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Eur J Immunol. Author manuscript; available in PMC 2010 November 19.
Published in final edited form as:
PMCID: PMC2988193
EMSID: UKMS33359

IL-4 induces expression of TARC/CCL17 via two STAT6 binding sites

Abstract

A cardinal feature of allergic disorders and immune responses is enhanced leukocyte trafficking. This is largely orchestrated by chemokines. The CC chemokine thymus- and activation-regulated chemokine (TARC/CCL17) selectively attracts Th2 cells via the G protein-coupled chemokine receptor CCR4. We show here that TARC/CCL17 is expressed by human T cells upon stimulation with IL-4. Mapping of the transcriptional start site revealed the presence of two putative STAT6 binding motifs in proximity to the start position. EMSA and chromatin immunoprecipitation experiments demonstrated that STAT6 was able to bind to both motifs. A fragment of the TARC/CCL17 promoter containing both sites was tested in reporter gene assays for IL-4 inducibility. The promoter was inducible in a STAT6-deficient cell line only after introduction of functional STAT6. When mutations were inserted into one of the STAT6 motifs, IL-4-induced promoter activation was reduced. With both sites mutated, inducibility was completely abrogated. These data demonstrate collectively that T cells serve as a source of TARC/CCL17 when stimulated with IL-4 and that STAT6 is essential for this.

Keywords: Allergy, Chemokines, Human, T cells, Transcription factors

Introduction

Chemokines are structurally related 8–12-kDa chemoattractant cytokines that regulate leukocyte trafficking and inflammatory diseases. Depending on the motif displayed by the first or first two of their conserved cysteines, they have been classified into C, CC, CXC or CX3C chemokine subfamilies. Chemokines have been shown to not only regulate hemopoietic cell migration but also to be involved in a number of other physiological and pathological processes [1]. The 8-kDa thymus- and activation-regulated chemokine (TARC/CCL17) was first isolated from phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) by Imai et al. in 1996 [2] by means of a signal sequence trap method [3]. The TARC/CCL17 gene is located on chromosome 16q13 in a cluster with MDC/CCL22 and Fractalkine/CX3CL1 [4]. It encodes a highly basic 94 amino acid residues long preprotein with a cleavage site between Ala23 and Ala24. The mature TARC/CCL17 protein is 71 amino acid residues in length and was shown to be constitutively expressed in thymus [2] and, upon activation with different inducers, in several cell types including PBMC [5], monocytes [6], dendritic cells [7], endothelial cells, and bronchial epithelial cells [8].

Leukocytes are activated by chemokines through seven-transmembrane G protein-coupled receptors. Based on the subfamily of chemokines they bind, chemokine receptors are named XCR1, CCR1–9, CXCR1–5 and CX3CR1 [9]. The specific functional high-affinity receptor for TARC/CCL17 is the CC chemokine receptor 4 (CCR4), which has also been shown to serve as receptor for CCL22/MDC (macrophage-derived chemokine). CCR4 is predominantly expressed on CD4+ T cells, but is also expressed on NKT cells, platelets, thymocytes and cutaneous lymphocyte-associated antigen (CLA)-positive T cells [10-12]. Tissue homing capabilities of T cells depend on expression of cell adhesion molecules and chemokine receptors; hence, T cell subsets not only differ in their cytokine expression profile but also in chemokine receptor expression [13]. After polarization into Th2 cells, CD4+ T cells express the chemokine receptors CCR3, CCR4 and CCR8, while Th1 cells preferentially express CXCR3 and CCR5 [14, 15]. The CCR4 ligand TARC/CCL17 has been shown to be strongly chemotactic for Th2 cells [6] and CLA+ CD4+ T cells of the memory/effector subtype [10, 16].

TARC/CCL17-mediated recruitment of Th2 cells and CLA+ CD4+ T cells was shown to play a major role in allergic diseases like atopic dermatitis [16, 17], allergic asthma [18-20], allergic rhinitis [21] and allergic contact dermatitis [22].

