By
§
From the * Laboratory of Host Defenses, The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis
in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane
domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously
reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium
flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type
G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60
clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is
unusual among known CC chemokines and known CC chemokine receptors. CCR8 may
regulate monocyte chemotaxis and thymic cell line apoptosis.
The chemokine superfamily consists of specific leukocyte chemoattractant proteins that can be sorted by
structure into four groups, designated C, CX3C, CC, and
CXC depending on the number and spacing of conserved
cysteines (1). The C and CX3C groups each have only
one known member, whereas the CC and CXC groups each have many members. CXC chemokines mainly target
neutrophils and T cells, and C and CX3C chemokines are
specific for T cells. CC chemokines target monocytes, eosinophils, basophils, and T cells with variable selectivity, but,
in most cases, they do not target neutrophils.
I-309 is a human CC chemokine first identified by molecular cloning in a search for genes expressed in activated
T cell lines (4). Like other CC chemokines, I-309 induces
chemotaxis in monocytes (5). Recently, I-309 was purified
from CD4+ T cells as a secreted factor that protects murine
thymic lymphoma cell lines from dexamethasone-induced
apoptosis (6). Other chemokines had little or no anti-apoptotic activity, suggesting a unique signaling pathway.
The first step in chemokine action involves binding to G
protein-coupled receptors on the cell surface. Seven CC
chemokine receptor subtypes, CCR1-7, have previously
been identified by molecular cloning (7-14c). They are all
expressed on leukocytes, and together they account for
binding sites for most of the known CC chemokines.
CCR1, CCR2, CCR3, and CCR5 bind overlapping sets of multiple CC chemokines, whereas only one high affinity
ligand has been identified for CCR4, CCR6, and CCR7.
None of these receptors has been shown to bind I-309. We
have previously reported the genomic DNA and deduced
protein sequence of an orphan receptor named CY6, cloned
by virtue of its sequence homology to known chemokine
receptors (GenBank no. U45983[GenBank], released April 2, 1996).
Subsequently, two groups published the same orphan sequence deduced from genomic clones, and named it TER1
and CKR-L1 (15, 16). Here, we show that CY6/TER1/
CKR-L1 encodes an I-309 receptor.
Genomic Cloning and Sequencing.
Genomic DNA from a healthy
donor was amplified by PCR using degenerate primers designed
from conserved sequences in the predicted third and seventh
transmembrane domains of CXCR2 (GenBank no. M73969[GenBank]) and
an orphan receptor named 9-6 (GenBank no. U45982[GenBank]). The
primer sequences included HincII sites for cloning purposes, and
are CC GTC GAC TGC ATI (T/A)(C/G)I GTI GA(C/T) (C/
A)GI TA (primer CY3), and CC GTC GAC AI IGG (A/G)TT
IA(A/G) (G/A)CA I(G/C)(A/T) (A/G)TG (primer CY7). The
reaction contained 1.3 µg template DNA, 1 µM of each primer,
200 µM dATP, dTTP, dCTP, and dGTP, 10 mM Tris-HCl, pH
8.3, 50 mM KCl, 2.5 mM MgCl2, and 2.5 U of DNA polymerase
(Perkin-Elmer Cetus; Norwalk, CT) in 100 µl, and was amplified for 33 cycles (93°C for 1.5 min, 50°C for 2 min, and 72°C
for 2 min), then given a final 7-min extension at 72°C. Products
were cloned into the HincII site of pUC18 and sequenced (17).
A novel sequence named CY6 was identified and used as a probe
to isolate a human genomic clone from a Mapping.
Fluorescence in situ hybridization (FISH) was carried out as previously described using the CY6 genomic clone as
probe (19). Radiation hybrid mapping was performed by PCR
using the Stanford G3 panel (Research Genetics, Huntsville, AL)
with primers CY6B and CY6 (GCTAGGATTACAGGCATGAGCCACA) to give a 341-bp product.
Creation of Cell Lines Expressing Chemokine Receptors.
The CY6
ORF was first amplified from the 1.9-kb genomic fragment using
primers 5 Cell Culture.
The promyelocytic cell line HL-60 clone 15 (CRL 1964; American Type Culture Collection, Rockville,
MD) was maintained and induced to differentiate to eosinophil-like cells by treatment with 0.5 µM butyric acid (Sigma) and 10 ng/ml IL-5 (R&D, Minneapolis, MN), as previously described
(20, 21). Human neutrophils and mononuclear cells were purified
from the peripheral blood of healthy donors. Mononuclear cells
were plated on tissue culture plastic for 18 h and the adherent and
nonadherent cells, enriched in monocytes and lymphocytes, respectively, were collected separately for RNA analysis.
