Cloning and Functional Expression of a Human Eosinophil CC Chemokine Receptor
1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês
10.1074/jbc.270.28.16491
ISSN1083-351X
AutoresChristophe Combadière, Sunil K. Ahuja, Philip M. Murphy,
Tópico(s)Chemokine receptors and signaling
ResumoEosinophils undergo chemotaxis, degranulate, and exhibit [Ca2+]i changes in response to the human CC chemokines macrophage inflammatory protein (MIP)-1α, 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-1α/RANTES receptor). When [Ca2+]i changes were monitored in stably transfected human embryonic kidney 293 cells, MIP-1α and RANTES were both potent agonists for CC CKR3 and CC CKR1. However, MIP-1β 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. Eosinophils undergo chemotaxis, degranulate, and exhibit [Ca2+]i changes in response to the human CC chemokines macrophage inflammatory protein (MIP)-1α, 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-1α/RANTES receptor). When [Ca2+]i changes were monitored in stably transfected human embryonic kidney 293 cells, MIP-1α and RANTES were both potent agonists for CC CKR3 and CC CKR1. However, MIP-1β 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. 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. 1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2270) Google Scholar). In particular, human MIP-1α,1 1The abbreviations used are: MIPmacrophage inflammatory proteinRANTESregulated on activation, normal T expressed and secretedMCPmonocyte chemoattractant proteinG proteinheterotrimeric guanine nucleotide-binding regulatory proteinIL-8interleukin-8ORFopen reading frameSSPEsaline/sodium/phosphate/EDTAHEKhuman embryonic kidneykbkilobase(s) or kilobase pair(s). RANTES, and MCP-3 and guinea pig eotaxin are potent eosinophil chemoattractants, whereas other known CC chemokines are not(2Kameyoshi Y. Dorschner A. Mallet A.I. Christophers E. Schroder J.-M. J. Exp. Med. 1992; 176: 587-594Crossref PubMed Scopus (651) Google Scholar, 3Rot A. Krieger M. Brunner T. Bischoff S.C. Schall T.J. Dahinden C.A. J. Exp. Med. 1992; 176: 1489-1497Crossref PubMed Scopus (595) Google Scholar, 4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 5Jose P.J. Griffiths-Johnson D.A. Collins P.D. Walsh D.T. Moqbel R. Totty N.F. Truong O. Hsuan J.J. Williams T.J. J. Exp. Med. 1994; 179: 881-886Crossref PubMed Scopus (769) Google Scholar). The best characterized CXC chemokine, interleukin-8 (IL-8), is a strong neutrophil chemoattractant but has only modest activity for eosinophils(1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2270) Google Scholar, 6Kernen P. Wymann M.P. von Tscharner V. Deranleau D.A. Tai P.-C. Spry C.J. Dahinden C.A. Baggiolini M. J. Clin. Invest. 1991; 87: 2012-2020Crossref PubMed Scopus (94) Google Scholar). macrophage inflammatory protein regulated on activation, normal T expressed and secreted monocyte chemoattractant protein heterotrimeric guanine nucleotide-binding regulatory protein interleukin-8 open reading frame saline/sodium/phosphate/EDTA human embryonic kidney kilobase(s) or kilobase pair(s). 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. (4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar) have proposed that eosinophils have specific receptors for RANTES and MCP-3 and a shared receptor for MIP-1α, RANTES, and MCP-3. Van Riper et al.(7Van Riper G. Nicholson D.W. Scheid M.P. Fischer P.A. Springer M.S. Rosen H. J. Immunol. 1994; 152: 4055-4063PubMed Google Scholar) have identified a shared functional binding site for MIP-1α 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 125I-human RANTES binding to guinea pig eosinophils, suggesting a shared receptor(5Jose P.J. Griffiths-Johnson D.A. Collins P.D. Walsh D.T. Moqbel R. Totty N.F. Truong O. Hsuan J.J. Williams T.J. J. Exp. Med. 1994; 179: 881-886Crossref PubMed Scopus (769) Google Scholar). 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(8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 10Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1247Crossref PubMed Scopus (141) Google Scholar, 11Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2755Crossref PubMed Scopus (654) Google Scholar). MIP-1α and RANTES are effective agonists for CC CKR1; however, its RNA is scarce in eosinophils(8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). Much higher expression is found in neutrophils, monocytes, and lymphocytes(10Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1247Crossref PubMed Scopus (141) Google Scholar, 12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). MCP-1 is an agonist for CC CKR2A and −2B, but it does not activate eosinophils(4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 11Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2755Crossref PubMed Scopus (654) Google Scholar). Moreover, CC CKR2 RNA is expressed in monocytes but not in eosinophils(12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). Together with the CXC chemokine receptors, the CC chemokine receptors form a subgroup of the rhodopsin superfamily of seven-transmembrane domain receptors(13Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1130) Google Scholar). Here we characterize the first eosinophil-selective member of this family. Construction of a λ gt11 cDNA library prepared from lipopolysaccharide-stimulated peripheral blood monocytes has been previously described(14Miki T. Matsui T. Heidaran M.A. Aaronson S.A. Gene (Amst.). 