(Received for publication, July 20, 1995; and in revised form, August 17, 1995)
From the
The CC chemokine monocyte chemoattractant protein-3 (MCP-3)
activates human monocytes, lymphocytes, basophils, and eosinophils.
MCP-3 has been reported to induce
[Ca]
changes in cells
transfected with the monocyte-selective MCP-1 receptor 2B (CC CKR2B)
and competes for
I-MCP-1 binding on CC CKR2B, suggesting
that it may mediate monocyte responses to MCP-3. However, we now show
that MCP-3 is a ligand and potent agonist for the macrophage
inflammatory protein-1
(MIP-1
)/regulated on activation,
normal T expressed, and secreted protein (RANTES) receptor CC CKR1
(rank order for [Ca
]
changes = MIP-1
> MCP-3 > RANTES), which
is expressed in monocytes > neutrophils > eosinophils.
I-MCP-3 bound directly to CC CKR1 and CC CKR2B (K
= 8 and 7 nM,
respectively). Binding to CC CKR1 was competed by all CC chemokines
tested except MCP-1. In contrast, binding to CC CKR2B was
competed only by MCP-3 and MCP-1. Both MCP-1 and MCP-3 were
equipotent agonists (EC
= 10 nM for
[Ca
]
changes). Thus,
MCP-3 is a functional ligand for both CC CKR1 and CC CKR2B, which
otherwise have distinct selectivities for CC chemokines. These data
suggest that monocyte responses to MCP-3 could be mediated by both CC
CKR2B and CC CKR1, whereas eosinophil responses to MCP-3 could be
mediated by CC CKR1.
The chemokine superfamily is a growing group of 8-10-kDa cytokines with relatively selective leukocyte chemoattractant activity (reviewed in (1) and (2) ). Chemokines form three subfamilies based on the number and arrangement of conserved cysteine residues. CC and CXC chemokines contain four conserved cysteines; the first two cysteines are separated by one amino acid in the case of CXC chemokines and are adjacent in the case of CC chemokines. Lymphotactin is a mouse thymocyte chemoattractant related to chemokines. However, it has only the second and fourth conserved cysteines and may be the first example of a new ``C'' chemokine subfamily(3) .
Whereas most CXC chemokines attract neutrophils, most CC chemokines
do not(1, 2) . Instead, CC chemokines are all monocyte
chemoattractants in vitro and have variable selectivity for
lymphocytes, basophils, and eosinophils. MCP-3 ()is a
particularly interesting CC chemokine originally purified from
osteosarcoma cell cultures(4) ; its cDNA has recently been
cloned(5) . When compared to other CC chemokines, the amino
acid sequence of MCP-3 is 71% identical to MCP-1, whereas it is only
25% identical to RANTES and 30% identical to MIP-1
. MCP-3 is one
of the most broadly active chemokines, potently inducing chemotaxis of
monocytes, basophils, eosinophils, and lymphocytes, as well as
degranulation of basophils, eosinophils, and
monocytes(4, 6, 7, 8, 9, 10, 11) .
The leukocyte receptors mediating MCP-3 responses have not yet been
fully defined. Sozzani et al.(7) have shown that
MCP-3 will compete for I-MCP-1 binding to monocytes,
suggesting a shared MCP-1/MCP-3 receptor. Uguccioni et al.(6) have shown that MCP-3 completely displaces
I-MIP-1
from monocytes, whereas RANTES has a lesser
effect and MCP-1 has little effect, suggesting a separate shared
MIP-1
/ RANTES/MCP-3 receptor on monocytes. By inspecting
cross-desensitization patterns for chemokine-induced calcium
transients, Dahinden et al.(8) have proposed that
MCP-3 activates three chemokine receptor subtypes on basophils and
eosinophils: 1) an MCP-1/MCP-3 receptor expressed on basophils, 2) a
RANTES/MCP-3 receptor on basophils and eosinophils, and 3) a shared
receptor for MIP-1
, RANTES, and MCP-3 on eosinophils. Van Riper et al.(12) have identified a shared functional
binding site for MIP-1
and RANTES on HL-60 cell-derived
eosinophil-like cells, but MCP-3 binding and MCP-3 responsiveness has
not yet been reported for these cells. Loetscher et al.(10) reported that MCP-1 completely desensitizes
[Ca
]
changes induced
by MCP-3 in CD4
T lymphocytes, again suggesting the
possibility of a shared receptor for these chemokines.
