Activated Platelets and Monocytes Generate Four Hydroxyphosphatidylethanolamines via Lipoxygenase

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.

15-oxo-Eicosatetraenoic Acid, a Metabolite of Macrophage 15-Hydroxyprostaglandin Dehydrogenase That Inhibits Endothelial Cell Proliferation

“…It is known that 15-LO-1 has a broad substrate Donnell, 2006). Furthermore, Maskrey et al (2007) showed that when IL-4-treated monocytes were stimulated with CI, they generated predominantly esterified 15-HETE, with manyfold less free 15-HETE (Maskrey et al, 2007). These studies suggest that CI treatment of the monocytes resulted in translocation of 15-LO-1 to the plasma membrane so that it was no longer present the cytosol.…”

A simplified procedure for semi-targeted lipidomic analysis of oxidized phosphatidylcholines induced by UVA irradiation

“…Because of the tremendous excess of unoxidized lipids and the presence of isobaric peaks, analysis of individual OxPLs by mass spectrometry is only feasible after chromatographic separation of lipids, typically by reversed-phase HPLC. Previous publications described mass spectrometry-based methods for analysis of oxidized cardiolipin (15)(16)(17), phosphatidylserine ( 15,16,18 ), phosphatidylethanolamine ( 6,(19)(20)(21)(22), and phosphatidylcholine ( 23-25 and references below). The results show that different PL classes have variable propensity for oxidation.…”

Directing eicosanoid esterification into phospholipids

“…At that time, neither the molecular species involved nor their biology was characterized. Later, in 2005, while the cellular regulation of 15-LOX turnover was being studied, 15-HETE was found to be acutely generated attached to four individual phospholipids in IL-4-treated human monocytes (8). Acute generation of eoxPL is a different process to incorporation of exogenous eicosanoids, as it is considerably faster and stimulated by inflammatory agonists.…”