Review: Structure–function and biological properties of the atypical chemokine receptor D6

Molecular Immunology 55 (2013) 87–93

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Review

Review: Structure–function and biological properties of the atypical chemokine receptor D6夽 Cinzia Cancellieri a,b,1 , Nicoletta Caronni a,b,1 , Alessandro Vacchini a,b , Benedetta Savino a,b , Elena M. Borroni a,b , Massimo Locati a,b , Raffaella Bonecchi a,b,∗ a b

Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Manzoni 56, I-20089 Rozzano (MI), Italy Humanitas Clinical and Research Center, Via Manzoni 56, I-20089 Rozzano (MI), Italy

a r t i c l e

i n f o

Article history: Received 29 June 2012 Received in revised form 1 August 2012 Accepted 7 August 2012 Available online 29 August 2012 Keywords: Chemokines Atypical chemokine receptors Inflammation GPCR

a b s t r a c t The atypical chemokine receptor D6 was initially called “silent” on the basis of lack of conventional signaling events that lead to directional cell migration. It has emerged that D6 is able to bind and drive to degradative compartments most inflammatory CC chemokines and that is able to convey G-protein independent signaling events to optimize its scavenging activity. We here summarize the knowledge available today on D6 structural and signaling properties and its essential role for the control of inflammatory cells traffic and proper development of the adaptive immune response. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction

2. D6 structure–function properties

Chemokines are key regulators of leukocyte migration and play fundamental roles both in physiological and pathological immune responses. In addition to the conventional signaling chemokine receptors, a new subclass of atypical chemokine receptors has recently emerged. This subfamily of atypical chemokine receptors (ACR) is characterized by lack of G protein-dependent signaling, induction of calcium fluxes, and directional cell migration, while, in most cases, internalization of the ligand to degradative compartments is observed. Differently from canonical receptors, which are mainly expressed by leukocytes, ACR are mainly expressed by non-leukocytic cells. Nonetheless, they play a key role in leukocyte recruitment by shaping chemokine gradients, essential to ensure directional cell migration.

Chemokine receptors belong to the class A rhodopsin-like family of seven transmembrane domain G protein-coupled receptors (GPCR) and consist of single polypeptide chains with three extracellular and three intracellular loops, an acidic aminoterminal extracellular domain involved in ligand binding and a serine/threonine-rich intracellular carboxi-terminal domain (Locati et al., 2005; Mantovani et al., 2006). The external interface contributes to the specificity of ligand recognition, whereas the conserved transmembrane sequences, the cytoplasmic loops and the C-terminal domain are involved in receptor signaling and internalization (Murphy et al., 2000). The seven transmembrane domain organization is well conserved in D6 and the overall sequence identity to canonical chemokine receptors is in the 30–35% range, similar to the identity rate observed among conventional receptors (Nibbs et al., 1997). Nevertheless, D6 presents alteration in conserved elements generally essential for G protein coupling and signal transduction that are supposed to be the main cause of apparently D6 signaling incompetence.

Abbreviations: ACR, atypical chemokine receptors; GPCR, G protein-coupled receptors; IBC, innate-like B cells; COPD, Chronic Obstructive Pulmonary Disease; LEC, lymphatic endothelial cells; CFA, complete Freund’s adjuvant; aPL, antiphospholipid; APS, antiphospholipid syndrome; DSS, dextran sulfate sodium; KS, Kaposi’s sarcoma; VEGF, vascular endothelial growth factor; MOG, oligodendroglial glycoprotein; DCs, dendritic cells; GvHD, graft versus host disease. 夽 This article belongs to Special Issue on Leukocyte Adhesion and Migration. ∗ Corresponding author at: Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Manzoni 56, I-20089 Rozzano (MI), Italy. Tel.: +39 02 82245117; fax: +39 02 82245101. E-mail address: [email protected] (R. Bonecchi). 1 These authors equally contributed to the study. 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.08.003

2.1. D6 structural motifs D6 binds with high affinity 14 inflammatory chemokines, all of them belonging to the CC subfamily of inflammatory chemokines and including almost entirely the ligands for CCR1–CCR5 receptors (Bonecchi et al., 2004). The binding site for these chemokines has not yet been identified. Studies were performed on D6 N-terminal