cDNAs for
three human leukocyte 7-transmembrane domain CC chemokine receptors
have been cloned: CC CKR1, CC CKR2A, and CC
CKR2B(13, 14, 15) . CC CKR1 has been shown
previously to be selective for MIP-1 and
RANTES(13, 14) ; its RNA is detectable in monocytes
> neutrophils > eosinophils(16) . Three of us recently
reported a human eosinophil CC chemokine receptor-like cDNA named CC
CKR3(17) . However, we have discovered that the cells whose
functional properties were described in this paper were not transfected
with the CC CKR3 cDNA as was claimed, but were inadvertently
transfected instead with another CC chemokine receptor cDNA designated
CC CKR5 ( (17) and correction). Thus, the agonists for CC CKR5
are MIP-1
, MIP-1
, and RANTES. (
)The only agonist
found so far for CC CKR3 is human eotaxin, an eosinophil-specific CC
chemokine. (
)CC CKR2A and CC CKR2B were originally shown to
be selective for MCP-1 by measuring calcium mobilization in Xenopus oocytes(15) . Subsequently, Franci et al.(18) showed that
I-MCP-1 binds to CC CKR2B,
and binding was competed by both MCP-1 and MCP-3 but not by other CC
chemokines(19) . Moreover, MCP-3 induced
[Ca
]
changes in cells
transfected with CC CKR2B. We have shown that a probe that recognizes
both CC CKR2A and CC CKR2B identifies 3.5-kb RNA in human monocytes but
not neutrophils or eosinophils(16) . We have also reported
preliminary evidence suggesting that MCP-3 is an agonist for CC CKR1;
however, its potency and direct binding capacity were not
known(17) . Here, we report that MCP-3 is a high affinity
ligand and high potency agonist for CC CKR1, and we provide a detailed
comparative characterization of its MCP-3 binding site with that on CC
CKR2B.
Figure 1:
Identification of
functional MCP-3 receptors. A, agonists for CC chemokine
receptors. [Ca]
was
monitored by ratio fluorescence of FURA-2-loaded HEK 293 cells stably
transfected with plasmids containing the cDNAs indicated at the top of each column. Cells were stimulated at the time
indicated by the arrows with 100 nM of the chemokine
indicated at the left of each row of tracings. The
tracings shown are from one experiment representative of at least three
experiments with one clone for CC CKR1 and two separate clones for CC
CKR2B. B and C, agonist potency for CC CKR1 and CC
CKR2B, respectively. The magnitude of the peak of the calcium transient
elicited by the indicated concentration of human chemokines from HEK
293 cells stably expressing CC CKR1 or CC CKR2B is shown. Each data
point represents the peak of one tracing. The data are from a single
experiment representative of at least three separate
experiments.
Fig. 1, B and C, show the
CC chemokine selectivity for each MCP-3 receptor. The rank orders of
potency were as follows: for CC CKR1, MIP-1 > MCP-3 >
RANTES; for CC CKR2B, MCP-1 = MCP-3 (EC
values
= 1 nM for MIP-1
, 10 nM for MCP-3, and 30
nM for RANTES (for CC CKR1) and 10 nM for both MCP-1
and MCP-3 (for CC CKR2B)).
Figure 2: Desensitization of calcium transients in HEK 293 cells stably transfected with CC CKR1. Ratio fluorescence was monitored from FURA-2-loaded cells before and during sequential addition of chemokines at 100 nM at the times indicated by the arrows. The first stimulus added (S1) for each tracing is indicated at the left of the row in which it is found. The second stimulus added (S2) is indicated at the top of the column in which the tracing is found. The tracings are from a single experiment representative of three separate experiments.
In the case of CC CKR2B, an initial
exposure to 100 nM MCP-1 abolished the response to a second
stimulation with MCP-1 and MCP-3 (Fig. 3). MCP-3 abolished the
response to a second stimulation with MCP-3 but only partially affected
the response to MCP-1. MIP-1 had no effect on the CC CKR2B
responses to MCP-1 and MCP-3. These results provide additional evidence
for a functional interaction of MCP-3 with both receptors, CC CKR1 and
CC CKR2B.
