Figure 8.

Analysis of expression of activation surface markers MHC class II and CD86 on macrophages with knockdown of Egr2 under inflammatory conditions in vitro and in vivo. Bone-marrow-derived macrophages (BMDMs) from B6 (A) or DsRed transgenic (B) mice were transfected with Egr2 siRNA or Control siRNA for 24 h, and after which the cells were used as unstimulated or activated in vitro with IL-4 or IFNγ for another 24 h-time period (A) or in vivo for 4 days in the model of thioglycollate-induced inflammation as described in Materials and Methods. (A) After in vitro incubation in media, IL-4 or IFNγ, the cells were washed, stained for surface markers F4/80, MHC class II and CD86 and F4/80⁺ gated macrophages were analyzed for the expression of MHC class II and CD86 by three-color follow cytometry as described in Materials and Methods. The expressions for MHC class II (top histograms) and CD86 (bottom histograms) of untreated (left histograms) or activated with IL-4 (middle histograms) or IFNγ (right histograms) macrophages transfected with Egr2 siRNA (solid line) vs. control siRNA (dotted line) are shown on representative histogram graphs. Staining with isotype-matched control mAbs is shown by shaded histograms. (B) The transfected DsRed-positive macrophages were injected i.p. into a group of 4-5 mice and peritoneal inflammation was induced by injection of thioglycollate medium as described in Materials and Methods. On day 4 after induction of inflammation, the cells were isolated by peritoneal lavage and cells were washed, stained for surface markers F4/80, MHC class II and CD86. F4/80⁺DsRed⁺ gated macrophages were analyzed for the expression of MHC class II and CD86 by four-color follow cytometry as described in Materials and Methods. The expressions for MHC class II (left histograms) and CD86 (right histograms) on F4/80⁺DsRed⁺ macrophages transfected with Egr2 siRNA (solid line) vs. control siRNA (dotted line) are shown on representative histogram graphs. Staining with isotype-matched control mAbs is shown by shaded histograms. (C) In (A,B), quantifications and statistics are shown in Table 2.