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Fig. 1. Structure–function differences between D6 and canonical chemokine receptors. The seven transmembrane domain organization is well conserved in the atypical chemokine receptor D6, the overall sequence identity to conventional chemokine receptors is in the 30–35% range. N-terminal domain of both receptors shows acidic amino acids, sulphated moieties and N-linked glycosylation sites which are important for ligand recognition. Moreover, the second transmembrane domain of both receptors includes a highly conserved TxP motif, relevant for receptor activation. The aspartic acid in the second transmembrane domain and the DRYLAIV motif in the third transmembrane domain, of relevance for G protein activation, are not conserved in D6. The C-terminal domain of conventional chemokine receptors is involved in receptor phosphorylation, ␤-arrestin interaction, receptor internalization and desensitization, whereas the C-terminal domain of D6 is required to prevent receptor degradation, allowing receptor recycling and progressive chemokine depletion but its phosphorylation status and association with ␤-arrestin is still under debate.

domain that, similarly to conventional chemokine receptors, contains several charged residues most likely involved in ligand recognition, including acidic amino acids and sulphated moieties (Fig. 1), presumably tyrosine-linked, but whether receptor sulphation is important for ligand binding remains to be demonstrated (Blackburn et al., 2004; Farzan et al., 1999; Seibert et al., 2002). Blackburn et al. also demonstrated that D6 is glycosylated on the predicted N-linked glycosylation site (Blackburn et al., 2004), but this posttranscriptional modification appears to be irrelevant for both receptor expression and ligand binding. Referring to the second transmembrane domain, the TxP (ThrXaa-Pro) motif that is a highly conserved structural determinant in chemokine receptors and plays an important role in receptor activation but not in ligand binding, is also present in D6 (Fig. 1) (Govaerts et al., 2001). On the contrary, the aspartic acid in position 92 in the same domain, present in most GPCRs and required for activation, is mutated into an asparagine in D6. Up to now the role of this motif change in D6 activity has not been assessed. At the boundary of the second intracellular loop with the third transmembrane domain the well conserved DRYLAIV motif is present as DKYLEIV in all mammalian D6 proteins (Fig. 1) (Hansell et al., 2011). Correction of this motif to DKYLAIV confers weak ligand-induced signaling activity (Nibbs et al., 2009), supporting the notion that altered DRY motif may be at least in part responsible for the lack of conventional signaling activities in the ACR subfamily. D6 presents differences with canonical chemokine receptors also in the C-terminal tail, which is longer (312-384 aa) and contains a serine cluster and a putative 8th helix after aa 340. These domains are dispensable for D6 internalization while required to prevent receptor degradation from entering late endosomes, allowing receptor recycling and progressive chemokine depletion (Fig. 1) (Graham, 2009; McCulloch et al., 2008). In fact, complete deletion of D6 C-tail (D6-326 aa) or truncation of the 8th helix (D6-340 aa) or mutation of serine cluster into alanine strongly reduces D6 stability by targeting to lysosomal compartment, presumably through ubiquitination of two lysine residues (142 and 324 aa) that are well conserved across all mammalian D6 sequences (McCulloch et al., 2008).

