I. Overview

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The aim of this article is to describe the nomenclature system for chemokine receptors, as approved by the Nomenclature Committee of the International Union of Pharmacology (NC-IUPHAR),² and to update their main molecular and biological properties. A general overview is given first, followed by a synopsis of key information about each receptor, with an emphasis on recent discoveries and new concepts.

Chemokine receptors are defined by their ability to signal on binding one or more members of the chemokine superfamily of chemotactic cytokines (Premack and Schall, 1996; Baggiolini et al., 1997; Yoshie et al., 1997; Luster, 1998; Zlotnik et al., 1999). To date, 18 human proteins have met this definition, and they have been designated CXCR1 through 5, CCR1 through 11, XCR1, and CX3CR1 based on their specific chemokine preferences, as described in subsequent sections. Together, chemokine receptors comprise a large branch of the rhodopsin family of cell surface, seven-transmembrane domain (7TMD), G protein-coupled receptors (GPCRs). In addition, D6 and Duffy (sometimes called the Duffy antigen receptor for chemokines, or DARC) are 7TMD mammalian chemokine-binding proteins that apparently do not signal and therefore are excluded from the systematic nomenclature (Horuk, 1994; Nibbs et al., 1997a).

To date, chemokine receptor-like sequences have been identified in mammals, birds (Gupta et al., 1998a), and fish (Daniels et al., 1999) but not in invertebrates, plants, yeast, or bacteria, suggesting a relatively recent origin. Common features include conserved structure [25-80% amino acid (aa) identity], coupling to the G_i class of G proteins, expression in leukocytes, and chemotactic signaling. The major shared biological function is leukocyte trafficking and dependent processes such as immune surveillance, innate and adaptive immune responses, and various forms of pathological inflammation (Springer, 1994; Foxman et al., 1997). Within this general area, however, each chemokine receptor appears to have a specific role, determined by its expression pattern on specific subsets of leukocytes, and by the temporal and spatial specificity of cognate ligand expression. Specific roles have also been delineated in hematopoiesis (Broxmeyer et al., 1996, 1999; Reid et al., 1999), angiogenesis (Salcedo et al., 1999), development (Forster et al., 1996; Nagasawa et al., 1996; Ma et al., 1998; Tachibana et al., 1998; Zou et al., 1998), and, counterintuitively, facilitation of certain infectious diseases.

With regard to the latter, two major themes have been defined. In the first, cellular chemokine receptors are exploited as cell entry and disease transmission factors by intracellular pathogens. Rigorously proved examples of this are the human immunodeficiency virus (HIV) coreceptor CCR5 in acquired immune deficiency syndrome (AIDS) and Duffy in the form of malaria caused by Plasmodium vivax; CXCR4 and other chemokine receptors also function as HIV coreceptors, but their importance in disease is not established (Horuk et al., 1994; Rucker et al., 1997; Berger et al., 1999). The second theme, which is not as well understood, involves herpesvirus- and poxvirus-encoded chemokines and chemokine receptors, evidently acquired as copied host genes, which may subvert the immune response or dysregulate cell growth (reviewed in Pease and Murphy, 1998).

Apart from Duffy in malaria and CCR5 in HIV/AIDS, disease indications have not yet been unequivocally established for chemokine receptors. Rapid progress in this area can be anticipated in the near future as receptor knockout mice and receptor-blocking agents are tested in animal models of disease. To date, only CXCR4 has been shown to be essential for life. Phenotypes of knockout mice for other chemokine receptors are more subtle in the absence of specific stresses (Gerard, 1999). Many types of chemokine and chemokine receptor-blocking agents of high and low selectivity have been discovered, including viral chemokine scavengers, viral chemokine antagonists, antagonistic chemokine variants, small molecules, ribozymes, intrakines, and monoclonal antibodies (mAbs) (Schwarz and Wells, 1999). Moreover, a novel HIV vaccine has been discovered in which CCR5 is a critical component (Lacasse et al., 1999). However, as of this writing, none of these has been tested in a clinical trial.

	II. Introduction

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To understand chemokine receptors, first their ligands must be explained. Chemokines are perhaps the most complex of GPCR ligands because of their large number, overlapping receptor specificity, and extensive phylogenetic divergence. To date, more than 40 different human chemokines have been identified, with the first identified in 1977, when Walz et al. (1977) sequenced native platelet factor 4, a procoagulant and angiostatic factor stored in platelet -granules. Subsequently, from 1984 through 1989, cDNAs for structurally related proteins, including IP-10 (see Table 1 for chemokine acronyms; synonyms and chemokine classes are given in Table 2), JE, Mig, RANTES (regulated on activation, normal T cell expressed and secreted), I-309, KC, and macrophage inflammatory protein-1 (MIP-1), were cloned by investigators looking mainly for cell differentiation- and activation-associated genes, establishing the existence of a gene family before identifying any functions (Wolpe and Cerami, 1989; Schall, 1991; Oppenheim et al., 1991).

The discovery of the neutrophil-targeted chemokine interleukin (IL)-8 represents a landmark in immunology because it was the first leukocyte subtype-selective chemoattractant to be found (Yoshimura et al., 1987; Walz et al., 1987). The discovery of IL-8 also focused the search for functions for other chemokines on leukocyte chemotaxis and stimulated a search for new family members. Interest in the field grew with subsequent reports of MCP-1, RANTES, and eotaxin, the first important monocyte-, T cell-, and eosinophil-directed chemokines, respectively (Matsushima et al., 1989; Yoshimura et al., 1989; Schall et al., 1990; Jose et al., 1994). Methods of chemokine discovery expanded to include purification of chemoattractant activity as well as cDNA cloning by signal sequence trapping, homology hybridization, and, most recently, bioinformatics and expressed sequence tag (EST) analysis (Tashiro et al., 1993, 1999; Wells and Peitsch, 1997). Chemokines are particularly easy to find in EST databases because their coding sequences are sufficiently small, typically 70 to 90 codons, to be captured by a single EST and because their conserved sequence motifs are easy to recognize (see later). As the number of family members expanded, various short-lived collective terms for them were used, including "the platelet factor-4 family" (Wolpe and Cerami, 1989), "the small inducible cytokine family" (Schall, 1991), and "the intercrines" (Oppenheim et al., 1991). Finally, in 1992 at the Third International Symposium on Chemotactic Cytokines in Baden, the term "chemokine," a neologism short for "chemotactic cytokines," was coined and accepted as the standard (Lindley et al., 1993).

With respect to leukocyte specificity, both broad- and narrow-spectrum chemokines have been identified. Together they cover the full spectrum of leukocytes, acting through a signaling pathway that includes a pertussis toxin-sensitive G protein (G_i/G_o), calcium flux, and chemotaxis. This fact pointed to use of GPCRs and suggested homology hybridization as a strategy to identify them (reviewed in Murphy, 1996), which has been highly successful.

