Prochemerin can be rapidly converted into a full ChemR23 agonist by proteolytic removal of a carboxy terminal hexa- or heptapeptide. This maturation step is mediated by neutrophil- derived serine proteases (elastase and cathepsin G) and serine proteases of the coagulation and fibrinolytic cascades, released or activated as a result of tissue injury, inflammation or infection [13,15,16]. Chemerin was also described as an adipokine, playing various roles in the regulation of adipocyte differentiation and the metabolism of lipids and carbohydrates [17–19]. In addition to its chemoattractant functions, chemerin was proposed to display anti-inflammatory properties on mouse peritoneal macrophages at low picomolar concentrations, damp- ening the production of pro-inflammatory cytokines and enhanc- ing the production of anti-inflammatory cytokines by these cells [20]. These observations might have explained our own in vivo results showing an anti-inflammatory role of chemerin in a LPS- induced acute lung injury model [7] and in a model of acute viral pneumonia [8]. We therefore studied ex vivo the anti-inflammatory properties of chemerin within a large range of concentrations on mouse and human macrophages, but failed to confirm any effects of chemerin on the production of pro- and anti-inflammatory cytokines by these cells following their activation by LPS and/or IFN-c. Methods Ethics statement The experiments using human samples and animals were carried out in strict accordance with the national, European (EU Directives 86/609/EEC) and international guidelines in use at the Universite´ Libre de Bruxelles and in accordance with the Helsinki Declaration. All procedures were reviewed and approved by the local ethic committee (Commission d’Ethique du Bien-Etre Animal, CEBEA) of the Universite´ Libre de Bruxelles (Permit Number: 222N and 341N). All efforts were made to minimize suffering. Written informed consent was obtained from all participants. Animals Eight to 12-week-old male C57BL/6 mice (Harlan Netherlands) were used throughout the study. ChemR23 knockout (ChemR232/2) mice were obtained from Deltagen and back- crossed for more than 10 generations onto the C57BL/6 background in a specific pathogen-free environment. Heterozy- gotes (ChemR23+/2) were then intercrossed to generate F2 ChemR232/2 mice and their wild-type (WT) controls. Mouse peritoneal macrophage preparations Mouse peritoneal macrophages were collected by using two different protocols. In a first set of experiments, macrophages were harvested 4 days following i.p. injection of a 2% thioglycolate broth. Following sacrifice, the exudate was collected by washing the peritoneal cavity with 265 ml of cold PBS (Invitrogen, Gibco, Gent, Belgium). Harvested cells were pooled, centrifuged, washed with PBS (Invitrogen) and resuspended in RPMI 1640 medium (Invitrogen) supplemented with 100 U/ml penicillin (Invitrogen), 100 mg/ml streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (FBS, Invitrogen). Cells were then seeded in 48- well tissue culture plates (Nunc, 4.105 cells in 500 ml) and allowed to adhere for 1 h at 37uC in a humidified atmosphere containing 5% CO2. Non-adherent cells were removed, and adherent cells were further used for the macrophage activation assay. The purity of the adherent macrophage population was evaluated by flow cytometry (F4-80+ Gr12 cells) to over 90%. In a second set of Chemerin Does Not Inhibit Inflammatory Responses experiments, WT and ChemR232/2 C57BL/6 mice (5 mice/ group) were injected i.p. with 1 ml of a Bio-Gel P100 poly- acrylamide bead suspension (2% w/v in PBS, Bio-Rad Labora- tories, Nazareth-Eke, Belgium), as described by Cash and colleagues [20]. Four days later, mice were sacrificed and the exudate was collected as above. Washed harvested cells were resuspended in Opti-MEM I medium (Gibco) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. The collected cells were either used directly for the chemotaxis assay, binding assay and flow cytometry analysis, or seeded in 48-well culture plates (Nunc, 4.105 cells in 500 ml), and cultured at 37uC in a humidified atmosphere containing 5% CO2. Non-adherent cells were removed after 2 or 24 hours by extensive washing and adherent cells were used for the macrophage activation assay and chemotaxis assay, respectively. The population of adherent cells was consistently composed of .90% F4-80+ Gr12 macrophages, as determined by flow cytometry analysis. Human macrophage preparations Human macrophages were prepared as previously described [21]. Briefly, leukocytes were recovered from buffy coats of healthy donors on a Ficoll (Lymphoprep Axis-Schield) density gradient and erythrocytes were lysed by ammonium chloride. Monocytes were purified by positive selection using CD14 microbeads (Miltenyi Biotec, Leiden, The Netherlands), were resuspended at the density of 106 cells/ml, and seeded in six-well plates (Nunc) in RPMI 1640 medium (Invitrogen) supplemented with 10% heat- inactivated FBS (Invitrogen). Macrophages were differentiated from monocytes in the presence of recombinant human M-CSF (50 ng/ml, Peprotech, London, UK) for 6 to 11 days. The purity of the cell preparations was evaluated by flow cytometry and over 85% of CD11b+/CD206+ macrophages were consistently ob- tained. Chemerin binding assay Chemerin binding assays were performed by incubating 2.105 mouse peritoneal macrophages, harvested after i.p. injection of Bio-Gel polyacrylamide beads, in binding buffer (50 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 250 nM sucrose, 0.5% BSA) with different concentrations of radioiodinated recombinant human chemerin (R&D Systems, Abingdon, UK; custom labeling performed by Perkin Elmer, Zaventem, Belgium). Non-specific binding was measured in the presence of a 100-fold excess of unlabeled chemerin. Samples were incubated for one hour and unbound tracer was removed by filtration through GF/B filters pre-soaked in 1% polyethyleneimine. Filters were counted in a c- scintillation counter. Binding parameters of saturation curves were determined with the PRISM software (Graphpad Softwares) using non-linear regression applied to one site or two sites binding models. The software compared the sum-of-square and the degree of freedom of each regression by using the F test and selected the most appropriate equation. Aequorin-based calcium mobilization assay Functional response of mouse ChemR23 to recombinant mouse chemerin was determined by using an aequorin-based calcium mobilization assay as previously described [13]. Briefly, CHO-K1 cells co-expressing mouse ChemR23, apoequorin and Ga16 (or control CHO-K1 cells co-expressing apoaequorin and Ga16), were incubated for 3 h in the dark in DMEM containing 5 mM coelenterazine H (Invitrogen, Molecular Probes). 5.104 cells in a volume of 50 ml were added to wells containing different concentrations of recombinant mouse chemerin and luminescence was recorded for 20 s in a Packard luminometer. Results (as PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e40043