Figure 2
Macrophages are the dominant cell population in bio-gel elicited PECs that migrate toward CB2 agonists.
(A) Flow cytometry analysis demonstrates that neutrophils (F4/80-, 7/4+) are the major cell population within bio-gel elicited PECs, whereas macrophages (F4/80+, 7/4-) and monocytes (F4/80+, 7/4+) form only a minor population. (B) Bio-gel elicited PECs were placed into petri dishes and allowed to adhere for 2 hours at 37 °C, 5% CO2 prior to washing with PBS. Cells were then left overnight at 37 °C, 5% CO2 and remaining adherent cells removed using PBS containing 10 mM EDTA. Flow cytometry analysis of these cells after staining with F4/80 and 7/4 demonstrates that this process strongly enriches macrophages and removes neutrophils. (C) Enriched macrophages (2 × 105) were placed into the upper chamber of a CIM-16 plate and allowed to migrate for 4 hours at 37 °C, 5% CO2 toward vehicle (0.3% DMSO), 5 nM chemokine or 10 μM cannabinoid. Real-time chemotaxis analysis demonstrates that JWH133 and HU308 caused strong directed migration of this macrophage population. Data show mean trace of 3-4 technical replicates per condition. (D) Flow cytometry analysis using F4/80 and 7/4 demonstrates that macrophages are the main cell population elicited by intraperitoneal injection of 4% thioglycollate. (E,F) Thioglycollate elicited macrophages (2 × 105) were placed into the upper chamber of a CIM-16 plate and allowed to migrate for 4 hours at 37 °C, 5% CO2 toward vehicle (0.3% DMSO) or 10 μM cannabinoid. (E) Representative real-time chemotaxis trace of thioglycollate elicited macrophage migration towards JWH133, HU308 or vehicle. Data show mean trace of 3-4 technical replicates per condition. (F) Quantification of chemotaxis demonstrates that JWH133 and HU308 significantly induced thioglycollate elicited macrophage chemotaxis. Data are mean ± SEM, n = 4-6 biological replicates with 3-4 technical replicates per condition. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction. * P < 0.05, ** P < 0.01, comparing all samples to vehicle control.
