Marijuana is a term that describes the dry leaves, stems, flowers, and seeds of the Cannabis sativa plant. It has been prohibited in the United States by federal law since the 1937 Marijuana Tax Act and the US Drug Enforcement Agency has classified it as an illegal schedule I drug . Schedule I drugs are considered the most dangerous class of drugs with potential for severe psychological and physiological dependence. Other schedule I drugs include heroin, ecstasy, and lysergic acid diethylamide . Marijuana can be smoked in hand-rolled cigarettes , in cigars that have been emptied out of its contents and refilled with marijuana , through vaporizers to avoid inhaling smoke, consumed in edibles, or brewed as tea. While the use of marijuana for medicinal, religious, and recreational purposes dates back 5000 years, the discovery of cannabinoid molecules and our understanding of how they interact with our endogenous human cannabinoid signalingsystem represents a relatively recent area of investigation [Aizpurua-Olaizola 2016, Herring 1998, Pertwee 2006]. Marijuana is composed of over 400 different compounds, including more than 100 different cannabinoids [Aizpurua-Olaizola 2016, Greydanus 2013]. Cannabinoids are the primary bioactive constituents of marijuana and the main psychoactive cannabinoid is delta-9-tetrahydrocannabinol . The THC content in the average illicit marijuana cigarette is reported by the Potency Monitoring Project to comprise approximately 12% by weight [ElSohly 2014]. Other cannabinoids also found in the Cannabis sativa plant include cannabidiol , cannabigerol , and cannabinol , but these are not considered to play a role in the psychoactive effects associated with marijuana consumption. Upon combustion and smoking, marijuana also liberates an array of polycyclic aromatic hydrocarbons including the known carcinogens benzopyrene and benzanthracene,grow rack which are components of the particulate phase of smoke [Roth 2001]. Toxic substances such as carbon monoxide, hydrogen cyanide, and nitrosamines are also released as part of the gas phase of marijuana smoke.

While all of these released constituents may have biologic and/or toxic consequences, the focus of this thesis work is on the cannabinoid constituents and specifically on the biology of the human type 2 cannabinoid receptor. The development of synthetic cannabinoids eventually led to the discovery of a human endogenous cannabinoid system that is comprised of at least two arachidonic acid-derived endocannabinoids, 2-arachidonoylglycerol and anandamide , their biosynthetic and degradative enzymes, and two cannabinoid receptors, CB1 and CB2. [Bisogno 2005, Cabral 2015]. The endocannabinoid system has been found to play a role in immunomodulation, metabolic regulation, bone growth, pain, cancer, and psychiatric disorders [Aizpurua-Olaizola 2016, Kleyer 2012]. Endocannabinoids are thought to be enzymatically produced and released “on demand” [Cabral 2015]. They bind and activate seven-transmembrane G protein-coupled receptors type I and type II and are linked to intracellular signaling cascades including adenylyl cyclase, cAMP, mitogen-activated protein kinase, and intracellular calcium [Howlett 2002, Maccarrone 2015]. Cannabinoid receptors, CB1 and CB2, share 44% amino acid homology and bind THC with relatively equal affinity [Cabral 2015, Shire 1996]. They are expressed in most organ systems, and their activation by marijuana smoke can have wide-ranging health effects [Grotenhermen 2003, Volkow 2014, Turcotte 2016]. The CB1 receptor is mostly found in the central nervous system and mediates the psychoactive components associated with cannabinoids. CB1 has been described to play a role in memory, pain regulation,stress response, and the regulation of metabolism [Busquets Garcia 2016, Cabral 2015]. More specifically, the highest concentration of CB2 is found in immune cells in addition to lower concentrations found in bone cells, keratinocytes, adipocytes, and renal tissue [Basu 2011, Mackie 2006]. The CB2 receptor is suggested to play a role in immunomodulatory mechanisms that regulate inflammation and also play a role in host defense [Basu 2011, Herring 1998, Turcotte 2016]. Despite the widespread use of marijuana and its increasing legalization across multiple states in the U.S., there is relatively little information known about the effects of cannabinoids on human immunity.

