STS resistance does not depend on immunoglobulins or Fc receptor-triggered pathways.
A. Pooled patient serum was subjected to serial depletion of immunoglobulins (Ig) using Protein G Plus/Protein A Agarose. Beads supernatant was collected after each depletion step. After 10 depletion steps, neutrophils from healthy volunteers (2 × 106/ml) were cultured in the presence of the Ig-depleted fractions and tested for intrinsic apoptosis resistance. Cells were treated with STS (0.2 μM) for 18 h and apoptosis was determined by flow cytometry. Data are presented as means ± SEM of four to eight independent experiments. *p<0.05, **p<0.01, ***p<0.001 (t-test). B. Serum depleted for Ig for 8 times was subjected to SDS-PAGE and the gel was stained with Coomassie Brillant Blue (left). In addition, immunoblot analysis using Ig-specific antibody was performed (right). Arrows indicate the light and heavy chain of Ig. C. Beads used for the 8th depletion step were washed with Tris-HCl (25 mM, pH 7.5) and Tris-NaCl (25 mM Tris-HCl pH 7.5, 1 M NaCl), respectively, and supernatants were collected. Neutrophils were cultured in the presence of Ig-depleted serum, Tris-eluted supernatant (each 1%) and STS (0.2 μM) as already described. The percentage of cells displaying fragmented DNA (% subG1) is depicted. Graphs show means ± SEM of four independent experiments. *p<0.05 (t-test). D. Neutrophils were cultured in the presence of three different concentrations of Fc receptor blocker, using a peptide based technology, and patient serum (1%) and further treated with STS (0.2 μM). Relative apoptosis rate vs. control sample without Fc receptor blocker and STS is displayed. Data (means ± SEM) from five independent experiments are presented. No significant differences in apoptosis could be found when compared to the STS-treated control without Fc blocker (t-test).