Figure 5.

Effect of viable and necrotic neutrophil augmentation on phagocytosis of FITC-labeled M. bovis BCG, and differential effect of necrotic neutrophil augmentation versus supernatant of necrotic neutrophils on cytokine release in blood. (A–C) Blood from six donors (four independent experiments) was augmented with viable neutrophils, necrotic neutrophils, or medium alone, infected with 2 × 10⁵ CFU FITC-labeled M. bovis BCG and incubated for 1 h at 37°C. 2 × 100 mcl aliquots were taken from each sample, incubated with CD14-PE and Viability Dye for 20 min, red blood cells were lysed, trypan blue was added to quench extracellular fluorescence and samples fixed in 2% paraformaldehyde before acquisition on a BD Fortessa flow cytometer. Results are presented as the percentage of BCG-FITC+ events (A), the percentage of BCG-FITC+ events associated with CD14+ cells (B), and the percentage of BCG-FITC+ events associated with neutrophils [as defined by forward and side scatter among CD14 negative events (C)]. (D,E) Blood from 17 donors (4 independent experiments) was augmented with necrotic neutrophils, the supernatant of necrotic neutrophils, or medium alone, infected with 2 × 10⁵ CFU M. bovis BCG and incubated for 96 h at 37°C. Samples were centrifuged and supernatants stored at −80°C until analysis by ELISA for tumor necrosis factor (TNF) (A) or interleukin-10 (B). Lines represent means, *p < 0.05 **p < 0.01, ***p < 0.001.