©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Human Eotaxin, an Eosinophil-selective CC Chemokine, and Identification of a Specific Eosinophil Eotaxin Receptor, CC Chemokine Receptor 3 (*)

(Received for publication, November 1, 1995; and in revised form, January 11, 1996)

Motoji Kitaura (1) Toshihiro Nakajima (1) Toshio Imai (1) Shigenori Harada (1) Christophe Combadiere (2) H. Lee Tiffany (2) Philip M. Murphy (2)(§) Osamu Yoshie (1)(¶)

From the  (1)Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan and (2)The Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma.


INTRODUCTION

Eosinophil accumulation in the blood and affected tissues is a classic characteristic of the inflammatory response to allergens and helminths(1) . Eosinophils are considered to be important effector cells for killing helminths, but are also considered to be responsible for tissue damage in hypersensitivity diseases. Because of the pathologic potential of eosinophils, identification and pharmacologic control of the specific factors regulating their accumulation in vivo is an important goal. In this regard, the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), (^1)RANTES, and monocyte chemoattractant protein (MCP)-3 have potent eosinophil chemotactic activity in vitro(2, 3, 4) , although so far only RANTES has been shown to recruit significant numbers of eosinophils when injected into animals(5) . MIP-1alpha, RANTES, and MCP-3 are ``broad spectrum'' chemokines, targeting neutrophils, monocytes, lymphocytes, and basophils, in addition to eosinophils, with different degrees of selectivity and potency(6, 7, 8, 9) .

Jose et al.(10, 11) recently purified a novel guinea pig CC chemokine and named it eotaxin for its ability to selectively recruit eosinophils but not neutrophils when instilled in the airways and skin of guinea pigs. The guinea pig eotaxin cDNA and a related mouse cDNA have been cloned(11, 12) . Eotaxin mRNA is increased in the lung in the guinea pig model of asthma and the related mouse mRNA is increased in interleukin-4-induced tumor suppression in the mouse(11, 12) . The effect on eosinophil recruitment in vivo appears to be direct since eotaxin also chemoattracts purified guinea pig eosinophils in vitro(10) . By analogy with other chemokines, eotaxin probably acts on eosinophils by binding to one or more subtypes of 7-transmembrane-domain G protein-coupled receptors (13) .

The ability of guinea pig eotaxin to induce calcium flux and chemotactic responses in human eosinophils (10) suggested that a human eotaxin-like signaling system exists. Here, we report the isolation of a cDNA and production of recombinant protein for a novel human CC chemokine named human eotaxin that is most closely related in sequence to human MCP-1 and guinea pig eotaxin. We show that human eotaxin is a selective agonist for human eosinophils and for CC chemokine receptor 3 (CC CKR3), a 7-transmembrane-domain G protein-coupled receptor selectively expressed in eosinophils(14, 15) . No other chemokines tested were agonists for CC CKR3, and eotaxin was not an agonist for the related receptors CC CKR1, CC CKR2B, and CC CKR5(^2)(16, 17, 18) .


EXPERIMENTAL PROCEDURES

Cloning of Human Eotaxin cDNA

To isolate a guinea pig eotaxin cDNA, we designed the following degenerate oligonucleotide primers based on the published amino acid (aa) sequence of guinea pig eotaxin protein(10) : +5`-gcgaattcAT(C/T/A)CC(C/G/T/A)AG(C/T)GCITG(C/T)TG(C/T)TT-3` and -5`-gcgaattcTT(C/G/T/A)GG(G/A)TCIGC(G/A)CA(G/T/A)ATCAT(C/T)TT-3`, where the lower case letters represent sequence included to facilitate subcloning of PCR products. The primers were used in an reverse transcriptase-PCR protocol to amplify from poly(A) RNA prepared from phytohemagglutinin-stimulated guinea pig thymocytes, a 148-bp fragment corresponding to nucleotides 79-227 of the guinea pig eotaxin open reading frame (ORF)(11) . A cDNA was then isolated from a phytohemagglutinin-activated guinea pig thymocyte library (19) using the fragment as probe. The guinea pig eotaxin cDNA, which is essentially identical to the one later reported(11) , was then used to screen a human lymphocyte genomic library (Stratagene, La Jolla, CA) under low stringency conditions (final wash in 0.5 times SSC at 50 °C). Hybridizing restriction fragments were subcloned into pBluescript II KS (Toyobo, Tokyo, Japan) and sequenced. A 217-bp genomic fragment corresponding to exon 3 of a novel human chemokine gene was then used to screen a human small intestine cDNA library (Clontech). A positive phage clone designated 141 was converted to plasmid following the manufacturer's instructions. The cDNA insert was excised by XhoI and XbaI digestion, subcloned into pBluescript II KS, and sequenced on both strands. Methods for analyzing human genomic DNA and RNA, and for chromosome mapping by PCR were as described(19) . The PCR primers specific for human eotaxin were: +5`-CCTCTCACGCCAAAGCTCACA-3` and -5`-TAGGCAACACTCAGGCTCTGG-3`.

