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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1899-1905
CCR5 Binds Multiple CC-Chemokines: MCP-3 Acts as a Natural Antagonist
By
Cédric Blanpain,
Isabelle Migeotte,
Benhur Lee,
Jalal Vakili,
Benjamin J. Doranz,
Cédric Govaerts,
Gilbert Vassart,
Robert W. Doms, and
Marc Parmentier
From IRIBHN and Service de Génétique Médicale,
Université Libre de Bruxelles, Campus Erasme, Bruxelles, Belgium;
and the Department of Pathology and Laboratory Medicine, University of
Pennsylvania, Philadelphia, PA.
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ABSTRACT |
CCR5 was first characterized as a receptor for MIP-1 , MIP-1 ,
and RANTES, and was rapidly shown to be the main coreceptor for
M-tropic human immunodeficiency virus (HIV)-1 strains and simian
immunodeficiency virus (SIV). Chemokines constitute a rapidly growing
family of proteins and receptor-chemokine interactions are known to be
promiscuous and redundant. We have therefore tested whether other
CC-chemokines could bind to and activate CCR5. All CC-chemokines
currently available were tested for their ability to compete with
[125I]-MIP-1 binding on a stable cell line expressing
recombinant CCR5, and/or to induce a functional response in these
cells. We found that in addition to MIP-1, MIP-1, and RANTES,
five other CC-chemokines could compete for
[125I]-MIP-1 binding: MCP-2, MCP-3, MCP-4, MCP-1, and
eotaxin binding was characterized by IC50 values of 0.22, 2.14, 5.89, 29.9, and 21.7 nmol/L, respectively. Among these ligands,
MCP-3 had the remarkable property of binding CCR5 with high affinity
without eliciting a functional response, MCP-3 could also inhibit the activation of CCR5 by MIP-1 and may therefore be considered as a
natural antagonist for CCR5. It was unable to induce significant endocytosis of the receptor. Chemokines that could compete with high
affinity for MIP-1 binding could also compete for monomeric gp120
binding, although with variable potencies; maximal gp120 binding
inhibition was 80% for MCP-2, but only 30% for MIP-1. MCP-3 could
compete efficiently for gp120 binding but was, however, found to be a
weak inhibitor of HIV infection, probably as a consequence of its
inability to downregulate the receptor.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CCR5 WAS CHARACTERIZED originally as a
receptor responding functionally to the CC-chemokines MIP-1 ,
MIP-1, and RANTES.1 CCR5 was further described as a
major coreceptor for human immunodeficiency virus (HIV), after the
demonstration that its three ligands constitute major HIV-suppressive
factors produced by CD8+ lymphocytes.2 Cellular
entry of primate lentivirus (HIV and simian immunodeficiency virus
[SIV]) is initiated by the interaction between the virus membrane
glycoprotein (gp120) and CD4. CD4 binding triggers conformational
changes in gp120 that enable it to interact with a coreceptor,
ultimately resulting in membrane fusion and viral entry.3
HIV tropism can be largely explained by coreceptor usage. CCR5 is the
major coreceptor for macrophage (M)-tropic HIV-1, HIV-2, and SIV
strains (therefore referred to as R5 strains), whereas CXCR4 is the
major coreceptor for T-tropic strains (now referred to as X4 strains).
Direct interaction between gp120 and CCR5 was shown to involve
conserved and variable regions of gp120.5-7 The strong
resistance to HIV-1 infection of individuals homozygous for a deletion
in the CCR5 coding region (CCR5 32), resulting in the production of a
truncated receptor not expressed at the cell surface,8-10
and HIV-1 inhibitory activities of MIP-1, MIP-1, and
RANTES,2-11 have highlighted the essential role of CCR5 in HIV-1 pathogenesis.
