Cells Expression in Murine Dendritic Cells and B Differential Regulation

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

This information is current as Hormas Ghadially, Xiao-Lan Ross, Claudia Kerst, Jun Dong, of September 24, 2021. Angelika B. Reske-Kunz and Ralf Ross J Immunol 2005; 174:5620-5629; ; doi: 10.4049/jimmunol.174.9.5620 http://www.jimmunol.org/content/174/9/5620 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells1

Hormas Ghadially,2 Xiao-Lan Ross, Claudia Kerst, Jun Dong, Angelika B. Reske-Kunz, and Ralf Ross3

The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5؅-flanking promoter region, and detected two distinct sites -for initiation of transcription by 5؅-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endog enous gene. Within the proximal promoter region, we identified potential binding sites for NF-␬B, Ikaros, and a putative GC box. All three regions bind proteins. The NF-␬B site was shown to specifically bind NF-␬B subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, TNF-␣-stimulated bone marrow-derived DC, and DC line XS106. Furthermore, promoter activity was affected by targeted mutagenesis of the NF-␬B site and transactivation with p50 and p65. The region Downloaded from harboring the putative Ikaros site contributes to promoter activity, but the binding protein does not belong to the Ikaros family. The GC box was shown to specifically bind Sp1 using extracts from LPS-stimulated B cells and A20/2J but not from DC and DC line XS106. Additionally, Sp1 transactivated the promoter in A20/2J but not in XS106 cells, and mutation of the Sp1 site dimin- ished transactivation. Furthermore, binding of the protein complex at the GC box is required for NF-␬B activity, and the spatial alignment of the binding sites is of critical importance for promoter activity. Thus, identical and distinct proteins contribute to expression of CCL22 in DC and B cells. The Journal of Immunology, 2005, 174: 5620–5629. http://www.jimmunol.org/

hemokines are a group of functionally and structurally cell-derived chemokine-1 or DC and B cell-derived chemokine related small proteins that are critically important in ini- (6–9). CCL22 has been shown to play an important role in C tiating and controlling the homing and migration of leu- various diseases and mouse disease models. For example, ele- kocytes (reviewed in Refs. 1 and 2). Other functions of individual vated levels of CCL22 have been found in affected tissue of chemokines or chemokine receptors include, e.g., the regulation of patients suffering from atopic dermatitis (10), atopic asthma angiogenesis (3) and hematopoiesis (4). Based on the relative po- (10), and contact hypersensitivity (11). Furthermore, CCL22 sition of cysteine residues, four families of chemokines are distin- has been shown to be critically involved in mouse models of by guest on September 24, 2021 guishable, namely CXC (␣), CC (␤), CX3C (␦), and C (␥). The allergen-induced lung inflammation (12), atopic dermatitis (13), genes of most of the CXC, as well as the CC chemokines, are and septic peritonitis (14). clustered and show a similar exon/intron structure, hinting at du- The major function of CCL22 appears to be the induction of plication of common ancestor genes during evolution. For most chemotactic migration of activated T cells (7–9, 15), predomi- chemokines, homologues have been identified in mice and hu- nantly of the Th2 type (16) and of CD4ϩCD25ϩ regulatory T cells 4 mans, but there are exceptions such as the dendritic cell (DC) - (17), through its specific receptor CCR4. However, another as yet chemokine 1 (no mouse homologue) (5) and IL-8 (no clear-cut unidentified receptor has also been implicated (18, 19). The murine murine homologue), suggesting relatively recent evolutionary re- CCL22 is expressed by activated B lymphocytes and DC (8, 9). arrangements and differences in the equipment of the immune sys- Murine epidermal Langerhans cells (LC), an exceptionally homo- tem of mice and humans. geneous population of immature DC, produce CCL22 mRNA only The CCL22 is present in both mice and humans. In humans, upon maturation (9), whereas other myeloid DC transcribe CCL22 CCL22 is also referred to as macrophage-derived chemokine or constitutively (8). Epidermal LC and other immature DC represent stimulated T cell chemotactic protein-1 and in mice as activated B sentinel cells of the immune system. Activation, e.g., by inflammatory stimuli, induces maturation into the most efficient APC known, which is outstanding in activating naive T cells (reviewed Clinical Research Unit Allergology, Department of Dermatology, Johannes Guten- in Ref. 20). Applying a differential screening approach, we iden- berg-University, Mainz, Germany tified numerous genes differentially expressed during matura- Received for publication July 1, 2004. Accepted for publication January 25, 2005. tion of LC (21, 22). We used regulatory sequences of one of The costs of publication of this article were defrayed in part by the payment of page these genes, namely the actin-bundling protein fascin, to tran- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. scriptionally target DC (23, 24). Furthermore, the CCL22 gene 1 This work was supported by the Deutsche Forschungsgemeinschaft, SFB548. was identified in this screening as the most abundantly ex- 2 Current address: Department of Vascular Biology and Thrombosis Research, Med- pressed gene induced during maturation of murine epidermal ical University of Vienna, Vienna, Austria. LC (9). The high expression level and the tight regulation of 3 Address correspondence and reprint requests to current address: Dr. Ralf Ross, expression, as indicated by the stage specificity, prompted us to Justus-Liebig-Universita¨t Giessen, Winchester Str. 2, D-35394 Giessen, Germany. isolate the gene and characterize the regulatory elements me- E-mail address: [email protected] diating expression in DC and B cells. Interestingly, DC and B 4 Abbreviations used in this paper: DC, dendritic cell; LC, Langerhans cell; BM-DC, bone marrow-derived DC; ORF, open reading frame; UTR, untranslated region; cells use—at least in part—the same promoter elements but EF1␣, elongation factor 1␣1. different protein complexes are formed.

Materials and Methods hybridizing with mouse CCL22 cDNA probe were identified by colony Animals filter hybridization. BALB/cJ mice were bred in the Central Animal Core Facility of the University DNA sequencing and sequence analysis of Mainz under specified pathogen-free conditions. Mice were used at 2–5 mo Nucleotide sequences were determined by cycle sequencing using the ABI of age. PRISM Dye Termination Cycle Sequencing Ready Reaction kit as recommended and analyzed on a PE 373A DNA sequencer (PerkinElmer). GC- Cells rich DNA templates were linearized, heat-denatured (98°C for 5 min) and Epidermal cell suspensions were prepared from pelts and used for LC mixed with 5% (v/v) DMSO before sequencing. enrichment either directly or following in vitro cultivation for 3 days as Potential transcription factor binding sites were identified by MatInspec- ͗ ͘ described previously (21). LC were enriched from epidermal cell suspen- tor V 2.2 ( http://transfac.gbf.de ). Transcription factors mentioned in the sions by immunomagnetic separation with Dynabeads M-450 (Dynal Bio- text are named according to the first corresponding publication. d,b d tech) loaded with anti-MHC class II mAb 2G9 (anti-mouse I-A , I-E , rat Construction of CCL22 promoter reporter gene plasmids IgG2a as described previously; Ref. 21). MHC class II-positive, bead-coupled LC and cells without attached beads were enumerated using a To assess activity of the CCL22 promoter, a fragment spanning a sequence Neubauer hemocytometer. A purity of ϳ92–95% was obtained as deter- ϳ4 kb long containing part of the 5Ј-nontranscribed region and the com- mined by the ratio of bead-rosetted to nonrosetted cells. Cell viability, plete 5Ј-untranslated region (UTR) was subcloned into promoterless firefly determined by trypan blue exclusion, was Ͼ95%. luciferase expression vector pGL3-Basic (Promega), resulting in reporter Bone marrow-derived DC (BM-DC) were cultured as previously de- construct pGL-CCL22. Correct orientation was confirmed by sequencing scribed (25) with modifications (26), and maturation was induced by add- with vector-specific primers (RVprimer3 and GLprimer2; Promega). 5Ј- ing either 50 ng/ml TNF-␣ (R&D Systems) or 1 ␮g/ml LPS (Sigma- Deletion constructs were obtained by restriction and religation or by nested

