Secondary Lymphoid-tissue Chemokine Is a Functional Ligand for the

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Dec 5, 1997 - 1, and MIP-1 (27, 28); CCR6 for LARC/MIP-3/exodus (29–. 31); CCR7 for ELC/MIP-3 (32); CCR8 for I-309 (33, 34); and. CX3CR1 for ..... SLC also has a unique extension of about 30 amino acids with two extra cysteine ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 12, Issue of March 20, pp. 7118 –7122, 1998 Printed in U.S.A.

Secondary Lymphoid-tissue Chemokine Is a Functional Ligand for the CC Chemokine Receptor CCR7* (Received for publication, December 5, 1997, and in revised form, January 3, 1998)

Ryu Yoshida‡, Morio Nagira‡, Motoji Kitaura, Noriko Imagawa, Toshio Imai, and Osamu Yoshie§ From the Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan

Secondary Lymphoid-tissue Chemokine (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. 1– 4). Based on the arrangement of the amino-terminal conserved cysteine residues, chemokines are grouped into two major subfamilies, CXC and CC. Molecules with a C or CX3C motif have also been described. CXC chemokines with the Glu-Leu-Arg (ELR) motif immediately prior to the CXC motif are potent chemoattractants for neutrophils, whereas those without the ELR motif are mostly directed to lymphocytes. On the other hand, CC chemokines are mainly directed to monocytes and also to eosinophils, basophils, and/or lymphocytes with variable selectivity. Notably, some recently * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Contributed equally to this work. § To whom correspondence should be addressed. Tel.: 81-6-382-2612; Fax: 81-6-382-2598; E-mail: [email protected].

identified CC chemokines are highly specific for lymphocytes (4). The specific effects of chemokines are mediated by a family of seven-transmembrane G-protein coupled receptors (1– 4). In humans, four CXC chemokine receptors (CXCR1 to 4), eight CC chemokine receptors (CCR1 to 8),1 and one CX3C chemokine receptor (CX3CR1) have been identified and defined for their ligand specificities: CXCR1 for IL-8 (5); CXCR2 for IL-8 and other CXC chemokines with the Glu-Leu-Arg motif (6 – 8); CXCR3 for IP-10 and Mig (9); CXCR4 for SDF-1/PBSF (10, 11); CCR1 for MIP-1a, RANTES, and MCP-3 (12–15); CCR2 for MCP-1, MCP-3, and MCP-4 (15–18); CCR3 for eotaxin, RANTES, MCP-2, MCP-3, MCP-4, and MPIF-2/eotaxin-2 (18 –24); CCR4 for TARC and MDC (25, 26); CCR5 for RANTES, MIP1a, and MIP-1b (27, 28); CCR6 for LARC/MIP-3a/exodus (29 – 31); CCR7 for ELC/MIP-3b (32); CCR8 for I-309 (33, 34); and CX3CR1 for fractalkine/neurotactin (35). Previously, we have described a novel human CC chemokine, termed Secondary Lymphoid-tissue Chemokine (SLC), which is mainly and constitutively expressed in the secondary lymphoid tissues such as lymph nodes, appendix, and spleen (36). Independently, the same chemokine has been reported with terms of 6Ckine and Exodus-2 (37, 38). 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 (36). Exodus-2 was also shown to be chemotactic for human T cells and B cells but not for monocytes or neutrophils (38). 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 (36). These findings suggest that lymphocytes express a class of receptors highly specific for SLC. Notably, the SLC gene (SCYA21) is mapped to chromosome 9p13 (36) where the gene for another lymphocyte-specific CC chemokine ELC (SCYA19) also exists (32), 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 (32). Here we demonstrate that SLC is also a high affinity functional ligand for CCR7.

1 The abbreviations and other trivial names used are: CCR, chemokine receptor; IL-8, interleukin 8; IP-10, interferon g-inducible 10-kDa protein; Mig, monokine induced by IFN-g; 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.

