(Received for publication, October 23, 1996, and in revised form, March 10, 1997)
From the Intramural Research Support Program and
§ Clinical Immunology Services, Monocyte chemotactic protein (MCP)-2 is a member
of the C-C chemokine subfamily, which shares more than 60% sequence
homology with MCP-1 and MCP-3 and about 30% homology with macrophage
inflammatory protein (MIP)-1 Monocyte chemotactic protein (MCP)1-2
is a C-C chemokine co-purified with MCP-1 and MCP-3 from human
osteosarcoma cells (1, 2). It shares over 60% amino acid identity with
MCP-1 and MCP-3 and has about 30% identity with other C-C chemokines
MIP-1 Recombinant human MCP-2 and other
chemokines, including Escherichia coli-derived MCP3, MCP1,
MIP1 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., Uppsala, Sweden) gradient as described
elsewhere (17). Elutriated monocytes (kind gifts from Dr. I. Espinoza-Gaetano, NCI) were also used in the study. The monocyte
preparations were >90% pure as assessed by morphological
criteria.
The CCR1 cDNA clone was isolated as described previously from a
natural killer-like cell line YT (12). The CCR2B cDNA was a kind
gift provided by Dr. I. F. Charo (San Francisco, CA; Ref. 15). The 293 human embryonic kidney epithelial cell line (HEK 293, a gift from Dr.
P. Gray, ICOS Corp., Bothell, WA) was grown in monolayers in
Dulbecco's modified Eagle's medium (BioWhittaker, Inc., Walkersville,
MD) with 10% fetal calf serum (Hyclone Laboratories, Logan, UT) and
antibiotics. The HEK 293 cells were either transfected with 5-20 µg
of CCR1 cDNA by using DOTAP transfection reagent (Boehringer
Mannheim) or with CCR2B cDNA by electroporation (1 × 107 cells, 950 microfarads, 0.2 KV). Stable transfectants
were established by adding G418 (800 µg/ml; Life Technologies, Inc.)
to maintain selection pressure and were designated CCR1/293 or
CCR2B/293 cells.
Binding assays
were performed by using a single concentration of
125I-labeled chemokines in the presence of increasing
concentrations of unlabeled ligands as described previously (12, 19).
The binding data were then analyzed with a Macintosh computer program LIGAND (P. Munson, Division of Computer Research and Technology, NIH,
Bethesda, MD). The rate of competition for binding by unlabeled chemokines was calculated as follows: % competition for binding = 1 Monocyte migration was evaluated by using
a 48-well microchamber technique as described (12, 18, 19). The
migration of HEK 293 cells transfected with cDNA clones was also
assessed by a 48-well microchamber technique (12). Data were presented as the chemotaxis index and were the representative of at least five
experiments performed. The chemotaxis index was calculated by:
chemotaxis index = 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 using the Student's t test.
Recombinant human MCP-2 (PeproTech, lot number 85411) was highly
effective in inducing human monocyte migration in vitro at an optimal concentration range of 6-12 nM (50-100 ng/ml)
(data not shown). This recombinant human MCP-2 did not induce
significant calcium mobilization in monocytes at concentrations below
60 nM (500 ng/ml) (not shown) as reported previously (3,
6), even though it did induce calcium flux at concentrations of 6-12
nM in a cultured murine T lymphoma cell line (data not
shown). 125I-MCP-2, prepared using the recombinant protein
of the same lot number, induced monocytes chemotaxis comparable with
that of the unlabeled ligand, indicating that radioiodination did not
significantly change the molecular structure of the MCP-2 and its
capacity to interact with receptors. It is therefore possible to
examine whether human leukocytes express specific binding sites for
MCP-2 and whether these binding sites are shared by multiple C-C
chemokines as suggested by various investigators (6-9). Fig.
