Figure 6.

IL-4–mediated M2 polarization does not use oxidative stress. (A) Murine alveolar macrophages were incubated in the presence or absence of LFN (200 μM) for 2 hours and then treated with either vehicle or IL-4 (20 ng/ml). Immunoblot analysis for phosphorylated STAT6 (p-STAT6) in cell lysates was performed. Figure is representative of three experiments. (B) Macrophages were cultured in the presence or absence of IL-4 with DMSO or LFN (200 μM). Nuclear fractions were isolated, and immunoblot analysis was performed for H3K27 me3. Figure is representative of three experiments. (C) Murine alveolar macrophages were incubated in the presence or absence of LFN (200 μM) for 2 hours and then chrysotile. Immunoblot analysis for p-STAT6 in cell lysates was performed. Each lane in the figure was run on the same gel. Figure is representative of three experiments. (D) Macrophages were treated with IL-4 (20 ng/ml) or chrysotile (10 μg/cm²). Rate of H₂O₂ generation was measured by pHPA assay (n = 8); *P < 0.05 versus vehicle and IL-4. Macrophages were transfected with STAT6_WT or STAT6_C528S. After 24 hours, cells were cultured in the presence or absence of IL-4 for 4 hours. Total RNA was isolated, and (E) fizz1, (F) ym1, (G) tnf-α, and (H) il-1β gene expression was measured by quantitative RT-PCR (n = 3); *P < 0.00001 versus (−) in both groups, **P < 0.0001 versus all other groups. (I) Macrophages were transfected as described previously here. Lysates were subjected to His pulldown and immunoblot for V5 and p-STAT6 were performed. Figure is representative of three replicates. (J) Macrophages were treated with DMSO or LFN (200 μM), and then stimulated with IL-4 (20 ng/ml) for 4 hours. Total RNA was isolated, and mannose receptor and tnf-α gene expression were measured by quantitative RT-PCR (n = 5); *P < 0.05 versus DMSO (−IL-4), **P < 0.05 versus DMSO (+IL-4). Emp, empty; IB, immunoblot; PD, pull-down.