Cannabinoids have been described to have anti-inflammatory effects on leukocytes. [Cabral 2015, Roth 2015, Volkow 2014]. In mouse studies, the CB2 receptor has been found to play a role in the responsiveness to infectious pathogens and play a role in immune homeostasis [Newton 1994, Newton 2009]. In human studies, alveolar macrophages from the lungs of marijuana smokers have been found to be deficient in the production of cytokines, nitric oxide, and mediation of bacteria killing [Baldwin 1997, Roth 2002, Shay 2003]. Human T cells activated in the presence of THC have also been found to result in a T helper type 2 -skewed pattern of cytokine production with limited proliferation [Yuan 2002]. With the highest levels of expression on immune cells, the CB2 receptor is suggested to mediate the immune regulating effects of cannabinoids [Cabral 2015, Roth 2015, Volkow 2014, Turcotte 2016]. In support of this statement, there are several studies done with animal models, including CB2 knock-out mice [Liu 2009, Turcotte 2016, Ziring 2006]. Although murine CB2 and human CB2 share 82% amino acid homology of the coding regions, there are significant differences in non-coding regions of their respective genes, suggesting that some inter-species differences likely exist with respect to regulation and expression [Liu 2009]. This potential difference argues that a combination of both animal models and human studies are required to understand the regulation and function of the CB2 receptor with respect to the immune system. Nonetheless, CB2 knock-out mice have been reported to exhibit higher levels of leukocyte recruitment and an over-production of pro-inflammatory cytokines [Buckley 2008]. While these mice do not exhibit obvious morphological differences they have also been noted to have abnormalities in the formation of several T cell and B cell subsets within lymphoid organs, making the CB2 receptor vital for the formation of T cell and B cells subsets involved in immune homeostasis [Turcotti 2016, Ziring 2006]. An increase in IgE production and allergic diseases would be expected in a model that is driven towards Th2 skewing [Agudelo 2008].

Surprisingly, THC treated CB2 knockout mice showed increased levels of IgE serum production, suggesting a role for CB2 receptor in the regulation of IgE [Newton 2012]. Immune suppression was also observed when THC was administered to tumor-bearing mice, which promoted tumor growth in a CB2-dependent manner [Zhu 2000]. Translating in vivo and in vitro experiments performed in animal or cell line models into an understanding of the biology in humans is also challenging because of the route of consumption, amount of exposure, and the pattern of use in marijuana users is entirely different, and there are often concurrent exposures of humans to tobacco, alcohol, and other substances that might affect the immune system in an additional or different manner.The previously described work suggests that cannabinoid receptors may be centrally involved in immune function, and therefore, the CB2 pathway may represent an attractive target for cannabinoid-based drugs. Cannabinoids have been promoted as a new class of drugs with the potential for beneficial anti-inflammatory, immunoregulatory, and anti-fibrotic effects [Atwood 2012, Pacher 2011, Turcotti 2016]. CB2 agonists have already been shown to reduce inflammation through the p38-MK2 pathway [Turcotti 2016]. There are currently multiple FDA-approved cannabinoid based medications. Marinol and Cesamet have been prescribed for the treatment of chemotherapy induced nausea and vomiting. Marinol has also been prescribed as an appetite stimulant and as a treatment for glaucoma by lowering intraocular pressure. Recently in July of 2016, SyndrosTM , an orally administered liquid formulation of dronabinol,greenhouse tables has also received FDA approval. It has been prescribed to treat anorexia associated weight loss in AIDS patients and chemotherapy induced nausea and vomiting. Also, Sativex, a sublingual spray that is composed of equal concentrations of THC and CBD, has received FDA approval to proceed with phase III clinical trials for the treatment of pain in patients with advanced cancer. It is also prescribed for the treatment of spasticity due to multiple sclerosis. CBD is of great therapeutic interest since it has been shown to have anti-emetic, anti-inflammatory, and anti-psychotic effects [Bergamaschi 2011, Cabral 2015, Turcotti 2016]. There have also been no effects observed on blood pressure, pulse, body temperature, or gastrointestinal and psychological function [Bergamaschi 2011]. Another cannabinoid formulation that contains only CBD, Epidiolex, is also undergoing phase III testing for the treatment of a rare genetic seizure disorder . Despite the Schedule I DEA classification