Figure 5
Figure 5. The stimulatory effect of adenosine on IL-10 production is transcriptional. (A) Adenosine enhances E coli–induced IL-10 mRNA accumulation. Peritoneal macrophages were pretreated for 2 hours with 5 μg/mL Actinomycin D before adding 100 μM adenosine and heat-killed E coli. IL-10 mRNA concentrations were measured by real-time polymerase chain reaction using RNA isolated 5 hours after stimulating with E coli and adenosine. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment. *P < .05 vs. E coli alone. (B) Actinomycin D inhibits the stimulatory effect of E coli and adenosine on IL-10 protein release. Supernatants were harvested from these treatments and IL-10 levels were measured using enzyme-linked immunosorbent assay. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment. **P < .005 vs. unstimulated control. ***P < .001 vs. E coli alone. (C) Adenosine up-regulates IL-10 promoter luciferase activity in RAW 264.7 macrophages exposed to E coli. To measure IL-10 promoter activity, cells were transiently transfected with an IL-10 promoter-luciferase reporter construct and an A2A receptor-expressing (pA2A-cytomegalovirus) or control (pRC-cytomegalovirus) plasmid. Cells were treated with E coli in the presence or absence of adenosine for 8 hours. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05 and ***P < .001 vs. control. (D) Sequences between −438 and −376 from the transcription start site in the IL-10 promoter are necessary for the stimulatory effect of adenosine on IL-10 promoter activity. RAW 264.7 cells were transfected with a series of IL-10 promoter deletion mutants that were inserted in the pGL2B luciferase reporter vector. Transfected cells were stimulated with E coli or E coli plus adenosine for 8 hours. Luciferase activities are expressed as the mean activity and SEM relative to the activity of the full-length promoter (−1538/ + 64; 100%) after E coli stimulation followed by normalization to protein concentration. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. (E) C/EBP consensus sequences in the 5′ flanking region between −410/−385 of the IL-10 promoter are crucial for the stimulatory effect of adenosine. RAW 264.7 cells were transfected with a C/EBP consensus mutant of the IL-10 promoter that was inserted in the pGL2B luciferase reporter vector. Transfected cells were treated with E coli in the presence or absence of adenosine for 8 hours. Luciferase activities were normalized to protein concentration. C/EBP MUT: mutant. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05, **P < .005 vs E coli alone, and ***P < .001 vs. control.

The stimulatory effect of adenosine on IL-10 production is transcriptional. (A) Adenosine enhances E coli–induced IL-10 mRNA accumulation. Peritoneal macrophages were pretreated for 2 hours with 5 μg/mL Actinomycin D before adding 100 μM adenosine and heat-killed E coli. IL-10 mRNA concentrations were measured by real-time polymerase chain reaction using RNA isolated 5 hours after stimulating with E coli and adenosine. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment. *P < .05 vs. E coli alone. (B) Actinomycin D inhibits the stimulatory effect of E coli and adenosine on IL-10 protein release. Supernatants were harvested from these treatments and IL-10 levels were measured using enzyme-linked immunosorbent assay. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment. **P < .005 vs. unstimulated control. ***P < .001 vs. E coli alone. (C) Adenosine up-regulates IL-10 promoter luciferase activity in RAW 264.7 macrophages exposed to E coli. To measure IL-10 promoter activity, cells were transiently transfected with an IL-10 promoter-luciferase reporter construct and an A2A receptor-expressing (pA2A-cytomegalovirus) or control (pRC-cytomegalovirus) plasmid. Cells were treated with E coli in the presence or absence of adenosine for 8 hours. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05 and ***P < .001 vs. control. (D) Sequences between −438 and −376 from the transcription start site in the IL-10 promoter are necessary for the stimulatory effect of adenosine on IL-10 promoter activity. RAW 264.7 cells were transfected with a series of IL-10 promoter deletion mutants that were inserted in the pGL2B luciferase reporter vector. Transfected cells were stimulated with E coli or E coli plus adenosine for 8 hours. Luciferase activities are expressed as the mean activity and SEM relative to the activity of the full-length promoter (−1538/ + 64; 100%) after E coli stimulation followed by normalization to protein concentration. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. (E) C/EBP consensus sequences in the 5′ flanking region between −410/−385 of the IL-10 promoter are crucial for the stimulatory effect of adenosine. RAW 264.7 cells were transfected with a C/EBP consensus mutant of the IL-10 promoter that was inserted in the pGL2B luciferase reporter vector. Transfected cells were treated with E coli in the presence or absence of adenosine for 8 hours. Luciferase activities were normalized to protein concentration. C/EBP MUT: mutant. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05, **P < .005 vs E coli alone, and ***P < .001 vs. control.

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