Induction of surface CCR4 and its functionality in mouse Th2 cells is regulated differently during Th2 development Yasunari Morimoto,*,† Yang Bian,* Ping Gao,* Yumi Yashiro-Ohtani,* Xu-Yu Zhou,* Shiro Ono,*,1 Hirokazu Nakahara,† Mikihiko Kogo,† Toshiyuki Hamaoka,* and Hiromi Fujiwara* *Department of Oncology, Graduate School of Medicine, and †Graduate School of Dentistry, Osaka University, Japan
Abstract: T helper cell type 1 (Th1) and Th2 cells express distinct sets of chemokine receptors. In contrast to Th1 chemokine receptors, it is largely unknown how Th2 chemokine receptors such as CC chemokine receptor 4 (CCR4) are induced during Th2 differentiation. Here, we investigated the induction of CCR4 surface expression and ligand responsiveness evaluated by functional assays such as chemokine binding and chemotaxis. This was done in comparison with those of a Th1 chemokine receptor, CXC chemokine receptor 3 (CXCR3). Resting T cells expressed neither CXCR3 nor CCR4. CXCR3 expression and ligand responsiveness were observed when resting T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of [interleukin (IL)12ⴙanti-IL-4] and then recultured without T cell receptor (TCR) stimulation. Unlike CXCR3, CCR4 was induced immediately after anti-CD3/anti-CD28 stimulation in the presence of (IL-4ⴙanti-interferon␥ⴙanti-IL-12). However, these CCR4-positive cells failed to exhibit chemokine binding and chemotaxis. Although the levels of surface CCR4 expression were not increased after the subsequent reculture in the absence of TCR stimulation, CCR4 responsiveness was induced in this stage of Th2 cells. The induction of CCR4 expression and the acquisition of CCR4 responsiveness did not occur in IL-4-deficient (IL4–/–) and signal transducer and activator of transcription (STAT)6–/– T cells. CCR4 expression and functionality were regained in IL-4–/– but not in STAT6–/– T cells by the addition of recombinant IL-4. Although surface expression and functionality of CCR4 are induced depending on the IL-4/STAT6 signaling pathway, the present results indicate that the functionality of CCR4 does not correlate with CCR4 expression but emerges at later stages of Th2 differentiation. J. Leukoc. Biol. 78: 753–761; 2005. Key Words: chemokine/chemokine receptor 䡠 Th1 䡠 CXCR3 䡠 IL-4 STAT6
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INTRODUCTION Upon antigen sensitization, naive CD4 T cells differentiate into two groups of polarized effector cells, T helper cell type 1 (Th1) 0741-5400/05/0078-753 © Society for Leukocyte Biology
and Th2 [1]. These two subsets of cells are defined by their cytokine production profiles; Th1 cells produce interleukin (IL)-2 and interferon-␥ (IFN-␥), whereas Th2 cells secrete IL-4, IL-5, and IL-6 [2– 6]. Th differentiation has also been documented to be determined by cytokines in the environment, such as IL-12/IFN-␥ for Th1 and IL-4 for Th2 [6, 7]. In addition to cytokine production, Th1 and Th2 cells acquire different migratory capacities [8]. The combined action of adhesion molecules and chemokines/chemokine receptors is considered to provide an address code for leukocyte trafficking to the relevant sites [9 –12]. Many recent studies have shown that chemokine receptors are differentially expressed on antigen-stimulated T cells depending on their polarization, and Th1 lymphocytes express CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 3 (CXCR3) and Th2 lymphocytes, CCR3, CCR4, and CCR8 [13–16]. For example, Th1 cells, recruited to sites of inflammation such as the synovial fluid of rheumatoid arthritis, express CCR5 and CXCR3 [17, 18]. Although the expression of particular chemokine receptors on Th1 and Th2 cells has been well-recognized, the mechanisms underlying the induction of a given chemokine receptor in antigen-sensitized, naive T cells remained to be defined. In this context, our recent studies have shown that CCR5 is induced on T cell receptor (TCR)-triggered CD4 and CD8 T cells, depending on the IL-12/signal transducer and activator of transcription (STAT)4 signaling pathway [19, 20], whereas the induction of CXCR3 requires IFN-␥ stimulation [21]. These observations indicated that the cytokines involved in Th1 differentiation are required for the acquisition of the capacities of Th1 cells, not only to produce Th1 cytokines but also to express Th1 chemokine receptors. In contrast to Th1 chemokine receptor induction, the mechanisms of Th2 chemokine receptor expression are largely unknown. Moreover, the patterns and regulation of CCR4 expression are complicated: For example, a part of human CCR4⫹ CD4 T cells coexpresses CXCR3/CCR5 and produces a Th1 cytokine IFN-␥ after short-term stimulation [22]. In this study, we investigated how CCR4 surface expression as well as CCR4 responsiveness to the relevant chemokines are induced in the
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Correspondence: Department of Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail:
[email protected] Received March 11, 2005; revised May 9, 2005; accepted May 10, 2005; doi: 10.1189/jlb.0305139.
