Emerging role of the cannabinoid receptor CB₂ in immune regulation: therapeutic prospects for neuroinflammation

Abstract

There is now a large body of data indicating that the cannabinoid receptor type 2 (CB₂) is linked to a variety of immune events. This functional relevance appears to be most salient in the course of inflammation, a process during which there is an increased number of receptors that are available for activation. Studies aimed at elucidating signal transduction events resulting from CB₂ interaction with its native ligands, and of the role of exogenous cannabinoids in modulating this process, are providing novel insights into the role of CB₂ in maintaining a homeostatic immune balance within the host. Furthermore, these studies suggest that the CB₂ may serve as a selective molecular target for therapeutic manipulation of untoward immune responses, including those associated with a variety of neuropathies that exhibit a hyperinflammatory component.

Cannabinoids are highly lipophilic molecules that have been shown to alter the functional activities of immune cells in vitro and in vivo. The term ‘exogenous cannabinoids’ describes cannabinoids that are extracted from the marijuana plant Cannabis sativa or are synthesised in the laboratory. The marijuana cannabinoids delta-9-tetrahydrocannabinol (Δ⁹-THC), cannabinol (CBN) and cannabidiol (CBD) have been the most studied exogenous cannabinoids. Δ⁹-THC acts as a partial receptor agonist: on binding to cannabinoid receptors it elicits a response that never reaches the maximum efficacy. This compound is the major psychoactive and immunomodulatory component in marijuana and primarily exerts immunosuppressive effects on immune cells at peripheral sites and within the central nervous system (CNS). Synthetic exogenous cannabinoids that have been used widely in research include CP55940, WIN55212-2, SR141716A and SR144528 (Fig. 1). CP55940 and WIN55212-2 are full receptor agonists: on binding to cannabinoid receptors they elicit responses that garner maximum efficacy.

Figure 1. Representative cannabinoids. (a) Exogenous cannabinoids. Δ⁹-THC is a partial agonist for CB₁ and CB₂; CP55940 and WIN55212-2 are full agonists for CB₁ and CB₂. (b) Endogenous cannabinoids. AEA and 2-AG show agonist behaviour at CB₁ and CB₂. (c) Cannabinoid receptor antagonists. SR141716A is an antagonist for CB₁; SR144528 is an antagonist for CB₂. Abbreviations: AEA, arachidonoylethanolamide (anadamide); CB₁, cannabinoid receptor 1; CB₂, cannabinoid receptor 2; CP55940, (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; SR141716A, 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide hydrochloride; SR144528, (1S-endo)-5-(4-Chloro-3-methylphenyl)-1-((4-methylphenyl)methyl)-N-(1,3,3-trimethylbicyclo(2.2.1)hept-2-yl)-1H-pyrazole-3-carboxamide; Δ⁹-THC, delta-9-tetrahydrocannabinol; WIN55212-2, (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone.

Endocannabinoids (‘endogenous cannabinoids’) constitute a second group of cannabinoids; these are found natively in vertebrate systems and are produced by a variety of cell types, including endothelial cells (Ref. 1), adipocytes (Ref. 2), glial cells (Ref. 3), macrophages (Ref. 4) and Purkinje cells (Ref. 5). These molecules are constituent elements of the ‘endocannabinoid system’, which also encompasses mediators responsible for their synthesis, metabolism and catabolism, as well as the cannabinoid receptors that serve as their molecular targets. Endocannabinoids are derivatives of integral components of cellular membranes and act as hydrophobic lipid messengers. As a result of their hydrophobicity, these molecules are not able to translocate in aqueous environments and, upon release, activate cannabinoid receptors locally or on nearby cells. Within the CNS, these bioactive lipids act as synaptic modulators (retrograde messengers); however, unlike other synaptic messengers such as the neurotransmitters acetylcholine and dopamine, endocannabinoids are not presynthesised and stored in vesicles but are produced ‘on demand’.

The first endocannabinoid to be identified was arachidonoylethanolamide (AEA; anandamide), which was isolated from porcine brain (Ref. 6). AEA is the amide component of arachidonic acid and ethanolamine. The second endocannabinoid to be identified was 2-arachidonoylglycerol (2-AG), which was isolated from canine gut (Ref. 7). 2-AG is an ester derivative of arachidonic acid and glycerol, and is synthesised from the hydrolysis of 1,2-diacylglycerol (DAG) by a DAG lipase. In the brain, 2-AG is more bioactive and abundant than AEA. Both AEA and 2-AG are transported across the cell membrane before being degraded by fatty acid amide hydrolase (FAAH), although 2-AG can also be degraded by monoacylglycerol lipase (MGL), a serine hydrolase (Refs 8, 9, 10). Several reports suggest that transport across cellular membranes is facilitated by carrier proteins, which have yet to be fully identified (Refs 11, 12, 13, 14). In addition, while cannabinoid receptors serve as the molecular targets of AEA and 2-AG, it has been reported that these two compounds also can exert their effects in a receptor-independent fashion (Refs 15, 16, 17, 18).

Cannabinoid receptors

CB₁

The initial evidence for the existence of a cannabinoid receptor came from pharmacological studies. Treatment of neuroblastoma cells with Δ⁹-THC, or with the synthetic compounds levonantradol and desacetyllevonantradol, resulted in inhibition of plasma membrane activity of adenylate cyclase, the enzyme that catalyses the conversion of ATP to cyclic AMP (cAMP) and pyrophosphate (Refs 19, 20). However, in contrast to levonantradol, dextronantradol was shown to have no effect on this activity, indicating that the inhibition was stereoselective – a requisite condition for a receptor-mediated action. Additional studies demonstrated that the putative cannabinoid receptor was coupled to an inhibitory guanine-nucleotide-binding complex (G_i/o), because treatment with pertussis toxin reversed the inhibitory effect on adenylate cyclase (Ref. 21). Radioligand binding assays and in situ mRNA hybridisation demonstrated that the receptor was distributed throughout the brain and was localised predominantly to the cerebellum, cerebral cortex, hippocampus, basal ganglia and spinal cord (Refs 6, 22, 23, 24). Subsequently, the gene encoding the receptor (CNR1) was isolated and cloned from a rat brain cDNA library (Ref. 22), revealing the sequence for a 473 amino acid long, seven-transmembrane G-protein-coupled protein with an extracellular, glycosylated N-terminus and an intracellular C-terminus (Fig. 2a). This receptor was referred to initially as the ‘neuronal’ or ‘central’ cannabinoid receptor and has since been designated cannabinoid receptor 1 (CB₁).

