspondence and reprint requests to Dr. Osamu Yoshie, Department of strated that a substantial fraction of plasma cells derived from hu- Microbiology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka- Sayama, Osaka 589-8511, Japan. E-mail address: [email protected] man bone marrow expresses CCR10 and efficiently migrates to its 4 ligands CCL27 and CCL28 (25). Furthermore, Butcher and his Abbreviations used in this paper: ASC, Ab-secreting cell; 1,25-(OH)2D3, 1,25-di- hydroxyvitamin D3; DC, dendritic cell; GALT, gut-associated lymphoid tissue; HEK, colleagues (26, 27) have demonstrated that IgA-ASCs present in human embryonic kidney; LCL, lymphoblastoid cell line; RA, all-trans retinoic acid; various mucosal tissues commonly express CCR10. We have also VDR, vitamin D receptor; VDRE, vitamin D response element. demonstrated that CCR10 plays an important role in the homing of Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 IgA-ASCs into the small intestine and colon (11). Consequently, it www.jimmunol.org The Journal of Immunology 2787 is now considered that the CCL28-CCR10 system comprises an Systems and Jackson ImmunoResearch Laboratories, respectively. The important element in the common mucosal immune system by pro- 1,25-(OH)2D3 was purchased from Cayman Chemical. All-trans retinoic acid (RA) was purchased from Sigma-Aldrich. 22-Oxa-1,25-(OH) D moting the wide distribution of locally induced IgA-ASCs to var- 2 3 (OCT), an analog of 1,25-(OH)2D3 and a VDR agonist, was provided by ious mucosal tissues in the body (2). However, the regulatory Chugai Pharmaceutical. TEI-9647, a VDR antagonist, was provided by mechanism of CCR10 expression in terminally differentiating B Teijin Institute for Bio-Medical Research. Unused transfusion blood sam- cells has not been determined yet. ples were provided by Osaka Red Cross Hospital. The use of the blood The 1,25-dihydroxyvitamin D (1,25-(OH) D ) is the biologi- samples was approved by the ethical committee of Kinki University School 3 2 3 of Medicine. PBMC were prepared using Ficoll-Paque. CD19ϩ B cells cally active metabolite of vitamin D3, which binds and activates were purified from PBMC by negative selection using BD IMag Human B the nuclear vitamin D receptor (VDR). The activated VDR dimer- Lymphocyte Enrichment Set-DM and BD IMagnet (BD Biosciences). The izes with another nuclear receptor, one of the retinoid X receptors, preparations were typically Ͼ90% CD19ϩ B cells. Induction of B cell and the heterodimer binds with high affinity to vitamin D response differentiation into plasma cells was performed essentially as described previously (36). Briefly, purified CD19ϩ B cells were cultured in the pres- elements (VDREs) in the promoter region of target genes (28). Ј ␮ ␮ ence of anti-IgM F(ab )2 at 5 g/ml, anti-CD40 at 1 g/ml, and IL-21 at Besides the role in calcium homeostasis, 1,25-(OH)2D3 is also 100 ng/ml for 6 days. On day 3, 1,25-(OH)2D3, OCT, TEI-9647, and RA known to exert potent immunomodulatory activities (29–31). For were added to the cultures, as indicated. example, 1,25-(OH)2D3 suppresses the production of Th1 cyto- kines such as IFN-␥ and IL-2, and consequently leads to the en- Flow cytometry hanced production of Th2 such as IL-4 and IL-5, thus FITC-labeled anti-CD38 (HIT2), PE-labeled anti-IgD (IA6–2), and PerCP- potentially promoting humoral immune responses (29, 30). The Cy5.5-labeled anti-CD19 (SJ25C1) were purchased from BD Biosciences.

