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

Abstract

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 Gi-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gi effects by concurrent Gs activation. Monitoring downstream signaling events, activation of p38 was mediated by Gi whereas ERK1/2 and Akt phosphorylation were mediated by Gi-coupled {beta}{gamma}. Activation of CREB integrated multiple components; Gs and {beta}{gamma} mediated [~]85% of the response, while [~]15% was attributed to Gi. 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-1, and TNF- 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.\n\nSignificance statementCannabinoid receptor 2 (CB2) is a G protein-coupled receptor which plays a complex role in immunomodulation and is a promising target in a range of disorders with immune system involvement. However, to date the majority of the studies in this field have been performed on cell lines, rodent models, or stimulated primary cells. Here we provide a detailed account of CB2-mediated signaling in primary human immune cells under conditions which closely mimic their in vivo state. We reveal a complex signaling system involving an unprecedented CB2 signaling pathway and leading to immunomodulatory functional outcomes. This work provides not only a critical foundation impacting CB2-targeted drug discovery, but reveals important wider considerations for GPCR signaling studies and model validity.\n\nTable of Contents Summary Figure\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC=\"FIGDIR/small/663831v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@599095org.highwire.dtl.DTLVardef@2331a9org.highwire.dtl.DTLVardef@917860org.highwire.dtl.DTLVardef@75e8f7_HPS_FORMAT_FIGEXP M_FIG C_FIG