Molecular Cloning of a Novel Human CC Chemokine EBI1-ligand Chemokine That Is a Specific Functional Ligand for EBI1, CCR7*

By searching the e xpressed s equence t ag (EST) data base, we identified partial cDNA sequences encoding a novel human CC chemokine. We determined the com- plete cDNA sequence that encodes a highly basic polypeptide of a total 98 amino acids with 20 to 30% identity to other human CC chemokines. We termed this novel chemokine from E BI1- L igand C hemokine as ELC (see below). The ELC mRNA was most strongly expressed in the thymus and lymph nodes. Recombinant ELC protein was expressed as a fusion protein with the Flag tag (ELC-Flag). For receptor-binding assays, re- combinant ELC protein fused with the secreted form of alkaline phosphatase (SEAP) was used. By stably ex- pressing five CC chemokine receptors (CCR1 to 5) and five orphan receptors, ELC-SEAP was found to bind spe- cifically to an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3, eotaxin, MIP-1 (cid:97) , MIP-1 (cid:98) Calcium Mobilization Assay— K562 cells stably expressing cloned chemokine receptors were suspended at 3 (cid:51) 10 6 cells/ml in Hank’s balanced salt solution supplemented with 1 mg/ml BSA and 10 m M HEPES, pH 7.4, and loaded with 1 (cid:109) M Fura-PE3-AM Fluorescence Labs) by incubation for 1 h atroom temperature in the dark. Loaded cells were washed twice with Hank’s balanced salt solution-BSA and resuspended in the same buffer at 2.5 (cid:51) 10 6 cells/ml. To measure intracellular calcium, 2 ml of the cell suspension was placed in a quartz cuvette in a Perkin-Elmer LS 50B spectrofluorimeter and stimulated with chemokines at 37 °C. Fluorescence was monitored at 340 nm ( (cid:108) ex1), 380 nm ( (cid:108) ex2), and 510 nm ( (cid:108) every 200 ms. To determine EC 50 for calcium mobilization, a dose-response curve was generated in each experiment by plotting percent maximum responses.

The chemokines are a group of approximately 70 -90 amino acid structurally related polypeptides that play important roles in inflammatory and immunological responses primarily by virtue of their ability to recruit selective leukocyte subsets (1,2). Some chemokines may also play roles in normal lymphocyte recirculation and homing (3,4). Furthermore, certain chemokines have been shown to have other biological activities such as suppression of hematopoiesis (5)(6)(7), stimulation of angiogenesis (8), suppression of angiogenesis (9,10), suppression of apoptosis (11), and suppression of human immunodeficiency virus infection (12)(13)(14). The chemokines are grouped into the CXC and CC subfamilies on the basis of the arrangement of the two NH 2 -terminal cysteine residues. One amino acid separates the two cysteine residues in the CXC chemokines, whereas the two cysteines are adjacent in the CC chemokines. Most CXC chemokines are potent attractants for neutrophils, whereas most CC chemokines are able to recruit monocytes, and also lymphocytes, basophils, and/or eosinophils with variable selectivity (1,2). Recently, a novel chemokine-like cytokine lymphotactin/SCM-1 1 has been reported, which carries only the second and the fourth of the four cysteine residues conserved in the chemokines and seems to act specifically on lymphocytes (15,16). This may suggest the existence of the C-type chemokine subfamily.
The expressed sequence tags (ESTs) consist of partial "single pass" cDNA sequences from various tissues (44). Analysis of the EST data bases is becoming a powerful approach to look for new members of gene families. Recently, we have identified a number of novel human CC chemokines by initially searching the EST data bases for homology with known CC chemokine members (38,45). Here we report a novel human CC chemokine that is expressed in various lymphoid tissues and turns out to be a specific high-affinity functional ligand for EBI1 (40). Thus, we have designated this novel CC chemokine ELC from EBI1-ligand chemokine. The ELC gene is mapped to chromosome 9p13 instead of 17q11.2 where the genes for most other CC chemokines are clustered. We now propose EBI1 to be designated as CCR7.
