Figure 2
PARylated HMGB1 inhibits phagocytosis of apoptotic cells by macrophages more effectively than non-PARylated HMGB1. (A) Phagocytosis assays were performed after preincubation of apoptotic thymocytes for 2 h with 1 μg/mL recombinant HMGB1 or PARylated HMGB1. *P < 0.05 and **P < 0.01 compared with control BSA-treated thymocytes. #P< 0.05 compared with HMGB1-treated thymocytes. (B) After preincubation of thymocytes for 2 h with PARylated or unmodified HMGB1, phagocytosis assays were performed for 5 and 30 min. The macrophages were then washed three times, and cell lysates were prepared to determine Rac-1 activation. As a negative control, macrophages were preincubated with media in the absence of thymocytes. White bars represent unmodified HMGB1, and gray bars represent PARylated HMGB1; *P< 0.05 versus PARylated HMGB1. (C) Phagocytosis assays were performed after preincubation of macrophages for 2 h with 1 μg/mL recombinant HMGB1 or PARylated HMGB1. *P < 0.05 compared with control BSA-treated macrophages. #P< 0.05 compared with HMGB1-treated macrophages. (D.1) Phagocytosis assays were performed after preincubation of macrophages for 2 h with 1 μg/mL PARylated HMGB1 or free PAR and compared with control BSA-treated macrophages. (D.2) PARP-1 protein was incubated in a poly(ADP-ribosyl)ation reaction containing NAD and activated DNA for 30 min. PARG was then added to the reaction for 30 min. Separation of free PAR from PARP-1 was performed using Amicon Ultra 0.5 filters. A portion (10%) of the reaction mixture was used for immunoblot analysis with antibodies to PAR. A representative gel is shown. A second independent experiment provided similar results. All phagocytosis experiments were performed at least three independent times with similar results. (E) Apoptotic thymocytes (107) preincubated for 2 h with 2 ig HMGB1, PARylated HMGB1 or BSA were administered intratracheally in 50 μL PBS into anesthesized mice. BAL fluid was collected 90 min later. The samples were resuspended in PBS with 1% BSA and stained with FITC-CD11b Ab (macrophage marker) and APC-CD 90.2 Ab (thymocyte marker). Flow cytometry was performed, and the phagocytosis index was calculated as the ratio of FITC+PKH26+APC− cells to all cells gated. n = 3 mice in each group. *P < 0.05 compared with control. #P< 0.05 compared with HMGB1-treated thymocytes. A second independent experiment provided similar results.