Figure 2. Human cannabinoid receptor amino acid sequences. (a) Amino acid sequence of the full-length human cannabinoid receptor 1 (CB₁). The putative asparagine-linked glycosylation sites are shown as Ψ. The seven transmembrane domains are highlighted and noted TM1 to TM7. The Genebank accession numbers are NM_016083, NM_001840 and NP_057167.2. (b) Amino acid sequence of the full-length human cannabinoid receptor 2 (CB₂). The putative asparagine-linked glycosylation site is shown as Ψ. The seven transmembrane domains are highlighted and noted TM1 to TM7. The Genebank accession numbers are NM_001841 and NP_001832.

CB₁ inhibits the phosphorylation of A-type potassium channels through the coupled inhibitory G protein (G_i/o), as explained in further detail below in the cannabinoid receptor signalling section. Inhibition of the phosphorylation of A-type potassium channels results in continuous potassium currents that may prevent neurotransmission (Refs 25, 26). N-type calcium channels also are inhibited by CB₁ through direct interaction with the inhibitory G protein (G_i/o). CB₁-mediated restriction of neurotransmission via potassium and calcium channels accounts for cognitive impairment and sedative-like effects experienced by marijuana users (Refs 26, 27).

CB₂

Following the identification of CB₁, a ‘peripheral’ or ‘non-neuronal’ cannabinoid receptor was cloned from a human promyelocytic cell line (HL60) cDNA library, and was designated cannabinoid receptor 2 (CB₂) (Ref. 28). The gene for this receptor (CNR2) was shown to encode a 360 amino acid long, seven-transmembrane G-protein-coupled receptor (Fig. 2b) with, like CB₁, an extracellular, glycosylated N-terminus and an intracellular C-terminus. Unlike CB₁, there is a considerable level of sequence variation for CB₂ among human, mouse and rat species, particularly when comparing rat and human sequences. There is 81% amino acid identity between rat and human CB₂, as compared with 93% amino acid identity between rat and mouse CB₂ (Ref. 29). The C-terminus in particular differs in rat as a result of intronic DNA (Ref. 30). Of note, it is the C-terminus of CB₂ that plays a critical role in regulating receptor desensitisation and internalisation (Ref. 31); thus, sequence variation within this region should be taken into consideration when investigating physiological, pharmacological and immunological responses of CB₂ in diverse species.

Another distinctive feature of CB₂ in comparison with CB₁ is that its distribution is predominantly in cells and tissues of the immune system, including the thymus, tonsils, B cells, T cells, macrophages, monocytes, natural killer (NK) cells and polymorphonuclear cells. B cells express the highest amounts of CB₂, followed by NK cells, macrophages and T cells, in that order (Refs 32, 33). Recent studies have demonstrated that CB₂ is also expressed within the CNS during various states of inflammation (Refs 34, 35, 36, 37), as discussed in detail later in the article. This expression of CB₂ has been localised primarily to microglia, the resident macrophages of the CNS. CB₂ expression is detected in these cells upon activation by various insults and stimuli, but measurable levels of CB₂ expression cannot be detected in resident, unstimulated microglia. In addition, during neuroinflammation, infiltrating immunocytes from peripheral non-neuronal sites that enter the brain as a result of breakdown of the blood–brain barrier contribute to the overall expression of CB₂. Table 1 lists select references for reports of the distribution of CB₁ and CB₂ in various immune tissues and cell types.

Table 1. Distribution of cannabinoid receptors in the immune system

^aCannabinoid receptor type not specified.

Other receptors

There is accumulating evidence that additional cannabinoid receptors exist, primarily from studies in which CB₁ knockout or CB₁–CB₂ double-knockout mice have been used to investigate the pharmacology and pharmacokinetics of Δ⁹-THC, AEA and cannabinoid analogues. Recently, it has been suggested that the G-protein-coupled receptor GPR55, first cloned and identified in silico from an expressed sequence tags (ESTs) database (Refs 38, 39, 40), may be a novel cannabinoid receptor. Similar to CB₁ and CB₂, GPR55 has seven conserved transmembrane sequences and is activated by plantonic and synthetic exogenous cannabinoids such as Δ⁹-THC, CBD, abnormal CBD, HU-210 and CP55940, and by the endogenous cannabinoids AEA, 2-AG and noladin ether (Ref. 41). Unlike CB₁ and CB₂, GPR55 is not activated by the synthetic agonist WIN55212-2, but is coupled to a G_α12/13 protein instead of a G_i/o protein (Ref. 41) and has been shown to increase intracellular calcium levels upon activation (Ref. 42). GPR55 expression has been identified in a variety of tissues including spleen, gastrointestinal tissues and brain (Ref. 39). However, the physiological and pharmacological functional relevance of GPR55 has yet to be elucidated.

Another receptor reported to be a candidate cannabinoid receptor is the transient receptor potential vanilloid 1 (TRVP1) receptor, a ligand-gated cation channel and a member of the transient receptor potential channel family (Ref. 43). TRVP1 receptors are inherently activated by naturally occurring compounds such as capsaicin, vanilloids and resiniferatoxin (Ref. 44). Its implied role as a cannabinoid receptor is based on the ability of the endogenous cannabinoid AEA, shown to be structurally similar to capsaicin, to bind to and activate this receptor (Refs 45, 46, 47, 48). Nevertheless, despite the various speculative reports of additional cannabinoid receptor subtypes, a novel cannabinoid receptor that meets rigid criteria pharmacologically and functionally has yet to be identified (Refs 49, 50, 51, 52).

Cannabinoid receptor signalling

Both CB₁ and CB₂ are involved in activating signalling cascades that include adenylate cyclase and cAMP, mitogen-activated protein kinase (MAPK), and modulation of levels of intracellular calcium (Refs 26, 53, 54, 55, 56). Upon cannabinoid receptor interaction with its cognate ligand, the receptor-coupled G protein exchanges the inactive guanine nucleotide GDP for its active form GTP, and the heterotrimeric G protein dissociates into α and βγ subunits (Fig. 3a). The βγ subunits are thought to take part in signalling pathways distinct from those of the α subunit, such as the regulation of phospholipase C (PLC) isoforms and activation of the MAPK signalling network (Ref. 25) (Fig. 3b). CB₂ partly exerts its effects through initiation of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP₃) signalling pathways that result in increased levels of intracellular calcium (Ref. 56). The α subunit binds to, and inhibits the activity of, adenylate cyclase, thereby preventing synthesis of the second messenger cAMP and negatively affecting downstream cAMP-dependent signalling events. As a decrease in cAMP production underlies a mechanism in which CB₁ prevents neurotransmitter release and maintains the homeostatic integrity of the CNS, decreased cAMP production also may represent a mode by which CB₂ signalling in response to endocannabinoids maintains immunological homeostasis or, alternatively, in response to exogenous cannabinoids such as Δ⁹-THC superimposes a perturbing immunosuppressive effect (Ref. 57).