2.2. D6 trafficking properties In resting conditions, D6 is predominantly located in intracellular perinuclear compartments and only 5% is detectable on the cell surface (Blackburn et al., 2004; Weber et al., 2004). D6 is constitutively internalized in Rab5-positive vesicles through clathrin-coated pits by a dynamin-dependent mechanism, and it is then targeted to early endosomes (Bonecchi et al., 2010; Weber et al., 2004). Internalized molecules are recycled back to plasma membrane via both a direct rapid recycling pathway (Rab4-positive vesicles) and a slower recycling pathway (Rab11-positive vesicles) involving D6 transit in the recycling endosomes before coming back to cell surface (Bonecchi et al., 2008). The trafficking properties of D6 are intimately linked to its activity in chemokine scavenging. Differently from conventional chemokine receptors, after ligand engagement D6 does not decrease but rather increases its expression on the cell surface, thus optimizing its degradatory activity. D6 ligands do not modify the receptor internalization rate, but induce a dose-dependent receptor up-regulation on the cell membrane, due to its mobilization from the intracellular pool, in particular by accelerating receptor recycling through the Rab11-positive vesicles (Bonecchi et al., 2008). Once internalized, ligands dissociate from the receptor and are targeted to degradation in Rab7-positive lysosomal compartments, while the receptor is free to recycle back to the cell surface (Fra et al., 2003; Weber et al., 2004), with mechanisms that are strictly dependent on cytoskeleton dynamics (Borroni et al., in preparation). Thus constitutive cycling and liganddependent receptor upregulation are mechanisms allowing rapid modulation of ligand uptake and degradation (Borroni et al., 2009). Internalization of conventional chemokine receptors leading to receptor desensitization requires ␤-arrestin recruitment at phosphorylated C-tail (Borroni et al., 2010; Shenoy and Lefkowitz, 2003). Referring to D6 both phosphorylation status and ␤-arrestin association are under debate. McCulloch et al. demonstrated that D6 is constitutively phosphorylated at the serine-rich motif in the C-tail but both constitutive internalization and recycling is independent from its phosphorylation status (Blackburn et al., 2004; McCulloch et al., 2008). D6 expression induces ␤-arrestin re-localization

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Fig. 2. Actin dynamics sustain D6 up-regulation and degradatory activity. In basal condition D6 is internalized in a Rab5- and dynamin-dependent way, and it recycles to the plasma membrane through a Rab4/Rab11-dependent mechanism. Active cofilin maintains actin cytoskeleton organization in stress fibers (here shown in brown), supporting constitutive internalization and recycling of D6 to the plasma membrane. Stimulation with active ligands, which are characterized by the presence of a proline residue in position 2 (here shown in purple), induces cofilin phosphorylation and its inactivation. In this way actin polymerize at plasma membrane in cortical actin (here shown in green), allowing D6 up-regulation and increasing its scavenging efficiency and chemokine degradation. On the other hand, D6 stimulation with neutral ligands (here shown in gray) does not change actin organization and cofilin activation state without affecting its distribution and scavenging efficiency. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

at the plasma membrane but D6 internalization is ␤-arrestinindependent and even in the complete absence of the C-tail, D6 still internalize but has limited degradation activity (McCulloch et al., 2008). On the other hand, Galliera et al. demonstrated that D6 is not phosphorylated in the tail, it is constitutively associated to ␤-arrestin, and the internalization is ␤-arrestin-dependent and receptor-phosphorylation independent (Galliera et al., 2004). All these data, although conflicting on the molecular mechanism, indicate that ␤-arrestin is required for optimal D6 function, probably acting on D6 recycling from intracellular compartments to the plasma membrane, as described for other ACR (Mahabaleshwar et al., 2012). 2.3. D6 signaling properties Though detailed structure–function analysis of ACR is not available yet, emerging evidences suggest that these receptors could activate a G-protein independent signaling pathway which controls chemokine gradient shaping (Shenoy and Lefkowitz, 2011). Observation of ligand-dependent modification of D6 intracellular traffic and more relevantly the identification of chemokines that bind D6 with high affinity but are not targeted to degradative compartments nor are able to upregulate D6 on cell surface, strongly suggested that a signaling event is present after ligand engagement (Savino et al., 2009). Further supporting this notion, analysis of the aminoacidic sequences of these two classes of D6 ligands revealed that a proline residue in position 2 is present in all ligands able to induce receptor activation (Savino et al., 2009), and only active ligands induce strong remodeling of actin and microtubules cytoskeleton which is required for the adaptive up-regulation of D6 allowing optimal degradation of chemokines (Borroni et al., in

