Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: a distinct target in the calcium dependent pathway.

Calcium (Ca2+) plays an essential role in lymphocyte activation and differentiation by affecting signaling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of tolerance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specifically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500 bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ionomycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regulation of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals.

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.