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(Received for publication, January 31, 1997, and in revised form, March 24, 1997)
From the ABSTRACT Liver and
activation-regulated chemokine
(LARC) is a recently identified CC chemokine that is expressed mainly
in the liver. LARC functions as a selective chemoattractant for
lymphocytes that express a class of receptors specifically binding to
LARC with high affinity. To identifiy the receptor for LARC, we
examined LARC-induced calcium mobilization in cells stably expressing
five CC chemokine receptors (CCR1-CCR5) and five orphan
seven-transmembrane receptors. LARC specifically induced calcium flux
in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan
receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably
expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with
secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji
cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1,
RANTES, MIP-1 INTRODUCTION The chemokines are a group of structurally related approximately
70-90-amino acid polypeptides involved in leukocyte recruitment and
activation (1, 2). The chemokines are grouped into two main
subfamilies, CXC and CC, on the basis of the arrangement of the
N-terminal two conserved cysteine residues. One amino acid separates
the two cysteines in the CXC chemokines while the two cysteines are
adjacent in the CC chemokines. Most CXC chemokines are potent
neutrophil attractants while most CC chemokines recruit monocytes and
also lymphocytes, basophils, and/or eosinophils with variable
selectivity. Recently, a novel lymphocyte-specific chemotactic
cytokine, lymphotactin/SCM-1,1 has been
reported, which carries only the second and the fourth of the four
cysteine residues conserved in all other chemokines (3, 4). This may
suggest the existence of the C type chemokine subfamily.
The specific effects of chemokines on leukocytes are known to be
mediated by a family of seven-transmenbrane G-protein-coupled receptors
(5, 6). In humans, four CXC chemokine receptors and five CC chemokine
receptors have been cloned and defined for their ligand specificity.
They are CXCR1 for IL-8 (7); CXCR2 for IL-8 and other CXC chemokines
with the ELR motif (8-10); CXCR3 for IP-10 and MIG (11); CXCR4 for
SDF-1/PBSF (12, 13); CCR1 for MIP-1 Recently, we have identified a novel CC chemokine, LARC
(liver and activation-regulated
chemokine), and mapped its gene to chromosome 2q33-q37 (28).
Expression of LARC mRNA was detected mainly in the liver among
various human tissues and also induced in several human cell lines by
phorbol 12-myristate 13-acetate. LARC was chemotactic for lymphocytes
but not for monocytes. LARC fused with the secreted alkaline
phosphatase (LARC-SEAP) bound specifically to lymphocytes with a
Kd of 0.4 nM. Notably, the binding of
LARC-SEAP was competed only by LARC and not by other chemokines so far
tested (28). These results indicated the presence of a class of
receptors specific for LARC on lymphocytes. In the present study, we
have demonstrated that an orpan receptor GPR-CY42 is the LARC receptor that is
selectively expressed on lymphocytes.
EXPERIMENTAL PROCEDURES Human hematopoietic cell lines were maintained in
RPMI 1640 supplemented with 10% fetal calf serum (FCS). 293/EBNA-1
cells were purchased from Invitrogen (San Diego, CA) and maintained in
Dulbecco's modified Eagle's medium supplemented with 10% FCS. Peripheral blood leukocytes were fractionated by surface markers as
described previously (25, 29). In brief, peripheral blood mononuclear
cells (PBMC) were isolated from venous blood obtained from healthy
adult donors using Ficoll-Paque (Pharmacia, Uppsala, Sweden).
Monocytes, B cells, and T cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD14, FITC-conjugated anti-CD19, and FITC-conjugated anti-CD3, respectively, and positively selected from PBMC by using MACS (Miltenyi Biotec, Bergisch, Germany). CD16+ CD3 Recombinant LARC, TARC, Eotaxin, and MCP-1 were
produced by using a baculovirus expression system and purified as
described previously (20, 28, 29). MIP-1 Cells stably
expressing CCR1 (14-17), CCR2B (17-19), CCR3 (20-23), CCR4 (24, 25),
CCR5 (26, 27, 45), V28/CMKBRL1 (30, 31), GPR-CY42
(GenBankTM accession number U45984[GenBank]), GPR-9-63
(GenBankTM accession number:U45982), EBI1 (32), and BLR1
(33) were described previously (25). In brief, the expression plasmids
based on pDREF-Hyg (29) were transfected into Raji cells by
electroporation and into 293/EBNA-1 cells by LipofectAMINE (Life
Technologies, Inc.). After selection with 250 µg/ml hygromycin for 1 to 2 weeks, drug-resistant cells were pooled and used for experiments.
K562 cells were transfected with the expression plasmids based on
pCAGG-Neo (25) by electroporation. After selection with 800µg/ml G418 for 1-2 weeks, clones expressing transfected receptors at high levels
were selected by binding assays and/or Northern blot analysis.
