Temporal variation in CB2R levels following T lymphocyte activation: evidence that cannabinoids modulate CXCL12-induced chemotaxis

Cannabinoids have long been proposed to affect the immune system, especially as one of the cannabinoid receptors, the cannabinoid receptor-2 (CB(2)R) has been found almost exclusively on immune cells. Here, using human in vitro activated peripheral blood-derived T lymphocytes we investigated the long-term changes in cannabinoid receptor protein expression following cellular activation and the effects of cannabinoids on migration. We report that resting T lymphocytes do not detectably express either the cannabinoid receptor-1 (CB(1)R) or CB(2)R at the protein level. However, CB(2)R protein expression is upregulated in a biphasic manner in T lymphocytes following activation by superantigen. The cannabinoids 2-AG and JWH-133 were found to elicit activation of downstream biochemical effectors (as assessed by the phosphorylation of the ERK1/2 MAP kinases). Neither 2-AG nor JWH-133 induced chemotaxis in day 5 activated T lymphocytes, when receptor expression was at its highest. Interestingly, both 2-AG and JWH-133 inhibited CXCL12-induced chemotaxis, suggesting a modulatory role for cannabinoids in activated T lymphocytes.     

The plant sterol guggulsterone attenuates inflammation and immune dysfunction in murine models of inflammatory bowel disease

Inflammatory bowel diseases (IBD) are chronic inflammatory and relapsing diseases of the gut that may manifest as either Crohn's disease (CD) or ulcerative colitis (UC). CD and UC are immunologically different diseases characterized by exacerbated Th1 and Th2 response. T-cell resistance against apoptosis contributes to inappropriate T-cell accumulation and the perpetuation of chronic mucosal inflammation. In the present study we have investigated the effect exerted by guggulsterone (GS) a plant derived steroid isolated from the gum resin of the Commiphora mukul tree, in two models of intestinal inflammation induced in mice by trinitro-benzene sulfonic acid (TNBS) and oxazolone. We provided evidence that E-GS protects mice against development of sign and symptoms of colon inflammation. E-GS effectively attenuated the severity of wasting disease and the fecal score and colon inflammation as assessed by measuring the macroscopic- and microscopic-damage scores. Administration Z-GS failed to ameliorate colon inflammation in TNBS-induced colitis and had a partial effect in oxazolone-induced colitis. In vitro, mechanistic studies carried out using CD4+ cells isolated from the intestinal lamina propria demonstrate that GS effectively regulates the function of effector T cells by modulating cell signaling activation pathway caused by CD3/CD28. The net biological effects resulting from exposure to GS includes attenuation of generation of interleukin-2 and -4 and interferon-γ as well as T cell proliferation. In conclusion, GS is an anti-inflammatory compound with the capacity to prevent and ameliorate T-cell-induced colitis. These data ground the use of GS, a natural cholesterol-lowering agent, in the treatment of chronic inflammatory diseases.     

Interleukin-1beta converting enzyme (caspase-1) in intestinal inflammation

An imbalance of T helper cell type 1 (Th1) versus type 2 (Th2) polarization in favor of Th1 cell subsets appears to be a key pathogenic mechanism in chronic inflammatory bowel disease (IBD), in particular in Crohn's disease. The interferon gamma-inducing factor interleukin (IL)-18 acts in strong synergism with the Th1 polarizing cytokine IL-12. Recent studies provide evidence for the participation of IL-18 in the pathogenesis of IBD: IL-18 expression is increased in inflamed lesions of Crohn's disease patients and neutralization of IL-18 in different models of experimental colitis resulted in a dramatic amelioration of disease severity. IL-18 and IL-1beta are cleaved and thereby activated by the interleukin-1beta converting enzyme (ICE). Activation of ICE also occurs during different types of infectious colitis, and ICE expression and subsequent release of IL-1beta and IL-18 significantly contribute to intestinal inflammation. ICE knockout mice as well as mice treated with the ICE inhibitor pralnacasan are protected against experimental mucosal inflammation. Thus, inhibition of ICE represents an intriguing new target that requires further investigation in animal models.     

