Carayon and associates generated and purified polyclonal rabbit anti-CB2 antibody directed against the C-terminal of human CB2. As fixation and permeabilization were required for antigen detection, their approach precluded a comparison between extracellular and intracellular staining. A fluorescent signal was detected from stained B cells and was inhibited by excess peptide, but the findings were much less convincing with respect to the staining of other cell types. More recently, Graham and coworkers evaluated polyclonal antibodies from several commercial manufacturers and reported that human B cells, T cells, monocytes, NK cells, and polymorphonuclear cells all express high levels of extracellular CB2. However, the staining patterns in their report were highly variable from manufacturer to manufacturer and from batch to batch. Furthermore, in the absence of appropriate control antibodies or the inclusion of known positive and negative controls, one cannot really draw conclusions about sensitivity and specificity. Based on these concerns we focused on a defined mAb with the ability to detect extracellular CB2 expression. In order to optimize and validate staining patterns, we constructed cell lines expressing defined levels of human CB2 and compared staining patterns to those observed with parental cells . During the optimization process, it was obvious that non-specific background staining could easily be mistaken for receptor expression if antibodies were not carefully titrated and appropriate isotype controls employed. By including the expression of a linked GFP reporter gene in our vector construct, we also possessed a mechanism for independently assessing expected CB2 staining patterns.
Perhaps the most important technical advancement was the inclusion of both intracellular and extracellular staining protocols. In this respect,hydroponic racks our studies were also aided by the use of an ImageStreamX® cytometer. Due to the impact of fixation and permeabilization on antibody staining, we could not use MFI to directly compare extracellular and intracellular protein levels by conventional flow cytometry. However, visual inspection of captured images readily identified the cytoplasmic compartment as the primary source of our CB2 signal. Imaging also allowed us to independently confirm the process of receptor internalization in response to ligand exposure. Given the controls and approaches employed, there should be little doubt regarding the performance characteristics of this flow cytometry approach. In summary, we describe a rapid and flexible approach for detecting and localizing human CB2 protein expression in cell lines and primary human cells. This approach uses commercially available reagents and should have wide applicability. In addition, for the first time, we report that CB2 receptor is primarily located at intracellular sites in PBL and that expression is not limited to the cell membrane as previously thought. Even in B cells, which express both extracellular and intracellular CB2, the majority of receptor protein is located within the cell. Our findings and related investigations carried out with CB2 suggest that there is trafficking between receptor locations and that intracellular receptors are likely to be biologically active. Future studies focused on understanding the role of differential CB2 receptor location on cannabinoid function are warranted.The expression of cannabinoid receptors by human leukocytes suggests that both endogenous ligands and inhaled marijuana smoke might exert immunoregulatory properties that are distinct from their effects on the brain .
Furthermore, while brain cells exclusively express cannabinoid receptor type 1 , leukocytes express both CB1 and CB2, with CB2 reported as the predominant subtype . Both CB1 and CB2 are transmembrane G-protein coupled receptors that inhibit the generation of cyclic adenosine monophosphate and can signal through a variety of pathways including PI3-kinase, MAP kinase, NF-κΒ, AP-1, and NF-AT . The resulting effects on host immunity have primarily been studied in animal models and suggest a coordinated down-regulation of cellular responses that can occur through altered trafficking, selective apoptosis, or functional skewing of antigen presenting cells and T cells away from T helper type 1 or Th17 response patterns and type 2 and signal through an endogenous human cannabinoid system to produce their biologic effects [Aizpurua-Olaizola 2016, Cabral 2015, Maccarrone 2015, Pacher 2006]. Expression of CB2 predominates in cells from the immune system [Castaneda 2013, Schmöle 2015], and cannabinoids have been described to exert potent immunosuppressive effects on antigen presenting cells [Klein 2006, Roth 2015], B cells and antibody production [Agudelo 2008, Carayon 1998], T cell responsiveness and cytokine production [Eisenstein 2015, Yuan 2002], and monocyte/macrophage function [Hegde 2010, Roth 2004]. However, the majority of these findings stem from studies employing agonists and antagonists with defined CB2 binding specificities, and only limited insight has been available regarding the actual expression patterns and dynamic regulation of CB2 protein. CB2 has traditionally been described as a seven transmembrane G protein-coupled receptor expressed on the cell surface and responsive to extracellular ligand binding.
