The biochemistry and biology of the atypical chemokine receptors

Immunology Letters 145 (2012) 30–38

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Review

The biochemistry and biology of the atypical chemokine receptors G.J. Graham a,∗,1 , M. Locati b,c,∗∗,1 , A. Mantovani b,c,1 , A. Rot d,1 , M. Thelen e,1 a

Institute of Infection, Immunity and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, UK Istituto Clinico Humanitas IRCCS, I-20089 Rozzano (Milan), Italy c Department of Translational Medicine, University of Milan, I-20089 Rozzano (Milan), Italy d MRC Centre for Immune Regulation, Institute of Biomedical Research, University of Birmingham, UK e Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland b

a r t i c l e

i n f o

a b s t r a c t

Keywords: Chemokines Chemokine receptors Scavenging receptors Atypical receptors Immunology

A subset of chemokine receptors, initially called “silent” on the basis of their apparent failure to activate conventional signalling events, has recently attracted growing interest due to their ability to internalize, degrade, or transport ligands and thus modify gradients and create functional chemokine patterns in tissues. These receptors recognize distinct and complementary sets of ligands with high affinity, are strategically expressed in different cellular contexts, and lack structural determinants supporting G␣i activation, a key signalling event in cell migration. This is in keeping with the hypothesis that they have evolved to fulfil fundamentally different functions to the classical signalling chemokine receptors. Based on these considerations, these receptors (D6, Duffy antigen receptor for chemokines (DARC), CCX-CKR1 and CXCR7) are now collectively considered as an emerging class of ‘atypical’ chemokine receptors. In this article, we review the biochemistry and biology of this emerging chemokine receptor subfamily. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

[5,6]. All atypical chemokine receptors clearly fail to sustain activation of these signalling pathways in response to ligands [7,8]. Triggering of classical downstream signalling requires coupling of chemokine receptors to pertussis toxin-sensitive G␣i proteins [5,6], as well as the presence of a highly conserved DRYLAIV motif in the second intracellular loop which is assumed to be essential for G protein coupling. As the second intracellular loop of atypical chemokine receptors has sequences divergent from this canonical sequence [9,10], this is now regarded as a characteristic feature of this subfamily of chemokine receptors. However, exchange of this sequence with the signalling motif provides conflicting data. With D6, this is associated with a reconstitution of weak signalling ability and with CXCR7 with no signalling competence (Graham et al., unpublished). Together these data indicate that additional sequence elements are missing from these receptors for effective G␣i coupling. It is of note that the International Union of Pharmacologists only permits assignment of systematic names to receptors on demonstration of signalling in response to ligand binding [11]. This is why most of the atypical receptors have not been assigned ‘systematic’ names. Ligand-induced chemokine receptor internalization is known to be independent of G protein coupling. In general receptor internalization is initiated through agonist-stimulated receptor phosphorylation, arrestin recruitment, and clathrin-mediated internalization. Several lines of evidence suggest that internalization of atypical chemokine receptors in principle follows this paradigm. Accordingly, ligand binding induces arrestin recruitment [10,12–16] and deletion of the C-terminus, an important site for

Chemokines are the fundamental regulators of in vivo leukocyte migration and their activity is regulated at a number of transcriptional, translational and post-translational levels. Over the past 15 years it has become apparent that there is a subfamily of chemokine receptors, collectively referred to as ‘atypical chemokine receptors’, which are characterized by their apparent inability to signal following ligand binding, and which have emerged as important regulators of chemokine function [1–4]. Some of these atypical receptors have clearly been shown to be able to scavenge chemokines and therefore to manipulate chemokine responses in vivo. In addition, others appear to be important for the local regulation of chemokine presentation and function. This review will focus on the biochemistry and biology of this ‘atypical chemokine receptor’ family. 2. Signalling properties of the atypical receptors All conventional chemokine receptors share conserved signalling pathways leading to calcium mobilization and chemotaxis

∗ Corresponding author. ∗∗ Corresponding author at: Department of Translational Medicine, University of Milan, I-20089 Rozzano (Milan), Italy. E-mail address: [email protected] (G.J. Graham). 1 All authors contributed equally and are listed in alphabetical order. 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.04.004