1A shows a typical binding profile of
125I-MCP-2 to monocytes with an estimated
Kd value of 2 nM. The binding of
125I-MCP-2 to monocytes was efficiently displaced by MCP-2
itself, and also by MCP-1 and MCP-3 (Fig. 1B), two C-C
chemokines with high degree of homology with MCP-2 (1-3). MIP-1
To identify receptors for MCP-2, we examined the binding and function
of recombinant MCP-2 to cells transfected to express C-C chemokine
receptors, including CCR1, which is a common receptor for MCP-3 (12,
26) and MIP-1
Our efforts to obtain reproducible calcium mobilization in chemokine
receptor-transfected 293 cells were not successful. We therefore
utilized chemotaxis assays as a functional indicator, which has been
shown to be a specific, sensitive, and physiologically relevant method
of assessing the action of chemokines on their receptors (12, 20, 21).
Fig. 3A shows that MCP-2 induced significant
migration of CCR1/293 cells in vitro. The MCP-2
concentrations required to elicit optimal cell response ranged from 6 to 12 nM (50-100 ng/ml), comparable with those of MIP-1
The capacity of MCP-2 to compete for MCP-1 binding sites and
cross-desensitize one another on native leukocytes pointed to the
possibility that MCP-2 also interacted with MCP-1 binding sites (6-9).
This is supported by the binding data of 125I-MCP-2 to
monocytes obtained in this study. Previous study showed no effect of
MCP-2 on CCR2B/293 cells based on the inability of MCP-2 to induce
calcium flux in these cells and to desensitize the effect of MCP-1 (16,
26). However, in this study, we observed that 125I-MCP-2
did specifically bind to CCR2B/293 cells (3 ± 1 nM,
Fig. 4A), although the binding affinity of
125I-MCP-2 to CCR2B/293 cells was relatively lower than
that of 125I-MCP-1 (0.5 ± 0.1 nM, Fig.
4B). 125I-MCP-2 binding was fully displaced by
both MCP-1 and MCP-3 (Fig. 4C), but only slightly by
MIP-1
We further examined the function of MCP-2 on CCR2B/293 cells by its
capacity to induce directional cell migration. CCR2B/293 cells showed
highly reproducible and potent chemotactic responses to MCP-2 as well
as to MCP-1 and MCP-3 (Fig. 3B). Furthermore, the
chemotactic activity of these three MCPs for CCR2B/293 cells was
attenuatated by each of the ligands (not shown), in agreement with
previously reported results obtained in monocytes (6). However, at
least 5-fold more MCP-2 was required to completely attenuate the
migration of CCR2B/293 cells to MCP-1, in correlation with the binding
competition pattern (see above). Thus our binding and chemotaxis data
indicate that CCR2B is also a functional receptor for MCP-2 and the
functional domains on CCR2B differentially interact with MCP-2 and
MCP-1.
The significance of MCP-2 production in pathophysiological conditions
has yet to be defined (3). MCP-2 is constitutively expressed in tumor
cells and is inducible by proinflammatory cytokines in mononuclear
cells and fibroblasts (1-3). MCP-2, like MCP-3, exhibits a broader
spectrum of targeted cells, including cells of dendritic phenotype as
we recently reported (22). 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 with
cloned CCR1 and CCR2B that MCP-2 is a functional ligand for at least
these two receptors. Other data based on native cells suggested the
possible existence of additional receptors used by MCP-2 (7, 8). This
possibility was also evidenced by our experiments with a murine T
lymphoma cell line, in which MCP-2 totally desensitized MCP-1-, MCP-3-, as well as MIP-1 Although there is an apparent redundancy in the binding and function of
chemokine and receptor family (for reviews, see Refs. 11, 23, and 24),
C-C chemokines have been implicated to be important mediators of many
pathological conditions such as chronic inflammation, immune diseases,
neoplasia, and atherosclerosis (11, 23, 24). Recently, several
chemokine receptors have been reported to function as HIV-1 fusion
co-factors, and some chemokines were able to interfere with the viral
replication (reviewed in Ref. 25). Investigation into the shared and
unique functional domains on both chemokine ligands and receptors will
prove important in the development of therapeutic approaches to
chemokine- and chemokine receptor-mediated pathology.
