title: Modulation of TNFalpha, IL-10 and IL-12p40 levels by a ceramide-1-phosphate analog, PCERA-1, in vivo and ex vivo in primary macrophages.
abstract: Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha), and thus as a putative drug for the treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFalpha production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFalpha production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFalpha, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFalpha and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-gamma. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFalpha production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFalpha and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.
authors.affiliation: Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel.
authors.forename: Dorit
authors.initials: D
authors.lastname: Avni
authors.forename: Meir
authors.initials: M
authors.lastname: Goldsmith
authors.forename: Orna
authors.initials: O
authors.lastname: Ernst
authors.forename: Roi
authors.initials: R
authors.lastname: Mashiach
authors.forename: Tove
authors.initials: T
authors.lastname: Tuntland
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meijler
authors.forename: Nathanael S
authors.initials: NS
authors.lastname: Gray
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
authors.forename: Tsaffrir
authors.initials: T
authors.lastname: Zor
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002518:Ceramides / Q000494:pharmacology*; D015232:Dinoprostone / Q000276:immunology / Q000378:metabolism; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D016753:Interleukin-10 / Q000819:agonists / Q000096:biosynthesis*; D053762:Interleukin-12 Subunit p40 / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis*
chemical_list: D002518:Ceramides; D053762:Interleukin-12 Subunit p40; D008070:Lipopolysaccharides; C541330:PCERA-1; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma; D015232:Dinoprostone