Download Cannabinoid receptors in microglia of the central nervous system

Cannabinoid receptors in microglia of the central nervous
system: immune functional relevance
G. A. Cabral1 and F. Marciano-Cabral
Department of Microbiology and Immunology, Virginia Commonwealth University, School of Medicine,
Richmond, Virginia
Abstract: Microglia, resident macrophages of the
brain, function as immune effector and accessory
cells. Paradoxically, they not only play a role in
host defense and tissue repair but also have been
implicated in a variety of neuropathological processes. Microglia, in addition to exhibiting phenotypic markers for macrophages, express CB1 and
CB2 cannabinoid receptors. Recent studies suggest
the existence of a third, yet-to-be cloned, non-CB1,
non-CB2 cannabinoid receptor. These receptors
appear to be functionally relevant within defined
windows of microglial activation state and have
been implicated as linked to cannabinoid modulation of chemokine and cytokine expression. The
recognition that microglia express cannabinoid receptors and that their activation results in modulation of select cellular activities suggests that they
may be amenable to therapeutic manipulation for
ablating untoward inflammatory responses in the
central nervous system. J. Leukoc. Biol. 78:
1192–1197; 2005.
Key Words: brain immune modulation 䡠 cytokines 䡠 marijuana
nitric oxide
䡠
Marijuana is a complex plant material, which can elicit a
variety of pharmacological and immunological effects. Its
major psychoactive component is ⌬-9-tetrahydrocannabinol
(THC), a compound that has been reported to account for the
majority of effects on the immune system [1, 2]. Several
modes of action have been proposed as accounting for the
effects of THC on immune cells. At high concentrations,
such as those exceeding micromolar levels, THC and other
cannabinoids may have direct effects on membranes, as they
are highly lipophilic [3]. However, stereospecificity and
structural requirements for biological activity indicate that
cannabinoids also act through specific receptors. To date,
two unique cannabinoid receptors have been identified. The
CB1 is located primarily in the brain and is responsible for
most, if not all, of the centrally mediated effects of cannabinoids [4, 5]. The CB2 is present primarily in cells of the
immune system but has been detected in adult human
uterine tissue and embryonic organs and adult rat retina [6,
7]. Both receptors are Gi/o protein-coupled, as evidenced by
inhibition of adenylyl cyclase [8], inhibition of N-type calcium channels [9], and increased binding of nonhydrolyz-
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Journal of Leukocyte Biology Volume 78, December 2005
able guanylyl-5⬘-O-(␥-thio)-triphosphate in the presence of
cannabinoids [10]. The CB1 differs from the CB2, however,
in that it also modulates Q-type calcium channels [11].
Recent studies suggest the existence of a third receptor, a
non-CB1/non-CB2 receptor [12–14].
Major targets of marijuana and exogenous cannabinoids
in the immune system are cells of macrophage lineage.
Ultrastructural abnormalities have been observed in alveolar macrophages of humans who have been heavy users of
marijuana [15] and in peritoneal macrophages of mice exposed in vitro to various concentrations of pure THC [16]. In
addition, various functional defects of alveolar or peritoneal
macrophages obtained from humans, rats, or mice following
in vivo or in vitro exposure to marijuana or THC have been
observed [17]. Microglia constitute a resident population of
macrophages in the brain, the spinal cord, and retina and
are morphologically, phenotypically, and functionally related to cells of macrophage lineage [18 –21]. The function
of quiescent microglia in normal brain is not well understood, but in pathological conditions, these cells play an
active role as immunoeffector/accessory cells. Microglia
migrate and proliferate during and after injury and inflammation [22–25]. Once activated, they produce various cytokines including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-␣ (TNF-␣) and express major histocompatibility
complex classes I and II antigens and the complement
receptor, CR3. Microglia are also phagocytic and can process antigens and exert cytolytic functions. Paradoxically,
these cells not only play a role in host defense and tissue
repair in the central nervous system [26, 27] but also have
been implicated in nervous system disorders such as multiple sclerosis [28], Alzheimer’s disease [29], Parkinson’s
disease [30], and AIDS dementia [31–33].
Microglia, as macrophage-like cells, undergo a process of
maturation, differentiation, and activation, which is characterized by differential gene expression and correlative acquisition
of specified functions [22–25, 34, 35]. This pattern of differential expression also applies to cannabinoid receptors (Fig.
