CXCR1 and CXCR2

CXCR1 and CXCR2 Iris Roth* and Caroline Hebert Department of Molecular Oncology, Genentech, 1 DNA Way South San Francisco, San Francisco, CA 94080, USA * corresponding author tel: 650 225 2176, fax: 650 225 8221, e-mail: [email protected] DOI: 10.1006/rwcy.2000.21001.

SUMMARY Chemokines induce cell migration and activation by binding to specific seven transmembrane (7TM) G protein-coupled cell surface receptors (GPCRs) on target cells (Murphy, 1994; Premack and Schall, 1996). Chemokine receptors, like all members of the GPCR superfamily, mediate signal transduction through G proteins. They have a single polypeptide chain and have 25±80% amino acid sequence identity among all family members. CXCR1 and CXCR2 were the first members of the chemokine receptor family to be cloned, and share a high degree of homology with receptors for C5a and formyl peptide. There is also a high level of homology between receptors from human, rabbit, rat, and mouse. Like the other chemokine receptor family members, CXCR1 and CXCR2 are expressed on a variety of cells and have a characteristically narrow ligand-binding profile, including a subgroup of human CXC chemokines defined by the conserved sequence motif glutamic acid-leucinearginine (ELR). The human ELR-containing CXC chemokines IL-8, growth-related oncogene (GRO), GRO , GRO , neutrophil-activating peptide-2 (NAP-2), epithelial cell-derived neutrophil activating peptide 78 (ENA-78), and granulocyte chemoattractant protein 2 (GCP-2) are 40±90% identical in amino acid sequence. CXCR1 and CXCR2 are distinguished by their different selectivities for these chemokine ligands, with CXCR1 displaying a relatively narrow selectivity and preference for IL-8. Both receptors are expressed on a wide variety of cell types, including neutrophils, monocytes, CD8‡ T cells, mast cells, basophils, natural killer cells, keratinocytes, fibroblasts, neurons, endothelial cells, and melanocytes. They are distinguished by their differential affinities for a group of chemokine ligands. CXCR1 and CXCR2 play an important role in acute

inflammation by transducing the signal for one ligand, IL-8. These receptors are attractive targets for the development of therapeutics for inflammatory disease, as GPCRs have proven to be good targets for small molecule antagonists. However, the relative contributions of each receptor to disease process have not yet been established, thus it is still not known which of the two must be targeted for drug development. Selective antagonists for CXCR1 and CXCR2 have begun to emerge, and these will be useful tools for further elucidating the role of each receptor in the pathophysiology of inflammatory diseases. Because the two receptors share a multitude of characteristics, including expression patterns and functional properties, they will be discussed together in this review. This chapter will include brief descriptions of their genetics, structure±function analysis, ligand-binding requirements, and signal transduction mechanisms, as well as expression patterns and roles in normal tissues as well as in disease states.

BACKGROUND

Discovery The discovery of IL-8 marked the emergence of the chemokine field. Previously described chemotactic factors, including the formyl peptide N-formyl methionyl-leucyl-phenylalanine (fMLP), complement factor C5a, platelet-activating factor (PAF), and leukotriene B4 (LTB4), were promiscuous, acting on a variety of leukocyte subsets (Locati and Murphy, 1999). IL-8, produced by lipopolysaccharide (LPS)stimulated monocytes, was active on neutrophils but not monocytes/macrophages. This chemoattractant was described by independent work in several laboratories and named neutrophil-activating factor (NAF)

1982 Iris Roth and Caroline Hebert (Walz et al., 1987), monocyte-derived neutrophilactivating peptide (MONAP) (Schroder et al., 1987), and monocyte-derived neutrophil chemotactic factor (MDNC) (Yoshimura et al., 1987). It was subsequently shown that this factor is produced by various cell types and has multiple targets, thus it was renamed interleukin 8 (IL-8) (Balkwill and Burke, 1989; Larsen et al., 1989). Lack of cross-desensitization suggested that this chemokine acts through selective receptors on neutrophils distinct from those for fMLP, C5a, PAF, and LTB4 (Peveri et al., 1988). Samanta et al. first demonstrated specific binding of [125I]IL-8 to the surface of human neutrophils (Samanta et al., 1989). Scatchard analysis yielded a curvilinear plot, which the authors interpreted as representing a single binding affinity receptor. Chemical crosslinking of ligand±receptor complexes identified two IL-8-binding proteins, thought to be noncovalently associated subunits of this receptor. Subsequent binding studies using different preparations of IL-8 resulted in conflicting interpretations. Besemer et al. also observed nonlinear Scatchard plots, but concluded that their data were consistent with two populations of receptors (Besemer et al., 1989). Others demonstrated a linear Scatchard plot and only one protein chemically crosslinked to IL-8 ± results consistent with the presence of only one class of receptors on neutrophils (Grob et al., 1990). The binding affinities ranged from 11 pM to 2 nM, with estimated receptor numbers ranging from 20,000 to 75,000 per cell. The search for chemokine receptors began when several groups utilized expression cloning strategies to identify the human formyl peptide receptor (FPR) (Boulay et al., 1990; Coats and Navarro, 1990; Murphy et al., 1990). One such cDNA clone was capable of conferring specific fMLP binding to transfected mammalian COS cells (Boulay et al., 1990). A putative rabbit homolog, F3R, was later cloned from a neutrophil cDNA library and shown to bind fMLP when expressed in Xenopus oocytes (Thomas et al., 1990). However, F3R was only 26% homologous to the clone previously identified as the human fMLP receptor. Unique clones for two distinct human IL-8 receptors were later independently described by Holmes and Murphy in the same issue of Science (Holmes et al., 1991; Murphy and Tiffany, 1991). Using an expression cloning strategy in COS-7 cells, Holmes et al. isolated the clone for CXCR1 from a human neutrophil cDNA library. COS cells transfected with this cDNA specifically bound IL-8 with a Kd of 3.6 nM, within the range of 0.8±4 nM reported for binding to neutrophils. The cDNA contains a single long open reading frame, which encodes a 350 amino

acid protein with seven hydrophobic domains. The IL-8 receptor described in this study is 79% identical to F3R at the amino acid level. Because F3R shares only a 26% identity with the human fMLP receptor, the authors suggested that this sequence might actually encode an IL-8 receptor. In an accompanying paper, Murphy and Tiffany screened a differentiated HL-60 cDNA library to identify the human homolog of F3R (Murphy and Tiffany, 1991). One clone shared a 69% amino acid identity to F3R, yet demonstrated a pattern of expression more like that of IL-8-binding sites than N-formyl peptide-binding sites. When expressed in Xenopus oocytes, this clone was found to respond to IL-8, but not to fMLP. Because specific binding was not saturated at the highest concentration of [125I]IL-8 tested, a dissociation constant could not be determined. Thus, the receptor encoded by this cDNA appeared to be a low-affinity IL-8 receptor (CXCR2). The structurally related ligands NAP-2 and melanoma growth-stimulatory activity (MGSA)/ growth-related oncogene (GRO) induced calcium flux in transfected oocytes, though with decreased potency compared to IL-8. These results correlated with the effectiveness of these chemokines to compete with IL-8 for binding to neutrophils, as demonstrated by Moser et al. (1991). Meanwhile, the original rabbit F3R clone was expressed in COS cells and found to confer saturatable specific binding of IL-8, not fMLP (Thomas et al., 1991). IL-8 bound neutrophils and F3Rtransfected cells with apparent Kd values of 1.2 and 1.4 nM, respectively. F3R antibodies specifically immunoprecipitated [125I]IL-8 bound to the membranes of F3R-transfected COS cells as well as to neutrophils. With these results, Navarro's laboratory and others then correctly identified the F3R protein as the rabbit homolog of CXCR1 (Thomas et al., 1991; Cerretti et al., 1993a).

Alternative names CXCR1: IL-8RA, IL-8R1, IL-8R CXCR2: IL-8RB, IL-8R2, IL-8R The official names CXCR1 and CXCR2 were assigned by a consensus nomenclature agreement at the second Gordon Conference on Chemotactic Cytokines in Plymouth, NH, USA in 1996.