Sensitivity to delta9-tetrahydrocannabinol is selectively enhanced in beta-arrestin2 -/- mice

Little is known about the roles of beta-arrestins in the regulation of brain CB1 cannabinoid receptors. This study investigated the role of beta-arrestin2 in cannabinoid behavioral effects using beta-arrestin2 -/- mice and their wild-type counterparts. A variety of cannabinoid ligands from different chemical classes that exhibit a variety of efficacies for activation of CB1 receptors were investigated, including Delta-tetrahydrocannabinol, CP55940, methanandamide, JWH-073, and O-1812. Delta-tetrahydrocannabinol produced both greater antinociception and greater decreases in body temperature in beta-arrestin2 -/- compared with beta-arrestin2 +/+ mice. No significant differences were, however, present in either assay for the other CB1 agonists. Antagonist radioligand binding indicated no difference in the density of cannabinoid CB1 receptors in the cerebellum, cortex, or hippocampus of beta-arrestin2 +/+ and -/- mice. These data demonstrate that beta-arrestin2 may regulate cannabinoid CB1 receptor sensitivity in an agonist-specific manner.     

Recent advances in the development of selective ligands for the cannabinoid CB(2) receptor

Two subtypes of the mammalian cannabinoid receptor have been identified and successfully cloned since 1990. The CB(1) receptor is primarily located in the central nervous system and the CB(2) receptor is almost exclusively expressed in cells of the immune system. The CB(1) and CB(2) receptors are both G-protein coupled receptors and are involved in the inhibition of adenylate cyclase. The CB(2) receptor is of particular importance due to its involvement in signal transduction in the immune system, making it a potential target for therapeutic immune intervention. A number of these selective ligands are derivatives of traditional dibenzopyran based cannabinoids. These include the very recently synthesized (2'R)-1-methoxy-3-(2'-methylbutyl)- Delta (8)-THC (JWH-359) which has a 224 fold selectivity for the CB(2) receptor, readily comparable to the well known 1-deoxy-3-(1',1'-dimethylbutyl)- Delta (8)-THC (JWH-133) which has 200 fold selectivity for the CB(2) receptor. Several 9-hydroxyhexahydrocannabinols have also been synthesized and are found to be selective high affinity ligands for the CB(2) receptor. These are 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, K(i) = 2.7 nM) and 1-deoxy-9beta-hydroxy-dimethylpentylhexahydrocannabinol (JWH-300, K(i) = 5.3 nM). CB(2) selective cannabi-mimetic indoles include 1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholine]ethyl)-5-methoxyindole (L768242), (R)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) and 1-propyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), which exhibit significant selectivity for the CB(2) receptor coupled with weak affinity for the CB(1) receptor. Bristol-Meyer Squibb has produced a phenylalanine derived cannabimimetic indole which possesses high CB(2) affinity (K(i) = 8 nM) and very low affinity for the CB(1) receptor (K(i) = 4000 nM). This review will discuss the current advances and recent results in the structure-activity relationships (SAR) of selective ligands for the cannabinoid CB(2) receptor.     

Mucosal T cell proliferation and apoptosis in inflammatory bowel disease

Both forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), represent prototypical conditions whose most salient features are the presence of chronic inflammation involving various parts of the intestinal tract and an increased risk of cancer, which is a complication directly related to the duration and activity of gut inflammation. Several factors have been implicated in the unrelenting mucosal inflammation of IBD, prominent among them being the presence of a persistently elevated number of activated T cells in the mucosa of CD and UC patients. These T cells display various defects of proliferation and apoptosis, and these abnormalities are credited with directly contributing to the pathogenesis of IBD and possibly the progression to colon cancer. This notion is supported by the observation that T cells are also prominently found infiltrating most tumors and are functionally impaired compared to T cells in the circulation. This establishes a parallel that may constitute a link between chronic intestinal inflammation and the development of malignancies in the inflamed intestine. This article will review some of the basic features of human intestinal mucosal T cells, examine the mechanisms underlying the processes of cell cycling and cell death, describe the defective proliferative and apoptotic function detected in CD and UC, and discuss the implications of modulating T cell apoptosis in IBD for therapeutic purposes and eventually decreasing the risk of cancer development.     

