In a Lung Disease Model an Anti-Inflammatory Activity Of

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model This information is current as of September 29, 2021. Souphalone Luangsay, Valérie Wittamer, Benjamin Bondue, Olivier De Henau, Laurie Rouger, Maryse Brait, Jean-Denis Franssen, Patricia de Nadai, François Huaux and Marc Parmentier

J Immunol 2009; 183:6489-6499; Prepublished online 19 Downloaded from October 2009; doi: 10.4049/jimmunol.0901037 http://www.jimmunol.org/content/183/10/6489 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inﬂammatory Activity of Chemerin in a Lung Disease Model1

Souphalone Luangsay,2* Vale´rie Wittamer,2* Benjamin Bondue,2* Olivier De Henau,* Laurie Rouger,* Maryse Brait,† Jean-Denis Franssen,† Patricia de Nadai,* Franc¸ois Huaux,‡ and Marc Parmentier3*

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 Downloaded from 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. Alto- http://www.jimmunol.org/ gether, 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. The Journal of Immunology, 2009, 183: 6489–6499.

uring an inflammatory process, damaged tissues release are the most potent APCs. They are equipped with a set of recep- mediators that contribute to the mounting of immune tors for danger signals, allowing them to adapt their behavior and D responses by regulating the trafficking of leukocyte pop- dictate the outcome of the resulting immune response (3–6). Given ulations. Chemokines and other chemoattractant molecules there- this pivotal position at the intersection of innate and adaptive im- by guest on September 29, 2021 fore play fundamental roles in the physiology of inflammatory munity, DCs are attractive targets for the development of thera- events, as well as in the pathological dysregulations of theses pro- peutic strategies shaping the immune responses in pathological cesses (1, 2). APCs are multifunctional immune effector cells that states such as cancer, inflammatory diseases, and graft rejection act as sentinels, capturing Ags and transporting them to lymphoid (7). Two main categories of DCs have been described, the con- tissues, where they activate naive T cells. Dendritic cells (DCs)4 ventional, or myeloid, DCs (mDCs) and the plasmacytoid DCs (pDCs), which have clearly distinct roles in the initiation of immunity against specific pathogens (8, 9). mDCs include resident *Institut de Recherche Interdisciplinaire en Biologie Humaine et Molećulaire, Uni- immature DCs that are found in most peripheral tissues, including versite´Libre de Bruxelles, Campus Erasme, Brussels, Belgium; †Euroscreen, Gosse- lies, Belgium; and ‡Laboratoire de Toxicologie Industrielle, UniversiteĆatholique de primary and secondary lymphoid organs. In nonlymphoid tissues, Louvain, Brussels, Belgium resident DCs are frequently located in close contact with the mu- Received for publication April 2, 2009. Accepted for publication September 11, 2009. cosal surfaces (respiratory tract, lung, intestine) where they can The costs of publication of this article were defrayed in part by the payment of page directly sample incoming pathogens through the epithelial barrier charges. This article must therefore be hereby marked advertisement in accordance (10). Following maturation, mDCs migrate to the local draining with 18 U.S.C. Section 1734 solely to indicate this fact. lymph nodes. pDCs are recruited directly to lymphoid tissues 1 This work was supported by the Interuniversity Attraction Poles Programme, Bel- through high endothelial venules (11–13). Whereas mDCs display gian State, Belgian Science Policy; the Actions de Recherche Concerteés of the Com- munaute´ Française de Belgique; the European Union (LSHB-CT-2005–518167/ a full battery of chemokine receptors, only a few receptors were INNOCHEM); the Fonds de la Recherche Scientifique Me´dicale of Belgium; the demonstrated to be functional for the recruitment of immature Walloon Region (Programme d’excellence “CIBLES”); The Fonds Ithier; the Fe´de´r- ation Belge contre le Cancer; and the Fondation Me´dicale Reine Elisabeth to pDCs, including CXCR4, CXCR3, and CCR9 (13–17). M.P. B.B. and O.D.H. are aspirants of the Belgian National Fund for Scientific Chemerin is a novel extracellular mediator that we identified as Research. the ligand of ChemR23, a G protein-coupled receptor expressed by 2 These authors contributed equally to this work. immature mDCs and pDCs, NK cells, and macrophages (18–21). 3 Address correspondence and reprint requests to Dr. Marc Parmentier, I.R.I.B.H.M. High amounts of active chemerin were found in various inflam- Universite´Libre de Bruxelles, Campus Erasme, Route de Lennik 808, B-1070, Brus- sels, Belgium. E-mail address: [email protected]. matory situations as in human (rheumatoid arthritis, inflammatory 4 Abbreviations used in this paper: DC, dendritic cell; AM, alveolar macrophage; ascites) and ChemR23-expressing cell recruitment was described BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; BMDC, bone in human inflammatory diseases (21, 22). Chemerin was charac- marrow-derived DC; KO, knockout; mDC, myeloid DC; pDC, plasmacytoid DC; terized as a strong chemoattractant factor acting at subnanomolar WT, wild type. concentrations (18, 22). The protein is secreted as an inactive pre- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 cursor, prochemerin, which is converted into a full agonist of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901037 6490 MOUSE CHEMERIN/CHEMR23 SYSTEM

