Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.     

Neutrophil-mediated maturation of chemerin: a link between innate and adaptive immunity

Dendritic cells and macrophages are professional APCs that play a central role in initiating immune responses, linking innate and adaptive immunity. Chemerin is a novel chemoattractant factor that specifically attracts APCs through its receptor ChemR23. Interestingly, chemerin is secreted as a precursor of low biological activity, prochemerin, which upon proteolytic removal of a C-terminal peptide, is converted into a potent and highly specific agonist of its receptor. Given the fact that APCs are often preceded by polymorphonuclear cells (PMN) in inflammatory infiltrates, we hypothesized that PMN could mediate chemerin generation. We demonstrate here that human degranulated PMNs release proteases that efficiently convert prochemerin into active chemerin. The use of specific protease inhibitors allowed us to identify the neutrophil serine proteases cathepsin G and elastase as responsible for this process. Mass spectrometry analysis of processed prochemerin showed that each protease generates specifically a distinct form of active chemerin, differing in their C terminus and initially identified in human inflammatory fluids. These findings strongly suggest that bioactive chemerin generation takes place during the early stages of inflammation, underscoring the functional contribution of chemerin as a bridge between innate and adaptive immunity.     

Chemerin/ChemR23 axis triggers an inflammatory response in keratinocytes through ROS-sirt1-NF-κB signaling

Psoriasis is a chronic disease which carries the emotional and social burden, promotes joint disability and raises comorbidity possibility in patients. Obesity is closely correlated with the occurrence of psoriasis and adipokines produced by adipose tissues were found to be critical culprits. Chemerin is one of them and its expression was increased in patients with psoriatic arthritis. In our hypothesis, chemerin might act on keratinocytes and promote an inflammatory response, which plays an essential role in psoriatic epidermis. To validate our hypothesis, HaCaT cells and primary human keratinocytes were treated with chemerin (5, 10, and 20 ng/mL for 24 hours). Enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion of inflammatory factors. Nuclear factor-κB (NF-κB) activation and p65 acetylation were evaluated by Western blot analysis. The expression and activity of sirtuin 1 (sirt1), a deacetylase act on p65, were also analyzed. The results showed that chemerin prompted inflammatory factors secretion, NF-κB activation and p65 acetylation through chemerin receptor 23 receptor. Chemerin constrained the expression and deacetylase activity of sirt1 through augment of reactive oxygen species (ROS) production. Additionally, chemerin exacerbated psoriasiform dermatitis in imiquimod-treated mice model. In conclusion, chemerin can seduce inflammatory response and promote NF-κB activation through inhibition of sirt1 activity by ROS production.     

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.     

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 activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades

Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the "rapid responder" leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation.     

Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

Introduction

Chemerin is a chemotactic agonist identified as a ligand for ChemR23 that is expressed on macrophages and dendritic cells (DCs). In this study, we analyzed the expression of chemerin and ChemR23 in the synovium of rheumatoid arthritis (RA) patients and the stimulatory effects of chemerin on fibroblast-like synoviocytes (FLSs) from RA patients.

Methods

Chemerin and ChemR23 expression in the RA synovium was ascertained by immunohistochemistry and Western blot analysis. Chemerin expression on cultured FLSs was analyzed by ELISA. ChemR23 expression on FLSs was determined by immunocytochemistry and Western blot analysis. Cytokine production from FLSs was measured by ELISA. FLS cell motility was evaluated by utilizing a scrape motility assay. We also examined the stimulating effect of chemerin on the phosphorylation of mitogen-activated protein kinase (MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), p38MAPK, c-Jun N-terminal kinase (JNK)1/2 and Akt, as well as on the degradation of regulator of NF-κB (IκBα) in FLSs, by Western blot analysis.