Figure 3: Desensitization of calcium transients in HEK 293 cells stably transfected with CC CKR2B. Ratio fluorescence was monitored from FURA-2-loaded cells before and during sequential addition of chemokines at 100 nM at the times indicated by the arrows. The first stimulus added (S1) for each tracing is indicated at the left of the row in which it is found. The second stimulus added (S2) is indicated at the top of the column in which the tracing is found. The tracings are from a single experiment representative of three separate experiments.
To directly assess the ability of MCP-3 to
bind to CC CKR1 and CC CKR2B and to investigate the relationship of
MCP-3 binding sites with those for other CC chemokines on the cloned
receptors, radioligand binding was performed. I-MCP-3
specifically bound to both CC CKR1 and CC CKR2B (Fig. 4).
I-MCP-3 bound to CC CKR1 could be competed by MCP-3,
MIP-1
, RANTES, and MIP-1
, whereas MCP-1 and IL-8 had no
effect. The K
values were 8, 10, 15, and 75
nM, respectively, for MCP-3, MIP-1
, RANTES, and
MIP-1
. In contrast,
I-MCP-3 bound to CC CKR2B could
only be competed by MCP-1 and MCP-3 (K
7
nM for each). Thus,
I-MCP-3 bound to both
receptors with similar affinity, but the chemokines able to displace
this binding differed from one receptor to the other, indicating that
the MCP-3 binding sites are distinct.
Figure 4:
Specific binding of chemokines to HEK 293
cells stably transfected with human CC chemokine receptor cDNAs. Cells
stably transfected with CC CKR1 (A) and CC CKR2B (B)
were incubated in duplicate with 0.1 nMI-human
MCP-3 in the presence of increasing concentrations of the unlabeled
chemokines identified in the inset at the bottom left of each panel. Average total binding was 6000 (A) and 8000 cpm (B). The data shown are the means of
values pooled from two experiments for each point for MIP-1
and
RANTES in A, two to five experiments for each point for MCP-3,
MCP-1, MIP-1
, and IL-8 in A, and two to four experiments
for each point for each chemokine in B. The standard error of
the mean was <15% in all cases. The K
values were determined using the program LIGAND from curves
fit to the pooled binding data(21) . The average number of
MCP-3 binding sites was 154,000 for CC CKR1 and 80,000 for CC
CKR2B.
To determine whether this signal is from CC CKR2A or CC CKR2B, oligonucleotides specific for the divergent C termini were used as hybridization probes on monocyte Northern blots. Each probe identified the same 3.5-kb band, suggesting that both receptor subtypes are expressed in monocytes (Fig. 5). When a phagocyte Northern blot was probed with a CC CKR1 cDNA probe, a different banding pattern was detected: large amounts of a 3-kb transcript in the monocyte sample, somewhat less in the neutrophil sample, and small amounts in the eosinophil sample(17) .
Figure 5:
Monocyte expression of CC CKR2A and CC
CKR2B. A Nytran blot containing 10 µg total RNA from peripheral
blood-derived human monocytes was hybridized sequentially with two
oligonucleotides specific for the CC CKR2A (A) or CC CKR2B (B) C-terminal segment. After each hybridization, the blot was
washed at 60 °C in 5 SSPE for 5 min, then exposed to XAR-2
film with an intensifying screen at -80 °C for 2 days.
Between hybridizations with probe A and probe B, the
blot was stripped of probe in a solution of 50% formamide and 6
SSPE at 70 °C for 30 min.
In the present work, we have identified two distinct MCP-3
receptor subtypes, CC CKR1 and CC CKR2B. This confirms and extends the
report by Franci et al.(18) regarding the selectivity
of CC CKR2B for MCP-3. The EC values for calcium
mobilization by MCP-3 are in the same range for CC CKR1 and CC CKR2B
expressed in HEK 293 cells as for those determined using human
monocytes, eosinophils, and CD4
and CD8
T lymphocytes (1-10
nM)(6, 8, 10) .
CC CKR1 and CC
CKR2B have three major similarities. First, they both bind MCP-3 with
similar affinity (K = 7 nM).