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course of mouse Th2 differentiation. The results show that CCR4 is not expressed on resting T cells but induced when they are stimulated with anti-CD3 plus anti-CD28 in the cytokine environment of Th2 polarization. However, ligand responsiveness of CCR4, as evaluated by chemotaxis assays, was not yet generated in such stimulated cells but emerged after the subsequent resting cultures without TCR stimulation. This contrasted with the observation that surface expression and ligand responsiveness of CXCR3 developed after resting cultures subsequent to TCR stimulation cultures toward Th1 polarization. As IFN-␥ was required for the induction of CXCR3 expression and function [21], surface expression and functionality of CCR4 were generated depending on the IL-4/ STAT6 signaling pathway. Thus, the results indicate that CCR4 exhibits the unique property in terms of Th2 differentiation stages, at which surface expression of this chemokine receptor occurs, and its functionality emerges.
Sunnyvale, CA), as described previously [19]. Labeled cells were separated from unlabeled cells by magnetic cell sorting using the MiniMACS (Miltenyi Biotec). The magnetically labeled cells were retained in a MiniMACS column inserted into a MiniMACS magnet, and the unlabeled cells passed through. Labeled cells were eluted after the column was removed from the magnet. The cells that passed though the column and were eluted from the column were used as CD62L– and CD62L⫹ T cell populations, respectively.
In vitro stimulation for Th1/Th2 polarization Anti-CD3 (1 g/ml) and anti-CD28 (5 g/ml) were coimmobilized to individual wells of 24-well culture plates (Corning 25820, Corning Glass Works, Corning, NY) in a volume of 0.625 ml. After 3 h, solutions were discarded, and plates were washed with phosphate-buffered saline (PBS) twice. Purified T cells were cultured in 2 ml RPMI-1640 medium supplemented with 10% fetal bovine serum and 2-mercaptoethanol at 2 ⫻ 106 cells/well for 3 days in the presence of 10 g/ml anti-IL-4 mAb plus 1 ng/ml rIL-12 or 20 g/ml anti-IFN-␥ mAb, 10 g/ml anti-IL-12 mAb, plus 10 ng/ml rIL-4. Cells were harvested, washed, and then recultured for an additional 3 days in cultures with either set of the above Th-polarization reagents but without TCR/CD28 stimulation.
Immunofluorescence staining for CXCR3 and CCR4 MATERIALS AND METHODS Mice BALB/c mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). IL-4-deficient (IL-4–/–) BALB/c mice (BALB/ctm2Nnt) [23], STAT6-deficient (STAT6–/–) BALB/c mice (BALB/c-Stat6tm1Gru) [24], and IFN-␥-deficient (IFN-␥–/–) BALB/c mice (BALB/c-Ifngtm1Ts) [25] were obtained from Jackson Laboratories (Bar Harbor, MA). These knockout mice were bred in our laboratory and used at 6 –9 weeks of age.
Reagents Mouse recombinant (mr)IL-12 was provided by Wyeth Institute (Cambridge, MA). mrIL-4 and rIFN-inducible protein 10 (rIP-10) were purchased from PeproTech EC Ltd. (London, UK). Mouse recombinant macrophage-derived chemokine (mrMDC) was from R&D Systems (Minneapolis, MN). Anti-CD3 (145-2C11) [26], anti-mouse CD28 (37.51) [27], anti-I-Ad/b (34-5-3S) [28], anti-IL-4 (11B11) [29], anti-IL-12 (C17.8) [30], and anti-mIFN-␥ (ATCC Clone R4-6A2) monoclonal antibodies (mAb) were purified from culture supernatants or ascitic fluids of respective hybridomas. Rabbit anti-mouse CXCR3 antibody (Zymed Laboratories, South San Francisco, CA), biotinylated goat anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA), goat anti-mouse CCR4 antibody (Capralogics, Hardwick, MA), fluroescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG (Jackson ImmunoResearch), phycoerythrin (PE)-conjugated anti-CD62 ligand (CD62L) mAb (BD PharMingen, San Diego, CA), FITC- and PE-conjugated streptavidin (Becton Dickinson, Mountain View, CA), and control rabbit IgG and control goat IgG (Biomeda, Foster City, CA) were also purchased.
