RESUMEN
delta9-Tetrahydrocannabinol (delta9-THC) is capable of modulating a variety of immune responses, but has not been evaluated in models of immune-based diabetes. The objectives of the present study were: (a) to investigate the effect of delta9-THC in an established model of multiple low dose streptozotocin (MLDSTZ)-induced autoimmune diabetes; and (b) to determine the contribution of the immune response in the MLDSTZ model. CD-1 mice were treated with 40 mg/kg STZ for 5 days in the presence or absence of delta9-THC treatment. delta9-THC administered orally in corn oil at 150 mg/kg for 11 days attenuated, in a transient manner, the MLDSTZ-induced elevation in serum glucose and loss of pancreatic insulin. MLDSTZ-induced insulitis and increases in IFN-gamma, TNFalpha and IL-12 mRNA expression were all reduced on Day 11 by co-administration of delta9-THC. In separate studies, six doses of delta9-THC, given after completion of STZ treatment, was found equally effective in attenuating mice from MLDSTZ-induced diabetes. Studies performed using B6C3F1 mice showed moderate hyperglycemia and a significant reduction in pancreatic insulin by MLDSTZ in the absence of insulitis. In addition, MLDSTZ produced a less pronounced hyperglycemia compared to CD-1 mice that was not attenuated by delta9-THC. These results suggest that MLDSTZ can initiate direct beta-cell damage, thereby augmenting the destruction of beta-cells by the immune system. Moreover, these results indicate that delta9-THC is capable of attenuating the severity of the autoimmune response in this experimental model of autoimmune diabetes.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Diabetes Mellitus Experimental/prevención & control , Dronabinol/farmacología , Inmunosupresores/farmacología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Complejo CD3/análisis , Citocinas/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , ARN Mensajero/análisis , EstreptozocinaRESUMEN
Cannabinoids can paradoxically regulate interleukin-2 (IL-2) expression either positively or negatively. This study investigated the mechanism responsible for cannabinol-mediated IL-2 modulation. In primary murine splenocytes and EL4.IL-2 T cells, the contrasting effects of cannabinol on IL-2 secretion depended on the magnitude but not the mode of T-cell activation. Suboptimal activation of T cells in the presence of cannabinol produced an enhancement of IL-2 secretion, which was paralleled by an increase in nuclear phospho-extracellular-regulated kinase (ERK) 1/2. In contrast, T cells activated with stimuli that were optimized to induce maximal IL-2 secretion elicited a marked suppression in the production of this cytokine when cultured in the presence of cannabinol. Moreover, cannabinol-mediated enhancement of IL-2 secretion by splenocytes was attenuated to various degrees by staurosporine, Ro-31-8220, and KN93. These results suggest that the enhancement of IL-2 secretion by cannabinol is associated with an increase in ERK mitogen-activated protein kinase, which is protein kinase C and calmodulin-kinase dependent.
Asunto(s)
Interleucina-2/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cannabinoides/farmacología , Línea Celular , Células Cultivadas , Activación Enzimática , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Bazo/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Cannabinoid compounds inhibit the cAMP signalling cascade in leukocytes. One of these compounds, cannabinol (CBN) has been shown to inhibit interleukin-2 (IL-2) expression and the activation of cAMP response element binding protein (CREB) and nuclear factor for immunoglobulin kappa chain in B cells (NF-kappaB) following phorbol-12-myristate-13 acetate (PMA) plus ionomycin (Io) treatment of thymocytes. Therefore, the objective of the present studies was to determine the role of cAMP and protein kinase A (PKA) in the CBN-mediated inhibition of IL-2, CREB, and NF-kappaB in PMA/Io-activated thymocytes. The inhibition of CREB/ATF-1 phosphorylation, or cAMP response element (CRE) or kappaB DNA binding activity produced by CBN in PMA/Io-activated thymocytes, could not be reversed by DBcAMP costimulation. Furthermore, DBcAMP failed to reverse the concentration-dependent inhibition of IL-2 protein secretion by CBN. Pretreatment of thymocytes with H89 produced a modest inhibition of PMA/Io-induced CREB/ATF-1 phosphorylation and CRE DNA binding activity but H89 had no effect on protein binding to a kappaB motif. Additionally, H89 modestly inhibited PMA/Io-induced IL-2 secretion. In light of the modest involvement of the cAMP pathway in CBN-mediated inhibition of CREB and IL-2 in PMA/Io-activated thymocytes, PD098059 (PD), the MEK inhibitor, was utilized to determine the role of ERK MAP kinases in thymocytes. ERKs play a critical role in IL-2 production but not for CREB phsophorylation. Collectively, these findings suggest that CBN may modulate several signalling pathways in activated T cells.
