Other than signalling receptors sustaining leucocyte recruitment during inflammatory reactions, the chemokine system includes ‘silent’ receptors with distinct specificity and tissue distribution. The best-characterized molecule of this subgroup is the CC chemokine receptor D6, which binds most inflammatory CC chemokines and targets them to degradation via constitutive ligand-independent internalization. Structure–function analysis and recent results with gene-targeted animals indicate that 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 and placenta to dampen inflammation in tissues and draining lymph nodes.

Introduction

Cell migration is a key element in the ontogenesis of lymphoid tissues and in the orientation of innate and adaptive immunity. Leucocyte trafficking is controlled by chemokines, small secreted proteins with chemotactic and cytokine-like activities [1]. These molecules are classified according to structural properties related to the number and position of conserved cysteine residues in two major (CXC and CC) and two minor (C and CX3C) subfamilies [2,3], and according to their production in homoeostatic (i.e. produced constitutively) and inflammatory (i.e. produced in response to inflammatory or immunological stimuli) situations [4]. Chemokine biological activities are mediated by chemokine receptors, a distinct subfamily of GPCRs (G-protein-coupled receptors) [5]. In these receptors, the acidic N-terminal extracellular domain is involved in ligand binding, cytoplasmic loops associate signal transducer molecules, and a serine/threonine-rich intracellular C-terminal domain provides the interacting surface for the internalization machinery. At present, 18 chemokine receptors have been molecularly defined, ten for CC chemokines [CCR1 (CC chemokine receptor 1)–CCR10], six for CXC chemokines [CXCR1 (CXC chemokine receptor 1)–CXCR6] and one for C chemokines and CX3C chemokines (XCR1 and CX3CR1 respectively) (Figure 1).

Leucocyte expression and ligand specificity of chemokine receptors

Figure 1
Leucocyte expression and ligand specificity of chemokine receptors

The ligand spectra of conventional chemokine receptors (right) and D6 (left) are shown. The central columns list chemokines, identified with one old acronym and with the new nomenclature in which the first part of the name identifies the family and ‘L’ stands for ‘ligand’ followed by a progressive number. Ba, basophils; BCA-1, B-cell-attracting chemokine-1; BRAK, breast and kidney chemokine; CTACK, cutaneous T-cell-attracting chemokine; ELC, Epstein–Barr virus-induced molecule 1 ligand CC chemokine; ENA-78, epithelial-cell-derived neutrophil-activating peptide 78; Eo, eosinophils; GCP-2, granulocyte chemotactic protein 2; GRO, growth-related oncogene; HCC, haemofiltrate CC chemokine; iDC, immature dendritic cells; IP-10, interferon-γ-inducible protein 10; I-TAC, interferon-inducible T-cell α-chemoattractant; MCP-1, monocyte chemotactic protein-1; mDC, mature dendritic cells; MDC, macrophage-derived chemokine; Mig, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; Mo, monocytes; Mø, macrophages; MPIF-1, myeloid progenitor inhibitory factor 1; NAP-2, neutrophil-activating peptide 2; NK, natural killer; PARC, pulmonary and activation-regulated chemokine; PMN, polymorphonuclear cell; RANTES, regulated upon activation, normal T-cell expressed and secreted; SCM-1β, single C motif-1β; SDF-1α/β, stromal cell-derived factor 1α/β; SLC, secondary lymphoid tissue chemokine; T act, activated T-cell; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; T muc, mucosal-homing T-cells; T naive, naïve T-cells; T reg, regulatory T-cells; T skin, skin-homing T-cells.

Figure 1
Leucocyte expression and ligand specificity of chemokine receptors

The ligand spectra of conventional chemokine receptors (right) and D6 (left) are shown. The central columns list chemokines, identified with one old acronym and with the new nomenclature in which the first part of the name identifies the family and ‘L’ stands for ‘ligand’ followed by a progressive number. Ba, basophils; BCA-1, B-cell-attracting chemokine-1; BRAK, breast and kidney chemokine; CTACK, cutaneous T-cell-attracting chemokine; ELC, Epstein–Barr virus-induced molecule 1 ligand CC chemokine; ENA-78, epithelial-cell-derived neutrophil-activating peptide 78; Eo, eosinophils; GCP-2, granulocyte chemotactic protein 2; GRO, growth-related oncogene; HCC, haemofiltrate CC chemokine; iDC, immature dendritic cells; IP-10, interferon-γ-inducible protein 10; I-TAC, interferon-inducible T-cell α-chemoattractant; MCP-1, monocyte chemotactic protein-1; mDC, mature dendritic cells; MDC, macrophage-derived chemokine; Mig, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; Mo, monocytes; Mø, macrophages; MPIF-1, myeloid progenitor inhibitory factor 1; NAP-2, neutrophil-activating peptide 2; NK, natural killer; PARC, pulmonary and activation-regulated chemokine; PMN, polymorphonuclear cell; RANTES, regulated upon activation, normal T-cell expressed and secreted; SCM-1β, single C motif-1β; SDF-1α/β, stromal cell-derived factor 1α/β; SLC, secondary lymphoid tissue chemokine; T act, activated T-cell; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; T muc, mucosal-homing T-cells; T naive, naïve T-cells; T reg, regulatory T-cells; T skin, skin-homing T-cells.

