Proc. Natl. Acad. Sci. USA Vol. 95, pp. 8205–8210, July 1998 Immunology

The CC 6Ckine binds the CXC CXCR3

HORTENSIA SOTO*†,WEI WANG*†,ROBERT M. STRIETER‡,NEAL G. COPELAND§,DEBRA J. GILBERT§, NANCY A. JENKINS§,JOSEPH HEDRICK*, AND ALBERT ZLOTNIK*¶

*DNAX Research Institute for Cellular and Molecular Biology, Palo Alto, CA 94304; ‡Department of Internal Medicine, Medical Center, University of Michigan, Ann Arbor, MI 48109; and §Mammalian Genetic Laboratory, Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute–Frederick Cancer Research and Development Center, Frederick, MD 21702

Edited by James P. Allison, University of California, Berkeley, CA, and approved May 12, 1998 (received for review February 20, 1998)

ABSTRACT We cloned the mouse homologue of the che- Here, we describe the cloning and characterization of mouse mokine receptor CXCR3, which is located in mouse chromo- CXCR3 (mCXCR3). We identified a new ligand for this some X. We screened a large panel of for their receptor, the recently described CC chemokine 6Ckine (5) ability to induce a calcium flux in mouse CXCR3-transfected [also called exodus 2 (12) or SLC (13)], which will hereafter be cells and identified a new ligand for this receptor, the recently referred to as 6Ckine. This constitutes an example of a CC reported CC chemokine 6Ckine. This represents an example chemokine that specifically binds a CXC receptor. of a CC chemokine, which binds to a CXC chemokine receptor. Like other ligands of this receptor, 6Ckine has angiostatic MATERIALS AND METHODS properties. 6Ckine is known to chemoattract T cells. In line with this, CXCR3 is expressed preferentially in Th1 cells and Cloning of mCXCR3. Part of the mouse CXCR3 chemokine ϩ Ϫ Ϫ in lymphoid organs of the IL-10؊/؊ mouse that develops receptor was amplified by PCR from a TCR␣␤ CD4 CD8 chronic colitis. Its ability to attract T cells as well as its thymocyte cDNA library, prepared in pJFE-14. Two degen- angiostatic properties suggest that 6Ckine may be an effective erate oligonucleotide primers corresponding to a conserved anti-tumor agent. motif of chemokine receptors (known as the DRY box) [GA(T͞C)(A͞C)GNTA(T͞C)CT(A͞G)GC(N)TA(CAG) GT(N) CA(T͞C)GC] and to the TMD-7 [GGGTCATGA Chemokines are a family of molecules composed of at least 50 ͞ ͞ ͞ ͞ ͞ different leukocyte chemotractants, which are produced dur- IGGGTTIAI(G A)CA(G A)C(T A)(G A)(T C)G] were ing and regulate leukocyte migration (1). These used in the PCR reactions. Fifty microliter reaction mixtures containing 1 ␮g of cDNA, 1ϫ GeneAmp buffer (Perkin– proteins are 68–120 amino acids in length and have been ␮ ␮ divided into four structural categories (2) based on the number Elmer), 1.5 mM MgCl2, 250 M each deoxynucleotide, 1 M and arrangement of their conserved cysteines: the CXC, CC, primers, and 2.5 units of AmpliTac DNA polymerase (Perkin– Elmer) were subjected to 30 cycles (94°C for 1 min, 55°C for and CX3C groups have four conserved cysteines, whereas the C chemokine (lymphotactin) has only two (3). A membrane 1 min, and 72°C for 2 min) on a Thermal Cycler (Perkin– bound chemokine with a CX C motif also has been described Elmer). All of the PCR products were cloned into the pCR 2.1 3 vector (Invitrogen) and sequenced. A PCR product of 237 (4). Most CXC chemokines are chemotactic for , nucleotides showed 80% nucleotide sequence identity with whereas the CC generally attract and ; human CXCR3. The 237-bp probe was used for screening of the C chemokine is specific for T and NK lymphocytes (5), and ϩ Ϫ Ϫ the ␣␤ TCRCD4 CD8 T-cell cDNA library. Four full length the CX C, like the CC chemokines, appears to be a potent 3 clones were isolated and sequenced to completion. chemoattractant for monocytes and lymphocytes but not for Tissue Distribution of mCXCR3. The tissue distribution of neutrophils. Leukocytes respond to chemokines through G mCXCR3 was analyzed by Northern analysis of multiple tissue protein-coupled receptors (GPCR). These receptors belong to Northern blots (CLONTECH) with 32P-labeled 237-bp PCR a large and functionally diverse superfamily of proteins con- product and with the full length clone, both of which were taining seven transmembrane domains (7TMD). A common labeled by random priming using a Prime-It (Stratagene). feature of signaling through the known chemokine receptors is The distribution of CXCR3 also was analyzed in cDNA that their signal transduction pathway results in the release of 2ϩ libraries by Southern blotting as described (14). intracellular Ca and is sensitive to pertussis toxin (6), Generation of mCXCR3-Transfected Cells. The mCXCR3 indicating that most chemokine receptors are linked to the Gi cDNA was amplified using primers containing 5Ј-ClaI and class proteins. Chemokine receptors have been divided into 3Ј-NotI restriction enzyme sequences. The PCR fragment was five separate classes: (i) CXC restricted, (ii) CC restricted, (iii) ligated into the same sites of an expression vector pFLAG– CC and CXC restricted, (iv) orphans (with ligands undefined), CMV3 (Kodak) containing a N-terminal FLAG fusion se- and (v) viral. In humans, five CXC chemokine receptors quence. The pFLAG–CMV3–mCXCR3 construct was used to (CXCR1–5) and nine CC chemokine receptors (CCR1–9) transfect HEK293 cells by LipofectAmine (GIBCO͞BRL). have been described so far based on their ligand specificity: ␣ Stable transfectants were generated by selection with 400 CXCR1 for IL-8, GRO- , NAP-2 (binding with different ␮g͞ml G418 for 2 wk. The mCXCR3-expressing cells were affinities), and GCP-2 (7); CXCR2 for IL-8, GRO-␣, NAP-2 (binding with the same affinity), and GCP-2 (8); CXCR3 for This paper was submitted directly (Track II) to the Proceedings office. IP-10 and MIG (9); CXCR4 for SDF-1 (10), and CXCR5 Abbreviations: bFGF, basic fibroblast growth factor; VEGF, vascular (BRL-1) for a new CXC chemokine (11). endothelial growth factor; GPCR, -coupled receptors; TMD transmembrane domain. The publication costs of this article were defrayed in part by page charge Data deposition: The mouse CXCR3 sequence reported in this paper has been deposited in the GenBank database (accession no. payment. This article must therefore be hereby marked ‘‘advertisement’’ in AF045146). accordance with 18 U.S.C. §1734 solely to indicate this fact. †H.S. and W.W. contributed equally to this study. © 1998 by The National Academy of Sciences 0027-8424͞98͞958205-6$2.00͞0 ¶To whom reprint requests should be addressed. e-mail: zlotnik@ PNAS is available online at http:͞͞www.pnas.org. dnax.org.

