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The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4

The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 23, Issue of June 6, pp. 15036 –15042, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4* (Received for publication, December 12, 1996, and in revised form, April 2, 1997) Toshio Imai‡, Masataka Baba, Miyuki Nishimura, Mayumi Kakizaki, Shin Takagi, and Osamu Yoshie From the Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan Thymus and activation-regulated chemokine (TARC) and IP-10 (4), is characterized by the presence of a single amino is a recently identified CC chemokine that is expressed acid separating the first two cysteines. The two cysteines are constitutively in thymus and transiently in stimulated adjacent in the CC chemokine subfamily, which includes RAN- peripheral blood mononuclear cells. TARC functions as TES (5), MCP-1 (6, 7), MCP-2 (8), MCP-3 (9), MCP-4 (10), a selective chemoattractant for T cells that express a MIP-1a (11), MIP-1b (12), I-309 (13), eotaxin (14, 15), HCC-1 class of receptors binding TARC with high affinity and (16), TARC (17), and LARC (18). The CXC chemokines prefer- specificity. To identify the receptor for TARC, we pro- entially attract and activate neutrophils, whereas the CC che- duced TARC as a fusion protein with secreted alkaline mokines usually attract and activate monocytes and also ba- phosphatase (SEAP) and used it for specific binding. By sophils, eosinophils, or lymphocytes with variable selectivity stably transfecting five orphan receptors and five (19). Recently, lymphotactin/single C motif 1 that carries only known CC chemokine receptors (CCR1 to -5) into K562 the second and the fourth of the four cysteine residues con- cells, we found that TARC-SEAP bound selectively to served in other chemokines has been identified, suggesting the cells expressing CCR4. TARC-SEAP also bound to K562 existence of the C type chemokine subfamily (20, 21). The cells stably expressing CCR4 with a high affinity (K 5 human genes for the CXC, CC, and C chemokines are clustered 0.5 nM). Only TARC and not five other CC chemokines on human chromosomes 4, 17, and 1, respectively (1, 22, 23). (MCP-1 (monocyte chemoattractant protein-1), RANTES Recent studies indicate that genes for certain chemokines are (regulated upon activation, normal T cells expressed present outside these clusters. For example, a CXC chemokine and secreted), MIP-1a (macrophage inflammatory pro- SDF-1/PBSF has been mapped to human chromosome 10 (24), tein-1a), MIP-1b, and LARC (liver and activation-regu- and CC chemokines TARC and LARC have been mapped to lated chemokine)) competed with TARC-SEAP for bind- human chromosomes 16 and 2, respectively (18, 25). In addi- ing to CCR4. TARC but not RANTES or MIP-1a induced migration and calcium mobilization in 293/EBNA-1 cells tion to chemotactic activity, some chemokines have a regula- stably expressing CCR4. K562 cells stably expressing tory activity on hematopoiesis and angiogenesis (26 –28). Re- CCR4 also responded to TARC in a calcium mobilization cently, it has been shown that three CC chemokines, MIP-1a, assay. Northern blot analysis revealed that CCR4 mRNA MIP-1b, and RANTES, block infection of macrophage-tropic was expressed strongly in human T cell lines and pe- strains of human immunodeficiency virus type 1, while a CXC ripheral blood T cells but not in B cells, natural killer chemokine, SDF-1/PBSF, blocks infection of T cell line-tropic cells, monocytes, or granulocytes. Taken together, TARC human immunodeficiency virus type 1 strains (29, 30). is a specific functional ligand for CCR4, and CCR4 is the The specific effects of chemokines on target cells are medi- specific receptor for TARC selectively expressed on T ated by seven-transmembrane G-protein-coupled receptors cells. (31). To date, at least five human CC chemokine receptors have been defined for ligand specificity. CCR1 is a receptor for MIP- 1a, RANTES, and MCP-3 (32–35); CCR2 is a receptor for Chemokines are small secreted polypeptides that play im- MCP-1 and MCP-3 (35, 36); CCR3 is a receptor for eotaxin, portant roles in a wide range of inflammatory and immunolog- RANTES, and MCP-3 (14, 37, 38); CCR4 is a receptor for ical processes by recruiting selected subsets of leukocytes (1, 2). MIP-1a, RANTES, and MCP-1 (39); and CCR5 is a receptor for The known chemokines are divided into two major subfamilies MIP-1a, MIP-1b, and RANTES (40 – 42). The specific ligands based on the spacing of the first two cysteines in the conserved for CCR1, CCR2, CCR3, and CCR5 were demonstrated by motif. The CXC chemokine subfamily, which includes IL-8 (3) specific binding and functional assays such as chemotaxis and calcium flux using cDNA-transfected mammalian cells. In the case of CCR4, however, only marginal levels of binding of * The costs of publication of this article were defrayed in part by the MIP-1a and RANTES were shown with HL-60 cells transfected payment of page charges. This article must therefore be hereby marked with CCR4 (43), while a chloride current induction in response “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. to MIP-1a, RANTES, and MCP-1 was demonstrated in CCR4 ‡ To whom correspondence and reprint requests should be addressed. cRNA-injected oocytes (39). Except for CCR3 that is almost Tel.: 81-6-382-2612; Fax: 81-6-382-2598; E-mail: toshio.imai@ exclusively expressed on eosinophils (38, 44), other receptors shionogi.co.jp. were reported to be expressed on monocytes and lymphocytes. The abbreviations used are: IL-8, interleukin 8; IP-10, interferon g-inducible 10-kDa protein; RANTES, regulated upon activation, nor- Notably, CCR4 that was originally cloned from a human baso- mal T cells expressed and secreted; MCP, monocyte chemoattractant philic cell line was shown to be expressed selectively in thymus protein; MIP, macrophage inflammatory protein; TARC, thymus and besides peripheral blood mononuclear cells (PBMCs) (39). activation-regulated chemokine; LARC, liver and activation-regulated chemokine; SDF-1, stromal cell-derived factor 1; PBSF, pre-B-cell growth-stimulating factor; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; PBMC, peripheral blood mononuclear cells; G- FCS, fetal calf serum; PCR, polymerase chain reaction; SEAP, secreted protein, heterotrimeric guanine nucleotide-binding regulatory protein; alkaline phosphatase; NK, natural killer. 15036 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Identification of CCR4 as Specific Receptor for TARC 15037 CAAG-39) and 39 TARC-XbaI primer (259-CGCTCTAGACATGTTCTT- Recently, we have isolated a novel CC chemokine, TARC GACTTTTTTACT-39). After digestion with SalI and XbaI, the TARC (thymus and activation-regulated chemokine) by an efficient cDNA was subcloned into SalI-XbaI sites of pDREF-SEAP(His) -Hyg signal sequence trap using an Epstein-Barr virus vector (17). vector (18) to express TARC as a soluble fusion protein linked through TARC is expressed transiently in phytohemagglutinin-stimu- five amino acid residues (Ser-Arg-Ser-Ser-Gly) with secreted placental lated PBMC and constitutively and selectively in thymus. alkaline phosphatase (SEAP) tagged with six histidine residues TARC induces chemotaxis in certain human T cell lines but not ((His) ). 293/EBNA-1 cells were transfected with the expression vector pDREF-TARC-SEAP(His) using LipofectAMINE (Life Technologies, in monocytes or granulocytes. Pretreatment of cells with Per- 6 Inc.). The transfected cells were incubated for 3– 4 days in Dulbecco’s tussis toxin abolishes cell migration induced by TARC. Radio- modified Eagle’s medium supplemented with 10% FCS. The superna- labeled recombinant TARC specifically binds to T cell lines and tant containing TARC-SEAP(His) was collected by centrifugation, fil- peripheral blood T cells but not to monocytes or granulocytes. tered (0.45 mm), and stored at 4 °C after adding 20 mM Hepes (pH 7.4) The binding of radiolabeled TARC to T cells is competed by and 0.02% sodium azide. For one-step affinity purification of TARC- TARC but not by other chemokines such as IL-8, MIP-1a, SEAP(His) , the supernatant was applied to 1 ml of Hisbond resin (Qiagen, Hilden, Germany). After washing, bound TARC-SEAP(His) RANTES, or MCP-1. These results indicate the existence of a 6 was eluted with 100 mM imidazol. The concentration of TARC-SEA- class of highly specific Pertussis toxin-sensitive G-protein-cou- P(His) was determined by a sandwich type enzyme-linked immunosor- pled receptors for TARC on T cells. In the present study, we bent assay as described previously (18). Briefly, 96-well microtiter have demonstrated that CCR4 is the specific high affinity func- plates (Nunc Maxsorb) were coated with antiplacental alkaline phos- tional receptor for TARC that is expressed selectively on T phatase monoclonal antibody (Medix Biotech, Foster City, CA). After cells. blocking with 1 mg/ml bovine serum albumin (BSA) in phosphate- buffered saline, the samples were titrated and incubated for1hat room EXPERIMENTAL PROCEDURES temperature. After washing, plates were incubated with biotinylated Cells—Human hematopoietic cell lines were maintained in RPMI rabbit antiplacental alkaline phosphatase diluted 1:500 for1hat room 1640 supplemented with 10% fetal calf serum (FCS). The murine pre-B temperature, washed again, and incubated with peroxidase-conjugated