We thank Dr. Joost J. Oppenheim for his
critical review of this manuscript and Dr. I. F. Charo (Gladstone
Institute of Cardiovascular Disease, San Francisco, CA) for providing
us with the CCR2B cDNA. The radiolabeled MCP-2 was a kind gift from
Dr. G. Brown of the DuPont NEN. The technical support from K. Bengali
and the secretarial assistance by Ms. T. Covell and Ms. C. Fogel are
gratefully appreciated. X. G. was supported in part by a fellowship
from The Office of the International Affairs, NCI.
Laboratory of Molecular Immunoregulation,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, regulated on activation of normal T
cell expressed (RANTES), and MIP-1
. Despite this considerable
sequence homology to other C-C chemokines, MCP-2 appears to have unique
functional properties in comparison with other C-C chemokines such as
MCP-1 and MCP-3. Although evidence obtained from studies on leukocytes suggested that MCP-2 may share the receptors with these C-C chemokines, the actual functional receptors for MCP-2 have not yet been identified. In this study, by using radioiodinated MCP-2, we identified high affinity binding sites for MCP-2 on human peripheral blood monocytes. The MCP-2 binding was competed for by MCP-1 and MCP-3, but less well by
MIP-1
or RANTES. In experiments using cells transfected with C-C
chemokine receptors, 125I-MCP-2 bound to human
embryonic kidney 293 cells transfected with CCR1 or CCR2B, known to
also bind MIP-1
/RANTES and MCP-1, respectively, but both shared by
MCP-3. The binding of 125I-MCP-2 to these
receptor-transfected cells was displaced completely by chemokines that
bind to these receptors. Both CCR1- and CCR2B-transfected 293 cells
showed significant migration in response to MCP-2, in addition to
responding to other specific chemokines. These results clearly
demonstrate that MCP-2, unlike MCP-1, uses both CCR1 as well as CCR2B
as its functional receptors, and this accounts for the unique
activities of MCP-2.
, RANTES, and MIP-1
(1, 2 and reviewed in Ref. 3). MCP-2 is
chemotactic for and activates a wide variety of inflammatory cells, and
its spectrum of action on leukocytes is similar to that of MCP-3, including monocytes, T lymphocytes, NK cells, basophils, mast cells,
and eosinophils (1, 3-9), but differs from MCP-1, which is not active
on eosinophils (10). Based on cross-desensitization of calcium
mobilization in leukocyte subsets by different chemokines, and the
competition by MCP-2 for binding sites of MCP-1 and MIP-1
on
monocytes, MCP-2 has been proposed to interact with multiple C-C
chemokine receptors, including those used by MCP-1 and MCP-3 (6, 7).
However, since leukocytes express a multiplicity of chemokine receptors
with promiscuous binding and functional properties (reviewed in Ref.
11), it is difficult to identify the receptors used by a given ligand
on these cells. A number of C-C and C-X-C chemokine receptors have been
cloned and functionally expressed on non-leukocytic cells. It has been
shown that CCR1 is a receptor for MCP-3 and MIP-1
/RANTES (12-14,
26), while CCR2B is shared mainly by MCP-1 and MCP-3 (15, 16, 26). Although CCR2B has been reported not to be functional for MCP-2 (16,
26), the conclusion was derived from the failure of synthetic MCP-2 to
induce calcium mobilization in CCR2B transfected HEK 293 cells and the
inability of MCP-2 to desensitize MCP-1 induced calcium flux (16, 26).