1989; 83: 137-146Crossref PubMed Scopus (54) Google Scholar). 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 [γ-32P]dCTP using a random primer labeling kit (Boehringer Mannheim). Plaque lifts were incubated in 106 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 (107) 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 (8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar) 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. Human genomic DNA was analyzed by restriction enzyme cleavage and Southern hybridization as described previously(8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar). 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 (15Ahuja S.K. Shetty A. Tiffany H.L. Murphy P.M. J. Biol. Chem. 1994; 269: 26381-26389Abstract Full Text PDF PubMed Google Scholar). RNA samples were analyzed by Northern blot hybridization exactly as described previously(15Ahuja S.K. Shetty A. Tiffany H.L. Murphy P.M. J. Biol. Chem. 1994; 269: 26381-26389Abstract Full Text PDF PubMed Google Scholar). 106 transfected cells were incubated in duplicate with 0.1-0.5 nM125I-labeled RANTES, MCP-1, MIP-1α, or MIP-1β (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. Cells (107/ml) were incubated in Hanks' buffered saline solution with Ca2+ and Mg2+ 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 × 106 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 λex1 = 340 nm, λex2 = 380 nm, and λem = 510 nm. The data were recorded as the relative ratio of fluorescence excited at 340 and 380 nm. Data were collected every 200 ms. 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(8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 10Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1247Crossref PubMed Scopus (141) Google Scholar, 11Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2755Crossref PubMed Scopus (654) Google Scholar, 16Holmes W.E. Lee J. Kuang W.-J. Rice G.C. Wood W.I. Science. 1991; 253: 1278-1280Crossref PubMed Scopus (917) Google Scholar, 17Murphy P.M. Tiffany H.L. Science. 1991; 253: 1280-1283Crossref PubMed Scopus (766) Google Scholar). 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 β2-adrenergic receptor(18Lefkowitz R.J. Cell. 1993; 74: 409-411Abstract Full Text PDF PubMed Scopus (404) Google Scholar). 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 (1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2270) Google Scholar). These cysteines are less frequently found in other G protein-coupled receptors(19Probst W.B. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (685) Google Scholar). 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. 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 (9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar).2 2J.-L. Gao and P. M. Murphy, unpublished data. 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(12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). CC CKR1 mRNA has also been detected in peripheral blood-derived lymphocytes and Staphylococcus aureus Cowan strain-activated tonsillar B cells (8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar, 10Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1247Crossref PubMed Scopus (141) Google Scholar) and in small amounts in heart, kidney, skeletal muscle, pancreas, and brain but in larger amounts in placenta, lung, and liver(12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). 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(12Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; (in press)PubMed Google Scholar). Thus, CC CKR1, −2, and −3 are differentially expressed in a cell type-specific pattern in human peripheral blood leukocytes. Since MIP-1α, RANTES, and MCP-3 are the only known human CC chemokines that activate eosinophils, they were the best candidate agonists for CC CKR3(2Kameyoshi Y. Dorschner A. Mallet A.I. Christophers E. Schroder J.-M. J. Exp. Med. 1992; 176: 587-594Crossref PubMed Scopus (651) Google Scholar, 3Rot A. Krieger M. Brunner T. Bischoff S.C. Schall T.J. Dahinden C.A. J. Exp. Med. 1992; 176: 1489-1497Crossref PubMed Scopus (595) Google Scholar, 4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 7Van Riper G. Nicholson D.W. Scheid M.P. Fischer P.A. Springer M.S. Rosen H. J. Immunol. 1994; 152: 4055-4063PubMed Google Scholar). Chemotactic responses to chemokines are associated with rapid and transient elevations of [Ca2+]i, 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 [Ca2+]i transients in response to MIP-1α, RANTES, and MIP-1β but not in response to MCP-1, MCP-2, MCP-3, IL-8, or γIP-10, all tested at 100 nM (Fig. 3A). The rank order of potency was MIP-1α > RANTES > MIP-1β (Fig. 3B). CC CKR3 is the first known MIP-1β receptor to be cloned. As previously reported, HEK 293 cells stably transfected with CC CKR1 also responded to MIP-1α and RANTES(9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 20Gao J.-L. Murphy P.M. J. Biol. Chem. 1994; 269: 28539-28542Abstract Full Text PDF PubMed Google Scholar). However, unlike CC CKR3, CC CKR1-transfected cells also responded to MCP-3 but not to MIP-1β 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(16Holmes W.E. Lee J. Kuang W.-J. Rice G.C. Wood W.I. Science. 