- Email: [email protected]
Cannabinoid receptors and T helper cells
Journal of Neuroimmunology 147 (2004) 91 – 94 www.elsevier.com/locate/jneuroim
Cannabinoid receptors and T helper cells Thomas W. Klein *, Cathy Newton, Kellie Larsen, Joe Chou, Izabella Perkins, Lily Lu, Liang Nong, Herman Friedman Department of Medical Microbiology and Immunology, MDC Box 10, University of South Florida, 12901 Bruce Downs Boulevard, Tampa, FL 33612, USA
Abstract We have reported that injection of marijuana cannabinoids, such as D9-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) g production and enhancement of IL-4 production suggesting THCinduced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL12Rh2 or GATA3 gene products. The results showed that THC suppressed IL-12Rh2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rh2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rh2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Th1 cells; Cannabinoid; IL-12
1. Introduction The marijuana cannabinoid, D9-tetrahydrocannabinol (THC), and other related cannabinoids and endocannabinoids are reported to modulate the immune response (Klein et al., 1998, 2001). However, the role of cannabinoid receptors and the molecular mechanisms involved in this modulation are unclear. Several years ago, we reported that THC injection into mice suppressed the development of cell-mediated, Th1 immunity to infection with the intracellular, bacterial pathogen, Legionella pneumophila (Lp) (Newton et al., 1994). This was followed by our report that THC injection suppressed Th1 immunity by inhibiting the mobilization of interferon (IFN) g and interleukin (IL)-12 as well as the expression of IL-12 receptors (IL-12R) and increasing the expression of the Th2-promoting cytokine IL-4 (Klein et al., 2000). It was also demonstrated, using cannabinoid receptor 1 (CB1) and 2 (CB2) antagonists, that
* Corresponding author. Tel.: +1-813-974-2502; fax: +1-813-9744151. E-mail address: [email protected] (T.W. Klein). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.019
both receptors were involved in some way in this THCinduced T helper cell biasing. Other laboratories have reported similar T helper biasing effects of cannabinoids as well as other neuroimmune axis modulating agents. For example, THC injection into mice reportedly suppressed, by a CB2 mechanism, the development of anti-tumor immunity by enhancing Th2 cytokines (Zhu et al., 2000) and THC treatment of cultured human PBMCs also shifted to Th2 immunity via a CB2 receptor mechanism (Yuan et al., 2002). In addition, cannabinoid treatment of mice primed with C. parvum injection and challenged with endotoxin induced a decrease in serum tumor necrosis factor (TNF) a and IL-12 and an increase in IL-10 (Smith et al., 2001). Drugs and endogenous mediators other than cannabinoids also appear to bias the activity of developing Th1 and Th2 cells. Some of these are drugs of abuse such as morphine (Roy et al., 2001), while others are endogenous mediators such as prostaglandins (Kuroda et al., 2000), glucocorticoids (Cohen et al., 2002), adrenergic mediators (Swanson et al., 2001) and chemokines (Braun and Kelsall, 2001). Because many of these agents affect cells through G protein-coupled receptors, it is possible that signaling mechanisms associated with
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T.W. Klein et al. / Journal of Neuroimmunology 147 (2004) 91–94
Fig. 1. The biasing of T helper cell development. Microbes stimulate different types of dendritic cells (DC) to produce cytokines that cause naı¨ve precursors of CD4+, T helper cells to differentiate into either T helper 1 (Th1) or T helper 2 (Th2) cells. These cells then regulate either cellmediated or humoral immunity. IFNg, IL, IL-12R, IL-4 receptor (IL-4R), signal transducers and activators of transcriptions 4 and 6 (STAT 4 and 6), GATA nucleotide sequence (GATA3), TNFh and immunoglobulin (Ig).