ChemR23 by proteolytic removal of the last six or seven amino binding assays were normalized for the nonspecific binding (0%) and the acids (23). Extracellular proteases, such as the neutrophil-derived specific binding in the absence of competitor (100%). Binding parameters cathepsin G and elastase, were shown to generate chemerin from were determined with the Prism software using nonlinear regression applied to a one-site competition model. prochemerin, suggesting that the processing takes place at sites of inflammation (24, 25). Indeed, increased chemerin production was Antibodies detected in psoriasis skin (26, 27) and in lupus erythematosus skin mAbs 489C (IgM) and 681 (IgG2ck) directed against the mouse ChemR23 lesions (22), as well as high expression of ChemR23 on pDCs, receptor were obtained following immunization of HsdCpd Wistar Uni- known to play a determinant role in skin inflammatory processes lever rats (Harlan Netherlands) with a peptide corresponding to the second (26, 28). The novel chemerin/ChemR23 system therefore consti- extracellular loop (acetyl-CAPESSPHPAHSQV-amide). tutes an attractive candidate for directing specific DC migration Mouse DC, macrophage, and NK cell preparations under inflammatory conditions. To investigate the role of Mouse BMDCs were generated as previously described (30). Briefly, ChemR23 and its ligand in physiological and pathophysiological BMDCs were recovered by flushing femurs and tibiae and cells were cul- situations, we initiated the study of animal disease models with the tured for 14 days in RPMI 1640 containing L-glutamine (Cambrex) sup- aim of potentially validating ChemR23 as a therapeutic target in plemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 50 ␮M specific diseases. As little information is presently available re- 2-ME (Sigma-Aldrich), 10% heat inactivated FBS (Jacques Boy Institute), and 20 ng/ml mouse recombinant GM-CSF (BioSource International) and garding this system in animal models, we performed a detailed used as immature DCs for subsequent experiments. Spleen DCs (CD11cϩ pharmacological and functional characterization of chemerin and DCs and pDCs) and NK cells were purified using the specific isolation kits its receptor in the mouse and described the distribution of both (Miltenyi Biotec) following the manufacturer’s instructions. The CD11cϩ b proteins at the tissue and cellular levels. Moreover, in a mouse DCs population was analyzed by flow cytometry for the CD11c and I-A Downloaded from model of acute lung injury, we demonstrated that chemerin can act DC markers (BD Pharmingen), NK cells were stained with CD49b (BD Pharmingen) and NKP46/NCR1 (R&D Systems) markers, and pDCs were not only as a strong chemoattractant to APCs but also display fluorescently stained with anti-mPDCA-1-allophycocyanin (Miltenyi Bio- potent and ChemR23-dependent anti-inflammatory properties tec) and anti-Ly-6C-PE (BD Pharmingen). Macrophages were harvested in vivo. from peritoneal exudates 5 days after intraperitoneal injection of 2 ml 4% thioglycolate (Sigma-Aldrich). Macrophages were obtained by adherence after 24 h in culture. The cell population was consistently composed of Materials and Methods Ͼ90% macrophages, as determined by flow cytometry analysis using anti- http://www.jimmunol.org/ Mice CD11b, I-Ab (BD Pharmingen), and F4/80 (Serotec). Eight to 12-wk-old C57BL/6 mice (Harlan Netherlands) were used Chemotaxis throughout these studies. ChemR23-deficient knockout (KO) mice were obtained from Deltagen and backcrossed for 10 generations into the Migration of macrophages and DCs was measured using a 48-well micro- C57BL/6 background in a specific pathogen-free environment. Wild-type chemotaxis Boyden chamber (NeuroProbe) with polycarbonate membranes ␮ (WT) littermates from F1 matings were used as controls for ChemR23 KO (5- m pores; NeuroProbe) as previously described (31). All conditions mice. All procedures were reviewed and approved by the local ethical were tested in triplicate. Controls were performed in the absence of che- committee (Commission d’Ethique du Bien Etre Animal, Universite´Libre moattractant in the lower wells. The results were expressed as migration de Bruxelles). ratio (mean cell number per well with chemoattractant over mean cell number per well without chemoattractant). by guest on September 29, 2021 RT-PCR for mouse chemerin and ChemR23 Immunostaining Total RNA was isolated from mouse organs (Qiagen) and RT-PCR was conducted using primer pairs (Eurogentec) for mouse chemerin (forward: Double immunofluorescence staining on mouse DCs was performed using ␭ 5Ј-GACAGAGCCCGAACTCA-3Ј, reverse: 5Ј-GGACTATCCGGCCTAG anti-CD11c (clone HL3, Armenian hamster IgG1 , dilution 1/50; BD b ␬ AA-3Ј), ChemR23 (forward: 5Ј-CGACTTCCTGTTCAACATCT-3Ј, re- Pharmingen), anti I-A (clone AF6–120.1, mouse IgG2a , dilution 1/30; verse: 5Ј-AAGACGATGGTAAGGTAGCA-3Ј), and mouse GAPDH (for- BD Pharmingen), and anti-mouse ChemR23 (clone 489C, rat IgM, dilution ward: 5Ј-ATGTCGTGGAGTCTACTGGT-3Ј, reverse: 5Ј-GTAGGAACA 1/100; Euroscreen SA) mAbs. Freshly isolated mouse NK cells were CGGAAGGCCAT-3Ј). stained directly on cytospin slides with anti-NKP46/NCR1 (goat IgG, dilution 1/100; R&D Systems) and anti-mouse ChemR23 (clone 489C). Ap- Peptides propriate Texas red-, Cy3-, and FITC-conjugated isotype-specific secondary Abs (Jackson ImmunoResearch Laboratories) were used to reveal the Peptides were synthesized as previously described (23) by solid phase primary Abs. Paraffin-embedded mouse tissues (5 ␮m) were incubated first chemistry using 9-fluorenyl-methoxycarbonyl as protecting group with an with either anti-mouse chemerin (goat IgG, dilution 1/200; R&D Systems) automated Symphony apparatus (Protein Technologies). The synthetic 15- or anti-mouse ChemR23 (clone 489C and 681, rat IgM and IgG2ck re- mer chemerin-derived peptide C15 (141AGEDPHGYFLPGQFA155) (29) spectively, dilution 1/2000 and 1/150 respectively), then with appropriate was purchased from Anygen. peroxidase-conjugated secondary Abs (Jackson ImmunoResearch Labora- tories), and revealed with diaminobenzidine (DakoCytomation). Images Calcium mobilization and binding assays were captured with an Axioplan 2 imaging fluorescence microscope The calcium mobilization response of mouse ChemR23 was analyzed on (Zeiss), equipped with a Diagnostic Spot digital camera and analyzed by CHO-K1 cells stably expressing mouse ChemR23 using an aequorin-based the Spot Advance soft imaging system and Adobe Photoshop 7.0. The lenses used were 20ϫ/0.5, ϱ/0.17; 40ϫ/0.75, ϱ/0.17; and 100ϫ/1.30, calcium mobilization assay as previously described (23). Briefly, cells were ϱ incubated for3hinthedark in DMEM containing 5 ␮M coelenterazine H /0.17. (Molecular Probes). Peptides at different concentrations were plated in 96- LPS-induced acute lung injury model well plates and a cell suspension (5 ϫ 104 cells) was added to the wells in a Packard luminometer. The parameters of the dose-response curves were Mice were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg; determined with the Prism software (GraphPad Software) using nonlinear Pfizer) and xylazine (10 mg/kg; Bayer). After visualization of the trachea’s regression applied to a sigmoidal dose-response model. Mouse bone mar- lumen, acute lung inflammation was induced by transoral instillation into the row-derived DCs (BMDCs) were loaded with 5 ␮M FURA-2 (Molecular trachea of PBS (controls) or 1 ␮gofEscherichia coli LPS (LPS 0111:B4; Probes) and Ca2ϩ mobilization was measured in a fluorescence spectrom- Sigma-Aldrich) in 50 ␮l PBS, containing or not 5 ␮g of mouse recombinant eter LS50B (PerkinElmer) in response to 30 nM mouse chemerin (R&D chemerin (R&D Systems). At selected time points, bronchoalveolar lavages Systems) and 1 mM ATP (Sigma-Aldrich). Competition binding assays (BAL) were obtained by flushing the lungs with sterile 0.9% NaCl and dif- were performed using the 125I-YHSFFFPGQFAFS peptide as tracer (Phoe- ferential cell counts were performed on cytospin preparations after Diff-Quick nix Pharmaceuticals) and variable concentrations of competitors, as de- staining (Dade Behring). Lungs were collected 18 h after LPS instillation for scribed previously (23). Total binding was measured in the absence of histological analysis and preparation of cell suspensions. Briefly, lungs were competitor and nonspecific binding was measured in the presence of a perfused with 10 ml PBS through the right ventricle, dissected, minced, and 100-fold excess of the unlabeled peptide. Data resulting from competition incubated with 2 mg/ml collagenase D and 0.02 mg/ml DNase I (Roche) for The Journal of Immunology 6491

FIGURE 1. Dendograms of ChemR23 and prochemerin from selected vertebrate species. The dendrograms were constructed by using ClustalW for amino acid sequences from a set of mammalian and vertebrate species. The identity scores between human and other species are given for ChemR23 and prochemerin.

