Figure 5
JWH133, HU308 and L-759,656 act as chemoattractants for CB2-/- primary murine macrophages.
Bio-gel elicited murine PECs from CB2-/- mice were placed into the upper chamber of a CIM-16 plate and allowed to migrate for 3-4 hours at 37 °C, 5% CO2 toward vehicle (0.3% DMSO), 5 nM CCL5 or 10 μM cannabinoid. (A) Representative trace showing that the chemotaxis-positive CB2 agonists remain chemoattractants for CB2-/- murine macrophages. (B) Quantification of chemotaxis demonstrates that CCL5 (5 nM), JWH133, HU308 and L-759,656 (all 10 μM) significantly induced CB2-/- macrophage chemotaxis. Data are mean ± SEM, n = 5-15 biological replicates with 3-4 technical replicates per condition. (C) Pre-treatment of CB2-/- bio-gel elicited macrophages with PTX (200 ng/ml, 90 min at 37 °C, 5% CO2) significantly reduced JWH133 and HU308-induced chemotaxis (both 10 μM). Data are mean ± SEM, n = 3 biological replicates with 2-3 technical replicates per condition. (D) Bio-gel elicited CB2-/- macrophages were stimulated with either vehicle (0.3% DMSO) or 10 μM cannabinoid. Relative levels of phosphorylated ERK1/2 and total ERK1/2 were determined by western blotting and densitometric analysis confirmed that JWH133 and HU308 significantly elicited ERK1/2 phosphorylation. Data are mean ± SEM, n = 6 biological replicates. A representative phospho-ERK1/2 blot is shown as an insert. (E,F) Bio-gel elicited CB2-/- macrophages were placed into a 96 well E-plate allowed to adhere for 2-3 hours at 37 °C, 5% CO2. Afterwards, cells were stimulated with either vehicle (0.3% DMSO) or cannabinoid (10 μM). (E) Representative trace showing the kinetics of CB2 agonist-induced cytoskeletal rearrangements. (F) Quantification demonstrates that JWH133 and HU308 significantly elicited cell spreading in CB2-/- murine macrophages. Data are mean ± SEM, n = 4-5 biological replicates with 3-4 technical replicates per condition. For B,D,F statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction. For C statistical analysis was conducted by two-way ANOVA with Sidak’s multiple comparisons correction * P < 0.05, ** P < 0.01, *** P < 0.001, comparing all samples to vehicle control.
