(Received for publication, April 18, 1995; and in revised form, June 23, 1995)
From the
Monocyte chemotactic protein-3 (MCP3) is a recently identified
and molecularly cloned C-C chemokine that is chemotactic for and
activates a great variety of inflammatory cell types. MCP3 has been
reported to interact with several C-C chemokine receptors, which can be
simultaneously or selectively expressed on leukocyte subpopulations. In
order to isolate receptor(s) for MCP3, a cDNA library was constructed
using mRNA from a human NK-like cell line, YT. These cells showed high
affinity binding sites for I-MCP3 and migrated in
response to MCP3. A chemokine receptor cDNA clone, designated YT4, was
sequenced and found to be identical to the known C-C CKR1 or macrophage
inflammatory protein-1
(MIP1
)/Rantes receptor. YT4 cDNA was
subcloned into a mammalian expression vector, and stable transfectants
were prepared using the embryonic kidney cell line 293. The
transfectants (YT4/293) showed high affinity binding for
I-MCP3 in addition to specifically binding
I-MIP1
and
I-Rantes. All three C-C
chemokines were able to cross-compete for binding sites on YT4/293
cells and induced directional migration of YT4/293 cells in
vitro, with MCP3 being the most potent chemoattractant. MCP3,
MIP1
, and Rantes were equally able to cross-attenuate the
migratory response of YT4/293 cells to one another. In contrast, MCP1
and MIP1
had very limited capacity to compete for MCP3 binding on
YT4/293 cells and had only a minor attenuating effect on MCP3-induced
migration. Since MCP3 has been reported to use MCP1 receptor(s), our
results with transfected 293 cells expressing only C-C CKR1 clearly
establish that C-C CKR1 is also a functional receptor for MCP3.
The newly identified C-C chemokine, monocyte chemotactic
protein-3 (MCP3) ()is chemotactic for and activates a
variety of inflammatory cells ((1, 2, 3, 4, 5, 6, 7, 8) ;
reviewed in (9) ). MCP3 has 71% amino acid identity to MCP1,
and both activate monocytes, T cells, and
basophils(1, 2, 3, 4, 5, 6, 8, 10) .
However, MCP3, unlike MCP1, also activates
eosinophils(6, 7) . On the other hand, MIP1
and
Rantes which are about 30% identical to MCP3, both also activate
eosinophils, in addition to
monocytes(6, 7, 8) , T
lymphocytes(10) , and basophils ((6, 7, 8) ; reviewed in Refs. 9, 11, and
12). Based on calcium flux desensitization experiments and competition
by MCP3 for binding of
I-MCP1 and
I-MIP1
on monocytes, MCP3 has been proposed to
interact with the receptors for MCP1, MIP1
, and
Rantes(4, 6, 7, 10) .
Several
chemokine receptors have been cloned and functionally expressed
(reviewed in (13) and (14) ). These include two
receptors for the C-X-C chemokine IL8 (15, 16) and receptors for the C-C chemokines MCP1
(MCP1R; (17) ) and MIP1/Rantes (C-C CKR1; Refs. 18 and
19). All of these receptors belong to the seven-transmembrane,
G-protein-coupled receptor superfamily(13, 14) .
Studies with cells transfected with the MCP1 receptor showed that MCP3
is able to induce calcium flux in cells expressing only the MCP1
receptor(20) . On the other hand, since MCP3 is able to
interact with the receptors for MIP1
and Rantes on
leukocytes(6, 7, 10) , MCP3 has also been
proposed to use the C-C CKR1, which is mainly activated by MIP1
and Rantes(18, 19) . In addition, ongoing studies of
the binding of
I-MCP3 to human monocytes and neutrophils
with
I-MCP3 suggest that MCP3 may also share a receptor
used by MIP1
as well as another unique receptor. (
)Since human leukocytes usually express multiple C-C
chemokine receptors, it is difficult to conclude from the interaction
of MCP3 with normal leukocytes which of these receptors it may be
using. To obtain more definitive data, we have therefore investigated
the binding and signaling of MCP3 through a single receptor. In this
report, by using human embryonic kidney cell line transfected with a
C-C CKR1 cDNA, we show that C-C CKR1 is indeed a functional receptor
shared by MCP3, MIP1
, and Rantes.