Conventional (i.e. signalling) chemokine receptors, like all other members of the GPCR family, mainly transduce intracellular signals through the activation of heterotrimeric G-proteins, and all chemokine receptors in particular mediate signalling through pertussis toxin-sensitive Gαi proteins. A significant body of evidence has been gathered on a subfamily of chemoattractant receptors unable to sustain signalling activities typically observed after chemokine receptor triggering, such as calcium fluxes and chemotaxis, and for this reason referred to as ‘silent’ chemoattractant receptors, although the ‘silence’ of these molecules (referring to conventional functional and biochemical responses) has epistemological limitations intrinsic to a definition based on a negative (lack of signalling) property [6]. The subfamily of silent chemoattractant receptors includes the chemokine receptors DARC (Duffy antigen receptor for chemokines) [7], D6 [8,9] and CCX-CKR [10], as well as the receptor for complement fractions C5L2 [11]. A detailed structure–function analysis of this receptor subfamily is not available yet, but it is interesting to note that structural determinants required for receptor signalling, which include a conserved motif [known as the DRY (Asp-Arg-Tyr) motif] in the second intracellular loop that is involved in G-protein coupling, are not conserved in these receptors.

The decoy receptor D6

The best-characterized silent chemokine receptor was cloned from placenta [8] and haemopoietic stem cells [9,12] and named D6. The D6 molecule is a typical chemokine receptor, but like DARC, it lacks sequence motifs that are critical for the G-protein coupling and signalling functions of chemokine receptors, such as the DRY motif in the second intracellular loop as well as the TXP (Thr-Xaa-Pro) motif in the second transmembrane domain. D6 binds a broad range of ligands that includes most of the inflammatory CC chemokines (agonists of CCR1–CCR5) (Figure 1). Nevertheless, D6 has some binding selectivity, in that among inflammatory CC chemokines, it interacts with the non-allelic variant CCL3L1 (CC chemokine ligand 3L1) but not with CCL3 [13], it distinguishes between the active form and the N-terminal CD26-processed inactive forms of CCL22 [14], and does not recognize homoeostatic CC chemokines as well as chemokines of other families [9].

D6 is expressed at very low levels by circulating leucocytes (E.M. Borroni, C. Buracchi, Y. Martinez de la Torre, E. Galliera, A. Vecchi, R. Bonecchi, A. Mantovani and M. Locati, unpublished work), but it is selectively expressed at high levels by endothelial cells of lymphatic afferent vessels in the skin, gut and lungs [15], and in the placenta, where it is present on invading trophoblast cells, on the apical side of syncytiotrophoblast cells and on decidual macrophages (E.M. Borroni, C. Buracchi, Y. Martinez de la Torre, E. Galliera, A. Vecchi, R. Bonecchi, A. Mantovani and M. Locati, unpublished work). In all cells tested so far, including the orthologous milieu of lymphatic endothelium [16] and trophoblast cells (E.M. Borroni, C. Buracchi, Y. Martinez de la Torre, E. Galliera, A. Vecchi, R. Bonecchi, A. Mantovani and M. Locati, unpublished work), D6 does not mediate conventional signalling activities, or facilitate chemokine transfer through the cell monolayer. Conversely, the presence of D6 consistently resulted in the degradation of appropriate ligands. In fact, this receptor seems to be particularly suited to function as a chemokine scavenger, as it is mainly localized in intracellular stores associated with early and recycling endosomes [17,18], it is constitutively internalized in a ligand-independent way through a β-arrestin-dependent clathrin-coated-pits-mediated pathway, and is rapidly recycled on the cell membrane [18,19]. Differently from conventional chemokine receptors, D6 internalization is a phosphorylation-independent event and requires a C-terminal region of acidic amino acids that is not found in other chemokine receptors that mediate constitutive interaction with β-arrestin [19]. D6 is particularly efficient at internalizing chemokines, which are then rapidly dissociated and degraded during vesicle acidification, leaving the receptor free to recycle to the cell surface [16,18]. Thus, in in vitro settings, D6 does not mediate signalling activities or support chemokine transcytosis, but behaves as a decoy receptor that scavenges inflammatory CC chemokines acting as ‘tapis roulant’ which cycles continuously and independently from ligand engagement.