Chitosan oligosaccharide as potential therapy of inflammatory bowel disease: therapeutic efficacy and possible mechanisms of action

Inflammatory bowel disease (IBD) results from intestinal epithelial barrier defect and dysregulated mucosal immune response. This study aimed to evaluate the therapeutic potential of chitosan oligosaccharide (COS), a biodegradation product of dietary fiber chitosan, in the treatment of IBD and to elucidate its possible mechanisms of action. Oral administration of COS protected against mortality and intestinal inflammation in a mouse model of acute colitis induced by 5% dextran sulfate sodium (DSS). The most effective dose range of COS was 10-20 mg/kg/day. In addition, nuclear factor kappa B (NF-κB) activation, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in colonic tissues were suppressed in mice receiving COS. Similar protective effect of COS against mortality and intestinal inflammation was observed in another mouse model of acute colitis induced by rectal instillation of 4% acetic acid. Importantly, COS administration after colitis induction was effective in ameliorating intestinal inflammation in both acute colitis models induced by 5% DSS and chronic colitis models induced by cycles of 2.5% DSS. In human colonic epithelial cells (T84 cells), COS treatment prevented NF-κB activation, production of TNF-α and IL-6, and loss of epithelial barrier integrity under both lipopolysaccharide (LPS) and TNF-α-stimulated conditions. Furthermore, binding of LPS to T84 cells, and TNF-α and oxidative stress-induced apoptosis of T84 cells were prevented by treatment with COS. These results suggest that COS may be effective in the treatment of IBD through inhibition of NF-κB signaling and apoptosis of intestinal epithelial cells.     

Andrographolide sulfonate ameliorates experimental colitis in mice by inhibiting Th1/Th17 response

Inflammatory bowel disease (IBD) is a chronic, relapsing and remitting condition of inflammation involves overproduction of pro-inflammatory cytokines and excessive functions of inflammatory cells. However, current treatments for IBD may have potential adverse effects including steroid dependence, infections and lymphoma. Therefore new therapies for the treatment of IBD are desperately needed. In the present study, we aimed to examine the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on murine experimental colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Andrographolide sulfonate was administrated through intraperitoneal injection to mice with TNBS-induced colitis. TNBS-induced body weight loss, myeloperoxidase activity, shortening of the colon and colonic inflammation were significantly ameliorated by andrographolide sulfonate. Both the mRNA and protein levels of pro-inflammatory cytokines were reduced by andrographolide sulfonate administration. Moreover, andrographolide sulfonate markedly suppressed the activation of p38 mitogen-activated protein kinase as well as p65 subunit of nuclear factor-κB (NF-κB). Furthermore, CD4⁺ T cell infiltration as well as the differentiation of Th1 (CD4⁺IFN-γ⁺) and Th17 (CD4⁺IL17A⁺) subset were inhibited by andrographolide sulfonate. In summary, these results suggest that andrographolide sulfonate ameliorated TNBS-induced colitis in mice through inhibiting Th1/Th17 response. Our study shows that water-soluble andrographolide sulfonate may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.     

Functional CB2 type cannabinoid receptors at CNS synapses

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.     

Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity

K2 and several similar purported "incense products" spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3-M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist (K(b)～40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad (e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice. In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products.     