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further selected by a fluorescence-activated cell sorter by using Interspecific Mouse Backcross Mapping. Interspecific back- biotin-conjugated M2 anti-FLAG antibody (Eastman Kodak) cross progeny were generated by mating (C57BL͞6J ϫ Mus ͞ to obtain a population in which Ͼ95% of the cells expressed spretus)F1 females and C57BL 6J males as described (16). A the receptor. total of 205 N2 mice were used to map the Cxcr3 locus. Calcium Flux Assay. HEK293 cells stably expressing Southern blot transfer and hybridization were performed as Ϸ ͞ mCXCR3 were suspended in 1 ϫ 107͞ml in DMEM with 10% described (17). The probe, an 1.1-kb NotI BamHI fragment ␣ 32 fetal calf serum. The cells were loaded with 3 ␮M Indo-1 of mCXCR3, was labeled with [ - P]dCTP and washing to a final stringency of 1ϫ standard saline citrate phosphate (SSCP; acetoxymethyl ester (Molecular Probes) at room temperature ϫ ϭ ͞ ͞ in the dark for 45 min. The loaded cells were washed and 1 SSCP 120 mM NaCl 5 mM sodium citrate 20 mM resuspended in Hank’s balanced salt buffer (2 mM CaCl ͞145 sodium phosphate, pH 6.8 ) and 0.1% SDS, 65°C. A fragment 2 of 14.0 kb was detected in EcoRI-digested M. spretus DNA. mM NaCl͞5mMKCl͞1 mM MgCl ͞5mMD-glucose͞20 mM 2 The presence or absence of the 7.8-kb EcoRI M. spretus- Hepes, pH 7.3) with 1% fetal calf serum at the same concen- specific fragment was followed in backcross mice. tration. Calcium mobilization of 1 ϫ 106 cells was measured in A description of the probes and restriction fragment length 2 ml of Hanks’ balanced salt solution with 1.6 mM CaCl in a 2 polymorphisms for the loci linked to Cxcr3 including Efnb1 and continuously stirring acrylic cuvette at 37°C in a Photon Pou3f4 has been reported previously (17). Recombination Technologies spectrofluorimeter (Princeton). The fluores- distances were calculated by using MAP MANAGER, version 2.6.5 cence was monitored as ratio of emission at 405 and 483 nm (17). order was determined by minimizing the number of at an excitation wavelength of 350 nm. All the chemokines recombination events required to explain the allele distribu- listed in Table 1 were tested in concentrations ranging from tion patterns. ␮ 100 nM to 1 M. Chromosomal Mapping of Human CXCR3. 150 ng of Corneal Micropocket Model of Angiogenesis. In vivo angio- Genomic DNA from a panel of 20 human–rodent somatic genic activity was assayed in the avascular cornea of Long hybrid cell lines (Bios, New Haven, CT) were used as a Evans rat eyes, as described (15). In brief, were template in a 50 ␮l of PCR reaction. The human CXCR3 combined with sterile Hydron (Interferon Sciences, New primer pairs were: 5Ј sense primer ATGGTCCTTGAGGT- Brunswick, NJ) casting solution, and 5 ␮l of aliquots were GAGTGACCAC and 3Ј antisense primer TCACAAGC- air-dried on the surface of polypropylene tubes. Before im- CCGAGTAGGAGGCCTC. Total human genomic DNA was plantation, pellets were rehydrated with normal saline. Ani- used as positive control. Chinese hamster and mouse genomic mals were anesthetized with an i.p. injection of ketamine (150 DNA were used as negative controls. mg͞kg) and atropine (250 ␮g͞kg). Rat corneas were anesthe- tized with 0.5% proparacaine hydrochloride ophthalmic solu- RESULTS tion followed by implantation of the Hydron pellet into an intracorneal pocket (1–2 mm from the limbus). Six days after Cloning of mCXCR3. Chemokine receptors belong to the implantation, animals were pretreated i.p. with 1,000 units of GPCR family and exhibit common structural features. We initially sought to identify