D6 Expression Research Articles

Enoxaparin Increases D6 Receptor Expression and Restores Cytoskeleton Organization in Trophoblast Cells from Preeclampsia.

D6 is a scavenger receptor for CC chemokines expressed in the human placenta. It prevents excessive leukocyte tissue infiltration by internalizing chemokines through cytoskeleton-dependent intracellular transport. In preeclampsia (PE), the D6 receptor is overexpressed in trophoblast cells, but functionally impaired, due to cytoskeleton destructuring. Low molecular weight heparin (LMWH) represents a potential treatment for PE based on its anti-thrombotic and anti-inflammatory properties. Here, we investigated the effect of enoxaparin on D6 expression, and cytoskeleton organization primary cytotrophoblast cell cultures were obtained from the placentae of women with PE (n = 9) or uncomplicated pregnancy (n = 9). We demonstrated that enoxaparin is able to (i) increase D6 expression, and (ii) improve cytoskeletal fiber alignment in trophoblast cells from PE patients.

Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

Ceramide kinase (CerK) and ceramide-1-phosphate (C1P) are involved in various cellular functions, while regulation of the enzyme activity has not been well elucidated. We herein investigated the effects of several glycerophospholipids on human recombinant CerK activity with CaCl2 and MgCl2 by measuring the formation of fluorescent labeled C1P in vitro. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl2, and 144±32 with 2 mM MgCl2 in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P2), and phosphatidic acid had no effect on CerK activity with CaCl2, although PI(4,5)P2 and phosphatidic acid bound to CerK in the lipid-protein overlay assay. The addition of cardiolipin (diphosphatidylglycerol) at concentrations up to 0.1 µM increased, whereas those more than 1 µM decreased CerK activity with CaCl2/MgCl2. In the lipid-protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl2-independent manner. Cardiolipin also bound to CerK in the multilamellar vesicle binding assay. A deviation from the normal range of cellular cardiolipin, both the decrease by phospholipase D6 expression and increase by an exogenous addition of the lipid, negatively regulated C1P formation in intact HepG2 cells. Our results revealed that cardiolipin bound to CerK and regulated the formation of C1P in vitro and in cells.

Chemokine decoy receptor D6 mimicking trap (D6MT) prevents allosensitization and immune rejection in murine corneal allograft model.

Although corneal allotransplantation is performed in the immune-privileged cornea, many grafts are still rejected after transplantation. This study examined the role of chemokine receptor D6 expression in a corneal allograft rejection, investigated the modulation of D6 expression in cells, and determined the effect of D6 on graft survival. Interestingly, D6 was highly expressed in CD45 -: cells and the corneal epithelium of accepted corneal allografts. From the mouse corneal allograft model, TGF-β was found to play a key role in D6 up-regulation, leading to reduced CCL2, CCL5, and CCL3. To modulate D6 chemokine binding, a D6MT was developed and showed effective chemokine trapping through SPR and FACS assays. By treating corneal allografts with D6MT, the allograft survival rate was improved, and (lymph) angiogenesis was reduced. Direct allosensitization and DC LN homing was drastically reduced in the mouse corneal allograft model. These findings suggest that TGF-β is a positive regulator of D6 expression, and it is a potential therapeutic target to enhance the survival of corneal allografts.

ERK-dependent downregulation of the atypical chemokine receptor D6 drives tumor aggressiveness in Kaposi sarcoma.

D6 is an atypical chemokine receptor acting as a decoy and scavenger for inflammatory CC chemokines expressed in lymphatic endothelial cells. Here, we report that D6 is expressed in Kaposi sarcoma (KS), a tumor ontogenetically related to the lymphatic endothelium. Both in human tumors and in an experimental model, D6 expression levels were inversely correlated with tumor aggressiveness and increased infiltration of proangiogenic macrophages. Inhibition of monocyte recruitment reduced the growth of tumors, while adoptive transfer of wild-type, but not CCR2(-/-) macrophages, increased the growth rate of D6-competent neoplasms. In the KS model with the B-Raf V600E-activating mutation, inhibition of B-Raf or the downstream ERK pathway induced D6 expression; in progressing human KS tumors, the activation of ERK correlates with reduced levels of D6 expression. These results indicate that activation of the K-Ras-B-Raf-ERK pathway during KS progression downregulates D6 expression, which unleashes chemokine-mediated macrophage recruitment and their acquisition of an M2-like phenotype supporting angiogenesis and tumor growth. Combined targeting of CCR2 and the ERK pathway should be considered as a therapeutic option for patients with KS.

