Expression, regulation, and function of atypical chemerin receptor CCRL2 on endothelial cells

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)β(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.     

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system – chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) – in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.     

Chemokine-like receptor 1 expression and chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood

Plasmacytoid dendritic cells (pDCs) are versatile cells of the immune response, secreting type I IFNs and differentiating into potent immunogenic or tolerogenic APCs. pDCs can express adhesion and chemokine receptors for lymphoid tissues, but are also recruited by unknown mechanisms during tissue inflammation. We use a novel mAb specific for serpentine chemokine-like receptor 1 (CMKLR1) to evaluate its expression by circulating leukocytes in humans. We show that CMKLR1 is expressed by circulating pDCs in human blood, whereas myeloid DCs (mDCs) as well as lymphocytes, monocytes, neutrophils, and eosinophils are negative. We identify a major serum agonist activity for CMKLR1 as chemerin, a proteolytically activated attractant and the sole known ligand for CMKLR1, and we show that chemerin is activated during blood coagulation and attracts pDC but not mDC in ex vivo chemotaxis assays. We conclude that CMKLR1 expression and chemerin-mediated chemotaxis distinguish circulating pDCs from mDCs, providing a potential mechanism for their differential contribution to or regulation of immune responses at sites of bleeding or inflammatory protease activity.     

The Expression and Regulation of Chemerin in the Epidermis

Chemerin is a protein ligand for the G protein-coupled receptor CMKLR1 and also binds to two atypical heptahelical receptors, CCRL2 and GPR1. Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein. Although chemerin was initially identified as a highly expressed gene in healthy skin keratinocytes that was downregulated during psoriasis, the regulation of chemerin and its receptors in the skin by specific cytokines and microbial factors remains unexplored. Here we show that chemerin, CMKLR1, CCRL2 and GPR1 are expressed in human and mouse epidermis, suggesting that this tissue may be both a source and target for chemerin mediated effects. In human skin cultures, chemerin is significantly downregulated by IL-17 and IL-22, key cytokines implicated in psoriasis, whereas it is upregulated by acute phase cytokines oncostatin M and IL-1β. Moreover, we show that human keratinocytes in vitro and mouse skin in vivo respond to specific microbial signals to regulate expression levels of chemerin and its receptors. Furthermore, in a cutaneous infection model, chemerin is required for maximal bactericidal effects in vivo. Together, our findings reveal previously uncharacterized regulators of chemerin expression in skin and identify a physiologic role for chemerin in skin barrier defense against microbial pathogens.     

Expression of chemokine-like receptor 1 (CMKLR1) on J744A.1 macrophages co-cultured with fibroblast and/or tumor cells: modeling the influence of microenvironment

Maturation of macrophages is influenced by the composition of surrounding microenvironment. Expression of CMKLR1, the receptor for chemerin, is potentially associated with the differentiation status of macrophages. In this study, CMKLR1 was determined on peritoneal and tumor-infiltrating macrophages. CMKLR1 expression was found to be associated with the fibroblast-assisted maturation of J744A.1 monocyte/macrophage cells in the co-cultures established to model tumor microenvironment, whereas the presence of tumor cells was able to upregulate CMKLR1 expression independent of macrophage maturation. In addition, macrophages cultured with tumor cells or in tumor cell-conditioned media responded to recombinant chemerin(17-156) peptide and increased the expression of proinflammatory IL-1β, TNF-α and IL-12 p40 cytokines. The native form of chemerin (prochemerin) supplied by fibroblasts did not induce a functional response. These observations may indicate a potential role for chemerin and CMKLR1 in the regulation of inflammatory responses in the tumor microenvironment.     

Chemerin and its receptors in leukocyte trafficking, inflammation and metabolism

Chemerin was isolated as the natural ligand of the G protein-coupled receptor ChemR23. Chemerin acts as a chemotactic factor for leukocyte populations expressing ChemR23, particularly immature plasmacytoid dendritic cells, but also immature myeloid DCs, macrophages and natural killer cells. Chemerin is expressed by epithelial and non-epithelial cells as an inactive precursor, present at nanomolar concentrations in plasma. Processing of the precursor C-terminus is required for generating bioactive forms of chemerin. Various proteases mediate this processing, including neutrophil serine proteases and proteases from coagulation and fibrinolytic cascades. ChemR23-expressing cells are recruited in human inflammatory diseases, such as psoriasis and lupus. In animal models, both pro-inflammatory and anti-inflammatory roles of chemerin have been reported. Recently, two other receptors for chemerin were described, GPR1 and CCRL2, but their functional relevance is largely unknown. Both chemerin and ChemR23 are also expressed by adipocytes, and the emerging role of chemerin as an adipokine regulating lipid and carbohydrate metabolism is an area of intense research.     

