Human immunodeficiency virus, type I (HIV-1)
cell-type tropism is dictated by chemokine receptor usage: T-cell line
tropic viruses use CXCR4, whereas monocyte tropic viruses primarily use CCR5 as fusion coreceptors. CC chemokines macrophage inflammatory protein (MIP)-1
, MIP-1
, and RANTES (regulated on activation normal T cell expressed and secreted) inhibit CD4/CCR5-mediated HIV-1
cell fusion. MCP-2 is also a member of the CC chemokine subfamily and
has the capacity to interact with at least two receptors including
CCR-1 and CCR2B. In an effort to further characterize the binding
properties of MCP-2 on leukocytes, we observed that MCP-2, but not
MCP-1, effectively competed with MIP-1
for binding to monocytes,
suggesting that MCP-2 may interact with CCR5. As predicted, MCP-2
competitively inhibited MIP-1
binding to HEK293 cells stably
transfected with CCR5 (CCR5/293 cells). MCP-2 also bound to and induced
chemotaxis of CCR5/293 cells with a potency comparable with that of
MIP-1
. Confocal microscopy indicates that MCP-2 caused remarkable
and dose-dependent internalization of CCR5 in CCR5/293
cells. Furthermore, MCP-2 inhibited the entry/replication of
HIV-1ADA in CCR5/293 cells coexpressing CD4. These results indicated that MCP-2 uses CCR5 as one of its functional receptors and
is an additional potent natural inhibitor of HIV-1.
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INTRODUCTION |
Members of the seven-transmembrane chemokine receptor superfamily
have been identified as co-receptors for HIV-11infection (1-8). HIV-1 cell type
tropism seems to be dependent on chemokine receptor usage, and T-cell
line tropic viruses use CXCR4, whereas monocyte tropic viruses
primarily use CCR5 as fusion coreceptors. A minority of HIV-1 strains
may also use other CC chemokine receptors as their fusion co-factors.
Dual tropic HIV-1 strains presumably interact with more than one type
of chemokine receptor (5, 8). The CC chemokines MIP-1
, MIP-1
, and
RANTES were able to inhibit the entry of monocyte tropic viruses (9), whereas the CXC chemokine SDF-1 abrogates CXCR4-mediated fusion by T
lymphotropic HIV-1 strains (6, 7).
Monocyte chemotactic protein (MCP)-2 is a CC chemokine co-purified with
MCP-1 and MCP-3 from human osteosarcoma cells (10-12). It shares over
60% amino acid identity with MCP-1 and MCP-3 and has about 30%
identity with the CC chemokines MIP-1
, RANTES, and MIP-1
(10-12). MCP-2, similar to MCP-3, is chemotactic for and activates a
wide variety of inflammatory cells, including monocytes, T lymphocytes,
NK cells, basophils, mast cells, and eosinophils (12), but differs from
MCP-1, which is not active on eosinophils (13). We recently reported
that MCP-2 uses CCR1 and CCR2B as its functional receptors (14), which
may account for its action on a greater variety of target cells. In the
course of studies on leukocyte activation by MCP-2, we observed that MCP-2 could competitively inhibit the binding to monocytes of 125I-MIP-1
, a CC chemokine that is believed thus far to
exclusively use CCR5 as a functional receptor on human leukocytes
(15-17). This prompted us to further investigate the effect of MCP-2
on cloned CCR5. We report that MCP-2 is also an efficient ligand for
CCR5 and a potent inhibitor of CD4/CCR5-mediated HIV-1
entry/replication.
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EXPERIMENTAL PROCEDURES |
Chemokines--
Recombinant human (rh) MCP-2 and other
chemokines were purchased from PeproTech Inc. (Rocky Hill, NJ).
Radioiodinated MCP-2 was a kind gift from Dr. G. Brown (NEN Life
Science Products). Other radioiodinated chemokines were purchased from
NEN. All radioiodinated chemokines have a specific activity of 2200 Ci/mmol.
Cells--
Human peripheral blood monocytes were isolated from
normal donors (National Institutes of Health Clinical Center,
Transfusion Department, Bethesda, MD) with an iso-osmotic Percoll
(Pharmacia Biotech Inc.) gradient as described elsewhere (18). The
monocyte preparations were >90% pure. The 293 human embryonic kidney
epithelial cell line stably transfected with FLAG-tagged CCR5
(CCR5/293) was generated and grown in monolayers as described (17).