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phosphorylation and arrestin binding, prevents receptor internalization [8,10]. Binding of arrestin to GPCRs and 7TMDs not only initiates receptor internalization, but also triggers intracellular signalling cascades independently of G protein activation [17]. In line with this general notion, it was shown that CXCR7 could activate the MAP kinase pathway but with typically delayed kinetics compared to G protein-mediated kinase activation [18,19]. 3. Discovery of the atypical receptors 3.1. D6 Cloned in 1997 from placenta [20] and haematopoietic stem cells [21], D6 is a CC-chemokine receptor encoded by a gene within the CCR cluster at the 3p21.3 region of the human genome [22]. One major difference between D6 and the other signalling receptors is that, in D6, the DRYLAIV motif is altered, in all mammalian D6 genes, to DKYLEIV. In 2003 D6 was shown to act as a chemokine decoy and scavenger receptor, thus becoming the first atypical chemokine receptor to be functionally characterized [7]. D6 is characterized by a broad ligand binding profile including most of the inflammatory CC-chemokines (agonists of CCR1 through CCR5), but it also has binding selectivity in that it does not recognize homeostatic CC-chemokines or CXC, XC or CX3C chemokines [21]. Among inflammatory CC-chemokines D6 selectively degrades the active forms of CCL22 and CCL14 but not their amino-terminal CD26-processed inactive forms [23,24]. This is also seen with other prototypic inflammatory CC chemokine ligands such as CCL3 [25] and suggests that CD26-processing may be a physiological mechanism for suppressing the recognition of inflammatory chemokines by D6. 3.2. CCX-CKR1 CCX-CKR1 was initially cloned in a study aimed at identifying novel receptors for the monocyte chemoattractant protein family [26]. Preliminary work suggested that this receptor was capable of mediating signalling and it was therefore named CCR11. Subsequent studies, however, failed to confirm either monocyte chemoattractant family binding or the signalling properties of the receptor which is therefore referred to most often as CCX-CKR1 or by its gene designation, CCRL1 [27,28]. In keeping with the other members of the family, this apparent lack of signalling competence is associated with alterations to the canonical DRYLAIV motif as in CCX-CKR1 this is altered to DRYVAVT. In many ways, CCXCKR1 can be regarded as being a homeostatic chemokine binding equivalent of D6 in that it selectively binds the homeostatic CCchemokines CCL19, CCL21 and CCL25 [27,28] suggesting a role in the regulation of chemokine-mediated aspect of the adaptive immune response. Human CCX-CKR1 does not bind inflammatory CC-chemokines but was shown to bind the homeostatic CXC chemokine, CXCL13, albeit with lower affinity [27,28]. Again in keeping with D6 and CXCR7, CCX-CKR1 appears to internalize and scavenge its ligands [29] though not as robustly as D6, leaving open the possibility that CCX-CKR1 may fulfil other potential functions [30]. Both mouse and human genomes carry a copy of the CCX-CKR1 gene within the major chromosomal locus incorporating most of the other CC-chemokine receptors (chromosome 9 in the mouse and chromosome 3 in human) [28]. Interestingly, in both species, there is differential expression of alternative forms of the receptor from these loci. These are discriminated between by the presence, or absence, of a 5 extension, which derives from the neighbouring acylCoA dehydrogenase gene at this locus. Interestingly, humans have an additional, highly similar, copy of CCX-CKR1, which is encoded by a gene located on chromosome 6. There appear