Structure Human CXCR1 and CXCR2 have a single polypeptide chain 350, and 355 or 360 amino acids in length, respectively. The receptors share 76% amino acid

CXCR1 and CXCR2 1983 identity to one another (Figure 3) (Holmes et al., 1991; Murphy, 1994). As members of the GPCR superfamily, CXCR1 and CXCR2 are 7TM receptors that signal via G proteins (Murphy, 1994; Premack and Schall, 1996). Their structure can be inferred from homology to another family member, rhodopsin, which signals light sensitivity in retinal cones (Luo et al., 1997). The seven hydrophobic regions of the receptors are embedded in the plasma membrane, giving it a serpentine appearance (Figure 1). The free N-terminal tail is extracellular, and the C-terminus is in the cytoplasm.

Main activities and pathophysiological roles CXCR1 and CXCR2 mediate a diversity of chemokine functions in a variety of cell types. In leukocytes they are important in mediating antimicrobial host defenses. Both receptors act to induce chemotaxis and calcium flux in different leukocyte subsets. In neutrophils, receptor activation also stimulates the release of granule enzymes as well as the generation of superoxide in respiratory burst (Loetscher et al., 1994; Hammond et al., 1995; Jones et al., 1996). In addition to their effects on immune cells, CXCR1 and/or CXCR2 may be important in regulating vasculogenesis and consequent tumor growth (Koch et al., 1992; Strieter et al., 1995). ELR-containing CXC chemokines signal via these receptors to induce endothelial cell chemotaxis in vitro as well as angiogenesis in

various animal models (Koch et al., 1992; Strieter et al., 1995).

GENE

Accession numbers Human CXCR1: L19592 Rat CXCR1-like: U71089 Gorilla CXCR1: X91110 Chimpanzee CXCR1: X91109 Human CXCR2: M99412 Mouse CXCR2: L23637 Rat CXCR2: U70988 Gorilla CXCR2: X91114 Rhesus CXCR2: X91116 Orangutan CXCR2: X91115 Chimpanzee CXCR2: X91113

Chromosome location and linkages The human genes for CXCR1 and CXCR2 (il8ra and il8rb, respectively), along with one pseudogene (il8rp), have been mapped to chromosome 2q34±35, 20 kb apart (Ahuja et al., 1992; Morris et al., 1992; Lloyd et al., 1993). The close clustering of the three genes, as well as their high degree of homology, suggests that they may have arisen by duplication of a common ancestral gene. The CXCR2 gene is approximately 12 kb in length while il8ra spans just 4 kb (Kelvin et al.,

Figure 1 Putative model of the proposed tertiary structure of CXCR1 and CXCR2. The transmembrane  helical domains are indicated by the tubular structures, numbered 1±7. Three extracellular loops are designated EL1-3. Two potential disulfide bridges, linking the extracellular N-terminal domain with the third extracellular loop, and the first and second extracellular loops, are shown in red. (Full colour figure can be viewed online.)

1984 Iris Roth and Caroline Hebert 1993; Ahuja et al., 1994). In both cases, the open reading frame (ORF) and 30 untranslated regions are contained in a single exon, while the 50 UTR is more complex. There is one pseudogene, named il8rp, contained in the CXCR1/CXCR2 gene cluster. This locus has an 87% identity to CXCR2, but contains multiple frameshifts and point mutations introducing stop codons (Ahuja et al., 1992; Mollereau et al., 1993). The 50 UTR of il8ra resides in the first two of three exons (Ahuja et al., 1994). Neutrophils contain two equally abundant mRNAs for CXCR1, 2.0 and 2.4 kb in length, the result of usage of two alternative polyadenylation signals (Figure 2). For il8rb, alternative splicing of 11 exons forms seven distinct mRNAs with one predominant splice variant, designated IL8RB3. Primer extension analysis identified two major transcriptional start sites for il8ra and 11 for il8rb. The promoters of both genes appeared to be very similar: a nonclassical TATA box and a GC-rich 50 flanking region was identified immediately upstream of the transcription start site (Sprenger et al., 1995). These minimal promoters were sufficient to induce strong

constitutive activity when cloned upstream from a chloramphenicol acetyltransferase (CAT) reporter gene. A granulocyte colony-stimulating factor (GCSF) responsive element was mapped within the first 118 nucleotides upstream of the transcription start site of il8rb (Sprenger et al., 1995). Expression analyses of additional regulatory regions suggested that both promoters are negatively controlled by silencer elements. Recent work has identified promoter elements of il8ra and demonstrated that the Ets family transcription factor PU.1 is a major regulator for activation of the CXCR1 promoter (Wilkinson and Navarro, 1999).

PROTEIN

Accession numbers Human CXCR1: P25024 Rabbit CXCR1: P21109 Rat CXCR1-like protein: P70612

Figure 2 Genomic organization of CXCR1 (a) and CXCR2 (b). Boxes, exons; lines connecting exons, introns; dotted lines, gaps in the sequence; H, sites for HindIII cleavage. Exons are numbered in the upper left of the corresponding box; introns are numbered in blue (CXCR1) or red (CXCR2) boxes, and their lengths are indicated in base pairs below the intron number. Splicing patterns are shown below each gene map, and the name for each mRNA form is given at the right of the splice pattern diagram. CXCR2 mRNA names are based on the number of the 50 -most exon. Horizontal lines for each mRNA indicate the corresponding exon in the gene, and internal tick marks indicate exon boundaries; dashed lines indicate splicing patterns. (Full colour figure can be viewed online.)

CXCR1 and CXCR2 1985 Gorilla CXCR1: P55919 Chimpanzee CXCR1: P55920 Human CXCR2: P25025 Mouse CXCR2: P35343 Rabbit CXCR2: P35344 Rat CXCR2: P35407 Gorilla CXCR2: Q28422 Rhesus CXCR2: Q28519 Chimpanzee CXCR2: Q28807 Bovine CXCR2: Q28003

Sequence See Figure 3.

Description of protein The human CXCR1 and CXCR2 proteins are highly homologous at the amino acid level (76%) (Figure 3). Both proteins also have a significant degree of homology to other chemokine receptors (25±30%). Residues important for ligand binding and receptor signaling have been elucidated using alanine scanning mutagenesis as well as the construction of receptor chimeras. Sequence differences between CXCR1 and CXCR2 appear to be clustered at the N-terminus, as well as in the second extracellular loop (Murphy, 1997). These regions may thus be important in conferring differential ligand specificity to each receptor. The key residues of CXCR1 important for ligand binding were defined by extensive site-directed mutagenesis, in which extracellular acidic residues were replaced with alanine (Figure 4) (Hebert et al., 1993; Leong et al., 1994). While it was originally thought that acidic residues at the N-terminus interact with

the basic ligand, IL-8, alanine substitutions at eight of the nine charged positions of the CXCR1 N-terminal domain had no effect on binding. Ligand binding was only affected when one residue, Asp11, was mutated to Ala. As predicted, IL-8 binding was retained when Asp11 was replaced with another acidic residue, such as Glu, or with Lys, found at position 11 in CXCR2 (Hebert et al., 1993). These data suggest that the acidic nature of the N-terminal segment of the IL-8 receptors is not functionally important. Additional residues in extracellular domains of CXCR1 were also shown to be important for IL-8 binding and subsequent signaling (Figure 4). Cells transformed with constructs containing alanine substitutions at residues Arg199 and Arg203, in the second extracellular loop, as well as Asp265, in the third extracellular loop, exhibited markedly diminished or absent IL-8 binding compared with wild-type CXCR1 (Leong et al., 1994). These positions are also conserved in the sequence of CXCR2 and are thus unlikely to play a role in receptor subtype specificity. Ala substitution of each of another two residues, Glu275 and Arg280, both in the third extracellular loop of CXCR1, also resulted in a dramatic loss of IL-8 binding and signaling (Hebert et al., 1993). These two residues are conserved in CXCR1 and CXCR2, as well as rabbit and mouse homologs of CXCR1. Glu275 and Arg280 are thought to interact with the positively charged Arg6 and Glu4 residues of IL-8, respectively. The Arg6 residue of IL-8 is itself critical for receptor binding: Ala or Lys substitution of this single residue causes a 1000-fold decrease in IL-8 binding affinity (Hebert et al., 1991). Site-directed mutagenesis studies demonstrated that binding occurs via determinants on multiple extracellular domains of CXCR1, in addition to the N-terminus. However, others have shown that an

