The chemokine CCL25 is selectively and constitutively expressed in the small intestinal epithelium and plays an important role in mediating lymphocyte recruitment to this site. In this study, we demonstrate that CCL25 expression in murine small intestinal epithelial cells is independent of signaling through the lymphotoxin β receptor and is not enhanced by inflammatory stimuli, pathways involved in driving the expression of most other chemokines. We define a transcriptional start site in the CCL25 gene and a region −141 to −5 proximal of exon 1 that is required for minimal promoter activity in the small intestinal epithelial cell lines, MODE-K and mICc12. These cell lines expressed far less CCL25 mRNA than freshly isolated small intestinal epithelial cells indicating that they are missing important factors driving CCL25 expression. The CCL25 promoter contained putative binding sites for the intestinal epithelial-associated Caudal-related homeobox (Cdx) transcription factors Cdx-1 and Cdx-2, and small intestinal epithelial cells but not MODE-K and mICc12 cells expressed Cdx-1 and Cdx-2. EMSA analysis demonstrated that Cdx proteins were present in nuclear extracts from freshly isolated small intestinal epithelial cells but not in MODE-K or mICcl2 cells, and bound to putative Cdx sites within the CCL25 promoter. Finally, cotransfection of MODE-K cells with Cdx transcription factors significantly increased CCL25 promoter activity as well as endogenous CCL25 mRNA levels. Together these results demonstrate a unique pattern of regulation for CCL25 and suggest a role for Cdx proteins in regulating CCL25 transcription.

Chemokines are a large family of low m.w. proteins primarily recognized for their role as leukocyte chemoattractants and in regulating leukocyte trafficking. They function through seven transmembrane G protein-coupled receptors to induce directed cellular migration and enhanced integrin-mediated adhesion, which are processes critical for leukocyte extravasation (1). Chemokines can be divided into two groups, inflammatory and homeostatic chemokines, based on their regulation and function (2). Inflammatory chemokines control the recruitment of effector leukocytes, including cells from both the innate and adaptive immune response, to sites of infection or inflammation, and can be induced in a wide variety of cells upon exposure to host or pathogen-derived inflammatory stimuli (2). Homeostatic chemokines, by contrast, are constitutively expressed in primary and secondary lymphoid organs and in tertiary tissues, such as the skin and intestine, where they control lymphocyte migration during hemopoiesis, initiation of immune responses, and immune surveillance of healthy peripheral tissues (2). The expression of homeostatic chemokines in lymphoid organs and the intestine is largely dependent on lymphotoxin (LT)4 β receptor signaling (3, 4, 5). The division of chemokines into inflammatory and homeostatic chemokines is, however, not absolute because many homeostatic chemokines can be up-regulated in response to inflammatory stimuli (6, 7), and inflammatory chemokines can target noneffector leukocytes at sites of leukocyte development (8).

The chemokine CCL25 is selectively and constitutively expressed in the small intestine and thymus, primarily by resident epithelial cells (9, 10, 11, 12). Its sole functional receptor, CCR9, is expressed on small intestinal lymphocytes, a subset of circulating gut tropic lymphocytes, and thymocytes (13, 14). Analysis of CCR9−/− mice, and in vivo studies using neutralizing anti-CCL25 Ab, or CCR9−/− TCR transgenic T cells have demonstrated a central role for CCL25/CCR9 in the generation of the small intestinal lymphocyte compartment (14, 15, 16, 17, 18, 19, 20, 21). Despite the importance of CCL25/CCR9 in small intestinal immunity, the mechanisms underlying the selective and constitutive expression of CCL25 in the small intestine are not understood.

In the current study, we have examined expression and regulation of CCL25 in small intestinal epithelial cells. Our results demonstrate that CCL25 displays a unique pattern of regulation compared with other inflammatory or homeostatic chemokines and suggest a role for the Caudal-related homeobox transcription factors in enhancing CCL25 promoter activity in the small intestine.

Germfree or conventional Swiss Webster mice were from Taconic Farms, and C57BL/6 mice were from the Microbiology, Immunology, and Glycobiology animal facility and the Biomedical Centre animal facility (Lund University, Lund, Sweden). Small intestinal tissue from athymic mice with a truncated common cytokine receptor γ-chain (CRγ−/Ynu/nu) and CRγ+nu/+ mice was provided by Dr. H. Ishikawa (Keio University School of Medicine, Tokyo, Japan), tissue from TNFR1−/− mice was provided by Dr. N. Lycke (University of Gothenburg, Gothenburg, Sweden), and tissue from LTα−/− and LTβ−/− mice was provided by Dr. D. Finke (University of Lausanne, Lausanne, Switzerland). All animal studies were approved by the local ethical committee.

Epithelial cells were removed from the small intestine with EDTA. Briefly, the small intestine was rinsed with ice-cold PBS, inverted, and cut into 5-cm fragments. Intestinal fragments were then incubated in PBS containing 30 mM EDTA for 30 min at 37°C on a rotating platform and EDTA was changed every 5 min. Murine small intestinal epithelial crypts were isolated and cultured as described (22). RT-PCR for cytokeratin 18 expression was performed to confirm the epithelial identity of cultured cells (data not shown). The murine small intestinal epithelial cell lines MODE-K provided by Dr. P. Ernst (University of Texas Medical Branch, Galveston, TX) and S1-H10 provided by Dr. J. I. Gordon (Washington University School of Medicine, St. Louis, MO), and the murine fibroblast cell line BALB/3T3 provided by Dr. C. Owman (Lund University, Lund, Sweden) were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS (Sigma-Aldrich), nonessential amino acids (Invitrogen Life Technologies), 1 mM sodium pyruvate (Invitrogen Life Technologies), and 50 U/ml penicillin and 50 mg/ml streptomycin (Invitrogen Life Technologies). The mouse intestinal mICc12 cells provided by Dr. A. Vandervalle (Institut National de la Santé et de la Recherche Médicale, Faculté X, Paris, France) were grown as described (23). Cells were grown to confluence in 24-well plates before addition of cytokines.

Small intestinal tissue sections (10 μm) were cut from Tissue-Tek OCT embedded tissue. The sections were placed on PEN membrane-coated slides (P.A.L.M. Microlaser Technologies), and fixed in 70% ethanol for 30 s and acetone for 4 min. Fixed sections were stained with Harris hematoxylin for 10 s (Sigma-Aldrich), washed, overlaid with 10% DMSO, and placed on dry ice or kept at −80°C until use. All aqueous solutions were made from diethyl pyrocarbonate-treated water and supplemented with vanadyl-ribonucleoside complex RNase Inhibitor (Sigma-Aldrich). Laser capture was performed on a Zeiss microscope equipped with a microcatapulting laser system (P.A.L.M. Microlaser Technologies). Total RNA was extracted from the catapulted samples using a Stratagene Microprep RNA kit.

Acetone-fixed small intestinal tissue sections (8-μm thick) were incubated with 1% H2O2 for 15 min to block endogenous peroxidase activity, followed by an avidin-biotin blocking kit (Vector Laboratories) to block endogenous streptavidin-biotin activity. Sections were incubated for 1 h with anti-CCL25 (at 5 μg/ml, clone 89827; R&D Systems) or with an isotype control Ab. Sections were then washed and incubated with biotinylated mouse anti-rat IgG2a secondary Ab (2.5 μg/ml, clone RG7/1.30; BD Biosciences), and the signal was amplified using streptavidin-HRP and tyramid-biotin (NEN/ PerkinElmer) according to the manufacturer’s instructions and visualized using streptavidin-Alexa Fluor 488 (2 μg/ml; Molecular Probes). Nuclei were stained using 4′,6′-diamidino-2-phenylindole.