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to be no differences in expression of these two receptors and their functional significance remains to be determined. 3.3. CXCR7 Over 20 years ago Vassart and colleagues identified the sequence of a putative G-protein coupled receptor (GPCR), named RDC1, from a canine cDNA library using a human probe that was amplified with degenerate primers for GPCRs [31]. Sequence similarity analysis and localization of the mouse gene in the genomic region incorporating CXCR2 and CXCR4 on chromosome 1 led to the conclusion that the orphan receptor RDC1 belonged to the chemokine receptor family [32]. In humans a similar arrangement of the same chemokine receptors is found on chromosome 2. Extensive phylogenetic analyses placed the orphan receptor RDC1 in close proximity to the chemokine receptors within the rhodopsin family of GPCRs [33]. Only later was RDC1 shown to bind, with high affinity, CXCL12, the unique ligand for the chemokine receptor CXCR4 and, with about 20 fold lower affinity, CXCL11 a ligand for the chemokine receptor CXCR3 [34,35]. This prompted the renaming of the receptor to CXCR7, despite lack of evidence of coupling to G proteins and cell activation. In line with the other atypical receptors CXCR7 displays alterations of the canonical DRYLAIV motif. The divergence of the CXCR7 sequence DRYLSIT is less pronounced, nevertheless, addition of ligands does not promote GTP␥S binding to heterotrimeric G proteins, indicative of lack of coupling of the receptor to G proteins [36]. Thus, CXCR7 should not be classified as GPCR, but rather as a seven transmembrane domain receptor (7TMD). 3.4. DARC DARC was discovered over 60 years ago as the Duffy blood group antigen (sometimes also referred to as Fy antigen) and named after the first patient, a polytransfused heamophiliac, shown to bear antibodies recognizing this antigen [37]. Initial molecular cloning of the Duffy antigen suggested a conformational structure of a membrane nine-spanner [38] and only subsequently it was modelled as a seven-spanner, akin to the classical GPCRs [39]. DARC is the most promiscuous among chemokine receptors; it binds at least 20 different CC and CXC-chemokines, though with a broad range of affinities [40–42]. All chemokine ligands of DARC belong to the “inflammatory” functional subset [39–43]. In addition to chemokines, DARC binds other molecules including, rather contentiously, a tetraspanin CD82 [44] as well as HIV-1 [45,46] and malarial parasites Plasmodium vivax and knowlesi, which invade erythrocytes after binding to DARC [47,48]. Unlike other atypical chemokine receptors, which carry various degrees of modification of the DRYLAIV consensus domain, DARC completely lacks this motif. Accordingly, no conventional GPCR signalling could be demonstrated downstream of DARC [39,49]. There are three major polymorphisms of the human Duffy gene. FY*A and FY*B, which encode the two antithetical “Duffy positive” antigens differing by a single base substitution 306 G→A in codon 44 encoding glycine in Fya and aspartic acid in Fyb [50]. The third polymorphism, leads to a Duffy “negative” phenotype characteristic of individuals of West African stock. This polymorphism is defined by a single T to C substitution at nucleotide −46 of FY*B within the erythroid GATA1 promoter region, which effectively blocks the transcription of DARC in the erythroid lineage only [51] and is often denoted as FY*B(ES), for ‘erythroid silent’. Thus, Duffy “negative” individuals still express DARC on non-erythroid cells in various tissues, most notably, endothelial cells [52]. As discussed in more detail bellow, DARC has disparate functions in the alternative cellular contexts in which it is expressed.

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4. Biochemical properties of the atypical receptors

5. Expression of the atypical receptors

Whilst most chemokine receptors are expressed on the cell surface and show only a limited tendency to spontaneously internalize, D6 and CXCR7 rapidly cycle between the plasma membrane and endosomes, where they are preferentially localized [8,13,53–57]. Investigations into D6 and CXCR7 trafficking revealed ligand-dependent and -independent cycling, suggesting that these receptors act as scavengers for their cognate ligands [8,13,55]. Consistent with this interpretation, their capacity to internalize chemokines does not become saturated and D6 function is therefore effectively catalytic in nature. Similar studies have, so far, proven difficult with CCX-CKR1 due to the lack of availability of high quality antibodies. New antibodies have now been reported and may well be useful in this regard [58]. In the case of CXCR7, the assumption that its scavenging activity titrates the availability of CXCL12 for its signalling receptor CXCR4 is also supported by its higher affinity for the common ligand CXCL12 [34,35]. This is not the case for D6, or CCXCKR1, whose binding affinities for their CC-chemokine ligands are in the same order of magnitude as for their cognate conventional receptors. Conversely, in case of D6, cycling properties have been reported to have profound impact on its scavenging properties. In contrast to conventional chemokine receptors, which are down-regulated by cognate ligands [59], D6 undergoes rapid Rab5- and Rab11-dependent redistribution to the cell membrane after ligand engagement and its redistribution corresponds to an increased chemokine degradation rate [55]. Interestingly, this process occurs at different efficiency with different ligands [24], suggesting the involvement of a presently unknown signalling pathway differently regulated by different D6 ligands. As D6 appears to show only limited regulation at the transcriptional level [60], this post-transcriptional regulation process represents a rapid mechanism allowing D6 to control inflammation. In contrast to other atypical chemokine receptors, DARC in unstimulated cells is primarily expressed on the surface membrane of both heterologous in vitro transfectants [61] as well as in nucleated cells in vivo [62]. Similarly to classical GPCRs, cognate ligands induce DARC internalization into the intracellular vesicles, caveolae in particular, followed by reappearance of DARC together with its chemokine cargo on the cell membrane [61]. The fact that DARC internalization is induced by ligand ligation [61] reflects a putative signalling event of yet unknown biochemical nature. In polarized cells, DARC transports chemokines primarily from the baso-lateral to the apical surface where chemokines remain associated with DARC on the tips of the cell microvilli [61]. No significant scavenging of chemokines has been demonstrated following their internalization by DARC [61]. Thus, DARC-mediated chemokine internalization may facilitate the removal of chemokines from some extracellular microenvironments and induce their translocation into other potentially hard to reach specialized tissue microdomains. No endocytosis from erythrocyte membranes has been recorded. Therefore chemokines bound by these cells, remain associated with DARC on the erythrocyte surface and can be removed by other chemokines with higher affinity for DARC [63] or by heparin or molecules of the activated coagulation cascade [64]. Similarly to CXCR7 and CXCR4, an atypical and classical chemokine receptor pair which has been shown to hetero-oligomerize [65–67] DARC and CCR5, which share several CC-chemokine ligands, also heterodimerize [68] with consequences for the intracellular signalling through CCR5. Thus, the expression of an atypical and classical chemokine receptor in cis-geometry may allow modification and fine-tuning of cellular responses to chemokines downstream of classical chemokine GPCRs.

5.1. D6 D6 displays a fairly restricted expression pattern. In general, it is expressed in barrier tissues including the skin, gut, lung and placenta [60,69–71]. In the skin gut and lung the predominant site of expression is on subsets of lymphatic endothelial cells and D6 is associated predominately with initial lymphatic vessels. In the placenta, D6 displays prominent expression on the syncytiotrophoblast layer and thus it is positioned precisely between the mother and fetus. In addition to these sites, D6 is also expressed in a range of leukocytes [72,73] with most marked expression being evident on subsets of B cells as well as on both plasmacytoid and myeloid dendritic cells. Whilst there is currently only limited information on the regulation of D6 expression, its expression is reduced by pro-inflammatory mediators such as LPS and is upregulated by treatment of cells with molecules associated with resolution of inflammation including TGF␤ [72]. Interestingly, we have recently published that peripheral blood leukocytes from patients with systemic sclerosis display a 10-fold elevation of D6 expression [74]. The nature of the factors regulating enhanced expression in these leukocytes, and the pathological significance of this upregulation, is currently unclear. Most recently, strong expression of D6 on murine innate-like B cells has been shown suggesting that these cells are likely to be major vehicles for in vivo D6 function [75,76]. 5.2. CCX-CKR1 Expression analyses of CCX-CKR1 have yielded conflicting results although there are some consistencies emerging from these studies [27,28]. At the level of detection by Northern blotting, both human and murine CCX-CKR1 are expressed in the heart and lung and possibly also in the gut. However, neither human nor murine CCX-CKR1 is expressed at detectable levels in leukocytes. Quantitative real-time PCR analyses reveal low-level expression in a range of other tissues but again confirm the undetectable expression of CCX-CKR1 in leukocytes. More recently a study utilizing CCX-CKR1 reporter mice [77] failed to demonstrate expression in leukocytes but also, strikingly, failed to demonstrate expression in heart tissues. This study did report expression in a variety of secondary lymphoid organs as well as in thymic epithelial cells and skin keratinocytes. The lack of consistency in the data reporting expression of CCX-CKR1 is, currently, a significant impairment to our analysis of its in vivo function. 5.3. CXCR7 High-throughput gene expression profiling of the transcriptome of various cell types indicated a broad expression pattern of CXCR7 including hematopoietic cells, mesenchymal cells and neuronal tissue [78]. Other studies employed northern blot analyses, RT-PCR and in situ hybridization to localize the expression [9,53,79]. Considerable discrepancy concerning the expression of CXCR7 mRNA in leukocytes has been reported. Several investigations report CXCR7 mRNA in leukocytes [80,81], in particular in the human B cell compartment [53,82]. In mice, transcripts from the CXCR7 gene are highly expressed in splenic marginal zone B cells and in transitional type 2 MZ precursors [66]. However, another study indicated that the receptor is not expressed in human and mouse leukocytes [83]. Similar controversial reports exist on the protein level in leukocytes, when standard FACS analysis was used to detect CXCR7 on leukocytes [53,34,84]. Nevertheless, Hartmann et al. show in a functional assay that on CD4 T cells CXCR7, although only weakly expressed, is required for rapid CXCR4-dependent signalling and that treatment of CXCR7 with a small molecule inhibitor developed