EST Data Base Search-The dbEST (44) was searched with various CC chemokine nucleotide sequences or amino acid sequences as queries using the data base search and analysis service Search Launcher (50) available on the World Wide Web. The program used was Basic Local Alignment Search Tool (51).
Isolation and Sequence of ELC cDNA-The full-length cDNA sequence was obtained by the rapid amplification of cDNA ends (RACE) method (52). In brief, 5Ј and 3Ј RACE polymerase chain reactions (PCR) were carried out using human fetal lung cDNA commercially available for RACE-PCR (CLONTECH, Palo Alto, CA). The cDNA was amplified by PCR with one of the gene-specific primers based on an EST sequence (GenBank TM accession number N71167) (5Ј RACE-primer, CTCTGAC-CACACTCACCCTCTCGCT; 3Ј RACE-primer, GAGCCCGGAGTC-CGAGTCAAGCATT) and an AP1 primer (CLONTECH), which is complementary to part of the cDNA adaptor ligated at both ends of the cDNA. PCR was performed in a 50-l reaction mixture containing 0.2 mM each of dNTPs, 10 pmol of each of the primers, 2.5 units of TAKARA LA Taq (Takara, Kyoto, Japan), 1 ϫ buffer supplied with the polymerase, and 0.55 g of TaqStart antibody (CLONTECH). The PCR conditions were 5 cycles of 94°C for 30 s and 72°C for 4 min, 5 cycles of 94°C for 30 s and 70°C for 4 min, and then 25 cycles of 94°C for 30 s and 68°C for 4 min. The amplification products were cloned into pCR-II vector (Stratagene, La Jolla, CA) by T-A ligation and sequenced on both strands using gene-specific and commercial primers.
Northern Blot Analysis-This was carried out as described previously (53). In brief, multiple tissue blots, and immune blots were purchased from CLONTECH. Filters were hybridized with the 32 P-labeled ELC cDNA probe at 65°C for 1 h in QuikHyb Hybridization Solution (Stratagene) containing denatured 100 g/ml salmon sperm DNA. After washing at 65°C for 30 min in 0.2 ϫ SSC and 0.1% SDS, filters were exposed to x-ray films at Ϫ80°C with an intensifying screen.
Production and Purification of ELC-Flag Fusion Protein-ELC was expressed as a fusion protein with the Flag tag (54). We originally constructed the pBluescriptKS-MCP1-Flag, encoding MCP-1 fused with the Flag tag, as follows. The SalI-MCP1-XbaI-Flag fragment was amplified from pCRScript-MCP1 by PCR using the LacZ␣-B primer (5Ј-AAAGGGGGATGTGCTGCAAGGCG) and the MCP1-XbaI-GG-Flag primer (5Ј-GTCCTTGTAGTCGCCGCCTCTAGAAGTCTTCGGAGTTT-GGGT). Then, the SalI-MCP1-XbaI-Flag-NotI fragment was amplified from the first PCR products by using the LacZ␣-B primer and the GG-Flag-NotI primer (5Ј-CGCGCGGCCGCTCACTTGTCATCGTCGT-CCTTGTAGTCGCCGCC). After digestion with SalI and NotI, the fragment was ligated into the SalI and NotI site of pBluescript KS vector. The MCP-1 coding sequence was removed from this vector by SalI and XbaI, and the ELC cDNA was subcloned in place of the MCP-1 cDNA. Then the DNA fragment encoding ELC-Flag was liberated by SalI and NotI, and inserted into pDREF-Hyg (53) to prepare the expression vector pDREF-ELC-Flag that expressed ELC fused at the COOH terminus with a 5 amino acid-linker (Ser-Arg-Ser-Ser-Gly) and the Flag tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (54). To produce the ELC-Flag protein, 293/EBNA-1 cells were transfected with pDREF-ELC-Flag using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) and cultured for 3-4 days. The culture supernatants were collected by centrifugation, filtered (0.22 m), and applied to Anti-FLAG ® M2 Affinity gel (Eastman Kodak Company, New Haven, CT) 2 times. After washing with 5 bed volumes of phosphate-buffered saline (PBS), proteins were eluted with 100 mM glycine-HCl, pH 3.0. Eluted fractions were immediately neutralized by adding 1/10 volume of 1 M Tris-HCl, pH 8.0, and analyzed by SDS-polyacrylamide electrophoresis and silver staining. The fractions containing the ELC-Flag protein were pooled, dialyzed against 20 mM Tris-HCl, pH 8.0, and injected into a reverse-phase high performance liquid chromatography column (4.6 ϫ 250 mm Cosmocil 5C4-AR-300)(Cosmo Bio, Tokyo, Japan) equilibrated with 0.05% trifluoroacetic acid. Proteins were eluted with a 0 -60% gradient of acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. Fractions containing ELC-Flag were pooled and lyophilized. Protein concentrations were determined by the BCA kit (Pierce, Rodkford, IL). NH 2terminal sequence analysis was performed on a protein sequencer (Shimazu, Tokyo, Japan).