1). Using an in vitro model of multistep activation, in which
1
Correspondence: Department of Microbiology and Immunology, Virginia
Commonwealth University, School of Medicine, 1101 E. Marshall Street,
Richmond, VA 23298-0678. E-mail: [email protected]
Received April 24, 2005; revised August 19, 2005; accepted August 22,
2005; doi: 10.1189/jlb.0405216
0741-5400/05/0078-1192 © Society for Leukocyte Biology
Fig. 1. Differential levels of CB2 mRNA are detected in neonatal rat brain
cerebral cortex microglia in relation to cell activation state. (A) Southern
blot of mutagenic reverse transcriptase-polymerase chain reaction (MRTPCR) products from total nucleic acid of microglia maintained in medium
(“responsive”) or treated (6 h) with 100 U/ml rat interferon-␥ (IFN-␥;
“primed”), 100 U/ml rat IFN-␥ plus 100 ng/ml lipopolysaccharide (LPS;
multisignal-activated), or 1 ␮g/ml LPS (“fully activated”). MRT-PCR was
performed as described [36]. RT primers were used, which introduced a
single base mismatch into the cDNA of CB1 or CB2 to generate a unique
MspI or HindIII restriction site, respectively. The upper band of the doublet
is amplified genomic DNA (gDNA). The lower band of the doublet (arrow)
represents an amplified cDNA product from mRNA. (Upper panel) CB1
mRNA was detected at low levels for all treatment groups. (Lower panel)
High levels of CB2 mRNA were detected for microglia maintained in
medium or IFN-␥ (100 U/ml), and low levels were detected for cells treated
with IFN-␥ plus LPS or with LPS alone. (B) Graphic representation of
relative levels of CB2 mRNA depicted by MRT-PCR. The graph represents
a single experiment performed in triplicate. The ordinate designated as
Relative OD Units represents densitometric analysis of cDNA-amplified
product based on area X pixel density and is represented relative to the
corresponding densitometry obtained for the gDNA-amplified product. A
significant decrease in levels of CB2 mRNA was recorded for cells treated
with LPS or with LPS plus IFN-␥ as compared with those for untreated
cells. Error bars are ⫾ SD, n ⫽ 3, **, P ⬍ 0.01. OD, Optical density.
microglia are driven sequentially from a “resting” state to
responsive, primed, and fully activated states, the CB1 was
found at constitutive low levels at all states of cell activation.
In contrast, the CB2 was found to be expressed inducibly and
at maximal levels when microglia were in responsive and
primed states. Collectively, the observations that expression of
CB1 is constitutive, and that of the CB2 is inducible, that the
two receptors are present at disparate levels, and that they
exhibit distinctive compartmentalization [36, 37] suggest that
the two receptors have discriminative, functional relevance in
microglia.
We have demonstrated that cannabinoids inhibit the production of inducible nitric oxide (iNO) by microglia, which are
fully activated, in a mode that is mediated, at least in part, by
the CB1 [36]. Table 1 lists select ligands, which have been
used to study cannabinoid receptor-associated functions. Pretreatment of microglia with the CB1/CB2 high-affinity binding
enantiomer CP55940 (Ki⫽0.9 nM) resulted in inhibition of
iNO (Fig. 2) elicited in response to bacterial LPS used in
combination with IFN-␥. A less-inhibitory effect was exerted
by the lower affinity-binding, paired enantiomer CP56667
(Ki⫽62 nM). The differential effect exerted by CP55940 versus
CP56667 is consistent with a role of a cannabinoid receptor in
the inhibition of iNO production, as selective binding affinity of
paired cannabinoid stereoisomers has been shown to correlate
with bioactivity in vivo and in vitro [5], and differential doserelated effects of one enantiomer versus its enantiomeric pair
are implicative of a functional linkage to a receptor. The
receptor, which was found as linked to the cannabinoid-mediated inhibition of iNO production, was CB1-based through the
application of antagonist experiments. Treatment of microglia
with the CB1-selective antagonist SR141716A prior to exposure to the agonist CP55940 blocked the CP55940-mediated
inhibition of iNO production.
Cannabinoid-mediated alteration of microglial activities,
as linked to the CB2, appears to be limited to that window of
cell activation encompassing the responsive and primed
states, for which the CB2 is expressed at high levels. Critical
activities of responsive and primed microglia include chemotaxis and antigen processing/presentation, respectively.
We have demonstrated that the partial agonist THC and the
full agonist CP55940 inhibit the processing of select antigens by murine peritoneal macrophages and that this effect
is mediated, at least in part, by the CB2 [47, 48]. These
observations suggest that a similar effect may occur for
microglia. There is, however, accumulating evidence that
the CB2 is expressed in vivo by microglia in the context of
a variety of inflammatory states [49]. We have shown that
Acanthamoeba culbertsoni (Fig. 3A), an opportunistic, human pathogen, which is the causative agent of granulomatous amebic encephalitis (GAE) [50], can induce increased
levels of CB2 in microglia in vitro (Fig. 3B). Comparable
results were obtained when total RNA from brain of mice
inoculated with A. culbertsoni was assessed for CB2 mRNA
(Fig. 3C). Histopathological analysis of brain sections revealed focal brain lesions containing amebae circumscribed
by cells exhibiting morphological features typical of microglia (Fig. 3D). Collectively, these results, although not definitive, are consistent with microglia as the source of inducible expression of CB2 in vivo and suggest a potential
target for ablating inflammation associated with GAE.