This was carried out as
described previously (25). In brief, cells were suspended at 3 × 106 cells/ml in Hank's balanced salt solution (HBSS)
containing 1 mg/ml of bovine serum albumin (BSA) and 10 mM
HEPES, pH 7.4, (HBSS-BSA) and incubated with 1 µM
fura-PE3-AM (Texas Fluorescence Labs) at room temperature for 1 h
in the dark. After washing twice with HBSS-BSA, cells were suspended in
HBSS-BSA at 2.5 × 106 cells/ml. 2 ml of the cell
suspension in a quartz cuvette was placed in a luminescence
spectrometer (Perkin-Elmer LS 50B) and fluorescence was monitored at
340 nm ( Cell migration was determined by using a
48-well microchemotaxis chamber as described previously (29). In brief,
each chemo-attractant was diluted in Hepes-buffered RPMI 1640 supplemented with 1% BSA and placed in lower wells (25 µl/well).
Cells suspended in RPMI 1640 with 1% BSA at 2 × 106
cells/ml were placed in upper wells (50 µl/well). Upper and lower wells were separated by a polyvinylpyrrolidone-free polycarbonate filter with 8-µm pores precoated with type IV collagen. Incubation was carried out at 37 °C for 4 h in 5% CO2, 95%
air. Filters were removed, washed, and stained with Diff-Quik. Migrated
cells were counted in five randomly selected high-power fields (400 ×)
per well. All determinations were done in triplicate.
This was carried out as described previously
(25, 28, 29). In brief, for displacement experiments, 2 × 105 cells were incubated for 1 h at 16 °C with 1 nM of SEAP(His)6 or LARC-SEAP(His)6
in the presence of 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. For saturation experiments, cells were
incubated for 1 h at 16 °C with increasing concentrations of
LARC-SEAP(His)6 in the presence or absence of 1 µM unlabeled LARC. After incubation, cells were washed
five 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 phosphatase. After brief centrifugation to remove
cell debris, alkaline phosphatase activity in 10 µl of lysate was
measured by chemiluminescent assay as described previously (28). All determinations were done in duplicate. The binding data were analyzed by the LIGAND program (34).
This was carried out as described
previously (28, 29). In brief, total RNA was prepared by using Trizol®
reagent (Life Technologies, Inc.). RNA samples were separated by
electrophoresis on a 1% agarose gel containing 0.66 M
formaldehyde, blotted onto a filter membrane (Hybond N+)
(Amersham Japan, Tokyo). Multiple tissue Northern blots and immune
blots were purchased from CLONTECH (Palo Alto, CA).
Hybridization was carried out with probes labeled with 32P
using Prime It II kit (Stratagene, La Jolle, CA) at 65 °C in QuickHyb solution (Stratagene). After washing at 55 °C with 0.2 × SSC and 0.1% SDS, filters were exposed to x-ray films at RESULTS To examine
interaction of LARC with each cloned receptor, we measured LARC-induced
calcium mobilization in a panel of K562 cells stably expressing the
five known CCRs (CCR1-CCR5) and five orphan chemokine receptors,
V28/CMKBRL1 (31, 32), EBI1 (33), BLR1 (34), GPR-CY42
(GenBankTM accession number U45984[GenBank]), and
GPR-9-63 (GenBankTM accession
number U45982[GenBank]). As shown in Fig. 1A, LARC specifically induced calcium flux in K562 cells expressing GPR-CY4 with
complete desensitization against a rapid successive treatment with
LARC. LARC did not induce calcium flux in parental K562 cells or those
expressing CCR1, CCR2B, CCR3, CCR4, CCR5, or four other orphan
receptors. On the other hand, MIP-1
Previously, we showed that
LARC induced chemotaxis in freshly isolated peripheral blood
lymphocytes with a maximal effect at 1 µg/ml (28). We therefore
examined whether LARC was capable of inducing migration of 293/EBNA-1
cells stably expressing GPR-CY4. As shown in Fig.
2A, LARC induced migration in cells stably
expressing GPR-CY4 with a typical bi-modal dose-response curve with a
maximum effect at 1 µg/ml and an EC50 of ~100 ng/ml
(~12 nM). LARC did not induce migration in cells
transfected with the vector alone. A checkerboard-type analysis
revealed that the migration of GPR-CY4-transfected 293/EBNA-1 cells
toward LARC was mostly chemotactic (Fig. 2B).
Previously, we showed that
LARC-SEAP(His)6 specifically bound to a single class of
receptors expressed on lymphocytes with a Kd of 0.4 nM (28). Importantly, the binding of
LARC-SEAP(His)6 was competed only by LARC and not by any
other chemokines so far tested, indicating that the LARC receptor is
not shared by other chemokines (28). We therefore examined the binding
of LARC-SEAP(His)6 to a panel of Raji cells stably
expressing GPR-CY4 and other cloned receptors.
