Figure 2.
BHA blocks M2 differentiation by inhibiting O2− generation. (A) Monocytes were either left untreated or pretreated with BHA for 1 h. Cells were then treated with GM-CSF or M-CSF and O2− generation was measured at the indicated times. (B) Monocytes were either left untreated or pretreated with BHA for 1 h and then differentiated for 6 days with GM-CSF or M-CSF, with or without H2O2 (0.001 mM). Representative light microscopy images of cells differentiated with GM-CSF or M-CSF are shown. (C, D) Monocytes were either left untreated or treated with apocynin (500 μM) or TEMPO (500 μM) for 1 h then differentiated for 6 days with GM-CSF or M-CSF. On day 6, GM-CSF-treated cells were treated with LPS and IFNγ (Μ1) for 24 h. M-CSF-treated cells were treated with IL-4 (M2) for 24 h. Flow analysis of M1 marker CD86 in GM-CSF-treated or polarized M1 cells (C) and M2 marker CD163 in M-CSF-treated or polarized M2 cells (D) are shown. Gray histogram represents unstained cells. (E, F) Detection of M1 cytokines (TNFα, IL-6) (E) and M2 cytokines (IL-10) and chemokines (CCL17, CCL18, CCL24) (F) by real-time PCR in M1 and M2 macrophages left untreated or pretreated with TEMPO or apocynin as compared to monocytes. G, GM-CSF; M, M-CSF; APO, apocynin. Error Bars: ± SEM, data from at least three independent experiments. *P < 0.05; **P < 0.01; #P < 0.001.