Serum AAT confers resistance to STS in human neutrophils.

A. Neutrophils from healthy volunteers (2 × 10⁶/ml) were cultured in medium supplemented with different concentrations of plasma-derived human AAT (range 0–2 mg/ml) and treated with STS (0.2 μM). After 18 h, apoptosis was quantified by propidium iodide staining and FACS analysis. Percentage of apoptotic cells displaying fragmented DNA is depicted. Results are presented as mean ± SEM of three independent experiments. *p<0.05 (t-test). B. Neutrophils cultured in medium supplemented with the albumin-depleted fraction B11 and the serum fraction depleted for Ig for 9 times (3%), which have been shown to not confer resistance to STS, were additionally pre-incubated with 2 mg/ml plasma-derived AAT. After 1 h, STS (0.2 μM) was added and cells were further cultured overnight. Data of three independent experiments are expressed as fold changes vs. control cells cultured in the presence of patient serum. No significant differences could be found. C. Pooled patient serum was first diluted to a final concentration of 3 mg protein/ml. Then, serum was reduced for AAT by using an AAT-specific antibody and AAT decrease was further confirmed by Elisa. Empty beads were used as control. AAT concentrations are depicted in the table on the right. Patient serum, AAT-reduced serum (each 1%) with or without plasma-derived human AAT (native AAT; 2 mg/ml) were added to the culture medium of neutrophils (2 × 10⁶/ml). After treatment with STS (0.2 μM) for 18 h, apoptosis rate was quantified. Fold changes in apoptosis vs. control cells are displayed. Graphs show results of four independent experiments. **p<0.01 vs. untreated control; §p<0.05 (one-way ANOVA with Newman keuls post-hoc test).