hours following drug treatment and antigen challenge, serum IL-12 and IFNg mobilization was decreased relative to drug vehicle controls and IL-4 was increased by drug treatment in spleen preparations (Klein et al., 2000). The expression of IL-12 receptor h2 (IL-12Rh2) is also important in the development of Th1 cells (Fig. 1) and the gene for this receptor is thought to be regulated by IL-12 and IFNg (Chang et al., 1999; Szabo et al., 1997). Because THC treatment decreased IL-12 production, we tested to see if this had an inhibitory effect on IL-12Rh2 gene activity. Mice were injected with THC 18 h prior to infection with Legionella. At 2 h following infection, spleens were removed and processed for measuring IL12Rh2 mRNA by RT-PCR. Fig. 2 shows that, like IL-12, THC treatment suppressed the activation of IL-12Rh2 gene activity. In addition to these findings, we also tested the role of CB1 and CB1 in this effect. Mice were injected with either the CB1 antagonist, SR141716A (SR1), or the CB2 antagonist, SR144528 (SR2), followed 30 min later by THC injection. These mice were then infected with Legionella 18 h later and the spleens tested for IL-12 receptor message. Fig. 2 shows that the CB1 but not CB2 antagonist attenuated the THC suppressive effect suggesting that the down-regulation of IL-12Rh2 gene was mediated by CB1 receptors.
these kinds of receptors are linked to the regulation of T helper biasing events (Braun and Kelsall, 2001). T helper cell biasing is regulated by the production and action of several key cytokines and their cognate receptors (Fig. 1). The production by dendritic cells of either IL-4 or IL-12 in response to antigen stimulation is one of many key steps in the development of Th1 and Th2 cells. IL-12 leads to the phosphorylation of STAT 4 and the subsequent activation of IFNg and IL-12 receptor genes. The up-regulation of these genes drives the development of Th1 cells (Fig. 1). IL-4, on the other hand, leads to phosphorylation of STAT 6 and the subsequent activation of genes such as IL-4 receptor that promote Th2 development. In addition, in the relative absence of IL-12, IL-4 stimulation increases the Th2-dependent transcription factor GATA3 that further promotes Th2 differentiation (Moser and Murphy, 2000). In the present report, to better understand possible cannabinoid receptor-mediated mechanisms of THC-induced T helper biasing, we studied the effect of THC treatment on the regulation of expression of the key T helper biasing mediators, IL-12 receptor and GATA3.
2. Results and discussion Our previous work suggested that THC injection into mice prior to Legionella antigen challenge not only suppressed the development of Th1 activity but also increased the development of Th2 activity. For example, within
Fig. 2. THC injection suppresses IL-12Rh2 mRNA by CB1-mediated mechanism. BALB/c mice were injected i.v. with either saline or THC (8 mg/kg) 18 h prior to infection with Legionella pneumophila (7 106 bacteria). Two hours after infection, spleens were removed, total RNA extracted and analyzed by semi-quantitative RT-PCR for IL-12 receptor and h-actin mRNA. In some experiments, CB1 (SR1) or CB2 (SR2) antagonists were injected 30 min prior to THC treatment.