Preparation of T cell populations Mouse lymph node cells were depleted of B cells and Ia⫹ antigen-presenting cells by immunomagnetic negative selection as described [31]. Briefly, lymph node cells were allowed to react with mouse anti-I-Ad/b mAb and then incubated with magnetic particles bound to goat anti-mouse IgG (Qiagen, Germany). A T cell population depleted of anti-I-Ad/b-labeled and surface Ig⫹ cells was obtained by removing cell-bound magnetic particles with a rare earth magnet (Advanced Magnetics, Cambridge, MA). Purity of the resulting T cell populations was examined by flow cytometry and found to be consistently ⬎95%. For the preparation of the CD4⫹ T cell population, lymph node cells were allowed to react with anti-I-Ad/b and rat anti-CD8 (ATCC Clone 2.43) mAb and then incubated with a mixture of magnetic particles bound to goat anti-mouse IgG (Qiagen) and goat anti-rat IgG (Qiagen). A CD4⫹ T cell population was obtained by removing cell-bound magnetic particles with a rare earth magnet. Purity of the resulting CD4⫹ T cell population was consistently ⬎95%. To obtain CD62L⫹ and CD62L– cells, purified T cells were labeled with magnetic microbeads conjugating anti-CD62L mAb (Miltenyi Biotec,
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CXCR3 or CCR4 was stained by incubation with rabbit anti-mouse CXCR3 or goat anti-mouse CCR4 antibody, followed by a mixture of biotinylated goat anti-rabbit IgG plus PE-conjugated streptavidin [21] or FITC-conjugated donkey anti-goat IgG, respectively. Stained cells were analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA).
Analyses of intracellular cytokine (IFN-␥ and IL-4) production by flow cytometry The procedure was essentially the same as that described previously [32]. Cultured T cells were stimulated with 50 ng/ml phorbol 12-myristate 13acetate and 500 ng/ml ionomycin in the presence of 2 M monensin for 4 or 24 h. The cells harvested (106) were incubated for 30 min at 4°C with allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb in 20 l staining buffer [Hank’s balanced salt solution, 0.1% bovine serum albumin (BSA) and 0.1% sodium azide (NaN3)]. Then, cells were washed with staining buffer and fixed for 20 min at room temperature with 500 l PBS containing 4% paraformaldehyde. After washing, the cells were incubated in PBS containing 0.5% BSA, 0.1% NaN3, and 0.5% saponin for cell membrane permeabilization. For intracellular staining, fixed cells were stained for 60 min at room temperature with rat anti-IFN-␥ (XMG1.2) FITC and rat anti-IL-4 (11B11) PE. PE-rat IgG1 and FITC-rat IgG1 antibodies were used as an isotype control. All staining reagents were purchased from BD PharMingen. Flow cytometric analysis was performed on a FACSCalibur, and cells were analyzed by CellQuest software (Becton Dickinson, San Jose, CA).
[125I]-labeled chemokine-binding assay A radiolabeled chemokine-binding assay was performed by referring to the previous papers [33, 34]. Briefly, T cells (2⫻106/tube) suspended in 100 l binding buffer [50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA (pH 7.4)] were preincubated with or without 100-fold excess amounts of cold chemokines in a microtube for 5 min at room temperature and were followed by incubation with 10 pmol [125I]-labeled IP-10 or MDC (specific activity 2000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) for 30 min. The binding reaction was terminated by washing out the free ligand with cold PBS, and the cell-associated radioactivity was measured by auto ␥ counter. The specific chemokine binding was expressed as actual counts calculated by subtracting the binding in the presence of cold chemokine [counts per minute (cpm)] from the binding in the absence of cold chemokine (cpm). The results are expressed as arithmetic means (SE) of triplicate cultures.
Chemotaxis assay Chemotaxis was assessed in 24-well Transwells equipped with 3 M pore polycarbonate membranes (Transwell, Corning Costar Corp., Cambridge, MA) as described in our previous paper [32]. Cells (2⫻105) suspended in 100 l RPMI 1640 supplemented with 10% fetal calf serum were transferred to the
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upper chamber. The lower chamber contained 600 l medium with various concentrations of chemokines. After 2 h, unless otherwise indicated, the upper chambers were removed, and the migrated cells to the lower chambers were counted by a FACSCalibur. All assays were done in triplicate, and the results were expressed as a percentage of migration by the following formula: % Migration ⫽ 100 ⫻ (cell number of lower chamber in the presence of chemokine)/(cell number applied to upper chamber). The results are expressed as arithmetic means (SE) of triplicate cultures.
Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was prepared from mAb/cytokine-treated T cells by StrataPrep™ Total RNA Miniprep kit (Stratagene Cloning Systems, La Jolla, CA). Total RNA (1 g) was reverse-transcribed into cDNA in a total volume of 20 l, using random primers and Superscrit™ II RNase H– RT (Life Technologies, Rockville, MD). PCR amplification was carried out in a total volume (25 l) of 1⫻ PCR buffer (Applied Biosystems, Foster City, CA) containing 0.5 l of the first strand cDNA, 0.25 mM of each deoxy-unspecified nucleoside 5⬘-triphosphate, 2 M each primer, and 25 U/ml Ampli Taq Gold™ DNA polymerase (Applied Biosystems). The following oligonucleotides were used: CCR4 sense primer 5⬘-ATCCTGAAGGACTTCAAGCTCCA-3⬘, CCR4 anti-sense primer 5⬘-AGGTCTGTGCAAGATCGTTTCATGG-3⬘, -actin sense primer 5⬘AGAAGAGCTATGAGCTGCCTGACG-3⬘, and -actin antisense primer 5⬘CTCTTTGATGTCACGCACGATTTC-3⬘. Cycle parameters were annealing 1 min at 55°C, elongation 1 min at 72°C, and denaturation 30 s at 94°C. Resulting PCR products were separated in 2% agarose gel and visualized by staining with SYBR™ Green I nucleic acid gelstain (FMC Bioproducts, Rockland, ME). Sequences of the CCR4 and -actin (for standardization) were amplified out of each cDNA batch with 21 and 18 amplification cycles, respectively.
RESULTS Induction of surface CXCR3 expression and CXCR3 responsiveness in developing Th1 cells Our earlier study demonstrated that surface CXCR3 was hardly detected on most of freshly prepared mouse T cells but induced
when they were stimulated with anti-CD3 plus anti-CD28 mAb and then recultured without these mAb [21]. CXCR3 induction was also shown to depend on IFN-␥ produced following antiCD3/anti-CD28 stimulation [21]. Here, purified mouse CD4⫹ T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of the Th1 (rIL-12 and anti-IL-4)- or Th2 (rIL-4, anti-IFN-␥, and anti-IL-12)-polarizing reagents for 3 days and then recultured for an additional 3 days in cultures containing Th-polarizing reagents but not including anti-CD3/anti-CD28 (Fig. 1). The cells harvested from TCR/CD28-stimulation culture and reculture were examined for intracellular expression of IFN-␥ or IL-4, according to the procedure described in our recent paper [32]. Figure 1 shows that only a minor portion of cells receiving TCR/CD28 stimulation in the Th1 or Th2 condition expresses intracellular IFN-␥ or IL-4, respectively, and that IFN-␥- and IL-4-producing cells increase strikingly after reculture. In this study, the cells after TCR/CD28 stimulation and reculture were thus designated Th1- or Th2-polarizing and Th1- or Th2-polarized ones, respectively. We examined CXCR3 expression on these Th1/Th2-polarizing and Th1/ Th2-polarized cells (Fig. 2). Upon TCR/CD28 stimulation, CXCR3 was negative on T cells during polarization toward either T cell subset. CXCR3 was induced on Th1-polarized cells, whereas CXCR3 induction was prevented in the Th2polarizing reculture, including anti-IFN-␥ mAb. These observations are consistent with our previous results [21]. We next examined whether CXCR3 induced on Th1 cells exhibits the responsiveness to its ligand chemokines. The functionality of CXCR3 was examined by chemotaxis assays (Fig. 3). The chemotaxis induced by IP-10, a CXCR3 ligand, was observed for polarized Th1 cells after reculture, but not for polarizing Th1 cells after TCR stimulation, which correlates with the time course of CXCR3 surface expression detected by flow cytom-
Fig. 1. Induction of IFN-␥- or IL-4-producing cells by TCR/CD28 stimulation in the respective Th1- or Th2-polarization culture. Purified lymph node CD4⫹ T cells (2⫻106/well) were stimulated for 3 days with immobilized anti-CD3 (1 g/ml) plus anti-CD28 (5 g/ml) in the presence of either set of Th1 [1 ng /ml rIL-12 plus 10 g/ml anti (␣)-IL-4]- or Th2-polarizing reagents (10 ng/ml rIL-4, 20 g/ml anti-IFN-␥, and 10 g/ml anti-IL-12). Stimulated cells were recultured for an additional 3 days in the absence of anti-CD3/anti-CD28 but in the continuous presence of Th1- or Th2-polarizing reagents. Freshly prepared CD4⫹ T cells and TCR/CD28-stimulated cells before or after reculture were submitted to the analysis of intracellular IFN-␥ or-IL-4 production as described in Materials and Methods. The results are representative of three independent experiments.