Two mechanisms have been proposed to account for the ability of
chemokines to inhibit HIV-1 infection. Firstly, the partial overlap of
chemokine and gp120 binding sites allows a direct competition for
access to the coreceptor.12 Secondly, receptor activation by full or partial agonists results in desensitization and
internalization of the receptor, and therefore in the reduction of
coreceptor surface expression.13,14 These mechanisms are
likely to be complementary, although their relative contributions
remain to be clarified in each case. The chemokine family has grown
rapidly over the recent years, and more than 20 CC-chemokines have been reported to date. For most of the recently described proteins, receptors functionally responding to them have been identified. Some
CC-chemokines however, such as LEC/ILINK and PARC/DC-CK1, have not yet
been shown to act through one of the currently characterized chemokine
receptors or related orphan receptors. Chemokine-receptor interactions
are well known to be promiscuous and redundant, because most receptors
are functionally activated by several chemokines, and most chemokines
can bind and activate more than one receptor. It was therefore likely
that some of the newly described chemokines would act through CCR5.
Defining CCR5 pharmacology could help to understand the relationship
between structural, functional, and anti-HIV properties of
CC-chemokines.
In this work, we have tested all currently available CC-chemokines for
their ability to bind to CCR5, to activate the receptor, to promote its
downregulation, and to inhibit CCR5-mediated HIV-1 entry. We have shown
that in addition to MIP-1, MIP-1, and RANTES, five other CC
chemokines could efficiently compete for MIP-1 binding on CCR5.
These were, with decreasing affinities: MCP-2, MCP-3, MCP-4, eotaxin,
and MCP-1. Among these, MCP-2 and MCP-4 were full agonists, whereas
MCP-3 could bind CCR5 with high affinity without eliciting a functional
response and had the ability to inhibit functional response to
MIP-1. MCP-3 induced no CCR5 endocytosis, could also compete for
gp120 binding, but was a weak inhibitor of HIV infection.
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MATERIALS AND METHODS |
Chemokines.
Recombinant human MIP-1, MIP-1, RANTES, MCP-1, MCP-2, MCP-3,
MCP-4, eotaxin, MIP-3, MIP-3, SLC, TARC, MDC, I309, and MPIF-2 were obtained from R&D Systems (London, UK). Recombinant TECK, MPIF-1,
PARC, and LEC were obtained from Peprotech (London, UK). HCC1, HCC2,
and HCC3 were kindly provided by W.G. Forssmann (IPF, Hannover,
Germany). [125I]-MIP-1 (specific activity: 2200 Ci/mmol) was obtained from New England Nuclear (Cambridge, MA). The
lyophilized chemokines were dissolved as 10 µmol/L solutions in
sterile phosphate-buffered saline (PBS) and stored at  20° C
in aliquots. They were diluted to the working concentrations
immediately before use.
Cell culture.
PM-1 were cultured in RPMI-1640 medium supplemented with 10% fetal
calf serum (Life Technologies, Merelbeke, Belgium), 100 units/mL
penicillin, and 100 g/mL streptomycin (Life Technologies). CHO-K1 cells
were cultured using HAM's F12 medium supplemented with 10% fetal calf
serum (Life Technologies), 100 units/mL penicillin, and 100 µg/mL
streptomycin (Life Technologies).
Expression of mutant receptors in CHO-K1 cells.
A plasmid encoding apoaequorin and G 16 under control of
the SR promoter15 was transfected into CHO-K1 cells, using Fugene 6 (Boerhinger Mannheim, Mannheim, Germany).
Zeocin (250 µg/mL; Invitrogen, Carlsbad, CA) selection
of transfectants was initiated 2 days after transfection. Individual
clones were isolated 3 weeks later, and the most responding clone was
selected on the basis of its functional response (luminescence signal)
to ionomycin A (100 nmol/L) and ATP (10 µmol/L). A construct encoding
wild-type CCR5 was further transfected using Fugene 6 in this
apoaequorin and G16-expressing cell line. Selection of
transfected cells was made for 14 days with 400 µg/mL G418 (Life
Technologies), and a clonal cell population expressing high CCR5 level
was used for binding and functional studies. The level of receptor
expression was measured by flow cytometry using antibodies directed
against the second extracellular loop (2D7) of CCR5 (Pharmingen, San
Diego, CA).
[125I]-MIP-1 binding assays.