RNA isolation, cDNA synthesis, and semiquantitative RT-PCR were performed as previously described (17). The following primers were used for RT-PCR: CXCL1 sense 5′-ATAGCCACACTCAAGAATGGTCG-3′ and antisense 5′-CACCCTTCTACTAGCACAGTGG-3′; CCL20 sense 5′-ACTGTTGCCTCTCGTACATAC-3′ and antisense 5′-GTGTCCAATTCCATCCCAA-3′; CXCL13 sense 5′-CAGAATGAGGCTCAGCACAGC-3′ and antisense 5′-TCTCTTACTCACTGGAGCTT-3′; HGPRT (hypoxanthine-guanine phosphoribosyltransferase) sense 5′-CACAGGACTAGAACACCTGC-3′ and antisense 5′-GCTGGTGAAAAGGACCTCT-3′. Primers for Cdx-1 were sense 5′-AGAGCGGCAGGTAAAGATCT-3′ and antisense 5′-CTACTCTCCAGAGCCAGTCT-3′, or sense 5′-GCGGAATTCACCATGTATGTGGGCTATGTGCTG-3′ and antisense 5′-GCGGAATTCTATGGCAGAAACTCCTCTTTCACA-3′, and for Cdx-2 were sense 5′-GCGGAATTCACCATGTACGTGAGCTACCTTCTG-3′ and antisense 5′-GCGGAATTCACTGGGTGACAGTGGAGTTTAAAACC-3′. Primers for β-actin were sense 5′-GGTGGGAATGGGTCAGAAGGACT-3′ and antisense 5′-CCACGCTCGGTCAGGATCTTCAT-3′. Primers for CCL25 were sense 5′-ATAGGCAATACACGCTACAAGC-3′ and antisense 5′-GCGGAATTCGTCTTCAAAGGCACCTTGGGCATGG-3′. Primers for cytokeratin 18 were sense 5′-AGATCGACAATGCCCGCCTTG-3′ and antisense 5′-AGACTTGGTGGTGACAACTGT-3′. Primers for Madcam-1 were sense 5′-CCTGAGTCTGAGGTAGCTGTGG-3′ and 5′-GAGTGCCTGTGTGTCTGACAGCAT-3′ antisense and for intestinal alkaline phosphatase sense 5′-GCCGTGAAAGTGCTAAGCAGG-3′ and antisense 5′-GGTCAGAGTGTCGCGTTCACTA-3′. CCL25 mRNA levels were determined by real-time RT-PCR as previously described (14).

Mouse small intestinal cells were isolated by EDTA treatment. Total RNA was prepared using Stratagene RNA Miniprep kit. For 5′ RACE total RNA was reverse transcribed onto magnetic beads (Dynal Biotech) using Superscript III (Invitrogen Life Technologies) according to manufacturer’s instructions. The single-stranded cDNA was tailed with dATP on the 3′ end using terminal transferase EC 2.7.7.31 (Roche). The second strand was synthesized with Pfx-polymerase (Invitrogen Life Technologies) and the following adaptor primer: 5′-GTCCGCGGCCGCGTAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTT-3′. The cDNA was removed from the magnetic beads and subjected to PCR using the first gene-specific primer 5′-GCGGAATTCTTTGATCCTGTGCTGGTAACCCAGG-3′ and the adaptor primer. The product was used in a consecutive PCR with a second gene-specific primer 5′-GCGGAATTCGTCTTCAAAGGCACCTTGGGCATGG-3′. The second PCR resulted in a product of 270 bp in length, which was cloned into pBluescript and seven obtained clones sequenced using BigDye (Applied Biosystems). For the 3′ RACE, 5 μg of cDNA (prepared as previously described) was reverse transcribed using Superscript III (Invitrogen Life Technologies). A PCR using the adaptor primer and a gene-specific primer (5′-CCGGCGGCCGCGGAGAACCCCAACAGTACAAGCG-3′) resulted in one gene-specific RACE product, which was cloned into pBluescript and eight obtained clones were sequenced.

The different promoter regions were amplified by PCR using the following reverse primers: PE1 5′-GCGCTCGAGCAATGCCTTTCTGGTCCTGAGAGCTGGT-3′ for PF1 and PF2 or PE2 5′-GCGCTCGAGAAGGTTAGATCTCCTCTCCAGATACC-3′ for PF3, and the following forward primers: 5′-GCGGAGCTCCGTATCACTCACTGCCCCACTGAAAGTGT-3′ for PF1 or 5′-GCGGAGCTCAAGGCCAGGACAGAGCAAGAGAGCAAGAA-3′ for PF2 and PF3 on mouse genomic DNA. PCR products were cut with SacI and XhoI, cloned into the pGL3-basic (Promega), and their identity verified by sequencing. Different parts of the promoter were deleted using restriction endonucleases. The HindIII digestion gave rise to the plasmids pGL3b-PF4 and pGL3b-PF5, whereas the PstI restriction resulted in the plasmid pGL3b-PF6. The constructs pGL3b-PF7-9 were constructed by ligating double-stranded oligonucleotides in the pGL3b vector. The following oligonucleotides were used: 5′-CTAGCTCTATCTGAAGGAGAGAGAAGTCCCAAGTCTCACAGAGTGGC-3′; 5′-TCGAGCCACTCTGTGAGACTTGGGACTTCTCTCTCCTTCAGATAGAG-3′; 5′-TCGAGAGGAGGAGCAGGAGGAGGGAGGGAGAGAAGATAGGGGGCAGGTCA-3′; 5′-GATCTGACCTGCCCCCTATCTTCTCTCCCTCCCTCCTCCTGCTCCTCCTC-3′; 5′-GATCTGCAGGGTGGGGCTCTGACTATAAAGAATGAAGCCAGTTCACTGA-3′; and 5′-AGCTTCAGTGAACTGGCTTCATTCTTTATAGTCGGAGCCCCACC CTGCA-3′. To construct the positive control plasmids 3xCdxA and 3xCdxA Mutant, the following oligonucleotides were cloned into pGL3-basic: 5′-TCGAGATTTATGCATTTATGATTTAT GGGCCCTATAT-3′; 5′-AGCTATATAGGGCCCATAAATCATAAAT GCATAAATC-3′; 5′-TCGAGATCTATGCATCTATGATCTATGG GCCCTATAT-3′; and 5′-AGCTATATAGGGCCCATAGATCATAGAT GCATAGATC-3′. All constructs were verified by sequencing.

Cells were transfected using Metafectene (Biontex) according to manufacturer’s recommendations, adding 1.5 μg of plasmid DNA complexed with 5 μl of Metafectene to 450,000 cells/well. For analysis of luciferase activity, cells were harvested 24 h posttransfection by adding passive lysis buffer and stored at −80°C until analysis. Luciferase assays were performed using the Dual Luciferase Assay kit (Promega) in a BMG LUMIstar Galaxy instrument. For cotransfection experiments using Cdx-1, 0.5 μg of pGL3-PF1 or pGL3-PF7 were cotransfected with 0.9 μg of pcDNA3Cdx-1 plasmid (provided by Dr. P. Soubeyran, Institut National de la Santé et de la Recherche Médicale, Marseille, France) (24) or empty pcDNA3 plasmid as control. For cotransfection experiments using Cdx-2, 0.5 μg of pGL3-PF1 or pGL3-PF7 were cotransfected with 0.5 μg of pTREtightCdx-2 (provided by Dr. T. Uesaka, Hiroshima University, Hiroshima, Japan) (25) and 0.5 μg of pTet-ON (BD Biosciences). Cdx-2 expression was initiated by adding doxycycline (Sigma-Aldrich) to a final concentration of 10 μM. All transfections performed included 50 ng of the pTK-RL Renilla plasmid (Promega) for the normalization of transfection efficiencies. Expression of Cdx-1 and Cdx-2 mRNA was verified by RT-PCR. To assess the effect of Cdx-1 and Cdx-2 on endogenous CCL25 mRNA expression, mICc12 cells were transfected with 1.5 μg of pcDNA3 Cdx-1, 1.5 μg of pcDNA3Cdx-2, or 0.75 μg of both, or 1.5 μg of pcDNA3-EGFP or of pcDNA3 as controls. Stable clones were selected in the presence of 250 μg/ml G-418 (Sigma-Aldrich) for 12 days. The percentage of pcDNA3-EGFP transfected cells expressing enhanced GFP after this time ranged from 65 to 85%. CCL25 and GAPDH mRNA copy number were determined as earlier described.

Putative transcription factor binding sites in the CCL25 promoter were identified using Transcription Element Search System (TESS) (〈www.cbil.upenn.edu/tess/〉) and MatInspector (〈www.genomatix.de/cgi-bin/matinspector/matinspector.pl〉). All reported hits were sorted by their matrix similarity (for TESS according to TRANSFAC and for MatInspector according to MatInspector matrices). All hits with a threshold over 0.8 were considered relevant. Factors predicted to interact with both human and murine sequences were checked for their predicted tissue distribution using Geneatlas (〈www.dsi.univ-paris5.fr/genatlas/〉), and transcription factors limited to cells of hemopoietic origin were excluded. The alignment of the human and mouse core promoter sequence was performed using ClustalX (26).