Figure 3 Alignment of the sequences of human CXCR1 and CXCR2. Identical residues are marked by an asterisk. Conservative substitutions are marked by a dot. Human CXCR1: 1 61 121 181 241 301

MSNITDPQMW MLVILYSRVG FYSGILLLAC NNSSPVCYEV MRVIFAVVLI NPIIYAFIGQ

DFDDLNFTGM RSVTDVYLLN ISVDRYLAIV LGNDTAKWRM FLLCWLPYNL NFRHGFLKIL

PPADEDYSPC LALADLLFAL HATRTLTQKR VLRILPHTFG VLLADTLMRT AMHGLVSKEF

MLETETLNKY TLPIWAASKV HLVKFVCLGC FIVPLFVMLF QVIQETCERR LARHRVTSYT

VVIIAYALVF NGWIFGTFLC WGLSMNLSLP CYGFTLRTLF NNIGRALDAT SSSVNVSSNL

LLSLLGNSLV KVVSLLKEVN FFLFRQAYHP KAHMGQKHRA EILGFLHSCL

FEDFWKGEDL LVILYSRVGR YSGILLLACI NVSPACYEDM RVIFAVVLIF PLIYAFIGQK

SNYSYSSTLP SVTDVYLLNL SVDRYLAIVH GNNTANWRML LLCWLPYNLV FRHGLLKILA

PFLLDAAPCE ALADLLFALT ATRTLTQKRY LRILPQSFGF LLADTLMRTQ IHGLISKDSL

PESLEINKYF LPIWAASKVN LVKFICLSIW IVPLLIMLFC VIQETCERRN PKDSRPSFVG

VVIIYALVFL GWIFGTFLCK GLSLLLALPV YGFTLRTLFK HIDRALDATE SSSGHTSTTL

Human CXCR2: 1 61 121 181 241 301

MEDFNMESDS LSLLGNSLVM VVSLLKEVNF LLFRRTVYSS AHMGQKHRAM ILGILHSCLN

1986 Iris Roth and Caroline Hebert Figure 3

antibody that maps to the N-terminal region of CXCR1 blocks IL-8 binding (Chuntharapai et al., 1994a). Similarly, an antibody that maps to the Nterminus of CXCR2 blocks binding of IL-8 and

(Continued )

MGSA/GRO (Hoch et al., 1996; Norgauer et al., 1996). While antibodies to the N-terminus inhibit binding, peptides corresponding to the N-termini of each receptor only weakly inhibit IL-8 binding to

CXCR1 and CXCR2 1987 Figure 4 Model of the secondary structures of human CXCR1 and CXCR2. As deduced by alanine scanning mutagenesis, residues in CXCR1 that are important for receptor expression or for ligand binding are indicated in green (Hebert et al. 1993; Leong et al. 1994). Residues involved in G protein signaling are shown in blue (Damaj et al., 1996b; Xie et al., 1997). Important structural determinants conserved in both CXCR1 and CXCR2 are indicated in red. (Full colour figure can be viewed online.)

CXCR1 and CXCR2 (Ki=17±2.2 mM, and Ki ˆ 150 mM, respectively; Gayle et al., 1993). In addition, a soluble peptide of the first 37 amino acids of CXCR1 is only capable of binding IL-8 with low affinity (Gayle et al., 1993). Another peptide corresponding to residues 1±40 of CXCR1 was also shown to bind IL-8 with a dissociation constant of 170  50 mM (Clubb et al., 1994). These results are in agreement with data from alanine-scanning mutagenesis of CXCR1, demonstrating that multiple extracellular domains of the receptor are involved in high-affinity ligand interactions. Using receptor chimeras, others have studied the selectivity determinants in CXCR1 and CXCR2 (LaRosa et al., 1992; Gayle et al., 1993; Ahuja et al., 1996; Wu et al., 1996). Experiments with human/ rabbit chimeric receptors first implicated the Nterminal domains in determining ligand specificities (LaRosa et al., 1992; Gayle et al., 1993). Chimeras containing the CXCR1 N-terminus were selective for IL-8, whereas those carrying the N-terminal domain of CXCR2 bound both IL-8 and MGSA/GRO with high affinity. Further studies utilized chimeric forms of both human receptors, yet observations of individual groups using the same constructs did not agree. For example, a chimera composed of the CXCR1 Nterminus displayed a CXCR2 phenotype in one study: MGSA/GRO and NAP-2 bound with high affinity and induced a strong calcium flux (Ahuja et al., 1996). The results of these studies by Ahuja et al.

suggest that, in contrast to previous reports, the Nterminal segment of CXCR1 is not a dominant determinant of receptor subtype specificity. In another report, the same chimeric receptor had a CXCR1 phenotype: MGSA/GRO did not bind transfected cells, nor did it induce chemotaxis (Wu et al., 1996). These observations reported by Wu et al. are more consistent with those previously reported by groups using the rabbit CXCR1 N-terminus in the chimera rather than the human CXCR1 (LaRosa et al., 1992; Gayle et al., 1993; Wu et al., 1996). More recently, Katancik et al. mapped the domains involved in binding of the ligands IL-8, NAP-2, and MGSA/GRO to CXCR2 using peptides corresponding to distinct regions of this receptor (Katancik et al., 1997). Peptides representing the N-terminus and first extracellular loop of CXCR2 both inhibited IL-8 binding to the receptor. However, NAP-2 binding was inhibited only by the peptide corresponding to the first extracellular loop, and MGSA/GRO binding was inhibited by portions of the N-terminus. These results suggest that distinct extracellular domains of CXCR2 mediate the binding of each ligand to this receptor. Additional residues in both CXCR1 and CXCR2 are important for maintaining correct tertiary structure of the receptors. Scanning mutagenesis demonstrated that amino acids Cys30 (in the N-terminus), Cys187 (in the second extracellular loop), and Cys277 (in the third extracellular loop) of CXCR1 are

1988 Iris Roth and Caroline Hebert sensitive to mutation to alanine (Leong et al., 1994). Cys30 and Cys277 are conserved in human CXCR1 and CXCR2, as well as rabbit and mouse IL-8 receptors, and may thus be critical for correct protein folding (Hoch et al., 1996). These two cysteine residues likely form a disulfide bridge, bringing Asp11, Glu275, and Arg280 into close spatial proximity to one another. These three charged residues, each critical for ligand binding, thus form a binding domain in CXCR1 where they likely make contact with oppositely charged residues in IL-8 (Hebert et al., 1993). An additional feature common to nearly all GPCRs includes Asp85, located in the second transmembrane domain of CXCR1. This residue is conserved in more than 90% of members of the GPCR superfamily, and is also thought to maintain correct tertiary structure of the receptor. An Asp residue in the second transmembrane domain is also conserved in almost all GPCRs and replacement of this residue leads to a loss of ligand binding (Probst et al., 1992). When Asp85 of CXCR1 is replaced with Ala, the receptor is not expressed on the cell surface, thus abolishing binding of IL-8 (Hebert et al., 1993). The domains in CXCR1 and CXCR2 involving ligand binding may not be important for receptor signaling. A detailed study of chimeric forms of both human CXCR1 and CXCR2 demonstrated that IL-8, GRO, and NAP-2 signal via interactions with multiple receptor domains (Ahuja et al., 1996). In this study, the authors demonstrated that all three ligands could elicit calcium flux via a CXCR2 chimeric receptor containing the N-terminus of human CC chemokine receptor 1 (CCR1), even though no IL-8 binding was detected. The structural determinants important for ligand binding and signaling have been further dissected using various blocking antibodies against the IL-8 receptors (Wu et al., 1996). Studies showed that an antibody against CXCR1 can block IL-8-mediated functional responses, but is ineffective at blocking ligand binding. Together, these findings suggest that the structural requirements for IL-8 binding and signaling are distinct. This is consistent with the idea that conformational changes of the receptor secondary to ligand binding are required to elicit biological responses (Gilman, 1987). Additional structural determinants are also important for receptor signaling (Figure 4). All chemokine receptors, including CXCR1 and CXCR2, contain a highly conserved DRYLAIVHA motif at the end of the third transmembrane domain (TM3) (Baggiolini et al., 1997). As the DRY motif is necessary for G protein activation in other receptors, this domain may be important for signaling and biological activity of CXCR1 and CXCR2 (O'Dowd et al., 1989). Additional motifs conserved among members of the chemokine

