(Received for publication, February 4, 1997)
From the Shionogi Institute for Medical Science,
2-5-1 Mishima, Settsu-shi, Osaka 566, Japan, the Departments of
§ Biochemistry and ¶ Internal Medicine, Kumamoto
University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan, and the
Laboratory of Genetic Resources, National Institute of Health,
1-23-1 Toyama, Shinjuku-ku, Tokyo 16, Japan
By searching the expressed
sequence tag (EST) data base, we identified
partial cDNA sequences encoding a novel human CC chemokine. We
determined the complete 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 EBI1-Ligand Chemokine 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, recombinant
ELC protein fused with the secreted form of alkaline phosphatase (SEAP)
was used. By stably expressing five CC chemokine receptors (CCR1 to 5)
and five orphan receptors, ELC-SEAP was found to bind specifically to
an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3,
eotaxin, MIP-1, MIP-1
, RANTES (regulated on activation normal T
cell expressed and secreted), thymus and activation-regulated chemokine
(TARC), or liver and activation-regulated chemokine (LARC), competed
with ELC-SEAP for EBI1. ELC-Flag-induced transient calcium mobilization
and chemotactic responses in EBI1-transfected cells. ELC-Flag also
induced chemotaxis in HUT78 cells expressing endogenous EBI1 at high
levels. By somatic hybrid and radiation hybrid analyses, the gene for
ELC (SCYA19) was mapped to chromosome 9p13 instead of
chromosome 17q11.2 where the genes for CC chemokines are clustered.
Taken together, ELC is a highly specific ligand for EBI1, which is
known to be expressed in activated B and T lymphocytes and strongly
up-regulated in B cells infected with Epstein-Barr virus and T cells
infected with herpesvirus 6 or 7. ELC and EBI1 may thus play roles in
migration and homing of normal lymphocytes, as well as in
pathophysiology of lymphocytes infected with these herpesviruses. We
propose EBI1 to be designated as CCR7.
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 (5-7), stimulation of angiogenesis (8), suppression of angiogenesis (9, 10), suppression of apoptosis (11), and suppression of human immunodeficiency virus infection (12-14). The chemokines are grouped into the CXC and CC subfamilies on the basis of the arrangement of the two NH2-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-11 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 specific effects of chemokines are mediated by a family of
7-transmembrane G-protein coupled receptors (17, 18). In humans, four
CXC chemokine receptors (CXCR1 to 4) and five CC chemokine receptors
(CCR1 to 5) have been defined for their ligand specificity: CXCR1 for
IL-8 (19); CXCR2 for IL-8 and other CXC chemokines with the ELR motif
(20-22); CXCR3 for IP-10 and MIG (23); CXCR4 for SDF-1/PBSF (13, 14);
CCR1 for MIP-1, RANTES and MCP-3 (24-27); CCR2 for MCP-1 and MCP-3
(27-29); CCR3 for eotaxin, RANTES, MCP-3 and MCP-4 (30-33); CCR4 for
TARC (34); CCR5 for RANTES, MIP-1
, and MIP-1
(35, 36).
Furthermore, there are a growing number of putative chemokine receptors
whose ligands remain to be identified. In this regard, we have recently
demonstrated that an orphan receptor
GPR-CY42/DRY63/CKR-L3
(37) is the specific receptor for a novel human CC chemokine LARC (38)
and, thus, have proposed CCR6 for its designation (39). Among the known
orphan receptors, EBI1, being designated from Epstein-Barr virus
(EBV)-induced gene 1 (40), is expressed in various lymphoid tissues and
activate B and T lymphocytes (40, 41). EBI1 is notable because it is
strongly up-regulated in B cells upon infection with EBV (40, 42), is
transactivated by EBV-encoded nuclear antigen 2 (EBNA-2) (42) and is
also up-regulated in CD4+ T cells upon infection with human
herpesvirus 6 (HHV-6) and HHV-7 (43).
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.
Human hematopoietic cell lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum. 293/EBNA-1 cells were purchased from Invitrogen (San Diego, CA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. K562 cells and 293/EBNA-1 cells stably expressing CCR1 (24-27), CCR2B (27-29), CCR3 (30-33), CCR4 (46), CCR5 (35, 36), V28/CMKBLR1 (47, 48), GPR-CY42 (GenBankTM accession number U45984[GenBank]), GPR-9-64 (GenBankTM accession number U45982[GenBank]), EBI1 (40), and Burkitt's lymphoma receptor 1 (BLR1) (49) were described previously (34).
