Modulation of Human Peripheral Blood Mononuclear Cell Signaling by Medicinal Cannabinoids

Abstract

Medical marijuana is increasingly prescribed as an analgesic for a growing number of indications, amongst which terminal cancer and multiple sclerosis. However, the mechanistic aspects and properties of cannabis remain remarkably poorly characterized. In this study we aimed to investigate the immune-cell modulatory properties of medical cannabis. Healthy volunteers were asked to ingest medical cannabis, and kinome profiling was used to generate comprehensive descriptions of the cannabis challenge on inflammatory signal transduction in the peripheral blood of these volunteers. Results were related to both short term and long term effects in patients experimentally treated with a medical marijuana preparation for suffering from abdominal pain as a result of chronic pancreatitis or other causes. The results reveal an immunosuppressive effect of cannabinoid preparations via deactivation of signaling through the pro-inflammatory p38 MAP kinase and mTOR pathways and a concomitant deactivation of the pro-mitogenic ERK pathway. However, long term cannabis exposure in two patients resulted in reversal of this effect. While these data provide a powerful mechanistic rationale for the clinical use of medical marijuana in inflammatory and oncological disease, caution may be advised with sustained use of such preparations.

Keywords: T cells; inflammatory signaling; kinome profiling; mTOR-S6; monocyte.

FIGURE 1

Potentiated signal transduction in PBMCs before and after the intake of medical cannabis. Blood was drawn from three self-reported cannabis-naive volunteers before and 2 h after the drinking of a medical cannabis preparation and stimulated for 10 min with lipopolysaccharide (100 ng/ml 10 min). Subsequently PBMC were isolated and investigated using peptide arrays exhibiting 976 different kinase substrates. Results were collapsed on elective signal transduction categories and expressed in gray scale (representing the fraction of peptides phosphorylated in either condition per pathway. White means no peptides were significantly phosphorylated and black means all phosphorylated substrates were found in one of the conditions). Some of the most prominently affected signal transduction elements have been highlighted in yellow. Note inhibition of pro-inflammatory signaling following medical cannabis use.

FIGURE 2

In vitro validation of direct THC effect on immune cells. PBMCs were isolated from healthy subjects (n = 6) and after in vitro pretreatment with THC (2 ng/mL) for 1 h, cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 10 min. Western blot analysis shows that unstimulated cells show relatively little activation of p38MAPK, AKT, S6 or ERK, while LPS induces phosphorylation of these kinases. Pretreatment of PBMCs with THC significantly reduces LPS-induced pro-inflammatory phosphorylation patterns. (A) Representative example is shown. (B) Quantification of phosphorylation levels of LPS-stimulated cells, normalized for total Actin levels are shown. pS6 and pERK levels are significantly reduced upon THC treatment of cells (^∗0.0357, ^∗∗0.0238). Phosphorylation of p38 was reduced upon THC treatment in 5/6 subjects. pAKT was detectable in only four subjects, in three of which phosphorylation was reduced in THC treated cells.

FIGURE 3

Differential activation of immune signaling using monocyte or T cell –specific triggers. Whole PBMC fractions were stimulated with either LPS (10 min) or αCD3/CD28 (10 min or 1h). (A) Western blot analysis shows that both stimuli induce a distinct set of phosphorylations. (B) Percentage of phospho-S6 positive cells as determined by intracellular FACS analysis, confirming that LPS does not trigger S6 phosphorylation in CD3⁺ T cells, whereas αCD3/CD28 generates a robust response in this cell type. In contrast, lipopolysaccharide does stimulate S6 phosphorylation in monocytes, with αCD3/CD28 also eliciting a response. (C) Representative example of FACS analysis of 4 independent experiments is shown.

FIGURE 4

THC reduces S6 phosphorylation in T cells and monocytes. (A) Basal levels of S6 phosphorylation were determined by intracellular flow cytometry. S6 phosphorylation was reduced in monocytes and T cells upon in vitro treatment of cells with THC for 1 or 3 h. (B) Mean percentage of pS6 positive CD3⁺T cells (upper panel) and monocytes (lower panel) of six independent experiments. (C) Whole PBMC fractions were pretreated in vitro with THC for 1h or left untreated, and subsequently stimulated with either LPS (10 min) or αCD3/CD28 (1 h). THC pretreated samples show less S6 phosphorylation upon LPS or αCD3/CD28 stimulation (representative example of 6 experiments is shown). (D). FACS analysis of LPS (10 min) or αCD3/CD28 (1 h) stimulated PBMCs, gated on monocyte and T cell fractions, respectively. Mean of four independent experiments is shown.

FIGURE 5

Intake of THC reduces S6 phosphorylation in patient PBMCs in the short term, but enhances S6 phosphorylation in the long term. (A) PBMCs were isolated from patients taking medicinal marijuana for pain-related complaints for 52 days. On day 52, samples were obtained pre-dose, and 1, 3, and 5 h after THC intake. A representative example of n = 4 is shown, demonstrating that the short term effects of THC intake include a decrease in S6 phosphorylation. (B) Mean percentage pS6 positive T cells and monocytes (n = 4). (C) For two patients, PBMCs were isolated pre-dosing on day 0, day 15 and day 52, as well as post-dose on day 52. Both patients show a clear increase of S6 phosphorylation during prolonged THC treatment, which is decreased upon short term treatment.