T.W. Klein et al. / Journal of Neuroimmunology 147 (2004) 91–94
Besides suppression of IL-12 and IFNg in response to Legionella challenge, we also previously reported that THC injection increased IL-4 production in the spleens of antigen challenged mice. GATA3 is an important signaling factor in the regulation of T helper cell development being found in Th2 cells but not Th1 (Das et al., 2001). Also, GATA3 and IL-12 appear to be mutually antagonistic in their action with the former increasing in activity in the absence of the latter (Ouyang et al., 1998). Because THC suppressed IL-12, we tested to see if the drug increased GATA3 injection following Legionella challenge. Spleens were harvested 2 h following Legionella infection and total RNA tested by RTPCR for GATA3 mRNA. Fig. 3 shows that GATA3 message is increased by THC injection suggesting that this Th2 biasing transcription factor is increased by drug treatment. To determine if cannabinoid receptors are involved in this effect, mice were pretreated with SR141716A (SR1) or SR144528 (SR2) as before prior to THC injection and Legionella challenge. As seen in Fig. 3, the CB2 antagonist but not the CB1 attenuated the THC effect suggesting that the marijuana cannabinoid increased GATA3 gene activity through CB2 but not CB1. Our previous work showed that THC injection prior to Legionella challenge suppressed the Th1 associated cytokines IL-12 and IFNg and that this effect was attenuated by antagonists to both CB1 and CB2. In the current report, other T helper biasing mechanisms were studied and the results suggested that CB1 and CB2 involvement was split between the suppression of IL-12Rh2 and the increase in GATA3 messages. Unlike with IL-12 and IFNg production, THC suppression of IL-12Rh2 appeared to be mediated by CB1 only while the drug-induced increase in GATA3 was
93
Fig. 4. Signaling factors generated by Gi protein coupling. Gi when activated splits into aGTP and h/g dimer subunits that can associate with and activate a variety of signaling factors. These downstream events activate genes involved in cytokine production and T cell activation. Protein kinase C (PKC), inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG), nuclear factor kappa B (NFkB), MAPK/ERK kinase (MEK), extracellular related kinase (ERK), phospholipase C (PLC) and activator protein 1 (AP1).
mediated by CB2 only. Cannabinoid receptors are G protein-coupled receptors and have both been shown to be coupled to Gai/o (Bayewitch et al., 1995) and to suppress adenylyl cyclase but activate MAP kinase (Bouaboula et al., 1999). It is now recognized that activation of G proteins through receptor ligation sets into motion a variety of signaling and gene activation pathways mediated by not only the Ga component but also the h/g component of the G protein (Braun and Kelsall, 2001). As seen in Fig. 4, various signaling factors can be either activated or suppressed by G protein activation and the dominating pathways can vary from cell to cell depending upon the endogenous tone of the receptors (Felder et al., 1998), the complement of neighboring receptors in the membrane (Bouaboula et al., 1999), and other unknown factors. Because we have seen a CB1mediated decrease in IL-12Rh2 gene activity but a CB2mediated increase in GATA3, it seems likely that the differences in regulation of these genes by the cannabinoid receptors stem from differences in the receptor and signaling factor milieu in Th1 and Th2 cells. CB1 receptors on Th1 cells and the activated Gi subunits could signal a decrease in the IL-12Rh2 gene product while CB2 receptor ligation on Th2 cells could signal an increase in the GATA3 gene. This cell-dependent dichotomy of signaling function is readily testable and will be the subject of future studies in this model.
Acknowledgements Fig. 3. THC injection increases GATA3 mRNA by CB2-mediated mechanism. See Fig. 2 for details.
This work was supported by NIDA grants DA03646, DA07245 and DA10683.
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References Bayewitch, M., Avidor-Reiss, T., Levy, R., Barg, J., Mechoulam, R., Vogel, Z., 1995. The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling. FEBS Lett. 375, 143 – 147. Bouaboula, M., Desnoyer, N., Carayon, P., Combes, T., Casellas, P., 1999. Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation. Mol. Pharmacol. 55, 473 – 480. Braun, M.C., Kelsall, B.L., 2001. Regulation of interleukin-12 production by G-protein-coupled receptors. Microbes Infect. 3, 99 – 107. Chang, J.T., Shevach, E.M., Segal, B.M., 1999. Regulation of interleukin (IL)-12 receptor h2 subunit expression by endogenous IL-12: a critical step in the differentiation of pathogenic autoreacitve T cells. J. Exp. Med. 189, 969 – 978. Cohen, M., Klein, E., Kuten, A., Fried, G., Zinder, O., Pollack, S., 2002. Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevated levels of stress hormones and decreased secretion of Th1 cytokines. Int. J. Cancer 100, 347 – 354. Das, J., Chen, C.-H., Yang, L., Cohn, L., Ray, P., Ray, A., 2001. A critical role for NF-kB in Gata3 expression and Th2 differentiation in allergic airway inflammation. Nat. Immunol. 2, 45 – 50. Felder, C.C., Joyce, K.E., Briley, E.M., Glass, M., Mackie, K.P., Fahey, K.J., Cullinan, G.J., Hunden, D.C., Johnson, D.W., Chaney, M.O., Koppel, G.A., Brownstein, M., 1998. LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation. J. Pharmacol. Exp. Ther. 284, 291 – 297. Klein, T., Newton, C., Friedman, H., 1998. Cannabinoid receptors and immunity. Immunol. Today 19, 373 – 381. Klein, T.W., Newton, C.A., Nakachi, N., Friedman, H., 2000. D9-Tetrahydrocannabinol treatment suppresses immunity and early IFN-g, IL-12, and IL-12 receptor h2 responses to Legionella pneumophila infection. J. Immunol. 164, 6461 – 6466.
