Specific agonists to TLR2 or TLR4 and purified mycobacterial-cell wall stimulated TRAIL/Apo-2L release from neutrophils. (A) Human neutrophils were isolated from blood of healthy donors and placed into culture (3 × 10⁶/mL) with or without IFN-α (1000 U/mL) for 20 hours, and then stimulated with either BCG (1 CFU/cell) or the TLR agonists Pam3CSK, poly I:C, or LPS, at the indicated concentrations, for an additional 8 hours. TRAIL/Apo-2L levels were then measured in the culture supernatant by ELISA. (B) Human neutrophils placed into culture (3 × 10⁶/mL) with BCG (1 CFU/cell), irradiated M tuberculosis whole cells, M tuberculosis whole-cell lysate (WCL), M tuberculosis cell wall (27 000g) fraction, M tuberculosis cytosol fraction, and LAM of M tuberculosis, at the indicated concentrations, for 24 hours. The culture supernatant was assayed for TRAIL/Apo-2L levels by ELISA. ELISA results in panels A-B are averaged from 3 different donors. (C) Neutrophils (3 × 10⁶/mL) were stimulated in vitro with BCG (1 CFU/cell) or E coli (1 CFU/cell), and TRAIL/Apo-2L levels were measured in the culture supernatant over a 24-hour period. Results are averaged from 3 different donors.