Cannabinoid Receptor 2 (CB₂) Signals via G‑alpha‑s and Induces IL‑6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

Cannabinoid receptor 2 (CB₂) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB₂ signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB₂-selective ligand (HU308). We observed a “lag” phase of unchanged cAMP concentration prior to development of classically expected Gα_i-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gα_i effects by concurrent Gα_s activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gα_i, whereas ERK1/2 and Akt phosphorylation were mediated by Gα_i-coupled βγ. Activation of CREB integrated multiple components; Gα_s and βγ mediated ∼85% of the response, while ∼15% was attributed to Gα_i. Responses to HU308 had an important functional outcomesecretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB₂. Additionally, signaling and functional effects were completely blocked by a CB₂-selective inverse agonist, giving additional evidence for CB₂ involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB₂ ligands as immunomodulatory therapeutics.