Klein, T.W., Newton, C.A., Friedman, H., 2001. Cannabinoids and the immune system. Pain Res. Manag. 6, 95 – 101. Kuroda, E., Sugiura, T., Zeki, K., Yoshida, Y., Yamashita, U., 2000. Sensitivity difference to the suppressive effect of prostaglandin E among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice. J. Immunol. 164, 2386 – 2395. Moser, M., Murphy, K., 2000. Dendritic cell regulation of Th1 – Th2 development. Nat. Immunol. 1, 199 – 205. Newton, C.A., Klein, T.W., Friedman, H., 1994. Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9tetrahydrocannabinol injection. Infect. Immun. 62, 4015 – 4020. Ouyang, W., Ranganath, S.H., Weindel, K., Bhattacharya, D., Murphy, T.L., Sha, W.C., Murphy, K.M., 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9, 745 – 755. Roy, S., Balasubramanian, S., Sumandeep, S., Charboneau, R., Wang, J., Melnyk, D., Beilman, G.J., Vatassery, R., Barke, R.A., 2001. Morphine directs T cells toward T(H2) differentiation. Surgery 130, 304 – 309. Smith, S.R., Terminelli, C., Denhardt, G., 2001. Modulation of cytokine responses in Corynebacterium parvum-primed endotoxemic mice by centrally administered cannabinoid ligands. Eur. J. Pharmacol. 425, 73 – 83. Swanson, M.A., Lee, W.T., Sanders, V.M., 2001. IFN-gamma production by Th1 cells generated from naive CD4+ T cells exposed to norepinephrine. J. Immunol. 166, 232 – 240. Szabo, S.J., Dighe, A.S., Gubler, U., Murphy, K.M., 1997. Regulation of the interleukin (IL)-12R h2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185, 817 – 824. Yuan, M., Kiertscher, S.M., Cheng, Q., Zoumalan, R., Tashkin, D.P., Roth, M.D., 2002. D9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells. J. Neuroimmunol. 133, 124 – 131. Zhu, L.X., Sharma, S., Stolina, M., Gardner, B., Roth, M.D., Tashkin, D.P., Dubinett, S.M., 2000. D-9-Tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373 – 380.
