No one has been able to characterize CB2 at a different locations of the cell other than the cell surface. If CB2 does exist at different locations, there is currently no evidence as to whether these receptors are functional or not. This gap in the field was mostly due to a lack of a set of reliable tools to measure cannabinoid receptor expression at the cell surface and at intracellular locations. There was also conflicting published results regarding cell surface receptor expression, which lacked reliable controls. In addition, these conflicting data result from the comparison of naïve versus activated cells, use of polyclonal versus monoclonal antibodies, and animal versus human CB2. In chapter 3, we describe in Castaneda et al. and Roth et al. how we have designed a novel cellular and molecular approach to investigate the expression, cellular distribution, and trafficking of the CB2 receptor in primary human cells in order to develop a better understanding of how these features impact cannabinoid-mediated signaling and biologic function. Interestingly, Kaplan and associates describe that there is also evidence that the CB1 receptor, despite its predominant presence in the central nervous system, can mediate many immune system effects,4×8 grow table with wheels including direct modulation of immune function by endogenous and exogenous cannabinoids in T cells and innate cells [Kaplan 2013]. With this novel flow cytometry approach, we have also proven that this innovative detection method has the same capability of detecting CB1 in human leukocytes in a reliable and specific manner.
With the new assay created in Castaneda et al. and further described in Roth et al., we strive to measure the distribution of cell surface and intracellular CB2 receptor expression in human immune leukocytes in order to further understand the role of the CB2 receptor in human immunity for the possible development of future therapeutics.While the use of Cannabis for medicinal, religious, and recreational purposes dates back 5,000 years, the identification of cannabinoids and the discovery of an endogenous cannabinoid ligand and receptor signaling pathway in human cells represents a relatively recent discovery . Cannabinoid receptor subtype 1 is highly expressed in the brain and well known for mediating the psychoactive effects of marijuana, while the highest expression of mRNA encoding for cannabinoid receptor subtype 2 exists in peripheral tissues and particularly within cells of the immune system . Both receptors are membraneassociated G-protein coupled receptors and bind Δ9 -tetrahydrocannabinol with relatively equal affinity . However, a number of other ligands have been identified, which express high selectivity for CB2 . Using these reagents, it has been shown that activation of CB2 receptor can regulate both innate and adaptive immunity including the ability to suppress anti-cancer responses and host defenses against pneumonia , promoteapoptosis of antigen presenting cells and T cells , alter cytokine production and antibody isotype switching , modulate the infectivity and replication of HIV virus , regulate the inflammatory aspects of atherosclerosis , and play a role in several autoimmune diseases . This body of work has lead to considerable interest in understanding the role that endogenous cannabinoids have in the immune system and in developing CB2-selective therapies .
However, the direct examination of CB2 protein on human cells has been limited by an inability to reliably detect and quantitate receptor protein. While mRNA profiles have suggested that there is differential expression of CB2 by B cells, T cells, and other leukocyte subsets, there have been very few studies evaluating differences in protein expression or cellular distribution. The current research focuses on the development and validation of a flow cytometry approach for measuring and tracking CB2 receptor protein in human cells. The findings provide a flexible method for receptor study in primary cells and new insights regarding the differential expression of CB2 receptors at intracellular versus extracellular locations in human B cells, T cells, and monocytes.Following informed consent, peripheral blood leukocytes were isolated by Ficoll-gradient centrifugation from the blood of healthy human donors. Cell subsets were identified by flow cytometry using fluorescent-labeled monoclonal antibodies directed against B cells , T cell subsets , and monocytes . Purities for each subset were confirmed by flow cytometry. The human embryonic kidney cell line 293T and lung cancer epithelial cell line A549 were maintained in culture as adherent monolayers in complete medium composed of DMEM or RPMI-1640, respectively , supplemented with 10 % fetal bovine serum and antibiotics. The 293T/CB2-GFP and A549/CB2-GFP cell lines were constructed by transducing the corresponding parental lines with a self-inactivating lentivirus expressing full-length human CB2 receptor cDNA and green fluorescent protein as previously described .