Expression of the chemokine decoy receptor D6 is decreased in colon adenocarcinomas.

Recruitment of immune cells to tumors is a complex process crucial for both inflammation-driven tumor progression and specific anti-tumor cytotoxicity. Chemokines control the directed migration of immune cells, and their actions are partly controlled by nonsignaling chemokine decoy receptors. The role of the receptors such as D6, Duffy antigen receptor for chemokines and ChemoCentryx chemokine receptor in immunity to tumors is still unclear. Using real-time PCR, we detected significantly decreased expression of D6 mRNA in colon tumors compared to unaffected mucosa. D6 protein was expressed by lymphatic endothelium and mononuclear cells in the colon lamina propria and detected by immunohistochemistry in two out of six tissue samples containing high D6 mRNA levels, whereas no staining was observed in any tissue samples expressing low mRNA levels. When examining the density of lymphatic vessels in colon tumors, we detected a marked increase in vessels identified by the lymphatic endothelial marker Lyve-1, excluding passive regulation of D6 due to decreased lymphatic vessel density. In parallel, the Treg-recruiting chemokine CCL22, which is sequestered by D6, was threefold increased in tumor tissue. Furthermore, we could show that low D6 expression correlated to more invasive tumors and that tumor location influences D6 expression, which is lower in the more distal parts of the colon. The data support that regulation of D6 by colon tumors results in altered levels of proinflammatory CC chemokines, thereby shaping the local chemokine network to favor tumor survival. This may have implications for the design of future immunotherapy for colon cancer.

SAT0037 Local expression of the atypical chemokine receptor D6 in systemic sclerosis

BackgroundThe atypical chemokine receptor D6 is an inflammatory CC-chemokine scavenger. It is involved in resolution of inflammatory responses and leukocyte trafficking from sites of injury to secondary lymphoid organs through...

An analysis of the function and expression of D6 on lymphatic endothelial cells

AbstractThe mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma–associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.

Atypical chemokine receptors predict lymph node metastasis and prognosis in patients with cervical squamous cell cancer

ObjectiveAtypical chemokine receptors (ACRs), including CCX-CKR, DARC, and D6, have been reported to be involved in cancer invasion and metastasis. The objective of this study was to investigate the prognostic importance of ACRs in patients with cervical squamous cell carcinoma (CSCC). MethodsThe expression of three ACRs was investigated by immunohistochemical (IHC) examination in a total of 317 cervical specimens including 40 normal cervical tissues, 50 cases of carcinoma in situ of cervix (CIS), and 227 cases of CSCC by immunohistochemistry. ResultsThe expression rate of DARC and CCX-CKR in CSCC, CIS, and normal cervix increased gradually (p<0.01). D6 expression is decreased in CSCC compared to either in CIS or in normal cervix (p<0.05). In addition, the expression of CCL2 and CCL19 was inversely associated with ACR expression (p<0.05), while that of LCA was positively correlated with ACR expression (p<0.05). Moreover, DARC expression, CCX-CKR expression, and ACR coexpression were negatively correlated with lymph node metastasis (P<0.01). D6 expression and ACR coexpression were negatively related to tumor size (p=0.018) and recurrence (p=0.028). In multivariate Cox regression analysis, CCX-CKR expression was a positive indicator for overall survival (p=0.008), and D6 expression was an independent predictor of both overall and recurrence-free survival (p=0.041) in CSCC. ConclusionsOur results suggest that the loss of ACRs may play important roles in the tumorigenesis and migration of cervical cancer. ACR expression may be considered as prognostic markers in patients with CSCC.