Aldrich). MHC class II-positive cells were enriched 24 h later by immu- deletion using the double-stranded Nested Deletion kit (Amersham Downloaded from nomagnetic separation as described above. Biosciences). B lymphocytes were purified from spleen cells using the Mouse B Cell Recovery kit (Cedarlane Laboratories). Purity as assessed by cytofluorom- Transient cell transfection and luciferase assay etry was Ͼ95%. For stimulation, B cells were cultivated for 2–3 days in the presence of 25 ␮g/ml LPS (Sigma-Aldrich) in IMDM (Invitrogen Life Cells were transfected using GenePORTER (Gene Therapy Systems) as ␮ Technologies) supplemented with 5% FCS (Sigma-Aldrich), 2 mM L-glu- recommended by the manufacturer. Briefly, 1 g of reporter DNA and 0.1 Ϫ ␮ ␮ tamine, 5 ϫ 10 5 M 2-ME, 100 IU/ml penicillin, and 100 ␮g/ml strepto- g of the coreporter (see next paragraph) were incubated with 4–8 lof GenePORTER in 250 ␮l of serum-free culture medium for 1 h before 4 ϫ http://www.jimmunol.org/ mycin (complete medium). Naive B cells were isolated by immunomag- 5 netic separation using anti-IgM-coupled Dynabeads. The murine B cell 10 cells were added. After 4 h, 2 ml of complete medium were added. lymphoma A20/2J (27) was cultivated as described previously and stimu- Cells were analyzed 24 h posttransfection. To this end, cells were har- lated with 25 ␮g/ml LPS for 24 h. The murine fibroblast line NIH 3T3 (28) vested, washed in PBS, and lysed in Passive Lysis Buffer (Promega). Cell was cultivated in complete medium. The murine immature and mature DC lysates were analyzed sequentially for firefly and Renilla luciferase activity lines XS52 and XS106, respectively (29), kindly donated by Dr. A. using the Dual-Luciferase Reporter Assay System (Promega) in a Turner Takashima (University of Texas Southwestern Medical Center, Dallas, luminometer TD-20/20 (Turner Design) as recommended by the TX), were cultivated in complete medium supplemented with 10% super- manufacturer. natant of NS 47 cells (30) and 5 and 10 ng/ml, respectively, of mouse To normalize absolute firefly luciferase reporter expression values for recombinant GM-CSF. GM-CSF was kindly provided by Drs. F. Seiler and differences in transfection efficiency and cell recovery, a Renilla luciferase expression construct was cotransfected. To avoid increased coreporter ac- D. Krumwieh (Behringwerke AG). Mouse rIL-4 was a kind donation by guest on September 24, 2021 by DNAX. tivity due to cytokine- or LPS-mediated stimulation of CMV promoter, pRL-EF1␣ was used as a coreporter construct (24). Renilla expression in ␣ ␣ ␣ Isolation of mRNA and RT-PCR pRL-EF1 is driven by the strong human elongation factor 1 1(EF1 ) gene promoter regulated mainly by GC box-binding transcription factors mRNA was isolated using the QuickPrep Micro mRNA Purification kit as (34). Reporter activities are calculated as ratio of firefly luciferase test recommended (Amersham Biosciences). Reverse transcription reaction construct activity divided by activity of cotransfected Renilla luciferase was performed as described previously (31). RNA concentration and effi- reporter construct. For transactivation studies using expression plasmids ciency of reverse transcription reaction were assessed by RT-PCR using for Sp1 and Sp3, respectively, luciferase activity was normalized to the primers specific for the housekeeping gene hypoxanthine phosphoribosyl- amount of protein in the cell lysate. transferase as reported previously (21). Hypoxanthine phosphoribosyl- Expression plasmids for NF-␬B p50, p65, and c-Rel were kindly pro- transferase-standardized amounts of cDNA were subjected to PCR with vided by Dr. S. Pettersson (Karolinska Institute, Stockholm, Sweden), and CCL22-specific primers as described previously (9). plasmids for the expression of NF-␬B p52 and RelB were kind gifts by Dr. F. Weih (Forschungszentrum Karlsruhe, Karlsruhe, Germany). Expression 5Ј-RACE plasmids for Sp1 and Sp3 (35) were kind gifts of Dr. G. Suske (Phillips- Universita¨t, Marburg, Germany). 5Ј-RACE was performed using the SMART cDNA Library Construction kit (Clontech) for first-strand synthesis. PCR was performed using the 5Ј- EMSA and site-directed mutagenesis SMART PCR primer (5Ј-AAGCAGTGGTATCAACGCAGAGT-3Ј; Clontech) and CCL22 gene-specific primer 1 (5Ј-ATGGCTACCCT Nuclear proteins were isolated by the method of Schreiber et al. (36), and GCGTCGTGTCCC-3Ј) or primer 2 (5Ј-GACGAGCACCAGGAGTGG- protein concentration was determined using the Roti-Quant Protein Assay 3Ј), respectively. Products were separated on an agarose gel, blotted on a kit (Roth). Complementary oligonucleotides were incubated for 10 min at nylon membrane (Roche), and hybridized with a digoxigenin-labeled probe 95°C and annealed by decreasing the temperature by 1°C/min to 4°C. Fol- derived from CCL22 cDNA. Signals were detected using the PCR DIG lowing agarose gel electrophoresis, double-stranded oligonucleotides were Probe Synthesis kit (Roche). Alternatively, products from a PCR using recovered from the gel by electroelution and end-labeled using T4 polynu- primer 2 and the 5Ј-SMART PCR primer were sequenced using primer 1. cleotide kinase (New England Biolabs). Radiolabeled DNA probe (25,000 cpm) was mixed with 1 ␮g of poly(dI:dC) (Amersham Biosciences), 10 ␮g Isolation and characterization of a murine genomic CCL22 clone of nuclear proteins, and binding buffer (25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM DTT, and 10% glycerol). Complex formation was allowed to A murine genomic library derived from a female C57BL/6 mouse (library proceed for 30 min at room temperature. For competition experiments, no. 703) (32) was obtained from the Resource Center/Primary Database. 50-fold molar excess of unlabeled, double-stranded oligonucleotides were The library was screened by hybridization with a probe containing the added to the binding reaction. For supershift experiments, nuclear proteins mouse CCL22 open reading frame (ORF) (9). Hybridization reactions and were incubated with 2 ␮g of specific Abs for 30 min on ice before the signal detection were performed as reported previously (33). binding reaction. Complexes were separated on 5% nondenaturing poly- P1 clones selected following library screening were used for DNA prep- acrylamide gels. Gels were dried afterward and exposed to Kodak MS films aration (NucleoBond Plasmid kit; Clontech). Hybridization with mouse (Eastman Kodak) at Ϫ80°C. CCL22 cDNA probe was verified by Southern blot analysis. For additional Site-directed mutagenesis was performed as described previously characterization, restriction fragments of P1 clone DNA were subcloned (37). The sequence of oligonucleotides (top strand) for EMSA and site- randomly into pZErO 2.1 (Invitrogen Life Technologies), and subclones directed mutagenesis were as follows: CCL22I, 5Ј-ACCGCTAAGGG 5622 REGULATION OF THE CCL22 GENE