Production of ELC-SEAP Fusion Protein-ELC was expressed as a fusion protein with the secreted form of alkaline phosphatase (SEAP) with a COOH terminus tag of 6 histidine residue, the (His) 6 tag, as described previously (38). In brief, the ELC cDNA was subcloned into pDREF-SEAP(His) 6 -Hyg (38) so that ELC was fused through a 5 amino acid linker (Ser-Arg-Ser-Ser-Gly) to SEAP with the (His) 6 tag. To produce the ELC-SEAP fusion protein, 293/EBNA-1 cells (Invitrogen) were transfected with pDREF-ELC-SEAP(His) 6 -Hyg by using Lipofectamine (Life Technologies, Inc.). After 3-4 days, the culture supernatants were collected by centrifugation, filtered (0.22 m), and added to 20 mM HEPES, pH 7.4, and 0.02% sodium azide. For the NH 2 -terminal sequence analysis, the fusion protein was affinity purified by nickelagarose chromatography (QIAGEN, Hilden, Germany). The concentration of ELC-SEAP was determined by a sandwich-type enzyme-linked immunosorbent assay as described previously (38). Briefly, 96-well microtiter plates (Maxsorb, Nunc, Roskilde, Denmark) were coated with 2 g/ml of monoclonal anti-placental alkaline phosphatase antibody (Medix Biotech, Foster City, CA) in 50 mM Tris-HCl, pH 9.5. After blocking nonspecific binding sites with 1 mg/ml bovine serum albumin (BSA) in PBS, the samples were titrated in PBS with 0.02% Tween-20. After incubation for 1 h at room temperature, the plates were washed, incubated with biotinylated rabbit anti-placental alkaline phosphatase antibody diluted 1:500 for 1 h at room temperature, washed again, and incubated for 30 min with peroxidase-conjugated streptavidin (Vector Laboratories, Burlingam, CA). After washing, bound peroxidase was detected by 3,3Ј-5,5Ј-tetramethylbenzidine. The reaction was stopped by adding H 2 SO 4 , and the absorbance at 450 nm was read. The enzymatic activity of SEAP and ELC-SEAP were determined by a chemiluminescence assay using the Great EscApe Detection kit (CLONTECH). Purified placental alkaline phosphatase (Cosmo Bio, Tokyo, Japan) was used to generate the standard curve. Alkaline phosphatase activity was expressed as relative light units, and 1 pmol of SEAP and ELC-SEAP employed in the present study corresponded to 1.45 ϫ 10 8 and 1.99 ϫ 10 8 relative light units, respectively.
