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Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 nM); the binding was
prevented completely by an excess of cold TCA-3, and only partially
competed (40%) by I-309. The identification of I-309 and TCA-3 as the
functional ligands for CCR8 receptors will help to unravel the role of
these proteins in physiologic and pathologic situations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, in which X is any amino acid residue, and CC or
ß-chemokines (1, 2, 3). Recently, lymphotactin, a chemokine with only
one cysteine residue in the N-terminal region (4), and
fractalkine/neurotactin, a chemokine with a CX3C motif
(5, 6), have been described. -chemokines exert their actions mainly
on neutrophils, while ß-chemokines stimulate a wide variety of cell
types, such as monocytes, eosinophils, basophils, and lymphocytes.
Chemokine receptors are a family of single-chain, G protein-coupled
proteins (7, 8, 9, 10). Four receptors for -chemokines have been reported.
Two of them bind IL-8 (11, 12): CXCR1, which also binds GCP-2, and
CXCR2, which also binds Gro-, Gro-ß, Gro-
, NAP-2, and GCP-2
(13, 14, 15). CXCR3 binds IP-10 and monokine induced by IFN- (16), and
SDF-1 is the only known ligand for CXCR4 (17, 18). Concerning receptors
for ß-chemokines, seven have been described to date. The identified
ligands for CCR1 are
MIP-1,3 RANTES, and MCP-3
(19, 20, 21). CCR2 is able to bind MCP-1, MCP-3, and MCP-4 (22, 23, 24, 25, 26).
Eotaxin, RANTES, MCP-3, and MCP-4 are bound by CCR3 (26, 27, 28, 29). CCR4
seems to bind only TARC (30), and CCR5 is a receptor for RANTES,
MIP-1, and MIP-1ß (27). The most recently identified ß-chemokine
receptors are CCR6 and CCR7, which are able to signal upon the specific
binding of LARC and ELC, respectively (31, 32).
Despite this degree of promiscuity, with most chemokine receptors being able to bind more than one chemokine, and many chemokines binding more than one receptor, some putative receptors still remain orphan (33, 34, 35, 36) and the receptors for some chemokines are not known (4, 5, 6, 37). Unraveling the specificity of interactions between chemokines and their receptors would be of great help for understanding the molecular basis of the variety and specificity of chemokine activities.
We have reported recently the cloning of CKR-L1 and CKR-L3, two human genes that are expressed in some lymphoid tissues and are new putative chemokine receptors (38). The ligand for CKR-L3, the ß-chemokine LARC, has been described very recently, and now this receptor is called CCR6 (31). However, no ligand for CKR-L1 has been reported to date. We now present evidence showing that CKR-L1 specifically binds the ß-chemokine I-309. Therefore, according to the current chemokine receptor nomenclature, we designate CKR-L1 as CCR8. We also report the cloning of the mCCR8 receptor and data showing its specific binding of TCA-3, the I-309 mouse homologue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human chemokines were obtained from PeproTech (London, UK) or R&D Systems (Minneapolis, MN). Recombinant mouse chemokines were bought from PharMingen (San Diego, CA) (TCA-3) or R&D Systems (C10). Human embryonic kidney 293 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and grown in DMEM supplemented with 10% FCS (Seralab, Crawley Down, U.K.) and antibiotics, at 37°C in a humidified 5% CO2 atmosphere. The CCR8 genes were cloned in pCIneo (Promega Corp., Madison, WI), and the corresponding 293 stable transfectants were obtained by G418 selection of calcium phosphate-transfected cells, as described (39). 293-EBNA cells (Invitrogen Corp., San Diego, CA) were used as host for transient transfections. To study the effect of pertussis and cholera toxins on I-309-mediated calcium mobilization and migration of CCR8 transfectants, the cells were cultured in the presence of 0.1 µg/ml pertussis toxin or 0.4 µg/ml cholera toxin for 16 h before the assays. The mouse thymic lymphoma BW5147 cell line was obtained from ATCC and was grown in RPMI 1640 supplemented with 10% FCS (Seralab), 2 mM glutamine, 1 mM sodium pyruvate, and antibiotics.