CCR5-expressing CHO-K1 cells were collected from plates with
Ca2+ and Mg2+-free PBS supplemented with 5 mmol/L EDTA, gently pelleted for 2 minutes at 1000g, and
resuspended in binding buffer (50 mmol/L Hepes pH 7.4, 1 mmol/L
CaCl2, 5 mmol/L MgCl2, 0.5% BSA). Competition binding assays were performed in Minisorb tubes (Nunc, Roskilde, Denmark), using 0.08 nmol/L 125I-MIP-1
(2200 Ci/mmol, New England Nuclear) as tracer, variable concentrations of competitors, and 40,000 cells in a final volume of
0.1 mL. Total binding was measured in the absence of competitor and
nonspecific binding was measured with a 100-fold excess of unlabelled
ligand. Samples were incubated for 90 minutes at 27°C, then bound
tracer was separated by filtration through GF/B filters presoaked in
1% BSA. Filters were counted in a -scintillation counter. Binding
parameters were determined with the PRISM software (Graphpad Software,
San Diego, CA) using nonlinear regression applied to a
one-site competition model.
Functional assays.
Functional response to chemokines was analyzed by measuring the
luminescence of aequorin as described.16 CCR5-,
apoaequorin-, and G16-expressing cells were collected from
plates with Ca2+ and Mg2+-free Dulbecco's
modified Eagle's medium (DMEM) supplemented with 5 mmol/L EDTA,
pelleted for 2 minutes at 1000g, resuspended in DMEM at a
density of 5 × 106 cells/mL and incubated for 2 hours
in the dark in the presence of 5 µmol/L coelenterazine H (Molecular
Probes, Eugene, OR). Cells were diluted 7.5-fold before use. Agonists
in a volume of 50 L DMEM were added to 50 µL of cell suspension
(33,000 cells) and luminescence was measured for 1 minute in a
Packard luminometer (Downers Grove, IL). For assaying
antagonistic activities, chemokines were added to cell suspensions 1 minute before measuring the functional response to 1 nmol/L MIP-1.
[125I]-gp120 binding assays.
Soluble JRFL gp120 was iodinated using Iodogen (Pierce, Rockford, IL)
to a specific activity of 1000 Ci/mmol by using 5 µg protein with 500 µCi. Na125I radiolabeled proteins were purified from free
Na125I by separation through a 0.3 mL Dowex column prepared
in a 1 mL syringe and pre-equilibrated in Env binding buffer (50 mmol/L Hepes, pH 7.4, 5 mmol/L MgCl2, 1 mmol/L CaCl2)
containing 1% BSA and 150 mmol/L NaCl. Env binding assays were
performed by resuspending cells in 75 µL of Env binding buffer
containing 5% BSA. 0.5 nmol/L of labeled protein, saturating amounts
of sCD4 (100 nmol/L), and the indicated concentrations of chemokines
were added to cells in 25 µL of binding buffer for a total volume of
100 µL. 2 × 105 293T cells transfected with CCR5
were incubated at room temperature for 1 hour unless specified.
Unbound radioactivity was removed by filtering cells through 25 mm
Whatman GF/C filters (Maidstone, UK) presoaked in 0.2%
polyethylenimine (Sigma, St Louis, MO), and washing two
times with 4 mL wash buffer (50 mmol/L Hepes pH 7.4, 500 mmol/L
NaCl, 5 mmol/L MgCl2, 1 mmol/L CaCl2).
Filters were counted in a Wallac 1470 Wizard gamma
counter (Wallac, Turku, Finland).
Infection assays.
Plasmids encoding the HIV-1 ADA and BaL envs were provided by John
Moore (Aaron Diamond AIDS Research Center, New York, NY). The NL4-3
luciferase virus backbone (pNL-Luc-E-R-) was provided by Ned Landau
(Aaron Diamond AIDS Research Center). Luciferase reporter viruses were
prepared as previously described by cotransfecting 293T cells with the
indicated env construct and the NL4-3 luciferase virus. Virus
supernatants were used to infect PM-1 cells, a T-cell line naturally
expressing both CD4 and CCR5. Inhibition of infection by chemokines was
assayed by adding 1 µg/mL of the chemokine at the time of virus
infection. Incubation was performed at 37°C, and 4-days
postinfection, the cells were lysed with 0.5% triton X-100 in PBS, and
the lysate was analyzed for luciferase activity.
CCR5 endocytosis assay.