Since MCP-2 was a poor inducer of calcium mobilization in monocytes
(6), and its chemotactic activity for monocytes was not Pertussis
toxin-sensitive, MCP-2 was proposed to use signaling pathways distinct
from other C-C chemokines (6). Despite this, MCP-2 was effective in
attenuating the MCP-1-induced monocyte migration, through homologous
deactivation, suggesting the utilization of the same MCP-1 receptor
domains by MCP-2 (6). However, this cannot be clearly established using
native cells, but requires the use of cells transfected to express only
a single chemokine receptor. In this study, by determining the binding properties of radiolabeled MCP-2 and performing sensitive chemotaxis assays to assess receptor functions, we demonstrate that CCR1 and CCR2B
are both functional receptors for MCP-2.
Chemokines
, MIP1
, and RANTES were purchased from PeproTech Inc. (Rocky
Hill, NJ). Radioiodinated MCP-2 was a kind gift from Dr. G. Brown
(DuPont NEN). Other radioiodinated chemokines were purchased from
DuPont NEN. All radioiodinated chemokines have a specific activity of
2200 Ci/mmol.
(counts/min obtained in the presence of unlabeled ligand/cpm obtained in the absence of unlabeled ligand) × 100%.
and
RANTES, which share about 30% homology with MCP-2, only partially
displaced 125I-MCP-2 binding (Fig. 1B). MIP-1
and C-X-C chemokine IL-8 did not compete for 125I-MCP-2
binding sites on monocytes. Our data obtained with monocytes support
the suggestion that MCP-2 interacts with receptors shared by multiple
C-C chemokines (6-9).
Fig. 1.
Binding of radiolabeled MCP-2 to human
monocytes. Monocytes were incubated with a fixed concentration of
radiolabeled MCP-2 (120 pM) in the presence of increasing
concentrations of unlabeled ligand. The radioactivity associated with
the cells were analyzed using the LIGAND program and plotted as shown
in A. B, displacement of 125I-MCP-2
binding to monocytes by unlabeled chemokines. , MCP-2;
, MCP-1;
, MCP-3;
, MIP-1
;
, RANTES.
[View Larger Version of this Image (0K GIF file)]
/RANTES (13, 14), and CCR2B, which is shared by MCP-1
and MCP-3 (15, 16, 26). The parental HEK 293 cells and cells
transfected with vector alone did not bind 125I-MCP-2, nor
did they migrate in response to any of the C-C chemokines, including
MCP-2 (Ref. 12 and data not shown). HEK 293 cells stably transfected
with CCR1 (CCR1/293 cells) showed a high level of specific binding for
125I-MCP-2 (Fig. 2A) and also
bound radiolabeled MIP-1
(Fig. 2B) and MCP-3 (Ref. 12).
The binding affinity of 125I-MCP-2 for CCR1/293 cells is
comparable with 125I-MIP-1
(Kd = 5 ±2 nM and 4 ± 0.8 nM, respectively, Fig.
2, A and B), but lower than
125I-MCP-3 (Kd = 0.7 nM,
Ref. 12). Cross-competition experiments, as shown in Fig.
2C, indicate that MCP-3, MIP-1
, and RANTES are all
effective competitors for 125I-MCP-2 binding to CCR1/293
cells with IC50 values of about 5 nM,
comparable with MCP-2 itself. MIP-1
had very limited effect. To our
surprise, MCP-1, which was a weak competitor for MCP-3, or
MIP-1
/RANTES binding to CCR1/293 cells (12, 13), displaced rather
effectively the 125I-MCP-2 binding to CCR1/293 cells (Fig.
2C), suggesting MCP-2 may share certain binding domains on
CCR1 that react with MCP-1. In fact, MCP-1 at high concentrations did
induce signaling in CCR1 (13) and partially displaced MCP-3 binding
(12). Unlabeled MCP-2 fully displaced radiolabeled MIP-1
and RANTES
binding to CCR1/293 cells (not shown). However, MCP-2 was a less potent
competitor for binding of radiolabeled MCP-3 to CCR1/293 cells with an
IC50 10 times higher than that of MCP-3 itself (15 nM versus 1.5 nM, not shown), in
agreement with the relatively lower binding affinity of MCP-2 to
CCR1/293 cells than MCP-3.