The following dsDNA oligonucleotides were used: Cdx 5′-TCTGAC TATAAAGAATGAAGCC-3′ and mutant Cdx 5′-TCTGACTGGG GAGAATGAAGCC-3′. The Cdx probe was 32P-labeled using T4 Polynucleotide kinase (Invitrogen Life Technologies) according to the manufacturer’s instructions. EMSA was performed in 15 μl of binding buffer (20 mM phosphate buffer (pH 6), 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Nonidet P-40, 0.1 mM NaCl, 100 μg/ml BSA, 4% Ficoll) containing 5–10 μg of nuclear protein extract, 2 μg of poly(dI:dC), and 20,000–30,000 cpm of 32P-labeled DNA probe. Reactions were allowed to proceed for 30 min at room temperature. Complexes were separated on a 6% nondenaturing polyacrylamide gel, and the gels were dried and analyzed by autoradiography. For competition experiments, a 200-fold molar excess of unlabelled Cdx or mutant Cdx oligonucleotide was added before the addition of labeled probe. For Ab blocking experiments, 2 μg of rabbit polyclonal anti-mouse Cdx Ab (CeMines) or 2 μg of rabbit serum (DakoCytomation) as control was added, and samples were incubated for 30 min at room temperature before the addition of probe.

Statistical analysis was performed using the Student’s unpaired t test with GraphPad InStat software.

CCL25 mRNA was constitutively and selectively expressed in the murine small intestine and thymus (Fig. 1,A) consistent with previous reports (9, 12, 14). In situ hybridization studies have demonstrated that epithelial cells constitutively express CCL25 mRNA (10, 12), and immunohistochemical staining of small intestinal sections with anti-CCL25 Ab showed predominant staining in small intestinal epithelial cells (Fig. 1,B) (20). To determine the contribution of epithelial cells to total CCL25 mRNA levels in the murine small intestine, epithelial cells were removed from small intestinal tissue with EDTA, and CCL25 mRNA expression assessed by quantitative real-time RT-PCR. Hematoxylin staining of small intestinal sections confirmed that villous and crypt epithelium were effectively removed by this procedure (Fig. 1,C). Small intestinal epithelial cells expressed CCL25 mRNA, whereas CCL25 mRNA was barely detected in intestinal tissue devoid of epithelial cells (Fig. 1,D). FACS sorted CD8+ intraepithelial lymphocytes (IEL) failed to express CCL25 mRNA (data not shown), excluding the possibility that any contaminating IEL in epithelial cell preparations are a significant source of CCL25 mRNA. CCL25 mRNA was expressed at high levels by both crypt and villous epithelium (Fig. 1, E and F). Previous immunohistochemical studies have suggested that lamina propria microvascular endothelial cells are a potential additional source of intestinal CCL25 (11, 20). However, CCL25 mRNA was not detected in laser capture microscopy samples taken from the lamina propria of EDTA-treated small intestinal tissue that contained microvascular endothelial cells (as assessed by a positive signal for MadCAM-1 mRNA) (Fig. 1 G), indicating that CCL25 detected on these cells may derive from epithelial cells. Indeed, cellular presentation of exogenous-derived chemokines has been previously described in other systems (27, 28, 29).

FIGURE 1.

Epithelial cells are the major source of CCL25 mRNA in the murine small intestine. A, CCL25 mRNA expression in different tissues as assessed by real-time RT-PCR. Data are mean ± SEM (n = 3–4 mice). B, CCL25 protein expression in small intestinal epithelium. Small intestinal tissue sections were stained with an anti-CCL25 or isotype control Ab, and cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole. C, Hematoxylin staining of murine proximal small intestine (PSI) before and after treatment with EDTA. Location of crypt (c) and villous (v) epithelial cells (∗) are indicated. These cells are not present in EDTA-treated tissue (arrow). D, CCL25 mRNA expression in the intestine. Intestinal epithelial cells (IEC), proximal small intestine (PSI), and proximal small intestine after treatment with EDTA (PSI + EDTA) are shown. CCL25 mRNA expression was determined by real-time RT-PCR. Data are mean (±SEM) for n = 3 mice. E, Epithelial cells from crypt and villous region were isolated from small intestinal sections by laser capture microscopy. F, CCL25 mRNA expression in crypt and villous epithelium as assessed by real-time RT-PCR. Results from one representative experiment of two performed. G, CCL25 mRNA expression in laser capture microscopy samples from EDTA-treated small intestinal tissue. cDNA derived from laser capture isolated lamina propria (lane 1); small intestine cDNA (lane 2); dH2O as template (lane 3) are shown. MadCAM-1 is expressed by microvascular endothelial cells, and intestinal alkaline phosphatase (IAP) is selectively expressed by small intestinal epithelial cells.

FIGURE 1.

Epithelial cells are the major source of CCL25 mRNA in the murine small intestine. A, CCL25 mRNA expression in different tissues as assessed by real-time RT-PCR. Data are mean ± SEM (n = 3–4 mice). B, CCL25 protein expression in small intestinal epithelium. Small intestinal tissue sections were stained with an anti-CCL25 or isotype control Ab, and cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole. C, Hematoxylin staining of murine proximal small intestine (PSI) before and after treatment with EDTA. Location of crypt (c) and villous (v) epithelial cells (∗) are indicated. These cells are not present in EDTA-treated tissue (arrow). D, CCL25 mRNA expression in the intestine. Intestinal epithelial cells (IEC), proximal small intestine (PSI), and proximal small intestine after treatment with EDTA (PSI + EDTA) are shown. CCL25 mRNA expression was determined by real-time RT-PCR. Data are mean (±SEM) for n = 3 mice. E, Epithelial cells from crypt and villous region were isolated from small intestinal sections by laser capture microscopy. F, CCL25 mRNA expression in crypt and villous epithelium as assessed by real-time RT-PCR. Results from one representative experiment of two performed. G, CCL25 mRNA expression in laser capture microscopy samples from EDTA-treated small intestinal tissue. cDNA derived from laser capture isolated lamina propria (lane 1); small intestine cDNA (lane 2); dH2O as template (lane 3) are shown. MadCAM-1 is expressed by microvascular endothelial cells, and intestinal alkaline phosphatase (IAP) is selectively expressed by small intestinal epithelial cells.

Close modal

Together, these results demonstrate that small intestinal epithelial cells are the major if not the sole source of the CCL25 mRNA in the murine small intestine.

Constitutive expression of homeostatic chemokines in secondary lymphoid organs and the small intestine is regulated by members of the LT/TNF family of cytokines (3, 4, 5). To determine whether these factors are required for high CCL25 mRNA in the small intestine, CCL25 mRNA expression was examined in the small intestine of LTα−/−, LTβ−/−, or TNFR1−/− mice. CXCL13 mRNA expression was reduced in the small intestine of LTα−/− and LTβ−/− mice (data not shown), consistent with a previous report (5). In contrast, CCL25 mRNA was expressed at similar levels in the small intestine of LTα−/−, LTβ−/−, and TNFR1−/− mice as in wild-type mice (Fig. 2,A). To determine whether intestinal CCL25 mRNA was enhanced after exposure to inflammatory stimuli, mice were injected i.v. with LPS and intestinal CCL25 mRNA levels determined 3 h later. This procedure has been shown to induce expression of multiple chemokine mRNA species in the small intestine, including CCL20, a chemokine constitutively expressed by small intestinal epithelial cells (30, 31). As expected, injection of LPS i.v. enhanced levels of CCL20 mRNA in the murine small intestine (Fig. 2,B); however, intestinal CCL25 mRNA levels remained unchanged (Fig. 2,B). In a second set of experiments, we determined whether proinflammatory cytokines could enhance CCL25 mRNA expression in the small intestinal epithelial cell lines MODE-K, S1-H10, or mICcl2. TNF-α and IFN-γ were chosen because these cytokines enhance inflammatory chemokine expression in a wide range of intestinal epithelial lines including MODE-K cells (32, 33, 34, 35). Addition of TNF-α (10–100 ng/ml) or IFN-γ (10–500 U/ml) alone to confluent MODE-K, S1-H10, or mICcl2 epithelial cell layers for 6 and 24 h failed to alter CCL25 mRNA levels (data not shown). Furthermore, whereas TNF-α and IFN-γ enhanced CXCL1 expression (Fig. 2,C) as previously described (33), this cytokine combination failed to enhance CCL25 mRNA expression (Fig. 2 C). Similarly these cytokines failed to induce CCL25 mRNA expression in human HT-29 and Caco-2 colonic epithelial lines, and FHs 74 Int cells (CCL-241; American Type Culture Collection), a morphologically epithelial-like cell line derived from the human small intestine (data not shown). Together, these results indicate that transcriptional regulation of CCL25 mRNA is unique compared with that of other homeostatic or inflammatory chemokines.

FIGURE 2.