In 1995, an NC-IUPHAR subcommittee on chemokine receptor nomenclature was organized. Recommendations developed at the second Gordon Conference on Chemotactic Cytokines held in Plymouth, NH, in 1996, were accepted unanimously by meeting participants, ratified by NC-IUPHAR in January 1997, and widely used since. The nomenclature is based on the subclassification of the chemokine superfamily, delineated in the next section. In 1998, a second nomenclature committee, led by O. Yoshie and A. Zlotnik, was formed to address the proliferation of chemokine aliases that has accompanied the codiscovery of chemokines by multiple groups using bioinformatics (Table 1). A nomenclature system that parallels the receptor nomenclature was proposed at the Keystone Symposium on Chemokine and Chemokine Receptors, January 18 to 23, 1999, in Keystone, CO (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 The chemokine family A systematic chemokine nomenclature, based on protein structure and a previous nomenclature for chemokine gene loci, was developed by A. Zlotnik and O. Yoshie to deal with the proliferation of synonyms that has attended chemokine discovery and was proposed at the Keystone Symposium on Chemokines and Chemokine Receptors, Keystone, CO, 1999. At present, the systematic names refer only to human chemokines, in part because of uncertainties regarding the identity of mouse orthologs. Nevertheless, the table also includes accession numbers and common names for putative mouse orthologs. In cases such as CCL6, CCL9, and CCL12, where a mouse chemokine lacks a known human ortholog, the standard name is reserved for the potential human counterpart, although it may not exist due to lineage-specific gene duplication. In many cases, the same common name applies to human and mouse counterparts. In others, species-specific names are preferentially used to convey substantially different properties, such as a major difference in sequence (e.g., human I-309 versus mouse TCA-3) or length (e.g., mouse JE versus MCP-1). The number in the systematic name for each chemokine matches that in an alias for the corresponding human gene name, and the roots for gene names correspond to protein roots as follows: SCYA = CCL, SCYB = CXCL, SCYC = XCL, and SCYD = CX3CL, where SCY denotes small cytokine; A, B, C, and D denote the chemokine classes in the gene locus; and L denotes "ligand" in the root of the protein name. Thus, for example, SCYB1 is a gene alias for the human chemokine CXCL1. Accession numbers are from the SwissProt database, when available; N.A. indicates not available in any database. A discussion of tissue and cell sources and regulation for the chemokines is beyond the scope of this article but can be found in Oppenheim et al. (2000).

Chemokines can be subclassified by structure according to the number and spacing of conserved cysteines into four major groups, given the preferred names CXC, CC, C, and CX3C, which are used in the systematic nomenclatures (Tables 2 and 3). Less commonly these groups are referred to by the Greek letters , , , and , respectively. CXC, CC, and CX3C chemokines all have four conserved cysteines, whereas C chemokines have only two, corresponding to the second and fourth cysteines in the other groups (Fig. 1). A small subgroup of CC chemokines has six cysteines. CXC and CX3C chemokines are distinguished by the presence of one (CXC) or three (CX3C) aa between the first and second cysteines, whereas the first two cysteines of CC chemokines are adjacent. Both the CC and CXC groups have many known members, whereas human lymphotactin  and  (Kelner et al., 1994) and fractalkine (Bazan et al., 1997) and their equivalents in other species are the only known examples of C and CX3C chemokines, respectively. A cDNA encoding a CXC chemokine-like protein has been discovered in lamprey, suggesting that the origin of the family, and possibly the division into subclasses, is ancient (Najakshin et al., 1999).

View this table: [in this window] [in a new window]   TABLE 3 Chemokine receptors: nomenclature, pharmacology and biology N.D., not determined; N.A., not available. Two splice variants affecting the ORF have been identified for each of the following receptors: CXCR4, CXCR5, CCR2, and CCR9; however, biological or pharmacological significance has not been determined. Other HIV coreceptors include US28 (Pleskoff et al., 1997), the leukotriene B4 receptor (Owman et al., 1998), and the orphans Apj (Choe et al., 1998), BOB/GPR15 (Deng et al., 1997; Farzan et al., 1997), STRL33/Bonzo/TYMSTR (Deng et al., 1997; Liao et al., 1997; Loetscher et al., 1997), GPR1 (Farzan et al., 1997), ROC1 (Shimizu et al., 2000); and ChemR23 (Samson et al., 1998).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structural classification of the chemokine family by signature cysteines. The number of members in each subclass is listed at the right of each structure. Underlines indicate gaps in the alignment; X, an amino acid other than cysteine; and dots, other amino acids. Spacing between cysteines is similar in all four groups. The N and C termini can vary in length.

Fractalkine is an interesting model for how chemokines may be presented to target cells. It has a multimodular structure consisting of a chemokine domain fused to a mucin-like stalk plus a transmembrane domain, which anchors the molecule to the plasma membrane, and a cytoplasmic domain. Consistent with this, it functions as an adhesion molecule by binding directly to CX3CR1 (Imai et al., 1997b). Fractalkine also induces cell migration as either a tethered or shed ligand. Although other chemokines lack a transmembrane domain and are secreted, they are able to use glycosaminoglycans for tethering to plasma membrane. This provides a mechanism for gradient formation under conditions of high blood flow. Once "posted" in this manner, chemokines may be "read" by passing leukocytes, which then activate ₂-integrins, bind to endothelium and transmigrate from blood to tissue (Tanaka et al., 1993). However, fractalkine is the only chemokine shown to act as a direct cell adhesion molecule.

CXC chemokines can be further subclassified by structure into ELR+ and ELR molecules based on the presence or absence of the tripeptide motif glutamic acid-leucine-arginine (ELR) N-terminal to the first cysteine. This provides the only strong functional correlate of the structural classification: the specificity of ELR+ CXC chemokines for neutrophils (Hebert et al., 1991). A second classification scheme based on function and expression pattern has also been proposed. It includes an inflammatory/inducible group, which is regulated by proinflammatory stimuli such as lipopolysaccharide and primary cytokines such as IL-1 and tumor necrosis factor, and which together orchestrate innate and adaptive immune responses; a homeostatic/constitutive group, which is important in lymphocyte and dendritic cell trafficking in immune surveillance (Cyster, 1999a,b); and an overlap group. Genes encoding inflammatory chemokines are typically found in two major clusters on human chromosomes 4 (CXC) and 17 (CC), whereas genes for homeostatic chemokines are located alone or in small clusters on chromosomes 1, 2, 5, 7, 9, 10, and 16. Homeostatic receptors include CXCR4, CXCR5, CCR4, CCR7, and CCR9. Inflammatory receptors include CXCR1, CXCR2, CXCR3, CCR1, CCR2, CCR3, CCR5, and CCR6.