Cannabinoid receptors and T helper cells Thomas W. Klein *, Cathy Newton, Kellie Larsen, Joe Chou, Izabella Perkins, Lily Lu, Liang Nong, Herman Friedman Department of Medical Microbiology and Immunology, MDC Box 10, University of South Florida, 12901 Bruce Downs Boulevard, Tampa, FL 33612, USA
Abstract We have reported that injection of marijuana cannabinoids, such as D9-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) g production and enhancement of IL-4 production suggesting THCinduced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL12Rh2 or GATA3 gene products. The results showed that THC suppressed IL-12Rh2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rh2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rh2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells. D 2003 Elsevier B.V. All rights reserved. Keywords: Th1 cells; Cannabinoid; IL-12
1. Introduction The marijuana cannabinoid, D9-tetrahydrocannabinol (THC), and other related cannabinoids and endocannabinoids are reported to modulate the immune response (Klein et al., 1998, 2001). However, the role of cannabinoid receptors and the molecular mechanisms involved in this modulation are unclear. Several years ago, we reported that THC injection into mice suppressed the development of cell-mediated, Th1 immunity to infection with the intracellular, bacterial pathogen, Legionella pneumophila (Lp) (Newton et al., 1994). This was followed by our report that THC injection suppressed Th1 immunity by inhibiting the mobilization of interferon (IFN) g and interleukin (IL)-12 as well as the expression of IL-12 receptors (IL-12R) and increasing the expression of the Th2-promoting cytokine IL-4 (Klein et al., 2000). It was also demonstrated, using cannabinoid receptor 1 (CB1) and 2 (CB2) antagonists, that
* Corresponding author. Tel.: +1-813-974-2502; fax: +1-813-9744151. E-mail address: [email protected] (T.W. Klein). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.019
both receptors were involved in some way in this THCinduced T helper cell biasing. Other laboratories have reported similar T helper biasing effects of cannabinoids as well as other neuroimmune axis modulating agents. For example, THC injection into mice reportedly suppressed, by a CB2 mechanism, the development of anti-tumor immunity by enhancing Th2 cytokines (Zhu et al., 2000) and THC treatment of cultured human PBMCs also shifted to Th2 immunity via a CB2 receptor mechanism (Yuan et al., 2002). In addition, cannabinoid treatment of mice primed with C. parvum injection and challenged with endotoxin induced a decrease in serum tumor necrosis factor (TNF) a and IL-12 and an increase in IL-10 (Smith et al., 2001). Drugs and endogenous mediators other than cannabinoids also appear to bias the activity of developing Th1 and Th2 cells. Some of these are drugs of abuse such as morphine (Roy et al., 2001), while others are endogenous mediators such as prostaglandins (Kuroda et al., 2000), glucocorticoids (Cohen et al., 2002), adrenergic mediators (Swanson et al., 2001) and chemokines (Braun and Kelsall, 2001). Because many of these agents affect cells through G protein-coupled receptors, it is possible that signaling mechanisms associated with
92
T.W. Klein et al. / Journal of Neuroimmunology 147 (2004) 91–94
Fig. 1. The biasing of T helper cell development. Microbes stimulate different types of dendritic cells (DC) to produce cytokines that cause naı¨ve precursors of CD4+, T helper cells to differentiate into either T helper 1 (Th1) or T helper 2 (Th2) cells. These cells then regulate either cellmediated or humoral immunity. IFNg, IL, IL-12R, IL-4 receptor (IL-4R), signal transducers and activators of transcriptions 4 and 6 (STAT 4 and 6), GATA nucleotide sequence (GATA3), TNFh and immunoglobulin (Ig).
hours following drug treatment and antigen challenge, serum IL-12 and IFNg mobilization was decreased relative to drug vehicle controls and IL-4 was increased by drug treatment in spleen preparations (Klein et al., 2000). The expression of IL-12 receptor h2 (IL-12Rh2) is also important in the development of Th1 cells (Fig. 1) and the gene for this receptor is thought to be regulated by IL-12 and IFNg (Chang et al., 1999; Szabo et al., 1997). Because THC treatment decreased IL-12 production, we tested to see if this had an inhibitory effect on IL-12Rh2 gene activity. Mice were injected with THC 18 h prior to infection with Legionella. At 2 h following infection, spleens were removed and processed for measuring IL12Rh2 mRNA by RT-PCR. Fig. 2 shows that, like IL-12, THC treatment suppressed the activation of IL-12Rh2 gene activity. In addition to these findings, we also tested the role of CB1 and CB1 in this effect. Mice were injected with either the CB1 antagonist, SR141716A (SR1), or the CB2 antagonist, SR144528 (SR2), followed 30 min later by THC injection. These mice were then infected with Legionella 18 h later and the spleens tested for IL-12 receptor message. Fig. 2 shows that the CB1 but not CB2 antagonist attenuated the THC suppressive effect suggesting that the down-regulation of IL-12Rh2 gene was mediated by CB1 receptors.
