Our findings suggest that cannabinoids are capable of altering the differentiation and activation of cells involved in human cell-mediated immunity. Functional coupling of cannabinoid receptors to G-protein activity was assessed by measuring forskolin-induced cAMP levels in CHO-CB2 cells and fresh human monocytes. CHO-CB2 cells were cultured overnight at 5×105 cells/well in a 6-well plate. The next day, DMSO was added and cells cultured for an additional 18 h. THC , JWH-015 , the combination of SR144528 and THC , or diluent alone, were then added to the wells and incubated for 15 min prior to an 18 h stimulation with 50 μM forskolin . For studies with monocytes, 5× 106 PBMC were placed into each well of a 24-well plate and monocytes allowed to adhere for 2 h in X-VIVO-15 medium. Non-adherent cells were then removed, wells rinsed, and the remaining monolayer treated in a manner identical to that described for CHO-CB2 cells. Forboth cell types, supernatants were harvested at the end of the forskolin stimulation and stored at −80 °C until assayed in duplicate for cAMP using a standard competitive enzyme immunoassay kit . The final reaction product was read in a plate reader at 405 nm and the amount of cAMP determined by regression analysis. DC and THC-DC were evaluated for their capacity to activate T cells in a standard MLR assay . Allogeneic CD45RA+ T cells were isolated by negative selection with specific antibody and immunomagnetic beads, then labeled using the Vybrant CDSE/CFSE Cell Tracer Kit according to the manufacturer’s protocol. DC were cultured with 2×105 T cells at 1:50 DC:T cell ratios in X-VIVO 15 medium in 96 well round-bottom plates at 37 °C in a humidified CO2 incubator. For some experiments, DC and THC-DC were matured by culture with 20 μg/ml SAC for 18–24 h prior to co-culture with the T cells. In other experiments, the co-cultures were supplemented with 2 ng/ml of either IL-7, IL-12 or IL-15. On day 5 of co-culture,vertical grow trays the T cells were collected and analyzed by FACS for proliferation and cell surface marker expression .
Cell-free supernatants were collected from the wells and assessed for cytokines by custom multiplex analysis .As an initial step in understanding the potential interaction between cannabinoids and human monocyte-derived DC, monocytes were evaluated for the expression of the CB1 and CB2 receptor subtypes by RT-PCR and flow cytometry . RT-PCR studies were carried out on monocytes that had been purified to >90% purity by either negative depletion or fluorescent cell sorting. mRNA encoding for both CB1 and CB2 were detected, although expression of CB2 predominated whether analyzed by standard RT-PCR or by an automated quantitative RT-PCR using cells from 4 different donors . Despite the presence of mRNA, standard flow cytometry failed to detect CB1 or CB2 receptor protein on the cell surface of monocytes even though antibodies were directed against their N-terminal epitopes. However, when cells were fixed and permeabilized, specific staining for both CB1 and CB2 was detected, consistent with the presence of intracellular protein . Intracellular background staining with isotype control mAb was minimal for CB1 but dimly-positive for CB2, likely reflecting the need for APC-labeled goat anti-mouse F2 as a secondary detection reagent. Due to these differences in fluorescent labels and staining protocols, the relative fluorescent intensity for CB1 and CB2 cannot be directly compared as measures of receptor concentration. The presence of functional CB2 receptor complexes was then assessed by measuring the impact of different cannabi-noids on forskolin-induced generation of cAMP . Using CHO-CB2 cells as a model, we confirmed that treatment with THC significantly inhibited the generation of cAMP, as did JWH-015 at p<0.01. Furthermore, the inhibition of cAMP by THC was blocked by pretreatment with SR144528, a selective CB2 receptor antagonist . The same assays were repeated using purified human monocytes . Again, an overall CB2 agonist treatment effect was present. Pretreatment with either THC or JWH-015 inhibited the forskolin-induced generation of cAMP and the effects of THC were blocked by SR144528 . While monocytes express both CB1 and CB2, the predominance of CB2 mRNA and the response of these cells to CB2-selective agents suggest that CB2 acts as the dominant cannabinoid signaling pathway. The differentiation of human monocytes into DC is associated with characteristic changes in cell surface proteins involved in antigen presentation .