cell line L1.2 was kindly provided by Dr. Craig Gerard (Harvard Med- streptavidin (Vector) for 30 min. After washing, bound peroxidase was ical School, Boston, MA) and maintained in RPMI 1640 supplemented reacted with 3,39-5,59-tetramethylbenzidine. Reaction was stopped by with 10% FCS. 293/EBNA-1 cells were purchased from Invitrogen (San adding 1 N H SO , and absorbance at 450 nm was measured. Alkaline 2 4 Diego, CA) and maintained in Dulbecco’s modified Eagle’s medium phosphatase activity was determined by a chemiluminescent assay supplemented with 10% FCS. PBMCs were isolated from venous blood using the Great EscApe detection kit (CLONTECH). Purified antipla- obtained from healthy adult donors using Ficoll-Paque (Pharmacia, cental alkaline phosphatase (Cosmo Bio) was used to generate a stand- Uppsala, Sweden). Monocytes were stained with fluorescein isothiocya- ard curve. The enzymatic activity was expressed as relative light nate-conjugated anti-CD14 and positively selected by MACS (Miltenyi units/s. The SEAP(His) and TARC-SEAP(His) used in the present 6 6 Biotec, Bergisch, Germany). B cells were stained with fluorescein iso- study had a specific activity of approximately 8.88 3 10 relative light thiocyanate-conjugated anti-CD19 and positively selected by MACS. T units/s and 1.23 3 10 relative light units/s per pmol, respectively. cells were stained with fluorescein isothiocyanate-conjugated anti-CD3 Binding Assay—For displacement experiments, cells were incubated and positively selected by MACS. Natural killer (NK) cells were sorted for1hat16 °C with 1 nM of SEAP(His) or TARC-SEAP(His) in the 6 6 by FACStar Plus (Beckton Dickinson, Mountain View, CA) as CD16 or presence of increasing concentrations of unlabeled chemokines in 200 ml 1 2 CD56 and CD3 cells with appropriate forward and side scatters. of RPMI 1640 containing 20 mM Hepes (pH 7.4), 1% BSA, and 0.02% 1 1 Purification of CD4 and CD8 T cells from PBMCs was carried out by sodium azide. For saturation experiments, cells were incubated for 1 h negative selection using Dynabeads (Dynal, Oslo, Norway) after incu- at 16 °C with increasing concentrations of TARC-SEAP(His) in the bation of PBMCs with anti-CD16, anti-CD14, anti-CD20, and anti-CD8 presence or absence of 1 mM unlabeled TARC. After incubation, cells or anti-CD4, respectively. Granulocytes were obtained from the pellet were washed five times and lysed in 50 mlof10mM Tris-HCl (pH 8.0), fraction of Ficoll-Paque gradient by dextran sedimentation and hypo- 1% Triton X-100. Samples were heated at 65 °C for 10 min to inactivate tonic lysis of erythrocytes. The purity of each cell population was always cellular phosphatases. Lysates were collected by centrifugation, and in the range of 95–99% as determined by flow cytometric analysis or by alkaline phosphatase activity in 25 ml of lysate was determined by the staining with Diff-Quik (Baxter Scientific Products, McGaw Park, IL). chemiluminescent assay described above. For direct binding experi- Construction of Receptor Expression Plasmids—The cDNA fragments ments, cells were incubated for1hat16 °C with 0.1 nM of I-RAN- 125 125 covering the open reading frames for various chemokine receptors and TES, I-MCP-1, I-MIP-1a, or TARC-SEAP(His) without or with orphan receptors were obtained as follows. Cloning of CCR3 cDNA was 200 nM of unlabeled chemokines in 200 ml of low salt binding buffer (50 described previously (14). EBI1 and BLR1 cDNA were isolated by mM Hepes, pH 7.5, 1 mM CaCl ,5mM MgCl , 0.5% BSA, and 0.05% 2 2 screening a phytohemagglutinin-stimulated human PBMC cDNA li- sodium azide). The cells were then washed five times with low salt brary. Other receptors were cloned from a human PBMC cDNA library binding buffer containing 0.5 M NaCl. All assays were done in duplicate. or human genomic library by polymerase chain reaction (PCR). The Binding data were analyzed by the LIGAND program (45). primers were designed using the sequences from the following Gen- Migration Assay—Cell migration was assayed by using a 48-well TM Bank submissions: CCR1 (L09320), CCR2B (U03905), CCR4 microchemotaxis chamber as described previously (17, 34). In brief, (X85740), CCR5 (X91492), CXCR4 (M99293), V28 (U20350), GPR-CY4 chemoattractants were diluted in Hepes-buffered RPMI 1640 supple- (U45984), GPR-9-6 (U45982), EBI1 (L31581), BLR1 (X68149). The frag- mented with 1% BSA and placed in lower wells (25 ml/well). Cells were ments were cloned into an expression vector pDREF-Hyg (17) for effi- suspended in the same medium at 2 3 10 cells/ml and added to upper cient expression in 293/EBNA-1 cells and Raji cells, or pCAGG-Neo wells (50 ml/well). The upper and lower wells were separated by a (kindly provided by T. Nakajima) for expression in K562 cells and L1.2 polyvinylpyrrolidone-free polycarbonate filter with 8-mm pores that was cells. precoated with type IV collagen (34). The chamber was incubated for 4 h Stable Transfection—For stable expression in Raji cells, cells were at 37 °C in 5% CO , 95% air. Filters were removed and stained with transfected by electroporation as described previously (17). For stable Diff-Quik. Each sample was assayed in triplicate, and migrated cells expression in 293/EBNA-1 cells, 1 3 10 cells were plated onto 100-mm were counted in five randomly selected high power fields (3 400) per dishes. After 12–20 h, cells were transfected with recombinant plasmids well. (10 mg each) using LipofectAMINE (Life Technologies, Inc.). After se- Calcium Mobilization Assay—Cells were suspended at 3 3 10 lection with 250 mg/ml hygromycin for 1–2 weeks, drug-resistant cells cells/ml in Hank’s balanced salt solution supplement with 1 mg/ml BSA were pooled and used for experiments. For stable expression in K562 and 10 mM HEPES, pH 7.4. Cell were incubated with 1 mM fura- cells and L1.2 cells, 1 3 10 cells in 500 ml of phosphate-buffered saline PE3-AM (Texas Fluorescence Labs) at room temperature for1hinthe were electroporated with 10 mg of linearized plasmid at 260 V, 960 dark. After washing twice, cells were resuspended at 2.5 3 10 cells/ml. microfarads using a Gene Pulser (Bio-Rad). After selection with 800 To measure intracellular calcium, cells in 2 ml were placed in a quartz mg/ml of G418 for 1–2 weeks, cells expressing transfected receptors at cuvette in a Perkin-Elmer LS 50B spectrofluorimeter. Fluorescence was high levels were identified by binding assays and/or Northern blot monitored at 340 nm (lex1), 380 nm (lex2), and 510 nm (lem) every 200 analysis and cloned by limiting dilution. ms. A dose-response curve was generated in each experiment, and Production of TARC-SEAP Fusion Protein—The DNA fragment en- results were expressed as percentage of maximum response. coding TARC was amplified from clone D3A (17) by PCR using 59 Northern Blot Analysis—Total RNAs were prepared from various SalI-TARC primer (159-CGCGTCGACAAAACCATGTGCTGTAC- subsets of peripheral blood leukocytes using Trizol (Life Technologies, 15038 Identification of CCR4 as Specific Receptor for TARC FIG.1. Expression of TARC-SEAP(His) fusion protein. A, sche- matic diagram of the SEAP(His) tag vector, pDREF-SEAP(His) , with 6 6 the inserted TARC cDNA fragment. pDREF-SEAP(His) contains the EF-1a promoter, the coding sequence for SEAP(His) , the hygromycin- resistant gene (hyg ), the EBNA-1 gene, and the Epstein-Barr virus origin for episomal replication (oriP). TARC cDNA was inserted be- tween SalI and XbaI sites and expressed as a fusion protein with SEAP FIG.2. Binding characteristics of TARC-SEAP(His) to Hut78 and the (His) tag. B, purified TARC-SEAP(His) fusion protein. TARC- 6 6 cells. A, displacement of TARC-SEAP(His) with unlabeled TARC. SEAP(His) was purified from culture supernatants of transfected 293/ Hut78 cells (4 3 10 cells) were incubated with 1 nM TARC-SEAP(His) EBNA-1 cells by metal affinity chromatography, subjected to electro- in the presence of the indicated concentrations of unlabeled TARC. phoresis on a 4 –20% polyacrylamide gel, and stained with Coomassie Representative results from three separate experiments are shown. B, Brilliant Blue. Positions of size markers (kDa) are shown on the right. 