RNA Analysis.
Total RNA was prepared using a commercial
kit (Stratagene, La Jolla, CA). Blots were prepared and hybridized
with 32P-labeled probes as previously described (21).
Intracellular [Ca2+] Measurements.
Cells (107/ml) were incubated in PBS, pH 7.4, and 2.5 µM Fura-2 AM (Molecular
Probes, Eugene, OR) for 30-60 min at 37°C in the dark. The
cells were subsequently washed twice with HBSS, and resuspended at 1 × 106 cells/ml. 106 cells were stimulated in a total
volume of 2 ml in a continuously stirred cuvette at 37°C in a fluorimeter (Photon Technology, Inc., South Brunswick, NJ). Recombinant human chemokine sources: SDF-1 Chemotaxis.
Cells were harvested and washed twice with
PBS, then resuspended in serum-free RPMI 1640. Cells were
loaded in a total volume of 25 µl into the upper compartment of
a microchemotaxis chamber (Neuroprobe, Cabin John, MD).
Chemoattractants were loaded in a final volume of 31 µl at indicated concentrations in the lower compartment. The two compartments were separated by a polyvinylpyrollidone-free polycarbonate filter with 5-µm pores. The chemotaxis chamber was incubated at 37°C, 100% humidity, and 5% CO2 for 4 h. The filter was then removed, and the number of cells migrating into
each bottom compartment was counted using a hemocytometer.
All conditions were tested in triplicate.
Taking advantage of the
observation that G protein-coupled receptor genes have
conserved sequences and often lack introns in the coding
region, we used degenerate PCR primers to amplify a
novel human genomic sequence named CY6 related to CC
chemokine receptors. A 1,953-bp fragment of a genomic
clone containing the CY6 sequence was then isolated and
sequenced. It extended to the 5 We have mapped the CY6 genomic fragment to human
chromosome 3p22-p23 by FISH. 49 cells were examined,
with 25 showing paired hybridization signal and 17 showing a single signal at 3p23-p22. Two-point linkage analysis
of the radiation hybrid data by the Stanford Radiation Hybrid server gives a LOD of 11.5 for linkage to D3S3527 at
a distance of 5.4 cR10000. This places the gene between the
Genethon markers D3S1260 and D3S3522, and between the gene for CCR4 and a cluster of genes for CCR1, 2, 3, and 5 (22). Using FISH, Napolitano et al. (15) reported
that TER1 maps to chromosome 3p21.
Using a multitissue Northern blot, mRNA was detected
at high levels in thymus, and at lower levels in spleen, but
not in 14 other tissues tested (data not shown). Using
Northern blots from the same supplier, the same RNA distribution pattern has been reported by Napolitano et al. and
Zaballos et al. (15, 16). Napolitano et al. (15) also detected
transcripts in the MOLT-4 T cell line and the NK3.3 NK
cell line, but not in primary NK cells, monocytes, neutrophils, or PHA/PMA-activated PBMCs. We were able to
detect a 4.6-kb mRNA band in total RNA from adherent monocytes, consistent with the size in thymus, but not in
neutrophil or lymphocyte samples (Fig. 1). Zaballos et al.
(16) also detected mRNA in monocyte/macrophages, as
well as in lymph node, and CD4+, CD8+, and CD19+
lymphocytes.
Because of the functional specificity that we now describe,
we have provisionally named the protein product of the
CY6/TER1 open reading frame (ORF) CC chemokine receptor 8 or CCR8. This is in keeping with a nomenclature
system accepted by consensus at the Second Gordon Conference on Chemotactic Cytokines (1997, Plymouth, NH).
To identify a specific agonist, we
screened a panel of chemoattractants for the ability to induce calcium flux in the mouse pre-B cell line 4DE4 before and after transfection with a plasmid encoding CCR8.