Binding Assay-This was carried out as described previously (38). In brief, 2 ϫ 10 5 cells were incubated for 1 h at 16°C with 1 M of SEAP or ELC-SEAP without or with increasing concentrations of unlabeled chemokines in 200 l of RPMI 1640 containing 20 mM HEPES, pH 7.4, 1% BSA, and 0.02% sodium azide. MCP-1, eotaxin, LARC, and TARC were prepared as described previously (30,38,53). MIP-1␣, MIP-1␤, MCP-2, MCP-3, and RANTES were purchased from Pepro Tech (Rocky Hill, NJ). After that, cells were washed 5 times and lysed in 50 l of 10 mM Tris-HCl, pH 8.0, and 1% Triton X-100. Samples were heated at 65°C for 10 min to inactivate cellular phosphatases and centrifuged to remove cell debris. AP activity in 10 l of lysate was determined by the chemiluminescence assay as described above. All samples were determined in duplicate. The binding data were analyzed by the LIGAND program (55).
Calcium Mobilization Assay-K562 cells stably expressing cloned chemokine receptors were suspended at 3 ϫ 10 6 cells/ml in Hank's balanced salt solution supplemented with 1 mg/ml BSA and 10 mM HEPES, pH 7.4, and loaded with 1 M Fura-PE3-AM (Texas Fluorescence Labs) by incubation for 1 h at room temperature in the dark. Loaded cells were washed twice with Hank's balanced salt solution-BSA and resuspended in the same buffer at 2.5 ϫ 10 6 cells/ml. To measure intracellular calcium, 2 ml of the cell suspension was placed in a quartz cuvette in a Perkin-Elmer LS 50B spectrofluorimeter and stimulated with chemokines at 37°C. Fluorescence was monitored at 340 nm (ex1), 380 nm (ex2), and 510 nm (em) every 200 ms. To determine EC 50 for calcium mobilization, a dose-response curve was generated in each experiment by plotting percent maximum responses.
Migration Assay-The cell migration assay was performed using a 48-well microchemotaxis chamber as described previously (53). In brief, chemokines were diluted in Hepes-buffered RPMI 1640 supplemented with 1% BSA and placed in lower wells (30 l/well). Cells suspended in RPMI 1640, 1% BSA at 2 ϫ 10 6 /ml (293/EBNA-1 cells) or at 8 ϫ 10 6 /ml (HUT78) were added to upper wells (50 l/well) that were separated from lower wells by a polyvinylpyrrolidone-free polycarbonate filter with 5-or 8-m pores precoated with type IV collagen. The chamber was incubated for 2 or 4 h at 37°C in 5% CO 2 , 95% air. Filters were removed and stained with Diff-Quik (Harleco, Gibbstown, NJ). Migrated cell were counted in five randomly selected high-power fields (ϫ 400) per well. All assays were done in triplicate. . PCR products were electrophoresed on 2% agarose gel. The product was 166-base pairs in length. Radiation hybrid mapping data were analyzed by accessing the server at http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl.

RESULTS
Cloning of ELC cDNA-By searching the EST data base (44) with nucleotide and amino acid sequences of various CC chemokines, we identified seven EST sequences potentially encoding a novel human CC chemokine (GenBank TM accession numbers T97490, D31180, D31431, W05519, W07401, N71167, and N80273) (Fig. 1). Later, we came to designate this novel CC chemokine as ELC from EBI1-ligand chemokine (see below), but we use this term hereinafter for the sake of convenience. To determine the full-length cDNA sequence, we carried out 5Ј and 3Ј RACE (52). The primers were designed from the EST sequence N71167 (Fig. 1). Since most ESTs were derived from fetal lung cDNA libraries, human fetal lung cDNA commercially prepared for RACE-PCR (CLONTECH) was used for the reaction. The full-length cDNA is 687 base pairs in length and contains a long open reading frame starting from the first methionine codon and encoding a highly basic polypeptide of a total 98 amino acids with a calculated molecular weight of 10,992 ( Fig. 2A). The nucleotide sequence around the first methionine codon conforms well to the consensus sequence of the eukaryotic translational initiation site (57). The 3Ј noncoding region contains a typical AATAAA polyadenylation signal but not the ATTTA motif for rapid mRNA degradation that is frequently found in the 3Ј noncoding regions of cytokines and chemokines (58).