Recent studies suggest that a third, yet-to-be cloned
receptor, a non-CB1, non-CB2 receptor [12–14], can also
play a role in cannabinoid mediation of microglial activities.
We have shown that the potent cannabinoid receptor agonist
levonantradol (Ki⫽1.06 nM) inhibits the inducible expression of mRNAs for the proinflammatory cytokines IL-1␣ and
TNF-␣ in a mode that is not blocked by the CB1 antagonist
SR141716A or the CB2 antagonist SR144528 (Fig. 4).
Similarly, the partial agonist THC (Ki⫽42 nM) and the
agonist CP55940 (Ki⫽0.9 nM) inhibited the induction of
Cabral and Marciano-Cabral Cannabinoid receptors and brain immunity
1193
TABLE 1.
Properties of Select Cannabinoid Receptor Ligands
Description
Receptor
target
THC
Partial agonist
CP55940
Agonist
CP56667
Stereoisomer of
CP55940
HU210
Agonist
HU211
Stereoisomer of
HU210
Levonantradol
Agonist
Dextronantradol
Stereoisomer of
levonantradol
SR141716A
Antagonist
CB1
SR144528
Antagonist
CB2
Liganda
Action
Comments
CB1/CB2
Activates
CB1/CB2
[38]
CB1/CB2
Activates
CB1/CB2
–
Has lower intrinsic activity
than full agonist and
produces lower maximum
effect; CB1 Ki ⫽ 40.7
nM; CB2 Ki ⫽ 36.4 nM
CB1/CB2 Ki ⫽ 0.9 nM
Pharmacologically less
active than CP55940;
CB1/CB2 Ki ⫽ 62 nM
CB1Ki ⫽ 0.0606 nM; CB2
Ki ⫽ 0.524 nM
Pharmacologically less
active than HU210; CB1
Ki ⬎ 10 ␮M
CB1/CB2 Ki ⫽ 1.06 nM
[5, 36]
Pharmacologically less
active than levonantradol;
CB1/CB2 Ki ⫽ 3100 nM
CB1 Ki ⫽ 5.6 nM; CB2 Ki
⫽ 1000 nM
[45]
CB1 Ki ⫽ 437 nM; CB2 Ki
⫽ 0.60 nM
[46]
–
CB1/CB2
–
CB1/CB2
–
Activates
CB1/CB2
–
Activates
CB1/CB2
–
Blocks
agonist
activation
of CB1
Blocks
agonist
activation
of CB2
References
[5, 39–41]
[11, 42, 43]
[44]
[45]
[39]
a
Ligand abbreviations: CP55940: (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; CP56667: (1S,3S,4S)3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; HU210: (6aR,10aR)-3-(1,1⬘-dimethylheptyl)-6a,7,10,10a-tetrahydro-1hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol; HU211: 3-(1,1-dimethylheptyl)-6aS,7,10,10aS-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]
pyran-9-methanol; SR141716A: N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR144528:
N{(1S)-endo-1,3,5-trimethylbicyclo[2.2.1]heptan-2-yl}-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide. Ki: molar association constant.
cytokine mRNAs for IL-1␣, IL-1␤, IL-6, and TNF-␣ in a
mode that was not blocked by the CB1 or the CB2 antagonist
(data not shown). Furthermore, enantiomeric selectivity for
the CB1/CB2 high-affinity ligands, as compared with the
paired, lower affinity counterparts, was not observed [51].
The “less bioactive” enantiomers CP56667 and HU211
exhibited inhibitory activity comparable with that of the
potent CB1/CB2 agonists CP55940 and HU210, respectively. A similar outcome was obtained when the stereoisomers levonantradol (Ki⫽1.06 nM) and dextranantradol
(Ki⫽3100 nM) were used. Collectively, the observations
that gene expression for proinflammatory cytokines is associated with microglia that are fully activated and express
low levels of CB2, that stereoselective paired cannabinoids
exert comparable inhibitory effects on the induction of
proinflammatory cytokine mRNAs, and that the CB1 and
CB2 selective antagonists SR141716A and SR144528 do not
block the inhibition of cytokine gene expression by the
agonists CP55940 and levonantradol indicate that cannabinoid-mediated modulation of proinflammatory cytokine gene
expression is not linked to the CB1 or the CB2. Whether
these results are indicative of the presence of a non-CB1,
non-CB2 receptor in microglia, which is functionally rele-
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Journal of Leukocyte Biology Volume 78, December 2005
vant when these cells are in a state of full activation, awaits
biochemical and molecular analysis.