preparation) (Fig. 2). Interestingly, dynamic actin turnover is also required to sustain D6 constitutive internalization and sorting to both Rab4/Rab11 recycling endosome, suggesting that a putative ligand-independent signaling event is triggered by the receptor in order to sustain its constitutive trafficking properties (Fig. 2). Studies on molecules able to regulate actin dynamics identified a signaling pathway that starts from D6 upon active ligand stimulation and that induces phosphorylation of cofilin (dos Remedios et al., 2003). This pathway is required for actin fibers rearrangements, D6 membrane up-regulation and chemokine scavenging (Borroni et al., in preparation) (Fig. 2). Interestingly, D6-mediated cofilin activation is G␣i-independent and ␤-arrestin-1 dependent. ␤-arrestin activities are not only restricted to chemokine receptor desensitization and internalization, as a negative regulator of GPCR activity through the uncoupling of G proteins, while they can also act as a signal transducer, allowing internalized receptors to continue signaling (Shenoy and Lefkowitz, 2011). These different activities of ␤-arrestin rely on its interaction with different intracellular domains of chemokine receptors: interaction with receptor C-terminal tail supports receptor internalization and desensitization, while binding to intracellular loops mediates signaling events (Cheng et al., 2000; Huttenrauch et al., 2002). As mentioned above, the association of D6 with ␤-arrestin is still under debate, but surely D6 requires a ␤-arrestin-dependent signaling pathway to regulate cofilin activity and optimize its chemokine scavenging activity (Borroni et al., in preparation). 3. Biological functions of the ACR D6 While there is a consensus about D6 expression by lymphatic endothelial cells (Nibbs et al., 2001), and placental trophoblasts

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Fig. 3. D6 in vivo activity. The atypical chemokine receptor D6 acts as central regulator for the correct orchestration of the immune response by containing the local inflammatory response and controlling the migration to secondary lymphoid organs of DC and a subset of myeloid population with immunosuppressive properties. These activities have been demonstrated by the use of D6-deficient mice in different disease models, here subdivided in two classes: inflammatory and tumoral models and models involving specific immune response.

(Madigan et al., 2010; Martinez de la Torre et al., 2007), its expression on leukocytes is still matter of debate. Hansell and Nibbs (2011) reported that D6 is expressed by all conventional IBC (innate-like B cells: MZ B cells, B1a, and B1b), while McKimmie et al. (2008) reported D6 expression by several leukocytes of innate immunity. D6 expression was also found in human alveolar macrophages of COPD patients (Bazzan et al., 2012), and scattered data on D6 expression on tissue macrophages were reported also by other groups (Martinez de la Torre et al., 2007; Vetrano et al., 2010). Up to now the role of D6 expression by leukocytes is not fully understood while the use of knock-out and chimeric mice have confirmed that D6 expressed by non-hematopoietic cell types has a non-redundant role as a chemokine scavenger both in homeostatic and inflammatory conditions (Fig. 3).

3.1. Role of D6 in homeostatic conditions In homeostatic conditions D6 KO mice present a selective increase in the number of Ly6Chigh circulating and spleen monocytes while in the bone marrow a slight but significant decrease of this population was observed, suggesting an increased rate of monocyte exit from this compartment. Moreover, Ly6Chigh monocytes derived from D6−/− mice, compared to wilde type (wt) cells, present a more immature phenotype, as indicated by lower expression of maturation markers (CD11b, CD115, F4/80) that was not due to an intrinsic defect of D6 in leukocytes but to the absence of D6 expression on the non-hematopoietic compartment, possibly the lymphatic vessels which express D6 at high levels (Savino et al., 2012). D6 deficiency, in resting conditions, leads to enhanced B1cell responses to CXCL13, reduction in B1 cells and lower levels of serum anti-PC Abs. These phenotypes might be linked, because the migratory properties of B1 cells profoundly influence their survival and Ab-secreting capacity (Hansell and Nibbs, 2011). Furthermore, D6 KO mice have increased serum concentration of CC chemokines, in particular CCL11 (Martinez de la Torre et al., 2007) and CCL2 (Savino et al., 2012). All these data point to a non-redundant role of D6 in resting conditions as a key regulator of chemokine levels and its impact on leukocyte distribution.

