Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.     

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner

Abstract

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.     

Hydrogen peroxide and tumour necrosis factor-α induce NF-κB-DNA binding in primary human T lymphocytes in addition to T cell lines

Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H₂O₂), have been implicated as second messengers in the activation of NF-κB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-α). The aim of the present study was to examine the effects of ROIs on NF-κB activation in primary human CD3+ T lymphocytes and human peripheral blood mononuclear cells (PBMCs). For comparison purposes, Jurkat T cells (subclones JR and JE6.1) were also investigated. Cells were incubated in the presence of either H₂O₂ or TNF-α and nuclear proteins were extracted. NF-κB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H₂O₂ required to activate NF-κB in human primary CD3+ T lymphocytes was as low as 1 μM. In contrast, much higher concentrations of H₂O₂ were required to activate NF-κB in PBMCs and in the JR subclone of Jurkat T cells. H₂O₂-induced NF-κB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-κB was activated by TNF-α in all four cell types tested. In PBMCs and Jurkat T cells (subclones JR and JE6.1), this activation could be inhibited by pre-treatment with the antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-l-cysteine (NAC). Our results support a role for ROIs in NF-κB-DNA binding in human primary T lymphocytes.     

Lipid raft assembly and Lck recruitment in TRAIL costimulation mediates NF-κB activation and T cell proliferation

The TNF-related apoptosis-inducing ligand was shown to provide a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell proliferation and cytokine production. Although a number of signaling pathways were linked to the TCR/CD3 complex, it is not known how these two receptors cooperate to induce T cell activation. In this study, we show that TRAIL-induced costimulation of T cells depends on activation of the NF-κB pathway. TRAIL induced the NF-κB pathway by phosphorylation of inhibitor of κB factor kinase and protein kinase C in conjunction with anti-CD3. Furthermore, we demonstrated that TRAIL costimulation induced phosphorylation of the upstream TCR-proximal tyrosine kinases, Lck and ZAP70. Ligation of the TRAIL by its soluble receptor, DR4-Fc, alone was able to induce the phosphorylation of Lck and ZAP70 and to activate the NF-κB pathway; however, it was insufficient to fully activate T cells to support T cell proliferation. In contrast, TRAIL engagement in conjunction with anti-CD3, but not TRAIL ligation alone, induced lipid raft assembly and recruitment of Lck and PKC. These results demonstrate that TRAIL costimulation mediates NF-κB activation and T cell proliferation by lipid raft assembly and recruitment of Lck. Our results suggest that in TRAIL costimulation, lipid raft recruitment of Lck integrates mitogenic NF-κB-dependent signals from the TCR and TRAIL in T lymphocytes.     

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Anti-osteoclastic effect of caffeic acid phenethyl ester in murine macrophages depends upon the suppression of superoxide anion production through the prevention of an active-Nox1 complex formation

This study investigated the anti-osteoclastic effect of caffeic acid phenethyl ester (CAPE) through suppression of Nox1-mediated superoxide anions production. The multi-nucleated cells were counted and followed by measuring their tartrate-resistant acid phosphatase (TRAP) activity. The superoxide anion production was determined by using fluorescent probe dihydroethidium (DHE). After one day of exposure to the receptor activator of nuclear factor-κB ligand (RANKL), the expression of the proteins involved in superoxide anion production was determined by western blotting. A potent anti-osteoclastic effect of CAPE was observed; the superoxide anion level reached a maximum value after one day of incubation. CAPE attenuated the expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) and Rac1, and mitigated the RANKL-induced translocation of p47^phox to the cell membrane. In addition, CAPE suppressed the expression of nuclear factor-kappa B (NF-κB p65), its translocation to the nucleus, and the activation of NF-κB inhibitor (IκBα) and its kinase (IKKβ). Furthermore, CAPE diminished the expression and activation of the c-jun N-terminal kinase (JNK) and the expression of protein-1 activators (AP-1) such as c-Fos and c-Jun. The expression of Nox1 was suppressed by CAPE through the down-regulation of IKKβ/IκBα/NF-κB and JNK/AP-1 signal pathway. This study provides evidence that the anti-osteoclastic effect of CAPE depends upon the attenuated superoxide anion production, which is closely related with interruption of an active Nox1 complex formation due to the attenuated catalytic subunit Nox1 expression resulting from suppression of the IKKβ/IκBα/NF-κB and JNK/AP-1 signaling pathway and the down-regulation of the p47^phox subunit translocation to the cell membrane.