- Email: [email protected]
Th2 cytokine balance in activated human T cells
Journal of Neuroimmunology 133 (2002) 124 – 131 www.elsevier.com/locate/jneuroim
D9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells Michael Yuan a, Sylvia M. Kiertscher a,b, Qingwen Cheng a, Richard Zoumalan a, Donald P. Tashkin a, Michael D. Roth a,b,* a
Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA b Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA Received 13 March 2002; received in revised form 23 September 2002; accepted 7 October 2002
Abstract Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and D9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625 – 5 Ag/ml). THC suppressed T cell proliferation, inhibited the production of interferon-g and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-g, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Human; Antigen presentation; Cytokines; T helper 1; T helper 2; Flow cytometry
1. Introduction D9-Tetrahydrocannabinol (THC) mediates its effects on the central nervous system by interacting with type 1 cannabinoid receptors (CB1) (Matsuda et al., 1990). In 1993, both CB1 and a second cannabinoid receptor, CB2, were identified on immune cells (Bouaboula et al., 1993; Munro et al., 1993). Unlike CB1, CB2 is not found in the brain and is expressed primarily on B cells, neutrophils, monocytes, NK cells and T cells (Munro et al., 1993; Galiegue et al., 1995). Both receptors belong to the G protein-coupled receptor superfamily, modulating intracellular cAMP and calcium, and are activated by THC as well as by endogenous compounds such as anandamide and 2arachidonyl glycerol (Howlett, 1995; Lee et al., 1995). While there have been several reports on the effects of cannabinoid * Corresponding author. Department of Medicine, CHS 37-131, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA. Tel.: +1-310-206-7389; fax: +1-310-267-2020. E-mail address: [email protected] (M.D. Roth).
receptor ligands on the immune system in general, there is relatively little information about the effects of THC on human T cells or on antigen-specific immune responses (reviewed in Klein et al., 1998; Berdyshev, 2000). In recent murine studies, cannabinoids were reported to down-regulate the production of T helper 1 (Th1)-associated cytokines, including IL-2, IL-12 and interferon-g (IFN-g), and increase the production of T helper 2 (Th2)-associated cytokines, including IL-4 and IL-10 (Newton et al., 1994; Klein et al., 2000; Zhu et al., 2000; Smith et al., 2000; Ouyang et al., 1998). Consistent with the induction of a Th2-dominant immune response (Mosmann and Coffman, 1989; O’Garra, 1998), THC was found to suppress both anti-tumor immunity and the immune response to infection in these model systems (Newton et al., 1994; Klein et al., 2000; Zhu et al., 2000). Similar functional impairments in cell-mediated immunity have been observed in marijuana smokers. Tindall et al. (1988) evaluated 386 HIV-positive individuals and observed a more rapid progression from HIV infection to AIDS in marijuana users. Marijuana use is also associated with a greater risk for bacterial pneumonia, opportunistic infections and Kaposi’s sarcoma in HIV-positive individuals (Newell et
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 ( 0 2 ) 0 0 3 7 0 - 3
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
al., 1985; Caiaffa et al., 1994). We previously recovered alveolar macrophages from the lungs of otherwise healthy marijuana smokers and documented deficiencies in phagocytosis, bacterial killing, tumor cytotoxicity and cytokine production when compared to cells recovered from the lungs of nonsmokers or tobacco smokers (Baldwin et al., 1997). Direct studies on T cells recovered from marijuana smokers have produced mixed results (Nahas et al., 1974; Lau et al., 1976). However, in the largest such study, T cell proliferation in the MLR and mitogen-stimulation assays was reduced by 40 –45% when examined in 51 marijuana smokers as compared to 81 healthy controls (Nahas et al., 1974). In order to link these clinical observations to the immunoregulatory properties of THC, we collected T cells from healthy volunteers and activated them in vitro in the presence or absence of THC to determine the impact on antigen-specific proliferation, cytokine release, and the generation of activated Th1 or Th2 cells. Whether added to mixed leukocyte reaction (MLR) assays or to T cells stimulated by immobilized anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), THC down-regulated the generation of T cells capable of expressing and releasing Th1 cytokines and altered Th1/Th2 cytokine balance.
2. Materials and methods 2.1. Cells and reagents Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from the blood of healthy human donors and cultured in complete medium composed of RPMI 1640 with L-glutamine (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA), penicillin – streptomycin – fungizone (Life Technologies, Grand Island, NY), and 10 mM HEPES (Sigma, St. Louis, MO). Dendritic cells (DC) were prepared by culturing the adherent fraction of PBMC with 800 U/ml GM-CSF (specific activity 1.13 107 U/mg; Schering-Plough Research Institute, Kenilworth, NJ) and 500 U/ml IL-4 (specific activity 8 106 U/mg; R&D Systems, Minneapolis, MN). After 7 days, DC were recovered by vigorous rinsing and purified by negative depletion using a mAb cocktail (anti-CD3, antiCD19 and anti-CD56; BD PharMingen, San Diego, CA) and anti-mouse Ig-conjugated immunomagnetic beads (Dynal, Lake Success, NY) as previously described (Kiertscher and Roth, 1996). T cells were prepared from nonadherent PBMCs using a cocktail of anti-CD14, anti-CD16 and anti-CD19 mAbs, followed by immunomagnetic depletion (BD PharMingen) (Roth, 1994). The purity of DC and T cell preparations were routinely z 95% as determined by flow cytometry. In some experiments, T cells were activated with either anti-CD3 mAb alone, or in combination with antiCD28 mAb (BD PharMingen). IL-12 was from Peprotech (Rocky Hills, NJ). A 50 mg/ml stock of THC in ethanol was
125
provided by the National Institute on Drug Abuse (NIDA, Bethesda, MD) and diluted serially in DMSO and complete medium prior to use (final ethanol concentration V 0.01% and DMSO concentration V 0.125%). The highly CB1selective antagonist AM251 (CB1ra; Alexis, San Diego, CA) (Paria et al., 1998) and the CB2-selective antagonist SR144528 (CB2ra; provided by Murielle Rinaldi-Carmona, Sanofi Recherche, Montpellier, France) (Rinaldi-Carmona et al., 1998) were used to block the CB1- or CB2 receptormediated effects of THC, respectively, and were diluted in a manner identical to THC. 2.2. MLR assays THC was evaluated for its effects on antigen-specific T cell proliferation in the MLR assay. Purified DCs (5 103) were co-cultured in complete medium with 1 105 purified allogeneic T cells (DC/TC ratio, 1:20) in 96-well roundbottomed plates at 37 jC in a humidified CO2 incubator. On day 5, wells were pulsed with 1.25 ACi/well titrated thymidine (Amersham, Arlington Heights, IL) and harvested 18 h later. Proliferation was determined as cpm using a liquid scintillation counter. In some experiments, anti-CD3 mAb (1 Ag/ml) was added to replicate culture wells on day 2 and culture supernatants harvested 24 h later for detection of IFN-g (BD PharMingen) and IL-4 (Biosource, Camarillo, CA) by ELISA. THC was added at the beginning of culture to experimental wells at concentrations ranging from 0.625 to 5.0 Ag/ml. Ethanol (0.01% final concentration) and DMSO (0.125% final concentration) were added as a diluent control to wells that did not contain THC. 2.3. Regulation of Th1/Th2 differentiation as measured by intracellular cytokine detection The capacity for THC (5 Ag/ml), IL-4 (500 U/ml) or IL-12 (1 ng/ml) to alter the maturation of T cells into either Th1 cells, capable of secreting IFN-g when subsequently activated, or Th2 cells, capable of producing IL-4 when activated, was evaluated using a short-term in vitro model for Th1/Th2 differentiation (Schylze-Koops et al., 1998; Elson et al., 1995). CD3+ T cells were cultured at 1 106 cells/ml in wells containing immobilized anti-CD3 (5 Ag/ml) and anti-CD28 (0.5 Ag/ml) mAbs. In contrast to the MLR assay, this approach pan-activated all T cells simultaneously. Potential Th1/Th2 modulators (THC, IL-4 or IL-12) were added at the initiation of the culture period and 50% of the medium was replenished with fresh medium, containing the corresponding modulating agents, on day 2. T cells were harvested on day 4, washed to remove THC or cytokines, and restimulated to determine their ability to secrete IFN-g and IL-4. Cytokine production following this secondary challenge was determined by intracellular cytokine analysis according to the Fast-Immune protocol (Becton Dickinson, San Jose, CA). This approach allowed each T cell to be evaluated for the presence and intensity of its cytokine
126
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
response. In brief, cells were restimulated with 0.75 Ag/ml calcium ionophore A23187 and 25 ng/ml PMA (Sigma). One hour later, 10 Ag/ml Brefeldin A (Sigma) was added to prevent cytokine secretion and cells were cultured an additional 4 h. Cells were then incubated with 0.02% EDTA in PBS (Irvine Scientific) to break-up cell clumps, washed, fixed with 4% paraformaldehyde (Sigma) and cryopreserved at 80 jC. On the day of analysis, cells were thawed and treated with FACS Lysing Solution (Becton Dickinson) and FACS Permeabilizing Solution (Becton Dickinson) as per manufacturer’s instructions. Cells were stained with antiIFN-g FITC, anti-IL-4 – PE and anti-CD8 – PerCP mAb (Becton Dickinson), and analyzed by three-color FACS analysis using a FACScan II flow cytometer and CellQuest Software (Becton Dickinson). Replicate samples were stained with fluorescent-labeled isotype-specific control mAbs (to irrelevant antigens) as a control for nonspecific intracellular staining. Events (30,000 – 50,000) were acquired for each sample and results expressed as the percentage of CD3+/CD8+ or CD3+/CD8 (i.e. CD4+) T cells staining for a given cytokine. PMA down-regulates expression of CD4, preventing direct detection of the CD3+/CD4+ population using fluorochrome-labeled anti-CD4 (Baran et al., 2001). Mean linear fluorescence intensity was used to describe the average cytokine expression of the positively stained populations. The role of the CB1 and CB2 receptors in the response to THC were evaluated by pretreating cells for 10 min with either AM251 (CB1ra, 0.05 –1.0 AM) or SR144528 (CB2ra, 1 AM) prior to addition of THC at the beginning of culture (Paria et al., 1998; Rinaldi-Carmona et al., 1998). The impact of THC on T cell proliferation was also evaluated using the same activation model. T cells were stimulated with immobilized anti-CD3/CD28 in the presence or absence of THC as described above, and pulsed with 1.25 ACi/well titrated thymidine (Amersham) on day 3. Cells were harvested 18 h later and proliferation determined by the level of radioactive incorporation.