Intestinal CCL25 mRNA expression is independent of LT and TNFR signaling, inflammatory stimuli, and the presence of intestinal microflora or lymphocytes. A, CCL25 mRNA levels in whole proximal small intestine of LTα−/− and LTβ−/− and TNFR1−/− mice as assessed by real-time RT-PCR. Results are the mean (±SEM) of three mice per group. B, LPS fails to enhance CCL25 mRNA expression in the murine small intestine. CCL20 and CCL25 mRNA levels were determined by semiquantitative and real-time RT-PCR, respectively, 3 h after administration of 200 μg of LPS (Escherichia coli, serotype O55:B5; Sigma-Aldrich) i.v. into C57BL/6 mice. For real-time RT-PCR results are the mean (±SEM) of three mice. For semiquantitative PCR, cDNA was serially diluted 1/10, and results are representative of three mice per group. C, TNF-α and IFN-γ fail to enhance CCL25 mRNA expression in MODE-K cells. MODE-K cells were stimulated with TNF-α (100 ng/ml; PeproTech) and IFN-γ (500 U/ml; PeproTech) for 24 h and CXCL1 and CCL25 mRNA expression determined by semiquantitative and real-time RT-PCR, respectively. For real-time RT-PCR results are the mean (±SEM) of triplicate wells and from one representative experiment of three performed. For semiquantitative PCR, cDNA was serially diluted 1/10. Results are from one representative experiment of three performed. D, CCL25 mRNA expression in whole small intestine of athymic mice with a truncated common cytokine receptor γ-chain (CRγ−/−) and germfree mice as determined by real-time RT-PCR. Results are representative from three mice per group (germfree and conventional (convent.)) or from three intestinal pieces from different sites along the small intestine (athymic CRγ−/− mice).

FIGURE 2.

Intestinal CCL25 mRNA expression is independent of LT and TNFR signaling, inflammatory stimuli, and the presence of intestinal microflora or lymphocytes. A, CCL25 mRNA levels in whole proximal small intestine of LTα−/− and LTβ−/− and TNFR1−/− mice as assessed by real-time RT-PCR. Results are the mean (±SEM) of three mice per group. B, LPS fails to enhance CCL25 mRNA expression in the murine small intestine. CCL20 and CCL25 mRNA levels were determined by semiquantitative and real-time RT-PCR, respectively, 3 h after administration of 200 μg of LPS (Escherichia coli, serotype O55:B5; Sigma-Aldrich) i.v. into C57BL/6 mice. For real-time RT-PCR results are the mean (±SEM) of three mice. For semiquantitative PCR, cDNA was serially diluted 1/10, and results are representative of three mice per group. C, TNF-α and IFN-γ fail to enhance CCL25 mRNA expression in MODE-K cells. MODE-K cells were stimulated with TNF-α (100 ng/ml; PeproTech) and IFN-γ (500 U/ml; PeproTech) for 24 h and CXCL1 and CCL25 mRNA expression determined by semiquantitative and real-time RT-PCR, respectively. For real-time RT-PCR results are the mean (±SEM) of triplicate wells and from one representative experiment of three performed. For semiquantitative PCR, cDNA was serially diluted 1/10. Results are from one representative experiment of three performed. D, CCL25 mRNA expression in whole small intestine of athymic mice with a truncated common cytokine receptor γ-chain (CRγ−/−) and germfree mice as determined by real-time RT-PCR. Results are representative from three mice per group (germfree and conventional (convent.)) or from three intestinal pieces from different sites along the small intestine (athymic CRγ−/− mice).

Close modal

The expression of CCL25 mRNA in the murine small intestine is increased between 2 and 3 wk of age (17), a time point correlating with increased numbers of IEL (17) and bacterial colonization of the intestine. Because the small intestinal epithelium is in intimate contact with intestinal microflora and IEL, we determined whether the presence of these components was important in maintaining high constitutive CCL25 expression in the murine small intestine. CCL25 mRNA was expressed at similar levels in the small intestine of athymic mice with a truncated common cytokine receptor γ-chain (CRγ−/Ynu/nu) (Fig. 2,D), which contains a negligible TCR IEL population (36). Furthermore, coincubation of confluent MODE-K monolayers for 6 h with 50–500,000 freshly isolated syngeneic CD8+ IEL failed to enhance epithelial CCL25 expression (data not shown). Finally, CCL25 mRNA was expressed at similar levels in the small intestine of germfree and conventional mice (Fig. 2 D). Thus constitutive CCL25 mRNA expression in the murine small intestine is independent on interactions with mature IEL, signaling through the cytokine receptor γ-chain or the presence of intestinal bacteria.

The findings suggesting that CCL25 expression is regulated in a manner different from other chemokines motivated us to search for regulatory elements involved in the transcriptional regulation of this gene. To identify the putative CCL25 transcriptional start site, 5′ RACE was performed on cDNA prepared from small intestinal epithelial cells (Fig. 3,A). Seven clones obtained from 5′ RACE were sequenced, three of which showed identity to clone A, three to clone B, and one to clone C (Fig. 3,A). These transcripts are a few base pairs longer at the 5′ end as compared with the presumed full-length CCL25 mRNA transcript previously described in mouse thymus (GenBank accession number NM_009138). Analysis of expressed sequence tags obtained in the mouse (GenBank) showed good agreement with these sequences (data not shown). Eight clones obtained from 3′ RACE were sequenced, four of which showed identity to clone 1 and four to clone 2 (Fig. 3,A). Thus, murine CCL25 mRNA appears to have two alternative polyadenylation sites. Having identified mRNA ends of the murine CCL25 message, we determined the genomic organization of the murine and human CCL25 gene (Fig. 3,B). Both murine and human CCL25 consists of six exons covering 10.5 and 9.9 kb of genomic DNA, respectively. Exon 1 of human CCL25 that was missing from the human CCL25 mRNA sequence (NM_005624) was identified from expressed sequence tags derived from the human small intestine (BX415301) (Fig. 3 B).

FIGURE 3.

Genomic structure and transcriptional start site of the mouse CCL25 gene. A, The transcriptional start site of the murine CCL25 transcript in small intestinal epithelial cells was identified by 5′ RACE. The 3′ RACE identified two different polyadenylation sites separated by 17 bp. The two sites of poly(A) addition (∗) are indicated. The sequenced CCL25 cDNA has been deposited (GenBank accession number DQ158256). B, Schematic genomic organization of the mouse (GenBank accession number NT_039455.2) and human (NM_005624 and BX415301) CCL25 gene locus. The exons are presented as boxes and numbered with roman numerals (I-VI) and the introns are numbered using Arabic numerals (1–5). Indicates translated (▪) and untranslated (□) regions.

FIGURE 3.

Genomic structure and transcriptional start site of the mouse CCL25 gene. A, The transcriptional start site of the murine CCL25 transcript in small intestinal epithelial cells was identified by 5′ RACE. The 3′ RACE identified two different polyadenylation sites separated by 17 bp. The two sites of poly(A) addition (∗) are indicated. The sequenced CCL25 cDNA has been deposited (GenBank accession number DQ158256). B, Schematic genomic organization of the mouse (GenBank accession number NT_039455.2) and human (NM_005624 and BX415301) CCL25 gene locus. The exons are presented as boxes and numbered with roman numerals (I-VI) and the introns are numbered using Arabic numerals (1–5). Indicates translated (▪) and untranslated (□) regions.

Close modal

To determine whether the region upstream of the predicted CCL25 transcriptional start site contained promoter activity, we generated a series of constructs comprising regions surrounding exon 1 (Fig. 4,A) fused to a luciferase reporter gene. Next we attempted to identify a cell line that constitutively expressed high levels of CCL25 mRNA for use in transfection studies. Screening of a wide range of murine and human epithelial cell lines including MODE-K, mICc12, S1-H10, HT-29, FHs 74 Int, Caco-2, and T-84 cells failed to identify any cell line constitutively expressing CCL25 mRNA levels comparable to that of freshly isolated epithelial cells (Fig. 4,B and data not shown). Furthermore freshly isolated epithelial cells showed a dramatic reduction in CCL25 mRNA expression after culture (Fig. 4,B). We therefore determined whether any of the constructs displayed basal promoter activity in murine MODE-K and mICc12 cells. The murine fibroblast line BALB/c 3T3 was used as a control cell line because these cells failed to express full-length CCL25 mRNA as assessed by RT-PCR using a 5′ primer in exon 1 and 3′ primer in exon 2 of CCL25 (data not shown). Constructs PF1 to PF3 increased luciferase activity ∼20 times compared with the control in MODE-K and mICcl2 cells (Fig. 4,C), but not in BALB/c 3T3 cells. Thus the area upstream of exon 1 contains the minimal CCL25 promoter, and intron 1 does not appear to contribute to promoter activity in these cell lines. Construct PF4 consistently induced greater luciferase expression compared with PF2, indicating that the region −583 to −311 contained elements that repressed promoter activity in these cell lines. In addition, PF6 failed to induce luciferase expression, demonstrating that a region between −45 and −167 is critical for promoter activity in these cells. Consistent with this finding PF7 covering region −141 to −5 had strong promoter activity, whereas PF8, covering region −99 to −5, and PF9, covering region −49 to −5, showed poor promoter activity (Fig. 4,C). Constructs containing region −141 to −99 alone or region −141 to −99 fused to region −49 to −5 showed no promoter activity (data not shown). Together these results demonstrate that the region upstream of exon 1 of the CCL25 gene contains the minimal murine CCL25 promoter including both activating and repressive elements (Fig. 4 D).