Chemokine-induced CCR5 endocytosis was performed as
described.13 Briefly, CHO-K1 cells stably expresssing human
CCR5 were collected from plates with 5 mmol/L EDTA in PBS, washed with
PBS, and incubated for 2 hours at 37°C with various chemokines at a 500 nmol/L concentration. Cells were washed two times with 3 mL of cold
PBS supplemented with 0.1% sodium azide and 0.1% BSA, and incubated
for 30 minutes at 4°C with phycoerythrin-conjugated anti-CCR5 2D7
Mab (Pharmingen). Cells were further washed, resuspended, and their
cell fluorescence was analyzed by fluorescence-activated cell sorter (FACS).
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RESULTS |
Promiscuous binding of CC-chemokines to CCR5.
We investigated the ability of all CC-chemokines available to date to
compete for [125I]-MIP-1 binding on CCR5 by using a
CHO-K1 cell line stably expressing the receptor. We first screened
chemokines for their ability to inhibit [125I]-MIP-1
binding at a concentration of 200 nmol/L. MIP-3, MIP-3, MIP-4,
HCC-1, HCC-2, HCC-3, MPIF-1, MPIF-2, TARC, TECK, SLC, LEC, MDC, and
I-309 had no significant effects on MIP-1 binding (data not shown).
In addition to the three classical ligands of CCR5, MIP-1, MIP-1,
and RANTES, five additional chemokines (MCP-1, MCP-2, MCP-3, MCP-4, and
eotaxin) did compete for the binding of the tracer and inhibited 80%
to 100% of MIP-1 binding at 200 nmol/L. For these ligands,
competition binding curves were established (Fig
1), allowing the determination of binding
affinity parameters (Table 1). On this
basis, the CCR5 ligands could be subdivided into high-affinity ligands
(IC50 < 1 nmol/L, RANTES = MCP-2 MIP-1 > MIP-1),
intermediate affinity ligands (1 nmol/L < IC50 < 10 nmol/L, MCP-3 > MCP4) and low-affinity ligands (IC50 > 10 nmol/L, MCP-1 = eotaxin). Since for some G-protein-coupled
receptors, the apparent affinity of ligands can vary depending on the
nature of the tracer,17 we have performed similar
competition binding assays, using three other iodinated chemokines as
tracers: RANTES, MIP-1, and MCP-2. The order of ligand affinities
were not significantly different as compared with the results obtained
with MIP-1 as tracer. As an example, the pIC50 obtained
in one experiment using [125I]-MCP-2 as tracer were 9.61 ± 0.09 for MCP-2, 9.17 ± 0.21 for MIP-1, 9.14 ± 0.15 for
RANTES, 9.03 ± 0.13 for MIP-1, and 7.82 ± 0.14 for MCP-3.
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| Fig 1.
Binding of CC-chemokines to CCR5. A CHO-K1 cell line
stably expressing human CCR5 and apoaequorin was established, and
characterized by saturation-binding assay as expressing 2 pmoles
receptor per mg protein. CC-chemokines were first tested for their
ability to compete with [125I]-MIP-1 binding at high
concentration (200 nmol/L), and competition curves were further
established for chemokines displaying CCR5-binding activity. Chemokines
that did not compete significantly at 200 nmol/L included MIP-3,
MIP-3, MIP-4, HCC-1, HCC-2, HCC-3, MPIF-1, MPIF-2, TARC, TECK, SLC,
LEC, MDC, and I-309. The results were analyzed by the Graphpad Prism
software, using a single-site model, and the data were normalized for
the nonspecific binding (0%) and the specific binding in the absence
of competitor (100%). All points were run in triplicate (error bars:
S.E.M.). The presented curves are representative of at least two
independent experiments. Table 1 presents the averaged values from the
various experiments.
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Functional activity of CCR5 ligands.
The same set of chemokines was tested for their ability to activate
CCR5, using a sensitive assay based on the use of apoaequorin as a
reporter system for intracellular calcium release. Activation of
chemokine receptors, including CCR5, is known to result in calcium
signalling. The release of calcium was coupled to the production of a
luminescent signal by coexpressing apoaequorin in the CCR5 cell line
and incubating the cells with coelenterazin before the assay to
reconstitute an active calcium-dependent enzyme complex.16
G16 was also coexpressed in the CCR5/apoaequorin cell line,
although this additional G protein is not necessary for the efficient
coupling of the receptor to the production of luminescence in this
assay (data not shown).
