Figure 8. Purified Hla induces eosinophil apoptosis.

Mixed granulocytes were cultured with a concentration response of purified S. aureus Hla and cell death assessed. A, B, In a single expt, cells were harvested after 1 (A) and 3 hours (B), stained with CD66c-PE, Annexin-FITC and ToPro-3 and analysed alongside Countbright beads by flow cytometry. Eosinophils were gated out as CD66c-low events. Charts show changes in total number of eosinophils (closed bar, right axis), percentage eosinophil Annexin positivity (open bars, left axis), neutrophil Annexin positivity (striped bars, left axis A only) and percentage eosinophil ToPro-3 positivity (shaded bars, left axis). C, D, In separate experiments, cells were harvested after 5 (C) or 24 hours (D) and eosinophils assessed by light microscopy for morphological changes of apoptosis. Neutrophil apoptosis was also assessed in these experiments (E). In separate experiments, eosinophils further purified by negative magnetic selection were also incubated with Hla for 5 (G) or 24 (H) hours and cells observed by light microscopy. Representative images shown in F. Data are shown as mean ±SEM from 4 (C–E) or 3 (G–H) independent experiments and statistically significant differences calculated by ANOVA with Dunnett's post-test (*p≥0.05, **p≥0.01, ** p≥0.001).