ATP-derived adenosine modulates inflammatory macrophage activation. (A) Wild-type BMDMs were stimulated with LPS in the absence or presence of 100 μM ATP or nonhydrolyzable ATPγs and TNF-α (black bars), IL-12/23p40 (striped bars), and IL-10 (gray bars) production was measured by ELISA 8 hours later. The means of 3 independent experiments ± SEM are shown. (B) Levels of TNF-α (black bars), IL-12/23p40 (striped bars), and IL-10 (gray bars) produced by wild-type BMDMs stimulated with LPS in the absence or presence of 100 μM ATP, ADP, AMP, adenosine (Ado), clodronate (Clod.), pyrophosphate, or phosphate. Cytokine levels were measured by ELISA after 8 hours. The means ± SD of triplicate determinations are shown and are representative of 3 independent experiments. Statistical analysis is relative to samples stimulated with LPS alone. (C) The kinetics of ¹⁴C-ADP hydrolysis by resting macrophages over time (left) was determined by TLC. Hydrolysis controls consist of apyrase alone for 5 min (Apy) or apyrase in the presence of alkaline phosphatase (Apy/AP) and ¹⁴C-ADP (right). Data are representative of at least 3 independent experiments. (D-F) Levels of TNF-α (D), IL-12/23p40 (E), and IL-10 (F) produced by wild-type BMDMs stimulated with LPS in the absence or presence of 100 μM ATP or adenosine in the absence or presence of 1 U/mL ADA. Cytokines were measured 8 hours after stimulation by ELISA. The means ± SD of triplicate determinations are shown and are representative of 3 independent experiments.