Chemokines that did not compete for MIP-1 binding on CCR5 at 200 nmol/L (MIP-3, MIP-3, MIP-4, HCC-1, HCC-2, HCC-3, MPIF-1, MPIF-2,
TARC, TECK, SLC, LEC, MDC, and I-309) were also unable to promote
intracellular calcium release in CCR5 transfected cells at all
concentrations tested (up to 100 nmol/L). The chemokines binding CCR5
with high affinities (IC50 < 1 nmol/L) activated the receptor with
high potency (Fig 2A). RANTES appeared as a slightly better agonist (EC50 of 1.3 nmol/L), whereas
MIP-1 (EC50: 3.2 nmol/L), MCP-2 (EC50: 3.6 nmol/L), and MIP-1 (EC50: 3.4 nmol/L) were less potent.
MCP-4 activated CCR5 with a potency (EC50: 103 nmol/L) in
relation to its binding affinity. For the low-affinity ligands MCP-1
and eotaxin, although mild stimulation of the receptor was observed at
the highest concentration tested (500 nM), a full dose response could
not be established, and the EC50 was estimated as in excess
of 500 nmol/L (Table 1).
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| Fig 2.
(A) Functional response of CCR5 to various CC-chemokines.
The functional activity of chemokines able to bind to CCR5 was assayed
by using a cell line coexpressing the receptor together with
G16 and apoaequorin. Light emission resulting from the
activation of the apoaequorin-coelenterazine complex in the presence of
intracellular calcium was recorded in a luminometer. The results were
analyzed by the Graphpad Prism software, using a single-site model, and
the data were normalized for basal luminescence (0%) and maximal
luminescence in the presence of 200 nmol/L MIP-1 (100%). All points
were run in duplicate (error bars: S.E.M.). The displayed curves
represent a typical experiment out of three performed independently.
(B) Inhibition of the MIP-1 functional response by MCP-3. The
antagonistic activity of MCP-3 was measured by preincubating the cells
for 1 minute with MCP-3, before the addition of 1 nmol/L MIP-1, and
recording of luminescence. All points were run in triplicate (error
bars: S.E.M.), and the results are representative of two independent
experiments.
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Interestingly, MCP-3 that is characterized by a better affinity for
CCR5 than MCP-4 (IC50: 2.1 nmol/L versus 5.8 nmol/L) did not result in intracellular calcium release up to 100 nmol/L, a
concentration that fully competed [125I]-MIP-1 binding
on the CCR5 cell line. We further tested whether MCP-3 could inhibit
the signaling mediated by MIP-1. Different concentrations of MCP-3
were added on the CCR5 cell line 1 minute before the addition of
MIP-1 (1 nmol/L). MIP-1-induced signalling decreased with
increasing MCP-3 concentrations (Fig 2B); 50 nmol/L MCP-3 reduced the
luminescent signal by 50%, whereas 100 nmol/L MCP-3 totally blunted
the functional response to MIP-1. From these results, MCP-3 can be
considered as a natural antagonist of CCR5.
Inhibition of gp120 binding and inhibition of HIV infection by
CC-chemokines.
The ability of high-affinity (MIP-1, MIP-1, RANTES, and MCP-2)
and intermediate-affinity (MCP-3 and MCP-4) CCR5 ligands to compete for
HIV-1 gp120 binding was investigated. Recombinant gp120 from the
M-tropic HIV-1 strain JR-FL was produced in 293T cells, purified,
iodinated, and used as tracer in competition-binding assays using the
CCR5-transfected HEK cells. As shown in Fig
3, chemokines that competed efficiently for
MIP-1 binding also inhibited gp120 binding, although with variable
efficacy, in accordance with previously reported data, using YU2
competition binding.6 The IC50 observed for
MIP-1, MIP-1, RANTES, and MCP-2 were found to be similar,
although the maximal inhibition varied for different chemokines. MCP-2
(600 nmol/L) inhibited over 80% of gp120 binding whereas MIP-1 did
not inhibit more than 50% at the same concentration. MCP-3 competed
for gp120 binding with an efficiency similar to that of MCP-2. In
contrast MCP-4 competed for gp120 binding with a much lower potency.
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| Fig 3.
Inhibition of gp120 binding by CC-chemokines.