Second, MCP-3 is an equipotent agonist when
[Ca
]
changes are used as a
measure of receptor activation (EC
= 10
nM). Third, RNA for both receptors is present in monocytes.
Therefore, both receptors could mediate chemotaxis and other monocyte
responses to MCP-3.
However, CC CKR1 and CC CKR2B have several fundamental differences. First, RNA for CC CKR1 but not CC CKR2B is expressed in eosinophils and neutrophils. Therefore, CC CKR1 may be a receptor that mediates chemotaxis and other eosinophil responses to MCP-3.
Second, aside from the common agonist MCP-3, completely
different sets of CC chemokines activate CC CKR1 and CC CKR2B. Thus,
MIP-1, RANTES, and MIP-1
are agonists for CC CKR1 and compete
for its
I-MCP-3 binding site, whereas none of them are
agonists for CC CKR2B and they do not compete for its
I-MCP-3 binding site. Conversely, MCP-1 is an agonist for
CC CKR2B and competes for its
I-MCP-3 binding site,
whereas it is not an agonist for CC CKR1 and competes very poorly if at
all for its
I-MCP-3 binding site.
Third, in the case
of CC CKR2B, the cross-desensitization pattern for MCP-1 and MCP-3 was
asymmetric (i.e. MCP-1 completely desensitizes responsiveness
to MCP-3, but MCP-3 only partially desensitizes responsiveness to
MCP-1), whereas in the case of CC CKR1, the cross-desensitization
pattern for MIP-1 and MCP-3 was symmetric (i.e. both
MIP-1
and MCP-3 responses can be completely cross-desensitized) ( Fig. 2and Fig. 3; (18) ). This is particularly
striking because MCP-1 and MCP-3 are equipotent agonists for CC CKR2B,
whereas MIP-1
is 10-fold more potent than MCP-3 for CC CKR1.
Franci et al.(18) have shown that
I-MCP-1 binding to CC CKR2B on HEK 293 cells can be fully
competed by unlabeled MCP-3, although the K
values
differ considerably. Our data indicate that
I-MCP-3
binding to CC CKR2B on HEK 293 cells can be almost completely competed
by MCP-1. At this point there is no strong evidence for a unique
binding site for MCP-1 on CC CKR2B that is not shared by MCP-3. Perhaps
the asymmetric cross-desensitization phenomenon is due to differences
in receptor internalization or phosphorylation or differences in
coupling to G proteins or other regulatory elements induced by MCP-1
and MCP-3.
These differences might result from the involvement of different ligand-receptor binding sites for MCP-1 and MCP-3 on CC CKR2B that are not detectable by equilibrium binding at 4 °C. This hypothesis could be tested by making chimeric MCP-1/MCP-3 chemokines and testing their desensitizing capacity for wild type MCP-1 and MCP-3. This could help to identify critical residues involved in binding and perhaps point to strategies for designing selective antagonists.
At present, receptor subtype-specific neutralizing antibodies and antagonists and genetically manipulated leukocytes are not available to precisely determine the functional role of each cloned CC chemokine receptor in leukocytes. Inferences must be made based on the concordance of binding, signal transduction, and RNA data for native leukocytes and cloned receptors. At present, there is a large amount of conflicting data that needs to be reconciled, as reviewed below.
In
the case of monocytes, unlabeled MCP-1 and MCP-3 compete for monocyte I-MCP-1 binding sites, just as they do for CC CKR2B, yet
the rank orders of competition are inverted: MCP-3>MCP-1 for
monocytes and MCP-1>MCP-3 for CC CKR2B(7, 18) .