Although most chemokine receptors recognize more than one chemokine, they are almost always restricted to a single subclass (Table 4). Thus, the nomenclature system is rooted by the chemokine subclass specificity of the receptor. Human CC and CXC chemokine receptor names consist of the root CCR or CXCR, respectively, followed by a number. The lymphotactin and fractalkine receptors are named XCR1 ["X" to distinguish it from complement receptor 1 (CR1)] and CX3CR1, respectively. The use of the letter "R" in receptor names is nonstandard for pharmacologists but is widely accepted practice for immunologists and was therefore authorized as an exception by NC-IUPHAR. Thus, these receptors are referred to as, for example, "CXCR1," and not "the CXCR1 receptor," which would be redundant. Splice variants, if pharmacologically distinct, are designated by a lowercase letter starting from the beginning of the alphabet subscripted in parentheses after the receptor name. Species orthologs are indicated by an appropriate species abbreviation followed by a space before the receptor name (Vanhoutte et al., 1998). By consensus agreement of the conferees at the 1996 Gordon Conference on Chemotactic Cytokines, new names are assigned by a committee composed of Phil Murphy (pmm{at}nih.gov), Craig Gerard (gerard_c{at}gonzo.tch.harvard.edu), and Tom Schall (tschall{at}chemocentryx.com).

Like the receptor names, systematic chemokine names, shown in Table 2 with their corresponding common names, are also built from cysteine subclass roots, followed by "L" for "ligand" and a number. In general, the numbers correspond to the same number used in the corresponding gene nomenclature, which takes the form "SCY" for "small cytokine," followed by "A", "B", "C," or "D" for "CC", "CXC", "C," or "CX3C" subclass, respectively, followed by the number.

Analysis of chemokine receptors presents problems not faced with other types of GPCRs. Most imposing is the large number of receptors and endogenous ligands and their overlapping specificities for each other and for leukocyte subtypes. In addition, both chemokines and their receptors may vary markedly in sequence among species, as much as 55% aa divergence in the case of certain chemokines. As a result, even though chemokine orthologs from different species usually cross-activate receptors, the receptors may have markedly different biology and pharmacology. Even the repertoire of chemokine and chemokine receptors may differ in different species. For example, mouse orthologs of IL-8 and CXCR1 have not been found, and there is persuasive evidence in the case of IL-8 that a mouse form does not exist (Modi and Yoshimura, 1999). Why these molecules are evolving so rapidly is unknown, but it is a property shared fairly selectively with the class of genes involved in immunity and inflammation (Murphy, 1993).

The sequences of chemokine receptors have 25 to 80% aa identity (Fig. 2), indicating a common ancestor. However, many other G protein-coupled peptide receptors also have ~25% aa identity to chemokine receptors, illustrating that the structural boundary is not sharp. Although they lack a single structural signature, there are several features that together are found more frequently among chemokine receptors than other types of GPCRs. These include a length of 340 to 370 aa; an acidic N-terminal segment; the sequence DRYLAIVHA, or a variation of it, in the second intracellular loop; a short basic third intracellular loop; and a cysteine in each of the four extracellular domains. A tyrosine sulfation motif is commonly found in the N terminus of chemokine receptors and has been shown to be critical for HIV coreceptor activity for CCR5 (Farzan et al., 1999).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Structural relationships among human chemokine receptors. The dendrogram was constructed by Marc Rothenberg using default parameters in the PILEUP algorithm of the University of Wisconsin Genetics Computer Group.

The three-dimensional structure of chemokine receptors is unknown, but a reasonable working model can be constructed for the transmembrane domains based on analogy with rhodopsin (Baldwin, 1993; Unger et al., 1997; Lomize et al., 1999). Models of the extracellular and intracellular domains are completely speculative, although in some cases domain-specific antibodies have verified the general location. Evidence has been reported that CCR2, CCR5, and CXCR4 form homodimers (Benkirane et al., 1997; Lapham et al., 1999; Rodriguez-Frade et al., 1999), and in the case of CCR2, a dimer has been implicated as the functional form of the receptor, which may be needed for signaling.

In contrast, many chemokine structures have been determined, including both CC and CXC subtypes, and a common fold is apparent (Clark-Lewis et al., 1995; Clore and Gronenborn, 1995). The N terminus before the first cysteine is structurally disordered, whereas the C terminus after the last cysteine is -helical. The remainder of the molecule is constrained by disulfide bonding between the first and third and the second and fourth cysteines and contains three -sheets separated by short loops arranged in the shape of a Greek key. The backbone structures are largely superimposable. Chemokines appear to act as monomers, despite the fact that in most cases they are dimers or higher-order multimers at high concentrations or in crystals (Baggiolini et al., 1997).

The N terminus is not usually important for high-affinity receptor binding but is typically critical for receptor triggering. Native chemokines purified from biological material often exist as families of peptides derived from the same gene that differ in the length of the N- and C-terminal domains, which in some cases has been attributed to the action of specific proteases such as CD26 (a prolylpeptidase) and cathepsin G (Walz and Baggiolini, 1990; Oravecz et al., 1997). The length of the N-terminal segment is important in determing whether a given chemokine binds to receptor, and if so, whether it functions as an agonist or antagonist. Truncation may also cause a switch in receptor specificity as in the cases of NAP-2 and MCP-1.

Each chemokine receptor has a distinct chemokine and leukocyte specificity (Table 4), but the specificities can overlap considerably, because some chemokines can bind multiple receptor subtypes, and some receptors can bind multiple chemokines. Mutagenesis has indicated that the ligand binding site of chemokine receptors is highly complex, being composed of multiple noncontiguous domains and at least two distinct subsites: one for docking and the other for triggering (Ahuja et al., 1996; Monteclaro and Charo, 1996; Crump et al., 1997). At least for CCR5 and CXCR4, the first two TMDs and associated loops, but not the N-terminal segment, appear to be dispensable for normal receptor expression and function (Ling et al., 1999). Multiple low-affinity interactions together provide the high-affinity binding energy. A conserved HIV gp120 glycoprotein structure has been solved that is involved in chemokine receptor binding (Rizzuto et al., 1998).

Inflammatory chemokines (mainly those encoded by the chromosome 4 and 17 clusters of genes) have highly promiscuous relationships with receptors. There are fewer homeostatic chemokines, but those that map to the same chromosome tend to bind to the same receptor [e.g., MDC and TARC at CCR4; EBI ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC) at CCR7]. Recently, the number of monogamous chemokine ligand-receptor relationships, which had previously been regarded as exceptional, has risen substantially (e.g., SDF-1 and CXCR4; TECK and CCR9; BLC and CXCR5; LARC and CCR6; lymphotactin and XCR1; fractalkine and CX3CR1).