Figure 3. Cannabinoid receptor signalling. (a) The adenylate cyclase (AC) pathway. G_iα signalling downregulates the pro-inflammatory immune response via adenylate cyclase. Upon cannabinoid receptor activation, the α subunit of the G_i protein interacts with adenylate cyclase to inhibit its activity. This results in a decrease of cAMP production, which leads to inactivation of protein kinase A (PKA). IκB-α remains unphosphorylated due to PKA inactivation, thus preventing activation, nuclear translocation and DNA binding of NF-κB and other transcription factors. These events ultimately lead to the downregulation of pro-inflammatory mediator gene expression. (b) The mitogen-activated protein kinase (MAPK) pathway. Following cannabinoid receptor activation, the βγ subunits of the G_i protein induce Ras-dependent MAP kinase signalling pathways, culminating in chemotaxis. The SRC kinase is activated upon interaction with G_i βγ subunits and phosphorylates tyrosine residues of the EGF receptor. The SHC–SOS–GRB2 trimer binds the EGF receptor via the SHC adaptor protein, while the guanine-nucleotide-exchange factor SOS activates Ras. Following Ras activation, two independent signalling pathways are initiated, leading to the activation of p38 (encoded by MAPK14) and ERK1 and 2 (encoded by the MAPK1 and MAPK2 genes, respectively). p38 as well as ERK1 and 2 can activate JNK, which induces chemotaxis of immune cells. Abbreviations: EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; MEK1 and MEK2, MAPK kinase encoded by MAP2K1 and MAP2K2 genes, respectively; MEKK1, MAPK kinase kinase encoded by MAP3K1 gene; MKK4, MAPK kinase 4 encoded by MAP2K4 gene; RAC1, Ras-related C3 botulinum toxin substrate 1; RAF1, v-Raf-1 murine leukaemia viral oncogene homologue; SHC, Src homology 2 domain-containing; SOS, son of sevenless; TNF, tumour necrosis factor.

Role of CB₂ in immune modulation

Effect of exogenous cannabinoids on host resistance and immunity

Exogenous cannabinoids have been shown to decrease host resistance to a variety of infectious agents. Administration of Δ⁹-THC to mice lessened their ability to resist infection with the bacterial agent Listeria monocytogenes and herpes simplex virus 2 (Refs 58, 59). In mice and guinea pig models of genital herpes, treatment with Δ⁹-THC increased incidence of viral lesions and recurrence (Refs 60, 61). In additon, cannabinoids compromised host resistance to Legionella pneumophila, Staphylococcus albus, Treponema pallidum, Friend leukaemia virus and Acanthamoeba (Refs 58, 62, 63, 64, 65, 66). These observations are consistent with the notion that exogenous cannabinoids can affect the activities of immune cells.

Indeed, in vitro studies using cells of human and rodent origin have demonstrated that cannabinoids alter the functionality of a diverse array of immune cells. Δ⁹-THC and the synthetic cannabinoids CP55940 and HU-210 inhibited cell-contact-dependent cytolysis of tumour cells, which is mediated by macrophages and macrophage-like cells (Refs 67, 68). Δ⁹-THC suppressed proliferation of B and T cells in response to cell-specific mitogens (Refs 67, 68, 69, 70), suppressed the cytolytic activity of NK cells (Ref. 69), and inhibited cell-killing activity, proliferation and maturation of cytotoxic T lymphocytes (CTLs) (Ref. 69). In addition, exogenous cannabinoids affected immune cell recruitment and chemotaxis to sites of infection and/or injury (Refs 71, 72). In murine models of granulomatous amoebic encephalitis (GAE) and atherosclerosis, macrophages and macrophage-like cells exposed to Δ⁹-THC displayed less migration to sites of infection (Refs 73, 74, 75, 76). Thus, the collective data suggest that exogenous cannabinoids such as Δ⁹-THC inhibit the functional activities of a variety of immunocytes – an outcome that is consistent with the role of these compounds in decreased host resistance to infectious agents.

Role of CB₂ in cell-mediated and humoral immunity

The recognition that select exogenous cannabinoids acted as anti-inflammatory agents and that immune cells also expressed cannabinoid receptors served as an impetus for studies aimed at defining a functional linkage between these two events. The preponderance of studies to date indicates that the cannabinoid receptor that is linked to modulation of the majority of immune functional responses is CB₂.

Effects of exogenous cannbinoids

The partial agonist Δ⁹-THC, as well as the full cannabinoid agonists CP55940 and WIN55212-2, has been reported to enhance human tonsillar B cell growth when used at nanomolar concentrations (Ref. 77) by a CB₂-mediated mechanism. Consistent with these results, the CB₂-selective antagonist SR144528 reversed the stimulating effects of CP55940 on human tonsillar B cell activation (Ref. 78). By contrast, several reports have indicated that cannabinoids suppress the antibody response of humans and animals (Refs 79, 80), at least in part through inhibition of adenylate cyclase by a pertussis-toxin-sensitive G-protein-coupled mechanism (Ref. 81). Furthermore, CB₂ was shown to be downregulated at the mRNA and protein levels during B cell differentiation (Ref. 78). Together, these observations suggest that the CB₂ plays a stimulatory role in B cell activation and growth, while exerting a suppressive role in B cell differentiation and antibody production.

Cannabinoids also have been reported to suppress various activities of T cells, apparently through CB₂. For example, in vivo administration of Δ⁹-THC to mice resulted in significant inhibition of NK cytolytic activity without affecting ConA-induced splenocyte proliferation (Ref. 82). Concomitant with this inhibition, levels of interferon γ (IFN-γ) were reduced significantly, and CB₁ and CB₂ antagonists reversed this effect. In view of these observations, it was suggested that both CB₁ and CB₂ were involved in the network that mediates NK cytolytic activity.

