CCL23 Expression Is Induced by IL-4 in a STAT6-Dependent Fashion Hermann Novak, Anke Müller, Nathalie Harrer, Claudia Günther, Jose M. Carballido and Maximilian Woisetschläger This information is current as of September 28, 2021. J Immunol 2007; 178:4335-4341; ; doi: 10.4049/jimmunol.178.7.4335 http://www.jimmunol.org/content/178/7/4335 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCL23 Expression Is Induced by IL-4 in a STAT6-Dependent Fashion

Hermann Novak, Anke Mu¨ller, Nathalie Harrer, Claudia Gu¨nther, Jose M. Carballido, and Maximilian Woisetschla¨ger1

The CCL23 is primarily expressed in cells of the myeloid lineage but little information about its regulation is available. In this study, it is demonstrated that IL-4 and IL-13 induced CCL23 expression in human peripheral blood . GM-CSF had no effect on its own but synergized with IL-4, but not IL-13. CCL23 promoter reporter gene constructs were sensitive to IL-4 stimulation in the presence of the STAT6. A canonical STAT6 binding site in the promoter region of the CCL23 gene was critical for the IL-4-inducible phenotype because reporter plasmids with a defective STAT6 binding site were unable to respond to IL-4 stimulation. In addition, two tandem copies of the STAT6 site conferred responsiveness to a heterologous minimal promoter. Furthermore, IL-4 inducibility of the CCL23 promoter was dependent on the absence of a Downloaded from negatively acting cis-element downstream of the STAT6 binding site. The negative function of this element was operative also on heterologous IL-4-inducible promoters. CCL23 was also expressed in skin from patients suffering from atopic dermatitis at higher levels than in normal individuals. However, no correlation between CCL23 expression in the serum and IgE levels as a diagnostic marker for was found. Collectively, these data suggest a link between the inducible phenotype of CCL23 expression in monocytes by the prototype Th2 molecule pair IL-4/STAT6 and the increased number of CCL23-expressing cells in skin of atopic dermatitis patients. The Journal of Immunology, 2007, 178: 4335–4341. http://www.jimmunol.org/

hemokines are small which regulate cell traf- ulation of CCL23 expression. In monocytes, IL-1␤ and IFN-␥, albeit ficking during homeostasis as well as during inflamma- at low levels, induce CCL23 expression (16). Maturation of mono- C tory processes. Chemokine subfamilies have been de- cyte-derived DC with agonistic CD40 Abs or IFN-␥ reduces CCL23 fined classified by the distance between the first two conserved RNA and expression. In contrast, treatment of immature DC cysteine residues with the CXC and the CC subfamily containing with IL-10 induces CCL23 production (15). the vast majority of all . CXC chemokines act mainly A major constituent of the IL-4 signal transduction pathway is on and activated T (1), while CC chemo- STAT6 (19, 20). This protein is a transcription factor that resides by guest on September 28, 2021 kines attract a wider number of cell types including monocytes (2, in the cytoplasm of quiescent cells in a latent state. Upon binding 3), lymphocytes (4–6), basophils (7, 8), and (9, 10). of IL-4 to its , the ␣-chain of the IL-4R becomes phos- CCL23 (MPIF-1, CK␤8, SCYA23), a member of the CC chemo- phorylated by the tyrosine kinases JAK1 and JAK3. The phos- kine family, was originally isolated from a human aortic endothelial phorylated tyrosine residues on the IL-4R serve as docking sites cell library (11) and from the human monocytic cell line THP-1 (12). for STAT6 molecules. Once recruited to the receptor, STAT6 also It is most closely related to MIP-1␤ and interacts with its receptor becomes phosphorylated by JAKs at a single tyrosine residue. Acti- CCR1, which is expressed on monocytes, dendritic cells (DC),2 lym- vated STAT6 dissociates from the receptor, dimerizes, and translo- phocytes, and endothelial cells. An amino-terminally truncated form cates into the nucleus, where it binds to specific sequences found of CCL23 is also expressed which can also bind to formyl peptide- within promoters of IL-4-regulated genes. STAT6 binding sites have receptor-like 1 receptor (13). Functionally, CCL23 has chemotactic been found in the promoter regions of various IL-4-inducible genes activity for monocytes (12, 14), DC (15, 16), lymphocytes, neutro- such as FIZZ1 (21), CD23 (22), eotaxin-1 (23, 24), eotaxin-3 (25), phils (11), osteoclast precursor cells (17), and endothelial cells (18). In IL-4R (26), and in Ig germline ␧ (27) and ␥-l promoters. The con- contrast, CCL23 reduces the proliferation of progenitor cells giving sensus binding sequence of STAT6 consists of the palindromic TTC Ј Ј rise to granulocyte and lineages (11), whereas it enhances sequence separated by four nucleotides 5 -TTC(N)4GAA-3 (28). angiogenesis of endothelial cells (18). Little is known about the reg- This study demonstrates that CCL23 production is specifically induced by IL-4 and IL-13 on human monocytes. The activating function of IL-4 is mediated by the STAT6 protein and is depen- Department of Autoimmunity and Transplantation, Novartis Institutes for Biomedical dent on the presence of a STAT6 binding site in the promoter Research, Vienna, Austria region of the CCL23 gene. CCL23-expressing cells are found at Received for publication August 23, 2006. Accepted for publication January 10, 2007. higher frequency in the epidermis of patients suffering from atopic The costs of publication of this article were defrayed in part by the payment of page dermatitis compared with normal individuals. The chemokine was charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. also detectable in plasma of normal and atopic individuals but no 1 Address correspondence and reprint requests to Dr. Maximilian Woisetschla¨ger, correlation with IgE as marker of atopy was found. Department of Autoimmunity and Transplantation, Novartis Institutes for Bio- medical Research, Brunnerstrasse, Vienna, Austria. E-mail address: maximilian. [email protected] Materials and Methods 2 Abbreviations used in this paper: DC, dendritic cell; Ct, cycle threshold; EF1-␣, Cell culture and elongation factor 1␣. Human primary monocytes were isolated by counterflow centrifugal Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 elutriation (29) from PBMC derived from healthy volunteer donors. The www.jimmunol.org 4336 REGULATION OF CCL23 EXPRESSION IN HUMAN MONOCYTES BY IL-4