1 h at 37°C. After lysis of erythrocytes, cells were stained for neutrophils (CD11bHi Gr-1Hi CD11cNeg) with FITC-CD11b, PerCpCy5.5-Gr-1, and ϩ

allophycocyanin-CD11c; stained for interstitial macrophages (F4/80 Downloaded from CD11bHi I-A/I-EInt CD11cNeg) and alveolar macrophages (F4/80ϩ CD11cHi I-A/I-EInt CD11bNeg/Lo) with FITC- F4/80 (Serotec), PE- I-A/I-E, PerCp-Cy5.5- CD11b, and allophycocyanin-CD11c; and stained for isotype controls (BD Pharmingen). All samples were analyzed using a dual- laser ﬂow cytometer (FACSCalibur) using the CellQuest software (BD Pharmingen). BAL ﬂuids (BALF) were assayed for TNF-␣, IL-6, IL-1␤, and KC/CXCL1 using cytometric bead array-based immunoassays (CBA

flex set; BD Biosciences), a dual-laser flow cytometer (FACSCalibur; BD http://www.jimmunol.org/ Biosciences) and the FCAP array software (BD Biosciences) for analysis, following the manufacturer’s instructions. Histology was obtained after lung insufflations with 1 ml of 4% paraformaldehyde and embedded in paraffin. Lung sections (5 ␮m) were stained with H&E and assessed by light microscopy. Statistical analysis Analysis of differences in BALF cytokines and differential cell counts was performed by one-way ANOVA using the Prism 4 software. The Student-

Newman-Keuls test was used for pairwise comparisons. For all tests, p Ͻ by guest on September 29, 2021 0.05 was considered as significant. FIGURE 2. Tissue distribution of mouse ChemR23 and prochemerin. Results A, ChemR23 and prochemerin transcripts were amplified by RT-PCR from The chemerin/ChemR23 system is well conserved in vertebrate a set of mouse tissues. The data are representative of three independent species experiments. MLN, Mesenteric lymph nodes; PP, Peyer’s patches. B, Im- The characterization of mouse chemerin and its receptor ChemR23 munohistochemical detection of chemerin in mouse ileum, lung bronchi- ϫ ϫ constitutes a necessary step to study the biological functions of this oles, and skin. Original magnifications are 400 on top row and 1000 on bottom row. C, Immunohistochemical detection of ChemR23 in the ileum new leukocyte chemoattractant system. Human and mouse of WT and ChemR23 KO mice, using the rat mAb 681C. Original mag- ChemR23 share 80% identity at the amino acid level. The mouse nifications are ϫ200 (inset, ϫ1000). ChemR23 gene, also called mcmklr1 or Dez, is localized on chromosome 5. The cDNA was originally cloned from a brain library (32), and the genomic organization of the gene was previously ment includes the splice acceptor site, the start codon, and the reported (33, 34). Prochemerin (Ensembl ID ENS- sequence encoding the first two transmembrane segments of the 5 MUSG00000009281) and ChemR23 (Ensembl ID ENS- receptor (supplemental data 1). The ChemR23 KO mice did not MUSG00000042190) amino acid sequences from vertebrate spe- display differences with their WT littermates in terms of breeding, cies were aligned with ClustalW and a dendrogram was growth, and survival in a specific pathogen-free environment. No constructed (Fig. 1). Orthologs of chemerin and ChemR23 were major changes in spleen and blood leukocyte populations were unambiguously found in primate, mammalian, bird, and fish spe- observed in physiological conditions (T and B cells, macrophages, cies. The six cysteines presumably involved in disulfide bridging DCs, NK, granulocytes; supplemental data 1). were strictly conserved in all species. In addition, the C-terminal Tissue and cell distribution of mouse chemerin and ChemR23 nonapeptide of the mature protein, previously shown to be important for the biological activity of human chemerin, was highly The expression of the mouse genes was determined and compared conserved (YFPGQFAFS) in all mammalian species. This sug- with what is known in human. By RT-PCR, preprochemerin tran- gests that the interaction with the cognate receptor and the mech- scripts were detected in most tissues tested (Fig. 2A). ChemR23 anisms allowing generation of bioactive chemerin are well con- transcripts were found at high levels in skin, lung, colon, ovary, served across species. testis, spleen, and mesenteric lymph nodes, and were detected in other tissues as well. This broad distribution parallels what has Phenotype of ChemR23 KO mice been described in human. The ChemR23 KO mice, in which part of the third exon is replaced by a Neo cassette, were obtained from Deltagen. The deleted seg- 5 The online version of this article contains supplemental material. 6492 MOUSE CHEMERIN/CHEMR23 SYSTEM

FIGURE 3. Expression of mouse ChemR23 by DCs and NK cells. Im- munofluorescence detection of ChemR23 on mouse CD11cϩ (A) and CD49bϩ cells (B) purified from spleen. DCs from WT mice were labeled for ChemR23 (489C monoclonal, red) and either MHCII or CD11c (green). NK cells from WT or ChemR23 KO mice were labeled for ChemR23 (489C monoclonal, green) and NKP46 (red). Original magnifications are ϫ400 except inset (ϫ1000). Downloaded from C, FACS analysis of ChemR23 expression on CD11cϪ and CD11cϩ cell populations prepared from mouse spleen by magnetic cell sorting. D, FACS analysis of ChemR23 on mature (MCHIIHi) and immature (MCHIILo) BMDCs from WT (left http://www.jimmunol.org/ panels) and ChemR23 KO mice (right panels). The cells were costained with anti-CD11c, anti-I-Ab, and anti- ChemR23 (489C) monoclonals. E, FACS analysis of ChemR23 expression on a pDC-enriched population prepared from mouse spleen. ChemR23 staining is seen on ϩ ϩ

mPDCA CD11c cells (pDCs). The by guest on September 29, 2021 displayed data are representative of at least three independent experiments.

