Erinn S. Raborn

CB2 receptors in the brain: role in central immune function

Recently, it has been recognized that the cannabinoid receptor CB2 may play a functionally relevant role in the central nervous system (CNS). This role is mediated primarily through microglia, a resident population of cells in the CNS that is morphologically, phenotypically, and functionally related to macrophages. These cells also express the cannabinoid receptor CB1. The CB1 receptor (CB1R) is constitutively expressed at low levels while the CB2 receptor (CB2R) is expressed at higher levels and is modulated in relation to cell activation state. The relatively high levels of the CB2R correspond with microglia being in ‘responsive’ and ‘primed’ states, suggesting the existence of a ‘window’ of functional relevance during which activation of the CB2R modulates microglial activities. Signature activities of ‘responsive’ and ‘primed’ microglia are chemotaxis and antigen processing, respectively. The endocannabinoid 2‐arachidonylglycerol has been reported to stimulate a chemotactic response from these cells through the CB2R. In contrast, we have shown in vivo and in vitro that the exogenous cannabinoids delta‐9‐tetrahydrocannabinol and CP55940 inhibit the chemotactic response of microglia to Acanthamoeba culbertsoni, an opportunistic pathogen that is the causative agent of Granulomatous Amoebic Encephalitis, through activation of the CB2R. It is postulated that these exogenous cannabinoids superimpose an inhibitory effect on pro‐chemotactic endocannabinoids that are elicited in response to Acanthamoeba. Furthermore, the collective results suggest that the CB2R plays a critical immune functional role in the CNS.

The Cannabinoid Delta-9-tetrahydrocannabinol Mediates Inhibition of Macrophage Chemotaxis to RANTES/CCL5: Linkage to the CB2 Receptor

The chemotactic response of murine peritoneal macrophages to RANTES/CCL5 was inhibited significantly following pretreatment with delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana. Significant inhibition of this chemokine directed migratory response was obtained also when the full cannabinoid agonist CP55940 was used. The CB2 receptor-selective ligand O-2137 exerted a robust inhibition of chemotaxis while the CB1 receptor-selective ligand ACEA had a minimal effect. The THC-mediated inhibition was reversed by the CB2 receptor-specific antagonist SR144528 but not by the CB1 receptor-specific antagonist SR141716A. In addition, THC treatment had a minimal effect on the chemotactic response of peritoneal macrophages from CB2 knockout mice. Collectively, these results suggest that cannabinoids act through the CB2 receptor to transdeactivate migratory responsiveness to RANTES/CCL5. Furthermore, the results suggest that the CB2 receptor may be a constituent element of a network of G protein-coupled receptor signal transductional systems, inclusive of chemokine receptors, that act coordinately to modulate macrophage migration.

Cannabinoids Inhibit Migration of Microglial-like Cells to the HIV Protein Tat

Microglia are a population of macrophage-like cells in the central nervous system (CNS) which, upon infection by the human immunodeficiency virus (HIV), secrete a plethora of inflammatory factors, including the virus-specified trans-activating protein Tat. Tat has been implicated in HIV neuropathogenesis since it elicits chemokines, cytokines, and a chemotactic response from microglia. It also harbors a β-chemokine receptor binding motif, articulating a mode by which it acts as a migration stimulus. Since select cannabinoids have anti-inflammatory properties, cross the blood–brain barrier, and target specific receptors, they have potential to serve as agents for dampening untoward neuroimmune responses. The aim of this study was to investigate the effect of select cannabinoids on the migration of microglial-like cells toward Tat. Using a mouse BV-2 microglial-like cell model, it was demonstrated that the exogenous cannabinoids Delta-9-tetrahydrocannabinol (THC) and CP55940 exerted a concentration-related reduction in the migration of BV-2 cells towards Tat. A similar inhibitory response was obtained when the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) was used. The CB2 receptor (CB2R) antagonist SR144528, but not the CB1 receptor (CB1R) antagonist SR141716A, blocked this inhibition of migration. Similarly, CB2R knockdown with small interfering RNA reversed the cannabinoid-mediated inhibition. In addition, the level of the β-chemokine receptor CCR-3 was reduced and its intracellular compartmentation was altered. These results indicate that cannabinoid-mediated inhibition of BV-2 microglial-like cell migration to Tat is linked functionally to the CB2R. Furthermore, the results indicate that activation of the CB2R leads to altered expression and compartmentation of the β-chemokine receptor CCR-3.