5 Hut78 cells (2 3 10 cells) were incubated with 1 nM TARC-SEAP(His) in the absence or presence of 200 nM unlabeled TARC, MCP-1, RAN- Inc.). RNA samples (5 mg each) were fractionated by electrophoresis on TES, MIP-1a, MIP-1b, or LARC. The percentage of inhibition of specific a 1% agarose gel containing 0.66 M formaldehyde. Gels were blotted binding by unlabeled ligands was calculated after subtracting nonspe- cific binding estimated with 1 nM SEAP(His) . Each histogram repre- onto a filter membrane (Hybond N ) (Amersham Japan, Tokyo). The sents the mean 6 S.E. from three separate experiments. probe was the SmaI-PstI fragment of clone D3A (17). Hybridization was carried out at 65 °C in QuickHyb solution (Stratagene) with probes labeled with P using Prime it II (Stratagene). After washing at 55 °C shown). These results may indicate that Hut78 expresses at with 0.2 3 SSC and 0.1% SDS, filters were exposed to x-ray films at least two types of TARC receptors, one highly specific for TARC 280 °C with an intensifying screen. and another shared by RANTES and LARC. At any rate, the RESULTS present results were highly consistent with those obtained previously by using I-labeled TARC. Thus, TARC-SEA- Use of TARC-Alkaline Phosphatase Fusion Protein for Recep- P(His) was proven to retain its ability as a specific high affin- tor Binding Assay—Chemokines often have a tendency for self- 6 ity ligand for TARC receptors. aggregation at physiological pH and tonicity that interferes CCR4 Is a High Affinity Specific Receptor for TARC—Since with their receptor studies (46, 47). To circumvent this prob- TARC was shown not to share the major class of its high lem, Luster et al. generated IP-10 fused with SEAP and showed affinity binding sites on T cells with other CC chemokines that that the fusion protein retained its ability to interact specifi- are known to bind to one or more CCRs (17), we first tested the cally with cell surface receptors without self-aggregation in binding of TARC-SEAP(His) to orphan receptors such as EBI1 physiological buffers (46). To prepare labeled TARC convenient 6 (48), BLR1 (49), V28 (50), GPR-9 – 6 (GenBank accession num- for receptor binding experiments, we decided to adopt this ber: HSU45982), GPR-CY4 (GenBank accession number: method and expressed TARC as a fusion protein with SEAP HSU45984), and LESTR/fusin (51). LESTR/fusin is now known with the (His) tag (Fig. 1A). The alkaline phosphatase activity as the receptor for SDF-1/PBSF (CXCR4) (52, 53). TARC-SEA- allowed quantitative determination of specific binding, P(His) showed no significant binding to Raji, 293/EBNA-1, or whereas the (His) tag in the C terminus was used for one-step K562 cells stably transfected with any of these orphan recep- affinity purification with a nickel agarose column. TARC-SEA- tors (data not shown). In parallel experiments, we also exam- P(His) was expressed in 293/EBNA-1 cells and purified as a ined the binding of TARC-SEAP(His) to the five CC chemokine single major protein with an apparent molecular mass of 74 6 receptors that are known to be shared by several CC chemo- kDa (Fig. 1B). Previously, we showed that I-labeled TARC kines (CCR1 to -5). Surprisingly, TARC-SEAP(His) specifi- specifically bound to a single class of receptors expressed on a 6 cally bound to CCR4 expressed on K562 (Fig. 3), Raji, and human T cell line Jurkat with a K of 2.1 nM (17). Importantly, 293/EBNA-1 (not shown). To further characterize the binding the binding was competed only by TARC and not by any other of TARC to CCR4, experiments were performed using K562 chemokines so far tested. To verify specificity and affinity of cells stably expressing CCR4. When the binding was performed TARC-SEAP(His) for TARC receptors, we carried out TARC- with increasing concentrations of TARC-SEAP(His) (Fig. 4A), SEAP(His) binding experiments using another human T cell a single class of receptors with a K of 0.5 nM and 29,000 line Hut78. The binding of TARC-SEAP(His) was fully com- d sites/cell was demonstrated (Fig. 4B). Competition experiments peted by unlabeled TARC with an IC of 0.5 nM (Fig. 2A). CC showed that unlabeled TARC fully competed with TARC-SEA- chemokines such as MCP-1, MIP-1a, or MIP-1b did not show P(His) for CCR4 (Fig. 4C). In contrast, other CC chemokines any significant competition with TARC-SEAP(His) (Fig. 2B). such as RANTES, MCP-1, MIP-1a, MIP-1b, and LARC showed RANTES and LARC, however, partially (,30%) inhibited TARC-SEAP(His) binding. Such partial inhibition by heterol- ogous chemokines was not, however, seen with Jurkat using We also confirmed that TARC-SEAP(His) induced calcium flux in either I-labeled TARC (17) or TARC-SEAP(His) (data not CCR4-transfected K562 cells with similar potency as TARC. 6 Identification of CCR4 as Specific Receptor for TARC 15039 FIG.3. Selective binding of TARC-SEAP(His) to CCR4-trans- fected cells. K562 cells were stably transfected with the vector or plasmids expressing indicated receptors. Clones expressing transfected receptors at high levels were selected by binding assays and/or North- ern blot analysis. Cells (1 3 10 cells) were incubated for1hat16 °C with 1 nM of TARC-SEAP(His) and washed, and bound TARC-SEA- P(His) was determined. Each histogram represents the mean 6 S.E. of total binding. 125 125 FIG.5. Direct binding of TARC-SEAP(His) , I-MCP-1, I- RANTES, and I-MIP-1a to endogenous receptors and CCR4. L1.2 cells transfected with CCR4 (5 3 10 cells) were incubated for 1 h at 16 °C with 0.1 nM of TARC-SEAP(His) (A), I-labeled MCP-1 (B), 125 125 I-labeled RANTES (C), or I-labeled MIP-1a (D) in the absence (filled bars) or presence (open bars) of unlabeled chemokines. For pos- itive controls, cells expressing endogenous receptors, Hut78 (A)or THP-1 (B–D), were used. Each histogram represents the mean 6 S.E. of total binding. Representative results from two separate experiments are shown. cally bound to CCR4-transfected L1.2 cells at levels about 20-fold higher than those obtained with Hut78 cells that ex- press endogenous TARC receptors at 1000 –2000 sites/cell (Fig. 2). In contrast, RANTES, MCP-1, or MIP-1a bound to CCR4- transfected L1.2 cells at marginal levels, if any, although these chemokines efficiently bound to THP-1 cells that express en- dogenous receptors at 1000 –5000 sites/cell (1). These results FIG.4. Binding characteristics of TARC-SEAP(His) to CCR4- further strengthen the possibility that TARC is the physiolog- transfected cells. A, saturable binding of TARC-SEAP(His) to CCR4- ical ligand for CCR4. transfected K562 cells. Cells (4 3 10 cells) were incubated for1hat TARC Induces Chemotaxis in CCR4-transduced Cells—Pre- 16 °C with the indicated concentrations of TARC-SEAP(His) . Nonspe- viously, we showed that TARC induced chemotaxis in certain cific binding determined by the addition of a 100-fold molar excess of human T cell lines (17). We therefore examined whether TARC TARC was subtracted. Representative results from three separate ex- periments are shown. B, Scatchard analysis of the binding data in panel also induced migration of cells expressing transfected CCR4. A. The calculated K is 0.5 nM. C, displacement of binding of TARC- 293/EBNA-1 cells were stably transfected with CCR4 or CCR1, SEAP(His) to CCR4-transfected K562 cells with unlabeled TARC. and migration of these cells to TARC, MIP-1a, and RANTES Cells (4 3 10 cells) were incubated with 1 nM TARC-SEAP(His) in the was examined. As shown in Fig. 6, TARC induced migration of presence of indicated concentrations of unlabeled TARC. Representa- tive results from three separate experiments are shown. D, competition cells transfected with CCR4 but not those transfected with of the binding of TARC-SEAP(His) to CCR4-transfected K562 cells by CCR1. On the other hand, MIP-1a and RANTES induced mi- unlabeled TARC and other chemokines. Cells (2 3 10 cells) were gration of cells transfected with CCR1 but not those transfected incubated for 1 h at 16 °C with 1 nM TARC-SEAP(His) in the absence with CCR4. Parental 293/EBNA-1 cells or those transfected or presence of 200 nM unlabeled TARC, MCP-1, RANTES, MIP-1a, MIP-1b, or LARC. The percentage of inhibition of specific binding by with the vector alone did not respond to TARC, MIP-1a,or unlabeled ligands was calculated after subtracting nonspecific binding RANTES (data not shown). Migration of CCR4-transfected 293/ estimated with 1 nM SEAP(His) . Each histogram represents the EBNA-1 cells to TARC was concentration-dependent, being mean 6 S.E. from three separate experiments. promoted at concentrations from 10 to 1000 ng/ml. Desensiti- zation was observed at 10 mg/ml (not shown). A checkerboard no significant competition with TARC-SEAP(His) for CCR4 analysis indicated that the migration of CCR4-transfected 293/ (Fig. 4D). Similar results were obtained using Raji or 293/ EBNA-1 cells toward TARC was mostly chemotactic but partly EBNA-1 cells stably transfected with CCR4 (data not shown). (;40%) chemokinetic (data not shown). Collectively, TARC but Collectively, the binding characteristics of TARC-SEAP(His) not MIP-1a or RANTES is a functional ligand for CCR4. to CCR4 were highly consistent with those obtained with the TARC Induces Calcium Mobilization in CCR4-transduced major class of the endogenous TARC receptors expressed on T Cells—We next examined induction of calcium mobilization in cells (Fig. 2) (17). K562 cells expressing CCR4, CCR1, or CCR2B (Fig. 7A). TARC We next compared direct binding of TARC-SEAP(His) , induced calcium flux in K562 cells expressing CCR4, whereas RANTES, MCP-1, and MIP-1a to CCR4-transfected cells (Fig. RANTES, MIP-1a, or MCP-1 did not. In addition, TARC, but 5). In these experiments, murine L1.2 cells transfected with not RANTES, MIP-1a, or MCP-1, was able to desensitize the CCR4 were used (38). As expected, TARC-SEAP(His) specifi- cells expressing CCR4 for subsequent stimulation with TARC 6 15040 Identification of CCR4 as Specific Receptor for TARC FIG.6. Chemotactic response of CCR4-transfected cells to TARC. Migration of CCR4- or CCR1-transfected 293/EBNA-1 cells to FIG.7. Calcium mobilization in CCR4-transfected cells stimu- TARC (closed circles), MIP-1a (closed triangle), RANTES (open lated with TARC. A, CCR4-, CCR1-, or CCR2B-transfected K562 cells squares), and medium (closed squares) were assayed in a 48-well che- were loaded with fura-PE3-AM and stimulated with TARC (100 nM), motaxis chamber. The assay was done in triplicate, and the number of MIP-1a (100 nM), RANTES (100 nM), and MCP-1 (100 nM). The arrow- migrating cells in five high power fields (3 400) were counted for each heads indicate the time of application of chemokine. Intracellular con- well. Each