LARC-SEAP(His)6 bound specifically to cells expressing GPR-CY4 but not to parental cells or those expressing five CCRs or four
other orphan receptors (data not shown). As shown in Fig. 3A, the binding of
LARC-SEAP(His)6 to GPR-CY4 was saturable when increasing
concentrations of LARC-SEAP(His)6 were incubated with Raji
cells expressing GPR-CY4. The Scatchard analysis revealed a
Kd of 0.9 nM and 28,800 sites/cell (Fig.
3B). Unlabeled LARC fully competed with
LARC-SEAP(His)6 for GPR-CY4 with an IC50 of 3.4 nM (Fig. 3C). Furthermore, no other CC
chemokines, MCP-1, RANTES, MIP-1
We have
shown that the endogenous class of LARC receptors is expressed
selectively on lymphocytes (28). Therefore, we examined the expression
pattern of GPR-CY4 in various human tissues and leukocyte subsets by
Northern blot analysis (Fig. 4). When blots for various
tissues were hybridized with the 32P-labeled GPR-CY4
cDNA probe (Fig. 4A), GPR-CY4 mRNA was found to be
expressed strongly in the spleen and weakly in the lymph nodes. Weak
expression was also detected in the testis (larger transcripts), small
intestine, and PBL. Notably, the mRNA expression was very low, if
any, in the liver where the LARC transcripts were mainly detected (28).
When blots specific for various lymphoid tissues were probed, GPR-CY4
mRNA was detected strongly in the spleen, lymph nodes, and
Appendix, and weakly in the fetal liver (Fig. 4B). When the
same lymphoid tissue blots were rehybridized with the
32P-labeled LARC cDNA probe, LARC mRNA was detected
moderately in the appendix and weakly in the lymph nodes, PBL, and
fetal liver (Fig. 4B). Thus, the constitutive expression of
GPR-CY4 and that of LARC overlap partly in the secondary lymphoid
tissues. We then examined the expression of GPR-CY4 mRNA in various
leukocyte subsets. T cells (both CD4+ and CD8+
T cells) and B cells were clearly positive, whereas NK cells, monocytes, or granulocytes were virtually negative even though some RNA
loading differences were noted (Fig. 5A). We
also examined the expression of GPR-CY4 mRNA in various human
hematopoietic cell lines. Only a T cell line, Hut102, weakly expressed
GPR-CY4, whereas other T cell lines (Molt-4, Jurkat, and Hut78),
monocytic cell lines (THP-1 and U937), B cell lines (Raji and Daudi),
an erythroleukemia cell line (K562), a promyelocytic cell line (HL-60), a basophilic cell line (KU812), and a megakaryocytic cell line (MEG-1)
were virtually negative (not shown). Collectively, the expression of
GPR-CY4 is mostly limited in the secondary lymphoid tissues and also in
T and B lymphocytes. The expression pattern of GPR-CY4 is thus highly
consistent to the lymphocyte-selective expression of the endogenous
LARC receptor described previously (28).
Loetscher et al. (35) have reported that CD45RO+
T cells express CCR1 and CCR2 only after prolonged treatment with IL-2. They further showed that activation of T cells with PHA, anti-CD3, or
anti-CD3 and anti-CD28 did not induce expression of CCR1 or CCR2 but
rather suppressed the effect of IL-2 (35). We therefore examined the
effect of IL-2 without or with PHA on expression of GPR-CY4 in
CD4+ and CD8+ T cells (Fig. 5B).
Expression of GPR-CY4 in both CD4+ and CD8+ T
cells was strongly up-regulated by IL-2. The effect of IL-2 was,
however, strongly suppressed by co-treatment with PHA. Thus, the
expression of GPR-CY4 in T cells is positively regulated by IL-2 but
negatively regulated by T-cell activation like those of CCR1 and CCR2
(35).
DISCUSSION LARC is a novel CC chemokine with 20~28% identity to other
cloned human CC chemokines (28). LARC is mainly expressed in the liver
and also induced in human cell lines, such as a monocytic cell line
U937, by phorbol myristate acetate. Thus, we designated this chemokine
as LARC from Liver and
Activation-Regulated Chemokine (28).
The present study has further demonstrated that LARC is constitutively
expressed at relatively low levels in tissues such as the lymph nodes,
Appendix, and fetal liver (Fig. 4B). It remains to be
explored what types of cells produce LARC in the liver and some
lymphoid tissues and what kinds of cytokines and stimulants regulate
LARC expression.
LARC is chemotactic for lymphocytes in vitro with a maximal
activity at 1 µg/ml (28). At high concentrations, LARC may also be
chemotactic for neutrophils. However, LARC is totally inactive on
monocytes (28). A similar relatively low potency in induction of
chemotaxis in lymphocytes has been noted for TARC (29) and SDF-1/PBSF
(36). Lymphocytes, especially resting ones, may be relatively
inefficient in chemotactic responses to these chemokines. In keeping
with the lymphocyte-selective activity of LARC, lymphocytes possess a
class of receptors binding LARC with a high affinity (Kd = 0.4 nM)(28). Furthermore, the
receptor expressed on lymphocytes is highly specific for LARC and not
shared by other CC chemokines so far tested (28). Interestingly, TARC
(29) and SDF-1/PBSF (36) are also the ones that possess receptors, CCR4
(25) and CXCR4 (12, 13), respectively, that are not shared by other
chemokines so far tested.
