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 (18)(2)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.
Highlights
12/15-LOX is expressed by monocytes/macrophages in response to interleukin-4 and -13 and oxidizes arachidonate at either C12 or C15 [5, 6]
Before the advent of high sensitivity mass spectrometry, this question was addressed by the addition of radiolabeled HETEs to cells followed by examination of their fate by radio-TLC [12,13,14,15,16,17]
The studies identify specific esterified eicosanoids that form after immune cell activation and, show the powerful nature of precursor scanning as a lipidomic tool for identifying novel endogenously generated lipid adducts
Summary
The straw-colored supernatant was collected and underlayed with Lymphoprep 2:1 (v/v, supernatant:Lymphoprep) and centrifuged 800 ϫ g for 20 min at 4 °C. The interface was collected and diluted 1:1 (v/v) with ice-cold PBS containing 0.4% (w/v) citrate, pH 7.4, and spun at 400 ϫ g, 10 min at 4 °C. The supernatant was discarded, and the cell pellet was washed 5ϫ with ice-cold PBS/citrate buffer, 400 ϫ g for 5 min at 4 °C. 1640 (10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine). 108 cells were seeded per T75 flask and incubated at 37 °C for 2 h to allow monocytes to adhere. The medium was replaced, and cells were cultured for 72 h with.
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