GACTTTACAATCCTCC-3Ј; CCL22Imut, 5Ј-ACCGCTAAGGATAC TTTACAATCCTCC-3Ј; NF-␬B, 5Ј-GATCGAGGGGACTTTCCCTAGC- 3Ј; Oct2A, 5Ј-GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCT GG-3Ј; CCL22II, 5Ј-ACCCACAGTTTGGGAGAGTGGCAGTCC-3Ј; CCL22IImut, 5Ј-ACCCACAGTTTGTAAGAGTGGCAGTCC-3Ј; Ikaros, 5Ј- GATCATCCATAGGGAAAATTATCCTAG-3Ј; CCL22 III, 5Ј-CGCCTTT GGATCCTCCCGCTGGGGGAAG-3Ј; CCL22IIImut, 5Ј-CGCCTTTGG ATCCTCCCGCTGTGAGAAG-3Ј; Sp1, 5Ј-ATTCGATCGGGGCGGG GCGAG-3Ј; and C/EBP, 5Ј-TGCAGATTGCGCAATCTGCA-3Ј. Abs against Sp4 and NF-␬B subunits were obtained from Santa Cruz Biotechnol- ogy. Antisera against Sp1 and Sp3 (35) were kind gifts by Dr. G. Suske (Phillips-Universita¨t, Marburg, Germany).

Statistical analysis of data Statistical analysis of the experimental data was performed by the Stu- dent’s t test. A value of p Ͻ 0.05 was considered statistically signiﬁcant.

Results Isolation of the murine CCL22 gene and identification of transcriptional initiation sites Downloaded from The murine CCL22 gene was isolated from a genomic library with a cDNA probe encompassing the CCL22 ORF. As only the exon/ intron boundaries of the gene had been reported previously (8), we subcloned and sequenced a continuous region of 11,728 bp, cov- ering the murine CCL22 gene, including 4,096 bp of the 5Ј-flanking region (NCBI accession no. AJ315897). Analysis of the se- http://www.jimmunol.org/ quence confirmed that CCL22 is, as with other CC chemokines, encoded by three exons (Fig. 1A). The exons are separated by two introns of 1114 and 2758 bp, respectively. The sequences of the splice junctions are highly homologous to the corresponding human sequence and identical to the splice junctions in mouse reported by Schaniel et al. (8). To identify the transcriptional initiation site(s), we performed 5Ј-RACE using mRNA of in vitro-cultured LC and LPS-activated

B cells, previously shown to express CCL22 (9). Two gene-spe- by guest on September 24, 2021 cific 3Ј-primers were used in combination with a common 5Ј- primer, and products were detected by hybridization with a probe Ј containing the first exon of the CCL22 gene. Two lengths of 5 - FIGURE 1. A, Schematic depiction of the murine CCL22 gene and UTR were identified in both cell types, indicating the presence of CCL22 gene-derived reporter gene constructs. Exons are shown as boxes: two distinct transcriptional start sites (Fig. 1B). Sequencing of the f represents translated regions, and Ⅺ represents untranslated regions. The RACE products revealed that the majority of the transcripts in both two transcriptional start sites are indicated by positions relative to the start cell types are initiated from a site 28 bp upstream of the translation of translation. Recognition sites of restriction enzymes are abbreviated as initiation codon (position 4070 of sequence AJ315897), giving rise follows: B, BamHI; E, EcoRI; H, HindIII; and S, SpeI. A luciferase reporter to a very short 5Ј-UTR. In both cell types, a fraction of the tran- gene construct of the 5Ј-flanking promoter region and a series of constructs Ј scripts are initiated from a site 124 bp upstream of the start codon obtained by successive 5 -truncation of the promoter region are represented below. B, Identification of the transcriptional start sites of the CCL22 gene. (position 3973 of sequence AJ315897), which corresponds to the 5Ј-RACE was performed on mRNA extracted from in vitro-cultured LC start of the cDNA sequenced by Schaniel et al. (NCBI accession and LPS-activated B cells (BC), respectively. Products were separated by no. AF052505; Ref. 8). Interestingly, a nearly perfect TATA box agarose gel electrophoresis and detected by Southern hybridization using a consensus sequence was found ϳ32 bp upstream of the major probe containing exon I of the CCL22 gene. C, Analysis of the promoter transcription initiation site, indicating that the majority of the tran- activity of the 5Ј-flanking region of the CCL22 gene. The indicated cell scripts of both, cultured LC and activated B cells, arise from a lines were transfected transiently with a reporter gene plasmid containing promoter containing a TATA box. 4.1 kb of the 5Ј-flanking region of the CCL22 gene and cotransfected with a reporter plasmid coding for renilla luciferase driven by the EF1␣ pro- The CCL22 promoter reflects the activity of the endogenous gene moter. Transcriptional activity was calculated as relative light unit relative to pGL3-Basic. Shown are the mean values (Ϯ SD) of two independent Based on our previous observation that CCL22 is expressed by experiments performed in triplicate. D, Deletion analysis of the CCL22 mature, but not immature, DC and by activated, but not resting, B promoter. Equimolar amounts of luciferase reporter plasmids with 5Ј-to cells, we searched for an adequate model system to study CCL22 3Ј-deletions of the CCL22 promoter were transfected transiently into the expression. By RT-PCR, we found that the mature DC line XS106 indicated cell lines and cotransfected with a reporter plasmid coding for expressed CCL22 but not the immature DC line XS52 and con- renilla luciferase driven by the EF1␣ promoter. A20/2J cells were activated ␮ firmed that LPS stimulation induced expression of CCL22 in the B with LPS (25 g/ml) immediately after transfection or were left untreated. lymphoma cell line A20/2J (data not shown). In addition, we used The numbers on the abscissae represent the length of the promoter fragment in kb. Transcriptional activity was calculated as relative light unit the CCL22-negative fibroblast cell line NIH 3T3. To analyze relative to pGL3-Basic. Shown are the mean values (Ϯ SD) of two inde- CCL22 promoter activity in these cell lines, a 4069-bp fragment pendent experiments performed in triplicate. directly upstream of the translational start site was inserted into the vector pGL3-Basic, driving expression of the adjacent firefly The Journal of Immunology 5623

luciferase ORF (pGL-CCL22). This expression plasmid was transfected transiently into the cell lines, and firefly luciferase activity was measured 24 h posttransfection. A plasmid coding for renilla luciferase driven by the human EF1␣ promoter was cotransfected and used to standardize transfection efficiency. As shown in Fig. 1C, pGL-CCL22 mediated robust reporter activity in the mature DC line XS106, in LPS-stimulated A20/2J cells, and, to a markedly lesser extent (ϳ4-fold less, p Ͻ 0.01), in unstimulated A20/2J cells. No activity was detected in XS52 and NIH 3T3. The enhancement of CCL22 expression by LPS stimulation of A20/2J cells was concentration dependent with a maximal induction at 25 ␮g/ml LPS (data not shown). As the activity of the CCL22 promoter fragment reflected the activity of the endogenous gene, we concluded that the major positive or negative regulatory elements necessary for stage-specific CCL22 expression in these cell types were present in pGL-CCL22. Boundaries of the CCL22 promoter

To deﬁne the extension of the CCL22 promoter and to detect reg- Downloaded from ulatory regions within the promoter, we generated serial 5Ј-deletion constructs of pGL-CCL22. Constructs were transfected into XS106 and A20/2J cells, respectively, and analyzed for activity by luciferase assay. A20/2J cells were either stimulated with LPS directly after transfection or were left untreated. As shown in Fig.