EST Data Base SearchThe 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 cDNAThe full-length
cDNA sequence was obtained by the rapid amplification of cDNA
ends (RACE) method (52). In brief, 5 and 3
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 (GenBankTM
accession number N71167[GenBank]) (5
RACE-primer, CTCTGACCACACTCACCCTCTCGCT; 3
RACE-primer, GAGCCCGGAGTCCGAGTCAAGCATT) 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-µl 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 × buffer supplied with
the polymerase, and 0.55 µg 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.
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 32P-labeled ELC cDNA probe at 65 °C for
1 h in QuikHyb Hybridization Solution (Stratagene) containing
denatured 100 µg/ml salmon sperm DNA. After washing at 65 °C for
30 min in 0.2 × SSC and 0.1% SDS, filters were exposed to x-ray
films at 80 °C with an intensifying screen.
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 (5
AAAGGGGGATGTGCTGCAAGGCG) and the
MCP1-XbaI-GG-Flag primer (5
-GTCCTTGTAGTCGCCGCCTCTAGAAGTCTTCGGAGTTTGGGT). 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
(5
-CGCGCGGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCGCCGCC). 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 µm), 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 × 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). NH2-terminal sequence analysis was performed
on a protein sequencer (Shimazu, Tokyo, Japan).
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 µm), and added to 20 mM HEPES, pH 7.4, and 0.02% sodium azide. For the
NH2-terminal sequence analysis, the fusion protein was
affinity purified by nickel-agarose 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 µg/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 3,3-5,5
-tetramethylbenzidine. The reaction was stopped by adding H2SO4, 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 × 108 and 1.99 × 108 relative light units, respectively.
This was carried out as described previously
(38). In brief, 2 × 105 cells were incubated for
1 h at 16 °C with 1 µM of SEAP or ELC-SEAP without or with increasing concentrations of unlabeled chemokines in
200 µl 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 µl 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 µl 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).
K562 cells stably expressing
cloned chemokine receptors were suspended at 3 × 106
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 µM 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 × 106 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 EC50 for calcium mobilization, a dose-response
curve was generated in each experiment by plotting percent maximum
responses.
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 µl/well). Cells suspended in RPMI 1640, 1% BSA at 2 × 106/ml (293/EBNA-1 cells) or at 8 × 106/ml (HUT78) were added to upper wells (50 µl/well) that were separated from lower wells by a polyvinylpyrrolidone-free polycarbonate filter with 5- or 8-µm pores precoated with type IV collagen. The chamber was incubated for 2 or 4 h at 37 °C in 5% CO2, 95% air. Filters were removed and stained with Diff-Quik (Harleco, Gibbstown, NJ). Migrated cell were counted in five randomly selected high-power fields (× 400) per well. All assays were done in triplicate.
Somatic Cell and Radiation Hybrid MappingDNAs of the
human × rodent somatic cell hybrids containing human
monochromosomes (National Institute of General Medical Science Mapping
Panel No. 2, Version 2, Coriell Cell Repositories, Camden, NJ) and of
93 radiation hybrids (56) (Gene Bridge 4 Mapping Panel, Reseach
Genetics, Huntsville, AL) were analyzed by PCR using ELC primers
(5-GAGCCCGGAGTCCGAGTCAAGCATT and 5
-CTCTGACCACACTCACCCTCTCGCT). The
PCR conditions were 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min in a 25-µl reaction buffer
containing 0.25 mM each of dNTP, 1 pmol each of the
primers, and 1.25 units of AmpliTaq Gold (Perkin-Elmer, Norwalk, CT).
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.
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 (GenBankTM accession numbers T97490[GenBank], D31180[GenBank],
D31431[GenBank], W05519[GenBank], W07401[GenBank], N71167[GenBank], and N80273[GenBank]) (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 5 and 3
RACE (52). The primers were designed
from the EST sequence N71167[GenBank] (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 3
noncoding region contains a typical AATAAA polyadenylation signal but not the ATTTA motif for rapid mRNA degradation that is frequently found in the 3
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 arrangement. 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.
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 NH2-terminal 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-CY42,
GPR-9-64, 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
NH2-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 IC50 of 18 nM.
In contrast, no other CC chemokines such as MCP-1 (66, 67), MCP-2 (63),
MCP-3 (64), eotaxin (30), MIP-1/LD78
(62), MIP-1
(60), RANTES
(61), TARC (53), and LARC (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
EC50 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).5
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 IC50 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 EC50 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+ 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+ 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.
We are grateful for Drs. Yorio Hinuma, Masakazu Hatanaka, and Retsu Miura for constant support and encouragement.