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Deficient apoptosis of activated T cells can result in immunological disorders. Molecules associated with energy and metabolisms are suggested to be involved in pathogenesis of immune diseases, but remain uninvestigated. In the present study we reported that glibenclamide exerted a new pharmacological effect on inflammatory responses by selectively triggering apoptosis of activated T cells. Glibenclamide demonstrated an inhibition on activated T lymphocytes, whereas showed no toxicity in the naive cells. This effect was mainly related with its ability to facilitate apoptosis in activated T cells with an up-regulation of cleaved-caspases and cleaved-PARP. Glibenclamide enhanced Fas expression and suppressed the expression of antiapoptotic cellular FLICE-inhibitory protein. The underlying mechanism of glibenclamide was not associated with its classical inhibitory effect on ATP-sensitive potassium channels, but due to a unique suppression on the phosphorylation of 5' adenosine monophosphate-activated protein kinase, which was augmented during T cell activation. An in vivo experiment further demonstrated that glibenclamide ameliorated T-cell-mediated contact hypersensitivity in mice. Altogether, these results suggest that AMPK inhibition by glibenclamide can regulate the survival and death of T lymphocytes and be beneficial for the treatment of autoimmune diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We attempted to clarify whether hypersecretion of Muc5b mucin from mouse nasal submucosal glands that is enhanced by interleukin (IL)-33 under allergic conditions can be ameliorated by administration of 2-APB. Immunohistochemistry was used to examine both the distribution of T cells in the nasal mucosa of an allergic rhinitis mouse model and expressions of IL-33 receptor ST2 and Muc5b protein in mouse submucosal gland cells. The amounts of protein and mRNA of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 in mouse nasal lavage fluid (NLF) and nasal mucosa were determined using enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction. Expressions of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 were up-regulated in the allergic state and IL-33 increased the levels of Muc5b, IL-4, IL-5 and IL-13, but did not influence proliferation of T cells; however, ST2 was diminished in nasal submucosal gland cells. 2-APB reduced proliferation of T cells and the Orai1 level in the nasal mucosa. It also reduced the concentrations of IL-4, IL-5 and IL-13 in NLF and nasal mucosa, and hypersecretion of Muc5b from glandular cells that was enhanced by IL-33, but did not affect IL-33 production. 2-APB decreased Muc5b mucin hypersecretion from submucosal gland that was enhanced by IL-33 in allergic mice by limiting Ca(2+) release-activated Ca(2+) channel activity in which Orai1 plays a crucial role in the gland cells and/or by controlling channel activation in T cells and proliferation of these cells.

The activation of Kv1.3 potassium channel has obligatory roles in immune responses of T lymphocytes. Stromal cell-derived factor-1alpha (SDF-1alpha) binds to C-X-C chemokine receptor type 4, activates phosphoinositide 3-kinase, and plays essential roles in cell migration of T lymphocytes. In this study, the effects of phosphoinositides and SDF-1alpha on Kv1.3 current activity were examined in the Jurkat T cell line using whole cell patch-clamp techniques. The internal application of 10 microM phosphatidylinositol 4,5-bisphosphate (PIP(2)) or 10 microM phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) significantly reduced Kv1.3 current, but that of 10 microM phosphatidylinositol-4-monophosphate (PIP) did not. The coapplication of 10 microg/ml anti-PIP(3) antibody with PIP(2) from the pipette did not change the reduction of Kv1.3 current by PIP(2), but the coapplication of the antibody with PIP(3) eliminated the reduction. The heat-inactivated anti-PIP(3) antibody had no effect on PIP(3)-induced inhibition. These results suggest that PIP(2) per se can reduce Kv1.3 current as well as PIP(3). External application of 1 muM Akt-kinase inhibitor VIII did not reverse the effect of intracellular PIP(3). External application of 10 and 30 ng/ml SDF-1alpha significantly reduced Kv1.3 current. Internal application of anti-PIP(3) antibody reversed the SDF-1alpha-induced reduction. These results suggest that, in Jurkat T cells, PIP(2), PIP(3), and SDF-1alpha reduce Kv1.3 channel activity and that the reduction by SDF-1alpha may be mediated by the enhancement of PIP(3) production. These novel inhibitory effects of phosphoinositides and SDF-1alpha on Kv1.3 current may have a significant function as a downregulation mechanism of Kv1.3 activity for the maintenance of T lymphocyte activation in immune responses.

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca(2+) influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.