Fig. 3.

Temporal macrophage polarization and lipid mediator pathway induction. Human monocytes were differentiated by GM-CSF or M-CSF (20 ng/ml, each) for 6 days. Cells were either polarized with 100 ng/ml LPS plus 20 ng/ml IFN-γ (M_GM-CSF) to obtain M1 or with 20 ng/ml IL-4 (M_M-CSF) to obtain M2. After the indicated times, cells were analyzed for a surface expression of polarization markers for M1 (CD54, CD80) and M2 (CD206, CD163) at 0–72 h intervals by flow cytometry; representative histograms from n = 4 separate donors, b expression of 5-LOX, 15-LOX-1, FLAP, and β-actin by western blot (upper panel), and densitometric analysis thereof (lower panels). Results are given as means ± S.E.M., n = 4 separate donors, *P < 0.05, **P < 0.01, ***P < 0.001 M1 vs. M2 as determined by two-tailed t test. c Lipid mediator formation after incubation of M1 and M2 with E. coli (O6:K2:H1; ratio 1:50) in PBS + Ca/Mg for 90 min at 37 °C after the indicated time points during polarization; formed lipid mediators were isolated by SPE and analyzed by LC–MS–MS. Results are given as means ± S.E.M., n = 4 (0 and 6 h), n = 5 (24 and 72 h), and n = 7 separate donors (48 h). *^,#P < 0.05; **^,##P < 0.01; ***^,###P < 0.001 vs. 0 h, data were log transformed for statistical analysis using one-way ANOVA with Bonferroni multiple comparisons test