Chemerin Does Not Inhibit Inflammatory Responses Figure 5. Effects of chemerin on the production of cytokines by stimulated human macrophages. Human macrophages were obtained as described in the Material and Methods section and were tested for their production of IL-6, TNF-a, IL-1b and IL-12p35 following stimulation by LPS, in the absence or presence of 100 nM recombinant human chemerin. At various time points, cells were collected and cytokine transcript levels were determined by quantitative RT-PCR. Results are expressed as fold increase over basal levels and represent the mean 6 SEM of 3 independent experiments. doi:10.1371/journal.pone.0040043.g005 (F4-802 Gr1+ cells), using ChemR232/2 cells as controls (Figure 2B). To further confirm the functional expression of ChemR23 by macrophages, we performed binding studies using radioiodinated chemerin and chemotaxis assays. In saturation binding experiments, chemerin bound to cells at a single binding site with a calculated Kd of about 1.2 nM (Figure 2C). This value is similar to those obtained for chemerin binding on CHO-K1 cell lines expressing recombinant mouse or human ChemR23 (1.1 nM and 0.9 nM, respectively) (data not shown). In contrast, very weak chemerin binding was detected on macrophages collected from ChemR232/2 mice, confirming that ChemR23 is by far the main chemerin receptor on these cells. Furthermore, macrophages collected after intraperitoneal injection of polyacrylamide beads and selected by adherence to plates, were also tested for their capacity to migrate in response to chemerin. As expected, the cells were shown to migrate in response to increasing concentrations of chemerin with a typical bell-shaped curve culminating for concentrations around 1 nM (Figure 2D); whereas cells collected from ChemR232/2 mice failed to migrate over background levels in response to chemerin. Taken together, these results demon- strated that peritoneal macrophages collected 4 days after intraperitoneal injection of polyacrylamide beads express a func- tional ChemR23 receptor. Finally, we assessed whether rm- chemerin used throughout our study was able to promote intracellular calcium release through the ChemR23 receptor. A CHO-K1 cell line expressing mouse ChemR23 was used in an aequorin-based calcium release assay. We determined that rm- chemerin wad indeed able to activate its receptor with an EC50 of 0.7 nM (Figure 2E). As a control, no calcium response was observed in wild-type CHO-K1 cells exposed to rm-chemerin (data not shown). Chemerin does not inhibit cytokine production following stimulation of mouse polyacrylamide-recruited peritoneal macrophages or human blood-derived macrophages According to the macrophage activation assay described by Cash et al. [20], peritoneal exudates obtained from polyacryl- amide beads-treated WT and ChemR232/2 mice were allowed to adhere for 2 hours. After extensive washing, cells were cultured in OptiMEM medium, in the presence of a range of rm-chemerin concentrations (from 10212 to 1026 M) for 1 hour and then stimulated by LPS/IFN-c. After 15 h of culture, levels of TNF-a, IL-1b, IL-6 and IL-10 in the supernatants were determined by ELISA. As previously observed for thioglycolate-recruited perito- neal macrophages stimulated by LPS alone, none of the induced cytokines was significantly modulated by the presence of chemerin at any concentration (Figure 3). As a control, peritoneal macrophages were also treated with 1 mM dexamethasone before stimulation by LPS/IFN-c. As shown in Figure 4, dexamethasone repressed the production of pro-inflammatory cytokines, particu- larly IL-6 and TNF-a. Finally, human macrophages differentiated from peripheral blood monocytes were also generated to assess the effect of chemerin on their LPS-induced cytokine production. As illustrated in Figure 5, 100 nM of recombinant human chemerin had no effect on the expression of the various cytokines tested. PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e40043