Adding to the complexity of the system, distinct receptor subtypes specific for the same chemokine and the same function can be coexpressed on the same cell (Morohashi et al., 1995), distinct chemokines acting at separate receptors coexpressed on the same cell can induce the same cellular response (Zaitseva et al., 1997), and the same receptor can sort signals from different ligands to distinct signaling pathways (Zhang et al., 1999). Also, chemokine receptors are not limited to leukocytes but in specific cases may also be expressed on endothelial cells, neurons, epithelial cells, and microglial cells of the brain (Hadley et al., 1994; He et al., 1997; Horuk et al., 1997; Gupta et al., 1998b; Salcedo et al., 1999). There is intense interest in understanding the biological roles of these receptors in these ectopic sites.

With rare exceptions (Blanpain et al., 1999), functional human chemokines are agonists at leukocyte receptors. In contrast, naturally occurring chemokine antagonists have only been found in viruses (Table 5). For example, the viral chemokines MC148R from the poxvirus Molluscum contagiosum virus (Damon et al., 1998) and vMIP-II from human herpesvirus 8 (HHV8) (Kledal et al., 1997) are broad-spectrum chemokine receptor antagonists, suggesting roles in immune evasion and the importance of normal chemokine signaling for antiviral host defense. Various orthopoxviruses (e.g., myxoma, vaccinia) deploy an alternative strategy to block chemokines, through two structurally unique classes of secreted, broadly specific chemokine scavengers, one of which also binds interferon- (Graham et al., 1997; Smith et al., 1997; Alcami et al., 1998). Neither has structural homology to chemokines, chemokine receptors, or any other proteins currently in the public databases. They may be good leads for development of novel anti-inflammatory agents, particularly for topical or single-use administration (Table 5).

Recently, a growing number of structurally diverse, naturally occurring, nonchemokine ligands for chemokine receptors has been identified. These include HIV tat at CCR2 (Albini et al., 1998) and CXCR4 (Xiao et al., submitted), HIV gp120 at various HIV coreceptors (Berger et al., 1999), a secreted domain of tyrosyl tRNA synthetase at CXCR1 (Wakasugi et al., 1999), and the human -defensin HBD2 at CCR6 (Yang et al., 1999).

A major new concept to emerge recently from studies of the leukocyte selectivities of chemokines is that interaction between antigen-loaded dendritic cells and antigen-specific T cells to achieve proper cell positioning in the periphery or in secondary lymphoid tissue for an adaptive immune response is not random but instead results in part from dynamic and coordinated changes in chemokine receptor expression. Moreover, the nature and strength of the immune response may be governed in part by specific chemokine receptor expression patterns. Thus, T lymphocytes and dendritic cells undergo highly dynamic regulation of chemokine receptors depending on whether the T cell is naïve or memory, Th1 or Th2, and resting or activated, and whether the dendritic cell is immature or mature (Sallusto et al., 1999a; Sozzani et al., 1999). For example, when naive T lymphocytes are activated and differentiate into memory/effector cells, they down-regulate receptors for constitutive chemokines such as CXCR4 and CCR7 and acquire receptors for inflammatory chemokines such as CCR3, CCR5, and CXCR3. Also, dendritic cell maturation after antigen loading is accompanied by a transition from expression of inflammatory to homeostatic chemokine receptors. Distinct selectivities for Th1 and Th2 polarized T lymphocytes have been reported for CC chemokine receptors, and actual chemokine receptor markers of these cell types have been claimed and debated (Sallusto et al., 1998; Annunziato et al., 1999). Moreover, homing of memory T cells to specific anatomic sites has been strongly correlated with specific chemokine receptor expression patterns (Campbell et al., 1999).

With this as a general introduction, the next sections are discussions of the molecular pharmacology and biology of individual chemokine receptor subtypes. Note that the voluminous literature correlating the presence of specific chemokines in disease has been extensively reviewed (Baggiolini et al., 1997) and is not repeated here. Instead, we emphasize direct tests of function of specific receptors in disease.

	III. CXC Chemokine Receptor Subtypes

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CXCR1 and CXCR2 were the first chemokine receptor subtypes to be defined. They are the only known mammalian receptors for ELR+ CXC chemokines, including IL-8, which binds to both receptors with similar high affinity; they do not bind other types of chemokines. They are also the major chemokine receptors expressed on neutrophils and are prototypic receptors for inflammatory/inducible chemokines. They appear to operate mainly in acute inflammation and innate immunity although a role in macrophage accumulation in atherosclerotic plaque has also been demonstrated for CXCR2 (Boisvert et al., 1998). They are considered together because of these shared properties.

CXCR1 cDNA was first cloned from rabbit neutrophils by homology hybridization using a probe based on conserved sequences in TMD 2 of known neuropeptide-specific GPCRs (Thomas et al., 1990). When expressed in frog oocytes, it appeared to be specific for formyl-methionyl-leucyl-phenylalanine, but this could not be reproduced in mammalian cells where IL-8 was a functional ligand (Thomas et al., 1991). Consistent with this, human CXCR1 cDNA was isolated independently from a neutrophil library by expression cloning using an ¹²⁵I-IL-8 binding assay in COS-7 cells (Holmes et al., 1991). CXCR2 cDNA was first cloned by homology hybridization from a dibutyryl cAMP-induced HL-60 cell library using an oligonucleotide probe corresponding to TMD2 of rabbit CXCR1 (Murphy and Tiffany, 1991); later, cDNAs were also isolated from a neutrophil library (Lee et al., 1992). The genes, designated il8ra and il8rb, are located 20 kb apart on human chromosome 2q35, and there is a linked pseudogene of CXCR2 named il8rp (Ahuja et al., 1992; White et al., 1994). The open reading frames (ORFs), which each occupy a single exon, are 350 codons for CXCR1 and either 355 or 360 codons for CXCR2 (both of two in-frame ATG codons are flanked by favorable Kozak sequences). CXCR1 and CXCR2 are 78% identical in aa sequence.

In addition to neutrophils and monocyte/macrophages, CXCR1 and CXCR2 have been detected on cytokine-activated eosinophils, basophils, T lymphocytes, mast cells, and dendritic cells, but important functional roles in vivo have not been clearly demonstrated (Chuntharapai et al., 1994; Hammond et al., 1995; Morohashi et al., 1995; Xu et al., 1995; Heath et al., 1997; Sozzani et al., 1997; Nilsson et al., 1999; Ochensberger et al., 1999; Petering et al., 1999). CXCR2, but not CXCR1, has been identified on brain Purkinje cells by mAb and radioligand binding, but function remains undefined there as well (Horuk et al., 1997).