1D, a reporter plasmid containing only the most proximal 250 bp http://www.jimmunol.org/ of the CCL22 promoter was able to drive most if not all of the luciferase activity in A20/2J cells. Moreover, full inducibility of the promoter by LPS was retained. Additionally, when 120 bp were removed from the 5Ј-end of the promoter fragment, nearly all activity was abrogated in nonactivated and LPS-activated A20/2J cells, indicating that pivotal regulatory elements are located within this region. In XS106 cells, deletion of the distal 2.5 kb of the promoter had little effect on reporter activity (Fig. 1D). However, additional de- by guest on September 24, 2021 letion of the remaining 1.6-kb promoter fragment to 1.4 kb resulted FIGURE 2. A, Schematic representation of the proximal region of the in a ϳ2-fold increase ( p Ͻ 0.001) in luciferase activity, hinting at murine CCL22 promoter. The proximal 250 bp of the CCL22 promoter was negatively regulating elements within this 200-bp region. Deletion analyzed using MatInspector software, and three putative binding sites for from 1.4 to 0.55 kb had no significant effect on reporter activity, transcription factors were identified. The sequences of the putative binding whereas an additional 5Ј-terminal deletion to 0.25 kb decreased sites are shown in the boxes, and the nucleotides changed by site directed promoter activity back to the level of the full-length promoter. mutagenesis are shown above the wild-type sequence. The position of the However, removal of additional 120 bp abrogated promoter activ- binding sites relative to the translation start codon is given in parentheses. ity nearly completely ( p Ͻ 0.001). The position of the translational start sites are indicated by arrows. B, Binding of proteins to the three sites in the proximal part of the CCL22 Thus, a small proximal promoter region of 250 bp is sufficient to promoter. EMSA were performed on oligonucleotides of the three boxes mediate at least a major part of promoter activity in both cell types. using nuclear extracts isolated from the indicated cell lines. A20/2J cells Ϫ Pivotal regulatory elements are located between positions 250 were activated with LPS (25 ␮g/ml) before the isolation. Data are repre- and Ϫ130 bp. Therefore, we searched for putative transcription sentative of three independent experiments. C, Serial deletion of the puta- factor binding sites within the most proximal 250 bp of the pro- tive binding sites in the proximal part of the CCL22 promoter. Equimolar moter using MatInspector software. Three regions harboring con- amounts of the indicated luciferase reporter plasmids were transfected tran- served transcription factor binding motifs were found, containing siently into the indicated cell lines and cotransfected with a reporter plas- a putative binding site for NF-␬B and Ikaros, as well as a G/C-rich mid coding for renilla luciferase driven by the EF1␣ promoter. A20/2J cells ␮ element (Fig. 2A). EMSA indicate that all three regions bind nu- were activated with LPS (25 g/ml) immediately after transfection. Tran- clear proteins (Fig. 2B). Furthermore, differences in the complexes scriptional activity was calculated as relative light unit relative to pGL3- Basic. Shown are the mean values (Ϯ SD) of two independent experiments obtained with nuclear extracts derived from unstimulated vs LPS- performed in triplicate. stimulated A20/2J cells and XS52 vs XS106 cells show that vari- able complexes are formed at box I and box III (Fig. 2B). To analyze the contribution of the three protein-binding regions to the activity of the CCL22 promoter, reporter plasmids were generated in which these three regions were deleted progressively (Fig. 2C). a slight but statistically not significant decrease of promoter activ- Deletion of box I, which contains the NF-␬B binding site, resulted ity. However, the additional deletion of box II resulted in a sig- Ͻ in loss of a major part of the inducibility of the promoter by LPS nificant decrease ( p 0.001). Most of the residual promoter ac- in A20/2J cells ( p Ͻ 0.001). Additional deletion of box II had little tivity was abrogated by the removal of all three boxes. These effect on the residual promoter activity, whereas removal of box III results indicate that all three regions contain cis-acting elements reduced the activity of the reporter plasmid nearly to background that contribute to the promoter activity in A20/2J and XS106 cells. level ( p Ͻ 0.002). In XS106 cells, the deletion of box I resulted in However, the relative contribution is cell type dependent. 5624 REGULATION OF THE CCL22 GENE

Three regulatory regions of the CCL22 promoter bind not affect complex formation and neither did an oligonucleotide transcription factors, including NF-␬B p50, p65, and Sp1 containing two base substitutions in the NF-␬B core sequence of EMSA and supershift assays were conducted to evaluate the spec- region I. ␬ iﬁcity of the protein complexes formed at box I–III and to identify To analyze whether indeed proteins of the NF- B family bind to the respective factors binding to these regions. this region, we conducted supershift assays. An anti-p50 antiserum At the distal box I, harboring a NF-␬B binding site, two DNA/ supershifted a complex formed with nuclear extracts of LPS-acti- protein complexes formed, using nuclear extracts from unstimu- vated A20/2J cells and XS106 cells, respectively (Fig. 3A). Addi- lated A20/2J cells (Fig. 2B). Stimulation of the cells induced the tion of anti-p65 antiserum resulted in decreased complex forma- formation of a third complex with a lower mobility. Similarly, tion in XS106 cells and A20/2J cells. Antisera or mAb directed three complexes were detected in the DC lines tested with the low against p52, RelB, or c-Rel had no substantial effect on complex mobility complex being prominent in the mature DC line XS106, formation (Fig. 3A). as compared with the immature DC line XS52. Competition ex- At box II, harboring an Ikaros binding site, three DNA/protein periments showed that complex formation was sequence speciﬁc complexes formed, regardless of whether the nuclear extracts were (Fig. 3A). An excess of unlabeled region I-oligonucleotide or an derived from unstimulated/LPS-stimulated A20/2J, XS52, or oligonucleotide with a NF-␬B consensus-binding sequence was XS106 cells (Fig. 2B). Excess of unlabeled region II-oligonucle- able to compete for complex formation, whereas an excess of an otide blocked complex formation (Fig. 3B). Notably, two base sub- unrelated oligonucleotide, harboring an octamer-binding motif, did stitutions in the Ikaros core sequence did not affect competition, and an Ikaros consensus binding site did not compete for complex

formation, both suggesting that proteins other than Ikaros bind to Downloaded from a distinct sequence of region II. This assumption was veriﬁed by supershift assays. Antiserum detecting all known differentially spliced Ikaros isoforms did not induce an additional shift (Fig. 3B). MatInspector detected solely the putative Ikaros site, which is ob- viously not involved. Thus, the binding site of region II or even the