These and other studies have indicated that cannabinoids not only exert direct effects on immune cells but also have effects beyond the initial target cell in that they alter the expression of chemokines and cytokines– key mediators in a complex network of cross-signalling among immune cells for homeostatic balance between pro-inflammatory (T helper 1; Th1) and anti-inflammatory (T helper 2; Th2) activities. For example, Δ⁹-THC treatment of BALB/c mice resulted in a decrease in levels of IFN-γ, interleukin (IL)-12 and IL-12 receptor β2 in response to Legionella pneumophila infection (Ref. 83). The use of cannabinoid receptor antagonists linked both CB₁ and CB₂ to decreased levels of IFN-γ and IL-12 and the suppression of Th1 immunity to Legionella. However, studies employing a tumour model implicated CB₂ as the receptor responsible for Δ⁹-THC-mediated inhibition of immunity by a cytokine-dependent pathway (Ref. 84). In these studies, using a weakly immunogenic mouse lung cancer model, Δ⁹-THC decreased tumour immunogenicity. At both the tumour site and in the spleens of Δ⁹-THC-treated mice, levels of the immune-inhibitory Th2 cytokines IL-10 and transforming growth factor (TGF) were augmented, whereas levels of the immune-stimulatory Th1 cytokine IFN-γ were downregulated. In vivo administration of the CB₂ antagonist SR144528 blocked the effects of Δ⁹-THC, suggesting that Δ⁹-THC promoted tumour growth by inhibiting antitumour immunity through a CB₂-mediated, cytokine-dependent pathway. Collectively, the results from several studies suggest that exogenous cannabinoids elicit a shift from a Th1 pro-inflammatory cytokine expression profile to a Th2 anti-inflammatory profile, and that CB₂ may be linked to this effect.

Effects of endogenous cannabinoids

Endocannabinoids also have been reported to affect immune function, mostly though CB₂. The effects of AEA and palmitoylethanolamide, as well as Δ⁹-THC, on the production of tumour necrosis factor (TNF)-α, IL-4, IL-6, IL-8, IL-10, IFN-γ, and p55 and p75 TNF-α soluble receptors have been examined (Ref. 85). AEA diminished production of IL-6 and IL-8 at low nanomolar concentrations and inhibited that of TNF-α, IFN-γ, IL-4 and p75 TNF-α soluble receptor at micromolar concentrations. Palmitoylethanolamide, at concentrations similar to those of AEA, inhibited the synthesis of IL-4, IL-6 and IL-8, and the production of p75 TNF-α soluble receptor. However, palmitoylethanolamide did not affect TNF-α and IFN-γ production. Neither AEA nor palmitoylethanolamide had an effect on IL-10 synthesis. Based on these observations, it was suggested that various endogenous fatty acid ethanolamides participated in the regulation of the immune response, and that the inhibitory properties of AEA and palmitoylethanolamide were due to activation of CB₂ since it has been shown that palmitoylethanolamide has a high affinity for the CB₂ receptor and does not bind the CB₁ receptor (Ref. 86). AEA also has been shown to exert an inhibitory effect on chemokine-elicited lymphocyte migration (Ref. 87). The inhibition of stromal derived factor 1 (SDF-1)-induced migration of CD8⁺ T cells was found to be mediated through the CB₂. However, there also are reports that AEA can exert potentiating effects. AEA acted as a synergistic growth factor for primary murine marrow cells and haematopoietic growth factor (HGF)-dependent cell lines (Ref. 88). AEA also augmented production of IL-6 by astrocytes infected with Theiler's murine encephalomyelitis virus (Ref. 89). However, in these studies the enhancing effect of AEA was shown to be blocked by the CB₁ antagonist SR141716A, suggesting involvement of CB₁ rather than CB₂ in the elevation of levels of this pleiotropic cytokine.

By contrast to AEA, 2-AG has been associated primarily with augmentation of immune responses. 2-AG stimulated the release of nitric oxide (NO) from human immune and vascular tissues and from invertebrate immunocytes via CB₁ (Ref. 90), and haematopoietic cells expressing CB₂ migrated in response to 2-AG (Ref. 91). Distinct profiles for CB₂ expression in lymphoid tissues have been reported to be dependent on the state of receptor activation, and it has been proposed that cell migration constitutes a function of CB₂ upon stimulation with 2-AG (Ref. 92). Furthermore, 2-AG induced the migration of human peripheral blood monocytes and promyelocytic leukaemia HL60 cells that have been differentiated into macrophage-like cells (Ref. 76), apparently through a CB₂-dependent mechanism. Subsequent studies showed that 2-AG apparently accelerated production of chemokines by the HL-60 cells (Ref. 93). In addition, rat microglia have been reported to synthesise 2-AG in vitro, an event that has been linked to increased proliferation through a CB₂-dependent mechanism (Ref. 94).

Role of CB₂ in macrophages and macrophage-like cells

A major target of the action of exogenous and endogenous cannabinoids appears to be cells of macrophage lineage. Cannabinoids suppress macrophage functions such as phagocytosis, bactericidal activity, and spreading (Refs 79, 95), interfere with macrophage cell-contact-dependent lysis of tumour cells, herpesvirus-infected cells and amoebae, and deplete macrophage-elicited soluble tumouricidal activity (Refs 67, 96). These observations are consistent with reports that Δ⁹-THC inhibits the synthesis of proteins associated with primed and activated macrophages (Ref. 97), alters cytokine secretion by activated macrophages (Refs 98, 99), and inhibits cytokine gene expression by microglia (Ref. 100). Cannabinoids also have been found to affect the production of NO by macrophages and macrophage-like cells (Refs 68, 101). Although it is now evident that cannabinoids exert a variety of effects on the activities of macrophage and macrophage-like cells, a picture is emerging as to the role of CB₂ in these processes and the state of cell activation under which it is functionally relevant.

Differential expression of CB₂

Macrophages and macrophage-like cells such as microglia undergo a maturation, differentiation and activation process characterised by differential gene expression and the acquisition of correlative distinctive functional capabilities (Refs 102, 103, 104). These cells can be driven sequentially in response to multiple signals from ‘resting’ to ‘responsive’, ‘responsive’ to ‘primed’, and ‘primed’ to ‘fully activated’ states, a process that mimics events in vivo. Using in vitro models, it has been shown that levels of CB₂ receptor mRNA and protein are modulated differentially in relation to cell activation state (Ref. 105). The CB₂ is not detected in resting cells, is present at high levels in responsive and primed cells, and is identified at greatly diminished levels in fully activated cells (Fig. 4). These observations suggest that the CB₂ is expressed ‘on demand’ and that the modulation of CB₂ levels is a feature common to cells of macrophage lineage as they participate in the inflammatory response and undergo differential gene expression and acquisition of distinctive functional properties. Furthermore, the relatively high levels of CB₂ recorded for macrophages when in responsive and primed states suggest that these cells, and possibly other immune cell types, exhibit a functionally relevant ‘window’ of CB₂ expression during which they are most susceptible to cannabinoid-mediated action. Furthermore, since CB₁ is expressed at low and constitutive levels in microglia as compared with CB₂, the kinetics of expression of the two receptors as linked to immune functional activities may be distinct.