new chemokine receptors expressed heparin (Elkins-Sinn, Cherry Hill, NJ), anesthetized with ␣␤ Ϫ Ϫ ketamine (150 mg͞kg), and perfused with 10 ml of colloidal by TCRCD4 CD8 T cells. To this end, we designed de- generate primers targeting the conserved DRY box and carbon via the left ventricle. Corneas were then harvested and seventh transmembrane (TM7) consensus sequences and am- photographed. No inflammatory response was observed in any ϩ Ϫ Ϫ plified a 237-bp product from ␣␤TCR CD4 CD8 cDNA of the corneas treated with the above reagents. Positive library. Sequencing of this PCR product indicated that it neovascularization responses were recorded only if sustained shared significant homology with GPCRs, but its sequence did directional ingrowth of capillary sprouts and hairpin loops not correspond to known receptors. This PCR product was toward the implant were observed. Negative responses were used as a probe for screening of the cDNA library from recorded when either no growth was observed or when only an ␣␤TCRϩCD4ϪCD8Ϫ T cells. A 1,626-bp clone encoding the occasional sprout or hairpin loop displaying no evidence of full-length receptor was identified subsequently. The full se- sustained growth was detected. Positive controls included basic quence of this clone indicated a high degree of nucleotide fibroblast growth factor (bFGF) and vascular endothelial sequence homology to human CXCR3 (hCXCR3), and its growth factor (VEGF). Angiostatic activity was demonstrated amino acid sequence was nearly identical (Fig. 1). This cDNA as the ability of a molecule to inhibit the angiogenic activity of has an ORF of 1,101 bp and encoded a 367 amino acid protein bFGF or VEGF. with a predicted molecular weight of 41,014. The predicted amino acid sequence exhibits the four highly conserved cys- Table 1. Chemokines tested in calcium flux experiment for teine residues in the extracellular region, three potential CXCR3 transfectants N-glycosylation sites: Asn22, Asn32, and Asn198 (also present Chemokine Calcium flux in the human version), and three threonine and eight serine ϩ residues that are present in the intracellular COOH-terminal IP-10 region, as potential phosphorylation sites which are common Mig ϩ ϩ among this family of receptors. The receptor also has the 6Ckine HCCXNP motif in the seventh TMD. IL-8 Ϫ ␣ Ϫ Expression Pattern. Northern blot analysis showed a 1.7-kb Gro mRNA present mainly in lung, , and heart and a very low Nap-2 Ϫ Ϫ but detectable amount in testis, kidney, skeletal muscle, and H-CC4 liver. A larger, 3.6-kb mRNA was detected in lung, spleen, and Mip-3␣ Ϫ ␤ Ϫ heart (Fig. 2A), suggesting that CXCR3 expression may not be Mip-3 specific. Southern blot analysis of various cDNA libraries TECK Ϫ Ϫ (Fig. 2B) indicated that CXCR3 mRNA is preferentially MDC transcribed in CD4ϩ Th1-polarized T cells and that this TARC Ϫ Ϫ difference (between Th1- and Th2-polarized populations) Eotaxin 2 increases with time. CXCR3 message also was detected in DC-CK-1 Ϫ ϩ ϩ ␣␤ ϩ Ϫ Ϫ Ϫ CD4 NK1.1 and TCR CD4 CD8 thymocytes (Fig. 2C) hI-309 and in CD8ϩ T cells (data not shown). Interestingly, CXCR3 Downloaded by guest on September 30, 2021 Immunology: Soto et al. Proc. Natl. Acad. Sci. USA 95 (1998) 8207

FIG. 1. Aligment of the mCXCR3 predicted amino acid sequence with hCXCR3. The arrowhead indicates potential N-linked glycosilation sites and the horizontal lines the putative TMD (TMD1–TMD7). The alignment was generated by using CLUSTALW (17). The sequence data are available from European Molecular Biology Laboratory (EMBL)͞GenBank under accession no. AF045146.