The ameliorating effect of cannabinoid type 2 receptor activation on brain,lung, liver and heart damage in cecal ligation and puncture-induced sepsis model in rats

Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response. It is already known that Cannabinoid Type 2 (CB2) receptors, which are commonly expressed in immune cells and in many other tissues, have an effect on the regulation of immune response. In the present study of ours, the effects of CB2 receptor agonist JWH-133 was investigated on cecal ligation and puncture (CLP)-induced polymicrobial sepsis model in rats. In the present study, Sprague-Dawley rats were divided into 5 groups (i.e. the Sham, CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg, and JWH-133 5 mg/kg Groups). Except for the Sham Group, the CLP-induced sepsis model was applied to all groups. The histopathological damage in brain, lung, liver and, heart was examined and the caspase-3, p-NF-κB, TNF-α, IL-1β and IL-6 levels were determined immunohistochemically. The serum TNF-α, IL-1β, IL-6, IL-10 levels were examined with the ELISA Method. The JWH-133 treatment decreased the histopathological damage in brain, heart, lung, and liver and reduced the caspase-3, p-NF-κB, TNF-α, IL-1β, IL-6 levels in these tissues. In addition to this, JWH-133 treatment also decreased the serum TNF-α, IL-1β, IL-6 levels, which were increased due to CLP, and increased the anti-inflammatory cytokine IL-10 levels. In the present study, it was determined that the CB2 receptor agonist JWH-133 decreases the CLP-induced inflammation, and reduces the damage in brain, lung, liver and heart. Our findings show the therapeutic potential of the activation of CB2 receptors with JWH-133 in sepsis.     

Involvement of central cannabinoid CB2 receptor in reducing mechanical allodynia in a mouse model of neuropathic pain

We sought to examine the involvement of central cannabinoid CB2 receptor activation in modulating mechanical allodynia in a mouse model of neuropathic pain. JWH133 was demonstrated to be a selective cannabinoid CB2 receptor agonist in mice, reducing forskolin-stimulated cAMP production in CHO cells expressing mouse cannabinoid CB2 and cannabinoid CB1 receptors with EC50 values of 63 nM and 2500 nM, respectively. Intrathecal administration of JWH133 (50 and 100 nmol/mouse) significantly reversed partial sciatic nerve ligation-induced mechanical allodynia in mice at 0.5 h after administration. In contrast, systemic (intraperitoneal) or local (injected to the dorsal surface of the hindpaw) administration of JWH133 (100 nmol/mouse) was ineffective. Furthermore, the analgesic effects of intrathecal JWH133 (100 nmol/mouse) were absent in cannabinoid CB2 receptor knockout mice. These results suggest that the activation of central, but not peripheral, cannabinoid CB2 receptors play an important role in reducing mechanical allodynia in a mouse model of neuropathic pain.     

In vivo treatment with a nonapeptide thymic hormone, facteur thymique serique (FTS), ameliorates chronic colitis induced by dextran sulphate sodium in mice

Facteur thymique serique (FTS), a thymic hormone with nonapeptide is involved in T cell differentiation in intestine. Here we investigated the effect of FTS on dextran sulphate sodium (DSS)-induced colitis. BALB/c mice were subcutaneously treated with 1 mug/mouse/day of FTS daily. FTS did not affect the course of acute colitis induced by DSS as assessed by survival rate, clinical activity of diseases, extent of tissue damage of colons. On the other hand, FTS significantly ameliorated chronic colitis induced by multiple cycles of DSS as reflected by lower lethality, weight loss, clinical scores and histological scores. The levels of interferon (IFN)-gamma, interleukin 1(IL-1)-beta, and IL-12p40 in the culture supernatants of lamina propria (LP) cells of colon without any stimulation and IFN-gamma by T cells in the LP T cells under T cell receptor (TCR) triggering were reduced in FTS-treated mice, whereas the levels of IL-10 by LP cells and LPT cells were higher in FTS-treated mice. Thus, FTS may serve to suppress inflammation in DSS-induced chronic colitis accompanied by increased IL-10 production.     