5% acryl-bis urea gels. A mRNA standard prepared from mitogen-activated peripheral blood mononuclear cells (BD PharMingen) was also run with each gel as a positive control. Gels were dried and exposed to a phosphor image screen and 2 h later read by a Molecular Dynamics Storm 860 PhosphoImager (Molecular Dynamics, Sunnyvale, CA). Cytokines bands were identified according to the position of the known cytokine mRNA standards and quantitated by ImageQuant for Macintosh v1.2 software (Molecular Dynamics). As for intracellular cytokine analysis, the role of the CB2 receptor was evaluated by pretreating cells for 10 min with SR144528 prior to the initial treatment with THC. 2.5. Statistical analysis Data from individual experiments are represented as the average value F S.D. for the indicated number of replicate wells. Pooled data from multiple experiments are represented as mean raw values, or as a percentage of control, F S.E. Differences between groups were determined by a two-tailed Student’s t-test for paired data with significant differences documented at p values of V 0.05 as indicated.
3. Results 3.1. THC inhibited T cell proliferation and altered the release of TH1/TH2 cytokines in the MLR T cells from normal donors were stimulated with allogenic DC in the presence or absence of THC and evaluated 6 days later for proliferation by [3H]-thymidine uptake. Exposure to THC produced a concentration-dependent decrease in the response to alloantigen (Fig. 1). The level of suppression varied somewhat among individuals, with 5.0 Ag/ml THC producing an average inhibition of 53 F 10% (range 28– 79%) compared to control T cells exposed to diluent alone
2.4. Analysis of cytokine mRNA The effects of THC on cytokine mRNA were examined using a ribonuclease protection assay capable of simultaneously quantitating mRNA encoding for IL-2, IL-4, IL-5, IL-10, IL-13, IL-14, IL-15, GM-CSF, IFN-g and housekeeping genes (Riboquantk hck1 probe set with GM-CSF substituted for IL-9; BD PharMingen). Purified T cells were activated for 4 days with immobilized anti-CD3 and antiCD28 mAbs, in the presence or absence of 5 Ag/ml THC, washed, and then stimulated with calcium ionophore and PMA as described above for intracellular cytokine detection. Four hours after final stimulation, total RNA was extracted by the Trizol method (Life Technologies) and 10 Ag from each condition incubated with [32P]-labeled cytokine mRNA probe sets and subjected to RNAse digestion according to the manufacturer’s instructions. Precipitates from the final reaction tubes were dissolved in 10 Al loading buffer and run on
Fig. 1. THC inhibited T cell proliferation in a concentration dependent manner. Purified human CD3+ T cells (1 105) were co-cultured at a 20:1 ratio with allogeneic DC for 5 days in the presence of THC at concentrations ranging between 0 and 5 Ag/ml. Cells were pulsed with [3H]-thymidine and harvested 18 h later to determine proliferation (cpm). Data presented as the average F S.D. for four replicate wells. Representative experiment out of six performed. *p V 0.01 compared to control (no THC).
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
127
Fig. 2. THC decreased secretion of IFN-g while increasing IL-4 release during the MLR assay. CD3+ T cells were co-cultured with allogeneic DC in the presence or absence of 5 Ag/ml THC as described in Fig. 1. Proliferation was determined after 5 days by [3H]-thymidine incorporation. Replicate wells from the same MLC were treated with 1 Ag/ml anti-CD3 mAb on day 2 to boost cytokine production and supernatants harvested 24 h later for determination of IFN-g and IL-4 concentration by ELISA. The percent change in proliferation, IFN-g release, IL-4 production, and Th1/Th2 cytokine ratio (ratio of IFNg to IL-4 in pg/ ml) resulting from treatment with THC was calculated for each set of experiments (n = 6 experiments). Graphs represent the average change F S.E. for all experiments comparing THC-treated wells to control wells. Average proliferation in control wells was 21,253 cpm. The average production of IFN-g in control wells was 2435 pg/ml and the average production of IL-4 was 52.6 pg/ml. *p V 0.05 compared to control.
( p V 0.01, n = 6). Supernatants were harvested from replicate wells during the MLR cultures and examined for the presence of IFN-g and IL-4 by ELISA. Whether simply collected from MLR cultures, or following an additional 24-h activation with soluble anti-CD3 mAb, supernatants from wells exposed to 5 Ag/ml of THC demonstrated a consistent change in the cytokine pattern (Fig. 2). On culture day 3, IFN-g concentrations were reduced on average by 51 F 18% ( p V 0.05), while IL-4 levels were increased on average to 105 F 11% of diluent control levels. This down-regulation of IFN-g and modest up-regulation of IL-4 resulted in a significant shift in the ratio of Th1 (IFN-g) to Th2 (IL-4) cytokine balance ( p V 0.05).
helper regulatory effects of THC were then compared to that of a conventional Th1 inducer, IL-12, and a conventional Th2 inducer, IL-4, using the same intracellular cytokine assay. As demonstrated in Fig. 4, the addition of IL-12 during T cell activation resulted in a marked increase in the number of cells capable of expressing IFN-g, as well as the MFI of expression. There was a relatively modest decrease in
3.2. THC down-regulated the development of Th1 cytokineproducing cells The effects of THC on proliferation and cytokine production during the MLR suggested an effect on the maturation of Th subsets following antigen stimulation. To evaluate this possibility further, T cells were pan-activated with immobilized anti-CD3 and anti-CD28 mAbs in vitro in the presence or absence of 5 Ag/ml THC. Four days later, activated cells were either pulsed with [3H]-thymidine to measure the impact on proliferation, or washed to remove THC and restimulated with PMA and calcium ionaphore to determine any changes in their capacity to produce Th1 or Th2 cytokines. On average, exposure to THC produced a 52 F 18% decrease in proliferation (n = 3 experiments) and a 58.7 F 17% reduction in the percentage of T cells capable of expressing intracellular IFN-g as determined by intracellular cytokine staining (n = 10 experiments, Fig. 3). THC reduced both the percentage of T cells expressing IFN-g and the average level of intracellular IFN-g detected per cell (mean linear fluorescence intensity, MFI), with obvious effects on both CD4+ and CD8+ subsets. The effect on the capacity to produce IL-4 was more variable, with some subjects demonstrating a reduction in number but an increase in average fluorescence intensity (Fig. 3), and most subjects demonstrating a slight increase in the percentage and/or MFI of cells stained for IL-4 (as shown in Figs. 4 and 5). The T
Fig. 3. THC decreased the percentage of IFN-g producing T cells and the relative concentration of IFN-g as determined by intracellular cytokine detection. T cells were stimulated with immobilized anti-CD3 (5 Ag/ml) and anti-CD28 (0.5 Ag/ml) mAb in the presence or absence of 5 Ag/ml THC. On day 4, cells were washed and restimulated with 0.75 Ag/ml Ca ionophore and 25 ng/ml PMA, and 10 Ag/ml brefeldin A was added after the first 1 h to prevent cytokine secretion. Four hours later, cells were fixed, permeabilized and stained with fluorescent-labeled anti-CD8, anti-IFN-g and IL-4 mAb. Events (30,000 – 50,000) were acquired by FACS analysis and gated to display cytokine production by total T cells (A), as well as by the CD4+ (B) and CD8+ (C) subsets. Numbers represent the % of cells staining for each cytokine and the mean fluorescence intensity (in parentheses). Representative experiment out of 10 performed.
128
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
Fig. 4. THC and IL-4 produce similar changes in the production of IFN-g and IL-4 by activated T cells. T cells were stimulated with anti-CD3 and antiCD28 and cultured in the presence of control medium, 1 ng/ml IL-12, 500 U/ ml IL-4, or 5 Ag/ml THC. Expression of intracellular IFN-g and IL-4 were detected after 4 days by FACS analysis as described in Fig. 3. Numbers represent the % of cells staining for each cytokine and the mean fluorescence intensity (in parentheses). Representative experiment out of four performed.
