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The 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{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: anandamide • platelet-activating factor • macrophages • human platelets
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
Deuterated 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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.
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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.
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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.
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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)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
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
24
25
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 
, 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-
B 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)
.
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ACKNOWLEDGMENTS |
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Received for publication March 7, 2001.
Revision received June 15, 2001.
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9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells. Eur. J. Pharmacol. 330,231-240[Medline]
production in murine macrophages, and in mice. Eur. J. Pharmacol. 406,R5-R7[Medline]
B. Biochem. Biophys. Res. Commun. 205,1137-1142[Medline]
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