Asunto(s)
Cannabinol/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Timo/citología , Timo/efectos de los fármacos , Animales , Western Blotting , Carcinógenos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Flavonoides/farmacología , Interleucina-2/biosíntesis , Ionomicina/farmacología , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación/efectos de los fármacos , Acetato de Tetradecanoilforbol , Timo/metabolismo , Factores de TiempoRESUMEN
The B-cell, a major cellular component of humoral immunity, has been identified as a sensitive target of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The actual molecular mechanism responsible for the immunotoxic effects produced by TCDD is unclear; however, many of the biological effects produced by TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). Using the CH12.LX B-cell line, the present studies show that inhibition of mu gene expression and IgM protein secretion by polychlorinated dibenzo-p-dioxin congeners follow a structure-activity relationship for AhR binding. Furthermore, these effects may be mediated by the two dioxin-responsive enhancer (DRE)-like sites that were identified within the Ig heavy chain 3'alpha-enhancer. Electrophoretic mobility shift assay-Western analysis demonstrated TCDD-induced binding of the AhR nuclear complex to both DRE-like sites as well as TCDD-induced binding of several nuclear factor-kappa B/Rel proteins to a kappa B site, which overlaps one of the DRE-like sites. Interestingly, kappa B binding in the AhR-deficient BCL-1 B-cells was also induced by TCDD, demonstrating an AhR-independent effect of TCDD on kappa B binding. Taken together, these results support an AhR/DRE-mediated mechanism for TCDD-induced inhibition of IgM expression.
Asunto(s)
Contaminantes Ambientales/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Inmunoglobulina M/biosíntesis , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/fisiología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión , Citocromo P-450 CYP1A1/biosíntesis , Elementos de Facilitación Genéticos/efectos de los fármacos , Elementos de Facilitación Genéticos/fisiología , Inducción Enzimática , Inmunoglobulina M/genética , Cadenas mu de Inmunoglobulina/biosíntesis , Cadenas mu de Inmunoglobulina/genética , Ligandos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Dibenzodioxinas Policloradas/análogos & derivados , Receptores de Hidrocarburo de Aril/metabolismo , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacosRESUMEN
Transforming growth factor-beta(1) (TGF-beta(1)) is a critical bifunctional regulator of inflammatory responses. Evidence strongly suggests that these regulatory consequences are, at least in part, a result of profound pleiotropic effects on T lymphocyte effector function. The mechanisms underlying the contradictory biological effects of TGF-beta(1) remain ambiguous. The objective of the present studies was to test the hypothesis that the concentration of TGF-beta(1) and the temporal relationship between activation of the T cell receptor (TCR) and the TGF-beta receptor regulate the effect of TGF-beta(1) on T lymphocyte activation and proliferation. Toward this end, we have quantified the concentration- and time-dependent effect of TGF-beta(1) on interleukin-2 (IL-2) protein secretion as an index of T lymphocyte activation and [3H]-thymidine incorporation as an index of cell proliferation in primary splenocytes and thymocytes. Our results suggest that TGF-beta(1) stimulates IL-2 production at low concentrations (0.1-1 pg/ml) and conversely inhibits IL-2 production at high concentrations (1-10 ng/ml) in CD3epsilon monoclonal antibody (mAb)+/-CD28 mAb-activated splenocytes. Additionally, concentrations of TGF-beta(1) that stimulate IL-2 production in CD3epsilon mAb+CD28 mAb-activated splenocytes concominantly inhibit splenocyte proliferation under similar conditions. Furthermore, we provide evidence suggesting that the effects of TGF-beta(1) on T lymphocytes are dependent upon the temporal relationship between activation of the TCR and the TGF-beta receptor. A time-dependent loss of a stimulatory effect and a concomitant gain of an inhibitory response by TGF-beta(1) on IL-2 production in response to CD3epsilon and CD28 mAbs is observed when TGF-beta(1) is added following T lymphocyte activation. In summary, these data unequivocally demonstrate that the orchestration of paradoxical effects of TGF-beta(1) on T-lymphocyte function is dependent upon the concentration of TGF-beta(1) and the temporal relationship between activation of signaling through the TCR and the TGF-beta receptor. Future mechanistic studies addressing the putative role that these factors play in modulating the effects of TGF-beta(1) on T lymphocyte activity will undoubtedly provide valuable insight towards the pharmacological intervention of inflammatory responses.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD28/inmunología , Complejo CD3 , Interleucina-2/biosíntesis , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Bazo/inmunología , Timidina/metabolismo , Timo/inmunología , Factor de Crecimiento Transformador beta/fisiología , Adyuvantes Inmunológicos/fisiología , Animales , Relación Dosis-Respuesta Inmunológica , Femenino , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Bazo/citología , Bazo/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Factores de Tiempo , TritioRESUMEN