Binding Assays with Radiolabeled Chemokines--
Binding assays
were performed by using a single concentration of radiolabeled
chemokines in the presence of increasing concentrations of unlabeled
ligands as described previously (14, 18). Cells (2 × 106/sample for monocytes and 1 × 106/sample for CCR5/293 cells) were suspended in 200 µl
of modified binding medium composed of RPMI 1640, 1 mg/ml bovine serum
albumin, 25 mM HEPES, and 0.05% sodium azide and incubated
in duplicates at room temperature for 40 min. After incubation, the
cells were pelleted through a 10% sucrose/phosphate-buffered saline
cushion, and the radioactivity associated with cell pellets was
determined in a
-counter (Clinigamma-Pharmacia, Gaithersburg, MD).
Experiments were also performed at 4 °C in the absence of sodium
azide and yielded similar binding and competition curves as obtained at room temperature. The binding data were then analyzed with a Macintosh computer program LIGAND (P. Munson, Division of Computer Research and
Technology, NIH, Bethesda, MD). The degree of competition for binding
by unlabeled chemokines was calculated as follows: % competition for
binding = 1
(cpm obtained in the presence of unlabeled
ligand/cpm obtained in the absence of unlabeled ligand) × 100.
Chemotaxis Assay--
The migration of HEK 293 cells transfected
with cDNA clones was assessed by a 48-well microchamber technique
(14, 19). Different concentrations of chemokines were placed in the
lower wells of the chamber. The CCR5/293 cells (50 µl, 0.5-1 × 106/ml) were loaded in the upper wells. The lower and upper
wells were separated by a polycarbonate filter
(polyvinylpyrrolidone-free, 10-µm pore size; Poretics, CA) precoated
with 20 µg/ml mouse collagen type IV or 50 µg/ml collagen type 1 for 2 h at 37 °C. The chamber was incubated at 37 °C for
5 h in humidified air with 5% CO2. At the end of the
incubation, after removal of nonmigrating cells, the filter was fixed
and stained with Diff-Quik (Biochemical Sciences, NJ). The cells
migrating across the filter were counted in three high power fields
under light microscopy in triplicates with all samples coded. The
chemotaxis index was calculated as the number of cells migrating to
chemokines/number of cells migrating to medium. The significance of the
difference between test and control groups was analyzed by paired
Student's t test.
Confocal Microscopy--
CCR5/293 cells were pretreated for
3 h at 37 °C with PMA (100 nM), RANTES, IL-8 (120 nM), or different concentrations of MCP-2. The cells were
centrifuged on to slides and permeabilized using 0.15% saponin in
phosphate-buffered saline. The slides were then stained with an
anti-FLAG monoclonal antibody (M1, Kodak, New Heaven, CT) followed by
incubation with FITC-labeled goat anti-mouse IgG F(ab')2
fragments. Slides were examined using a Zeiss 310 Confocal Laser
Scanning Microscope (Carl Zeiss). Nomarski and FITC (488 nm, green)
images were prepared for each specimen, and fluorescent images were
superimposed on Nomarski images.
HIV-1 Inhibition Assay--
CCR5/293 cells were co-transfected
using electroporation with a CD4 expression vector (fragment excised
from pTB4, NIH AIDS Research and Reference Reagent Program, Bethesda,
MD) and cloned into a plasmid vector, pSVZeo (Invitrogen, CA). 24 h after electroporation, the cells (CCR5/CD4/293) were plated at a
density of 105/well in 24-well plate for 24 h. The
culture media were removed, and the cells were preincubated for 30 min
with 800 ng/ml recombinant human MCP-2 or RANTES. The monocyte tropic
HIV-1ADA was added at a final multiplicity of infection
between 1 and 0.1. Cultures were continued for 24 h and washed,
and total genomic DNA was isolated and purified (20). HIV-1
entry/reverse transcription was monitored by polymerase chain reaction
(PCR) using primers specific for HIV-1gag region to detect late reverse
transcription products (21). Two primers are as follows: M667
corresponds to nucleotide positions 496-516 in the
HIV-1JR-CSF sequence; M661 (antisense) corresponds to the
positions 695-672. PCR products were resolved on 2% agarose gels and
photodocumented after ethidium bromide staining. No PCR products were
detected in CCR5/CD4/293 cells infected by the lymphocyte tropic virus
HIV-1RF nor in CCR5/293 cells without CD4 coexpression incubated with
HIV-1ADA.
