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. 2012 Jan;26(1):376–386. doi: 10.1096/fj.11-190934

Figure 1.

Figure 1.

Adenosine (ado) augments alternative macrophage activation. A) Adenosine up-regulates IL-4-induced arginase activity in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated with 5 ng/ml IL-4 and treated with 100 μM adenosine for 8 h, and then arginase activity was measured from the cell lysates. Results are representative of ≥3 experiments; n = 6/experiment. ***P < 0.001 vs. control (con) group, ##P < 0.01 vs. IL-4. B) Adenosine augments IL-4-stimulated arginase-1 mRNA accumulation in RAW 264.7 cells. RAW 264.7 macrophages were treated with 100 μM adenosine and 5 ng/ml IL-4, and arginase-1 mRNA levels were measured by real-time PCR using RNA isolated 3 h after stimulating with IL-4 and adenosine. Results are representative of ≥3 experiments; n = 6/experiment. ***P < 0.001 vs. con, ###P < 0.001 vs. IL-4. C) Adenosine or NECA enhances IL-4-stimulated arginase-1 protein expression in RAW 264.7 cells. RAW 264.7 cells were challenged with IL-4 in the presence or absence of adenosine or NECA for 8 h, and arginase-1 protein level was determined from cell extracts using Western blotting with antibodies raised against arginase-1. β-Actin was utilized as internal control. Blots are representative of 3 separate experiments. D) Adenosine increases arginase activity of IL-4-challenged peritoneal macrophages. Peritoneal macrophages were obtained from C57BL/6 mice, and were treated with 5 ng/ml IL-4 and 100 μM adenosine. After 24 h of incubation, arginase activity was determined from whole-cell lysates. Results are representative of ≥3 experiments; n = 6/experiment. ***P < 0.001 vs. con, ##P < 0.01 vs. IL-4. E) Adenosine up-regulates IL-4-induced arginase-1 mRNA accumulation in peritoneal macrophages. Peritoneal macrophages were obtained from male C57BL/6 mice and were treated with 5 ng/ml IL-4 and 100 μM adenosine. After 4, 8, 12, or 24 h of incubation, arginase-1 mRNA abundance was measured using real-time PCR. Results are representative of ≥3 experiments; n = 6/experiment. *P < 0.05 vs. IL-4. F) Adenosine enhances IL-4-induced TIMP-1 release by mouse peritoneal macrophages. Peritoneal macrophages were stimulated with IL-4 and treated with 100 μM adenosine for the indicated times, and then TIMP-1 was measured from the supernatants using ELISA. Results are representative of ≥3 experiments; n = 4/experiment. **P <0.01, ***P < 0.001 vs. IL-4. G) Adenosine increases IL-4-induced TIMP-1 mRNA accumulation. Peritoneal macrophages were stimulated with 100 μM adenosine and 5 ng/ml IL-4, and TIMP-1 mRNA levels were measured by real-time PCR using RNA isolated 8 h after adenosine/IL-4. Results are representative of ≥3 experiments; n = 6/experiment. **P < 0.01 vs. con, ##P < 0.01 vs. IL-4. H) Adenosine up-regulates IL-4-induced TIMP-1 production by both TLR4-WT and -KO macrophages. Peritoneal macrophages were obtained from TLR4-KO and WT mice and were treated with IL-4 or IL-4 and adenosine for 24 h, after which procedure TIMP-1 production was determined from the supernatants. Results are representative of ≥2 experiments; n = 4/experiment. ***P < 0.001, ##P < 0.01 vs. corresponding IL-4-treated groups. I, J) Adenosine (I) or NECA (J) augments IL-4-stimulated mgl-1 mRNA accumulation in RAW 264.7 cells. RAW 264.7 macrophages were treated with 100 μM adenosine or 3 μM NECA and 5 ng/ml IL-4, and mgl-1 mRNA levels were measured by real-time PCR using RNA isolated 3 h after stimulating with IL-4 and adenosine or NECA. Results are representative of ≥3 experiments; n = 4/experiment. **P < 0.01, ***P < 0.001 vs. con; #P < 0.05, ##P < 0.01 vs. IL-4. Results are shown as means ± se.