IL-4-producing cells in response to IL-12. In contrast, IL-4 significantly down-regulated the capacity of restimulated T cells to express IFN-g while having little effect, or a modest increase, in the number of cells capable of producing IL-4. In every experiment, the effects of THC closely modeled the response observed with IL-4, suggesting activity as a modulator of Th1/Th2 differentiation. 3.3. A CB2ra, but not a CB1ra, protected T cells from the effects of THC Specific CB1ra (AM251) and CB2ra (SR144528) were used to determine the role of cannabinoid receptors in mediating the response to THC (Fig. 5). Pretreating T cells with AM251 in the range of 0.05 –1.0 AM did not prevent THC from altering cytokine production as measured by the intracellular cytokine assay. In contrast, the addition of 1 AM SR144528 prior to treatment with THC led to increases in the percentage of IFN-g+ cells and a decrease in the percentage of IL-4+ cells as compared to cells treated with THC alone. Pretreatment with SR144528 protected 94.4 F 2% of the THC-sensitive cells from losing IFN-g
Fig. 6. THC up-regulated Th2 cytokine mRNA and down-regulated Th1 cytokine mRNA in a CB2-dependent manner. T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence or absence of 5 Ag/ml THC, and then restimulated with calcium ionaphore and PMA as described in Fig. 4. (A) Total RNA was harvested after 4 h, incubated with [32P]labeled cyokine RNA probes, treated with ribonuclease and the products resolved on a 5% acryl-bis urea gel (Riboquantk). The radiolabeled probe set and a known cytokine standard mRNA prep were run with each gel as a control and the location of bands for specific cytokine genes are shown. (B) Cytokines bands were quantitated using a phosphoimager and quantitative imaging software. Relative expressions of cytokine genes from THCtreated cells are expressed as a % of control, normalized for expression of GAPDH. Representative experiment out of two performed.
expression, but only partially blocked the average decrease in IFN-g signal intensity (seven experiments). Treatment of T cells with SR144528 alone, in the absence of THC, had little effect on IFN-g or IL-4 expression (data not shown). 3.4. THC regulated the expression of Th1 and Th2 cytokine mRNA We hypothesized that if THC was altering Th differentiation, it would also regulate the expression pattern for
Fig. 5. The CB2 antagonist SR144528, but not the CB1 antagonist AM251, prevented the effects of THC on intracellular expression of IFN-g and IL-4. T cells were stimulated with anti-CD3 and anti-CD28, and intracellular IFN-g and IL-4 detected as described in Fig. 4. Control T cells were compared to T cells treated with 5 Ag/ml THC, or T cells pretreated for 10 min with either AM251 (CB1ra, 50 nM) or SR144528 (CB2ra, 1 AM) prior to the addition of THC. Numbers represent the % of cells staining for each cytokine. Representative experiment out of seven performed.
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
Fig. 7. CB2ra protected against the effects of THC on cytokine mRNA. Induction of cytokine mRNA following T cell activation was determined using a ribonuclease protection assay as described in Fig. 6. T cells were cultured during the initial activation period with diluent alone, with 5 Ag/ml THC, or with the combination of 1 AM SR144528 and 5 Ag/ml THC. Cytokines bands were quantitated using a phosphoimager and quantitative imaging software. Relative expressions of cytokine genes under different conditions are expressed as a % of control, normalized for expression of GAPDH. Representative experiment out of two performed.
cytokine-specific mRNA. T cells were activated in the same manner as for the intracellular cytokine assay and mRNA extracted at either 2, 4 or 8 h after the secondary stimulation with calcium ionophore and PMA. Cytokine mRNA for both Th1 (IL-2, IFN-g) and Th2 (IL-4, IL-5) cytokines were then directly compared using a ribonuclease protection assay. Consistent with the pattern of changes obtained by ELISA and by FACS analysis, mRNA encoding for IFN-g and IL-2 was reduced by 21 –48% in cells treated with 5 Ag/ ml THC, and mRNA encoding for IL-4 and IL-5 was increased by 1.5- to 11.2-fold (Fig. 6). Pretreatment with 1 AM SR144528 prevented the majority of these THCmediated effects (Fig. 7).
4. Discussion This report demonstrates that THC can regulate human T cell activation. Not only did THC suppress the proliferative response to alloantigen, it shifted the balance of activationinduced cytokines and the differentiation of activated T cells in favor of a Th2 cytokine response. Marijuana, and its primary psychoactive constituent, THC, were first proposed as immune modulators in the 1970s when abnormal T cell responses were observed in THC-treated animals (Elson et al., 1995; Nahas et al., 1973) and in PBMC collected from chronic marijuana smokers (Nahas et al., 1974). However, these findings were not uniformly observed (Lau et al., 1976; Rachelefsky et al., 1976), and it was not until the discovery of CB1 and CB2 receptors that interactions between cannabinoids and the immune system began to be investigated in detail (Matsuda et al., 1990; Bouaboula et al., 1993; Munro
129
et al., 1993; Galiegue et al., 1995; Howlett, 1995). The CB1 receptor is expressed at high levels in the central nervous system and at much lower levels in peripheral tissues. In contrast, the CB2 receptor is expressed only in peripheral tissues and primarily in cells of hematopoietic origin. Of the two cannabinoid receptor subtypes, mRNA encoding for the CB2 receptor is present in mouse spleen at levels 10- to 100fold higher than mRNA encoding for CB1 (Galiegue et al., 1995; Schatz et al., 1997). CB2 receptor is also preferentially expressed in human leukocytes, with B cells expressing several-fold higher levels of CB2 receptor protein than T cells (Galiegue et al., 1995; Marchand et al., 1999; Nong et al., 2002). In contrast to our results with T cells, activation of the CB2 receptor enhances the proliferation of CD40-activated human B cells (Galiegue et al., 1995; Derocq et al., 1995). The capacity for THC to suppress antigen-induced T cell proliferation, modulate cytokine balance in favor of a Th2 profile and stimulate B cell proliferation suggests a coordinated role for cannabinoids in promoting humoral over cell-mediated immunity. The production of cytokines by stimulated T cells plays a critical role in amplifying and targeting immunity. When activated T cells mature into Th1 cells, they secrete IL-2 and IFN-g and potentiate cell-mediated immunity by enhancing T cell proliferation and effector functions, up-regulating MHC expression on targets, promoting IgG2a isotype switching, and activating effector cells such as NK cells and macrophages (Mosmann and Coffman, 1989; O’Garra, 1998). In contrast, when activated T cells acquire a Th2 phenotype, they secrete IL-4, IL-5 and/or IL-10, and potentiate allergic/atopic antibody responses while suppressing T cell proliferation and cell-mediated immunity. Other investigators have utilized animal models to evaluate the effects of THC on cytokine production and the generation of Th1/ Th2 phenotypes. Newton et al. (1994) observed that mice treated with THC prior to infection with Legionella pneumophila exhibited reduced antigen-specific T cell proliferation, along with a shift in antibody response from a predominantly IgG2a isotype to an IgG1 isotype. Splenocytes cultured with THC in vitro secreted lower levels of IFN-g and higher levels of IL-4 than control cells. Similarly, Klein et al. (2000) found that THC skewed both the immediate and delayed cytokine response toward a Th2 profile, and that THC-treated mice failed to exhibit protective immunity when rechallenged with L. pneumophila. Splenocytes from tumor-bearing mice treated with THC demonstrated decreased production of IFN-g, but increased production of both IL-10 and TGF-h (Zhu et al., 2000). In addition, THC promoted the rapid growth of subcutaneously implanted tumors. This growth could be prevented by treatment with neutralizing antibody against IL-10, or a selective CB2ra. Taken together, these in vivo models suggest a direct role for altered Th1/Th2 cytokine balance in the immunosuppressive consequences of THC. Our in vitro results with human T cells confirm and complement these previous reports. We found that THC
130
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
produced a concentration-dependent inhibition of T cell proliferation. In addition, THC decreased the secretion of IFN-g during the MLR response, while having a modest effect on increasing the release of IL-4, resulting in a significant shift in Th1/Th2 balance. To examine the impact of THC on Th maturation and cytokine production at the single-cell level, we activated T cells with anti-CD3/antiCD28 mAbs in the presence of THC. As in the MLR assay, THC inhibited activation-induced T cell proliferation. Activation in the presence of THC also resulted in fewer T cells capable of producing IFN-g and more cells producing IL-4, as well as changes in the amount of cytokine secreted per cell. By modeling the effects of THC to those mediated by standard Th1/Th2 inducers, we confirmed that the response to THC was very similar to that produced by the prototypic Th2 stimulator, IL-4. We observed a similar, but more prominent pattern in the levels of cytokine mRNA, with down-regulation of mRNA encoding for IL-2 and IFN-g and up-regulation of mRNA encoding for IL-4 and IL-5. These effects on cytokine mRNA, as well as those on cytokine protein, were significantly abrogated by pretreatment with a CB2ra. However, the CB2ra did not completely block the effects of THC, making it possible that stimulation of CB1, or activation of receptor-independent pathways, also contributed to the impact on cytokines. These results suggest that cannabinoids have a capacity to act as Th2 inducers (Schylze-Koops et al., 1998; O’Garra, 1998). This conclusion is consistent with the clinical finding of increased IgE levels in marijuana smokers (Rachelefsky et al., 1976), the down-regulation of effector functions in alveolar macrophages recovered from the lungs of chronic marijuana users (Baldwin et al., 1997), and the variety of clinical reports suggesting that marijuana is a risk factor for opportunistic infections (Tindall et al., 1988; Newell et al., 1985). There are several mechanisms by which THC might regulate T cell responses. At the receptor level, the effects of THC on cytokine production were blocked primarily by a CB2ra. A primary role for CB2 in regulating Th1/Th2 balance was also reported by Zhu et al. (2000) in their mouse tumor model. However, others have demonstrated that both CB1ra and CB2ra can block the in vivo consequences of THC (Klein et al., 2000; Massi et al., 2000). In vivo, THC may act on CB1 receptors in the central nervous system to stimulate the release of corticotropin-releasing factor and adrenocorticotropic hormone (Manzanares et al., 1999). Activation of the hypothalamic –pituitary –adrenal axis in this manner could indirectly suppress lymphocyte activation and promote Th2 cytokine responses (Elenkov et al., 1999). It is also possible that THC acts through CB1 receptors on other leukocytes, such as NK cells or B cells, to modulate the production of T helper regulatory cytokines. These potential effects were excluded from our studies by employing highly purified T cells in all of our assays. Both the CB1 and CB2 receptors are pertussis toxin sensitive GTP-binding proteins that inhibit adenylate cyclase and down-regulate intracellular cAMP (Howlett, 1995; Kaminski et al., 1994). Down-regu-
lation of cAMP by THC inhibits the activation of protein kinase A with downstream effects on cAMP response elements, activation of AP-1 promoter sites and NF-nB/RelB pathways (Kaminski, 1998). Other signaling pathways may be affected including mitogen-activated protein kinases (Faubert and Kaminski, 2000). In addition, Klein et al. (2000) found that exposure to THC down-regulated the expression of IL-12 receptors. As such, it is possible that the effects of THC are mediated in part by effects on cytokine receptor expression. While many of these pathways are known to be involved in T cell activation and cytokine production, the mediator changes responsible for the effects of THC on human T cells remain to be determined. In summary, our results demonstrate that THC can interact with CB2 receptors on human T cells to suppress activation and skew cytokine production in favor of a Th2 response. These coordinated effects suggest a potential role for endogenous endocannabinoids in regulating the balance between humoral and cellular immunity.