receptor family are: TD(X)YLLNLA(X2)DLLF(X2) TLP(X)W in TM2, PLL(X)M(X2)CY in TM5, W(X)PYN in TM6, and HCC(X)NP(X)IYAF(X) G(X2) FR in TM7. A serine/threonine-rich C-terminal region is also a common feature of GPCRs, including CXCR1 and CXCR2 (Probst et al., 1992). This region may be a target for phosphorylation by serine threonine kinases. Phosphorylation of these residues at the C-terminus of the -adrenergic and rhodopsin receptors, both GPCR family members, leads to receptor desensitization (Lefkowitz et al., 1990; Palczewski and Benovic, 1991). A series of truncation mutants further defined key determinants important for receptor signaling. Because expression of CXCR2 requires an intact Nterminal tail, the role of this domain could not be determined (Gayle et al., 1993; Ahuja et al., 1996). Analysis of truncated forms of CXCR2 suggests that signaling requires the C-terminal sequence between amino acids 317 and 324 (Ben-Baruch et al., 1995a). Cells transfected with truncated mutant receptors bound IL-8 with the same high affinity as those transfected with wild-type receptors, but were unable to chemotax in response to IL-8. In addition, the Cterminus-truncated mutant of CXCR2 is not phosphorylated by high doses of IL-8, as is the wild-type receptor (Ben-Baruch et al. 1997). Phosphorylation analysis of the wild-type receptor further demonstrates that IL-8 induced higher levels of phosphorylation than did NAP-2. The greater ability of IL-8 to induce receptor phosphorylation may thus also contribute to its more potent stimulation of chemotaxis compared with NAP-2. Chemokine receptor structure, including that of CXCR1 and CXCR2, is unknown. However, a model can be constructed based on analogy with another GPCR, bacteriorhodopsin, whose crystal structure has been solved (Strader et al., 1994; Luo et al., 1997; Unger et al., 1997; Lomize et al., 1999). The hydrophobic regions of the receptor traverse the plasma membrane, separating extracellular and cytoplasmic loops of the protein and thus giving it a serpentine appearance (Figure 1). CXCR1 and CXCR2 are probably in the cell membrane with the N-terminal tail extracellular, and the C-terminus in the cytoplasm, with the seven hydrophobic domains embedded in the membrane as  helices. The resulting structure comprises a total of three extracellular and three intracellular loops connecting each of the seven transmembrane domains, and free N- and C-termini. CXCR1 has five potential N-linked glycosylation sites (Strosberg, 1991). The molecular size predicted from the amino acid sequence is approximately 40 kDa (Holmes et al., 1991). However, the receptor is

CXCR1 and CXCR2 1989 glycosylated, resulting in an observed mass of 55± 69 kDa (Samanta et al., 1989). CXCR2 is also glycosylated at its one N-linked glycosylation site, to give a final mass of approximately 60 kDa (Horuk, 1994).

Relevant homologies and species differences The human CXCR1 and CXCR2 proteins are highly homologous at the amino acid level (77%) with the highest homology over the membrane-spanning regions and significant divergence at both N- and C-termini (Kelvin et al., 1993). Both proteins also have a significant degree of homology to other chemokine receptors: sequence alignment of the human receptors for the three neutrophil chemoattractants IL-8, fMLP, and C5a shows a 29±34% amino acid identity between these receptors. Genes encoding CXCR1 and CXCR2 homologs from four nonhuman primates have been cloned and sequenced (Alvarez et al., 1996). Both gorilla and chimpanzee CXCR1 homologs show 98±99% similarity to human CXCR1 (Figure 5). Rhesus and orang utan CXCR1 homologs are pseudogenes, in which a 2 bp insertion has generated a sequence with several stop codons. However, the CXCR2 genes from all four primate species are 95±99% identical to their human homolog. Human and rabbit CXCR1 proteins share a 84% amino acid identity (Holmes et al., 1991). The affinity profile and ligand specificity of rabbit CXCR1 is Figure 5 Graphical representation of the sequence homology among CXCR1 and CXCR2 proteins from different animal species.

much like that of the human receptor: IL-8 (Kd  4 nM) > MGSA > NAP-2 (Prado et al., 1994). No binding was observed with radiolabeled GRO or PF4 (Thomas et al., 1994). IL-8 also stimulates calcium flux of transfected cells, while fMLP has no effect. Rabbit CXCR2 has an 80% amino acid identity to human CXCR2, 74% to rabbit CXCR1, and 73% to human CXCR1 (Prado et al., 1994). Rabbit CXCR2 binds IL-8, NAP-2, and MGSA with apparent Ki values of 4, 120, and 320 nM, respectively. IL-8 induced calcium flux and desensitization in mammalian cells transfected with rabbit CXCR2. A mouse IL-8 receptor homolog was cloned by screening a cDNA library for sequences homologous to human and rabbit receptors (Bozic et al., 1994; Harada et al., 1994). The cDNA corresponding to human CXCR2 hybridizes strongly with two restriction fragments in mouse genomic DNA, suggesting that there are two candidate murine homologs of the human IL-8 receptors (Bozic et al., 1994). However, only one murine IL-8 receptor-like protein has been isolated to date, most closely resembling human CXCR2 in its binding characteristics (Lee et al., 1995). Mouse CXCR2 binds the murine counterpart of human GRO proteins, macrophage inflammatory protein 2 (MIP-2), with high affinity (Kd  5 nM), activating both calcium flux and a chemotactic response in neutrophils (Bozic et al., 1994; Lee et al., 1995). Another murine GRO homolog, KC, is approximately 10-fold less potent at triggering these responses. Mouse CXCR2 is 359 amino acids in length, and shares a 68 and 71% homology to human CXCR1 and CXCR2, respectively (Cerretti et al., 1993b). The mRNA for this receptor is expressed in peritoneal neutrophils, and southern blotting analysis suggests that this is a single-copy gene (Cerretti et al., 1993b; Harada et al., 1994). A murine form of IL-8 has not been identified, but human IL-8 is an agonist for mouse CXCR2 (Kd  400 nM) (Bozic et al., 1994; Suzuki et al., 1994). Two rat genes, CXCR1-like and CXCR2, have also been cloned (Dunstan et al., 1996). Both genes are approximately 70% identical to their human homologs at the amino acid level (Dunstan et al., 1996; Gobl et al., 1997). Both rat genes also share a 65 and 86% identity, respectively, with murine CXCR2. Southern blot analysis indicates that rat CXCR1-like and CXCR2 are each single-copy genes. CXCR2 mRNA is detected in adult rat lung, spleen, and neutrophils. CXCR1-like mRNA is expressed in adult rat lung and primary rat macrophages, but not in neutrophils. Murine MIP-2 induces calcium flux in mammalian cells expressing the rat CXCR2 receptor, but not the CXCR1-like protein. Hybridization analysis of mouse genomic DNA suggests that the rat

1990 Iris Roth and Caroline Hebert CXCR1-like gene is homologous to the second candidate murine IL-8 receptor homolog (Bozic et al., 1994; Dunstan et al., 1996). Since the rat CXCR1-like protein is nearly equally homologous to both human CXCR1 and CXCR2 sequences, these unique rat and murine genes may represent a third class of receptors, distinct from CXCR1 and CXCR2.

