Valérie Wittamer

Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin

Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123+ plasmacytoid DCs and by CD1a+ DC-SIGN+ DCs in the interfollicular T cell area. ChemR23+ DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin+ endothelial cells were surrounded by ChemR23+ plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/chemerin axis in directing plasmacytoid DC trafficking.

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.

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

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.

Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways.

CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophage-tropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of Gi-mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [35S]guanosine 5′-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.

Characterization of the Mononuclear Phagocyte System in Zebrafish

The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation

Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein‐coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy‐terminal peptide. This maturation step is mediated by the neutrophil‐derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil‐derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy‐terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C‐terminus. This effect was shown to be specific to bioactive chemerin (chemerin‐157 and to a lesser extent, chemerin‐156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.

An evolutionarily conserved program of B-cell development and activation in zebrafish

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.