High fat diet exacerbates dextran sulfate sodium induced colitis through disturbing mucosal dendritic cell homeostasis

Epidemiological studies have shown that fat rich western diet contributes to the high incidence of inflammatory bowel disease (IBD). Moreover, accumulated data indicated that fat dietary factor might promote the change of the composition and metabolism in commensal flora. But, the exact mechanisms for fatty diet in gut inflammation are not well demonstrated. In this study, we found that high fat diet (HFD) promoted inflammation and exacerbated the disease severity of dextran sulfate sodium (DSS) induced colitis in mice. Compared with low fat diet (LFD)/DSS mice, shorter colon length, more epithelial loss and crypt destruction and more Gr-1⁺ myeloid inflammatory cells infiltration in colons were observed in HFD/DSS cohorts. Interestingly, such HFD mediated inflammation accompanied with the dys-regulation of hematopoiesis, and more hematopoiesis stem and progenitor cells were detected in colon and spleen. We further analyzed the effects of HFD and DSS treatment on mucosal DC subsets, and found that DSS treatment in LFD mice mainly dramatically increased the percentage of CD11c⁺ CD103⁻ CD11b⁺ DCs in lamina propria (LP). While, in HFD/DSS mice, HFD pre-treatment not only increased the percentage of CD11c⁺ CD103⁻ CD11b⁺ DCs, but also decreased CD11c⁺ CD103⁺ CD11b⁺ in both LP and mesenteric lymph nodes (MLN) in mice with colitis. This disequilibrium of mucosal dendritic cells in HFD/DSS mice may depend on the reduced levels of buytrate and retinoic acid. Thus, this study declared the effects of HFD on gut microenviroment, and further indicated its potential role in the development of DSS induced colitis.     

Cannabinoid CB(2) receptors modulate ERK-1/2 kinase signalling and NO release in microglial cells stimulated with bacterial lipopolysaccharide

BACKGROUND AND PURPOSE

Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs in chronic immune inflammatory diseases. In the present study, we characterized the signal transduction pathways affected by CB₂ receptors in quiescent and lipopolysaccharide (LPS)-stimulated murine microglia.

EXPERIMENTAL APPROACH

We examined the effects of the synthetic CB₂ receptor ligand, JWH-015, on phosphorylation of MAPKs and NO production.

KEY RESULTS

Stimulation of CB₂ receptors by JWH-015 activated JNK-1/2 and ERK-1/2 in quiescent murine microglial cells. Furthermore, CB₂ receptor activation increased p-ERK-1/2 at 15 min in LPS-stimulated microglia. Surprisingly, this was reduced after 30 min in the presence of both LPS and JWH-015. The NOS inhibitor l-NAME blocked the ability of JWH-015 to down-regulate the LPS-induced p-ERK increase, indicating that activation of CB₂ receptors reduced effects of LPS on ERK-1/2 phosphorylation through NO. JWH-015 increased LPS-induced NO release at 30 min, while at 4 h CB₂ receptor stimulation had an inhibitory effect. All the effects of JWH-015 were significantly blocked by the CB₂ receptor antagonist AM 630 and, as the inhibition of CB₂ receptor expression by siRNA abolished the effects of JWH-015, were shown to be mediated specifically by activation of CB₂ receptors.

CONCLUSIONS AND IMPLICATIONS

Our results demonstrate that CB₂ receptor stimulation activated the MAPK pathway, but the presence of a second stimulus blocked MAPK signal transduction, inhibiting pro-inflammatory LPS-induced production of NO. Therefore, CB₂ receptor agonists may promote anti-inflammatory therapeutic responses in activated microglia.     

Sch35966 is a potent, selective agonist at the peripheral cannabinoid receptor (CB2) in rodents and primates