transcription factor binding to this site has not been identified http://www.jimmunol.org/ previously. Box III harbors a GC-rich sequence with putative binding sites for transcription factors of the Sp and AP-2 family. Nuclear proteins from unstimulated and LPS-activated A20/2J, as well as XS106, cells formed three distinct complexes, whereas proteins from XS52 cells formed only one complex (Fig. 2B). Competition experiments showed that formation of two of the complexes was specific, whereas the formation of the complex with the highest mobility was nonspecific. An unlabeled oligonucleotide with a by guest on September 24, 2021 two-base substitution in the G-rich sequence did not compete for complex formation, whereas an oligonucleotide with a known Sp1- binding sequence did compete for complex formation using nuclear extracts of A20/2J and XS106 cells (data not shown). Binding of factors of the Sp and AP-2 family to box III was analyzed by supershift assays using polyclonal antisera against Sp1, Sp3, Sp4, AP-2␣, AP-2␤, and AP-2␥ (Fig. 3C). The anti-Sp1 antiserum supershifted complex 2 formed with nuclear proteins derived from LPS-stimulated A20/2J cells but not from XS106 cells. The other antisera did not have an effect on complex formation with nuclear proteins of LPS-stimulated A20/2J cells or XS106 cells. As region III also harbors a sequence with some similarity to the C/EBP consensus-binding sequence, we used mAb or antisera against the C/EBP isoforms ␣, ␤, ␥, ␦, ⑀, and ␨ for supershift assays (Fig. 3C). No effect on complex formation with nuclear proteins of LPS-stimulated A20/2J cells or XS106 cells was observed (Fig. 3C). An antiserum cross-reacting with the C/EBP isoforms ␣, ␤, ␦, and ⑀ did not have an effect either (data not shown). In conclusion, Sp1 binds to box III using extracts of LPS-stimulated A20/2J cells but not of XS106 cells. The corre- FIGURE 3. Identification of proteins binding to the binding sites in the sponding factor derived from XS106 cells was not identifiable be- CCL22 promoter. EMSA were performed using oligonucleotides of box I cause antisera against all known factors with specificity for the (A), box II (B), and box III (C), and nuclear extracts were isolated from the putative binding sites of box III had no effect in supershift assays. indicated cell lines in the presence or absence of a molar excess of a cold competitor or in the presence or absence of antisera. Antisera were added to the nuclear proteins 30 min before adding the hot probe. A20/2J cells were Effect of site-directed mutagenesis of transcription factor activated with LPS (25 ␮g/ml) before the isolation. Competitors: A, wild-type binding sites on CCL22 promoter activity box I (wt), consensus-binding sequence of Oct2a (Oct2a), consensus-binding sequence of NF-␬B (NF-␬B), and mutated box I (mut); and B, wild-type box To further elucidate the importance of the identified binding sites II (wt), consensus-binding sequence of Ikaros (Ikaros), and mutated box II of the CCL22 promoter and the relative contribution of the respec- (mut). Data are representative of two independent experiments. tive transcription factors, single nucleotides of the binding sites The Journal of Immunology 5625 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. A, Effect of mutations in the transcription factor binding sites of boxes I, II, and III on the activity of the CCL22 promoter. Base pairs in the binding sites were changed to the sequence indicated in Fig. 2A using site-directed mutagenesis. Equimolar amounts of the indicated luciferase reporter plasmids were transfected transiently into the indicated cell lines and cotransfected with a reporter plasmid coding for Renilla luciferase driven by the EF1␣ promoter. A20/2J cells were activated with LPS (25 ␮g/ml) immediately after transfection. Transcriptional activity was calculated as relative light unit relative to pGL3-Basic. Shown are the mean values (Ϯ SD) of two independent experiments performed in triplicate. B, Effect of mutations in the binding sites on the inducibility of the CCL22 promoter by Sp1 and NF-␬B. Equimolar amounts of the indicated luciferase reporter plasmids were transfected transiently into the indicated cell lines and cotransfected with a plasmid facilitating the expression of the indicated transcription factor. Activity measured after transfection with a plasmid containing no promoter (pGL3-Basic) was set to 1. For experiments using NF-␬B expression plasmids, transfection efﬁciency was normalized to the activity of the coreporter vector pRL-EF1␣, whereas for experiments using Sp expression plasmids, activity was normalized to the protein content of the cell lysate. Shown are the mean values (Ϯ SD) of two independent experiments performed in triplicate. C, Effect of the overexpression of NF-␬B subunits and Sp proteins, respectively, on the activity of the CCL22 promoter. The wild-type luciferase reporter plasmid was transfected transiently into the indicated cell lines and cotransfected with a plasmid facilitating the expression of the indicated transcription factor. Transcriptional activity was calculated as relative light unit relative to pGL3-Basic. For experiments using NF-␬B expression plasmids, transfection efﬁciency was normalized to the activity of the coreporter vector pRL-EF1␣, whereas for experiments, using Sp expression plasmids activity was normalized to the protein content of the cell lysate. Shown are the mean values (Ϯ SD) of two independent experiments performed in triplicate. D, Effect of the substitution of box II and the alteration of the spatial alignment, respectively, on the activity of the CCL22 promoter. Box II was substituted with an unrelated sequence of the same length, or short sequences were inserted into the wild-type reporter plasmid as shown. Equimolar amounts of the indicated luciferase reporter plasmids were transfected transiently into the indicated cell lines and cotransfected with a reporter plasmid coding for Renilla luciferase driven by the EF1␣ promoter. A20/2J cells were activated with LPS (25 ␮g/ml) immediately after transfection. Transcriptional activity was calculated as relative light unit relative to pGL3-Basic. Shown are the mean values (Ϯ SD) of two independent experiments performed in triplicate. were substituted in luciferase reporter constructs (see Fig. 2A), and ulated A20/2J cells ( p Ͻ 0.001 and p Ͻ 0.002, respectively), ab- promoter activity was analyzed by luciferase assays. rogating inducibility of the promoter (Fig. 4A). In XS106 cells, Substitution of either two nucleotides of the NF-␬Borofthe mutation of the Sp1 binding site had a strong effect on promoter Sp1 binding site drastically reduced promoter activity in LPS-stim- activity as well ( p Ͻ 0.001), indicating that exactly this site is 5626 REGULATION OF THE CCL22 GENE