these kinds of receptors are linked to the regulation of T helper biasing events (Braun and Kelsall, 2001). T helper cell biasing is regulated by the production and action of several key cytokines and their cognate receptors (Fig. 1). The production by dendritic cells of either IL-4 or IL-12 in response to antigen stimulation is one of many key steps in the development of Th1 and Th2 cells. IL-12 leads to the phosphorylation of STAT 4 and the subsequent activation of IFNg and IL-12 receptor genes. The up-regulation of these genes drives the development of Th1 cells (Fig. 1). IL-4, on the other hand, leads to phosphorylation of STAT 6 and the subsequent activation of genes such as IL-4 receptor that promote Th2 development. In addition, in the relative absence of IL-12, IL-4 stimulation increases the Th2-dependent transcription factor GATA3 that further promotes Th2 differentiation (Moser and Murphy, 2000). In the present report, to better understand possible cannabinoid receptor-mediated mechanisms of THC-induced T helper biasing, we studied the effect of THC treatment on the regulation of expression of the key T helper biasing mediators, IL-12 receptor and GATA3.
2. Results and discussion Our previous work suggested that THC injection into mice prior to Legionella antigen challenge not only suppressed the development of Th1 activity but also increased the development of Th2 activity. For example, within
Fig. 2. THC injection suppresses IL-12Rh2 mRNA by CB1-mediated mechanism. BALB/c mice were injected i.v. with either saline or THC (8 mg/kg) 18 h prior to infection with Legionella pneumophila (7 106 bacteria). Two hours after infection, spleens were removed, total RNA extracted and analyzed by semi-quantitative RT-PCR for IL-12 receptor and h-actin mRNA. In some experiments, CB1 (SR1) or CB2 (SR2) antagonists were injected 30 min prior to THC treatment.
T.W. Klein et al. / Journal of Neuroimmunology 147 (2004) 91–94
Besides suppression of IL-12 and IFNg in response to Legionella challenge, we also previously reported that THC injection increased IL-4 production in the spleens of antigen challenged mice. GATA3 is an important signaling factor in the regulation of T helper cell development being found in Th2 cells but not Th1 (Das et al., 2001). Also, GATA3 and IL-12 appear to be mutually antagonistic in their action with the former increasing in activity in the absence of the latter (Ouyang et al., 1998). Because THC suppressed IL-12, we tested to see if the drug increased GATA3 injection following Legionella challenge. Spleens were harvested 2 h following Legionella infection and total RNA tested by RTPCR for GATA3 mRNA. Fig. 3 shows that GATA3 message is increased by THC injection suggesting that this Th2 biasing transcription factor is increased by drug treatment. To determine if cannabinoid receptors are involved in this effect, mice were pretreated with SR141716A (SR1) or SR144528 (SR2) as before prior to THC injection and Legionella challenge. As seen in Fig. 3, the CB2 antagonist but not the CB1 attenuated the THC effect suggesting that the marijuana cannabinoid increased GATA3 gene activity through CB2 but not CB1. Our previous work showed that THC injection prior to Legionella challenge suppressed the Th1 associated cytokines IL-12 and IFNg and that this effect was attenuated by antagonists to both CB1 and CB2. In the current report, other T helper biasing mechanisms were studied and the results suggested that CB1 and CB2 involvement was split between the suppression of IL-12Rh2 and the increase in GATA3 messages. Unlike with IL-12 and IFNg production, THC suppression of IL-12Rh2 appeared to be mediated by CB1 only while the drug-induced increase in GATA3 was
93
Fig. 4. Signaling factors generated by Gi protein coupling. Gi when activated splits into aGTP and h/g dimer subunits that can associate with and activate a variety of signaling factors. These downstream events activate genes involved in cytokine production and T cell activation. Protein kinase C (PKC), inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG), nuclear factor kappa B (NFkB), MAPK/ERK kinase (MEK), extracellular related kinase (ERK), phospholipase C (PLC) and activator protein 1 (AP1).