BACKGROUND AND PURPOSE: The peripheral cannabinoid receptor (CB(2)) is expressed on peripheral immune cells and is thought to have a role in the immunosuppressive effects of cannabinoids. Historically, there have been few potent, CB(2)-selective agonists to assess the contribution of CB(2) to this phenomenon. The studies presented here describe the synthesis of 8,10-bis[(2,2-dimethyl-1-oxopropyl)oxy]-11-methyl-1234-tetrahydro-6H-benzo[beta]quinolizin-6-one (Sch35966), which binds with low nanomolar potency to CB(2) in both primates and rodents. EXPERIMENTAL APPROACH: The affinity, potency and efficacy of Sch35966 and other cannabinoid ligands at CB(2) was assessed using competition binding assays vs [(3)H]CP55,940, [(35)S]GTPgammaS exchange, cAMP accumulation and cell chemotaxis assays. KEY RESULTS: We showed that Sch35966 has >450-fold selectivity for CB(2) binding vs the central cannabinoid receptor (CB(1)) in primates (humans and cynomolgus monkeys) and rodents (rats and mice). Sch35966 is an agonist as it effectively inhibited forskolin-stimulated cAMP synthesis in CHO-hCB(2) cells, stimulated [(35)S]GTPgammaS exchange and directed chemotaxis in cell membranes expressing CB(2). In all species examined, Sch35966 was more potent, more efficacious and more selective than JWH-015 (a commonly used CB(2)-selective agonist). CONCLUSIONS AND IMPLICATIONS: Taken together, the data show that Sch35966 is a potent and efficacious CB(2)-selective agonist in rodents and primates.     

Role of cannabinoid CB1 receptors and tumor necrosis factor-alpha in the gut and systemic anti-inflammatory activity of SR 141716 (rimonabant) in rodents

(1) We investigated the effect of the cannabinoid CB1 receptor antagonist, SR 141716, on indomethacin-induced small intestine inflammation and Escherichia coli lipopolysaccharide (LPS)-induced plasma TNF-alpha (TNF) release in comparison to the cannabinoid CB2 receptor antagonist, SR 144528, in rodents. (2) In rats, indomethacin induced significant ulcer formation in the small intestine; this was accompanied by an increase in tissue TNF levels and myeloperoxidase (MPO) activity. SR 141716 prevented the ulcers and the rise in TNF levels (ID50 3.3, 0.4 mg kg-1, respectively) and MPO activity. SR 144528 prevented intestinal ulcers only. (3) The effect of SR 141716 against indomethacin-induced ulcers and increase of plasma TNF levels after LPS was also studied in wild-type and CB1 receptor knockout mice. Indomethacin induced intestinal ulcers in mice, but not tissue TNF production and MPO activity. SR 141716 reduced the ulcers to a similar extent in wild-type and CB1 receptor knockout mice. In rats and wild-type mice, but not in CB1 receptor knockout mice, SR 141716 inhibited the LPS-induced increase in plasma TNF levels. (4) These findings provide evidence that the indomethacin model of intestinal lesions differs in rat and mouse and support the existence of several mechanisms for the antiulcer activity of SR141716, the most important involving the inhibition of TNF production. The potent anti-inflammatory activity of SR141716 in rodents indicated its potential therapeutic interest in chronic immune-inflammatory diseases.     

Activation of cannabinoid CB2 receptor negatively regulates IL-12p40 production in murine macrophages: role of IL-10 and ERK1/2 kinase signaling

1 Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs for the treatment of many disease conditions. In the present study, we investigated the effects of the synthetic CB(2) ligand, JWH-133 on the production of interleukins (ILs), IL-12 and IL-10 by lipopolyssacharide (LPS) or Theiler's virus (TMEV)-activated macrophages. 2 JWH-133 evoked a concentration-related inhibition (10 nM-5 microM) of LPS/IFN-gamma induced IL-12p40 release. The effect of JWH-133 (100 nM) was significantly blocked by the CB2 antagonist SR-144528 (1 microM). Macrophages infected with TMEV increased IL-12p40 production and activation of CB2 receptors by JWH-133 (100 nM) inhibited it. 3 The inhibitory effect of JWH-133 (100 nM) on IL-12p40 production may involve extracellular-regulated kinase (ERK1/2) signaling: (i) JWH-133 induced a greater and sustained activation of ERK1/2 kinase in comparison with the level of activation observed following LPS; (ii) the inhibition of ERK1/2 by the specific inhibitor PD98059 increased LPS-induced IL-12p40 production in the presence or absence of JWH-133 suggesting a negative regulation of ERK pathway on IL-12p40 biosynthesis. 4 Activation of CB2 receptors by JWH-133 (10 nM-5 microM) enhanced IL-10 release by LPS/IFN-gamma-activated macrophages and addition of SR144558 (1 microM) totally blocked the effect of JWH (100 nM). 5 Inhibition of ERK by PD98059 significantly suppressed IL-10 production by LPS-activated macrophages. Endogenous IL-10 plays a modulatory role in IL-12 production. Blocking IL-10 with neutralizing antibody resulted in increased IL-12p40 secretion by LPS-activated macrophages in the absence or presence of JWH-133. In contrast, the addition of exogenous mIL-10 reduced the secretion of IL-12p40 in response to LPS.     