In calcium flux and chemotaxis assays, CXCR2 is relatively nonselective for IL-8 versus all other ELR+ CXC chemokines studied (<10-fold range in EC₅₀), whereas CXCR1 is highly selective for IL-8 (>50-fold difference in EC₅₀) (Lee et al., 1992; Loetscher et al., 1994; Ahuja and Murphy, 1996); GCP-2 is an equipotent agonist at both CXCR1 and CXCR2 (Wuyts et al., 1997). Thus, GRO, NAP-2, and ENA-78 are selective ligands for CXCR2. Recently, a selective nonchemokine endogenous ligand was identified for CXCR1: the N-terminal cytokine module of human tyrosyl tRNA synthetase, which contains an ELR motif and functions as a neutrophil chemoattractant in vitro. Its biological function is not established but could involve inflammatory signaling by apoptotic cells (Wakasugi and Schimmel, 1999). Consistent with coexpression of CXCR1 and CXCR2 on neutrophils, IL-8 effectively blocks binding of other ELR+ CXC radioligands to human neutrophils and interferes with signaling (calcium flux), but conversely, other ELR+ CXC chemokines can only partially block IL-8 binding to neutrophils and subsequent calcium flux (Moser et al., 1991; Ahuja and Murphy, 1996). The receptors appear to function independently.

Antagonists at CXCR2 include N-terminal truncations of IL-8 and GRO (Hesselgesser et al., 1995), selective neutralizing monoclonal and polyclonal antibodies (Hammond et al., 1995; Green et al., 1996; Jones et al., 1996), a small peptide of undefined selectivity (Hayashi et al., 1995), and SB 225002 [N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea], a selective small molecule, nonpeptide inhibitor of CXCR2 (White et al., 1998) (Fig. 3). The latter is a potent antagonist of ¹²⁵I-IL-8 binding with an IC₅₀ value of 22 nM and has >150-fold selectivity over CXCR1. In vitro, SB 225002 potently inhibits human and rabbit neutrophil chemotaxis induced by both IL-8 and GRO, and in vivo it selectively blocks IL-8-induced neutrophil margination in rabbits.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Structures of nonpeptide small molecule antagonists of specific chemokine receptors. Note that a basic nitrogen is a common feature. (Figure courtesy of Kurt Jarnagin.)

In vivo roles of IL-8 and related ligands have been extensively studied, but specific receptor roles are less well defined. The mouse chemokines MIP-2 and KC are human GRO homologs specific for mouse CXCR2 (Bozic et al., 1994; Lee et al., 1995). CXCR2 knockout mice fail to mobilize neutrophils to chemically irritated peritoneum in vivo, and / neutrophils do not migrate in vitro in response to KC or MIP-2, indicating that CXCR2 is the dominant neutrophil receptor for these chemokines (Cacalano et al., 1994).

Unexpectedly, CXCR2 / mice have massive expansion of neutrophils and B cells throughout the hematopoietic system when derived in specific pathogen-free conditions but not in germ-free conditions (Moore et al., 1995). The explanation may reside in part in the fact that CXCR2 is a negative regulator of hematopoiesis (Broxmeyer et al., 1996). Alternatively, Cacalano et al. (1994) speculated that the inability to properly survey tissues and eliminate external pathogens in the knockouts may result in the release of cytokines that stimulate neutrophil and B cell production. However, the animals have not been reported to have increased susceptibility to infectious disease from either environmental or challenge pathogens.

The defect in neutrophil-mediated inflammation in these mice is consistent with the effects of CXCR2 ligand neutralization in mouse (KC, MIP-2) and rabbit (IL-8) in diverse models of acute inflammation (skin, airway, pleura, glomeruli) (Sekido et al., 1993; e.g., Broaddus et al., 1994). These results suggest indications for IL-8 receptor antagonists in diseases such as psoriasis, coronary artery reperfusion injury, and acute glomerulonephritis. Still, it is important to point out that rodents are poor models of the human IL-8 signaling system: they lack IL-8, a mouse counterpart of CXCR1 has not been identified, and rat CXCR1 is expressed in macrophages not neutrophils (Dunstan et al., 1996). Nevertheless, IL-8 receptor function in the monocyte/macrophage lineage may be more important than was initially appreciated. In particular, IL-8 can trigger firm adhesion of human monocytes to vascular endothelium under flow conditions (Gerszten et al., 1999), and CXCR2 is critical for macrophage accumulation in atherosclerotic lesions of LDL receptor-deficient mice (Boisvert et al., 1998).

CXCR1 and CXCR2 have been reported to carry out different functional roles in human neutrophils in vitro. CXCR1 appears to be dominant for chemotaxis, superoxide production, and phospholipase D activation in response to IL-8 (Hammond et al., 1995; Jones et al., 1996), as well as for chemotaxis to NAP-2 at high concentrations (>1 µM) (Ludwig et al., 1997), whereas CXCR2 appears to mediate neutrophil chemotaxis to NAP-2 (and GRO) at low concentrations. Calcium flux and degranulation are mediated through both receptors. However, cell migration may be more important than cell activation for IL-8 receptor function in vivo, as suggested by the accumulation of unactivated neutrophils and the lack of inflammatory pathology at sites of KC transgene expression in mice; this may be a general property of chemokines (Lira et al., 1994).

Despite abundant evidence that IL-8 is important in acute inflammation, proof of concept is still lacking for differential roles of CXCR1 versus CXCR2 in vivo and in human disease. As suggested earlier, major obstacles include the inadequacy of small animal models and the lack of adequate selective small molecule antagonists. Other major unanswered questions about these receptors include their structure, the relative roles of CXCR1 and CXCR2 in ELR+ CXC chemokine-induced angiogenesis and modulation of myelopoiesis (Broxmeyer et al., 1997), and the putative function of CXCR2 in brain (Horuk et al., 1997).

Two functional viral homologs of CXCR2 have been identified, ECRF3 of Herpesvirus saimiri (Ahuja et al., 1993) and KSHV GPCR of KSHV (HHV8) (Arvanitakis et al., 1997), which are quite different from CXCR2 and are reviewed in a later section.

CXCR3 is the first chemokine receptor identified that is highly induced by T cell activation. The ORF was first identified in incomplete form in 1995 on a genomic clone isolated by polymerase chain reaction-based homology hybridization. The gene was named GPR9 and was originally mapped incorrectly to human chromosome 8p11.2-12 (Marchese et al., 1995) and later mapped correctly to Xq13 (Loetscher et al., 1998a). A full-length cDNA was independently isolated from an IL-2-activated T cell library (Loetscher et al., 1996). The ORF is interrupted by one intron in the region encoding the N-terminal segment and predicts a polypeptide 368 aa in length. The deduced protein sequence of human CXCR3 is ~30% identical with CXCR1 and CXCR2.