Regulation of the activator protein-1 (AP-1) complex is very intricate because it involves phosphorylation state, protein-protein, and protein-DNA interactions. In these studies, the regulation of AP-1 activity, with emphasis on c-fos and c-jun regulation, was investigated using cannabinol (CBN) in primary mouse splenocytes in vitro. Cannabinoid compounds exhibit immunosuppressive actions that are putatively mediated through Gi-protein coupled receptors that negatively regulate adenylate cyclase. However, recent studies suggest that cannabinoids modulate other signaling cascades. Indeed, we demonstrate that CBN inhibited binding to AP-1-containing sites from the interleukin-2 promoter. This inhibition of binding was, in part, due to decreased nuclear expression of c-fos and c-jun. We further determined that the effects of CBN were due to posttranslational modifications of these phosphoproteins and showed that CBN inhibited the activation of ERK MAP kinases. Thus, cannabinoid-induced immunosuppression involves disruption of the ERK signaling cascade.
Asunto(s)
Cannabinol/farmacología , Inmunosupresores/farmacología , Interleucina-2/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Nucleares , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Factor de Transcripción AP-1/antagonistas & inhibidores , Animales , Sitios de Unión , Western Blotting , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Interleucina-2/biosíntesis , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción NFATC , Regiones Promotoras Genéticas , Organismos Libres de Patógenos Específicos , Bazo/citología , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/química , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We previously reported that immunosuppressive cannabinoids inhibited interleukin (IL)-2 steady-state mRNA expression and secretion by phorbol-12-myristate-13-acetate plus ionomycin-activated mouse splenocytes and EL4 murine T-cells. Here we show that inhibition of IL-2 production by cannabinol, a modest central nervous system-active cannabinoid, is mediated through the inhibition of IL-2 gene transcription. Moreover, electrophoretic mobility shift assays demonstrated that cannabinol markedly inhibited the DNA binding activity of nuclear factor of activated T-cells (NF-AT) and activator protein-1 (AP-1) in a time- and concentration-dependent manner in activated EL4 cells. The inhibitory effects produced by cannabinol on AP-1 DNA binding were quite transient, showing partial recovery by 240 min after cell activation and no effect on the activity of a reporter gene under the control of AP-1. Conversely, cannabinol-mediated inhibition of NF-AT was robust and sustained as demonstrated by an NF-AT-regulated reporter gene. Collectively, these results suggest that decreased IL-2 production by cannabinol in EL4 cells is due to the inhibition of transcriptional activation of the IL-2 gene and is mediated, at least in part, through a transient inhibition of AP-1 and a sustained inhibition of NF-AT.
Asunto(s)
Cannabinol/farmacología , Proteínas de Unión al ADN/farmacología , Interleucina-2/genética , Factor de Transcripción AP-1/antagonistas & inhibidores , Factores de Transcripción/farmacología , Animales , Aductos de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC , Proteínas Nucleares/farmacología , Plásmidos , Timoma , Factores de Tiempo , Factor de Transcripción AP-1/fisiología , Transcripción Genética/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
Cannabinol (CBN), an immunosuppressive cannabinoid and ligand for the peripheral cannabinoid receptor CB2, inhibits the cAMP signaling cascade in forskolin-stimulated thymocytes. The objective of the present studies was to further characterize the mechanism of CBN immune modulation by investigating its effects on interleukin-2 (IL-2) secretion, cAMP response element (CRE), and kappaB DNA binding activity in phorbol ester (phorbol-12-myristate-13-acetate, PMA) plus calcium ionophore (PMA/Io)-activated thymocytes. PMA/Io treatment induced CRE and kappaB DNA binding activity that was attenuated in the presence of CBN. A concomitant and concentration-related inhibition of IL-2 also was produced by CBN in PMA/Io-activated thymocytes. PMA/Io induced two CRE DNA binding complexes, a major complex consisting of a cAMP response element-binding protein (CREB)-1 homodimer, and a minor CREB-1/activating transcription factor (ATF)-2 complex. Both CRE complexes were inhibited by CBN. Conversely, two kappaB DNA binding complexes were observed, but only one was PMA/Io-inducible. However, the DNA binding activity of both complexes was diminished in the presence of CBN. The PMA/Io-inducible kappaB complex was a p65/c-Rel heterodimer. Analysis of up-stream regulation revealed a decrease in phosphorylated CREB/ATF nuclear proteins in PMA/Io-activated thymocytes after CBN treatment. Similarly, CBN prevented the phosphorylation-dependent degradation of the nuclear factor-kappaB inhibitory protein IkappaB-alpha. These results provide a potential link between the CBN-mediated inhibition of thymocyte function, including IL-2 production, and the inhibition of two critical transcription factor families, CREB/ATF and NF-kappaB/Rel.