purity of these preparations assessed by CD14 staining was typically CCL23p-XEmut were introduced by two sequential PCR as described Ͼ96%. Cells were cultured at a density of 1 ϫ 106/ml in 96-well plates at previously (31) using the mutant primers 5Ј-GAGAATAAATGGGAGT Ј Ј 37°C with 5% CO2 in IMDM supplemented with 2% heat-inactivated FCS TTATTTTTGAAGGAATAATAAGAT-3 and 5 -ATCTTATTATTC (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 ␮g/ml CTTCAAAAATAAACTCCCATTTATTATC-3Ј. Plasmid STAT6mBG streptomycin. was created by ligating a double-stranded oligonucleotide with XhoI/ ␬ ␬ HEK293 cells were cultured at 37°C with 5% CO2 in DMEM supple- BglII overhangs into mBG-LUC NF- B1, replacing the two NF- B mented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 sites with two copies of the CCL23 STAT6 site. The sequence of the ␮g/ml streptomycin. Purified human rIL-4 and GM-CSF were obtained oligonucleotides used are: 5Ј-TCGAGATGGGAGTTTTCTTTTGAAG from Novartis and were used at a concentration of 40 and 50 ng/ml, re- GAAATGGGAGTTTTCTTTTGAAGGAAA-3Ј and 5Ј-GATCTTTCCTT spectively. Commercially available IL-13 (PeproTech) was used at 50 ng/ CAAAAGAAAACTCCCATTTCCTTCAAAAGAAAACTCCCATC-3Ј.By ml. LPS (Sigma-Aldrich) was used at 500 ng/ml. a similar strategy, plasmid STAT6mut-mBG was created using the oligonu- cleotides 5Ј-TCGAGATGGGAGTTTATTTTTGAAGGAAATGGGAGTAG ELISA for CCL23 TTTATTTTTGAAGGAAA-3Ј and 5Ј-GATCTTTCCTTCAAAAATAAAC Ј Human CCL23 protein levels were measured with a standard sandwich TCCCATTTCCTTCAAAAATAAACTCCCATC-3 . ELISA. Microtiter wells (Maxisorp Immuno plates; Nunc) were coated The CCL23 promoter-negative regulatory element was cloned as a dou- with 0.5 ␮g/ml anti-human CCL23 Ab MAB371 (R&D Systems) over- ble-stranded oligonucleotide with KpnI/MluI overhangs into the constructs ϩ EOT3 (25) and mBG-LUC NF-␬B1/IL4RE (30) upstream of the STAT6 night at 4°C. Samples were added in different dilutions for 90 min at Ј 37°C. As standard, recombinant CCL23, aa 22–120 was used (R&D Sys- sites using the following sequences: 5 -CCCTTTCCCAGAGACTAAGCGCA GGGCTGAGAAGGA-3Ј and 5Ј-CGCGTCCTTCTCAGCCCTGCGCTTA tems). Afterward, the immune complexes were detected by incubation with Ј 0.5 ␮g/ml biotinylated CCL23 Ab (R&D Systems) for 90 min at 37°C. The GTCTCTGGGAAAGGGGTAC-3 . complexes were made visible by adding streptavidin-linked HRP. The Transient transfection of HEK293 cells lower limit of sensitivity of the assay was 300 pg/ml. Transient transfection of HEK293 cells was achieved using calcium phos- Downloaded from IgE determination phate coprecipitation (24). Briefly, the transfectants were washed 24 h after transfection and cultured in fresh medium in the presence or absence of 40 Total IgE was determined in human plasma samples using an Immuno- ng/ml IL-4 for another 24 h before luciferase assays were conducted in CAP Total IgE (Pharmacia) on the Immuno-CAP-250 device (Pharma- duplicates according to the instructions of the manufacturer using the Pro- cia) as recommended by the supplier. mega Luciferase Assay System (Promega). Immunohistology Preparation of nuclear extracts and EMSA http://www.jimmunol.org/ Human skin biopsies (4-mm punch biopsies) were obtained from healthy Nuclear extracts from unstimulated human primary blood monocytes or volunteers and from patients with atopic dermatitis after informed consent. cells that had been stimulated for 4 h with IL-4, GM-CSF, or both were Tissue samples were embedded in OCT medium (Tissue-Tek) and kept at prepared according to the method described by Andrews and Faller (32). A Ϫ80°C. Cryostat sections of 5 ␮m were fixed with dried acetone (Merck ␮ double-stranded oligonucleotide probe containing the STAT6 binding site 1.00299) and stained with 10 g/ml mouse IgG1 anti-human CCL23 mAb in the CCL23 promoter was end-labeled using [␣-32P]dCTP (Amersham MPIF-1 (clone 69512.111; R&D Systems) or with control mouse serum. Ex- ϩ Biosciences) and Klenow polymerase (Roche Diagnostics). The nucleo- pression of CCL23 was detected by using the DakoCytomation EnVision protein-binding reaction was done as described previously (33) using 5 ␮g System-HRP according to manufacturer’s instructions. Samples were subse- of nuclear extracts. For oligonucleotide competition experiments, extracts quently counterstained with hematoxylin (Vector Laboratories), mounted us- were preincubated with various amounts of unlabeled competitor oligonucle- ing Faramount aqueous mounting medium (DakoCytomation), and analyzed