FIGURE 4.

Identification of the CCL25 minimal promoter. A, Schematic representation of the promoter constructs, their names, length and position in respect to the first and second exon of the mouse CCL25 gene. B, CCL25 mRNA expression in freshly isolated murine small intestinal epithelial cells (SIEC), MODE-K and mICcl2 cells, and in primary small intestinal epithelial cells before and after culture as assessed by real-time RT-PCR. Results are representative from one experiment of three performed for the cell lines and from one experiment of two performed for cultured epithelial cells. C, Nine constructs of varying length were transiently transfected into mICc12, MODE-K, and BALB/3T3 cells. The empty plasmid pGL3-basic was used as a negative control and the cotransfected Renilla plasmid was used for standardization. Data are mean ± SD from one representative experiment of three to six performed. ∗, p < 0.05, ∗∗, p < 0.01 and ∗∗∗, p < 0.001. D, Summary schematic of the CCL25 promoter depicting positive (+) and negative (−) regulatory elements.

FIGURE 4.

Identification of the CCL25 minimal promoter. A, Schematic representation of the promoter constructs, their names, length and position in respect to the first and second exon of the mouse CCL25 gene. B, CCL25 mRNA expression in freshly isolated murine small intestinal epithelial cells (SIEC), MODE-K and mICcl2 cells, and in primary small intestinal epithelial cells before and after culture as assessed by real-time RT-PCR. Results are representative from one experiment of three performed for the cell lines and from one experiment of two performed for cultured epithelial cells. C, Nine constructs of varying length were transiently transfected into mICc12, MODE-K, and BALB/3T3 cells. The empty plasmid pGL3-basic was used as a negative control and the cotransfected Renilla plasmid was used for standardization. Data are mean ± SD from one representative experiment of three to six performed. ∗, p < 0.05, ∗∗, p < 0.01 and ∗∗∗, p < 0.001. D, Summary schematic of the CCL25 promoter depicting positive (+) and negative (−) regulatory elements.

Close modal

To identify potential transcriptional factors that may contribute to the high CCL25 expression in normal small intestinal epithelial cells, we examined the CCL25 promoter for putative transcription factor binding sites. A consensus TATA box could be identified ∼25 bp upstream of the transcriptional start site (Fig. 5). Additional binding sites were predicted for a number of transcription factors, including Cdx-1 and Cdx-2, Krueppel-like factor, GATA, MAZ, and TFII-I (Fig. 5 and data not shown). Binding sites for IRF-3 and classical NF-κB dimers, such as RelA:p50 implicated in the induction of inflammatory chemokines, or binding sites for nonclassical NF-κB dimers RelB:p52, implicated in driving homeostatic chemokine gene expression (7, 37, 38, 39, 40, 41, 42, 43, 44, 45), were not detected within a region covering −2000 to +25 bp of the CCL25 promoter, supporting the notion that these factors are not directly involved in the regulation of CCL25 expression.

FIGURE 5.

Predicted transcription factor binding sites in the mouse CCL25 promoter. Alignment of the core promoter regions of the mouse and human CCL25 gene. Conserved nucleotides (∗) and mouse exons (underlined) are indicated, the transcriptional start site is marked (+1), and putative relevant transcription factor binding sites are shown in italic and labeled. Restriction enzyme sites are shown in bold italic and labeled.

FIGURE 5.

Predicted transcription factor binding sites in the mouse CCL25 promoter. Alignment of the core promoter regions of the mouse and human CCL25 gene. Conserved nucleotides (∗) and mouse exons (underlined) are indicated, the transcriptional start site is marked (+1), and putative relevant transcription factor binding sites are shown in italic and labeled. Restriction enzyme sites are shown in bold italic and labeled.

Close modal

The presence of putative Cdx-1 and Cdx-2 binding sites within the murine CCL25 promoter was of interest because Cdx-1 and Cdx-2 expression is restricted to the gut epithelium in adult mice (46), and these transcription factors have been implicated in regulating expression of intestinal specific genes (47, 48, 49, 50, 51, 52). Importantly, although freshly isolated small intestinal epithelial cells expressed Cdx-1 and Cdx-2 mRNA (Fig. 6,A), MODE-K and mICcl2 cells, and cultured primary small intestinal epithelial failed to express Cdx mRNA (Fig. 6). Thus expression of Cdx mRNA correlated with the cells ability to express high levels of CCL25 mRNA.

FIGURE 6.

Small intestinal epithelial cell lines and cultured primary epithelial cells express reduced levels of Cdx. Cdx-1 and Cdx-2 expression in small intestinal epithelial cells (SIEC) (A) and in mICcl2 and MODE-K cells (B), as determined by RT-PCR. Results are representative of one experiment from two performed for the cultured cells and one from three performed on the cell lines.

FIGURE 6.

Small intestinal epithelial cell lines and cultured primary epithelial cells express reduced levels of Cdx. Cdx-1 and Cdx-2 expression in small intestinal epithelial cells (SIEC) (A) and in mICcl2 and MODE-K cells (B), as determined by RT-PCR. Results are representative of one experiment from two performed for the cultured cells and one from three performed on the cell lines.

Close modal

To determine whether small intestinal Cdx protein could interact with Cdx binding motifs within the CCL25 promoter, nuclear extracts were prepared from primary small intestinal epithelial cells, MODE-K and mICcl2 cells, and incubated with a 32P-labeled probe covering the Cdx-binding site predicted within the TATA box (Fig. 5). A single complex was observed when labeled Cdx probe was incubated with nuclear extracts from primary small intestinal epithelial cells, and this complex was competed away with unlabeled probe, but not unlabeled mutant probe (Fig. 7,A). The complex formation was also inhibited by the addition of anti-Cdx, but not control Ab, to the nuclear extracts before addition of labeled probe (Fig. 7,A). In contrast, this complex was not observed using nuclear extracts from MODE-K cells (Fig. 7 A), mICcl2 cells, or the thymocyte cell line 2017 (data not shown) (53). Thus, Cdx is present in freshly isolated epithelial cells, but not MODE-K or mICcl2 cells, and epithelial cell derived Cdx can bind to the putative Cdx binding motif within the TATA box of the CCL25 promoter.

FIGURE 7.

Cdx proteins bind to the CCL25 promoter and enhance promoter activity in small intestinal epithelial cell lines. A, EMSA is performed using nuclear protein extracts from small intestinal epithelial cells (SIEC) and MODE-K cells as indicated. Labeled probe and nuclear extract only (lanes 1 and 6), with excess of unlabeled probe (lanes 2 and 7), with excess of mutant probe (lanes 3 and 8), anti-Cdx Ab (lanes 4 and 9), and control Ab (lanes 5 and 10). Results are representative from one experiment of three performed. Cotransfection of Cdx-2 (pTREtightCdx-2) (B) or Cdx-1 (C) together with the pGL3b-PF1 or pGL3b-PF7 increased luciferase activity in MODE-K cells. Results are from one representative experiment of three performed. Bars indicate mean ± SEM. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. D, Transfection of Cdx-2, or Cdx-1 and Cdx-2 enhanced the endogenous CCL25 transcript level. Stable transfectants of mICc12 cells expressing Cdx-2, Cdx-1, or both were generated as described in Materials and Methods. The endogenous CCL25 mRNA level was determined by quantitative real-time PCR. Data are mean ± SEM (n = 3–4 wells). ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 7.