Acknowledgements This work was funded by NIDA/National Institutes of Health grant #DA03018-16. S.M.K. is supported by a Young Investigator Award from the U.S. Army Medical Research and Material Command under DAMD17-98-18181.
References Baldwin, G.C., Tashkin, D.P., Buckley, D.M., Park, A.N., Dubinett, S.M., Roth, M.D., 1997. Marijuana and cocaine impair alveolar macrophage function and cytokine production. Am. J. Respir. Crit. Care Med. 156, 1606 – 1613. Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate – ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303 – 313. Berdyshev, E.V., 2000. Cannabinoid receptors and the regulation of immune response. Chem. Phys. Lipids 108, 169 – 190. Bouaboula, M., Rinaldi, M., Carayon, P., Carillon, C., Delpech, B., Shire, D., Le Fur, G., Casellas, P., 1993. Cannabinoid-receptor expression in human leukocytes. Eur. J. Biochem. 214, 173 – 180. Caiaffa, W.T., Vlahov, D., Graham, N.M., Astemborski, J., Solomon, L., Nelson, K.E., Munoz, A., 1994. Drug smoking, Pneumocystis carinii pneumonia, and immunosuppression increase risk of bacterial pneumonia in human immunodeficiency virus-seropositive injection drug users. Am. J. Respir. Crit. Care Med. 150, 1493 – 1498. Derocq, J.M., Segui, M., Marchand, J., Le Fur, G., Casellas, P., 1995. Cannabinoids enhance human B-cell growth at low nanomolar concentrations. FEBS Lett. 369, 177 – 182. Elenkov, I.J., Webster, E.L., Torpy, D.J., Chrousos, G.P., 1999. Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: acute and chronic effects. Ann. N.Y. Acad. Sci. 876, 1 – 11. Elson, L.H., Nutman, T.B., Metcalf, D.D., Prussin, C., 1995. Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131 and T helper 0 cells within the human CD4+CD27 lymphocyte subpopulation. J. Immunol. 154, 4294 – 4301. Faubert, B.L., Kaminski, N.E., 2000. AP-1 activity is negatively regulated by cannabinol through inhibition of its protein components, c-fos and c-jun. J. Leukoc. Biol. 67, 259 – 266. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G., Casellas, P., 1995. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur. J. Biochem. 232, 54 – 61. Howlett, A.C., 1995. Pharmacology of cannabinoid receptors. Annu. Rev. Pharmacol. Toxicol. 35, 607 – 634. Kaminski, N.E., 1998. Regulation of the cAMP cascade, gene expression and immune function by cannabinoid receptors. J. Neuroimmunol. 83, 124 – 132. Kaminski, N.E., Koh, W.S., Yang, K.H., Lee, M., Kessler, F.K., 1994. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism. Biochem. Pharmacol. 48, 1899 – 1908. Kiertscher, S.M., Roth, M.D., 1996. Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4. J. Leukoc. Biol. 59, 208 – 218. Klein, T.W., 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. Delta9-tetrahydrocannabinol treatment suppresses immunity and early IFN-gamma, IL-12, and IL-12 receptor beta2 responses to Legionella pneumophila infection. J. Immunol. 164, 6461 – 6466. Lau, R.J., Tubergen, D.G., Barr Jr., M., Domino, E.F., Benowitz, N., Jones, R.T., 1976. Phytohemagglutinin-induced lymphocyte transformation in humans receiving delta9-tetrahydrocannabinol. Science 192, 805 – 807. Lee, M., Yang, K.H., Kaminski, N.E., 1995. Effects of putative cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, on immune function in B6C3F1 mouse splenocytes. J. Pharmacol. Exp. Ther. 275, 529 – 536. Manzanares, J., Corchero, J., Fuentes, J.A., 1999. Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats. Brain Res. 839, 173 – 179. Marchand, J., Bord, A., Penarier, G., Laure, F., Carayon, P., Casellas, P., 1999. Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting: application to peripheral cannabinoid receptors. Cytometry 35, 227 – 234. Massi, P., Fuzio, D., Vigano, D., Sacerdote, P., Parolaro, D., 2000. Relative involvement of cannabinoid CB(1) and CB(2) receptors in the delta(9)tetrahydrocannabinol-induced inhibition of natural killer activity. Eur. J. Pharmacol. 387, 343 – 347. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., Bonner, T.I., 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561 – 564. Mosmann, T.R., Coffman, R.L., 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7, 145 – 173. Munro, S., Thomas, K.L., Abu-Shaar, M., 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365, 61 – 65. Nahas, G.G., Zagury, D., Schwartz, I.W., Nagel, M.D., 1973. Evidence for
131
the possible immunogenicity of delta9-tetrahydrocannabinol (THC) in rodents. Nature 243, 407 – 408. Nahas, G.G., Suciu-Foca, N., Armand, J.-P., Morishima, A., 1974. Inhibition of cellular mediated immunity in marihuana smokers. Science 183, 419 – 420. Newell, G.R., Mansell, P.W., Wilson, M.B., Lynch, H.K., Spitz, M.R., Hersh, E.M., 1985. Risk factor analysis among men referred for possible acquired immune deficiency syndrome. Prev. Med. 14, 81 – 91. Newton, C.A., Klein, T.W., Friedman, H., 1994. Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta9-tetrahydrocannabinol injection. Infect. Immun. 62, 4015 – 4020. Nong, L., Newton, C., Cheng, Q., Friedman, H., Roth, M.D., Klein, T.W., 2002. Altered cannabinoid receptor mRNA expression in peripheral blood mononuclear cells from marijuana smokers. J. Neuroimmunol. 127, 169 – 176. O’Garra, A., 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 81, 275 – 283. Ouyang, Y., Hwang, S.G., Han, S.H., Kaminski, N.E., 1998. Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonylglycerol is mediated through down-regulation of the nuclear factor of activated T cells. Mol. Pharmacol. 53, 676 – 683. Paria, B.C., Ma, W., Andrenyak, D.M., Schmid, P.C., Schmid, H.H., Moody, D.E., Deng, H., Makriyannis, A., Dey, S.K., 1998. Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors. Biol. Reprod. 58, 1490 – 1495. Rachelefsky, G.S., Opelz, G., Mickey, M.R., Lessin, P., Kiuchi, M., Silverstein, M.J., Stiehm, E.R., 1976. Intact humoral and cell-mediated immunity in chronic marijuana smoking. J. Allergy Clin. Immunol. 58, 483 – 490. Rinaldi-Carmona, M., Barth, F., Millan, J., Derocq, J.M., Casellas, P., Congy, C., Oustric, D., Sarran, M., Bouaboula, M., Calandra, B., Portier, M., Shire, D., Brelie`re, J.C., Le Fur, G., 1998. SR144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. J. Pharmacol. Exp. Ther. 284, 644 – 650. Roth, M.D., 1994. Interleukin-2 induces the expression of CD45RO and the memory phenotype by CD45RA+ peripheral blood lymphocytes. J. Exp. Med. 179, 857 – 864. Schatz, A.R., Lee, M., Condie, R.B., Pulaski, J.T., Kaminski, N.E., 1997. Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol. Appl. Pharmacol. 142, 278 – 287. Schylze-Koops, H., Lipsky, P.E., Davis, L.S., 1998. Human memory T cell differentiation into Th2-like effector cells is dependent on IL-4 and CD28 stimulation and inhibited by TCR ligation. Eur. J. Immunol. 28, 2517 – 2529. Smith, S.R., Terminelli, C., Denhardt, G., 2000. Effects of cannabinoid receptor agonist and antagonist ligands on production of inflammatory cytokines and anti-inflammatory interleukin-10 in endotoxemic mice. J. Pharmacol. Exp. Ther. 293, 136 – 150. Tindall, B., Cooper, D.A., Donovan, B., Barnes, T., Philpot, C.R., Gold, J., Penny, R., 1988. The Sydney AIDS Project: development of acquired immunodeficiency syndrome in a group of HIV seropositive homosexual men. Aust. N.Z. J. Med. 18, 8 – 15. Zhu, L.X., Sharma, S., Stolina, M., Gardner, B., Roth, M.D., Tashkin, D.P., Dubinett, S.M., 2000. Delta9-tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373 – 380.
