©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Functional Expression of a Human Eosinophil CC Chemokine Receptor (*)

Christophe Combadiere , Sunil K. Ahuja , Philip M. Murphy (§)

From the (1)Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Eosinophils undergo chemotaxis, degranulate, and exhibit [Ca] changes in response to the human CC chemokines macrophage inflammatory protein (MIP)-1, regulated on activation, normal T expressed and secreted (RANTES), and monocyte chemoattractant protein-3 (MCP-3), but the receptors involved have not been defined. We have isolated a human cDNA encoding the first eosinophil-selective chemokine receptor, designated CC chemokine receptor 3 (CC CKR3). CC CKR3 is a seven-transmembrane domain G protein-coupled receptor most closely related to the previously reported monocyte- and neutrophil-selective receptor CC CKR1 (also known as the MIP-1/RANTES receptor). When [Ca] changes were monitored in stably transfected human embryonic kidney 293 cells, MIP-1 and RANTES were both potent agonists for CC CKR3 and CC CKR1. However, MIP-1 was also an agonist for CC CKR3 but not CC CKR1; MCP-3 was an agonist for CC CKR1 but not CC CKR3. CC CKR3 may be one of the host factors responsible for selective recruitment of eosinophils to sites of inflammation.


INTRODUCTION

The types of white blood cells that appear in inflammatory infiltrates can differ markedly depending in part on the identity of the inflammatory irritant and the duration of irritation. The host factors responsible for these differences are likely to be cell type-specific or selective chemoattractants and chemoattractant receptors. CC and CXC chemokines are two related families of relatively selective leukocyte chemoattractants. CC chemokines are all potent monocyte chemoattractants in vitro but differ in their capacity to activate neutrophils, lymphocytes, basophils, and eosinophils (reviewed in Ref. 1). In particular, human MIP-1,()RANTES, and MCP-3 and guinea pig eotaxin are potent eosinophil chemoattractants, whereas other known CC chemokines are not(2, 3, 4, 5) . The best characterized CXC chemokine, interleukin-8 (IL-8), is a strong neutrophil chemoattractant but has only modest activity for eosinophils(1, 6) .

The receptors responsible for CC chemokine action on eosinophils have not been defined. By inspecting cross-desensitization patterns for chemokine-induced calcium transients, Dahinden et al. (4) have proposed that eosinophils have specific receptors for RANTES and MCP-3 and a shared receptor for MIP-1, RANTES, and MCP-3. Van Riper et al.(7) have identified a shared functional binding site for MIP-1 and RANTES on HL-60 cell-derived eosinophil-like cells, but MCP-3 binding to this site has not yet been tested. Eotaxin competes for I-human RANTES binding to guinea pig eosinophils, suggesting a shared receptor(5) .

cDNAs for three human leukocyte CC chemokine receptors have been cloned, CC CKR1, CC CKR2A, and CC CKR2B (also known as MCP-1 receptors A and B), but their properties are not fully consistent with eosinophil responses to CC chemokines(8, 9, 10, 11) . MIP-1 and RANTES are effective agonists for CC CKR1; however, its RNA is scarce in eosinophils(8, 9, 12) . Much higher expression is found in neutrophils, monocytes, and lymphocytes(10, 12) . MCP-1 is an agonist for CC CKR2A and -2B, but it does not activate eosinophils(4, 11) . Moreover, CC CKR2 RNA is expressed in monocytes but not in eosinophils(12) . Together with the CXC chemokine receptors, the CC chemokine receptors form a subgroup of the rhodopsin superfamily of seven-transmembrane domain receptors(13) . Here we characterize the first eosinophil-selective member of this family.