To identify more precisely which cell populations express DC populations were either purified directly from spleen by chemerin in the various tissues, we tested its distribution by im- magnetic bead sorting, or were generated from BMDCs (30, 35). munohistochemistry, focusing on a set of tissues playing key roles Double immunofluorescent staining was performed on cryosec- in immune defense mechanisms and frequently involved in inflam- tions of spleen CD11cϩ cells, demonstrating coexpression of matory diseases. Similar results were obtained with a rat mAb ChemR23 and the DC markers MHC class II (I-Ab) and CD11c (R&D Systems) and two goat polyclonal Abs (R&D Systems and (Fig. 3A). FACS analysis confirmed ChemR23 expression in the Santa Cruz Biotechnology). As shown in Fig. 2B, staining for majority of CD11cϩ cells, although two distinct peaks chemerin was obtained in the epithelial cell layer of the small (ChemR23Lo and ChemR23Hi) were seen (Fig. 3C). A small frac- intestine; the ciliated epithelium of the lung bronchioles; and the tion (Ͻ 10%) of the CD11cϪ cells was also weakly positive for granular keratinocyte layer, external root sheath, and sebaceous ChemR23 (Fig. 3C). These cells could correspond to macrophages glands in the skin. and NK cells previously shown to be ChemR23ϩ in human (18, We also generated rat mAbs directed against mouse ChemR23. 21, 22). The monoclonals 489C and 681 were validated by FACS and im- BMDCs were prepared from WT and ChemR23 KO mice, and munohistochemistry using mouse ChemR23-expressing CHO-K1 tested by FACS for a number of DC and maturation state markers cells, and further used to study the distribution of the receptor on (CD11c, CD11b, I-Ab, CD40, CD86). No difference was seen be- tissue sections and in leukocyte populations. The specificity was tween the two groups in terms of number of BMDCs recovered checked by using samples originating from ChemR23 KO mice as and maturation states (data not shown). After 14 days of culture, a controls. In the small intestine, scattered positive cells were found majority of immature DCs (75%, CD11cϩ MHCIILo) was recov- in the lamina propria of the villi (Fig. 2C) using the two mono- ered, of which ϳ40% coexpressed ChemR23 at a high level (Fig. clonals. No staining was obtained for ChemR23 KO mice. 3D). The CD11cϩ MHCIIHi population of mature dendritic cells The Journal of Immunology 6493

Table I. Nomenclature of peptides and parameters of their binding and functional properties on mouse ChemR23a

Functional Parameters

Peptide Nomenclature Peptide Sequence pEC50 pIC50 Rec m-chemerin 1–157 9.34 Ϯ 0.06 9.63 Ϯ 0.11 m-prochemerin 138–163 IAQAGEDPHGYFLPGQFAFSRALRTK Յ5 Յ6 m-prochemerin 138–157 IAQAGEDPHGYFLPGQFAFS 7.71 Ϯ 0.06 7.83 Ϯ 0.05 m-prochemerin 138–158 IAQAGEDPHGYFLPGQFAFSR Յ5 Յ6 m-prochemerin 150–157 LPGQFAFS 7.11 Ϯ 0.03 7.18 Ϯ 0.07 m-prochemerin 149–157 FLPGQFAFS 8.83 Ϯ 0.07 8.11 Ϯ 0.03 m-prochemerin 148–157 YFLPGQFAFS 8.50 Ϯ 0.12 8.27 Ϯ 0.08 m-prochemerin 147–157 GYFLPGQFAFS 8.53 Ϯ 0.07 8.42 Ϯ 0.15 m-prochemerin 146–157 HGYFLPGQFAFS 8.33 Ϯ 0.01 8.41 Ϯ 027 m-prochemerin 141–155 (C15) AGEDPHGYFLPGQFA Յ5 Յ6

a The sequence of all peptides tested in the present study is displayed, as well as the functional (pEC50) and binding (pIC50) parameters of their activity on mouse ChemR23-expressing CHO-K1 cells. Data are given as mean Ϯ SEM and were calculated from at least three independent experiments. was essentially negative for ChemR23. As expected, no ChemR23 Ca2ϩ was observed in response to chemerin (Fig. 4D). We next staining was seen on BMDCs derived from ChemR23 KO mice tested the chemotactic properties of chemerin on mouse APCs. As Downloaded from (Fig. 3D). shown in Fig. 4, E and F, mouse chemerin promoted a dose-de- We further purified pDCs from the population of mouse spleen pendent migration of immature BMDCs and macrophages, with a DCs and used CD11c, Ly-6C, and mPDCA as pDC markers. typical bell-shaped curve culminating for concentrations of 3 and FACS costaining demonstrated that the whole population of 1 nM, respectively. The migration index was significantly higher mPDCAϩ cells (pDCs) expressed ChemR23 at high level on their for BMDCs than for macrophages. The efficacy of chemerin in ϩ surface (Fig. 3E). It appears therefore that the ChemR23 popu- promoting BMDC migration was higher than that of CCL5 (CCR1 http://www.jimmunol.org/ lation corresponds to the pDC subset. As in human, ChemR23 is and CCR5 ligand, peak at 10 nM) and CXCL12 (CXCR4 ligand, more highly expressed on pDCs than on mDCs or macrophages. peak at 10 nM; supplemental data 3), as observed previously in Only a fraction of macrophages (CD11bϩ F4/80ϩ) differenti- human (22). As expected, BMDCs and macrophages prepared ated from bone marrow were positive for ChemR23 (ϳ10%) in from ChemR23 KO mice failed to migrate in response to FACS, while relatively weak staining was obtained for purified chemerin, while the response to CCL5 and CXCL12 was kept NK cells (CD49bϩ) from WT but not from KO mice (supplemen- (supplemental data 3). Altogether, these data demonstrate that tal data 2). Immunofluorescence staining confirmed expression of chemerin is a potent and efficient chemoattractant factor for im- ChemR23 in purified NK cells (Fig. 3B). The staining was mostly mature DCs and macrophages, and that this effect is exclusively cytoplasmic, which is not unusual for chemoattractant receptors mediated by ChemR23. We also tested the influence of chemerin by guest on September 29, 2021 for which intracellular pools are often seen in the endosomal onto the maturation of DCs by analyzing maturation markers by compartments. FACS. It was found that chemerin does not promote DC maturation in vitro, nor prevents the maturation initiated by LPS (data not Biological activity of mouse chemerin shown). The pharmacological properties of the mouse chemerin/ChemR23 system were evaluated using CHO-K1 cell lines coexpressing Pharmacological characterization of synthetic peptides derived from the chemerin C terminus mouse ChemR23 and apoaequorin (23). The pIC50 and pEC50 values derived from binding and calcium mobilization assays are dis- Previous studies with the human chemerin/ChemR23 system dem- played in Table I. Competition binding assays were performed onstrated that accurate proteolytic processing of the precursor using the 125I-YHSFFFPGQFAFS peptide as tracer (Fig. 4A). Re- prochemerin is required for the generation of bioactive chemerin. combinant mouse chemerin was shown to bind ChemR23 with This processing affects the C-terminal part of the protein and only Ϯ ϭ high affinity (IC50 of 0.25 0.05 nM, n 3). The activation of two C-terminal variants, lacking the last six or seven amino acids ChemR23 was monitored using the aequorin-based calcium mo- of the precursor, are able to exert biological activity on ChemR23 bilization assay as previously described (23). Recombinant mouse (24). To investigate whether this precise activation mechanism is chemerin activated the receptor with low nanomolar potency conserved between the human and mouse species, we compared Ϯ (EC50 of 0.4 0.06 nM; Fig. 4B). No calcium response of re- the activity of different recombinant C-terminal variants of mouse combinant chemerin was observed on WT CHO-K1 cells (not chemerin on ChemR23-expressing cells. The conditioned medium shown). We also tested human recombinant chemerin on mouse of CHO-K1 cells expressing mouse mature chemerin (form 1–157, ChemR23 and mouse chemerin on human ChemR23, both in bind- lacking the last six amino acids of prochemerin) activated ing and functional assays. No significant differences were obtained ChemR23 in the aequorin assay, whereas a variant lacking two in terms of IC50 and EC50 (not shown), suggesting a strong con- additional amino acids (form 1–155) was inactive (supplemental servation of the ligand-receptor interface between human and data 4). However, Western blotting demonstrated that the two re- mouse. combinant chemerin variants were present in similar amounts in We further evaluated the biological activity of chemerin on the conditioned medium, confirming the weak bioactivity of mouse DCs and macrophages, the two major APC populations chemerin 1–155. These observations strongly suggest that, as expressing ChemR23. As displayed in Fig. 4C, recombinant mouse shown for human chemerin, accurate C-terminal processing of the chemerin (30 nM) was able to promote a strong intracellular Ca2ϩ precursor is essential for the regulation of the bioactivity of mouse response in immature BMDCs generated from WT animals. In chemerin. contrast, while BMDCs generated from ChemR23-deficient mice We have previously demonstrated that human chemerin could responded similarly to ATP (1 ␮M), no increase of intracellular be trimmed-down to its C-terminal nonapeptide (chemerin 6494 MOUSE CHEMERIN/CHEMR23 SYSTEM

