Chemerin Does Not Inhibit Inflammatory Responses Peritoneal cells from WT or ChemR232/2 mice, collected following i.p. Bio-Gel injection, were analysed by flow cytometry, directly or after selection by adherence to plates. Cells were stained for leukocyte markers (F4-80 and Gr1) and expression of ChemR23 on F4-80+ Gr12 (Mw) and F4-802 Gr1+ (PMN) cells was evaluated using flow cytometry. Histograms represent the fluorescence observed following ChemR23 staining on peritoneal cells from WT (black histogram) or ChemR232/2 (white histogram) mice, compared to the isotype-matched control (dotted line). One representative experiment out of 3 is shown. (C) Total peritoneal cells from WT and ChemR232/2 mice, collected following i.p. Bio-Gel injection, were incubated with increasing concentrations of radiolabeled chemerin (N). Non-specific binding was determined in the presence of a 100-fold excess of unlabeled chemerin (*), and specific binding (%) was calculated as the difference. One representative experiment out of 3 is shown. (D) Peritoneal macrophages from WT or ChemR232/2 mice, collected following i.p. Bio-Gel injection, were tested for their ability to migrate in response to recombinant mouse chemerin. In the left panel (mean 6 SEM for 3 independent experiments performed in quadruplicate), the whole populations of cells recovered was used. In the right panel (mean 6 SD for an experiment performed in triplicate), macrophages selected by overnight adherence were used. Results are expressed as migration index. (E) The biological activity of mouse recombinant chemerin was measured on mouse ChemR23-expressing CHO-K1 cells using the aequorin-based intracellular Ca2+ mobilization assay. Results are expressed as the percentage of the response to ATP and represent the mean 6 SD of duplicated data points. One representative experiment out of 3 is shown. doi:10.1371/journal.pone.0040043.g002 of peritoneal macrophages was measured using a 48-well microchemotaxis Boyden chamber with polycarbonate mem- branes (5 mm pores, NeuroProbe, MD, USA). The cell suspension (5.104 cells in 50 ml) was placed in the upper chamber. The lower wells contained 30 ml of medium with different concentrations of chemoattractant, and the chamber was incubated at 37uC for 90 min. All conditions were tested in triplicate. Controls were performed in the absence of chemoattractant in the lower wells. The results were expressed as migration ratio (mean cell number per well with chemoattractant over mean cell number per well without chemoattractant). Macrophage activation assay Murine macrophages obtained by the thioglycolate procedure were preincubated for 1 h with 1026 to 10212 M recombinant mouse (rm)-chemerin (R&D Systems) in RPMI 1640 medium (Invitrogen), before challenging them with 100 ng/ml LPS (E. coli strain 055:B5; Sigma-Aldrich, Bornem, Belgium). Murine macro- phages obtained by the Bio-Gel procedure were preincubated for 1 h with 1026 to 10212 M rm-chemerin (R&D Systems) in Opti- MEM I medium (Invitrogen), before challenging them with 100 ng/ml LPS (E. coli strain 055:B5; Sigma-Aldrich) and 20 ng/ ml rm-IFN-c (Peprotec). At selected time points after stimulation, supernatants were harvested for cytokine determination and total RNA was extracted from adherent cells for gene expression analysis. Similarly, human macrophages were preincubated for 30 min with 1028 M of recombinant human chemerin (R&D Systems) and subsequently matured for 2, 4, 6, or 8 h by incubation with 100 ng/ml LPS. Cytokine assays Supernatants of cultured macrophages were harvested and levels of interleukin (IL)-1b (detection limit of 31.2 pg/ml), IL-6 (detection limit of 15.6 pg/ml), IL-10 (detection limit of 31.2 pg/ ml) and tumor necrosis factor (TNF)-a (detection limit of 15.6 pg/ ml) were determined by enzyme-linked immunosorbent assays (BD Biosciences, Erembodegem, Belgium). In some experiments, total RNA was extracted from cultured macrophages and purified using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). After DNase I treatment (Ambion), samples were reverse transcribed into cDNA using random hexamers (Roche, Brussels, Belgium) as primers and the Superscript III polymerase (Invitrogen). RT products were analyzed by quantitative real-time RT-PCR. The sequence of primer pairs used for mouse and human genes is provided as supplemental data (Tab. S1 and S2). Raw data were normalized for each sample using YWHAZ and CANX or GAPDH transcripts as references. Flow cytometry analysis Peritoneal macrophages were incubated for 10 min at 4uC with anti-CD16/CD32 monoclonal antibodies (BD Biosciences) to block non-specific binding to Fc receptors. Then, cells were stained with PE-conjugated anti-mouse CMKLR1 (e-Bioscience, clone bz194, Vienna, Austria), FITC-conjugated anti-mouse F4- 80 (AbD Serotec, Dusseldorf, Germany), PerCp-conjugated anti- mouse Gr1 (BD Biosciences) monoclonal antibodies or the corresponding isotype-matched controls (BD Biosciences). All samples were analyzed using a FC500 flow cytometer (Beckman- Coulter). Statistical analysis Statistical analysis was performed using nonparametric t tests or tests for multiple comparisons (Kruskal-Wallis test and the Dunn’s multiple comparison post-test), using the GraphPad Prism 5 software. For all tests, a p value #0.05 was considered as significant. Results Chemerin does not inhibit the LPS-induced production of pro-inflammatory cytokines by thioglycolate-recruited peritoneal macrophages Previous experiments have shown that peritoneal macrophages collected from WT mice following intraperitoneal injection of thioglycolate express a functional ChemR23 receptor [7]. There- fore, to further evaluate the potential anti-inflammatory effect of chemerin reported by others [20], peritoneal macrophages were recovered from WT and ChemR232/2 mice 4 days after intraperitoneal injection of thioglycolate and tested in a time- course experiment. Macrophages were stimulated by 100 ng/ml LPS in the presence or absence of 100 nM rm-chemerin, and the expression and release of cytokines were determined at different time points respectively by quantitative RT-PCR (qRT-PCR) and ELISA. As shown in Figure 1A, qRT-PCR analysis performed 2, 4, 6, 8, 12, 16 and 24 hours after LPS stimulation showed an important time-dependent upregulation of TNF-a, IL-6 and IL-10 transcripts, with a peak observed at 4 to 6 hours for the three cytokines. This LPS-induced upregulation was not affected by the presence of 100 nM chemerin in the medium. At the protein level, LPS-stimulated macrophages released high levels of TNF-a, IL-6 and IL-10 with, as expected, peak values obtained at later time- points (i.e. 8–12 hours) than for the corresponding transcripts. Similarly, no significant differences were observed in terms of cytokine levels between cells treated or not with 100 nM of chemerin (Figure 1B). PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e40043