Interleukin-4 (IL-4), mainly produced by Th2 T cells, mast cells, basophils and eosinophils, appears to be a crucial factor in allergic responses. IL-4 induces differentiation of allergen-specific Th2 cells and class switching towards IgE production, and it promotes activation of eosinophils, basophils and mast cells [23]. On IL-4 receptor (IL-4R) engagement, receptor-associated Janus kinases (JAK) get activated and subsequently phosphorylate tyrosines of the IL-4Rα chain. These provide docking sites for monomeric STAT6 molecules, which, after becoming phosphorylated themselves, form the biologically active dimers [23]. Dimerized STAT6 molecules translocate to the nucleus and bind to IFN-γ-activated sequence (GAS) motifs with the consensus TTC(N4)GAA. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N4 motif) distinguishes it from other STAT molecules, which preferentially bind to N3 motifs [24]. STAT6 is critical for the activation of several IL-4-responsive genes, including those for MHC class II molecules, CD23, Eotaxin-1 and -3, germ-line IgE transcripts, and suppressor of cytokine signaling (SOCS)-1 [25-29].

TARC/CCL17 expression is induced by a variety of stimuli including IL-4 [8, 30-32]. IL-4-dependent regulation of TARC/CCL17 expression has been shown for airway smooth muscle cells [33], the keratinocyte cell line HaCaT [34] and fibroblasts; the mechanism of this regulation, however, remained uncharacterized.

In the present study, we show that TARC/CCL17 expression can be induced in human T cells by IL-4. This is due to IL-4-induced STAT6 binding to the TARC/CCL17 promoter. We identified two adjacent GAS motifs located ~ 150 bp upstream of the transcriptional start site (TSS) as the STAT6 binding elements.

Results

IL-4 induces expression of TARC/CCL17 in human primary T cells

TARC/CCL17 expression has been shown to be inducible by IL-4 in various cell types. To test whether its expression can be induced by IL-4 in human T cells, primary T cells were isolated and stimulated with IL-4 for different times. TARC/CCL17 mRNA and protein expression were quantified by real-time PCR and ELISA, respectively.

An up-regulation of TARC/CCL17 mRNA expression was detectable after 2 h of IL-4 stimulation and continued to rise after longer periods of IL-4 stimulation (Fig. 1). IL-4-stimulated cells showed an approximately 200-fold induction when compared to cells of time point zero, albeit induction rates varied among individual donors. Upon cultivation, TARC/CCL17 mRNA levels increased also in non-stimulated T cells. This accounted for an approximately threefold increase of TARC/CCL17 transcripts after 24 h when compared to freshly isolated cells.

Figure 1
IL-4 induces expression of TARC/CCL17 mRNA in T cells. Human primary T cells of four different donors were stimulated with IL-4 or cultivated without induction for the indicated times. mRNA levels were measured by real-time PCR. Fold changes in the mRNA ...

To quantify TARC/CCL17 expression on the protein level, the cell culture supernatants were tested for TARC/CCL17 protein by ELISA. IL-4 induced TARC/CCL17 protein expression in a time-dependent manner. Detectable levels were measured after 24 h of induction, steadily increasing to approximately 300 pg/mL by 96 h of induction (Fig. 2).

Figure 2
IL-4 induces TARC/CCL17 secretion in T cells. Human primary T cells of five different donors were stimulated with IL-4 or were left unstimulated (nil) for the indicated times. The TARC/CCL17 protein concentration in the culture medium was measured by ...

As seen in the real-time PCR experiment, T cells from some donors produced detectable levels of TARC/CCL17 after 96 h of cultivation without induction. To exclude that this resulted from endogenously produced IL-4, an IL-4-neutralizing antibody was added to the culture medium in one experiment. This, however, did not alter non-stimulated TARC/CCL17 production of the examined donor (data not shown). The reasons for individual differences in protein production between donors remain unknown.