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by ChemoCentryx (CCX733), abolished CXCR4-dependent rapid integrin activation [85]. In humans, expression of CXCR7 in memory B cells correlates with the ability of the cells to differentiate into plasmablasts and to efficiently produce antibodies [53]. In contrast, Berahovich et al. used a CXCL12-binding signature to conclude that CXCR7 is not expressed on leukocytes. The assay takes advantage of the fact that 125 I-CXCL12 binding to CXCR7 can be competed with CXCL11, which does not bind to CXCR4. In addition whilst CXCL12 binds to both CXCR4 and CXCR7, binding to CXCR7 is preferentially inhibited by the small molecule CCX771, but not by AMD3100, a selective antagonist of CXCR4 [83]. However, the assay appears not to detect any binding sites for CXCL12 in bone marrow cells, where prominent expression of CXCR4 and CXCL12 binding is widely accepted [86]. 5.4. DARC In addition to the paradigmatic expression by erythrocytes of the Duffy antigen “positive” individuals, DARC is expressed most prominently on the vascular endothelial cells, primarily in postcapillary and collective venules and small veins within different organs and tissues [52]. These importantly include the sinusoids in the spleen and the high endothelial venules of the lymph nodes [87–89]. Capillary and arterial endothelial cells as well as the endothelium in human umbilical veins, often used for in vitro studies, are normally completely devoid of DARC [62] and accordingly fail to bind chemokines [90]. However, under pathological conditions of infection, inflammation and transplant rejection DARC is upregulated in venules and its expression spreads to other segments of the vasculature [91–96]. It is unknown if such upregulation and extension of DARC expression in these diseases is a prerequisite for their pathogenesis or takes place only as a consequence of ongoing inflammation. It may be explained by the fact that major inflammatory cytokines can induce DARC expression e.g. in umbilical vein endothelial cells [97]. The afferent lymphatics are known to express D6 but not DARC [49]. Curiously subsequent segments of lymphatic vascular tree, the precollectors, which are characterized by weak expression of podoplanin, were shown to express DARC [98]. It is unknown if in chemokines mediate cell migration at this microanatomical site and how DARC expression may influence it. In addition to these endothelial sites, Purkinje neurons of the cerebellum [99] and epithelial cells of kidneys and lungs [100] were shown to express DARC with completely unexplored functional consequences. Moreover, the latter findings are based on immunoreactivity using a polyclonal antibody and have been subsequently contested [101]. 6. Biological functions of the atypical receptors The most prominent biological function of conventional chemokine receptors is their ability to induce directional cell migration. In contrast, a common unifying feature of the atypical chemokine receptors is their inability to sustain such function leading to the idea that they have evolved to perform fundamentally different roles. 6.1. D6 In the case of D6, gene-targeted animals are viable and do not display obvious phenotypes under resting conditions [102,103]. However, after challenge with different types of inflammatory stimuli, D6-deficient animals reproducibly demonstrate a defect in their ability to resolve inflammation. In the absence of D6 a significant increase in the concentration of its ligands, in plasma and inflamed tissues, and the development of exacerbated and prolonged inflammatory responses have been observed. Importantly,