Calcium mobilization assays
Variations in the intracellular concentration of calcium in stable transfected cells were analyzed by fluorimetry essentially as described by Didsbury et al. (40). Briefly, 293/CCR8-transfectant cells were loaded with 3 to 5 µM Indo-1-AM (Molecular Probes, Leiden, The Netherlands) at a density of 107 cells/ml in HBSS containing 20 mM HEPES (pH 7.3) and 0.1% BSA. After washing, the cells were resuspended in the same buffer at a density of 1 to 3 x 106 cells/ml, and samples of 1.5 ml were placed in a cuvette in a heated holder equilibrated at 37°C, under stirring, in an SLM-8000C spectrofluorimeter. A wavelength of 350 nm was used for excitation; samples of chemokines in 15 µl were injected into the cuvette, while dual-fluorescence emission was detected continuously at 405 nm and over 500 nm. The ratio of fluorescence emission at each wavelength, F405/F>500, was recorded as a measure of calcium concentration. Calcium mobilization in transiently transfected cells was analyzed as described elsewhere (41). Briefly, plasmid pcytAEQ (Molecular Probes), encoding apoaequorin, was cotransfected into 293-EBNA cells along with each plasmid encoding the receptors to be tested. Twenty-four hours after transfection, the cells were loaded with hcp-coelenterazine to reconstitute the aequorin protein as a luminescent calcium probe. The loaded cells were analyzed for luminescence in the same way described for the fluorescence analysis, except that the samples were not excited and filters were not used for collecting the emitted light.
Chemotaxis assays
The migration of 293 cells stable transfected with hCCR8 was studied in a 48-well microchamber (Neuro Probe, Cabin John, MD) essentially as described elsewhere (21). Chemokines in different concentrations were loaded in the lower wells (27 µl/well), and cells (50 µl/well, 106 cells/ml) in the upper wells. Polyvinylpirrolidone-free filters with 10-µm pores (Poretics, Livermore, CA), precoated for 2 h at 37°C with type VI collagen (Sigma Chemical Co., St. Louis, MO), were used. The chamber was incubated for 5 to 6 h at 37°C in a humidified atmosphere with 5% CO2. After incubation, filters were removed and the cells present in the upper part were wiped off. The filters were then fixed and stained with a crystal violet solution (0.5% crystal violet, 20% methanol). Blue spots developed at positions in which cell migration had occurred. Between 5,000 and 50,000 cells, the color intensity of the spots was checked to be directly proportional to the number of cells present in the filter. This allowed the quantification of the migration results by densitometry of the spots (National Institutes of Health Image software). A migration index was calculated by the following ratio: densitometric result of samples of cells migrated to chemokines/densitometric result of samples of cells migrated to buffer.
Cloning of mCCR8
A quantity amounting to 6 x 105
plaque-forming units from a 129 SVJ mouse genomic DNA library in
GEM-12 vector was plated and screened with a 32P-labeled
DNA probe corresponding to the complete ORF of the hCCR8 gene. Positive
plaques were rescreened until plaque-purified clones were obtained.
Clone 301 was shown to contain a complete ORF homologue to that of
the hCCR8 gene. The identified mCCR8 sequence was subcloned into pCIneo
to generate the corresponding expression vector.
Northern blot analysis
Total RNA from normal adult mouse tissues or the BW5147 murine T cell lymphoma was extracted using Tri-reagent (Sigma Chemical Co.), as recommended by the supplier. Poly(A)+ RNA was purified from the total RNA samples using oligo(dT) cellulose. Poly(A)+ RNA was fractionated by electrophoresis on a denaturing formaldehyde-agarose gel, transferred to a Nylon Hybond N+ membrane, and UV cross-linked. The membrane was prehybridized and then hybridized in Rapid Hyb buffer (Amersham, Little Chalfont, U.K.), as recommended by the supplier.
