Secondary Lymphoid-tissue Chemokine Is a Functional Ligand for the CC Chemokine Receptor CCR7

Secondary Lymphoid-tissueChemokine (SLC) is a recently identified CC chemokine that is constitutively expressed in various lymphoid tissues and is a potent and specific chemoattractant for lymphocytes. The SLC gene and the gene encoding another lymphocyte-specific CC chemokine, EBI1-ligand chemokine (ELC), form a mini-cluster at human chromosome 9p13. Here, we show that SLC is a high affinity functional ligand for chemokine receptor 7 (CCR7) that is expressed on T and B lymphocytes and a known receptor for ELC. SLC induced a vigorous calcium mobilization in murine L1.2 cells stably expressing human CCR7. SLC tagged with the secreted form of alkaline phosphatase (SLC-SEAP) showed specific binding to CCR7 that was fully competed by SLC with an IC50 of 0.5 nm. SLC also induced a vigorous chemotactic response in CCR7-expressing L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nm. When assessed using CCR7-transfected L1.2 cells, SLC and ELC were essentially equivalent in terms of cross desensitization in calcium mobilization via CCR7, cross-competition in binding to CCR7, and induction of chemotaxis via CCR7. SLC and ELC were also shown to fully share receptors expressed on cultured normal T cells known to express CCR7. Notably, however, SLC was somehow less efficient in cross-desensitization against ELC in calcium mobilization and in cross-competition with ELC for binding when assessed using cultured normal T cells. Thus, SLC and ELC, even though sharing only 32% amino acid identity, constitute a genetically and functionally highly related subgroup of CC chemokines. Secondary Lymphoid-tissueChemokine (SLC) is a recently identified CC chemokine that is constitutively expressed in various lymphoid tissues and is a potent and specific chemoattractant for lymphocytes. The SLC gene and the gene encoding another lymphocyte-specific CC chemokine, EBI1-ligand chemokine (ELC), form a mini-cluster at human chromosome 9p13. Here, we show that SLC is a high affinity functional ligand for chemokine receptor 7 (CCR7) that is expressed on T and B lymphocytes and a known receptor for ELC. SLC induced a vigorous calcium mobilization in murine L1.2 cells stably expressing human CCR7. SLC tagged with the secreted form of alkaline phosphatase (SLC-SEAP) showed specific binding to CCR7 that was fully competed by SLC with an IC50 of 0.5 nm. SLC also induced a vigorous chemotactic response in CCR7-expressing L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nm. When assessed using CCR7-transfected L1.2 cells, SLC and ELC were essentially equivalent in terms of cross desensitization in calcium mobilization via CCR7, cross-competition in binding to CCR7, and induction of chemotaxis via CCR7. SLC and ELC were also shown to fully share receptors expressed on cultured normal T cells known to express CCR7. Notably, however, SLC was somehow less efficient in cross-desensitization against ELC in calcium mobilization and in cross-competition with ELC for binding when assessed using cultured normal T cells. Thus, SLC and ELC, even though sharing only 32% amino acid identity, constitute a genetically and functionally highly related subgroup of CC chemokines. Chemokines constitute a group of small, mostly basic, heparin-binding cytokines with common structural features that mediate recruitment of leukocytes into sites of inflammation and immune responses. Chemokines are also considered to play roles in homeostatic recirculation and homing of lymphocytes (for review, see Refs. 1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1977) Google Scholar, 2Ben-Baruch A. Michiel D.F. Oppenheim J.J. J. Biol. 