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in D6−/− animals this pathological inflammatory response can be prevented by blocking inflammatory CC chemokines. Overall these observations demonstrate the non-redundant role of D6 in the control of inflammatory chemokines in vivo and together with biochemical evidence obtained in vitro support the notion of its role as chemokine scavenger receptor. As mentioned, D6-mediated attenuation of inflammation by means of chemokine scavenging has been demonstrated in various animal models and in several organs. Initial studies, using a range of models of cutaneous inflammation [102,103], demonstrated a clear role for D6 in the resolution of the cutaneous inflammatory response. These studies prompted further analyses of roles for D6 in the resolution of inflammatory responses in other tissues. Consistent with D6 expression in lung lymphatic vessels [69], D6−/− mice showed increased inflammation in an allergen-induced airway disease model [104] and increased lethality rate after intranasal administration of Mycobacterium tuberculosis [105]. D6 has also been shown to control inflammation in the liver, as demonstrated using a murine model of acute toxic injury [106]. Interestingly this result is in broad agreement with genetic evidence in humans of the impact of a D6 single nucleotide polymorphism in the development of liver inflammation in a cohort of HCV-infected patients [107]. D6 also appears to be involved in the control of inflammation in the colon, and though conflicting results have been reported in animal models [108,109], evidence for D6 up-regulation under inflammatory conditions in colitic mice and in colon samples of inflammatory bowel disease patients have been reported [108,109]. Finally, D6 is expressed at high levels in syncytiotrophoblast cells where it has been found to reduce inflammation-induced, and anti-allogeneic immunity-induced, fetal loss in mice [71,70]. Consistent with the view that trophoblast D6 scavenges maternal chemokines at the fetomaternal interface and helps to ensure fetal survival, loss of D6 immunoreactivity was also observed in arresting versus viable littermate attachment sites in the porcine uterus [110]. Interestingly, in certain conditions the uncontrolled local inflammation observed in D6−/− mice has been shown to impair the development of an appropriate specific immune response. In the EAE model of encephalomyelitis, the absence of D6 was in fact associated with an increased tissue inflammation, but as a consequence CD11c+ dendritic-like cells were found to accumulate in peripheral tissue, causing a blunted adaptive immune response and protection from disease development [111]. More recent data from studies examining the biological roles for D6 on lymphatic endothelial cells further support a role for D6 at this site in the coordination of innate and adaptive immune responses [158]. In line with its recognized role in the control of the inflammatory response, recent data show that D6−/− mice also have increased susceptibility to tumor development. In the absence of D6, a significant increase in chemokine levels and inflammatory cell infiltration was demonstrated in both the phorbol ester-induced skin carcinogenesis model [102] and the azoxymethane/sodium dodecylsulfate model of colon cancer [109]. Furthermore, transgenic expression of D6 in keratinocytes was able to confer significant protection from phorbol ester-induced papilloma formation [102]. In humans, D6 is expressed in different tumors, and in human breast cancer D6 expression is inversely correlated with clinical stages and lymph nodes metastasis and positively with disease-free survival rate [112]. These observations indicate that D6 expressed by tumor cells or lymphatic vessels of the tumor stroma acts as a tumor suppressor gene by negative regulation of chemokine availability. D6 is also prominently expressed on tumor cells in Kaposi’s sarcoma [69], which is likely to be in keeping with the lymphatic endothelial phenotype of these tumor cells. Finally, in keeping with the ability of transgenic D6 to ameliorate cutaneous inflammatory responses and tumor development, transgenic D6 expression in pancreatic islet cells is also able to suppress the development of diabetes in the NOD

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mouse model [113]. Together, these studies suggest a therapeutic potential for D6 in a range of inflammatory contexts. 6.2. CCX-CKR1 Currently there is limited information on the in vivo biological functions for CCX-CKR1. CCX-CKR1-deficient mice have been generated and are healthy and viable suggesting no clear role for CCX-CKR1 in development [77,114]. There have been two studies focusing on CCX-CKR1 deficient mice. In the first, a phenotype indicating a role for CCX-CKR1 in basal trafficking of dendritic cells to lymph nodes and of embryonic thymic precursors to the developing thymus has been reported [77]. More recently, a report has provided evidence for in vivo scavenging activity for CCXCKR1 by demonstrating elevated levels of CCL21 in the serum of the knockout mice and elevated levels of CCL19 and 21 in lymph nodes of the knockout mice [114]. This study also has demonstrated an important role for this atypical receptor in immune responses [114]. Specifically, in the EAE model, CCX-CKR1 deficient mice displayed a more rapid onset of disease. This appeared not to relate to increased T cell activation in the lymph nodes of the CCX-CKR1 deficient mice but develop as a consequence of increased Th17 response in these mice which is also accompanied by increased splenic IL-23 expression and elevated expression of the Th17 associated chemokine receptor CCR6 [115]. Beyond these preliminary mouse studies there is limited information on CCX-CKR1 in pathologies. There is evidence of expression in hepatocytes and in hepatic tumors from cell line studies using a novel monoclonal antibody [58] and studies into the expression of the atypical chemokine receptors in breast cancer [116] suggests that coexpression of CCX-CKR1 along with D6 and DARC is significantly associated with disease-free survival. This is taken to suggest that multiple loss of the atypical chemokine receptors, including CCXCKR1, may be an important driver of the development of aggressive tumors. 6.3. CXCR7 Targeted disruption of the CXCR7 gene in mice leads to a lethal phenotype. Animals die at birth, or shortly after, due to a heart valve defect [66,117,118]. The same phenotype is seen in animals with a conditional deletion of CXCR7 in endothelial cells, implying a critical function for the receptor in these cells. Because the phenotype is not observed in mice that do not express CXCL11, it is plausible that CXCL12 is the critical target of CXCR7 scavenging [66]. The lethal consequences of CXCR7 deletion were unexpected as ‘knock out’ of the genes encoding CXCR4 and CXCL12 in mice resulted in very similar phenotypes leading to the general acceptance of a monogamous interaction between chemokine and receptor [119–121]. This finding suggested that the lack of CXCR7 compromises CXCR4-dependent signalling. A key observation in this respect was that in cells lacking CXCR7, CXCR4 protein is also absent, despite mRNA expression. This is a consequence of CXCR4 down regulation being mediated by the excess of extracellular CXCL12 which accumulates in the absence of CXCR7 [54]. Although these findings do not exclude a direct interaction between CXCR4 and CXCR7 [36,66], they support the notion of a crucial role for CXCR7 in regulating the availability of CXCL12 for CXCR4, consistent with the higher affinity of CXCR7, than CXCR4, for CXCL12 and the highly dynamic cell surface expression of the receptor. The first evidence for a functional role of CXCR7 in vivo was obtained from studies in zebrafish showing that the abrogation of primordial germ cell migration upon deletion of CXCL12 was not mirrored upon elimination of CXCR4, but required deletion of both receptors CXCR4 and CXCR7 [122,123]. It was then shown that CXCR7 expression on stromal cells is necessary to sustain the CXCL12

