Characterization of Endocannabinoid-Metabolizing Enzymes in Human Peripheral Blood Mononuclear Cells under Inflammatory Conditions.

Brittany Szafran,Barbara Kaplan,Kelly Andrzejewski,Abdolsamad Borazjani,Jung Lee,Peter Morrison,Matthew Ross,Grace Zimmerman

doi:10.3390/molecules23123167

Abstract

Endocannabinoid-metabolizing enzymes are downregulated in response to lipopolysaccharide (LPS)-induced inflammation in mice, which may serve as a negative feedback mechanism to increase endocannabinoid levels and reduce inflammation. Increased plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and decreased fatty acid amide hydrolase (FAAH) activity in peripheral lymphocytes from individuals diagnosed with Huntington’s disease (HD) suggests that a similar negative feedback system between inflammation and the endocannabinoid system operates in humans. We investigated whether CpG- (unmethylated bacterial DNA) and LPS-induced IL-6 levels in peripheral blood mononuclear cells (PBMCs) from non-HD and HD individuals modulated the activities of endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and carboxylesterase (CES). Baseline plasma IL-6 levels and 2-arachidonoylglycerol (2-AG) hydrolytic activity in PBMC lysates were not different in HD and non-HD individuals. Inhibition of MAGL and CES1 activity in PBMCs using the inhibitors JZL184 and WWL113, respectively, demonstrated that MAGL was the dominant 2-AG hydrolytic enzyme in PBMCs, regardless of disease state. Correlative analyses of 2-AG hydrolytic activity versus enzyme abundance confirmed this conclusion. Flow cytometric analysis of PBMCs showed that MAGL and CES1 were primarily expressed in monocytes and to a lesser extent in lymphocytes. In conclusion, these data suggest that IL-6 did not influence 2-AG hydrolytic activity in human PBMCs; however, monocytic MAGL was shown to be the predominant 2-AG hydrolytic enzyme.

Highlights

The endocannabinoid system comprises arachidonoyl-containing endocannabinoids, their cognateG-protein coupled receptors, and biosynthetic and catabolic enzymes that regulate the levels of these lipid signaling molecules
We examined IL-6 levels in plasma obtained from Huntington’s disease (HD) and non-HD individuals, assessed IL-6 levels and endocannabinoid-metabolizing enzyme activity in peripheral blood mononuclear cells (PBMCs) isolated from HD and non-HD individuals that were stimulated with inflammogens, and characterized the expression of monoacylglycerol lipase (MAGL) and CES1 in
IL-6 levels were determined in plasma obtained from non-HD and HD individuals (Figure 1A); levels were determined obtained from non-HD

Summary

Introduction

The endocannabinoid system comprises arachidonoyl-containing endocannabinoids, their cognateG-protein coupled receptors, and biosynthetic and catabolic enzymes that regulate the levels of these lipid signaling molecules. Molecules 2018, 23, 3167 and are hydrolytically catabolized by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively, yielding pro-inflammatory arachidonic acid (AA) [3,4]. Other enzymes known to hydrolyze and inactivate 2-AG include carboxylesterases (CES1 and CES2 in human, Ces2g in mice; human and mouse gene symbols for carboxylesterases are CES and Ces, respectively), α,β-hydrolase domain (ABHD) 6, and ABHD12 [5,6,7]. Inactivation of one or more of these enzymes can increase steady-state endocannabinoid levels and is one proposed mechanism to limit or reduce inflammation [9,10,11]. Previous work from our lab demonstrated that lipopolysaccharide (LPS) treatment of mice induced pro-inflammatory mediators such as interleukin (IL)-6 and reduced the metabolism of endocannabinoids in the spleen by Ces2g [9]. Several studies using in vivo and ex vivo approaches have shown that

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