Neutrophils contain a cleaved form of TRAIL/Apo-2L that retains tumoricidal activity when released into culture supernatant after BCG stimulation. (A) Freshly isolated neutrophils were lysed and TRAIL/Apo-2L was detected by immunoblotting. Full-length TRAIL/Apo-2L from Ad5-TRAIL–infected cells and recombinant soluble TRAIL/Apo-2L protein were used as reference. SDS-PAGE was performed under nonreducing conditions. (B) Cell-mediated tumoricidal activity of BCG-stimulated neutrophils (PMN). Neutrophils were cultured for 20 hours in the absence or presence of BCG (1 CFU/cell), and then added to ⁵¹Cr-labeled RT-4 target cells at the indicated effector-target cell ratios for 16 hours. Inclusion of the pan-caspase inhibitor zVAD-fmk (20 μM) inhibited killing of RT-4 target cells. (C-F) Conditioned medium from unstimulated or BCG-stimulated neutrophils was added to RT-4 cells, after which cell death and caspase-8 activation were determined. (C) Crystal violet staining was performed to determine cell death after 24 hours, and a portion of the BCG-stimulated neutrophil supernatant was immunodepleted of TRAIL/Apo-2L, as described in “Materials and methods,” and the resultant killing returning to baseline levels. (D) RT-4 cells were harvested after 8 hours and caspase-8 cleavage was determined by immunoblotting. (E) RT-4 cells were harvested after 6 hours and assayed for caspase-8 activity using carboxyfluorescein-labeled LETD fluoromethyl ketone. (F) The addition of the pan-caspase inhibitor zVAD-fmk (20 μM) blocked cell death back to control levels. Data are representative of 3 independent experiments showing similar results.