In the present study, we have demonstrated that an orphan receptor
GPR-CY4 (GenBankTM accession number U45984[GenBank]) is the LARC
receptor expressed on lymphocytes. Recently, the same receptor was also
deposited in the data base as DRY64
(GenBankTM accession number U60000[GenBank]). It was also reported
as an orphan receptor CKR-L3 (37). LARC induced calcium mobilization and chemotactic responses specifically in K562 cells and 293/EBNA-1 cells stably expressing GPR-CY4 (Figs. 1 and 2). LARC fused with SEAP
bound specifically to Raji cells stably expressing GPR-CY4 with a
Kd of 0.9 nM (Fig. 3). Binding of
LARC-SEAP to GPR-CY4 was blocked only by LARC and not by any other
chemokines so far tested (Fig. 3). GPR-CY4 was found to be expressed
strongly in the secondary lymphoid tissues such as the spleen, lymph
nodes, and Appendix, and also in the fetal liver (Fig. 4). A very
similar result was reported for the tissue expression of CKR-L3 (37). Furthermore, GPR-CY4 was expressed highly selectively in peripheral blood lymphocytes, namely both CD4+ and CD8+ T
cells and B cells (Fig. 5A). Collectively, these results
clearly indicate that GPR-CY4 is the receptor that specifically binds LARC with high affinity and is expressed selectively on lymphocytes. We
propose GPR-CY4 to be designated as CCR6.
Compared with the high affinity binding of LARC to CCR6
(Kd = 0.9 nM), LARC needed much higher
concentrations to induce intracellular calcium mobilization
(EC50 = ~50 nM) or chemotactic responses
(EC50 = ~12 nM) in cells stably transfected
with CCR6. At present, we do not know the exact causes of such
discrepancies, but these may be due in part to differences in assay
conditions such as temperature, duration of incubation, etc.
Furthermore, Monteclaro and Charo (46) recently demonstrated a two-step
mechanism for activation of CCR1 by MCP-1 in which high affinity
binding of MCP-1 with the amino terminus of CCR1 allows subsequent low affinity interactions of MCP-1 with the extracellular
loops/transmembrane domains of CCR1 that lead to receptor activation
and signaling. A similar two-step mechanism may explain high affinity
binding versus low signaling potency of LARC to CCR6.
As in the cases of CCR1 and CCR2 (35), IL-2 strongly induces the
expression of CCR6 in resting T cells while PHA activation blocked the
inducing effect of IL-2 (Fig. 5B). Thus, not antigenic stimulation per se, but subsequent IL-2-mediated expansion
may enhance T-cell responsiveness to LARC. In mice, repeated injection of IL-2 was shown to induce massive lymphocyte infiltration in the
liver and lung (38). Since LARC is expressed rather selectively in the
liver and lung (28), LARC may be involved in the IL-2-induced lymphocyte infiltration in these organs.
Most CC chemokines are known to interact with multiple shared receptors
(1, 2, 5, 6). For example, MIP-1 In this regard, the CXC chemokine SDF-1/PBSF is also the one that acts
via its specific receptor CXCR4 (12, 13), is constitutively expressed
in various tissues (40), and is mapped to chromosome 10q (40) instead
of chromosome 4 where the genes for other CXC chemokines are known to
be clustered (1, 2). Furthermore, the recently described C type
chemokine lymphotactin/SCM-1 (3, 4) that also acts selectively on
lymphocytes is distinctly mapped to human chromosome 1q23 (41). By
generating gene-targeted mice, SDF-1/PBSF has been shown to be
essential for B cell lymphopoiesis in the fetal liver and for
myelopoiesis and B cell lymphopoiesis in the bone marrow during
embryonic development (42). Thus, during embryogenesis, SDF-1/PBSF may
be involved in generation of B cell progenitors in the fetal liver and
in colonization of hematopoietic precursor cells into the bone marrow
(42). Recently, gene-targeted mice lacking a putative CXC chemokine
receptor BLR1 that is expressed on mature B cells and a subpopulation
of CD4+ T cells have been shown to have anatomical defects
such as lack of inguinal lymph nodes, impaired development of Peyer's
patches, and defective formation of primary follicles and germinal
centers in the spleen (43). When injected into wild type mice, B cells lacking BLR1 failed to migrate from the T cell-rich zone into B cell
follicles in the spleen and Peyer's patches (43). Thus, BLR1 may be
involved in B cell migration within specific anatomic compartments in
the spleen and Peyer's patches. Likewise, TARC and LARC with their
respective receptors, CCR4 and CCR6, may play roles not only in
inflammatory and immunological responses but also in the normal
lymphocyte trafficking and microenvironmental homing that are essential
for development and maintenance of various lymphoid tissues.