D6−/− mice have been generated, and the results obtained in different animal models are consistent with a role of D6 as a chemokine scavenger in vivo. D6−/− mice have an exacerbated inflammatory response induced following application of phorbol ester to the skin [20] or by subcutaneous injection of complete Freund's adjuvant [21]. In the first model, inflammation is initiated by tumour necrosis factor and is then sustained by pro-inflammatory chemokines, with a prominent inflammatory infiltrate that includes T-cells, mast cells and neutrophils. Keratinocyte proliferation and neovascularization were also observed, leading to the development of psoriasiform-like lesions. In the second model, D6−/− animals showed increased inflammatory response at early time points characterized by prominent necrosis and neovascularization that evolved into macroscopic granuloma-like lesions and hyperplasia of draining lymph nodes. Increased levels of inflammatory CC chemokines were detected locally in both models, and pre-treatment with blocking antibodies specific for chemokines prevented the development of lesions. Interestingly, the increased local inflammation observed in D6-deficient mice resulted in an impaired specific immune response and protection in an encephalomyelitis model [22]. In this model, subcutaneous immunization led to a marked local inflammatory infiltrate in D6−/− mice, with CD11c+ cells becoming ‘trapped’ in aggre gates associated with micro-abscesses, and this altered response protects mice from developing the disease. Our recent results also highlight an important role of D6 in placental biology. Exposure of D6-deficient pregnant mice to lipopolysaccharide (LPS) or phospholipid-specific auto-antibodies resulted in increased leucocyte infiltration of the placenta and a consequent increase in the rate of foetal loss, which could be prevented by blocking inflammatory chemokines (E.M. Borroni, C. Buracchi, Y. Martinez de la Torre, E. Galliera, A. Vecchi, R. Bonecchi, A. Mantovani and M. Locati, unpublished work). In conclusion, in vitro and in vivo results strongly support a decoy function for D6, which both in lymphatic vessels (Figure 2) and in the placenta acts as a chemokine scavenger and prevents excessive inflammation.

Role of the chemokine decoy receptor D6 in lymphatic vessels

Figure 2
Role of the chemokine decoy receptor D6 in lymphatic vessels

D6 expressed on lymphatic vessels plays a role in dampening concentrations of inflammatory chemokines in inflamed tissues and tune their access to draining lymph nodes.

Figure 2
Role of the chemokine decoy receptor D6 in lymphatic vessels

D6 expressed on lymphatic vessels plays a role in dampening concentrations of inflammatory chemokines in inflamed tissues and tune their access to draining lymph nodes.

Concluding remarks

The concept of a receptor was originally formulated at the end of the 19th century as a receptive substance that binds a ligand, usually with high affinity and specificity, and elicits a cellular response [6]. Almost one century later, the first decoy receptor [the IL-1 (interleukin-1) type II receptor] was identified, and defined as a receptor structurally incapable of transducing signal but able to recognize the agonist with high affinity and specificity [23]. After the initial observation in the IL-1 system, decoy receptors have emerged as a general strategy conserved in evolution from Drosophila to human to tune the action of cytokines and growth factors [6,24]. Decoy receptors are now recognized as a general strategy to negatively regulate primary inflammatory cytokines though competition with signalling receptors for the ligand [6]. In addition, in some cases (i.e. the IL-1 decoy type II receptor), decoy receptors target the agonist for endocytosis and degradation, thus acting as scavengers [25]. In vitro and in vivo evidence suggests that ‘silent’ chemoattractant receptors may operate as decoy receptors for chemotactic agents, represent a new strategy to tune, shape and tame innate and adaptive immune responses, and may represent a target for therapeutic intervention.

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

     
  • CCL

    CC chemokine ligand

  •  
  • CCR

    CC chemokine receptor

  •  
  • CXCR

    CXC chemokine receptor

  •  
  • DARC

    Duffy antigen receptor for chemokines

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • IL-1

    interleukin-1

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