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In humans, four CXC chemokine receptors (CXCR1 to 4), eight CC chemokine receptors (CCR1 to 8), 1The abbreviations and other trivial names used are: CCR, chemokine receptor; IL-8, interleukin 8; IP-10, interferon γ-inducible 10-kDa protein; Mig, monokine induced by IFN-γ; SDF, stroma-derived factor; PBSF, pre-B cell stimulatory factor; MIP, macrophage inflammatory protein; RANTES, regulated upon activation, normal T cell-expressed and secreted; MCP, monocyte chemoattractant protein; MPIF, myeloid progenitor inhibitory factor; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine; LARC, liver and activation-regulated chemokine; SEAP, the secreted form of alkaline phosphatase; PHA, phytohemagglutinin; IL-2, interleukin 2; PAGE, polyacrylamide gel electrophoresis; EBI, EBV-induced gene; HHV, human herpesvirus; SLC, secondary lymphoid-tissue chemokine; ELC, EBI1-ligand chemokine; RACE, rapid amplification of cDNA ends; MES, 4-morpholineethanesulfonic acid; EBV, Epstein-Barr virus. 1The abbreviations and other trivial names used are: CCR, chemokine receptor; IL-8, interleukin 8; IP-10, interferon γ-inducible 10-kDa protein; Mig, monokine induced by IFN-γ; SDF, stroma-derived factor; PBSF, pre-B cell stimulatory factor; MIP, macrophage inflammatory protein; RANTES, regulated upon activation, normal T cell-expressed and secreted; MCP, monocyte chemoattractant protein; MPIF, myeloid progenitor inhibitory factor; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine; LARC, liver and activation-regulated chemokine; SEAP, the secreted form of alkaline phosphatase; PHA, phytohemagglutinin; IL-2, interleukin 2; PAGE, polyacrylamide gel electrophoresis; EBI, EBV-induced gene; HHV, human herpesvirus; SLC, secondary lymphoid-tissue chemokine; ELC, EBI1-ligand chemokine; RACE, rapid amplification of cDNA ends; MES, 4-morpholineethanesulfonic acid; EBV, Epstein-Barr virus. and one CX3C chemokine receptor (CX3CR1) have been identified and defined for their ligand specificities: CXCR1 for IL-8 (5Holmes W.E. 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Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T.J. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar). Previously, we have described a novel human CC chemokine, termedSecondary Lymphoid-tissue Chemokine (SLC), which is mainly and constitutively expressed in the secondary lymphoid tissues such as lymph nodes, appendix, and spleen (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Independently, the same chemokine has been reported with terms of 6Ckine and Exodus-2 (37Hedrick J.A. Zlotnik A. J. Immunol. 1997; 159: 1589-1593PubMed Google Scholar, 38Hromas R. Kim C.H. Klemsz M. Krathwohl M. Fife K. Cooper S. Schnizlein-Bick C. Broxmeyer H.E. J. Immunol. 1997; 159: 2554-2558PubMed Google Scholar). We have demonstrated that recombinant SLC, while not active on peripheral blood monocytes or neutrophils, is a potent chemoattractant for peripheral blood lymphocytes and induces a vigorous calcium mobilization in cultured normal T cells (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Exodus-2 was also shown to be chemotactic for human T cells and B cells but not for monocytes or neutrophils (38Hromas R. Kim C.H. Klemsz M. Krathwohl M. Fife K. Cooper S. Schnizlein-Bick C. Broxmeyer H.E. J. Immunol. 1997; 159: 2554-2558PubMed Google Scholar). Consistent with its lymphocyte-specific activities, SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) bound specifically to lymphocytes, and its binding was fully displaced only by SLC among ten CC chemokines so far tested (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). These findings suggest that lymphocytes express a class of receptors highly specific for SLC. Notably, the SLCgene (SCYA21) is mapped to chromosome 9p13 (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar) where the gene for another lymphocyte-specific CC chemokine ELC (SCYA19) also exists (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), suggesting their divergence from a common ancestral gene. We have shown that ELC is a high affinity functional ligand for CCR7 that is expressed on T and B lymphocytes (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Here we demonstrate that SLC is also a high affinity functional ligand for CCR7. A murine pre-B cell line L1.2 (39Gallatin W.M. Weissman I.L. Butcher E.C. Nature. 