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Fig. 2. Surface CXCR3 expression is induced only in Th1 cells. According to the protocol in Figure 1, Th1- or Th2-polarizing (cells after TCR/CD28 stimulation) or polarized CD4⫹ T cells (cells after reculture) were prepared. Freshly prepared CD4⫹ T cells and TCR/CD28-stimulated T cells before or after reculture were stained with rabbit anti-mouse CXCR3 antibody and then with biotinylated goat anti-rabbit IgG followed by PE-conjugated streptavidin (solid lines). The dashed lines represent negative staining with control antibodies. The results are representative of five independent experiments.
etry using anti-CXCR3 antibody. Thus, the functional CXCR3 is induced on polarized Th1 cells.
Induction of surface CCR4 and expression of CCR4 mRNA in developing Th2 cells We examined whether CCR4 expression and responsiveness are induced on CD4⫹ T cells differentiating toward Th2. As shown in Figure 4, resting T cells did not express CCR4, whereas CCR4 was induced strongly and only transiently during Th2 and Th1 polarization, respectively. Notably, there was an essential difference between CCR4 (Fig. 4) and CXCR3 expression (Fig. 2) regarding their time courses. Unlike
CXCR3, CCR4 was induced on CD4⫹ T cells harvested immediately after TCR/CD28 stimulation. To verify the time course of CCR4 surface expression, the induction of CCR4 mRNA was examined in resting and developing Th1 and Th2 cells (Fig. 5). CCR4 mRNA was not detected in resting T cells. However, marginal levels of CCR4 mRNA were detected only on polarizing but not on polarized Th1 cells. Consistent with surface CCR4 expression, TCR/ CD28-stimulated (polarizing) Th2 cells before reculture expressed CCR4 mRNA. This contrasted with the observations that surface CXCR3 (Fig. 2 and ref. [21]) and CXCR3 mRNA [21] were induced after TCR/CD28-stimulated cells were recultured.
Functionality of CCR4 expressed in developing Th2 cells
Fig. 3. IP-10-induced chemotaxis is observed in Th1 cells harvested after reculture. Th1/Th2-polarizing or -polarized cells prepared similarly to those in Figures 1 and 2 were submitted to chemotaxis assays with 10 nM IP-10. Percent migration ⫽ 100 ⫻ (cell number in lower chamber with IP-10)/(cell number applied to upper chamber). The spontaneous migration in the absence of chemokines was less than 1%. The results are representative of three independent experiments.
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To determine whether CCR4 expressed in developing Th2 cells is functional, its chemokine responsiveness was examined in chemokine binding and chemotaxis assays. Resting T cells and Th1 (control) and Th2 cells at various developing stages were incubated with [125I]-labeled MDC in the presence or absence of excess amounts of unlabeled MDC. The MDC-binding capacity is expressed as a ⌬ cpm, which is calculated by subtracting cpm in the presence of cold inhibitor from cpm in the absence of cold inhibitor. As shown in Figure 6A, enhanced MDC-binding capacity was not detected in developing Th1 cells but induced in polarized Th2 cells. It should be noted that polarizing Th2 cells, although expressing comparable levels of surface CCR4 with those of polarized Th2 cells (Fig. 4), failed to exhibit the MDC-binding capacity. In chemotaxis assays, cell migration was elicited by a CCR4 ligand, MDC. Resting T cells did not exhibit MDC responsiveness (Fig. 6B). Although MDC-induced chemotaxis was only marginally induced in polarizing Th2 cells, polarized Th2 cells exhibited high levels of chemotaxis (Fig. 6B). These observations are consistent with the results obtained in chemokinebinding assays (Fig. 6A). Th2-polarization cultures were also http://www.jleukbio.org
Fig. 4. Surface CCR4 expression is induced on developing Th2 cells. Purified CD4⫹ T cells were stimulated in Th1 or Th2 polarization culture. Cells after TCR stimulation (Th1- or Th2-polarizing cells) and after reculture (Th1- or Th2-polarized cells) were stained with goat anti-mouse CCR4 antibody and followed by FITC-conjugated donkey anti-goat IgG (solid lines). As a control, the cells were stained with control goat antibody followed by FITC-conjugated donkey anti-goat IgG (dashed lines). The results are representative of five independent experiments.
conducted using freshly isolated CD62L⫹ (naive) and CD62L– (memory) resting T cells. Polarized Th2 cells generated from either subset developed CCR4 responsiveness, although the latter subset of T cells exhibited relatively higher levels of CCR4 functionality (Fig. 6C).
