Figure 5.

p38 activation is required for the stimulatory effect of adenosine on alternatively activated macrophages. A) Adenosine increases p38 activation. Macrophages were treated with IL-4 in the presence or absence of adenosine (ado) for 20 min. p38 activation was determined from cell extracts using immunoblotting with antibodies raised against the active, doubly phosphorylated form of p38. β-Actin was used as internal control (con). Blots are representative of 3 separate experiments. B) Inhibition of the p38 pathway prevents the stimulatory effect of adenosine on arginase activity in IL-4-activated macrophages. RAW 264.7 macrophages were pretreated for 30 min with SB203580 (p38 pathway inhibitor) before adding 100 μM adenosine and 5 ng/ml IL-4. After 8 h of incubation, arginase activities were assessed. ***P < 0.001 vs. IL-4; ^##P < 0.01 vs. IL-4/adenosine. C) p38 pathway inhibition prevents the stimulatory effect of adenosine on TIMP-1 production by IL-4-stimulated macrophages. Peritoneal macrophages were pretreated for 30 min with SB203580 before adding adenosine and IL-4. After 24 h, supernatants were collected. TIMP-1 levels were assessed from the supernatants using ELISA. ***P < 0.001 vs. IL-4 alone; ^##P < 0.01 vs. ado + IL-4. Results are shown as means ± se and are representative of ≥3 experiments; n = 6/experiment.