Cannabinoid Inhibition of Macrophage Migration to the Trans-Activating (Tat) Protein of HIV-1 Is Linked to the CB2 Cannabinoid Receptor

Macrophages and macrophage-like cells are important targets of HIV-1 infection at peripheral sites and in the central nervous system. After infection, these cells secrete a plethora of toxic factors, including the viral regulatory trans-activating protein (Tat). This protein is highly immunogenic and also serves as a potent chemoattractant for monocytes. In the present study, the exogenous cannabinoids δ-9-tetrahydrocannabinol (THC) and (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55940) were shown to significantly inhibit migration of human U937 macrophage-like cells to the Tat protein in a concentration-related manner. The CB1 receptor-selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) had no effect on Tat-mediated migration. In contrast, the CB2 receptor-selective agonist (1R,3R)-1-[4-(1,1-dimethylheptyl)-2,6-dimethoxyphenyl]-3-methylcyclohexanol (O-2137) exerted a concentration-related inhibition of U937 cell migration in response to Tat. Pharmacological blockage of CB1 receptor signaling using the antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide hydrochloride (SR141716A) had no effect on CP55940-mediated inhibition of macrophage migration to Tat, whereas treatment with the CB2 receptor antagonist (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 (SR144528) reversed the CP55940-mediated inhibition of migration. In addition, THC had no inhibitory effect on U937 migration to Tat after small interfering RNA knockdown of the CB2 receptor. Collectively, the pharmacological and biochemical knockdown data indicate that cannabinoid-mediated modulation of macrophage migration to the HIV-1 Tat protein is linked to the CB2 cannabinoid receptor. Furthermore, these results suggest that the CB2 cannabinoid receptor has potential to serve as a therapeutic target for ablation of HIV-1-associated untoward inflammatory response.

Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins

AIMS The aim of this study was to assess the effect of select cannabinoids on human immunodeficiency virus type 1 (HIV-1) transactivating (Tat) protein-enhanced monocyte-like cell adhesion to proteins of the extracellular matrix (ECM). MAIN METHODS Collagen IV, laminin, or an ECM gel was used to construct extracellular matrix layers. Human U937 monocyte-like cells were exposed to Tat in the presence of ∆(9)-tetrahydrocannabinol (THC), CP55,940, and other select cannabinoids. Cell attachment to ECM proteins was assessed using an adhesion assay. KEY FINDINGS THC and CP55,940 inhibited Tat-enhanced attachment of U937 cells to ECM proteins in a mode that was linked to the cannabinoid receptor type 2 (CB2R). The cannabinoid treatment of Tat-activated U937 cells was associated with altered β1-integrin expression and distribution of polymerized actin, suggesting a modality by which these cannabinoids inhibited adhesion to the ECM. SIGNIFICANCE The blood-brain barrier (BBB) is a complex structure that is composed of cellular elements and an extracellular matrix (ECM). HIV-1 Tat promotes transmigration of monocytes across this barrier, a process that includes interaction with ECM proteins. The results indicate that cannabinoids that activate the CB2R inhibit the ECM adhesion process. Thus, this receptor has potential to serve as a therapeutic agent for ablating neuroinflammation associated with HIV-elicited influx of monocytes across the BBB.

Acanthamoeba culbertsoni elicits soluble factors that exert anti-microglial cell activity.

ABSTRACT Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.

Delta-9-tetrahydrocannabinol, the major psychoactive component in marijuana, inhibits macrophage chemotaxis to Acanthamoeba.