Preparation of Recombinant Eotaxin Protein

The clone 141 ORF was amplified by PCR using specific primers incorporating NotI and XbaI cloning sites, and cloned into the baculovirus transfer vector pVL1392 to make pVLEOS-1. Cotransfection of Tn5B1-4 insect cells with BacPAK6 (CLONTECH) and pVLEOS-1 DNA and isolation of recombinant viruses were carried out according to the manufacturer's instructions. All of the following protein purification steps were carried out in 20 mM sodium phosphate buffer at pH 7.5 containing the indicated [NaCl]. Growth medium was collected from Tn5B1-4 cells 48 h after infection with recombinant baculovirus. The medium was dialyzed against 150 mM NaCl, and applied to HiTrap Heparin (Pharmacia Biotech) pre-equilibrated with 150 mM NaCl. The column was washed with 10 volumes of 400 mM NaCl, and bound protein was eluted with 600 mM NaCl. Eluted fractions were diluted with 2 volumes of NaCl-free buffer and applied onto HiTrap SP (Pharmacia Biotech) pre-equilibrated with 300 mM NaCl. The column was washed with 300 mM NaCl and eluted with a linear NaCl gradient from 0.3 to 1 M. Fractions containing recombinant protein were pooled, diluted with 4 volumes of buffer, and concentrated in a Centricon 3 (Amicon). The protein concentration was determined by the BCA kit (Pierce), and purity was checked by silver staining after SDS-PAGE. The purity was >95%. The signal peptide cleavage site was determined by N-terminal sequencing.

[Ca](i) Measurements

Induction of clone 15 HL60 cells was as described previously(20) . Leukocytes were purified from venous blood of normal donors by sedimentation using 1.5% dextran T70 (Pharmacia) in 0.9% NaCl. Eosinophils were negatively selected with anti-CD16-coated immunomagnetic beads (Dynal) after Ficoll-Paque (Pharmacia) separation as described previously(21) . Neutrophils and monocytes were purified as described(22) . The purity of each leukocyte subset, determined by standard cytologic criteria, was >95%. Isolated cells were resuspended in HACM buffer (20 mM Hepes, 125 mM NaCl, 5 mM KCl, 0.5 mM glucose, 0.025% bovine serum albumin, 1 mM CaCl(2), and 1 mM MgCl(2)). Leukocytes were loaded with 0.3 nmol of fura-2/AM (Molecular Probes) per 10^6 cells in 0.5 ml of HACM buffer for 30 min at 37 °C. Cells were washed with HACM buffer, resuspended in HACM buffer at 5 times 10^6 cells/ml, and placed in a continuously stirred cuvette at 37 °C. Cells were excited at 340 and 380 nm in a fluorimeter (Perkin-Elmer) and the relative ratio of fluorescence emitted at 510 nm was recorded every 20 ms. The procedures for creating stable human embryonic kidney (HEK) 293 cell lines expressing CC CKR1, CC CKR2B, CC CKR3, or CC CKR5 were exactly as described(14, 23) .^2 [Ca](i) was measured in fura-2/AM-loaded transfectants as described(14, 23) .^2 Where indicated, fura-2-loaded CC CKR3 tranfectants, 5 times 10^6/ml, were incubated in Bordetella pertussis toxin (List, Campbell, CA), 250 ng/ml in HBSS at 37 °C for 2 h. Cells were washed twice with HBSS and then stimulated. Except for eotaxin, all chemokines were from R& (Minneapolis, MN).