125I-TCA-3 binding to 293/mCCR8 cells
Recombinant mouse TCA-3 (PharMingen) was iodinated as described previously (42). Briefly, 2 µg TCA-3 in 50 µl 0.1 M borate buffer, pH 8.5, was incubated in ice for 15 min with 1 mCi 125I-labeled Bolton-Hunter reagent (Amersham). The reaction was stopped by adding 250 µl of 0.5 M glycine in the same buffer, and the nonincorporated 125I was removed by chromatography in a Sephadex G-25 PD10 column (Pharmacia, Uppsala, Sweden). The sp. act. of radiolabeled TCA-3 was about 8 x 108 cpm/µg. The binding assays using 125I-TCA-3 were performed essentially as described (43). 293-EBNA cells were transfected transiently with an expression plasmid encoding mCCR8. After 48 h, 106 cells were mixed with different concentrations of 125I-TCA-3 in the presence or absence of a 100-fold molar excess of cold TCA-3, in a final volume of 300 µl of buffer A (RPMI 1640 containing 1% BSA and 20 mM HEPES, pH 7.4). After incubating the mixtures for 2 h at 4°C, cell suspensions were overlaid onto tubes containing 700 µl of cold FCS and they were spun. Cell pellets were washed with 900 µl of ice-cold buffer A and, finally, the radioactivity associated to cell pellets was counted in a gamma counter (Wallac Oy, Turku, Finland). In binding competition assays, 106 cpm/sample of 125I-TCA-3 was competed for 2 h at 4°C with a 170-fold molar excess of unlabeled TCA-3, or 250-fold molar excess of I-309, JE, mMCP-5, or C10, in 300 µl of buffer A.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction of I-309 with hCCR8 was studied by measuring
changes in intracellular calcium concentration of 293 cell clones
stable transfected with a hCCR8 expression plasmid (38) or with the
void vector (pCIneo), upon addition of I-309 at different
concentrations. Figure 1
A
depicts the results obtained with 293/hCCR8-transfectant cells. The
addition of 0.1 nM I-309 did not result in detectable calcium
mobilization. Higher I-309 concentrations (0.3–1 nM) induced a
significant wave of calcium mobilization, and peaks of increasing
intensity were obtained upon stimulation with higher concentrations of
the chemokine. As expected, a second addition of 100 nM I-309 to a
sample previously stimulated with the same concentration of chemokine
did not result in calcium mobilization. Other - or ß-chemokines
such as IL-8, Gro-, Gro-ß, Gro-, ENA-78, IP-10, monokine
induced by IFN-, NAP-2, PF-4, MCP-1, MCP-2, MCP-3, MCP-4, RANTES,
MIP-1, MIP-1ß, HCC1, eotaxin, and lymphotactin did not elicit a
significant calcium mobilization upon addition to hCCR8 transfectants
(38, and results not shown). I-309 did not provoke calcium mobilization
in the 293 control cells transfected with the void vector (Fig. 1B).
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C, calcium mobilization induced by I-309
was abolished completely by pretreatment of the cells with pertussis
toxin, while the cells were still able to mobilize calcium in response
to another receptor-mediated, pertussis-insensitive stimulus such as
the addition of 50 µM ATP. Conversely, pretreatment of 293/hCCR8
cells with cholera toxin did not decrease their I-309-induced calcium
response (not shown). I-309-induced chemotaxis of 293/hCCR8 cells
Figure 2A shows that
I-309 induced the specific migration of 293/hCCR8 cells. The
I-309-dependent migration followed a dose-response curve typical of
chemokines, with a maximum between 5 and 10 nM. Control 293 cells
transfected with the vector alone did not significantly migrate to
I-309. The effect of a pretreatment with toxins on the I-309-induced
migration of 293/hCCR8 cells was also tested. As shown in Figure 2B, pertussis toxin practically abolished the migration
induced by I-309, while cholera toxin did not significantly affect
migration.
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By screening of a 129 SVJ mouse genomic library, we have isolated
the murine homologue of hCCR8. A DNA probe consisting in the complete
ORF of the hCCR8 gene was used to identify a positive clone, in which
an ORF encoding a protein of 353 amino acid residues was identified.
The comparison between this deduced amino acid sequence and that
corresponding to the hCCR8 showed that the overall identity between
both proteins was 71% (Fig. 3). In the
extracellular N-terminal region, in which the murine protein is two
amino acids shorter than the human one, the identity is 50%. The N8
amino acid residue of mCCR8 is a potential site for N-glycosylation.