Expression of CB2 was regulated by a hCMV promoter with the expression of GFP linked through an internal ribosomal entry site. Transduced cells were sorted by flow cytometry for GFP-expressing clones, and aliquots of the expanded cell lines were cryopreserved for subsequent use.In order to assess trafficking between extracellular and intracellular CB2 receptors, we employed two complementary approaches to assess for ligand-induced receptor internalization. Using the 293T/CB2-GFP cell line as a model, we assessed changes in expression of extracellular CB2 in response to treatment with THC. Incubating cells with a 4 μM concentration of THC for up to 80 min at 37 °C was associated with a time-dependent decrease in cell surface CB2 expression . Similarly, exposing cells for 40 min to increasing concentrations of THC from 0 to 8 μM resulted in a concentration-dependent decrease in surface staining by antiCB2 mAb . THC-dependent changes did not occur when cells were maintained at 4 °C confirming an energy-dependent process. In a second approach, imaging flow cytometry was used to assess the impact of THC exposure on receptor location . Viable 293T/CB2-GFP cells were stained with antiCB2 mAb and secondary APC-labeled GAM and then incubated at 37 °C with either 8 μM THC or diluent alone for 40 min. While conventional flow cytometry demonstrated no change in overall fluorescent signal , fluorescent imaging demonstrated trafficking and coalescence of the fluorescent signal within the cytoplasm in response to THC. Cells labeled with anti-CB2 mAb and incubated at 37 °C, in the absence of THC, did show evidence of antibody-induced capping and early vacuolization, but extensive trafficking and coalescence of the CB2 label within the cytoplasm occurred only in the presence of THC. This visual assessment was confirmed by using quantitative measurement of the intracellular to extracellular fluorescent ratios for the two conditions, which demonstrated a significant intracellular shift in response to THC.Having validated the capacity for flow cytometry to assess cell surface and total cellular CB2 expression in cell lines, we assessed whether this approach could detect CB2 expression in primary human PBL. Blood samples were obtained from healthy non-smoking subjects in order to avoid any impact of exogenous THC exposure on receptor expression. As demonstrated in Fig. 4a, cell surface staining with the anti-CB2 mAb was only observed in B cells. There was no difference in fluorescence staining between the anti-NK1.1 and anti-CB2 mAb when T cells and monocytes were examined. However,grow tray stand the CB2 expression pattern was entirely different after fixation and permeabilization . In addition to B cells, a fluorescent signal for CB2 was detected in all T cells and monocytes. In contrast to studies with 293T/CB2-GFP cells where fixation and permeabilization was associated with a decrease in the MFI for CB2 staining, there was a marked increase in CB2 fluorescent intensity when B cells were stained after fixation and permeabilization. This finding would suggest that a much higher percentage of CB2 protein is expressed in the cytoplasm of B cells as compared to expression on the cell surface. Furthermore, while T cells failed to exhibit any CB2 expression on their cell surface, they exhibited high levels of intracellular fluorescence. Intracellular staining of monocytes was also consistently positive for cytoplasmic CB2 protein, and there were no consistent differences in the level of expression between B cells, T cells, and monocytes. The fluorescent staining intensity exhibited by these different subsets, broken down into cell surface staining and total cellular staining, are summarized in Table 1. Visual confirmation of antibody binding location was obtained using the ImageStreamX® imaging cytometer . As in our cell lines, intracellular staining revealed diffuse cytoplasmic staining in B cells, T cells, and monocytes. There was no fluorescent signal when cells were stained with anti-NK1.1 mAb.
As had been observed when cells from the 293T/CB2- GFP line were exposed to THC, the extracellular expression of CB2 by CD20+ B cells was also down-regulated when PBMC were exposed to THC in the range of 0.5 to 2.0 μM . No change was observed for monocytes or T cells, which did not demonstrate extracellular CB2 staining under any conditions.Expression of CB2 protein by flow cytometry was correlated with mRNA expression using a quantitative real-time RTPCR assay. Labeled probes for CB2 and a housekeeping gene, GAPDH, were quantitated simultaneously in the same well, allowing the relative level of CB2 expression to be described by the relative differences in cycle times . Immunomagnetic selection was used to isolate B cell, T cell, and monocyte subsets with purity confirmed by flow cytometry at an average of 83.2±5.3 % for B cells, 96.5± 3.5 % for T cells, and 94.5±2.4 % for monocytes . Similar to the results for total cellular CB2 expression, all of these subsets expressed similar levels of CB2 mRNA with no statistically-significant difference noted between groups .The CB2 gene was cloned from a human leukemia cell line in 1993 and found to encode for a GPCR that bound cannabinoids with high affinity, but unlike CB1, it was expressed primarily in lymphoid organs by lymphocytes, monocytes, and polymorphonuclear cells . The functional consequences of cannabinoids on immunity have turned out to be extensive with the capacity to regulate chemotaxis, phagocytosis, bacterial killing, antigen processing and presentation, T cell activation and cytokine production, and B cell differentiation and isotype switching . This has led to considerable interest in developing therapeutic drugs based on their interaction with CB2 receptor . However, there is relatively little information regarding the expression and distribution of CB2 protein on target cells. In this study, we constructed cell lines expressing different levels of human CB2 and used a commercial anti-CB2 mAb to develop a sensitive and specific flow cytometry assay for detecting CB2 protein. This mAb was developed using gene-modified cells expressing full length human CB2 as the immunogen. It readily detects CB2 expressed on the cell membrane, and in our hands, cell staining was not blocked by pre-incubation with a 50-mer N-terminal peptide , suggesting that it may be directed against one of the extracellular loops of the GPCR structure. With its high throughput and the capacity for multiplexing, this assay should provide an important tool forprobing CB2 receptor status in cells of interest. More importantly, with the addition of cell permeabilization and imaging flow cytometry, our findings challenge the longstanding notion that CB2 functions primarily as a cell surface receptor . GPCR have the capacity to traffic between different cell compartments where they can interact with different adaptor proteins and signaling pathways . When examining primary human B cells, our studies identified CB2 protein at both extracellular and intracellular locations. However, while B cells, T cells, and monocytes expressed similar levels of CB2 mRNA, CB2 protein expression was restricted entirely to intracellular sites in T cells and monocytes. In an analogous manner, a number of research groups have recently described a primary intracellular distribution of CB1 protein within different sets of neurons . Cannabinoids are highly lipophilic molecules, and it has been shown that both extracellular and intracellular CB1 receptors can mediate signaling and biologic responses when exposed to ligands . Others have also recently begun to evaluate CB2 receptor internalization and trafficking . Using gene-modified cell lines and epitope-tagged CB2 molecules, a complex relationship between CB2 ligand exposure, receptor internalization, and cell signaling has been reported . In this setting, our results suggest a similar paradigm for the expression of native human CB2 by PBL and make it likely that the differential expression of CB2 at extracellular and intracellular sites plays an important role in the immune responses to cannabinoids. This differential expression of CB2 may also be linked to the variety of signaling pathways that have been associated with CB2 activation . While others have used flow cytometry to evaluate CB2 receptor expression on cells , there are several features which distinguish our assay from past studies with human PBL.