FIGURE 4. Pharmacological and functional characterization of the mouse chemerin/ChemR23 system. A, Competition binding assay on mouse ChemR23-expressing CHO-K1 cells, using the 125I-YHSFFFPGQFAFS peptide as tracer and recombinant mouse chemerin (rec m-chemerin) as competitor. B, Functional response of mouse ChemR23-expressing CHO-K1 cells to recombinant mouse chemerin in an aequorin-based intracellular Ca2ϩ mobili- Downloaded from zation assay. The curves represent the mean Ϯ SEM of triplicate data points. C and D, Recording of Ca2ϩ ﬂux in immature BMDCs from WT (C) and ChemR23 KO mice (D) in response to 30 nM mouse recombinant chemerin

and 1 mM ATP. E and F, Chemotaxis http://www.jimmunol.org/ of BMDCs (E) and macrophages (F)to mouse recombinant chemerin. The displayed data are representative of at least three independent experiments. by guest on September 29, 2021

149–157, YFPGQFAFS, also called chemerin-9) while keeping a A second set of peptides was synthesized to explore the effect of low nanomolar affinity for its receptor (18). Such synthetic N-terminal truncations, starting from the active m-chemerin 138– chemerin-derived peptides have been used to characterize the 157 peptide. As shown in Fig. 5B and Table I, shortening the pharmacology of the human chemerin/ChemR23 system and to peptide to nine amino acids (149–157) slightly increased the po- identify key residues contributing to the biological activity of Ϯ tency (EC50 of 4.8 0.8 nM). However, further removal of chemerin. With the aim of investigating the pharmacological prop- Phe149, resulting in the octapeptide m-chemerin 150–157, de- erties of the mouse counterpart, we have tested synthetic peptides Ϯ creased the potency by one order of magnitude (EC50 of 78 6 derived from the C-terminal end of the mouse chemerin. All the nM). In conclusion, these results identified the nonapeptide peptides started after the last cysteine presumably involved in di- 149FLPGQFAFS157 as the shortest C-terminal peptide derived sulfide bonding. We synthesized peptides corresponding to the in- from mouse chemerin retaining high potency on ChemR23. active mouse prochemerin (m-prochemerin 138–163), the active The binding properties of these peptides were next investigated mouse chemerin (m-chemerin 138–157), a 21-amino acid peptide in the competition binding assay (18). The pIC values derived with one additional amino acid (m-chemerin 138–158), and m- 50 from the binding curves are displayed in Table I for the peptides chemerin 141–155 corresponding to the synthetic 15-mer tested in this assay. As shown in Fig. 5C, the bioactive m-chemerin chemerin-derived peptide C15 used by Cash et al (29) (Table I). 138–157 peptide inhibited binding of the tracer with an IC of These peptides were tested for their ability to trigger intracellular 50 Ϯ calcium release through mouse ChemR23, using the aequorin- 14.8 1.7 nM, whereas the functionally inactive peptides based assay. As shown in Fig. 5A and Table I, m-prochemerin m-prochemerin 138–163 and m-chemerin 138–158 did not signif- ␮ 138–163, m-chemerin 138–158, and m-chemerin 141–155 (29) icantly compete with the tracer up to concentrations of 1 M. The were devoid of biological activity up to the concentration of 1 ␮M. whole set of N-terminally truncated peptides (Table I) was also In contrast, m-chemerin 138–157 activated ChemR23 with high tested in the binding assay (Fig. 5D). The nonapeptide m-chemerin Ϯ 149–157, the shortest active peptide, bound the receptor with an potency (EC50 of 19.7 2.9 nM). These observations demonstrate that generation of active mouse chemerin also requires a C-termi- apparent affinity of 7.8 Ϯ 0.6 nM. In contrast, the shorter peptide nal proteolytic cleavage of the precursor and highlight the required m-chemerin 150–157 had a 10-fold lower affinity, indicating that accuracy of the processing, as a single additional residue abolishes the loss of bioactivity is secondary to a decrease in affinity for the biological activity of the peptide. ChemR23. The Journal of Immunology 6495 Downloaded from

FIGURE 5. Structure-function analysis of mouse chemerin and its C-terminal peptides. A, The biological activity of mouse recombinant mature chemerin (rec m-chemerin; ‚), the 26-amino acid C-terminal peptide of mouse prochemerin (m-prochemerin 138–163; छ), the corresponding 20-amino acid C-terminal peptide of processed mouse chemerin (m-chemerin 138–157; F), a 21-amino acid peptide corresponding to active chemerin with one additional amino acid (m-chemerin 138–158; Ⅺ), and a 15-amino acid peptide corresponding to position 141–155 as previously described (Ref. 29; m-chemerin 141–155; E) were measured on mouse ChemR23-expressing CHO-K1 cells using the aequorin-based Ca2ϩ release assay. B, Biological activity in the same http://www.jimmunol.org/ assay for the chemerin-derived peptides corresponding to positions 150–157 (f), 149–157 (Ⅺ), 148–157 (F), 147–157 (E) and 146–157 (छ). C and D, Competition binding assay for the protein and peptides tested in A and B, using a ChemR23-expressing CHO-K1 cell line and 125I-YHSFFFPGQFAFS as tracer. The curves represent the mean Ϯ SEM of triplicate data points and are representative of at least three independent experiments.

