Figure 7

Neutrophil activation triggered by mtDNA and fMLF. (A, B) C57BL/6 mice were injected with normal saline, fMLF (1 μM, 200 μl), mtDNA (5 μg), mitochondria (200 μg) and sacrificed 24 h after injection. n = 3 per group. HE staining of representative lung sections was performed. Scale bar, 50 μm (A). The esterase staining of neutrophils and the staining of Gr-1⁺ cells were counted in ten HPFs (B). (C-F) Isolated murine neutrophils (5 × 10⁶) were treated in vitro for 2 h with fMLF (1 μM), mtDNA (5 μg/ml) and mitochondria (200 μg/ml). The release of MPO and elastase in the supernatant of the treated neutrophils was determined (C). MMP-8 was determined by immunoblot in supernatants of the treated neutrophils (F: fMLF, D: mtDNA, D+F: mtDNA and fMLF, Mito: mitochondria). The relative intensity of MMP-8 was quantified (D). Activation of p38 MAPK in neutrophils induced by mtDNA. Bar graph shows the relative intensity of pp38 to p38 (E). Neutrophil chemotaxis assays were performed using a modified Boyden chamber with fMLF (1 μM) and mtDNA (5 μg/ml). The numbers of cells that migrated were counted in ten HPFs (F). Data are mean ± SEM; n = 3.^*P < 0.05,^***P< 0.001 by Student's t-test.