The stimulatory effect of IL-4 appeared to depend also on the cytokine concentration. Cultivation of T cells with increasing quantities of IL-4 led to a corresponding increase in TARC/CCL17 mRNA levels. Maximum TARC/CCL17 production was reached at approximately 5 ng/mL IL-4 (Fig. 3).

Figure 3
IL-4-induced TARC/CCL17 mRNA expression is dose dependent. TARC/CCL17 mRNA expression was analyzed by real-time PCR in human primary T cells stimulated with increasing amounts of IL-4 for 8 h. The data were normalized to non-stimulated cells. One of two ...

Mapping the TARC/CCL17 promoter TSS

To identify the genomic location of the TARC/CCL17 promoter and possible IL-4-responsive elements in it, we had to determine the TSS. To determine the physical location of the TSS, we carried out a 5′ RACE. The semi-nested PCR resulted in a single band when subjected to agarose gel electrophoresis. This band was gel-excised and cloned into a plasmid vector. Several clones were sequenced. Three of the insert-containing clones featured a well-readable sequence which allowed an accurate determination of the TSS. All three clones map the TSS to position 64 ± 1 bp upstream of the translational start site (Fig. 4C). No clones with sequence parts of a further upstream lying putative exon were found.

Figure 4
(A) Genomic organization of the TARC/CCL17 locus. Filled boxes upstream of exon 1 represent GAS motifs A and B. The numbers indicate the positions of the GAS motifs relative to the translational start site. (B) Nucleotide sequences of motif A, motif B, ...

Identification of two STAT6 binding sites in the TARC/CCL17 gene promoter

Visual inspection of the TARC/CCL17 promoter revealed the presence of two potential STAT6 binding sites of the sequence TTC(N4)GAA. These are located at positions −177 to −187 (motif A) and −213 to −223 (motif B) relative to the translational start site (for structure see Fig. 4A, B). To test these motifs for interaction with STAT6, we carried out EMSA. Nuclear extracts were prepared from IL-4-stimulated T cells and incubated with radio-labeled double-stranded DNA oligonucleotide probes containing the putative STAT6 binding sites (probes A and B). IL-4 induced the formation of a nucleoprotein complex with the two probes (Fig. 5). As substitution of two nucleotides in the inverted repeat region of the STAT6 binding site has been shown to abrogate STAT6 binding [28], mutated oligonucleotides (probes MA and MB) featuring these substitutions were tested in the EMSA. These mutated oligonucleotides were unable to interact with the IL-4-induced factor. Intact canonical STAT6 binding motifs were thus necessary for interaction with the IL-4-induced factor.

Figure 5
STAT6 binds to the motifs A and B in the TARC/CCL17 promoter. (A) An EMSA was conducted with a radio-labeled probe containing the wild-type motif A or the mutated motif A (MA). Cold competitors, using the original probe, the mutant oligonucleotide (MA), ...

To test the specificity of the binding reaction, cold competitors were added in 50-fold molar excess. The addition of unlabeled probe resulted in a loss of factor binding to the labeled oligonucleotides. Additionally, we added mutated cold oligonucleotide probes MA and MB. These were unable to block binding to the radio-labeled probes. Further, we used oligonucleotides containing a known STAT6 binding site of the Eotaxin-3/CCL26 promoter [27] as a control. These completely blocked binding of the radio-labeled probes when added as cold competitors.

To identify the IL-4-induced factor, nuclear extracts were pre-incubated with antibodies directed against STAT6. This resulted in disappearance of the nucleoprotein complex and formation of two supershift bands. To test the specificity of antibody binding, nuclear extracts were also incubated with antibodies against the NF-κB subunit p52. No bandshift was observed with this antibody.

These data suggest that IL-4 stimulates interaction of STAT6 with the N4 motifs in the TARC/CCL17 promoter.