Expression of the Atypical Chemokine Receptor D6 in Human Alveolar Macrophages in COPD

D6 is an atypical chemokine receptor involved in chemokine degradation and resolution of acute inflammatory responses in mice. Emerging evidence suggests that D6 might behave differently in human chronic inflammatory conditions. We, therefore, investigated the involvement of D6 in the immune responses in COPD, a chronic inflammatory condition of the lung. D6 expression was quantified by immunohistochemistry in surgical resected lung specimens from 16 patients with COPD (FEV(1), 57% ± 6% predicted) and 18 control subjects with normal lung function (nine smokers and nine nonsmokers). BAL was also obtained and analyzed by flow cytometry, immunofluorescence, and molecular analysis for further assessment of D6 involvement. D6 expression in the lung was mainly detected in alveolar macrophages (AMs). The percentage of D6(+) AMs was markedly increased in patients with COPD as compared with both smoker and nonsmoker control subjects (P < .0005 for both). D6 expression was detected at both transcript and protein level in AMs but not in monocyte-derived macrophages. Finally, D6 expression was positively correlated with markers of immune activation (CD8(+) T lymphocytes, IL-32, tumor necrosis factor-α, B-cell activating factor of the tumor necrosis factor family, phospho-p38 mitogen-activated protein kinase) and negatively with lung function (FEV(1), FEV(1)/FVC). D6 is expressed in AMs from patients with COPD, and its expression correlates with the degree of functional impairment and markers of immune activation. Upregulation of D6 in AMs could indicate that, besides its known scavenger activity in acute inflammation, D6 may have additional roles in chronic inflammatory conditions possibly promoting immune activation.

P155 Control of LY6CHIGH monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6

The atypical chemokine receptor (ACR) D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. The aim of this project is to understand if altered antigen presentation and development of specific response in D6−/− mice are related to modified trafficking and activities of myeloid cells. Leukocytes populations of D6−/− mice were analyzed by FACS in basal and inflammatory conditions (Complete Freund’s adjuvant injection or intravenous CCL2 administration). T cell priming were studied in vitro and in vivo and GVHD models were developed in D6−/− mice. Mice lacking D6 expression in the stromal/lymphatic compartment have a selective increase in the number of Ly6C high monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6C high and Ly6G + CD11b + cells (Gr1 + myeloid cells) accumulate in increased number in secondary lymphoid organs of D6 −/− mice in a CCR2-dependent manner. Ly6C high monocytes derived from D6 −/− mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses and partially protect mice from the development of Graft-versus-Host Disease (GvHD). D6 differentially regulates the traffic of monocyte subsets and controls their differentiation into suppressor cells by the control of CCR2 ligands.

P041 G protein-independent signalling by the chemokine scavenger receptor D6 is required for its chemokine scavenging activity

Introduction Chemokine receptors include a set of molecules referred to as “atypical” chemokine receptors (ACR) because unable to induce directional cell migration and elicit conventional GPCR signalling. As no signaling activity has been observed after ACR engagement by their ligands, they are presently considered “silent” receptors. D6 is an ACR with scavenger activity on inflammatory CC chemokines with a non-redundant role for appropriate regulation of innate immunity and inflammation. Methods D6 expression was evaluated by FACS and confocal microscopy. Chemokine degradation was measured by ELISA test. Protein phosphorylation levels were detected by western blot. Results Our previous data showed that, differently from CCR5, ligand induces D6 up-regulation on plasma membrane through a rapid mobilization of receptor from recycling endosomes, thus improving its scavenging performance. Here we report that in cells expressing D6 or CCR5, ligand engagement causes a massive actin cytoskeleton reorganization and changes receptors colocalization with actin filaments in a completely opposite fashion. Both receptors signal to actin cytoskeleton by phosphorylating cofilinA through the Rac1-PAK1-LIMK1-dependent pathway. However, opposite to CCR5, D6 activates this pathway via a beta-arrestin-dependent, G protein-independent mechanism. Inhibition of each component of this signaling cascade completely abrogates D6 adaptive upregulation and scavenging activity. Conclusion D6, and possibly ACR in general, are signaling receptors exerting their regulatory functions on inflammation and immunity via the activation of a distinct signaling pathway.