D9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells Michael Yuan a, Sylvia M. Kiertscher a,b, Qingwen Cheng a, Richard Zoumalan a, Donald P. Tashkin a, Michael D. Roth a,b,* a
Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA b Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA Received 13 March 2002; received in revised form 23 September 2002; accepted 7 October 2002
Abstract Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and D9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625 – 5 Ag/ml). THC suppressed T cell proliferation, inhibited the production of interferon-g and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-g, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Human; Antigen presentation; Cytokines; T helper 1; T helper 2; Flow cytometry
1. Introduction D9-Tetrahydrocannabinol (THC) mediates its effects on the central nervous system by interacting with type 1 cannabinoid receptors (CB1) (Matsuda et al., 1990). In 1993, both CB1 and a second cannabinoid receptor, CB2, were identified on immune cells (Bouaboula et al., 1993; Munro et al., 1993). Unlike CB1, CB2 is not found in the brain and is expressed primarily on B cells, neutrophils, monocytes, NK cells and T cells (Munro et al., 1993; Galiegue et al., 1995). Both receptors belong to the G protein-coupled receptor superfamily, modulating intracellular cAMP and calcium, and are activated by THC as well as by endogenous compounds such as anandamide and 2arachidonyl glycerol (Howlett, 1995; Lee et al., 1995). While there have been several reports on the effects of cannabinoid * Corresponding author. Department of Medicine, CHS 37-131, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690, USA. Tel.: +1-310-206-7389; fax: +1-310-267-2020. E-mail address: [email protected] (M.D. Roth).
receptor ligands on the immune system in general, there is relatively little information about the effects of THC on human T cells or on antigen-specific immune responses (reviewed in Klein et al., 1998; Berdyshev, 2000). In recent murine studies, cannabinoids were reported to down-regulate the production of T helper 1 (Th1)-associated cytokines, including IL-2, IL-12 and interferon-g (IFN-g), and increase the production of T helper 2 (Th2)-associated cytokines, including IL-4 and IL-10 (Newton et al., 1994; Klein et al., 2000; Zhu et al., 2000; Smith et al., 2000; Ouyang et al., 1998). Consistent with the induction of a Th2-dominant immune response (Mosmann and Coffman, 1989; O’Garra, 1998), THC was found to suppress both anti-tumor immunity and the immune response to infection in these model systems (Newton et al., 1994; Klein et al., 2000; Zhu et al., 2000). Similar functional impairments in cell-mediated immunity have been observed in marijuana smokers. Tindall et al. (1988) evaluated 386 HIV-positive individuals and observed a more rapid progression from HIV infection to AIDS in marijuana users. Marijuana use is also associated with a greater risk for bacterial pneumonia, opportunistic infections and Kaposi’s sarcoma in HIV-positive individuals (Newell et
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 ( 0 2 ) 0 0 3 7 0 - 3
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
al., 1985; Caiaffa et al., 1994). We previously recovered alveolar macrophages from the lungs of otherwise healthy marijuana smokers and documented deficiencies in phagocytosis, bacterial killing, tumor cytotoxicity and cytokine production when compared to cells recovered from the lungs of nonsmokers or tobacco smokers (Baldwin et al., 1997). Direct studies on T cells recovered from marijuana smokers have produced mixed results (Nahas et al., 1974; Lau et al., 1976). However, in the largest such study, T cell proliferation in the MLR and mitogen-stimulation assays was reduced by 40 –45% when examined in 51 marijuana smokers as compared to 81 healthy controls (Nahas et al., 1974). In order to link these clinical observations to the immunoregulatory properties of THC, we collected T cells from healthy volunteers and activated them in vitro in the presence or absence of THC to determine the impact on antigen-specific proliferation, cytokine release, and the generation of activated Th1 or Th2 cells. Whether added to mixed leukocyte reaction (MLR) assays or to T cells stimulated by immobilized anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), THC down-regulated the generation of T cells capable of expressing and releasing Th1 cytokines and altered Th1/Th2 cytokine balance.
2. Materials and methods 2.1. Cells and reagents Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from the blood of healthy human donors and cultured in complete medium composed of RPMI 1640 with L-glutamine (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA), penicillin – streptomycin – fungizone (Life Technologies, Grand Island, NY), and 10 mM HEPES (Sigma, St. Louis, MO). Dendritic cells (DC) were prepared by culturing the adherent fraction of PBMC with 800 U/ml GM-CSF (specific activity 1.13 107 U/mg; Schering-Plough Research Institute, Kenilworth, NJ) and 500 U/ml IL-4 (specific activity 8 106 U/mg; R&D Systems, Minneapolis, MN). After 7 days, DC were recovered by vigorous rinsing and purified by negative depletion using a mAb cocktail (anti-CD3, antiCD19 and anti-CD56; BD PharMingen, San Diego, CA) and anti-mouse Ig-conjugated immunomagnetic beads (Dynal, Lake Success, NY) as previously described (Kiertscher and Roth, 1996). T cells were prepared from nonadherent PBMCs using a cocktail of anti-CD14, anti-CD16 and anti-CD19 mAbs, followed by immunomagnetic depletion (BD PharMingen) (Roth, 1994). The purity of DC and T cell preparations were routinely z 95% as determined by flow cytometry. In some experiments, T cells were activated with either anti-CD3 mAb alone, or in combination with antiCD28 mAb (BD PharMingen). IL-12 was from Peprotech (Rocky Hills, NJ). A 50 mg/ml stock of THC in ethanol was
125
provided by the National Institute on Drug Abuse (NIDA, Bethesda, MD) and diluted serially in DMSO and complete medium prior to use (final ethanol concentration V 0.01% and DMSO concentration V 0.125%). The highly CB1selective antagonist AM251 (CB1ra; Alexis, San Diego, CA) (Paria et al., 1998) and the CB2-selective antagonist SR144528 (CB2ra; provided by Murielle Rinaldi-Carmona, Sanofi Recherche, Montpellier, France) (Rinaldi-Carmona et al., 1998) were used to block the CB1- or CB2 receptormediated effects of THC, respectively, and were diluted in a manner identical to THC. 2.2. MLR assays THC was evaluated for its effects on antigen-specific T cell proliferation in the MLR assay. Purified DCs (5 103) were co-cultured in complete medium with 1 105 purified allogeneic T cells (DC/TC ratio, 1:20) in 96-well roundbottomed plates at 37 jC in a humidified CO2 incubator. On day 5, wells were pulsed with 1.25 ACi/well titrated thymidine (Amersham, Arlington Heights, IL) and harvested 18 h later. Proliferation was determined as cpm using a liquid scintillation counter. In some experiments, anti-CD3 mAb (1 Ag/ml) was added to replicate culture wells on day 2 and culture supernatants harvested 24 h later for detection of IFN-g (BD PharMingen) and IL-4 (Biosource, Camarillo, CA) by ELISA. THC was added at the beginning of culture to experimental wells at concentrations ranging from 0.625 to 5.0 Ag/ml. Ethanol (0.01% final concentration) and DMSO (0.125% final concentration) were added as a diluent control to wells that did not contain THC. 2.3. Regulation of Th1/Th2 differentiation as measured by intracellular cytokine detection The capacity for THC (5 Ag/ml), IL-4 (500 U/ml) or IL-12 (1 ng/ml) to alter the maturation of T cells into either Th1 cells, capable of secreting IFN-g when subsequently activated, or Th2 cells, capable of producing IL-4 when activated, was evaluated using a short-term in vitro model for Th1/Th2 differentiation (Schylze-Koops et al., 1998; Elson et al., 1995). CD3+ T cells were cultured at 1 106 cells/ml in wells containing immobilized anti-CD3 (5 Ag/ml) and anti-CD28 (0.5 Ag/ml) mAbs. In contrast to the MLR assay, this approach pan-activated all T cells simultaneously. Potential Th1/Th2 modulators (THC, IL-4 or IL-12) were added at the initiation of the culture period and 50% of the medium was replenished with fresh medium, containing the corresponding modulating agents, on day 2. T cells were harvested on day 4, washed to remove THC or cytokines, and restimulated to determine their ability to secrete IFN-g and IL-4. Cytokine production following this secondary challenge was determined by intracellular cytokine analysis according to the Fast-Immune protocol (Becton Dickinson, San Jose, CA). This approach allowed each T cell to be evaluated for the presence and intensity of its cytokine
126
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
response. In brief, cells were restimulated with 0.75 Ag/ml calcium ionophore A23187 and 25 ng/ml PMA (Sigma). One hour later, 10 Ag/ml Brefeldin A (Sigma) was added to prevent cytokine secretion and cells were cultured an additional 4 h. Cells were then incubated with 0.02% EDTA in PBS (Irvine Scientific) to break-up cell clumps, washed, fixed with 4% paraformaldehyde (Sigma) and cryopreserved at 80 jC. On the day of analysis, cells were thawed and treated with FACS Lysing Solution (Becton Dickinson) and FACS Permeabilizing Solution (Becton Dickinson) as per manufacturer’s instructions. Cells were stained with antiIFN-g FITC, anti-IL-4 – PE and anti-CD8 – PerCP mAb (Becton Dickinson), and analyzed by three-color FACS analysis using a FACScan II flow cytometer and CellQuest Software (Becton Dickinson). Replicate samples were stained with fluorescent-labeled isotype-specific control mAbs (to irrelevant antigens) as a control for nonspecific intracellular staining. Events (30,000 – 50,000) were acquired for each sample and results expressed as the percentage of CD3+/CD8+ or CD3+/CD8 (i.e. CD4+) T cells staining for a given cytokine. PMA down-regulates expression of CD4, preventing direct detection of the CD3+/CD4+ population using fluorochrome-labeled anti-CD4 (Baran et al., 2001). Mean linear fluorescence intensity was used to describe the average cytokine expression of the positively stained populations. The role of the CB1 and CB2 receptors in the response to THC were evaluated by pretreating cells for 10 min with either AM251 (CB1ra, 0.05 –1.0 AM) or SR144528 (CB2ra, 1 AM) prior to addition of THC at the beginning of culture (Paria et al., 1998; Rinaldi-Carmona et al., 1998). The impact of THC on T cell proliferation was also evaluated using the same activation model. T cells were stimulated with immobilized anti-CD3/CD28 in the presence or absence of THC as described above, and pulsed with 1.25 ACi/well titrated thymidine (Amersham) on day 3. Cells were harvested 18 h later and proliferation determined by the level of radioactive incorporation.