3.2. Protective role of D6 during inflammatory conditions D6−/− mice develop exacerbated inflammatory responses in different experimental diseases sustained by increased levels of inflammatory CC chemokines detected both locally and in draining lymph nodes. Because D6 is expressed by lymphatic endothelial cells (LEC) at sites of primary antigen exposure, in particular in the skin, two different models of cutaneous inflammation have been performed: phorbol ester skin painting (Jamieson et al., 2005) and subcutaneous injection of complete Freund’s adjuvant (CFA) (Martinez de la Torre et al., 2005). In the first model, D6−/− mice develop psoriasis-like lesions, characterized by an aberrant recruitment of T lymphocytes and mast cells, huge proliferation of keratinocytes and neovascularization. This increased inflammatory response is TNF␣-dependent and it is sustained by the accumulation of inflammatory chemokines that are not efficiently cleared from inflamed sites in the absence of D6. In the second model, after subcutaneous injection of CFA, D6-deficient mice show a severer evolution of the disease with earlier lesions, prominent necrosis and neovascularization. At shorter times (e.g. day 7) inflammation evolves in macroscopic granuloma-like lesions in a significant percentage of D6−/− animals, and only in a minority of wild-type littermates. Interestingly, differences are not evident at later time points (e.g. day 21). In both models D6-deficient mice have an exacerbated inflammatory response and increased levels of inflammatory CC chemokines detected locally, demonstrating that the increased inflammatory response is caused by the inefficient control of the chemokine system in absence of D6. Moreover, D6 has a central role in controlling the inflammation at the maternal–fetal interface. In fact D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. D6-deficient mice were studied in two models of inflammation-induced fetal loss by Martinez de la Torre et al. (Martinez de la Torre et al., 2007). The first model consists in treating pregnant mice with LPS, mimicking a clinical condition frequently associated with abortion and preterm delivery in humans; the second one was performed by injecting in pregnant

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mice antiphospholipid (aPL) autoantibodies purified from patients affected by the antiphospholipid syndrome (APS). In both cases D6−/− mice resulted to have an increased rate of fetal loss due to higher levels of inflammatory CC chemokines and increased leukocytes infiltrate in placenta. Furthermore, D6 has been demonstrated to suppress fetal resorption after embryo transfer into fully allogenic recipients, whereas it is not required for syngeneic or semiallogeneic fetal survival in unchallenged mice (Madigan et al., 2010). Madigan et al. also found that in humans the level of D6-binding chemokines in maternal plasma decreases during pregnancy, even in woman with pre-eclampsia, a disease associated with increased maternal inflammation. Finally, in a model of spontaneous fetal loss in swine, D6 was found expressed in endometrial epithelium, uterine glands, and trophoblast, and a notable loss in D6 immunoreactivity was observed in arresting versus viable littermate attachment sites (Wessels et al., 2007). These evidences highlight a role of chemokines in fetal abortion induced by inflammatory stimuli and suggest D6 as responsible for controlling chemokines distribution and bioavailability in the decidua and gestational membranes. Wiederholt et al. analyzed D6 expression in liver and found that genetic variations in gene sequence were associated with the grade of hepatic inflammation in patients with chronic hepatitis C infection (Wiederholt et al., 2008). Conversely Berres et al. studied the action of D6 in acute CCl4 -induced liver damage, demonstrating the importance of post-translational chemokine regulation within the liver. In this model D6-deficient mice resulted to have prolonged liver damage associated with increased levels of intrahepatic inflammatory chemokines and increased infiltration of CD45+ leukocytes, mainly T and NK cells (Berres et al., 2009). Whereas in the organs mentioned above the role of D6 as regulator of inflammation is well established, data in literature regarding colon-associated inflammatory pathologies remain controversial. Vetrano et al. reported that mice lacking D6 are significantly more susceptible to dextran sulfate sodium (DSS) colitis than wildtype mice and they fail to resolve colitis, because of a greater inflammation and accumulation of CC chemokines, associated with increased leukocyte infiltration and weight loss. Using bone marrow chimeras the ability of D6 to regulate colitis was tracked to the stromal/lymphatic compartment, with no contribution of hematopoietic cells (Vetrano et al., 2010). Opposite results in the same murine model of colitis were published by Bordon et al., who reported that D6-deficient mice have reduced susceptibility to colitis and suggested that D6 could contribute to the development of colitis by regulating IL-17A secretion by T cells in the inflamed colon. Despite the opposite results obtained in DSS colitis model, both groups showed that D6 is expressed by stromal cells in resting colon and that its expression is up-regulated in human colon sample from patients with inflammatory bowel disease (Bordon et al., 2009). D6 regulates pulmonary chemokine levels, inflammation, and airway responsiveness during allergen-induced airway disease. In fact allergen-challenged D6-deficient mice show more inflammation and infiltrating cells, primarily eosinophils and DCs whereas, unexpectedly, they have less airway reactivity to methacoline (Whitehead et al., 2007). In the same way, D6−/− mice after intranasal administration of low doses of Mycobacterium tuberculosis result to have dramatic local and systemic inflammatory response with increased number of mononuclear cells infiltrating tissues and lymph nodes which exerts in early death. The hypothesis proposed suggests that the absence of the receptor impairs CC chemokines removal from many different organs and tissues, which results in the influx of immune cells, leading to exacerbated production of pro-inflammatory cytokines and tissue damage (Di Liberto et al., 2008).

