Species-specific features of DARC, the primate receptor for Plasmodium vivax and Plasmodium knowlesi

The DARC (Duffy antigen/receptor for chemokines) gene, also called Duffy or FY, encodes a membrane-bound chemokine receptor. Two malaria parasites, Plasmodium vivax and Plasmodium knowlesi, use DARC to trigger internalization into red blood cells. Although much has been reported on the evolution of DARC null alleles, little is known about the evolution of the coding portion of this gene or the role that protein sequence divergence in this receptor may play in disease susceptibility or zoonosis. Here, we show that the Plasmodium interaction domain of DARC is nearly invariant in the human population, suggesting that coding polymorphism there is unlikely to play a role in differential susceptibility to infection. However, an analysis of DARC orthologs from 35 simian primate species reveals high levels of sequence divergence in the Plasmodium interaction domain. Signatures of positive selection in this domain indicate that species-specific mutations in the protein sequence of DARC could serve as barriers to the transmission of Plasmodium between primate species.     

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Duffy antigen receptor for chemokines mediates trans-infection of HIV-1 from red blood cells to target cells and affects HIV-AIDS susceptibility

Duffy antigen receptor for chemokines (DARC) expressed on red blood cells (RBCs) influences plasma levels of HIV-1-suppressive and proinflammatory chemokines such as CCL5/RANTES. DARC is also the RBC receptor for Plasmodium vivax. Africans with DARC -46C/C genotype, which confers a DARC-negative phenotype, are resistant to vivax malaria. Here, we show that HIV-1 attaches to RBCs via DARC, effecting trans-infection of target cells. In African Americans, DARC -46C/C is associated with 40% increase in the odds of acquiring HIV-1. If extrapolated to Africans, approximately 11% of the HIV-1 burden in Africa may be linked to this genotype. After infection occurs, however, DARC-negative RBC status is associated with slower disease progression. Furthermore, the disease-accelerating effect of a previously described CCL5 polymorphism is evident only in DARC-expressing and not in DARC-negative HIV-infected individuals. Thus, DARC influences HIV/AIDS susceptibility by mediating trans-infection of HIV-1 and by affecting both chemokine-HIV interactions and chemokine-driven inflammation.     

Sequence,evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.     

The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon

Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania.     

Targeting the Plasmodium vivax Duffy-binding protein

Plasmodium vivax requires interaction with the Duffy antigen receptor for chemokines (DARC) to enable its invasion of human erythrocytes. Interaction with DARC is mediated by the P. vivax Duffy-binding protein (PvDBP) and is essential for junction formation, which is a key step in the invasion process. The receptor-binding domain of PvDBP maps to a conserved cysteine-rich region, referred to as region II (PvRII). Here, we review data on the interaction of PvRII with DARC and explore the potential of targeting this crucial receptor-ligand interaction to develop new intervention strategies against P. vivax.     

Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells

Background

The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.

Methodology/Principal Findings

We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated ¹²⁵I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. ¹²⁵I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. ¹²⁵I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.

Conclusions/Significance

These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.     

Silent chemoattractant receptors: D6 as a decoy and scavenger receptor for inflammatory CC chemokines

The chemokine system includes at least three “silent” receptors, DARC, D6 and CCX CKR, with distinct specificity and tissue distribution. D6 binds most inflammatory, but not homeostatic, CC chemokines and shuttles in a ligand-independent way from the plasma membrane to endocytic compartments where chemokines are targeted to degradation. In vitro and in vivo evidence, including results with gene-targeted mice, is consistent with the view that D6 acts as a decoy and scavenger for inflammatory CC chemokines. Thus, D6 has unique functional and structural features, which make it ideally adapted to act as a chemokine decoy and scavenger receptor, strategically located on lymphatic endothelium to dampen inflammation in tissues and draining lymph nodes.     

Molecular evolution of a malaria resistance gene (DARC) in primates

Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a ~3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.     

Expression of Duffy antigen receptor for chemokines (DARC) is down-regulated in colorectal cancer

