The Endocannabinoid Metabolite Prostaglandin E 2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP This information is current as Receptors of September 27, 2021. Caroline Turcotte, Simona Zarini, Stéphanie Jean, Cyril Martin, Robert C. Murphy, David Marsolais, Michel Laviolette, Marie-Renée Blanchet and Nicolas Flamand J Immunol published online 3 March 2017 Downloaded from http://www.jimmunol.org/content/early/2017/03/03/jimmun ol.1601767 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 3, 2017, doi:10.4049/jimmunol.1601767 The Journal of Immunology The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors Caroline Turcotte,* Simona Zarini,† Ste´phanie Jean,* Cyril Martin,* Robert C. Murphy,† David Marsolais,* Michel Laviolette,* Marie-Rene´e Blanchet,* and Nicolas Flamand* The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl- ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase- derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study’s aim was to define the effects of Downloaded from these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by http://www.jimmunol.org/ immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo. The Journal of Immunology, 2017, 198: 000–000. cute and chronic inflammation are characterized by leu- Historically, COX-2 was perceived as a proinflammatory enzyme, kocyte infiltration, proinflammatory mediator production, because its expression is induced by inflammatory stimuli and leads to by guest on September 27, 2021 A and tissue destruction. Non-steroidal anti-inflammatory PGE2 synthesis. This was eventually challenged by Gilroy et al. (2) drugs have long been used to limit pain and inflammatory dam- who suggested that COX-2 could have anti-inflammatory properties. It age, notably by blocking the production of PGs and thromboxane. was then shown in several murine models that COX-2 blockade Moreover, cyclooxygenase-2 (COX-2) inhibitors, which prevent the worsens inflammation or delays its resolution, at least in mice (3–5). In biosynthesis of PGE2, have been helpful at decreasing inflammation addition to PGs, COX-2 participates in the synthesis of other lipids that and inflammatory pain (1). modulate nociception and inflammation, notably PG-glycerol (PG-G) and PG-ethanolamide (PG-EA). These endocannabinoid derivatives are products of the COX-2–dependent oxygenation of 2-arachidonoyl- *Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Que´bec, De´partement de Me´decine, Faculte´ de Me´decine, Universite´ Laval, Quebec glycerol (2-AG) and anandamide, respectively. PG-EAs, or pros- City, Quebec G1V 4G5, Canada; and †Department of Pharmacology, University of tamides, preferentially activate the prostamide receptor over PG Colorado Denver, Aurora, CO 80045 receptors (6). In contrast, PGE2-G can bind to several PGE2 re- ORCIDs: 0000-0002-9430-6515 (R.C.M.); 0000-0002-8935-6657 (D.M.); 0000- 0001-5944-7200 (N.F.). ceptors to a lesser extent than PGE2 (7). Moreover, PGE2-G in- Received for publication October 17, 2016. Accepted for publication February 1, duces key signaling events that are not mimicked by PGE2 (8, 9). 2017. PGE2 is a well-established inhibitor of human neutrophil func- This work was supported by Canadian Institutes of Health Research MOP-97930 (to N.F.), tions such as leukotriene B4 (LTB4) biosynthesis, reactive oxygen the Natural Sciences and Engineering Research Council of Canada (to N.F.), and the Fonds species (ROS) production, and migration (10–14). These effects sur les Maladies Respiratoires J.-D. Be´gin–P.-H. Lavoie (to N.F.). C.T. is the recipient of a doctoral award from the Canadian Institutes of Health Research and was supported by a are the consequence of elevated cyclic cAMP and involve the EP2 research award from the Canadian Consortium for the Investigation of Cannabinoids. receptor. Given that PGE2-G is produced by COX-2 and binds to D.M., M.L., M.