Comparison of eight 15-lipoxygenase (LO) inhibitors on the biosynthesis of 15-LO metabolites by human neutrophils and eosinophils

Abstract

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.

Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome

“…Inhibitory effects of 3887 against 5‐LOX (Supplemental Fig. S2 D ) or against 15‐LOX‐2 (29) were not evident. The formation of the 15‐LOX–derived SPM—that is, LXA 4 , RvD2, ‐D4, ‐D5, PD1, and AT‐PD1, their derivatives 5,15‐diHETE and 10S,17S‐diHDHA, as well as MaR1—were efficiently suppressed by 3887 (IC 50 ≈ 100–200 nM, Fig.…”

Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2‐arachidonoyl‐glycerol and its congeners in human myeloid leukocytes

“…Human AMs were obtained by bronchoalveolar lavage (BAL) of healthy volunteers as previously described . The BAL fluid containing leukocytes was recovered and centrifuged (4°C, 350 g , 10 minutes).…”

“…Human AMs were obtained by bronchoalveolar lavage (BAL) of healthy volunteers as previously described. 25 The BAL fluid containing leukocytes was recovered and centrifuged (4°C, 350g, 10 minutes). Supernatants were discarded and cells were washed twice with cold HBSS then their viability was assessed by trypan blue exclusion.…”

“…The organic phase (top) was then collected and evaporated to dryness under a stream of nitrogen. Samples were reconstituted in 25 . A 25 µL of aliquot was injected onto an RP-HPLC column (Kinetex C8, 150 × 2.1 mm, 2.6 µm, Phenomenex).…”

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

“…One proposed pathway is that h5-LOX, expressed in macrophages (39,40) and PMN's (24), produces 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-docosahexaenoic acid (7S-HDHA) from DHA, and then h15-LOX-1 reacts with 7S-HDHA to produce RvD5 by oxidation at C17. This is a reasonable assumption since h15-LOX-1 oxygenates C17 of DHA efficiently; however, h15-LOX-2 is expressed at higher levels than h15-LOX-1 in both neutrophils and macrophages (4,13,41), so it is possible that h15-LOX-2, and not h15-LOX-1, may be involved in the formation of RvD5 from 7S-HDHA (Scheme 1). It should be noted that LOXs produce the hydroperoxy product, but the reducing environment of the cell quickly reduces them to the hydroxy products (42), such as 7S,14S-diHDHA and 7S,17S-diHDHA (RvD5).…”