The deduced polypeptide sequence contains a highly hydrophobic amino-terminal region characteristic of a signal peptide with a putative cleavage site between Ser-21 and Gly-22 ( Fig.  2A) (59). The predicted mature protein of 77 amino acids has a molecular weight of 8,800 and an isoelectric point of 10.11. There is no potential N-glycosylation site. The predicted mature protein shows significant homology to other human CC chemokines (Fig. 2B). All the four cysteine residues conserved in the CC chemokine subfamily are present in a proper ar-  rangement. In addition, several other amino acid residues such as Phe-62, Val-79, and Leu-86 that are conserved in all other CC chemokines are present. The amino acid identity of the mature protein is 31% with MIP-1␤ (60), 30% with RANTES (61) and LARC (38), 28% with MIP-1␣/LD78␣ (62), 26% with TARC (53), 23% with MCP-2 (63), MCP-3 (64) and I309 (65), and 21% with MCP-1 (66, 67) (Fig. 2B). Thus, the cDNA encodes a novel member of the CC chemokine subfamily.
Expression of ELC mRNA in Human Tissues-We determined the expression of mRNA in various human tissues (Fig.  3). The mRNA was found to be constitutively expressed at high levels in thymus and lymph nodes, at intermediate levels in colon and trachea, and at low levels in spleen, small intestine, lung, kidney, and stomach. Among lymphoid tissues, the mRNA was expressed at high levels in lymph nodes, thymus, and appendix. Spleen also contained the mRNA at low levels, but peripheral blood leukocytes, bone marrow, and fetal liver were virtually negative.
Production of Recombinant ELC Protein-To obtain ELC protein, we first tried the baculovirus expression system that we successfully employed for TARC and LARC (38, 53). But we were unable to get the ELC protein secreted from the cells, probably because the signal sequence of ELC was not recognized in insect cells (not shown). Previously, we found no adverse effect of the COOH-terminal Flag tag (54) on the activity of MCP-1 in comparison with non-tagged MCP-1 (data not shown). So we decided to produce the ELC protein tagged with the Flag sequence in the COOH terminus. 293/EBNA-1 cells were transfected with pDREF-ELC-Flag vector, and the ELC-Flag fusion protein in the culture supernatants was purified by anti-Flag affinity chromatography and reverse-phase high performance liquid chromatography. Recombinant ELC-Flag was eluted from the reverse-phase column as a single peak (Fig.  4A). When analyzed by SDS-polyacrylamide gel electrophoresis and silver staining, the purified protein migrated as a single band of approximately 12 kDa (Fig. 4B). Analysis of the NH 2terminal amino acid sequence demonstrated that the mature ELC-Flag started at Gly-22 of the predicted sequence, as expected (not shown).
Specific Binding of ELC to EBI1-We first examined the binding of ELC to the five human CC chemokine receptors (CCR1 to 5) and five orphan receptors, V28/CMKBLR1 (47,48), GPR-CY4 2 , GPR-9 -6 4 , EBI1 (40), and BLR1 (49). To prepare labeled ELC convenient for binding assay, we generated an expression vector encoding the ELC fused with SEAP tagged with (His) 6 (38). Alkaline phosphatase activity was useful for quantitative tracing, and the (His) 6 tag in the COOH terminus was used for affinity purification by nickel-agarose. ELC-SEAP was secreted by 293/EBNA-1 transfected with the expression vector as a protein with an apparent molecular mass of 73 kDa (not shown). Analysis of the NH 2 -terminal amino acid sequence of ELC-SEAP purified by nickel-agarose affinity chromatography revealed that the secreted ELC-SEAP started properly at Gly-22. K562 cells stably expressing CCR1 to 5 and four orphan receptors (BLR1 not included) were reacted with ELC-SEAP. As shown in Fig. 5, ELC-SEAP was found to bind specifically to EBI1 (40). No such binding was seen with K562 cells transfected with the vector only or those transfected with CCR1 to 5 or other three orphan receptors. Similar results were obtained by using 293/EBNA-1 cells stably transfected with the same set of cloned receptors including BLR1 (data not shown). As shown in Fig. 6, by displacement experiments, ELC-Flag fully competed with ELC-SEAP for EBI1 with an IC 50 (38) were capable of competing with ECL-SEAP for EBI1 (Fig. 6B). These results indicated that ELC is a highly specific high affinity ligand for EBI1.