Anti-inflammatory role of chemerin in an LPS-induced mouse BALFs and digested lungs from WT and ChemR23 KO mice 18 h model of acute lung injury after the LPS challenge. LPS induced a stronger inflammatory re- To determine in vivo the physiological role of chemerin, we next sponse in ChemR23 KO mice as compared with WT mice, with an investigated whether chemerin is able to regulate inflammation increase of neutrophils in BALFs and lung cell suspensions (Fig. by guest on September 29, 2021 in a mouse acute lung injury model. LPS mimics the symptoms 6B). Moreover, in WT mice receiving LPS plus chemerin, neutro- of acute lung injury, which is characterized by the accumulation phils were decreased significantly by 70% (BALF) and 50% (lung in the lungs of neutrophils producing inflammatory mediators. tissue) as compared with mice receiving LPS only. ChemR23 KO We used whole chemerin to investigate the consequences of mice did not respond to chemerin and displayed similarly high ChemR23 activation as synthetic chemerin-derived peptides neutrophil levels (Fig. 6B). Both WT and ChemR23-deficient mice such as the human nonapeptide (149YFPGQFAFS157) were showed increased macrophage levels in response to LPS, but mac- shown to have a short-lived and poor activity in cell cultures rophage recruitment was higher in BALFs and total lungs from KO (i.e., chemotactic) due to their rapid degradation by proteases. mice as compared with WT mice. FACS analysis revealed that Mice were injected transorally with LPS alone (1 ␮g) or in F4/80ϩ CD11bϩ interstitial macrophages and, to a lower extent, combination with a single dose of recombinant chemerin (5 F4/80ϩ CD11cϩ alveolar macrophages (AM) increased after LPS ␮ g). Cell counts and cytokine levels were measured in BAL and treatment in WT and KO mice (supplemental data 5). KO mice histological analyses and leukocyte typing by flow cytometry showed a significantly lower proportion of AM as compared with following tissue digestion were performed on lung at various WT mice (15% vs 27%, supplemental data 5), likely resulting from time points. As shown in Fig. 6A, histological examination of the relative dilution of resident macrophages by newly recruited lung tissues revealed a marked inflammation at 18 h in LPS- cells. No effect of chemerin was observed on the recruitment of treated mice, with hyperplasia of the bronchiolar epithelium, total lung macrophages in WT or KO mice (Fig. 6B), but in LPS- and peribronchiolar, perivascular, and alveolar infiltration by treated WT mice, chemerin increased the macrophage number in neutrophils. Such infiltrate was not found in control animals BALFs, whereas no effect was seen on mice deficient for receiving PBS and coadministration of chemerin with the LPS ChemR23. challenge resulted in a decrease in pathological signs and cell infiltration (Fig. 6A). Neutrophils were counted in the BAL at 6, We also measured a set of inflammatory mediators in BALFs of 12, 24, and 72h. A peak of neutrophils was reached between 12 WT and KO mice as hallmarks of the lung inflammatory response. ␣ ␤ and 24 h after LPS (Fig. 6A). Chemerin decreased neutrophil The results for KC/CXCL1, IL-6, TNF- , and IL-1 are displayed counts in BAL by 70% 12 h after the challenge, and this effect in Fig. 6C. PBS or chemerin alone did not promote any change in persisted at 24 (53% inhibition) and 72 h (63% inhibition) cytokine levels over baseline, whereas LPS treatment resulted in a within the resolution phase, during which the neutrophil counts strong increase of these mediators. The increase of all four cyto- decreased progressively in all groups. kines was much milder in the group of WT mice receiving To confirm these observations and determine whether the anti- chemerin in addition to LPS (78% reduction for KC/CXCL1, IL-6, inflammatory effects of chemerin are mediated through ChemR23, and IL-1␤, 73% for TNF-␣). In ChemR23 KO mice, chemerin the leukocyte populations recruited to lung were analyzed in both treatment had no effect on inflammatory cytokines and chemokines 6496 MOUSE CHEMERIN/CHEMR23 SYSTEM

FIGURE 6. Anti-inflammatory effect of chemerin in an LPS-induced acute lung injury model. Mice received an intratracheal instillation of PBS (solvent), LPS (1 ␮g), chemerin (5 ␮g), or LPS (1 ␮g) and chemerin (5 ␮g). A, Histological examination (H&E staining) of lung tissues 18 h after instillation of PBS (a), LPS (b), or LPS and chemerin (c). Original magnifications are ϫ50 (inset: ϫ400). LPS-induced lung inflammation is

characterized by a perivascular in- Downloaded from ﬂammation with marked edema (black arrow), and an alveolar and peribronchiolar inﬁltrate rich in neutrophils (white arrow). d, Effect of chemerin coadministration on the neutrophil count in BAL performed at

different time points in WT mice after http://www.jimmunol.org/ LPS challenge. B, Effect of chemerin on neutrophil and macrophage counts in BALFs and lung cell suspensions in WT (gray) and ChemR23 KO mice (black) 18 h after LPS and/or chemerin challenge. C, Chemokine (KC/CXCL1) and cytokine (IL-1␤, IL-6, TNF-␣) levels were determined in BALFs 18 h after LPS and/or chemerin challenge in WT and by guest on September 29, 2021 ChemR23 KO mice. The data shown are the mean Ϯ SEM for groups of ten ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .animals .p Ͻ 0.001 ,ءءء