In chemotaxis
``attenuation'' experiments, 6 nM (50 ng/ml) of
chemokines were placed in the lower wells of the chamber, and the cells
were placed in the upper wells in the presence of 6 nM chemokines. The assays were performed as described above. After
subtraction of the background migration (to medium alone), the
percentage of inhibition was calculated as 1 - (migration of
cells incubated with chemokines/migration of cells incubated with
medium) 100.
The significance of the difference between test and control groups was analyzed using Student's t test.
Figure 1:
Binding of YT4/293 cells for
radiolabeled MIP1 and Rantes. Aliquots of YT4/293 cells were
incubated with
I-MIP1
(A) or
I-Rantes (B) in the presence of increasing
concentrations of unlabeled chemokines. One representative experiment
out of five performed is shown.
, MIP1
;
, MIP1
;
, Rantes;
, MCP1. LOG(T), concentration of
unlabeled chemokines.
YT4/293 cells also specifically bound I-MCP3 with 21,000 (± 2400) sites/cell with high
affinity (K
= 0.7 nM) (Fig. 2A). The binding affinity of
I-MCP3
was consistently higher (0.5-1.8 nM) than that obtained
with
I-MIP1
(5-9 nM, Fig. 2B) on these cells, suggesting that MCP3 may have
a better interaction with C-C CKR1 than MIP1
. These results
suggest that C-C CKR1 is also a receptor for MCP3.
Figure 2:
Scatchard analyses of YT4/293 cell binding
for I-MCP3 and
I-MIP1
. YT4/293 cells
were incubated with
I-MCP3 (A) or
I-MIP1
(B) in the presence of increasing
concentrations of unlabeled MCP3 or MIP1
, respectively. Data from
one representative experiment out of five performed are shown. Insets, Scatchard plots of the binding data. LOG(Total), concentration of unlabeled
chemokines.
Figure 3:
Competition for binding of C-C chemokines
on YT4/293 cells by unlabeled MCP3. YT4/293 cells were incubated with
radiolabeled chemokines MIP1 (A), Rantes (B), or
MIP1
(C) in the presence of unlabeled chemokines at
increasing concentrations. Five experiments were performed with similar
results.
, MCP3;
, Rantes;
, MIP1
and MIP1
in panelsA and C, respectively. LOG(T), concentration of unlabeled
chemokines.
Figure 4:
Competition of I-MCP3
binding on YT4/293 cells by unlabeled chemokines. YT4/293 cells were
incubated with
I-MCP3 in the presence of increasing
concentrations of unlabeled chemokines. Five experiments were performed
with similar results.
, MCP3;
, MIP1
;
,
MIP1
;
, Rantes;
, MCP1. LOG(T),
concentration of unlabeled chemokines.
Figure 5:
Migration of YT4/293 cells in response to
chemokines. Different concentrations of the chemokines MCP3 (A), MIP1 (B), Rantes (C), and
MIP1
(D) were placed in the lower wells of the chemotaxis
chamber; the YT4/293 cells resuspended in medium were placed in the
upper wells. The upper and lower wells were separated by a
polyvinylpyrrolidone-free polycarbonate filter precoated with Collagen
type IV. The data were expressed as chemotaxis indices and were the
mean (± S.E.) values of three to five experiments performed. *, p < 0.002;**, p < 0.05, compared with migration
in response to control medium.
MCP3 activates a variety of inflammatory cells through a
number of different binding sites for C-C chemokines expressed on these
cells. Previous studies based on desensitization of calcium flux
predicted that MCP3 would be able to interact with at least three types
of receptors: 1) MCP1 receptor on monocytes and
basophils(4, 6, 7) , 2) selective Rantes
receptor on basophils and eosinophils(6, 7) , and 3)
selective MIP1 receptor on basophils, eosinophils, and neutrophils (6) . Results obtained from binding studies using
I-MCP1 and
I-MIP1
on monocytes
suggested that MCP3 may also interact with C-C CKR1, the
MIP1
/Rantes receptor. By using 293 cells transfected with the MCP1
receptor, it has been shown that MCP3 was able to signal through MCP1
receptor(20) . However, all of these observations were based on
cross-desensitization of calcium mobilization between MCP3 and other
C-C chemokines or on the ability of MCP3 to compete for
I-MCP1 and
I-MIP1
binding. No data
concerning direct binding of radiolabeled MCP3 to native cells or to
cells transfected with cloned receptors has been reported. Recently, by
using
I-MCP3, we have characterized the binding behavior
of MCP3 on human monocytes and neutrophils and provided evidence that
in addition to interacting with binding sites for MCP1, MIP1
, and
Rantes, MCP3 also interacts with a binding site for MIP1
as well
as with as yet undefined unique site(s) not shared by other C-C
chemokines.
