Cannabinoids as novel anti-inflammatory drugs
Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of Δ9 -tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found in the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, the administration of endocannabinoids or the use of inhibitors of enzymes that break down the endocannabinoids led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivo can constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components.
Cannabis, commonly known as marijuana, is a product of the Cannabis sativa plant, and the active compounds from this plant are collectively referred to as cannabinoids. For several centuries, marijuana has been used as alternative medicine in many cultures, and, recently, its beneficial effects have been shown in: the treatment of nausea and vomiting associated with cancer chemotherapy; anorexia, and cachexia seen in HIV/AIDS patients; and in neuropathic pain and spasticity in multiple sclerosis [1–4]. Cannabinoid pharmacology has made important advances in recent years after the discovery of cannabinoid receptors (CB1 and CB2). Cannabinoid receptors and their endogenous ligands have provided an excellent platform for the investigation of the therapeutic effects of cannabinoids. It is well known that CB1 and CB2 are heterotrimeric Gi/o-protein-coupled receptors and that they are both expressed in the periphery and the CNS. However, CB1 expression is predominant in the CNS, especially on presynaptic nerves, and CB2 is primarily expressed in immune cells [5,6].
Arachidonic acid metabolites have been shown to exhibit properties similar to compounds found in Cannabis sativa. These metabolites are hence referred to as endocannabinoids. These ubiquitous endogenous cannabinoids act as natural ligands for the cannabinoid receptors expressed in mammalian tissue, thus constituting an important lipid-signaling system termed the endocannabinoid system. The endocannabinoid system is an important biological regulatory system that has been shown to be highly conserved from lower invertebrates to higher mammals . Other than the lipid transmitters that serve as ligands for the cannabinoid receptors, the endocannabinoid family also comprises the enzymes for biosynthesis and degradation of the ligands. The endocannabinoids include Narachidonoylethanolamine, anandamide (AEA), 2-arachidonoyl glycerol (2-AG), Narachydonoyldopamine, Nolan ether, and virodhamine. AEA was discovered by Devane et al. and is an amide formed from arachidonic acid and ethanolamine . AEA binds to brain CB1 with high affinity and mimics the behavioral actions of exogenous cannabinoid Δ9 - tetrahydrocannabinol (THC) when injected into rodents. 2-AG was discovered independently three years later by Mechoulam et al.  and Sugiura et al. . It was found to exist in much higher concentrations in serum and brain than in AEA. 2-AG has similar affinities for both CB1 and CB2 receptors, as does AEA, but it exhibits higher efficacy. Endocannabinoids are derivatives of arachidonic acid conjugated with either ethanolamine or glycerol. They are synthesized on demand from phospholipid precursors residing in the cell membrane in response to a rise in intracellular calcium levels. Inside cells, endocannabinoids are catalytically hydrolyzed by the aminohydrolase fatty acid amide hydrolase (FAAH), which degrades AEA into arachidonic acid and ethanolamine . 2- AG is hydrolyzed into AEA and glycerol by either FAAH or by monoacylglycerol lipase (MAGL). Fatty acid-binding proteins (FABPs) have been reported to play an important role as intracellular carriers in the transport of AEA from the plasma membrane to FAAH for their subsequent inactivation . Studies to date indicate that the main pharmacological function of the endocannabinoid system is in neuromodulation: controlling motor functions, cognition, emotional responses, homeostasis, and motivation. However, in the periphery, this system is an important modulator of the ANS, immune system, and microcirculation . Some well-known natural and synthetic cannabinoids and endocannabinoids are depicted in Table 1.
Cannabinoids are potent anti-inflammatory agents, and they exert their effects through induction of apoptosis, inhibition of cell proliferation, suppression of cytokine production, and induction of T-regulatory cells (Tregs). In this review, we provide an in-depth description of all four different mechanisms, and we further discuss the immunosuppressive properties of cannabinoids in the context of inflammatory and autoimmune disease states triggered by cellular rather than humoral components of the immune system.