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SLC Is a Ligand for CCR7

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EXPERIMENTAL PROCEDURES

Cells—A murine pre-B cell line L1.2 (39) 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 (25). 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. Chemokines—Eotaxin, SLC, TARC, LARC, and MCP-1 were produced by a baculovirus expression system and purified to essential homogeneity as described previously (19, 36, 40, 41). MIP-1a 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 aminoterminal (His)6 tag and the enterokinase cleavage site (Asp-Asp-Asp-AspLys) linked to mature ELC, a DNA fragment termed EK-ELC-XhoI was generated from pCRII-59-RACE-ELC (32) by polymerase chain reaction using EK-ELC primer (159-TCCGACGACGACGACAAGGGCACCAATGATGCTGAA) and ELC-XhoI primer (259-ACGTCTCGAGTTAACTGCTGCGGCGCTTCAT). Then, a DNA fragment termed NheI-EK-ELC-XhoI was generated from EK-ELC-XhoI by polymerase chain reaction using NheI-EK primer (159-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-b-D-thiogalactopyranoside (Sigma) following the standard protocols. The pellet was collected by centrifugation and suspended by a lysis buffer (0.5% Sarkosyl, 20 mM Tris-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 3 250 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. 1A). The protein concentration was determined by a BCA kit (Pierce, Rockford, IL), and the purity was analyzed by SDS-PAGE and silver staining (Fig. 1B). Endotoxin levels were always ,4 pg/mg of ELC as determined by the Limulus amoebocyte lysate assay (QCL-1000) (BioWhittaker, Walkersville, MD). Calcium Mobilization Assay—Calcium mobilization in response to chemokines was determined as described previously (36). In brief, cells were loaded with 1 mM 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 3 106 cells/ml (L1.2 lines) or 1.25 3 106 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). Binding Assay—SLC and ELC fused with the secreted form of placental alkaline phosphatase (SLC-SEAP and ELC-SEAP) were produced as described previously (32, 36). For binding experiments, 2 3 105 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 ml 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 ml of 10 mM Tris-HCl, pH 8.0, and 1% Triton X-100. Cell

FIG. 1. Purification of recombinant ELC produced by E. coli. A, the elution profile of ELC from a reverse-phase high performance liquid chromatography. ELC was eluted as indicated by the arrow. B, analysis of purified ELC. Purified ELC was analyzed by SDS-PAGE and silver staining. Positions of size markers are shown on the right (kDa). lysates were heated at 65 °C for 10 min to inactivate cellular phosphatases and centrifuged to remove cell debris. AP activity in 10 ml of lysate was determined by a chemiluminescence method as described previously (32, 36). All assays were done in duplicate. Chemotaxis Assay—The chemotaxis assay was performed essentially as described previously (36). 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 ml were placed in the upper compartments of transwell chambers with 3 mm 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. RESULTS

SLC Induces Calcium Mobilization in CCR7-Transfected L1.2 Cells—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 (32). 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. SLC Binds to CCR7—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

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FIG. 2. Calcium mobilization in CCR7-transfected L1.2 cells by SLC and ELC. Transfected L1.2 cells stably expressing CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, or CCR7 were loaded with Fura 2-AM and stimulated with 10 nM each chemokine as indicated. Intracellular concentrations of calcium were monitored by fluorescence ratio R340/380. Representative results from three separate experiments are shown.

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. SLC Stimulates Chemotaxis in CCR7-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. SLC and ELC Share Receptors on Cultured Normal T Cells— CCR7 is expressed on T and B lymphocytes (42– 45). 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. 5A). On the other hand, ELC fully desensitized T cells against subsequent stimulation with an equal amount of SLC (Fig. 5B). As expected, SLC desensitized T cells against subsequent stimulation with an equal amount of SLC (Fig. 5C). SLC at 100 nM, however, fully desensitized T cells against ELC at 10 nM (Fig. 5D). 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

FIG. 3. Competition of SLC and ELC with SLC-SEAP and ELCSEAP for binding to CCR7. Transfected L1.2 cells (2 3 105 cells) stably expressing CCR7 were incubated with 1 nM SLC-SEAP or ELCSEAP as indicated without or with increasing concentrations of SLC (closed circle) and ELC (open circle) at 16 °C for 1 h. After washing, amounts of cell-bound SLC-SEAP or ELC-SEAP were determined enzymatically. All assays were done in duplicate. Representative results from two separate experiments are shown.

FIG. 4. Chemotaxis induction of CCR7 transfectants by SLC and ELC. Parental L1.2 cells (closed squares) or transfected L1.2 cells stably expressing CCR7 (circles) were placed in upper wells and allowed to migrate toward SLC (closed circles and closed squares) or ELC (open circles) in the lower wells. Migrated cells were collected and counted by flow cytometry. The assay was done in duplicate. Representative results from two separate experiments are shown.

and ELC were compared for their ability to compete with ELCSEAP 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 IC50 of 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). DISCUSSION

Previously, we have shown that ELC is a high affinity functional ligand for CCR7 (32). 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 (32). In the present study, we have demonstrated that SLC is also a high affinity functional

SLC Is a Ligand for CCR7

FIG. 5. Calcium mobilization in cultured normal T cells by SLC and ELC. Normal T cells expanded from peripheral blood mononuclear cells by stimulation with PHA for 2 days and subsequent cultivation with IL-2 for a week were loaded with Fura 2-AM and stimulated with SLC or ELC as indicated by arrowheads. Intracellular concentrations of calcium were monitored by fluorescence ratio R340/380. Panels A, B, and C, stimulated with SLC and ELC at 10 nM; panel D, stimulated with SLC at 100 nM and ELC at 10 nM. Representative results from three separate experiments are shown.