point represents the mean 6 S.E. from three separate centration of calcium were monitored by the fluorescence ratio (F340/ experiments. F380). Representative results from at least two separate experiments are shown. B, the concentration dependence of calcium mobilization induced by TARC. CCR4-transfected K562 cells were loaded with fura- PE3-AM and stimulated with the indicated concentrations of TARC. (Fig. 7A and data not shown). On the other hand, K562 cells Results are expressed as percentage of maximum response. Each point expressing CCR1 responded to MIP-1a but not to TARC, represents the mean 6 S.E. from three separate experiments. whereas K562 cells expressing CCR2B responded to MCP-1 but not to TARC. Parental K562 cells or those transfected with the vector alone did not show any response to TARC, RANTES, MIP-1a, or MCP-1 (data not shown). Responses to TARC were detectable above 1 nM, and maximum values were obtained at 100 nM with an EC of8nM (Fig. 7B). We further confirmed that TARC at 100 nM did not induce calcium fluxes in CCR1, CCR2B, CCR3, or CCR5-transfected K562 cells, while MIP-1a induced calcium fluxes in CCR1- and CCR5-transfected cells, MCP-1 in CCR2B-transfected cells, and eotaxin in CCR3-trans- fected cells (data not shown). Similar results were obtained by using 293/EBNA-1 cells. These observations again demon- strated that TARC is a functional ligand for CCR4. CCR4 Is Expressed Mainly in CD4 T Cells—Previously, we showed that high levels of binding sites for TARC were de- tected on certain T cell lines and peripheral blood T cells but not on peripheral monocytes or granulocytes (17). To determine FIG.8. Northern blot analysis for CCR4 mRNA expression. the cell types that express CCR4, we performed Northern blot Total RNA samples (5 mg/lane) were subjected to Northern blot analysis using the P-labeled CCR4 probe. The autoradiographs of the filter analysis. As shown in Fig. 8A, CCR4 mRNA was strongly (top) and photographs of the gel stained with ethidium bromide (bottom) expressed in T cell lines such as Hut78, Hut102, and Jurkat, all are shown. Positions of size markers (kb) are shown on the left. A, having been shown to display high levels of TARC binding sites expression of CCR4 mRNA in various human cell lines. Molt4, Jurkat, (17). A basophilic cell line KU812 that was originally used to and Hut78 are human T-cell leukemia virus type 1-negative T cell lines. Hut102 is a human T-cell leukemia virus type 1-positive T cell line. Raji clone CCR4 cDNA (39) and a megakaryocytic cell line MEG-1 and Daudi are Epstein-Barr virus-positive B cell lines. U937 and THP-1 were also found to express CCR4 mRNA. On the other hand, are monocytoid cell lines. K562 is an erythroid cell line. HL60 is a CCR4 mRNA was undetectable in THP-1, U937, Raji, K562, promyelocytic cell line. KU812 is a basophilic cell line. MEG1 is a and HL-60, all having been shown to possess negligible, if any, megakaryocytic cell line. B, expression of CCR4 mRNA in various 1 1 human peripheral blood leukocytes: CD4 T cells (T4), CD8 T cells binding sites for TARC (17). In normal peripheral blood mono- (T8), total T cells (T), B cells (B), NK cells (NK), monocytes (Mo), and nuclear cells (Fig. 8B), CCR4 mRNA was expressed in T cells, granulocytes (Gr). especially CD4 T cells, but not in B cells, NK cells, monocytes, DISCUSSION or granulocytes. The expression pattern of CCR4 is thus highly consistent with the T cell-selective expression of the endoge- Although a number of chemokines are known to act on T cells nous TARC receptor described previously (17). (54 – 61), TARC appears to be the first CC chemokine highly Identification of CCR4 as Specific Receptor for TARC 15041 selective for T cells (17). High levels of constitutive expression pressed on T cells, CCR4 appears to be the first CC chemokine receptor highly selective for T cells. By Northern blot analysis, of TARC have been detected only in thymus and not in spleen. High levels of specific binding sites for TARC have been de- CCR4 mRNA was detected highly selectively in some T cell lines and peripheral blood T cells, especially CD4 T cells, but tected on some T cell lines and peripheral blood T cells but not not in B cells, NK cells, monocytes, or granulocytes (Fig. 8). on monocytes or granulocytes. TARC has been shown to induce Previously, Power et al. (39) also demonstrated by Northern chemotaxis in certain human T cell lines. Furthermore, a class blot analysis that CCR4 was expressed strongly in thymus and of TARC receptors expressed on T cells is highly specific for peripheral blood leukocytes but very weakly in spleen. How- TARC and not shared by any other CC or CXC chemokines that ever, they further demonstrated by reverse transcriptase-PCR are known to act on T cells (17). Here we have presented analysis that CCR4 was expressed not only in T cells but also several lines of evidence indicating that CCR4 is the major in B cells and monocytes. Taken together, T cells are the cells class of receptors for TARC that is selectively expressed on T that express CCR4 at high levels, but other types of leukocytes cells and not shared by other CC or CXC chemokines. may also express CCR4 at low levels detectable by reverse CCR4 was originally cloned by Power et al. from a human transcriptase-PCR. The selective expression of CCR4 on T cells basophilic cell line KU-812 (39). The CC chemokines, MIP-1a, is thus consistent with the fact that T cells are the major target RANTES, and MCP-1, were presumed to be the functional of TARC. Furthermore, the fact that TARC and CCR4 are both ligands for CCR4, because among various chemokines, only constitutively and strongly expressed in thymus further sup- these were able to activate a calcium-dependent chloride chan- ports their important roles in trafficking and education of thy- nel in Xenopus laevis oocytes injected with CCR4 cRNA. How- mocytes within the thymus. ever, induction of chemotaxis or calcium flux in CCR4-trans- Notably, we also detected expression of CCR4 in a basophilic fected mammalian cells by MIP-1a, RANTES, MCP-1, or any cell line KU812 and a megakaryocytic cell line MEG-1 (Fig. 8). other chemokines has not been demonstrated. Here we have In fact, CCR4 was originally cloned from KU812, and, by using shown that introduction of the CCR4 cDNA into Raji, 293/ reverse transcriptase-PCR, Power et al. (39) demonstrated ex- EBNA-1, and K562 cells induced a class of high affinity binding pression of CCR4 in fresh basophils, especially after brief treat- sites for TARC (Figs. 3 and 4). Binding of TARC to CCR4 was ment with IL-5. They also mentioned that platelets contain competed only by TARC and not by any other chemokines high levels of CCR4 mRNA (39, 64). We have also detected high including MIP-1a, RANTES, and MCP-1 (Fig. 4). Binding of 125 levels of TARC-binding sites on platelets (data not shown). I-labeled RANTES, MCP-1, or MIP-1a to CCR4 were mar- Thus, it is clear that cells of the megakaryocyte/platelet lineage ginal, if any, while they bound to endogenous receptors ex- also express CCR4. It remains to be seen whether TARC affects pressed on THP-1 cells efficiently. (Fig. 5). Furthermore, only differentiation and/or function of basophils and megakaryo- TARC but not RANTES, MCP-1, or MIP-1a induced chemo- cytes/platelets besides T cells. taxis in CCR4-transfected 293/EBNA-1 and induced calcium It is also noteworthy that high levels of CCR4 expression are flux in CCR4-transfected 293/EBNA-1 and K562 (Figs. 6 and observed only in certain human T cell lines and peripheral 7). Collectively, these results have clearly demonstrated that blood T cells of especially the CD4 type. CCR4 may thus be TARC is the specific functional ligand for CCR4. The discrep- expressed selectively in particular subsets of T cells and/or in ancy between Power et al. (39) and the present study is prob- particular stages of differentiation and/or activation of T cells. ably due to the assay system used in the former study, i.e. Cultured CD45RO T cells were shown to express CCR1 and activation of a calcium-dependent chloride channel in Xenopus CCR2 in a strictly IL-2-dependent manner (65). Both CD4 and laevis oocytes injected with CCR4 cRNA. Besides CCR4, how- CD8 T cells were shown to constitutively express CCR5 (41). ever, some T cells may also express a class of receptors for Activated T cells were shown to express CXCR3, the first T TARC that is shared by RANTES and LARC (Fig. 2). cell-selective CXC chemokine receptor shared by IP-10 and Mig Compared with the high affinity value obtained from binding (66). It is thus important to determine phenotypes of T cells of TARC-SEAP(His) to CCR4 (K 5 0.5 nM), the potency of 6 d that express CCR4 and conditions that regulate CCR4 expres- TARC in induction of chemotaxis in CCR4-transfected 293/ sion. 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The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4

Imai, Toshio; Baba, Masataka; Nishimura, Miyuki; Kakizaki, Mayumi; Takagi, Shin; Yoshie, Osamu