Abstract

Duffy antigen receptor for chemokines (DARC) is a silent chemokine receptor which selectively binds angiogenic chemokines without inducing conventional signaling responses. DARC has been reported to inhibit the development of multiple cancers through clearance of angiogenic chemokines. However, its role in colorectal cancer (CRC) remains unclear. We investigated the expression of DARC in CRC and explored correlation of DARC expression with clinical pathological features and microvessel density (MVD). The protein expression levels of DARC were detected by immunohistochemistry in 90 CRC and 64 paired unaffected tissues. The mRNA levels of DARC were detected by quantitative real-time PCR in 15 CRC and paired unaffected tissues. MVD in CRC was also assessed by immunohistochemistry of CD34. We found that the mRNA and protein expression levels of DARC were significantly lower in CRC than in the unaffected tissues (p?<?0.05). The DARC protein expression levels were positively correlated with DARC mRNA expression levels in both CRC (p?<?0.001) and unaffected tissues (p?<?0.001). We also found that DARC expression was significantly correlated with tumor differentiation (p?<?0.001), lymph node metastasis (p?<?0.01) and TNM stage (p?<?0.05). Moreover, we observed a strong negative relationship between DARC expression and MVD in CRC (p?<?0.001). We showed that DARC expression is down-regulated in CRC and associated with clinical pathological features and MVD of CRC. DARC might be involved in tumorigenesis, progression, angiogenesis, and metastasis of CRC.     

Inhibition of a malaria host–pathogen interaction by a computationally designed inhibitor

Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.     

Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant (K(D)) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cysteines 5-8. Here, we have used site-directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.     

Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.     

Molecular characterization of CCR6: Involvement of multiple domains in ligand binding and receptor signaling

The CC chemokine receptor 6 (CCR6) is selectively expressed on memory T cells, B cells, and dendritic cells and appears to be involved in the initiation of a memory immune response. The only chemokine ligand for CCR6 is CCL20/MIP-3. In the present study, we attempted to define the extracellular domains (ECDs) of CCR6 responsible for CCL20/MIP-3 binding using a domain-swapping approach in which the ECDs of CCR6 were substituted with the corresponding CCR5 domains to generate various CCR6/CCR5 chimeras. These chimeras were tested for receptor expression, ligand binding, and functional activity as evaluated by calcium flux and chemotaxis. All chimeras showed respectable surface expression; however only one, substituted with extracellular loop 1 from CCR5, showed reduced functional activity. The general failure of functionality of the CCR6/CCR5 chimeras may imply that characteristics of each ECD are critical for coordination among all the ECDs of CCR6. Additionally, of interest, a chimera containing all of the ECDs from CCR5 in the context of CCR6 neither responded to CCR5 ligands nor served as a coreceptor for macrophage-tropic HIV-1. These results suggest that not only ECDs but also transmembrane and intracellular domains of CCR5 are involved in both ligand binding and coreceptor activity.     

Contribution of Duffy antigen to chemokine function

In addition to classical G protein-coupled receptors (GPCRs), a group of alternative, “silent” chemokine receptors has recently been identified. These serpentine molecules are not coupled to G proteins and subsequent signaling cascades, but can efficiently internalize their cognate chemokine ligands, thus act as “interceptors” (internalizing receptors). Here we discuss a mechanism by which a member of this family, Duffy antigen (DARC), contributes to chemokine-induced leukocyte emigration. Cumulative experimental evidence suggests that DARC on venular endothelium mediates chemokine internalization at the abluminal surface followed by transcytosis and transfer of the chemokine cargo onto the luminal surface. DARC is also expressed on the erythrocyte surface of DARC positive individuals. Erythrocyte DARC binds plasma chemokines which results, on one hand, in impediment of the chemokines loss from the circulation and, on the other hand, in neutralization of chemokines in the blood. This leads to leukocyte protection from inadvertent “desensitization” and enhancement of leukocyte recruitment.     

Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.     

Plasmodium simium,a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBP_II) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.     

Studies of a Murine Monoclonal Antibody Directed against DARC: Reappraisal of Its Specificity

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is ²²FEDVW²⁶, with ²²F and ²⁶W being the most important residues. In addition, we demonstrated that ³⁰Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that ²⁵V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.     

Shaping the gradient by nonchemotactic chemokine receptors

Chemokines are a class of inflammatory mediators which main function is to direct leukocyte migration through the binding to G protein-coupled receptors (GPCRs). In addition to these functional, signal-transducing chemokine receptors other types of receptors belonging to the chemokine GPCR family were identified. They are called atypical or decoy chemokine receptors because they bind and degrade chemokines but do not transduce signals or activate cell migration. Here there is the summary of two recent papers that identified other nonchemotactic chemokine receptors: the Duffy antigen receptor for chemokines (DARC) that mediates trancytosis of chemokines from tissue to vascular lumen promoting chemokine-mediated leukocyte transmigration and chemokine (CC motif) receptor-like 2 (CCRL2) that neither internalizes its ligands nor transduces signals but presents bound ligands to functional signaling receptors improving their activity. Collectively these nonchemotactic chemokine receptors do not directly induce cell migration, but appear nonetheless to play a nonredundant role in leukocyte recruitment by shaping the chemoattractant gradient, either by removing, transporting or concentrating their cognate ligands.Key words: Chemokine, chemokine receptor, leukocyte recruitment, chemotaxis, transcytosis