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

Abstract

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

Figure 1

CBD suppressed cytokine production in PMA/Io-stimulated B6C3F1 splenocytes. A-B.) Splenocytes (8 × 10⁵ cells) were treated with CBD (0.1-15 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (A.) or IFN-γ (B.) was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. C-D.) Splenocytes (5 × 10⁶ cells) were treated with CBD (0.5-10 μM) for 30 min, followed by cellular activation with PMA/Io for 6 hr. Real time PCR was performed for IL-2 and IFN-γ. Fold change was calculated as compared to NA. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).

Figure 2

CBD suppressed CD25 cell surface expression in PMA/Io-stimulated B6C3F1 splenocytes. Splenocytes (8 × 10⁵ cells) were treated with CBD (0.2-20 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Cells were harvested and stained with fluorescent antibodies directed against CD3 (FITC) or CD25 (PE). Cells were gated on FSC/SSC. Numbers denote percent gated events. Results are representative of three separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).

Figure 3

CBD suppressed cytokine production in wild type C57BL/6 and CB1^-/-/CB2^-/- splenocytes. Splenocytes (8 × 10⁵ cells) were treated with CBD (0.2-10 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (A-B.) or IFN-γ (C-D.) in wild type C57BL/6 and CB1^-/-CB2^-/- was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. * or ** denotes values that are significantly different from the respective vehicle control at p < 0.05 or 0.01. Data are presented as % VH control in B and D. Results are representative of two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).

Figure 4

CBD suppressed cytokine production in B6C3F1 splenic T cells. A-B.) Splenocytes (8 × 10⁵ cells) were treated with CBD (0.1-15 μM) for 30 min, followed by cellular activation with immobilized anti-CD3 plus soluble anti-CD28 for 24 hr. Supernatants were harvested and the amount of IL-2 (A.) or IFN-γ (B.) was determined by ELISA. C-D.) T cells purified from splenocytes (8 ×10⁵ cells) were treated with CBD (0.1-2 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (C.) or IFN-γ (D.) was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).

Figure 5

CBD suppressed cellular proliferation in B6C3F1 splenocytes in response to various stimuli. A-D.) Splenocytes (2 × 10⁵ cells) were treated with CBD (0.2-10 μM) for 30 min, followed by cellular activation. 18 hours prior to harvest, cells were pulsed with 1 μCi ³H-thymidine. Cells were harvested onto glass fiber filters and tritium incorporation was measured with a liquid scintillation counter. Splenocytes were activated with A.) PMA/Io for 48 hr; B.) LPS for 72 hr; C.) immobilized anti-CD3 plus soluble anti-CD28 for 48 hr; D.) mitomycin C-treated allogeneic lymphocytes for 96 hr. The data are expressed as the mean CPM ± SE of quadruplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least three separate experiments. R, responders; S, stimulators; NA, naïve (untreated); VH, vehicle (0.1% ethanol).

Figure 6

Effect of CBD on the IgM AFC response in B6C3F1 splenocytes. A-B.) Splenocytes (5 × 10⁶ cells for sRBC; 2.5 × 10⁶ cells for LPS) were treated with CBD (1-20 μM) or THC (20 μM) for 30 min, followed by stimulation with A.) sRBC for 5 days or B.) LPS for 72 hr. Cells were then incubated in a Bellco stainless steel tissue culture chamber pressurized to 5.5 psi with a gas mixture consisting of 10% O₂, 7% CO₂ and 83% N₂. The culture chamber was placed at 37°C with constant rocking for the duration of the culture period. Enumeration of the AFC response was performed as described in Materials and Methods. The data are expressed as the mean AFC/10⁶ viable cells ± SE of quadruplicate cultures. C.) B6C3F1 mice received CBD (25-100 mg/kg) or THC (50 mg/kg) by oral gavage for 3 days. On day 2, in addition to drug, mice received a single i.p. injection of sRBC (5 × 10⁸ cells/mouse). Mice were sacrificed on day 6, after which the AFC response was enumerated as described in Materials and Methods. The data are expressed as the mean AFC/10⁶ viable or recovered cells ± SE. Results are pooled from two separate experiments. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01.

Figure 7

Effect of CBD on the in vitro AFC response in wild type C57BL/6 and CB1^-/-/CB2^-/- splenocytes. Splenocytes (5 × 10⁶ cells) from C57BL/6 or CB1^-/-/CB2^-/- were treated with CBD (1-20 μM) or THC (20 μM) for 30 min, followed by stimulation with sRBC for 5 days. Cells were then cultured and the AFC were enumerated as stated in Figure 1. The data are expressed as the mean AFC/10⁶ viable cells ± SE of quadruplicate cultures for one representative experiment (A.) or % VH results for CBD from four experiments are pooled, with the exception of the 15 μM group, which represents a single experiment (B.). * or ** denotes values that are significantly different from the respective vehicle control at p < 0.05 or 0.01.

Figure 8

CBD suppressed IL-2 production and AP-1 and NFAT activity in PMA/Io-stimulated human Jurkat T cells. A.) Jurkat cells (5 × 10⁴ cells) were treated with CBD (0.1-10 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. B-C.) Jurkat cells (5 × 10⁵ cells) were transiently transfected with either AP-1-luciferase (B.) or NFAT-luciferase (C.). Three hours later, cells were treated with CBD (1-10 μM) for 30 min, followed by cellular activation with PMA/Io for 21 hr. Luciferase activity was determined as described in Materials and Methods. The data are expressed as the mean CPS of triplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).