©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Monocyte Chemoattractant Protein-3 Is a Functional Ligand for CC Chemokine Receptors 1 and 2B (*)

(Received for publication, July 20, 1995; and in revised form, August 17, 1995)

Christophe Combadiere (1) Sunil K. Ahuja (1) Jo Van Damme (2) H. Lee Tiffany (1) Ji-Liang Gao (1) Philip M. Murphy (1)(§)

From the  (1)Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and the (2)Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-1alpha (MIP-1alpha)/regulated on activation, normal T expressed, and secreted protein (RANTES) receptor CC CKR1 (rank order for [Ca] changes = MIP-1alpha > 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.


INTRODUCTION

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 (^1)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-1alpha. 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-1alpha from monocytes, whereas RANTES has a lesser effect and MCP-1 has little effect, suggesting a separate shared MIP-1alpha/ 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-1alpha, RANTES, and MCP-3 on eosinophils. Van Riper et al.(12) have identified a shared functional binding site for MIP-1alpha 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-1alpha 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-1alpha, MIP-1beta, and RANTES. (^2)The only agonist found so far for CC CKR3 is human eotaxin, an eosinophil-specific CC chemokine. (^3)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.


EXPERIMENTAL PROCEDURES

Creation of Cell Lines Stably Expressing CC Chemokine Receptors

The following previously reported cDNAs were used: 1) the p4 cDNA encoding CC CKR1 (13) and 2) the CC CKR3 cDNA(16) . The libraries, probe, and methods used to clone a 1.3-kb CC CKR2B cDNA have been detailed previously(16) . The sequence of the CC CKR2B cDNA matched that previously reported by Charo et al.(15) . The CC CKR3 cDNA was subcloned between the NheI and XhoI sites of the mammalian expression vector pREP9 (Invitrogen) as described previously(16) ; the CC CKR2B cDNA was subcloned between the BamHI and HindIII sites of pREP9 using the same methods described previously(16) . Human embryonic kidney (HEK) 293 cells (10^7) grown to log phase in Dulbecco's modified Eagle's medium and 10% fetal bovine serum were electroporated with 20 µg of plasmid DNA, and G418-resistant colonies were picked and expanded. The p4 cDNA encoding CC CKR1 was subcloned between the NotI and XhoI sites of pCEP4 (Invitrogen), and transfected HEK 293 cells were selected in 250 µg/ml of hygromycin.

Receptor Binding Assay

I-MCP-3 (specific activity, 2200 Ci/mmol) was obtained from DuPont NEN. The methods used for binding were identical to those described previously for CC chemokines (16) . Briefly, 20,000 cpm of I-MCP-3 were added to 2 million stably transfected cells in binding medium (RPMI 1640 with 1 mg/ml bovine serum albumin and 25 mM HEPES, pH 7.4) in a total volume of 200 µl, in the presence or absence of increasing concentrations of unlabeled human IL-8, MIP-1alpha, MIP-1beta, MCP-1, MCP-2, MCP-3, and RANTES. MCP-2 and MCP-3 were synthesized chemically as described previously(20) . All other chemokines used were recombinant proteins purchased from Peprotech (Rocky Hill, NJ). Recombinant MCP-3 from Peprotech was used in replicate experiments, giving results similar to those obtained with chemically synthesized MCP-3. After a 2-h incubation period at 4 °C, the suspension was centrifuged through 10% sucrose in phosphate-buffered saline, and the pelleted cells were counted in a counter. The binding data were analyzed with the program LIGAND to determine the binding constant and number of binding sites(21) . Cells expressing CC CKR1 or CC CKR2B typically bound 30-40% of the added I-MCP-3 radioligand. Control HEK 293 cells transfected and selected using identical conditions but expressing IL-8 receptor B bound 10% of the added radioligand, which was not displaced by excess unlabeled MCP-3, MIP-1alpha, or MCP-1. All binding assays were performed in duplicate, and the individual values were always within 10% of the average.

Receptor Activation Assay

Receptor activation was assessed by real time measurement of [Ca](i) changes in HEK 293 cell transfectants loaded with FURA-2 upon stimulation with chemokines. The materials and methods were exactly as described previously(13) .

RNA Analysis

Total RNA was prepared using an RNA isolation kit (Stratagene) from human peripheral blood neutrophils isolated by Hypaque-Ficoll differential centrifugation (>95% neutrophils), from human blood monocytes separated from lymphocytes by adherence to plastic for 18 h, and from human blood eosinophils isolated from a healthy individual with a >10-year history of stable hypereosinophilia (>99% eosinophils) as described previously(22) . RNA samples were analyzed by Northern blot hybridization exactly as described previously(22) . The cDNA probe was a 1.3-kb cDNA containing the entire open reading frame for CC CKR2B and was labeled with [alpha-P]dCTP using a random primed labeling kit (Boehringer Mannheim). Two oligonucleotides specific for each type of CC CKR2, corresponding to the divergent region in the 5`-end of the cDNA, were labeled with [-P]ATP using the 5`-end labeling kit (Boehringer Mannheim) and were hybridized separately to blots of monocyte RNA for 12 h as described previously(22) .


