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,vertical grow rack 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.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 heat killed 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 subtype 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 ,cannabis grow racks 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. In our studies, immuno regulatory effects on human monocyte-derived DC were observed at lower THC concentrations , more akin to peak levels that occur in the blood of marijuana smokers , and had no effect on cell recovery or surface staining by Annexin-V.

Instead of apoptosis, we observed broad-ranging effects of THC on the expression of MHC class II and costimulatory molecules, and the capacity for antigen uptake and IL-12 production. Furthermore, DC that had been exposed to THC during their in vitro differentiation were impaired in their capacity to activate T cells – including both CD4+ and CD8+ responders. T cell proliferation and the acquisition of a memory/effector phenotype were both impaired as was the release of Th1 cytokines. These effects of THC on the capacity for monocyte-derived DC to stimulate T cells are almost identical to the direct effects of THC on T cell activation , suggesting a coordinated immuno regulatory effect. It is interesting that other immuno suppressive factors, including IL-10 and TGF-β3, share this capacity to act in a coordinated manner on both DC and T cells . As is the case with IL-10–/– knockout mice , CB1–/–/CB2–/– double-knockout mice exhibit elevated levels of activated T cells and respond to antigen challenges by producing a higher number of activated effector cells and stronger IFN-γ responses . Collectively, these findings suggest an intrinsic role for endocannabinoid signaling as a homeostatic regulator of T cell activation. There are a number of critical features that develop during the transition from monocytes into DC that enable them to activate antigen specific T cells . Among these are high levels of antigen expression in the context of cell-surface MHC, the upregulation of adhesion and costimulatory molecules, and the elaboration of immuno stimulatory cytokines.Exposure to THC during the differentiation of monocyte-derived DC impaired antigen uptake and prevented the normal upregulation of MHC class II. These findings are consistent with earlier reports by McCoy et al. , where THC was found to impair the presentation of whole hen egg lysozyme, which required uptake and processing, but not the presentation of its immunodominant peptides, which bound directly to existing cell surface MHC. Dendritic cells that present antigen in the absence of adequate costimulatory molecules cannot fully activate T cells and may contribute to the development of T cell anergy . The inhibitory effects of THC on the expression of CD40, CD86 and other costimulatory molecules likely contributed to the failure of THC-DC to stimulate T cell proliferation. Finally, the relative production of IL-10 and IL-12 by DC plays a central role in their capacity to activate either Th1 or Th2 responses. In our studies, THC-DC produced only limited amounts of IL-12 but normal levels of IL-10. Lu et al. reported a similar suppressive effect of THC on the expression of MHC and costimulatory molecules and on production of IL-12 by mouse bone marrow-derived DC that had been infected with Legionella pneumophila. While these findings add to other compelling evidence that cannabinoids can exert important immuno suppressive effects, clinical evidence that marijuana smoking significantly impairs immune function in humans is limited. One explanation may be that inhaled THC never produces sufficient systemic levels, or that exposures may not be sustained for a sufficient period of time. to mediate immuno suppressive effects . Another explanation may be that the effects are short-lived or counterbalanced by the presence of other immune regulatory factors. The study of purified cells in vitro culture does not adequately replicate the complex environment that occurs during an immune challenge in vivo. In this study we hypothesized that the processes of DC activation and cytokine exposure that occur in response to an infectious challenge might modulate the impact of THC. Exposing DC and THC-DC to heat-killed and fixed SAC for 18– 24 h enhanced their capacity for Tcell activation; an effect that was more pronounced with THC-DC than with control DC. Adding IL-12 and IL-15 to the DC:T cell co-culture also enhanced T cell activation and proliferation, but these effects occurred equally with control and THC-DC.