EXPERIMENTAL PROCEDURES

Gene and cDNA Cloning

Construction of a gt11 cDNA library prepared from lipopolysaccharide-stimulated peripheral blood monocytes has been previously described(14) . The library was screened by plaque hybridization with the full-length open reading frame (ORF) of CC CKR2B produced by polymerase chain reaction and labeled with [-P]dCTP using a random primer labeling kit (Boehringer Mannheim). Plaque lifts were incubated in 10 cpm/probe/ml of hybridization buffer for 12 h and then were washed at 55 °C in 5 SSPE for 15 min. cDNA inserts of purified phage DNA were excised by SalI and SfiI double digestion, blunt ended with Pfu DNA polymerase, subcloned into the EcoRV site of pBluescript II SK (Stratagene, La Jolla, CA), and sequenced on both strands. cDNAs were then subcloned between the NheI and XhoI sites of the mammalian expression vector pREP9 (Invitrogen, San Diego, CA). Human embryonic kidney (HEK) 293 cells (10) 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 (8) encoding CC CKR1 was subcloned into the NotI and XhoI sites of pCEP4 (Invitrogen), and transfected HEK 293 cells were selected in 250 µg/ml hygromycin.

DNA Analysis

Human genomic DNA was analyzed by restriction enzyme cleavage and Southern hybridization as described previously(8) .

RNA Analysis

Total RNA was prepared using an RNA isolation kit (Stratagene, La Jolla, CA) 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 (15). RNA samples were analyzed by Northern blot hybridization exactly as described previously(15) .

Ligand Binding Analysis

10 transfected cells were incubated in duplicate with 0.1-0.5 nMI-labeled RANTES, MCP-1, MIP-1, or MIP-1 (specific activity, 2200 Ci/mmol, DuPont NEN) and varying concentrations of unlabeled recombinant human chemokines (Peprotech, Rocky Hill, NJ) in 200 µl of binding medium (RPMI 1640 with 1 mg/ml bovine serum albumin and 25 mM HEPES, pH 7.4). After incubation for 2 h at 4 °C, cells were pelleted through a 10% sucrose/phosphate-buffered saline cushion, and specific binding was determined by the difference in counts in the presence and absence of a 500-fold molar excess of unlabeled chemokine.

Intracellular [Ca] Measurements

Cells (10/ml) were incubated in Hanks' buffered saline solution with Ca and Mg supplemented with 10 mM HEPES, pH 7.4 (HBSS) containing 2.5 µM Fura-2/AM (Molecular Probes, Eugene, OR) for 60 min at 37 °C in the dark. Cells were washed twice with HBSS and resuspended at 2 10 cells/ml. Two ml were placed in a continuously stirred cuvette at 37 °C in a fluorimeter (Photon Technology Inc., South Brunswick, NJ). Fluorescence was monitored at = 340 nm, = 380 nm, and = 510 nm. The data were recorded as the relative ratio of fluorescence excited at 340 and 380 nm. Data were collected every 200 ms.


RESULTS AND DISCUSSION

Sequence Analysis of CC CKR3

Out of 6 positive human monocyte cDNA clones that cross-hybridized with a CC CKR2B ORF probe at low stringency, 2 were for CC CKR1, 1 was for CC CKR2B, 2 were for novel putative chemokine receptors, and 1 was for a cDNA unrelated to chemokine receptors. Here we detail the properties of one of the two novel putative chemokine receptors, which we have designated CC CKR3.

The CC CKR3 cDNA is 1.6 kb in length. The ORF encodes a predicted protein of 355 amino acids that is identical in length and 63% identical in sequence with CC CKR1. CC CKR3 has 51% identity with CC CKR2B (Fig. 1) but only 31% identity with the CXC chemokine receptors and IL-8 receptors A and B(8, 9, 10, 11, 16, 17) . The amino acid positions that differ between CC CKR1 and CC CKR3 are found mostly in the putative extracellular domains and adjacent portions of the transmembrane domains (Fig. 1); the predicted intracellular C-terminal segments are also divergent but have a high content of serine residues that may be sites for receptor phosphorylation as they are in rhodopsin and the -adrenergic receptor(18) . Like CC CKR1 and all other known chemokine receptors, the CC CKR3 sequence is acidic in the N-terminal segment before the first putative transmembrane domain, containing a net charge of -4. The second extracellular loop is also highly acidic (net charge of -5), whereas for CC CKR1 the corresponding region has a net charge of +3. Like all other known chemokine receptors, CC CKR3 has conserved cysteine residues in the N-terminal segment and the third predicted extracellular loop that could form a disulfide bond (13). These cysteines are less frequently found in other G protein-coupled receptors(19) . Unlike other chemokine receptors, CC CKR3 lacks a consensus sequence for N-linked glycosylation. The proline in a proline-cysteine motif conserved in the N-terminal segment of other chemokine receptors is leucine in CC CKR3.