Untransfected 4DE4 cells did not respond to any agonists
tested except for the CXC chemokine SDF-1 (Fig. 2 A). 4DE4 cells transfected with the CCR8 plasmid exhibited
[Ca2+]i transients in response to SDF-1 and I-309, but not
in response to the following tested at 50 nM or greater: the
CC chemokines HCC-1, MIP-1
The threshold for the calcium flux response of CCR8-expressing cells to I-309 was 0.1 nM, and the EC50 was 2 nM (Fig. 2 B). These values are similar to those observed
for other chemokine receptors (7). When the cells were
pretreated with other ineffective chemokines or with SDF-1,
there was no effect on the magnitude or kinetics of the
I-309-induced calcium flux response, suggesting that other
chemokines are not antagonists at CCR8 (data not shown). In contrast, when cells were sequentially stimulated with
the same concentration of I-309, no response to the second
application was observed (Fig. 2 A), suggesting homologous desensitization of the receptor.
I-309 was able to induce transmigration of 4DE4 cells
expressing CCR8, but not untransfected cells, across a filter
in a modified Boyden chamber assay of chemotaxis (Fig. 3).
The I-309 dose-response curve for chemotaxis was bell-shaped, which is typical for this response. I-309 was both
highly potent (EC50 of 2 nM and an optimal concentration
of 5 nM) and highly efficacious (~40% of input cells migrated across the filter at the optimal concentration).
Checkerboard analysis indicated that the response was chemotactic and not chemokinetic (data not shown).
The clone 15 variant of HL-60 cells can be induced by butyric acid and IL-5 treatment to differentiate
within 2 d to cells having many of the characteristics of peripheral blood eosinophils, including expression of eosinophil-specific granule proteins (20, 21). Using Northern blot
analysis, we were unable to detect mRNA for CCR8 in
the uninduced cells, and the cells did not respond to I-309
in either the calcium flux or chemotaxis assays (Fig. 4, A-C).
However, when the cells were cultured in the presence of
butyric acid and IL-5, a 4.6-kb band was detected by
Northern blot using a CCR8 ORF probe (Fig. 4 A).
Induction of CCR8 mRNA correlated with acquisition
of calcium flux responses to I-309 (Fig. 4 B). The EC50 was
1 nM (Fig. 4 C), similar to the value observed for I-309-
induced calcium flux and chemotaxis in 4DE4 cells expressing recombinant CCR8. Induced cells also responded
to MIP-1
Identification of CCR8 is an essential first step in understanding the mechanism of action of I-309. The ability of
recombinant CCR8 to support chemotaxis in transfected
pre-B cells suggests that endogenous CCR8 may mediate
the chemotactic activity of I-309 in monocytes. The pattern of constitutive CCR8 mRNA expression in tissues
that we and others (15, 16) have observed is unique relative
to known chemokine receptors, and suggests a role for CCR8 specifically in thymus. This is consistent with the
ability of I-309 to inhibit dexamethasone-induced apoptosis in mouse thymic cell lines (6). Together, these observations suggest the importance of future studies to test the
role of I-309 in thymocyte migration and development in
vivo. This may be accomplished by targeted genetic disruption of mouse CCR8, which has not yet been identified, or of TCA3, which appears to be the mouse homologue of I-309. Like I-309, TCA3 induces monocyte
chemotaxis. It has also been shown to induce degranulation, production of reactive nitrogen intermediates, and
upregulation of adhesion molecules in monocytes, but, unlike I-309, has been reported to have parallel activities on
neutrophils. TCA3 can also suppress the growth of certain
tumors in both immunocompetent and immunodeficient
mice (23).
Like other chemokines, I-309 is likely to provide directional information for orderly leukocyte trafficking in vivo
(26). Like other chemokines, if it is dysregulated, I-309 has
the potential to cause inappropriate inflammation and tissue
injury. In this regard, our identification of an I-309 receptor may be useful in future research aimed at evaluating this
pathway for development of potential anti-inflammatory
therapies.
Received for publication 2 April 1997 and in revised form 23 April 1997.
Laboratory of Cell Biology and § Section on Genetics,
the Krebs Institute, Department of Molecular
Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, United
Kingdom; the ¶ Biological Carcinogenesis and Development Program, Program Resources,
Incorporated/Dyncorp, National Cancer Institute, Frederick Cancer Research and Development Center,
Frederick, Maryland 21702-1201
library (18). A 1.9-kb
fragment containing the CY6 ORF was isolated by cutting an
EcoRI site in the vector and a genomic XbaI site, subcloned into
pUC18, and sequenced completely on both strands. The 5
end
of CY6 RNA was obtained using Clontech Marathon-Ready human thymus cDNA and nested primers from the coding region, named CY6A (CCAGAAGACTGAATACAAACAGGAGGCAA) and CY6B (GTCTGAATAAGTTCCGCATCACAGGGGCTT). The cDNA template was amplified using 10 pmol of
CY6A and adaptor primer AP1 (30 cycles of 90°C for 1 min,
60°C for 1min, and 72°C for 2 min). Product from this reaction
was reamplified using CY6B and AP2 primers. The 200-250-bp
product was gel-purified, digested with NotI and EcoRV (which
cuts immediately 5
of the CY6B primer), cloned into Bluescript,
and sequenced.