This is a very common structural feature of chemokine receptors,
although it is not present in hCCR8. When the amino acid sequence of
mCCR8 is compared with those of other murine ß-chemokine receptors,
the highest identity (43%) corresponds to mCCR4 (Fig. 3). Other mouse
ß-chemokine receptors share 40% (mCCR2 and MCCR5) and 37% (mCCR1
and mCCR3) identity with mCCR8 (Fig. 3). Given that the coding sequence
of mCCR8 was obtained from genomic DNA and 5' ends of the coding
sequences of several chemokine receptors have been shown recently to be
interrupted by introns, we have used an anchored PCR-based strategy to
analyze the 5' end of cDNA from the mCCR8 gene. Sequence analysis of
PCR products did not detect an alternative ATG codon (data not shown).
Similarly, the hCCR8 genomic coding sequence is coincident with that of
the cDNA (data not shown).
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The tissue distribution of mCCR8 was investigated by Northern
blotting of poly(A)+ RNA obtained from different mouse
organs. Using the complete mCCR8 ORF as a probe, two transcripts of
about 2.2 and 1.5 kb were detected in thymus (Fig. 4). We also detected a strong expression
of mCCR8 in the BW5147 mouse thymic lymphoma cell line (Fig. 4). The
message was not detectable in the rest of tissues tested.
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Figure 5 shows the results obtained
in calcium mobilization experiments when 293-EBNA transient
transfectant cells expressing either the human or murine CCR8 receptor
were stimulated with either I-309 or its murine homologue, TCA-3.
Addition of 10 nM I-309 or 10 nM TCA-3 to the transfectants expressing
mCCR8 provoked calcium mobilization, while addition of 100 nM C10,
another murine chemokine, did not provoke any response. However, the
cells expressing hCCR8 were only able to respond to I-309 and remained
unresponsive upon addition of 100 nM TCA-3. As expected, control cells
transfected with the void vector did not respond to the addition of
I-309 or TCA-3.
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Ligand-binding assays were performed with
125I-TCA-3 on human 293-EBNA cells transiently
transfected with an expression plasmid encoding mCCR8. The
transfectants bound radiolabeled TCA-3 with high affinity (Fig. 6). Nonspecific binding, defined as that
noninhibited by a 100-fold molar excess of unlabeled TCA-3, was
approximately 25% of total binding. The specific binding data were
plotted and showed the typical hyperbolic curve, which tended to a
plateau at 125I-TCA-3 concentrations over 2 nM (Fig. 6A). Scatchard analysis of these data was used to
estimate an equilibrium dissociation constant
(Kd) of 2 nM (Fig. 6B).
125I-TCA-3 did not bind significantly to 293 cells
transfected with the void vector (data not shown). Competitive binding
inhibition assays using other chemokines were also conducted. Figure 6C shows that a 170-fold molar excess of unlabeled TCA-3
completely inhibited the binding of 106 cpm of
125I-TCA-3, which was competed only partially by a 250-fold
molar excess of I-309. No inhibition of 125I-TCA-3 binding
was obtained with the same molar excess of other mouse chemokines
tested, such as C10, JE, or mMCP-5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrate that CKR-L1 is a specific receptor
for I-309, a human ß-chemokine that is produced by activated T
lymphocytes (46). We therefore propose to refer to CKR-L1 as to hCCR8.
At nanomolar concentrations, I-309 was able to mobilize intracellular
calcium in 293 cells transfected with hCCR8. The stimulation of
293/hCCR8 cells with 100 nM I-309 resulted in the complete
desensitization of the cells to a second similar stimulus. In addition,
293/hCCR8 cells were chemoattracted by nanomolar concentrations of
I-309. Chemokine receptors are coupled to heterotrimeric G proteins,
which are signal-transduction mediators for this kind of receptors.
Heterotrimeric G proteins constitute a family of effector proteins,
some of which can be inhibited by pertussis or cholera toxins. As shown
in Figures 1C and 2B, the I-309-induced
mobilization of intracellular calcium and migration on hCCR8
transfectants were inhibited clearly by pertussis toxin, suggesting
that hCCR8 is coupled to the Gi class of
G subunits (47). All of these data agree with the
reported behavior of other chemokine receptors and support the
conclusion that I-309 is a functional ligand for hCCR8. Indeed, while
this manuscript was under revision, two other groups have also reported
that I-309 is a specific ligand for this receptor (48, 49).