Affinity for ligand(s) IL-8 and GCP-2 are equipotent agonists at both CXCR1 and CXCR2, while the affinity for each receptor for other ligands is distinct. The rank order of potency of all agonists of CXCR1 and CXCR2 has been determined based on mean effective concentration values (EC50) in calcium flux assays (Ahuja and Murphy, 1996; Baggiolini et al., 1997; Van Damme et al., 1997; Wuyts et al., 1997, 1998; Wolf et al., 1998). In the case of CXCR2, ligand potency is as follows: GRO is the most potent (1 nM) > IL-8 (4 nM)  GCP-2 (3 nM)  GRO (5 nM)  GRO (4 nM)  NAP-2 (7 nM) > ENA-78 (11 nM). For CXCR1, potency was far more selective, with IL-8 and GCP-2 being the most potent agonists (4 and 3 nM, respectively) > ENA-78 (40 nM)  NAP-2 (45 nM) GRO (63 nM)  GRO (65 nM)  GRO . All six agonists for CXCR2 competed for highaffinity [125I]IL-8, [125I]GRO, [125I]NAP-2, and [125I]ENA-78 binding to CXCR2. GRO, GRO , GRO , NAP-2, and ENA-78 each competed weakly for high-affinity IL-8 binding to CXCR1. Although the affinity of CXCR1 for GRO and NAP-2 is almost 100-fold lower than that for IL-8, these ligands can act as low-potency agonists in chemotaxis assays (Loetscher et al., 1994; Ahuja and Murphy, 1996). CXCR1 has also recently been shown selectively to bind the N-terminal cytokine module of human tyrosyl tRNA synthetase with an affinity similar to that of IL-8 (Wakasugi and Schimmel, 1999). This ELR-containing enzyme is secreted under apoptotic conditions and may be involved in inflammatory signaling by apoptotic cells. Selective blocking antibodies to CXCR1 and CXCR2 have been used to determine the role of each receptor in mediating specific neutrophil functions. Responses such as chemotaxis, calcium flux, and the release of neutrophil granule enzymes appear to be independently mediated through both CXCR1 and CXCR2, while the respiratory burst and PLD activation depend exclusively on stimulation through CXCR1 (Loetscher et al., 1994; Hammond et al., 1995; Jones et al., 1996). This is consistent with the ability of GRO and NAP-2 to induce calcium flux and

degranulation, but not PLD activation, in human neutrophils (L'Heureux et al., 1995). IL-8-induced neutrophil chemotaxis and priming were predominantly mediated by CXCR1, whereas priming by GROandENA-78ismediatedbyCXCR2(Hammond et al., 1995; Green et al., 1996). The biological significance of the differential activity of CXCR1 and CXCR2 is unclear. For ligands other than IL-8, the two receptors may be involved in differential spatial transduction of chemotactic signals: At low chemokine concentrations, chemotaxis may be preferentially mediated by CXCR2. Near the site of inflammation, where chemokine concentrations are high and CXCR2 is unable to signal, CXCR1 might be more important. However, both receptors bind IL-8 with similar affinity, thus this mechanism would not be useful for IL-8 gradients. In this case, CXCR2 may mediate chemotaxis at more distal points, while CXCR1 induces respiratory burst at high chemokine concentrations in the inflammatory focus. In fact, the two receptor subtypes are differentially desensitized in human neutrophils. Higher concentrations of IL-8 are required for internalization of CXCR1 than CXCR2, thus downmodulating receptor expression on the cell surface (Chuntharapai and Kim, 1995; Sabroe et al., 1997).

Cell types and tissues expressing the receptor Using monoclonal antibodies specific for CXCR1 and CXCR2, their expression pattern on various peripheral blood leukocytes have been studied by flow cytometry (Figure 6) (Chuntharapai et al., 1994b; Oin et al., 1996). A wide range of donor variation in expression levels was observed. All neutrophils, all monocytes, and 5±25% of CD8‡ T cells and CD56‡ NK cells expressed both receptors (Chuntharapai et al., 1994b). No CD20‡ B cells or CD4‡ T cells expressed CXCR1 or CXCR2. Neutrophils expressed the highest level of both CXCR1 and CXCR2 at an approximately equal ratio, while CXCR2 expression prevailed on other leukocyte subsets, including monocytes and lymphocytes. Consistent with this report, Gerszten et al. recently demonstrated the prevalence of CXCR2 expression on elutriated monocytes (Gerszten et al., 1999). Relative prevalence varied among donors, with CXCR1 expressed on 23±90% of monocytes, while 22±93% of monocytes expressed CXCR2. These receptors on monocytes are functional, as antibodies to both receptors inhibited IL-8-induced monocyte chemotaxis as well as calcium flux.

CXCR1 and CXCR2 1991 Figure 6 Double-color flow cytometric analysis of the distribution of CXCR1 and CXCR2 and human peripheral CD4‡, CD8‡, CD56‡, and CD20‡ B cells. Cells were stained simultaneously with a F-mAb to each receptor and a PE-mAb detecting each leukocyte subset.

CXCR1 and CXCR2 are also expressed by certain subsets of lymphocytes. The mRNA for both receptors can be detected in T cells by RT-PCR, but not by northern blotting, suggesting that expression levels are quite low (Moser et al., 1993; Xu et al., 1995). Using monoclonal antibodies and flow cytometry, 7±42% of CD8‡ T cells and 39±76% of CD56‡ NK cells, but no CD20‡ B cells or CD4‡ T cells were shown to express CXCR2 (Chuntharapai et al., 1994b). Expression of both CXCR1 and CXCR2 was also demonstrated on a subset of T cells that were both CD8‡ and CD56‡ (Oin et al., 1996). Activation of T cells in vitro with anti-CD3 antibodies did not affect expression of these receptors. CXCR1 and CXCR2 expression has also been demonstrated on the human leukemic mast cell line, HMC-1 (Lippert et al., 1998; Nilsson et al., 1999). The mRNA for both receptors was detected by RTPCR, and flow cytometry demonstrated the presence of both proteins on the cell surface. The ligands IL-8, GRO, and NAP-2 all induced calcium flux, as well as chemotaxis of this cell line, demonstrating that the receptors are functionally active. Using

postembedding immunoelectron microscopy, the expression of CXCR1 was shown on the cytoplasmic membrane of isolated human skin mast cells, whereas CXCR2 was found in mast cell-specific granules. However, using the RNAase protection assay, Nilsson et al. (1999) detected only CXCR2 mRNA in HMC-1 cells. Flow cytometry analysis also documented the surface expression of CXCR2. Both reports suggest that ligands for CXCR1 and/or CXCR2 may play an important role in mast cell recruitment during the initiation of an inflammatory response. Controversial results have been published regarding the role of IL-8 in eosinophil activation and chemotaxis, particularly in allergic disease. Using a purified population of eosinophils, Petering et al. recently demonstrated that IL-8 does not induce chemotaxis of or calcium flux in these cells (Petering et al., 1999). Furthermore, RT-PCR experiments showed that eosinophils do not express the mRNA for either CXCR1 or CXCR2. As little as 5% neutrophil contamination was sufficient to produce a measurable IL-8-induced calcium flux in an eosinophil preparation. This suggests that previous reports of