5% acryl-bis urea gels. A mRNA standard prepared from mitogen-activated peripheral blood mononuclear cells (BD PharMingen) was also run with each gel as a positive control. Gels were dried and exposed to a phosphor image screen and 2 h later read by a Molecular Dynamics Storm 860 PhosphoImager (Molecular Dynamics, Sunnyvale, CA). Cytokines bands were identified according to the position of the known cytokine mRNA standards and quantitated by ImageQuant for Macintosh v1.2 software (Molecular Dynamics). As for intracellular cytokine analysis, the role of the CB2 receptor was evaluated by pretreating cells for 10 min with SR144528 prior to the initial treatment with THC. 2.5. Statistical analysis Data from individual experiments are represented as the average value F S.D. for the indicated number of replicate wells. Pooled data from multiple experiments are represented as mean raw values, or as a percentage of control, F S.E. Differences between groups were determined by a two-tailed Student’s t-test for paired data with significant differences documented at p values of V 0.05 as indicated.
3. Results 3.1. THC inhibited T cell proliferation and altered the release of TH1/TH2 cytokines in the MLR T cells from normal donors were stimulated with allogenic DC in the presence or absence of THC and evaluated 6 days later for proliferation by [3H]-thymidine uptake. Exposure to THC produced a concentration-dependent decrease in the response to alloantigen (Fig. 1). The level of suppression varied somewhat among individuals, with 5.0 Ag/ml THC producing an average inhibition of 53 F 10% (range 28– 79%) compared to control T cells exposed to diluent alone
2.4. Analysis of cytokine mRNA The effects of THC on cytokine mRNA were examined using a ribonuclease protection assay capable of simultaneously quantitating mRNA encoding for IL-2, IL-4, IL-5, IL-10, IL-13, IL-14, IL-15, GM-CSF, IFN-g and housekeeping genes (Riboquantk hck1 probe set with GM-CSF substituted for IL-9; BD PharMingen). Purified T cells were activated for 4 days with immobilized anti-CD3 and antiCD28 mAbs, in the presence or absence of 5 Ag/ml THC, washed, and then stimulated with calcium ionophore and PMA as described above for intracellular cytokine detection. Four hours after final stimulation, total RNA was extracted by the Trizol method (Life Technologies) and 10 Ag from each condition incubated with [32P]-labeled cytokine mRNA probe sets and subjected to RNAse digestion according to the manufacturer’s instructions. Precipitates from the final reaction tubes were dissolved in 10 Al loading buffer and run on
Fig. 1. THC inhibited T cell proliferation in a concentration dependent manner. Purified human CD3+ T cells (1 105) were co-cultured at a 20:1 ratio with allogeneic DC for 5 days in the presence of THC at concentrations ranging between 0 and 5 Ag/ml. Cells were pulsed with [3H]-thymidine and harvested 18 h later to determine proliferation (cpm). Data presented as the average F S.D. for four replicate wells. Representative experiment out of six performed. *p V 0.01 compared to control (no THC).
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
127
Fig. 2. THC decreased secretion of IFN-g while increasing IL-4 release during the MLR assay. CD3+ T cells were co-cultured with allogeneic DC in the presence or absence of 5 Ag/ml THC as described in Fig. 1. Proliferation was determined after 5 days by [3H]-thymidine incorporation. Replicate wells from the same MLC were treated with 1 Ag/ml anti-CD3 mAb on day 2 to boost cytokine production and supernatants harvested 24 h later for determination of IFN-g and IL-4 concentration by ELISA. The percent change in proliferation, IFN-g release, IL-4 production, and Th1/Th2 cytokine ratio (ratio of IFNg to IL-4 in pg/ ml) resulting from treatment with THC was calculated for each set of experiments (n = 6 experiments). Graphs represent the average change F S.E. for all experiments comparing THC-treated wells to control wells. Average proliferation in control wells was 21,253 cpm. The average production of IFN-g in control wells was 2435 pg/ml and the average production of IL-4 was 52.6 pg/ml. *p V 0.05 compared to control.
( p V 0.01, n = 6). Supernatants were harvested from replicate wells during the MLR cultures and examined for the presence of IFN-g and IL-4 by ELISA. Whether simply collected from MLR cultures, or following an additional 24-h activation with soluble anti-CD3 mAb, supernatants from wells exposed to 5 Ag/ml of THC demonstrated a consistent change in the cytokine pattern (Fig. 2). On culture day 3, IFN-g concentrations were reduced on average by 51 F 18% ( p V 0.05), while IL-4 levels were increased on average to 105 F 11% of diluent control levels. This down-regulation of IFN-g and modest up-regulation of IL-4 resulted in a significant shift in the ratio of Th1 (IFN-g) to Th2 (IL-4) cytokine balance ( p V 0.05).
helper regulatory effects of THC were then compared to that of a conventional Th1 inducer, IL-12, and a conventional Th2 inducer, IL-4, using the same intracellular cytokine assay. As demonstrated in Fig. 4, the addition of IL-12 during T cell activation resulted in a marked increase in the number of cells capable of expressing IFN-g, as well as the MFI of expression. There was a relatively modest decrease in
3.2. THC down-regulated the development of Th1 cytokineproducing cells The effects of THC on proliferation and cytokine production during the MLR suggested an effect on the maturation of Th subsets following antigen stimulation. To evaluate this possibility further, T cells were pan-activated with immobilized anti-CD3 and anti-CD28 mAbs in vitro in the presence or absence of 5 Ag/ml THC. Four days later, activated cells were either pulsed with [3H]-thymidine to measure the impact on proliferation, or washed to remove THC and restimulated with PMA and calcium ionaphore to determine any changes in their capacity to produce Th1 or Th2 cytokines. On average, exposure to THC produced a 52 F 18% decrease in proliferation (n = 3 experiments) and a 58.7 F 17% reduction in the percentage of T cells capable of expressing intracellular IFN-g as determined by intracellular cytokine staining (n = 10 experiments, Fig. 3). THC reduced both the percentage of T cells expressing IFN-g and the average level of intracellular IFN-g detected per cell (mean linear fluorescence intensity, MFI), with obvious effects on both CD4+ and CD8+ subsets. The effect on the capacity to produce IL-4 was more variable, with some subjects demonstrating a reduction in number but an increase in average fluorescence intensity (Fig. 3), and most subjects demonstrating a slight increase in the percentage and/or MFI of cells stained for IL-4 (as shown in Figs. 4 and 5). The T
Fig. 3. THC decreased the percentage of IFN-g producing T cells and the relative concentration of IFN-g as determined by intracellular cytokine detection. T cells were stimulated with immobilized anti-CD3 (5 Ag/ml) and anti-CD28 (0.5 Ag/ml) mAb in the presence or absence of 5 Ag/ml THC. On day 4, cells were washed and restimulated with 0.75 Ag/ml Ca ionophore and 25 ng/ml PMA, and 10 Ag/ml brefeldin A was added after the first 1 h to prevent cytokine secretion. Four hours later, cells were fixed, permeabilized and stained with fluorescent-labeled anti-CD8, anti-IFN-g and IL-4 mAb. Events (30,000 – 50,000) were acquired by FACS analysis and gated to display cytokine production by total T cells (A), as well as by the CD4+ (B) and CD8+ (C) subsets. Numbers represent the % of cells staining for each cytokine and the mean fluorescence intensity (in parentheses). Representative experiment out of 10 performed.
128
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
Fig. 4. THC and IL-4 produce similar changes in the production of IFN-g and IL-4 by activated T cells. T cells were stimulated with anti-CD3 and antiCD28 and cultured in the presence of control medium, 1 ng/ml IL-12, 500 U/ ml IL-4, or 5 Ag/ml THC. Expression of intracellular IFN-g and IL-4 were detected after 4 days by FACS analysis as described in Fig. 3. Numbers represent the % of cells staining for each cytokine and the mean fluorescence intensity (in parentheses). Representative experiment out of four performed.