1983; 304: 30-34Crossref PubMed Scopus (1234) Google Scholar) was maintained in RPMI 1640 supplemented with 10% fetal calf serum. L1.2 cells stably expressing transfected human CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, and CCR7 were generated as described previously (25Imai T. Baba M. Nishimura M. Kakizaki M. Takagi S. Yoshie O. J. Biol. Chem. 1997; 272: 15036-15042Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Peripheral blood mononuclear cells were isolated from EDTA-treated venous blood obtained from healthy adult donors by using Ficoll-Paque (Pharmacia Biotech Inc., Uppsala, Sweden). T cells were expanded by stimulation with phytohemagglutinin (PHA) (Life Technologies, Inc.) for 2 days and further cultivation with 200 units/ml interleukin 2 (IL-2) for a week. Eotaxin, SLC, TARC, LARC, and MCP-1 were produced by a baculovirus expression system and purified to essential homogeneity as described previously (19Kitaura M. Nakajima T. Imai T. Harada S. Combadiere C. Tiffany H.L. Murphy P.M. Yoshie O. J. Biol. Chem. 1996; 271: 7725-7730Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 40Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 41Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). MIP-1α and RANTES were purchased from Peprotech (Rocky Hill, NJ). ELC was produced in Escherichia coli and purified as follows. To express a fusion protein consisting of the amino-terminal (His)6 tag and the enterokinase cleavage site (Asp-Asp-Asp-Asp-Lys) linked to mature ELC, a DNA fragment termed EK-ELC-XhoI was generated from pCRII-5′-RACE-ELC (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) by polymerase chain reaction using EK-ELC primer (+5′-TCCGACGACGACGACAAGGGCACCAATGATGCTGAA) and ELC-XhoI primer (−5′-ACGTCTCGAGTTAACTGCTGCGGCGCTTCAT). Then, a DNA fragment termed NheI-EK-ELC-XhoI was generated from EK-ELC-XhoI by polymerase chain reaction using NheI-EK primer (+5′-GCGCTAGCAGCAGCGGATCCGACGACGACGACAAG) and ELC-XhoI primer. After digestion with NheI and XhoI, the DNA fragment was cloned into an expression vector pRSET A to generate pRSET A-(His)6-EK-ELC. BL21(DE3)pLysS strain (Novagen) was transformed with the expression vector and induced by isopropyl-β-d-thiogalactopyranoside (Sigma) following the standard protocols. The pellet was collected by centrifugation and suspended by a lysis buffer (0.5% Sarkosyl, 20 mmTris-HCl, pH 8.0, and 150 mm NaCl). After sonication and centrifugation, the supernatants were applied to a TARON metal affinity resin column (CLONTECH). Eluted fractions were analyzed by staining with Coomassie Blue after SDS-polyacrylamide gel electrophoresis (PAGE). The fractions containing the ELC-fusion protein were pooled, dialyzed against 0.5% acetic acid, lyophilized, and dissolved in distilled water. The solution was applied to a 1-ml HiTrap SP column (Pharmacia) equilibrated with 50 mm MES, pH 6.0, and eluted with a 25-ml linear gradient of 0.4–1.4 m NaCl in 50 mm MES, pH 6.0, at a rate of 0.5 ml/min on a fast protein liquid chromatography (Pharmacia). Fractions were analyzed by staining with Coomassie Blue after SDS-PAGE, and fractions containing the ELC fusion protein were pooled, dialyzed against 0.5% acetic acid, lyophilized, and dissolved in distilled water. After digestion with enterokinase in 10 mm MES, pH 6.0, and 0.1% Tween 20 at 4 °C, the solution was injected into a reverse-phase high performance liquid chromatography column (Cosmocil 5C4-AR-300, 4.6 × 250 mm) (Cosmo Bio, Tokyo, Japan) equilibrated with 0.05% trifluoroacetic acid and eluted with a 0–60% linear gradient of acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. The peak fraction containing ELC was lyophilized (Fig. 1 A). The protein concentration was determined by a BCA kit (Pierce, Rockford, IL), and the purity was analyzed by SDS-PAGE and silver staining (Fig.1 B). Endotoxin levels were always <4 pg/μg of ELC as determined by the Limulus amoebocyte lysate assay (QCL-1000) (BioWhittaker, Walkersville, MD). Calcium mobilization in response to chemokines was determined as described previously (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In brief, cells were loaded with 1 μm Fura 2-AM (Molecular Probe, Eugene, OR) in RPMI 1640 supplemented with 1% fetal calf serum and 10 mm HEPES, pH 7.4, for 1 h at 37 °C in the dark. Loaded cells were washed four times with phosphate-buffered saline containing 1% fetal calf serum, 1 mm CaCl2, and 1 mm MgCl2, and resuspended in the same buffer at 2.5 × 106 cells/ml (L1.2 lines) or 1.25 × 106 cells/ml (cultured normal T cells). To monitor intracellular calcium concentration, 2 ml of the cell suspension in a quartz cuvette was placed in a spectrofluorimeter (LS 50B, Perkin-Elmer) and stimulated with each chemokine at 37 °C. Emission fluorescence at 510 nm was monitored upon excitation at 340 (F340) and 380 nm (F380) at every 200 ms. Data were expressed by the ratio of F340 to F380(R340/380). SLC and ELC fused with the secreted form of placental alkaline phosphatase (SLC-SEAP and ELC-SEAP) were produced as described previously (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). For binding experiments, 2 × 105 L1.2 cells stably expressing CCR7 or cultured normal T cells were incubated for 1 h at 16 °C with 1 nm of SLC-SEAP or ELC-SEAP without or with increasing concentrations of unlabeled competitors in 200 μl of RPMI 1640 containing 20 mm HEPES, pH 7.4, 1% bovine serum albumin (BSA), and 0.02% sodium azide. Cells were washed and lysed in 50 μl of 10 mm Tris-HCl, pH 8.0, and 1% Triton X-100. Cell lysates were heated at 65 °C for 10 min to inactivate cellular phosphatases and centrifuged to remove cell debris. AP activity in 10 μl of lysate was determined by a chemiluminescence method as described previously (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). All assays were done in duplicate. The chemotaxis assay was performed essentially as described previously (36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In brief, parental L1.2 and L1.2 stably expressing CCR7 were suspended in the assay buffer (RPMI 1640 supplemented with 10 mm HEPES, pH 7.4, 1% BSA). The cells in 100 μl were placed in the upper compartments of transwell chambers with 3 μm pore size (Costar), while 0.6 ml of the buffer without or with chemokines were placed in the lower compartments. After 4 h at 37 °C, cells which migrated into lower chambers were collected and counted on a FACStar Plus (Becton Dickinson, Mountain View, CA). Results are expressed as percent input cells that migrated through the filter. All assays were done in duplicate. To investigate the ability of SLC to induce signaling through the known CC chemokine receptors, we examined calcium mobilization in murine L1.2 cells stably expressing human CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, and CCR7 upon stimulation with SLC. As shown in Fig. 2, SLC did not induce significant calcium flux in parental L1.2 or those expressing CCR1, CCR2B, CCR3, CCR4, CCR5, or CCR6. We confirmed that L1.2 expressing each receptor responded to an appropriate ligand with a vigorous calcium flux. Strikingly, however, SLC induced a vigorous calcium flux in L1.2 cells expressing CCR7, which is a known receptor for a CC chemokine ELC (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Thus, SLC is another functional ligand for CCR7. Furthermore, SLC fully desensitized CCR7-expressing L1.2 cells against subsequent stimulation with an equal amount of ELC. Conversely, ELC fully desensitized the same cells against subsequent stimulation with an equal amount of SLC. Thus, SLC and ELC are essentially equivalent in terms of calcium mobilization via CCR7 expressed on transfected L1.2 cells. We next compared the binding of SLC and ELC to CCR7. As shown in Fig. 3, SLC-SEAP bound specifically to CCR7-expressing L1.2 cells at high levels. The binding was fully competed by unlabeled SLC and ELC with an IC50 of 0.5 nm and 1.0 nm, respectively. Conversely, the binding of ELC-SEAP to CCR7 was fully competed by SLC and ELC with an IC50 of 8.2 and 6.1 nm, respectively. Thus, SLC and ELC are essentially equivalent in terms of cross-competition in binding to