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3.3. D6 and tumor biology Chemokines are crucial for cancer-related inflammation, which can promote tumor development and progression (Mantovani et al., 2010). Because of its non-redundant role in regulating the inflammation, D6 has been studied in different mouse model of cancer. Firstly, D6 role in tumor has been investigated in inflammation-induced carcinogenesis. D6-deficient mice were found to be more susceptible to tumor development in a phorbol ester-induced skin tumorigenesis model (Nibbs et al., 2007) and in an azoxymethane/sodium dodecylsulphate model of colon cancer (Vetrano et al., 2010). In both model, D6−/− mice resulted in a significant increase of inflammatory chemokines and leukocytes infiltrating the tumor. Nibbs et al. also found that transgenic expression of D6 in the basal epidermal layer of a susceptible mouse strain increases keratinocyte sequestration of inflammatory CC chemokines and offers considerable protection from papilloma formation (Nibbs et al., 2007). These data suggest that D6 activity in chemokines clearance is an effective method of tumor suppression. Furthermore, D6 is expressed by different tumors, including large granular lymphocytes leukemia cells (Daibata et al., 2004), malignant vascular tumors (Nibbs et al., 2001), Kaposi’s sarcoma (Savino et al., unpublished results) and breast cancers (Wu et al., 2008). In human breast cancer in particular D6 is differentially expressed and regulated by several cytokines including IL-1␤ and TNF-␣. A study from Wu et al. revealed that D6 is inversely correlated to lymph node metastasis and clinical disease stage, but positively correlated to disease-free survival rate in cancer patients. More importantly, D6 has an inhibitory effect on tumor biology, both in vitro and in vivo. In fact the over-expression of D6 in breast cancer cell lines is associated with decreased chemokine levels (e.g., CCL2 and CCL5), vessel density, and tumor-associated macrophage infiltration (Wu et al., 2008). D6 has been also studied in an in vivo model of Kaposi’s sarcoma obtained by subcutaneous injection in nude mice of in vitro generated D6-trasfected KS-IMM cell line. In this model, D6 overexpression was found to significantly reduce the tumor growth, associated with lower levels of inflammatory chemokines inside the tumor, lower number of tumor associated macrophages but increased monocytes, decreased amount of VEFG and reduced angiogenesis. These data clearly suggest that D6-mediated regulation of inflammatory chemokines bioavailability accounts for inhibition of monocyte differentiation and VEGF-A production at tumor site, resulting in reduced tumor growth (Savino et al., in preparation).

3.4. Role of D6 in the immune response D6-deficient mice have been studied in a wide range of disease mice models (Fig. 3), in particular in inflammatory ones. In all models, D6 showed a protective role by damping inflammation and innate immune response. Interestingly, in two different models D6 has been found to exert a role also in development of the specific immune response. D6−/− mice resulted protected in a model of experimental autoimmune encephalomyelitis, induced by myelin oligodendroglial glycoprotein (MOG) peptide 35–55 administration in CFA. The absence of D6 increased tissue inflammation and induced abnormal accumulation of CD11c+ dendritic cells (DCs) in cellular aggregates in the inflamed skin. The local retention of DCs resulted in poor priming of encephalitogenic T cells with reduced T cell proliferation and IFN␥ production. Thus, the impaired encephalitogenic responses protect D6-deficient mice from the development of encephalomyelitis (Liu et al., 2006). This essential role for correct DC migration to lymph nodes was further demonstrated by Lee