Results

Chemerin was expressed on endothelial cells and synovial lining and sublining cells. ChemR23 was expressed on macrophages, immature DCs and FLSs and a few mature DCs in the RA synovium. Chemerin and ChemR23 were highly expressed in the RA synovium compared with osteoarthritis. Chemerin and ChemR23 were expressed on unstimulated FLSs. TNF-α and IFN-γ upregulated chemerin production. Chemerin enhanced the production of IL-6, chemokine (C-C motif) ligand 2 and matrix metalloproteinase 3 by FLSs, as well as increasing FLS motility. The stimulatory effects of chemerin on FLSs were mediated by activation of ERK1/2, p38MAPK and Akt, but not by JNK1/2. Degradation of IκB in FLSs was not promoted by chemerin stimulation. Inhibition of the ERK1/2, p38MAPK and Akt signaling pathways significantly suppressed chemerin-induced IL-6 production. Moreover, blockade of the p38MAPK and Akt pathways, but not the ERK1/2 pathway, inhibited chemerin-enhanced cell motility.

Conclusions

The interaction of chemerin and ChemR23 may play an important role in the pathogenesis of RA through the activation of FLSs.     

Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

Chemerin is a leukocyte chemoattractant and adipokine with important immune and metabolic roles. Chemerin, secreted in an inactive form prochemerin, undergoes C-terminal proteolytic cleavage to generate active chemerin, a ligand for the chemokine-like receptor-1 (CMKLR1). We previously identified that adipocytes secrete and activate chemerin. Following treatment with the obesity-associated inflammatory mediator TNFα, unknown adipocyte mechanisms are altered resulting in an increased ratio of active to total chemerin production. Based on these findings we hypothesized adipocytes produce proteases capable of modifying chemerin and its ability to activate CMKRL1. 3T3-L1 adipocytes expressed mRNA of immunocyte and fibrinolytic proteases known to activate chemerin in vitro. Following treatment with a general protease inhibitor cocktail (PIC), the TNFα-stimulated increase in apparent active chemerin concentration in adipocyte media was amplified 10-fold, as measured by CMKLR1 activation. When the components of the PIC were investigated individually, aprotinin, a serine protease inhibitor, blocked 90% of the TNFα-associated increase in active chemerin. The serine proteases, elastase and tryptase were elevated in adipocyte media following treatment with TNFα and their targeted neutralization recapitulated the aprotinin-mediated effects. In contrast, bestatin, an aminopeptidase inhibitor, further elevated the TNFα-associated increase in active chemerin. Our results support that adipocytes regulate chemerin by serine protease-mediated activation pathways and aminopeptidase deactivation pathways. Following TNFα treatment, increased elastase and tryptase modify the balance between activation and deactivation, elevating active chemerin concentration in adipocyte media and subsequent CMKLR1 activation.     

NMR assignment of human chemerin, a novel chemoattractant

Chemerin is a potent chemoattractant for cells expressing the GPCR CMKLR1, and is thought to play important roles in cell migration and recruitment to sites of tissue damage and inflammation. Here we report the NMR assignments of the 15.6 kDa active form of uniformly ¹⁵N, ¹³C labeled chemerin.     

The chemerin/ChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages ex vivo

Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/-) mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.     

Chemerin induced by Treponema pallidum predicted membrane protein Tp0965 mediates the activation of endothelial cell via MAPK signaling pathway

Chemerin, a chemoattractant protein, is involved in endothelial dysfunction and vascular inflammation in pathological conditions. In a recent study, we observed the upregulation of chemerin in endothelial cells following in vitro treatment with Treponema pallidum. Here, we investigated the role of chemerin in endothelial cells activation induced by the T. pallidum predicted membrane protein Tp0965. Following stimulation of human umbilical vein endothelial cells (HUVECs) with Tp0965, chemerin and its receptor chemerin receptor 23 (ChemR23) were upregulated, companied with elevated expression of Toll-like receptor 2. Furthermore, chemerin from HUVECs activated endothelial cells via chemerin/ChemR23 signaling in an autocrine/paracrine manner, characterized by upregulated expression of intercellular adhesion molecule 1, E-selectin, and matrix metalloproteinase-2. Activation of endothelial cells depended on the mitogen-activated protein kinase signaling pathway. In addition, Tp0965-induced chemerin promoted THP-1-derived macrophages migration to endothelial cells, also via the chemerin/ChemR23 pathway. The RhoA/ROCK signaling pathway was also involved in THP-1-derived macrophages migration in response to chemerin/ChemR23. Our results highlight the role of Tp0965-induced chemerin in endothelial cells dysfunction, which contributes to the immunopathogenesis of vascular inflammation of syphilis.     