-GCTCTAGATCTGTGACCAGGTCCCGCTGCC
(upper strand), which contains an XbaI site (underlined) and nucleotides
4 to
25 relative to the ATG initiator, and 5
-CGGAATTCATATTTAGTCTTCATTGATCCT (lower strand),
which contains an XhoI site (underlined) and 21 nucleotides downstream of the stop codon. The PCR product was subcloned into
pcDNA3 (Invitrogen, San Diego, CA). Using the same methodology, we created Flag epitope-tagged constructs in pcDNA3 for
CCR1, CCR3, and CCR5 using the p4 (GenBank no. L10918[GenBank]), clone 3 (GenBank no. U28694[GenBank]), and 8.5 (GenBank no. U57840[GenBank]) cDNAs, respectively, as templates. The nucleotide sequences
were confirmed on both strands. 4DE4 pre-B cell lymphoma cells
(gift of L. Staudt, NCI, Bethesda, MD) were grown in RPMI
1640 containing 10% FCS and 50 µM 2-mercaptoethanol. Human embryonic kidney (HEK) 293 cells were grown in DMEM
with 10% FCS containing streptomycin 100 µg/ml and penicillin
100 U/ml. 1-1.5 × 107 cells in log phase were electroporated using a GenePulser (Bio-Rad Laboratories, Hercules, CA) with 20 µg
of plasmid DNA. HEK 293 cell colonies resistant to 2 g/L G-418
(GIBCO BRL, Gaithersburg, MD) were isolated and expanded
in the same media supplemented with 2 g/L G-418. 4DE4 cells
were cultured in 1 g/L G-418 and expanded. For CCR1 and
CCR3, mixed populations of 4DE4 cells resistant to G-418 were
enriched for receptor-expressing cells by chemotaxis in response
to appropriate agonists through a ChemoTx chemotactic chamber
(Neuroprobe, Inc., Cabin John, MD) with a 5-µm pore size. Clones
were obtained by limiting dilution, and receptor expression confirmed by FACS® using the anti-Flag mAb Bio M5 according to
the instructions of the manufacturer (Kodak, Rochester, NY).
, HCC-1, and
I-309, R&D; NAP-2, Bachem (Philadelphia, PA); the BB10010 variant of MIP-1
, a gift from L. Czaplewski (British Biotech, Inc., Oxford, UK); all others, Peprotech (Rocky Hill, NJ). C3a was a gift of C. Hammer. fMLP and recombinant human C5a
were from Sigma Chem. Co. (St. Louis, MO). The data were recorded every 200 ms as the relative ratio of fluorescence emitted
at 510 nm after sequential excitation at 340 and 380 nm. For
some experiments, cells were incubated with 250 ng/ml pertussis
toxin, 2 µg/ml cholera toxin, or 2 µM herbimycin A for 4 h before functional assay.
Cloning of the Gene for CCR8.
-end of the phage insert,
and contained a 1,065-bp ORF, and 250 and 620 bp of 5
and 3
sequence, respectively. The deduced protein sequence is most closely related to CCR1-5 (39-43% identity) with lower identity (25-30%) to CXC chemokine receptors. To confirm the initiation codon, we first identified
thymus as a rich natural source of CY6 mRNA and used it
to amplify the 5
-UTR sequence by anchored PCR. The
sequence revealed 120 bases 5
of the putative ATG initiator (GenBank no. AF005210) with an in-frame terminator 15 bases 5
of the ATG and residing at the 3
end of an upstream exon, strongly supporting this codon as the initiator.
Fig. 1.
RNA distribution
of CCR8. Northern blots containing total RNA from the
sources indicated above each
lane were hybridized with a
CCR8 ORF probe under high
stringency conditions. M, monocyte/macrophages (PBMCs that
remained adherent to plastic after
18-h overnight culture); L, lymphocytes (nonadherent PBMCs); N, neutrophils. The blots
were exposed for 3 d to x-ray
film using an intensifying screen.