Cdx proteins bind to the CCL25 promoter and enhance promoter activity in small intestinal epithelial cell lines. A, EMSA is performed using nuclear protein extracts from small intestinal epithelial cells (SIEC) and MODE-K cells as indicated. Labeled probe and nuclear extract only (lanes 1 and 6), with excess of unlabeled probe (lanes 2 and 7), with excess of mutant probe (lanes 3 and 8), anti-Cdx Ab (lanes 4 and 9), and control Ab (lanes 5 and 10). Results are representative from one experiment of three performed. Cotransfection of Cdx-2 (pTREtightCdx-2) (B) or Cdx-1 (C) together with the pGL3b-PF1 or pGL3b-PF7 increased luciferase activity in MODE-K cells. Results are from one representative experiment of three performed. Bars indicate mean ± SEM. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. D, Transfection of Cdx-2, or Cdx-1 and Cdx-2 enhanced the endogenous CCL25 transcript level. Stable transfectants of mICc12 cells expressing Cdx-2, Cdx-1, or both were generated as described in Materials and Methods. The endogenous CCL25 mRNA level was determined by quantitative real-time PCR. Data are mean ± SEM (n = 3–4 wells). ∗, p < 0.05; ∗∗, p < 0.01.

Close modal

To determine whether Cdx could enhance the activity of the CCL25 promoter in epithelial cell lines, the PF1 CCL25 promoter construct or the PF7 CCL25 promoter construct (which contained a single Cdx binding site corresponding to that predicted within the TATA box) were cotransfected with expression plasmids encoding Cdx-1 or Cdx-2 into MODE-K cells. In initial experiments functionality of the Cdx expression plasmids in MODE-K cells was confirmed using a promoter construct containing a TATA box proceeded by three consensus Cdx binding sites or three mutant Cdx sites, as a negative control, upstream of the luciferase gene. Cotransfection with the Cdx expression plasmids leads to a 3-fold increase in luciferase activity from the reporter construct containing 3 CdxA consensus sites but not from the mutant reporter (data not shown). MODE-K cells transfected with constructs encoding either Cdx-2 or Cdx-1 significantly enhanced CCL25 promoter activity compared with their relevant controls (Fig. 7, B and C). In contrast neither Cdx expression construct enhanced CCL25 promoter activity in BALB/c 3T3 cells (data not shown). Furthermore, stable ectopic expression of Cdx-2, or the combination of Cdx-1 and Cdx-2 in mICc12 cells, leads to an increase in endogenous CCL25 mRNA expression in these cells (Fig. 7 D). Together these results suggest a role for Cdx transcription factors in enhancing CCL25 mRNA transcription in small intestinal epithelial cells; however, because transfection with Cdx failed to enhance CCL25 mRNA expression to levels observed in freshly isolated small intestinal epithelial cells, they also suggest that other factors in addition to Cdx are responsible for maintaining the high constitutive expression of CCL25 in these cells.

Despite the importance of CCL25 in small intestinal immunity (14, 15, 16, 17, 18, 19, 20), the mechanisms underlying the tissue selective expression and regulation of this chemokine in the small intestine are unknown. In the present study we demonstrate that epithelial cells are the major source of CCL25 mRNA in the small intestine, and that constitutive CCL25 mRNA expression is independent of the presence of intestinal bacteria and lymphocytes. CCL25 expression was not regulated by the LTRβ and TNFR1 signaling pathways and was not enhanced by inflammatory mediators suggesting a unique pattern of regulation compared with homeostatic and inflammatory chemokines, respectively. The CCL25 promoter contained several putative binding sites for Cdx transcription factors and primary small intestinal epithelial cells that expressed high CCL25 mRNA levels expressed Cdx-1 and Cdx-2, whereas epithelial cell lines and cultured primary epithelial cells that expressed low levels of CCL25 mRNA failed to express Cdx-1 and Cdx-2. Finally EMSA and transfection studies suggested a role for Cdx transcription factors in contributing to the high CCL25 expression levels in small intestinal epithelial cells.

Chemokines have been broadly separated into homeostatic and inflammatory chemokines with partially distinct mechanisms of regulation. The LTα1β2 heterotrimer binds to the LTβR (3), signaling through which is required for maintaining constitutive expression of the homeostatic chemokines CXCL12, CCL21, CCL19, and CXCL13 in lymph nodes, spleen, and intestine (4, 5) as well as the epithelial-derived chemokine CCL20 (37). LTβR mediated expression of homeostatic chemokines functions through the alternative NF-κB pathway, involving translocation of RelB:p52 dimers into the nucleus (44). In this study we show that in contrast to homeostatic chemokine promoters (37, 43, 44, 54), putative binding sites for RelB:p52 dimers are not present in the CCL25 promoter and that small intestinal CCL25 mRNA expression is independent of LTα and LTβ signaling. In addition, transcriptional binding sites for NF-κB and IRF-3, both of which are involved in the induction of inflammatory chemokines (40, 45, 54), were not present in the CCL25 promoter, and intestinal epithelial CCL25 mRNA levels remained unaltered in two inflammatory models known to induce epithelial expression of inflammatory chemokines (30, 31, 32, 33, 34, 35). This inability of inflammatory mediators to induce CCL25 mRNA expression, is in apparent odds with the enhanced CCL25 expression reported in small intestinal crypts of Crohn’s disease patients in areas of lymphocytic infiltration; however, no quantitative data were presented in this study (55). Together, our results demonstrate that CCL25 is not regulated as other homeostatic or inflammatory chemokines.

We were unable to identify a cell line that could model freshly isolated small intestinal epithelial cells in their levels of CCL25 mRNA expression. Nevertheless constructs encompassing the area immediately upstream of exon 1 of the CCL25 gene showed promoter activity in MODE-K and mICc12 cells. Constructs lacking region −311 to −511 showed enhanced promoter activity, indicating that this area contained potential suppressor elements. We were also able to identify important activating elements required for minimal promoter activity in these cells. A region between −45 to −167 appeared critical for minimal promoter activity, and constructs containing region −99 to −141 had significantly higher promoter activity than constructs lacking this sequence. Cdx-1 and Cdx-2 are clearly not involved in driving this activity because MODE-K and mICc12 cells failed to express these transcription factors and the region −99 to −141 contained no Cdx binding sites. However, two binding sites for the TFII family of general transcription factors, which make up part of the initiation complex involved in gene transcription (56), were located within this region and are thus likely to be critical for minimal promoter activity in these cells.

The far higher levels of CCL25 mRNA transcription in primary small intestinal epithelial cells compared with epithelial cell lines suggested that critical components required for driving CCL25 promoter activity were missing or not functional within the cell lines. Furthermore, because cultured primary small intestinal epithelial cells showed reduced CCL25 transcription, the intestinal environment appears important in maintaining the expression and/or activity of these components. Several results from the current study suggest that Cdx transcription factors are one such component. Firstly analysis of the CCL25 promoter predicted several binding sites for Cdx proteins. Secondly, Cdx was expressed by primary small intestinal epithelial cells but not cultured primary epithelial cells or epithelial cell lines. Thirdly, Cdx present in nuclear extracts from freshly isolated small intestinal epithelial cells, which has previously been shown to interact with TATA boxes of several intestinal specific genes (49, 50, 51, 52), bound to the Cdx site predicted in the CCL25 TATA box. Finally, transfection of Cdx expression plasmids caused a significant enhancement CCL25 promoter construct activity in MODE-K and mICc12 cells and enhanced CCL25 mRNA expression in mICc12 cells.

Nevertheless, because transfection of epithelial cell lines with Cdx expression plasmids failed to induce CCL25 mRNA expression to the levels observed in small intestinal epithelial cells, additional factors other than Cdx must be involved in maintaining high CCL25 transcription levels in these cells. Consistent with this, Cdx transcription factors are expressed by small intestinal and colonic epithelium (46), and colonic epithelial cells express ∼50 times less CCL25 mRNA than small intestinal epithelial cells (Fig. 1 A). The mechanisms driving selective gene expression in the small intestine vs colon are poorly understood. For the small intestinal specific gene sucrase isomaltase, tissue selectivity is achieved through the combination of HNF-1α, GATA-4, and Cdx-2 (48), together with active suppression of this gene in the colon (57). Promoter analysis using MatInspector predicted a binding site for this repressor (CLOX/CDP) within the CCL25 promoter. Similarly, regulation of the chemokine CCL20 in colonic epithelial cells is obtained through the combined effects of NF-κB, Sp1, and ESE-1, an enterocyte specific transcription factor (42). In this regard, the CCL25 promoter contains putative binding sites for Krueppel-like factors, a family of transcription factors implicated in driving expression of intestinal genes (58), indicating that CCL25 may belong to an intestine restricted gene battery. We also detected potential binding sites for broadly expressed transcription factors such as GATA, MAZ, Sp1, and TFII-I (56, 59, 60, 61, 62, 63) and although they are unlikely to directly participate in the tissue specific gene regulation of CCL25, they may play important roles in overall promoter function. Of note, CCL25 was recently identified as an Egr-1 target gene in human endothelial cells after expression of this transcriptional activator in these cells (64). However, we failed to identify Egr-1 binding sites within the CCL25 promoter indicating that this effect may be mediated indirectly through the activation of additional transcriptional activators.