1,25-(OH) D also promotes innate immunity by directly inducing PE-labeled anti-IgA1 was purchased from Southern Biotechnology Asso- Downloaded from 2 3 ciates. PerCP-Cy5.5-labeled control mouse IgG1 was purchased from BD the gene expression of antimicrobial peptides cathelicidin and Biosciences. Other labeled isotype-matched control mouse Abs were pur- ␤-defensin 2 in various human cell types (32, 33). For example, chased from Beckman Coulter. Allophycocyanin-conjugated anti-CCR10 1,25-(OH)2D3 is involved in the enhanced killing of intracellular (314305) and allophycocyanin-conjugated rat IgG2a isotype control were Mycobacterium tuberculosis by TLR-triggered human macro- purchased from R&D Systems. Cells were suspended in ice-cold PBS con- phages via the induction of cathelicidin (33). Moreover, if supplied taining 2% FCS and 0.05% sodium azide (staining buffer) and treated with normal human serum for 20 min to block FcRs. Cells were incubated for as an adjuvant supplement in systemic immunization, 1,25- 30 min with a mixture of FITC-CD38, PE-IgD, PerCP-Cy5.5-CD19, and http://www.jimmunol.org/ (OH)2D3 has been shown to promote common mucosal immune allophycocyanin-CCR10; a mixture of FITC-CD38, PE-IgA1, PerCP- responses, as evidenced by the increased mucosal secretion of IgA Cy5.5-CD19, and allophycocyanin-CCR10; or a mixture of appropriate and IgG Abs (29, 34). Recently, Sigmundsdottir et al. (35) have isotype-matched control IgGs. After washing, the cells were immediately analyzed on FACSCalibur (BD Biosciences) with appropriate gatings and reported that 1,25-(OH)2D3 induces CCR10 expression in acti- quantified with respect to the isotype-matched control IgGs. vated human T cells. Thus, 1,25-(OH)2D3 may play an important role in skin immunity by enabling T cells to migrate to the skin via Chemotaxis assay CCL27, the CCR10 ligand constitutively expressed by the epider- Assays were performed using a 96-well ChemoTx chamber (NeuroProbe) mal keratinocytes (35). Because 1,25-(OH)2D3 is also known to with 8-␮m pores, as described previously (25). Cells were suspended in promote mucosal immunity (29, 34), we hypothesized that 1,25- RPMI 1640 containing 10% FCS and 10 mM HEPES (chemotaxis me- by guest on September 28, 2021 dium), and placed over the lower wells containing chemotaxis medium (OH)2D3 might also induce CCR10 expression in terminally dif- ferentiating B cells and thus enable them to migrate to various only or CCL27 (R&D Systems). After4hat37°C, input cells and cells in the lower wells were stained with FITC-labeled anti-CD38 and PC5-la- mucosal tissues via CCL28, which is widely produced by the mu- beled CD19 (Beckman Coulter). CD19ϩCD38ϩ cells were counted on cosal epithelial cells (11, 19–21). In the present study, we have FACSCalibur. Results are presented as percentage of input cells that mi- demonstrated that 1,25-(OH)2D3 efficiently induces CCR10 in ter- grated to lower wells. minally differentiating human B cells in vitro. Furthermore, we RT-PCR have revealed that the human CCR10 promoter is directly acti- vated by Ets-1 and VDR in the presence of 1,25-(OH)2D3. Template cDNAs were generated from total RNAs from various human B cell lines using TRIzol reagent, as described previously (22). From the cultured B cells, the dead cells were first removed by using MACS Dead Materials and Methods Cell Removal Kit (Milteny Biotec), and template cDNAs were generated Cell lines using Cells to-cDNA II kit (Applied Biosystems). For semiquantitative RT-PCR, template cDNAs equivalent to 20 ng of total RNAs were ampli- BJAB and Ramos (EBV-negative Burkitt’s lymphoma cell lines), Daudi fied in a solution of 20 ␮l containing 10 pmol each primer, 0.2 mM dNTP, and Raji (EBV-positive Burkitt’s lymphoma cell lines), and BCL-SM and and1UofEx-Taq polymerase (Takara Bio). The amplification conditions BCL-TOS (EBV-immortalized B cell lymphoblastoid cell lines (LCLs)) consisted of denaturation at 94°C for 30 s (5 min for the first cycle), an- were described previously (22). BALL-1 (a human B acute lymphoblastic nealing at 60°C for 30 s, and extension at 72°C for 30 s (5 min for the last leukemia cell line) was obtained from the Health Science Research Re- cycle) for 36 cycles for CCR10, VDR, Blimp-1, and BCL-6; 33 cycles for sources Bank. All of these cell lines were cultured in RPMI 1640 supple- XBP-1; 32 cycles (B cell lines) or 36 cycles (myeloma cell lines) for Ets-1; mented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glu- and 28 cycles for GAPDH. The primers used for RT-PCR were as follows: tamine, 1 mM sodium pyruvate, 1% nonessential amino acid, 100 U/ml ϩ5Ј-GGGTAGCGACTTCTTGTTTG-3Ј and Ϫ5Ј-GTTAATGGAGTCAA penicillin, 100 ␮g/ml streptomycin, and 50 ␮M 2-ME. Human myeloma CCCAGC for Ets-1; ϩ5Ј-ATGGCCATCTGCATCGTCTC-3Ј and Ϫ5Ј-GC cell lines KMS-12BM and KMS-12PE were obtained from the Health Sci- ACCGCACAGGCTGTCCTA-3Ј for VDR; ϩ5Ј-TGCTGGATACTGCC ence Research Resources Bank. Human myeloma cell lines L-363, OPM-2, GATCTACTG-3Ј and Ϫ5Ј-TCTAGATTCGCAGCCCTAGTTGTC-3Ј for AMO-1, SK-MM-2, and KARPAS-620 were obtained from Deutsche CCR10; ϩ5Ј-GAGTAAAGAATACATACCAAAGGG-3Ј and Ϫ5Ј-CATT Sammlung von Mikroorganismen und Zellkulturen. All of the myeloma TTTCTCAGTGCTCGGTTGCT-3Ј for Blimp-1; ϩ5Ј-CAAGAAGTTTCT cell lines were cultured in RPMI 1640 supplemented with 15% FCS, 2 mM AGGAAAGGCCGG-3Ј and Ϫ5Ј-GATTGATCACACTAAGGTTGCA L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. Human TT-3Ј for BCL-6; ϩ5Ј-CCTTGTAGTTGAGAACCAGG-3Ј and Ϫ5Ј-GG embryonic kidney (HEK) 293T cells were cultured in DMEM supple- GGCTTGGTATATATGTGG-3Ј for XBP-1; ϩ5Ј-GCCAAGGTCATCCA mented with 10% FCS, 1% nonessential amino acids, 2 mM L-glutamine, TGACAACTTTGG-3Ј and Ϫ5Ј-GCCTGCTTCACCACCTTCTTGATG 100 U/ml penicillin, and 100 ␮g/ml streptomycin. TC-3Ј for GAPDH. Real-time PCR was performed on 7900 HT Fast Real- Induction of human B cell differentiation Time PCR System (Applied Biosystems) using TaqMan Gene Expression Assays for CCR10, VDR, and GAPDH. The PCR conditions were essen- Human rIL-21 was purchased from Invitrogen Life Technologies. Anti- tially as described previously (11). Gene expression was quantified by us- Ј CD40/TNFRSF5 (82111) and anti-IgM F(ab )2 were purchased from R&D ing Sequence Detection System Software (Applied Biosystems). 2788 INDUCTION OF CCR10 BY VITAMIN D3

Promoter-reporter plasmids and transcription factor expression man B cells. CD19ϩ B cells purified from PBMC (Ͼ90%) were vectors treated with anti-IgM and anti-CD40, which mimics B cell acti- The proximal CCR10 promoter region (from Ϫ1toϪ1500 bp upstream vation via Ag and T cell interactions, and cultured in the presence from the start codon) was amplified by nested PCR from human genomic of 100 ng/ml IL-21 for 6 days. As reported previously (36), ϩ DNA using Pyrobest DNA polymerase (Takara Bio) and the following whereas freshly isolated peripheral blood CD19 B cells con- primers: ϩ5Ј-ACCTCAGCCAGTGCTAGCACCATCA-3Ј and Ϫ5Ј-AC tained very few IgDϪCD38ϩ cells representing plasma cells (Ͻ1% TCCCACCTGCTCTGTGGCCT-3Ј for the first PCR, and ϩ 5Ј-TTTGTC Ϫ ϩ Ј Ϫ Ј from 6 donors), a dramatic increase in the percentage of IgD CD38 GACTCCCTGCCACAAAACCCAGCCCCA-3 and 5 -TTTAAGCTT ϳ CTCTGGCTACACAGGTTTCTGAGGT-3Ј for the second PCR. The cells was observed after 6 days of culturing (25 40% from 6 amplified fragments were digested at the SalI/HindIII sites (underlined se- donors) (Fig. 1A). We confirmed that IL-21 was essential for the quences) and cloned into the XhoI/HindIII sites of pGL3-basic luciferase efficient induction of these cells (data not shown). When added to reporter plasmid (Promega) to generate pGL3-CCR10 (Ϫ1500/Ϫ1). Seri- the cultures on day 3, 1,25-(OH) D further increased the percent- Ј Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ 2 3 ally 5 -truncated promoter fragments ( 976/ 1, 694/ 1, 514/ 1, Ϫ ϩ ϳ Ϫ348/Ϫ1, Ϫ171/Ϫ1, Ϫ137/Ϫ1, Ϫ126/Ϫ1, Ϫ110/Ϫ1, Ϫ56/Ϫ1) were also age of IgD CD38 cells (40 65% from 6 donors) and also, as PCR amplified and inserted into the XhoI/HindIII sites of pGL3-basic. reported previously (38), up-regulated the levels of CD38 expres- Site-directed mutagenesis was performed by using the overlap PCR sion (mean Ϯ SD of mean fluorescence intensity from 6 donors: method (37). The plasmids with mutated sequences were as follows: pGL3- Ϯ Ϯ 1463 183 with 1,25-(OH)2D3 vs 768 102 without 1,25- CCR10 (Ϫ171⌬Ets-1/Ϫ1), from ACAGGAAGGA to ACAGGTAGGA; Ͻ (OH)2D3, p 0.001) (Fig. 1A). We then examined CCR10 ex- pGL3-CCR10 (Ϫ171⌬Ikaros/Ϫ1), from GAATGGGAAAGT to GAAT ϩ Ϫ ϩ Ϫ ⌬ Ϫ pression in the resulting CD19 IgD CD38 cells. As shown in GGCTAAGT; pGL3-CCR10 ( 171 VDRE/ 1), from GGGTCTACGG ϩ Ϫ ϩ GGTCA to GGTACTACGGGGTCA. An Ets-1 expression vector Fig. 1B, a minor proportion of cells in the CD19 IgD CD38

