Julie Gardener
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Activation Of PAF Receptors Results In Enhanced Synthesis Of 2-Arachidonoylglycerol (2-AG) In Immune cells
EVGUENI V. BERDYSHEV1, PATRICIA C. SCHMID, RANDY J. KREBSBACH and HARALD H. O. SCHMIDThe Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA
1Correspondence: The Hormel Institute, University of Minnesota, 801 16th Ave., N.E., Austin, MN 55912, USA. E-mail: eberdyshev@hotmail.com
(The FASEB Journal. 2001;15:2171-2178.)
© 2001 FASEB
ABSTRACT
The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, 2-arachidonoylglycerol (2-AG) or N-arachidonoylethanolamine (20:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of 2-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and 2-monoacylglycerols, including 2-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of 2-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN52021 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U73122 and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced 2-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that 2-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.–Berdyshev, E. V., Schmid, P. C., Krebsbach, R. J., Schmid, H. H. O. Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells.
INTRODUCTION
IT IS NOW well recognized that the activation of cannabinoid receptors can provide signals resulting in the down-regulation of various cell functions. In neurobiological responses, cannabinoids are known to decrease pain, cause long-term impairment of learning and memory functions, diminish motivation, and delay behavioral responses (1) . In the periphery, cannabinoids are known to impair immune response through the disruption of macrophage-T cell cooperation, down-regulation of proinflammatory cytokine production and NO synthase activity in macrophages, resulting in the loss of general resistance to bacterial and viral infections (reviewed in refs 2 , 3 ).
Since the discovery of the endogenous cannabinoid receptor agonists anandamide (N-arachidonoylethanolamine, 20:4n-6 NAE) (4) and 2-arachidonoylglycerol (2-AG) (5 , 6) , much progress has been made in our understanding of cannabinoid receptor-mediated cell signaling (7; reviewed in ref 3 ). Nevertheless, some important questions remain unresolved. Because 2-AG is often present at higher levels than anandamide and because anandamide is generated by the same pathway as are the major saturated and monounsaturated NAEs, which may have different signaling functions (8 , 9) , it is possible that 2-AG is the true 'endocannabinoid' as proposed by Sugiura et al. (10) . Furthermore, the nature of the physiological signal leading to an enhanced generation of endocannabinoids is not well understood.
Much more information is available on the up-regulation of the immune response than on its negative control. In most cases, there is little information about how the positive signals are controlled and which factors return cells to their normal nonactivated state. Taking into account the fact that cannabinoid receptors may provide such a negative signal (11 12 13) , we addressed the hypothesis that endocannabinoids may be rapidly generated in response to proinflammatory stimulation of immune cells, thus providing a negative feedback control over the proinflammatory response. Here we show that the potent bioactive phospholipid, platelet-activating factor (PAF), which plays an important role in the onset and propagation of the proinflammatory immune response, stimulates the rapid, PAF receptor-mediated generation and release of 2-AG, but not anandamide, by human platelets and mouse P388D1 macrophages.
MATERIALS AND METHODS
Materials
Lyophilized PAF (a mixture of 16:0 and 18:0 PAF) was from Sigma (St. Louis, MO). PAF was dissolved in minimum essential medium (MEM) containing 0.1% fetal bovine serum (FBS) at a concentration of 1 x 10-5 M and kept in aliquots at -70°C. PAF aliquots were diluted just before use with phosphate-buffered saline (PBS), pH 7.4, containing 0.1% BSA or MEM-FBS medium for experiments with platelets and P388D1 macrophages, respectively. U-73122, wortmannin (both were first solubilized in DMSO), and D609 (dissolved in the MEM-FBS) were from Sigma-RBI (Natick, MA). Methylarachidonoylfluorophosphonate (MAFP) was from BIOMOL (Plymouth Meeting, PA). BN52021 (lyophilized) was a generous gift from the Institut Henri Beaufour (Les Ulis, France).
Cell culture and treatment
Human blood was obtained from healthy volunteers by venipuncture using heparinized tubes. Platelet-rich plasma (PRP) was obtained by blood centrifugation at 200 g for 10 min. Cells were counted with a hemocytometer and cell concentration was adjusted to ∼2 x 108 cells/ml with PBS (pH 7.4). Platelets were kept at 37°C for 10 min before treatment, treated with PAF or BN52021, followed by PAF, with periodic tube shaking by hand. After treatment, PRP was subjected to lipid extraction (14) and processed for monoacylglycerol and N-acylethanolamine (NAE) determination by GC/MS (see below). In some experiments, PRP was centrifuged at 400 g for 10 min at room temperature or treated with 1.5 x 10-8 M PAF for 30 s, then centrifuged at 400 g for 10 min at room temperature; lipids were extracted separately from plasma and platelets.