was more abundant in a cDNA library from resting models in which Th1 cells are believed to play a role. As shown ␣␤TCRϩCD4ϪCD8Ϫ than in a corresponding cDNA library in Fig. 2B, CXCR3 is up-regulated in cDNA libraries from from CD3-activated ␣␤TCRϩCD4ϪCD8Ϫ (Fig. 2C). The latter mesenteric lymph nodes and Peyer’s patches from the IL-10Ϫ/Ϫ phenomenon (down-regulation upon activation) also was ob- mouse, suggesting the presence of Th1 cells in these organs. In served in a Th1 cell clone (D1.1) after activation with Con A fact, polarization of the Th cell subsets is likely to occur mainly (data not shown). Conversely, CXCR3 is up-regulated upon in the secondary lymphoid organs (18). activation in pro-T thymocytes (Fig. 2C). This indicates that In addition, mCXCR3 also was detected in a cDNA CXCR3 is differentially regulated in various T cell subsets. library (Fig. 2B), in line with a recent report that found Given the correlation of CXCR3 expression with the Th1 CD4 chemotactic activity of 6Ckine for B cells (13, 19). CXCR3 subset (18), we then investigated its expression in animal expression also was detected in endothelial cells (Fig. 2B). 6Ckine Binds to CXCR3. To study calcium mobilization, we generated stable transfectants in HEK293 cells with p-flag-

FIG. 2. Distribution of mRNA of mCXCR3. (A) Multiple tissue Northern blot was hybridized with a full-length mCXCR3 cDNA probe, and two bands of 1.7 and 3.6 kb were detected in lung, spleen, and heart. (B) Southern blot analysis of different cDNA libraries: Th1 1 wk, naive cells from spleen were polarized for 7 days with IFN␥ and anti-IL-4; Th2 1 wk, naive cells were polarized for 7 days with IL-4 and anti-IFN␥. Th1 3 wk: naive cells from Rag1Ϫ/Ϫ ϫ DO-11.10-transgenic mice were stimulated with ovalbumin-(323–339) peptide in the pres- FIG. 3. Calcium flux and desensitization analysis. (A) Calcium ence of irradiated splenic APC and IL-12 and anti-IL-4 for 3 wk. Th2 mobilization and dose response of Mig, IP-10, and 6Ckine with mouse 3 wk: naive cells from transgenic mice (similar to Th1 3 wk) were CXCR3 stable-transfected HEK293 cells. Arrow indicates the time stimulated with anti-IL-12 plus IL-4 for 3 wk; IL-10Ϫ/Ϫ mice Peyer’s point of addition of the indicated ligands. Each chemokine was loaded patches; Peyer’s patches; IL-10Ϫ/Ϫ mice mesenteric lymph nodes; from 1 nmol to 1 ␮mol in concentration. (B) Cross desensitization mouse mesenteric lymph nodes; B cells derived from spleen; and among Mig, IP-10, and 6Ckine. Arrows indicate the first and the mouse endothelial cells. (C) CD4ϩNK1.1ϩ-activated T cells; second addition of the indicated chemokines. The chemokines were ␣␤TCRϩCD4ϪCD8Ϫ resting T cells; ␣␤TCRϩCD4ϪCD8Ϫ -activated loaded at 1 ␮mol in concentration to induce a maximum calcium T cells; pro-T-resting cells; and pro-T-activated cells. mobilization response. Downloaded by guest on September 30, 2021 8208 Immunology: Soto et al. Proc. Natl. Acad. Sci. USA 95 (1998)

CMV3-mCXCR3. As reported for hCXCR3, the CXC che- Table 2. The angiogenic response of 6Ckine, bFGF, VEGF, or in mokines Mig and IP10 induced calcium flux in the CXCR3 combination using the rat cornea micropocket assay transfectants, whereas nontransfected HEK293 did not show for neovascularization this response (Fig. 3A). Mig and IP-10 induced rapid receptor Corneas positive for desensitization after successive treatment with the respective Condition neovacularization (percentage) chemokines (Fig. 3B). We then tested 15 ligands, including many recently described chemokines that had not been tested Control (vehicle alone) 0 of 4 (0%) previously against CXCR3 transfectants (Table 1). Surpris- bFGF (5 nM) 4 of 4 (100%) ingly, the recently described CC chemokine 6Ckine also in- VEGF (5 nM) 4 of 4 (100%) duced a Ca2ϩ flux in the CXCR3-transfected cells. Cross- 6Ckine (10 nM) 2 of 12 (17%) bFGF (5 nM) ϩ 6CKine (10 nM) 1 of 6 (17%) desensitization experiments showed that Mig and IP-10 de- ϩ sensitize the 6Ckine response; however, the reverse VEGF (5 nM) 6CKine (10 nM) 1 of 6 (17%) desensitization pattern induced only partial desensitization. This phenomenon has been documented between IP-10 and analysis by using progeny derived from