Journal of Biological Chemistry – Jun 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 23, Issue of June 6, pp. 15036 –15042, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4* (Received for publication, December 12, 1996, and in revised form, April 2, 1997) Toshio Imai‡, Masataka Baba, Miyuki Nishimura, Mayumi Kakizaki, Shin Takagi, and Osamu Yoshie From the Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan Thymus and activation-regulated chemokine (TARC) and IP-10 (4), is characterized by the presence of a single amino is a recently identified CC chemokine that is expressed acid separating the first two cysteines. The two cysteines are constitutively in thymus and transiently in stimulated adjacent in the CC chemokine subfamily, which includes RAN- peripheral blood mononuclear cells. TARC functions as TES (5), MCP-1 (6, 7), MCP-2 (8), MCP-3 (9), MCP-4 (10), a selective chemoattractant for T cells that express a MIP-1a (11), MIP-1b (12), I-309 (13), eotaxin (14, 15), HCC-1 class of receptors binding TARC with high affinity and (16), TARC (17), and LARC (18). The CXC chemokines prefer- specificity. To identify the receptor for TARC, we pro- entially attract and activate neutrophils, whereas the CC che- duced TARC as a fusion protein with secreted alkaline mokines usually attract and activate monocytes and also ba- phosphatase (SEAP) and used it for specific binding. By sophils, eosinophils, or lymphocytes with variable selectivity stably transfecting five orphan receptors and five (19). Recently, lymphotactin/single C motif 1 that carries only known CC chemokine receptors (CCR1 to -5) into K562 the second and the fourth of the four cysteine residues con- cells, we found that TARC-SEAP bound selectively to served in other chemokines has been identified, suggesting the cells expressing CCR4. TARC-SEAP also bound to K562 existence of the C type chemokine subfamily (20, 21). The cells stably expressing CCR4 with a high affinity (K 5 human genes for the CXC, CC, and C chemokines are clustered 0.5 nM). Only TARC and not five other CC chemokines on human chromosomes 4, 17, and 1, respectively (1, 22, 23). (MCP-1 (monocyte chemoattractant protein-1), RANTES Recent studies indicate that genes for certain chemokines are (regulated upon activation, normal T cells expressed present outside these clusters. For example, a CXC chemokine and secreted), MIP-1a (macrophage inflammatory pro- SDF-1/PBSF has been mapped to human chromosome 10 (24), tein-1a), MIP-1b, and LARC (liver and activation-regu- and CC chemokines TARC and LARC have been mapped to lated chemokine)) competed with TARC-SEAP for bind- human chromosomes 16 and 2, respectively (18, 25). In addi- ing to CCR4. TARC but not RANTES or MIP-1a induced migration and calcium mobilization in 293/EBNA-1 cells tion to chemotactic activity, some chemokines have a regula- stably expressing CCR4. K562 cells stably expressing tory activity on hematopoiesis and angiogenesis (26 –28). Re- CCR4 also responded to TARC in a calcium mobilization cently, it has been shown that three CC chemokines, MIP-1a, assay. Northern blot analysis revealed that CCR4 mRNA MIP-1b, and RANTES, block infection of macrophage-tropic was expressed strongly in human T cell lines and pe- strains of human immunodeficiency virus type 1, while a CXC ripheral blood T cells but not in B cells, natural killer chemokine, SDF-1/PBSF, blocks infection of T cell line-tropic cells, monocytes, or granulocytes. Taken together, TARC human immunodeficiency virus type 1 strains (29, 30). is a specific functional ligand for CCR4, and CCR4 is the The specific effects of chemokines on target cells are medi- specific receptor for TARC selectively expressed on T ated by seven-transmembrane G-protein-coupled receptors cells. (31). To date, at least five human CC chemokine receptors have been defined for ligand specificity. CCR1 is a receptor for MIP- 1a, RANTES, and MCP-3 (32–35); CCR2 is a receptor for Chemokines are small secreted polypeptides that play im- MCP-1 and MCP-3 (35, 36); CCR3 is a receptor for eotaxin, portant roles in a wide range of inflammatory and immunolog- RANTES, and MCP-3 (14, 37, 38); CCR4 is a receptor for ical processes by recruiting selected subsets of leukocytes (1, 2). MIP-1a, RANTES, and MCP-1 (39); and CCR5 is a receptor for The known chemokines are divided into two major subfamilies MIP-1a, MIP-1b, and RANTES (40 – 42). The specific ligands based on the spacing of the first two cysteines in the conserved for CCR1, CCR2, CCR3, and CCR5 were demonstrated by motif. The CXC chemokine subfamily, which includes IL-8 (3) specific binding and functional assays such as chemotaxis and calcium flux using cDNA-transfected mammalian cells. In the case of CCR4, however, only marginal levels of binding of * The costs of publication of this article were defrayed in part by the MIP-1a and RANTES were shown with HL-60 cells transfected payment of page charges. This article must therefore be hereby marked with CCR4 (43), while a chloride current induction in response “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. to MIP-1a, RANTES, and MCP-1 was demonstrated in CCR4 ‡ To whom correspondence and reprint requests should be addressed. cRNA-injected oocytes (39). Except for CCR3 that is almost Tel.: 81-6-382-2612; Fax: 81-6-382-2598; E-mail: toshio.imai@ exclusively expressed on eosinophils (38, 44), other receptors shionogi.co.jp. were reported to be expressed on monocytes and lymphocytes. The abbreviations used are: IL-8, interleukin 8; IP-10, interferon g-inducible 10-kDa protein; RANTES, regulated upon activation, nor- Notably, CCR4 that was originally cloned from a human baso- mal T cells expressed and secreted; MCP, monocyte chemoattractant philic cell line was shown to be expressed selectively in thymus protein; MIP, macrophage inflammatory protein; TARC, thymus and besides peripheral blood mononuclear cells (PBMCs) (39). activation-regulated chemokine; LARC, liver and activation-regulated chemokine; SDF-1, stromal cell-derived factor 1; PBSF, pre-B-cell growth-stimulating factor; CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; PBMC, peripheral blood mononuclear cells; G- FCS, fetal calf serum; PCR, polymerase chain reaction; SEAP, secreted protein, heterotrimeric guanine nucleotide-binding regulatory protein; alkaline phosphatase; NK, natural killer. 15036 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Identification of CCR4 as Specific Receptor for TARC 15037 CAAG-39) and 39 TARC-XbaI primer (259-CGCTCTAGACATGTTCTT- Recently, we have isolated a novel CC chemokine, TARC GACTTTTTTACT-39). After digestion with SalI and XbaI, the TARC (thymus and activation-regulated chemokine) by an efficient cDNA was subcloned into SalI-XbaI sites of pDREF-SEAP(His) -Hyg signal sequence trap using an Epstein-Barr virus vector (17). vector (18) to express TARC as a soluble fusion protein linked through TARC is expressed transiently in phytohemagglutinin-stimu- five amino acid residues (Ser-Arg-Ser-Ser-Gly) with secreted placental lated PBMC and constitutively and selectively in thymus. alkaline phosphatase (SEAP) tagged with six histidine residues TARC induces chemotaxis in certain human T cell lines but not ((His) ). 293/EBNA-1 cells were transfected with the expression vector pDREF-TARC-SEAP(His) using LipofectAMINE (Life Technologies, in monocytes or granulocytes. Pretreatment of cells with Per- 6 Inc.). The transfected cells were incubated for 3– 4 days in Dulbecco’s tussis toxin abolishes cell migration induced by TARC. Radio- modified Eagle’s medium supplemented with 10% FCS. The superna- labeled recombinant TARC specifically binds to T cell lines and tant containing TARC-SEAP(His) was collected by centrifugation, fil- peripheral blood T cells but not to monocytes or granulocytes. tered (0.45 mm), and stored at 4 °C after adding 20 mM Hepes (pH 7.4) The binding of radiolabeled TARC to T cells is competed by and 0.02% sodium azide. For one-step affinity purification of TARC- TARC but not by other chemokines such as IL-8, MIP-1a, SEAP(His) , the supernatant was applied to 1 ml of Hisbond resin (Qiagen, Hilden, Germany). After washing, bound TARC-SEAP(His) RANTES, or MCP-1. These results indicate the existence of a 6 was eluted with 100 mM imidazol. The concentration of TARC-SEA- class of highly specific Pertussis toxin-sensitive G-protein-cou- P(His) was determined by a sandwich type enzyme-linked immunosor- pled receptors for TARC on T cells. In the present study, we bent assay as described previously (18). Briefly, 96-well microtiter have demonstrated that CCR4 is the specific high affinity func- plates (Nunc Maxsorb) were coated with antiplacental alkaline phos- tional receptor for TARC that is expressed selectively on T phatase monoclonal antibody (Medix Biotech, Foster City, CA). After cells. blocking with 1 mg/ml bovine serum albumin (BSA) in phosphate- buffered saline, the samples were titrated and incubated for1hat room EXPERIMENTAL PROCEDURES temperature. After washing, plates were incubated with biotinylated Cells—Human hematopoietic cell lines were maintained in RPMI rabbit antiplacental alkaline phosphatase diluted 1:500 for1hat room 1640 supplemented with 10% fetal calf serum (FCS). The murine pre-B temperature, washed again, and incubated with peroxidase-conjugated cell line L1.2 was kindly provided by Dr. Craig Gerard (Harvard Med- streptavidin (Vector) for 30 min. After washing, bound peroxidase was ical School, Boston, MA) and maintained in RPMI 1640 supplemented reacted with 3,39-5,59-tetramethylbenzidine. Reaction was stopped by with 10% FCS. 293/EBNA-1 cells were purchased from Invitrogen (San adding 1 N H SO , and absorbance at 450 nm was measured. Alkaline 2 4 Diego, CA) and maintained in Dulbecco’s