Figure 4

Chemotaxis towards CB₂ selective agonists is blocked by pertussis toxin, but not by CB₂ or CB₁ inverse agonists.

Bio-gel elicited PECs were incubated with either vehicle, (A) 200 ng/ml pertussis toxin (PTX) for 90 min or (B) 1 μM SR144528 for 30 min at 37 °C, 5% CO₂. Following treatment, cells were allowed to migrate for 3-4 hours at 37 °C, 5% CO₂ toward vehicle, 5 nM chemerin, 5 nM CCL5 or 10 μM cannabinoid. For experiments using SR144528, 1 μM was added in the lower chamber alongside the indicated chemoattractant. Data are mean ± SEM, (A) n = 4-11, (B) n = 3-10 biological replicates with 3-4 technical replicates per condition. (C,D) CHO-K1 cells expressing human CB₂ were incubated for 30 min at 37 °C, 5% CO₂ with either vehicle or SR144528 (1 μM - final assay concentration). Following treatment, vehicle, JWH133 (C) or HU308 (D) was added in assay buffer containing forskolin (FSK – 20 μM final assay concentration) to give the indicated final concentrations. The cells were then incubated for 30 min at 37 °C, 5% CO₂ before the addition of the detection reagents. Data are mean ± SEM, n = 6. (E) Enriched macrophages were stimulated with either vehicle, or 10 μM cannabinoid in the presence and absence of 1 μM SR144528. Relative levels of phosphorylated ERK1/2 and total ERK1/2 were determined by western blotting. Blot is representative of n = 3 independent biological replicates. (F) Bio-gel elicited murine PECs were incubated with either vehicle or 1 μM SR141716A for 30 min at 37 °C, 5% CO₂. Following treatment, cells were allowed to migrate for 3-4 hours at 37 °C, 5% CO₂ toward vehicle or 10 μM cannabinoid. SR141716A at 1 μM was added in the lower chamber alongside the indicated cannabinoid. Data are mean ± SEM, n = 4 biological replicates with 3-4 technical replicates per condition. For all data, statistical analysis was conducted by two-way ANOVA with Sidak’s multiple comparisons correction. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, for indicated comparisons. ††† P < 0.001, compared to vehicle + FSK alone.