Radioligand Binding Assay

Human eotaxin 5 µg was iodinated with chloramine T using Iodo-Beads (Pierce) following the manufacturer's instructions. The iodinated protein migrated as a single 12-kDa band on an 8-16% acrylamide gel, and had a specific activity of 1400 Ci/mmol. 10^6 transfected cells expressing CC CKRs were incubated with the radioligand in the presence or absence of excess unlabeled eotaxin 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 or 37 °C, cells were pelleted through a 10% sucrose, phosphate-buffered saline cushion and counted in a -counter.


RESULTS

Cloning of a cDNA for Human Eotaxin

Of 24 clones isolated from the human genomic library, 21 were identical to previously described members of the MCP subgroup of CC chemokines, and 3 were for a novel chemokine gene. A single cDNA (clone 141) corresponding to the novel gene was then isolated from a human small intestine cDNA library. Clone 141 contained an insert of 859 bp (Fig. 1). An ORF of 291 bp encoding a 97-aa polypeptide starts with the first ATG, which is in a sequence context that conforms to the consensus rules for eukaryotic translational initiation sites(24) . The 3`-untranslated region ends with a poly(A) tail of 52 nucleotides starting at position 808 and contains a single polyadenylation signal of a rare type (ATTAAA)(25) . Two potential mRNA destabilization signals (ATTTA) are present in tandem. The first 23 aa of the polypeptide are hydrophobic and probably constitute a leader sequence. The following 74 aa are colinear with mature native guinea pig eotaxin, and contain four cysteine residues with the first two adjacent, the signature of a CC chemokine. Based on the properties detailed below, we have named the molecule human eotaxin.


Figure 1: Nucleotide and deduced aa sequences of the human eotaxin cDNA. The probable signal sequence is underlined. The stop codon is indicated by an asterisk. A potential mRNA destabilization signal is double underlined. The polyadenylation signal is underlined in bold. The nucleotide and deduced aa sequences are available from DDBJ/GenBank/EMBL under the accession number D49372.



Homology of Human Eotaxin with Other Chemokines

The human eotaxin precursor sequence is 70 and 69% identical to human MCP-1 and MCP-3(6, 26, 27, 28) , respectively, and 62 and 59% identical to guinea pig and mouse eotaxin, respectively (11, 12) (Fig. 2). This is a high degree of cross-species sequence divergence for orthologous proteins, even for chemokines which as a rule tend to be quite divergent. Rothenberg et al.(12) have pointed out three sequence features common to guinea pig eotaxin and a putative mouse eotaxin that differ from MCP-1, -2, and -3(12) . First, the MCPs all have glutamine at aa 1 of the mature protein, whereas the corresponding residue is histidine for both guinea pig and mouse eotaxin; human eotaxin has glycine at this position. Second, mature guinea pig eotaxin and the deduced mouse eotaxin-like sequence have a gap of 2 aa N-terminal to the CC motif relative to the MCPs; human eotaxin has a gap in the same location. Third, guinea pig and mouse eotaxin have three consecutive lysine residues in the region C-terminal to the fourth cysteine, whereas the MCPs have only 1 or 2 lysines in the corresponding region; human eotaxin has all three conserved lysines (Fig. 2). These criteria suggest a closer sequence relationship of human eotaxin with guinea pig and mouse eotaxin than with the MCPs.


Figure 2: Sequence alignment of human eotaxin with closely related chemokines(10, 11, 12, 25, 26, 27) . Boxed aa, identities; dashes, gaps; gp, guinea pig; mu, mouse; arrowhead, signal peptide cleavage site of recombinant human eotaxin. Numbers on top of the aligned sequence enumerate the human eotaxin sequence and are left justified. The % identity of each whole and mature sequence relative to human eotaxin is at the bottom right.



Genes Related to Human Eotaxin

A guinea pig eotaxin cDNA probe hybridized with at least five restriction fragments of human genomic DNA at low stringency (Fig. 3A). We have determined that four of them correspond to genes for MCP-1, MCP-2, MCP-3 (data not shown), and human eotaxin (Fig. 3A, lanes 3 and 4), respectively. The fifth band could represent a gene even more closely related to guinea pig eotaxin than the human eotaxin gene. PCR analysis of genomic DNA from rodent-human somatic cell hybrid mapping panels (19) with primers specific for human eotaxin revealed that the presence of the gene correlated only with hybrids containing human chromosome 17 with 0% discordance (Fig. 4). Thus, the gene is located on human chromosome 17 as are all the other mapped CC chemokine genes(2) .