modified Eagle’s medium phosphatase activity was determined by a chemiluminescent assay supplemented with 10% FCS. PBMCs were isolated from venous blood using the Great EscApe detection kit (CLONTECH). Purified antipla- obtained from healthy adult donors using Ficoll-Paque (Pharmacia, cental alkaline phosphatase (Cosmo Bio) was used to generate a stand- Uppsala, Sweden). Monocytes were stained with fluorescein isothiocya- ard curve. The enzymatic activity was expressed as relative light nate-conjugated anti-CD14 and positively selected by MACS (Miltenyi units/s. The SEAP(His) and TARC-SEAP(His) used in the present 6 6 Biotec, Bergisch, Germany). B cells were stained with fluorescein iso- study had a specific activity of approximately 8.88 3 10 relative light thiocyanate-conjugated anti-CD19 and positively selected by MACS. T units/s and 1.23 3 10 relative light units/s per pmol, respectively. cells were stained with fluorescein isothiocyanate-conjugated anti-CD3 Binding Assay—For displacement experiments, cells were incubated and positively selected by MACS. Natural killer (NK) cells were sorted for1hat16 °C with 1 nM of SEAP(His) or TARC-SEAP(His) in the 6 6 by FACStar Plus (Beckton Dickinson, Mountain View, CA) as CD16 or presence of increasing concentrations of unlabeled chemokines in 200 ml 1 2 CD56 and CD3 cells with appropriate forward and side scatters. of RPMI 1640 containing 20 mM Hepes (pH 7.4), 1% BSA, and 0.02% 1 1 Purification of CD4 and CD8 T cells from PBMCs was carried out by sodium azide. For saturation experiments, cells were incubated for 1 h negative selection using Dynabeads (Dynal, Oslo, Norway) after incu- at 16 °C with increasing concentrations of TARC-SEAP(His) in the bation of PBMCs with anti-CD16, anti-CD14, anti-CD20, and anti-CD8 presence or absence of 1 mM unlabeled TARC. After incubation, cells or anti-CD4, respectively. Granulocytes were obtained from the pellet were washed five times and lysed in 50 mlof10mM Tris-HCl (pH 8.0), fraction of Ficoll-Paque gradient by dextran sedimentation and hypo- 1% Triton X-100. Samples were heated at 65 °C for 10 min to inactivate tonic lysis of erythrocytes. The purity of each cell population was always cellular phosphatases. Lysates were collected by centrifugation, and in the range of 95–99% as determined by flow cytometric analysis or by alkaline phosphatase activity in 25 ml of lysate was determined by the staining with Diff-Quik (Baxter Scientific Products, McGaw Park, IL). chemiluminescent assay described above. For direct binding experi- Construction of Receptor Expression Plasmids—The cDNA fragments ments, cells were incubated for1hat16 °C with 0.1 nM of I-RAN- 125 125 covering the open reading frames for various chemokine receptors and TES, I-MCP-1, I-MIP-1a, or TARC-SEAP(His) without or with orphan receptors were obtained as follows. Cloning of CCR3 cDNA was 200 nM of unlabeled chemokines in 200 ml of low salt binding buffer (50 described previously (14). EBI1 and BLR1 cDNA were isolated by mM Hepes, pH 7.5, 1 mM CaCl ,5mM MgCl , 0.5% BSA, and 0.05% 2 2 screening a phytohemagglutinin-stimulated human PBMC cDNA li- sodium azide). The cells were then washed five times with low salt brary. Other receptors were cloned from a human PBMC cDNA library binding buffer containing 0.5 M NaCl. All assays were done in duplicate. or human genomic library by polymerase chain reaction (PCR). The Binding data were analyzed by the LIGAND program (45). primers were designed using the sequences from the following Gen- Migration Assay—Cell migration was assayed by using a 48-well TM Bank submissions: CCR1 (L09320), CCR2B (U03905), CCR4 microchemotaxis chamber as described previously (17, 34). In brief, (X85740), CCR5 (X91492), CXCR4 (M99293), V28 (U20350), GPR-CY4 chemoattractants were diluted in Hepes-buffered RPMI 1640 supple- (U45984), GPR-9-6 (U45982), EBI1 (L31581), BLR1 (X68149). The frag- mented with 1% BSA and placed in lower wells (25 ml/well). Cells were ments were cloned into an expression vector pDREF-Hyg (17) for effi- suspended in the same medium at 2 3 10 cells/ml and added to upper cient expression in 293/EBNA-1 cells and Raji cells, or pCAGG-Neo wells (50 ml/well). The upper and lower wells were separated by a (kindly provided by T. Nakajima) for expression in K562 cells and L1.2 polyvinylpyrrolidone-free polycarbonate filter with 8-mm pores that was cells. precoated with type IV collagen (34). The chamber was incubated for 4 h Stable Transfection—For stable expression in Raji cells, cells were at 37 °C in 5% CO , 95% air. Filters were removed and stained with transfected by electroporation as described previously (17). For stable Diff-Quik. Each sample was assayed in triplicate, and migrated cells expression in 293/EBNA-1 cells, 1 3 10 cells were plated onto 100-mm were counted in five randomly selected high power fields (3 400) per dishes. After 12–20 h, cells were transfected with recombinant plasmids well. (10 mg each) using LipofectAMINE (Life Technologies, Inc.). After se- Calcium Mobilization Assay—Cells were suspended at 3 3 10 lection with 250 mg/ml hygromycin for 1–2 weeks, drug-resistant cells cells/ml in Hank’s balanced salt solution supplement with 1 mg/ml BSA were pooled and used for experiments. For stable expression in K562 and 10 mM HEPES, pH 7.4. Cell were incubated with 1 mM fura- cells and L1.2 cells, 1 3 10 cells in 500 ml of phosphate-buffered saline PE3-AM (Texas Fluorescence Labs) at room temperature for1hinthe were electroporated with 10 mg of linearized plasmid at 260 V, 960 dark. After washing twice, cells were resuspended at 2.5 3 10 cells/ml. microfarads using a Gene Pulser (Bio-Rad). After selection with 800 To measure intracellular calcium, cells in 2 ml were placed in a quartz mg/ml of G418 for 1–2 weeks, cells expressing transfected receptors at cuvette in a Perkin-Elmer LS 50B spectrofluorimeter. Fluorescence was high levels were identified by binding assays and/or Northern blot monitored at 340 nm (lex1), 380 nm (lex2), and 510 nm (lem) every 200 analysis and cloned by limiting dilution. ms. A dose-response curve was generated in each experiment, and Production of TARC-SEAP Fusion Protein—The DNA fragment en- results were expressed as percentage of maximum response. coding TARC was amplified from clone D3A (17) by PCR using 59 Northern Blot Analysis—Total RNAs were prepared from various SalI-TARC primer (159-CGCGTCGACAAAACCATGTGCTGTAC- subsets of peripheral blood leukocytes using Trizol (Life Technologies, 15038 Identification of CCR4 as Specific Receptor for TARC FIG.1. Expression of TARC-SEAP(His) fusion protein. A, sche- matic diagram of the SEAP(His) tag vector, pDREF-SEAP(His) , with 6 6 the inserted TARC cDNA fragment. pDREF-SEAP(His) contains the EF-1a promoter, the coding sequence for SEAP(His) , the hygromycin- resistant gene (hyg ), the EBNA-1 gene, and the Epstein-Barr virus origin for episomal replication (oriP). TARC cDNA was inserted be- tween SalI and XbaI sites and expressed as a fusion protein with SEAP FIG.2. Binding characteristics of TARC-SEAP(His) to Hut78 and the (His) tag. B, purified TARC-SEAP(His) fusion protein. TARC- 6 6 cells. A, displacement of TARC-SEAP(His) with unlabeled TARC. SEAP(His) was purified from culture supernatants of transfected 293/ Hut78 cells (4 3 10 cells) were incubated with 1 nM TARC-SEAP(His) EBNA-1 cells by metal affinity chromatography, subjected to electro- in the presence of the indicated concentrations of unlabeled TARC. phoresis on a 4 –20% polyacrylamide gel, and stained with Coomassie Representative results from three separate experiments are shown. B, Brilliant Blue. Positions of size markers (kDa) are shown on the right. 5 Hut78 cells (2 3 10 cells) were incubated with 1 nM TARC-SEAP(His) in the absence or presence of 200 nM unlabeled TARC, MCP-1, RAN- Inc.). RNA samples (5 mg each) were fractionated by electrophoresis on TES, MIP-1a, MIP-1b, or LARC. The percentage of inhibition of specific a 1% agarose gel containing 0.66 M formaldehyde. Gels were blotted binding by unlabeled ligands was calculated after subtracting nonspe- cific binding estimated with 1 nM SEAP(His) . Each histogram repre- onto a filter membrane (Hybond N ) (Amersham Japan, Tokyo). The sents the mean 6 S.E. from three separate experiments. probe was the SmaI-PstI fragment of clone D3A (17). Hybridization was carried out at 65 °C in QuickHyb solution (Stratagene) with probes labeled with P using Prime it II (Stratagene). After washing at 55 °C shown). These results may indicate that Hut78 expresses at with 0.2 3 SSC and 0.1% SDS, filters were exposed to x-ray films at least two types of TARC receptors, one highly specific for TARC 280 °C with an intensifying screen. and another shared by RANTES and LARC. At any rate, the RESULTS present results were highly consistent with those obtained previously by using I-labeled TARC. Thus, TARC-SEA- Use of TARC-Alkaline Phosphatase Fusion Protein for Recep- P(His) was proven to retain its ability as a specific high affin- tor Binding Assay—Chemokines often have a tendency for self- 6 ity ligand for TARC receptors. aggregation at physiological pH and tonicity that interferes CCR4 Is a High Affinity Specific Receptor for TARC—Since with their receptor studies (46, 47). To circumvent this prob- TARC was shown not to share the major class of its high lem, Luster et al. generated IP-10 fused with SEAP and