[View Larger Version of this Image (64K GIF file)]
, RANTES, MIP-1
,
MCP-1, MCP-2, MCP-3, MCP-4, and eotaxin; the CXC
chemokines IL-8,
IP-10, NAP-2, GRO-
, GRO-
, GRO-
, and ENA-78; the C chemokine lymphotactin;
and the nonchemokine leukocyte chemoattractants fMLP,
C3a, and C5a (data not shown). I-309 did not induce calcium flux in 4DE4 cell lines expressing CCR1 or CCR3,
or in HEK 293 cells stably expressing CCR5 (Fig. 2 A; data
not shown). The CCR1, CCR3, and CCR5 cell lines all responded appropriately to previously described agonists
(Fig. 2 A; data not shown).
Fig. 2.
I-309 is an agonist
for CCR8. (A) Receptor specificity and homologous densensitization. [Ca2+]i was monitored by
ratio fluorescence of Fura-2-loaded
pre-B cells or HEK 293 cells stably transfected with plasmids encoding CC chemokine receptors
as indicated adjacent to each
tracing. Cells were stimulated
with chemokines 50 nM at the
times indicated by arrowheads.
Data are representative of at least
three experiments with CCR8-expressing cells. (B) Potency.
The amplitude of the peak of the
calcium transient elicited by the
indicated concentration of I-309
in CCR8 transfectants is shown.
Data are representative of two
separate experiments.
[View Larger Version of this Image (18K GIF file)]
Fig. 3.
CCR8 is a chemotactic receptor. Untransfected
pre-B cells (open squares) and cells
stably expressing CCR8 (closed
circles) were incubated in a microchemotaxis chamber and tested
with the indicated concentrations
of I-309. The number of input
cells was 350,000/well. Data are
the mean ± SEM of triplicate determinations, and are from a single experiment representative of
two separate experiments. Checkerboard analysis indicated that the
activity was chemotactic, not chemokinetic (data not shown).
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
An HL-60 clone 15 cell line model of endogenous
CCR8 expression and function.
HL-60 clone 15 cells were cultured for 6 d in the presence of
butyric acid 0.5 µM and for the
final 4 d in the presence of IL-5
10 ng/ml, which induces differentiation to an eosinophilic phenotype. (A) CCR8 mRNA expression. A Northern blot containing 10 µg total RNA
from undifferentiated (U) and
differentiated (D) cells was hybridized with a CCR8 ORF
probe (top) and washed under
high stringency conditions. The
blot was then exposed to x-ray
film using an intensifying screen
for 20 h. The corresponding region of the ethidium bromide-
stained gel is shown in the lower panel. (B) Calcium flux response
to I-309. Fura-2-loaded undifferentiated (top tracing, U) and
differentiated (lower tracing, D)
cells were stimulated with I-309
50 nM. (C) Potency of I-309 for
calcium flux. Data are from a
single experiment representative
of three separate experiments.
(D) Distinct receptor usage by
I-309 and other CC chemokines.
Differentiated cells were stimulated with the indicated chemokines 50 nM and Fura-2 fluoresence was monitored.
[View Larger Version of this Image (33K GIF file)]
, RANTES, MCP-3, and eotaxin, consistent
with the induction of CCR1 and CCR3 mRNA (Tiffany, H.L., and P.M. Murphy, unpublished observations), but
none of these chemokines was able to desensitize I-309-
induced calcium flux, consistent with their lack of agonist
and antagonist activity at CCR8 (Fig. 4 D). I-309-induced
calcium flux in both differentiated HL-60 clone 15 cells
and 4DE4 cells expressing CCR8 was abolished by treatment with pertussis toxin, but not by cholera toxin or herbimycin A, suggesting specific coupling of the receptor to G proteins of the Gi class in both cell types (Fig. 5). Although the HL-60 clone 15 cells are a useful model system
for studying endogenous CCR8, it is important to point
out that we have not been able to demonstrate CCR8
mRNA or I-309 responsiveness in primary human eosinophils, even when stimulated with IL-5.
Fig. 5.
CCR8 couples to a Gi-type G protein. [Ca2+]i was measured
as the relative fluorescence emitted by Fura-2-loaded pre-B cells stably
transfected with CCR8 or HL-60 clone 15 cells differentiated with butyric acid and IL-5 after 4-h treatment with the inhibitors indicated to the
right of each tracing. I-309 50 nM was added at the time indicated by the
arrow. The results are from a single experiment representative of three
separate experiments.
