Figure 3.

A_2B and, to a lesser extent, A_2A receptors mediate the stimulatory effect of adenosine on IL-4-induced arginase activity and TIMP-1 production. A) Effect of adenosine receptor agonists on IL-4-induced arginase activity in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated with IL-4 and treated with adenosine receptor agonists for 8 h, and then arginase activity was measured from the cell lysate. ***P < 0.001 vs. vehicle. B) Effect of adenosine receptor agonists on IL-4-stimulated TIMP-1 release by peritoneal macrophages. Peritoneal macrophages were treated with IL-4 and adenosine receptor agonists for 24 h, and TIMP-1 concentrations were determined in the supernatants. *P < 0.05, ***P < 0.001 vs. vehicle. C, D) Adenosine (C) and NECA (D) enhance TIMP-1 production by IL-4-challenged WT and, to a lesser extent, A_2A-KO mice. Both adenosine and NECA are ineffective in A_2B-KO peritoneal macrophages. A_2A-KO, A_2B-KO, and WT peritoneal macrophages were stimulated with 5 ng/ml IL-4 and treated with adenosine (C) or NECA (D) for 24 h, and then TIMP-1 levels were measured from the supernatants using ELISA. *P < 0.05, **P < 0.01 vs. IL-4. E) Pharmacological A_2B receptor blockade using PSB0778 prevents the NECA enhancement of arginase activity in IL-4-stimulated RAW 264.7 macrophages. RAW 264.7 macrophages were pretreated for 30 min with 10–1000 nM PSB0778; following this time period, cells were treated with 300 nM NECA immediately before IL-4 stimulation. Cells were incubated for an additional 8 h, at which point cell lysates were prepared for arginase measurements. **P < 0.01 vs. IL-4; ^#P < 0.05, ^##P < 0.01 vs. IL-4/NECA. F) Pharmacological A_2B receptor blockade inhibits the NECA augmentation of TIMP-1 production by IL-4-induced WT and A_2A-KO macrophages. WT and A_2A-KO peritoneal macrophages were pretreated for 30 min with 100 nM PSB0778; following this time period, cells were treated with 300 nM NECA immediately before IL-4 stimulation. Cells were incubated for an additional 24 h, at which point supernatants were harvested. TIMP-1 levels were measured from the supernatants using ELISA. ***P < 0.001 vs. vehicle; ^###P < 0.001 vs. IL-4; ^$$$P < 0.001 vs. IL-4/NECA. Results are shown as means ± se and are representative of ≥3 experiments; n = 6/experiment.