Figure 3: Human eotaxin gene, RNA, and recombinant protein. A, gene. Southern blots containing 20 µg of human genomic DNA per lane digested with EcoRI (lanes 1 and 3) or HindIII (lanes 2 and 4) were hybridized at low stringency (final wash: 0.2 times SSC at 55 °C) with the guinea pig eotaxin full-length ORF probe (left) or at high stringency with a 217-bp probe from exon 3 of the human eotaxin gene (right). The location of size standards in kilobase pairs (kbp) is indicated at the left of each panel. Asterisks and arrowheads indicate restriction fragments of the human eotaxin gene. B, RNA distribution. Lanes contain 2 µg of poly(A) RNA from the following tissues: 1, heart; 2, brain; 3, placenta, 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon; and 16, peripheral blood leukocytes. The RNA was analyzed by Northern blot hybridization using a clone 141 ORF cDNA probe. The filter was washed in 0.2 times SSC at 55 °C and autoradiographed at -80 °C overnight using an intensifying screen. C, recombinant protein. A silver-stained gel containing 3 µg of recombinant protein produced in insect cells analyzed by SDS-PAGE is shown.




Figure 4: Chromosomal mapping of the human eotaxin gene. A total of 20 rodent-human somatic cell hybrids were analyzed for the presence of the human eotaxin gene by using specific PCR. Karyotyping symbols are as follows: +, chromosome present in the cell line; M, chromosome present but at low frequency; D, chromosome present but with multiple deletions; blank, chromosome absent. D% is the percent discordance between the presence of a chromosome and the PCR detection of the human eotaxin gene.



Eotaxin mRNA Distribution

Northern blot analysis of eotaxin mRNA in various human tissues revealed that two distinct transcripts, 1.1 and 0.85 kilobases long, are expressed constitutively at high levels in colon and small intestine, at intermediate levels in heart, and at low levels in kidney, pancreas, and some other organs (Fig. 3B).

Production of Recombinant Human Eotaxin

Recombinant human eotaxin protein was produced in insect cells using a baculovirus vector. After purification, the protein appeared as a single band of 12 kDa by SDS-PAGE (Fig. 3C). Its sequence began at aa 24 of the ORF, indicating cleavage of a 23-aa leader in insect cells.

Eosinophil Selectivity of Human Eotaxin

We monitored [Ca](i) by fura-2 fluorescence to identify cell types responsive to eotaxin (Fig. 5). This response is strongly associated with the chemotactic responses of leukocytes to chemokines(2, 13) . Human eotaxin was active on normal eosinophils (EC = 2 nM; n = 2), but not on normal neutrophils or monocytes (Fig. 5). Clone 15 HL60 cells treated with butyric acid also exhibited calcium flux responses when stimulated with eotaxin. These cells resemble normal peripheral blood eosinophils in morphology, and express eosinophil-specific granule proteins such as eosinophil-derived neurotoxin and eosinophil cationic protein(20) . Moreover, they respond to MIP-1alpha, RANTES, and MCP-3 (data not shown), the same CC chemokines that activate peripheral blood-derived eosinophils. Van Riper et al.(29) have reported that a related HL60 cell derivative expresses functional MIP-1alpha and RANTES binding sites when treated with butyric acid. The other myeloid cell lines tested have properties that resemble leukocyte subtypes other than eosinophils, and they failed to respond to eotaxin. Instead they responded to chemokines that target the corresponding normal myeloid cell types (Table 1). The butyric acid-treated clone 15 HL60 cells responded to eotaxin at concentrations as low as 1 nM (not shown).


Figure 5: Eosinophil selectivity of recombinant human eotaxin. [Ca] was monitored by ratio fluorescence in the fura-2-loaded leukocyte subtypes indicated to the left of each row of tracings. Arrows mark the time of addition of chemokines at the indicated concentration. The tracings shown are from one experiment representative of at least three experiments.