Figure 4. Cannabinoid receptor mRNA levels and macrophage activation state. Levels of cannabinoid receptor 2 (CB₂) mRNA are modulated differentially in relation to macrophage activation state, as reported in Ref. 105. CB₂ is detected at low levels in ‘resting’ cells (0), is present at high levels in ‘responsive’ (R) and ‘primed’ (P) cells, and is identified at greatly diminished levels in cells that have been subjected to multistep activation (MA) or direct activation (DA). The resting state was achieved by growing cells on an agar substratum, whereas the responsive state was obtained by growing cells on a plastic surface. Cells were treated with interferon (IFN)-γ (100 U/ml), IFN-γ (100 U/ml) plus lipopolysaccharide (LPS) (100 ng/ml), or LPS (100 ng/ml) to obtain prime, multistep activated, or directly activated states, respectively. Southern blot analysis was performed on mutagenic reverse transcriptase PCR (MRT-PCR) products of total RNA from mouse peritoneal macrophages (Ref. 92). The upper band of each doublet represents amplified genomic DNA used as an internal quantitative standard. The lower band of each doublet represents amplified product from mRNA.

Chemotaxis as a signature activity of ‘responsive’ macrophages

Chemotaxis and antigen presentation are signature activities of macrophages and macrophage-like cells when in responsive and primed states of activation – states that are associated with early stages of the inflammatory response. Chemotaxis describes the ability of cells to migrate towards an increasing concentration gradient of stimulating agent (Refs 106, 107, 108, 109) and is distinct from chemokinesis, which represents stimulus-dependent random cellular motion (Refs 110, 111, 112, 113). During chemotaxis, macrophage interaction with a chemoattractant results in the initiation of a rapid and directed movement that is associated with a complex array of cellular events that includes changes in ion fluxes, alterations in integrin avidity, production of superoxide anions, and secretion of lysosomal enzymes (Ref. 114). Classical chemoattractants include bacterial-derived N-formyl peptides, the complement fragment peptides C5a and C3a, and lipids such as leukotriene B₄ and platelet-activating factor (Refs 115, 116, 117, 118). Chemokines – cytokines of 8 to 17 kDa molecular mass that are selective for leukocytes in vitro and elicit accumulation of inflammatory cells in vivo – represent a second group of chemoattractants (Refs 119, 120, 121, 122). As in the case of cannabinoid binding to cannabinoid receptors, the specific effects of chemokines on target cells are mediated by G-protein-coupled receptors (Refs 114, 123). Ligation of chemokines to their cognate receptors initiates a series of signal transduction events that results in leukocyte trafficking during infection, inflammation, tissue injury and tumour development (Ref. 123).

The current data indicate that cannabinoids act through CB₂ to alter macrophage migration, with exogenous cannabinoids such as Δ⁹-THC exerting an inhibitory effect and, conversely, endocannabinoids such as 2-AG eliciting a stimulatory effect. For example, in vivo and in vitro treatment of rat peritoneal macrophages with CP55940 resulted in decreased migration in vitro to the peptide formal-methionyl-leucine-phenylalanine (fMLP) in a mode that is linked primarily to CB₂ (Ref. 72). Also, the chemotactic response of mouse macrophages to fMLP was decreased by CBD (Ref. 124), which binds weakly to CB₂. CB₂ was implicated in this response since the CB₂-selective antagonist SR144528 prevented the decrease in migration. In contrast to events observed for Δ⁹-THC, 2-AG triggered migration of microglia, and CB₂ was involved in this effect (Ref. 3). Recently, a combined pharmacological and genetic approach (employing macrophages from knockout mice) demonstrated that Δ⁹-THC and CP55940 inhibited mouse peritoneal macrophage chemotaxis to CCL5 (RANTES) through CB₂ (Ref. 71). The Δ⁹-THC and CP55940 deactivation of migratory responsiveness to the chemokine CCL5, an event that is mediated through activation of the cognate G-protein-coupled chemokine receptor CCR5, suggested that signalling through CB₂ leads to ‘cross-talk’ between that receptor and CCR5. These and other studies implicate the CB₂ in a network of G-protein-coupled receptor signal transduction systems, inclusive of chemokine receptors, that act co-ordinately to modulate macrophage migration.

Antigen processing as a signature activity of ‘primed’ macrophages

It has also been shown that CB₂ is involved in cannabinoid-mediated inhibition of processing of antigens by macrophages. In studies examining the effect of Δ⁹-THC on the processing of intact lysozyme by macrophages, Δ⁹-THC impaired the ability of a macrophage hybridoma to function as an antigen-presenting cell based on its ability to secrete IL-2 upon stimulation of a soluble-protein-antigen-specific helper T cell hybridoma (Ref. 70). Δ⁹-THC exposure resulted in a significant reduction in the T cell response to the native form of lysozyme after pretreatment of the macrophages with nanomolar drug concentrations. However, Δ⁹-THC did not affect IL-2 production when the macrophages presented a synthetic peptide of the antigen to T cells, suggesting that the drug interfered with antigen processing, not peptide presentation. The cannabinoid inhibition of the T cell response to native lysozyme was stereoselective, consistent with the involvement of a cannabinoid receptor: bioactive CP55940 diminished T cell activation whereas the relatively inactive stereoisomer CP56667 did not. The macrophage hybridoma expressed mRNA for CB₂ but not for CB₁. Furthermore, the CB₁-selective antagonist SR141716A did not reverse the suppression caused by Δ⁹-THC, whereas the CB₂-selective antagonist SR144528 completely blocked the Δ⁹-THC suppression of the T cell response. Collectively, these results implicated macrophages as the target of cannabinoid inhibition of antigen processing through CB₂.

CB₂ and neuroinflammation

CB₂ localisation in the CNS

Early studies to define the functional relevance of CB₁ and CB₂ suggested that CB₁ was compartmentalised to the CNS while the expression of CB₂ was limited to cells and tissues of the immune system. The development of phenotypically normal CB₂-knockout mice (Ref. 125) was a major breakthrough that contributed to elucidation of the role of CB₂ in immune modulation, including that in the CNS. In addition to the CB₂-knockout mouse strain developed by Buckley and colleagues (Ref. 125), Deltagen (San Mateo, CA, USA) developed a CB₂-knockout mouse strain that is commercially available through Jackson Laboratories (Bar Harbor, ME, USA) (Ref. 126); these CB₂-knockout mice strains have mutations in the C-terminus and the N-terminus of CB₂, respectively. The tissues from these mice have been employed extensively in studying CB₂ function and CB₂-mediated responses. Macrophage function in helper T cell activation in the knockout mouse developed by Buckley and colleagues is not sensitive to the inhibitory effects of Δ⁹-THC, in contrast with macrophage function in wild-type counterparts (Ref. 125).