mating of [(C57BL͞ ϫ ϫ ͞ ͞ Mig (9). Taken together, these data suggest that the affinity or 6J M. spretus)F1 C57BL 6J] mice (16). C57BL 6J and M. binding avidity of CXCR3 is highest for Mig and lower for spretus DNAs were digested with several enzymes and analyzed IP-10 and 6Ckine or that these ligands interact with CXCR3 by Southern blot hybridization for restriction fragment length through different binding sites. polymorphisms by using a CXCR3 cDNA probe. The 7.8-kb 6Ckine Exhibits Angiostatic Activity. Because two other EcoRI M. spretus restriction fragment length polymorphism known CXCR3 ligands (Mig and IP-10) exhibit angiostatic was used to follow the segregation of the Cxcr3 locus in activity, we decided to examine the potential angiostatic backcross mice. The mapping results indicate that Cxcr3 is activity of 6ckine in vivo. To this end, hydron pellets alone, located in the central region of the mouse pellets containing 6Ckine (10 nM), bFGF (5 nM), or VEGF (5 linked to Efnb1 and Pou3f4. (Fig. 5). The ratios of the total ϩ nM) or pellets containing combinations of 6Ckine bFGF or number of mice exhibiting recombinant chromosomes to the ϩ 6Ckine VEGF, were embedded into the normally avascular total number of mice analyzed for each pair of loci and the rat cornea and assessed for a neovascular response (Fig. 4). most likely gene order are: centromere–Efnb1–3͞172–Cxcr3– Both bFGF and VEGF induced positive corneal angiogenic 6͞174–Pou3f4. The recombination frequencies [expressed as responses in four of four corneas tested, without evidence for genetic distances in centimorgans (cM) Ϯ the standard error] significant leukocyte infiltration (assessed by light microscopy) are –Efnbl–1.7 Ϯ Cxcr3–3.5 Ϯ 1.4–Pou3f4. (Table 2). In contrast, hydron pellets alone (n ϭ 4 corneas), or We have compared our interspecific map of the X chromo- pellets containing 6Ckine (n ϭ 12 corneas for each chemo- some with a composite mouse linkage map that reports the kine), only resulted in a positive neovascular response in 17% map location of many uncloned mouse mutations (provided of the corneas tested for each variable. When 6Ckine was added in combination with bFGF or VEGF (Fig. 4 and Table from the Mouse Genome Database, a computerized database 2, respectively), it inhibited either the bFGF or VEGF-induced angiogenesis in five of six corneas (n ϭ 6 corneas for each manipulation). These results indicate that 6Ckine has angio- static activity. CXCR3 Maps to Chromosome X. The mouse chromosomal location of Cxcr3 was determined by interspecific backcross

FIG.5. Cxcr3 maps to the central region of the mouse chromosome X. Cxcr3 was placed on the mouse chromosome X by interspecific backcross analysis. The segregation patterns of Cxcr3 and flanking in 161 backcross animals that were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL͞6J ϫ M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL͞6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial X chromosome-linkage map showing the location of Cxcr3 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of the loci in human chromosomes, where known, are show to the right. References for the FIG. 4. Angiostatic activity of 6Ckine. Angiogenic response in the human map positions of loci cited in this study can be obtained from rat corneal micropocket assay to bFGF (C) (5 nM) and VEGF (E)(5 Genome Database (GDB), a computerized database of human linkage nM) were inhibited by 6Ckine (10 nM) (D and F, respectively). Control information maintained by the William H. Welcher Medical Library (A) was DMEM with 0.1% BSA alone or 6Ckine alone (B). of The Johns Hopkins University (Baltimore, MD). Downloaded by guest on September 30, 2021 Immunology: Soto et al. Proc. Natl. Acad. Sci. USA 95 (1998) 8209