Asunto(s)
Cannabinol/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteínas de Unión al ADN , Interleucina-2/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Linfocitos T/metabolismo , Factor de Transcripción Activador 1 , Animales , Western Blotting , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Densitometría , Depresión Química , Ensayo de Inmunoadsorción Enzimática , Femenino , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos , FN-kappa B/metabolismo , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismoRESUMEN
The yolk protein precursor vitellogenin (Vtg) is secreted by the liver of female as well as male fish, in response to estrogenic compounds. In this study, an in vitro assay was developed for measuring Vtg induction, using cultured primary hepatocytes from genetically uniform strains of carp (Cyprinus carpio). Vtg production was measured by indirect competitive ELISA, using a polyclonal antiserum against goldfish Vtg that cross-reacts with carp Vtg. Vtg was dose-dependently induced by 17beta-estradiol (E2) in hepatocytes of both sexes. E2 had a lowest observed effect concentration (LOEC) for Vtg induction of 2 nM, an EC50 between 50 and 150 nM, and a maximum response at 2 microM. The plasticizer and xenoestrogen bisphenol-A induced Vtg secretion by hepatocytes of both sexes at 50 and 100 microM. This carp hepatocyte (CARP-HEP) assay can also be used to detect antiestrogenic activity, which was measured as the reduction of E2-stimulated Vtg synthesis. Two well-known antiestrogenic compounds, tamoxifen and 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), were tested. TCDD caused a reduction in Vtg synthesis in female hepatocytes at concentrations <0.1 nM, making it approximately 10,000-fold more potent than tamoxifen. Carp hepatocytes were also sensitive to induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD). Depending on the exposure time, 18 or 96 h, EROD EC50 values for TCDD were 27 or 6 pM, respectively. The CARP-HEP assay, using the 96-well plate format, offers good possibilities to screen large numbers of compounds for (anti)estrogenic properties. In addition, it can simultaneously determine aryl hydrocarbon receptor agonist properties, measured as CYP1A induction.
Asunto(s)
Carpas/metabolismo , Antagonistas de Estrógenos/farmacología , Estrógenos/farmacología , Hígado/metabolismo , Vitelogeninas/biosíntesis , Xenobióticos/farmacología , Animales , Compuestos de Bencidrilo , Células Cultivadas , Citocromo P-450 CYP1A1/biosíntesis , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Femenino , Hígado/citología , Masculino , Fenoles/farmacología , Dibenzodioxinas Policloradas/farmacología , Tamoxifeno/farmacologíaRESUMEN
Phospholipase A2 (PLA2) has been postulated to play a role in the regulation of cytokine expression. Therefore, the objective of the present study was to investigate the effects of PLA2 inhibitors p-bromophenacyl bromide (BPB) and arachidonyl trifluoromethyl ketone (AACOCF3) on interleukin-2 (IL-2) expression in murine primary splenocytes. Pretreatment of the splenocytes with both BPB and AACOCF3 suppressed phorbol 12-myristate 13-acetate plus ionomycin-induced IL-2 secretion in a concentration-dependent manner. Inhibition > 90% of IL-2 secretion was observed at 1 microM BPB and 10 microM AACOCF3 compared to the respective vehicle control. Likewise, IL-2 steady-state mRNA expression was inhibited by both PLA2 inhibitors in a concentration-dependent fashion with > 90% inhibition at 1 microM BPB and 20 microM AACOCF3. Taken together, these data demonstrated that PLA2 inhibitors BPB and AACOCF3 are robust inhibitors of IL-2 expression at both the mRNA and protein levels in murine splenocytes. Moreover, these findings suggest that drugs and chemicals which inhibit PLA2 may have marked effects on T-cell function.