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RESULTS AND DISCUSSION |
Recombinant human MCP-2 is a potent monocyte chemoattractant and
has been shown to activate at least two promiscuous CC chemokines receptors, CCR1 and CCR2B (14). The binding of 125I-labeled
MCP-2 to human monocytes was of high affinity and was efficiently
competed by MCP-1, which is a ligand for CCR2B. MCP-3, which uses both
CCR1 and CCR2B, also efficiently competed with MCP-2 for binding to
monocytes. MIP-1
and RANTES, two ligands for both CCR1 and CCR5,
partially competed with MCP-2 for binding to monocytes, whereas
MIP-1
, which only uses CCR5, had no effect (14). We further observed
that although unlabeled MIP-1
did not competitively inhibit MCP-2
binding to monocytes, the binding of 125I-MIP-1
was
completely inhibitable by unlabeled MCP-2 (IC50 = 2.9 nM versus 1.7 nM for unlabeled
MIP-1
itself, Fig. 1). In contrast,
the binding of 125I-MIP-1
to monocytes was only
partially but significantly displaced by unlabeled MCP-1 at high
concentrations (IC50 = 425 nM), in agreement
with our earlier observations (22). There was no significant difference
between the competition curves yielded at room temperature (Fig.
1A) and at 4 °C (Fig. 1B). The uni-directional
inhibition of MIP-1
binding to monocytes by MCP-2 prompted us to
investigate whether MCP-2, in addition to using CCR1 and CCR2B as
functional receptors, also uses CCR5. HEK293 cells stably expressing
CCR5 (CCR5/293) showed a high level of specific binding for
125I-MIP-1
that was competitively inhibited by MCP-2
either at room temperature (Fig.
2A) or at 4 °C (Fig.
2B). Unlabeled MIP-1
also completely inhibited
125I-MIP-1
binding (Fig. 2). These results suggest that
MCP-2 has the capacity to interact with CCR5 on HEK293 cells with an
efficacy similar to known CCR5 ligands such as MIP-1
and
MIP-1
.

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Fig. 1.
Competition of 125I-MIP-1
binding to human monocytes by MCP-2. Aliquots of monocytes (2 × 106/200 µl) were incubated with 0.12 nM of
125I-MIP-1 in the presence of different concentrations
of unlabeled chemokines. The binding assays were performed at room
temperature (A) or at 4 °C in the absence of 0.05%
sodium azide (B). Curves shown are from one experiment. Six
experiments were performed yielding similar results. Unlabeled MCP-1 at
10 nM and more showed partial but significant competition
of 125I-MIP-1 binding (p < 0.05 compared with binding in the absence of unlabeled ligand, Student's
t test).
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Fig. 2.
Competition of 125I-MIP-1
binding to CCR5/293 cells by MCP-2. Aliquots of CCR5/293 cells
(1 × 106/200 µl) were incubated with 0.12 nM of 125I-MIP-1 in the presence of
increasing concentrations of unlabeled chemokines. The binding assays
were performed at room temperature (A) or at 4 °C in the
absence of sodium azide (B). Results from one experiment out
of five performed are shown.
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Further support for the ability of MCP-2 to interact with CCR5 was
obtained with binding studies using 125I-MCP-2. As shown in
Fig. 3, 125I-MCP-2
specifically bound to CCR5/293 cells with an estimated Kd in the 5 nM range (Fig.
3A). The binding of 125I-MCP-2 to CCR5/293 cells
was efficiently inhibited by unlabeled MIP-1
and RANTES (Fig.