mediated by CB2 only. Cannabinoid receptors are G protein-coupled receptors and have both been shown to be coupled to Gai/o (Bayewitch et al., 1995) and to suppress adenylyl cyclase but activate MAP kinase (Bouaboula et al., 1999). It is now recognized that activation of G proteins through receptor ligation sets into motion a variety of signaling and gene activation pathways mediated by not only the Ga component but also the h/g component of the G protein (Braun and Kelsall, 2001). As seen in Fig. 4, various signaling factors can be either activated or suppressed by G protein activation and the dominating pathways can vary from cell to cell depending upon the endogenous tone of the receptors (Felder et al., 1998), the complement of neighboring receptors in the membrane (Bouaboula et al., 1999), and other unknown factors. Because we have seen a CB1mediated decrease in IL-12Rh2 gene activity but a CB2mediated increase in GATA3, it seems likely that the differences in regulation of these genes by the cannabinoid receptors stem from differences in the receptor and signaling factor milieu in Th1 and Th2 cells. CB1 receptors on Th1 cells and the activated Gi subunits could signal a decrease in the IL-12Rh2 gene product while CB2 receptor ligation on Th2 cells could signal an increase in the GATA3 gene. This cell-dependent dichotomy of signaling function is readily testable and will be the subject of future studies in this model.
Acknowledgements Fig. 3. THC injection increases GATA3 mRNA by CB2-mediated mechanism. See Fig. 2 for details.
This work was supported by NIDA grants DA03646, DA07245 and DA10683.
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References Bayewitch, M., Avidor-Reiss, T., Levy, R., Barg, J., Mechoulam, R., Vogel, Z., 1995. The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling. FEBS Lett. 375, 143 – 147. Bouaboula, M., Desnoyer, N., Carayon, P., Combes, T., Casellas, P., 1999. Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation. Mol. Pharmacol. 55, 473 – 480. Braun, M.C., Kelsall, B.L., 2001. Regulation of interleukin-12 production by G-protein-coupled receptors. Microbes Infect. 3, 99 – 107. Chang, J.T., Shevach, E.M., Segal, B.M., 1999. Regulation of interleukin (IL)-12 receptor h2 subunit expression by endogenous IL-12: a critical step in the differentiation of pathogenic autoreacitve T cells. J. Exp. Med. 189, 969 – 978. Cohen, M., Klein, E., Kuten, A., Fried, G., Zinder, O., Pollack, S., 2002. Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevated levels of stress hormones and decreased secretion of Th1 cytokines. Int. J. Cancer 100, 347 – 354. Das, J., Chen, C.-H., Yang, L., Cohn, L., Ray, P., Ray, A., 2001. A critical role for NF-kB in Gata3 expression and Th2 differentiation in allergic airway inflammation. Nat. Immunol. 2, 45 – 50. Felder, C.C., Joyce, K.E., Briley, E.M., Glass, M., Mackie, K.P., Fahey, K.J., Cullinan, G.J., Hunden, D.C., Johnson, D.W., Chaney, M.O., Koppel, G.A., Brownstein, M., 1998. LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation. J. Pharmacol. Exp. Ther. 284, 291 – 297. Klein, T., Newton, C., Friedman, H., 1998. Cannabinoid receptors and immunity. Immunol. Today 19, 373 – 381. Klein, T.W., Newton, C.A., Nakachi, N., Friedman, H., 2000. D9-Tetrahydrocannabinol treatment suppresses immunity and early IFN-g, IL-12, and IL-12 receptor h2 responses to Legionella pneumophila infection. J. Immunol. 164, 6461 – 6466.
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