Asunto(s)
Acetofenonas/farmacología , Ácidos Araquidónicos/farmacología , Inhibidores Enzimáticos/farmacología , Interleucina-1/genética , Fosfolipasas A/antagonistas & inhibidores , Bazo/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica/efectos de los fármacos , Ratones , Fosfolipasas A2 , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/metabolismo , Factores de TiempoRESUMEN
These studies characterized the profile of AhR and ARNT expression in primary splenocytes and purified splenic B cells after cellular activation with lipopolysaccharide (LPS). LPS treatment of mouse splenocytes markedly increased the magnitude of both AhR and ARNT steady state mRNA expression. AhR mRNA expression peaked at 8 hr post-LPS activation and was increased by approximately 5-fold compared with freshly isolated splenocytes (i.e., time 0). ARNT mRNA expression began to increase at 8 hr postactivation, peaked at approximately 48 hr and was increased by approximately 4-fold when compared with nonactivated splenocytes at time 0. Western blotting also demonstrated an increase in the relative magnitude of both the AhR and ARNT proteins in LPS activated splenocytes. Likewise, the presence of the AhR, ARNT and cytochrome P450IA1 (CYP1A1) proteins were also detected in purified primary splenic B cells, and the magnitude of protein expression was enhanced in LPS activated splenic B cells at 12 and 24 hr relative to time matched controls for each of these proteins. In summary, these findings suggest that on LPS activation the magnitude of AhR and ARNT mRNA and protein increases in both splenocytes and purified primary splenic B cells. Moreover, because the increase in the relative magnitude of CYP1A1 protein in response to LPS occurred in the absence of exogenous AhR ligand, these results suggest that B-cell activation is sufficient to induce AhR nuclear translocation and binding to dioxin-responsive elements in the promoter region of AhR-responsive genes.
Asunto(s)
Linfocitos B/efectos de los fármacos , Proteínas de Unión al ADN , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptores de Hidrocarburo de Aril/biosíntesis , Bazo/efectos de los fármacos , Factores de Transcripción/biosíntesis , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Linfocitos B/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Ratones , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/metabolismo , Factores de Transcripción/genéticaRESUMEN
Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Interleucina-2/biosíntesis , FN-kappa B/biosíntesis , Proteínas Nucleares , Factor de Transcripción AP-1/biosíntesis , Factores de Transcripción/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Animales , Expresión Génica/efectos de los fármacos , Interleucina-2/genética , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The objective of this article is to discuss the putative role of cannabinoid receptors in immune modulation by cannabinoid compounds. The primary focus is on the signal transduction events that are initiated following ligand binding to cannabinoid receptors and how these events lead to detrimental effects on the normal responsiveness of immunocompetent cells. Toward this end, signalling events are traced from the cannabinoid receptor to the transcription factors which are adversely regulated in the presence of cannabinoid compounds during leukocyte activation. Moreover, this aberrant regulation of transcription factors is discussed in the context of altered gene expression and the impact this has on leukocyte function. Lastly, an important goal of this article is to dispel a long standing myth that the cyclic adenosine 3':5'-monophosphate (cAMP) cascade is a negative regulatory pathway for immunocompetent cells. This chapter examines two major immunologic cell-types which are well established as exhibiting altered function following cannabinoid treatment, helper T-cells and the macrophage. Not discussed are the effects of cannabinoids on B-cell function. This is primarily due to the rather refractory nature of B-cells to inhibition by cannabinoids in spite of the fact that this cell-type expresses functional cannabinoid receptors [Schatz, A.R., Koh, W.S., Kaminski, N.E., 1993. Delta9-tetrahydrocannabinol selectively inhibits T-cell dependent humoral immune responses through direct inhibition of accessory T-cell function. Immunopharmacol., 26, pp. 129-137.]. One cautionary note, although the focus of this article is on cannabinoid receptor mediated signalling events, immune modulation by cannabinoid compounds is likely multi-factorial presumably involving receptor as well as receptor-nonrelated events. Effects on leukocytes by cannabinoids which are believed to be mediated by receptor-nonrelated events are outside the scope of this paper and will not be discussed. One last introductory point is that even though their is presumably little overlap in the genes which are regulated by cannabinoids in leukocytes as compared to other cell-types (e.g., neural cells), the major signalling pathways involved in cellular regulation are ubiquitous. With that in mind, it is likely that their is a considerable amount of similarity in the signalling pathways regulated by cannabinoids in cell-types of different lineage, given that they express cannabinoid receptors. In this context, signalling events observed in leukocytes can provide important insight into which genes may be modulated by cannabinoid in other cell types.
