Figure 5. Macrophage ApoE production enhances muscle debris phagocytosis.
(a) Quantification of in vivo phagocytosis, by assessment of the numbers of infiltrated necrotic myofibers versus total necrotic myofibers infiltrated or not on day 4 post injury. Scale bar, 100 μm. Data are means+s.e.m. of six mice per strain in two independent experiments. (b) Phagocytosis activity of mononuclear phagocytes extracted from injured-muscle of WT, Cx3cr1−/−, Ccl2−/− and Cx3cr1−/− Ccl2−/− mice on day 4 post injury was assessed by incubating muscle cells for 4 h with red fluorescent microspheres. This parameter is expressed as the number of cells that engulfed at least one bead per 10,000 cells. Results are means+s.e.m. of six mice in three independent experiments. (c) Phagocytosis activity of Ccl2−/− muscle mononuclear phagocytes in the absence or presence of exogenous human APOE3 protein. Results are mean±s.e.m. of five mice in two independent experiments. (d) Quantification on day 4 post injury of Ccl2−/− myofiber removal in the absence (n=2) or presence (n=4) of exogenous human APOE3 protein. (e) Phagocytosis activity of mononuclear phagocytes from injured-muscle of WT, Cx3cr1−/− and Ccl2−/− mice on day 4 post injury was assessed 16 h after incubation of muscle cells with CX3CL1 or CCL2 chemokines. The numbers of muscle cells that engulfed at least one fluorescent microsphere were compared between treated and untreated cells for all mice strains. (f) Effect of CX3CL1 and CCL2 on IL-1, IL-1 receptor antagonist (Ra) and iNOS (nitric oxide synthase) expression was measured by real-time PCR in mononuclear phagocytes extracted from WT mice incubated during 16 h with CX3CL1 (100 nM) or CCL2 (100 nM), and compared with untreated cells. Data are mean+s.e.m. of six mice per strain in three independent experiments. NS, not significant, *P<0.05, **P<0.01 and ***P<0.001.