(pcDNA-Ets-1) was provided by M. Ouchida (Okayama University, Grad- cells spontaneously expressed CCR10 (8ϳ18% from 6 donors), Downloaded from uate School of Medicine, Okayama, Japan). VDR was amplified from the although their surface CCR10 levels were relatively low (see be- ϩ Ј cDNA of 1,25-(OH)2D3-treated KARPAS-620 cells using the 5 -TTT Ј Ϫ Ј low). 1,25-(OH)2D3 dramatically increased the proportion of GATATCTCCTTCAGGGATGGAGGCAAT-3 and 5 -TTTCTCGAG ϩ ϩ Ϫ ϩ ACAGGCTGTCCTAGTCAGGAG-3Ј, digested at the EcoRV/XhoI sites CCR10 cells in the CD19 IgD CD38 cells in a dose-depen- (underlined sequences), and cloned into pcDNA3 to generate dent manner (34ϳ52% from 6 donors at 100 nM). The surface pcDNA3-VDR. levels of CCR10 expression were also greatly enhanced by 1,25-

Ϯ http://www.jimmunol.org/ Luciferase reporter assay (OH)2D3 (mean SD of mean fluorescence intensity from 6 do- nors: 524 Ϯ 158 at 100 nM vs 86 Ϯ 30 at 0 nM, p Ͻ 0.001). At Transient transfection was performed using DMRIE-C transfection reagent 1000 nM 1,25-(OH) D , however, the frequencies of CCR10ϩ (Invitrogen Life Technologies), following the manufacturer’s protocol. 2 3 KARPAS-620 cells (3ϳ4 ϫ 105) and HEK293T cells (1ϳ2 ϫ 105) were cells were mostly similar to those obtained at 100 nM (data not cotransfected with 0.5ϳ1 ␮g of pSV-␤-galactosidase and 3 ␮g of pGL3- shown). To further confirm that the observed up-regulation in basic or pGL3-basic inserted with various CCR10 promoter fragments. In CCR10 expression was truly mediated by 1,25-(OH) D , we also ␮ 2 3 some experiments, HEK293T cells were further cotransfected with 1.5 g examined the effects of OCT (a 1,25-(OH) D analog) and TEI- of pcDNA3-Ets-1 and/or 1.5 ␮g of pcDNA3-VDR. The cells were treated 2 3 9674 (a VDR antagonist) on CCR10 expression in IL-21-induced with either medium only or 100 nM 1,25-(OH)2D3 starting 6 h posttrans- ϩ Ϫ ϩ fection. After 48 h, the cells were lysed for luciferase assays using the CD19 IgD CD38 cells. As shown in Fig. 1C, OCT increased

ϩ by guest on September 28, 2021 Luciferase Assay System Kit (Promega). Luciferase activity was normal- the number of CCR10 cells as efficiently as 1,25-(OH)2D3 did, ized by ␤-galactosidase activity, which served as an internal control for whereas TEI-9647 effectively suppressed 1,25-(OH)2D3- and transfection efficiency. OCT-induced increases in CCR10ϩ cells. By real-time PCR, we NoShift transcription factor assays further verified the strong increases in CCR10 mRNA in the B cell

cultures treated with 1,25-(OH)2D3 (Fig. 1D). Furthermore, as Anti-Ets-1 (C-20) and anti-VDR (C-20) were purchased from Santa Cruz ϩ ϩ Biotechnology. Transcription factors binding to specific DNA sequences shown in Fig. 1E, 1,25-(OH)2D3-treated CD19 CD38 cells ex- were identified by using NoShift Transcription Factor Assay Kit (EMD hibited much enhanced chemotactic responses toward CCL27, the Biosciences), which is an ELISA-like colorimetric assay alternative to the CCR10-specific ligand (14). Collectively, these results EMSA. In brief, nuclear extracts were prepared from cells treated with clearly indicate that 1,25-(OH)2D3 is a potent inducer of CCR10 medium only or with 100 nM 1,25-(OH)2D3 for 24 h by using NucBuster Protein Extraction Kit (EMD Biosciences). Nuclear proteins were first expression in IL-21-induced terminally differentiating human B bound to the double-stranded, synthetic, biotinylated oligonucleotides, and cells. then the transcription factor-oligonucleotide complexes were captured on a Recently, Mora et al. (39) have reported that dendritic cells streptavidin-coated plate. The bound complexes were reacted with specific (DCs) derived from mucosal tissues, but not those derived from Abs, followed by HRP-conjugated anti-rabbit IgG (EMD Biosciences). Colors were developed with 3,3Ј,5,5Ј-tetramethylbenzidine, and their ab- nonmucosal tissues, strongly induce IgA secretion and CCR9 ex- sorbance value was measured at 450 nm. The synthetic oligonucleotides pression in activated B cells. All-trans retinoic acid (RA) is partly used were as follows (differences underlined): biotinylated Ets-1 and com- responsible for the specific function of mucosal tissue DCs. Fur- petitor Ets-1, ACTAGCGGGACAGGAAGGAAGAGGCG; mutant Ets-1, thermore, B cells treated with RA and cocultured with DCs mi- ACTAGCGGGACAGGTAGGAAGAGGCG; biotinylated VDRE and grated to CCL25, the CCR9 ligand (4), but not to CCL28, the competitor VDRE, GGATTAGGGTCTACGGGGTCACTTTCC; mutant VDRE, GGATTAGGTACTACGGGGCTACTTTCC. CCR10 ligand (19, 20). The latter observation led the authors to conclude that CCR10 expression was not induced by RA (39). Statistical analysis However, this does not formally exclude the induction, to a certain Statistical significance was determined using Student’s t test. We consid- extent, of CCR10 expression in terminally differentiating B cells ered p Ͻ 0.05 as statistically significant. by RA. We therefore examined the effect of RA on the surface expression of CCR10 in IL-21-induced CD19ϩCD38ϩ cells. As Results shown in Fig. 1F, RA at 100 nM, but not at 10 nM (data not Induction of CCR10 expression in terminally differentiating shown), slightly increased the number of CCR10ϩ cells in the ϩ Ϫ ϩ human B cells by 1,25-(OH)2D3 resulting CD19 IgD CD38 cells; however, its effect was much