IL-4-producing cells in response to IL-12. In contrast, IL-4 significantly down-regulated the capacity of restimulated T cells to express IFN-g while having little effect, or a modest increase, in the number of cells capable of producing IL-4. In every experiment, the effects of THC closely modeled the response observed with IL-4, suggesting activity as a modulator of Th1/Th2 differentiation. 3.3. A CB2ra, but not a CB1ra, protected T cells from the effects of THC Specific CB1ra (AM251) and CB2ra (SR144528) were used to determine the role of cannabinoid receptors in mediating the response to THC (Fig. 5). Pretreating T cells with AM251 in the range of 0.05 –1.0 AM did not prevent THC from altering cytokine production as measured by the intracellular cytokine assay. In contrast, the addition of 1 AM SR144528 prior to treatment with THC led to increases in the percentage of IFN-g+ cells and a decrease in the percentage of IL-4+ cells as compared to cells treated with THC alone. Pretreatment with SR144528 protected 94.4 F 2% of the THC-sensitive cells from losing IFN-g
Fig. 6. THC up-regulated Th2 cytokine mRNA and down-regulated Th1 cytokine mRNA in a CB2-dependent manner. T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence or absence of 5 Ag/ml THC, and then restimulated with calcium ionaphore and PMA as described in Fig. 4. (A) Total RNA was harvested after 4 h, incubated with [32P]labeled cyokine RNA probes, treated with ribonuclease and the products resolved on a 5% acryl-bis urea gel (Riboquantk). The radiolabeled probe set and a known cytokine standard mRNA prep were run with each gel as a control and the location of bands for specific cytokine genes are shown. (B) Cytokines bands were quantitated using a phosphoimager and quantitative imaging software. Relative expressions of cytokine genes from THCtreated cells are expressed as a % of control, normalized for expression of GAPDH. Representative experiment out of two performed.
expression, but only partially blocked the average decrease in IFN-g signal intensity (seven experiments). Treatment of T cells with SR144528 alone, in the absence of THC, had little effect on IFN-g or IL-4 expression (data not shown). 3.4. THC regulated the expression of Th1 and Th2 cytokine mRNA We hypothesized that if THC was altering Th differentiation, it would also regulate the expression pattern for
Fig. 5. The CB2 antagonist SR144528, but not the CB1 antagonist AM251, prevented the effects of THC on intracellular expression of IFN-g and IL-4. T cells were stimulated with anti-CD3 and anti-CD28, and intracellular IFN-g and IL-4 detected as described in Fig. 4. Control T cells were compared to T cells treated with 5 Ag/ml THC, or T cells pretreated for 10 min with either AM251 (CB1ra, 50 nM) or SR144528 (CB2ra, 1 AM) prior to the addition of THC. Numbers represent the % of cells staining for each cytokine. Representative experiment out of seven performed.
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
Fig. 7. CB2ra protected against the effects of THC on cytokine mRNA. Induction of cytokine mRNA following T cell activation was determined using a ribonuclease protection assay as described in Fig. 6. T cells were cultured during the initial activation period with diluent alone, with 5 Ag/ml THC, or with the combination of 1 AM SR144528 and 5 Ag/ml THC. Cytokines bands were quantitated using a phosphoimager and quantitative imaging software. Relative expressions of cytokine genes under different conditions are expressed as a % of control, normalized for expression of GAPDH. Representative experiment out of two performed.
cytokine-specific mRNA. T cells were activated in the same manner as for the intracellular cytokine assay and mRNA extracted at either 2, 4 or 8 h after the secondary stimulation with calcium ionophore and PMA. Cytokine mRNA for both Th1 (IL-2, IFN-g) and Th2 (IL-4, IL-5) cytokines were then directly compared using a ribonuclease protection assay. Consistent with the pattern of changes obtained by ELISA and by FACS analysis, mRNA encoding for IFN-g and IL-2 was reduced by 21 –48% in cells treated with 5 Ag/ ml THC, and mRNA encoding for IL-4 and IL-5 was increased by 1.5- to 11.2-fold (Fig. 6). Pretreatment with 1 AM SR144528 prevented the majority of these THCmediated effects (Fig. 7).
4. Discussion This report demonstrates that THC can regulate human T cell activation. Not only did THC suppress the proliferative response to alloantigen, it shifted the balance of activationinduced cytokines and the differentiation of activated T cells in favor of a Th2 cytokine response. Marijuana, and its primary psychoactive constituent, THC, were first proposed as immune modulators in the 1970s when abnormal T cell responses were observed in THC-treated animals (Elson et al., 1995; Nahas et al., 1973) and in PBMC collected from chronic marijuana smokers (Nahas et al., 1974). However, these findings were not uniformly observed (Lau et al., 1976; Rachelefsky et al., 1976), and it was not until the discovery of CB1 and CB2 receptors that interactions between cannabinoids and the immune system began to be investigated in detail (Matsuda et al., 1990; Bouaboula et al., 1993; Munro
129
et al., 1993; Galiegue et al., 1995; Howlett, 1995). The CB1 receptor is expressed at high levels in the central nervous system and at much lower levels in peripheral tissues. In contrast, the CB2 receptor is expressed only in peripheral tissues and primarily in cells of hematopoietic origin. Of the two cannabinoid receptor subtypes, mRNA encoding for the CB2 receptor is present in mouse spleen at levels 10- to 100fold higher than mRNA encoding for CB1 (Galiegue et al., 1995; Schatz et al., 1997). CB2 receptor is also preferentially expressed in human leukocytes, with B cells expressing several-fold higher levels of CB2 receptor protein than T cells (Galiegue et al., 1995; Marchand et al., 1999; Nong et al., 2002). In contrast to our results with T cells, activation of the CB2 receptor enhances the proliferation of CD40-activated human B cells (Galiegue et al., 1995; Derocq et al., 1995). The capacity for THC to suppress antigen-induced T cell proliferation, modulate cytokine balance in favor of a Th2 profile and stimulate B cell proliferation suggests a coordinated role for cannabinoids in promoting humoral over cell-mediated immunity. The production of cytokines by stimulated T cells plays a critical role in amplifying and targeting immunity. When activated T cells mature into Th1 cells, they secrete IL-2 and IFN-g and potentiate cell-mediated immunity by enhancing T cell proliferation and effector functions, up-regulating MHC expression on targets, promoting IgG2a isotype switching, and activating effector cells such as NK cells and macrophages (Mosmann and Coffman, 1989; O’Garra, 1998). In contrast, when activated T cells acquire a Th2 phenotype, they secrete IL-4, IL-5 and/or IL-10, and potentiate allergic/atopic antibody responses while suppressing T cell proliferation and cell-mediated immunity. Other investigators have utilized animal models to evaluate the effects of THC on cytokine production and the generation of Th1/ Th2 phenotypes. Newton et al. (1994) observed that mice treated with THC prior to infection with Legionella pneumophila exhibited reduced antigen-specific T cell proliferation, along with a shift in antibody response from a predominantly IgG2a isotype to an IgG1 isotype. Splenocytes cultured with THC in vitro secreted lower levels of IFN-g and higher levels of IL-4 than control cells. Similarly, Klein et al. (2000) found that THC skewed both the immediate and delayed cytokine response toward a Th2 profile, and that THC-treated mice failed to exhibit protective immunity when rechallenged with L. pneumophila. Splenocytes from tumor-bearing mice treated with THC demonstrated decreased production of IFN-g, but increased production of both IL-10 and TGF-h (Zhu et al., 2000). In addition, THC promoted the rapid growth of subcutaneously implanted tumors. This growth could be prevented by treatment with neutralizing antibody against IL-10, or a selective CB2ra. Taken together, these in vivo models suggest a direct role for altered Th1/Th2 cytokine balance in the immunosuppressive consequences of THC. Our in vitro results with human T cells confirm and complement these previous reports. We found that THC
130
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131
produced a concentration-dependent inhibition of T cell proliferation. In addition, THC decreased the secretion of IFN-g during the MLR response, while having a modest effect on increasing the release of IL-4, resulting in a significant shift in Th1/Th2 balance. To examine the impact of THC on Th maturation and cytokine production at the single-cell level, we activated T cells with anti-CD3/antiCD28 mAbs in the presence of THC. As in the MLR assay, THC inhibited activation-induced T cell proliferation. Activation in the presence of THC also resulted in fewer T cells capable of producing IFN-g and more cells producing IL-4, as well as changes in the amount of cytokine secreted per cell. By modeling the effects of THC to those mediated by standard Th1/Th2 inducers, we confirmed that the response to THC was very similar to that produced by the prototypic Th2 stimulator, IL-4. We observed a similar, but more prominent pattern in the levels of cytokine mRNA, with down-regulation of mRNA encoding for IL-2 and IFN-g and up-regulation of mRNA encoding for IL-4 and IL-5. These effects on cytokine mRNA, as well as those on cytokine protein, were significantly abrogated by pretreatment with a CB2ra. However, the CB2ra did not completely block the effects of THC, making it possible that stimulation of CB1, or activation of receptor-independent pathways, also contributed to the impact on cytokines. These results suggest that cannabinoids have a capacity to act as Th2 inducers (Schylze-Koops et al., 1998; O’Garra, 1998). This conclusion is consistent with the clinical finding of increased IgE levels in marijuana smokers (Rachelefsky et al., 1976), the down-regulation of effector functions in alveolar macrophages recovered from the lungs of chronic marijuana users (Baldwin et al., 1997), and the variety of clinical reports suggesting that marijuana is a risk factor for opportunistic infections (Tindall et al., 1988; Newell et al., 1985). There are several mechanisms by which THC might regulate T cell responses. At the receptor level, the effects of THC on cytokine production were blocked primarily by a CB2ra. A primary role for CB2 in regulating Th1/Th2 balance was also reported by Zhu et al. (2000) in their mouse tumor model. However, others have demonstrated that both CB1ra and CB2ra can block the in vivo consequences of THC (Klein et al., 2000; Massi et al., 2000). In vivo, THC may act on CB1 receptors in the central nervous system to stimulate the release of corticotropin-releasing factor and adrenocorticotropic hormone (Manzanares et al., 1999). Activation of the hypothalamic –pituitary –adrenal axis in this manner could indirectly suppress lymphocyte activation and promote Th2 cytokine responses (Elenkov et al., 1999). It is also possible that THC acts through CB1 receptors on other leukocytes, such as NK cells or B cells, to modulate the production of T helper regulatory cytokines. These potential effects were excluded from our studies by employing highly purified T cells in all of our assays. Both the CB1 and CB2 receptors are pertussis toxin sensitive GTP-binding proteins that inhibit adenylate cyclase and down-regulate intracellular cAMP (Howlett, 1995; Kaminski et al., 1994). Down-regu-
lation of cAMP by THC inhibits the activation of protein kinase A with downstream effects on cAMP response elements, activation of AP-1 promoter sites and NF-nB/RelB pathways (Kaminski, 1998). Other signaling pathways may be affected including mitogen-activated protein kinases (Faubert and Kaminski, 2000). In addition, Klein et al. (2000) found that exposure to THC down-regulated the expression of IL-12 receptors. As such, it is possible that the effects of THC are mediated in part by effects on cytokine receptor expression. While many of these pathways are known to be involved in T cell activation and cytokine production, the mediator changes responsible for the effects of THC on human T cells remain to be determined. In summary, our results demonstrate that THC can interact with CB2 receptors on human T cells to suppress activation and skew cytokine production in favor of a Th2 response. These coordinated effects suggest a potential role for endogenous endocannabinoids in regulating the balance between humoral and cellular immunity.