Chemerin158K protein is the dominant chemerin isoform in synovial and cerebrospinal fluids but not in plasma

Chemerin is a chemoattractant involved in immunity that may also function as an adipokine. Chemerin circulates as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C terminus, involving proteases involved in coagulation, fibrinolysis, and inflammation. However, the key proteolytic steps in prochemerin activation in vivo remain to be established. Previously, we have shown that C-terminal cleavage of chem163S by plasmin to chem158K, followed by a carboxypeptidase cleavage, leads to the most active isoform, chem157S. To identify and quantify the in vivo chemerin isoforms in biological specimens, we developed specific ELISAs for chem163S, chem158K, and chem157S, using antibodies raised against peptides from the C terminus of the different chemerin isoforms. We found that the mean plasma concentrations of chem163S, chem158K, and chem157S were 40 ± 7.9, 8.1 ± 2.9, and 0.7 ± 0.8 ng/ml, respectively. The total level of cleaved and noncleaved chemerins in cerebrospinal fluids was ～10% of plasma levels whereas it was elevated ～2-fold in synovial fluids from patients with arthritis. On the other hand, the fraction of cleaved chemerins was much higher in synovial fluid and cerebrospinal fluid samples than in plasma (～75%, 50%, and 18% respectively). Chem158K was the dominant chemerin isoform, and it was not generated by ex vivo processing, indicating that cleavage of prochemerin at position Lys-158, whether by plasmin or another serine protease, represents a major step in prochemerin activation in vivo. Our study provides the first direct evidence that chemerin undergoes extensive proteolytic processing in vivo, underlining the importance of measuring individual isoforms.     

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.     

