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. Author manuscript; available in PMC: 2013 Jan 16.
Published in final edited form as: J Immunol. 2011 Apr 20;186(11):6427–6436. doi: 10.4049/jimmunol.1003449

FIGURE 6.

FIGURE 6

PMNs require MMP9 for intracellular antimicrobial activity. A, S. pneumoniae was incubated with APMA-activated MMP2 and MMP9 in HBSS–10 mM HEPES for 60 min at 37°C. Bacteria alone (Input) and APMA-containing buffer used to activate MMP2 and MMP9 (Vehicle) serve as controls. Data are expressed as CFU (representative of three independent experiments; mean ± SEM). B, Peritoneal PMNs (Mmp9−/−, Mmp2−/−, and WT) were incubated with S. pneumoniae at a 1:2 ratio at 37°C for 60 min, and S. pneumoniae CFU were determined as described in Fig. 4 (n = 4 mice per group; mean ± SEM). *p = 0.0006 (WT and Mmp9−/−); NS, p = 0.3 (Mmp2−/− and WT) (Student t test). C, Peritoneal PMNs (Mmp9+/+ and Mmp9−/−) were incubated with S. pneumoniae in HBSS–10 mM HEPES without serum for 20 min at 37°C and washed with PBS three times (at time 0). S. pneumoniae (CFU) in lysed PMNs were determined at indicated time points (0, 1, and 2 h). Data are representative of three independent experiments (n = 3 mice per group; mean ± SEM). *p < 0.05. D and E, Superoxide production as a function of time (D) and total superoxide production (E) for Mmp9+/+ and Mmp9−/− PMNs were measured after incubation with IgG opsonized S. pneumoniae and IgG opsonized latex beads (3 μM) in PBS without antibiotics at 37°C for 1.5 h. Data are representative of three independent experiments (n = 4 mice per group; mean ± SEM). *p < 0.05 (versus Mmp9+/+ PMNs with IgG opsonized S. pneumoniae incubation), **p < 0.001 (versus Mmp9+/+ PMNs with IgG opsonized latex beads incubation) (t test). F, Flow cytometry of apoptosis analysis after 60-min coincubation of S. pneumoniae and peritoneal PMNs of WT and Mmp9−/− mice at 37°C; assay with annexin V/PI staining to measure the percentage of early apoptotic cells (annexin V+/PI−) and late apoptotic death/necrotic cells (annexin V+/PI+). Numbers in quadrants indicate percentage of cells in each. G, Average frequency of late apoptotic death/necrotic (annexin V+/PI+) Mmp9−/− PMN cells with S. pneumoniae incubation. Data are representative of three independent experiments (n = 4 mice per group; mean ± SEM). *p < 0.05 (versus Mmp9+/+ PMNs coincubation with S. pneumoniae). SP, S. pneumoniae.