3B) but less effectively by MCP-1 and MCP-3, which in
contrast were able to completely displace MCP-2 binding to CCR1 and
CCR2B (14). Thus, MCP-2 possesses binding domains for interaction with
CCR5 in addition to CCR1 and CCR2B. This was supported by the
observations that although MCP-1, MCP-2 and MCP-3 are all functional
ligands for CCR2B, the binding of MCP-1 on CCR2B was poorly competed
for by MCP-2 or MCP-3 (14, 23) suggesting differential utilization of
certain binding domains on a receptor by multiple ligands. Because
promiscuity is a common feature of chemokines and their receptors (24,
25) and receptor activation appears to be dependent on the relative
affinity to interact with a ligand, structure analyses and mutagenesis
studies are required to more precisely determine the functional
epitopes on both ligands and receptors.

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Fig. 3.
Binding of 125I-MCP-2 to CCR5/293
cells and competition by CC chemokines. A, Scatchard
analysis. B, Cross-competition. CCR5/293 cells were
incubated with 0.12 nM of 125I-MCP-2 in the
presence of different concentrations of unlabeled MCP-2. The binding
assays were performed at room temperature. The data are from one
representative experiments out of three performed and were analyzed
using the Macintosh computer program LIGAND. MCP-1 and MCP-3 at 10 nM and more showed partial but significant competition of
125I-MIP-1 binding (p < 0.05 compared
with binding in the absence of unlabeled ligand, Student's
t test). B/T, bound/total; B/F, bound/free.
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We next determined the functional role of MCP-2 on CCR5. Both rhMCP-2
and rhMIP-1
are poor Ca2+ mobilizers in monocytes (18,
26), and we were not able to observe significant Ca2+ flux
in CCR5/293 cells with these two ligands, although a RANTES (120 nM)-induced signal was obtained (data not shown). We
therefore utilized chemotaxis assays, which in our previous studies
were demonstrated to be very sensitive and reproducible in assessing the activity of a chemokine on a given receptor (14, 19). CCR5/293
cells showed a significant chemotactic response to MCP-2 and MIP-1
(Fig. 4), yielding a typical bell-shaped
dose response. MCP-2 was similar in potency to MIP-1
with an almost
6-fold increase over medium control at 1.2-6 nM
concentration range, but it was less efficacious (EC50:
MCP-2, 0.12 nM; MIP-1
, 0.04 nM,
respectively) in inducing CCR5/HEK293 cell migration. Other chemokines
known to activate CCR5, such as MIP-1
and RANTES also bound to and induced considerable chemotactic migration of CCR5/293 cells. Although
MCP-1 partially displaced MCP-2 binding to CCR5/293 cells, it only
bound to and induced migration of CCR2B/293 but not CCR5/293 cells
(data not shown). Likewise, MCP-3 also partially displaced MCP-2
binding to CCR5/293 cells; we did not detect significant binding of
125I-MCP-3 to the same cells, nor did it induce migration
of CCR5/293 cells at a wide range of concentrations (data not shown).
Further proof of the activation of CCR5 by MCP-2 is provided by
confocal microscopy (Fig. 5) in which
MCP-2 markedly and dose-dependently induced CCR5
internalization in CCR5/293 cells. Another CCR5 ligand RANTES and PMA,
a potent protein kinase C activator, similarly caused internalization
of CCR5. In contrast, CXC chemokine IL-8, which uses CXCR1 and CXCR2 as
functional receptors, did not induce CCR5 internalization (Fig. 5).

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Fig. 4.
Migration of CCR5/293 cells induced by
MIP-1 and MCP-2. The figure presents results (means ± S.D.) from a typical experiment out of five performed. Chemotaxis Index
values over 2 are statistically significant (p < 0.05)
as evaluated by paired Student's t test, and * indicates
p < 0.001.
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Fig. 5.
Internalization of CCR5 by pretreatment of
CCR5/293 cells with MCP-2. The CCR5/293 cells were treated with
different concentrations of MCP-2 and other reagents and then were
centrifuged on to slides and permeabilized. The slides were stained
with anti-FLAG monoclonal antibody followed by FITC-labeled goat
anti-mouse IgG. Nomarski and FITC images were prepared for each
specimen using a Zeiss 310 Confocal Laser Scanning Microroscope.
Fluorescent images were superimposed on Nomarski images. The images
represent untreated cells (A) and cells treated with IL-8
(B), PMA (C), RANTES (D), and 10-1000
ng/ml MCP-2 (E-G).