1992 Iris Roth and Caroline Hebert eosinophil expression of IL-8 receptors may have been the result of experiments with impure populations of cells. IL-8 has also been proposed as a stimulus for IgEindependent basophil activation. [125I]IL-8 binding studies revealed approximately 9600 receptors with a mean Kd value of 0.15 nM on human basophils (Krieger et al., 1992). NAP-2 weakly competed for IL-8 binding, and both chemokines led to a transient rise of cytosolic free calcium in these cells. GRO, , and have also been shown to induce calcium flux and chemotaxis of human basophils (Geiser et al., 1993). However, these studies only indirectly imply the presence of CXCR1 and/or CXCR2 on the cell surface. A more recent study demonstrated the expression of intermediate levels of CXCR1 and CXCR2 on basophils (Ochensberger et al., 1999). However, receptor agonists did not stimulate cell functions such as cytokine and leukotriene release. There is a lack of consensus regarding endothelial cell expression of either CXCR1 or CXCR2. The mRNA for GRO is upregulated when this chemokine is added to human umbilical vein endothelial cell (HUVEC) cultures in vitro, implying that these cells express the GRO receptor, CXCR2 (Wen et al., 1989). More indirect evidence comes from experiments that show IL-8-induced albumin flux across HUVEC monolayers in the absence of neutrophils, an effect that is inhibited by cycloheximide (Biffl et al., 1995). Additionally, IL-8 appears to be chemotactic for HUVECs in an in vitro chemotaxis assay (Koch et al., 1992). IL-8 was also shown to bind to the surface of endothelial cells at different anatomical locations; however the identity of these binding sites is unknown (Rot et al., 1996). Studies demonstrating direct expression of CXCR1 or CXCR2 on endothelial cells have been inconclusive. Schonbeck et al. found that IL-8 binds to both HUVEC and saphenous vein endothelial cells, and used RT-PCR techniques to demonstrate the expression of CXCR1 on these cells (Schonbeck et al., 1995). Others were unable to show IL-8 binding, IL-8-induced calcium flux, or IL-8-induced endothelial cell proliferation using both large-vessel and microvascular endothelial cells (Petzelbauer et al., 1995). A recent report by Murdoch et al. demonstrates endothelial cell expression of the mRNAs for CXCR1 and CXCR2, but low levels of surface expression of only CXCR1 protein (Murdoch et al., 1999). RT-PCR and immunocytochemical techniques have also detected CXCR1 and CXCR2 expression in normal melanocytes as well as melanoma cells (Moser et al., 1993; Norgauer et al., 1996). Keratinocytes have also been shown to possess binding sites for IL-8 (Kemeny et al., 1994b). Receptors for IL-8

are also expressed throughout the central nervous system (reviewed in Hesselgesser and Horuk, 1999a; Mennicken et al., 1999). While CXCR1 expression was not detected, CXCR2 is expressed at high levels by subsets of neurons in diverse regions of the brain and spinal cord (Horuk et al., 1997).

Regulation of receptor expression Bacterial LPS has been shown to downmodulate neutrophil CXCR1 and CXCR2 expression via the stimulation of tyrosine kinase activity (Khandaker et al., 1998). However, others have demonstrated that serum-activated LPS acts to induce the surface expression of IL-8 receptors through de novo protein synthesis (Manna et al., 1995). IL-8 receptor expression is also upregulated by formyl peptide via degranulation of the neutrophil secretory vesicle (Manna and Samanta, 1995). CXCR1 mRNA expression, IL8 binding, and neutrophil chemotactic responses are upregulated by G-CSF as well (Lloyd et al., 1995). Conversely, treatment of neutrophils with tumor necrosis factor  (TNF) decreases cell surface CXCR1 and CXCR2 expression (Jawa et al., 1999). Others have demonstrated that TNF downregulates the expression of CXCR2, but CXCR1 levels are unaffected (Asagoe et al., 1998). Transcription of the CXCR2 gene in human T lymphocytes is downregulated by incubating cells at 37 C, and is restored by co-culturing T cells with monocytes (Xu et al., 1995). CXCR1 and CXCR2 expression on the cell surface is also regulated endogenously by receptor internalization. More than 90% of [125I]IL-8-bound neutrophil receptors is endocytosed within 10 minutes at 37 C, and the receptors are expressed on the cell surface 10 minutes later (Besemer et al., 1989; Grob et al., 1990; Samanta et al., 1990). Inhibitory lysosomotropic agents, such as ammonium chloride, inhibit receptor internalization as well as chemotaxis, suggesting that chemotactic activity is dependent on receptor recycling (Samanta et al., 1990). The expression of all GPCRs, including the chemokine receptors CXCR1 and CXCR2, is strictly regulated by desensitization (Baggiolini et al., 1994; Ben-Baruch et al., 1995b). In homologous desensitization, exposure to high concentrations of ligand renders the cell unresponsive to the same ligand (Kelvin et al., 1993; Ben-Baruch et al., 1995b). In contrast, heterologous desensitization is a reversible loss of responsiveness to multiple ligands. Homologous desensitization after stimulation with IL-8 or GRO is mediated by phosphorylation of serines and threonines in the C-terminus of CXCR1

CXCR1 and CXCR2 1993 and CXCR2, respectively (Baggiolini et al., 1994; Mueller et al., 1994; Richardson et al., 1995; Schraufstatter et al., 1998). Ser346 and Ser348 are the primary sites of phosphorylation of CXCR2 (Schraufstatter et al., 1998). Alanine substitution at these residues prevents receptor desensitization after repeated exposure to IL-8; however, receptor internalization was unaffected. Heterologous desensitization also involves C-terminus phosphorylation, as phosphorylation-deficient mutant CXCR1 is resistant to cross-desensitization by receptors to fMLP, C5a, or PAF (Richardson et al., 1995, 1998). Phosphorylation at these resides in the -adrenergic and rhodopsin receptors, both GPCR family members, results in arrestin binding, thus sterically inhibiting the binding of G proteins to the receptor and preventing signaling (Zhao et al., 1995; Lefkowitz, 1998). Receptor desensitization is likely an important mechanism in the detection of changes in ligand concentration, allowing cells to move toward a chemokine gradient.

SIGNAL TRANSDUCTION Two groups of G proteins, the heterotrimeric G proteins and monomeric low molecular weight G proteins, mediate signal transduction through chemokine receptors. Like all members of the GPCR superfamily, CXCR1 and CXCR2 act as guanine nucleotide exchange factors for the heterotrimeric G proteins (Figure 7) (Kupper et al., 1992; Strader et al., 1994). Heterotrimeric G proteins associate with the intracellular domains of GPCRs when in their inactive, or guanosine diphosphate (GDP)-bound,

state. Upon ligand binding, the GDP is exchanged for guanosine triphosphate (GTP), thus activating the G protein. The active G protein subsequently dissociates into its  and subunits to stimulate effector molecules. In neutrophils, the free  subunit can activate both phospholipase C (PLC) 1 and PLC 2, while the free subunit preferentially activates PLC 2 (BenBaruch et al., 1995b). The activated PLCs catalyze the hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messengers inositol-1,4,5-trispohsphate (IP3) and 1,2-diacylglycerol (DAG) (Baggiolini and Clark-Lewis, 1992; Baggiolini et al., 1993). IP3 diffuses into the cytosol and mobilizes intracellular calcium, and DAG activates protein kinase C (PKC; Thelen et al., 1988; Murphy, 1994). IL-8-induced activation of PKC and the rise in cytosolic calcium further induce PLC and phospholipase D (PLD), which yield DAG and phosphatidic acid, respectively, and result in a positive feedback loop (Sozzani et al., 1994a; Howard et al., 1996). Others have been able to detect PLD activation only in IL-8-stimulated T lymphocytes, but not in neutrophils (Bacon et al., 1995). Additional signaling pathways, such as the MAPK cascade and the serine/threonine and tyrosine kinases, are also activated following G protein activation (Grinstein and Furuya, 1992; Grinstein et al., 1994; Thompson et al., 1994; Jones et al., 1995). Experiments using neutrophils as well as transfected cells suggest that both CXCR1 and CXCR2 physically couple to many G protein subtypes, including Gi2, Gi3, G14, G15, and G16, but not to Gq or G11 (Wu et al., 1993; Damaj et al., 1996a). Furthermore, Gi2 peptides or antisera can effectively

Figure 7 Schematic representation of chemokine signal transduction. Abbreviations: DAG, diaglycerol; IP3, inositol-1,4,5-trisphosphate; PA, phosphatidic acid; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; [Ca2‡]i, intracellular calcium.