In conclusion, our results demonstrate that CCL25 displays a unique regulation pattern for chemokines, and points to a complex network of transcription factors, involving Cdx, responsible for maintaining the high and tissue selective expression of CCL25 in the murine small intestinal epithelium.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the Swedish Medical Research Council; the Crafoordska, Österlund, Åke Wiberg, Richard and Ruth Julins, Nanna Svartz and Kocks Foundations; the Swedish Medical Society; the Royal Physiographic Society; the Swedish Foundation for Strategic Research “Microbes and Man” program and INGVAR II program; and a Crohns and Colitis Foundation of America project grant (to W.A.). K.K. is supported by a SWEGENE Postdoctoral Fellowship.

4

Abbreviations used in this paper: LT, lymphotoxin; IEL, intraepithelial lymphocyte.

1
Kunkel, E. J., D. J. Campbell, E. C. Butcher.
2003
. Chemokines in lymphocyte trafficking and intestinal immunity.
Microcirculation
10
:
313
-323.
2
Moser, B., M. Wolf, A. Walz, P. Loetscher.
2004
. Chemokines: multiple levels of leukocyte migration control.
Trends Immunol.
25
:
75
-84.
3
Mebius, R. E..
2003
. Organogenesis of lymphoid tissues.
Nat. Rev. Immunol.
3
:
292
-303.
4
Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, J. L. Browning, J. D. Sedgwick, J. G. Cyster.
1999
. Lymphotoxin αβ and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen.
J. Exp. Med.
189
:
403
-412.
5
Kang, H. S., R. K. Chin, Y. Wang, P. Yu, J. Wang, K. A. Newell, Y. X. Fu.
2002
. Signaling via LTβR on the lamina propria stromal cells of the gut is required for IgA production.
Nat. Immunol.
3
:
576
-582.
6
Ogawa, H., M. Iimura, L. Eckmann, M. F. Kagnoff.
2004
. Regulated production of the chemokine CCL28 in human colon epithelium.
Am. J. Physiol.
287
:
G1062
-G1069.
7
Homey, B., H. Alenius, A. Muller, H. Soto, E. P. Bowman, W. Yuan, L. McEvoy, A. I. Lauerma, T. Assmann, E. Bunemann, et al
2002
. CCL27-CCR10 interactions regulate T cell-mediated skin inflammation.
Nat. Med.
8
:
157
-165.
8
Romagnani, P., F. Annunziato, E. Lazzeri, L. Cosmi, C. Beltrame, L. Lasagni, G. Galli, M. Francalanci, R. Manetti, F. Marra, et al
2001
. Interferon-inducible protein 10, monokine induced by interferon γ, and interferon-inducible T-cell α chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) αβ+ CD8+ single-positive T cells, TCRγδ+ T cells, and natural killer-type cells in human thymus.
Blood
97
:
601
-607.
9
Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, A. Zlotnik.
1997
. TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development.
Immunity
7
:
291
-301.
10
Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. I. Roberts, E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, et al
2000
. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.
J. Exp. Med.
192
:
761
-768.
11
Papadakis, K. A., J. Prehn, V. Nelson, L. Cheng, S. W. Binder, P. D. Ponath, D. P. Andrew, S. R. Targan.
2000
. The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system.
J. Immunol.
165
:
5069
-5076.
12
Wurbel, M. A., J. M. Philippe, C. Nguyen, G. Victorero, T. Freeman, P. Wooding, A. Miazek, M. G. Mattei, M. Malissen, B. R. Jordan, et al
2000
. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9.
Eur. J. Immunol.
30
:
262
-271.
13
Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al
1999
. Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis.
J. Exp. Med.
190
:
1241
-1256.
14
Svensson, M., J. Marsal, A. Ericsson, L. Carramolino, T. Broden, G. Marquez, W. W. Agace.
2002
. CCL25 mediates the localization of recently activated CD8αβ+ lymphocytes to the small-intestinal mucosa.
J. Clin. Invest.
110
:
1113
-1121.
15
Wurbel, M. A., M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, B. Malissen.
2001
. Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor γδ+ gut intraepithelial lymphocytes.
Blood
98
:
2626
-2632.
16
Uehara, S., A. Grinberg, J. M. Farber, P. E. Love.
2002
. A role for CCR9 in T lymphocyte development and migration.
J. Immunol.
168
:
2811
-2819.
17
Marsal, J., M. Svensson, A. Ericsson, A. H. Iranpour, L. Carramolino, G. Marquez, W. W. Agace.
2002
. Involvement of CCL25 (TECK) in the generation of the murine small-intestinal CD8α α+CD3+ intraepithelial lymphocyte compartment.
Eur. J. Immunol.
32
:
3488
-3497.
18
Pabst, O., L. Ohl, M. Wendland, M. A. Wurbel, E. Kremmer, B. Malissen, R. Forster.
2004
. Chemokine receptor CCR9 contributes to the localization of plasma cells to the small intestine.
J. Exp. Med.
199
:
411
-416.
19
Johansson-Lindbom, B., M. Svensson, M. A. Wurbel, B. Malissen, G. Márquez, W. Agace.
2003
. Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant.
J. Exp. Med.
198
:
963
-969.
20
Hieshima, K., Y. Kawasaki, H. Hanamoto, T. Nakayama, D. Nagakubo, A. Kanamaru, O. Yoshie.
2004
. CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells.
J. Immunol.
173
:
3668
-3675.
21
Onai, N., M. Kitabatake, Y. Y. Zhang, H. Ishikawa, S. Ishikawa, K. Matsushima.
2002
. Pivotal role of CCL25 (TECK)-CCR9 in the formation of gut cryptopatches and consequent appearance of intestinal intraepithelial T lymphocytes.
Int. Immunol.
14
:
687
-694.
22
Booth, C., J. A. O’Shea, C. S. Potten.
1999
. Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium.
Exp. Cell Res.
249
:
359
-366.
23
Bens, M., A. Bogdanova, F. Cluzeaud, L. Miquerol, S. Kerneis, J. P. Kraehenbuhl, A. Kahn, E. Pringault, A. Vandewalle.
1996
. Transimmortalized mouse intestinal cells (m-ICc12) that maintain a crypt phenotype.
Am. J. Physiol.
270
: (6 Pt. 1):
C1666
-C1674.
24
Soubeyran, P., F. Andre, J. C. Lissitzky, G. V. Mallo, V. Moucadel, M. Roccabianca, H. Rechreche, J. Marvaldi, I. Dikic, J. C. Dagorn, J. L. Iovanna.
1999
. Cdx1 promotes differentiation in a rat intestinal epithelial cell line.
Gastroenterology
117
:
1326
-1338.
25
Uesaka, T., N. Kageyama.
2004
. Cdx2 homeodomain protein regulates the expression of MOK, a member of the mitogen-activated protein kinase superfamily, in the intestinal epithelial cells.
FEBS Lett.
573
:
147
-154.
26
Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, T. J. Gibson.
1998
. Multiple sequence alignment with Clustal X.
Trends Biochem. Sci.
23
:
403
-405.
27
Middleton, J., S. Neil, J. Wintle, I. Clark-Lewis, H. Moore, C. Lam, M. Auer, E. Hub, A. Rot.
1997
. Transcytosis and surface presentation of IL-8 by venular endothelial cells.
Cell
91
:
385
-395.
28
Palframan, R. T., S. Jung, G. Cheng, W. Weninger, Y. Luo, M. Dorf, D. R. Littman, B. J. Rollins, H. Zweerink, A. Rot, U. H. von Andrian.
2001
. Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues.
J. Exp. Med.
194
:
1361
-1373.
29
Baekkevold, E. S., T. Yamanaka, R. T. Palframan, H. S. Carlsen, F. P. Reinholt, U. H. von Andrian, P. Brandtzaeg, G. Haraldsen.
2001
. The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.
J. Exp. Med.
193
:
1105
-1112.
30
Tanaka, Y., T. Imai, M. Baba, I. Ishikawa, M. Uehira, H. Nomiyama, O. Yoshie.
1999
. Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans.
Eur. J. Immunol.
29
:
633
-642.
31
Widney, D. P., Y. R. Xia, A. J. Lusis, J. B. Smith.
2000
