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Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

ABSTRACT: Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

SUBMITTER: Matheu MP

PROVIDER: S-EPMC2732399 | biostudies-other | 2008 Oct

REPOSITORIES: biostudies-other

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Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

Matheu Melanie P MP Beeton Christine C Garcia Adriana A Chi Victor V Rangaraju Srikant S Safrina Olga O Monaghan Kevin K Uemura Marc I MI Li Dan D Pal Sukumar S de la Maza Luis M LM Monuki Edwin E Flügel Alexander A Pennington Michael W MW Parker Ian I Chandy K George KG Cahalan Michael D MD

Immunity 20081002 4

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated ...[more]

PMID: 18835197

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Project description:BACKGROUND:In T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases. METHODS:We combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin. RESULTS:The peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-? and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. CONCLUSIONS:The Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.

Project description:Background: Fibroblast proliferation is a critical feature during heart failure development. Previous studies reported regulatory T-lymphocytes (Tregs)' protective role against myocardial fibrosis. However, notably, Tregs also secrete fibrogenic cytokine TGF-? when activated. This study aimed to clarify the intriguing link between Tregs and fibrosis, the role of Tregs Kv1.3 potassium channel (regulating T-lymphocytes activation) in the fibrosis process, and how selective aldosterone receptor antagonist Eplerenone affects Tregs and fibrosis through its action on Kv1.3 channel. Methods and Results: After co-incubation with Tregs, cardiac fibroblast proliferation (CCK-8 assay) and levels of collagen I, III, and Matrix metalloproteinase2 (ELISA) significantly elevated. Cell viability assays, Kv1.3 channel mRNA (RT-qPCR), and protein expression (In-Cell Western Blotting) revealed Tregs were activated/proliferated when co-cultured with fibroblasts. Treg intracellular TGF-? level increased by 5.8-fold, far more than that of intracellular IL-10, extracellular TGF-? and IL-10 (ELISA). And 30 ?M eplerenone suppressed Tregs proliferation by 82.77% and furthermore, suppressed intracellular TGF-? level to a significantly greater extent than that of intracellular IL-10, extracellular TGF-? and IL-10. Moreover, the Kv1.3 current (whole-cell patch clamp) of Tregs in congestive heart failure patients and rats (induced by coronary artery ligation and exhaustive exercise) elevated by >4-fold than that of healthy volunteers and control rats, whereas 30 ?M eplerenone suppressed the current by >60% in control Tregs. In addition, docking calculations (AutoDock software 4.0 suite) showed eplerenone has higher H-bond energy with Kv1.3 channel than other selective blockers. Conclusion: Immuno-regulation in the late stage of CHF activates Tregs proliferation via the upregulation of Kv1.3 channels, which promotes cardiac fibrosis by primarily secreting TGF-?. Taken together, eplerenone's high affinity to Kv1.3 channel enables it to antagonize the Kv1.3 channels directly to suppress Tregs proliferation, which in turn may play an immuno-regulatory role during CHF.

Project description:Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

Dataset Information

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

Publications

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

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