Chemerin Does Not Inhibit Inflammatory Responses Figure 3. Dose-response effects of chemerin on the production of cytokines by activated peritoneal macrophages. Peritoneal macrophages from WT (white bars) or ChemR232/2 (black bars) mice, collected following i.p. Bio-Gel injection and selected by adherence, were tested for their production of pro-inflammatory (IL-6, IL-1b and TNF-a) and anti-inflammatory (IL-10) cytokines in response to stimulation by LPS and IFN-c, in the presence or not of graded concentrations of recombinant chemerin (from 10212 to 1026 M). After 15 h of culture, supernatants were collected and cytokine levels were determined by ELISA. Results are expressed as the percentage of the LPS/IFN-c-induced levels (left panels) or as pg/ml (right panels) and represent the mean 6 SEM of 3 to 4 independent experiments. doi:10.1371/journal.pone.0040043.g003 Murine peritoneal macrophages obtained from polyacrylamide beads-treated mice express a functional ChemR23 receptor As we did not observe any anti-inflammatory activity of chemerin on LPS-stimulated peritoneal macrophages collected after thioglycolate injection, similar experiments were conducted according to the protocol published by Cash et al. [20], using macrophages collected after intraperitoneal injection of poly- acrylamide beads and stimulated by LPS and IFN-c. Macrophages (F4-80+ Gr12 cells) represented about 45% of the total cells harvested, and their purity was increased to over 90% following selection by adherence to plates. The purity of the cell population and the expression of ChemR23 were also evaluated after two plating times (2 or 24 hours) without significant differences (data not shown). The total number of cells and the percentage of F4- 80+ Gr12 macrophages were not different between WT and ChemR232/2 mice. To validate this protocol, we first confirmed by qRT-PCR the presence of ChemR23 transcripts in the macrophage population prepared from WT mice. We showed also that stimulation by LPS did not significantly affect ChemR23 expression in these cells (Figure 2A). The same observation was made following stimulation by IFN-c (data not shown). Using flow cytometry, surface expression of ChemR23 was then evaluated on cells collected by peritoneal lavage before and after plating. Using a PE-conjugated anti-mouse ChemR23 mAb, we confirmed the expression of the receptor on macrophages but not neutrophils Figure 4. Production of cytokines by activated peritoneal macrophages. Peritoneal macrophages from WT (white bars) or ChemR232/2 (black bars) mice, collected following i.p. Bio-Gel injection and selected by adherence, were tested for their production of pro-inflammatory (IL-6, IL- 1b and TNF-a) and anti-inflammatory (IL-10) cytokines in response to stimulation by LPS and IFN-c, in the presence or not of graded concentrations of recombinant chemerin (from 10212 to 1026 M) or 1026 M dexamethasone (Dex). After 15 h of culture, supernatants were collected and cytokine levels were determined by ELISA. The results, expressed as pg/ml, represent the mean 6 SD of 2 independent experiments. doi:10.1371/journal.pone.0040043.g004 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e40043