CC CKR3 Is a Human Eotaxin Receptor

Sequential stimulation experiments indicated that the response of normal human eosinophils to eotaxin underwent homologous desensitization, whereas the response was unaffected by prestimulation with MIP-1alpha or MCP-3, suggesting a unique receptor (not shown). To date, six human CC CKRs have been cloned and characterized (Table 2)(15, 16, 17, 18, 30) .^2 They all are 7-transmembrane domain receptors and have 45-75% aa identity. Each is a receptor for a unique set of one or more CC chemokine agonists, but the compositions of the sets overlap in ways that are not predictable from the sequence relationships. Thus, CC CKR1 is a receptor for MIP-1alpha, RANTES, and MCP-3(16, 17, 23, 31) ; CC CKR2A for MCP-1(18) ; CC CKR2B for MCP-1 and MCP-3(18, 23, 31, 32) ; CC CKR4 for MIP-1alpha, RANTES, and MCP-1(30) ; and CC CKR5 for MIP-1alpha, MIP-1beta, and RANTES.^2 RNA for all of these is detectable in monocytes(23, 30, 33) .^2 Evidence for eosinophil expression in the form of RNA detection on Northern blots has only been obtained for CC CKR1(33) .



In contrast, the related orphan receptor CC CKR3 is expressed selectively in eosinophils, making it an excellent candidate for an eotaxin receptor(14, 15) . When calcium flux was measured, three independent HEK 293 cell lines stably transfected with CC CKR3 responded to eotaxin, but not to any other chemokines tested at 100 nM (Fig. 6A). Conversely, MIP-1alpha, RANTES, MIP-1beta, MCP-1, and MCP-3 were agonists for their appropriate receptors, CC CKR1, CC CKR2B, and CC CKR5, whereas eotaxin was not (Fig. 6A and data not shown).


Figure 6: CC CKR3 is a human eotaxin receptor. A, specificity. Each tracing represents [Ca] levels of fura-2-loaded HEK 293 cell lines expressing the CC CKR indicated to its left, measured as relative fluorescence over time. Arrows mark the time of addition of chemokines at 100 nM coded as follows: a, MIP-1alpha; b, RANTES; c, MIP-1beta, d, MCP-1; e, MCP-3; f, human eotaxin. The data are from one experiment representative of at least three experiments with 4 separate clones for CC CKR3. CC CKR3 transfectants were tested individually with the chemokines shown also with negative results (not shown). B, potency. Each data point represents the peak of the calcium flux response to eotaxin in CC CKR3-transfected HEK 293 cells. The data are from a single experiment representative of three separate experiments with two independent cell lines expressing CC CKR3. C, mechanism. CC CKR3 transfectants were incubated in pertussis toxin at the concentration indicated at the left of each tracing, and then stimulated with 25 nM eotaxin (left arrow) and 2 µM ATP (right arrow).



Sequential stimulation experiments indicated that the eotaxin response underwent homologous desensitization, whereas the response was unaffected by prestimulation with other chemokines (Fig. 6A). Conversely, eotaxin did not affect the response of CC CKR1, CC CKR2B, or CC CKR5 to their respective agonists. The EC for induction of the calcium flux response by eotaxin in CC CKR3 transfected cells was 5 nM, consistent with the sensitivity of the native receptor to eotaxin (Fig. 6B). When stable CC CKR3 transfectants were pretreated with pertussis toxin, 250 ng/ml, the calcium flux response to eotaxin but not to ATP was abolished, suggesting that a G(i) type G protein is involved in eotaxin signal transduction (Fig. 6C).

We infer by analogy with the agonists for other G protein-coupled receptors that eotaxin induces calcium flux responses in transfected HEK 293 cells by binding specifically to CC CKR3 on the plasma membrane. However, so far we have been unable to show specific I-human eotaxin binding. When we tested 10^6 cells and concentrations of the radioligand ranging from 0.1 to 5 nM at 4 or 37 °C, total binding was the same for untransfected and CC CKR3-transfected HEK 293 cells. Using identical radiolabeling and binding conditions, and radioligands similar in specific activity to the I-eotaxin used here, we have been able to show specific binding of I-MIP-1alpha, -MIP-1beta, -MCP-1, -RANTES, and -MCP-3 to their respective receptors CC CKR1, CC CKR2B, CC CKR5, a mouse MIP-1alpha receptor, and a human cytomegalovirus CC chemokine receptor (Refs. 23, 34, and 35, and data not shown).^2 We conclude that the levels of expression of CC CKR3 and/or its affinity for eotaxin are low, preventing detection of the binding site.