required, although it is not a known factor of the Sp1 family that Functional interaction of region I and III depends on the spatial binds to this region, as shown. Mutation of the NF-␬B site resulted alignment within the CCL22 promoter in a moderate but statistically not significant reduction of promoter Interaction between members of the NF-␬B family and Sp1 was activity in XS106 cells as well (Fig. 4A). reported before, e.g., for the promoter of VCAM-1 or HIV-long- In accordance with the finding that Ikaros and AP-2 do not bind terminal repeat (38, 39). Likewise, importance of interaction of the to the respective sites of the CCL22 promoter, base substitutions in protein complexes formed at box I and III of the CCL22 promoter these sites had no effect on promoter activity in both cell lines. was suggested by the finding that mutation of the Sp1 binding site ϩ Effect of overexpression of transcription factors on CCL22 markedly affected inducibility by overexpression of p50 p65 promoter activity (Fig. 4B). To demonstrate that the spatial alignment of box I and III is important for the functional interaction of the protein com- Next, we examined the effect of overexpression of transcription plexes, we inserted small DNA fragments of 18 and 24 bp, respec- factors on the activity of the CCL22 promoter. CCL22 promoter/ tively. To exclude that effects were caused by disruption of a so-far luciferase reporter constructs were cotransfected with expression undetected regulatory element or by disruption of a possible in- ␬ vectors coding for the NF- B proteins p50, p52, p65, RelB, and teraction between box II and either boxes I or III, one fragment c-Rel, either separately or as combination of two factors, to allow was inserted between boxes I and II and the other fragment be- for formation of functional units of homo- and heterodimers, re- tween box II and III. Promoter activity of both modified reporter spectively. In A20/2J cells, overexpression of p65 alone increased constructs was markedly reduced in LPS-stimulated A20/2J cells ϳ Ͻ promoter activity 2-fold ( p 0.001; Fig. 4C). Overexpression and XS106 cells (all p Ͻ 0.001; Fig. 4D). Moreover, both inser- Ͻ Downloaded from of c-Rel had a lesser effect ( p 0.05), and overexpression of p50, tions reduced promoter activity to the same extent compared with p52, and RelB had no marked effect. Overexpression of both, p50 the wild-type construct. This reduction was more pronounced in ϳ and p65, resulted in an 10-fold increase of promoter activity A20/2J cells than in XS106 cells. Ͻ ␬ ( p 0.001), being by far the most effective combination of NF- B Keeping in mind the importance of the spatial alignment of box proteins in activation of the CCL22 promoter. In contrast to A20/2J I and III, we replaced box II by an unrelated sequence of exactly cells, XS106 cells require, as reported above, no stimulation to the same length to evaluate contribution of region II to promoter show strong CCL22 promoter activity. Nevertheless, a combina- activity. The sequence was selected to minimize sequence simi- http://www.jimmunol.org/ Ͻ tion of p50 and p65 further enhanced promoter activity ( p larity to avoid that it competes with the wild-type sequence for 0.001), whereas all other combinations did not augment the al- transcription factor binding. A marked reduction ( p Ͻ 0.001) of ready high expression level (Fig. 4C). To investigate the effect of promoter activity was observed in XS106 cells (Fig. 4D). In ac- ␬ overexpression of NF- B subunits on the endogenous CCL22 cordance with the data presented above (Fig. 2C), the relative loss mRNA production, A20/2J cells were transfected transiently with of activity by replacement of box II was less pronounced in LPS- ␬ NF- B expression plasmids and analyzed for CCL22 mRNA ex- stimulated A20/2J cells ( p Ͻ 0.02; Fig. 4D). pression by RT-PCR. To assure comparable transfection efficiency, cells were cotransfected with a renilla luciferase coreporter, and samples displaying similar coreporter activity were Distinct proteins from primary DC and B cells bind to region I by guest on September 24, 2021 chosen for RNA isolation. Although overexpression of p50 alone and region III of the CCL22 promoter was not sufficient to induce CCL22 mRNA production, overex- To investigate whether the same transcription factors are involved pression of p65 or of p50 together with p65 induced endogenous in primary cells as in the cell lines tested, EMSA and supershift CCL22 mRNA production (data not shown). Taken together, these assays were performed with nuclear extracts from primary B cells experiments show that p50 and p65 not only bind to region I of the and BM-DC. CCL22 promoter, as indicated by EMSA, but induce expression Stimulation of primary B cells with LPS enhanced complex for- acting probably as heterodimers. mation (data not shown), an antiserum against NF-␬B p50 induced The transcription factor Sp1, shown to bind to region III in a supershift, and an antiserum against p65 resulted in decreased EMSA using nuclear extracts from LPS-stimulated A20/2J cells, complex formation, using nuclear extracts from LPS-activated B was analyzed likewise. Cotransfection of expression plasmids cod- cells (Fig. 5). ing for Sp1 resulted in an ϳ6-fold increase in luciferase reporter To investigate complex formation in DC, we used nuclear ex- activity of CCL22 reporter constructs in A20/2J cells ( p Ͻ 0.001; tracts of BM-DC instead of LC because in BM-DC analysis is not Fig. 4C). However, in XS106 cells, cotransfection of expression hampered by the rapid changes of CCL22 expression observed in plasmids coding for Sp1 had no effect on promoter activity, again LC (Refs. 8 and 9 and our unpublished results). We obtained dis- indicating that Sp1 is involved in induction of expression in tinct stages of maturation in BM-DC by stimulation with TNF-␣ A20/2J but not in XS106 cells. Overexpression of Sp3 had no and LPS, respectively; the latter representing a stronger maturation significant effect on promoter activity of reporter constructs in both stimulus than TNF-␣. Three distinct complexes were formed using cell lines (Fig. 4C). Furthermore, overexpression of either Sp1 or nuclear extracts from TNF-␣-stimulated BM-DC. An antiserum Sp3 was not sufficient to induce endogenous CCL22 mRNA pro- against NF-␬B p50 induced a supershift, and an antiserum against duction in A20/2J cells (data not shown). p65 resulted in decreased formation of the preexisting complexes, Transactivation of the CCL22 promoter by overexpression of whereas a new low molecular mass complex appears (Fig. 5). In Sp1 or NF-␬B p50 ϩ p65 in A20/2J cells or of NF-␬B p50 ϩ p65 addition, an antiserum against RelB reduced complex formation. in XS106 cells was abrogated by introducing a mutation in the However, when extracts from LPS-stimulated BM-DC were used, respective binding sites (all p Ͻ 0.001; Fig. 4B). Interestingly, addition of an antiserum against p50 supershifted both complexes, mutation of the Sp1 site affected inducibility of the promoter by whereas the addition of an antiserum against p65 had little effect overexpression of p50 ϩ p65 in A20/2J and XS106 cells (both p Ͻ on complex formation. Instead, addition of an antiserum against 0.001; Fig. 4B), hinting at an interaction between the protein com- RelB induced a marked decrease in formation of the lower mobil- plexes formed at region I and region III. Mutation of the AP-2 site ity complex, indicating that p50/RelB rather than p50/p65 het- had, as expected, no effect on inducibility by overexpression of erodimers from nuclear extracts of LPS-stimulated BM-DC bind to Sp1 in A20/2J cells (data not shown). region I. The Journal of Immunology 5627

effect indicates that in this cell line, which expresses CCL22 constitutively, this NF-␬B site is of minor importance. In LPS-stimulated DC, p65 is replaced by RelB. A slightly decreased complex formation was already observed with antisera against RelB in TNF-␣-stimulated DC (Fig. 5). Because LPS represents the stronger maturation stimulus compared with TNF-␣, this may suggest that RelB replaces p65 during maturation of DC. However, in contrast to p65 overexpression of RelB does not enhance CCL22 promoter activity in A20/2J and XS106 cells, either alone or in combination with other proteins of the NF-␬B family. Thus, replacement of p65 by RelB may rather reduce than enhance CCL22 expression. Accordingly, RelB had been shown to be the most actively translocated NF-␬B protein in maturing DC (41). FIGURE 5. Identiﬁcation of proteins isolated from LPS-activated B Saccani et al. (42) recently demonstrated that p65-containing ␣ cells and TNF- - or LPS-matured BM-DC binding to box I and III, re- dimers are replaced completely by RelB-containing dimers at the spectively. EMSA were performed on oligonucleotides of box I and box human CCL22 promoter during maturation of DC. A similar re- III, respectively, using nuclear extracts isolated from the indicated cells in the presence or absence of a molar excess of a cold competitor or in the placement of p65 by RelB at the IL-12p40 promoter correlated presence or absence of antisera. Antisera were added to the nuclear proteins with a transcriptional shutdown of the promoter and a drop of 30 min before adding hot probe. Data are representative of two independent polymerase II recruitment. However, they observed that RNA Downloaded from experiments. polymerase II levels at the CCL22 promoter peaked when RelB recruitment was maximal and that expression of the I␬B␣ super- repressor, which affects translocation of p65- and c-Rel- but not RelB-containing dimers, rather enhanced than suppressed CCL22 We next conducted EMSA with proteins from primary cells us- transcription (42). This may suggest that the human and mouse

