Abstract
BackgroundCannabinoid receptor 2 (CB2) is expressed predominantly in the immune system, particularly in plasma cells, raising the possibility that targeting the CB2 pathway could yield an immunomodulatory effect. Although the role of CB2 in mediating immunoglobulin class switching has been reported, the effects of targeting the CB2 pathway on immunoglobulin secretion per se remain unclear.MethodsHuman B cell line SKW 6.4, which is capable of differentiating into IgM-secreting cells once treated with human IL-6, was employed as the cell model. SKW 6.4 cells were incubated for 4 days with CB2 ligands plus IL-6 (100 U/ml). The amount of secreted IgM was determined by an ELISA. Cell proliferation was determined by the 3H-Thymidine incorporation assay. Signal molecules involved in the modulation of IgM secretion were examined by real-time RT-PCR and Western blot analyses or by using their specific inhibitors.ResultsWe demonstrated that CB2 inverse agonists SR144528 and AM630, but not CB2 agonist HU308 or CB1 antagonist SR141716, effectively inhibited IL-6-induced secretion of soluble IgM without affecting cell proliferation as measured by thymidine uptake. SR144528 alone had no effects on the basal levels of IgM in the resting cells. These effects were receptor mediated, as pretreatment with CB2 agonist abrogated SR144528-mediated inhibition of IL-6 stimulated IgM secretion. Transcription factors relevant to B cell differentiation, Bcl-6 and PAX5, as well as the protein kinase STAT3 pathway were involved in the inhibition of IL-6-induced IgM by SR144528.ConclusionsThese results uncover a novel function of CB2 antagonists and suggest that CB2 ligands may be potential modulators of immunoglobulin secretion.
Highlights
Cannabinoid receptor 2 (CB2) is expressed predominantly in the immune system, in plasma cells, raising the possibility that targeting the CB2 pathway could yield an immunomodulatory effect
To explore the role of CB2 receptor signaling in the immunoglobulin production in plasma cell, we employed the human B cell line SKW 6.4 to investigate the effects of CB2 ligands on cytokine-induced immunoglobulin M (IgM) production
IL-6 induces IgM secretion in a concentration-dependent manner Upon stimulation with IL-6, 12-O-tetradecanoylphorbol13-acetate (TPA) or lipopolysaccharide (LPS), B cells are activated, rapidly proliferate and initiate differentiation processes leading to increased antibody secretion, e.g., immunoglobulin M (IgM) [19,21,22,25,26]
Summary
Reagents and cell culture Recombinant human IL-6 was purchased from GenScript (Piscataway, NJ). Cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 100 U/mL penicillin–streptomycin (Sigma-Aldrich) at 37°C with 5% CO2. Determination of IgM secretion Supernatants of SKW 6.4 cell cultures were harvested by centrifugation for the IgM enzyme-linked immunosorbent assay (ELISA). Each supernatant sample containing IgM was added into the well of the microtiter plates that have been coated with anti-human IgM antibodies. Anti-IgM antibody conjugated with horseradish peroxidase was added to the well and the plates were allowed to incubate for another 0.5 hr. The cells were cultured in 96-well culture plates in RPMI-1640 medium containing 10% FBS with or without drugs for 72 hours at 37°C with 5% CO2. Results were considered significantly different for P < 0.05
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