assigned to marijuana , there is obvious evidence that strategies focused on regulating CB2 signaling might represent promising treatments for autoimmune or chronic inflammatory diseases. Understanding the expression and function of the human CB2 receptor may provide an important key to unlocking further cannabinoid-based drug development. The CB2 receptor has traditionally been described as a cell surface GPCR. GPCRs respond to a wide variety of stimuli and play crucial roles in neurotransmission, cellular metabolism, secretion, differentiation, growth, inflammation, and immune responses. GPCR activation is initiated by ligand binding, an event that usually occurs at the cell surface. Ligand binding induces a conformational change that activates heterotrimeric G-protein signaling and a subsequent cascade of events leading to internalization of the receptor and linkage with other signaling pathways [Jean-Alphonse 2011, Syrovatkina 2016]. The CB2 receptor has been reported to exhibit a complex pharmacology , signaling and trafficking pattern [Aizpurua-Olaizola 2016, Basu 2011, Howlett 2005]. The characterization of THC has led to the synthesis of cannabinoid analogs classified as synthetic cannabinoids, which are used to study structure-activity relationships, characterize cannabinoid-mediated bioactivity, and contribute to the understanding of mechanism of action by which endocannabinoids and phytocannabinoids exert their effects on the immune system [Cabral 2015].

The development of new ligands that can mimic the protective effects of cannabinoids has proven particularly difficult due to the constant discovery of multiple endogenous ligands, targets, and sites of interaction. Further research is needed to understand the mechanism of action of cannabinoids since the patterns of activation and induction of intracellular signaling differs with each compound. As demonstrated in CB2 transfected CHO cells, human HL-60, human bronchial epithelial cells, murine microglial cells, and a murine macrophage cell line, CB2 signaling is initiated through its interaction with heterotrimeric Gi-proteins and the inhibition of adenylyl cyclase [Turcotte 2016]. CB2 signaling has been linked to phosphorylation of MAP kinase, phosphorylation of AKT, modulation of intracellular calcium, and generation of intracellular ceramide [Basu 2011, Brown 2012, Chen 2012, Cudaback 2010, Howlett 2005, Turcotte 2016]. The mechanisms responsible for this signaling diversity have not been adequately explained. In studies with other GPCRs, it is often the process of receptor internalization that allows the receptor to become associated with an array of adaptor and signaling molecules [Calebiro 2010, Jean-Alphonse 2011]. The finding that CB1 receptor is expressed at intracellular sites and can mediate signaling adds further support for CB2 to play a role in mediating intracellular signaling [Rozenfeld 2011]. Rab proteins direct receptor trafficking to specific intracellular organelles, and CB2 receptors have been suggested to internalize via Rab-mediated endocytosis and initiate downstream intracellular signaling [Calebiro 2010, Grimsey 2011]. In artificial cell constructs, CB2 has been observed to undergo both constitutive and ligand-based internalization and traffic through endosomal and lysosomal compartments [Atwood 2010, Grimsey 2011, Kleyer 2012]. Blocking internalization or shifting the use of adaptor proteins has been observed to shift intracellular versus extracellular GPCR distribution [Grimsey 2011]. The dynamic balance between CB2 receptors at the cell surface and at possible intracellular sites might play a vital role in understanding cannabinoid receptor biology. The availability of cell surface receptors for ligand interaction can determine the responsiveness of a cell and further induction of intracellular signaling. Receptor availability for ligand binding is a very important feature in order to understand drug action and how the CB2 receptor can be exploited for therapeutic purposes. There is great diversity in the trafficking of GPCRs, and it is vital to understand the specific pathways involved with CB2. Localization of receptors at the cell membrane has been described to determine signaling via G protein pathways. Kleyer and associates also describe that the amount of cannabinoid receptor on the surface can directly determine receptor function. Interestingly, they also describe that cannabinoid receptors in primary human cells do not only internalize upon agonist interaction. They describe movement of the receptors between cytoplasm and cell membranes by ligand independent trafficking mechanisms, such as triggering by hydrogen peroxide that is present during inflammation and triggering by nonspecific protein tyrosine phosphatase inhibitors [Kleyer 2012].