-R.B., and N.F. are members of the inflammation group of the Respiratory some of the EP receptors (7), because PGE inhibits neutrophil Health Network of the Fonds de Recherche du Que´bec-Sante´. 2 functions, and because PG-Gs were shown to regulate inflammation Address correspondence and reprint requests to Dr. Nicolas Flamand, Centre de Recherche de l’ Institut Universitaire de Cardiologie et de Pneumologie de Que´bec, (15), we undertook experiments to assess whether PGE2-G and Universite´ Laval, 2725 Chemin Sainte-Foy, Room A2142, Quebec City, QC G1V PGE2-EA would also modulate human neutrophil functions. 4G5, Canada. E-mail address: [email protected] Abbreviations used in this article: AA, arachidonic acid; 2-AG, 2-arachidonoyl-glycerol; COX-2, cyclooxygenase 2; EA, ethanolamide; G, glycerol; LC-MS/MS, liquid Materials and Methods chromatography–tandem mass spectrometry; LTB4, leukotriene B4;MAFP,methyl Materials arachidonoyl fluorophosphonate; PKA, cAMP-dependent protein kinase; qPCR, quantitative PCR; ROS, reactive oxygen species; SA, serinol-amide. AH6809, 19-OH-PGB2, 2-AG, butaprost (free acid), CAY 10598, H-89, JZL184, L-902 688, methyl arachidonoyl fluorophosphonate (MAFP), Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 PGE2, PGE2-EA, PGE2-G, PGE2-serinol amide (SA), PGD2-G, PGD2-SA, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601767 2 PGE2-G INHIBITS NEUTROPHIL FUNCTIONS PGF2a-G, sulprostone, tetrahydrolipstatin, WWL70, and the primary Ab Migration assay for MAG lipase were purchased from Cayman Chemical (Ann Arbor, m MI). Thapsigargin was obtained from Tocris Bioscience (Ellisville, MO). Migration assays were performed using 3 m pore inserts (Becton Dick- m DMSO was purchased from Sigma-Aldrich (St Louis, MO). Protease inson), as recommended by the manufacturer. In brief, 700 l of pre- inhibitor mixture tablets and adenosine deaminase were purchased from warmed (37˚C) HBSS containing 1.6 mM CaCl2 and 100 nM LTB4 were m Roche (Laval, QC, Canada). Aprotinin, leupeptin, and WWL113 were added in the lower chamber and 200 l of prewarmed neutrophil sus- 3 6 purchased from Sigma-Aldrich. DFP was purchased from BioShop pensions [37˚C, 2.5 10 cells/ml in HBSS containing 1.6 mM CaCl2 and Canada (Burlington, ON, Canada). The HRP-linked anti-mouse IgG and 5% (w/v) FBS] were added in the upper chamber of the transmigration anti-rabbit IgG secondary were obtained from Cell Signaling Technology apparatus. Neutrophils were allowed to migrate for 2 h at 37˚C. The upper (Beverly, MA). Primary Abs for ABHD6, ABHD12, and PPT1 were chambers were then removed and migrated cells in the lower chamber of purchased from Abcam (Toronto, ON, Canada). The LYPLA2 primary the migration apparatus were counted using a Scepter 2.0 handheld auto- Ab was purchased from Abnova (Taipei City, Taiwan), the ABHD16A mated cell counter. In experiments where PGE2, PGE2-G, and the type IV primary Ab was purchased from Thermo Fisher Scientific (Waltham, phosphodiesterase RO 20-1724 were used, they were added to both upper MA), and the primary Ab for CES1 was purchased from R&D Systems and lower chamber for 5 min before the addition of LTB4 in the lower (Minneapolis, MN). PMSF, RO 20-1724, palmostatin B, and the ECL chamber of the migration assay. detection kit were purchased from EMD Millipore (Billerica, MA). The magnetic bead–conjugated anti-CD16 mAb and MACS were purchased Bacterial growth assays from Miltenyi Biotec (Auburn, CA). HBSS and RPMI 1640 were ob- Escherichia coli (#25922; American Type Culture Collection) was grown tained from Wisent Laboratories (St-Bruno, QC, Canada). Dextran and overnight at 37˚C in tryptic soy broth.