To evaluate the effects of THC on this aspect of differentiation, adherent PBMC were cultured for 7 days with GM-CSF and IL-4 and examined for the expression of typical monocyte and DC markers by flow cytometry . Exposure to THC did not prevent the normal down-regulation of CD14, but did inhibit the upregulation of other cell surface markers characteristic of antigen presenting cells including CD11c, HLA-DR, CD40 and CD86. The effects were concentration-dependent, with 0.5 μg/ml THC inhibiting expression of all of these markers by 40–60%. Interestingly, the response profiles were not uniform for every protein. THC produced a uniform decrease in the expression of CD11c and CD40 on all of the cells but resulted in two distinct subsets with respect to the expression of HLA-DR and CD86 – one population that did not express these markers and one that expressed relatively normal levels . In the latter case, the relative proportions of these two subsets depended upon the concentration of THC, with higher levels of THC resulting in fewer marker-positive cells. Cannabinoids have been reported to promote the apoptosis of mouse bone marrow-derived DC under certain conditions . In order to assure that the phenotypic changes observed in our studies were not the result of poor viability, DC that had been differentiated in the presence of either THC or JWH-015 were stained with propidium iodide and Annexin-V-FITC. There was no significant impact of either cannabinoid on the number of recovered cells or on the frequency of apoptotic or dead cells . In addition to their high level expression of major histo-compatibility complex and costimulatory molecules, monocyte-derived DC are usually characterized by their capacity for antigen uptake, as well as their secretion of cytokines that promote cell mediated immunity. Receptor-mediated endocytosis was measured by the uptake of FITC-dextran and was dramatically suppressed in cells that had been exposed to THC . The production of IL-10 and IL-12 was also assessed by stimulating cells with SAC and measuring cytokines released into the culture media at 48 h following stimulation. Interestingly, while the production of IL-12 was significantly suppressed , the secretion of IL-10, which can bias T cell activation toward T helper type 2 and/or T regulatory phenotypes, was not altered . This differential effect on cytokine production is consistent with an immuno regulatory effect rather than a global suppression of DC function. The capacity for DC to stimulate the activation and proliferation of antigen-specific T cells represents a final integrated measure of their function. DC that had been generated in the presence or absence of THC were recovered from the 7 day culture of adherent PBMC, purified by negative depletion, and cultured with allogeneic Tcells in a MLR assay . In order to avoid direct effects on responder T cells, no further THC was added during the 5 days of DC:T cell co-culture. Proliferation was monitored by labeling cells with CFSE, which also allowed the phenotype of responder cells to be tracked with each cell division over time. While control DC were potent stimulators of both CD4+ and CD8+ respond-er T cells,vertical marijuana grow the proliferative response to THC-DC was severely blunted . THC-treated DC induced T cell proliferation that ranged from only 17 to 32% of control values . Furthermore, while control DC promoted the conversion from naïve CD45RA+ T cells to activated CD45RO+ clones expressing the high affinity IL-2 receptor , this functional transformation into effector/memory T cells was almost completely absent when T cells were stimulated with THC-DC .
There was a similar impact on the generation of effector cell cytokines as measured by the release of IFN-γ and TNF-α into the culture supernatant . A number of factors can help restore function to impaired antigen presenting cells or enhance their capacity to stimulate T cell responses. Given our findings with THC-DC, we hypothesized that a combination of DC activation and cytokine replacement might be effective for this purpose. In initial experiments, DC and THC-DC were exposed to heatkilled and fixed SAC for 18–24 h prior to co-culture with T cells. The goal was to replicate bacterial activation signals that might occur during an immune challenge in vivo. In other experiments, the co-cultures were supplemented with IL-7, IL-12 or IL-15 to replace key cytokines known to be involved in the proliferation and differentiation of effector/memory T cells. As demonstrated in Fig. 6, pre-treating control DC with SAC enhanced their capacity to stimulate T cell proliferation and maturation. In addition, exposing THC-DC to SAC restored some of their capacity to generate mature responder T cells. This effect correlated with the upregulation of HLA-DR, CD80 and CD86 on THC-DC . In addition, supplementing the co-cultures with IL-7 helped SAC-stimulated DC to further promote the expansion and phenotypic maturation of effector T cells, a synergistic effect that was not observed with either IL-12 or IL-15. When assessed in a limited number of experiments, IL-7 also increased the production of IFN-γ and TNF-α, consistent with a restoration of their effector/memory function . The human CB2 receptor was first cloned from a human myeloid cell line and has been reported as the predominant cannabinoid receptor sub-type expressed by immune cells . Consistent with this, we found that expression of CB2 mRNA predominated over CB1 when fresh human monocytes were purified and assayed by semi-quantitative RT-PCR techniques. However, neither cannabinoid receptor could be detected on the extracellular surface of monocytes when stained with mAbs known to be specific for their N-terminal sequences. We recently reported that CB2 may exist as an intracellular protein in immune cells and others have suggested that CB1 may also function as an intracellular receptor . Consistent with these observations, the addition of an initial fixation and permeabilization step resulted in positive staining by both anti-CB1 and anti-CB2 mAbs, but not by their respective isotype controls. Functional receptor protein was confirmed by assaying the capacity for cannabinoids to inhibit forskolin-induced changes in cAMP. Addition of THC, a pan-agonist with equal affinity for CB1 and CB2, blocked forskolin-induced cAMP in both transduced CHO-CB2 cells and in fresh human monocytes. In addition, this effect was recapitulated by exposure to JWH-015, a selective CB2 agonist, and the effects of THC were completely blocked by SR144528, a selective CB2 antagonist. These findings confirm reports that CB2 predominates as the functional cannabinoid receptor pathway in human monocytes and add the caveat that receptor expression occurs at an intracellular location rather than on the cell surface. Monocytes act as myeloid precursors that can differentiate along a number of functionally distinct pathways depending upon their interaction with cytokines, growth factors, infectious signals and other regulatory mediators . When driven to differentiate into monocyte-derived DC under the influence of GM-CSF and IL-4 , their function can also be modulated by a variety of factors . Concurrent exposure to IL-6 and macrophage-colony stimulating factor can divert differentiation toward macrophages instead of DC . Transforming growth factor -β and IL-23 promote the development of DC that promote Th17 biased responses . IL-10 promotes tolerogenic and Th2-promoting features , while a variety of toll-like receptor ligands and immuno stimulatory cytokines will promote DC that stimulate effector/memory T cells . In this setting, we hypothesized that exposure to THC during the process of DC differentiation would provide valuable insight regarding its immuno regulatory properties. Further, given the immuno suppressive effects that cannabinoids have on antigen-specific T cell responses in animals in vivo and on human T cell activation in vitro , we hypothesized that cannabinoids might render DC tolerogenic or otherwise skew their stimulatory activity. Only a few studies have examined the interaction of cannabinoids with DC and in most cases the focus has been on murine models or on the effects of cannabinoids on differentiated DC . Do et al. suggested that THC can impair immune responses by inducing DC apoptosis. However, they studied mouse bone marrow-derived DC and apoptosis occurred primarily when THC concentrations exceeded 5 μM.