In summary, microglia are macrophage-like cells that undergo a multistep process to full activation during inflammation. This multistep process is associated with differential gene
expression and the acquisition of correlative functional activities. The differential expression of genes in relation to cell
activation also applies to cannabinoid receptors. The CB1 is
expressed constitutively and at low levels throughout multistep
activation, indicating a potential for this receptor to be functionally relevant for a broad spectrum of cannabinoid-mediated
effects. However, as it is expressed at low levels, it may exhibit
less “sensitivity” to the action of nonselective CB1/CB2 agonists as compared with the CB2. In contrast, the CB2 is expressed inducibly and is present at high levels as compared
with the CB1 when microglia are in responsive and primed
states of activation. A signature, functional activity attributed
to microglia, when in a responsive state, is chemotaxis, and
these observations are consistent with reports of cannabinoid
effects on cell migration, which is linked to the CB2 [52].
Recent pharmacological data suggest the existence of a third
cannabinoid receptor, a non-CB1, non-CB2 receptor, which
may play a role in cannabinoid-mediated inhibition of proin-
http://www.jleukbio.org
Fig. 2. CP55940-mediated inhibition of iNO release by microglia is blocked
by the CB1 receptor-selective antagonist SR141716A. Microglia were pretreated (1 h) with 5 ⫻ 10⫺7 M SR141716A prior to exposure (8 h) to 5 ⫻ 10– 6
M CP55940 or CP56667 and LPS plus IFN-␥ activation (24 h). Culture
supernatants were assayed for nitrite using the Griess reagent. The CP55940
inhibition was stereoselective, as the paired enantiomer CP56667 was less
bioactive. Results (mean⫾SEM of triplicate wells) are expressed as percent
inhibition versus vehicle control (*, P⬍0.01, vs. SR141716A). Nitrite accumulation in LPS plus IFN-␥-treated vehicle control cultures was 29.3 ⫾ 3.5
(␮M/106 cells). VEH, Vehicle (0.01% ethanol).
Fig. 4. The CB1 and CB2 receptor-specific antagonists do not block the
inhibitory effects of levonantradol (Ki⫽1.06 nM) on cytokine mRNA expression. Microglia were treated (4 h) with levonantradol or pretreated (1 h)
with 1 ⫻ 10⫺6 M SR141716A or SR144528 prior to exposure (3 h) to 1 ⫻
10– 6 M levonantradol. Cells then were treated (6 h) with LPS (100 ng/ml),
and levels of cytokine mRNA were determined using the RiboQuant
multiprobe RPA (PharMingen). The ordinate designates that the data are
presented as the percent LPS-treated (100 ng/ml) vehicle control. Results
are expressed as the mean percent cytokine mRNA expression of triplicate
cultures versus that for the LPS-treated vehicle mean ⫾ SEM (*, P⬍0.01;
two-tailed Student’s t-test). SR1: CB1-selective antagonist SR141716A;
SR2: CB2-selective antagonist SR144528; VEH: 0.01% ethanol.
Fig. 3. Augmentation of CB2 mRNA levels in response
to A. culbertsoni. (A) Scanning electron micrograph of an
A. culbertsoni trophozoite. (B) Graphic representation of
relative levels of cannabinoid receptor mRNA. Microglia
were incubated with A. culbertsoni at a microglia:ameba
ratio of 10:1. Levels of mRNA for the CB1 receptor
remained unaffected, but those for the CB2 receptor
exhibited a time-dependent augmentation as determined
by RNase protection assay (RPA; PharMingen, San Diego, CA). The ordinate designated as Relative OD Units
represents densitometric analysis based on area X pixel
density relative to that of constitutively expressed L32
ribosomal (L32) mRNA product for each sample. The
graph represents a representative single experiment. (C)
RPA detection of mRNA for CB1 and CB2 receptors from
whole brain homogenates of mice infected intranasally
with A. culbertsoni (1⫻105 50% lethal dose). As expected, an excess of mRNA for the CB1 was obtained
from whole brain homogenates. An apparent increase in
levels of CB1 mRNA was noted for homogenates of brain
obtained at 14 and 21 days. mRNA levels for the CB2
receptor exhibited a time-related increase following infection with amebae. Infection was confirmed by isolation
of amebae in culture from mouse brains. (D) Hematoxylin
and eosin-stained murine brain section demonstrating
multiple foci of amebae surrounded by cells (arrows),
which morphologically resemble microglia. (A, D) Original bars, 10 ␮m.
Cabral and Marciano-Cabral Cannabinoid receptors and brain immunity
1195
flammatory cytokine production, an activity that may be associated with microglia when fully activated.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes
of Health/National Institute on Drug Abuse Awards R01
DA05832, R01 DA015608, and 2P50 DA05274.
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