An N3 GAS motif located 10 bp downstream of motif A did not exhibit STAT6 binding (data not shown).

To investigate whether STAT6 binds to the TARC/CCL17 promoter in vivo, unstimulated and IL-4-stimulated T cells were subjected to chromatin immunoprecipitation (ChIP). Cross-links of the precipitated DNA-protein complexes were reversed. Samples were treated with proteinase K followed by DNA purification and PCR analysis using primers flanking a 214-bp region containing the two putative STAT6 motifs of the TARC/CCL17 promoter. A PCR product was obtained only after precipitating from IL-4-stimulated cells with anti-STAT6 antibody (Fig. 5C). In experiments conducted without (not shown) or with IgG control antibody, only a weak background signal was observed. These findings suggest that STAT6 binds to the TARC/CCL17 promoter of IL-4-stimulated T cells in vivo.

STAT6 mediates promoter activation by IL-4

To test the relevance of the two N4 motifs in IL-4-induced promoter activation, transient transfection experiments with TARC/CCL17 reporter constructs were conducted. A 913-bp DNA fragment containing the two GAS N4 motifs of the TARC/CCL17 promoter was cloned 5′ of the firefly luciferase gene and a thiokinase TATA box. In addition, mutants of this construct featuring nucleotide exchanges in the motifs were prepared. By using the same mutations as in the EMSA studies, we obtained three constructs, two mutants featuring single exchanges in one of the two motifs and one double mutant with both motifs mutated. The constructs were termed MA, MB and MAB, referring to the mutated motif.

The role of STAT6 in transcriptional activation of these constructs was examined by transient transfection in HEK293 cells. These cells lack functional STAT6 but contain an otherwise intact IL-4 signal transduction cascade [35]. To restore STAT6-dependent transcriptional activation, STAT6 has to be ectopically expressed in these cells. In the absence of functional STAT6, cells transfected with the wild-type reporter construct did not respond to IL-4 stimulation. Upon cotransfection of STAT6 expression vector, luciferase activity increased >8-fold compared to non-stimulated cells after 12 h of IL-4 stimulation (Fig. 6). Transfection of the single mutants resulted in a ~70% decrease in IL-4 inducibility. The double mutant was not IL-4 inducible at all. These data demonstrate that both motifs are functional and necessary for the full promoter activation of TARC/CCL17.

Figure 6
IL-4 induces luciferase expression of reporter constructs featuring 913 bp of the TARC/CCL17 promoter. HEK293 cells were transiently transfected with wildtype or mutated constructs as indicated. The cells were stimulated with IL-4 (IL-4) or were left ...

The experiments were repeated with a construct shortened in the 5′ region, featuring 239 bp of the TARC/CCL17 promoter. This resulted in an increase in IL-4 inducibility to a >13-fold higher luciferase activity compared to non-stimulated cells, which could imply the presence of negative control elements upstream of the STAT6 sites (Fig. 7). However, transfections of the mutated 239-bp constructs resulted in induction rates similar to the longer versions.

Figure 7
IL-4 induces luciferase expression of reporter constructs featuring 239 bp of the TARC/CCL17 promoter. HEK293 cells were transiently transfected with wild-type or mutated constructs, as indicated. The cells were stimulated with IL-4 (IL-4) or were left ...

To exclude effects of the vector backbone, the empty luciferase vector was also tested for IL-4 inducibility. However, no IL-4-dependent changes in luciferase activity were seen with the empty vector (data not shown). We also tested the promoter fragments in TATA box-less vectors. This, however, resulted in unsteady IL-4 inducibility, and background activity of those constructs was very low (not shown). This might be due to a low activity of the TARC/CCL17 promoter in systems other than T cells.