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The identification of CCR5 as the principal co-receptor for cellular
entry by monocyte tropic HIV-1 strains (2-5, 8) and the inhibition of
viral fusion and replication by the CCR5 ligands MIP-1
, MIP-1
,
and RANTES (9) led us to question whether MCP-2 similarly could inhibit
HIV-1 entry and replication. The susceptibility to HIV-1 infection of
CCR5/CD4/293 cells was determined in the presence or the absence of
MCP-2 (Fig. 6). Preincubation of
CCR5/CD4/293 cells with either 800 ng/ml MCP-2 or RANTES completely
inhibited HIV-1 entry and subsequent reverse transcription. No reverse
transcription products were detected in CCR5/CD4/293 cells treated with
HIV-1RF, a T lymphocyte tropic strain (data not shown). CCR5/293 cells without CD4 were not infected by HIV-1ADA. Therefore, the
ability of MCP-2 to internalize CCR5 confers on it the ability to
interrupt virus entry, with an antiviral activity equivalent to RANTES. MCP-2 also appears to be a natural inhibitor of CD4/CCR5-mediated HIV-1
entry/replication in host cells in addition to MIP-1
, MIP-1
, and
RANTES (6, 7, 9).

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Fig. 6.
Inhibition of HIV-1ADA
entry/replication in CCR5/CD4/HEK293 cells by MCP-2. CCR5/293
cells coexpressing CD4 were preincubated in the presence or the absence
of chmeokines for 30 min at 37 °C followed by 24 h of
incubation with HIV-1ADA. After washing, cellular genomic
DNA was extracted, and HIV-1 reverse transcription products were
monitored by PCR with primers corresponding to HIV-1gag region. Cells, CCR5/CD4/293 cells without virus; Cells + Virus, CCR5/CD4/293 cells incubated with HIV-1ADA;
RANTES, cells pretreated with 800 ng/ml RANTES then with
HIV-1ADA; MCP-2, cells pretreated with 800 ng/ml
MCP-2 and then with HIV-1ADA; PCR-control,
CEM-SS cells without HIV-1 infection; PCR+control, CEM-SS
cells infected with HIV-1RF. Results are from one
experiment out of three performed.
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MCP-2 is constitutively expressed in tumor cells and is inducible by
pro-inflammatory cytokines in mononuclear cells and fibroblasts (10,
11). MCP-2 exhibits a broader spectrum of targeted cells, including
cells of dendritic phenotype (27). Therefore, MCP-2 may play an
important role in recruiting/activating immune cells at inflammatory
and neoplastic foci. In the present study, we demonstrated for the
first time using cloned CCR5 that MCP-2 is a highly efficacious ligand
for this receptor in addition to CCR1 and CCR2B (14). In our
preliminary study, HEK293 cells transfected with CCR3, which is a
receptor for CC chemokines eotaxin and MCP-4 (28, 29), also were
induced to migrate significantly and reproducibly in response to MCP-2
in addition to eotaxin and MCP-4 (data not shown). Thus, MCP-2 appears
to use CCR1, CCR2B, CCR5, and possibly CCR3, suggesting it has a more
promiscuous functional pattern than other known CC chemokines.
Despite an apparent redundancy in chemokines and their receptor family
(24, 25, 30, 31), CC chemokines have been implicated as important
mediators of many pathological conditions such as chronic inflammation,
immune diseases, neoplasia, and atherosclerosis (30, 31). CCR5 ligands
MIP-1
, MIP-1
, and RANTES are also major HIV-1 inhibitors produced
by activated mononuclear cells (9). Our current observations extend the
functional scope of MCP-2 as a potent inhibitor of monocyte tropic
HIV-1 infection in CD4+/CCR5+ cells.
Investigation into the shared and unique functional domains on MCP-2 in
comparison with other chemokine ligands will be important in the
development of therapeutic approaches to chemokine- and chemokine
receptor-mediated pathological states.
We thank K. Bengali and N. Dunlop for
technical support. Radiolabeled MCP-2 was a kind gift from Dr. G. Brown
of NEN Life Science Products. The secretarial assistance by C. Fogle is
greatly appreciated.