RESULTS

MCP-3 Is an Agonist for CC CKR1 and CC CKR2B

To test whether the cloned CC chemokine receptors are functional receptors for MCP-3, we created stable HEK 293 cell lines expressing CC CKR1 and CC CKR2B and measured [Ca](i) changes upon stimulation with a panel of CC chemokines. This functional response has been consistently correlated with chemotactic responses of leukocytes to chemokines and is a convenient measure of receptor activation(1, 2) . CC CKR1 transfectants responded to MCP-3 (Fig. 1A). Control stimulations with MIP-1alpha, RANTES, MIP-1beta, MCP-1, and IL-8 gave results concordant with previously published data(13, 14, 15, 17, 18, 19) . That is, CC CKR1 transfectants responded to MIP-1alpha, RANTES, and very weakly to MIP-1beta (Fig. 1A). As we have previously reported, CC CKR3 transfectants gave no response to MCP-3(16) . As previously reported by Franci et al.(18) , CC CKR2B transfectants responded selectively to MCP-1 and MCP-3.


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-1alpha > MCP-3 > RANTES; for CC CKR2B, MCP-1 = MCP-3 (EC values = 1 nM for MIP-1alpha, 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)).

Desensitization Patterns for Cloned MCP-3 Receptors

[Ca](i) transients in response to sequential stimulation with chemokines are sometimes useful for dissecting ligand-receptor relationships(8, 14, 17, 18, 19, 23, 24) . When CC CKR1 transfectants were sequentially stimulated with chemokines at 100 nM, the following results were obtained (Fig. 2). MIP-1alpha and MCP-3 each abolished the response to a second stimulation with MIP-1alpha, RANTES, or MCP-3. RANTES abolished the response to a second stimulation with RANTES but reduced by only 50% the MIP-1alpha and MCP-3 responses. MIP-1beta, which was a poor agonist, had little or no effect on a second stimulation with MIP-1alpha, RANTES, or MCP-3.


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-1alpha 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.



Binding of I-MCP-3 to CC CKR1 and CC CKR2B

The fact that MCP-1 completely desensitizes the CC CKR2B response to MCP-3, but MCP-3 only partly affects the CC CKR2B response to MCP-1, suggests that the sets of CC CKR2B binding sites for MCP-1 and MCP-3 are not identical. This is in contrast to the reciprocal desensitization phenomenon observed for CC CKR1 with respect to MIP-1alpha and MCP-3.

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-1alpha, RANTES, and MIP-1beta, whereas MCP-1 and IL-8 had no effect. The K(i) values were 8, 10, 15, and 75 nM, respectively, for MCP-3, MIP-1alpha, RANTES, and MIP-1beta. In contrast, I-MCP-3 bound to CC CKR2B could only be competed by MCP-1 and MCP-3 (K(i) 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-1beta and RANTES in A, two to five experiments for each point for MCP-3, MCP-1, MIP-1alpha, 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.



mRNA Distribution of CC CKR2A and CC CKR2B

Two MCP-1 receptor subtypes have been described, CC CKR2A and CC CKR2B, whose sequences are identical until amino acid 313(15) . The divergent region is in the predicted C-terminal segment of the receptors, and both subtypes are products of the same gene. (^2)Much less is known about CC CKR2A than CC CKR2B. In particular, it is not yet known whether CC CKR2A is an MCP-3 receptor. We have previously shown that a full-length CC CKR2B open reading frame probe that cross-hybridizes with CC CKR2A recognizes a 3.5-kb class of transcripts in monocyte RNA samples but not in neutrophil or eosinophil samples(16) .

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 times 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 times SSPE at 70 °C for 30 min.




DISCUSSION

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(i) = 7 nM). Second, MCP-3 is an equipotent agonist when [Ca](i) 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-1alpha, RANTES, and MIP-1beta 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-1alpha and MCP-3 was symmetric (i.e. both MIP-1alpha 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-1alpha 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(i) 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](i) 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-1alpha and I-MIP-1beta to monocytes can be competed equally effectively by unlabeled MIP-1alpha and MIP-1beta, whereas I-MIP-1alpha but not I-MIP-1beta binds to CC CKR1, and unlabeled MIP-1beta 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](i) 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-1alpha 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](i) 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](i) 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-1alpha and RANTES also induce [Ca](i) 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-GROalpha to Duffy-transfected cells. Second, the agonist rank order for IL-8 receptor B expressed in HEK 293 cells is IL-8=GROalpha = NAP-2, whereas when it is expressed in Xenopus oocytes it is IL-8GROalpha = NAP-2 (30) . (^3)

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-1alpha>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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Laboratory of Host Defenses, NIAID, National Institutes of Health, Bldg. 10, Rm. 11N113, Bethesda, MD 20892. Tel.: 301-480-2037; Fax: 301-402-0789.

(^1)
The abbreviations used are: MCP, monocyte chemoattractant protein; kb, kilobase(s) or kilobase pairs; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T expressed, and secreted protein; IL-8, interleukin-8; HEK, human embryonic kidney.

(^4)
C. Combadiere and P. M. Murphy, unpublished data.

(^5)
S. K. Ahuja and P. M. Murphy, manuscript in preparation.

(^2)
C. Combadiere, S. K. Ahuja, and P. M. Murphy, submitted for publication.

(^3)
M. Kitaura, T. Nakajima, T. Imai, S. Harada, C. Combadiere, H. L. Tiffany, P. M. Murphy, and O. Yoshie, submitted for publication.


ACKNOWLEDGEMENTS

-We thank S. Mawhorter for providing purified eosinophils.

Note Added in Proof-Power et al. (36) have recently reported a cDNA for a basophil CC chemokine receptor selective for MIP-1alpha, RANTES, and MCP-1.


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