Ligand binding has been shown to initiate both receptor internalization [Atwood 2012] and a diverse number of intracellular signaling cascades, including adenylyl cyclase, cAMP, mitogen-activated protein kinase, and intracellular calcium [Howlett 2005, Jean-Alphonse 2011, Maccarrone 2015]. However, after using a highly sensitive and specific monoclonal anti-CB2 antibody and fluorescent imaging, we were surprised to find that CB2 was expressed exclusively in the intracellular compartment of human monocytes, dendritic cells, and T cells without detectable cell surface staining [Castaneda 2013, Roth 2015]. Only human B cells expressed CB2 on the cell surface, which internalized in response to ligand exposure, as well as within the intracellular compartment [Castaneda 2013]. These findings challenge our understanding of the CB2 receptor and identify the need for additional insight. It is not yet clear whether cannabinoids routinely bind and activate intracellular CB2, but there is at least one report providing direct experimental evidence for this [Brailoiu 2014]. It is also not clear why B cells exhibit a receptor expression pattern that is distinct from other leukocytes or whether this is a unique feature in cells obtained from peripheral blood or related to the specific stage of cell activation or differentiation. B cell activation has been suggested to play a role in the pattern of CB2 expression in a prior report [Carayon 1998]. In order to better understand CB2 expression patterns exhibited by human B cells, this report examines cells obtained from three different tissue sources , evaluates the relationship between defined B cell subsets and CB2 expression patterns,indoor garden table and uses an in vitro model for activating B cells in order to examine changes in CB2 expression as they correlate to the life cycle of functional B cell responses. Following informed consent, peripheral blood leukocytes were isolated by Ficoll-gradient centrifugation from the blood of healthy human donors. Human umbilical vein cord blood leukocytes were obtained from anonymous donors through the UCLA Virology Core and isolated in the same manner. Fresh human tonsillar tissue was also obtained in an anonymous manner through the UCLA Translational Pathology Core from patients undergoing routine elective tonsillectomies. Tonsillar tissue was handled in a sterile manner, minced, and then extruded through a sterile 100 uM filter to produce single cells. Filtered cells were then rinsed with PBS and processed in the same manner as PBL. Cell subsets were identified by flow cytometry using fluorescent-labeled monoclonal antibodies directed against T cells , B cells , and B cell subsets . The human B cell non-Hodgkin’s lymphoma cell line, SUDHL-4 was cryopreserved, and when needed, it was cultivated in suspension in complete medium composed of RPMI-1640 supplemented with 10% fetal bovine serum , 50 uM 2-mercaptoethanol , and 1% antibiotic-antimycotic solution . CB2 on the extracellular membrane was detected as previously described [Castaneda 2013].