Since murine models are valuable tools to study the biologic functions of new genes, we decided to search for a putative mCCR8 receptor. A potential seven-transmembrane receptor 71% similar to hCCR8 was cloned, and the ligand-binding characteristics shown by this protein strongly indicate that this clone is indeed the mCCR8 receptor. Among murine ß-chemokine receptors, mCCR8 shows the highest degree of amino acid homology to mCCR4. This is also the case when hCCR8 is compared with the other human chemokine receptors. Northern blot analysis of the RNA expression of mCCR8 showed that, like its human counterpart, this receptor is expressed mainly in thymus. In the mouse, two transcripts of 2.2 and 1.5 kb are detected, whereas a single CCR8 transcript of 4.3 kb is present in humans (38).
As expected for a murine homologue of hCCR8, TCA-3, the murine
homologue of I-309, is able to induce calcium mobilization in
transiently transfected 293-EBNA/mCCR8 cells. Moreover,
125I-TCA-3 binds to these transient transfectants with a
Kd of 2 nM. A similar Kd
value has been reported for the binding of 125I-TCA-3 to
mouse monocyte/macrophage cell lines and mouse mesangial cells (43).
I-309 was also able to induce a calcium mobilization response in
transiently transfected 293-EBNA/mCCR8 cells. This was consistent with
the binding competition results, which showed that I-309 was able to
cause a 40% inhibition of the specific binding of
125I-TCA-3 to mCCR8. In addition, these data agree with
previous reports describing the activity of I-309 on murine cells (50).
However, TCA-3 was not able to induce calcium mobilization on transient
transfectant 293-EBNA cells expressing hCCR8 (Fig. 5), and the same
result was obtained with stable transfected 293/hCCR8 cells (results
not shown). Nevertheless, this somewhat more species-restricted ligand
specificity of hCCR8 must be interpreted cautiously, since TCA-3 has
been reported to stimulate calcium mobilization on human THP-1 cells
(51). Whether this apparent inconsistency reflects differences in the
quality of the protein preparations (insect cell-produced TCA-3 was
used in our experiments; TCA-3 produced in CHO cells was used in 51 , in the sensitivity of the techniques used to measure calcium
mobilization, or the existence of a human TCA-3-sensitive receptor
different to hCCR8 remains to be established. It must be noted,
however, that Samson et al. (45) did not detect hCCR8 transcripts in
THP-1 cells by Northern blot analysis, which suggests that, indeed, a
different receptor might be involved.
I-309 has been reported to chemoattract human monocytes, but not
neutrophils, in in vitro migration assays, and to increase cytoplasmic
free calcium concentration in human peripheral blood monocytes, but not
in lymphocytes or neutrophils (52). The sensitivity of monocytes to
I-309 and the insensitivity of neutrophils are consistent with our
previous observations of hCCR8 RNA expression in monocytes/macrophages,
but not in granulocytes. However, the reasons for the lack of
lymphocyte response are not clear, since hCCR8 RNA seems to also be
expressed in peripheral blood CD4+ and CD8+
lymphocytes (38). As suggested, the apparent lack of a standard
chemokine functionality of I-309 on lymphocytes might reflect a
differential coupling of hCCR8 to signal-transduction molecules in
different cell types (52). Actually, an effect of I-309 on lymphocytic
cells has been reported: I-309 protects murine BW5147 T cell lymphoma
against dexamethasone-induced apoptosis, an activity that is also
shared by TCA-3 (50). Interestingly, this protection against apoptosis
showed by I-309 is not exerted by other chemokines, and it is abolished
completely in the presence of pertussis toxin (50), suggesting that
CCR8 might be the receptor involved. As shown in Figure 4, Northern
analysis of poly(A)+ RNA from BW5147 cells shows a strong
expression of mCCR8 transcripts. Indeed, we have performed Scatchard
analysis of the specific binding of 125I-TCA-3 to BW5147
cells (data not shown), and obtained a Kd of 1.1
nM, close to that estimated for the TCA-3/mCCR8 interaction.
As reported before (51), the pattern of murine blood cells responsive to TCA-3 does not exactly match that of human cells responsive to I-309. In several assays, TCA-3 has been shown to stimulate neutrophils (53, 54), whereas, as indicated above, I-309 seems to be inactive on human neutrophils. This is interesting because ß-chemokine activation of neutrophils is not usual, and now the availability of mCCR8 will help to determine the molecular basis for this difference between the human and mouse systems.
