PMC

Production of oxygen radicals by peritoneal macrophages after infection with S. pyogenes. Uninfected macrophages (A) or S. pyogenes-infected macrophages (B) were incubated with NBT for 45 min and examined by light microscopy. NBT precipitates as a blue-purple formazan when reduced by superoxide (red arrows). (C) Macrophages infected with green fluorescence-labeled S. pyogenes and incubated with NBT for 45 min. Production of ROS by macrophages is evidenced by black precipitation. (D) Colocalization of ROS and S. pyogenes (red arrows). Bars represent 15 μm in panels A to C and 5 μm in panel D.

Effects of S. pyogenes on the regulation of Nos2 and Arg2 gene transcription. (A) RT-PCR analysis of inflammatory gene transcription (Tnf-α, Mip-1α, and Mip-1β) in resident macrophages unstimulated (0 h) or stimulated with IFN-γ plus LPS for 1 h, 4 h, 6 h, or 16 h. Rsp9 expression served as an internal control (B) RT-PCR analysis of Nos2 gene transcription in resident macrophages uninfected or after 1 h, 4 h, or 16 h of infection with S. pyogenes. Macrophages stimulated with IFN-γ plus LPS were used as a positive control. Rsp9 expression served as an internal control. (C) RT-PCR analysis of Arg2 gene transcription in resident macrophages uninfected or after 1 h, 4 h, or 16 h of infection with S. pyogenes. Macrophages stimulated with IFN-γ plus LPS were used as a negative control. Rsp9 expression served as an internal control.

Levels of bacteria in the blood of mice at 24 h after intravenous infection with S. pyogenes. Wild-type, p47^phox−/− (A), or iNOS^−/− (B) mice were inoculated with 10⁵ CFU of S. pyogenes in 0.2 ml of PBS via a lateral tail vein. Viable bacterial counts were determined in the blood of infected mice by collecting blood samples from the tail vein at 24 h postinoculation. Each column represents the mean ± standard deviation of 10 mice per group. P < 0.0001 (ANOVA).

Transcriptional profile of resident murine macrophages after 1 h of infection with S. pyogenes. (A) Expression pattern of cDNAs representing differentially regulated genes analyzed by microarray. Gene regulation was expressed as signal log ratios (calculated by Affymetrix MAS5 software) from the comparison of infected versus uninfected control macrophages. Induced gene expression by S. pyogenes is indicated in red, whereas suppressed gene expression is indicated in green. The degree of red intensity represents the level of induction, whereas that of green intensity represents the level of repression. (B) After 1 h infection with S. pyogenes, more than 400 genes were found to be differentially expressed with respect to the uninfected state; 70% were induced while 30% were repressed.

Confirmation of microarray data by RT-PCR and protein expression. (A) RT-PCR analysis of selected gene transcription in resident macrophages uninfected or infected with S. pyogenes. Uninfected samples were loaded in lanes 1, and infected samples were loaded in lanes 2. β-Actin expression served as a control. (B) IL-6 protein expression by S. pyogenes-infected macrophages. Resident macrophages were isolated from the peritoneal cavity of mice after 1 h of infection with S. pyogenes and cultured in vitro for 2 h. Noninfected macrophages were used as a control. The levels of IL-6 in the supernatants were determined by ELISA. Each column represents the mean ± standard deviation of triplicate samples obtained from three independent experiments. P < 0.0001 (ANOVA).

Roles of iNOS and phagocyte oxidase in the killing of S. pyogenes by peritoneal macrophages. (A) Killing activity of S. pyogenes by peritoneal macrophages isolated from wild-type or p47^phox−/− mice. (B) Killing activity of S. pyogenes by peritoneal macrophages from wild-type or iNOS^−/− mice. Macrophages were isolated from the peritoneal cavity of mice after 1 h of intraperitoneal infection with 5 × 10⁷ CFU of S. pyogenes and were cultured at 37°C, 5% CO_2. At several time points, the macrophages were lysed with dH₂O, and surviving bacteria were enumerated by plating serial dilutions in blood agar. Data presented are means ± standard deviations for triplicate samples from one experiment representative of three independent determinations.