Lipidomic analysis of RvE1 in human plasma. (a) Representative MS/MS selected ion chromatogram at m/z 291 and (b) MS³ selected ion chromatogram at m/z 229. (c) Scheme of RvE1 generation from EPA. Human endothelial cells expressing COX-2 treated with aspirin transform EPA by abstracting hydrogen at C16 to give R insertion of molecular oxygen to yield 18R-H(p)EPE. Alternatively, cytochrome P450 monooxygenase can convert EPA to 18R-HEPE (reference 28). They are further converted via sequential actions of a leukocyte 5-LO–like reaction, which leads to formation of RvE1.

Properties of RvE1. (a) Total organic synthesis of RvE1. Precursors 1–3 were prepared in isomerically pure form from starting materials with known stereochemistry and coupled sequentially to form acetylenic intermediate compound 4, which was selectively hydrogenated to form isomerically pure RvE1. (b) MS/MS spectrum of synthetic RvE1. (c) Inhibition of leukocyte trafficking in murine dorsal air pouch. 18R-HEPE and RvE1 (biogenic and synthetic) are at 100 ng/mouse, and dexamethasone is at 10 μg/mouse. Values represent mean ± SEM from five different mice. *, P < 0.05 (vs. vehicle control).

RvE1 receptor. (a) Phylogenetic tree representing amino acid sequence similarities between the human LXA₄ receptor (ALX) and related GPCRs. (b) Functional screening for RvE1 receptors. HEK293 cells cotransfected with pNF-κB-luciferase and pcDNA3-GPCRs were exposed to RvE1 (10 nM) and TNF-α. (c) Amino acid sequence alignment of human ChemR23 with ALX. Asterisks indicate conserved amino acids. Putative transmembrane domains are lined and labeled I–VII. (d) RvE1 inhibits luciferase activity in a concentration-dependent manner on cells transfected with pcDNA3-ChemR23 (closed circles) but not pcDNA3 (open circles). (e) Ligand specificity for ChemR23. Cells transfected with pcDNA3-ChemR23 were exposed to 100 nM of each compound. Results are expressed as percent inhibition of luciferase activity and represent the mean ± SEM from n = 3 (b) or n = 4 (d and e); *, P < 0.05. (f) Actions of RvE1 (closed circles), RvE1 derivative (6,14-diacetylenic-RvE1; open rectangle), and 18R-HEPE (closed diamonds) on [³⁵S]-GTP-γS binding to membrane expressing ChemR23. Results are expressed as a percentage of vehicle control with the mean ± SEM (n = 3). *, P < 0.05.

Labeled RvE1 and specific binding. (a) Elution profile of the [³H]-labeled RvE1 on reverse phase HPLC. Tracing was recorded at 270 nm absorbance and chromatographic peak at the same retention time as standard RvE1 is indicated by the asterisk. The inset shows the online UV spectrum of the product. (b) [³H]-RvE1–specific binding to ChemR23. Human ChemR23–transfected (closed circles) or mock-transfected (open inverted triangles) CHO cells (10⁶ cells) were incubated with indicated concentrations of [³H]-RvE1 in the presence or absence of 10 μM of unlabeled RvE1. Saturation curve and Scatchard plot (inset) are representative of n = 3. (c) Binding specificity. Human ChemR23-transfected CHO cells (10⁶ cells) were incubated with 10 nM of [³H]-RvE1 in the presence of 10 μM of RvE1, EPA, 18R-HEPE, or chemerin peptide. Results are expressed as percent competition of [³H]-RvE1–specific binding. Results represent the mean ± SEM from duplicates of n = 3∼5. p-values were obtained by Student's t test comparing heteroligand (i.e., EPA, 18R-HEPE, or chemerin) with homoligand (i.e., RvE1). *, P < 0.01; ^#, P = 0.07.

Receptor expression. (a) RT-PCR analysis of human peripheral blood leukocytes and glioma (DBTRG-05MG), monocytic (THP-1), lung epithelial (A549), hepatoma (HepG2), embryonic kidney (HEK293) cell lines, and brain and liver. (b) RT-PCR analysis of human peripheral blood monocytes exposed to either buffer alone, 10 ng/ml TNF-α, or 25 ng/ml IFN-γ for 6 h (gray bars) and 24 h (black bars). Expression levels were quantified by a National Institutes of Health image, normalized by GAPDH levels, and expressed as fold increase over vehicle-treated cells. (c) MAP kinase activation in human peripheral blood monocytic cells were exposed to RvE1 for 5 or 15 min and HEK-ChemR23 cells were treated with 100 nM RvE1 (E) or vehicle (V). (d) Pertussis toxin (PTX) blocks RvE1-induced ERK activation and NF-κB inhibition in HEK-ChemR23 cells. (e) EAR with HEK-ChemR23 cells exposed to vehicle (red line), 100 nM RvE1 (blue line), 10 μM chemerin peptide, or 10 μM ATP using Cytosensor microphysiometer. These responses were observed from three separate experiments. (f) Actions of RvE1 (red) and chemerin peptide (black) on [³⁵S]-GTP-γS binding to HEK293 cell membranes expressing human ChemR23. (g) Inhibition of TNF-α induced NF-κB luciferase activities by RvE1 (red) and chemerin peptide (black) on HEK293 cells expressing human ChemR23.

RvE1 regulates DCs. (a) RvE1 inhibits DC IL-12 production in vitro stimulated by pathogen extract (STAg). CD11c⁺ DCs incubated with vehicle (open circles) or RvE1 (closed circles) before STAg or no STAg (open squares). (b) Reduction of ChemR23 expression by siRNA eliminates RvE1 signaling. Expression of ChemR23 and GAPDH mRNA from DCs treated with either scramble or ChemR23-specific siRNAs (inset). Spleen cells transfected with siRNAs were treated with vehicle (ethanol, 0.1% vol/vol) or RvE1 (1.0 μg/ml). 8 h later, cells were stimulated with 10 μg/ml STAg and IL-12p40 was measured. Bars represent mean ± SD (n = 3), *, P < 0.05 (control vs. specific siRNA). (c) RvE1 blocks IL-12 production in vivo. Mice administered with either 100 ng RvE1 or vehicle were challenged intraperitoneally with PBS or STAg, and IL-12p40 secretion from splenic CD11c⁺ DCs was measured. (d–g) RvE1 blocks trafficking of CD11c⁺ DCs in spleen with pathogen extract challenge. Spleens from mice given 10 μg RvE1 or vehicle were stained for CD11c. (d) PBS plus vehicle (e) STAg plus vehicle (f) PBS plus RvE1 (g) STAg plus RvE1. Arrows indicate CD11c positive DCs accumulated in T cell–enriched area.