CXCR3 binds three highly potent, inflammatory/inducible, ELR-negative CXC chemokine agonists, I-TAC, Mig, and IP-10 (Loetscher et al., 1998a; Cole et al., 1998; Weng et al., 1998), all of which chemoattract and induce calcium flux in activated T cells, tumor-infiltrating lymphocytes, and CXCR3-transfected cells. The rank order of binding affinity is I-TAC > Mig ~ IP-10. Curiously, the human CC chemokines eotaxin and MCP-4 also bind to CXCR3-transfected cells but with much lower affinity (K_i ~ 60 nM) and without activating the receptor (Weng et al., 1998). Also, the mouse CC chemokine SLC/6Ckine has been reported to induce calcium flux through mouse CXCR3 (Soto et al., 1998), but this was not observed with human 6Ckine with either human or mouse CXCR3 (87% aa identity) (Jenh et al., 1999). A CXCR3-specific mAb named 1C6 has been reported that blocks human IP-10, but not human Mig, binding to CXCR3 (Qin et al., 1998).

CXCR3 is expressed on a portion of circulating blood T cells, B cells, and natural killer (NK) cells (Qin et al., 1998). Although freshly isolated T cells respond to Mig, curiously they are relatively less responsive to IP-10. Expression and responsiveness are both markedly increased by T cell activation (Rabin et al., 1999), classifying CXCR3 as an inflammatory/inducible type of chemokine receptor. CXCR3 has been detected preferentially on Th1 T cell lines and clones in vitro but could not discriminate between Th1- (Crohn's disease) and Th2- (systemic sclerosis) dominant responses in vivo and therefore may not be a practical marker of Th1 cells, as had been suggested (Bonecchi et al., 1998; Sallusto et al., 1998, 1999b; Annunziato et al., 1999). Blood T cells expressing CXCR3 are mostly CD45RO+ memory cells and express high levels of 1-integrins. Virtually all T cells in rheumatoid arthritis synovial fluid and in various inflamed tissues, such as in ulcerative colitis, chronic vaginitis, and sarcoidosis, express CXCR3, particularly in perivascular regions, whereas fewer T cells within normal lymph nodes are positive (Agostini et al., 1998; Qin et al., 1998). CXCR3 is also consistently detected in functional form on transformed B cells from CLL patients (Trentin et al., 1999).

The biological role of CXCR3 is not yet known, and it has not been established as a disease target, although a role in Th1 dominant diseases have been anticipated. Antagonists and gene knockouts have not been reported. In addition to T cell chemotaxis, CXCR3 ligands are angiostatic factors in vivo, but mechanisms are not defined.

CXCR4 is the first chemokine receptor shown to be an HIV-1 coreceptor (Feng et al., 1996) and the only one shown to be essential for life, at least in mice (Ma et al., 1998; Tachibana et al., 1998; Zou et al., 1998). Four groups identified it based on "orphan receptor" cloning strategies, whereas Feng et al. (1996) rediscovered the cDNA by expression cloning of its HIV-1 coreceptor activity and named the protein "fusin." Specificity for the homeostatic CXC chemokine SDF-1 was established shortly thereafter (Bleul et al., 1996; Oberlin et al., 1996), and fusin was renamed CXCR4.

The ORF is interrupted by one intron in the region encoding the N-terminal segment and predicts a polypeptide 352 aa in length. A splice variant of unclear significance has been found, which affects the length of the N-terminal portion of the molecule upstream of TMD1, but not affinity for ligand (Heesen et al., 1997; Frodl et al., 1998; Gupta and Pillarisetti, 1999).

CXCR4 is unusually widely expressed on most hematopoietic cell types, including neutrophils, monocytes, T lymphocytes, B cells, B cell precursors, CD34⁺ progenitor cells from blood and bone marrow, blood-derived dendritic cells, Langerhans cells, T cells and macrophages, and both immature and mature T cells in thymus (Bleul et al., 1997; Zaitseva et al., 1997, 1998). It is also expressed at high levels on vascular endothelial cells (Gupta et al., 1998b), neurons from both the central and peripheral nervous systems (Hesselgesser et al., 1997), and microglia and astrocytes (He et al., 1997). In blood-derived T cells, CXCR4 is preferentially expressed on the naive, unactivated CD26low CD45RA+ CD45R0 subset (Bleul et al., 1997), and expression is rapidly up-regulated by phytohemagglutinin and IL-2 (Loetscher et al., 1996) and down-regulated by SDF-1 (Amara et al., 1997).

CXCR4 has also been implicated in platelet formation. Although there is agreement over whether it is expressed throughout platelet development, there is some disagreement about its function (Power et al., 1995a; Hamada et al., 1998; Wang et al., 1998; Kowalska et al., 1999). SDF-1-induced transendothelial migration by mature marrow megakaryocytes and megakaryocyte progenitors has been reported by at least one group but not consistently confirmed. The receptor is on mature platelets but appears to be functionally uncoupled.

The SDF-1 gene is alternately spliced to form SDF-1 and SDF-1, which differ by a 4-aa extension at the C terminus (Shirozu et al., 1995). These variants, originally isolated from bone marrow stromal cells, are functionally indistinguishable and are the only known endogenous ligands and agonists for CXCR4, inducing calcium flux and chemotaxis in transfected and primary cells in vitro. Genetic disruption of SDF-1 and CXCR4 in the mouse gives the same phenotype (Nagasawa et al., 1996; Ma et al., 1998; Tachibana et al., 1998; Zou et al., 1998), suggesting that they make up a monogamous signaling unit in vivo. The animals die in the perinatal period, the only known chemokine system components for which this is true, and have ventricular septal defects, defective gastric vasculogenesis and cerebellar development, abnormal bone marrow myelopoiesis, and defective B cell, but normal T cell, lymphopoiesis. Functions of CXCR4 in the adult are not defined. In one study, human stem cell engraftment was reported to be regulated by CXCR4 in NOD/SCID mice (Peled et al., 1999). Both SDF-1 and CXCR4 have highly conserved sequences (e.g., 98 and 94% aa identity between human and mouse, respectively), which is highly atypical for chemokines and chemokine receptors, which are among the most rapidly evolving proteins in mammals (Murphy, 1993).