Specificity of CCR4 functionality induced in polarized Th2 cells We examined the specificity of two CCR4 functions, i.e., ligand (MDC)-mediated chemotaxis and MDC binding. Polarized Th1 and Th2 cells were prepared by reculturing TCR/CD28-stimulated cells in the presence of the respective Th1- and Th2polarizing reagents. These cells were submitted to chemotaxis assays and binding assays using chemokines for CXCR3 (IP-
Fig. 5. Time course in the induction of CCR4 mRNA expression in developing Th2 cells. Total RNA was obtained from polarizing and polarized Th1 and Th2 cells prepared similarly to those in Figure 3 and subjected to RT-PCR for the determination of CCR4 mRNA expression. The results are representative of three similar experiments.
10) or CCR4 (MDC; Fig. 7). CCR4 functions as detected by MDC-induced chemotaxis and MDC binding were observed selectively in Th2 cells. Conversely, IP-10 responses were induced preferentially in Th1 cells.
Role for IL-4/STAT6 in the induction of surface CCR4 and CCR4 responsiveness Previously, Zhang et al. [35] demonstrated the requirement of STAT6 for CCR4 mRNA induction under Th2 conditions. Considering the time-different induction of CCR4 surface expression and function, we examined whether CCR4 surface induction and functionality depend on the IL-4/STAT6 signaling and whether the presence of IL-4 is required for the differentiation of surface CCR4⫹ Th2-polarizing cells to Th2polarized cells. CD4⫹ T cells from IL-4–/– and wild-type (WT) mice were stimulated with anti-CD3/anti-CD28 in the absence (Fig. 8A) or presence (Fig. 8B) of Th2-polarizing reagents and then recultured. Without Th2-polarizing reagents (designated Th0), detectable, albeit slight levels of surface CCR4 were induced in WT T cells with the capacity to produce endogenous IL-4, whereas IL-4–/– T cells failed to express CCR4 (Fig. 8A). Even in IL-4–/– T cells, however, surface CCR4 expression was elicited in Th2 conditions including exogenous rIL-4 (Fig. 8B, middle panels). Further, the results (Fig. 8B, bottom panels) show that STAT6–/– T cells, generated in the Th2polarizing culture, do not express CCR4. To determine the requirement for IL-4/STAT6 in the induction of CCR4 functionality, WT, IL-4–/–, or STAT6–/– CD4⫹ T cells were stimulated with anti-CD3/anti-CD28 and recultured in the Th0 (Fig. 9A, left panel)- or Th2-polarizing condition (Fig. 9, A, right panel, and B). Recultured cells were stimulated with MDC in chemotaxis assays. WT and IL-4–/– T cells cultured in the Th0 condition exhibited only slight and negligible levels of chemotaxis, whereas high levels of chemotaxis were induced in both types of cells generated in the Th2 condition (Fig. 9A, right). In contrast to IL-4–/– T cells, STAT6–/– T cells generated in the Th2 condition failed to
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Fig. 6. CCR4 function is induced only in polarized Th2 cells. Resting CD4⫹ T cells and polarizing and polarized Th1 or Th2 cells were examined for the binding to [125I]-radiolabeled MDC in the presence or absence of excess amounts of cold IP-10 or MDC as described in Materials and Methods (A) and chemotaxis assays with 10 nM MDC as in Figure 3. (B). The specific chemokine binding ⫽ (the binding cpm without cold inhibitor) – (the binding cpm with cold inhibitor). (C) Purified CD4⫹ T cells were separated into CD62L⫹ (naı¨ve) and CD62L– (memory) cells. These two populations were stimulated with anti-CD3 plus anti-CD28 in the Th2-polarizing condition for 3 days and then placed in resting cultures. After an additional 3 days, they were subjected to chemotaxis assays with 10 nM MDC. The results of binding assays and chemotactic assays are representative of three and four independent experiments, respectively.
exhibited MDC responsiveness (B). These results correlate with the patterns of surface CCR4 expression in WT, IL-4–/–, and STAT6–/– T cells harvested from recultures and indicate the requirement of IL-4/STAT6 for the induction of surface CCR4 and CCR4 functionality. We finally examined whether the induction of CCR4 functionality requires the presence of IL-4 in recultures responsible for the differentiation of Th2-polarizing cells to Th2-polarized cells. WT and IL-4–/– T cells were stimulated with anti-CD3/ anti-CD28 in the Th2 condition. These polarizing cells were submitted to recultures with or without IL-4. Recultured cells were examined for the expression of surface CCR4 and MDC responsiveness in chemotaxis assays (Fig. 10). All groups of recultured cells expressed CCR4, although the levels of CCR4 expression were slightly lower in IL-4–/– T cells recultured in the absence of IL-4 than other groups of cells. However, CCR4 functionality was observed in WT and IL-4–/– T cells recul-
tured in the presence of IL-4. The levels in these cells were much higher than those of WT and IL-4–/– cells recultured in the absence of IL-4. These results indicate that CCR4 functionality emerges depending on the presence of IL-4 in the reculture step required for Th2 differentiation.