Figure 1: Alignment of amino acid sequences deduced from cDNAs for CC CKR1, CC CKR2B, and CC CKR3. Verticalbars indicate identical residues for each adjacent sequence position. Arabicnumbers enumerate the CC CKR3 sequence and are left-justified. Dashes indicate gaps that were inserted to optimize the alignment. The locations of predicted membrane-spanning segments I-VII are noted. Openboxes designate predicted sites for N-linked glycosylation. The DNA and proteins sequences have been deposited in GenBank and have been given the accession number U28694.



Hybridization of the CC CKR3 cDNA to total human genomic DNA digested with PstI, EcoRI, and HindIII revealed one strongly hybridizing band in each case (Fig. 2A), suggesting that it is the product of a small, single-copy gene. The 1.8-kb HindIII band was also seen in genomic DNA hybridized under low stringency conditions with a CC CKR1 cDNA probe due to cross-hybridization to CC CKR3 (9).()


Figure 2: Analysis of the CC CKR3 gene and RNA. A, gene. 5 µg of total human genomic DNA was digested with the restriction enzymes indicated at the top of each lane, gel-fractionated, and blot-hybridized with the CC CKR3 cDNA. The blot was washed at 65 °C in 0.2 SSPE for 1 h and then exposed to XAR-2 film with an intensifying screen at -80 °C for 5 days. Size markers in kb are indicated at the left. B, distribution of mRNA encoding CC CKR3 in human leukocytes. A Nytran blot containing 10 µg of total RNA from peripheral blood-derived human neutrophils (N), monocytes (M), and eosinophils (E) was hybridized with a CC CKR3 cDNA probe. The blot was washed at 65 °C in 0.2 SSPE for 1 h and then exposed to XAR-2 film with an intensifying screen at -80 °C for the times indicated at the bottom of each panel. The position of ribosomal bands is indicated at the left.



Distribution of CC CKR3 RNA

Two size classes of CC CKR3 mRNA were detected in human peripheral blood-derived eosinophils (Fig. 2B). The major species is 1.6 kb, similar in length to the CC CKR3 cDNA that we cloned. A minor 4-kb species is also present. Faint 1.6-kb bands were visible in neutrophil and monocyte samples only after prolonged exposures. When the same blot was probed with CC CKR1 cDNA, a reciprocal banding pattern was detected: large amounts of a 3-kb transcript in neutrophil and monocyte lanes and trace amounts in the eosinophil lane(12) . CC CKR1 mRNA has also been detected in peripheral blood-derived lymphocytes and Staphylococcus aureus Cowan strain-activated tonsillar B cells (8, 10) and in small amounts in heart, kidney, skeletal muscle, pancreas, and brain but in larger amounts in placenta, lung, and liver(12) . We have not been able to detect CC CKR3 transcripts in these solid organs (not shown). When the same phagocyte blot was probed with the CC CKR2B ORF, a 3.5-kb transcript was seen only in the monocyte lane(12) . Thus, CC CKR1, -2, and -3 are differentially expressed in a cell type-specific pattern in human peripheral blood leukocytes.