in BAL. Altogether, these data clearly demonstrate that chemerin precursor is permanently available and that the activation of di- can exert an anti-inflammatory activity, which is strictly dependent verse proteolytic systems results in the local generation of active on ChemR23. chemerin able to recruit pDCs, mDCs, macrophages, and NK cells (24, 25, 40). However, how similar the human system is to the Discussion mouse system in terms of pharmacology, precursor processing, Chemerin was identified as the natural ligand of the previously and distribution of ChemR23 among leukocyte populations is in- orphan ChemR23 receptor (18). No other ligand has so far been completely known. described for ChemR23, with the exception of resolvin E1 (36, We first analyzed the conservation of chemerin and ChemR23 in 37), which in our study did not act on ChemR23 either in func- mice and other species. Both the receptor and ligand are relatively tional or in binding assays (unpublished data). GPR1 was de- well conserved between mammalian species (respectively 59% scribed recently as an additional receptor for chemerin (38). GPR1 and 80% identity for chemerin and ChemR23 between human and expression was previously found in AM (39) but was not reported mouse). Importantly, the C-terminal end of chemerin, which is in other leukocyte populations so far and the signaling cascades essential for the activation of ChemR23, is particularly well con- activated by GPR1 remain elusive. served in mammalian species. It appears therefore that the The biologically inactive precursor of chemerin, prochemerin, is chemerin-ChemR23 system has been well conserved in evolution, expressed by most tissues in humans and is present at nanomolar suggesting important and stable functions all along the vertebrate concentrations in plasma. It appears therefore that a large pool of lineage. The Journal of Immunology 6497

We have studied the pharmacology of the mouse chemerin- between two opposite effects: on one side, the direct recruitment of ChemR23 system in vitro and have tested whether the bioactivity macrophages by chemerin, on the other, a reduction of macro- of chemerin was dependent, as in human, on the precise proteolytic phage recruitment by other factors as a result of the anti- maturation of its precursor. The affinity of the mouse receptor for inflammatory effects of chemerin. mouse chemerin was similar to that of the human receptor (around Cash et al (29) recently reported an anti-inflammatory activity of 1 nM). The generation of the most active chemerin form requires chemerin and chemerin-derived peptides. Despite the similar con- the proteolytic removal of the last six or seven amino acids from clusions reached by their and our present studies, there are signif- the mouse C terminus prochemerin. This is strictly parallel to what icant differences. Cash et al describe that mouse chemerin and a we observed previously for the human system (23). Moreover, the synthetic 15-mer chemerin-derived peptide (C15, 141AGEDPHG shortest peptide retaining similar bioactivity of full-size chemerin YFLPGQFA155) inhibit macrophage activation in vitro at picomo- is a C terminus nonapeptide of mature chemerin (149FLPGQFAF lar concentrations. The C15 peptide (but not full-sized chemerin) S157), which differs slightly from the equivalent human peptide was also reported by Cash et al to display in vivo anti- (149YFPGQFAFS157). inflammatory properties in a mouse acute peritonitis model (29). Using RT-PCR, immunohistochemistry, and FACS, we ana- These data are difficult to reconcile with our own results because lyzed the expression of preprochemerin and ChemR23 in a set of the C15 peptide lacks the amino acid F156, which is essential for mouse tissues and leukocyte populations. As described previously the activity of chemerin-derived peptides on ChemR23, both in for human systems (18), preprochemerin transcripts can be found human and mouse (23, 24, 31). We have tested the functional in all tissues. The protein was also detected at high levels by im- activity of the C15 peptide in an aequorin-based calcium release munohistochemistry in a number of epithelial cell types, including assay on CHO-K1 cells expressing mouse ChemR23, but could not Downloaded from keratinocytes, enterocytes, and bronchiolar epithelial cells. detect any activity of this peptide, while recombinant chemerin and ChemR23 transcripts were found at variable levels in all tissues, other peptides were fully active. Moreover, the very low concentra- but most of these signals might be attributed to the presence of tions (10Ϫ12–10Ϫ10 M range) of chemerin and C15 peptide tested by resident DCs, macrophages, and NK cells. Cash et al differ considerably from all previous reports showing that The functionality of ChemR23 in macrophages and immature chemerin and its C-terminal peptides are active on ChemR23 in the Ϫ10 Ϫ8 Ϫ9 Ϫ7 DCs was demonstrated by measuring calcium mobilization and 10 –10 M and 10 –10 ranges, respectively (18, 21, 22, 41). http://www.jimmunol.org/ chemotaxis. These data are consistent with the observations made We also tested the activity of recombinant chemerin on LPS-treated previously in human models (18, 22). It was, however, reported DCs and peritoneal and lung macrophages, and could not find, besides that mouse ChemR23 was not present on mouse mDCs and pDCs their chemotactic response, any positive or negative effect on activa- (40, 41). These observations are presumably the result of the poor tion and maturation of these cells in terms of cytokine production or sensitivity of the Abs used in these studies, as in our study, chemokine receptor expression (supplemental data 6). Also, in con- ChemR23 was both present and functional in these cells. More- trast to our study, Cash et al did not observe a difference between WT over, the specificity of the labeling and the involvement of and KO mice in terms of neutrophil and monocyte infiltration in their ChemR23 in the chemerin-mediated chemotaxis were clearly dem- mouse acute peritonitis model (29), whereas an increased inflamma- onstrated for both cell populations by the lack of labeling and tory response was seen in our model, suggesting a role of endog- by guest on September 29, 2021 migration when the cells were prepared from ChemR23 enously generated chemerin in the lung inflammatory model. knock-out mice. Besides the anti-inflammatory function in lung homeostasis, The expression of chemerin in bronchiolar and other epithelial other functions of the chemerin-ChemR23 system must be consid- cells lining mucosal barriers and the presence of ChemR23ϩ cells ered. Overexpression of chemerin and recruitment of ChemR23ϩ in the stroma of the same tissues suggest a role for this system in cells were described in human skin diseases and chemerin was the regulation of defense mechanisms against pathogens or other proposed to recruit immature NK cells and favor their interaction types of tissue aggression. In this context, chemerin instillation in with DCs (21, 22, 26). The chemerin/ChemR23 system might LPS-treated mice increased the mobilization of airway macro- therefore play a role in the bidirectional crosstalk between NK phages in BALFs and inhibited neutrophil recruitment and the re- cells and DCs and potentially influence the polarity of the immune lease of inflammatory cytokines (IL-6, TNF-␣, IL-1␤) and che- responses (52–57). The presence of chemerin on high endothelial mokines (KC/CXCL1). These effects were strictly dependent on venules in human lymph nodes and tonsils suggests a role in the ChemR23, as KO mice did not respond to chemerin administra- recruitment of cell populations to secondary lymphoid organs (22). tion. Furthermore, ChemR23-deficient mice exhibited increased Also, expression of both the ligand and receptor in human adipo- neutrophil and macrophage counts in BALFs and lung tissue after cytes has suggested a role of chemerin as an autocrine/paracrine LPS challenge, as compared with control mice, and their inflam- adipokine regulating adipogenesis and lipolysis (58–60). matory response was unaffected by chemerin. Mobilization of In conclusion, the mouse chemerin-ChemR23 system appears macrophages in BAL was increased in WT mice receiving very well conserved between human and mouse and, based on the chemerin in addition to LPS. As macrophages express ChemR23, available sequence data, likely in other mammalian species as we suggest that the recruitment of macrophages by either endog- well. The pharmacology of the receptor is very similar in the two enous or exogenous chemerin plays a role in the modulation of species, as well as the C-terminal determinants of the ligand re- lung inflammation. Indeed, AMs are known regulators of local quired for bioactivity. Mouse ChemR23 is expressed in the same immunological homeostasis, and their immunosuppressive activi- leukocyte populations as in humans, namely immature pDCs and ties have been reported in several studies (42–45), including mDCs, macrophages and NK cells, and the ligand precursor dis- down-regulation of local T cell function (46) and Ag presentation plays the same broad distribution. We have in the present work capacity by DCs (44). The mechanism underlying their suppres- demonstrated the expression of mouse chemerin in epithelial bar- sive activity may be explained in part by their ability to produce riers and provided the demonstration of its anti-inflammatory role NO (47, 48) and prostaglandin E2, which contribute to the down- in vivo, through the ChemR23 receptor. Mouse appears therefore regulation of cell-mediated immunity during lung inflammation as an excellent model to help understand the physiological role of (49–51). However, macrophage counts in the lung of WT mice chemerin and ChemR23 in immune and inflammatory responses, were not affected by chemerin administration, suggesting a balance as well as in models of human diseases. 6498 MOUSE CHEMERIN/CHEMR23 SYSTEM