P388D1 mouse macrophages were obtained from ATCC (Manassas, VA). Cells were cultured in RPMI 1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and supplemented with 10% FBS. Cells were grown to 95% confluence, then treated with PAF with or without preincubation with BN52021, U-73122, wortmannin, or D609. After treatment, the medium was collected and cells were scraped off the dish in a mixture of methanol-2.5% aqueous sodium chloride (2:1, v/v) and transferred into a glass tube. Cells and media were subjected to lipid extraction (14) and processed for monoacylglycerol and NAE determination by GC/MS.
Gas chromatography-mass spectrometry
d4-derivatives of NAE, including 20:4n-6 NAE (anandamide) (15 , 16) and d5-derivatives of 2-AG, 2-oleoylglycerol (2-OG) and 2-linoleoylglycerol (2-LG) (17) were prepared as described previously and used as internal standards to quantify anandamide and monoacylglycerols, respectively. Internal standards (0.1 µg of each compound) were added during lipid extraction. Anandamide and monoacylglycerols were isolated as a single fraction by solid phase extraction on silica gel at 4°C in order to minimize acyl migration from the secondary to the primary hydroxyl group of glycerol (17) . Both lipid classes were converted to tert.-butyldimethylsilyl (tBDMS) derivatives and analyzed by GC/MS in selected ion monitoring mode as described (15 16 17) .
Data analysis
Each experiment was repeated two to four times. Results are expressed as the mean ± SE of at least three independent measurements. Analysis of statistical significance was done using Student's t test.
RESULTS
Stimulation of human platelets with PAF results in enhanced synthesis of 2-AG but not anandamide
Platelets are known to possess receptors for PAF and to respond to PAF treatment with irreversible or reversible aggregation, depending on the strength of stimulation (18 , 19) . As expected, human platelets in PRP responded to PAF treatment with aggregation (data not shown). After 5 min of treatment with PAF, we found a twofold increase in the level of 2-AG and a fourfold increase in the level of 1-AG content in PRP (Fig. 1 ). The levels of oleoylglycerols (2-OG and 1-OG) and linoleoylglycerols (2-LG and 1-LG) were also increased (Fig. 2 ), but no significant changes in anandamide were detected (Fig. 1) . To confirm that the effect of PAF is mediated by PAF receptors, we preincubated PRP with BN52021, a natural PAF receptor antagonist (20) , for 2 min before challenging the cells with PAF. This treatment resulted in complete blockage of PAF-induced aggregation (data not shown) and 2-AG generation and an almost complete inhibition of 1-AG synthesis (Fig. 3 ). Pretreatment of PRP with BN52021 had no effect on the level of anandamide.
Figure 1. PAF stimulates the synthesis of 2-AG and 1-AG in human platelets. Human PRP (1 ml, ∼2x108 cells/ml) in polypropylene tubes was stimulated with 1.5 x 10-8 M PAF at 37°C. One or 5 min after stimulation began, methanol and chloroform were added to stop it and initiate lipid extraction. Monoacylglycerols and NAEs were isolated by SPE, derivatized with tBDMS, and analyzed by GC/MS as described in Materials and Methods. Data for one of four representative experiments are shown. *P < 0.05, ***P < 0.001 against corresponding controls.
Figure 2. PAF stimulates the synthesis of 2-OG, 1-OG, 2-LG, and 1-LG in human platelets. Human platelets in PRP were treated and monoacylglycerols were analyzed as described in legend to Fig. 1 . Data for one of four representative experiments are shown. **P < 0.01, ***P < 0.001 against corresponding controls.
Figure 3. PAF receptor mediates PAF-induced stimulation of 2-AG and 1-AG synthesis in human platelets. Human PRP (1 ml, ∼2x108 cells/ml) in polypropylene tubes was preincubated with 2 x 10-4 M BN52021 for 2 min at 37°C, then stimulated with 1.5 x 10-8 M PAF for 5 min. 2-AG, 1-AG, and anandamide were analyzed as described in Materials and Methods. Data for one of three representative experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001 against corresponding controls.