CC-chemokines were tested for their ability to compete with the binding
of [125I]-gp120 from the M-tropic HIV-1 strain JR-FL to
CCR5-expressing 293 cells, in the presence of soluble CD4 (100 nmol/L).
The displayed curves are representative of two independent experiments.
The data were normalized for the nonspecific binding (0%) and the
specific binding in the absence of competitor (100%). The data shown
are the mean and S.E.M. derived from two independent experiments.  0 nmol/L,  20 nmol/L,  100 nmol/L,  600 nmol/L.
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The various CC-chemokines were tested for their ability to inhibit
infection of PM-1 cells, a human T-cell line naturally expressing CD4
and CCR5, by using luciferase reporter viruses pseudotyped with the Env
proteins of two HIV-1 (ADA and BaL) R5 strains (Fig
4). All high-affinity CCR5 ligands
(MIP-1, MIP-1, RANTES, and MCP-2) displayed potent anti-HIV-1
inhibitory activities (60% to 80% inhibition at 1 µg/mL).
Interestingly, MCP-2 displayed a greater inhibitory activity than
MIP-1 and MIP-1. In contrast, MCP-3 exhibited no significant
inhibitory effects (20% inhibition), at concentrations competing
efficiently for gp120 binding (1 g/mL). This suggested that competition
for binding was not sufficient to inhibit viral entry. We therefore
tested the ability of the various CCR5 ligands to promote receptor
internalization. The reduction of cell surface CCR5 immunoreactivity
was measured by FACS analysis, in response to 500 nmol/L chemokines,
using the 2D7 monoclonal antibody (MoAb). Possibly in line with its
lack of significant agonistic activity, MCP-3 did not reduce surface expression of CCR5, as compared with 30% reduction for MCP-4, 50% to
60% for MIP-1, MIP-1, MCP-2, and RANTES, and over 80% for
AOP-RANTES (Fig 5).
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| Fig 4.
Inhibition of HIV infection by CC-chemokines. Inhibition
of viral entry by chemokines was assayed by infecting PM-1 cells with
viruses pseudotyped with the env protein of the M-tropic HIV-1 strains
ADA () and BaL (). Chemokines were used at a 1 µg/mL
concentration, and the luciferase activity resulting from viral
infection was measured. The data were normalized for basal luciferase
activity (0%) and maximal activity in the absence of chemokines
(100%). Each condition was run in triplicates, and the displayed
results represent the mean of two independent experiments (error bars:
S.E.M.).
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| Fig 5.
Chemokine-induced internalization of CCR5.
Internalization of CCR5 in the presence of various chemokines was
estimated by FACS analysis of CHO-K1 cells expressing CCR5, using the
2D7 MoAb. The cells were incubated for 2 hours with the chemokines (500 nmol/L) before the test. The data (mean fluorescence) were normalized
for the fluorescence of untransfected CHO-K1 cells (0%) and maximal
fluorescence in the absence of chemokines (100%), and the displayed
results represent the mean of three independent experiments (error
bars: S.E.M.).
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DISCUSSION |
CCR5 has been characterized originally as a receptor responding to the
three CC-chemokines MIP-1, MIP-1, and RANTES.1 CCR5
was further identified as a coreceptor for HIV-1, HIV-2, and SIV
strains.3 Most M-tropic strains that are found early in
infection are strictly dependent on CCR5 for entry, and the essential
role of CCR5 in HIV transmission was shown by the strong resistance to
HIV infection of individuals homozygous for a 32 bp deletion in the
CCR5 coding region.8,9 The absence of obvious phenotype
associated with this defective CCR5 variant and the potent in vitro and
ex vivo HIV-suppressive activities of CCR5 ligands and antibodies makes
CCR5 an ideal target for therapeutic intervention.
Since we first characterized the pharmacology of CCR5, many new
CC-chemokines have been identified or made available. Some of these
chemokines have been shown to interact with receptors sharing high
sequence similarities and/or common ligands with CCR5. To evaluate
whether the range of CCR5 ligands had to be expanded in the context of
the known redundancy between chemokine receptors, we tested all
CC-chemokines described to date for their ability to bind and/or
activate the human CCR5 receptor expressed in recombinant cell lines.