Recently, Ettinger et al. (36) have reported that IL-21 efficiently weaker than that of 1,25-(OH)2D3. In combination, RA at 100 nM, induces the differentiation of activated human B cells into but not at 10 nM (data not shown), also enhanced the 1,25- ϩ Ϫ ϩ ϩ CD19 IgD CD38 plasma cells. By using this in vitro system, (OH)2D3-induced increases in the number of CCR10 cells in the we examined CCR10 expression in terminally differentiating hu- CD19ϩIgDϪCD38ϩ cells. This is probably because RA inhibits The Journal of Immunology 2789 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Effects of 1,25-(OH)2D3 on CCR10 expression in terminally differentiating human B cells. A, IL-21-induced terminal differentiation of activated B cells. CD19ϩ B cells were prepared from PBMCs obtained from healthy donors (n ϭ 6) by negative selection (purity, Ͼ90%). An aliquot of cells was analyzed for the surface expression of IgD and CD38 (left). The remaining cells were cultured in the presence of anti-IgM (5 ␮g/ml), anti-CD40 ␮ (1 g/ml), and IL-21 (100 ng/ml) for 6 days. 1,25-(OH)2D3 was added at a concentration of 100 nM on day 3. The cells were stained for CD19, IgD, and CD38 (right). CD19ϩIgDϪCD38ϩ cells represent plasma cells. The representative results obtained from six donors are shown. B, Induction of CCR10 by ϩ 1,25-(OH)2D3. CD19 B cells were cultured as described in A. 1,25-(OH)2D3 was added on day 3, as indicated. The cells were stained for CD19, IgD, CD38, and CCR10. The representative results obtained from three donors are shown. C, Effects of OCT (an analog of 1,25-(OH)2D3) and TEI-9647 (a VDR ϩ ␮ antagonist) on surface expression of CCR10. CD19 B cells were cultured as described in A. 1,25-(OH)2D3 (10 nM), OCT (10 nM), and TEI-9647 (1 M) were added on day 3, as indicated. Data represent mean Ϯ SEM from three cultures. The representative results from three donors are shown. D, Real-time ϩ PCR for CCR10. CD19 B cells were cultured as described in A. 1,25-(OH)2D3 was added at a concentration of 100 nM on day 3. Data represent mean Ϯ SEM from three cultures. The representative results from three donors are shown. E, Chemotactic response of CD19ϩCD38ϩ cells to CCL27. ϩ CD19 B cells were cultured as described in A. 1,25-(OH)2D3 was added at a concentration of 100 nM on day 3. Chemotaxis assays 2790 INDUCTION OF CCR10 BY VITAMIN D3

the 1,25-(OH)2D3-mediated feedback induction of CYP24A1, which encodes 1,25-(OH)2D3-24-hydroxylase, the enzyme that in- activates 1,25-(OH)2D3 (data not shown) (40). In contrast, we ob- served no significant increase in the number of CCR9ϩ cells in IL-21-induced CD19ϩIgDϪCD38ϩ cells upon treatment with RA

or 1,25-(OH)2D3, alone or in combination (data not shown). As reported by Mora et al. (39), RA may require the presence of DCs for its efficient induction of CCR9 in activated B cells. Next, we investigated whether CCR10 expression was coregu- lated with IgA expression in terminally differentiating B cells. As

shown in Fig. 1G, 1,25-(OH)2D3 did not significantly increase the IgA content in the culture supernatants of IL-21-induced CD19ϩIgDϪCD38ϩ cells. However, the potent growth-inhibitory

effect of 1,25-(OH)2D3 might paradoxically reduce the total amount of IgA produced by IL-21-induced CD19ϩIgDϪCD38ϩ cells as a whole (41, 42). We therefore also examined surface IgA expression in the resulting CD19ϩCD38ϩ cells. Although surface IgAϩ cells were detected only at low levels in IL-21-induced ϩ ϩ CD19 CD38 cells, their levels were significantly increased upon Downloaded from

treatment with 1,25-(OH)2D3 (Fig. 1H). Furthermore, the increase ϩ in the number of surface IgA cells by 1,25-(OH)2D3 was more pronounced in the CCR10ϩ fraction than in the CCR10Ϫ fraction (mean percentage Ϯ SD from 3 donors: IgAϩCCR10Ϫ cells, from 2.0 Ϯ 1.1 to 3.8 Ϯ 1.9; IgAϩCCR10ϩ, from 0.7 Ϯ 0.6 to 6.5 Ϯ ϩ

0.3). Nevertheless, the majority of CCR10 cells remained nega- http://www.jimmunol.org/

tive for surface IgA. Thus, 1,25-(OH)2D3 alone may not be suffi- cient in inducing IgA expression in terminally differentiating hu- man B cells. In contrast, RA had no significant effect on surface IgA expression in IL-21-induced CD19ϩCD38ϩ cells, including the CCR10ϩ fraction (data not shown). This may again be due to its requirement of DCs for the efficient induction of IgA expression in activated B cells (39).

