Chemokines play a key role in directing and driving leucocyte trafficking. The efficient regulation of leucocyte recruitment by chemokines requires their appropriate localization in functional micro-anatomical domains, as well as setting limits to their effects in space and time. Both processes are influenced by silent chemokine receptors (interceptors), including DARC (Duffy antigen receptor for chemokines). Increasing experimental evidence suggests that DARC is involved in accumulation of extravascular chemokines in endothelial cells, chemokine transcytosis and presentation on their luminal surface, leading to leucocyte adhesion and emigration. Additionally, DARC is expressed on erythrocytes and can act as a sink for chemokines in blood. This limits the dissemination of chemokines through blood into distant organs and tissues as well as reducing their effects on the circulating leucocytes.
- Duffy antigen
- endothelial cell
- leucocyte trafficking
Chemokines are small secreted proteins which share considerable homology, most importantly, a conserved tetracysteine motif [1–3]. Based on the position of the first two cysteine residues, chemokines are divided into two major subclasses. The cysteine residues in CXC chemokines are separated by one non-conserved amino acid, whereas in CC chemokines, the first two cysteine residues are adjacent. Besides these two major subclasses, two additional small chemokine groups exist. The XC chemokines lack the first consensus cysteine, whereas CX3C chemokine is characterized by three non-conserved amino acids between the first two cysteine residues [1–3]. Chemokines exert their biological role through specific G-protein-coupled receptors with seven transmembrane domains. Signalling is mainly mediated through the dissociation of the βγ G-protein subunits from the pertussis toxin-sensitive Gαi protein, leading to generation of a multitude of intracellular effectors, ultimately leading to actin polymerization and directed cell migration [1,4,5]. In addition to classical signalling receptors, chemokines bind to ‘silent’ receptors known also as interceptors (internalizing receptors). Two interceptors, DARC (Duffy antigen receptor for chemokines) and D6, bind ‘inflammatory’ chemokines, whereas a receptor described alternatively as CCRL1, CCR10, CCR11 or CCX-CKR binds only ‘homoeostatic’ ones . Chemokine interceptors are seven-transmembane molecules with high homology to the classical G-protein-coupled chemokine receptors. However, they either lack completely or exhibit an altered DRYLAIV motif in the second intracellular loop and therefore cannot couple with G-proteins and subsequent signalling cascades. Nevertheless, interceptors impact significantly on chemokine homoeostasis [7–15]. D6 is expressed on lymphatic ECs (endothelial cells) , placental syncytiotrophoblasts and also blood leucocytes. This interceptor internalizes almost all inflammatory CC chemokines and targets them for efficient degradation into the lysosomes. DARC binds both CC and CXC chemokines [17,18]. It is expressed on venular ECs, cerebellar neurons and erythrocytes of Duffy antigen-positive individuals . Here, we summarize experimental results which suggest that DARC plays a complex and important, but not entirely clear, role in chemokine homoeostasis.
Molecular and cell biology of DARC
The Fy gene that encodes DARC is located in the 1q22–q23 region of chromosome 1. Four different alleles were described: FYA, FYB, FYB(ES) and FYB(WK) . These alleles lead to five phenotypes: Fy(a+b−), Fy(a−b+), Fy(a+b+), Fy(a−b+wK) and Fy(a−b−). Five different antigenic epitopes have been described on DARC: Fy(a), Fy(b), Fy3, Fy5 and Fy6. The FYB(ES) allele with a mutation in the promoter region results in the lack of DARC on erythrocytes, the Fy(a–b–) or Duffy-negative phenotype. This phenotype is prevalent in individuals of African ancestry. Duffy antigen is the erythrocyte receptor for the malaria parasites Plasmodium vivax and Plasmodium knowlesi. Therefore the Duffy-negative phenotype protects from malaria infection by these parasite strains , explaining its evolutionary evolvement. The extracellular N-terminal domain of DARC, which carries the Fya/b and Fy6 blood group antigens is sufficient for the binding of the malaria parasites P. vivax and P. knowlesi . The four extracellular domains of DARC are essential for its interaction with chemokines; however, the exact molecular determinants of chemokine binding to DARC that may explain its unique ligand promiscuity are not entirely clear .
DARC on erythrocytes
The Duffy blood group antigen was discovered in 1950 and named after a polytransfused haemophiliac patient who displayed high antibody titres against the erythrocytes of several individuals (for a review, see ). In the early 1990s, Duffy antigen was described to be a receptor for IL-8 (interleukin 8)  and other inflammatory chemokines [25,26] and subsequently named DARC. Darbonne et al.  suggested that DARC on erythrocytes acts as a chemokine sink, thereby limiting the stimulation of leucocytes by IL-8 in the blood. This is supported by results from DARC-deficient mice  demonstrating an increased number of neutrophils in organs of DARC−/− mice following intraperitoneal injection of lipopolysaccharide. The major drawback of this study is that it does not distinguish between the function of DARC on erythrocytes and ECs. A recent report investigates the possible role of DARC on erythrocytes in prostate tumour growth . It proposes a connection between DARC-negative phenotype in humans and 60% greater incidence of prostate cancer and increased mortality, which is observed in this group of individuals. Again, DARC−/− mice were used to suggest that erythrocyte DARC is clearing angiogenic CXC chemokines from the prostate tumour circulation and thus acts as an anti-angiogenic principle. However, also in this animal model, the contribution of EC DARC in clearing angiogenic chemokines cannot be excluded. Such an anti-angiogenic effect of endothelial DARC has been demonstrated previously using DARC transgenic mice . The binding of chemokines to erythrocyte DARC, in addition to their neutralization, also prevents their diffusion from blood into the lungs and the kidneys and possibly other organs and tissues. Thus DARC acts also as a chemokine reservoir maintaining plasma chemokine levels (, and M. Pruenster and A. Rot, unpublished work). It is still unclear what may be the function of this chemokine reservoir on the erythrocyte surface, how long chemokines remain bound to erythrocytes and what happens to these chemokines at the end of the erythrocyte lifespan.