Agonists for CC CKR3

Since MIP-1, RANTES, and MCP-3 are the only known human CC chemokines that activate eosinophils, they were the best candidate agonists for CC CKR3(2, 3, 4, 7) . Chemotactic responses to chemokines are associated with rapid and transient elevations of [Ca], which can be used as a convenient indicator of receptor usage. Neither untransfected nor mock-transfected and selected HEK 293 cells responded to any of the chemokines tested. In contrast, when three independent HEK 293 cell clones stably transfected with CC CKR3 cDNA were tested, all three exhibited [Ca] transients in response to MIP-1, RANTES, and MIP-1 but not in response to MCP-1, MCP-2, MCP-3, IL-8, or IP-10, all tested at 100 nM (Fig. 3A). The rank order of potency was MIP-1 > RANTES > MIP-1 (Fig. 3B). CC CKR3 is the first known MIP-1 receptor to be cloned. As previously reported, HEK 293 cells stably transfected with CC CKR1 also responded to MIP-1 and RANTES(9, 20) . However, unlike CC CKR3, CC CKR1-transfected cells also responded to MCP-3 but not to MIP-1 at 100 nM (Fig. 3A). CC CKR1 is the first known MCP-3 receptor to be cloned. The relationship of CC CKR1 to CC CKR3 is analogous to the relationship of IL-8 receptor A to IL-8 receptor B, in that both are pairs of receptors with highly related sequences and an overlapping but non-identical set of agonists restricted to either CC or CXC chemokines(16, 17) . However, IL-8 receptors A and B are both expressed selectively at high levels in the same cell type, the neutrophil(15) .


Figure 3: Agonists for CC CKR3. A, selectivity for CC CKR3 versus CC CKR1. [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 the chemokine indicated at the left of each row of tracings, at 100 nM. The tracings shown are from one experiment representative of at least three experiments with three separate clones for CC CKR3 and one clone for CC CKR1. B, rank order of potency of agonists for CC CKR3. The magnitude of the peak of the calcium transient elicited by the indicated concentration of human chemokines from HEK 293 cells stably expressing the CC CKR3 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.



Desensitization of CC CKR3

After activation, chemokine receptors have altered sensitivity to repeated stimulation with the activating agonist and other agonists(1, 4, 9, 20) . When CC CKR3 transfectants were sequentially stimulated with 100 nM of various combinations of MIP-1, MIP-1, RANTES, and MCP-1, the following results were obtained (Fig. 4). MCP-1 had no effect on the responses to MIP-1, MIP-1, or RANTES. MIP-1, MIP-1, and RANTES completely desensitized the homologous response. MIP-1 and RANTES completely cross-desensitized the response to MIP-1, but MIP-1 only partially cross-desensitized the response to MIP-1 and RANTES. Finally, RANTES and MIP-1 partially cross-desensitized the responses to each other. This provides additional evidence for a functional interaction of MIP-1, MIP-1, and RANTES with the same receptor, CC CKR3.


Figure 4: Desensitization of calcium transients in HEK 293 cells stably transfected with CC CKR3. 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.



It is important to note that the [Ca] changes in response to CC chemokines in eosinophils differ significantly from those obtained with CC CKR1 and CC CKR3 in transfected HEK 293 cells (4, 9) (Fig. 4). MIP-1 more effectively desensitizes the CC CKR1 and CC CKR3 RANTES responses than the eosinophil RANTES response. Conversely, RANTES more effectively desensitizes the eosinophil MIP-1 response than the CC CKR1 and CC CKR3 MIP-1 responses. Finally, MIP-1 is a potent agonist for CC CKR3 but not for eosinophils. Neutrophil and monocyte responses to MIP-1 and RANTES also differ from those found for the cloned receptors. For neutrophils, responses to MIP-1 and RANTES can be completely cross-desensitized, yet the magnitude of the neutrophil RANTES response is half that of the neutrophil MIP-1 response at saturating concentrations, whereas for CC CKR1 and CC CKR3 transfectants they are similar(8, 9) (Fig. 3). RANTES attenuates both the CC CKR1 and CC CKR3 MIP-1 response in transfected cells but not the monocyte MIP-1 response (9, 21).