Asunto(s)
AMP Cíclico/metabolismo , Regulación de la Expresión Génica/inmunología , Receptores de Droga/genética , Receptores de Droga/inmunología , Transducción de Señal/inmunología , Animales , Cannabinoides/genética , Cannabinoides/inmunología , Humanos , Receptores de CannabinoidesRESUMEN
The immune system has been identified as a sensitive target for the toxic effects produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Furthermore, the B cell has been identified as a sensitive cellular target of TCDD by previous cell-type fractionation studies from this laboratory. The mechanism responsible for the immunotoxic effects produced by TCDD is unclear; however, many of the biological effects of TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). Here, we describe two B cell lines that differ considerably in their expression of the AhR and in their sensitivity to TCDD. Our results demonstrated a marked expression of the AhR protein in the CH12.LX B cell line but not in the BCL-1 B cell line. Transcripts for the AhR were not detected by reverse transcriptase-polymerase chain reaction in the BCL-1 cells. The AhR nuclear translocator (ARNT) protein was highly expressed in both cell lines. In addition, the AhR and ARNT are functional in CH12.LX cells as demonstrated by TCDD-induced CYP1A1 induction. TCDD did not induce CYP1A1 in BCL-1 cells. Furthermore, TCDD treatment resulted in suppression of lipopolysaccharide (LPS)-induced IgM secretion in CH12.LX cells. Conversely, TCDD-induced inhibition of IgM secretion was not demonstrated in LPS-stimulated BCL-1 cells, implicating a role for the AhR in the inhibition of B cell effector function. LPS-induced differentiation of the CH12.LX cells also resulted in a marked induction of Ahr expression which was not induced in LPS-stimulated BCL-1 cells. These studies have implicated the AhR as a critical factor in TCDD-induced inhibition of IgM secretion and have demonstrated an induction of AhR gene and protein expression after B cell activation.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN , Inmunoglobulina M/metabolismo , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Carcinoma Hepatocelular , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Linfoma de Células B/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Factores de Transcripción/biosíntesis , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
2-Arachidonyl-glycerol (2-Ara-Gl) recently was identified as a putative endogenous ligand for cannabinoid receptor types CB1 and CB2 by competitive binding. More recent immune function assays demonstrated that 2-Ara-Gl possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-Gl was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-Gl to mouse splenocyte cultures suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 secretion and steady state mRNA expression in a dose-dependent manner. 2-Ara-Gl also produced a marked inhibition of IL-2 promotor activity as determined by transient transfection of EL4.IL-2 cells with a pIL-2-CAT construct. 2-Ara-Gl at 5, 10, 20, and 50 microM suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 promotor activity by 18%, 28%, 39%, and 54%, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2 by 2-Ara-Gl, the DNA-binding activity of transcription factors, nuclear factor of activated T cells (NF-AT), nuclear factor for immunoglobulin kappa chain in B cells (NF-kappa B/Rel), activator protein-1(AP-1), octamer, and cAMP-response element binding protein was evaluated by electrophoretic mobility shift assay in mouse splenocytes. In addition, a reporter gene expression system for p(NF-kappa B)3-CAT, p(NF-AT)3-CAT, and p(AP-1)3-CAT was used in transiently transfected EL4.IL-2 cells to determine the effect of 2-Ara-Gl on promoter activity for each of the specific transcription factors. 2-Ara-Gl reduced both the NF-AT-binding and promoter activity in a dose-dependent manner and, to a lesser degree, NF-kappa B/Rel-binding and promoter activity. No significant effect was observed on octamer- and cAMP-response element-binding activity. AP-1 DNA-binding activity was not inhibited by 2-Ara-Gl, but a modest inhibition of promoter activity was observed.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Ácidos Araquidónicos , Regulación hacia Abajo/efectos de los fármacos , Glicéridos/farmacología , Interleucina-2/antagonistas & inhibidores , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Cannabinoides/metabolismo , Células Cultivadas , Secuencia de Consenso , Cruzamientos Genéticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Endocannabinoides , Femenino , Glicéridos/metabolismo , Factor C1 de la Célula Huésped , Interleucina-2/biosíntesis , Interleucina-2/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Factores de Transcripción NFATC , Factor 1 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genéticaRESUMEN