HIV-1 strains able to use CXCR4 for cell entry in vitro are named X4 strains (Berger et al., 1998). They are typically isolated late in the course of infection and correlate more or less with T cell line cytotropism and the syncytium-inducing methods of classification used before the discovery of HIV-1 coreceptors (reviewed in Berger et al., 1999). The importance of CXCR4 in HIV pathogenesis has been suggested but not proved by the detection of X4 HIV in CCR5-deficient HIV-positive individuals (Michael et al., 1998), and the discovery of a single nucleotide polymorphism in the 3'-UTR of SDF-1 (SDF1-3'A) that is associated with slowed progression to AIDS (Winkler et al., 1998). Direct studies of the effect of this polymorphism on SDF-1 production in vivo have not been reported, but any effect could conceivably modulate the extent of X4 HIV interaction with CXCR4.

gp120 from HIV-1 envelope glycoprotein can bind to CXCR4 in the presence of CD4 (Lapham et al., 1999), and X4 virus entry is dependent on CD4 (Feng et al., 1996). However, CD4 independent association of gp120 to CXCR4 has also been demonstrated. Purified X4 gp120 can function as a CXCR4-dependent monocyte chemoattractant, perhaps to recruit more targets, and can induce apoptosis of the human neuronal cell line hNT (Hesselgesser et al., 1998a). Consistent with this, chemokines can regulate hippocampal neuronal signaling and gp120 neurotoxicity (Meucci et al., 1998). These findings may be relevant to the pathogenesis of HIV encephalitis and AIDS dementia. Interaction of gp120 with CXCR4 on macrophages can also induce apoptosis of CD8⁺ T cells, suggesting a coreceptor mechanism of CTL suppression (Herbein et al., 1998).

Several mAbs have been developed that bind CXCR4, including the prototype 12G5, which blocks HIV infection (Endres et al., 1996). Several small molecules and peptides, including some originally identified in HIV drug discovery programs, have been shown to selectively block chemokine receptor and/or HIV coreceptor activities of CXCR4. They include SDF-1 derived peptides (Loetscher et al., 1998b, Heveker et al., 1998); the synthetic peptide T22 ([Tyr⁵,12,Lys⁷]polyphemusin II), which consists of 18 aa residues and an analog of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus) (Murakami et al., 1997); the related synthetic peptides T134 and T140 (Tamamura et al., 1998; Xu et al., 1999); the polyarginine ALX40-4C (Doranz et al., 1997); the peptoid CGP64222 (Daelemans et al., 2000); and the bicyclam AMD3100 (Schols et al., 1997; Donzella et al., 1998; Bridger et al., 1999) (Fig. 3). The distamycin analog 2,2'-[4,4'-[[aminocarbonyl]amino]bis[N,4'-di[pryrrole-2-carboxamide-1,1'-dimethyl]]-6,8-naphthalenedisulfonic acid] hexasodium salt (NSC 651016) also blocks X4 viral use of CXCR4, but it has a broad specificity for multiple other chemokine receptors (Howard et al., 1998). CXCR4 has also been blocked with intrakines, which are modified forms of SDF-1 delivered by gene therapy that remain in the endoplasmic reticulum and block surface expression of newly synthesized CXCR4 (Chen et al., 1997).

In addition, the HIV protein Tat, which has a highly basic domain but lacks a chemokine fold, can block both SDF-1-induced calcium flux at CXCR4 and X4 HIV entry of target cells (Xiao et al., submitted). The inability of Tat to affect CCR5 function suggests a possible mechanism for restriction of HIV to R5 strains early in infection but cannot explain the appearance of X4 strains late in infection during immune system collapse. Moreover, it conflicts with the reported ability of Tat to up-regulate CXCR4 and serve as a vaccine target in nonhuman primates (Gallo, 1999).

The clinical development of CXCR4 blocking agents in HIV infection will have to confront safety questions of whether the virus will evolve to use other coreceptors and whether one or more of the phenotypes seen in CXCR4 knockout mice will occur. To date, CXCR4 has not been established as a therapeutic target for other diseases.

CXCR5 was the first chemokine receptor shown to be involved in lymphocyte homing and development of normal lymphoid tissue (Forster et al., 1996) and the first B cell selective chemokine receptor (Gunn et al., 1998a; Legler et al., 1998). Two cDNAs for CXCR5 were cloned independently by two groups as orphans and named, according to the source, monocyte-derived receptor 15 (MDR15; Barella et al., 1995) and Burkitt's lymphoma receptor 1 (BLR1; Dobner et al., 1992). The ORF of MDR15 has 327 codons and is 45 codons shorter at the N terminus than BLR1 due to alternative splicing of the gene. Distinct pharmacology has not been demonstrated for the two forms. The aa sequence is ~40% identical with CXCR1 and CXCR2.

Using a mAb directed to the N terminus of BLR1, CXCR5 has been detected on all peripheral blood and tonsillar B cells but only on a fraction of cord blood and bone marrow B cells. It is also present on a small subset of peripheral blood CD4⁺ (14%) and CD8⁺ (2%) T cells, which are also CD45R0+, IL-2R, CD44^high, and L-selectin^low, suggesting a memory phenotype. In contrast, in secondary lymphatic tissue, the majority of CD4⁺ cells are positive, and in cord blood, T cells are negative (Forster et al., 1994). The murine homolog of CXCR5 has been cloned, and specific transcripts found in a pattern similar to the human receptor, including expression on mature B cells and a subpopulation of T helper cells, as well as in secondary lymphatic organs and to a lesser extent in brain, specifically in the granule and Purkinje cell layer of the cerebellum (Kaiser et al., 1993). RNA in situ hybridization localizes transcripts to primary follicles and to the mantle zone of secondary follicles. Like other chemokine receptors, CXCR5 is dynamically regulated on T cells. After T cell receptor (TCR) stimulation, CXCR5 is up-regulated on memory/effector T cells, whereas IL-2 causes down-regulation (Sallusto et al., 1999b). Up-regulation of CXCR5 on antigen-activated T cells implies a role for movement of Th cells to B cell follicles (Ansel et al., 1999).

To date, B cell-attracting chemokine 1 (BCA-1, also known as BLC) is the only known agonist for CXCR5 (Gunn et al., 1998a; Legler et al., 1998). Conversely, CXCR5 is the only known receptor for BCA-1. Signaling includes chemotaxis and Ca²⁺ mobilization. BCA-1, a member of the homeostatic class of chemokines, is B cell selective and constitutively expressed in secondary lymphoid organs. It has weak effects on small numbers of T cells and macrophages. Consistent with this, CXCR5 knockout mice have a severe defect in normal B cell migration and localization (Forster et al., 1996). The animals lack inguinal lymph nodes, have few Peyer's patches, and have abnormal primary lymphoid follicles and no functional germinal centers in spleen. Nevertheless, immunoglobulin levels are normal. Disease phenotypes have not been reported.

Thus, although the biological importance of this receptor is established, evidence is lacking for its significance as a therapeutic target in disease. No CXCR5 antagonists or neutralizing mAbs have been developed yet. Recently, CXCR5 was reported to have coreceptor activity selective for HIV-2 (Kanbe et al., 1999).

	IV. CC Chemokine Receptor Subtypes

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CCR1 was the first CC chemokine receptor identified and the first shown to have a functional viral homolog, US28 of human cytomegalovirus (Gao et al., 1993; Neote et al., 1993; Gao and Murphy, 1994). The gene is on human chromosome 3p21 in a cluster with CCR2, CCR3, CCR4, CCR5, CCR8, CCR9, XCR1, CX3CR1, and several orphans (Samson et al., 1996c). The ORF is on a single exon, and the predicted polypeptide is 355 aa in length.