1994 Iris Roth and Caroline Hebert block calcium flux responses to IL-8 in cells expressing CXCR1 and CXCR2 (Thelen et al., 1988; Damaj et al., 1996a). The structural determinants on CXCR1 and CXCR2 involved in mediating G protein signaling have been described (Damaj et al., 1996b; Xie et al., 1997). Using site-directed mutagenesis, key residues of CXCR1 were shown to be involved in the interaction of the receptor with Gi2 and G16 (Figure 4). Activation of G16 was abolished by alanine substitutions at both Lys158 and Arg159 in the second intracellular loop of CXCR1, suggesting that either of these two basic residues is sufficient for G16 coupling (Xie et al., 1997). Four residues in the second intracellular loop (Tyr136, Leu137, Ile139, and Val140) and one residue in the third intracellular loop (Met241) were shown to be crucial for Gi2 coupling and transduction of an IL-8-induced calcium flux (Damaj et al., 1996). Other residues in the second and third intracellular loops were also found to affect CXCR1 signaling, but to a lesser extent. An intrinsic GTPase activity regulates G protein signaling by hydrolyzing GTP to GDP. The resulting inactive GDP-bound G protein then reforms a complex with unoccupied receptors. GTPase-activating proteins known as regulators of G protein signaling (RGS) modulate this activity for heterotrimeric G proteins (Koelle, 1997). Studies with cotransfected HEK293 cells show that several RGS proteins modulate IL-8 signaling through CXCR2 (Bowman et al., 1998). In addition to signaling via the heterotrimeric G proteins, CXCR1 and CXCR2 signaling also activates monomeric, low molecular weight G proteins of the Ras and Rho families (Laudanna et al., 1996). Rho proteins are usually involved in cell motility through regulation of actin-dependent processes such as membrane ruffling, pseudopod formation, and assembly of focal adhesion complexes. In mouse preB cells transfected with CXCR1, IL-8 signals via RhoA to stimulate adhesion to fibrinogen (Laudanna et al., 1996). Studies suggest that different neutrophil functions are activated by distinct signaling pathways. The assembly or activation of NADPH oxidase and the subsequent generation of superoxide anions in respiratory burst are associated with tyrosine phosphorylation and DAG±PLD interactions, respectively (Qualliotine-Mann et al., 1993; Richard et al., 1994). IL-8 stimulates the activation of PLD only at concentrations required for stimulating respiratory burst, not at the 10- to 100-fold lower concentrations that mediate chemotaxis (Sozzani et al., 1994a). Chemotaxis can be triggered by agonists for receptors coupled to the Gi subfamily of G proteins,

but not by agonists for receptors coupled to Gs and Gq (Wu et al., 1993). Furthermore, treatment of neutrophils with pertussis toxin inhibits IL-8-directed chemotaxis by preventing the activation of Gi. These observations suggest that chemotaxis is mediated by the specific pertussis-sensitive  subunit of Gi proteins. However, recent evidence demonstrates that chemotaxis does not require activation of Gi, and that the free subunit transmits the necessary chemotactic signal (Neptune et al., 1999). There is evidence that neutrophil chemotaxis results from PLC activation and subsequent calcium flux (Murphy, 1994). However, others have shown that cell migration does not require an increase in intracellular calcium (Sha'afi et al., 1986; Sham et al., 1993; Sozzani et al., 1994b). More recent reports suggest that IL-8-induced neutrophil chemotaxis is dependent on phosphatidylinositol 3-kinase (PI-3 kinase) activity, as migration can be inhibited by wortmannin and the PI-3 kinase inhibitor LY294002 (Knall et al., 1997). PI-3 kinase signaling further activates the Ras/Raf/extracellularly responsive kinase (ERK) pathway in neutrophils and in cells transfected with CXCR1 and CXCR2 (Knall et al., 1996; Shyamala and Khoja, 1998). However, ERK activation does not play a role in mediating IL-8directed chemotaxis, as the ERK kinase inhibitor, PD098059, has no effect on neutrophil migration (Knall et al., 1997).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors CXCR1 and CXCR2 mediate a diversity of chemokine functions in a variety of cell types. Both CXCR1 and CXCR2 induce chemotaxis and calcium flux of cells expressing these receptors. Both receptors also stimulate the release of granule enzymes in neutrophils. The generation of superoxide in the neutrophil respiratory burst is mediated exclusively by CXCR1 (Loetscher et al., 1994; Hammond et al., 1995; Jones et al., 1996). CXCR1 and/or CXCR2 also mediate the angiogenic effects of ELR-containing CXC chemokines. These ligands induce endothelial cell proliferation and chemotaxis in vitro as well as angiogenesis in various animal models (Koch et al., 1992; Strieter et al., 1995).

CXCR1 and CXCR2 1995 CXCR1 and CXCR2 are important in mediating antimicrobial host defenses. Their function in directing leukocyte recruitment and activation leads to clearance of infective agents. In addition to their role in regulating immune responses, CXCR1 and/or CXCR2 are also involved in stimulating vasculogenesis. CXC chemokines are divided into two groups that either contain or lack the ELR motif at their Nterminus. ELR-containing chemokines are angiogenic, as shown by their effects on endothelial cell proliferation and chemotaxis in vitro as well as angiogenesis in animal models, including the rat cornea and tumor regression in vivo (Koch et al., 1992; Strieter et al., 1995; Arenberg et al., 1997). Chemokines that lack the ELR motif inhibit the mitogenic effects of angiogenic chemokines, as well as the major angiogenic mediators vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). ELR-containing CXC chemokines are ligands for CXCR1 and CXCR2. It is unclear which of these receptors are expressed on endothelial cells, and therefore which are responsible for the angiogenic effects. Bioassays A variety of assays are available to measure the biological activities of chemokine receptors and their ligands. Ligand-binding assays are initially useful for analyzing receptor-binding affinities. Cells expressing receptors are incubated with 125I-labeled ligand, as well as varying concentrations of unlabeled ligand to compete for binding sites. After incubation, the cells are washed and emissions are counted. Data are then analyzed using Scatchard analysis to determine Kd values (Scatchard, 1949). More recently, the binding of nonradioactive, recombinant His-tagged ligands can be visualized by flow cytometry using monoclonal antibodies against the His-tag (Wilken et al., 1999). Following receptor binding, chemokine receptor activation is classically associated with changes in intracellular calcium concentration (Murphy, 1994). Intracellular calcium flux can be readily detected by monitoring the response of fluorescent calcium-binding dyes. Compounds such as Fura-2, Indo-1, and Fluo-3 (Molecular Probes, Eugene, OR) show changes in their spectral characteristics (fluorescence intensity or excitation or emission spectra) upon calcium binding (reviewed in Kao, 1994; McColl and Naccache, 1997). As the spectral characteristics of the three probes are unique, each may be useful under distinct experimental conditions or with different recording equipment, such as fluorescence microscopy, flow cytometry, and fluorescence spectroscopy.

Several assays measure the ability of chemokine ligands to stimulate activated primary neutrophils, cells which express both CXCR1 and CXCR2 (Baly et al., 1997). As part of the host response to invading pathogens, neutrophils migrate to the site of infection, where they undergo oxidative burst then the release of degradative granular enzymes. The oxidative burst can be monitored in vitro in response to chemokine ligands (Baly et al., 1997). One method involves the detection of hydrogen peroxide (H2O2), generated during respiratory burst. Neutrophils are incubated with dichlorofluorescein diacetate (DCFHDA), a stable, nonfluorescent, nonpolar compound, and the DCFH-DA diffuses into the cells where it is deacetylated by intracellular esterases. The resulting nonfluorescent compound, DCFH, is polar, thus trapped within the cell and serves as a substrate for hydrogen peroxide. During the oxidative burst, hydrogen peroxide catalyzes the generation of the polar and highly fluorescent 20 ,70 -dichlorofluorescein (DCF), which can be readily measured by flow cytometry. Additional assays detect the enzymes elastase and -glucuronidase, both released from intracellular azurophilic storage granules upon stimulation of activated neutrophils (Baly et al., 1997). Enzyme concentrations are determined by measuring their proteolytic activity on synthetic substrates, such as MeOSuc-Ala-Ala-Pro-Val-PNA for elastase and 4methylumbelliferyl- -D-glucuronide for -glucuronidase. The two enzymes differ in the time course of their release, their assay stability, and their susceptibility to interfering substances in the test samples. Following stimulation with IL-8, elastase release continues for 3 hours or more, while the release of -glucuronidase is complete after 15 minutes. One benefit to measuring -glucuronidase release is that enzymatic activity can be readily stopped by diluting samples in a high-pH glycine solution. This stable end-point reduces the risk of assay drift when large numbers of samples are assayed. Lastly, unlike that of -glucuronidase, elastase activity is extremely sensitive to inhibition by plasma at concentrations as low as 0.05% (v/v). A recent report describes a method for measuring granule enzyme release in a 96-well microplate format (Tiberghien et al., 1999). Cell migration in response to chemokine ligands is also studied in vitro. Boyden first described the measurement of chemotaxis using membrane filters in 1962. Since then, a variety of methods have been used to assay chemotaxis with membrane filters (reviewed in Frevert et al., 1998; Wilkinson, 1998). New assays for chemotaxis utilize a rapid fluorescence-based 96well plate format. In this technique, cells expressing chemokine receptors are labeled with a fluorescent