Identification of the LARC receptor CCR6 now enables us to define the
exact types of cells that respond to LARC. Generation of gene-targeted
mice lacking LARC and CCR6 will be useful to address their in
vivo functions.
FOOTNOTES ACKNOWLEDGEMENTS We are grateful to Drs. Yorio Hinuma and
Masakazu Hatanaka for constant support and encouragement.
REFERENCES
Volume 272, Number 23,
Issue of June 6, 1997
pp. 14893-14898
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,,,,,
Shionogi Institute for Medical Science,
2-5-1 Mishima, Settsu-shi, Osaka 566, Japan and the Departments of
§ Biochemistry and ¶ Internal Medicine, Kumamoto
University Medical School, Honjo, Kumamoto 860, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, MIP-1
, and TARC) competed with LARC-SEAP for
binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was
expressed mainly in speen, lymph nodes, Appendix, and fetal liver among
various human tissues. Among various leukocyte subsets, GPR-CY4
mRNA was detected in lymphocytes (CD4+ and
CD8+ T cells and B cells) but not in natural killer cells,
monocytes, or granulocytes. Expression of GPR-CY4 mRNA in
CD4+ and CD8+ T cells was strongly up-regulated
by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC
expressed selectively on lymphocytes, and LARC is a unique functional
ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.
, RANTES, and MCP-3 (14-17);
CCR2 for MCP-1, MCP-3, and MCP-4 (17-19, 44); CCR3 for Eotaxin,
RANTES, MCP-3, and MCP-4 (20-23, 44); CCR4 for TARC (24, 25); CCR5 for
RANTES, MIP-1, and MIP-1 (26, 27, 45). Furthermore, there are a
growing number of putative chemokine receptors whose ligands remain to be identified.
and CD56+
CD3 cells with appropriate forward and side scatters were
sorted on a FACStar Plus (Beckton Dickinson, Mountain View, CA) as
natural killer (NK) cells. CD4+ T cells and
CD8+ T cells were purified from PBMC by negative selection
using Dynabeads (Dynal, Oslo, Norway) after incubation with anti-CD16,
-CD14, -CD20, and -CD8, or -CD16, -CD14, -CD20, and -CD4, respectively. Granulocytes were obtained from the pellet fraction of Ficoll-Paque gradient by dextran sedimentation and hypotonic lysis of erythrocytes. The purity of each cell population was always >95% as determined by
flow cytometry or by staining with Diff-Quik (Baxter Scientific Products, McGaw Park, IL).
and MIP-1 were purchased from Pepro Tech (Rocky Hill, NJ). LARC fused with the secreted form of
alkaline phosphate tagged with six histidine residues, LARC-SEAP(His)6, was prepared and purified as described
previously (28). In brief, the LARC cDNA was subcloned into the
SEAP(His)6 vector (pDREF-SEAP(His)6-Hyg)(28),
making the expression vector pDREF-LARC-SEAP. 293/EBNA-1 cells were
transfected with pDREF-LARC-SEAP using LipofectAMINE (Life
Technologies, Inc., Gaithersburg, MD) and cultured for 3-4 days in
DMEM containing 10% FCS. The culture supernatants were centrifuged,
filtered (0.45 µm), added to 20 mM HEPES, pH 7.4, and
0.02% sodium azide, and stored at 4 °C. The concentration of
LARC-SEAP was determined by a sandwitch-type enzyme-linked
immunosorbent assay as described previously (28).
ex1), 380 nm (ex2) and 510 nm
(em) every 200 ms. To determine EC50, a
dose-response curve was generated in each experiment by plotting data
as percent maximum response.
80 °C with an intensifying screen.
, MIP-1, MCP-1, eotaxin, or
TARC did not induce calcium flux in K562 cells expressing GPR-CY4 (not
shown). These chemokines, however, properly induced calcium flux in
K562 cells expressing their respective CCRs even after treatment with
LARC (Fig. 1A). Similar results were obtained by using a
panel of 293/EBNA-1 cells stably expressing these cloned receptors
(data not shown). As shown in Fig. 1B, 293/EBNA-1 cells stably expressing GPR-CY4 responded to LARC in calcium mobilization with an EC50 of ~50 nM. These results clearly
demonstrated that LARC was a specific functional ligand for
GPR-CY4.
Fig. 1.
Calcium mobilization in cells expressing
GPR-CY4 by LARC. A, K562 cells stably transfected with
indicated cloned receptors were loaded with fura-PE3-AM and stimulated
with indicated chemokines at 100 nM (LARC and TARC) or 10 nM (MIP-1, MCP-1, eotaxin, and MIP-1).