In addition, CB₂-knockout mice have been utilised to study the specificity of various CB₂ antibodies. However, CB₂ localisation within the CNS has proven to be an elusive target. While some laboratories have reported detection of CB₂ in the brain (Refs 126, 127, 128), other laboratories have not been able to identify this protein, raising concern about the reliability and specificity of the CB₂ antibodies used. In the studies performed that identified the CB₂ protein in brainstem neurons, a polyclonal antibody against the C-terminus was used to detect the receptor, and the CB₂-knockout strain developed by Buckley and colleagues and wild-type mice were used as the knockout and positive controls, respectively, to confirm the specificity of the polyclonal CB₂ antibody (Refs 126, 127). The knockout control was appropriate for those experiments since this knockout strain has a deletion in the C-terminus of the CB₂ protein. In other studies, on brain of wild-type mice, an antibody that was raised against the C-terminus of the CB₂ protein was used to demonstrate receptor expression (Refs 126, 128, 129, 130). The investigators identified the CB₂ protein in various brain regions using an antibody specific for the N-terminus of the CB₂ protein; however, a knockout control using tissues from CB₂-knockout mice was not used to confirm the specificity of this antibody (Refs 126, 128). Similar potentially confounding issues have been raised for CB₁ antibodies. Grimsey and colleagues demonstrated that various CB₁-specific antibodies used in immunostaining and western blot analyses displayed a multitude of variability in expression profiles – an outcome that was attributed to possible conformational changes, dimerisation with other G-protein-coupled receptors, or post-translational modifications (Ref. 131). It was postulated that such factors, separately or combined, could result in epitope masking or insufficient binding of antibody (Ref. 131). Collectively, these studies highlight the importance of employing specific cannabinoid receptor antibodies whose specificity can be confirmed using appropriate knockout controls, particularly when investigating the complex arena of the CNS. While localisation of the CB₂ protein in the CNS may have been elusive, in vitro studies have shown that in addition to expression in microglia (Ref. 105), CB₂ has since been identified in neurons, oligodendrocytes and other glial cells in vitro and in intact tissue (Refs 34, 35, 36). This receptor can be induced on demand during early inflammatory events (Refs 35, 37) and is linked to attenuation of pro-inflammatory cytokine production by microglia, as discussed earlier (Refs 132, 133, 134, 135, 136).

Neuroinflammatory disorders: CB₂ as a therapeutic target?

Like macrophages at peripheral sites, microglia are able to phagocytose and process antigens, and upon activation produce pro-inflammatory factors including the cytokines IL-1, IL-6 and TNF-α (Refs 137, 138, 139). Pro-inflammatory mediators released from microglia are cytotoxic and also can secondarily activate astrocytes, leading to a further induction of the expression of inflammatory factors. The resultant ‘storm’ of pro-inflammatory mediators contributes to breakdown of the blood–brain barrier and plays a critical role in promoting influx into the CNS of immunocytes from peripheral non-neuronal sites that also express CB₂. Microglia are believed to play a major role in many neuropathogenic diseases and disorders such as Alzheimer disease, multiple sclerosis, amyotrophic lateral sclerosis, and HIV (human immunodeficiency virus) encephalitis.

Alzheimer disease

Alzheimer disease is the most common neurodegenerative disorder that causes senile dementia. The defining neuropathological features of the disease are the presence of extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles in the brain; as neurodegeneration progresses, there is accelerated neurofibrillary tangle formation, neuroinflammation and neuronal loss.

It has been reported that cannabinoids can be neuroprotective in Alzheimer disease by inhibiting the activation of microglia (Ref. 35) induced by amyloid plaques consisting of extracellular aggregates of amyloid β (Aβ) peptides (Refs 140, 141). Recently, the CB₁/CB₂ agonist CP55940 and the CB₂ agonist JWH-015 were found to protect and rescue peripheral blood lymphocytes from Aβ- and H₂O₂-induced apoptosis by two alternative mechanisms (Ref. 142). A receptor-independent pathway was implicated through the demonstration that the reactive oxygen species dihydrorhodamine was not oxidised into fluorescent rhodamine 123 as a result of cannabinoid inhibition of Aβ-generated H₂O₂, while a receptor-dependent pathway was implicated through the activation of the transcription factor NF-κB and downregulation of the transcription factor p53, which can initiate apoptosis. The downregulation of p53 involved phosphoinositide 3-kinase, as demonstrated through the use of the phosphoinositide 3-kinase inhibitor LY294002 (Ref. 142). Collectively, these results suggested that cannabinoids have potential as neuroprotective compounds in Alzheimer disease.

Multiple sclerosis

Multiple sclerosis is a chronic, inflammatory demyelinating disease of the human CNS that primarily affects adults (Refs 143, 144, 145). The disease is characterised by T-cell-mediated degeneration of the myelin sheath that covers axons, resulting in an inflammatory process that stimulates other immune cells to secrete pro-inflammatory mediators and antibodies, breakdown of the blood–brain barrier, activation of macrophages, and production of ‘cytotoxic’ proteins such as metalloproteinases (Refs 146, 147).

A significantly greater density of CB₂-immunoreactive microglia/macrophages has been identified in affected regions of human multiple sclerosis post-mortem spinal cord (Ref. 148). However, most studies aimed at assessment of effects of cannabinoids on multiple sclerosis, and the role of CB₂ in this process have involved the use of mouse models, particularly the experimental autoimmune encephalomyelitis (EAE) model, which exhibits a CD4⁺-T-cell-mediated autoimmune disease (Ref. 144). Δ⁹-THC markedly inhibited neurodegeneration in the EAE model and reduced the associated induced elevated level of glutamate in cerebrospinal fluid (Ref. 149). CB₂ mRNA expression and protein internalisation were upregulated significantly in activated microglia of mice experiencing EAE, implicating CB₂ in this disease (Ref. 150). The cannabinoid WIN55212-2 ameliorated EAE and diminished cell infiltration of the spinal cord. WIN55212-2 was found to induce encephalitogenic T cell apoptosis, partly through CB₂ (Ref. 151). More recently, it has been proposed that CB₂ plays a protective role in EAE pathology by targeting myeloid progenitor trafficking, which may contribute to microglial activation in the CNS (Ref. 152). In Theiler's virus infection of murine CNS, another mouse model for human multiple sclerosis, improved neurological deficits, concomitant with reduced microglial activation, major histocompatibility complex class II expression and T cell infiltration, were observed following treatment of mice with the synthetic cannabinoids WIN55212-2, ACEA (a CB₁-selective agonist) and JWH-015 (a CB₂-selective agonist) (Refs 153, 154). In the Theiler's model of multiple sclerosis, clinical signs and axonal damage in the spinal cord were reduced by the AMPA (amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamatergic receptor antagonist NBQX (Ref. 155). The cannabinoid HU-210 ameliorated symptoms, accompanied by a reduction of axonal damage. Furthermore, the HU-210-mediated reduction in AMPA-induced excitotoxicity in vivo and in vitro was linked to CB₁ and CB₂.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is characterised pathologically by progressive degeneration of cortical motor neurons (upper motor neurons) and clinically by muscle wasting, weakness and spasticity that progresses to complete paralysis (Ref. 156). A pathological hallmark of amyotrophic lateral sclerosis is neuroinflammmation, a process that is mediated by pro-inflammatory cytokines, prostaglandins and NO (Ref. 157).