maintained at the Jackson Laboratory, Bar Harbor, ME). If 6Ckine binds CXCR3, it should share biological activities Cxcr3 maps in a region of the composite map that lacks mouse with IP-10 and Mig. Other chemokines have suppressive mutations with a phenotype consistent with an alteration in effects in hematopoietic progenitors (27), and 6Ckine also this locus (data not shown). exhibits this characteristic (12). Our results suggest that the The corresponding syntenic human region is located in inhibitory effect of 6Ckine may be mediated by CXCR3 as chromosome X. However, this prediction was not consistent well. with a previous report in which human CXCR3 (GPR9) was The binding of 6Ckine to CXCR3 suggested that it could mapped by fluorescence in situ hybridization to chromosome have angiostatic effects, as has been reported for IP-10 and 8 (20). To clarify this, we performed PCR analysis by using Mig (28, 29). Previous work showed that IP-10 and Mig inhibit human CXCR3 primers under high stringency conditions on angiogenesis induced by IL-8 or by bFGF in the rat corneal hamster͞human hybrid lines. The results showed a specific micropocket assay and that IP-10 or Mig block the endothelial band in hybrids containing human chromosome X (data not cell to IL-8 or to bFGF (29). To determine whether shown), confirming that human Cxcr3 is located in human 6Ckine could inhibit the angiogenic activity of bFGF or VEGF chromosome X. in vivo, 6Ckine was used in the rat corneal micropocket assay. As predicted, 6Ckine showed a strong angiostatic effect (Fig. 4). This represents an example of a CC chemokine with DISCUSSION angiostatic properties. The ELR-CXC chemokines favor an- Chemokines have been classified in four classes, CXC, CC, C, giogenesis, unlike the non-ELR-CXC chemokines (PF-4, IP- and CX3C, and the chemokines receptors have been subdi- 10, and Mig), which are angiostatic (29). It also is known that vided on the basis of their ligands into CXCR and CCR. So far the ELR motif in CXC chemokines has a functional role in the chemokine receptors described only bind ligands from a chemokine-mediated angiogenesis (29). By analogy, it is likely single chemokine class (CXC or CC), with the only exception that the AQD motif before the first two cysteines of 6Ckine being the Duffy antigen͞receptor for chemokines (DARC), may be connected with the angiostatic properties of 6Ckine, as which does not induce a calcium flux but binds certain CC and has been demonstrated for the TVR and DLQ motifs present CXC chemokines (6). Recently, the discovery of new chemo- in IP-10 and PF4, respectively (29). kines has accelerated by using genomic and bioinformatics- The binding of IP-10 and Mig to CXCR3 has not been studied from the structural point of view. Structural studies based searches (14). This way a CC chemokine (6Ckine) was ␣ identified (5, 12, 19). While screening for new chemokine with IL-8 and GRO showed that the flexible NH2-terminal receptors in a cDNA library from ␣␤TCRϩCD4ϪCD8Ϫ thy- region is the most critical receptor binding site for these CXC mocytes, we isolated a cDNA encoding the mouse homologue chemokines, and that a second binding site exists in the loop of CXCR3. We then observed that 6Ckine is a ligand for that follows the two disulfides (30). Whether any of these conclusions applies to the binding of 6Ckine to CXCR3 CXCR3. Given that 6Ckine is a CC chemokine, this represents remains to be determined. an example of a CC chemokine binding a CXC receptor. This The ability of 6Ckine to chemoattract T cells, including Th1 is a particular feature of 6Ckine because none of the other CC cells, and its angiostatic activity make it a strong candidate to ligands tested bind CXCR3 (Table 2). Recently, 6ckine also test in cancer immunotherapy. IP-10 is known to be expressed, has been shown to bind CCR7 (21, 22) but not other CCRs along with IL-6 and TNF␣, at higher levels by regressing tested (CCR1–CCR6) (22). We have observed that CCR7 tumors, compared with progressing tumors (31). Experiments expression in T cells is either very low or absent (data not to demonstrate the IP-10 antitumor response were performed shown). In contrast, we found significant expression of CXCR3 by using genetically engineered tumor cells that secrete high in various T cells populations (Fig. 2). The latter observation levels of IP-10. Although this chemokine did not have adverse suggests that the chemotactic effects of 6ckine in T cells are effects on the growth of these tumor cell cultures, it elicited a mainly mediated through CXCR3. In fact, the other two powerful host-mediated antitumor effect in