Subcellular distribution of TRAIL /Apo-2L in neutrophils. (A) Intracellular neutrophil TRAIL /Apo-2L levels decrease after BCG stimulation. Neutrophils (10⁶/mL) were stimulated in vitro with BCG (1 CFU/cell) for 4 hours, then intracellular TRAIL/Apo-2L levels were determined as described in “Materials and methods.” (B) Inhibition of protein and RNA synthesis does not reduce TRAIL/Apo-2L release from BCG-stimulated neutrophils. Neutrophils (3 × 10⁶/mL) were treated with cycloheximide (10 μg/mL; CHX-BCG) or actinomycin-D (1 μg/mL; ActD-BCG) for 1 hour before stimulation with BCG (1 CFU/cell). TRAIL/Apo-2L levels were measured in the culture supernatant at 4, 12, and 24 hours after adding BCG. (C) Neutrophils (200 × 10⁶) were isolated from peripheral blood and equally aliquoted into 2 groups: one placed into culture for 4 hours without any stimulus, and the other stimulated with BCG for 4 hours. The cells were then fractionated by nitrogen cavitation and density gradient centrifugation as described in “Materials and methods.” The amount of TRAIL/Apo-2L protein in each fraction was quantitated by ELISA. Similar results were obtained in two independent experiments.