CCR7 expressed on transfected L1.2 cells. We next compared the ability of SLC and ELC to induce chemotaxis. As shown in Fig. 4, both SLC and ELC induced vigorous migration in CCR7-transfected L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nm. The potency and efficiency of SLC and ELC in induction of chemotaxis via CCR7 were very similar. Untransfected L1.2 cells failed to migrate toward SLC or ELC (not shown). These results again demonstrate that SLC and ELC are essentially equivalent as agonists for CCR7 expressed on transfected L1.2 cells. CCR7 is expressed on T and B lymphocytes (42Birkenbach M. Josefsen K. Yalamanchili R. Lenoir G. Kieff E. J. Virol. 1993; 67: 2209-2220Crossref PubMed Google Scholar, 43Schweickart V.L. Raport C.J. Godiska R. Byers M.G. Eddy Jr., R.L. Shows T.B. Gray P.W. Genomics. 1994; 23: 643-650Crossref PubMed Scopus (82) Google Scholar, 44Burgstahler R. Kempkes B. Steube K. Lipp M. Biochem. Biophys. Res. Commun. 1995; 215: 737-743Crossref PubMed Scopus (81) Google Scholar, 45Hasegawa H. Utsunomiya Y. Yasukawa M. Yanagisawa K. Fujita S. J. Virol. 1994; 68: 5326-5329Crossref PubMed Google Scholar). To test whether SLC and ELC also share receptors on lymphocytes, we next examined induction of calcium mobilization in cultured peripheral blood T cells that were initially stimulated with PHA and subsequently expanded with IL-2. As shown in Fig. 5, SLC and ELC at 10 nm induced vigorous calcium mobilization in cultured normal T cells (A and B). In contrast to the results with CCR7-transfected L1.2 cells (Fig. 2), however, SLC only partially desensitized cultured normal T cells against subsequent stimulation with an equal amount of ELC (Fig. 5 A). On the other hand, ELC fully desensitized T cells against subsequent stimulation with an equal amount of SLC (Fig. 5 B). As expected, SLC desensitized T cells against subsequent stimulation with an equal amount of SLC (Fig. 5 C). SLC at 100 nm, however, fully desensitized T cells against ELC at 10 nm (Fig.5 D). These results suggest that, even though SLC and ELC fully share a class of receptors expressed on normal T cells, they may exhibit different binding affinities. To test this possibility, SLC and ELC were compared for their ability to compete with ELC-SEAP for binding to cultured normal T cells. As shown in Fig. 6, both SLC and ELC were indeed capable of fully competing with ELC-SEAP for binding to T cells. However, SLC and ELC exhibited an IC50of 10 and 1.6 nm, respectively. Thus, even though SLC and ELC fully share receptors expressed on cultured normal T cells, most probably CCR7, SLC appeared to bind to CCR7 expressed on these cells with an affinity somehow lower than that of ELC in contrast to CCR7 expressed on transfected L1.2 cells (Fig. 3). Previously, we have shown that ELC is a high affinity functional ligand for CCR7 (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). ELC binds specifically to CCR7 with high affinity and induces vigorous calcium mobilization and efficient chemotaxis in CCR7-transfected human cells such as K562 and HEK293/EBNA-1 (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). In the present study, we have demonstrated that SLC is also a high affinity functional ligand for CCR7. SLC binds to CCR7 with high affinity and induces calcium mobilization and chemotaxis in CCR7-transfected murine L1.2 cells (Figs. Figure 2, Figure 3, Figure 4). Quite remarkably, SLC and ELC are almost equivalent as ligands for CCR7 when CCR7 is expressed on L1.2 cells. This was revealed by full cross-desensitization against each other in calcium mobilization via CCR7 (Fig. 2), efficient cross competition in each binding to CCR7 (Fig. 3), and a very similar potency and efficiency in induction of chemotaxis via CCR7 (Fig. 4). SLC, however, was much less efficient in desensitizing cultured normal T cells against ELC (Fig. 5). Even though SLC fully competed with ELC-SEAP for binding to cultured normal T cells, SLC appears to bind to these cells with an affinity lower than that of ELC (Fig. 6). Collectively, the cell background may