Immune suppression by cannabinoids has been widely demonstrated in a variety of experimental models. The identification of two major types of G-protein-coupled cannabinoid receptors expressed on leukocytes, CB1 and CB2, has provided a putative mechanism of action for immune modulation by cannabinoid compounds. Ligand binding to both receptors negatively regulates adenylate cyclase, thereby lowering intracellular cyclic AMP (cAMP) levels. In the present studies, we demonstrated that cannabinol (CBN), a ligand that exhibits higher binding affinity for CB2, modulates immune responses and cAMP-mediated signal transduction in mouse lymphoid cells. Direct addition of CBN to naive cultured splenocytes produced a concentration-dependent inhibition of lymphoproliferative responses to anti-CD3, lipopolysaccharide, and phorbol-12-myristate-13-acetate/ionomycin stimulation. Similarly, a concentration-related inhibition of the in vitro anti-sheep red blood cell IgM antibody forming cell response was also observed by CBN. Evaluation of cAMP signaling in the presence of CBN showed a rapid and concentration-related inhibition of adenylate cyclase activity in both splenocytes and thymocytes. This decrease in intracellular cAMP levels produced by CBN resulted in a reduction of protein kinase A activity, consequently leading to an inhibition of transcription factor binding to the cAMP response element and kappaB motifs in both cell preparations. Collectively, these results demonstrate that CBN, a cannabinoid with minimal CNS activity, inhibited both cAMP signal transduction and immune function, further supporting the involvement of CB2 receptors in immune modulation by cannabimimetic agents.
Asunto(s)
Cannabinol/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , AMP Cíclico/fisiología , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Adenilil Ciclasas/metabolismo , Animales , Formación de Anticuerpos/efectos de los fármacos , Colforsina/farmacología , Medios de Cultivo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Inmunoglobulina M/biosíntesis , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Ratones , Ratones Endogámicos , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
Previous studies have demonstrated that repeated (14 day) administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances the suppression of humoral immunity in DBA/2 (Ah-low responder) mice relative to the effect seen with identical cumulative doses after a single treatment (cumulative doses of 4.2, 14.0, and 42 mg/kg). In the present studies, we have explored this phenomenon further by determining the status of several specific parameters, which might account for the increase in antibody suppression in the DBA/2 strain following repeated TCDD exposures. Included in these studies was the induction of hepatic and splenic microsomal 7-ethoxyresorufin-o-deethylase (EROD; P4501A1) activity and biodistribution of the administered TCDD into various target organs and tissues. Changes in lymphocyte subpopulations within the spleen were also assessed by flow cytometry following both single and repeated dosing. All studies made use of direct comparisons between DBA/2 (Ah-low responder) and B6C3F1 (Ah-high responder) female mice. Results of these studies demonstrate that the enhanced suppression of humoral immunity in DBA/2 mice following repeated exposure to TCDD is not directly associated with increases in liver microsomal EROD activity and does not appear to be correlated with changes in the pattern of biodistribution or amount of TCDD within the liver or spleen of these animals. In contrast, the most significant changes that occurred following repeated dosing in either strain were observed in the levels of microsomal EROD activity and immune cell ratios within the spleen. This effect was characterized as an increase in microsomal EROD activity, and a corresponding reduction in the numbers of a non-B/non-T cell population in the spleen.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Citocromo P-450 CYP1A1/biosíntesis , Hígado/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Bazo/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Inducción Enzimática , Femenino , Hígado/enzimología , Hígado/patología , Ratones , Ratones Endogámicos DBA , Tamaño de los Órganos/efectos de los fármacos , Bazo/enzimología , Bazo/patología , Timo/efectos de los fármacos , Factores de TiempoRESUMEN
Immune modulation by cannabinoids has been widely established over the past three decades. In spite of this, the mechanism of action responsible for immune modulation and other well described biological effects attributed to cannabinoid compounds has been elusive. The identification and cloning of two novel G protein coupled receptors, CB1 and CB2, both of which bind cannabimimetic agents has served as the basis for a putative mechanism of action. CB1, which is also referred to as the central cannabinoid receptor is the primary form expressed within the central nervous system (CNS). Conversely, the peripheral cannabinoid receptor, CB2, does not appear to be expressed within the CNS but is the predominant form of the receptor expressed within the immune system. Both CB1 and CB2 negatively regulate adenylate cyclase activity through a pertussis toxin sensitive GTP-binding protein. Recent investigations addressing the mechanism by which cannabinoids disrupt leukocyte function have demonstrated that in the presence of cannabinoids the cAMP signaling cascade is markedly inhibited as evidenced by decreased adenylate cyclase and protein kinase A activity and decreased DNA binding by cAMP response element binding proteins. The focus of this discussion will be on the effects cannabinoids elicit on events within the cAMP cascade and related signaling pathways critical to the regulation of cytokine genes.
