Indeed, TNF-a, IL-1b, IL-10 and IL-12p35 transcript levels, evaluated by qRT-PCR 2, 4, 6, and 8 hours after LPS stimulation, were not significantly affected by the presence of chemerin in the medium, whereas a strong increase was monitored in all cases, with peak values obtained 2 (for TNF-a) or 4 hours (for IL-1b, IL- 10 and IL-12p35) after stimulation. Discussion In this study, we demonstrate that bioactive chemerin does not exert any modulation of the cytokine inflammatory response of mouse and human macrophages stimulated by LPS or LPS and IFN-c, despite the large concentration range tested. Recently, Cash and colleagues reported data suggesting that recombinant chemerin at picomolar concentrations could behave in vitro as a potent anti-inflammatory mediator on mouse peritoneal macrophages stimulated by LPS and IFN-c, in a proteolysis- dependent manner [20]. These in vitro observations were somehow in line with our own in vivo results showing an anti-inflammatory role of the chemerin/ChemR23 axis in a model of acute lung injury induced by LPS [7] and a model of acute viral pneumonia [8]. We therefore addressed the hypothesis of this putative direct anti-inflammatory effect of chemerin on macrophage populations. Surprisingly, while mouse peritoneal macrophages expressed a fully functional ChemR23 and while recombinant chemerin induced chemotaxis of peritoneal macrophages collected from WT but not ChemR232/2 mice, we were not able to reproduce the in vitro anti-inflammatory effects of chemerin. Our first experiments were performed with macrophages recruited to the peritoneal cavity using a different procedure than in Cash’s report and macro- phages were stimulated in a medium containing fetal calf serum. We therefore performed another set of experiments that were conducted using experimental settings identical to those reported by Cash and colleagues. Macrophages were obtained following injection of polyacrylamide beads and stimulated in a serum-free medium allowing the potential proteolysis of chemerin by macrophage-derived proteases. However, even in these experi- mental settings, no inhibition of the production of the pro- inflammatory mediators TNF-a, IL-6 and IL-1b, and no enhancement of the production of the anti-inflammatory cytokine IL-10 were found when stimulated macrophages were exposed to murine chemerin in a concentration range from 10212 to 1026 M. We specifically used low picomolar concentrations of chemerin, which are in our hands inactive even on cell lines expressing high levels of recombinant human or mouse ChemR23, because Cash and colleagues reported anti-inflammatory activities for such low concentrations of chemerin. Indeed, such a high potency for chemerin differs considerably from all previous experiments showing that chemerin is active on ChemR23 in the 10210 to 1028 M range and peptides derived from its C-terminus in the 1029 to 1027 M range [4,13,14]. We confirmed in the present study that low picomolar concentrations of recombinant chemerin did not trigger any signalling in the sensitive aequorin-based calcium mobilizing assay, which was characterized by a somewhat References 1. Martin TR, Frevert CW (2005) Innate immunity in the lungs. Proc Am Thorac Soc 2: 403–411. 2. Taylor PR, Martinez-Pomares L, Stacey M, Lin H-H, Brown GD et al. (2005) Macrophage receptors and immune recognition. Annu Rev Immunol 23: 901– 944. 3. Serhan CN, Chiang N, Van Dyke TE (2008) Resolving inflammation: dual anti- inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 8: 349–361. 4. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E et al. (2003) Specific recruitment of antigen presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 198: 977–985. Chemerin Does Not Inhibit Inflammatory Responses classical EC50 of 0.7 nM. In addition, binding of 125I-chemerin on peritoneal macrophages yielded a Kd value of about 1.2 nM. Finally, no migration of peritoneal macrophages was observed for low picomolar concentrations of chemerin. These results are in agreement with other previous reports showing that mouse DCs and lung interstitial macrophages express ChemR23 and exhibit chemotactic responses to chemerin, but are not affected by chemerin up to concentrations of 1026 M in their production of cytokines or expression of chemokine receptor following their activation [7]. Discrepancies between our in vitro observations and those of Cash and collaborators [20] might in part be explained by the genetic background of the mice used in the respective studies. Throughout our experiments, we used peritoneal macrophages from WT and KO mice generated from the same line backcrossed on the C57BL/6 background for over 10 generations (see Methods section). Cash et al. used C57BL/6 mice for most experiments, which should therefore be comparable, but also in some experiments ChemR23 KO mice in the 129SvEv background. In order to extend our observations to human cells, we also evaluated the activity of recombinant human chemerin on human macrophages differentiated from peripheral blood monocytes and following stimulation by LPS. As for mouse cells, no anti- inflammatory activities were identified for chemerin in this in vitro setting. As a conclusion, our results rule out a direct anti-inflammatory effect of chemerin on macrophages. As a consequence, the anti- inflammatory effects of the chemerin/ChemR23 system observed in our models of viral pneumonia and LPS-induced acute lung injury should be explained by other mechanisms. We presently favor an hypothesis following which chemerin exerts its anti- inflammatory properties through its activity on non-leukocytic cells expressing ChemR23, such as epithelial or endothelial cells. This hypothesis is supported by experiments performed on chimeric mice showing that the anti-inflammatory activity of chemerin is not mediated by bone marrow-derived leukocyte populations [8], as well as data showing high expression of ChemR23 in non-leukocytic lung cell populations [22]. Supporting Information Table S1 Sequence of murine primers used for quanti- tative RT-PCR. (DOC) Table S2 Sequence of human primers used for quanti- tative RT-PCR. (DOC) Author Contributions Conceived and designed the experiments: BB ODH SL TD PdN JYS MP OV. Performed the experiments: BB ODH SL TD PdN JYS OV. Analyzed the data: BB ODH SL TD PdN JYS MP OV. Wrote the paper: BB ODH JYS MP OV. 5. Samson M, Edinger AL, Stordeur P, Rucker J, Verhasselt V et al. (1998) ChemR23, a putative chemoattractant receptor, is expressed in monocyte- derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains. Eur J Immunol 28: 1689–1700. 6. Parolini S, Santoro A, Marcenaro F, Luini W, Massardi L et al. (2007) The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues. Blood 109: 3625–3632. 7. Luangsay S, Wittamer V, Bondue B, De Henau O, Rouger L et al. (2009) Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e40043