. The murine chemokine CXCL11 (IFN-inducible T cell α chemoattractant) is an IFN-γ- and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia.
J. Immunol.
164
:
6322
-6331.
32
Song, F., K. Ito, T. L. Denning, D. Kuninger, J. Papaconstantinou, W. Gourley, G. Klimpel, E. Balish, J. Hokanson, P. B. Ernst.
1999
. Expression of the neutrophil chemokine KC in the colon of mice with enterocolitis and by intestinal epithelial cell lines: effects of flora and proinflammatory cytokines.
J. Immunol.
162
:
2275
-2280.
33
Izadpanah, A., M. B. Dwinell, L. Eckmann, N. M. Varki, M. F. Kagnoff.
2001
. Regulated MIP-3α/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity.
Am. J. Physiol.
280
:
G710
-G719.
34
Dwinell, M. B., N. Lugering, L. Eckmann, M. F. Kagnoff.
2001
. Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells.
Gastroenterology
120
:
49
-59.
35
Berin, M. C., M. B. Dwinell, L. Eckmann, M. F. Kagnoff.
2001
. Production of MDC/CCL22 by human intestinal epithelial cells.
Am. J. Physiol.
280
:
G1217
-G1226.
36
Oida, T., K. Suzuki, M. Nanno, Y. Kanamori, H. Saito, E. Kubota, S. Kato, M. Itoh, S. Kaminogawa, H. Ishikawa.
2000
. Role of gut cryptopatches in early extrathymic maturation of intestinal intraepithelial T cells.
J. Immunol.
164
:
3616
-3626.
37
Rumbo, M., F. Sierro, N. Debard, J. P. Kraehenbuhl, D. Finke.
2004
. Lymphotoxin β receptor signaling induces the chemokine CCL20 in intestinal epithelium.
Gastroenterology
127
:
213
-223.
38
Sugita, S., T. Kohno, K. Yamamoto, Y. Imaizumi, H. Nakajima, T. Ishimaru, T. Matsuyama.
2002
. Induction of macrophage-inflammatory protein-3α gene expression by TNF-dependent NF-κB activation.
J. Immunol.
168
:
5621
-5628.
39
Wickremasinghe, M. I., L. H. Thomas, C. M. O’Kane, J. Uddin, J. S. Friedland.
2004
. Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis.
J. Biol. Chem.
279
:
27199
-27210.
40
Ohmori, Y., T. A. Hamilton.
1993
. Cooperative interaction between interferon (IFN) stimulus response element and κB sequence motifs controls IFNγ- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter.
J. Biol. Chem.
268
:
6677
-6688.
41
Matsukura, S., F. Kokubu, H. Kuga, M. Kawaguchi, K. Ieki, M. Odaka, S. Suzuki, S. Watanabe, H. Takeuchi, M. Adachi, et al
2003
. Differential regulation of eotaxin expression by IFN-γ in airway epithelial cells.
J. Allergy Clin. Immunol.
111
:
1337
-1344.
42
Kwon, J. H., S. Keates, S. Simeonidis, F. Grall, T. A. Libermann, A. C. Keates.
2003
. ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3α gene expression in Caco-2 human colonic epithelial cells.
J. Biol. Chem.
278
:
875
-884.
43
Bonizzi, G., M. Bebien, D. C. Otero, K. E. Johnson-Vroom, Y. Cao, D. Vu, A. G. Jegga, B. J. Aronow, G. Ghosh, R. C. Rickert, M. Karin.
2004
. Activation of IKKα target genes depends on recognition of specific κB binding sites by RelB:p52 dimers.
EMBO J.
23
:
4202
-4210.
44
Dejardin, E., N. M. Droin, M. Delhase, E. Haas, Y. Cao, C. Makris, Z. W. Li, M. Karin, C. F. Ware, D. R. Green.
2002
. The lymphotoxin-β receptor induces different patterns of gene expression via two NF-κB pathways.
Immunity
17
:
525
-535.
45
Sakaguchi, S., H. Negishi, M. Asagiri, C. Nakajima, T. Mizutani, A. Takaoka, K. Honda, T. Taniguchi.
2003
. Essential role of IRF-3 in lipopolysaccharide-induced interferon-β gene expression and endotoxin shock.
Biochem. Biophys. Res. Commun.
306
:
860
-866.
46
Duprey, P., K. Chowdhury, G. R. Dressler, R. Balling, D. Simon, J. L. Guenet, P. Gruss.
1988
. A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine.
Genes Dev.
2
:
1647
-1654.
47
Beck, F..
2004
. The role of Cdx genes in the mammalian gut.
Gut
53
:
1394
-1396.
48
Boudreau, F., E. H. Rings, H. M. van Wering, R. K. Kim, G. P. Swain, S. D. Krasinski, J. Moffett, R. J. Grand, E. R. Suh, P. G. Traber.
2002
. Hepatocyte nuclear factor-1α, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription: implication for the developmental regulation of the sucrase-isomaltase gene.
J. Biol. Chem.
277
:
31909
-31917.
49
Gautier-Stein, A., C. Domon-Dell, A. Calon, I. Bady, J. N. Freund, G. Mithieux, F. Rajas.
2003
. Differential regulation of the glucose-6-phosphatase TATA box by intestine-specific homeodomain proteins CDX1 and CDX2.
Nucleic Acids Res.
31
:
5238
-5246.
50
Lambert, M., S. Colnot, E. Suh, F. L’Horset, C. Blin, M. E. Calliot, M. Raymondjean, M. Thomasset, P. G. Traber, C. Perret.
1996
. cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box.
Eur. J. Biochem.
236
:
778
-788.
51
Barley, N. F., S. R. Prathalingam, P. Zhi, S. Legon, A. Howard, J. R. Walters.
1999
. Factors involved in the duodenal expression of the human calbindin-D9k gene.
Biochem. J.
341
:
491
-500.
52
Suh, E., Z. Wang, G. P. Swain, M. Tenniswood, P. G. Traber.
2001
. Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium.
Am. J. Physiol.
280
:
G149
-G156.
53
Petersson, K., F. Ivars, M. Sigvardsson.
2002
. The pTα promoter and enhancer are direct targets for transactivation by E box-binding proteins.
Eur. J. Immunol.
32
:
911
-920.
54
Weih, F., J. Caamano.
2003
. Regulation of secondary lymphoid organ development by the nuclear factor-κB signal transduction pathway.
Immunol. Rev.
195
:
91
-105.
55
Papadakis, K. A., J. Prehn, S. T. Moreno, L. Cheng, E. A. Kouroumalis, R. Deem, T. Breaverman, P. D. Ponath, D. P. Andrew, P. H. Green, et al
2001
. CCR9-positive lymphocytes and thymus-expressed chemokine distinguish small bowel from colonic Crohn’s disease.
Gastroenterology
121
:
246
-254.
56
Roy, A. L..
2001
. Biochemistry and biology of the inducible multifunctional transcription factor TFII-I.
Gene
274
:
1
-13.
57
Boudreau, F., E. H. Rings, G. P. Swain, A. M. Sinclair, E. R. Suh, D. G. Silberg, R. H. Scheuermann, P. G. Traber.
2002
. A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.
Mol. Cell. Biol.
22
:
5467
-5478.
58
Hinnebusch, B. F., A. Siddique, J. W. Henderson, M. S. Malo, W. Zhang, C. P. Athaide, M. A. Abedrapo, X. Chen, V. W. Yang, R. A. Hodin.
2004
. Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor.
Am. J. Physiol.
286
:
G23
-G30.
59
Song, J., H. Ugai, H. Nakata-Tsutsui, S. Kishikawa, E. Suzuki, T. Murata, K. K. Yokoyama.
2003
. Transcriptional regulation by zinc-finger proteins Sp1 and MAZ involves interactions with the same cis-elements.
Int. J. Mol. Med.
11
:
547
-553.
60
Williams, L. J., A. B. Abou-Samra.
2000
. The transcription factors SP1 and MAZ regulate expression of the parathyroid hormone/parathyroid hormone-related peptide receptor gene.
J. Mol. Endocrinol.
25
:
309
-319.
61
Okamoto, S., K. Sherman, G. Bai, S. A. Lipton.
2002
. Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation.
Brain Res. Mol. Brain Res.
107
:
89
-96.
62
Patient, R. K., J. D. McGhee.
2002
. The GATA family (vertebrates and invertebrates).
Curr. Opin. Genet. Dev.
12
:
416
-422.
63
Duncan, D. D., A. Stupakoff, S. M. Hedrick, K. B. Marcu, G. Siu.
1995
. A Myc-associated zinc finger protein binding site is one of four important functional regions in the CD4 promoter.
Mol. Cell. Biol.
15
:
3179
-3186.
64
Fu, M., X. Zhu, J. Zhang, J. Liang, Y. Lin, L. Zhao, M. U. Ehrengruber, Y. E. Chen.
2003
. Egr-1 target genes in human endothelial cells identified by microarray analysis.
Gene
315
:
33
-41.