Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes

Abstract

Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10,17S-docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1/neuroprotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10,17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Additional isomers identified included trace amounts of Delta15-trans-PD1 (isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer I), present in exudates. 18O2 labeling showed that 10S,17S-diHDHA (isomer I) carried 18O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (approximately 50%) human neutrophil transmigration, whereas Delta15-trans-PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (approximately 40% at 1 ng/mouse) in peritonitis. The rank order at 1- to 10-ng dose was PD1 approximately PD1 methyl ester >> Delta15-trans-PD1 > 10S,17S-diHDHA (isomer I). 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (isomer VI) proved > or = PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.