otide on ice for 30 min before the radiolabeled probe was added. The following by guest on September 28, 2021 in an Axioplan2 microscope (Zeiss) equipped with a digital Spot camera (Vis- oligonucleotides were used in these experiments: CCL23, 5Ј-TCGAGGAGA itron Systems). ATAAATGGGAGTTTTCTTTTGAAGGAATAATAAGATA-3Ј and 5Ј-GA Total RNA isolation, cDNA synthesis, and RT-PCR TCTATCTTATTATTCCTTCTTCAAAAGAAAACTCCCATTTATTCT CC-3Ј and eotaxin-3, 5Ј-TGTTCCCAACCACAGAATTCTCTGGAATTG Total RNA was isolated using the Absolutely RNA Miniprep kit (Strat- TTTTCAGGGCCGT-3Ј and 5Ј-GAGACGGCCCTGAAAACAATTCCA agene) according to the instructions of the manufacturer. cDNA was syn- GAGAATTCTGTGGTTGGGA-3Ј. thesized from 0.7 ␮g of RNA by reverse transcription using iScript reverse transcriptase (Bio-Rad) in a total volume of 20 ␮l according to the instruc- Results tions of the manufacturer. DC have been shown to express CCL23 (15). Because these cells The expression of CCL23 was analyzed with the commercially available can be generated from monocytes by IL-4 and GM-CSF, it was SYBR Green RT-PCR kit (Applied Biosystems.) in an ABI7900 machine with the following PCR parameters: one cycle of 2 min at 50°C and 10 min determined whether CCL23 production was regulated in mono- at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 62°C. All cytes during the differentiation process into DC. Monocytes were templates were analyzed in duplicates and the average was used for further incubated with IL-4, GM-CSF, or the combination of the two cy- calculation. The average cycle threshold (Ct) values of the CCL23 samples tokines and the expression of CCL23 gene transcripts was mea- were normalized to the average Ct values of the housekeeping gene elongation factor 1-␣ (EF1-␣) using the following equation: 2(CtЈЈEF1-␣ЈЈ Ϫ CtЈЈCL23ЈЈ ϫ sured by quantitative RT-PCR. The constitutive production of 1000. The following primer pairs were used: CCL23, 5Ј-ATGCTTGTTACT CCL23 mRNA was almost undetectable in unstimulated cells. GCCCTTGG-3Ј and 5Ј-GGGTCATCTGAGGACCAATC-3Ј and EF1-␣,5Ј- IL-4, but not GM-CSF induced the synthesis of significant TTTGAGACCAGCAAGTACTATGTGACT-3Ј and 5Ј-TCAGCCTGAGA amounts of CCL23 transcripts within 24 h. The combination of the Ј TGTCCCTGTAA-3 . cytokines had a synergistic effect in all 10 donors analyzed (Fig. Recombinant plasmids 1A). The mean factor of induction at 24 h with IL-4/GM-CSF was Ϯ Various DNA fragments containing the human CCL23 promoter were 375 91-fold depending on the donor. Because IL-13 is known to amplified from human genomic DNA using the upstream PCR primer share most of the biological activities with IL-4, monocytes were 5Ј-CTGACTCGAGGCATCTCATTGACCTGGCTGTCC-3Ј along with stimulated with IL-13 alone or in combination with GM-CSF for 3 the reverse primers 5Ј-AGTCAAGCTTGACTCCTGGGCTCACTGCTT days to assess whether this cytokine was also able to promote Ј Ј C-3 (CCL23p-XH), 5 -AGTCAGATCTAATAGTCCTCGTCCAGTG CCL23 expression. The results demonstrated that, like IL-4, IL-13 TC-3Ј (CCL23p-XE plus 30), 5Ј-AGTCAGATCTCCTTCTCAGCCC TGCGCTTA-3Ј (CCL23p-XE plus 60), and 5Ј-AGTCAGATCTAGGA alone was sufficient to induce CCL23 expression. The response to TGAGCACATGGGAGCT-3Ј (CCL23p-XE plus 90). All amplicons IL-13 was always significantly lower than after IL-4 induction. were cloned into the luciferase reporter vector mBG-LUC NF-␬B1 (30), However, in contrast to IL-4, GM-CSF had no synergistic effects replacing the two tandem NF-␬B sites with the respective CCL23 pro- (Fig. 1B). When monocytes were stimulated with LPS plus IFN-␥, moter piece. A 421-bp XhoI/EcoRV promoter subfragment (CCL23p- XE), a 558-bp XhoI/XbaI fragment (CCL23p-XX), and a 881-bp XhoI/ no activation of CCL23 expression was observed (data not shown). AccI fragment (CCL23p-XA) were ligated into the same vector A kinetic study showed that maximal CCL23 expression was backbone. Site-directed mutations in the STAT6 site in construct reached at day 3 in IL-4 plus GM-CSF-stimulated monocytes (Fig. The Journal of Immunology 4337

FIGURE 1. CCL23 induction in monocytes. A rep- resentative experiment of 10 is shown. A, CCL23 steady-state mRNA expression in monocytes is depicted relative to the housekeeping gene EF1-␣. B, GM-CSF synergizes with IL-4 but not with IL-13 to stimulate CCL23 expression in monocytes. Error bars, SDs. C, Time course of IL-4- and GM-CSF-induced CCL23 RNA and protein expression by monocytes. Error bars, SDs. Downloaded from

1C). Cell supernatants from the same experiment were investigated factor STAT6. This factor is well described to mediate IL-4 re- for CCL23 protein by ELISA. CCL23 became detectable at day 3 sponsiveness to a variety of genes, including the chemokines and increased at day 4. The slower kinetics of the protein com- eotaxin-1 and eotaxin-3 (24, 25). Indeed, a canonical STAT6 bind- http://www.jimmunol.org/ pared with the RNA may be partially explained by the moderate ing site was identified between position Ϫ698 and Ϫ689 relative to sensitivity of the ELISA (ϳ300 pg/ml). The same explanation the transcriptional start site. In fact, this 5Ј-TTCTTTTGAA-3Ј site likely applies for the fact that no CCL23 protein was measurable differed by only two nucleotides in the central variable region from in IL-13-stimulated cells. This argument is based on the fact that the functional STAT6 site found in the eotaxin-3 promoter (Fig. the amount of CCL23 protein in IL-4/GM-CSF-induced cells is 2A). Various DNA segments encompassing the regulatory region ϳ2- to 3-fold above the lower limit of detection of the ELISA. upstream of the CCL23 gene and including the putative STAT6 Because IL-13 stimulation was always significantly lower than IL- site were cloned in a reporter plasmid in which basal expression of 4/GM-CSF at the RNA level, correspondingly lower concentra- the luciferase gene is driven by the TATA box of the mouse ␤-globin by guest on September 28, 2021 tions of CCL23 protein would not be detectable by the ELISA. gene (30) (Fig. 2A). These CCL23 reporter constructs were transiently To investigate the mechanism by which IL-4 regulates CCL23 transfected into HEK293 cells in the presence of a STAT6 expression expression in monocytes, the putative promoter region of the vector (34). HEK293 cells have been shown earlier to harbor a de- CCL23 gene was scanned for binding sites of the transcription fective STAT6 protein while all other components of the IL-4 signal