[View Larger Version of this Image (23K GIF file)]
Address corespondence to Philip M. Murphy, M.D., Building 10, Room 11N113, National Institutes of
Health, Bethesda, MD 20892. Phone: 301-496-2877; FAX: 301-402-4369; E-mail: pmm{at}nih.gov; or Tom I. Bonner, Ph.D. Building 36, Room 7A07, National Institutes of Health, Bethesda, MD 20892. Phone: 301-496-8906; FAX: 301-402-1748; E-mail: tibonner{at}helix.nih.gov
1.
Baggiolini, M.,
B. Dewald, and
B. Moser.
1994.
Interleukin-8
and related chemotactic cytokines CXC and CC chemokines.
Adv. Immunol.
55:
97-179
[Medline].
2.
Bazan, J.F.,
K.B. Bacon,
G. Hardiman,
W. Wang, and
T.J. Schall.
1997.
A new class of membrane-bound chemokine
with a CX3C motif.
Nature (Lond.).
385:
640-644
[Medline].
3.
Kelner, G.S.,
J. Kennedy,
K.B. Bacon,
S. Kleyensteuber,
D.A. Largaespada,
N.A. Jenkins,
N.G. Copeland,
J.F. Bazan,
K.W. Moore,
T.J. Schall, and
A. Zlotnick.
1994.
Lymphotactin: a cytokine that represents a new class of chemokine.
Science (Wash. DC).
266:
1395-1398
[Medline].
4.
Miller, M.D.,
S.D. Wilson,
M.E. Dorf,
H.N. Seuanez,
S.J. O'Brien, and
M.S. Krangel.
1990.
Sequence and chromosomal location of the I-309 gene. Relationship to genes encoding a family of inflammatory cytokines.
J. Immunol.
145:
2737-2744
5.
Miller, M.D., and
M.S. Krangel.
1992.
The human cytokine
I-309 is a monocyte chemoattractant.
Proc. Natl. Acad. Sci.
USA.
89:
2950-2954
[Abstract].
6.
Van Snick, J.,
F. Houssiau,
P. Proost,
J. Van Damme, and
J.-C. Renauld.
1996.
I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasone-induced
apoptosis.
J. Immunol.
157:
2570-2576
[Abstract].
7.
Neote, K.,
D. DiGregorio,
J.Y. Mak,
R. Horuk, and
T.J. Schall.
1993.
Molecular cloning, functional expression, and
signaling characteristics of a C-C chemokine receptor.
Cell.
72:
415-425
[Medline].
8.
Gao, J.-L.,
D.B. Kuhns,
H.L. Tiffany,
D. McDermott,
X. Li,
U. Francke, and
P.M. Murphy.
1993.
Structure and functional expression of the human macrophage inflammatory
protein-1 /RANTES receptor.
J. Exp. Med.
177:
1421-1427
[Abstract].
9.
Charo, I.,
S.J. Myers,
A. Herman,
C. Franci,
A.J. Connolly, and
S.R. Coughlin.
1994.
Molecular cloning and functional
expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal
tails.
Proc. Natl. Acad. Sci. USA.
91:
2752-2756
[Abstract].
10.
Kitaura, M.,
T. Nakajima,
T. Imai,
S. Harada,
C. Combadiere,
H.L. Tiffany,
P.M. Murphy, and
O. Yoshie.
1996.
Molecular cloning of human eotaxin, an eosinophil-selective
CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3.
J. Biol. Chem.
271:
7725-7730
11.
Ponath, P.,
S. Qin,
T.W. Post,
J. Wang,
L. Wu,
N.P. Gerard,
W. Newman,
C. Gerard, and
C.R. Mackay.
1996.
Molecular
cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils.
J. Exp. Med.
183:
2437-2448
[Abstract].
12.
Power, C.A.,
A. Meyer,
K. Nemeth,
K.B. Bacon,
A.J. Hoogewerf,
A.E.I. Proudfoot, and
T.N.C. Wells.
1995.
Molecular cloning and functional expression of a novel CC
chemokine receptor cDNA from a human basophilic cell
line.
J. Biol. Chem.
270:
19495-19500
13.
Samson, M.,
O. Labbe,
C. Mollereau,
G. Vassart, and
M. Parmentier.
1996.
Molecular cloning and functional expression of a new human CC-chemokine receptor gene.