1996 Iris Roth and Caroline Hebert probe, such as calcein-AM (Molecular Probes, Eugene, OR) and added above a porous filter in a disposable 96-well plate chemotaxis chamber (ChemoTx, Neuro Probe, Gaithersburg, MD). Chemokine ligand is added below the filter, and cell migration into the lower chamber is measured using total fluorescence as a marker for cell number. This fluorescence end-point assay is more rapid and more sensitive, and is a significant advance over traditional chemotaxis assays.

Phenotypes of receptor knockouts and receptor overexpression mice Disruption of the murine CXCR2 gene demonstrates that this expression of this chemokine receptor is essential for normal myeloid and lymphoid development. CXCR2 knockout mice fail to mobilize neutrophils in response to intraperitoneal thioglycollate in vivo (Cacalano et al., 1994). Neutrophils isolated from these animals also fail to chemotax in response to KC or MIP-2 in vitro, indicating that CXCR2 is the dominant neutrophil receptor for these chemokines (Lee et al., 1995). When raised in a specific pathogen-free environment, knockout mice have a massive expansion of neutrophils and B cells in bone marrow, lymphoid tissue, and blood (Cacalano et al., 1994). However, neutrophil counts are not increased when mice are raised in a germ-free environment. This may be due to the fact that CXCR2 is a negative regulator of hematopoiesis (Broxmeyer et al., 1996). Alternatively, Cacalano et al. have speculated that the inability to survey tissues properly and completely to eliminate external pathogens in the knockouts may result in the release of cytokines that stimulate neutrophil and B cell production (Cacalano et al., 1994). Regardless, this defect does not appear to alter susceptibility to environmental or challenge pathogens. Another unexpected observation is an elevation of IL-4 and IgE plasma levels in these CXCR2 knockout mice.

Human abnormalities Individuals without functional CXCR1 or CXCR2 have not been identified. However, a recent study using FACS analysis suggests that both receptors are downregulated in neutrophils from patients suffering from pulmonary tuberculosis and in those seropositive for HIV-1 (Meddows-Taylor et al., 1998). The reduced expression of IL-8 receptors also resulted in an impairment of both chemotaxis and calcium flux in

response to IL-8. Exposure of neutrophils to hypoxia/ reoxygenation also caused a downregulation of CXCR1 expression at both the mRNA and protein level (Grutkoski et al., 1999). CXCR2 surface expression is downregulated by 50% on neutrophils from patients with sepsis (Cummings et al., 1999). While neutrophil chemotaxis to IL-8 and the bacterial peptide fMLP were unaffected, cells from septic patients had a markedly suppressed migratory response to the CXCR2 ligands, ENA-78 and GRO. Increased expression of epidermal IL-8 receptors has been observed in psoriasis and other inflammatory and hyperproliferative skin diseases (reviewed in Kemeny et al., 1994b). For example, RT-PCR and immunohistochemical techniques have detected CXCR1 and CXCR2 expression on fibroblasts and smooth muscle cells of burn lesions (Nanney et al., 1995). Using immunohistochemistry and in situ hybridization techniques, CXCR2 but not CXCR1 was localized to suprabasal lesional psoriatic keratinocytes (Kulke et al., 1998). Keratinocyte IL-8 receptors are not expressed in normal skin, and thus may be useful targets in the treatment of inflammatory skin diseases (Schulz et al., 1993; Kemeny et al., 1994a; Kulke et al., 1998). Chemokines and their receptors, including CXCR1 and CXCR2, may be involved in pathologies of the central nervous system (reviewed in Hesselgesser and Horuk, 1999b). CXCR2 is widely expressed in the normal central nervous system, and also in neuritic plaques surrounding amyloid deposits in Alzheimer's disease (Horuk et al., 1997; Xia et al., 1997; Xia and Hyman, 1999). As the receptor for GRO, CXCR2 may also mediate this chemokine's effects on melanoma cell growth. CXCR2 expression on various melanoma cell lines was demonstrated by RT-PCR as well as by flow cytometry using specific anti-CXCR2 antibodies (Norgauer et al., 1996). CXCR2 may be involved in syncytium formation in HIV infection and the direct cell-to-cell transfer of virus. The envelope protein of a T cell line-adapted HIV-2 strain has been shown to utilize CXCR2 to induce fusion. Although less efficient than CXCR4 and CCR3, both also used by the well-characterized HIV-1 strain, fusion with CXCR2 expressing cells was specific and was inhibited by antibodies against CXCR2 (Bron et al., 1997).

THERAPEUTIC UTILITY Antibody neutralization studies have demonstrated that IL-8 is a key mediator of neutrophil-mediated acute inflammation in the rabbit (Sekido et al., 1993; Broaddus et al., 1994; Folkesson et al., 1995).

CXCR1 and CXCR2 1997 However, the selectivity of the two known rabbit IL-8 receptors for different rabbit chemokines has not been defined, thus their relative importance in inflammatory models is also not yet understood. Potential disease targets include many neutrophilmediated inflammatory processes such as ischemia± reperfusion injury, adult respiratory distress syndrome, and certain forms of glomerulonephritis and dermatitis. Recent evidence suggests that IL-8 may also play a role in Alzheimer's disease, as well as in the growth of tumors via its angiogenic effects. Because of ligand redundancy, chemokine receptors may be better targets for development of therapeutic blocking agents. However, it is still not known whether it will be required to block both receptor subtypes, or whether inhibition of just one will suffice. Regardless, the abnormal phenotype of CXCR2 knockout mice calls into question the safety of therapeutically blocking these receptors.

Effects of inhibitors (antibodies) to receptors Selective neutralizing monoclonal and polyclonal antibodies against CXCR1 and CXCR2 have been described (Hammond et al., 1995; Green et al., 1996; Jones et al., 1996). N-terminal truncations of IL-8 and GRO are also effective antagonists of CXCR2 signaling (Baggiolini et al., 1997). Similarly, Nterminally modified analogs of IL-8 block IL-8induced calcium mobilization in cells transfected with CXCR1 or CXCR2 (Jones et al., 1997). Analogs of GRO and PF4 bound only CXCR2 with high affinity, and blocked calcium flux at this receptor alone. These two analogs had no effect on IL-8elicited superoxide generation and release of granule enzymes in neutrophils, as these functional responses are induced via CXCR1 alone. As such proteins have some limitations as therapeutics, the search for small molecule antagonists of chemokine receptors has been an active area of pharmaceutical research in the past few years. A potent and selective nonpeptide small molecule inhibitor of CXCR2 was recently described (Ponath, 1998; White et al., 1998). SB 225002 (N-(2-hydroxy-4nitrophenyl)-N 0 -(2-bromophenyl)urea) is an antagonist of [125I]IL-8 binding to CXCR2 with an IC50=22 nM and 150-fold selectivity over CXCR1. SB 225002 potently inhibits the chemotaxis of both rabbit and human neutrophils to IL-8 and GRO in vitro. In vivo, this molecule selectively blocks IL-8induced neutrophil migration in rabbits, suggesting that CXCR2 plays an important role in this process.

Using receptor-specific antibodies, others have shown that CXCR1 is the key receptor involved in neutrophil chemotaxis in vitro (Quan et al., 1996). Regardless, this compound will be useful in defining the role of CXCR2 in neutrophil-mediated inflammatory diseases.

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