1991; 253: 1278-1280Crossref PubMed Scopus (917) Google Scholar, 17Murphy P.M. Tiffany H.L. Science. 1991; 253: 1280-1283Crossref PubMed Scopus (766) Google Scholar). However, IL-8 receptors A and B are both expressed selectively at high levels in the same cell type, the neutrophil(15Ahuja S.K. Shetty A. Tiffany H.L. Murphy P.M. J. Biol. Chem. 1994; 269: 26381-26389Abstract Full Text PDF PubMed Google Scholar). After activation, chemokine receptors have altered sensitivity to repeated stimulation with the activating agonist and other agonists(1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2270) Google Scholar, 4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 20Gao J.-L. Murphy P.M. J. Biol. Chem. 1994; 269: 28539-28542Abstract Full Text PDF PubMed Google Scholar). When CC CKR3 transfectants were sequentially stimulated with 100 nM of various combinations of MIP-1α, MIP-1β, RANTES, and MCP-1, the following results were obtained (Fig. 4). MCP-1 had no effect on the responses to MIP-1α, MIP-1β, or RANTES. MIP-1α, MIP-1β, and RANTES completely desensitized the homologous response. MIP-1α and RANTES completely cross-desensitized the response to MIP-1β, but MIP-1β only partially cross-desensitized the response to MIP-1α and RANTES. Finally, RANTES and MIP-1α partially cross-desensitized the responses to each other. This provides additional evidence for a functional interaction of MIP-1α, MIP-1β, and RANTES with the same receptor, CC CKR3. It is important to note that the [Ca2+]i changes in response to CC chemokines in eosinophils differ significantly from those obtained with CC CKR1 and CC CKR3 in transfected HEK 293 cells (4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar) (Fig. 4). MIP-1α more effectively desensitizes the CC CKR1 and CC CKR3 RANTES responses than the eosinophil RANTES response. Conversely, RANTES more effectively desensitizes the eosinophil MIP-1α response than the CC CKR1 and CC CKR3 MIP-1α responses. Finally, MIP-1β is a potent agonist for CC CKR3 but not for eosinophils. Neutrophil and monocyte responses to MIP-1α and RANTES also differ from those found for the cloned receptors. For neutrophils, responses to MIP-1α and RANTES can be completely cross-desensitized, yet the magnitude of the neutrophil RANTES response is half that of the neutrophil MIP-1α response at saturating concentrations, whereas for CC CKR1 and CC CKR3 transfectants they are similar(8Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Crossref PubMed Scopus (339) Google Scholar, 9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar) 2 (Fig. 3). RANTES attenuates both the CC CKR1 and CC CKR3 MIP-1α response in transfected cells but not the monocyte MIP-1α response (9Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 21Wang J.M. McVicar D.W. Oppenheim J.J. Kelvin D.J. J. Exp. Med. 1993; 177: 699-705Crossref PubMed Scopus (106) Google Scholar). 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 GROα does not effectively cross-desensitize [Ca2+]i 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 GROα, which are thought to be mediated by IL-8 receptor B(22Moser B. Schumacher C. von Tscharner V. Clark-Lewis I. Baggiolini M. J. Biol. Chem. 1991; 266: 10666-10672Abstract Full Text PDF PubMed Google Scholar, 23Schraufstatter I.U. Barritt D.S. Ma M. Oades Z.G. Cochrane C.G. J. Immunol. 1993; 151: 6418-6428PubMed Google Scholar). Since MIP-1α, RANTES, and MIP-1β are agonists for CC CKR3, they must bind to it. Nevertheless we have not yet been able to demonstrate specific binding of 125I-MIP-1α 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 125I-MIP-1α to CC CKR1-transfected HEK 293 cells (Fig. 5). This suggests that MIP-1α, MIP-1β, 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(5Jose P.J. Griffiths-Johnson D.A. Collins P.D. Walsh D.T. Moqbel R. Totty N.F. Truong O. Hsuan J.J. Williams T.J. J. Exp. Med. 1994; 179: 881-886Crossref PubMed Scopus (769) Google Scholar). Human eotaxin has not been identified yet. The high affinity binding of 125I-MIP-1α 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. The present and previous studies strongly suggest that normal human monocytes and eosinophils respond to MIP-1α and RANTES via two MIP-1α/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-1α and RANTES (24McColl S.R. Hachicha M. Levasseur S. Neote K. Schall T.J. J. Immunol. 1993; 150: 4550-4560PubMed Google Scholar). CC CKR1 may also be responsible in part for the responses of monocytes and eosinophils to MCP-3(4Dahinden C.A. Geiser T. Brunner T. von Tscharner V. Caput D. Ferrara P. Minty A. Baggiolini M. J. Exp. Med. 1994; 179: 751-758Crossref PubMed Scopus (316) Google Scholar, 25Van Damme J. Proost P. Lenaerts J.P. Opdenakker G. J. Exp. Med. 1992; 176: 59-65Crossref PubMed Scopus (345) Google Scholar). Since RNA for CC CKR1 is present in neutrophils, we predict that MCP-3 can elicit [Ca2+]i changes in neutrophils, although this has not been tested yet. The functional significance for eosinophils of the response of CC CKR3 to MIP-1β 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. 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(26Gao J.-L. Murphy P.M. J. Biol. Chem. 1995; 270 (in press)Google Scholar). We thank S. Mawhorter for providing purified eosinophils and H. Lee Tiffany for excellent technical assistance.
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