Figure 3
Only a subset of CB2 agonists are primary macrophage chemoattractants.
(A-C) Bio-gel elicited murine PECs (4 × 105) 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 chemokine or 10 μM cannabinoid. (A) Chemotaxis was measured as maximum cell index minus starting cell index and data are displayed as a fold change compared to cells migrating towards vehicle. Only CCL5, JWH133, HU308, L-759,656 and L-759,633 acted as chemoattractants. 2-AG was used at a concentration of 15 μM. Data are mean ± SEM, n = 3-26 biological replicates with 3-4 technical replicates per condition. (B and C) Representative traces of chemotaxis positive and chemotaxis negative CB2 agonists. Data show mean trace of 3-4 technical replicates per condition. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction. ns P > 0.05, *** P < 0.001, comparing all samples to vehicle control. (D,E) Bio-gel elicited PECs (5 × 104) 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. (D) Concentration response quantification demonstrates that L-759,656 caused cytoskeletal rearrangement in a concentration dependent manner, with an EC50 value of 7.3 μM. In contrast, (D) CP55,940, AM1241, (E) GP1a and WIN55,212-2 failed to elicit a response at any concentration (JWH133 curve is shown for comparison). Data are mean ± SEM, n = 3 biological replicates with 2-3 technical replicates per condition.