CBN delayed the onset of symptoms in mice suffering from experimentally induced amyotrophic lateral sclerosis without affecting survival (Ref. 158), and treatment of mice with WIN55212-2 after onset of symptoms delayed overall disease progression (Ref. 159). Also, the CB₂ agonist AM-1241 prolonged survival in a G93A-SOD1 mutant transgenic mouse model of amyotrophic lateral sclerosis when administered at onset of disease symptoms (Ref. 160). mRNA and receptor binding of CB₂ were selectively upregulated in spinal cords of these mice in a fashion that paralleled disease progression. Daily injections of AM-1241 initiated at onset of symptoms increased the survival interval after disease onset by 56%. Collectively, the results suggested that the CB₂ agonist extended the interval for motor neuron degeneration and prolonged function in these affected mice.

HIV encephalitis

HIV encephalitis, also known as acquired immune deficiency syndrome (AIDS)–dementia complex, results in progressive memory loss, intellectual deterioration, behavioural changes, and motor deficits (Ref. 161). The neuropathology of HIVE is characterised by neuronal loss, glial activation, presence of multinucleated giant cells, perivascular mononuclear infiltration, and in some cases vacuolar myelopathy and myelin pallor (Ref. 161). The production of pro-inflammatory cytokines such as TNF-α by activated monocytes and microglia, and neurotoxins such as glutamate and NO, is the primary cause of brain damage associated with this disorder. In addition, HIV-specific gene products such as the transactivator Tat and the envelope glycoprotein gp120 that are released from infected monocytes and microglia contribute to neuropathology.

The simian immunodeficiency model comes closest to replicating events that are associated with HIV infection of the human CNS. Examination of brains of macaques with simian immunodeficiency virus (SIV)-induced encephalitis has led to the suggestion that the endocannabinoid system participates in the development of HIV-induced encephalitis (Ref. 162). In this infectivity model, expression of CB₂ was induced in perivascular macrophages, microglial nodules, and T cells. It was proposed that activation of CB_2, expressed by perivascular macrophages, which play a critical role in viral entry into the CNS (Refs 163, 164), likely led to reduction of their antiviral response, thus favouring the entry of infected monocytes into the CNS (Ref. 162). In addition, the endogenous cannabinoid-degrading enzyme FAAH was reported as overexpressed in perivascular astrocytes as well as in astrocytic processes reaching cellular infiltrates (Ref. 162). Furthermore, activation of CB₂ resulted in inhibition of the transendothelial migration of Jurkat T cells and primary human T cells by interfering with the CXCL12–CXCR4 chemokine receptor system (Ref. 165). These observations suggest that activation of CB₂ can alter the activation of other G-protein-coupled receptors such as CXCR4, which functions as a coreceptor for T-lymphotropic HIV. A similar observation in terms of a linkage to CB₂ has been made for the chemokine receptor CCR5, which acts as the coreceptor for monotropic HIV (Ref. 71). Activation of CB₂ with Δ⁹-THC, CP55940 or with the CB₂-selective compound O-2137 inhibited activation of CCR5 by its native chemokine ligand CCL5. Collectively, these results indicate that CB₂ as a G_i/o-protein-coupled receptor ‘crosstalks’ with several other G-protein-coupled receptors, especially chemokine receptors, to alter heterologous signal transduction pathways. Furthermore, these interactions might have implications for HIV infection, particularly for those receptors such as CXCR4 and CCR5 that act in a coreceptor capacity for HIV. Additionally, possible therapeutic implications of ‘crosstalk’ between cannabinoid receptors and other cellular receptors has come from studies suggesting that CB₂, along with CB₁, could play a role in linking the endocannabinoid sytem with the modulation of neural stem cell proliferation through bidirectional ‘crosstalk’ with TNF receptors (Ref. 166).

Summary of cannabinoid receptors in neuropathological disorders

In summary, cannabinoid receptors appear to play an important role in neuropathological diseases. CB₁ has been reported to be critical for the overall homeostatic balance and regulation of the CNS, while CB₂ has been implicated as playing a functionally relevant role during neuroinflammation. Microglia, as resident macrophages in the CNS, not only play a role in host defence and tissue repair but also have been implicated as contributive to, if not causative of, a variety of inflammatory neuropathological processes. In these cells CB₁ appears to be present at constitutive and relatively low levels while CB₂ is expressed inducibly during the inflammatory process and at relatively high levels. Immune responses during the early phase of neuropathological processes appear to involve preponderantly CB₂, and levels and functional relevance of this receptor may be amplified as disease progresses to later stages of inflammation. The recognition that immunocytes resident within the brain express CB₂, during the inflammatory process suggests the existence of a temporal window during which these cells may be susceptible to therapeutic manipulation through the use of CB₂-selective agonists. That is, selective targeting of the CB₂ could result in dampening of untoward immune responses such as elicitation of a chemokine/cytokine ‘storm’ within the CNS that would result in breakdown of the blood–brain barrier, influx of immunocytes from peripheral non-neuronal sites, and further inflammation.

Clinical implications/applications

Cannabinoids, as ligands that signal through cannabinoid receptors, may be particularly useful as agents for therapeutic manipulation of hyperinflammatory immune responses within the CNS. These compounds are highly lipophilic and thus readily penetrate the blood–brain barrier, a challenge that is posed to a variety of agents that have therapeutic potential. Furthermore, through the application of appropriately engineered molecules, it may be possible to specifically target CB₂, a condition that would obviate generation of untoward psychotropic effects that could be engendered if the CB₁ were activated also.

