Figure 4

MFG-E8 deficiency leads to accumulation of death cells. (a) Apoptotic leukocytes in BALF and peritoneum of Mfge8^−/− and WT mice on 5 days after pristane exposure were stained with Annexin V/PI and determined by flow cytometry (n=6–8 mice per group). (b) Infiltration of death cells into the Mfge8^−/− and WT lungs after a 2-week pristane exposure was identified. Representative images were shown, original magnification × 200. The average TUNEL-positive cells were counted at more than five random microscopic fields. (c) For further estimate apoptosis, the lung tissues were subjected to western blotting to detect MFG-E8, Bcl-2, Bcl-xL and Bax protein levels. Mouse Bcl-2, Bcl-xL and Bax were detected as ~27, ~29 and ~20 kD protein, respectively, in SDS-PAGE. Results were normalized with β-actin, and Bcl-2/Bax ratios were evaluated and displayed. Data are presented as means±S.E.M., **P<0.01,***P<0.001 versus PBS treatment; ^#P<0.05 versus WT with pristane treatment. (d) Apoptotic BMDNs were stained with Annexin V/PI and detected by flow cytometry. (e) Peritoneal macrophages from the Mfge8^−/− mice showed impairment of phagocytosis. Isolated peritoneal macrophages from Mfge8^−/− and WT mice were stained with PI and apoptotic BMDNs were stained with green fluorescence PKH67. Representative images were shown, original magnification × 400. The phagocytic index was evaluated by phagocytotic macrophages/total macrophages per field. The average phagocytosis was counted at more than five random microscopic fields. Data are means±S.E.M. from more than two independent experiments, *P<0.05