cAMP regulates IL-10 production by normal human T lymphocytes at multiple levels: a potential role for MEF2

Signal transduction by the cAMP/cAMP-dependent protein kinase A (PKA) pathway is triggered through multiple receptors and is important for many processes in a variety of cells. In T cells, the engagement of the TCR-CD3 complex induces cAMP, a second messenger that controls immune response. IL-10, produced by a variety of lymphocyte subpopulations, is an important regulator of this response exerting a wide range of immunomodulatory actions. Elevation of cAMP has been shown to increase IL-10 production by monocytes. However, the mechanism of cAMP mediated regulation of IL-10 production by T lymphocytes remains unclear. In this study using normal peripheral T lymphocytes stimulated either through the TCR-CD3 complex or the TCR-CD3 and the CD28 molecule, we show that IL-10 is produced mainly by memory T lymphocytes after either way of stimulation and is drastically inhibited (70-90%) by cAMP elevating agents. cAMP mediated inhibition was reversed by the use of the specific PKA inhibitor Rp-8-Br-cAMP but not by the addition of exogenous rhIL-2, indicating that the inhibitory effect depends on PKA activation and is not secondary to IL-2 inhibition. Inhibition is taking place at both transcriptional and posttranscriptional level. Transfection of a luciferase reporter plasmid carrying the IL-10 promoter in T cells, revealed that TCR/CD28-induced activation was inhibited by 60% by cAMP elevation. The most sensitive part to cAMP mediated inhibition was a fragment of 135 bp upstream of TATA box, which contains multiple binding sites for MEF-2. Overexpression of MEF-2 in the same cells increased IL-10 promoter activity by 2.5-fold. Stimulation through TCR/CD28 increased MEF-2 binding in its corresponding binding sites which was inhibited by 80% in the presence of cAMP elevating agents. These results suggest that the inhibitory effect of cAMP on IL-10 production by normal peripheral T lymphocytes is cell type and stimulus specific, exerted on multiple levels and involves MEF2 transcription factor.