Figure 4.

TGF-b1 shifts CD11c-DNR macrophages from canonical to alternate Smad signaling and increases Aβ phagocytosis in vitro. (a) Primary microglia (MG) or macrophages (MΦ) from wild-type or CD11c-DNR mice went untreated or were treated for 30 min with a dose range of recombinant TGF-β1 (1, 5 or 10 ng/ml as indicated) with or without 50 ng/ml of LPS. Cell lysates were western blotted for phosphorylated (p) and total Smad2/3 proteins as an indicator of canonical TGF-β-activated signaling. (b) Primary macrophages were treated as above and western blotted for phosphorylated and total Smad1/5/8 or p21-activated kinase 2 (Pak2; both activated in the alternate Smad signaling pathway), or extracellular signal-regulated kinase (Erk) 1/2. (c) Primary macrophages were pulsed for 4 h with 2 μg/ml of preaggregated Aβ₄₈₈ and chased for 15 min before analysis by confocal microscopy with antibodies to CD11b or CD11c (merged images are shown on the right). (d) Quantification of confocal images (n = 3 randomly-selected fields per group) was performed, and Aβ₄₈₈₋ labeled area is shown on the left. Numbers of Aβ₄₈₈ phagocytic cells per field are shown in the middle graph. Data are represented as group means ± s.d. Cell lysates were prepared from macrophages treated in parallel with 2 μg/ml of unlabeled human synthetic Aβ_1–42, and 2 ng of the peptide (cell-free) was western blotted side-by-side with antibody 6E10 (right). Data shown in (a–d) are representative of three to four independent experiments in which similar results were obtained.