We have shown that, in addition to MIP-1, MIP-1, and RANTES, CCR5
could bind five other CC-chemokines at physiological concentration. MCP-2 interacted with the receptor with a high affinity
(IC50 < 1 nmol/L) comparable to that of MIP-1,
MPI-1, and RANTES. MCP-3 and MCP-4 displayed intermediate binding
affinities (1 nmol/L < IC50 < 10 nmol/L), whereas MCP-1
and eotaxin were characterized by relatively low affinities
(IC50 of 20 to 30 nmol/L). In functional assays, all
high-affinity ligands were shown to be potent agonists (EC50 < 10 nmol/L). MCP-4 exhibited an agonistic activity
in relation to its binding affinity. By contrast, MCP-3 did not show
detectable functional activity at concentrations that completely
competed for [125I]-MIP-1 binding, but could inhibit
MIP-1-induced signaling of CCR5, showing its ability to function as
a natural CCR5 antagonist. MCP-1 and eotaxin displayed modest
functional activities at high concentrations (above 50 nmol/L),
suggesting that these two chemokines are unlikely to mediate biological
activities through CCR5 in vivo. While this work was in progress, MCP-2
and MCP-4 have been described as agonists of CCR5.18,19 Our
results show that full-length natural chemokines can bind to receptors
without activating it. Truncated chemokines such as truncated RANTES,
have also been shown to act as antagonists.20 MCP-3
appears, to our knowledge, as the first full-length natural chemokine
displaying antagonistic activity on CCR5. Interestingly, MCP-3 was
described as adopting preferentially a CXC-like dimer
conformation,21 in contrast to other CC-chemokines. Whether
this structural difference may be correlated with the antagonist
activity of the chemokine on CCR5 is unclear at this stage.
CCR5, which is able to bind seven chemokines, appears therefore as a
fairly promiscuous receptor. With the exception of MIP-1, which so
far does not activate other known receptors at low nmol/L concentrations, all other CC-chemokines binding to CCR5 also act through one or several other receptors. MIP-1 is the most potent agonist of CCR1 (IC50: 5 to 10 nmol/L). RANTES binds and
activates CCR1 and CCR3. MCP-2 has a wide spectrum of receptor usage as it activates also CCR1, CCR2, and CCR3. It was, however, shown recently
that the biological activity of MCP-2 on activated T cells was mediated
essentially through CCR5, although these cells express other functional
receptors for MCP-2.19 MCP-4 binds to CCR2 and CCR3, in
addition to CCR5, with similar affinities for all three receptors.
MCP-3 is an agonist for CCR1, CCR2, and CCR3. Noteworthy, its binding
affinity for CCR1 and CCR2 (around 10 nmol/L) is comparable to that of
CCR5.22 The antagonistic activities of MCP-3 on CCR5,
therefore, takes place at concentrations at which MCP-3 activates other
receptors, and may therefore have a functional relevance in vivo. MCP-1
and eotaxin are respectively active on CCR1 and CCR3.
The physiological significance of these overlapping activities of
inflammatory chemokines is unclear. This lack of specificity could
simply reflect the recent amplification of receptor and chemokine
genes, as suggested by their genomic clustering, that have not evolved
yet into nonredundant systems. It may also play a role in the
coordinated recruitment of leukocyte subsets to orchestrate various
types of immune responses. By contrast, constitutive chemokines,
involved in the trafficking of leukocyte populations to specific
compartments of lymphoid organs, appear as more specific in their
interactions with receptors. TARC and MDC bind exclusively to CCR4, ELC
and SLC to CCR7.
Primary sequence similarity among chemokines does not appear to
correlate with their receptor usage. For example HCC-1 is much more
similar to MIP-1 than MCP-2, but did not bind to or activate CCR5.
Understanding the structural determinants that underlie the binding
affinity and specificity of chemokines for receptors, as well as their
agonistic properties could provide great insight for the design of
chemokine analogs of higher affinity and efficacy. The N-terminal
region of CC-chemokines has been shown to be important for receptor
activation as well as receptor specificity.23-26 The
sequence comparison of chemokines acting on a single receptor could
allow to expand the understanding of structure-function relationships
of chemokines.