FIGURE 2. A, The schematic structure of four dif- ferent CCL23 promoter reporter gene constructs is shown. The numbers refer to the number of nucleotides relative to the start site of transcription (bent arrow). TATA indicates the minimal promoter from the mouse ␤-globin gene linked to the firefly luciferase reporter (Luc). The black box marks the position of the STAT6 site. The nucleotide sequence of the STAT6 site in the CCL23 promoter is shown along with known STAT6 sites from the eotaxin-3 and the IgE germline promoter. Shaded nucleotides highlight and the two arrows indi- cate the position of the inverted repeat triplet in the STAT6 site. B, IL-4 inducibility of CCL23 reporter plasmids in transiently transfected HEK293 cells. The induction factor represents the ratio of luciferase ex- pression in IL-4-treated vs uninduced cells. One repre- sentative experiment of four is shown. 4338 REGULATION OF CCL23 EXPRESSION IN HUMAN MONOCYTES BY IL-4

FIGURE 4. STAT6 protein interacts with its binding site in IL-4-in- duced monocytes. EMSA using STAT6 binding site containing double- stranded oligonucleotides from the eotaxin-3 or the CCL23 promoter as probes. The numbers indicate the molar excess of competitor oligonucle- otides relative to the radiolabeled probe. The position of the STAT6-con- taining nucleoprotein complex is shown. Downloaded from FIGURE 3. The CCL23 STAT6 site is required for IL-4 induction of the To directly demonstrate binding of STAT6 to the 5Ј-TTCTTTT CCL23 promoter. Top, The schematic structure of the CCL23p-XE re- GAA-3Ј element, EMSA experiments were conducted. Incubation porter plasmid is shown. The black box denotes the position of the STAT6 of a radiolabeled double-stranded oligonucleotide containing the site. Below, the sequence of the wild-type (CCL23p-XE) and mutated putative STAT6 binding site with nuclear extracts derived from STAT6 site (CCL23p-XEmut) is indicated. Left graph, The IL-4 response unstimulated primary monocytes or cells stimulated with IL-4, of the two CCL23 promoter plasmids. Right graph, The cytokine induc- ibility of the STAT6 site in the absence of additional CCL23 promoter GM-CSF, or IL-4 plus GM-CSF revealed the formation of two http://www.jimmunol.org/ elements. One representative of six experiments is shown. distinct bands detectable with all four extracts. In extracts from IL-4- and IL-4 plus GM-CSF- treated monocytes, a third more slowly migrating nucleoprotein complex was observed (Fig. 4, left panel). This band was specifically induced by IL-4 because it was transduction pathway are intact. Therefore, HEK293 cells are a suit- absent in extracts derived from unstimulated or GM-CSF-treated able tool to study the involvement of STAT6 in IL-4-driven cellular cells. Moreover, its migratory behavior was identical to the responses (35). The transfectants were stimulated with IL-4 and the STAT6-containing complex formed when the same extracts were response of the reporter plasmids to the cytokine was monitored by incubated with the functional STAT6 binding site identified in the measuring the enzymatic activity of luciferase. The results demon- eotaxin-3 promoter (25) (Fig. 4, left panel). These data strongly by guest on September 28, 2021 strated that only plasmid XE harboring a short XhoI/EcoRV pro- indicated that STAT6 interacted with the IL-4-responsive element. moter fragment was inducible with IL-4 while the other three re- Competition experiments were used to compare the relative porter constructs (XH, XA, and XX) with longer promoter strength of the interaction of STAT6 to the CCL23 vs the eotaxin-3 sequences at the 3Ј end were not responsive (Fig. 2B). The sensi- binding site. Titration of different amounts of unlabeled CCL23 or tivity of the XE plasmid toward IL-4 was dependent on the pres- eotaxin-3 STAT6 binding site oligonucleotides showed that a ence of cotransfected STAT6 (Fig. 2B, right panel). These data 3-fold molar excess of the eotaxin-3 STAT6 site double-stranded showed that the 421-bp promoter segment in XE contained cis- oligonucleotide efficiently competed for binding to the labeled acting sequences mediating IL-4 responsiveness. In addition, se- eotaxin-3 STAT6 probe (Fig. 4, right panel). In contrast, a 10-fold quences between the EcoRV and the XbaI site must contain cis- higher concentration was needed to achieve a similar degree of acting elements which acted in a dominant negative fashion by competition with the CCL23 STAT6 sequence. These data sug- suppressing the cytokine response. gested that the CCL23 STAT6 motif bound STAT6 with low af- To explore whether the putative STAT6 site was functionally finity compared with the eotaxin-3 recognition site. involved in IL-4-mediated activation of the XE construct, two As shown in Fig. 2, a dominant negative acting regulatory ele- point mutations were introduced into the 5Ј-located direct repeat ment was located between the EcoRV and the XbaI site in the TTC (Fig. 3, upper panel). Such mutations have been shown ear- CCL23 promoter. To map this element in more detail, additional lier to abrogate the function of bona fide STAT6 binding sites (25). reporter constructs were made with progressive extensions at the Transient transfection of the mutant reporter plasmid XEmut along 3Ј end (Fig. 5, upper panel). IL-4 inducibility of these plasmids with the STAT6 expression vector in HEK293 cells revealed that was analyzed in transiently transfected HEK293 cells in the pres- IL-4 was unable to induce promoter activation, demonstrating that ence of the STAT6 expression vector. Construct XE ϩ 30 led to a this site was critically involved in the cytokine response (Fig. 3, small but statistically significant decrease in cytokine responsive- lower left panel). To further prove the function of this site as IL-4 ness compared with construct XE, suggesting that this stretch of response element in the absence of any other CCL23 promoter DNA contributed to the negative regulatory function. In contrast, sequences, two tandem wild-type or mutated copies were cloned IL-4 could not induce luciferase expression in cells transfected upstream of a heterologous TATA box in the vector mBG-luc (30). with the plasmids XE ϩ 60 and XE ϩ 90 (Fig. 5, lower left panel), Transfectants carrying plasmid STAT6mBG responded to IL-4 demonstrating that the 30 additional nucleotides in XE ϩ 60 vs with an 8-fold increase in luciferase expression while cells trans- XE ϩ 30 (top of Fig. 5), were critically involved in repression of the fected with STAT6mut-mBG did not respond to cytokine stimu- IL-4 response. To explore whether the negative function of this lation (Fig. 3, lower right panel). Again, IL-4 stimulation of DNA stretch was specific for the CCL23 gene, a single copy of this STAT6mBG was dependent on the presence of cotransfected sequence was cloned into two reporter plasmids that had been STAT6 (data not shown). shown earlier to be inducible with IL-4 in a STAT6-dependent The Journal of Immunology 4339