DISCUSSION Polarized Th1 and Th2 cells have been shown to differentially express chemokine receptors that allow their proper positioning in the immune response [11–14]. However, little is known regarding the dynamics of chemokine receptor expression during the early stages of naive T cell activation and commitment to Th1 or Th2 polarization. Here, we investigated cell-surface expression and functionality of the representative Th2 chemokine receptor CCR4 in comparison with those of a Th1 che-
Fig. 7. Specificity of chemokine binding and responsiveness by Th1 and Th2 cells. Polarized Th1 and Th2 cells were examined for the chemotactic responsiveness to 10 nM IP-10 or 10 nM MDC (A) and binding to [125I]-radiolabeled IP-10 or MDC (B). The results of chemotactic and binding assays are representative of three independent experiments.
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Fig. 8. The induction of surface CCR4 in developing Th2 cells is affected by IL-4/STAT6 deficiency. Purified WT and IL-4–/– CD4⫹ T cells were stimulated with anti-CD3/anti-CD28 and then placed in resting cultures in the absence of Th2polarizing reagents (A). Purified WT, IL-4–/–, and STAT6–/– CD4⫹ T cells were stimulated with antiCD3/anti-CD28 and then placed in resting cultures in the presence of Th2-polarizing reagents (B). Cells after anti-CD3/anti-CD28 stimulation and resting cultures were stained for CCR4 as above described. The results are representative of three independent experiments.
mokine receptor CXCR3. The following observations were made: Surface CXCR3 was not detected on most naive/resting T cells but expressed selectively in polarized Th1 cells concurrently with the emergence of CXCR3 responsiveness. Although CCR4 was induced preferentially in developing Th2 cells, surface expression of this chemokine receptor dissociated with the induction of its functionality. Namely, surface CCR4 and CCR4 mRNA were detectable in polarizing Th2 cells harvested immediately after TCR/CD28 stimulation in Th2 culture conditions. However, these cells did not show CCR4 functionality, as observed in chemokine binding and chemotaxis assays. CCR4 functionality was detected when TCR/CD28-stimulated cells were placed in resting cultures for additional days. Thus, CCR4 surface expression and function-
ality emerged in developing Th2 cells in different time courses, although the induction of both events depended on the IL-4/ STAT6 signaling pathway. It has been reported that among a number of CCRs and CXCRs, Th1 cells predominantly express CXCR3 and CCR5 [17, 18]; Th2 cells selectively express CCR3 [15], CCR4 [13, 14], and CCR8 [16]. Thus, these chemokine receptors have been implicated as a set of differentiation markers for Th1 and Th2 cells. However, recent studies showed that CCR4 mRNA is also expressed by in vitro-polarized [36] or -polarizing Th1 cells [37]. Moreover, a portion of freshly isolated human CD4⫹ memory T cells is CCR4-positive [22, 38], but major fractions of these CCR4⫹ memory T cells coexpress the Th1-associated chemokine receptors CXCR3 and CCR5 and readily express
Fig. 9. Requirement for IL-4/STAT6 in the acquisition of CCR4 responsiveness. Purified WT and IL-4–/– CD4⫹ T cells were stimulated with anti-CD3/anti-CD28 and then placed in resting cultures in the presence (Th2 condition) or absence (Th0 condition) of Th2-polarizing reagents during both culture periods (A). Polarized Th2 cells induced by stimulation and resting cultures in the Th2 condition were prepared from purified WT, IL-4–/–, and STAT6–/– T cells (B). Cells harvested from resting cultures were subjected to chemotaxis assays with 10 nM MDC. The results are representative of three independent experiments.