Chemerin, a novel peroxisome proliferator-activated receptor gamma (PPARgamma) target gene that promotes mesenchymal stem cell adipogenesis

Chemerin is an adipocyte-secreted protein that regulates adipogenesis and the metabolic function of mature adipocytes via activation of chemokine-like receptor 1 (CMKLR1). Herein we report the interaction of peroxisome proliferator-activated receptor γ (PPARγ) and chemerin in the context of adipogenesis. Knockdown of chemerin or CMKLR1 expression or antibody neutralization of secreted chemerin protein arrested adipogenic clonal expansion of bone marrow mesenchymal stem cells (BMSCs) by inducing a loss of G(2)/M cyclins (cyclin A2/B2) but not the G(1)/S cyclin D2. Forced expression of PPARγ in BMSCs did not completely rescue this loss of clonal expansion and adipogenesis following chemerin or CMKLR1 knockdown. However, forced expression and/or activation of PPARγ in BMSCs as well as non-adipogenic cell types such as NIH-3T3 embryonic fibroblasts and MCA38 colon carcinoma cells significantly induced chemerin expression and secretion. Sequence analysis revealed a putative PPARγ response element (PPRE) sequence within the chemerin promoter. This PPRE was able to confer PPARγ responsiveness on a heterologous promoter, and mutation of this sequence abolished activation of the chemerin promoter by PPARγ. Chromatin immunoprecipitation confirmed the direct association of PPARγ with this PPRE. Treatment of mice with rosiglitazone elevated chemerin mRNA levels in adipose tissue and bone marrow coincident with an increase in circulating chemerin levels. Together, these findings support a fundamental role for chemerin/CMKLR1 signaling in clonal expansion during adipocyte differentiation as well as a role for PPARγ in regulating chemerin expression.     

Signal Relay by CC Chemokine Receptor 2 (CCR2) and Formylpeptide Receptor 2 (Fpr2) in the Recruitment of Monocyte-derived Dendritic Cells in Allergic Airway Inflammation

Chemoattractant receptors regulate leukocyte accumulation at sites of inflammation. In allergic airway inflammation, although a chemokine receptor CCR2 was implicated in mediating monocyte-derived dendritic cell (DC) recruitment into the lung, we previously also discovered reduced accumulation of DCs in the inflamed lung in mice deficient in formylpeptide receptor Fpr2 (Fpr2^−/−). We therefore investigated the role of Fpr2 in the trafficking of monocyte-derived DCs in allergic airway inflammation in cooperation with CCR2. We report that in allergic airway inflammation, CCR2 mediated the recruitment of monocyte-derived DCs to the perivascular region, and Fpr2 was required for further migration of the cells into the bronchiolar area. We additionally found that the bronchoalveolar lavage liquid from mice with airway inflammation contained both the CCR2 ligand CCL2 and an Fpr2 agonist CRAMP. Furthermore, similar to Fpr2^−/− mice, in the inflamed airway of CRAMP^−/− mice, DC trafficking into the peribronchiolar areas was diminished. Our study demonstrates that the interaction of CCR2 and Fpr2 with their endogenous ligands sequentially mediates the trafficking of DCs within the inflamed lung.     

Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin

Interferon α-producing plasmacytoid dendritic cells (pDC) are crucial contributors to pro-inflammatory or tolerogenic immune responses and are important in autoimmune diseases such as psoriasis. pDC accumulate in the lesional skin of psoriasis patients, but are rarely found in the affected skin of patients with atopic dermatitis (AD). While homeostatic chemokine CXCL12 and inducible pro-inflammatory CXCR3 chemokine ligands may regulate pDC influx to psoriatic skin, the mechanism responsible for selective pDC recruitment in psoriasis vs. AD remains unknown. Circulating pDC from normal donors express a limited number of chemoattractant receptors, including CXCR3 and CMKLR1 (chemokine-like receptor 1). In this work, we demonstrate that circulating pDC from normal donors as well as psoriasis and AD patients express similar levels of CXCR3 and responded similarly in functional migration assays to CXCL10. We next found that blood pDC from normal, AD, and psoriasis patients express functional CMKLR1. In contrast to normal skin, however, lesional skin from psoriasis patients contains the active form of the CMKLR1 ligand chemerin. Furthermore, in affected skin from psoriatic patients the level of active chemerin was generally higher than in AD skin. Taken together, these results indicate that local generation of active chemerin may contribute to pDC recruitment to psoriatic skin.     

Development of a Membrane-anchored Chemerin Receptor Agonist as a Novel Modulator of Allergic Airway Inflammation and Neuropathic Pain

The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gα_i, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149–157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21–157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.     

Calcitonin Gene-Related Peptide Regulates Type IV Hypersensitivity through Dendritic Cell Functions

Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2⁺ DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2⁺ DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2⁻ DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

Comparative modeling of CCRL1, a key protein in masked immune diseases and virtual screening for finding inhibitor of this protein

Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1 and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1- CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1- CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7 causes immune potentiaiton.     

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

The Formylpeptide Receptor 2 (Fpr2) and Its Endogenous Ligand Cathelin-related Antimicrobial Peptide (CRAMP) Promote Dendritic Cell Maturation

Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2^−/−) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2^−/− mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2^−/− mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2^−/− DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP^−/− mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

A Novel CMKLR1 Small Molecule Antagonist Suppresses CNS Autoimmune Inflammatory Disease

Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-¹ (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1⁺ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.     

Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC)

We recently reported the identification of a chemokine (CTACK), which has been renamed CCL27 according to a new systematic chemokine nomenclature. We report that CCL27 binds the previously orphan chemokine receptor GPR-2, as detected by calcium flux and chemotactic responses of GPR-2 transfectants. We renamed this receptor CCR10. Because of the skin-associated expression pattern of CCL27, we focused on the expression of CCL27 and CCR10 in normal skin compared with inflammatory and autoimmune skin diseases. CCL27 is constitutively produced by keratinocytes but can also be induced upon stimulation with TNF-alpha and IL-1beta. CCR10 is not expressed by keratinocytes and is instead expressed by melanocytes, dermal fibroblasts, and dermal microvascular endothelial cells. CCR10 was also detected in T cells as well as in skin-derived Langerhans cells. Taken together, these observations suggest a role for this novel ligand/receptor pair in both skin homeostasis as well as a potential role in inflammatory responses.     

The role of ChemR23 in the induction and resolution of cigarette smoke-induced inflammation

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11b(hi)CD103(-) and CD11b(lo)CD103(+) dendritic cells (DCs), and CD4(+) and CD8(+) T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11b(hi)CD103(-) DCs, and activated CD4(+) T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8(+) T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.     

Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism

Obesity is an alarming primary health problem and is an independent risk factor for type II diabetes, cardiovascular diseases, and hypertension. Although the pathologic mechanisms linking obesity with these co-morbidities are most likely multifactorial, increasing evidence indicates that altered secretion of adipose-derived signaling molecules (adipokines; e.g. adiponectin, leptin, and tumor necrosis factor alpha) and local inflammatory responses are contributing factors. Chemerin (RARRES2 or TIG2) is a recently discovered chemoattractant protein that serves as a ligand for the G protein-coupled receptor CMKLR1 (ChemR23 or DEZ) and has a role in adaptive and innate immunity. Here we show an unexpected, high level expression of chemerin and its cognate receptor CMKLR1 in mouse and human adipocytes. Cultured 3T3-L1 adipocytes secrete chemerin protein, which triggers CMKLR1 signaling in adipocytes and other cell types and stimulates chemotaxis of CMKLR1-expressing cells. Adenoviral small hairpin RNA targeted knockdown of chemerin or CMKLR1 expression impairs differentiation of 3T3-L1 cells into adipocytes, reduces the expression of adipocyte genes involved in glucose and lipid homeostasis, and alters metabolic functions in mature adipocytes. We conclude that chemerin is a novel adipose-derived signaling molecule that regulates adipogenesis and adipocyte metabolism.