Biochemistry.
35:
3362-3367
[Medline].
14.
Combadiere, C.,
S.K. Ahuja,
H.L. Tiffany, and
P.M. Murphy.
1996.
Cloning and functional expression of CC CKR5,
a human monocyte CC chemokine receptor selective for
MIP-1 , MIP-1
, and RANTES.
J. Leukocyte Biol.
60:
147-152
[Abstract].
14a.
Imai, T.,
M. Baba,
M. Nishimura,
M. Kakizaki,
S. Takagi, and
O. Yoshie.
1997.
The T cell-directed CC chemokine
TARC is a highly specific biological ligand for CC chemokine receptor 4.
J. Biol. Chem.
272:
15036-15042
14b.
Baba, M.,
T. Imai,
M. Nishimura,
M. Kakizaki,
S. Takagi,
K. Hieshima,
H. Nomiyama, and
O. Yoshie.
1997.
Identification of CCR6, the specific receptor for a novel lymphocyte-directed CC chemokine LARC.
J. Biol. Chem.
272:
14893-14898
14c.
Yoshida, R.,
T. Imai,
K. Hieshima,
J. Kusuda,
M. Baba,
M. Kitaura,
M. Nishimura,
M. Kakizaki,
H. Nomiyama, and
O. Yoshie.
1997.
Molecular cloning of a novel human CC
chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7.
J. Biol. Chem.
272:
13803-13809
15.
Napolitano, M.,
A. Zingoni,
G. Bernardini,
G. Spinetti,
A. Nista,
C.T. Storlazzi,
M. Rocchi, and
A. Santoni.
1996.
Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues.
J. Immunol.
157:
2759-2763
[Abstract].
16.
Zaballos, A.,
R. Varona,
J. Gutierrez,
P. Lind, and
G. Marquez.
1996.
Molecular cloning and RNA expression of two
new human chemokine receptor-like genes.
Biochem. Biophys. Res. Commun.
227:
846-853
[Medline].
17.
Song, Z.-H.,
W.S. Young,
M.J. Brownstein, and
T.I. Bonner.
1994.
Molecular cloning of a novel candidate G protein-
coupled receptor from rat brain.
FEBS Lett.
351:
375-379
[Medline].
18.
Lawn, R.M.,
E.F. Fritsch,
R.C. Parker,
G. Blake, and
T. Maniatis.
1978.
The isolation and characterization of linked
delta- and beta-globin genes from a cloned library of human
DNA.
Cell.
15:
1157-1174
[Medline].
19.
Tory, K.,
F. Latif,
W. Modi,
L. Schmidt,
M.H. Wei,
H. Li,
P. Cobler,
M.L. Prcutt,
J. Delisio,
L. Geil, et al
.
1992.
A genetic linkage map of 96 loci on the short arm of human chromosome 3.
Genomics.
13:
275-286
[Medline].
20.
Fischkoff, S.A..
1988.
Graded increase in probability of eosinophilic differentiation of HL-60 promyelocytic leukemia cells
induced by culture under alkaline conditions.
Leuk. Res.
12:
679-686
[Medline].
21.
Tiffany, H.L.,
F. Li, and
H.F. Rosenberg.
1995.
Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor
cells.
J. Leukocyte Biol.
58:
49-54
[Abstract].
22.
Samson, M.,
P. Soularue,
G. Vassart, and
M. Parmentier.
1996.
The genes encoding the human CC-chemokine receptors CC-CKR1 to CC-CKR5 (CMKBR1-CMKBR5) are
clustered in the p21.3-p24 region of chromosome 3.
Genomics.
36:
522-526
[Medline].
23.
Luo, Y.,
J. Laning,
S. Devi,
J. Mak, and
M. Dorf.
1994.
Biologic activities of the murine beta-chemokine TCA3.
J. Immunol.
153:
4616-4624
24.
Devi, S.,
J. Laning,
Y. Luo, and
M.E. Dorf.
1995.
Biologic
activities of the beta-chemokine TCA3 on neutrophils and
macrophages.
J. Immunol.
154:
5376-5383
25.
Laning, J.,
H. Kawasaki,
E. Tanaka,
Y. Luo, and
M.E. Dorf.
1994.
Inhibition of in vivo tumor growth by the beta
chemokine, TCA3.
J. Immunol.
153:
4625-4635
26.
Springer, T.A..
1994.
Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.
Cell.
76:
301-314
[Medline].