Centrifugation of PRP results in the secretion of 2-AG into the plasma
To investigate the possible secretion of 2-AG synthesized by platelets into plasma, we performed sedimentation of platelets by PRP centrifugation at 400 g for 10 min. Unfortunately, even such gentle treatment resulted in the activation of 2-AG synthesis and its secretion into plasma together with 1-AG (Fig. 4 ). The same level of stimulation was seen when combining PRP stimulation with PAF and subsequent PRP centrifugation at 400 g (Fig. 4) . These data suggest that the sedimentation of human platelets by centrifugation is a stimulant sufficient to induce full activation of 2-AG synthesis, which cannot be further increased by additional platelet stimulation with PAF.
Figure 4. PRP centrifugation results in stimulation of 2-AG and 1-AG synthesis and secretion by human platelets. PRP was centrifuged at 400 g for 10 min and lipids were extracted from platelets and plasma. PRP was stimulated with 1.5 x 10-8 M PAF for 30 s, centrifuged at 400 g for 10 min, and lipids were extracted from platelets and plasma. 2-AG and 1-AG were analyzed as described in Materials and Methods. Data for one of two representative experiments are shown.[/B]
Mouse P388D1 macrophages respond to PAF stimulation by selective synthesis and release of 2-AG
Macrophages play a major role in initiation of the proinflammatory response. PAF is one of many biologically active molecules synthesized by macrophages, which also express the PAF receptor (21 , 22) . When we tested the response of mouse P388D1 macrophages to PAF, we found that these cells increased production of 2-AG, but not of anandamide (Fig. 5 see Fig. 7 ) or any other saturated and monounsaturated NAEs, which comprised 95—97% of the total (data not shown). In contrast to platelets, stimulation of macrophages with PAF resulted in preferential induction of the synthesis of 2-AG but not of 1-AG, and the content of 2-OG, 1-OG, and 2-LG, 1-LG in macrophages remained almost unchanged (Fig. 6 ). The level of 2-AG was highest 30—45 s after cell stimulation and declined gradually thereafter (Figs. 5 , 7) . The 2-AG produced as a result of stimulation with PAF was immediately released into the medium (Fig. 7 ), where its level also declined in time. A short preincubation of the cell with MAFP (1 µM for 5 min), an inhibitor of fatty acid amidohydrolase and acyl hydrolases, decreased synthesis and release of 2-AG, but had no effect on anandamide content in cells or the medium. The effect of PAF was completely blocked by preincubation with the PAF receptor antagonist BN52021 (Table 1 ), which confirms the link between PAF-induced 2-AG synthesis and activation of PAF receptors.
Figure 5. PAF specifically stimulates 2-AG synthesis in mouse P388D1 macrophages. Mouse P388D1 macrophages were grown to 95% confluence and stimulated with 3 x 10-8 M PAF. At the times indicated, the medium was poured off, lipids were extracted from attached macrophages, and 2-AG, 1-AG, and anandamide were analyzed as described in Materials and Methods. Data for one of three representative experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001 against corresponding controls.
Figure 7. P388D1 macrophages release 2-AG into the medium upon stimulation with PAF. Mouse P388D1 macrophages were grown to 95% confluence. Cells were stimulated with 3 x 10-8 M PAF or preincubated with 1 µM MAFP for 15 min, then stimulated with 3 x 10-8 M PAF for up to 20 min. Lipids were extracted from the medium, separated from the attached cells, and 2-AG and anandamide were analyzed as described in Materials and Methods. Data for one of two representative experiments are shown.
Figure 6. PAF does not efficiently stimulate the synthesis of OG and LG in P388D1 macrophages. Mouse P388D1 macrophages were grown to 95% confluence and stimulated with 3 x 10-8 M PAF. The medium was poured off, lipids were extracted from attached macrophages, and monoacylglycerols were analyzed as described in Materials and Methods. Data for one of three representative experiments are shown. *P < 0.05, **P < 0.01 against corresponding controls.
Table 1. The effect of PAF receptor antagonist, phospholipase C, and PI3-kinase inhibitors on PAF-induced 2-AG synthesis by P388D1 macrophagesa
Enhanced 2-AG generation is due to the activation of phosphatidylinositol- and phosphatidylcholine-specific phospholipases C
The very short time needed to activate 2-AG synthesis after macrophage stimulation with PAF suggested a possible link to the known PAF receptor-mediated activation of phosphatidylinositol (PI)- and phosphatidylcholine (PC) -specific phospholipases C (23 24 25 26) . Incubation of macrophages with the inhibitor of PI-specific phospholipase C, U-73122, resulted in a strong inhibition of 2-AG synthesis induced by PAF (Table 1) . The inhibitor of PC-specific phospholipase, C D609, also decreased 2-AG production, but at a higher inhibitor concentration and to a lesser extent (Table 1) .