OPPORTUNISTIC pathogens of the genus Acanthamoeba are causative agents of granulomatous amoebic encephalitis and cutaneous lesions (Martinez and Visvesvara 1997). There is accumulating evidence of increased incidence of these infections in immune suppressed individuals including those infected with the human immunodeficiency virus (HIV) or with AIDS (MarcianoCabral and Cabral 2003; Paltiel et al. 2004). Marijuana is the most widely used illicit drug in the United States and it is reasonable to anticipate that many of these individuals use it recreationally or therapeutically for treatment of nausea, loss of appetite, or chronic pain (Robson 2005; Felder, Dickason-Chesterfield, and Moore 2006). Marijuana and its principle psychoactive component delta9-tetrahydrocannabinol (THC) also are immune suppressive and modulate inflammatory responses (Cabral and Staab 2005). Delta9-tetrahydrocannabinol has been reported to inhibit macrophage functions including migration, nitric oxide production, lysis of bacteria and tumor cells, and inducible expression of cytokines (Cabral and Staab 2005; Klein, Friedman, and Specter 1998). It is not surprising, therefore, that THC alters host resistance to infection and has been shown to exacerbate infections due to bacteria and viruses (Cabral and Dove-Pettit 1998; Cabral and Staab 2005). Recently, we have demonstrated that (B6C3)F1 mice treated intraperitoneally with THC and subjected to instillation of A. culbertsoni intranasally experienced a higher incidence of mortalities as compared with similarly infected vehicle control animals (Marciano-Cabral et al. 2001). The mode through which THC alters the host response to Acanthamoeba has not been fully elucidated, but various studies indicate that macrophages play a major effector role against amebae (Marciano-Cabral and Toney 1998; Marciano-Cabral and Cabral 2003; Van Klink et al. 1996). These cells, along with other immunocytes are present in granulomas that surround amebae in tissues of immune competent patients and experimental animals and, in that capacity, may prevent dissemination of amebae (Marciano-Cabral and Cabral 2003). Thus, in order to obtain insight into processes that are operative within the host that are affected by THC and that account for increased mortality, we examined migratory responsiveness of macrophages to Acanthamoeba. Treatment of macrophages with THC in vitro or in vivo resulted in inhibition of chemotaxis to whole cell lysates of Acanthamoeba as well as to cell-free medium in which Acanthamoeba were maintained.

Sex Differences and Drug Dose Influence the Role of the α7 Nicotinic Acetylcholine Receptor in the Mouse Dextran Sodium Sulfate-Induced Colitis Model

Introduction α7 nicotinic acetylcholine receptors (nAChRs) play an important role in vagus nerve-based cholinergic anti-inflammatory effects. This study was designed to assess the role of α7 nAChRs in dextran sodium sulfate (DSS)-induced colitis in male and female mouse. We first compared disease activity and pathogenesis of colitis in α7 knockout and wild-type mice. We then evaluated the effect of several α7 direct and indirect agonists on the severity of disease in the DSS-induced colitis. Methods Male and female adult mice were administered 2.5% DSS solution freely in the drinking water for 7 consecutive days and the colitis severity (disease activity index) was evaluated as well as colon length, colon histology, and levels of tumor necrosis factor-alpha colonic levels. Results Male, but not female, α7 knockout mice displayed a significantly increased colitis severity and higher tumor necrosis factor-alpha levels as compared with their littermate wild-type mice. Moreover, pretreatment with selective α7 ligands PHA-543613, choline, and PNU-120596 decreased colitis severity in male but not female mice. The anti-colitis effects of these α7 compounds dissipated when administered at higher doses. Conclusions Our results suggest the presence of a α7-dependent anti-colitis endogenous tone in male mice. Finally, our results show for the first time that female mice are less sensitive to the anti-colitis activity of α7 agonists. Ovarian hormones may play a key role in the sex difference effect of α7 nAChRs modulation of colitis in the mouse. Implications Our collective results suggest that targeting α7 nAChRs could represent a viable therapeutic approach for intestinal inflammation diseases such as ulcerative colitis with the consideration of sex differences.