gradient required for primordial germ cell migration. Germ cells arrive at sites where CXCL12 is translated from mRNA, but do not migrate directionally until the scavenger activity for CXCR7 creates a guidance cue [124]. Overall, these results suggest a functional role for CXCR7 in the formation of a functional gradient of its ligands, and though its expression and function on many tissues remain to be resolved, the above paradigm most likely will apply. As for D6, CXCR7 expression has also been reported on barrier tissues, such as placenta [125]. A recent study revealed an important function of mammalian CXCR7 acting as scavenger on brain microvessel endothelium. Perivascular CXCL12 constitutes, under normal physiological conditions, a localization cue beneath the blood–brain barrier. During inflammation, IL-17 and IL-1dependent upregulation of CXCR7 on the abluminal surface of the microvessel endothelium leads to marked depletion of perivascular CXCL12 and therefore to a loss of the localization cue for leukocytes. In EAE it was shown that the upregulation of CXCR7 is essential for inflammatory leukocytes to infiltrate the CNS. Accordingly, inhibition of CXCR7 with the small molecule antagonist CCX771 reduces abluminal CXCL12 depletion and thereby markedly ameliorates EAE, suggesting that the receptor could be a potential therapeutic target [126]. The marked expression of CXCR7 in ganglia and the central nervous system (CNS) [9,127] suggests a functional relevance in these tissues. Two recent studies examined the role of CXCR7 in the CNS and revealed a regulatory function for the migration of interneurons to the subventricular and intermediate zone [54,128]. By far the majority of studies have focused on the potential role of CXCR7 expression in tumor cells. There is broad consensus that CXCR7 is expressed on tumors of hematopoietic origin, such as lymphomas [8,53,83,85] and of mesenchymal origin, such as sarcomas [129–133] as well as prostate and breast cancer [134,135]. The function of the receptor in the neoplasms, however, remains unclear. Some studies suggest moderate signalling activity by CXCR7 in the transformed cells compatible with arrestin-dependent intracellular pathways, and one report even suggests coupling to G␣i proteins [136]. The significance of the signalling for tumor cell growth and survival in vivo is not clear. The concomitant expression of multiple chemokine receptors on tumor cells often prevents unequivocal attribution of the signalling to a specific receptor. More interesting, at this point, is the observation that CXCR7 expression on tumor cells is frequently accompanied by the expression of CXCR4. It is well known, that the CXCL12/CXCR4 axis is important for tumor growth, survival and metastasis [137]. It is conceivable that CXCR7 expression in the same context as CXCR4 may regulate the availability of CXCL12 for the tumor cells. CXCR7 upregulation could reduce CXCL12-mediated tumor cell retention thus promoting spread or preventing down-regulation of survival promoting CXCR4, thereby playing an ambivalent role depending on the tumor context. Little is known of the dynamics of CXCR7 cell surface expression on tumor cells and its ability to scavenge CXCL12 in vivo. Thus, further studies will be required to resolve the function of CXCR7 in health and specific pathologies where, in some instances, attenuation of CXCL12 scavenging could be beneficial. Currently, the physiological relevance of CXCL11 scavenging by CXCR7 is not known, but could be relevant for the regulation of CXCR3 mediated immune responses. 6.4. DARC Based on the in vitro findings of chemokine binding to Duffypositive but not -negative erythrocytes, the initial functional role ascribed to DARC was that of a chemokine “sink” able to absorb excessive chemokines in blood [138]. This function was supported by subsequent studies in which DARC knockout mice failed to

