Barbara L. F. Kaplan

The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT).

Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-gamma (IFN-gamma) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-gamma production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-gamma. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1(-/-)/CB2(-/-) mice, it was determined that suppression of IL-2 and IFN-gamma and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1(-/-)/CB2(-/-) mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response.

The role of CB1 in immune modulation by cannabinoids.

There is clear evidence that CB(2), historically referred to as the peripheral cannabinoid receptor, mediates many of the immune modulatory effects of cannabinoids. However, cannabinoid receptors cannot be classified simply as central or peripheral since CB(2) has been shown to play a role in the central nervous system (CNS) and CB(1) mediates many immune system effects. Although Cnr1 mRNA and CB(1) protein expression is lower than Cnr2 mRNA or CB(2) protein expression in cells of the immune system, several studies have shown direct modulation of immune function via CB(1) by endogenous and exogenous cannabinoids in T cells, innate cells, and to a lesser extent, B cells. In addition, indirect, but CB(1)-dependent, mechanisms of immune modulation exist. In fact, the mechanism by which cannabinoids attenuate neuroinflammation via CB(1) is likely a combination of immune suppression and neuroprotection. Although many studies demonstrate that agonists for CB(1) are immune suppressive and anti-inflammatory, CB(1) antagonists also exhibit anti-inflammatory properties. Overall, the data demonstrate that many of the immune modulatory effects of cannabinoids are mediated via CB(1).

Effects of targeted deletion of cannabinoid receptors CB1 and CB2 on immune competence and sensitivity to immune modulation by Δ9‐tetrahydrocannabinol

The role of cannabinoid receptors, CB1 and CB2, in immune competence and modulation by Δ9‐tetrahydrocannabinol (Δ9‐THC) was investigated in CB1−/−/CB2−/− mice. Immunofluorescence analysis of splenic leukocytes showed no significant differences in the percentage of T cell subsets, B cells, or macrophages between wild‐type and CB1−/−/CB2−/− mice. Lymphoproliferative control responses to PHA, phorbol ester plus ionomycin, or LPS and sensitivity to suppression by Δ9‐THC showed no profound differences between the two genotypes, although some differences were observed in control baseline responses. Likewise, similar control responses and sensitivity to Δ9‐THC were observed in mixed lymphocyte responses (MLR) and in IL‐2 and IFN‐γ production in both genotypes. Conversely, humoral immune responses showed a markedly different profile of activity. Δ9‐THC suppressed the in vivo T cell‐dependent, anti‐sheep RBC (anti‐sRBC) IgM antibody‐forming cell (AFC) response in wild‐type but not in CB1−/−/CB2−/− mice, and the in vitro anti‐sRBC IgM response in CB1−/−/CB2−/− splenocytes was too low to rigorously assess CB1/CB2 involvement in modulation by Δ9‐THC. Conversely, comparable in vitro IgM AFC control responses to LPS and CD40 ligand (CD40L) activation were observed in the two genotypes. Interestingly, LPS‐induced IgM responses were refractory to suppression by Δ9‐THC, regardless of genotype, and CD40L‐induced IgM responses were only suppressed by Δ9‐THC in wild‐type but not in CB1−/−/CB2−/− B cells. Collectively, we demonstrate differential involvement of CB1 and/or CB2 in immune modulation by Δ9‐THC and in some control responses. Moreover, CB1/CB2 involvement was observed in humoral responses requiring CD40‐initiated signaling for suppression by Δ9‐THC.

2-Arachidonoyl-glycerol suppresses interferon-γ production in phorbol ester/ionomycin-activated mouse splenocytes independent of CB1 or CB2

2‐Arachidonoyl‐glycerol (2‐AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin‐2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2‐AG on interferon‐γ (IFN‐γ) expression and associated upstream signaling events. Pretreatment of splenocytes with 2‐AG markedly suppressed phorbol 12‐myristate 13‐acetate plus calcium ionophore (PMA/Io)‐induced IFN‐γ secretion. In addition, 2‐AG suppressed IFN‐γ steady‐state mRNA expression in a concentration‐dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1−/−/CB2−/− knockout mice were used. No difference in the magnitude of IFN‐γ suppression by 2‐AG in wild‐type versus CB1/CB2 null mice was observed. Time‐of‐addition studies revealed that 2‐AG treatment up to 12 h post‐cellular activation resulted in suppression of IFN‐γ, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2‐AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin‐induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN‐γ production. Indeed, Io partially attenuated the 2‐AG‐induced suppression of PMA/Io‐stimulated IFN‐γ production. Taken together, these data demonstrate that 2‐AG suppresses IFN‐γ expression in murine splenocytes in a CB receptor‐independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.