Induction of Calcium Mobilization in EBI1-Transfected
Cells-We next examined whether ECL-Flag was capable of inducing calcium mobilization in cells expressing EBI1. As shown in Fig. 7A, ELC-Flag induced calcium flux in K562 cells stably expressing EBI1 with complete desensitization for a rapid successive stimulation with ELC-Flag. ELC-Flag did not induce any calcium flux in parental K562 cells or those transfected with the vector alone (data not shown). The dose-response curve revealed an EC 50 of 0.9 nM.
Induction of Chemotaxis in EBI1-Expressing Cells-We next examined the chemotactic responses of cells expressing EBI1 to ELC-Flag. As shown in Fig. 8A, 293/EBNA-1 cells stably transfected with EBI1 but not with the vector alone responded to ELC-Flag with a typical bimodal dose-response curve with a maximal effect at 300 ng/ml. We also tested a human T cell line HUT78 that expressed endogenous EBI1 at high levels (data not shown) (41) for chemotactic responses to ELC-Flag. As shown in Fig. 8B, ELC-Flag induced chemotactic responses in HUT78 cells with a typical bimodal dose-response pattern with a maximal effect at 300 ng/ml. Thus, not only 293/EBNA-1 cells stably transfected with EBI1 but also HUT78 cells expressing endogenous EBI1 responded to ELC-Flag by cell migration.
Chromosaml Mapping of the ELC Gene-The chromosomal location of the ELC gene was investigated by PCR using a DNA panel of somatic cell hybrids, each containing a single human chromosome. Unlike other CC chemokines, the ELC gene was localized on chromosome 9 (Fig. 9A). To map the ELC gene on chromosome 9 more precisely, the radiation hybrid mapping was carried out. The results showed that the gene was located 164 centi-Ray away from the top of the chromosome and between the chromosomal markers D9S1978(WI-8765) and AFM326VD1 that are mapped at 9p13 (68) (Fig. 9B)

DISCUSSION
The EST data bases (44) are useful sources for identification of new members of gene families including chemokines (38,45). In the present study, we have described a novel human CC chemokine termed ELC from EBI1-ligand chemokine. ELC shows homologies to other CC chemokines with 20 -30% identity (Fig. 2). ELC is constitutively expressed in various lymphoid tissues such as thymus, lymph nodes, appendix, and spleen (Fig. 3). ELC-SEAP bound specifically to K562 cells stably transfected with EBI1 (Fig. 5). This was also confirmed by using 293/EBNA-1 cells stably transfected with EBI1 (not shown). The binding of ELC-SEAP to EBI1-transfected K562 cells was competed only by ELC-Flag with an IC 50 of 18 nM and not by other CC chemokines so far tested (Fig. 6). ELC-Flag induced transient calcium mobilization in EBI1-transfected K562 cells with an EC 50 of 0.9 nM (Fig. 7). ELC-Flag induced chemotactic responses in 293/EBNA-1 cells stably transfected with EBI1 and HUT78 cells expressing endogenous EBI1 at high levels (41) with a typical bimodal dose-response curve with a maximal effect at 300 ng/ml (Fig. 8). Collectively, ELC is a specific high affinity biological ligand for EBI1 (40). Since EBI1 was also shown to be constitutively expressed in various lymphoid tissues and on activated T and B lymphocytes (40,41), ELC and EBI1 may play roles not only in inflammatory and immunological responses but also in normal lymphocyte recirculation and homing. It remains to be seen what types of cells produce ELC in various lymphoid tissues and what kinds of cytokines regulate ELC production. We propose EBI1 to be designated as CCR7.