Arrowheads indicate time of application of chemokines. Intracellular calcium concentration was monitored by fluorescence ratio
(F340/F380). Representative results from at least three separate
experiments are shown. B, dose-response curve of calcium mobilization by LARC. 293/EBNA-1 cells stably transfected with GPR-CY4
were loaded with fura-PE3-AM and stimulated with indicated concentrations of LARC. Results were expressed as percent maximum responses. Each point represents mean ± S.E. from
three separate experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
Chemotactic response of GPR-CY4-transfected
cells to LARC. A, 293/EBNA-1 cells stably transfected with
GPR-CY4 (closed circle) or the vector alone (open
triangle) were tested for in vitro migration to
indicated concentrations of LARC by using a 48-well chemotaxis chamber.
The assay was done in triplicate, and the number of migrating cells in
five high power fields (400 ×) were counted for each well.
Representative results from three separate experiments are shown. Each
point represents mean ± S.E. B, a
checkerboard-type analysis of cell migration. In the chemotaxis assay
using a 48-well chemotaxis chamber, LARC was added to top and/or bottom
wells as indicated at 100 ng/ml. The assay was done in triplicate, and
the number of migrating cells in five high power fields (400 ×) were
counted for each well. Representative results from three separate
experiments are shown. Each histogram represents mean ± S.E.
[View Larger Version of this Image (17K GIF file)]
, MIP-1, or TARC, were capable of
competing with LARC-SEAP(His)6 for GPR-CY4 (Fig.
3D). These binding characteristics were highly consistent
with those obtained from the endogenous class of LARC receptors
expressed on lymphocytes (28).
Fig. 3.
Binding characteristics of
LARC-SEAP(His)6 to GPR-CY4-transfected cells. A,
specific binding of LARC-SEAP(His)6 to GPR-CY4. Raji cells
stably transfected with GPR-CY4 (2 × 105 cells) were
incubated at 16 °C for 1 h with increasing concentrations of
LARC-SEAP(His)6. Specific binding was determined by
subtracting nonspecific binding measured in the presence of 1 µM of LARC. Representative results from three separate
experiments are shown. B, Scatchard analysis of the binding
data in panel A. The calculated Kd is 0.9 nM. C, displacement of binding of
LARC-SEAP(His)6 to GPR-CY4 by LARC. Raji cells stably
expressing GPR-CY4 (2 × 105 cells) were incubated
with 1 nM of LARC-SEAP(His)6 in the presence of
increasing concentrations of unlabeled LARC. Representative results
from three separate experiments are shown. The calculated IC50 is 3.4 nM. D, competition of
LARC-SEAP(His)6 binding to GPR-CY4 by various CC
chemokines. Raji cells stably transfected with GPR-CY4 (2 × 105 cells) were incubated with 1 nM
LARC-SEAP(His)6 in the presence of indicated chemokines at
200 nM. Each histogram represents mean ± S.E. from
three separate experiments.
[View Larger Version of this Image (19K GIF file)]
Fig. 4.
Tissue expression of GPR-CY4 mRNA.
A, expression of GPR-CY4 mRNA in various human tissues.
Multi-tissue Northern blot filters (2 µg of poly(A)+
RNA/lane) (CLONTECH) were hybridized with the
32P-labeled GPR-CY4 cDNA probe. B,
expression of GPR-CY4 and LARC in various human lymphoid tissues.
Immune blot filters (2 µg/lane of poly(A)+ RNA)
(CLONTECH) were hybridized with the
32P-labeled GPR-CY4 probe. The same filters were
rehybridized with the 32P-labeled LARC cDNA
probe.
[View Larger Version of this Image (24K GIF file)]
Fig. 5.
Selective expression of GPR-CY4 mRNA in
lymphocytes. A, expression of GPR-CY4 mRNA in human
peripheral blood leukocyte subsets. Total RNA samples were prepared
from freshly isolated CD4+ T cells (T4),
CD8+ T cells (T8), total T cells (T),
B cells (B), NK cells (NK), monocytes
(Mo), and granulocytes (Gr), separated by agarose
gel electrophoresis (5 µg/lane), blotted onto filters, and hybridized with the 32P-labeled GPR-CY4 cDNA probe. The
autoradiograph of the filter (upper panel) and the
photograph of the gel stained with ethidium bromide (lower
panel) are shown. Positions of size markers (in kilobases) are
indicated on the left. B, effect of IL-2 and PHA on GPR-CY4 mRNA expression in CD4+ and CD8+
T cells. Total RNA samples were prepared from CD4+ T cells
(T4) and CD8+ T cells (T8) before or
after culture for 5 days in the presence of recombinant IL-2 at 400 units/ml without or with PHA (1/100), separated by agarose gel
electrophoresis (5 µg/lane), blotted onto filters, and hybridized
with the 32P-labeled GPR-CY4 cDNA probe. The
autoradiograph of the filter (upper panel) and the
photograph of the gel stained with ethidium bromide (lower
panel) are shown. Positions of size markers (in kilobases) are
indicated on the left.
[View Larger Version of this Image (72K GIF file)]
binds to CCR1 and CCR5 (14, 15, 26,
27, 45), while RANTES binds to CCR1, CCR3, and CCR5 (14, 15, 21, 22,
26, 27, 45). Eotaxin apparently interacts only with CCR3 (20-22), but CCR3 also binds RANTES, MCP-3, and MCP-4 (21-23, 44). Thus, each chemokine may recruit multiple types of cells even if they express different types of receptors, whereas each cell may respond to multiple
types of chemokines even by expressing a single type of receptor. The
exact physiological meanings of such redundant and complex
relationships between chemokines and their receptors are still unclear,
but such partially overlapping specificities may have advantages in
acute inflammatory responses where similar leukocyte subsets have to be
rapidly recruited in a wide variety of settings and microenvironments
even if there are considerable differences in the local pattern and
spectrum of chemokine production. In this context, LARC (28) and also
the recently identified T-cell-directed CC chemokine TARC (29) are
quite unique because they interact with highly specific receptors, CCR6
(this paper) and CCR4 (25), respectively. In fact, LARC and TARC have a
number of features in common that are unique among the known CC
chemokines. They are constitutively expressed in certain organs and
lymphoid tissues, with LARC mainly in the liver (28) and also in some secondary lymphoid tissues (Fig. 4B), whereas TARC is mainly
expressed in the thymus and also most probably in some secondary
lymphoid tissues (29). Both LARC and TARC act selectively on
lymphocytes, LARC on both T and B cells (28, Fig. 5), whereas TARC acts
mainly on CD4+ T cells (25, 29). Even though the genes for
other CC chemokines are known to be clustered on human chromosome
17q11.2 (1, 2), the genes for LARC and TARC are mapped distinctly to
chromosome 2q33-q37 and chromosome 16q13, respectively (28, 39). Thus, LARC and TARC may constitute a new group of CC chemokines that have
more specialized functions in lymphocyte trafficking and immune
responses than other CC chemokines clustered on chromosome 17.
To whom correspondence should be addressed: Shionogi Institute
for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan. Tel.:
81-6-382-2612; Fax: 81-6-382-2598; E-mail: osamu.yoshie{at}shionogi.co.jp.
1
The abbreviations and trivial names used are:
SCM-1, single C motif 1; G-protein, heterotrimeric guanine
nucleotide-binding regulatory protein; CXCR, CXC chemokine receptor;
IL-8, interleukin 8; IP-10, interferon 
-inducible protein 10; Mig,
monokine induced by interferon ; SDF-1, stroma derived factor 1;
PBSF, pre-B cell stimulatory factor; CCR, CC chemokine receptor; MIP,
macrophage inflammatory protein; RANTES, regulated on activation,
normal T cell expressed and secreted; MCP, monocyte chemoattractant
protein; TARC, thymus and activation-regulated chemokine; LARC, liver
and activation-regulated chemokine; SEAP, secreted alkaline
phosphatase; FCS, fetal calf serum; PBMC, peripheral blood mononuclear
cells; NK, natural killer; BSA, bovine serum albumin; EBI1, EBV-induced gene 1; BLR1, Burkitt's lymphoma receptor 1; IL-2, interleukin 2; PHA,
phytohemagglutinin; HBSS, Hank's balanced salt solution; PBL,
peripheral blood leukocytes.
2
Deposited by L. L. Lautens, W. Modi, and T. I. Bonner.
3
Deposited by L. L. Lautens, H. L. Tiffany, J.-L.
Gao, W. Modi, P. M. Murphy, and T. I. Bonner.
