They even describe these methods to be responsible for externalization of the cannabinoid receptor in T cells. A phenomenon that we have never seen before. Previous studies have demonstrated that intracellular CB1 receptors located in lysosomes and mitochondria are able to induce intracellular signaling, suggesting that intracellular stores of CB1 receptor are functional [Kleyer 2012, Rozenfeld 2011]. Understanding CB2 receptor trafficking could help determine cell-type specific effects of cannabinoids and the regulation of the CB2 receptor in different immune cells. While GPCRs are integral membrane proteins, there has been increasing interest in their expression and function at sites other than the extracellular membrane [Jean-Alphonse 2011]. Relatively little is known about the expression and distribution of CB2 protein in human leukocytes. CB2 has been traditionally described as a cell membrane GPCR expressed primarily by B cells while CB2 mRNA has been identified in most leukocytes but with expression levels to also be the highest in B cells [Carayon 1998, Mackie 2006, Sanchez Lopez 2015]. However, experimental data supporting the expression of the CB2 receptor in human leukocytes has been limited and sometimes contradictory, mostly due to the lack of reliable tools for detecting CB2 protein in cells of interest. CB2 protein detection had been particularly difficult due to non-specific staining of primary antibodies and use of CB2 polyclonal antibodies that can be cross-reactive to other proteins [Graham 2010]. In contrast to our preliminary findings, Graham and associates have suggested that CB2 was highly expressed on all PBMC,flood tray but they used commercial polyclonal rabbit or goat antibodies from different companies without any controls.
Staining was different for every manufacturer, from batch to batch, and from subject to subject.Cannabinoids have also been variably described to promote or suppress B cell proliferation and to bias immunoglobulin class switching, suggesting cannabinoids might play a role in B cell activation, differentiation, and maturation [Agudelo 2008, Basu 2013, Newton 1994, Newton 2009, Ngaotepprutaram 2013]. Previous evidence from mouse studies has also suggested that exposure to cannabinoids can bias the response toward a Th2 response through a mechanism involving CB2 receptors [Agudelo 2008, Carayon 1998]. Detailed studies in CB2-knockout mice revealed deficient B cell subsets in several lymphoid organs [Buckley 2012, Ziring 2006]. The CB2 receptor has also been identified to have chemotactic effects, and cannabinoids have been described to have a role in B cell trafficking within mouse lymph nodes [Basu 2011]. The specific role of CB2 in the biologic function of B cells, especially in humans, remains to be studied in detail. The primary function of B cells is to secrete antibodies and thereby provide the humoral component of immunity. Additionally, B cells play important roles in presenting antigens and by secreting cytokines, therefore exerting an influence over the other arms of the immune system [Hoffman 2016]. However, in their naïve state, B cells exhibit limited function and must be activated by exposure to antigens and co-stimulatory signals. Activation in this manner promotes the naïve B cell to enlarge, clonally expand, differentiate, and eventually undergo isotype switching, which results in the production of mature antibodies and differentiation into memory or plasma B cells [Hoffman 2016]. Different antibody isotypes are elicited in response to different pathogens and directly influenced by the nature of cost imulatory signals and the local cytokine environment [Hoffman 2016]. Isotype switching in human B cells can be induced by CD40L when combined with IL-21 and modulated by several other cytokines including IL-2, IL-4, IL-10 [Avery 2008, Moens 2014].
The natural process of activation, expansion and differentiation is antigen- specific and directly enhances the capacity for a host to respond rapidly and effectively to future immune challenges by that same antigen [Hoffman 2016]. Dysregulated activation and isotype switching is also important as it can lead to autoimmune disorders or immunodeficiency [Hoffman 2016]. Some B cell activating signals can also promote allergic or suppressive immune reactions [Hoffman 2016, Taylor 2006]. Employing a mouse model where-in animals were infected with the legionella pneumophila organism, a number of studies were carried out to study the impact of cannabinoid exposure on the host antibody response to this infection [Cabral 2009, Newton 2004]. This approach demonstrated that exposure to THC and to other CB2-specific ligands during B cell activation is associated with Ig class switching and the generation of allergic or immunosuppressive responses [Agudelo 2008, Moens 2014, Newton 1994, Newton 2009]. IL- 4 is a potent B cell activating cytokine. In the presence of IL-4, CB2 message has been demonstrated to increase in B cells and to skew isotype switching. Carlisle and associates described that CB2 might be particularly responsive to agonists when in a responsive or activated state. The immunomodulatory activity of CB2 might be dependent on the activation of the target as well [Carlisle 2002]. Persidsky and associates found that activation of CB2 blocks monocyte migration and reduces secretion of pro-inflammatory cytokine, TNF-α [Persidsky 2015]. Further investigation regarding the effects of cannabinoids and the role of the CB2 receptor on B cell activation and isotype switching is vital in order to characterize potential mediators in controlling isotype switching and assuring the appropriate immune response is induced following antigen exposure. In conclusion, there are conflicting data regarding the effects of cannabinoids on B cell function.