ing region III as a probe. As had been observed for the B cell line CCL22 promoters differ with respect to RelB activation. http://www.jimmunol.org/ A20/2J, extracts from LPS-activated splenic B cells formed three Remarkably, the factor Sp1 binds to box III in B cells and the B complexes, of which complex 2 was supershifted using an anti-Sp1 cell line A20/2J but not in DC and the DC line XS106. Moreover, antiserum. When proteins isolated from LPS-matured BM-DC Sp1 is able to transactivate the promoter in A20/2J but not in were used, three complexes were detected, of which none was XS106 cells, indicating that either the concentration of the factor affected by the addition of antisera against Sp1 or Sp3 (Fig. 5). replacing Sp1 in XS106 is not limiting the expression or Sp1 cannot substitute for this factor in XS106 cells. Nevertheless, exactly Discussion the same site is required for activation of CCL22 expression in The mouse CC chemokine CCL22 is involved critically in regu- XS106 cells because substitution of two nucleotides of the Sp1 lation of immune responses (8–12, 14, 15). Of interest are its tight binding site disrupts activity of the promoter in XS106 cells. Fur- by guest on September 24, 2021 regulation of expression and high expression levels in mature DC thermore, the complexes formed at box I and box III interact in and activated B cells. Thus, we characterized the CCL22 gene and both cell lines because a change of the spatial alignment of the its regulation. CCL22 is clustered with CCL17 and CX3CL1 on boxes affects activity of the promoter in both cases (Fig. 4D). human chromosome 16q13 (40), apart from the common CC che- Given these close similarities and the fact that neither Sp1 nor mokine locus on chromosome 17q11.2, where most CC chemo- another known member of the Sp1 family binds to the promoter in kines are clustered. Likewise, in mice these three chemokines are XS106 cells and DC, a so-far unknown member of the Sp1 family clustered separately on a short chromosomal fragment spanning may be active in DC. The fact that LPS did not induce an addi- Ͻ130 kb (19). The separate location and the extensive differences tional complex to be formed at region III by proteins isolated from to other CC chemokines on amino acid levels (9) indicate a diver- A20/2J indicates that Sp1 is already present in the nucleus of these gent evolution of this locus. Taking into account that the expres- cells before LPS activation. sion profiles of CCL22 and CCL17 are similar, this may suggest a The functional interaction of Sp1 and NF-␬BattheCCL22 pro- common regulation of gene expression of these two chemokines. moter, indicated (1) by the importance of the spatial alignment of Our data indicate that in case of the CCL22 gene, the regulatory the binding sites within the promoter and (2) by the fact that a elements required to mediate a strong and highly specific expres- functional Sp1 site is required for NF-␬B transactivation, was sion are included in a 4.1-kb promoter region, with the major el- demonstrated for other promoters previously (38, 39). The fact that ements active in DC and B cells being located in a small proximal the distance between the NF-␬B and the Sp1 binding sites is con- 250-bp region. Interestingly, the same regulatory elements within siderably greater in the CCL22 promoter may suggest involvement this proximal region (box I–III) are required in all cells analyzed, of additional proteins. Such proteins may bind to the promoter and there are similarities but there are also differences in the fac- themselves, e.g., at box II or even adjacent to the binding sites for tors involved. The data obtained from EMSA, supershift analysis, Sp1 or NF-␬B. site-directed mutagenesis, and transactivation studies are conclu- Likely candidates are C/EBP proteins because region III harbors sive in suggesting that the NF-␬B proteins p50 and p65 act to- a sequence with similarity to the C/EBP consensus-binding se- gether, probably as heterodimers, to activate the CCL22 gene by quence, and interaction of NF-␬B and C/EBP was demonstrated binding to box I of the promoter in nearly all cells tested, namely previously (43). The fact that we did not detect binding of C/EBP the B cell line A20/2J, the DC line XS106, primary LPS-stimu- isoforms ␣, ␤, ␥, ␦, ⑀, and ␨ in supershift assays (Fig. 3C) does not lated B cells, and TNF-␣-stimulated DC. Moreover, overexpres- completely rule out an involvement because unstable higher order sion of p50 and p65 enhanced the endogenous CCL22 expression complexes may remain unnoticed. C/EBP isoforms ␣, ␤, and ␦ in A20/2J cells synergistically, as determined by RT-PCR (data not were detected in nuclear extracts of A20/2J, and XS106 cells and shown). However, the fact that the mutation as well as the deletion cotransfection of C/EBP ␣ ϩ ␦ increased promoter activity in of the NF-␬B site had only a small and statistically not significant A20/2J cells ϳ5-fold, indicating that heterodimers of C/EBP ␣ and 5628 REGULATION OF THE CCL22 GENE