To confirm that 2-AG is generated from PI through activation of PI turnover, macrophages were preincubated with 100 nM wortmannin, which is known to block PI-3 kinase. This inhibitor was found to decrease slightly PAF-stimulated 2-AG production (Table 1) .
DISCUSSION
How cells maintain their physiological homeostasis and control their response on stimulation by a variety of factors remains one of the most important questions in cell physiology. In immune response, this knowledge is important in order to understand how cells control their activation state and what happens when this control fails, as it does, for example, during endotoxic shock (27) .
The increasing knowledge about cannabinoid receptor-dependent signaling suggests that the endocannabinoid system, which includes CB1/CB2 receptors and their endogenous ligands, may function as a down-regulatory control system, possibly linked to a proinflammatory immune response. If such a link exists, endocannabinoids could be generated as a consequence of proinflammatory cell stimulation, be secreted, and then stimulate CB1/CB2 receptors expressed by immune and adjacent cells with a resulting down-regulation of the initially induced proinflammatory response. Of two endocannabinoids, 2-AG is the first candidate to serve as an 'immediate response' signaling molecule, as it may be generated from plasma membrane phospholipids (28) at the site of the arrival of a proinflammatory signal. In contrast, the biosynthesis of anandamide and other NAEs occurs mainly in endoplasmic reticulum (8) , which makes them less likely to serve as the immediate regulators of cell reactivity.
PAF is one of the most potent endogenous proinflammatory autocoids and is linked by positive feedback relationships with multiple proinflammatory cytokines (29) . PAF receptors are linked to Gi- and Gs-proteins (22 , 30) , and the activation of PAF receptors results in stimulation of PI turnover (22 23 2425 26) . As PI turnover generates diacylglycerols containing arachidonic acid at the sn-2 position (31 , 32) , only one additional step will lead to 2-AG formation through lipase activity. Indeed, biosynthesis of 2-AG through this pathway was proposed to operate in neuronal cells (28) ; the conversion of 1-acyl-2-arachidonoylglycerol by neuroblastoma cell homogenate to 2-AG was also demonstrated (33) . Although ionomycin- or lipopolysaccharide (LPS) -induced formation of 2-AG was demonstrated for both circulating and cultured macrophages (34 , 35) , no information has yet been presented regarding the biosynthetic pathways of 2-AG formation in immune cells. Our finding that PAF receptor-mediated 2-AG formation in mouse P388D1 macrophages is stimulated within seconds (Figs. 5 , 7) clearly shows that proinflammatory stimulation through PAF receptors is accompanied by the immediate formation of this potent endogenous ligand for cannabinoid receptors. Most important, 2-AG formation in macrophages is highly selective since the stimulated synthesis of 1(3)-AG, and especially that of other 2- or 1(3)-acylglycerols, is significantly less pronounced (Figs. 5 , 6) .
The discovery of the immediate release of 2-AG into the medium by macrophages upon stimulation with PAF (Fig. 7) is particularly important. This finding suggests that macrophages can quickly provide 2-AG locally or into the circulation, where it can bind to cannabinoid receptors expressed by a variety of cells. It becomes clear that 2-AG may be regarded as an autacoid important for macrophage—immune cell and macrophage—endothelial cell cross-talk with potential systemic significance. Attention should be directed to the endothelial cell—macrophage interaction, where PAF is known to serve as one of the first chemotactic signals used by endothelial cells to initiate macrophage attachment (22) . On the other hand, endothelial cells were shown to express cannabinoid receptors (36) , generate and release 2-AG (36) , and respond to 2-AG by dilation (37) . All this suggests that macrophages and endothelial cells are important elements of a complex regulatory cellular network that is able to use the endocannabinoid system to control its functions.
