Alveolar macrophages require the second stimulation by ATP to release mature IL-1β after LPS. (A) Human alveolar macrophages were stimulated for 24 hours with RPMI, LPS (100 ng/mL), Pam3Cys (10 μg/mL), or S epidermidis (10⁶ organisms/mL). (B) Alveolar macrophages were stimulated with LPS (1 μg/mL) or a combination of LPS and ATP (1 mM). Intracellular pro-IL-1β after LPS stimulation after 4 hours, and the extracellular IL-1β release after stimulation with LPS or LPS/ATP, was assessed by ELISA. The stimulation experiments were performed in cells harvested from 5 volunteers (means ± SEM, *P < .05 compared with LPS stimulation alone). (C) Diagram representing the IL-1β activation pathways in monocytes and macrophages. Caspase-1 is constitutively activated in monocytes, and these cells release mature IL-1β after single stimulation with TLR ligands. IL-1β secretion is induced by endogenously released ATP. In contrast, macrophages need a double stimulation: one stimulus (TLR ligands) induces transcription, and a second stimulus (ATP) induces IL-1β secretion.