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et al. (2011) that have shown that D6 prevents excessive leukocyte adherence on lymphatic surfaces. The defect in the adaptive immune response has been found also in a model of graft versus host disease (GvHD) where D6-deficient mice resulted to be partially protected. In this model the inhibition of the development of adaptive immune response is caused by the accumulation, under inflammatory conditions, of Ly6Chigh monocytes in secondary lymphoid organs of D6−/− mice. This cell population belongs to a heterogeneous population of myeloid cells with immunoregulatory function presently alluded to as myeloidderived suppressor cells. In fact, Ly6Chigh monocytes derived from D6−/− mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses and partially protect mice from the development of GvHD (Savino et al., 2012). 4. Concluding remarks Data resumed here indicate that D6 plays a central regulatory function for the correct orchestration of the adaptive immune response via at least two parallel mechanisms: the containment of the local inflammatory response that allows the successful migration of dendritic cells to draining lymph nodes and the control of the migration to secondary lymphoid organs of myeloid population with immunosuppressive properties. To carry out this regulatory function, D6 has evolved by diversifying its structure from canonical chemokine receptors. It is not able to sustain cell migration but on the contrary has maintained a ␤-arrestin-dependent signaling that allows the receptor to regulate its surface expression to the extracellular concentrations of chemokine ligands to rapidly and efficiently drive them to degradation. Acknowledgements Research activities in the lab are supported by Ministero dell’Istruzione dell’Università e della Ricerca (PRIN and FIRB projects), Ministero della Salute (Ricerca Finalizzata), the Italian Association for Cancer Research (AIRC), Regione Lombardia (LIIN project), research grants Fondazione Cariplo and the European Community’s Seventh Framework Programme [FP7-2007-2013] under grant agreement HEALTH-F4-2011-281608 (TIMER). References Bazzan, E., Saetta, M., Turato, G., Borroni, E.M., Cancellieri, C., Baraldo, S., Savino, B., Calabrese, F., Ballarin, A., Balestro, E., et al., 2012. Expression of the atypical chemokine receptor D6 in human alveolar macrophages in Chronic Obstructive Pulmonary Disease. Chest. 1378, 11-3220. Berres, M.L., Trautwein, C., Zaldivar, M.M., Schmitz, P., Pauels, K., Lira, S.A., Tacke, F., Wasmuth, H.E., 2009. The chemokine scavenging receptor D6 limits acute toxic liver injury in vivo. Biological Chemistry 390, 1039–1045. Blackburn, P.E., Simpson, C.V., Nibbs, R.J., O’Hara, M., Booth, R., Poulos, J., Isaacs, N.W., Graham, G.J., 2004. Purification and biochemical characterization of the D6 chemokine receptor. The Biochemical Journal 379, 263–272. Bonecchi, R., Borroni, E.M., Anselmo, A., Doni, A., Savino, B., Mirolo, M., Fabbri, M., Jala, V.R., Haribabu, B., Mantovani, A., et al., 2008. Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation. Blood 112, 493–503. Bonecchi, R., Locati, M., Galliera, E., Vulcano, M., Sironi, M., Fra, A.M., Gobbi, M., Vecchi, A., Sozzani, S., Haribabu, B., et al., 2004. Differential recognition and scavenging of native and truncated macrophage-derived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor. Journal of Immunology 172, 4972–4976. Bonecchi, R., Savino, B., Borroni, E.M., Mantovani, A., Locati, M., 2010. Chemokine decoy receptors: structure–function and biological properties. Current Topics in Microbiology and Immunology 341, 15–36. Bordon, Y., Hansell, C.A., Sester, D.P., Clarke, M., Mowat, A.M., Nibbs, R.J., 2009. The atypical chemokine receptor D6 contributes to the development of experimental colitis. Journal of Immunology 182, 5032–5040. Borroni, E.M., Buracchi, C., Savino, B., Pasqualini, F., Russo, R.C., Nebuloni, M., Bonecchi, R., Mantovani, A., Locati, M., 2009. Role of the chemokine scavenger receptor

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