panel, the cytokine response of EOT3–30/60 and (IL4RE)2Luc- 30/60 was significantly reduced compared with the parental plas- mids. It should be noted, however, that the repressing effect was only partial and not complete as observed with the CCL23 XE plasmid. These data demonstrated that the negative function of this 30-nt DNA element was not limited to the CCL23 gene but could be transferred to other IL-4/STAT6-driven genes. The IL-4/STAT6 molecule pair is well known in Th2 cell-me- diated immune reactions and is critically involved in atopic dis- eases like atopic dermatitis or allergic asthma (36). Therefore, the possibility that CCL23 expression may be associated with atopy was investigated. To determine whether CCL23 was expressed in blood and whether differences in expression existed between healthy and atopic individuals, plasma from 57 individuals was analyzed for CCL23 protein by ELISA. Fifteen samples were pos- itive for CCL23 (26% of all samples) with concentrations varying over a wide range between 8.7 ng and 300 pg, which was the lower limit of detection (Table I). Based on the moderate sensitivity of the ELISA, it is quite possible that the percentage of donors con- Downloaded from taining biologically relevant concentrations of CCL23 is much higher than 26%. Serum IgE levels higher than 100 kU/L serve as one of several parameters indicative of atopy in adults (37). Thus, the same plasma samples were analyzed for IgE concentrations FIGURE 5. Fine mapping of the negative regulatory cis-acting element. (Table I). Thirteen of 57 donors had IgE plasma levels higher than http://www.jimmunol.org/ Top, The schematic structures of four CCL23 promoter reporter constructs 100 kU/L. Among those, three samples had measurable CCL23 is shown. The nucleotide sequence shown indicates the negative regulatory bioreactivity (23%). Among the 44 individuals with IgE concen- element. Below, The left graph demonstrates the IL-4 response of the four trations Ͻ100 kU/L, 12 were positive for CCL23 (27%). Thus, the promoter plasmids. Right graph, The effect of the negative element on percentage of CCL23-positive samples was very similar in both cytokine induction of the eotaxin-3 and the IgE germline promoter. One the high- and low-IgE groups, indicating no correlation between representative of three experiments is shown. Statistical significance was serum CCL23 and elevated IgE levels. determined using a Student two-tailed t test for pairs. To determine whether CCL23 is expressed in skin and whether differences could be detected in tissue derived from patients suf- fashion. Plasmid EOT3 contains the human eotaxin-3 promoter fering from atopic dermatitis, skin samples were assayed for the by guest on September 28, 2021 (25), whereas plasmid (IL4RE)2Luc contains two copies of the presence of CCL23-producing cells by immunohistochemistry. STAT6 site identified in the human IgE germline gene promoter CCL23 immunoreactivity was not or only very sparsely observed (30). In both cases, the negative CCL23 promoter element was in the skin of normal human subjects (Fig. 6C). In contrast, placed upstream of the STAT6 site. As shown in Fig. 6, lower right CCL23-immunoreactive cells appeared more frequently in the

FIGURE 6. CCL23 immunoreactivity in skin of normal human subjects (C) and lesional skin of atopic dermatitis patients (B and D). D, An enlarged image of the region marked by the rectangle in B. Arrows, The position of CCL23-immunoreactive cells. A, Isotype control staining of atopic skin. One representative donor of three is shown. 4340 REGULATION OF CCL23 EXPRESSION IN HUMAN MONOCYTES BY IL-4

Table I. No correlation between CCL23 and total IgE expression in locations, even in introns (41). The four central thymidines of the a human plasma CCL23 STAT6 site 5Ј-TTC TTTT GAA-3Ј, which separate the palindromic TTC half-sites, are not found in other STAT6 sites. Sample Size No. of CCL23-Positive Donors An alignment of functional STAT6 sites (40) resulted in a refined Total no. of donors 57 15 (26%) consensus sequence, 5Ј-TTC C(T/C)(G/A)G GAA-3Ј. It is likely Atopic donors 13 3 (23%) that the low-affinity binding of STAT6 to the CCL23 STAT6 site Healthy volunteers 44 12 (27%) compared with the site found in the eotaxin-3 promoter is due to a Human plasma samples were analyzed for CCL23 and total IgE. The percentages the differences in the variable central positions. in parentheses indicate the number of CCL23-positive donors in the sample. Downstream of the STAT6 site, another negatively acting reg- ulatory DNA element was mapped. The presence of this sequence suppressed the IL-4 response mediated via the STAT6 site. The atopic epidermis skin (Fig. 6, B and D). Immunoreactivity was function of the element was not gene specific since it attenuated limited to nonparenchymal polygonal cells distributed nonuni- the IL-4-driven activation of the eotaxin-3 and the IgE germline formly in the spinal cell layer of the epidermis. Dermis of both promoter. It will be interesting to determine whether the negative normal and atopic skin specimens was completely devoid of element also affects the activation of other genes which are not CCL23 immunoreactive cells. Isotype control Ab did not stain any regulated by IL-4. Bioinformatics predicted the possible interac- structures in normal (data not shown) or atopic skin (Fig. 6A). tion of the transcription factors H-APF-1 and P3A with this se- quence. H-APF-1 was originally described as -specific tran- Discussion Downloaded from This study extends the distribution of expression of CCL23 to scription factor which is involved in the IL-6-driven induction of human skin and blood. A sizeable proportion of the individuals the C-reactive protein gene promoter (42). P3A transcription fac- tested expressed the chemokine in these two organs. So far, tors were first described in sea urchins (43). Interestingly, these CCL23 transcripts have been observed in pancreas, skeletal muscle proteins exert a negative regulatory function on (12), , and liver (11). The expression of CCL23 in blood is (44). The mechanistic interplay between the negative regulatory