Most CC chemokine are potent attractants of monocytes and known to act via shared receptors (1,2,17,18). Their human genes are also clustered on chromosome 17q11.2 (1,2,45). Recently, however, we have identified two novel human CC chemokines, TARC (53) and LARC (38), that have notable differences from other standard CC chemokines. TARC, which is constitutively expressed mainly in the thymus and also in some other lymphoid tissues, acts selectively on T cells, especially CD4 ϩ T cells but not on monocytes (53), and binds to a class of receptors highly specific for TARC, namely CCR4 (34). Similarly, LARC, which is constitutively expressed mainly in the liver and in some other lymphoid tissues, acts selectively on both T and B lymphocytes but not on monocytes (38), and binds to a class of receptors highly specific for LARC, namely CCR6 (39). Importantly, CCR4 and CCR6 are monospecific for TARC and LARC, respectively, and not shared by any other chemokines so far tested (34,39). Furthermore, the genes for TARC and LARC are not present on chromosome 17 but distinctly mapped to chromosome 16q13 (69) and chromosome 2q33-37 (38), respectively. In this regard, ELC is another example of such a new category of CC chemokines. ELC functions via EBI1/CCR7 that is selectively expressed on activated T and B lymphocytes (40). EBI1/CCR7 is monospecific for ELC and not shared by any other CC chemokine so far tested (Fig. 6). Furthermore, the ELC gene is distinctly mapped to chromosome 9p13 ( Fig. 9) instead of chromosome 17. Collectively, TARC, LARC, and ELC may thus constitute a new category of CC chemokines that induce migration and activation of selective subsets of lymphocytes in particular lymphoid tissue microenvironments via respective specific receptors. The generation of gene-targeted mice lacking ELC and EBI1/CCR7 will be useful to elucidate their in vivo functions.
EBI1 and EBI2, being designated from EBV-induced genes 1 and 2, were isolated through their strong up-regulation in EBV-negative Burkitt's lymphoma cells upon infection with EBV (40). Similarly, BLR1 and BLR2, designated from Burkitt's lymphoma receptors 1 and 2, were isolated through the induction by EBV-infection (42,49). BLR2, which is identical to EBI1, was further shown to be induced by the EBV-encoded transactivator EBNA-2 (42). Strikingly, BLR1, EBI1/BLR2, and EBI2 are all predicted to encode seven transmembrane G-protein-coupled receptors. BLR1 and EBI1 are most homologous to the chemokine receptors, whereas EBI2 is most related to the thrombin receptor. Human herpesvirus 6 (HHV-6) and HHV-7 were also shown to induce EBI1 in CD4 ϩ T cells upon infection (43). Furthermore, herpesviruses such as cytomegalovirus (70), herpesvirus saimiri (71), HHV6 (72), HHV7 (73), and HHV8/Kaposi's sarcoma-associated herpesvirus (74) are all known to encode G-protein-coupled receptors homologous to chemokine receptors. A murine cytomegalovirus defective in the open reading frame M33 encoding a putative chemokine receptor revealed severely restricted growth in the salivary glands of infected mice (75). A chemokine receptor encoded by HHV8 was found to be constitutively active and to stimulate proliferation of transfected cells, making it a candidate viral oncogene (76). Taken together, these results suggest that virally encoded putative chemokine receptors play important roles in infection and life cycle of herpesviruses especially in vivo. The roles of ELC and EBI1 in EBV-infected B cells and HHV6-or HHV7-infected T cells are not known at present but may have biological activities on infected cells such as growth promotion, protection from apoptosis, and/or migration into specific anatomical locations in vivo. Identification of ELC as a specific ligand for EBI1 now enables us to examine the possible roles of ELC and EBI1 in infection and life cycle of these herpesviruses. Such studies may lead to a new strategy against herpesvirus infection.