Fig. 2.
Binding of radiolabeled MCP-2 and MIP-1 to
HEK 293 cells transfected with CCR1 (CCR1/293 cells). A and
B, binding isotherms of 125I-MCP-2
(A) and 125I-MIP-1
(B). C,
displacement of MCP-2 binding to CCR1/293 cells by unlabeled
chemokines.
, MCP-2;
, MCP-3;
, MCP-1;
, MIP-1
;
,
RANTES; ×, MIP-1
.
[View Larger Version of this Image (0K GIF file)]
and RANTES (12), but higher than effective MCP-3 concentrations (12). A
complete cross-attenuation of migration of CCR1/293 cells was achieved by placing equal concentrations of MCP-2, MCP-3, or MIP-1
in upper
and/or lower wells of the chemotaxis chamber (not shown).
Fig. 3.
Migration of CCR1/293 (A) and
CCR2B/293 (B) cells induced by chemokines. The figure
presents results from a typical experiment out of eight performed.
Chemotaxis index values over 2 are statistically significant
(p < 0.05).
[View Larger Version of this Image (0K GIF file)]
(Fig. 4C), not by RANTES or MIP-1
(not shown).
Unlabeled MCP-2 was able to completely displace the binding of
125I-MCP-3 on CCR2B/293 cells with slightly higher
IC50 (5 versus 3 nM) than MCP-3
itself. However, unlabeled MCP-2 appeared to be a poor competitor
(IC50 > 50 nM) for 125I-MCP-1
binding to CCR2B/293 cells. This may be explained by a lower affinity
of MCP-2 binding to CCR2B/293 cells than MCP-1. Or alternatively, there
exists differential occupancy of binding domains on CCR2B by MCP-1 and
MCP-2. Studies on the binding pattern of C-X-C chemokines, IL-8, and
NAP-2, on IL-8 receptor type B (CXCR2) showed that IL-8 completely
displaced NAP-2 binding, whereas IL-8 binding was only partially
displaced by NAP-2 (21), even though both ligands are functional for
CXCR2. Computer modeling suggested that IL-8 possesses multiple binding
domains, only some of these may be shared by NAP-2 (21).
Fig. 4.
Binding of radiolabeled MCP-2 and MCP-1 to
CCR2B/293 cells. A and B, binding isotherms of
125I-MCP-2 (A) and 125I-MCP-1
(B). C, displacement of 125I-MCP-2
binding to CCR2B/293 cells by unlabeled chemokines. , MCP-2;
,
MCP-3;
, MCP-1;
, MIP-1
.
[View Larger Version of this Image (0K GIF file)]
-induced calcium flux but not the vice
versa.2 Efforts are being made to identify
additional receptors for MCP-2. Although MCP-2 is a poor
Ca2+ flux inducer in human monocytes (Refs. 3 and 6 and
data not shown), we were able to obtain significant Ca2+
flux in murine T lymphoma cells induced by MCP-2, suggesting that
different cell types may exhibit agonist-dependent
Ca2+ signaling capacity, but the end result in terms of
cell migration is the same. Our current data obtained with CCR2B/293
cells again show that chemotaxis assay provides a powerful approach to
defining the ligand-receptor interaction with 293 cells transfected to express a single chemokine receptor, as we have reported previously for
both C-C and C-X-C chemokines (12, 20, 21).
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: IRSP,
SAIC-Frederick, NCI-FCRDC, Bldg. 560, Rm. 31-19, Frederick, MD 21702. Tel.: 301-846-1347; Fax: 301-846-7042.
1
The abbreviations used are: MCP, monocyte
chemotactic protein; MIP, macrophage inflammatory protein; CCR, C-C
chemokine receptor; IL, interleukin.
2
J. M. Wang, S. Sozzani, and D. B. Kuhns,
unpublished observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.