remarkable because only a small subset of chemokines can be element and the STAT6 site in its genomic context during IL-4 http://www.jimmunol.org/ readily detected in plasma such as CCL14 and CCL15 (38). treatment of monocytes needs further consideration. At least two Cultivation of blood monocytes with IL-4 plus GM-CSF re- scenarios are possible: first, the negative element is only functional sulted in activation of CCL23 expression both on the RNA and the in HEK293 cells due to an interacting repressory protein specifi- protein level. CCL23 production was maximal at 3 days. This time cally expressed in the cell line but not in monocytes. Second, the course is reminiscent of the one required for IL-4/GM-CSF-driven negative function in monocytes is needed for active repression of differentiation into monocyte-derived DC in vitro. Thus, it may be CCL23 gene expression in the absence of cytokine stimulation. speculated whether CCL23 may be a marker of this particular cell Treatment with IL-4 leads to STAT6 activation which, upon bind- population. It may be of interest if other cytokine mixtures used to ing to its cognate site, overrides the negative effect via a factor generate other DC subtypes (39) also induce CCL23 expression. expressed in monocytes but not in HEK293 cells. Comparative by guest on September 28, 2021 The sustained expression of CCL23 is in contrast to the transient studies in monocytes and HEK293 cells are required to discrimi- induction of chemokine expression upon IL-1␤ treatment which nate between these possibilities. peaks at 8 h and is rapidly lost thereafter (16). Although GM-CSF Besides CCL23, IL-4 and IL-13 can also regulate the expression had no inducing effect on its own, it synergized with IL-4. A num- of other chemokines, especially members of the CC chemokine ber of possible explanations may account for the mechanistic basis subfamily. Eotaxin-1 and eotaxin-3 production has been shown to of the synergistic effect. GM-CSF receptor engagement could in- be inducible by these Th2-associated cytokines in a STAT6-de- duce synergy at the transcriptional level by activating a costimu- pendent fashion by human fibroblasts (23–25). CCL17 and CCL22 latory transcription factor which cooperates with STAT6. Such a are induced by IL-4 and IL-13 in a variety of cell types (reviewed mechanism has been described earlier for the IgE germline gene in Ref. 45), although an involvement of STAT6 has not been de- which requires the cooperation between STAT6 and other fac- scribed. Similarly, CCL18 can be up-regulated in monocytes and tors such as NF-␬B and PU.1 for full gene activation by IL-4 DC by IL-4 (46, 47). This type of cytokine regulation appears to be plus CD40 signaling (30, 34). It is also possible that GM-CSF relevant for diseases in which Th2 cells and their derived cytokines signaling leads to the derepression or shutdown of a negative- play a major role, such as atopic dermatitis (48). Therefore, an acting factor. Alternatively, GM-CSF may act at the posttran- association of CCL23 expression with elevated IgE levels was in- scriptional level by stabilizing CCL23 mRNA. This possibility vestigated. Increased serum IgE level is one of several parameters appears less likely because no synergistic effect of GM-CSF used to define the atopic phenotype in adults but is not an absolute was found with IL-13. IL-4 inducibility of the CCL23 gene was dependent on a STAT6 criterion. Unlike CCL18 or CCL22, however, no increase in binding site in the regulatory region of the gene. This claim is plasma levels of CCL23 in atopic individuals (as defined by ele- Ͼ primarily based on the results in the HEK293 cell system. Indirect vated serum IgE 100 kU/L) was observed. The same was true support that STAT6 is also involved in regulating CCL23 expres- with the subgroup of plasma samples derived from individuals sion in primary monocytes comes from the fast kinetics of CCL23 who were positively diagnosed for atopy irrespective of serum IgE induction in monocytes where mRNA was clearly detectable al- levels (data not shown). However, increased numbers of CCL23- ready at 4 h upon IL-4/GM-CSF treatment (data not shown). This positive cells were found in the epidermis of skin derived from fast kinetics is typically found for a number of STAT6-regulated atopic dermatitis patients. Because CCL23 has been shown to have genes (21, 24, 25). The STAT6 site is located between Ϫ698 and chemotactic activity toward monocytes and DC (12, 14–16), it can Ϫ689 relative to the transcriptional start site. This is remarkable be speculated whether this chemokine is involved in the migration because the majority of STAT6 sites are found in the proximal of these cell types during the course of this disease. In any case, promoter region within 250 bp relative to the transcriptional start CCL23 expression in skin may have the potential to be developed site (40). However, STAT6 sites have also been identified in other as novel biomarker for atopic dermatitis in the future. The Journal of Immunology 4341