␦ may activate the promoter (data not shown). However, in XS106 2. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and cells, overexpression of C/EBP isoforms ␣, ␤, and ␦, either sep- their role in immunity. Immunity 12:121. 3. Strieter, R. M., P. J. Polverini, S. L. Kunkel, D. A. Arenberg, M. D. Burdick, arately or as combination of two factors, had no effect on reporter J. Kasper, J. Dzuiba, J. Van Damme, A. Walz, D. Marriott, et al. 1995. The activity (data not shown). functional role of the ELR motif in CXC chemokine-mediated angiogenesis. An additional difference in regulation of the CCL22 promoter in J. Biol. Chem. 270:27348. 4. Broxmeyer, H. E. 2001. Regulation of hematopoiesis by chemokine family mem- A20/2J and XS106 cells was indicated by luciferase assays fol- bers. Int. J. Hematol. 74:9. lowing 5Ј-terminal deletion of the promoter region, hinting at an 5. Adema, G. J., F. Hartgers, R. Verstraten, E. de Vries, G. Marland, S. Menon, J. Foster, Y. Xu, P. Nooyen, T. McClanahan, K. B. Bacon, and C. G. Figdor. enhancing promoter element located between 550 and 250 bp up- 1997. A dendritic-cell-derived C-C chemokine that preferentially attracts naive T stream of the translational start site, which is active in XS106 cells cells. Nature 387:713. but not in A20/2J cells. Because we detected a putative binding site 6. Godiska, R., D. Chantry, C. J. Raport, S. Sozzani, P. Allavena, D. Leviten, A. Mantovani, and P. W. Gray. 1997. Human macrophage-derived chemokine for GATA proteins in this region, we overexpressed GATA-1, (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic GATA-2, and GATA-3 but did not detect an effect on reporter cells, and natural killer cells. J. Exp. Med. 185:1595. gene expression in A20/2J and XS106 cells (data not shown). 7. Chang, M., J. McNinch, C. Elias, III, C. L. Manthey, D. Grosshans, T. Meng, T. Boone, and D. P. Andrew. 1997. Molecular cloning and functional character- Interestingly, a very recent study by Nakayama et al. (44) in- ization of a novel CC chemokine, stimulated T cell chemotactic protein (STCP-1) vestigated the roles of two adjacent NF-␬B sites and a single AP-1 that specifically acts on activated T lymphocytes. J. Biol. Chem. 272:25229. site of the human CCL22 promoter in induction by EBV-encoded 8. Schaniel, C., E. Pardali, F. Sallusto, M. Speletas, C. Ruedl, T. Shimizu, T. Seidl, ␬ J. Andersson, F. Melchers, A. G. Rolink, and P. Sideras. 1998. Activated murine LPM-1 in human B cells. Although we identified only one NF- B B lymphocytes and dendritic cells produce a novel CC chemokine which acts site in the murine CCL22 promoter, a putative AP-1 site is also selectively on activated T cells. J. Exp. Med. 188:451. present in the murine promoter (position 97–100 relative to the 9. Ross, R., X. L. Ross, H. Ghadially, T. Lahr, J. Schwing, J. Knop, and Downloaded from A. B. Reske-Kunz. 1999. Mouse Langerhans cells differentially express an acti- translation start site). However, because this site is situated be- vated T cell-attracting CC chemokine. J. Invest. Dermatol. 113:991. tween the two transcriptional start sites, it is unlikely that AP-1 is 10. Schmidt-Weber, C. B., J. G. Wohlfahrt, and K. Blaser. 2001. DNA arrays in involved in the regulation of the murine CCL22 promoter. How- allergy and immunology. Int. Arch. Allergy Immunol. 126:1. 11. Greaves, D. R., T. Hakkinen, A. D. Lucas, K. Liddiard, E. Jones, C. M. Quinn, ever, it cannot be ruled out that AP-1 is involved in the initiation J. Senaratne, F. R. Green, K. Tyson, J. Boyle, et al. 2001. Linked chromosome of transcription of only a fraction of the products. 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic le- Remarkably, no site with substantial homology to an Ikaros http://www.jimmunol.org/ sions. Arterioscler. Thromb. Vasc. Biol. 21:923. binding site is present in the human CCL22 promoter region, but 12. Gonzalo, J. A., Y. Pan, C. M. Lloyd, G. Q. Jia, G. Yu, B. Dussault, C. A. Powers, a very G-rich element, similar to the Sp1 site identified here, is A. E. Proudfoot, A. J. Coyle, D. Gearing, and J. C. Gutierrez-Ramos. 1999. Mouse monocyte-derived chemokine is involved in airway hyperreactivity and located 175 bp upstream of the translation start codon of the hu- lung inflammation. J. Immunol. 163:403. man CCL22 promoter. 13. Vestergaard, C., H. Yoneyama, M. Murai, K. Nakamura, K. Tamaki, Although the CCL22 promoter reflects the specificity of the en- Y. Terashima, T. Imai, O. Yoshie, T. Irimura, H. Mizutani, and K. Matsushima. 1999. Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting dogenous gene, there may be additional levels of regulation of atopic dermatitis-like lesions. J. Clin. Invest. 104:1097. CCL22 expression not analyzed in detail yet. Saccani and Natoli 14. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff, (45) detected, for example, acetylation of histones H3 and H4 at and S. L. Kunkel. 2000. Pivotal role of the CC chemokine, macrophage-derived

chemokine, in the innate immune response. J. Immunol. 164:5362. by guest on September 24, 2021 the human CCL22 locus during activation, a common mechanism 15. Tang, H. L., and J. G. Cyster. 1999. Chemokine up-regulation and activated T cell of gene activation on chromatin level (46). Enhancer elements are attraction by maturing dendritic cells. Science 284:819. often located far upstream, downstream, or in introns, especially in 16. Andrew, D. P., M. S. Chang, J. McNinch, S. T. Wathen, M. Rihanek, J. Tseng, J. P. Spellberg, and C. G. Elias, III. 1998. STCP-1 (MDC) CC chemokine acts the first intron. The latter was tested by incorporating a 1.2-kb specifically on chronically activated Th2 lymphocytes and is produced by mono- downstream region, including the first intron of the CCL22 gene cytes on stimulation with Th2 cytokines IL-4 and IL-13. J. Immunol. 161:5027. into a luciferase reporter construct, but no regulatory sequences 17. Iellem, A., M. Mariani, R. Lang, H. Recalde, P. Panina-Bordignon, F. Sinigaglia, and D. D’Ambrosio. 2001. Unique chemotactic response profile and specific were detected (data not shown). expression of chemokine receptors CCR4 and CCR8 by CD4ϩCD25ϩ regulatory The usage of two transcriptional initiation sites 28 and 124 bp T cells. J. Exp. Med. 194:847. upstream of the translational start codon, respectively, offers ad- 18. Struyf, S., P. Proost, S. Sozzani, A. Mantovani, A. Wuyts, E. De Clercq, D. Schols, and J. Van Damme. 1998. Enhanced anti-HIV-1 activity and altered ditional possibilities for regulation of CCL22 expression. Quanti- chemotactic potency of NH2-terminally processed macrophage-derived chemo- tative differences in the usage of these start sites were detected in kine (MDC) imply an additional MDC receptor. J. Immunol. 161:2672. cultured LC and LPS-activated B cells. The longer 5Ј-UTR con- 19. Schaniel, C., F. Sallusto, C. Ruedl, P. Sideras, F. Melchers, and A. G. Rolink. 1999. Three chemokines with potential functions in T lymphocyte-independent tains two small ORF (mini ORF) (position Ϫ120 to Ϫ96 and Ϫ116 and -dependent B lymphocyte stimulation. Eur. J. Immunol. 29:2934. to Ϫ80, respectively). Such mini ORF in the 5Ј-UTR have been 20. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of reported to interfere with translation of the corresponding proteins immunity. Nature 392:245. 21. Ross, R., X. L. Ross, J. Schwing, T. Langin, and A. B. Reske-Kunz. 1998. The (47), raising the possibility that CCL22 production is controlled actin-bundling protein fascin is involved in the formation of dendritic processes not only on the transcriptional but also on the translational level. in maturing epidermal Langerhans cells. J. Immunol. 160:3776. However, the significance of these mini ORF in the 5Ј-UTR of a 22. Ross, R., A. Endlich, K. Kumpf, J. Schwing, and A.B. Reske-Kunz. 1998. Mat- uration of epidermal Langerhans cells in vitro is accompanied by down-regula- proportion of the CCL22 transcripts is not clear. tion of 4F2 (CD98) as determined by differential display. J. Invest. Dermatol. Considering the pivotal role of DC in initiation of novel immune 110:57. responses and the promising strategies to transcriptionally target 23. Ross, R., S. Sudowe, J. Beisner, X. L. Ross, I. Ludwig-Portugall, J. Steitz, T. Tu¨ting, J. Knop, and A. B. Reske-Kunz. 2003. Transcriptional targeting of den- DC for therapeutic use (23, 24, 48), thorough knowledge of gene dritic cells for gene therapy using the promoter of the cytoskeletal protein fascin. regulation in DC will help to manipulate DC on this level. Gene Ther. 10:1035. 24. Bros, M., X. L. Ross, A. B. Reske-Kunz, and R. Ross. 2003. The human fascin gene promoter is highly active in mature DC due to a stage-specific enhancer. Disclosures J. Immunol. 171:1825. 25. Scheicher, C., M. Mehlig, R. Zecher, and K. Reske. 1992. Dendritic cells from The authors have no financial conflict of interest. mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor. J. Immunol. Methods 154: 253. References 26. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and 1. Ono, S. J., T. Nakamura, D. Miyazaki, M. Ohbayashi, M. Dawson, and M. Toda. G. Schuler. 1999. An advanced culture method for generating large quantities 2003. Chemokines: roles in leukocyte development, trafficking, and effector func- of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods tion. J. Allergy Clin. Immunol. 111:1185. 223:77. The Journal of Immunology 5629

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