Asunto(s)
Cannabinoides/toxicidad , AMP Cíclico/fisiología , Sistema Inmunológico/efectos de los fármacos , Inmunosupresores/toxicidad , Receptores de Droga/fisiología , Animales , Humanos , Receptores de CannabinoidesRESUMEN
Delta9-Tetrahydrocannabinol (delta9-THC) binding to cannabinoid receptors induces an inhibition in adenylate cyclase activity through the engagement of a pertussis toxin-sensitive GTP-binding protein. In this study we investigated the ramifications of decreased cyclic AMP (cAMP) formation by delta9-THC on signaling events through the cAMP pathway distal to adenylate cyclase in mouse splenocytes. Delta9-THC treatment produced a marked and concentration-related decrease in forskolin-inducible protein kinase A (PKA) activity. This decrease in kinase activity was due to an inhibition in cAMP formation and not through a direct effect on the kinase as evidenced by the fact that PKA activity could not be modulated directly by delta9-THC in the presence of exogenous cAMP. One of the primary roles of PKA in this signaling pathway is to activate transcription factors for subsequent binding to cAMP response elements (CRE) present in the promoter region of cAMP-responsive genes. In the present studies, we observed that forskolin treatment of splenocytes resulted in a rapid activation of trans-acting factor binding to the CRE, which peaked at 30-60 min and whose binding was repressed concentration dependently in the presence of delta9-THC. As with forskolin, mitogenic stimulation including anti-CD3 mAb or phorbol ester plus ionomycin treatment of splenocytes induced CRE binding activity, which was maximal around 60 min and was suppressed by delta9-THC treatment. In conclusion, these data indicate that cAMP-mediated signal transduction is inhibited by delta9-THC and consequently leads to a decrease in the activation of transcription factors that bind to CRE regulatory sites.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Dronabinol/farmacología , Alucinógenos/farmacología , Animales , Anticuerpos Monoclonales , Complejo CD3/inmunología , Colforsina/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Ionomicina/farmacología , Ratones , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Acute treatment of B6C3F1 mice with a hepatotoxic dose (500 mg/kg) of carbon tetrachloride (CCl4) produced a marked but transient increase in transforming growth factor beta 1 (TGF-beta 1) mRNA expression in the liver within 24 hr. We have previously shown that an identical dose of CCl4 also produces a marked increase in serum TGF-beta 1 concentrations which peak at 48 hr and produce a marked inhibition of the anti-sRBC IgM antibody forming cell (AFC) response. Similar increases in TGF-beta 1 transcripts scripts in the liver were also induced by an acute hepatotoxic dose (600 mg/kg) of acetaminophen. No increase in TGF-beta 1 mRNA expression was detected in the spleen following treatment with either agent. Direct addition of TGF-beta 1 (0.05-1.0 ng/ml) to naive splenocyte cultures produced a marked and dose-related inhibition of the anti-sRBC IgM AFC response. Under the same conditions, TGF-beta 1 induced a marked decrease in IL-4 and IL-5 mRNA expression in sRBC-sensitized splenocytes. Concomitantly, TGF-beta 1 induced a rapid increase in NF-kappa B/Rel trans-acting factor binding within the first 24 hr post-sRBC sensitization of splenocytes. Conversely, NF-kappa B/Rel binding activity was inhibited on Days 2 through 4 in sRBC-sensitized splenocytes in the presence of TGF-beta 1. The increase in NF-kappa B/Rel binding within 24 hr following sRBC sensitization is consistent with the positive influence TGF-beta 1 exerts on Th1 cytokines such as IL-2 and IFN-gamma. Conversely the decrease in NF-kappa B/Rel binding at the later time period during the AFC response (Days 2-4) coincides with the inhibitory effects TGF-beta 1 exerted on IgM production by B cells.