showed affinity binding sites on T cells with other CC chemokines that that the fusion protein retained its ability to interact specifi- are known to bind to one or more CCRs (17), we first tested the cally with cell surface receptors without self-aggregation in binding of TARC-SEAP(His) to orphan receptors such as EBI1 physiological buffers (46). To prepare labeled TARC convenient 6 (48), BLR1 (49), V28 (50), GPR-9 – 6 (GenBank accession num- for receptor binding experiments, we decided to adopt this ber: HSU45982), GPR-CY4 (GenBank accession number: method and expressed TARC as a fusion protein with SEAP HSU45984), and LESTR/fusin (51). LESTR/fusin is now known with the (His) tag (Fig. 1A). The alkaline phosphatase activity as the receptor for SDF-1/PBSF (CXCR4) (52, 53). TARC-SEA- allowed quantitative determination of specific binding, P(His) showed no significant binding to Raji, 293/EBNA-1, or whereas the (His) tag in the C terminus was used for one-step K562 cells stably transfected with any of these orphan recep- affinity purification with a nickel agarose column. TARC-SEA- tors (data not shown). In parallel experiments, we also exam- P(His) was expressed in 293/EBNA-1 cells and purified as a ined the binding of TARC-SEAP(His) to the five CC chemokine single major protein with an apparent molecular mass of 74 6 receptors that are known to be shared by several CC chemo- kDa (Fig. 1B). Previously, we showed that I-labeled TARC kines (CCR1 to -5). Surprisingly, TARC-SEAP(His) specifi- specifically bound to a single class of receptors expressed on a 6 cally bound to CCR4 expressed on K562 (Fig. 3), Raji, and human T cell line Jurkat with a K of 2.1 nM (17). Importantly, 293/EBNA-1 (not shown). To further characterize the binding the binding was competed only by TARC and not by any other of TARC to CCR4, experiments were performed using K562 chemokines so far tested. To verify specificity and affinity of cells stably expressing CCR4. When the binding was performed TARC-SEAP(His) for TARC receptors, we carried out TARC- with increasing concentrations of TARC-SEAP(His) (Fig. 4A), SEAP(His) binding experiments using another human T cell a single class of receptors with a K of 0.5 nM and 29,000 line Hut78. The binding of TARC-SEAP(His) was fully com- d sites/cell was demonstrated (Fig. 4B). Competition experiments peted by unlabeled TARC with an IC of 0.5 nM (Fig. 2A). CC showed that unlabeled TARC fully competed with TARC-SEA- chemokines such as MCP-1, MIP-1a, or MIP-1b did not show P(His) for CCR4 (Fig. 4C). In contrast, other CC chemokines any significant competition with TARC-SEAP(His) (Fig. 2B). such as RANTES, MCP-1, MIP-1a, MIP-1b, and LARC showed RANTES and LARC, however, partially (,30%) inhibited TARC-SEAP(His) binding. Such partial inhibition by heterol- ogous chemokines was not, however, seen with Jurkat using We also confirmed that TARC-SEAP(His) induced calcium flux in either I-labeled TARC (17) or TARC-SEAP(His) (data not CCR4-transfected K562 cells with similar potency as TARC. 6 Identification of CCR4 as Specific Receptor for TARC 15039 FIG.3. Selective binding of TARC-SEAP(His) to CCR4-trans- fected cells. K562 cells were stably transfected with the vector or plasmids expressing indicated receptors. Clones expressing transfected receptors at high levels were selected by binding assays and/or North- ern blot analysis. Cells (1 3 10 cells) were incubated for1hat16 °C with 1 nM of TARC-SEAP(His) and washed, and bound TARC-SEA- P(His) was determined. Each histogram represents the mean 6 S.E. of total binding. 125 125 FIG.5. Direct binding of TARC-SEAP(His) , I-MCP-1, I- RANTES, and I-MIP-1a to endogenous receptors and CCR4. L1.2 cells transfected with CCR4 (5 3 10 cells) were incubated for 1 h at 16 °C with 0.1 nM of TARC-SEAP(His) (A), I-labeled MCP-1 (B), 125 125 I-labeled RANTES (C), or I-labeled MIP-1a (D) in the absence (filled bars) or presence (open bars) of unlabeled chemokines. For pos- itive controls, cells expressing endogenous receptors, Hut78 (A)or THP-1 (B–D), were used. Each histogram represents the mean 6 S.E. of total binding. Representative results from two separate experiments are shown. cally bound to CCR4-transfected L1.2 cells at levels about 20-fold higher than those obtained with Hut78 cells that ex- press endogenous TARC receptors at 1000 –2000 sites/cell (Fig. 2). In contrast, RANTES, MCP-1, or MIP-1a bound to CCR4- transfected L1.2 cells at marginal levels, if any, although these chemokines efficiently bound to THP-1 cells that express en- dogenous receptors at 1000 –5000 sites/cell (1). These results FIG.4. Binding characteristics of TARC-SEAP(His) to CCR4- further strengthen the possibility that TARC is the physiolog- transfected cells. A, saturable binding of TARC-SEAP(His) to CCR4- ical ligand for CCR4. transfected K562 cells. Cells (4 3 10 cells) were incubated for1hat TARC Induces Chemotaxis in CCR4-transduced Cells—Pre- 16 °C with the indicated concentrations of TARC-SEAP(His) . Nonspe- viously, we showed that TARC induced chemotaxis in certain cific binding determined by the addition of a 100-fold molar excess of human T cell lines (17). We therefore examined whether TARC TARC was subtracted. Representative results from three separate ex- periments are shown. B, Scatchard analysis of the binding data in panel also induced migration of cells expressing transfected CCR4. A. The calculated K is 0.5 nM. C, displacement of binding of TARC- 293/EBNA-1 cells were stably transfected with CCR4 or CCR1, SEAP(His) to CCR4-transfected K562 cells with unlabeled TARC. and migration of these cells to TARC, MIP-1a, and RANTES Cells (4 3 10 cells) were incubated with 1 nM TARC-SEAP(His) in the was examined. As shown in Fig. 6, TARC induced migration of presence of indicated concentrations of unlabeled TARC. Representa- tive results from three separate experiments are shown. D, competition cells transfected with CCR4 but not those transfected with of the binding of TARC-SEAP(His) to CCR4-transfected K562 cells by CCR1. On the other hand, MIP-1a and RANTES induced mi- unlabeled TARC and other chemokines. Cells (2 3 10 cells) were gration of cells transfected with CCR1 but not those transfected incubated for 1 h at 16 °C with 1 nM TARC-SEAP(His) in the absence with CCR4. Parental 293/EBNA-1 cells or those transfected or presence of 200 nM unlabeled TARC, MCP-1, RANTES, MIP-1a, MIP-1b, or LARC. The percentage of inhibition of specific binding by with the vector alone did not respond to TARC, MIP-1a,or unlabeled ligands was calculated after subtracting nonspecific binding RANTES (data not shown). Migration of CCR4-transfected 293/ estimated with 1 nM SEAP(His) . Each histogram represents the EBNA-1 cells to TARC was concentration-dependent, being mean 6 S.E. from three separate experiments. promoted at concentrations from 10 to 1000 ng/ml. Desensiti- zation was observed at 10 mg/ml (not shown). A checkerboard no significant competition with TARC-SEAP(His) for CCR4 analysis indicated that the migration of CCR4-transfected 293/ (Fig. 4D). Similar results were obtained using Raji or 293/ EBNA-1 cells toward TARC was mostly chemotactic but partly EBNA-1 cells stably transfected with CCR4 (data not shown). (;40%) chemokinetic (data not shown). Collectively, TARC but Collectively, the binding characteristics of TARC-SEAP(His) not MIP-1a or RANTES is a functional ligand for CCR4. to CCR4 were highly consistent with those obtained with the TARC Induces Calcium Mobilization in CCR4-transduced major class of the endogenous TARC receptors expressed on T Cells—We next examined induction of calcium mobilization in cells (Fig. 2) (17). K562 cells expressing CCR4, CCR1, or CCR2B (Fig. 7A). TARC We next compared direct binding of TARC-SEAP(His) , induced calcium flux in K562 cells expressing CCR4, whereas RANTES, MCP-1, and MIP-1a to CCR4-transfected cells (Fig. RANTES, MIP-1a, or MCP-1 did not. In addition, TARC, but 5). In these experiments, murine L1.2 cells transfected with not RANTES, MIP-1a, or MCP-1, was able to desensitize the CCR4 were used (38). As expected, TARC-SEAP(His) specifi- cells expressing CCR4 for subsequent stimulation with TARC 6 15040 Identification of CCR4 as Specific Receptor for TARC FIG.6. Chemotactic response of CCR4-transfected cells to TARC. Migration of CCR4- or CCR1-transfected 293/EBNA-1 cells to FIG.7. Calcium mobilization in CCR4-transfected cells stimu- TARC (closed circles), MIP-1a (closed triangle), RANTES (open lated with TARC. A, CCR4-, CCR1-, or CCR2B-transfected K562 cells squares), and medium (closed squares) were assayed in a 48-well che- were loaded with fura-PE3-AM and stimulated with TARC (100 nM), motaxis chamber. The assay was done in triplicate, and the number of MIP-1a (100 nM), RANTES (100 nM), and MCP-1 (100 nM). The arrow- migrating cells in five high power fields (3 400) were counted for each heads indicate the time of application of chemokine. Intracellular con- well. Each point represents the mean 6 S.E. from three separate centration of calcium were monitored by the fluorescence ratio (F340/ experiments. F380). Representative results from at least two separate experiments are shown. B, the concentration dependence of calcium mobilization induced by TARC. CCR4-transfected K562 cells were loaded with fura- PE3-AM and stimulated with the indicated concentrations of TARC. (Fig. 7A and data not shown). On the other