affect the structure and/or function of CCR7 possibly through differential coupling of G proteins (46Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). This may cause differences in binding affinity and efficiency of cross-desensitization between SLC and ELC depending on cell types. SLC and ELC share only 32% amino acid identity (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). SLC also has a unique extension of about 30 amino acids with two extra cysteine residues in its carboxyl terminus. As shown in Fig. 7, however, certain amino acid residues marked by closed circles are shared only by SLC and ELC among the known human CC chemokines. Since the amino-terminal region preceding the first conserved cysteine residue, the amino-terminal loop region, the β-turn region containing the third cysteine, and the residues preceding the fourth cysteine have been shown to be important for the receptor specificity of IL-8 (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1977) Google Scholar), the residues shared only by SLC and ELC in such regions may be involved in their specific binding to CCR7. In addition to sharing CCR7, SLC and ELC constitute a mini-cluster at human chromosome 9p13 and have very similar patterns of tissue-expression (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Thus, genetically and functionally, SLC and ELC are highly related chemokines and possibly redundant to some extent. In fact, a similar functional redundancy is also noted for other sets of chemokines derived from mini-clusters. IP-10 and Mig form a mini-cluster at human chromosome 4q21.21 (47Farber J.M. J. Leukocyte Biol. 1997; 61: 246-257Crossref PubMed Scopus (688) Google Scholar). They are both highly inducible by IFN-γ (47Farber J.M. J. Leukocyte Biol. 1997; 61: 246-257Crossref PubMed Scopus (688) Google Scholar) and share CXCR3 (9Loetscher M. Gerber B. Loetscher P. Jones S.A. Piali L. Clark-Lewis I. Baggiolini M. Moser B. J. Exp. Med. 1996; 184: 963-969Crossref PubMed Scopus (1055) Google Scholar). TARC (40Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) and MDC (48Godiska R. Chantry D. Raport C.J. Sozzani S. Allavena P. Leviten D. Mantovani A. Gray P.W. J. Exp. Med. 1997; 185: 1595-1604Crossref PubMed Scopus (416) Google Scholar) form a mini-cluster at chromosome 16q13 (49Nomiyama H. Imai T. Kusuda J. Miura R. Callen D.F. Yoshie O. Cytogenet. Cell Genet. 1998; (in press)PubMed Google Scholar). They are both expressed mainly in the thymus (40Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 48Godiska R. Chantry D. Raport C.J. Sozzani S. Allavena P. Leviten D. Mantovani A. Gray P.W. J. Exp. Med. 1997; 185: 1595-1604Crossref PubMed Scopus (416) Google Scholar) and share CCR4 (26Imai T. Chantry D. Raport C.J. Wood C. Nishimura M. Godiska R. Yoshie O. Gray P.W. J. Biol. Chem. 1998; 273: 1764-1768Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Thus, the members of each mini-cluster, while relatively independent from the majority of chemokines encoded by the traditional gene clusters at chromosome 4q12-q13 (CXC chemokines) (50Tunnacliffe A. Majumdar S. Yan B. Poncz M. Blood. 1992; 79: 2896-2900Crossref PubMed Google Scholar) and 17q11.2 (CC chemokines) (51Naruse K. Ueno M. Satoh T. Nomiyama H. Tei H. Takeda M. Ledbetter D.H. Van Coillie E. Opdenakker G. Gunge N. Sakaki Y. Iio M. Miura R. Genomics. 1996; 34: 236-240Crossref PubMed Scopus (107) Google Scholar), obviously originate from a common ancestral gene and may have evolved to serve overlapping if not identical functions in vivo. Even though SLC and ELC are almost equivalent as ligands for CCR7, there are some notable differences in their tissue patterns of expression (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). For example, ELC was found to be much more strongly expressed in the thymus than SLC. Besides mostly overlapping expression in various lymphoid tissues, SLC but not ELC was also expressed in heart, pancreas, thyroid gland, etc. Such differences in tissue distribution may indicate their differential roles in vivo. Furthermore, as shown