We found no significant changes in anandamide concentrations in macrophages or in the medium during our experiments, even when the cells were preincubated with the fatty acid amidohydrolase inhibitor MAFP (Fig. 7) . This is consistent with our observation that treatment of mouse peritoneal macrophages with the ionophore A23187 (in the absence of exogenous ethanolamine) does not result in enhanced NAE synthesis. In contrast, other groups were able to demonstrate enhanced anandamide production as a result of ionophore or LPS treatment of macrophages (34 , 38) ; this discrepancy remains unexplained. Although one report (39) suggested a correlation between stimulated arachidonic acid release and anandamide production elicited in RAW264.7 mouse macrophages by a variety of treatments, including PAF, the rationale and biochemical explanation for this observation have yet to be provided. Our experiments clearly show that the response of macrophages to a specific proinflammatory stimulation through the PAF receptor does not involve activation of NAE biosynthesis. However, activation of PI- and PC-specific phospholipases C appeared to mediate the effect of PAF. It is known that activation of PAF receptors leads to stimulation of PI-specific phospholipase C (23 24 25 , 40) and, under some conditions, to PC-specific phospholipase C (23 , 40) . In our experiments, both PI- and PC-specific phospholipase C inhibitors as well as a PI3-kinase inhibitor were found to inhibit or decrease PAF-induced 2-AG generation (Table 1) . Although more experiments are needed with a broader range of inhibitor concentrations to clarify the roles of certain enzymes in 2-AG biosynthesis and eliminate possible nonspecific effects, our data suggest a link between PAF receptor-dependent phospholipase C activation and 2-AG generation in P388D1 macrophages.
Our finding that platelets respond to PAF with synthesis and release of 2-AG is important. In the bloodstream, thrombocytes are in contact with both immune and endothelial cells and thus provide an element of a joint response triggered by intercellular communication. The fact that PAF (which can be secreted by macrophages or presented to macrophages by endothelial cells) (22) is a potent activator of 2-AG synthesis in platelets strengthens the possible role of thrombocytes not only in positive but also in negative regulation of immune response. In contrast to macrophages, which respond to PAF with the selective synthesis and release of 2-AG, the production of all monoacylglycerols is stimulated to about the same extent in platelets (Figs. 1 , 2) . Thus, platelets do not possess a mechanism for selective 2-AG synthesis and provide 2-AG along with all other monoacylglycerols. This may serve to protect 2-AG from lipase degradation (41) . In accordance with previous findings (34 , 42) , we did not find anandamide in platelets. The extremely low levels of anandamide we detected in PRP are most likely due to the presence of anandamide in plasma and not in platelets, as PAF stimulation did not change the total anandamide content in PRP and the same amount of anandamide was present in PRP as in plasma after PRP stimulation with PAF and centrifugation.
Our finding that human platelets respond with 2-AG production to even gentle centrifugation may help to explain the known property of human thrombocytes to lose their sensitivity to PAF after washing (43) . Considering that 2-AG in platelets may also be produced through PI turnover and that 2-AG production is stimulated in platelets during centrifugation, it is logical to suggest that such a treatment depletes platelet PI from participating in the PAF-induced signaling cascade and renders platelets refractory to stimulation with PAF.
We doubt that 2-AG production is aimed at platelet stimulation, as was recently proposed (44) , because the 2-AG concentrations used in that study (50—400 µM) were far beyond physiological relevance. On the contrary, it is known that activation of the CB2 receptor by cannabinoids or 2-AG results in down-regulation of proinflammatory cytokine production (2 , 3 , 13) . As was shown for mouse splenocytes and cultured cell lines, cannabinoid receptor activation causes the inhibition of NF-AT and AP-1 nuclear factor binding to corresponding binding sites, thus blocking the up-regulation of interleukin-2 (IL-2) transcription in these cells (11 , 45 , 46) . It is possible that a similar mechanism functions in macrophages, resulting in inhibition of the synthesis and secretion of tumor necrosis factor α, IL-6, and IL-8 (47 48 49) . It is logical to suggest that PAF activity itself may also be negatively controlled through the inhibition of transcription of PAF receptors, as the PAF receptor is transcriptionally regulated by NF-κB and AP-1 (50) . Thus, 2-AG and cannabinoid receptors may occupy a central place in the immediate negative control of proinflammatory immune response, serving control functions at the transcriptional level and targeting the binding of major nuclear factors to corresponding consensus binding sites.
In summary, the present findings document for the first time a direct and specific link between 2-AG production by macrophages and platelets and cell activation through the PAF receptor. The PAF receptor-mediated activation of PI- and PC-specific phospholipases C mediates the rapid and specific enhancement of 2-AG synthesis in macrophages, whereas platelets may play a role as the amplifier of the response by providing additional 2-AG on stimulation. The fact that PAF receptor activation does not stimulate rapid anandamide and NAE synthesis, either in macrophages or in platelets, supports the hypothesis that 2-AG is the primary physiologically important endocannabinoid (10 , 51 , 52) .
ACKNOWLEDGMENTS
This work was supported by U.S. Public Health Service grant GM45741 (H.H.O.S.), INTAS grant 97—1297 (E.V.B.), and The Hormel Foundation.
Received for publication March 7, 2001. Revision received June 15, 2001.
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Source: Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells
