(Received for publication, January 31, 1997, and in revised form, March 24, 1997)
From the 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
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, 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.
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, 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.
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.
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 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).
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 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).
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.
Calcium Mobilization AssayThis 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 (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.
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.
Binding AssayThis 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).
Northern Blot analysisThis 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 80 °C with an intensifying screen.
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, 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.
Induction of Chemotaxis by LARC
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).
Binding of LARC
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, 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).
Selective Expression of GPR-CY4 in T and B Cells
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).
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 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.
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.
We are grateful to Drs. Yorio Hinuma and Masakazu Hatanaka for constant support and encouragement.