Elevated Expression of the Chemokine-Scavenging Receptor D6 Is Associated with Impaired Lesion Development in Psoriasis

D6 is a scavenging-receptor for inflammatory CC chemokines that are essential for resolution of inflammatory responses in mice. Here, we demonstrate that D6 plays a central role in controlling cutaneous inflammation, and that D6 deficiency is associated with development of a psoriasis-like pathology in response to varied inflammatory stimuli in mice. Examination of D6 expression in human psoriatic skin revealed markedly elevated expression in both the epidermis and lymphatic endothelium in "uninvolved" psoriatic skin (ie, skin that was more than 8 cm distant from psoriatic plaques). Notably, this increased D6 expression is associated with elevated inflammatory chemokine expression, but an absence of plaque development, in uninvolved skin. Along with our previous observations of the ability of epidermally expressed transgenic D6 to impair cutaneous inflammatory responses, our data support a role for elevated D6 levels in suppressing inflammatory chemokine action and lesion development in uninvolved psoriatic skin. D6 expression consistently dropped in perilesional and lesional skin, coincident with development of psoriatic plaques. D6 expression in uninvolved skin also was reduced after trauma, indicative of a role for trauma-mediated reduction in D6 expression in triggering lesion development. Importantly, D6 is also elevated in peripheral blood leukocytes in psoriatic patients, indicating that upregulation may be a general protective response to inflammation. Together our data demonstrate a novel role for D6 as a regulator of the transition from uninvolved to lesional skin in psoriasis.

Control of murine Ly6Chigh monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6

AbstractThe atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. Here we report that mice lacking D6 expression in the nonhematopoietic compartment have a selective increase in the number of Ly6Chigh monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6Chigh monocytes accumulate in increased number in secondary lymphoid organs of D6−/− mice in a CCR2-dependent manner. 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. Thus, control of CCR2 ligands by D6 regulates the traffic of Ly6Chigh monocytes and controls their immunosuppressive potential.

An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis

ObjectivesSystemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to sites of tissue damage; their removal...

Cardiac Allograft Rejection: Examination of the Expression and Function of the Decoy Chemokine Receptor D6

Inflammatory cell recruitment during allograft rejection is driven by a group of inflammatory cytokines termed chemokines. Chemokines are presented on the surface of the vascular endothelium where they ligate specific receptors expressed on the surface of leukocytes. Recently, a group of nonsignaling chemokine receptors have been described. These bind and internalize chemokines but do not drive leukocyte migration. It is believed that these compete with classical signaling receptors to modulate inflammation. This study describes the first examination of the human decoy chemokine receptor D6 during rejection; D6 binds at least 12 potent proinflammatory chemokines. The expression of D6 by graft infiltrating leukocytes was examined in cardiac allografts by confocal microscopy on biopsy sections (n=19). Cytokine regulation of D6 was examined in vitro, and a chemokine scavenging assay was performed using the prototypical transplant-associated chemokine CCL5/RANTES. D6 expression was found to be higher in the biopsies taken from more severe cardiac allograft rejection (P<0.01) and was predominantly localized to graft infiltrating CD45(+)CD68(+) leukocytes. In vitro studies demonstrated that the transforming growth factor-beta strongly increased the expression of D6 by monocytes, which significantly enhanced D6-mediated chemokine scavenging (by 85%, P<0.05). We present the first examination of the biology of D6 during rejection and identify a transplant-associated cytokine that is able to regulate its expression. These data suggest an exciting new mechanism for the antiinflammatory actions of transforming growth factor-beta. Understanding the expression patterns of D6 may provide important insight into the regulation and control of inflammatory cell recruitment during allograft rejection.

The lymphatic system controls intestinal inflammation and inflammation-associated colon cancer through the chemokine decoy receptor D6

Background and aimsInflammatory CC chemokines have long been associated with cancer, but unequivocal evidence of a role in clinically relevant models of carcinogenesis is lacking. D6, a promiscuous decoy receptor...

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.

Hemopoietic Cell Expression of the Chemokine Decoy Receptor D6 Is Dynamic and Regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.

Chemokine Decoy Receptor D6 Plays a Negative Role in Human Breast Cancer

Chemokine binding protein D6 is a promiscuous decoy receptor that can inhibit inflammation in vivo; however, the role it plays in cancer is not well known yet. In this study, we showed for the first time that human breast cancer differentially expressed D6 and the expression could be regulated by some cytokines. More importantly, overexpression of D6 in human breast cancer cells inhibits proliferation and invasion in vitro and tumorigenesis and lung metastasis in vivo. This inhibition is associated with decreased chemokines (e.g., CCL2 and CCL5), vessel density, and tumor-associated macrophage infiltration. Furthermore, D6 expression is inversely correlated to lymph node metastasis as well as clinical stages, but positively correlated to disease-free survival rate in cancer patients. Therefore, D6 plays a negative role in the growth and metastasis of breast cancer.