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control the levels of inflammatory chemokines in blood after an LPS challenge leading to an overt leukocyte activation in the circulation and in the tissues [139,140]. However, by protecting blood leukocytes from surplus of plasma chemokines, DARC may also prevent leukocyte desensitization for their subsequent migratory responses. Thus, by reducing the systemic chemokine “noise”, DARC may also enhance leukocyte emigration in response to the chemokines associated with discreet tissue sites (Rot, unpublished). Coexistence of these two different scenarios may explain the often conflicting outcomes and varying phenotypes observed in DARC knockout mice when studied in different chemokine-driven experimental inflammatory models [63,139,141–144]. Additional function of DARC on human and murine erythrocytes involves retaining inflammatory chemokines in blood and serving as their long term blood reservoir, with yet unclear teleological purpose [145,146]. The existence of human Duffy-negative polymorphism provides a unique possibility to study the contribution of erythrocyte DARC to human health and disease. Recently two genome-wide association studies correlated the FY*B(ES) polymorphism with the development of benign ethnic neutropenia [147] and asthma and high IgE [148]. In both cases the molecular and cellular pathomechanistic aspects remained entirely unexplored. Another association study tied the differences in plasma chemokine levels within the cohort of Duffy-positive individuals with their polymorphic FY*A or FY*B status [64], but leaving open entirely different potential mechanisms including differential chemokine binding by the two polymorphic DARC variants or different levels of their erythrocyte or endothelial cell expression. Because a common malarial parasite, P. vivax, requires DARC to bind to erythrocytes and invade them, Duffy-negative individuals are relatively resistant to vivax malaria suggesting that FY*B(ES) polymorphism may have evolved under the selective pressure from this infection [48]. DARC also binds HIV-1 leading to the transmission of the virion to the susceptible blood cells [45,46]. This mechanism potentially explains how HIV infected Duffy “negatives” may exhibit a slower progression to AIDS despite their higher infection rates [46]. These differences in HIV progression between Duffy negative and positive individuals were more significant between the leukopenic patient cohorts [149], but could not be observed in several other studies using different cohorts. Venular endothelial cells, the other major cellular site of DARC expression were shown to bind, transport and present inflammatory chemokines of both CC and CXC families thus enabling effective leukocyte emigration at this segment of circulatory tree [150–153]. It was suggested early on, that DARC might participate in these chemokine–endothelial cell interactions [90,154]. Recently this was clearly shown by demonstrating that DARC functions as a transcytosis receptor for its chemokine ligands and, in polarized cells, leads to chemokine immobilization on the apical cell surfaces [61]. DARC expression and over-expression are required for optimal pro-migratory function of chemokines in transcellular leukocyte migration assays and in vivo, respectively [61]. In addition to supporting the pro-emigratory functions of chemokines, their transendothelial transport by DARC may also provide means for chemokine elimination from the tissues, though in the majority of vascular beds this may happen by passive chemokine diffusion between the endothelial cells. Nevertheless, it was suggested that DARC on endothelial cells might function as a chemokine rheostat on the blood–tissue interface by supporting the placement and function of suboptimal concentrations of chemokines, but eliminating their excess [49,62]. Also the removal of extravascular, abluminally positioned chemokines may be a potential mechanism by which DARC can negatively regulate chemokine-induced angiogenesis [155] as well as suppress angiogenesis in experimental tumors [156,157].

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7. Conclusions Atypical chemokine receptors have emerged as a functionally diverse new subfamily of chemokine receptors which by different means modify chemokine availability and signalling and via chemokine removal or transcytosis contribute to the formation and maintenance of functional chemokine patterns in tissues. Though being unable to directly support cell migration, these receptors play important and often non-redundant roles in inflammatory and immune responses controlling leukocyte mobilization from the bone marrow, extravasation from the blood vessels into the inflamed tissue, and leukocyte traffic to lymph nodes.

Acknowledgments All authors were supported by European Union ‘Framework 6’ funding (INNOCHEM; LSHB-CT-2005-518167). In addition, work in GJG’s laboratory is supported by grants from the MRC. AR is supported by MRC grants G0802838 and G9818340 and the MRC Centre for Immune Regulation, respectively. MT is supported by Gottfried und Julia Bangerter-Rhyner-Stiftung, Basel and the Helmut Horten Foundation. ML and AM are supported by the Italian Association for Cancer Research (AIRC), Regione Lombardia (LIIN project), Fondazione Cariplo (NOBEL project) and Ministero dell’Università e della Ricerca (PRIN and FIRB projects).

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