Furthermore, MCP-1 and MCP-3 completely cross-desensitize monocyte
[Ca
]
transients elicited by
each other, whereas we and Franci et al.(18) found
asymmetric desensitization for MCP-1 and MCP-3 for CC CKR2B, which is
selectively expressed in monocytes (6, 7, 16; Fig. 3and Fig. 5). Wang et al.(25) have shown that
binding of
I-MIP-1
and
I-MIP-1
to
monocytes can be competed equally effectively by unlabeled MIP-1
and MIP-1
, whereas
I-MIP-1
but not
I-MIP-1
binds to CC CKR1, and unlabeled MIP-1
very weakly competes for the binding(14) . Wang et al.(26) and Van Riper et al.(27) also
characterized a THP-1 cell RANTES receptor. MCP-1 was able to
completely desensitize THP-1 cell
[Ca
]
transients elicited by
RANTES(26) , whereas MCP-1 has no effect on CC CKR1 RANTES
responses (Fig. 2). Moreover, MCP-1 effectively competes for
I-RANTES binding to THP-1 cells but poorly competes for
the CC CKR1 binding site for
I-RANTES(14, 26, 27) .
In
the case of eosinophils and basophils, MCP-3 binding has not yet been
evaluated. In the case of eosinophils, MIP-1 has little effect on
the RANTES-induced calcium response, whereas it is able to completely
desensitize the CC CKR1 RANTES response(8, 28) . Like
CC CKR2B expressed in HEK 293 cells, basophil
[Ca
]
transients elicited by
MCP-1 and MCP-3 are asymmetrically cross-desensitized; however, the
asymmetries are inverted: for basophils, MCP-1 partially affects the
response to MCP-3, and MCP-3 completely desensitizes the response to
MCP-1, whereas for CC CKR2B, the converse is true ( (8) and (18) ; Fig. 3).
The most concordant response pattern
for a cloned CC chemokine receptor and a native leukocyte receptor is
found in lymphocytes. Cross-desensitization of
[Ca]
transients induced by
MCP-1 and MCP-3 for CD4
and CD8
human
lymphocytes resembles that observed for CC CKR2B ( (10) and (18) ; Fig. 3). MIP-1
and RANTES also induce
[Ca
]
transients in lymphocytes,
but cross-desensitization patterns have not been defined (10) .
The differences summarized above for cloned and native leukocyte CC
chemokine receptors could be due to the presence of additional as yet
undiscovered CC chemokine receptors on leukocytes or else to different
properties of the cloned receptors in leukocytes relative to their
properties when expressed in heterologous cell types. Two chemokine
receptor examples of the latter are known. First, the native Duffy
antigen on erythrocytes binds I-IL-8 with high affinity,
whereas binding of
I-IL-8 to the cloned Duffy antigen
expressed in HEK 293 cells is not detectable(29) . IL-8 binding
can be inferred by the ability of unlabeled IL-8 to compete for
I-GRO
to Duffy-transfected cells. Second, the
agonist rank order for IL-8 receptor B expressed in HEK 293 cells is
IL-8=GRO
= NAP-2, whereas when it is expressed in Xenopus oocytes it is IL-8
GRO
= NAP-2 (30) . (
)
All known chemokine receptors, except
for the Duffy antigen, are restricted to binding either CC or CXC
chemokines, and all known chemokine receptors, except for IL-8 receptor
A, are selective for multiple
chemokines(13, 14, 15, 17, 18, 19, 30, 31, 32, 33, 34, 35) .
Nevertheless, within these simple boundaries, great complexity in the
ligand-receptor relationships is the rule. For example, it is
counterintuitive that MCP-3, which is 70% identical in amino acid
sequence to MCP-1 and only 25% identical to RANTES, is a potent agonist
for CC CKR1 (agonist rank order, MIP-1>MCP-3>RANTES) and CC
CKR2B (MCP-1 = MCP-3), whereas MCP-1 is a potent agonist only
for CC CKR2B. In summary, we have defined the chemokine ligands and
agonists and the leukocyte expression patterns for both CC CKR1 and CC
CKR2B. Both are functional receptors for multiple CC chemokines;
however, MCP-3 is common to both. Monocyte responses to MCP-3 are
likely to be the sum of inputs from both CC CKR1 and CC CKR2B and
perhaps other monocyte CC chemokine receptors such as CC CKR2A.
Additional work will be required to determine precisely the
contribution of each of these receptors to chemokine responses in
vivo by the cells that express them and to reconcile the
discrepancies between native leukocyte receptors and cloned receptors
with respect to chemokine binding and signal transduction.
Note Added in Proof-Power et al. (36) have recently reported a cDNA for a basophil CC chemokine
receptor selective for MIP-1, RANTES, and MCP-1.