High-affinity chemokine analogs could be valuable in the frame of
inflammatory diseases and HIV infection. The blockade of CCR5
coreceptor function by different approaches was shown to potently
inhibit HIV infection in vitro and ex vivo.2,11,27-28 Natural or chemically modified chemokines as well as anti-CCR5 MoAb
have been described as CCR5 antagonists with HIV-suppressive activity.
The ability of additional chemokines acting on CCR5 to compete for HIV
gp120 binding was therefore tested, as well as their HIV inhibitory
properties in vitro. CCR5 high-affinity agonists (MIP-1, MIP-1,
MCP-2, and RANTES) as well as ligands with intermediate affinity (MCP-3
and MCP-4) were able to compete for M-tropic HIV-1 gp120 binding on a
CCR5 cell line although with variable efficacy. Interestingly, the
maximal inhibition of gp120 binding was higher for MCP-2 (85% of gp120
total binding) than for other chemokines such as MIP-1 (50% of
gp120 total binding). This result may suggest that gp120 can bind to
CCR5 conformations not equally accessible for all chemokines. The same
mechanism has been proposed earlier to explain the greater potency of
AOP-RANTES to mediate HIV inhibition of infection.12
Inhibition of infection by the various chemokines was tested using
pseudotyped viruses. MIP-1, MIP-1, and RANTES inhibited HIV (ADA,
BaL) infection with similar efficacies. MCP-2 exhibited a greater
potency to inhibit infection by HIV ADA. Interestingly, whereas MCP-3
could compete for HIV gp120 binding on CCR5 with high affinity, it did
not inhibit HIV infection significantly. This may be due to the absence
of agonistic properties of this ligand. When tested in a downregulation
assay based on FACS analysis of cell-surface CCR5, MCP-3 did not induce
downmodulation of the receptor, as compared with 60% for agonists such
as MIP-1, or over 80% for AOP-RANTES. Receptor endocytosis is
classically linked to the phosphorylation of its agonist-bound active
conformation,29 again showing the pure antagonistic nature
of this ligand. It was suggested that potency of a chemokine or its
derivatives to mediate inhibition of HIV infection not only depended on
the competition for the virus-binding site,12 but could
also be correlated to the downmodulation of coreceptor surface
expression.13 The low efficacy of MCP-3 in an infection
inhibition assay could be due to its lack of downmodulation effect on
CCR5. Other agents, such as AOP-RANTES and truncated RANTES, described
as CCR5 functional antagonists in chemotaxis assay but presenting
partial agonistic properties in other functional assays (calcium flux,
microphysiometer), have been described as potent HIV suppressors and
efficient downregulators of receptor surface expression.
In conclusion, we have analyzed the pharmacology of CCR5 using binding
and functional assays. A number of new chemokines able to interact with
CCR5 were described, including MCP-2, MCP-3, and MCP-4. Among them,
MCP-3 displayed the original property of acting as a natural antagonist
of CCR5, at concentrations similar to those necessary to activate CCR1,
CCR2, and CCR3. We also showed that inhibition of HIV infection
required efficient activation of the coreceptor, in agreement with the
hypothesis that receptor downregulation is the main mechanism of
chemokine-mediated HIV suppressive activity.
| |
ACKNOWLEDGMENT |
Expert technical assistance was provided by M.J. Simons.
| |
FOOTNOTES |
Submitted February 22, 1999; accepted May 20, 1999.
Supported by the Actions de Recherche Concertées of the
Communauté Française de Belgique, the French Agence
Nationale de Recherche sur le SIDA, the Belgian programme on
Interuniversity, Poles of Attraction initiated by the Belgian State,
Prime Minister's Office, Science Policy Programming, the BIOMED and
BIOTECH programmes of the European Community (grants BIO4-CT98-0543 and
BMH4-CT98-2343), the Fonds de la Recherche Scientifique Médicale
of Belgium, and the Fondation Médicale Reine Elisabeth. The
scientific responsibility is assumed by the authors. C.B. is Aspirant
of the Belgian Fonds National de la Recherche Scientifique and C.G. is
a fellow of the Fonds pour la Recherche dans l'Industrie et
l'Agriculture. R.W.D. was supported by NIH NIAID R01-40880.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Marc Parmentier, MD, IRIBHN, ULB
Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium; e-mail:
mparment{at}ulb.ac.be.
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TOP
ABSTRACT
INTRODUCTION