2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties.Blocking 2-AG hydrolysis to enhance CB 2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), / -hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs). We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E 2 -glycerol [PGE 2 -G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG ≥ 15-HETE-G >> PGE 2 -G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), Palmostatin B, 4 ′ -carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, Nmethyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4 ′ -(aminocarbonyl)[1,1 ′ -biphenyl]-4-yl ester carbamic acid (WWL70), 4 ′ -[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1 ′ -biphenyl]-4-carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE 2 -G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE 2 -G, and 15-HETE-G by neutrophils and the hydrolysis of PGE 2 -G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling(ABPP) probe to label hydrolases in leukocytes, we found that they express many MAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of ∼52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple

The endocannabinoid (eCB) 2‐arachidonoyl‐gycerol (2‐AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2‐AG hydrolysis inhibition might represent an interesting anti‐inflammatory strategy that would simultaneously increase the levels of 2‐AG and decrease those of eicosanoids. Accordingly, 2‐AG hydrolysis inhibition increased 2‐AG half‐life in neutrophils. Under such setting, neutrophils, eosinophils, and monocytes synthesized large amounts of 2‐AG and other monoacylglycerols (MAGs) in response to arachidonic acid (AA) and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000‐fold more potent than G protein‐coupled receptor (GPCR) agonists. Triascin C and thimerosal, which, respectively, inhibit fatty acyl‐CoA synthases and acyl‐CoA transferases, prevented the UFA‐induced MAG biosynthesis, implying glycerolipid remodeling. 2‐AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While GPCR agonists poorly induced 2‐AG biosynthesis, they inhibited that induced by AA by 25%‐50%, suggesting that 2‐AG biosynthesis is decreased when leukocytes are surrounded by a pro‐inflammatory entourage. Our data strongly indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2‐AG and other MAGs and that hijacking the immune system with 2‐AG hydrolysis inhibitors might diminish inflammation in humans.

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The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15–30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

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