15-Deoxy-Δ12,14-prostaglandin J2-Glycerol Ester, a Putative Metabolite of 2-Arachidonyl Glycerol, Activates Peroxisome Proliferator Activated Receptor γ

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative capable of suppressing interleukin (IL)-2 production by activated T cells. 2-AG-mediated IL-2 suppression is dependent on cyclooxygenase-2 (COX-2) metabolism and peroxisome proliferator activated receptor γ (PPARγ) activation. The objective of the present studies was to examine whether 15-deoxy-Δ12,14-PGJ2-glycerol ester (15d-PGJ2-G), a putative metabolite of 2-AG, can mimic the actions of 2-AG on IL-2 regulation through PPARγ activation. 15d-PGJ2-G bound PPARγ-ligand binding domain in a PPARγ competitive binding assay. 15d-PGJ2-G treatment activated PPARγ in a reporter assay, and PPARγ activation was attenuated when a PPARγ antagonist, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907), was present. 15d-PGJ2-G treatment suppressed IL-2 production by activated Jurkat cells, which was partially attenuated when pretreated with T0070907. Moreover, IL-2 suppression was pronounced when 15d-PGJ2-G was present 30 min before or after T-cell activation. Concordant with IL-2 suppression, 15d-PGJ2-G treatment decreased nuclear factor of activated T cells (NFAT) transcriptional activity in transiently transfected Jurkat cells. It is noteworthy that T0070907 alone markedly increased NFAT reporter activity, suggesting the existence of endogenous PPARγ activation and modulation of NFAT. Because COX-2 metabolism of 2-AG is important for IL-2 suppression, the effect of 2-AG on COX-2 and PPARγ mRNA expression was investigated. 2-AG treatment decreased the up-regulation of COX-2 mRNA after T-cell activation, which suggests negative feedback limiting COX-2-mediated metabolism of 2-AG. PPARγ mRNA expression was increased upon activation, and 2-AG treatment produced a modest decrease in PPARγ mRNA expression. Collectively, our findings suggest that 15d-PGJ2-G activates PPARγ to decrease NFAT transcriptional activity and IL-2 expression in activated T cells.

Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection

We and others have reported that simultaneous targeted deletion of CB1 and CB2 resulted in exacerbation of immune reactivity, suggesting a role of endocannabinoids in down‐regulating immune function. In this study, we demonstrate that APC function is enhanced specifically in the absence of CB1 and CB2 signaling, resulting in an exacerbated immune response phenotype. After influenza infection, CB1−/−CB2−/− mice showed more pronounced pulmonary damage, increased inflammatory cell infiltrate, inflammation, and a greater cellular immune responses compared with WT mice, as evidenced by transcriptome analysis, more robust T cell activation, and effector cell cytokine production. After direct activation in vitro, there were no differences in the percentages of cytokine‐producing CD4+ T cells between CB1−/−CB2−/− and WT mice. However, untreated CB1−/−CB2−/− mice routinely had fewer naïve T cells compared with WT, suggesting dysregulation of APC immune homeostasis. Moreover, bmDCs and AM isolated from CB1−/−CB2−/− mice exhibited a more mature phenotype, with and without TLR stimulation, and bmDCs elicited T cells more robustly than WT mice. Collectively, these findings implicate a role for CB1 and CB2 on APCs in regulating immune responses and immune homeostasis.

Induction of the Aryl Hydrocarbon Receptor-Responsive Genes and Modulation of the Immunoglobulin M Response by 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin in Primary Human B Cells

Past studies in rodent models identified the suppression of primary humoral immune responses as one of the most sensitive sequela associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Yet, the sensitivity of humoral immunity to TCDD in humans represents an important toxicological data gap. Therefore, the objectives of this investigation were two-fold. The first was to assess the induction of known aryl hydrocarbon receptor (AHR)-responsive genes in primary human B cells as a measure of early biological responses to TCDD. The second was to evaluate the direct effect of TCDD on CD40 ligand-induced immunoglobulin M (IgM) secretion by human primary B cells. The effects of TCDD on induction of AHR-responsive genes and suppression of the IgM response were also compared with B cells from a TCDD-responsive mouse strain, C57BL/6. AHR-responsive genes in human B cells exhibited slower kinetics and reduced magnitude of induction by TCDD when compared with mouse B cells. Evaluation of B-cell function from 12 donors identified two general phenotypes; the majority of donors exhibited similar sensitivity to suppression by TCDD of the IgM response as mouse B cells, which was not attributable to decreased B-cell proliferation. In a minority of donors, no suppression of the IgM response by TCDD was observed. Although donor-to-donor variation in sensitivity to TCDD was observed, human B cells from the majority of donors evaluated showed impairment of effector function by TCDD. Collectively, data presented in this series of studies demonstrate that TCDD impairs the humoral immunity of humans by directly targeting B cells.

A COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol, mediates suppression of IL-2 secretion in activated Jurkat T cells.