vivo, which is T cell chemokines known to bind CXCR3, Mig and IP-10, are known dependent (32, 33). Similarly, Mig has antitumor activity in to preferentially affect T cells (23). Ϫ/Ϫ vivo (34). The antitumor effect of IP-10 is also evident in SCID The high expression of CXCR3 in Rag-1 suggests mice, suggesting that these chemokines also attenuate tumor that this receptor is expressed by thymic progenitors or stromal growth through inhibition of angiogenesis (35). Taken to- cells and therefore could be involved in T cell development. It gether, these observations strongly suggest that 6Ckine has has been suggested that hCXCR3 mediates selective lympho- antitumor activity like the other CXCR3 ligands. cyte recruitment (9); these data correlate with mCXCR3 ϩ We conclude that the angiostatic activity of 6Ckine is likely distribution in CD4 -derived cells. Human CXCR3 is not to be mediated through its interaction with CXCR3. Origi- expressed in (9), but one of its known ligands, nally, it was described that a specific cell surface heparan IP-10, has been reported to be a chemoattractant for both sulfate proteoglycan receptor on endothelial cells, which binds macrophages and T lymphocytes. It was recently described that IP-10 and PF4 (36), could be the potential endothelial receptor hCXCR3 is highly expressed in 12 days polarized Th1 but not for CXC angiostatic chemokines. Nevertheless, it is not known in Th2 cells (18). We obtained similar results for mCXCR3 by whether this receptor represents CXCR3. It has suggested that Northern and Southern analyses (Fig. 2D) and also observed the heparan sulfate containing proteoglycan and CXCR3 are that the difference in mCXCR3 mRNA expression between the same molecule (1). In vitro and in vivo systems have Th1- and Th2-polarized cells increases after 3 wk (Fig. 2). We demonstrated (35) that the CXC chemokines behave as either therefore analyzed the expression of CXCR3 in some models angiogenic or angiostatic factors, depending on the presence of of mouse inflammatory disease, and found it up-regulated in the ELR motif. In addition, it has been suggested that IP-10 Ϫ Ϫ the mesenteric lymph nodes and Peyer’s patches of IL-10 / and Mig may be distal mediators of the angiostatic effects of mice, supporting the conclusion that Th1 cells play a role in the interferons (35, 34). It remains to be determined whether Ϫ Ϫ development of enterocolitis in IL-10 / mice (24, 25). Re- 6Ckine expression is up-regulated by IFN␥. cently, 6Ckine has been shown to be produced by vascular GPCR genes are distributed throughout the human genome endothelium (13) and able to favor the initial binding of (37). Certain chemokine receptors are genetically clustered. circulating cells with the endothelium (26). Taken together, The CC receptors (CCR1–5, CCR8) are located in human these observations strongly suggest a critical role for 6Ckine in (38), hCCR6 gene maps to (39); the recruitment of T cells and other leukocytes from the whereas CXCR1, CXCR2, and CXCR4 are clustered on circulation. human . We found that mouse and human Downloaded by guest on September 30, 2021 8210 Immunology: Soto et al. Proc. Natl. Acad. Sci. USA 95 (1998)

CXCR3 map to chromosome X, representing a new locus for 17. Avraham, B. B., Cho, B. C., Gilbert, D. J., Fijii, H., Okamoto, K., chemokine receptors. Nevertheless, it is not the only one Shimazaki, T., Ito, T., Shoji, H., Wakamatsu, Y., Kondoh, H., et GPCR that maps in that chromosome because several opsin al. (1993) Genomics 18, 131–133. (photoreceptor proteins) genes are clustered on the X chro- 18. Bonecchi, R., Bianchi, G., Bordignon, P. P., D’Ambrosio, D., mosome (37). The central region of the mouse X chromosome Lang, R., Borsatti, A., Sozzani, S., Avellana, P., Gray, A. P., Mantovani, A., et al. (1998) J. Exp. Med. 187, 129–134. is syntenic with a region in the long arm of the human X 19. Nagira, M., Imai, T., Hieshima, K., Kusuda, J., Ridanpaa, M., chromosome (summarized in Fig. 5), suggesting that the Takagi, S., Nishimura, M., Kakizaki, M., Nomiyama, H. & human homologue of Cxcr3 maps to Xq, as well. Yoshie, O. (1997) J. Biol. Chem. 272, 19518–19524. 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