Discussion

The chemokine TARC/CCL17 is thought to play a major role in recruiting Th2-type T cells to sites of allergic inflammation. A couple of IL-4-inducible cellular sources of TARC/CCL17 have been identified. It is an interesting question whether IL-4 stimulation enables T cells themselves to secrete significant amounts of this chemokine. Albeit they were shown to express TARC/CCL17 mRNA and to express TARC/CCL17 protein upon allergen challenge [36, 37], Tcells have thus far not been demonstrated to express TARC/CCL17 after stimulation with IL-4. Hence, the present work was intended to study the IL-4 inducibility of the expression of TARC/CCL17 in T cells and to characterize the regulatory promoter elements necessary for this.

Based on our real-time PCR and ELISA data, this study provides evidence for IL-4-dependent activation of TARC/CCL17 expression in human primary T cells. Increased levels of TARC/CCL17 mRNA became detectable as early as 2 h after IL-4 stimulation. This swift induction appears to be typical, as similar kinetics are seen with many IL-4-induced genes including SOCS-1 [29, 38], FIZZ1 [39], Eotaxin/CCL11 [28] and Eotaxin-3/CCL26 [27]. Most other cell types that were examined for IL-4 inducibility of TARC/CCL17 require simultaneous stimulation with TNF-α to up-regulate its expression [8, 21, 30, 33, 40-42]. Such cooperative effects of IL-4 and TNF-α stimulation were shown before for other genes. Yet, single stimulation with IL-4 appears to be sufficient for T cells. For other cell types, IL-4 treatment appears to have a suppressing effect on TARC/CCL17 expression [34, 40]. More studies will thus be necessary to reveal cell type-specific regulatory mechanisms leading to TARC/CCL17 production. The T cells of some donors featured elevated TARC/CCL17 production. Increased TARC/CCL17 production capacity was noticed before for asthmatic/atopic individuals [36, 43] and may account for this observation.

The rapid induction of TARC/CCL17 mRNA in response to IL-4 stimulation is characteristic for a STAT6-mediated effect, as STAT6 is phosphorylated within minutes in IL-4-treated cells. A role for STAT6 in IL-4-stimulated TARC/CCL17 expression was proposed earlier [44]. While our work was being prepared, a study using a murine asthmatic model on a STAT6 knockout background was published, which showed that STAT6-deficient lungs exhibit a greatly diminished expression of TARC/CCL17 [45]. In our EMSA data, we demonstrate that STAT6 binds to two N4 motifs in the human TARC/CCL17 promoter upon IL-4 stimulation. The motifs are located −167 to −213 bp upstream of the translational start site. Fragments of the TARC/CCL17 promoter containing these motifs were tested in a reporter gene assay using the cell line HEK293, which lacks functional STAT6. The promoter fragments showed IL-4 inducibility only after ectopic expression of functional STAT6. This provides additional evidence that STAT6 is central for IL-4-induced activation of the human TARC/CCL17 gene. The close proximity of the two STAT6 binding motifs to each other (A to B, 26 bp) suggests possible cooperative binding effects. Our reporter gene data obtained with mutated versions of the promoter constructs hint at such positive cooperation, as the single mutants are clearly less well inducible than the wild-type promoter. This, however, has to be investigated in more detail. The oligomerization of STAT dimers was shown before to alter the binding affinities for single GAS motifs in regions containing clustered GAS motifs. This allows binding to sites that, if present as a single motif, do not exhibit STAT binding [46]. This might be also of relevance for the N3 GAS motif that is located only 10 bp downstream of motif A. Although we did not see IL-4-induced complex formation with the former, it might be involved in promoter activation by other cytokines. Additional studies to reveal possible interactions/cooperation of STAT in transcriptional regulation of TARC/CCL17 could thus prove rewarding. Another transcription factor that is probably involved in transcriptional regulation of TARC/CCL17 is NF-κB. Along with other stimuli, TNF-α provides a potent activator of the NF-κB signaling pathway. The promoter of TARC/CCL17 features putative binding sites for NF-κB. This is an interesting subject in the context of the cooperative effects IL-4 and TNF-α have on TARC/CCL17 activation in various cell types. Further studies will be necessary to investigate this.