for IP-10 and Mig (47Farber J.M. J. Leukocyte Biol. 1997; 61: 246-257Crossref PubMed Scopus (688) Google Scholar), the inducibility of SLC and ELC may be different in certain in vivo situations. The carboxyl-terminal extension of SLC with two extra cysteine residues may also have unique biological functions in vivo through interaction with other molecules. Besides CCR7, there may be still other receptors either for SLC or ELC. Targeted disruption of the SLC and ELC genes may be helpful to elucidate their respective physiological and pathological roles in vivo. CCR7 was originally identified through its strong up-regulation in an EBV-negative Burkitt's lymphoma cell line upon infection with EBV (thus originally termed EBI1 from Epstein-Barr virus-induced gene 1) (42Birkenbach M. Josefsen K. Yalamanchili R. Lenoir G. Kieff E. J. Virol. 1993; 67: 2209-2220Crossref PubMed Google Scholar). EBI1/CCR7 was shown to be transactivated by EBV-encoded nuclear antigen-2 (EBNA-2) (44Burgstahler R. Kempkes B. Steube K. Lipp M. Biochem. Biophys. Res. Commun. 1995; 215: 737-743Crossref PubMed Scopus (81) Google Scholar), and also to be up-regulated in CD4+ T cells upon infection with HHV6 and HHV7 (45Hasegawa H. Utsunomiya Y. Yasukawa M. Yanagisawa K. Fujita S. J. Virol. 1994; 68: 5326-5329Crossref PubMed Google Scholar). EBI1/CCR7 is expressed at high levels in various lymphoid tissues and on peripheral blood T and B lymphocytes (43Schweickart V.L. Raport C.J. Godiska R. Byers M.G. Eddy Jr., R.L. Shows T.B. Gray P.W. Genomics. 1994; 23: 643-650Crossref PubMed Scopus (82) Google Scholar). Circulating lymphocytes emigrate from blood into interfollicular regions of the secondary lymphoid tissues in a search for antigens presented on the reticular network of cells. Homing of lymphocytes into specific secondary lymphoid tissues is regulated by multi-step interactions between circulating lymphocytes and high endothelial venules (HEV) via specific combinations of adhesion molecules (52Butcher E.C. Picker L.J. Science. 1996; 272: 60-66Crossref PubMed Scopus (2507) Google Scholar, 53Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6380) Google Scholar). Several lines of evidence also indicate that G-protein-coupled receptors are essential for lymphocyte homing (54Bargatze R.F. Butcher E.C. J. Exp. Med. 1993; 178: 367-372Crossref PubMed Scopus (252) Google Scholar, 55Cyster J.G. Goodnow C.C. J. Exp. Med. 1995; 182: 581-586Crossref PubMed Scopus (164) Google Scholar). Chemokines, which elicit integrin activation and diapedesis via respective G-protein-coupled receptors, are thus likely to play important roles in directed migration of various lymphocyte classes and subsets from blood into and within lymphoid tissues, but their identity still remains mostly unknown. SLC and ELC are constitutively expressed in various lymphoid tissues where lymphocytes expressing CCR7 also exist abundantly (32Yoshida R. Imai T. Hieshima K. Kusuda J. Baba M. Kitaura M. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 13803-13809Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 36Nagira M. Imai T. Hieshima K. Kusuda J. Ridanpaa M. Takagi S. Nishimura M. Kakizaki M. Nomiyama H. Yoshie O. J. Biol. Chem. 1997; 272: 19518-19524Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 43Schweickart V.L. Raport C.J. Godiska R. Byers M.G. Eddy Jr., R.L. Shows T.B. Gray P.W. Genomics. 1994; 23: 643-650Crossref PubMed Scopus (82) Google Scholar). Thus, SLC and ELC may be the ones that are involved in the homeostatic lymphocyte recirculation and homing. They may also facilitate tissue accumulation of lymphocytes in various immune responses. Furthermore, they may affect tissue localization of lymphocytes infected by lymphotropic herpesviruses such as EBV, HHV6, and HHV7. Thus, SLC, ELC, and CCR7 may define new targets for drug development aiming at controlling various immune responses and/or suppressing persistent infections with certain lymphotropic herpesviruses. We are grateful for Dr. Yorio Hinuma and Dr. Masakazu Hatanaka for constant support and encouragement.