Delphinidin Activates NFAT and Induces IL-2 Production Through SOCE in T Cells

Delphinidin is an anthocyanidin that possesses antioxidant and anti-inflammatory effects; however, some reports suggest that delphinidin has pro-inflammatory properties. For this reason, we assessed the effect of delphinidin on cytokine production in T cells. We demonstrated that delphinidin increased the cytosolic-free Ca²⁺ concentration by releasing Ca²⁺ from intracellular stores and increasing Ca²⁺ entry. The putative Ca²⁺ release activated Ca²⁺ (CRAC) channel inhibitors BTP2 and gadolinium reduced the calcium entry stimulated by the anthocyanidin. Delphinidin induced nuclear factor of activated T cells (NFAT) translocation and NFAT-Luc activity in Jurkat cells and was dependent on the CRAC channel and calcineurin pathway. Delphinidin increased the mRNA expression and production of IL-2 in Jurkat cells and was inhibited by BTP2 and cyclosporine A. Using peripheral blood lymphocytes, we demonstrated that delphinidin increased the production of IL-2 and IFN-γ and was inhibited by BTP2. Taken together, our results suggest that delphinidin exerts immunostimulatory effects on T cells by increasing cytokine production through CRAC channel and NFAT activation.     

Interaction of the retinoblastoma gene product,RB, with cyclophilin A negatively affects cyclosporin-inhibited NFAT signaling

The retinoblastoma susceptibility gene product, p105Rb (RB), is generally believed to be an important regulator in the control of cell growth, differentiation, and apoptosis. Several cellular factors that form complexes with RB and exert their cellular regulatory functions have been identified, such as the newly identified RB:cyclophilin A (CypA) complex. The physical interactions between RB and CypA were demonstrated by glutathione S-transferase affinity matrix binding assays and immunoprecipitation, followed by Western blot analyses. The N-terminal region of CypA mediated the interaction with RB, whereas the region upstream of the A-pocket of RB was required for binding to CypA. Ectopic expression of RB into Jurkat cells partially blocks the function of cyclosporin (CsA) to inhibit nuclear factor for activation of T cell (NFAT) activation by phorbol ester (PMA) plus ionomycin A (IA), suggesting that RB may prevent CsA inhibition of T lymphocyte activation. These results are further evidenced by the effect of RB on both calcineurin (CN) and NFAT binding activity in vitro, suggesting that the interaction of RB with CypA interferes with the CsA:CypA complex and blocks CsA-inhibited CN activity. These data reveal the functional link between RB and CypA and their involvement in T cell activation signaling.     

The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

The Ca²⁺/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K_i value of 3.8 μm. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.     

Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506–FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKK-K) besides the calcineurin–NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co-expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin-2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin-dependent NFAT pathway and calcineurin-independent activation pathway for JNK and p38.     

Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells

Activation of T lymphocytes requires protein kinase C theta (PKC-theta) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-theta on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.     

The calcium/NFAT pathway: role in development and function of regulatory T cells

Calcium signals are essential for diverse cellular functions in the immune system. Sustained Ca²⁺ entry is necessary for complete and long-lasting activation of calcineurin/NFAT pathways. A growing number of studies have emphasized that Ca²⁺/calcineurin/NFAT pathway is crucial for both development and function of regulatory T cells.