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH₂-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH₂-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.Chemerin is a recently discovered chemoattractant molecule that is predicted to share structural similarity with cystatins (cysteine protease inhibitors) and cathelicidin precursors (antibacterial peptides) (1). Chemerin is present in circulating blood and several human inflammatory fluids (1). Even though chemerin is not similar to CXC and CC chemokines based on primary amino acid sequence, it functions like a chemokine in that it induces leukocyte migration and intracellular calcium mobilization. Chemerin receptor chemokine-like receptor 1 (CMKLR1,³ also named ChemR23) is a G protein-coupled receptor specifically expressed by circulating human plasmacytoid dendritic cells, natural killer cells, and tissue macrophages (1–5). In their capacity as antigen-presenting cells, plasmacytoid dendritic cells and macrophages can influence the activation of many other cell types, including monocytes, myeloid dendritic cells, B cells, T cells, and natural killer cells; thus chemerin appears to be an important chemoattractant in both innate and adaptive immune responses (2, 6, 7).Chemerin circulates in blood in an inactive prochemerin form at low nanomolar concentrations (∼3 nm) (4). Its chemotactic activity is released following proteolytic cleavage of its carboxyl-terminal amino acids by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades (4, 8). These include factor XIIa, VIIa, plasmin, neutrophil elastase, and mast cell tryptase. Of interest, staphopain B, a cysteine protease secreted by Staphylococcus aureus, also cleaves prochemerin and converts it into a potent chemoattractant (9). Interestingly, the cleavage sites in the labile carboxyl terminus (NH₂-YFPGQFAFSKALPRS-COOH) are not conserved, and the cleavage products generated by chemerin-activating proteases display different potencies in bioactivity assays. Based on synthetic peptides, the 9-mer NH₂-YFPGQFAFS-COOH is the most active, but it is still not as active as intact cleaved chemerin protein, indicating that the amino-terminal part of chemerin is required for maximal activity (4, 10).Plasma carboxypeptidases CPN and CPB cleave the basic amino acids arginine or lysine from the carboxyl terminus of proteins or peptides such as bradykinin and complement proteins C3a and C5a. CPN is a constitutively active zinc metalloprotease present in plasma at a concentration of about 100 nm and is considered the major anaphylatoxins inhibitor (11), generating inactive “desArg” forms of C3a and C5a. In contrast, CPB exists in plasma as a proenzyme, proCPB, or thrombin-activable fibrinolysis inhibitor (TAFI) at a concentration of about 50 nm and is activated by thrombin in complex with thrombomodulin on the vascular endothelial surface. CPB inhibits fibrin degradation by removing carboxyl-terminal lysines from partially digested fibrin, which prevents further incorporation of fibrinolytic plasminogen and tissue plasminogen activator (12, 13). CPB is thermolabile and has a half-life of ∼15 min at 37 °C (14). We have shown that CPB also has broad substrate reactivity and is able to cleave and inactivate bradykinin, C3a, C5a, and thrombin-cleaved osteopontin (15–17). CPN and CPB may play complementary roles, with the former being constitutively active and capable of regulating systemic anaphylatoxins, and the latter activated locally at sites of vascular injury to provide site-specific anti-inflammatory control. Peptidases can also modulate the biological activity of certain chemokines (4). For example, dipeptidyl peptidase (DPP-IV/CD26), a serine protease, inactivates CXCL9, CXCL10, CXCL11, and CXCL12 by cleaving these chemokines in the amino terminus (18, 19).Platelets store a variety of potent cytokines and chemokines within α-granules that are released upon cell activation. Platelet degranulation products, particularly the leukocyte chemoattractants, which include CXCL4 (platelet factor 4), β-thromboglobulin, CCL5 (RANTES), CCL7 (monocyte chemotactic protein 3), and CXCL12 (stromal-derived factor 1), may contribute to host defense and also play a role in pathophysiologic conditions (20, 21). For example, platelet factor 4 forms complexes with heparin in blood or some glycosaminoglycans on platelet surfaces to form the major antigen implicated in heparin-induced thrombocytopenia (22, 23). Platelets not only store CXCL12 but also express its receptor CXCR4, a coreceptor for cellular entry of human immunodeficiency virus, type 1, suggesting that platelets may be involved in host defense (24).In this study, we found that plasma CPN or CPB can function in concert with plasmin to elicit and augment the chemotactic activity of prochemerin. Furthermore, we show that platelets could store and release partially active chemerin upon activation. Thus circulating CPN/CPB and platelets may contribute to regulating the bioactivity of leukocyte chemoattractant chemerin and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.     

The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency

Chemerin is a novel protein identified as the natural ligand of ChemR23 (chemerinR), a previously orphan G protein-coupled receptor expressed in immature dendritic cells and macrophages. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active, but converted into a full agonist of chemerinR by proteolytic removal of the last six amino acids. In the present work, we have synthesized a number of peptides derived from the C-terminal domain of human prochemerin and have investigated their functional properties as agonists or antagonists of human chemerinR. We found that the nonapeptide (149)YFPGQFAFS(157) (chemerin-9), corresponding to the C terminus of processed chemerin, retained most of the activity of the full-size protein, with regard to agonism toward the chemerinR. Extension of this peptide at its N terminus did not increase the activity, whereas further truncations rapidly resulted in inactive compounds. The C-terminal end of the peptide appeared crucial for its activity, as addition of a single amino acid or removal of two amino acids modified the potency by four orders of magnitude. Alanine-scanning mutagenesis identified residues Tyr(149), Phe(150), Gly(152), Phe(154), and Phe(156) as the key positions for chemerinR activation. A modified peptide (YHSFFFPGQFAFS) was synthesized and iodinated, and a radioligand binding assay was established. It was found that the ability of the various peptides to activate the chemerin receptor was strictly correlated with their affinity in the binding assay. These results confirm that a precise C-terminal processing is required for the generation of a chemerinR agonist. The possibility to restrict a medium sized protein to a nonapeptide, while keeping a low nanomolar affinity for its receptor is unusual among G protein-coupled receptors ligands. The identification of these short bioactive peptides will considerably accelerate the pharmacological analysis of chemerin-chemerinR interactions.     