The principal potential cellular target in the CNS for these compounds – given its involvement in early stages of the inflammatory response in generation of a cascade of inflammatory factors and its expression of CB₂ – is the microglial cell. During activation, microglia, as macrophage-like cells, also upregulate an array of cell-surface receptors that may be critical in regeneration and/or degeneration of the CNS. Included among these are immunoglobulin (Ig) superfamily receptors, complement receptors, Toll-like receptors, cytokine/chemokine receptors, and opioid receptors. These cells, in addition to expressing both the CB₁ and the CB₂ in vitro (Refs 105, 167), also produce the endocannabinoids 2-AG as well as AEA, although the latter is generated in lesser quantities (Ref. 94). Thus, microglia appear to harbour a fully constituted system of endogenous cannabinoid ligands and cognate receptors. Activation of CB₂ on these cells appears to promote migration and proliferation. 2-AG induces migration of microglia and this occurs through the CB₂ and abnormal-CBD-sensitive receptors, which subsequently leads to activation of the extracellular-signal-regulated kinase (ERK)1 and 2 (encoded by the MAPK1 and MAPK2 genes, respectively) signal transduction pathway (Ref. 3). Furthermore, it has been shown that microglia express CB₂ at the leading edge of lamellipodia, consistent with an involvement in cell migration. There is accumulating evidence that CB₂ also is expressed in the CNS in vivo. The expression of CB₂ in microglial, astrocyte and neuronal subpopulations has been identified in a variety of neurodegenerative disease models (Ref. 37). This expression of CB₂ in vivo has been attributed, in large measure, to microglia. In several neurodegenerative diseases, upregulation of microglial CB₂ has been observed (Refs 148, 162, 168, 169, 170, 171). In studies investigating the expression profile of FAAH and the CB₂ in postmortem brain tissues from Alzheimer disease patients, it was observed that congregated microglia associated with neuritic plaques selectively overexpressed CB₂ (Ref. 172). In addition, CB₂-positive microglia have been identified dispersed within active multiple sclerosis plaques and in the periphery of chronic active plaques (Ref. 169).

The collective findings support the concept that, in addition to CB₁, CB₂ has a functionally relevant role in the CNS. This role appears to play out during the inflammatory process associated with a variety of neuropathies. In this context, it has been proposed that the role of the CB₂ in immunity in the CNS is primarily one that is anti-inflammatory (Ref. 173). Since microglia exhibit phenotypic and functional properties of macrophages and inducibly express CB₂ at maximal levels when in responsive and primed states, there may be a window of functional relevance for this receptor, comparable to that for macrophages at peripheral sites. That is, antigen processing and/or chemotaxis by these cells may also be susceptible to cannabinoids acting via CB₂.

Indeed, studies using a mouse model of GAE, a chronic progressive human infection of the CNS that is caused by the opportunistic pathogen Acanthamoeba, revealed a paucity of cells expressing the Mac-1 (CD11b) protein marker for macrophages and macrophage-like cells at focal sites containing Acanthamoeba in the brains of infected mice treated with Δ⁹-THC as compared with vehicle-treated Acanthamoeba-infected controls (Ref. 65). These observations indicated that microglia (and possibly macrophages introduced from peripheral sites) either did not migrate to infected areas or were selectively targeted by the Acanthamoeba and destroyed. Treatment of neonatal rat cerebral cortex microglial cultures with Δ⁹-THC resulted in inhibition of the migratory response to Acanthamoeba-conditioned medium, which harbours proteases and other factors released from amoebae that serve as chemotactic stimuli (Ref. 57). In addition, treatment with the potent CB₁/CB₂ agonist CP55940 resulted in a significant concentration-related decrease in microglial migration in response to conditioned medium. The highly selective CB₂ ligand O-2137 exerted a profound and significant inhibition in the microglial migratory response to conditioned medium while treatment with the CB₁-selective ligand ACEA had a minimal effect. Finally, treatment of microglia with the CB₁ antagonist SR141716A did not block the inhibitory effect of CP55940 while treatment with the CB₂-specific antagonist SR144528 resulted in a reversal of the inhibitory effect of CP55940. These collective results indicated that the cannabinoid-mediated inhibition of the conditioned-medium-stimulated microglial response to A. culbertsoni in mouse brain was linked, at least in part, to CB₂. The mode by which Δ⁹-THC and other exogenous cannabinoids such as CP55940 signal through CB₂ to inhibit the chemotactic response of microglia to Acanthamoeba remains to be defined. However, it is known that Acanthamoeba produces proteases, phospholipases and other factors (Ref. 174) that may act on phospholipids in microglial membranes, generating cleavage products (Ref. 175). It is postulated that bioactive lipid mediators thus generated include the endocannabinoid 2-AG that serves to drive chemotaxis by autocrine and/or paracrine activation of CB₂. The exogenous cannabinoid Δ⁹-THC may alter this chemotactic response, as well as chemotactic responses to other stimuli, by superimposing an inhibitory effect through activation of CB₂. That is, Δ⁹-THC could inhibit the synthesis and/or, release of 2-AG or, by virtue of its relative long half-life as compared with that of 2-AG, pre-empt this endocannabinoid from ligating to CB₂.

Summary, research in progress, and outstanding research questions

There is currently a large body of data indicating that the CB₂ plays a functionally relevant role during inflammation. This role is especially evident for cells of myeloid lineage, including macrophages and macrophage-like cells, as well as microglia that are resident in the CNS. These latter cells are morphologically, phenotypically, and functionally related to macrophages. The CB₂ is differentially expressed by macrophages and macrophage-like cells, with highest levels detected when these cells are in responsive and primed states, suggesting the existence of a window of functional relevance during which activation of the CB₂ modulates macrophage activities. Signature activities of responsive and primed macrophages are chemotaxis and antigen processing, respectively. The endocannabinoid 2-AG, elicited from macrophages and microglia during the activation process, stimulates a chemotactic response from these cells through the CB₂. By contrast, exogenous cannabinoids such as Δ⁹-THC and CP55940 inhibit the chemotactic response as well as antigen processing of antigens, through activation of the CB₂. Furthermore, exogenous cannabinoids such as Δ⁹-THC may superimpose an inhibitory effect on pro-chemotactic endocannabinoids.

Although in recent years major advances have been made regarding the functional relevance of the CB₂, a number of outstanding research questions remains. Principal among these is definition of the mechanism through which exogenous cannabinoids such as Δ⁹-THC superimpose an inhibitory effect on endocannabinoid-mediated immune functional activities. In this context, are differential signal transductional pathways involved following CB₂ activation by Δ⁹-THC versus endocannabinoids? Do exogenous cannabinoids by virtue of their relatively long half-life as compared with endocannabinoids persist in cells so as to affect receptor-mediated endocytosis and recycling of receptor–ligand complexes? In addition, what is the extent of the ability of the CB₂ to ‘crosstalk’ with other G-protein-coupled receptors, especially chemokine receptors such as CXCR4 and CCR5, which also serve as coreceptors for HIV? Do the endocannabinoids AEA and 2-AG exert differential effects on immune function, thereby acting in an immune homeostatic role? That is, does AEA act in an anti-inflammatory capacity while 2-AG acts as a pro-inflammatory agent as is typical for other bioactive lipids such as select prostaglandins that exert pro-inflammatory versus anti-inflammatory activities? These are but a few of the salient questions that await resolution.