Targeting Cannabinoid Receptor 2 on Peripheral Leukocytes to Attenuate Inflammatory Mechanisms Implicated in HIV-Associated Neurocognitive Disorder

HIV infection affects an estimated 38 million people. Approximately 50% of HIV patients exhibit neurocognitive dysfunction termed HIV-Associated Neurocognitive Disorder (HAND). HAND is a consequence of chronic low-level neuroinflammation due to HIV entry into the brain. Initially, monocytes become activated in circulation and traffic to the brain. Monocytes, when activated, become susceptible to infection by HIV and can then carry the virus across the blood brain barrier. Once in the brain, activated monocytes secrete chemokines, which recruit virus-specific CD8⁺ T cells into the brain to further promote neuroinflammation. HAND is closely linked to systemic inflammation driven, in part, by HIV but is also due to persistent translocation of microorganisms across the GI tract. Persistent anti-viral responses in the GI tract compromise microbial barrier integrity. Indeed, HIV patients can exhibit remarkably high levels of activated (CD16⁺) monocytes in circulation. Recent studies, including our own, show that HIV patients using medical marijuana exhibit lower levels of circulating CD16⁺ monocytes than non-cannabis using HIV patients. Cannabis is a known immune modulator, including anti-inflammatory properties, mediated, in part, by ∆⁹-tetrahydrocannabinol (THC), as well as less characterized minor cannabinoids, such as cannabidiol (CBD), terpenes and presumably other cannabis constituents. The immune modulating activity of THC is largely mediated through cannabinoid receptors (CB) 1 and 2, with CB1 also responsible for the psychotropic properties of cannabis. Here we discuss the anti-inflammatory properties of cannabinoids in the context of HIV and propose CB2 as a putative therapeutic target for the treatment of neuroinflammation.

HIV-associated neurocognitive disorder is a systemic inflammatory disease leading to activation of plasmacytoid dendritic cells, monocytes and T cells. Monocyte and CD8 T cell migration across the BBB and interaction with astrocytes promotes neurotoxic inflammatory mediators release. CB2 ligands are proposed as therapeutics capable of suppressing systemic and localized inflammation.

     

Liquid chromatography-tandem mass spectrometry analysis of urine specimens for K2 (JWH-018) metabolites

Marijuana is the most widely used drug of abuse all over the world. The major active constituent of the drug is Δ?- tetrahydrocannabinol (Δ?-THC). Δ?-THC exerts its psychological activities by interacting with the cannabinoid receptors (CB? and CB?) in the brain. JWH-018, HU-210, and CP-47497, with CB? agonist activity (similar to Δ?-THC), have been used by the drug culture to spike smokable herbal products to attain psychological effects similar to those obtained by smoking marijuana. The products spiked with these CB? agonists are commonly referred to as "Spice" or "K2". The most common compound used in these products is JWH-018 and related compounds (JWH-073 and JWH-250). Little work has been done on the detection of these synthetic cannabimimetic compounds in biological specimens. This report investigated the metabolism of JWH-018 by human liver microsomes, identification of the metabolites of JWH-018 in urine specimen of an individual who admitted use of the drug, and reports on the quantitation of three of its urinary metabolites, namely the 6-OH-, the N-alkyl OH (terminal hydroxyl)-, and the N-alkyl terminal carboxy metabolites using liquid chromatography-tandem mass spectrometry. The concentrations of these metabolites are determined in several forensic urine specimens.