Apoptotic effects of cannabinoids on immune cell populations
One major mechanism of immunosuppression by cannabinoids is the induction of cell death or apoptosis in immune cell populations. Under normal conditions, apoptosis is required in order to maintain homeostasis, and it involves morphological changes (i.e., cell shrinkage, nuclear fragmentation, and membrane blebbing) as well as molecular changes (i.e., induction of caspases and cytochrome c leakage) . The extrinsic pathway of apoptosis is initiated with the ligation of death receptors (i.e., CD95) on the cell surface, leading to the activation of major caspases, such as caspase 3, 8, and 10. The intrinsic pathway of apoptosis is initiated via mitochondria and caspase 9; cytochrome c and caspase 3 are the major players in the induction of cell death [14,15]. Macrophages and T cells. This study also showed that the process was mediated via the activation of Bcl-2 and caspases . It was difficult to demonstrate the apoptotic effects of THC on lymphocytes in vivo, and our laboratory speculated that this might be due to the rapid clearance of dead cells by phagocytic cells. Therefore, we exposed C57BL/6 mice to 10 mg/ kg body weight THC and, after several time points (4, 6, 24, and 72 h), obtained lymphocytes from the thymus and spleen of these animals. The cells were incubated for 12– 24 h ex vivo, and since phagocytosis was excluded in the cultures, we detected significant levels of THC-induced apoptosis in T cells, B cells, and macrophages . We have also demonstrated that THC induced higher levels of apoptosis in naive lymphocytes when compared with mitogen-activated lymphocytes because activated cells downregulated the levels of CB2 on their cell surface . Several studies also reported THC-induced apoptosis in antigen-presenting cells. In bone marrow-derived dendritic cells (DCs), THC induced apoptosis via ligation of both CB1 and CB2 and activation of caspases such as caspase 2, 8, and 9. In vivo, THC administration decreased the number of splenic DCs, as well as MHCII expression by DCs [18,19]. Furthermore, THC increased Bcl-2 and caspase one activity in naive and lipopolysaccharide (LPS)-activated macrophages isolated from the peritoneal cavity of mice .
Other natural and synthetic cannabinoid compounds (CBD, AEA, academic acid [AjA], and JWH-015), whose structures are depicted in Table 1, have also been shown to induce apoptosis in murine and human T lymphocytes. Cannabidiol, the nonpsychoactive ingredient in cannabis, induced apoptosis in CD4+ and CD8+ T cells at 4–8-μM concentrations by increasing reactive oxygen species (ROS) production as well as caspase 3 and 8 activity .
Ajulemic acid, a side-chain synthetic analog of Δ(8)-THC-11-oic acid, has been shown to induce apoptosis in human peripheral blood T lymphocytes via the intrinsic pathway at concentrations of 1, 3, and 10 μM . In addition, the use of synthetic CB2 agonist JWH-015 treatment in vitro led to cell death via both the death-receptor pathway and the intrinsic pathway. When JWH-015 was administered in vivo, the antigen-specific response to Staphylococcal enterotoxin A was inhibited significantly . It is important to note that, unlike in immune cells, cannabinoids can protect from apoptosis in nontransformed cells of the CNS, which can play a protective role in autoimmune conditions such as multiple sclerosis. Cannabinoids protect against apoptosis of oligodendrocytes via CB1 and CB2 receptors by signaling through the PI3K/AKT pathway. In vivo and in vitro exposure to arachidonic-2-ethylamide (ACEA) and WIN55,212-12 protected the cells, while pretreatment with CB1 receptor antagonist SR141716A and CB2 receptor antagonist SR144528 blocked the action of these cannabinoids . In a different study by Jackson et al., 3D mouse brain aggregate cell cultures were compared between wild-type mice and CB1 receptor knockout mice. IFN-γ treatment led to a decrease in the neurofilament-H expression in knockout cultures but not in wild-type cultures. In addition, caspase three activations were higher in knockout cultures, indicating a protective role of CB1 in neuronal cells .