Identification of the major regulatory elements in the human by guest on September 28, 2021 CCR10 promoter

Next, we examined the effect of 1,25-(OH)2D3 on CCR10 expres- sion in a human myeloma cell line KARPAS-620. As shown in

Fig. 2A, 1,25-(OH)2D3 up-regulated CCR10 expression in KAR

PAS-620 cells. 1,25-(OH)2D3 also up-regulated VDR expression, FIGURE 2. Effect of 1,25-(OH)2D3 on CCR10 expression in KARPAS- as reported previously (43). By real-time PCR, it was determined 620 cells. KARPAS-620 cells were cultured with medium only or with 100 nM 1,25-(OH)2D3 for 2 days. A, Semiquantitative RT-PCR analysis for that 1,25-(OH)2D3 up-regulated the levels of CCR10 mRNA and VDR mRNA in KARPAS-620 cells several folds (Fig. 2B). 1,25- expression of CCR10, VDR, and GAPDH. The representative results from two separate experiments are shown. B, Real-time PCR analysis for (OH) D also up-regulated the surface expression of CCR10 in 2 3 CCR10 and VDR. Data represent mean Ϯ SEM from three cultures. The KARPAS-620 cells (Fig. 2C). We also confirmed that OCT (an representative results from two separate experiments are shown. C, Effects analog of 1,25-(OH)2D3) increased the surface expression of of OCT and TEI-9647 on the surface expression of CCR10 in KARPAS- CCR10, whereas TEI-9647 (a VDR antagonist) suppressed the up- 620 cells. Cells were cultured for 2 days with medium only or with 1,25- ␮ regulation of surface CCR10 induced by 1,25-(OH)2D3 or OCT. (OH)2D3 (10 nM), OCT (10 nM), and/or TEI-9647 (1 M), as indicated. These results were highly concomitant with those obtained from Cells were stained with anti-CCR10 and analyzed by flow cytometry. Data IL-21-induced normal CD19ϩCD38ϩ cells (Fig. 1). Thus, KAR represent mean Ϯ SEM from three cultures. The representative results from PAS-620 cell line can be used as a model for the analysis of the two separate experiments are shown. transcriptional regulation of CCR10 expression in terminally dif- ferentiating human B cells. We transfected a luciferase reporter plasmid pGL3 or pGL3- and a human embryonic kidney cell line HEK293T. As shown in CCR10 (Ϫ1500/Ϫ1), which was inserted with the human CCR10 Fig. 3A, compared with the control pGL3, pGL3-CCR10 (Ϫ1500/ promoter fragment from Ϫ1500 to Ϫ1 bp, into KARPAS-620 cells Ϫ1) showed a high basal promoter activity only in KARPAS-620

were performed using 400 nM CCL27. Migrated CD19ϩCD38ϩ cells were counted by flow cytometry after surface staining to obtain percentage of input of CD19ϩCD38ϩ cells. Data represent mean Ϯ SEM from triple assays. The representative results from three donors are shown. F, Effect of RA on surface ϩ expression of CCR10. CD19 B cells were cultured as described in A. RA and 1,25-(OH)2D3 were added on day 3, as indicated. Cells were stained for CD19, IgD, CD38, and CCR10. The representative results from three donors are shown. G, ELISA for IgA. CD19ϩ B cells were cultured as described in A. IgA contents in the culture supernatants were determined by ELISA. Data represent mean Ϯ SEM from three cultures. The representative results from ϩ three donors are shown. H, Surface IgA expression. CD19 B cells were cultured as described in A. 1,25-(OH)2D3 was added at a concentration of 100 nM on day 3. Cells were stained for CD19, CD38, CCR10, and IgA. The representative results from three donors are shown. The Journal of Immunology 2791

FIGURE 3. Identification of the major regulatory elements in the human CCR10 promoter. Cells were transfected with pGL3 or pGL3 carrying the indicated human CCR10 promoter fragments. Starting 6 h posttransfection, cells were treated with me- dium only or with 100 nM 1,25-(OH)2D3. Luciferase assays were performed 48 h post- Downloaded from transfection. All data represent mean Ϯ SD from triplicate assays. The representative re- sults from at least three separate experiments are shown. A, Cell specificity. KARPAS-620 cells and HEK293T cells were transfected, as indicated. B, A schematic depiction of po- tential transcriptional elements in the http://www.jimmunol.org/ CCR10 promoter between Ϫ1500 and Ϫ1 bp (left) and between Ϫ137 and Ϫ110 bp (right). C, Deletion analysis of the CCR10 promoter. KARPAS-620 cells were trans- fected, as indicated. The fold induction by

1,25-(OH)2D3 is shown in parenthesis. D, Mutation analysis of the CCR10 promoter. KARPAS-620 cells were transfected, as in-

dicated. E, Reconstitution experiments. by guest on September 28, 2021 HEK293T cells were transfected with pGL3 or pGL3-CCR10 (Ϫ1500/Ϫ1) and also with an expression vector for Ets-1, that of VDR, or both, as indicated.

cells. Furthermore, 1,25-(OH)2D3 strongly up-regulated the pro- moter activity and induction by 1,25-(OH)2D3 with the promoter moter activity of pGL3-CCR10 (Ϫ1500/Ϫ1) in KARPAS-620 fragment from Ϫ126 to Ϫ1 bp in comparison with that from Ϫ137 Ϫ cells, but not in HEK293T cells. Thus, the CCR10 promoter frag- to 1 bp, whereas the induction by 1,25-(OH)2D3 was much re- ment from Ϫ1500 to Ϫ1 bp was proven to be specifically active duced with the promoter fragment from Ϫ110 to Ϫ1 bp, and there and responsive to 1,25-(OH)2D3 only in KARPAS-620 cells. was essentially no promoter activity with the promoter fragment As shown in Fig. 3B (left), the on-line program TFSEARCH from Ϫ56 to Ϫ1 bp. These results suggested the presence of the (www.cbrc.jp/reserach/db/TFSEARCH.html) revealed potential major regulatory elements to lie within the promoter region be- binding sites for various transcription factors in the promoter re- tween Ϫ171 and Ϫ56 bp; a possible negative regulatory element gion from Ϫ1500 to Ϫ1 bp. To identify the actual regulatory el- within Ϫ137 to Ϫ126 bp; a major VDRE within Ϫ126 to Ϫ110 ements in the CCR10 promoter, we next generated a series of bp; and a critical promoter element within Ϫ110 to Ϫ56 bp. As pGL3 reporter plasmids carrying progressively 5Ј-truncated pro- shown in Fig. 3B (left), TFSEARCH revealed an Ikaros site to lie moter fragments and transfected them to KARPAS-620 cells. As within Ϫ136 to Ϫ125 bp and an Ets-1 site within Ϫ71 to Ϫ62 bp; shown in Fig. 3C, we observed dramatic increases in basal pro- however, no potential VDRE was found to lie within Ϫ126 to 2792 INDUCTION OF CCR10 BY VITAMIN D3