Disclosures 23. Matsukura, S., C. Stellato, J. R. Plitt, C. Bickel, K. Miura, S. N. Georas, V. Casolaro, and R. P. Schleimer. 1999. Activation of eotaxin gene transcription All of the authors are with Novartis Pharma AG: N. Harrer, J. M. Carballido, by NF-␬B and STAT6 in human airway epithelial cells. J. Immunol. 163: and M. Woisetschla¨ger are full-time employees; A. Mu¨ller and C. Gu¨nther are 6876–6883. Postdoctoral Fellows, and H. Novak is a PhD student. 24. Hoeck, J., and M. Woisetschla¨ger. 2001. STAT6 mediates eotaxin-1 expression in IL-4 or TNF-␣-induced fibroblasts. J. Immunol. 166: 4507–4515. 25. Hoeck, J., and M. Woisetschla¨ger. 2001. Activation of eotaxin-3/CCL26 gene References expression in human dermal fibroblasts is mediated by STAT6. J. Immunol. 167: 1. Schall, T. J., and K. B. Bacon. 1994. Chemokines, leukocyte trafficking, and 3216–3222. inflammation. Curr. Opin. Immunol. 6: 865–873. 26. Kotanides, H., and N. C. Reich. -4-induced STAT6 recognizes and activates a target site in the promoter of the interleukin-4 receptor gene. J. Biol. 2. Van Damme, J., P. Proost, J. P. Lenaerts, and G. Opdenakker. 1992. Structural Chem. 271: 25555–25561. and functional identification of two human, tumor-derived monocyte chemotactic 27. Albrecht, B., S. Peiritsch, and M. Woisetschlager. 1994. A bifunctional control proteins (MCP-2 and MCP-3) belonging to the chemokine family. J. Exp. Med. element in the human IgE germline promoter involved in repression and IL-4 176: 59–65. activation. Int. Immunol. 6: 1143–1151. 3. Uguccioni, M., M. D’Apuzzo, M. Loetscher, B. Dewald, and M. Baggiolini. 28. Seidel, H. M., L. H. Milocco, P. Lamb, J. E. Darnell, Jr., R. B. Stein, and 1995. Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, J. Rosen. 1995. Spacing of palindromic half sites as a determinant of selective MIP-1␣ and MIP-1␤ on human monocytes. Eur. J. Immunol. 25: 64–68. STAT (signal transducers and activators of transcription) DNA binding and tran- 4. Schall, T. J., K. Bacon, K. J. Toy, and D. V. Goeddel. 1990. Selective attraction of scriptional activity. Proc. Natl. Acad. Sci. USA 92: 3041–3045. monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. 29. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human Nature 347: 669–671. blood: isolation of mononuclear cells by one centrifugation, and of granulocytes 5. Taub, D. D., K. Conlon, A. R. Lloyd, J. J. Oppenheim, and D. J. Kelvin. 1993. ϩ ϩ by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Preferential migration of activated CD4 and CD8 T cells in response to Suppl. 97: 77–89. MIP-1␣ and MIP-1␤. Science 260: 355–358. 30. Messner, B., A. M. Stu¨tz, B. Albrecht, S. Peiritsch, and M. Woisetschla¨ger. 1997. 6. Loetscher, P., M. Seitz, I. Clark-Lewis, M. Baggiolini, and B. Moser. 1994. Cooperation of binding sites for STAT6 and NF␬B/ in the interleukin-4 in-

Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants Downloaded from duced upregulation of the human IgE germline promoter. J. Immunol. 159: for human CD4ϩ and CD8ϩ T lymphocytes. FASEB J. 8: 1055–1060. 3330–3337. 7. Alam, R., P. A. Forsythe, S. Stafford, M. A. Lett-Brown, and J. A. Grant. 1992. ␣ 31. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. inflammatory protein-1 activates basophils and mast cells. J. Exp. Site-directed mutagenesis by overlap extension using the polymerase chain re- Med. 176: 781–786. action. Gene 77: 51–59. 8. Tanimoto, Y., K. Takahashi, and I. Kimura. 1992. Effects of cytokines on human 32. Andrews, N. C., and D. U. Faller. 1991. A rapid micropreparation technique for basophil . Clin. Exp. Allergy 22: 1020–1025. extraction of DNA: binding proteins from limiting numbers of mammalian cells. 9. Rot, A., M. Krieger, T. Brunner, S. C. Bischoff, T. J. Schall, and C. A. Dahinden. Nucleic Acids Res. 19: 2499. 1992. RANTES and macrophage inflammatory protein 1␣ induce the migration ␧ ␧ 33. Ko¨hler, I., and E. P. Rieber. 1993. Allergy-associated I and Fc receptor II http://www.jimmunol.org/ and activation of normal human granulocytes. J. Exp. Med. 176: (CD23b) genes activated via binding of an interleukin-4-induced transcription 1489–1495. factor to a novel responsive element. Eur. J. Immunol. 23: 3066–3071. 10. Baggiolini, M., and C. A. Dahinden. 1994. CC chemokines in allergic inflam- 34. Stu¨tz, A. M., and M. Woisetschla¨ger. 1999. Functional synergism of STAT6 with mation. Immunol. Today 15: 127–133. either NF-␬B or PU. 1 to mediate IL-4 induced activation of IgE germline gene 11. Patel, V. P., B. L. Kreider, Y. Li, H. Li, K. Leung, T. Salcedo, B. Nardelli, transcription. J. Immunol. 163: 4383–4391. V. Pippalla, S. Gentz, R. Thotakura, et al. 1997. Molecular and functional char- 35. Lu, B., M. Reichel, D. A. Fisher, J. F. Smith, and P. Rothman. 1997. Identifi- acterization of two novel human C-C chemokines as inhibitors of two distinct cation of a STAT6 domain required for IL-4-induced activation of transcription. classes of myeloid progenitors. J. Exp. Med. 185: 1163–1172. J. Immunol. 159: 1255–1264. 12. Youn, B. S., S. M. Zhang, H. E. Broxmeyer, S. Cooper, K. Antol, M. Fraser, Jr., 36. Pernis, A. B., and P. B. Rothman. 2002. JAK-STAT signaling in asthma. J. Clin. ␤ ␤ and B. S. Kwon. 1998. Characterization of CK 8 and CK 8-1: two alternatively Invest. 109: 1279–1283. ␤ spliced forms of human -chemokine, chemoattractants for neutrophils, mono- 37. Bousquet, J., Y. Coulomb, H. Arrendal, M. Robinet-Levy, and F. B. Michel. cytes, and lymphocytes, and potent agonists at CC 1. Blood 1982. Total serum IgE concentrations in adolescents and adults using the phad- by guest on September 28, 2021 91: 3118–3126. ebas IgE PRIST technique. Allergy 37: 397–406. 13. Elagoz, A., D. Henderson, P. S. Babu, S. Salter, C. Grahames, L. Bowers, 38. Forssmann, U., H. J. Magert, K. Adermann, S. E. Escher, and W. G. Forssmann. M. O. Roy, P. Laplante, E. Grazzini, S. Ahmad, and P. M. Lembo. 2004. A 2001. Hemofiltrate CC chemokines with unique biochemical properties: HCC- ␤ truncated form of CK 8-1 is a potent agonist for human formyl peptide-receptor- 1/CCL14a and HCC-2/CCL15. J. Leukocyte Biol. 70: 357–366. like 1 receptor. Br. J. Pharmacol. 141: 37–46. 39. Shortman, K., and Y.-J. Liu. 2002. Mouse and human dendritic cell subtypes. 14. Nardelli, B., H. L. Tiffany, G. W. Bong, P. A. Yourey, D. K. Morahan, Y. Li, Nat. Rev. Immunol. 2: 151–161. P. M. Murphy, and R. F. Alderson. 1999. Characterization of the signal trans- 40. Ehret, G. B., P. Reichenbach, U. Schindler, C. M. Horvath, S. Fritz, M. Nabholz, duction pathway activated in human monocytes and dendritic cells by MPIF-1, a and P. Bucher. 2001. DNA binding specificity of different STAT proteins: com- specific for CC chemokine receptor 1. J. Immunol. 162: 435–444. parison of in vitro specificity with natural target sites. J. Biol. Chem. 276: 15. Nardelli, B., D. K. Morahan, G. W. Bong, M. A. Semenuk, B. L. Kreider, and 6675–6688. G. Garotta. 1999. Dendritic cells and MPIF-1: chemotactic activity and inhibition 41. Schjerven, H., P. Brandtzaeg, and F.-E. Johansen. 2003. Hepatocyte NF-1 and of endogenous chemokine production by IFN-␥ and CD40 ligation. J. Leukocyte STAT6 cooperate with additional DNA-binding factors to activate transcription Biol. 65: 822–828. of the human polymeric Ig receptor gene in response to IL-4. J. Immunol. 170: 16. Forssmann, U., M. B. Delgado, M. Uguccioni, P. Loetscher, G. Garotta, and 6048–6056. M. Baggiolini. 1997. CK␤8, a novel CC chemokine that predominantly acts on 42. Majello, B., R. Arcone, C. Toniatti, and G. Ciliberto. 1990. Constitutive and monocytes. FEBS Lett. 408: 211–216. IL-6-induced nuclear factors that interact with the human C-reactive protein pro- 17. Votta, B. J., J. R. White, R. A. Dodds, I. E. James, J. R. Connor, moter. EMBO J. 9: 457–465. E. Lee-Rykaczewski, C. F. Eichman, S. Kumar, M. W. Lark, and M. Gowen. 43. Hoog, C., F. J. Calzone, A. E. Cutting, R. J. Britten, and E. H. Davidson. 1991. 2000. CK␤-8 [CCL23], a novel CC chemokine, is chemotactic for human oste- Gene regulatory factors of the sea urchin embryo: II. Two dissimilar proteins, oclast precursors and is expressed in bone tissues. J. Cell. Physiol. 183: 196–207. P3A1 and P3A2, bind to the same target sites that are required for early territorial 18. Hwang, J., K. N. Son, C. W. Kim, J. Ko, D. S. Na, B. S. Kwon, Y. S. Gho, and gene expression. Development 112: 351–364. J. Kim. 2005. Human CC chemokine CCL23, a ligand for CCR1, induces endo- 44. Zeller, R. W., R. J. Britten, and E. H. Davidson. 1995. Developmental utilization thelial cell migration and promotes angiogenesis. Cytokine 30: 254–263. of SpP3A1 and SpP3A2: two proteins which recognize the same DNA target site 19. Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, and W. E. Paul. 1999. The in several sea urchin gene regulatory regions. Dev. Biol. 170: 75–82. IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 45. Romagnani, S. 2002. Cytokines and chemoattractants in allergic inflammation. 17: 701–738. Mol. Immunol. 38: 881–885. 20. Jiang, H., M. B. Harris, and P. Rothman. 2000. IL-4/IL-13 signaling beyond 46. Gu¨nther, C., C. Bello-Fernandez, T. Kopp, J. Kund, N. Carballido-Perrig, JAK/STAT. J. Allergy Clin. Immunol. 105: 1063–1070. S. Hinteregger, S. Fassl, C. Schwa¨rzler, G. Lametschwandtner, G. Stingl, et al. 21. Stu¨tz, A. M., L. A. Pickart, A. Trifilieff, T. Baumruker, E. Prieschl-Strassmayr, 2005. CCL18 is expressed in atopic dermatitis and mediates skin homing of and M. Woisetschlager. 2003. The Th2 cell cytokines IL-4 and IL-13 regulate human memory T cells. J. Immunol. 174: 1723–1728. found in inflammatory zone 1/resistin-like molecule ␣ gene expression by a 47. Kodelja, V., C. Muller, O. Politz, N. Hakij, C. E. Orfanos, and S. Goerdt. 1998. STAT6 and CCAAT/enhancer-binding protein-dependent mechanism. J. Immu- Alternative macrophage activation-associated CC-chemokine-1, a novel struc- nol. 170: 1789–1796. tural homologue of macrophage inflammatory protein-1␣ with a Th2-associated 22. Park, H. J., Y. S. Choi, and C. E. Lee. 1997. Identification and activation mech- expression pattern. J. Immunol. 160: 1411–1418. anism of the interleukin-4-induced nuclear factor binding to the CD23(b) pro- 48. Pivarcsi, A., and B. Homey. 2005. Chemokine networks in atopic dermatitis: moter in human B lymphocytes. Mol. Cells 7: 755–761. traffic signals of disease. Curr. Allergy Asthma Rep. 5: 284–290.