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Fig. 10. Role for IL-4 in the induction of CCR4 functionality. WT and IL-4–/– T cells were stimulated with anti-CD3/anti-CD28 in the Th2 condition and then recultured in the Th0 or Th2 condition. Recultured cells were examined for CCR4 expression (A) and MDC responsiveness (B). The results are representative of two independent experiments.
the Th1 cytokine IFN-␥ after short-term stimulation [38]. Thus, it appears that unlike the preferential expression pattern of CXCR3 and CCR5, the patterns and regulation of CCR4 expression are more complex. Our results obtained in the mouse model showed that resting mouse T cells, including naive and memory T cells, express neither surface CCR4 nor CCR4 mRNA and that these are detected in cells that have started to differentiate toward Th2. Surface CCR4 or CCR4 mRNA was only marginally or hardly detected on polarizing or polarized Th1 cells. Thus, the results obtained with mouse T cells regarding CCR4 expression are different from the observation described above about human T cells [22, 36 –38]. The discrepancy in chemokine receptor expression between mouse and human T cells is not limited to CCR4, as there is also a considerable difference in CXCR3 expression [21, 39, 40]: Only a minor portion of resting mouse T cells expresses CXCR3 (ref. [17] and this study), whereas ⬃40% of resting human T cells are CXCR3-positive [39, 40]. Thus, cell-surface expression of CXCR3 and CCR4 appears to be regulated differently in mouse and human T cells. A more important aspect of the present study concerns the dissociation between CCR4 surface expression and the induction of CCR4 functionality. Regarding a Th1 chemokine receptor CXCR3, receptor surface expression and functionality were simultaneously induced when CD4⫹ T cells stimulated with anti-CD3/anti-CD28 in the Th1 culture condition (Th1polarizing cells) were placed in resting cultures for additional days and harvested as Th1-polarized cells. In contrast, surface CCR4 and CCR4 mRNA were expressed by cells receiving TCR/CD28 stimulation in the Th2 culture condition. However, these Th2-polarizing cells exhibited only marginal levels of CCR4 responsiveness. Full functionality of CCR4 was detected when they were harvested as Th2-polarized cells after additional days of resting cultures. Th2-polarizing cells exhibited comparable levels of surface CCR4 and CCR4 mRNA with those detected on Th2-polarized cells. Therefore, there may be a configurational difference in CCR4 proteins on both types of cells, and such a difference could not be discriminated by 760
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anti-CCR4 antibodies used here. According to this view, the lack of CCR4 function in Th2-polarizing cells may be explained by postulating that CCR4 responsiveness is generated along with the structural maturation of CCR4 during their differentiation into Th2-polarized cells. Further, the present study shows a requirement for CCR4 induction, particularly for the induction of CCR4 functionality, during Th2 differentiation. Th2 polarization is induced based on the exposure of responding cells to IL-4 along with the blockade of IL-12/IFN-␥ signaling [6, 7]. Therefore, it is possible that the induction of CCR4 expression and responsiveness depends on IL-4 and its signaling molecules such as STAT6. In fact, a previous study of Zhang et al. [35] demonstrated the requirement of STAT6 for CCR4 mRNA expression. Consistent with their report, our present results showed that CCR4 surface expression and functionality also depended on IL-4/STAT6 signaling, although these were induced at different polarization stages; although surface CCR4⫹ Th2-polarizing cells failed to display CCR4 function, they could acquire CCR4 functionality during differentiation into Th2-polarized cells. Notably, our present results showed that IL-4 is required for the induction of CCR4 functionality during such a differentiation period. However, the mechanism underlying IL-4mediated induction of CCR4 functionality remains unclear. Our present results illustrate that functional CCR4 is induced in polarized Th2 cells through the IL-4/STAT6 signaling pathway. Regarding the induction of Th1-type chemokine receptors on developing Th1 cells, our previous studies demonstrated that mouse CCR5 is induced on TCR-triggered T cells as a result of the IL-12/STAT4 signaling pathway [19, 20]. Mouse CXCR3 induction was also shown to depend on IFN-␥ action [21]. It is possible that IFN-␥ signaling occurs through the involvement of IFN-␥-activated STAT1. Taken together, surface expression and function of Th1 (CCR5/CXCR3)- and Th2 (CCR4)-associated chemokine receptors are induced through the signaling of cytokines responsible for the polarization of either Th subset. It is unclear how IL-4-stimulated STAT6, IL-12-activated STAT4, or IFN-␥-activated STAT1 http://www.jleukbio.org
promotes CCR4, CCR5, or CXCR3 gene expression, respectively. Thus, further studies will be required to investigate the mechanisms by which cytokines/STAT proteins responsible for promoting Th2 and Th1 development work to induce the expression of Th2 (CCR4) and Th1 chemokine receptors (CCR5/ CXCR3) as well as their functional maturation.
ACKNOWLEDGMENT The authors thank Mrs. Mami Yasuda for secretarial assistance.
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