The factors that account for these differences are not clear but could include distinct genes for novel CC chemokine receptors. Alternatively, cell type-specific accessory factors could exist that modulate agonist and desensitization properties for the receptors that are already known. It is known, for example, that GRO does not effectively cross-desensitize [Ca] changes in response to IL-8 in HEK 293 cells transfected with IL-8 receptor B, whereas IL-8 completely cross-desensitizes neutrophil responses to GRO, which are thought to be mediated by IL-8 receptor B(22, 23) .

Since MIP-1, RANTES, and MIP-1 are agonists for CC CKR3, they must bind to it. Nevertheless we have not yet been able to demonstrate specific binding of I-MIP-1 and -RANTES to CC CKR3-transfected HEK 293 cells using as much as 0.5 nM radioligand on 2 million transfected cells. We have used the same conditions to demonstrate specific binding of I-MIP-1 to CC CKR1-transfected HEK 293 cells (Fig. 5). This suggests that MIP-1, MIP-1, and RANTES activate CC CKR3 via low affinity binding interactions. It is possible that CC CKR3 is more selective for another, as yet untested, CC chemokine such as eotaxin(5) . Human eotaxin has not been identified yet. The high affinity binding of I-MIP-1 and -RANTES to a shared site reported for HL-60-derived eosinophils is most likely due to a receptor different from CC CKR3, perhaps CC CKR1.


Figure 5: Specific binding of chemokines to HEK 293 cells stably transfected with human CC chemokine receptor cDNAs. Cells stably transfected with the indicated human cDNAs were incubated in duplicate with 0.1 nMI-human MIP-1 in the presence or absence of 50 nM human MIP-1 at 4 °C. The total counts bound for CCCKR3 were not significantly different from untransfected cells (not shown). Data are representative of two separate experiments with three separate CC CKR3 clones.



The present and previous studies strongly suggest that normal human monocytes and eosinophils respond to MIP-1 and RANTES via two MIP-1/RANTES receptors, CC CKR1 and CC CKR3. The relative RNA distributions suggest that CC CKR1 functions principally, but not exclusively, in monocytes, and CC CKR3 functions principally, but not exclusively, in eosinophils. RNA for both CC CKR1 and CC CKR3 is expressed in neutrophils, but the functional importance is unclear since these cells do not chemotax or degranulate to MIP-1 and RANTES (24). CC CKR1 may also be responsible in part for the responses of monocytes and eosinophils to MCP-3(4, 25) . Since RNA for CC CKR1 is present in neutrophils, we predict that MCP-3 can elicit [Ca] changes in neutrophils, although this has not been tested yet. The functional significance for eosinophils of the response of CC CKR3 to MIP-1 is unclear at present.

While the distinctive RNA distribution patterns of the cloned CC chemokine receptors are a useful starting point, additional functional studies will be required to fully delineate the importance of these receptors for chemotaxis, degranulation, and other responses by specific types of leukocytes during inflammatory responses in vivo. Nevertheless, based on the data reported here, it is reasonable to hypothesize that CC CKR3 may be an important host factor regulating eosinophil accumulation at body sites undergoing allergic reactions, metazoan infestation, or other types of inflammation where eosinophils are present in large numbers.


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: Laboratory of Host Defenses, NIAID, Bldg. 10, Rm. 11N113, NIH, Bethesda, MD 20892. Tel.: 301-480-2037; Fax: 301-402-0789.

The abbreviations used are: MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T expressed and secreted; MCP, monocyte chemoattractant protein; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; IL-8, interleukin-8; ORF, open reading frame; SSPE, saline/sodium/phosphate/EDTA; HEK, human embryonic kidney; kb, kilobase(s) or kilobase pair(s).

J.-L. Gao and P. M. Murphy, unpublished data.


ACKNOWLEDGEMENTS

We thank S. Mawhorter for providing purified eosinophils and H. Lee Tiffany for excellent technical assistance.

Note Added in Proof-The gene symbol for the CC CKR3 gene is CMKBR3 (P. McAlpine, personal communication). A mouse gene designated scya3r-rs2 has been identified that appears to be the orthologue to CMKBR3(26) .


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.