These conflicting results can be attributed to inconsistencies in studies done with mixed immune cell populations versus isolated B cells, comparison of naïve versus activated cells, and peripheral blood B cells versus tonsillar or splenic B cells. Through the work of this dissertation, we hope to better understand how CB2 receptor location, the dynamic balance between extracellular and intracellular receptors, activation, and isotype switching link to the biologic effects of human leukocytes. We hypothesize that CB2 is an immunoregulatory molecule and its expression may be directly tied to the level and/or type of cell activation. Further research on the immunotoxic effects of marijuana and its effects on cannabinoid receptor biology are needed in order to develop a clear understanding of the balance between extracellular and intracellular CB2 receptors and the impact of intracellular location on cannabinoid-mediated signaling. These results will be of vital interest to the field of cannabinoid receptor biology and directly relevant to understanding the potential toxic effects of cannabinoids on immune function and how the cannabinoid/CB2 pathway can be exploited for immunotherapeutic purposes. Cannabinoids, the primary bioactive constituents of marijuana, activate CB1 and CB2 receptors and signal through an endogenous cannabinoid system to produce their biologic effects. Expression of the CB2 receptor predominates in cells from the immune system. However, there is little information known as to how the CB2 receptor influences human immunity and host defense, the specific location of CB2 receptors in human leukocytes, and the impact of cannabinoids on its distribution. While it is important to understand the immunotoxic effects that might result from marijuana smoking and exposure to cannabinoids, it is equally important to understand how the CB2 receptor might be exploited to control inflammation and regulate adaptive immunity from a therapeutic perspective. The CB2 receptor has traditionally been described as a cell membrane GPCR expressed primarily by B cells. However, as reliable methods for imaging the CB2 receptor did not exist, we hypothesized that a monoclonal antibody raised against the N-terminus of CB2 could be combined with conventional and imaging flow cytometry to study CB2 protein expression. Previous evidence suggested that T cells, which do not express surface CB2, can mediate the effects of cannabinoids through intracellular CB2 receptor expression. Therefore, this led us to also hypothesize that intracellular receptors must be able to mediate ligand-induced signaling and biological consequences. After employing a new approach with conventional and imaging flow cytometry, grow table we determined that B cells express CB2 on the cell surface and at intracellular locations, while T cells, monocytes, and dendritic cells only express CB2 at intracellular sites. Cell surface CB2 was responsive to THC by rapidly internalizing when exposed to the ligand. The distribution of this internalized CB2 did not appear to account for the pre-existing distribution of intracellular CB2. The reasons as to why CB2 is expressed on the cell surface of certain cells and not expressed on the cell surface in other cells remains unknown. After concluding with these findings, we hypothesize the cellular CB2 receptor location is a key feature that links location to specific biologic outcomes, and the expression of CB2 at extracellular versus intracellular sites may play an important function in mediating the biologic and toxic effects of cannabinoids. The expression of GPCRs at different cellular locations can promote functional heterogeneity with respect to downstream signaling and function. As such, we hypothesized that this differential expression of CB2 by leukocytes is likely a highly-regulated event and plays an important role in cannabinoid function. Activation of CB2 has been linked to many different signaling pathways and cellular events [Agudelo 2008, Carayon 1996, Ngaotepprutaram 2013].
By focusing on B cells, the only leukocyte discovered to express both CB2 at the cell surface and at intracellular locations, we hypothesize that B cell activation plays a key role in CB2 expression and in mediating the biologic function of B cells, such as isotype switching and antibody production. In order to investigate the impact of B cell activation on CB2 expression, we have designed an in vitro activation model where we can induce activation and differentiation in naïve mature B cells and track CB2 expression across the different stages of differentiation and maturation. We determined that cells found to be in an activated state and cells activated in vitro lacked cell surface CB2 and expressed high intracellular CB2. This finding allowed us to directly link the acquisition of an activated phenotype to the loss of surface CB2. The intracellular location of CB2 and the specific role of different receptors on biologic function remains to be determined but will likely be very informative in understanding cannabinoid biology. The experiments detailed in this dissertation are the first steps in determining what major factor controls the distribution of CB2 in human leukocytes and how this relates to biologic function. Ultimately, we hypothesize that CB2 receptor expression, location, and trafficking are all critical features that link cannabinoids to specific signaling and functional consequences on human leukocytes. Understanding how these features are linked to immune regulation could lead to the development of novel therapeutics by targeting specific biologic outcomes, such as apoptosis, cytokine production, and isotype switching. By the conclusion of these studies, we will have established a clear understanding of the differential expression patterns of CB2 by human B cells and how it relates biologic function. CB2 mRNA has been described to be expressed by most leukocytes, with expression levels to be greatest in B cells, less in monocytes, and low in T cells [Carayon 1998, Mackie 2006]. However, experimental data supporting this in humans has been limited. At the protein level, CB2 has traditionally been described as a cell membrane GPCR expressed primarily by B cells, but our recent findings challenge this description. Preliminary data from our laboratory suggests there is no CB2 receptor cell surface expression in T cells, but there is previous evidence that states that T cells have reduced T cell proliferation, activation, and cytokine production when exposed to THC [Cabral 2015, Roth 2002, Volkow 2014]. These findings led us to hypothesize that CB2 receptors must be present at intracellular locations, and these receptors must be capable of mediating ligand induced signaling and biologic function. The distribution of the CB2 receptor in human leukocytes and the reason as to why extracellular CB2 is not expressed in T cells is not known. It is not clear whether a difference in distribution of CB2 represents variable rates of internalization and recycling or whether cell-specific differences related to activation and maturation result in these differential expression patterns. It is also not clear what role these CB2 expression patterns have in mediating the biologic and toxic effects of cannabinoids on immune function. We hypothesize that the presence of CB2 at different cellular locations is an important feature that promotes functional heterogeneity with respect to downstream signaling and biologic responses. At this point in cannabinoid receptor biology, relatively little is known about the expression and distribution of CB2 protein.