ligand for CCR7. SLC binds to CCR7 with high affinity and induces calcium mobilization and chemotaxis in CCR7-transfected murine L1.2 cells (Figs. 2– 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 (46). 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 (32, 36). 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 aminoterminal loop region, the b-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 (1), 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 (32, 36). Thus, genetically and

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FIG. 6. Competition of SLC and ELC with ELC-SEAP for binding to cultured normal T cells. Normal T cells expanded from peripheral blood mononuclear cells by stimulation with PHA for 2 days and subsequent cultivation with IL-2 for a week were incubated with 1 nM ELC-SEAP without or with increasing concentrations of SLC (closed circles) or ELC (open circles) at 16 °C for 1 h. After washing, amounts of ELC-SEAP were determined enzymatically. All assays were done in duplicate. Representative results from two separate experiments are shown.

FIG. 7. Amino acid alignment of human SLC and ELC. The residues highly conserved among the known human CC chemokines are boxed. The residues shared only by SLC and ELC among the known human CC chemokines are marked by closed circles.

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 (47). They are both highly inducible by IFN-g (47) and share CXCR3 (9). TARC (40) and MDC (48) form a mini-cluster at chromosome 16q13 (49). They are both expressed mainly in the thymus (40, 48) and share CCR4 (26). 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) (50) and 17q11.2 (CC chemokines) (51), 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 (32, 36). 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 (47), the inducibility of SLC and ELC may be different in certain in vivo situations. The carboxylterminal extension of SLC with two extra cysteine residues

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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 EpsteinBarr virus-induced gene 1) (42). EBI1/CCR7 was shown to be transactivated by EBV-encoded nuclear antigen-2 (EBNA-2) (44), and also to be up-regulated in CD41 T cells upon infection with HHV6 and HHV7 (45). EBI1/CCR7 is expressed at high levels in various lymphoid tissues and on peripheral blood T and B lymphocytes (43). 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 (52, 53). Several lines of evidence also indicate that G-protein-coupled receptors are essential for lymphocyte homing (54, 55). 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 (32, 36, 43). 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. Acknowledgments—We are grateful for Dr. Yorio Hinuma and Dr. Masakazu Hatanaka for constant support and encouragement. REFERENCES 1. Baggiolini, M., Dewald, B., and Moser, B. (1997) Annu. Rev. Immunol. 15, 675–705 2. Ben-Baruch, A., Michiel, D. F., and Oppenheim, J. J. (1995) J. Biol. Chem. 270, 11703–11706 3. Rollins, B. (1997) Blood 90, 909 –928 4. Yoshie, O., Imai, T., and Nomiyama, H. (1997) J. Leukocyte Biol. 62, 634 – 644, 1997 5. Holmes, W. E., Lee, J., Kuang, W.-J., Rice, G. C., and Wood, W. I. (1991) Science 253, 1278 –1280 6. Murphy, P. M., and Tiffany, H. L. (1991) Science 253, 1280 –1283 7. Lee, J., Horuk, R., Rice, G. C., Bennett, G. L., Camerato, T., and Wood, W. I. (1992) J. Biol. Chem. 267, 16283–16287 8. Geiser, T., Dewald, B., Ehrengruber, M. U., Clark-Lewis, I., and Baggiolini, M. (1993) J. Biol. Chem. 268, 15419 –15424 9. Loetscher, M., Gerber, B., Loetscher, P., Jones, S. A., Piali, L., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1996) J. Exp. Med. 184, 963–969 10. Bleul, C. C., Farzan, M., Choe, H., Parolin, C., Clark-Lewis, I., Sodroski, J., and Springer, T. A. (1996) Nature 382, 829 – 833 11. Oberlin, E., Amara, A., Bachelerie, F., Bessia, C., Virelizier, J.-L., ArenzanaSeisdedos, F., Schwartz, O., Heard, J.-M., Clark-Lewis, I., Legler, D. F., Loetscher, M., Baggiolini, M., and Moser, B. (1996) Nature 382, 833– 835 12. Neote, K., DiGregorio, D., Mak, J. Y., Horuk, R., and Schall, T. J. (1993) Cell 72, 415– 425 13. Gao, J.-L., Kuhns, D. B., Tiffany, H. L., McDermott, D., Li, X., Francke, U., and Murphy, P. M. (1993) J. Exp. Med. 177, 1421–1427 14. Ben-Baruch, A., Xu, L., Young, P. R., Bengali, K., Oppenheim, J. J., and Wang, J. M. (1995) J. Biol. Chem. 270, 22123–22128 15. Combadiere, C., Ahuja, S. K., Van Damme, J., Tiffany, H. L., Gao, J.-L., and

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