Using a polyclonal rabbit antibody, Su et al. (1996) identified CCR1 on human peripheral blood lymphocytes and monocytes. A majority of CD3⁺, CD4⁺, CD8⁺, and CD16⁺ lymphocytes were positive. Among CD4⁺ peripheral blood T cells, CD45RO+ cells expressed greater amounts of CCR1 than CD45RO cells, suggesting selective expression on the memory subtype. Expression studies using an anti-CCR1 mAb have not been reported.

CCR1 binds multiple inflammatory/inducible CC chemokines with similar high affinity, including MIP-1, RANTES, MCP-2, MCP-3, leukotactin-1/MIP-5, MPIF-1 and HCC-1 (Neote et al., 1993; Gao et al., 1993; Youn et al., 1997; Gong et al., 1997; Tsou et al., 1998; Zhang et al., 1999; Nardelli et al., 1999). MIP-1 and MCP-1 bind with much lower affinity and are poor agonists (Neote et al., 1993). HCC-1 may be selective. Mouse CCR1 (80% aa identity) binds human and mouse MIP-1 with high affinity; agonists include mouse and human MIP-1 and human leukotactin-1/MIP-5 (Gao and Murphy, 1995; Post et al., 1995; Zhang et al., 1999). A closely related mouse orphan named MIP-1-RL1 (65% aa identity) has also been cloned, but it has no human counterpart (Gao and Murphy, 1995).

CCR1 signaling includes calcium flux, inhibition of adenylyl cyclase, and chemotaxis (Myers et al., 1995; Pease et al., 1998). Coupling to both G_i and G14, but not G_q/11 or G16, has been reported in transfected COS cells (Kuang et al., 1996). Signaling can be blocked efficiently by RANTES variants that have been modified at the N terminus, including truncated forms (Arenzana-Seisdedos et al., 1996; Struyi et al., 1998), Met-RANTES (Proudfoot et al., 1996), and amino-oxypentane (AOP)-RANTES (Simmons et al., 1997); however, none of these is selective for CCR1 over the other RANTES receptors, CCR3 and CCR5. High CCR1 selectivity has been reported by Berlex Biosciences for 4-hydroxypiperidines (K_i = 40-4000 nM) (Hesselgesser et al., 1998b; Ng et al., 1999) (Fig. 3), particularly 2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitrite, which inhibits MIP-1 binding to CCR1 (K_i ~ 40 nM) and blocks MIP-1-induced extracellular acidification, Ca²⁺ mobilization, and chemotaxis of peripheral blood mononuclear cells; effects in disease have not been reported yet. Other small molecule CCR1 antagonists have also been disclosed but have either lower potency or selectivity than the Berlex Biosciences compound.

Clear disease indications have not yet been identified for CCR1. Nevertheless, there is a fair amount now known about its biology from the phenotype of CCR1 knockout mice. The receptor is dispensable for growth, development, and reproduction, and the mice do not acquire spontaneous infections from environmental pathogens. It is the dominant receptor used by MIP-1 for induction of mouse neutrophil chemotaxis and calcium flux in vitro, mobilization of neutrophils and hematopoietic progenitor cells in vivo, and regulation of hematopoietic progenitor cell proliferation (Gao et al., 1997; Broxmeyer et al., 1999). Consistent with this, MIP-1 functions as a negative regulator of hematopoiesis (reviewed in Broxmeyer et al., 1997), and in vitro anti-CCR1 antibodies block MIP-1 inhibition of colony formation by burst-forming unit-erythroid from purified human CD34⁺ bone marrow cells (Su et al., 1997). In this regard, BB10010, an agonistic variant of MIP-1 (British Biotech, Inc.), has been tested in phase I and II clinical trials as a stem cell protective agent in patients undergoing chemotherapy. The agent was safe in the doses tested, but only small therapeutic effects were noted on myelopoiesis, perhaps because of insufficient stress on the bone marrow by the chemotherapy regimens tested (Clemons et al., 1998; Marshall et al., 1998). Another CCR1 agonist, MPIF-1 (Human Genome Sciences, Rockville, MD), has recently undergone phase I trial for the same indication.

Consistent with a role in neutrophils, CCR1 / mice have reduced alveolitis in a pancreatitis-alveolitis mouse model (Gerard et al., 1997), as well as increased lethality when infected with Aspergillus fumigatus, an organism controlled primarily by neutrophils (Gao et al., 1997). However, this is an example where mouse and human orthologs may differ in biological function, because the major CCR1 agonists MIP-1 and RANTES are poor agonists for human neutrophils (Coulin et al., 1997; Youn et al., 1997; Zhang et al., 1999). CCR1 also regulates granuloma formation and Th1/Th2 cytokine balance in response to Schistosome eggs deposited in mouse lung, but it is not a dominant receptor for MIP-1-induced macrophage chemotaxis in vitro (Gao et al., 1997). Nevertheless, CCR1 deficiency did not reduce neutrophil accumulation in a nephrotoxic nephritis mouse model; disease was actually exacerbated with increased accumulation of macrophages and CD4⁺ and CD8⁺ T cells, as well as enhanced effector immune responses (Topham et al., 1999). However, CCR1 deficiency suppressed development of acute and chronic cardiac allograft rejection in several mouse models (Gao et al., 2000). Thus CCR1 can modulate inflammatory responses either positively or negatively, depending on the context, through effects on multiple leukocyte subtypes. The phenotype of MIP-1 knockout mice includes protection from coxsackievirus myocarditis, influenza A alveolitis, and acute experimental allergic encephalomyelitis; however, specific roles for CCR1 are not defined (Cook et al., 1995; Kennedy et al., 1998).

CCR2 is the only leukocyte MCP-1 receptor identified so far, and it is important in inflammation, including atherosclerosis. The ORF is on two alternatively spliced exons that encode two distinct polypeptides 360 (CCR2_(a)) and 374 (CCR2_(b)) aa in length (Charo et al., 1994; Wong et al., 1997). The two have an identical sequence until aa 313, which is located in the C-terminal cytoplasmic region, and similar functional properties. Both RNAs are detectable in monocytes, blood-derived DC and NK cells and T lymphocytes but not in resting neutrophils or eosinophils. CCR2_(b) appears to be the predominant form. mAbs have identified functional CCR2 in monocytes, activated memory T cells, B cells, and basophils (Frade et al., 1997; Rabin et al., 1999). In vivo, chronic inflammation may potentiate neutrophil migration to MCP-1 (Johnston et al., 1999).

Signaling through CCR2 in transfected cells includes calcium mobilization, inhibition of adenylyl cyclase, and chemotaxis (M