As the IL-4-induced expression of TARC/CCL17 appears to decrease after longer stimulation periods, a negative regulatory mechanism could be involved. Likely participants in this mechanism are members of the SOCS family [47]. Proteins of this family recently gained much attention for being involved in negative regulation of signaling of many different cytokines, including IL-4. Particularly SOCS-1 and SOCS-3 are interesting candidates for negatively regulating IL-4-induced TARC/CCL17 expression. These proteins were recently demonstrated to be able to inhibit IL-4-induced expression of Eotaxin-3/CCL26 [48], another chemokine. Further, SOCS-1 and SOCS-3 were shown to be inducible by IL-4 [29, 38, 49], and SOCS-3 appears to be involved in the regulation of allergic responses [50]. The prominent role TARC/CCL17 plays in allergic diseases calls for additional studies to elucidate the negative regulatory mechanisms of its expression.

In summary, our study reveals a new source of IL-4-induced TARC/CCL17 expression and identifies the responsible mechanism. Taken into consideration the Th2 cell-chemoattractant activity of TARC/CCL17, our findings indicate a regulatory feedback loop for T cell recruitment. As TARC/CCL17 is added to the list of STAT6-regulated genes, our study further emphasizes the potential of this transcription factor as a therapeutic target in allergic disease.

Materials and methods

Purification of primary human T cells

Human peripheral blood T cells were prepared from healthy adult donors. PBMC were isolated over Ficoll-Paque Plus (Amersham, Arlington Heights, IL) density gradient centrifugation, washed two times with RPMI 1640 medium and incubated for 1 h in plastic tissue culture dishes at 37°C in a humidified atmosphere containing 5% CO2. Nonadherent lymphocytes were collected and Tcells were further purified by means of nylon wool chromatography (Polysciences, Eppelheim, Germany). Purity was assessed by staining for CD3 followed by FACS analysis. A purity of ≥90% was routinely achieved.

Cell culture and ELISA

Human primary T cells were cultured in RPMI 1640 + 10% FCS (heat inactivated), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Recombinant human IL-4 was obtained from Novartis (Basel, Switzerland). Human TARC/CCL17 levels were measured by a commercially available ELISA (R&D Systems, Minneapolis, MN).

RNA isolation and real-time PCR analysis

Total RNA from T cells was isolated using the TRIzol reagent (Life Technologies, Gaithersburg, MD). Total RNA (3 μg) was reverse transcribed using RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas, Germany) according to the manufacturer’s instructions. Real-time PCR analysis was done on a Rotorgene 2000 (Corbett Research, Sydney, Australia) using iQ SYBR Green Supermix (Bio-Rad). Intron-spanning primers used for real-time analysis of TARC/CCL17 expression were 5′-CCAGGGATGCCATCGTTTTTGTAACTGTGC-3′ (forward primer) and 5′-CCTCACTGTGGCTCTTCTTCGTCCCTGGAA-3′ (reverse primer), and 5′-GGCACCATTGAAATCCTGAGTGATGTG-3′ (forward primer) and 5′-TTGCGGACACCCTCCAGGAAGC-3′ (reverse primer) for large ribosomal protein P0, which was used as reference gene. The specificity of the PCR was checked by recording a melting curve, by carrying out agarose gel electrophoresis and by sequencing the amplicons using an ABI-prism automated sequencing machine (Applied Biosystems, Foster City, CA). Induction ratios (x) were calculated using the formula x = 2−ΔΔCt, where Ct represents the threshold cycle of a given gene and ΔCt represents the difference between the Ct values of the gene in question (TARC/CCL17) and the Ct value of the reference gene (large ribosomal protein P0). ΔΔCt is the difference between the ΔCt values of the samples induced with IL-4 and the ΔCt of the non-induced sample. The mean induction ratios of all replicate analyses were calculated.

