Δ9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells
Introduction
Δ9-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 2-arachidonyl glycerol Howlett, 1995, Lee et al., 1995. While there have been several reports on the effects of cannabinoid 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-γ (IFN-γ), 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 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.
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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
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 μg/ml THC producing an average inhibition of 53±10% (range 28–79%) compared to control T cells exposed to diluent alone (p≤0.01, n=6). Supernatants were harvested
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 activation-induced 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
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 DAMD17-98-1-8181.
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