Previous studies from this laboratory have demonstrated that a COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol (2-AG), inhibits IL-2 secretion in activated T cells through PPARgamma activation independent of the cannabinoid receptors, CB1/CB2. Because numerous cyclooxygenase (COX) products have been shown to activate PPARgamma, the primary purpose of the present studies was to determine the role of COX metabolism in the inhibition of IL-2 secretion by 2-AG. Pretreatment with nonselective and COX-2-specific inhibitors completely abrogated 2-AG-mediated suppression of IL-2 secretion. In contrast, pretreatment with COX-1-specific inhibitors had no effect upon 2-AG-mediated inhibition of IL-2 secretion. Interestingly, the current studies also demonstrate that while the potency of 2-AG is comparable between human Jurkat T cells and murine splenocytes, anandamide (AEA) is markedly more potent in suppressing IL-2 production in Jurkat T cells compared to murine splenocytes. Additionally, the present studies also demonstrate that COX-2 protein is readily detectable in resting Jurkat T cells, which is in contrast to resting murine splenocytes in which COX-2 protein is virtually undetectable. Furthermore, COX-2 protein and mRNA levels are significantly increased over basal levels by 2h following activation of Jurkat cells, whereas increases in COX-2 protein in murine splenocytes are not observed until 4h after cellular activation. These studies suggest that the potency of AEA in the suppression of IL-2 secretion may correlate with COX-2 protein levels in different T cell models. The present studies are also significant in that they demonstrate 2-AG-mediated inhibition of IL-2 secretion is dependent upon COX-2 metabolism.

Cannabinoids inhibit the activation of ERK MAPK in PMA/Io-stimulated mouse splenocytes

The mechanism of action of immune suppression by cannabinoids involves suppression of interleukin-2 (IL-2) production in phorbol ester plus calcium ionophore (PMA/Io)-stimulated lymphocytes. This decrease in IL-2 was due to inhibition of activator protein-1 (AP-1) and nuclear factor of activated T cells (NF-AT) transcription factors, both of which depend on proteins that are regulated by the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (ERK MAPK). Thus, the objective of the present study was to characterize the effects of cannabinoid compounds on ERK MAPK under conditions where IL-2 expression was suppressed. Using the MEK inhibitor PD098059 in order to assess the role of ERK MAPK in PMA/Io-stimulated splenocytes (SPLC), it was determined that IL-2 production and expression of c-fos and c-jun nuclear protein expression depended on activation of ERK MAPK. In response to PMA/Io, expression of nuclear phosphorylated ERK MAPK was rapidly induced, peaked at approximately 15 min, and was sustained for up to 240 min. Pretreatment with cannabinol (CBN) inhibited expression of phosphorylated ERK MAPK at several time points up to 240 min post cellular activation. Furthermore, WIN-55212-2, a synthetic cannabinoid, inhibited expression of phosphorylated ERK MAPK at 240 min post cellular activation. CBN did not induce activation of ERK MAPK in the absence of PMA/Io. Collectively, these studies suggest that cannabinoid-induced inhibition of IL-2 in PMA/Io-stimulated splenocytes might be due, in part, to inhibition of ERK MAPK activation.

Δ9-Tetrahydrocannabinol Impairs the Inflammatory Response to Influenza Infection: Role of Antigen-Presenting Cells and the Cannabinoid Receptors 1 and 2

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) has potent immune modulatory properties and can impair pathogen-induced immune defenses, which in part have been attributed to ligation of the cannabinoid receptors 1 (CB(1)) and 2 (CB(2)). Most recently, dendritic cells (DC) were identified for their potential to enhance influenza-induced immunopathology in mice lacking CB(1) and CB(2) (CB(1) (-/-)CB(2) (-/-)). This study focused on the modulation of the inflammatory immune response to influenza by Δ(9)-THC and the role of CB(1) and/or CB(2) as receptor targets for Δ(9)-THC. C57Bl/6 (wild type) and CB(1) (-/-)CB(2) (-/-) mice were administered Δ(9)-THC (75 mg/kg) surrounding the intranasal instillation of A/PR/8/34 influenza virus. Three days post infection (dpi), Δ(9)-THC broadly decreased expression levels of mRNA induced by the innate immune response to influenza, suppressed the percentage of interferon-gamma (IFN-γ)-producing CD4(+) and interleukin-17-producing NK1.1(+) cells, and reduced the influx of antigen-presenting cells (APC), including inflammatory myeloid cells and monocytes/macrophages, into the lung in a CB(1)- and/or CB(2)-dependent manner. Δ(9)-THC had little effect on the expression of CD86, major histocompatibility complex I (MHC I), and MHC II by APC isolated from the lung. In vitro studies demonstrated that lipopolysaccharide (LPS)-induced maturation was suppressed by Δ(9)-THC in bone marrow-derived DC (bmDC). Furthermore, antigen-specific IFN-γ production by CD8(+) T cells after coculture was reduced by Δ(9)-THC treatment of bmDC in a CB(1)- and/or CB(2)-dependent manner. Collectively, these studies suggest that Δ(9)-THC potently suppresses myeloid cell immune function, in a manner involving CB(1) and/or CB(2), thereby impairing immune responses to influenza infection.