12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid is a natural ligand for leukotriene B4 receptor 2

Abstract

Activated blood platelets and macrophages metabolize prostaglandin H₂ into thromboxane A₂ and 12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid (12-HHT) in an equimolar ratio through the action of thromboxane synthase. Although it has been shown that 12-HHT is abundant in tissues and bodily fluids, this compound has long been viewed as a by-product lacking any specific function. We show that 12-HHT is a natural ligand for leukotriene B₄ (LTB₄) receptor-2 (BLT2), a G protein–coupled receptor that was originally identified as a low-affinity receptor for LTB₄. BLT2 agonistic activity in lipid fractions from rat small intestine was identified as 12-HHT using high-performance liquid chromatography and mass spectrometry. Exogenously expressed BLT2 in mammalian cells was activated by synthetic 12-HHT, as assessed by guanosine 5′-O-(3-thio) triphosphate binding, the activation of intracellular signaling pathways, and chemotaxis assay. Displacement analysis using [³H]LTB₄ showed that 12-HHT binds to BLT2 with a higher affinity than LTB₄. Lipid extracts from cyclooxygenase 1–deficient mice failed to activate BLT2. Bone marrow–derived mast cells (BMMCs) isolated from wild-type mice migrated toward a low concentration of 12-HHT, whereas BMMCs from BLT2-deficient mice did not. We conclude that 12-HHT is a natural lipid agonist of BLT2 in vivo and induces chemotaxis of mast cells.