In this study, we show that adenosine augments IL-10 production in murine macrophages without altering IL-10 mRNA accumulation or IL-10 promoter activity. In addition, adenosine stimulates reporter activity from IL-10 reporter constructs containing regions of the 3′-UTR of the IL-10 gene. Therefore, we propose that adenosine enhances IL-10 production by a posttranscriptional mechanism.
Despite its biological importance, relatively little is known about the regulation of IL-10 gene expression. Most previous studies of IL-10 gene expression focused on the mechanisms of LPS-induced transcriptional activation of the IL-10 gene through the IL-10 promoter (6
). Using quantitative real-time PCR, we found that LPS stimulation increased IL-10 mRNA levels by ~2000-fold, while levels of secreted IL-10 protein were increased by only ~10-fold. These observations suggest that additional regulatory levels of IL-10 biosynthesis are operational in murine macrophages. A growing body of evidence suggests that regulation of gene expression may be accomplished through posttranscriptional mechanisms, which include regulation of mRNA stability, and translational regulation (42
). The discrepancy between IL-10 mRNA and protein levels was observed when measuring both IL-10 mRNA and protein accumulation at the same relatively late, 5-h time point, indicating that a destabilizing effect is unlikely to have a role in reducing IL-10 protein levels relative to mRNA concentrations. The 3′-UTR of the IL-10 mRNA has been shown previously to destabilize IL-10 mRNA in the EL4 T lymphocyte cell line (13
). In contrast, we found that the 3′-UTR of the IL-10 mRNA does not exert the same destabilizing effect in RAW 264.7 macrophages, because inserting the 3′-UTR of the IL-10 mRNA downstream of the luciferase coding region in the pGL3-control vector failed to decrease luciferase mRNA levels. On the other hand, there was a dramatic, ~100-fold drop in luciferase protein levels secondary to insertion of the 3′-UTR of the IL-10 mRNA after the luciferase coding region, confirming the translational regulation of IL-10. Considering that IL-10 mRNA was induced ~2000-fold at 5 h after LPS stimulation, while IL-10 protein expression was elevated by ~10-fold at this point, the 100-fold decrease in translation can explain why IL-10 protein is induced ~200-fold less than IL-10 mRNA. It is, however, important to note that other mechanisms, including transcriptional changes and changes in IL-10 mRNA stability level, might also have influenced the steady-state mRNA levels measured at this time point. The exact contribution of these processes will require further studies to measure transcriptional activity using nuclear run-on assays and to assess the course of mRNA degradation. The 3′-UTR of the TNF-α gene confers a similar translational repressive effect on TNF-α mRNA (27
). Interestingly, although the translational repression caused by the TNF-α 3′-UTR is relieved by LPS (this study and Refs. 27
), we find that LPS does not relieve the translational repressive effect of the 3′-UTR of the IL-10 mRNA.
In contrast, adenosine receptor activation by either the endogenous ligand adenosine, or by the stable adenosine analog NECA is able to relieve, at least in part, the translational repressive effect of the 3′-UTR of the IL-10 mRNA. The magnitude of this increase in translational activity (~2-fold) after adenosine receptor activation is similar to the degree of adenosine- or NECA-induced augmentation of IL-10 protein production. This observation supports the view that the stimulatory effect of adenosine receptor activation on LPS-induced IL-10 production is due to a translational effect involving the 3′-UTR of the IL-10 mRNA. Our data also demonstrate that the translational stimulatory effect of adenosine receptor occupancy is specific for the 3′-UTR of IL-10, because adenosine failed to alter reporter activity from the 3′-UTR of the TNF-α mRNA. In a recent study using DNA microarray analysis (33
), we reported that adenosine did not modulate expression of LPS-induced mRNAs in RAW 264.7 macrophages, nor did it alter mRNA expression in LPS-untreated, naive cells. Although the mRNA for TNF-α was among the many cytokine mRNAs whose expression was not changed by adenosine treatment in LPS-activated macrophages, levels of both secreted and intracellular TNF-α were substantially reduced following adenosine treatment (24
). Similar to this observation, an earlier study reported that adenosine had no effect on mRNA levels of TNF-α in LPS/IFN-γ-stimulated RAW 264.7 macrophages, despite its inhibitory effect on secreted TNF-α concentrations (51
). Together, these data indicate that adenosine appears to regulate cytokine production by posttranscriptional mechanisms.
We also found that treatment of cells with adenosine exerted similar posttranscriptional stimulatory effects on the AU1, AU2, and AU3 subregions of the IL-10 3′-UTR to that observed on the full IL-10 3′-UTR (AU4). AU1, AU2, and AU3 contain related potential regulatory nonamer sequences (40
) that might be involved in the posttranscriptional regulatory action of adenosine. AUAUUUAUU and CUAUUUAUU are present in AU1, GUAUUUAUU is found in AU2, and UUAUUUAUA is a part of AU3. These nonamers are thought to control posttranscriptional regulatory events via interactions with specific binding proteins (40
). Our results provide insight into the possible role of proteins that bind to the IL-10 3′-UTR in mediating the stimulatory effect of adenosine on the translational regulatory processes involving the IL-10 3′-UTR. We demonstrate that the AU2 and AU3 regions are able to form two different complexes with protein extracts taken from RAW cells, and that adenosine intensifies this complex formation. In addition, deletion of the GUAUUUAUU sequence from the AU2 region abrogated protein binding to AU2, identifying this nonamer motif as a key element in the translational regulation of the AU2 region. Further studies will be required to ascertain the identity of the proteins that bind to this region, and the mechanisms by which adenosine augments complex formation.
Our results indicate that A2B
receptors mediate the stimulatory effect of adenosine on IL-10 production in RAW 264.7 macrophages. Several lines of evidence support this conclusion. First, the order of potency of agonists (NECA > IB-MECA > CGS-21680) is typical of A2B
). Second, the selective A2B
receptor antagonist, alloxazine, prevented the adenosine-induced augmentation of IL-10 production. Finally, we detected A2B
receptors in membrane protein fractions from RAW 264.7 cells. Our data are partially contradictory to those of a previous study which demonstrated that the A2A
receptor agonist CGS-21680 potentiated IL-10 production by human monocytes and that this effect was reversed by a selective A2A
). In contrast, because in this previous study NECA was both a more potent (EC50
~100 nM for NECA vs EC50
~500 nM for CGS-21680) and more efficacious (~2.5-fold maximal increase with NECA vs ~1.5-fold maximal increase with CGS-21680) agonist than CGS-21680, a possible role for A2B
receptors in the enhancement of IL-10 production in human monocytes can also not be ruled out.
Previous studies have proposed the concept that the differential transcriptional regulation of IL-10 vs TNF-α and other proinflammatory cytokines might ensure the belated induction of IL-10 compared with proinflammatory cytokines (6
). In addition, this differential transcriptional regulation might serve to enable macrophages to uncouple IL-10 production from that of proinflammatory cytokines permitting differential responses to extracellular stimuli. Our results showing the differential posttranscriptional regulation of IL-10 and TNF-α in response to adenosine uncover a novel mechanism for uncoupling IL-10 production from that of TNF-α.