4
Deposited by R. McCoy and D. H. Perlmutter.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y. Isegawa, Z. Ping, K. Nakano, N. Sugimoto, and K. Yamanishi Human Herpesvirus 6 Open Reading Frame U12 Encodes a Functional beta -Chemokine Receptor J. Virol., July 1, 1998; 72(7): 6104 - 6112. [Abstract] [Full Text] [PDF]
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T. Yoshida, T. Imai, M. Kakizaki, M. Nishimura, S. Takagi, and O. Yoshie Identification of Single C Motif-1/Lymphotactin Receptor XCR1 J. Biol. Chem., June 26, 1998; 273(26): 16551 - 16554. [Abstract] [Full Text] [PDF]
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K. E. Cole, C. A. Strick, T. J. Paradis, K. T. Ogborne, M. Loetscher, R. P. Gladue, W. Lin, J. G. Boyd, B. Moser, D. E. Wood, et al. Interferon-inducible T Cell Alpha Chemoattractant (I-TAC): A Novel Non-ELR CXC Chemokine with Potent Activity on Activated T Cells through Selective High Affinity Binding to CXCR3 J. Exp. Med., June 15, 1998; 187(12): 2009 - 2021. [Abstract] [Full Text] [PDF]
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B.-S. Youn, S. M. Zhang, H. E. Broxmeyer, S. Cooper, K. Antol, M. Fraser Jr, and B. S. Kwon Characterization of CKbeta 8 and CKbeta 8-1: Two Alternatively Spliced Forms of Human beta -Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1 Blood, May 1, 1998; 91(9): 3118 - 3126. [Abstract] [Full Text] [PDF]
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R. Yoshida, M. Nagira, M. Kitaura, N. Imagawa, T. Imai, and O. Yoshie Secondary Lymphoid-tissue Chemokine Is a Functional Ligand for the CC Chemokine Receptor CCR7 J. Biol. Chem., March 20, 1998; 273(12): 7118 - 7122. [Abstract] [Full Text] [PDF]
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 I. M. Hayes, N. J. Jordan, S. Towers, G. Smith, J. R. Paterson, J. J. Earnshaw, A. G. Roach, J. Westwick, and R. J. Williams Human Vascular Smooth Muscle Cells Express Receptors for CC Chemokines Arterioscler. Thromb. Vasc. Biol., March 1, 1998; 18(3): 397 - 403. [Abstract] [Full Text] [PDF]
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D. F. Legler, M. Loetscher, R. S. Roos, I. Clark-Lewis, M. Baggiolini, and B. Moser B Cell-attracting Chemokine 1, a Human CXC Chemokine Expressed in Lymphoid Tissues, Selectively Attracts B Lymphocytes via BLR1/CXCR5 J. Exp. Med., February 16, 1998; 187(4): 655 - 660. [Abstract] [Full Text] [PDF]
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I. Goya, J. Gutierrez, R. Varona, L. Kremer, A. Zaballos, and G. Marquez Identification of CCR8 as the Specific Receptor for the Human {beta}-Chemokine I-309: Cloning and Molecular Characterization of Murine CCR8 as the Receptor for TCA-3 J. Immunol., February 15, 1998; 160(4): 1975 - 1981. [Abstract] [Full Text] [PDF]
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T. Imai, D. Chantry, C. J. Raport, C. L. Wood, M. Nishimura, R. Godiska, O. Yoshie, and P. W. Gray Macrophage-derived Chemokine Is a Functional Ligand for the CC Chemokine Receptor 4 J. Biol. Chem., January 16, 1998; 273(3): 1764 - 1768. [Abstract] [Full Text] [PDF]
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 J. J. Campbell, J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, and E. C. Butcher Chemokines and the Arrest of Lymphocytes Rolling Under Flow Conditions Science, January 16, 1998; 279(5349): 381 - 384. [Abstract] [Full Text]
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M. Wong and E. N. Fish RANTES and MIP-1alpha Activate Stats in T Cells J. Biol. Chem., January 2, 1998; 273(1): 309 - 314. [Abstract] [Full Text] [PDF]
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D. R. Greaves, W. Wang, D. J. Dairaghi, M. C. Dieu, B. d. Saint-Vis, K. Franz-Bacon, D. Rossi, C. Caux, T. McClanahan, S. Gordon, et al. CCR6, a CC Chemokine Receptor that Interacts with Macrophage Inflammatory Protein 3alpha and Is Highly Expressed in Human Dendritic Cells J. Exp. Med., September 15, 1997; 186(6): 837 - 844. [Abstract] [Full Text] [PDF]
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M. Nagira, T. Imai, K. Hieshima, J. Kusuda, M. Ridanpaa, S. Takagi, M. Nishimura, M. Kakizaki, H. Nomiyama, and O. Yoshie Molecular Cloning of a Novel Human CC Chemokine Secondary Lymphoid-Tissue Chemokine That Is a Potent Chemoattractant for Lymphocytes and Mapped to Chromosome 9p13 J. Biol. Chem., August 1, 1997; 272(31): 19518 - 19524. [Abstract] [Full Text] [PDF]
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H. L. Tiffany, L. L. Lautens, J.-L. Gao, J. Pease, M. Locati, C. Combadiere, W. Modi, T. I. Bonner, and P. M. Murphy Identification of CCR8: A Human Monocyte and Thymus Receptor for the CC Chemokine I-309 J. Exp. Med., July 7, 1997; 186(1): 165 - 170. [Abstract] [Full Text] [PDF]
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J. M. Perez-Canadillas, A. Zaballos, J. Gutierrez, R. Varona, F. Roncal, J. P. Albar, G. Marquez, and M. Bruix NMR Solution Structure of Murine CCL20/MIP-3alpha , a Chemokine That Specifically Chemoattracts Immature Dendritic Cells and Lymphocytes through Its Highly Specific Interaction with the beta -Chemokine Receptor CCR6 J. Biol. Chem., July 20, 2001; 276(30): 28372 - 28379. [Abstract] [Full Text] [PDF]
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