FIGURE 5. RT-PCR analysis of human B cell lines. Total RNA was isolated from the indicated cell lines as follows: BALL-1 (a human B acute

lymphoblastic leukemia cell line), BJAB and Ramos (EBV-negative Bur- Downloaded from kitt’s lymphoma cell lines), Daudi and Raji (EBV-positive Burkitt’s lym- FIGURE 4. NoShift transcription factor assays. A, Specific binding of phoma cell lines), BCL-SM and BCL-TOS (EBV-transformed LCLs), Ets-1 to the Ets-1 site. Nuclear extracts were prepared from CCR10-ex- KMS-12BM, KMS-12PE, L-363, OPM-2, AMO-1, SK-MM-2, and KAR pressing human myeloma cell lines KARPAS-620 and KMS-12BM. Nu- PAS-620 (myeloma cell lines). The isotype of secretory Ig is indicated in clear proteins that bound to the biotinylated oligonucleotide covering the parenthesis. NS, nonsecretor. Semiquantitative RT-PCR was performed for Ets-1 site were captured on an avidin-coated plate and detected by anti- CCR10, VDR, Ets-1, Blimp-1, BCL-6, XBP-1, and GAPDH. The repre- Ets-1 Ab. Specificity was demonstrated by adding the wild-type Ets-1 oli-

sentative results from three separate experiments are shown. http://www.jimmunol.org/ gonucleotide (competitor) or the mutant Ets-1 oligonucleotide (mut com- petitor) at a 10-fold excess. B, Specific binding of 1,25-(OH)2D3-activated VDR to the VDRE. Nuclear extracts were prepared from CCR10-express- ing human myeloma cell lines KARPAS-620 and KMS-12BM treated for we used the NoShift transcription factor assay, which is an ELISA- like colorimetric assay alternative to the EMSA. As shown in Fig. 24 h with medium only or with 100 nM 1,25-(OH)2D3. Nuclear proteins that bound to the biotinylated oligonucleotide covering the VDRE site were 4A, the specific binding of Ets-1 to the Ets-1 element was dem- captured on an avidin-coated plate and detected by anti-VDR Ab. Speci- onstrated by using the nuclear extracts from CCR10-expressing ficity was demonstrated by adding the wild-type VDRE oligonucleotide human myeloma cell lines KARPAS-620 and KMS-12BM. Fur- (competitor) or the mutant VDRE oligonucleotide (mut competitor) at a thermore, as shown in Fig. 4B, the specific binding of VDR to the

10-fold excess. putative VDRE was also confirmed by using the nuclear extracts by guest on September 28, 2021

from these myeloma cell lines treated with 1,25-(OH)2D3.

Ϫ110 bp. Yet, as shown in Fig. 3B (right), a close inspection of the Coexpression of Ets-1 and VDR in CCR10-expressing human nucleotide sequence revealed a possible VDRE within the region B cell lines from Ϫ126 to Ϫ110 bp based on the criteria of tandem direct Ets-1 is known to be ubiquitously expressed in B cells (46), repeats of 6 nt separated by 3 nt (44, 45). We, therefore, generated whereas activated B cells express VDR (47). Therefore, we exam- three reporter plasmids with a mutation in each potential element ined the correlation between CCR10 expression and those of Ets-1 in the background of the promoter fragment from Ϫ171 to Ϫ1bp and VDR in various human B cell lines. As shown in Fig. 5, the and transfected them and several control plasmids into KARPAS- CCR10-expressing cell lines (myeloma cell lines and EBVϩ 620 cells. As shown in Fig. 3D, the mutation at the Ikaros site LCLs) consistently expressed both Ets-1 and VDR. Although Ets-1 strongly up-regulated the basal promoter activity and the induction was also expressed in other B cell lines, the expression of VDR by 1,25-(OH)2D3. The mutation in the potential VDRE strongly highly correlated with CCR10 expression. The CCR10-expressing ϩ reduced the induction by 1,25-(OH)2D3, and the mutation at the myeloma cell lines and EBV LCLs also commonly expressed Ets-1 site almost completely abrogated the basal promoter activity. Blimp-1, the master regulator for plasma cell differentiation (1). These results demonstrated that the potential Ikaros site functioned XBP-1, another transcription factor essential for plasma cell de- as a negative regulatory element, the postulated VDRE was mainly velopment (1), was also commonly expressed in the CCR10-ex- ϩ responsible for the induction by 1,25-(OH)2D3, and the Ets-1 site pressing myeloma cell lines and EBV LCLs, although its expres- was essential for the basic promoter activity. sion was also observed in other B cell lines. In contrast, BCL-6 To further elucidate the critical roles of Est-1 and VDR in the was only expressed in Burkitt’s lymphoma cell lines, supporting activation of the CCR10 promoter, we performed a reconstitution their origin from germinal center B cells (1). It is also noteworthy experiment using HEK293T cells. As shown in Fig. 3E, the ex- that the isotype of Ig produced by the CCR10-expressing myeloma pression of Ets-1, but not that of VDR, strongly induced the basal cell lines was mostly IgG, supporting the notion that CCR10 ex- promoter activity of pGL3-CCR10 (Ϫ1500/Ϫ1). Furthermore, the pression and IgA production are independently regulated. expression of both Ets-1 and VDR led to the strong up-regulation of the promoter activity by 1,25-(OH)2D3. Discussion In the B cell lineage, CCR10 is considered to play an essential role Specific binding of Ets-1 and 1,25-(OH)2D3-activated VDR to in the common mucosal immune system by guiding IgA-ASCs to the respective elements in the CCR10 promoter various mucosal tissues via its ligand CCL28, which is constitu-