Acknowledgements This work was funded by NIDA/National Institutes of Health grant #DA03018-16. S.M.K. is supported by a Young Investigator Award from the U.S. Army Medical Research and Material Command under DAMD17-98-18181.
References Baldwin, G.C., Tashkin, D.P., Buckley, D.M., Park, A.N., Dubinett, S.M., Roth, M.D., 1997. Marijuana and cocaine impair alveolar macrophage function and cytokine production. Am. J. Respir. Crit. Care Med. 156, 1606 – 1613. Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate – ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303 – 313. Berdyshev, E.V., 2000. Cannabinoid receptors and the regulation of immune response. Chem. Phys. Lipids 108, 169 – 190. Bouaboula, M., Rinaldi, M., Carayon, P., Carillon, C., Delpech, B., Shire, D., Le Fur, G., Casellas, P., 1993. Cannabinoid-receptor expression in human leukocytes. Eur. J. Biochem. 214, 173 – 180. Caiaffa, W.T., Vlahov, D., Graham, N.M., Astemborski, J., Solomon, L., Nelson, K.E., Munoz, A., 1994. Drug smoking, Pneumocystis carinii pneumonia, and immunosuppression increase risk of bacterial pneumonia in human immunodeficiency virus-seropositive injection drug users. Am. J. Respir. Crit. Care Med. 150, 1493 – 1498. Derocq, J.M., Segui, M., Marchand, J., Le Fur, G., Casellas, P., 1995. Cannabinoids enhance human B-cell growth at low nanomolar concentrations. FEBS Lett. 369, 177 – 182. Elenkov, I.J., Webster, E.L., Torpy, D.J., Chrousos, G.P., 1999. Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: acute and chronic effects. Ann. N.Y. Acad. Sci. 876, 1 – 11. Elson, L.H., Nutman, T.B., Metcalf, D.D., Prussin, C., 1995. Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2
M. Yuan et al. / Journal of Neuroimmunology 133 (2002) 124–131 and T helper 0 cells within the human CD4+CD27 lymphocyte subpopulation. J. Immunol. 154, 4294 – 4301. Faubert, B.L., Kaminski, N.E., 2000. AP-1 activity is negatively regulated by cannabinol through inhibition of its protein components, c-fos and c-jun. J. Leukoc. Biol. 67, 259 – 266. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G., Casellas, P., 1995. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur. J. Biochem. 232, 54 – 61. Howlett, A.C., 1995. Pharmacology of cannabinoid receptors. Annu. Rev. Pharmacol. Toxicol. 35, 607 – 634. Kaminski, N.E., 1998. Regulation of the cAMP cascade, gene expression and immune function by cannabinoid receptors. J. Neuroimmunol. 83, 124 – 132. Kaminski, N.E., Koh, W.S., Yang, K.H., Lee, M., Kessler, F.K., 1994. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism. Biochem. Pharmacol. 48, 1899 – 1908. Kiertscher, S.M., Roth, M.D., 1996. Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4. J. Leukoc. Biol. 59, 208 – 218. Klein, T.W., 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. Delta9-tetrahydrocannabinol treatment suppresses immunity and early IFN-gamma, IL-12, and IL-12 receptor beta2 responses to Legionella pneumophila infection. J. Immunol. 164, 6461 – 6466. Lau, R.J., Tubergen, D.G., Barr Jr., M., Domino, E.F., Benowitz, N., Jones, R.T., 1976. Phytohemagglutinin-induced lymphocyte transformation in humans receiving delta9-tetrahydrocannabinol. Science 192, 805 – 807. Lee, M., Yang, K.H., Kaminski, N.E., 1995. Effects of putative cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, on immune function in B6C3F1 mouse splenocytes. J. Pharmacol. Exp. Ther. 275, 529 – 536. Manzanares, J., Corchero, J., Fuentes, J.A., 1999. Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats. Brain Res. 839, 173 – 179. Marchand, J., Bord, A., Penarier, G., Laure, F., Carayon, P., Casellas, P., 1999. Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting: application to peripheral cannabinoid receptors. Cytometry 35, 227 – 234. Massi, P., Fuzio, D., Vigano, D., Sacerdote, P., Parolaro, D., 2000. Relative involvement of cannabinoid CB(1) and CB(2) receptors in the delta(9)tetrahydrocannabinol-induced inhibition of natural killer activity. Eur. J. Pharmacol. 387, 343 – 347. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., Bonner, T.I., 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561 – 564. Mosmann, T.R., Coffman, R.L., 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7, 145 – 173. Munro, S., Thomas, K.L., Abu-Shaar, M., 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365, 61 – 65. Nahas, G.G., Zagury, D., Schwartz, I.W., Nagel, M.D., 1973. Evidence for
131
the possible immunogenicity of delta9-tetrahydrocannabinol (THC) in rodents. Nature 243, 407 – 408. Nahas, G.G., Suciu-Foca, N., Armand, J.-P., Morishima, A., 1974. Inhibition of cellular mediated immunity in marihuana smokers. Science 183, 419 – 420. Newell, G.R., Mansell, P.W., Wilson, M.B., Lynch, H.K., Spitz, M.R., Hersh, E.M., 1985. Risk factor analysis among men referred for possible acquired immune deficiency syndrome. Prev. Med. 14, 81 – 91. Newton, C.A., Klein, T.W., Friedman, H., 1994. Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta9-tetrahydrocannabinol injection. Infect. Immun. 62, 4015 – 4020. Nong, L., Newton, C., Cheng, Q., Friedman, H., Roth, M.D., Klein, T.W., 2002. Altered cannabinoid receptor mRNA expression in peripheral blood mononuclear cells from marijuana smokers. J. Neuroimmunol. 127, 169 – 176. O’Garra, A., 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 81, 275 – 283. Ouyang, Y., Hwang, S.G., Han, S.H., Kaminski, N.E., 1998. Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonylglycerol is mediated through down-regulation of the nuclear factor of activated T cells. Mol. Pharmacol. 53, 676 – 683. Paria, B.C., Ma, W., Andrenyak, D.M., Schmid, P.C., Schmid, H.H., Moody, D.E., Deng, H., Makriyannis, A., Dey, S.K., 1998. Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors. Biol. Reprod. 58, 1490 – 1495. Rachelefsky, G.S., Opelz, G., Mickey, M.R., Lessin, P., Kiuchi, M., Silverstein, M.J., Stiehm, E.R., 1976. Intact humoral and cell-mediated immunity in chronic marijuana smoking. J. Allergy Clin. Immunol. 58, 483 – 490. Rinaldi-Carmona, M., Barth, F., Millan, J., Derocq, J.M., Casellas, P., Congy, C., Oustric, D., Sarran, M., Bouaboula, M., Calandra, B., Portier, M., Shire, D., Brelie`re, J.C., Le Fur, G., 1998. SR144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. J. Pharmacol. Exp. Ther. 284, 644 – 650. Roth, M.D., 1994. Interleukin-2 induces the expression of CD45RO and the memory phenotype by CD45RA+ peripheral blood lymphocytes. J. Exp. Med. 179, 857 – 864. Schatz, A.R., Lee, M., Condie, R.B., Pulaski, J.T., Kaminski, N.E., 1997. Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol. Appl. Pharmacol. 142, 278 – 287. Schylze-Koops, H., Lipsky, P.E., Davis, L.S., 1998. Human memory T cell differentiation into Th2-like effector cells is dependent on IL-4 and CD28 stimulation and inhibited by TCR ligation. Eur. J. Immunol. 28, 2517 – 2529. Smith, S.R., Terminelli, C., Denhardt, G., 2000. Effects of cannabinoid receptor agonist and antagonist ligands on production of inflammatory cytokines and anti-inflammatory interleukin-10 in endotoxemic mice. J. Pharmacol. Exp. Ther. 293, 136 – 150. Tindall, B., Cooper, D.A., Donovan, B., Barnes, T., Philpot, C.R., Gold, J., Penny, R., 1988. The Sydney AIDS Project: development of acquired immunodeficiency syndrome in a group of HIV seropositive homosexual men. Aust. N.Z. J. Med. 18, 8 – 15. Zhu, L.X., Sharma, S., Stolina, M., Gardner, B., Roth, M.D., Tashkin, D.P., Dubinett, S.M., 2000. Delta9-tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373 – 380.