hand, K562 cells Results are expressed as percentage of maximum response. Each point expressing CCR1 responded to MIP-1a but not to TARC, represents the mean 6 S.E. from three separate experiments. whereas K562 cells expressing CCR2B responded to MCP-1 but not to TARC. Parental K562 cells or those transfected with the vector alone did not show any response to TARC, RANTES, MIP-1a, or MCP-1 (data not shown). Responses to TARC were detectable above 1 nM, and maximum values were obtained at 100 nM with an EC of8nM (Fig. 7B). We further confirmed that TARC at 100 nM did not induce calcium fluxes in CCR1, CCR2B, CCR3, or CCR5-transfected K562 cells, while MIP-1a induced calcium fluxes in CCR1- and CCR5-transfected cells, MCP-1 in CCR2B-transfected cells, and eotaxin in CCR3-trans- fected cells (data not shown). Similar results were obtained by using 293/EBNA-1 cells. These observations again demon- strated that TARC is a functional ligand for CCR4. CCR4 Is Expressed Mainly in CD4 T Cells—Previously, we showed that high levels of binding sites for TARC were de- tected on certain T cell lines and peripheral blood T cells but not on peripheral monocytes or granulocytes (17). To determine FIG.8. Northern blot analysis for CCR4 mRNA expression. the cell types that express CCR4, we performed Northern blot Total RNA samples (5 mg/lane) were subjected to Northern blot analysis using the P-labeled CCR4 probe. The autoradiographs of the filter analysis. As shown in Fig. 8A, CCR4 mRNA was strongly (top) and photographs of the gel stained with ethidium bromide (bottom) expressed in T cell lines such as Hut78, Hut102, and Jurkat, all are shown. Positions of size markers (kb) are shown on the left. A, having been shown to display high levels of TARC binding sites expression of CCR4 mRNA in various human cell lines. Molt4, Jurkat, (17). A basophilic cell line KU812 that was originally used to and Hut78 are human T-cell leukemia virus type 1-negative T cell lines. Hut102 is a human T-cell leukemia virus type 1-positive T cell line. Raji clone CCR4 cDNA (39) and a megakaryocytic cell line MEG-1 and Daudi are Epstein-Barr virus-positive B cell lines. U937 and THP-1 were also found to express CCR4 mRNA. On the other hand, are monocytoid cell lines. K562 is an erythroid cell line. HL60 is a CCR4 mRNA was undetectable in THP-1, U937, Raji, K562, promyelocytic cell line. KU812 is a basophilic cell line. MEG1 is a and HL-60, all having been shown to possess negligible, if any, megakaryocytic cell line. B, expression of CCR4 mRNA in various 1 1 human peripheral blood leukocytes: CD4 T cells (T4), CD8 T cells binding sites for TARC (17). In normal peripheral blood mono- (T8), total T cells (T), B cells (B), NK cells (NK), monocytes (Mo), and nuclear cells (Fig. 8B), CCR4 mRNA was expressed in T cells, granulocytes (Gr). especially CD4 T cells, but not in B cells, NK cells, monocytes, DISCUSSION or granulocytes. The expression pattern of CCR4 is thus highly consistent with the T cell-selective expression of the endoge- Although a number of chemokines are known to act on T cells nous TARC receptor described previously (17). (54 – 61), TARC appears to be the first CC chemokine highly Identification of CCR4 as Specific Receptor for TARC 15041 selective for T cells (17). High levels of constitutive expression pressed on T cells, CCR4 appears to be the first CC chemokine receptor highly selective for T cells. By Northern blot analysis, of TARC have been detected only in thymus and not in spleen. High levels of specific binding sites for TARC have been de- CCR4 mRNA was detected highly selectively in some T cell lines and peripheral blood T cells, especially CD4 T cells, but tected on some T cell lines and peripheral blood T cells but not not in B cells, NK cells, monocytes, or granulocytes (Fig. 8). on monocytes or granulocytes. TARC has been shown to induce Previously, Power et al. (39) also demonstrated by Northern chemotaxis in certain human T cell lines. Furthermore, a class blot analysis that CCR4 was expressed strongly in thymus and of TARC receptors expressed on T cells is highly specific for peripheral blood leukocytes but very weakly in spleen. How- TARC and not shared by any other CC or CXC chemokines that ever, they further demonstrated by reverse transcriptase-PCR are known to act on T cells (17). Here we have presented analysis that CCR4 was expressed not only in T cells but also several lines of evidence indicating that CCR4 is the major in B cells and monocytes. Taken together, T cells are the cells class of receptors for TARC that is selectively expressed on T that express CCR4 at high levels, but other types of leukocytes cells and not shared by other CC or CXC chemokines. may also express CCR4 at low levels detectable by reverse CCR4 was originally cloned by Power et al. from a human transcriptase-PCR. The selective expression of CCR4 on T cells basophilic cell line KU-812 (39). The CC chemokines, MIP-1a, is thus consistent with the fact that T cells are the major target RANTES, and MCP-1, were presumed to be the functional of TARC. Furthermore, the fact that TARC and CCR4 are both ligands for CCR4, because among various chemokines, only constitutively and strongly expressed in thymus further sup- these were able to activate a calcium-dependent chloride chan- ports their important roles in trafficking and education of thy- nel in Xenopus laevis oocytes injected with CCR4 cRNA. How- mocytes within the thymus. ever, induction of chemotaxis or calcium flux in CCR4-trans- Notably, we also detected expression of CCR4 in a basophilic fected mammalian cells by MIP-1a, RANTES, MCP-1, or any cell line KU812 and a megakaryocytic cell line MEG-1 (Fig. 8). other chemokines has not been demonstrated. Here we have In fact, CCR4 was originally cloned from KU812, and, by using shown that introduction of the CCR4 cDNA into Raji, 293/ reverse transcriptase-PCR, Power et al. (39) demonstrated ex- EBNA-1, and K562 cells induced a class of high affinity binding pression of CCR4 in fresh basophils, especially after brief treat- sites for TARC (Figs. 3 and 4). Binding of TARC to CCR4 was ment with IL-5. They also mentioned that platelets contain competed only by TARC and not by any other chemokines high levels of CCR4 mRNA (39, 64). We have also detected high including MIP-1a, RANTES, and MCP-1 (Fig. 4). Binding of 125 levels of TARC-binding sites on platelets (data not shown). I-labeled RANTES, MCP-1, or MIP-1a to CCR4 were mar- Thus, it is clear that cells of the megakaryocyte/platelet lineage ginal, if any, while they bound to endogenous receptors ex- also express CCR4. It remains to be seen whether TARC affects pressed on THP-1 cells efficiently. (Fig. 5). Furthermore, only differentiation and/or function of basophils and megakaryo- TARC but not RANTES, MCP-1, or MIP-1a induced chemo- cytes/platelets besides T cells. taxis in CCR4-transfected 293/EBNA-1 and induced calcium It is also noteworthy that high levels of CCR4 expression are flux in CCR4-transfected 293/EBNA-1 and K562 (Figs. 6 and observed only in certain human T cell lines and peripheral 7). Collectively, these results have clearly demonstrated that blood T cells of especially the CD4 type. CCR4 may thus be TARC is the specific functional ligand for CCR4. The discrep- expressed selectively in particular subsets of T cells and/or in ancy between Power et al. (39) and the present study is prob- particular stages of differentiation and/or activation of T cells. ably due to the assay system used in the former study, i.e. Cultured CD45RO T cells were shown to express CCR1 and activation of a calcium-dependent chloride channel in Xenopus CCR2 in a strictly IL-2-dependent manner (65). Both CD4 and laevis oocytes injected with CCR4 cRNA. Besides CCR4, how- CD8 T cells were shown to constitutively express CCR5 (41). ever, some T cells may also express a class of receptors for Activated T cells were shown to express CXCR3, the first T TARC that is shared by RANTES and LARC (Fig. 2). cell-selective CXC chemokine receptor shared by IP-10 and Mig Compared with the high affinity value obtained from binding (66). It is thus important to determine phenotypes of T cells of TARC-SEAP(His) to CCR4 (K 5 0.5 nM), the potency of 6 d that express CCR4 and conditions that regulate CCR4 expres- TARC in induction of chemotaxis in CCR4-transfected 293/ sion. Obviously, elucidation of the physiological roles of the EBNA-1 (EC 5 10 nM) or in calcium flux in CCR4-transfected TARC/CCR4 system will be greatly facilitated by generation of ,8nM) appeared to be considerably less. Pre- K562 cells (EC mutant mice lacking the respective genes. viously, we observed that TARC induced chemotactic responses Acknowledgments—We thank Dr. Craig Gerard for providing L1.2, in two human T cell lines Hut78 and Hut102 with an EC of and T. Nakajima and M. Kitaura for valuable help. We also thank Dr. about 2 nM (17). Types and/or efficiency of G-proteins coupling Y. Himuna and Dr. M. Hatanaka for constant support. to CCR4 may be different depending on the cell background. SDF-1/PBSF is another chemokine that was reported to re- REFERENCES quire high concentrations in induction of chemotaxis in lym- 1. Baggiolini, M., Dewald, B., and Moser, B. (1994) Adv. Immunol. 55, 97–179 2. Ben-Baruch, A., Michiel, D. F., and Oppenheim, J. J. (1995) J. Biol. 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