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Supplemental data 1. Schematic genomic construction of the ChemR23 knock-out mouse. (A) The mouse ChemR23 gene is located on chromosome 5, region F and spans three exons. The first and second exons are non-coding, and the whole coding region is localized in the third exon. Homologous recombination was performed by Deltagen. In the recombinant gene, a Neo cassette is inserted in the third exon, replacing codons 55 to 104 of the ChemR23 open reading frame, which encode the first two transmembrane segments of the receptor. Screening of animals is performed by PCR, using 5’-TGATCTTGCACATGGCCTTCCCGAA-3’as forward ChemR23 primer (F), 5’-GGGTGGGATTAGATAAATGCCTGCTCT-3’ as forward Neo primer (N), and 5’-TACAGCTTGGTGTGCTTCCTCGGTC-3’ as common reverse primer (R). The size of the products analyzed by agarose gel electrophoresis were 204 bp for the WT allele and 424 bp for the KO allele, both bands being present for heterozygotes (HZ). (B) FACS analysis of cell populations isolated from spleen and blood in WT and ChemR23 KO mice. No difference was observed in terms of leukocyte populations between the two groups.

Supplemental data 2. FACS analysis of mouse ChemR23 expression on NK cells and macrophages isolated from WT and ChemR23 KO mice. (A) Spleen NK cells were enriched as described in the Material and Methods section, and ChemR23 expression was tested on CD49b-positive cells. (B) Mononuclear phagocyte progenitor cells were prepared from bone marrow and differentiated into macrophages in the presence of GM-CSF for 10 days. ChemR23 expression was tested by FACS on the CD11b+F4/80+ cells.

Supplemental data 3. Migration of dendritic cells in response to CCL5 and CXCL12. Chemotaxis assays were performed using mouse BMDCs from WT and ChemR23 KO mice in response to increasing concentrations of mouse recombinant CCL5 and CXCL12, as described in the Material and Methods section.

Supplemental data 4. Biological activity of recombinant C-terminal variants of mouse chemerin.

CHO-K1 cells were stably transfected with plasmids encoding the presumably bioactive form of mouse chemerin (1-157) and a presumably inactive C-terminally truncated variant, lacking the last eight amino acids of prochemerin (1-155). The coding region of mouse preprochemerin cDNA (amino acids 1-163) was cloned from mouse ovary cDNA (Clontech Laboratories) using 5'-CAGGAATTCAGCATGCGACGGCTGCTGA-3’ as forward and 5’-GCTCTAGATTAGCTGCGGGGCAGGGCCTT-3’ as reverse primers. Constructs encoding a chemerin precursor without the “pro” terminal hexapeptide (amino acids 1-157) or further truncated variants of chemerin (amino acids 1-155) were amplified using respectively 5’- GTACTCTAGACTAGGAGAAGGCAAACTGTC-3’ as forward and 5’- GACTTCTAGACTAGGCAAACTGTCCAGGTAG-3’ as reverse primers. The resulting fragments were, cloned into pEFIN3 vector and sequenced. The plasmids were transfected in CHO-K1 cells, and G418-resistant cells were selected for over two weeks. For the characterization of chemerin variants, cells were grown to 70% confluence, and further incubated with serum-free DMEM-F12 (GIBCO BRL) which was collected after three days of culture. Conditioned media were tested for activity on CHO-K1 cells expressing mouse ChemR23 using the aequorin-based calcium mobilization assay and (pro)chemerin production was quantified by Western blotting, using mouse chemerin purchased from R&D Systems as standard. The mouse ChemR23 coding sequence was cloned into pEFIN3 and sequenced. The construct was transfected into CHO-K1 cells expressing apoaequorin, and G418-resistant clones were characterized for ChemR23 expression by RT-PCR.

Supplemental data 5. Analysis by flow cytometry of lung macrophage subpopulations. (A) Macrophage counts in lung cell suspensions 18 hours after trans-oral instillation of PBS, chemerin (5 µg), LPS (1 µg), or LPS and chemerin in WT and ChemR23-/- mice. Alveolar macrophages (AM) are F4/80+CD11cHiI-A/I-EIntCD11bNeg/Lo cells whereas interstitial macrophages (IM) are F4/80+CD11bHiI-A/I-EIntCD11cNeg. (B) Percentage of alveolar macrophages in the total macrophage (alveolar and interstitial) population.

Supplemental data 6. Activity of recombinant mouse chemerin on antigen presenting cells (A) Mouse macrophages isolated from lung and (B) the peritoneal cavity were incubated for 6 hours with medium alone, LPS (0,1 µg) or with LPS (0,1 µg) and chemerin (100 nM). Conditioned media were tested for TNF-α, IL-6 and IL-10 by ELISA. No significant differences were observed in cytokine levels according to the presence or absence of chemerin. (C) Bone marrow-derived dendritic cells (BMDCs) were incubated with increasing concentrations of recombinant chemerin (10-12 to 102 nM) with or without LPS (0,1 µg) for 16 hours and conditioned media were assessed for TNF-α by ELISA. (D) The expression of ChemR23 and chemokine receptors (CCR5, CCR7, CXCR4 and CXCR3) were measured in mouse BMDCs by quantitative real time PCR in basal conditions and after incubation with LPS (0,1 µg) and/or chemerin (20 nM).