Jose et al.(10) reported that I-human RANTES bound to guinea pig eosinophils, and that guinea pig eotaxin could partially compete for the binding site, suggesting a common receptor for eotaxin and RANTES. However, we have been unable to show specific I-RANTES binding to CC CKR3-transfected cells, and RANTES at concentrations as high as 100 nM is not an agonist for CC CKR3 capable of inducing a calcium flux response in transfected HEK 293 cells.


DISCUSSION

The present work establishes the cDNA and deduced aa sequences, chromosome location, RNA distribution, functional expression, and receptor selectivity for human eotaxin. Previous results in guinea pig and mouse and our data in human together indicate that an eotaxin signaling system is broadly conserved in mammalian species. The only biological function currently established for eotaxin is regulation of eosinophil trafficking during allergic airway and skin inflammation in the guinea pig(10) . While our data also indicate high eosinophil selectivity for human eotaxin, more work will be needed to determine whether additional eotaxin functions exist and whether those established in the guinea pig have been conserved during mammalian evolution.

Several features of human eotaxin and its receptor CC CKR3 are unusual when compared to other chemokines and chemokine receptors. First, most chemokine receptors have multiple chemokine agonists, whereas CC CKR3 appears to be monospecific for eotaxin (Table 2). Conversely, most chemokines recognize more than one CC CKR, whereas eotaxin appears to be monospecific for CC CKR3. Second, all CC chemokines except eotaxin can activate monocytes. Third, all CC CKRs except for CC CKR3 are expressed in monocytes, whereas only CC CKR1 and CC CKR3 are expressed in eosinophils. Taken together, these features suggest that CC CKR3 is a specific factor responsible for eotaxin's high cellular selectivity for eosinophils.

Fourth, chemokines are highly inducible genes and are typically not expressed at high constitutive levels, whereas human eotaxin RNA is constitutively present at quite high levels in small intestine and colon. Eosinophils are present in large numbers in the normal gastrointestinal tract and play a major role in host defense against helminthic parasites(36) ; perhaps eotaxin regulates this function. Eotaxin may also contribute to pathogenesis of certain inflammatory bowel diseases associated with increased numbers of eosinophils(37) . It is intriguing that eotaxin RNA is also present at relatively high levels in heart. Eotaxin may have unknown functions in normal heart and may play important roles in eosinophilic endomyocardial disease, a grave complication of chronic eosinophilia(37) .

Fifth, the sequence of human eotaxin is much closer to the MCPs than would be expected given their functional divergence, and much further from guinea pig eotaxin than would be expected for orthologous genes. Even the signal peptides of human eotaxin and the MCPs are similar. It is likely that human eotaxin is the orthologue of guinea pig eotaxin. However, it could be a member of the MCP branch that has evolved to be selective for eosinophils, converging functionally with the eotaxins. Human eotaxin, like most other CC chemokines, is highly basic, containing a net charge of +11. CC CKR3, like most other chemokine receptors, has an acidic N-terminal extracellular segment, but unlike most other chemokine receptors, also has an acidic second extracellular loop. It will be interesting to study whether these charge distributions are binding determinants.

Based on the data reported here, it is reasonable to hypothesize that eotaxin and CC CKR3 regulate eosinophil accumulation at body sites undergoing allergic reactions, metazoan infestation, or other types of inflammation where eosinophils are present in large numbers. Additional studies will be needed to show this, and to determine the importance of these molecules relative to other known eosinophil chemoattractants, including the broader spectrum CC chemokines MIP-1alpha, RANTES, and MCP-3, and the other known eosinophil chemokine receptor CC CKR1.


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 may be addressed: The Laboratory of Host Defenses, NIAID, Bldg. 10, Rm. 11N113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-480-2037; Fax: 301-402-0789; pmm{at}d10.niaid.pc.niaid.nih.gov.

To whom correspondence may be addressed: Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan. Tel.: 06-382-2612; Fax: 06-382-2598; osamu.yoshie{at}shionogi.co.jp.

(^1)
The abbreviations used are: MIP, macrophage inflammatory protein; aa, amino acid; bp, base pair(s); RANTES, regulated on activation, normal T expressed and secreted; MCP, monocyte chemoattractant protein; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; ORF, open reading frame; CC CKR, CC chemokine receptor; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis.

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


ACKNOWLEDGEMENTS

We thank Mayumi Kakizaki and Yumi Kubo for excellent technical assistance. We are also grateful to Drs. Yorio Hinuma and Masakazu Hatanaka for constant support and encouragement.


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