Resolution of inflammation by N-arachidonoylglycine

Abstract

N-arachidonoylglycine (NAgly) is an endogenous signaling lipid that is a member of the eicosanoid super family and is related to anandamide. It shows anti-inflammatory activity in vivo in the mouse peritonitis model where it reduces migration of inflammatory leukocytes following injection of pro-inflammatory agents into the peritoneal cavity. Using cell culture models, including GPR18 transfected HEK-293 cells, evidence is presented that the orphan receptor GPR18 is involved in this action. Increases in free arachidonic acid, and robust stimulation of anti-inflammatory eicosanoids were observed at low micromolar concentrations. These included 15-deoxy-delta-13,14-PGJ(2) and lipoxin A(4) both of which are believed to mediate the resolution stage of inflammation. It was further shown that NAgly might act via GPR18 activation in promoting the number of Trypan Blue stained cells, a possible indicator of programmed cell death. Thus, we hypothesize that NAgly induces the death of inflammatory cells, a process that is considered to be important for the resolution of inflammation.

Fig. 1. NAgly inhibits leukocyte migration in the mouse peritonitis model

The indicated treatments were administered by mouth and after 30 min, the mice were injected i.p. with 1 ml (sterile filtered) 8% BBL Fluid Thioglycollate Medium. Cells were harvested from the peritoneal cavity after 3 hours, exposed to lysing buffer for 2 minutes to remove erythrocytes, suspended in PBS/BSA and differential cell counts obtained. Controls were safflower oil and Palmitoylglycine (PALgly). N=8.

Fig. 2. Stimulation of arachidonic acid release from C6 glioma cells by NAgly

Cells were grown and treated as described previously (Pestonjamasp and Burstein, 1998). See also Methods Section. Following a 2 hr labeling period with ¹⁴C-arachidonic acid, the media were changed to RPMI+0.1% BSA and the cells treated with NAgly. Release into the media was measured by LSC of aliquots of medium. Values shown are the means ∓ SD. N = 4 Control: DMSO treated cells gave values of 190, 250 and 385 dpm respectively. Note: The cells were treated with non-radioactive NAgly.

Fig. 3. Stimulation by NAgly of levels of PGJ and LXA ₄ in HEK393 GPR18 transfected cells

Treatments were carried out in 48 well plates with 20,000 cells / 0.5 ml RPMI/FCS media/well. Cells were incubated for 20 hrs at 37°C and 5% CO₂. Media were changed to 0.5 ml of serum free RPMI and TNFα (10 nM) added, treated for 2 hrs and 0.1 ml removed for the assays. Assays were performed with PGJ or LXA₄ ELISA kits (Assay Design, Inc.). Controls: 1% DMSO vehicle. N=4

Fig. 4. Correlation of GPR18 mRNA levels with PGJ stimulation by NAgly

See Methods section for the procedures used. For the relative comparison of mRNA expression levels, the data from real time PCR were analyzed with a delta-delta Ct method and normalized to the amount of HRPT1 cDNA as an endogenous control. Controls for PGJ assays were 1% DMSO vehicle. N=3

Fig. 5. FACS analysis following NAgly treatment of HEK293 GPR18 transfected cells

Two T-75 flasks were seeded with 100,000 HEK293 GPR18 transfected cells/ml, TNFa (10 ng/ml final conc.) was added to the cells in DMEM + 10% FBS and incubated overnight. Cells were then treated with 5uM NAGLY in DMSO (A) or DMSO only (B), and incubated at 37°C and 5% CO₂ for 2.5 hrs. Cells from each flask were divided into 12 tubes/group, stained according to the protocol for BD Pharmingen FITC Annexin V Apoptosis Detection Kit II (Cat. # 556570) and analyzed by FACS. Panels A and B are representative data. Panel C shows the mean fluorescence values obtained for late-stage apoptotic cells (upper right) and necrotic cells (upper left). N=3