No abstract

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase-and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H] ؊ ) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H 2 18 O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Background: Lipoxygenases (LOXs) generate eicosanoids in inflammation.Results: Monocyte/macrophage LOXs generate novel phospholipid-esterified eicosanoids containing ketoeicosatetraenoic acid or hydroperoxyeicosatetraenoic acid. They activate peroxisome proliferator-activated receptor-γ transcriptional activity and are found in cystic fibrosis bronchoalveolar fluid.Significance: LOXs generate esterified eicosanoids in vitro and in vivo.Conclusion: These new lipids represent new families of bioactive mediators.

In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 ± 0.2 ng or 18.5 ± 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.