5′ RACE for determination of the TARC/CCL17 promoter region

For determination of the TARC/CCL17 promoter region, total RNA was isolated from human primary T cells using a nucleospin-RNA II RNA isolation kit (Macherey-Nagel, Düren, Germany) and reverse transcribed to cDNA using the genespecific primer 5′-CTGCATTCTTCACTCTCTTGT-3′ with RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas) according to the manufacturer’s instructions. After cDNA synthesis, RNA was degraded using an RNase mix containing RNase H and RNase A (MBI Fermentas). cDNA was purified using the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI) according to the manufacturer’s instructions. dC-tailing was conducted using TdT transferase (MBI Fermentas) according to the manufacturer’s instructions and C-tailed cDNA was purified using Sephadex G-25 columns (Amersham). PCR amplification was conducted using the primers 5′-GACTACTGTGCGCTAGCCT GGGIIGGGIIGGGIIGGGIG-3′ (contains an Nhe I restriction site, underlined) as forward and 5′-TCCCTTGAAGTACTCCAGGCAGCACTCCCG-3′ as reverse primer. Semi-nested PCR was conducted using 5′-GACTACTGTGCGCTAGCCTGGGIIGGGIIGGGIIGGGIG-3′ as forward and 5′-TCGAGCTGCGTGGATGTGCTGCAGAGAAG-3′as reverse primer. The PCR product was purified using the Wizard SV Gel and PCR Clean-up System (Promega), digested with Nhe I and Pst I (naturally occurring in the TARC/CCL17 coding sequence) and cloned into the vector pGL3 Basic (Promega) for subsequent sequencing.

Preparation of nuclear extracts and EMSA

Nuclear extracts from non-stimulated T cells or from cells that had been stimulated for 30 min with 50 ng/mL IL-4 were prepared according to the method described by Andrews and Faller [51]. One double-stranded oligonucleotide probe containing the GAS motif of the TARC/CCL17 promoter between positions (relative to the translational start site) −167 and −196 (5′-GAGCTAGACTTCTCCTGAATCAT-3′ was used as forward oligonucleotide, and 5′-GAGATGATTCAGGAGAAGTCTAG-3′ was used as reverse oligonucleotide) and one probe containing the second GAS motif between positions −205 and −232 (5′-GAGTGCCCATTCTCTGGAAATCCA-3′ was used as forward oligonucleotide, and 5′-TTGTGGATTTCCAGAGAATGGGCA-3′ was used as reverse oligonucleotide) were end-labeled using [32P]dCTP (Amersham) and Klenow Polymerase (MBI Fermentas). A double-stranded oligonucleotide containing the STAT6 binding site of the Eotaxin-3 promoter between positions −86 and −45 [27] was used for competition assays.

The nucleoprotein binding reaction was performed using 5 μg nuclear extracts. For oligonucleotide competition assays, a 50-fold molar excess of cold oligonucleotide was added to the binding reaction 30 min before the radio-labeled probe. For supershift experiments, extracts were pre-incubated with 2 μg antibody for 20 min before the radio-labeled probe was added. All antibodies used in the supershift experiments were from Santa Cruz Biotechnology.

ChIP

T cells (107) were either stimulated with IL-4 for 1 h or left unstimulated. Cross-linking was performed in PBS mixed with formaldehyde (final concentration 0.4%). Cells were then washed with PBS, resuspended in 500 μL lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1) and sonicated with a Branson Sonifier 250 sonicator. Sheared chromatin was diluted tenfold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl), precleared with BSA and salmon sperm-preblocked protein G beads and taken for ChIP with 4 μg polyclonal anti-STAT6 antibody (rabbit, SC-981; Santa Cruz Biotech) or an IgG control antibody. Immunoprecipitation was carried out at 4°C overnight. After addition of preblocked protein G beads and 30 min of incubation at 4°C, beads were washed once with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris pH 8.1), and twice with TE buffer. Beads were then incubated in elution buffer (1% SDS, 0.1 M NaHCO3). Following cross-linking reversal and proteinase K digestion, eluted DNA was purified using the Wizard SV Gel and PCR Clean-up System (Promega). The presence of selected promoter sequences was assessed via PCR using the following primers: 5′-CAGCTGTGCGTGGAGGCTTTTCA-3′ (forward) and 5′-TCCTTCCCTAGACCAGTGAAGTTCGAAGA-3′ (reverse). The product (214 bp) spanned both of the proposed STAT6 binding motifs in the TARC/CCL17 promoter region.