To prove the specific binding of Ets-1 and 1,25-(OH)2D3-activated tively expressed by epithelial cells in various mucosal tissues (2, VDR to the respective elements in the human CCR10 promoter, 11, 25–27). In the present study, by using IL-21-induced terminal The Journal of Immunology 2793 differentiation of human B cells in vitro (36), we have demon- It is known that IgA-ASCs in the intestinal tissues frequently strated that 1,25-(OH)2D3 robustly induces CCR10 expression in express CCR9 and/or CCR10 (26, 27). Recently, Mora et al. (39) terminally differentiating human B cells (Fig. 1). Furthermore, we have demonstrated that DCs in the gut-associated lymphoid tissues have demonstrated that Ets-1 and 1,25-(OH)2D3-activated VDR (GALT), but not those in the nonmucosal lymphoid tissues, induce ␣ ␤ cooperatively activate the CCR10 promoter via an Ets-1 site at the expression of 4 7 and CCR9 in activated B cells. Moreover, Ϫ71 to Ϫ62 bp and a major VDRE at Ϫ124 to Ϫ110 bp (Fig. 3). DCs in GALT efficiently induce IgA secretion in activated B cells We have also demonstrated that CCR10-expressing human my- (39). In this context, DCs in GALT produce RA, the active me- eloma cell lines and EBVϩ LCLs consistently express both Ets-1 tabolite of vitamin A, which plays an essential role in the induction and VDR at high levels (Fig. 5). Previous studies have demon- of gut tropism and IgA secretion in activated B cells by mucosal strated that 1,25-(OH)2D3, if supplied as an adjuvant supplement, DCs (39). However, RA alone is not sufficient and requires the promotes common mucosal immune responses even upon systemic presence of DCs for the observed effects (39). Thus, additional immunization (29, 34). Based on the present results, 1,25-(OH)2D3 signals via cytokines and/or surface molecules provided by DCs may promote mucosal immunity partly by inducing CCR10 ex- may be required for the induction of gut tropism and IgA secretion pression in terminally differentiating B cells. However, we ob- by RA (39). In the present study, we have demonstrated that, at served only a marginal increase in IgA expression in terminally high concentrations, RA moderately induces CCR10 expression in differentiating human B cells by 1,25-(OH)2D3 (Fig. 1). Therefore, terminally differentiating human B cells (Fig. 1). RA also enhances 1,25-(OH)2D3 alone may not be sufficient to promote mucosal IgA 1,25-(OH)2D3-mediated CCR10 induction in terminally differen- responses in vivo. Furthermore, Chen et al. (42) have recently tiating B cells (Fig. 1). However, we did not observe the induction

␤ Downloaded from reported that 1,25-(OH)2D3 inhibits the ongoing proliferation of of CCR9 or 7 integrin in terminally differentiating B cells by RA activated B cells by inducing their apoptosis, and thereby signif- or 1,25-(OH)2D3 in the present culture conditions (data not icantly inhibits the generation of plasma cells and memory B cells shown). This appears to be partly in agreement with the conclusion in vitro, although the up-regulation of the genetic programs in- by Mora et al. (39) that RA requires the presence of DCs for the ␣ ␤ volved in B cell differentiation was not strongly affected. There- efficient induction of 4 7 and CCR9 in activated B cells. fore, the effect of 1,25-(OH)2D3 on the terminal differentiation of The 1,25-(OH)2D3 has been shown to induce the expression of

B cells may be critically dependent on the timing of their exposure the antimicrobial peptides cathelicidin and ␤-defensin 2 in various http://www.jimmunol.org/ to 1,25-(OH)2D3. types of human cells (32, 33). Because CCL28, the CCR10 ligand

Vitamin D3 (cholecalciferol) is generated in the skin from 7-de- expressed by various mucosal epithelial cells (11, 19–21), is also hydrocholesterol in response to sun exposure (48). Vitamin D3 is a chemokine with a potent antimicrobial activity (21), 1,25- then transported to the liver and converted to 25-hydroxyvitamin (OH)2D3 might induce not only the CCR10 expression in termi-

D3, which is the main circulating vitamin D3 metabolite (48). The nally differentiating B cells, but also the expression of CCL28 and ␣ key enzyme 25-hydroxyvitamin D3-1 -hydroxylase, which is en- other antimicrobial peptides in mucosal tissue cells, thus possibly coded by CYP27B1, catalyzes the synthesis of the active form orchestrating both innate and acquired immunity in the mucosal

1,25-(OH)2D3 from 25-hydroxyvitamin D3 mainly in the kidney tissues. However, we did not observe 1,25-(OH)2D3 to exert any (48). Among the hemopoietic cells, macrophages and mature DCs strong effect on the constitutive expression of CCL28 in two hu- by guest on September 28, 2021 express CYP27B1 at high levels, whereas B cells express man colon epithelial cell lines T84 and Caco-2 (data not shown), CYP27B1 only at low levels (31, 49). Thus, macrophages and even though both of the cell lines strongly up-regulated the mature DCs, especially those in mucosal tissues, may produce expression of CYP24A1 in response to 1,25-(OH)2D3 (40). Fur-

1,25-(OH)2D3 to induce CCR10 expression in terminally differen- thermore, we detected CCL28 mRNA in the intestinal tissues of tiating B cells in vivo. VDR-deficient mice (53) at levels comparable to those of con- Upon activation, B cells are known to up-regulate VDR (47). trol wild-type mice (data not shown). However, only after a strong activation, such as the concomitant Recently, Sigmundsdottir et al. (35) have demonstrated that cross-linking of Ig receptor and ligation of CD40, do activated B 1,25-(OH)2D3 induces CCR10 expression in activated human T cells become responsive to 1,25-(OH)2D3 (50). Thus, the elevated cells. The authors have suggested the presence of a possible expression and function of VDR in terminally differentiating B VDRE(s) within the CCR10 promoter region from –96 to –54 bp, cells may in part explain the plasma cell stage-specific expression by demonstrating the specific binding of the complex of VDR and of CCR10. The stage-specific expression of CCR10 may also be its heterodimeric partner retinoid X receptor to this region by a regulated by a negative regulatory element(s) in the CCR10 pro- gel-shift assay (35). They have also noted that the region is well moter. We have indeed mapped a negative regulatory element in conserved between humans and mice (35). However, they did not close vicinity to the VDRE (Fig. 3). Sequence analysis of this perform the promoter analysis to prove the functional importance region has revealed a putative Ikaros site immediately upstream of of the suggested region for the induction by 1,25-(OH)2D3.Inthe the VDRE (Fig. 3). The Ikaros family of zinc finger transcription present study, we have mapped a strong VDRE at Ϫ124 to Ϫ110 factors (Ikaros, Aiolos, and Helios) is an important regulator of bp in the human CCR10 promoter, which thus locates in the up- lymphoid development and differentiation (51). Ikaros and Aiolos stream of the region suggested by Sigmundsdottir et al. (35). We have been shown to function as strong transcriptional repressors also observed a weak up-regulation of the promoter activity by