Since human leukocytes usually express
multiple C-C chemokine receptors, they exhibit a complex pattern of
shared as well as selective utilization of the receptors by different
C-C chemokines (4, 5, 6, 7, 8, 10, 25, 27) .
In order to more precisely evaluate the C-C receptor promiscuity, it is
crucial to examine the binding and signaling of each C-C chemokine
through a single isolated receptor. Using this approach, cells
transfected with the MCP1 receptor clearly were shown to bind and
signal in response to MCP1 but not to MIP1, MIP1
, or
Rantes(17) . It was subsequently shown that MCP3 was also able
to induce calcium flux in MCP1 receptor transfectants confirming the
use of the MCP1 receptor(20) .
In an effort to characterize
the binding sites for MCP3 on human cell lines, we observed that YT
cells exhibited a high level of specific binding for I-MCP3. The
I-MCP3 binding on YT cells was
progressively less well competed for by unlabeled MCP3, Rantes,
MIP1
, MIP1
, and MCP1, with MCP1 being the least potent
competitor for MCP3 binding. No
I-MCP1 binding was
detected on YT cells. The YT cells also migrated in response to various
C-C chemokines but not to MCP1. Thus the binding and biological
activity of MCP3 on YT cells was separable from that of MCP1.
A cDNA library was therefore constructed with the mRNA extracted
from YT cells, and several cDNA clones with seven-transmembrane domain,
G-protein-coupled receptor features were obtained. One of these clones
was identical to the C-C CKR1(18) , known as MIP1
/Rantes
receptor(19) , which has been shown to be mainly activated by
MIP1
and Rantes, although MCP1 and MIP1
could also weakly
signal through C-C CKR1. Since it was proposed that MCP3 might interact
with receptors for MIP1
and
Rantes(6, 7, 10) , we decided to examine this
possibility by using cells transfected to express only C-C CKR1 binding
sites. Our binding and functional studies with YT4/293 cells confirmed
that C-C CKR1 was very well activated by MIP1
and Rantes.
Furthermore, our study demonstrated that C-C CKR1 exhibited even higher
affinity binding for
I-MCP3. MCP3 was also the most
potent C-C chemokine in inducing the migration of C-C CKR1-transfected
cells.
MCP3 not only exhibited better binding and activation of
cells transfected with C-C CKR1, it also competed more effectively for I-Rantes and
I-MIP1
binding on C-C
CKR1 than unlabeled Rantes and MIP1
. This may be attributable to
the possibility that MCP3 may have more binding domains for C-C CKR1
than either Rantes or MIP1
, or that MCP3 may have a better
interaction with the same number of domains. Studies with IL8 and its
receptors have shown that more than one site either on the ligand or on
the receptor are responsible for the binding and
signaling(28, 29) . Accordingly, studies with mutants
derived from MCP3 ligand and cloned C-C chemokine receptors may provide
more information about the greater capacity of MCP3 to interact with
multiple C-C chemokine receptors. It will also be interesting to
investigate whether MCP3 can interact with two recently identified
promiscuous chemokine receptors, Duffy Antigen (30, 31) and the cytomegalovirus DNA open reading
frame, US28(19, 32) .
Overall, we have shown that
293 embryonic kidney cells stably transfected to express C-C CKR1,
known as MIP1/Rantes receptor, bind
I-MCP3 with
higher affinity (0.5-1.8 nM) than the affinity for
I-MIP1
(5-9 nM). MCP3, of all the C-C
chemokines tested, was the most potent chemoattractant for C-C
CKR1-transfected 293 cells (YT4/293 cells). Since MCP3 acts on a
variety of inflammatory cells and utilizes multiple receptors for its
function, characterization and isolation of the shared as well as
unique receptors for MCP3 will provide further insights into the
pathophysiological roles of MCP3. C-C chemokines are mediators of a
number of pathological conditions such as chronic inflammation, tumor,
allergy, and atherosclerosis (reviewed in Refs. 9, 11, and 12). Since
the binding and signaling of MCP3 is most promiscuous, the development
of compounds or peptides that can block MCP3 binding to the receptor
may prove to be useful in the treatment of diseases mediated by a
number of C-C chemokines.