Unlike most chemokine receptors, which are able to bind several chemokines, hCCR8 only binds 1 of 20 tested. This fact indicates a remarkable specificity, although we presently do not exclude the possibility that new hCCR8 and mCCR8 ligands will appear as the number of members of the chemokine family keeps on growing. In any case, the identification of I-309 and TCA-3 as the functional ligands for CCR8 receptors will help to unravel the role these proteins play in different physiologic and pathologic situations. In this regard, we are generating CCR8 knockout mice as a valuable tool to study the in vivo functions of this receptor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gabriel Márquez, Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco. 28049-Madrid. Spain. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; CRK, chemokine receptor-like; h, human; 125I-TCA-3, 125I-labeled TCA-3; m, murine; MCP, monocyte-chemotactic protein; ORF, open reading frame.
Received for publication August 18, 1997. Accepted for publication November 3, 1997.
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/RANTES receptor. J. Exp. Med. 177:1421./RANTES, is also a functional receptor for MCP3. J. Biol. Chem. 270:22123.This article has been cited by other articles:
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J. Roland, B. J. Murphy, B. Ahr, V. Robert-Hebmann, V. Delauzun, K. E. Nye, C. Devaux, and M. Biard-Piechaczyk Role of the intracellular domains of CXCR4 in SDF-1-mediated signaling Blood, January 15, 2003; 101(2): 399 - 406. [Abstract] [Full Text] [PDF]
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G. Spinetti, G. Bernardini, G. Camarda, A. Mangoni, A. Santoni, M. C. Capogrossi, and M. Napolitano The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway J. Leukoc. Biol., January 1, 2003; 73(1): 201 - 207. [Abstract] [Full Text] [PDF]
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C. D. Chung, F. Kuo, J. Kumer, A. S. Motani, C. E. Lawrence, W. R. Henderson Jr., and C. Venkataraman CCR8 Is Not Essential for the Development of Inflammation in a Mouse Model of Allergic Airway Disease J. Immunol., January 1, 2003; 170(1): 581 - 587. [Abstract] [Full Text] [PDF]
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C. A. Murphy, R. M. Hoek, M. T. Wiekowski, S. A. Lira, and J. D. Sedgwick Interactions Between Hemopoietically Derived TNF and Central Nervous System-Resident Glial Chemokines Underlie Initiation of Autoimmune Inflammation in the Brain J. Immunol., December 15, 2002; 169(12): 7054 - 7062. [Abstract] [Full Text] [PDF]
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T. Nakajima, N. Inagaki, H. Tanaka, A. Tanaka, M. Yoshikawa, M. Tamari, K. Hasegawa, K. Matsumoto, H. Tachimoto, M. Ebisawa, et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison Blood, December 1, 2002; 100(12): 3861 - 3868. [Abstract] [Full Text] [PDF]
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T. Chtanova, R. A. Kemp, A. P. R. Sutherland, F. Ronchese, and C. R. Mackay Gene Microarrays Reveal Extensive Differential Gene Expression in Both CD4+ and CD8+ Type 1 and Type 2 T Cells J. Immunol., September 15, 2001; 167(6): 3057 - 3063. [Abstract] [Full Text] [PDF]
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T. Ruckes, D. Saul, J. Van Snick, O. Hermine, and R. Grassmann Autocrine antiapoptotic stimulation of cultured adult T-cell leukemia cells by overexpression of the chemokine I-309 Blood, August 15, 2001; 98(4): 1150 - 1159. [Abstract] [Full Text] [PDF]
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H. R. Luttichau, J. Gerstoft, and T. W. Schwartz MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8 J. Leukoc. Biol., August 1, 2001; 70(2): 277 - 282. [Abstract] [Full Text] [PDF]
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S. W. Chensue, N. W. Lukacs, T.-Y. Yang, X. Shang, K. A. Frait, S. L. Kunkel, T. Kung, M. T. Wiekowski, J. A. Hedrick, D. N. Cook, et al. Aberrant in Vivo T Helper Type 2 Cell Response and Impaired Eosinophil Recruitment in Cc Chemokine Receptor 8 Knockout Mice J. Exp. Med., March 5, 2001; 193(5): 573 - 584. [Abstract] [Full Text] [PDF]