(52). The Ikaros family genes are transcribed as large numbers of 1,25-(OH)2D3 with the reporter plasmids missing the major isoforms through extensive alternative splicing. Shorter isoforms VDRE, but carrying the region suggested by Sigmundsdottir et al. tend to behave as dominant-negative isoforms upon heterodimer- (35) (see pGL3-CCR10 (Ϫ110/Ϫ1) in Fig. 3C and pGL3-CCR10 ization with longer isoforms and even with the isoforms of other (Ϫ171⌬VDRE) in Fig. 3D). Therefore, the region suggested by family members (51). Thus, the mechanisms of transcriptional reg- Sigmundsdottir et al. (35) may also contain a weak VDRE. ulation involving the Ikaros family are highly complex. Future Unexpectedly, the VDRE that we have identified in the human studies are necessary to identify the actual transcription factor(s) CCR10 promoter is not present in the mouse CCR10 promoter. In binding to the putative Ikaros site in the CCR10 promoter. Such fact, by aligning the human and mouse CCR10 promoter regions studies may provide further insight into the transcriptional regu- from Ϫ1500 to Ϫ1 bp, there are only three short stretches spanning lation of the plasma cell stage-specific gene expression. from Ϫ1258 to Ϫ996 bp, from Ϫ711 to Ϫ659 bp, and from Ϫ100 2794 INDUCTION OF CCR10 BY VITAMIN D3 to Ϫ1 bp that are homologous between the two species (the nu- thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. cleotide numbers are from the human sequence). Therefore, the J. Exp. Med. 195: 269–275. 10. Pabst, O., L. Ohl, M. Wendland, M. A. Wurbel, E. Kremmer, B. Malissen, and major regulatory elements that we have found in the region from R. Forster. 2004. CCR9 contributes to the localization of Ϫ137 to Ϫ110 bp are totally missing in the mouse sequence. plasma cells to the small intestine. J. Exp. Med. 199: 411–416. Furthermore, our preliminary studies demonstrated that 1,25- 11. Hieshima, K., Y. Kawasaki, H. Hanamoto, T. Nakayama, D. Nagakubo, A. Kanamaru, and O. Yoshie. 2004. CC chemokine ligands 25 and 28 play es- (OH)2D3 was mostly unable to induce CCR10 expression in sential roles in intestinal extravasation of IgA antibody-secreting cells. J. Immu- B220lowCD138ϩ terminally differentiating mouse B cells gen- nol. 173: 3668–3675. ϩ 12. Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, erated in vitro by culturing naive B cells with anti-IgM anti- N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, and A. Zlotnik. 1997. CD40 ϩ IL-21 (54), anti-IgM ϩ IL-5 ϩ IL-6 (39), or LPS ϩ TECK: a novel CC chemokine specifically expressed by thymic dendritic cells IL-5 ϩ TGF-␤ (55) (data not shown). Moreover, we detected and potentially involved in T cell development. Immunity 7: 291–301. 13. Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. I. Roberts, CCR10 mRNA in the intestinal tissues of VDR-deficient mice (53) E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, et al. 2000. Lym- at levels comparable to those in control wild-type mice (data not phocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine shown). The B220lowIgAϩ fraction of the mesenteric lymph node (TECK) expression distinguish the small intestinal immune compartment: epi- thelial expression of tissue-specific chemokines as an organizing principle in obtained from VDR-deficient mice also contained CCR10-ex- regional immunity. J. Exp. Med. 192: 761–768. pressing cells at levels comparable to those from wild-type mice 14. Homey, B., W. Wang, H. Soto, M. E. Buchanan, A. Wiesenborn, D. Catron, (data not shown). Notably, Sigmundsdottir et al. (35) also men- A. Muller, T. K. McClanahan, M. C. Dieu-Nosjean, R. Orozco, et al. 2000. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 tioned in their discussion that 1,25-(OH)2D3 induced CCR10 ex- (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ pression in mouse T cells much less efficiently than in human T ILC). J. Immunol. 164: 3465–3470. cells. Therefore, there may be a substantial species difference in 15. Morales, J., B. Homey, A. P. Vicari, S. Hudak, E. Oldham, J. Hedrick, R. Orozco, Downloaded from N. G. Copeland, N. A. Jenkins, L. M. McEvoy, and A. Zlotnik. 1999. CTACK, the role of 1,25-(OH)2D3 in the CCR10 expression in skin-homing a skin-associated chemokine that preferentially attracts skin-homing memory T T cells and terminally differentiating B cells between humans and cells. Proc. Natl. Acad. Sci. USA 96: 14470–14475. mice. However, it may not be so surprising that humans and noc- 16. Ishikawa-Mochizuki, I., M. Kitaura, M. Baba, T. Nakayama, D. Izawa, T. Imai, H. Yamada, K. Hieshima, R. Suzuki, H. Nomiyama, and O. Yoshie. 1999. Mo- turnal animals such as mice have evolved differently with regard to lecular cloning of a novel CC chemokine, -11 receptor ␣-locus che- their requirement for the sunlight exposure and the resulting 1,25- mokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by molluscum contagiosum virus. FEBS Lett. 460: (OH)2D3. The regulatory mechanism of CCR10 expression in ter- 544–548. http://www.jimmunol.org/ minally differentiating mouse B cells thus remains to be 17. Homey, B., H. Alenius, A. Muller, H. Soto, E. P. Bowman, W. Yuan, L. McEvoy, elucidated. A. I. Lauerma, T. Assmann, E. Bunemann, et al. 2002. CCL27-CCR10 interac- tions regulate T cell-mediated skin inflammation. Nat. Med. 8: 157–165. 18. Hudak, S., M. Hagen, Y. Liu, D. Catron, E. Oldham, L. M. McEvoy, and Acknowledgments E. P. Bowman. 2002. Immune surveillance and effector functions of CCR10ϩ We thank Namie Sakiyama for her excellent technical assistance; Dr. skin homing T cells. J. Immunol. 169: 1189–1196. 19. Wang, W., H. Soto, E. R. Oldham, M. E. Buchanan, B. Homey, D. Catron, Mamoru Ouchida (Okayama University, Graduate School of Medicine, N. Jenkins, N. G. Copeland, D. J. Gilbert, N. Nguyen, et al. 2000. Identification Okayama, Japan) for providing Ets-1/pcDNA3; Chugai Pharmaceutical for of a novel chemokine (CCL28), which binds CCR10 (GPR2). J. Biol. Chem. 275: providing OCT and VDR-KO mice; and TEIJIN Institute for Bio-Medical 22313–22323.

Research for providing TEI-9647. We are also very grateful to Dr. Shigeaki 20. Pan, J., E. J. Kunkel, U. Gosslar, N. Lazarus, P. Langdon, K. Broadwell, by guest on September 28, 2021 M. A. Vierra, M. C. Genovese, E. C. Butcher, and D. Soler. 2000. A novel Kato (University of Tokyo) for allowing us to use VDR-KO mice provided chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal by Chugai Pharmaceutical. tissues. J. Immunol. 165: 2943–2949. 21. Hieshima, K., H. Ohtani, M. Shibano, D. Izawa, T. Nakayama, Y. Kawasaki, Disclosures F. Shiba, M. Shiota, F. Katou, T. Saito, and O. Yoshie. 2003. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial The authors have no financial conflict of interest. activity. J. Immunol. 170: 1452–1461. 22. Nakayama, T., R. Fujisawa, D. Izawa, K. Hieshima, K. Takada, and O. Yoshie. 2002. 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