Cloning of TARC/CCL17 reporter constructs

A 913-bp DNA fragment covering the region from −146 to −1044 bp (relative to the translational start site) of the human TARC/CCL17 promoter was amplified from genomic DNA (Roche, Basel, Switzerland) using the primers 5′-TAGAAGCTTTAGAGTCACAGAGAAGCCAGTTCACCAA-3′ and 5′-GGATCCAGACCAGTGAAGTTCGAAGAATTTGAGA-3′ (containing restriction sites for Hind III and Bam HI, respectively; underlined). The fragment was gel-excised using the concert kit (Life Technologies). The purified PCR product was digested and cloned into the ptataLUC+ vector using the Hind III and Bam HI sites. A shorter construct featuring the region from −146 to −385 of the human TARC/CCL17 promoter was generated by digesting the first construct with Hind III and Nsi I. A restriction site for Nsi I is endogenously present in the sequence of the TARC/CCL17 promoter. The digested construct was subjected to agarose gel electrophoresis and the vector band was excised. The fragment was blunted using T4 polymerase and religated. The plasmids were analyzed by digestion with restriction enzymes and DNA sequencing.

Nucleotide substitutions were inserted into the two STAT6 binding motifs on both promoter constructs. The motifs were mutated from TTC(N)4GAA to TAT(N)4GAA. Two single mutants (MA and MB) and one double mutant (MAB) were prepared for each construct. The mutagenesis was carried out with the Quikchange kit (Stratagene, La Jolla, CA), and the constructs were checked by DNA sequencing of the inserts.

Plasmids for transfection experiments were purified with midi-prep kits (Promega, Mannheim, Germany).

Transient transfection of HEK293 cells

The day before transfection, 5 × 104 cells were seeded into 24-well cell culture plates in fresh medium. Cells were transfected using the calcium phosphate coprecipitation method. Briefly, 1 μg plasmid DNA (reporter construct) and 0.25 μg of expression vector pcDNA 3.1 (Invitrogen, Carlsbad, CA), containing the coding sequence of STAT6, were mixed with 7 μL 2 M CaCl2 and filled up to a total volume of 50 μL with water. This mixture was added dropwise to 50 μL 2 × Hepes-buffered saline (280 mM NaCl, 1.5 mM Na2HPO4, 50 mM Hepes, pH 7.05) while vortexing at the same time. After addition, the mixture was vortexed for another 10 s and incubated at room temperature for 30 min. After incubation, the mixture was vortexed for 5 s and added to the cells.

At 24 h after transfection, medium was exchanged and cells were cultured in the presence or absence of 50 ng/mL IL-4 for 12 h before luciferase assays were conducted in duplicate, using the Promega Luciferase Assay System (Promega) according to the manufacturer’s instructions.

Acknowledgements

D.H. was supported by the Doctoral Scholarship Programme of the Austrian Academy of Sciences (DOC-programme). This work was partially supported by the University of Salzburg, Schwerpunkt “Biomedizin und Gesundheit”. This work was partially supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF, P18409).

Abbreviations

CCR4
CC chemokine receptor 4
ChIP
chromatin immunoprecipitation
CLA
cutaneous lymphocyte-associated antigen
GAS
IFN-γ-activated sequence
TARC/CCL17
thymus- and activation-regulated chemokine/CC chemokine ligand 17
TSS
transcriptional start site

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