DARC on ECs
In order to reach the sites of inflammation, circulating leucocytes must cross biological barriers, the endothelium in particular. This process involves the formation of transient adhesive contacts between leucocytes and the ECs and manifests in leucocyte tethering and rolling. Next, the integrin-mediated step of firm leucocyte adhesion is induced by chemokines that are immobilized on the luminal endothelial surface and bind to their cognate receptors on leucocytes . Chemokines produced in the extravascular tissue can diffuse into the bloodstream through the junctions between ECs. Alternatively, they can be internalized by the venular ECs and transcytosed across them . It was assumed that EC DARC may be the molecule responsible for the transcytosis of chemokines . To investigate chemokine transport by DARC in vitro, we used DARC-transfected MDCK (Madin–Darby canine kidney) cell monolayers and could detect enhanced chemokine transcytosis compared with control mock-transfected cells. The transport was unidirectional, from the basolateral to the apical side only; transport from the apical to the basolateral side did not take place (M. Pruenster and A. Rot, unpublished work). Interestingly, during basolateral to apical transport, very high amounts of the transcytosed chemokine remained bound on the MDCK surface over a long period of time (M. Pruenster and A. Rot, unpublished work). Immobilization of chemokines on the endothelial surface is one of the central requirements for the establishment of a chemokine-induced firm leucocyte adhesion. Glycosaminoglycans, heparan sulfate in particular , bind chemokines with high affinity and are thought to immobilize and present chemokines on the EC surface [32–35]. Whether endothelial DARC may also play a role in presentation of chemokines to the circulating leucocytes is currently under investigation. Following firm adhesion to the endothelium, leucocytes spread and may initiate their extravasation into the tissue. Traditionally, chemokines were thought to be directly responsible for the leucocyte emigration step, which was shown to occur via two different routes: between the ECs (intercellular) or through them (transcellular) . It is not clear at all (i) what the mechanism of chemokine-induced leucocyte transmigration is, (ii) whether this is a chemokine gradient-dependent event, (iii) where such chemokine gradients may be formed and (iv) whether EC DARC plays any role in this process. However, we were able to show that the expression of DARC by the MDCK cell monolayer grown on transwell inserts may impact on the number of trans-migrating leucocytes. The expression of DARC enhanced the migration of monocytes to CCL2 by almost an order of magnitude (M. Pruenster and A. Rot, unpublished work). Migration of neutrophils across a DARC-transfected HUVEC (human umbilical-vein EC) monolayer has been investigated previously . However, in these experiments, the contribution of DARC to the chemokine-induced transendothelial migration could be unmasked only by using blocking anti-DARC antibodies. The contribution of DARC to leucocyte emigration was confirmed in vivo, as DARC-deficient mice show reduced leucocyte recruitment in response to local application of cognate chemokines (, and M. Pruenster and A. Rot, unpublished work). This is partially due to the lack of DARC on the ECs. We have generated chimaeric mice that lack DARC on ECs and express it only on erythrocytes. In these mice, we found significantly reduced recruitment of leucocytes into chemokine injection sites compared with the wild-type mice (M. Pruenster and A. Rot, unpublished work). Based on our findings, we suggest that DARC on ECs is involved in chemokine binding, internalization, transport and surface retention. Furthermore, DARC expression leads to the increased chemokine-induced leucocyte migration across the biological barriers. The up-regulation of DARC expression by ECs has been demonstrated in a multitude of human inflammatory and infectious diseases [37–42]. However, the cause–consequence relationship of DARC expression with the pathological changes, as well as the suggestions of direct DARC contribution to the inflammatory disease pathomechanisms, remains purely speculative.
In contrast with the other well-characterized interceptor, D6, which primarily functions as a chemokine decoy/scavenger, DARC has a more multifaceted, complex role in the chemokine homoeostasis (Figure 1). It mediates chemokine transcytosis and contributes to chemokine retention on the cell surfaces. These two functions signify the contribution of EC DARC to the pro-emigratory activities of chemokines on the endothelium–blood interface. DARC on erythrocytes acts, on one hand, as a chemokine sink, by preventing activation as well as desensitization of leucocytes in systemic circulation. On the other hand, erythrocyte DARC acts as a long-term blood reservoir of chemokines, preventing their loss into distant organs and tissues.
M.P. and A.R. are supported by the EU 6th Framework Program collaborative grant INNOCHEM, LSHB-CT-2005-518167.
Control of Immune Responses: A Focus Topic at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by B. Foxwell (Imperial College London, U.K.), G. Graham (Glasgow, U.K.), R. Nibbs (Glasgow, U.K.) and S. Ward (Bath, U.K.).
Abbreviations: DARC, Duffy antigen receptor for chemokines; EC, endothelial cell; IL-8, interleukin 8; MDCK, Madin–Darby canine kidney
- © 2006 The Biochemical Society