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L. Kremer, L. Carramolino, I. Goya, A. Zaballos, J. Gutierrez, M. del Carmen Moreno-Ortiz, C. Martinez-A., and G. Marquez The Transient Expression of C-C Chemokine Receptor 8 in Thymus Identifies a Thymocyte Subset Committed to Become CD4+ Single-Positive T Cells J. Immunol., January 1, 2001; 166(1): 218 - 225. [Abstract] [Full Text] [PDF]
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N. S. Haque, J. T. Fallon, M. B. Taubman, and P. C. Harpel The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium Blood, January 1, 2001; 97(1): 39 - 45. [Abstract] [Full Text] [PDF]
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G. Bernardini, G. Spinetti, D. Ribatti, G. Camarda, L. Morbidelli, M. Ziche, A. Santoni, M. C. Capogrossi, and M. Napolitano I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo Blood, December 15, 2000; 96(13): 4039 - 4045. [Abstract] [Full Text] [PDF]
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 R. M. Scoggins, J. R. Taylor Jr., J. Patrie, A. B. van't Wout, H. Schuitemaker, and D. Camerini Pathogenesis of Primary R5 Human Immunodeficiency Virus Type 1 Clones in SCID-hu Mice J. Virol., April 1, 2000; 74(7): 3205 - 3216. [Abstract] [Full Text]
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C.-R. Yu, K. W. C. Peden, M. B. Zaitseva, H. Golding, and J. M. Farber CCR9A and CCR9B: Two Receptors for the Chemokine CCL25/TECK/Ck{beta}-15 That Differ in Their Sensitivities to Ligand J. Immunol., February 1, 2000; 164(3): 1293 - 1305. [Abstract] [Full Text] [PDF]
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D. J. Dairaghi, R. A. Fan, B. E. McMaster, M. R. Hanley, and T. J. Schall HHV8-encoded vMIP-I Selectively Engages Chemokine Receptor CCR8. AGONIST AND ANTAGONIST PROFILES OF VIRAL CHEMOKINES J. Biol. Chem., July 30, 1999; 274(31): 21569 - 21574. [Abstract] [Full Text] [PDF]
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M. J. Endres, C. G. Garlisi, H. Xiao, L. Shan, and J. A. Hedrick The Kaposi's Sarcoma-related Herpesvirus (KSHV)-encoded Chemokine vMIP-I is a Specific Agonist for the CC Chemokine Receptor (CCR)8 J. Exp. Med., June 21, 1999; 189(12): 1993 - 1998. [Abstract] [Full Text] [PDF]
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A. Zaballos, J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez Cutting Edge: Identification of the Orphan Chemokine Receptor GPR-9-6 as CCR9, the Receptor for the Chemokine TECK J. Immunol., May 15, 1999; 162(10): 5671 - 5675. [Abstract] [Full Text] [PDF]
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A. Zingoni, H. Soto, J. A. Hedrick, A. Stoppacciaro, C. T. Storlazzi, F. Sinigaglia, D. D'Ambrosio, A. O'Garra, D. Robinson, M. Rocchi, et al. Cutting Edge: The Chemokine Receptor CCR8 Is Preferentially Expressed in Th2 But Not Th1 Cells J. Immunol., July 15, 1998; 161(2): 547 - 551. [Abstract] [Full Text] [PDF]
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).
B, Stable 293/hCCR8-transfectant cells were treated with
pertussis toxin (PTX) or cholera toxin (CTX), as indicated in
Materials and Methods, and in vitro migration tests were
then performed. I-309 (10 nM) was used as chemoattractant in these
assays. Tests were done in triplicate or tetraplicate, and a migration
index was estimated, as described in Materials and Methods.
The data represent mean ± SE. Representative results from at
least three different experiments are shown.
) and nonspecific (
) binding were obtained
by incubating 293/mCCR8 cells with increasing amounts of
125I-TCA-3, in the absence or presence of a 100-fold molar
excess of cold TCA-3. B, Scatchard analysis of the specific
binding data. C, Competition for the binding of
106 cpm of 125I-TCA-3 to mCCR8. A 170-fold
molar excess of cold TCA-3 or a 250-fold molar excess of the rest of
the indicated chemokines was used. In the competition experiments,
assays were done in duplicate; the data represent the mean of one
representative experiment from three independent experiments
performed.