Proteolytic cleavage of chemerin protein is necessary for activation to the active form, Chem157S, which functions as a signaling molecule in glioblastoma

Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ～50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.     

Anti-TNF Therapy Reduces Serum Levels of Chemerin in Rheumatoid Arthritis: A New Mechanism by Which Anti-TNF Might Reduce Inflammation

Background

Chemerin is a specific chemoattractant for macrophages and dendritic cells (DC). In addition, it can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules and is able to activate fibroblast-like synoviocytes (FLS), suggesting a role in the pathogenesis of rheumatoid arthritis (RA). Chemerin is also an adipocytokine that has been related to the inflammatory state of endothelial cells and as such could be involved in the changes in endothelial cells in RA and perhaps increased cardiovascular morbidity. We investigated whether anti-Tumor Necrosis Factor (TNF) treatment affects chemerin levels.

Materials and Methods

49 patients with active RA (disease activity score evaluated in 28 joints (DAS28) ≥3.2) were started on adalimumab therapy. Blood was drawn from patients while fasting at baseline and 16 weeks after initiation of treatment. Chemerin serum levels were measured by ELISA and related to disease activity, mediators of inflammation and known risk factors for cardiovascular disease.

Results

Adalimumab therapy reduced chemerin serum levels, which was correlated with the reduction in DAS28 (r = 0.37, p = 0.009). In addition, the decrease in chemerin serum levels after anti-TNF treatment was associated with the decrease in serum levels of IL-6 (r = 0.39, p = 0.033) and macrophage migration inhibitory factor (MIF) (r = 0.31, p = 0.049). Baseline chemerin serum levels were not related to traditional risk factors for atherosclerosis, except perhaps for smoking (p = 0.07).

Conclusions

This exploratory study shows that adalimumab therapy lowers chemerin levels, which is associated with the reduction in disease activity parameters, and inflammatory mediators IL-6 and MIF. This suggests a possible involvement of chemerin in the migration/retention of macrophages in the synovium.

Trial Registration Nederlands Trial Register

NTR 857     

Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect

Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-κB p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-α (5 ng/ml, 20 min-6 h). Inhibitor of NF-κB or p38 significantly inhibited the TNF-α-induced VCAM-1 expression. Chemerin also inhibited TNF-α-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-α-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-α-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-α-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-α-induced VCAM-1 expression and monocytes adhesion in vascular endothelial cells. The effect is mediated via inhibiting activation of NF-κB and p38 through stimulation of Akt/eNOS signaling and NO production.     

Chemerin及ChemR23与糖尿病大血管病变

Chemerin是2007年新确认的一种脂肪因子,其主要功能受体为ChemR23。近期研究发现chemerin可能是联系肥胖、糖尿病及动脉粥样硬化的潜在因子,有望为糖尿病及其血管并发症的预防及治疗提供新的靶点。然而,chemerin及其受体ChemR23参与糖尿病及其大血管病变的具体机制尚不明确。本文将就目前研究中chemerin及其受体ChemR23与糖尿病及其大血管病变的关系作一综述,并从免疫及炎症反应、氧化应激、自噬、糖脂代谢和胰岛素抵抗等方面,分析chemerin分别对巨噬细胞、血管内皮细胞、脂肪细胞及骨骼肌细胞的影响,从而进一步阐述chemerin及其受体ChemR23参与糖尿病及其大血管病变的具体生物学机制。     

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.