In summary, cells were pre-treated with human AB Serum followed by a 30 min incubation with unlabeled primary mouse IgG2 mAb directed against either human CB2 or isotype-matched mAb against an irrelevant antigen, mouse NK1.1 . After washing, cells were incubated with an APC-labeled goat antimouse F2 mAb for 30 min. To identify different leukocyte subsets, cells were incubated with lineage-specific fluorescent-labeled mAb for 20 min and washed. All cells were then fixed with 1% paraformaldehyde and washed. Samples were protected from light and stored at 4oC until analyzed. In order to detect total cellular CB2 expression , cell suspensions were fixed , permeabilized , and blocked with human AB serum. Staining with primary unlabeled mAb and secondary APC-labeled GAM were carried out as already detailed except for the use of a 60 minute incubation time and the presence of permeabilizing solution. After washing, leukocytes were further stained with fluorescent-labeled antibodies as indicated for individual experiments, fixed, and stored for analysis. In order to identify total cellular CB2 expression in specific B cells subsets, cells were prestained with B cell subset markers prior to fixation, permeabilization, and staining for CB2. This step prevented the detection of intracellular subset markers , which can otherwise result in misclassification. After staining, cells were fixed with 1% paraformaldehyde, washed, and cryopreserved in PBS with 2% human AB serum and 10% dimethyl sulfoxide . On the day of CB2 analysis, cells were rapidly thawed at 37oC, treated with permeabilizing solution and stained for 30 min with Alexa Fluor ® 647-labeled mouse IgG2a mAb directed against either human CB2 or isotype-matched mAb against an irrelevant antigen, mouse NK1.1 and with fluorescent-labeled antibodies directed against CD20 and CD3. The concept of CB2 as a simple GPCR expressed on the surface of human leukocytes [Graham 2010, Klein 2003] is being challenged by a number of recent findings, including our imaging studies that employ a mAb against the N-terminal domain of CB2 to detect protein expression [Castaneda 2013, Roth 2015]. Using a combination of multi-parameter flow cytometry and flow-based imaging, we observed that CB2 can be expressed on the cell surface, as expected, but is also present within the cytoplasm. Furthermore, the expression pattern for CB2 was not uniform across cell types. The intracellular expression, rather than the extracellular expression, was the predominant form [Castaneda 2013]. While peripheral blood B cells expressed both cell surface and intracellular CB2, T cells, monocytes, and dendritic cells exhibited only the intracellular form of CB2. Even though cell surface CB2 can rapidly internalize when exposed to a ligand, the distribution of this internalized CB2 did not appear to account for the pre-existing distribution of intracellular CB2. The biologic basis underlying these different CB2 expression patterns has not yet been fully delineated, but there is growing evidence that the presence of GPCRs at different cellular locations is an important feature of these receptors that promotes functional heterogeneity with respect to downstream signaling and biologic responses [Flordellis 2012, Gaudet 2015]. Along these lines, there is growing evidence that intracellular forms for both CB1 and CB2 are common and exert distinct biologic effects [Brailoiu 2011, Bernard 2012, Gómez-Cañas 2016]. In this setting, understanding the distribution, regulation, and dynamic balance between cell surface and intracellular CB2 receptors is likely to provide important insight regarding cannabinoid receptor biology. The unique expression of CB2 on the surface of peripheral blood B cells led us to question whether this represented an intrinsic and stable feature of B cells in general or was more characteristic of those in peripheral blood. B cells were therefore obtained from three sources for comparison including umbilical vein cord blood, adult peripheral blood, and tonsils. B cell subsets from these different sources were characterized as either naïve mature, activated, or memory B cells based on their expression of IgD, IgM, CD27 and CD38 [Ettinger 2005]. When analyzed in this manner, it became clear that all naïve and memory B cells, regardless of source, expressed both cell surface and intracellular CB2. On the other hand, B cells with an activated phenotype expressed only the intracellular form of CB2, and in most cases the level of intracellular CB2 was higher than that observed in naïve or memory B cells obtained from the same sample. Prior studies had noted that IgD- /CD38+ germinal center B cells, consistent with the activated tonsillar B cells studied here, express a different pattern of CB2 protein staining than other B cells. However, they were using a polyclonal rabbit antibody that targeted a C-terminal CB2 peptide sequence and concluded that their findings represented the transition of CB2 from an inactive to an “activated/phosphorylated” state [Carayon 1998, Rayman 2004]. It is plausible that their findings actually mirrored ours, but features related to receptor localization were not appreciated due to technical limitations. Given the unique CB2 signature of the activated B cell population, we entertained two possible hypotheses based on the existing literature.