Cannabinoid action on cytokines
Cytokines are the signaling proteins synthesized and secreted by immune cells upon stimulation. They are the modulating factors that balance the initiation and resolution of inflammation. One of the possible mechanisms of immune control by cannabinoids during inflammation is the dysregulation of cytokine production by immune cells and disruption of the well-regulated immune response . Furthermore, cannabinoids may affect immune responses and host resistance by perturbing the balance between the cytokines produced by T-helper subsets, Th1 and Th2. In vitro studies were performed to compare the effect of THC and cannabinol on cytokine production by human T, B, CD8+, NK, and eosinophilic cell lines. However, the results were variable, depending on the cell line and the concentration used . Both pro-inflammatory and anti-inflammatory effects of THC were demonstrated in this study, proposing that different cell populations have varied thresholds of response to cannabinoids. Generally, TNF-α, GM-CSF, and IFN-γ levels decreased with drug treatment. Interestingly, while the anti-inflammatory cytokine IL-10 decreased following THC treatment, there was an increase in the pro-inflammatory cytokine IL-8. In other studies, cannabinoid CP55,940 at nanomolar concentrations was shown to have a stimulatory effect on several cytokines in the human promyelocytic cell line HL-60 . At the molecular level, THC has also been shown to inhibit LPS-stimulated mRNA expression of IL-1α, IL-1β, IL-6, and TNF-α in cultured rat microglial cells; however, the effect was independent of the cannabinoid receptors . In a different study, mice were challenged with Corynebacterium parvum, in vivo, following the administration of the synthetic cannabinoids WIN55,212-2 and HU210. The animals were then challenged with LPS. The results showed decreased levels of TNF-α and IL-12 but increased levels of IL-10 in the serum . This effect was shown to be CB1 receptor-dependent.
During chronic inflammation, IL-6 suppression can decrease tissue injury . AjA has been reported to prevent joint-tissue injury in animal models of adjuvant arthritis . Recent studies showed that the addition of AjA to human monocyte-derived macrophages in vitro reduced the secretion of IL-6 from activated cells, suggesting that AjA may have a value for the treatment of joint inflammation in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and osteoarthritis . It has been observed that the CB2 agonist HU-308 attenuated the hepatic ischemia/reperfusion injury by decreasing the levels of TNF-α, MIP-1α, and MIP-2 in the serum and in liver homogenates . Recent in vitro studies have also shown the potent anti-inflammatory effect of synthetic cannabinoids (CP55,940 and WIN55,212-2). Both CP55,940 and WIN55,212-2 downregulated IL-6 and IL-8 cytokine production from IL-1β-stimulated rheumatoid fibroblast-like synoviocytes (FLS) via a non-CB1/CB2-mediated mechanism .
Endocannabinoids have also been reported to affect the cytokine biology of various cell systems. Antiproliferative effects of endocannabinoids on cancer cell lines are well established and are discussed in the later section of the review. However, AEA has also been reported to increase cytokine-induced proliferation. Mouse bone marrow cells, when cultured in the presence of IL-3 and AEA, were observed to produce more hematopoietic colonies than with IL-3 alone . Significant suppression of IL-2 expression by 2-AG and the nonhydrolyzable 2-AG ether was observed in leukocytes via activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) . Furthermore, in undifferentiated and macrophage-like differentiated HL-60 cells, 2-AG induced CB2-dependent acceleration in the production of IL-8 . In Theiler’s virus immune-mediated demyelinating disease, inactivation of endocannabinoids through the use of two selective inhibitors of their transport; (R)-N-oleoyl-(1′-hydroxy benzyl)-2′-ethanolamine] (OMDM2) and [(S)-Noleoyl-(1′-hydroxy benzyl)-2′-ethanolamine (OMDM1) led to decreased production of the pro-inflammatory cytokines IL-1β and IL-12 . On a contrary note, cytokines have also been shown to affect the endocannabinoid system. IL-12 and IFN-γ have been shown to reduce FAAH activity and protein expression of FAAH, whereas IL-4 or IL-10 stimulated FAAH activity . Table 2 provides a summary of the effect of cannabinoids on cytokines and chemokines in various cell models [26,28,29,32–34,37,40,41].