Seyeon Bae

α-Enolase Expressed on the Surfaces of Monocytes and Macrophages Induces Robust Synovial Inflammation in Rheumatoid Arthritis

α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/β, IFN-γ, and PGE2 via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.

Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production†

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over‐expression of cyclooxygenase (COX)‐2 and type I insulin‐like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX‐2 expression and IGF‐I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX‐2 expression and IGF‐I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK‐MEL‐2 without induction of apoptosis. At that moment, IGF‐II production was decreased, followed by the inhibition of COX‐2 activity. IGF‐IR expression was also down‐regulated by vitamin C treatment. It coincided with the result from the inhibition of COX‐2 by NS‐398 and COX‐2 siRNA. In addition, the decreased IGF‐IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C‐induced IGF‐II and IGF‐IR down‐regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX‐2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK‐MEL2 via the down‐regulation of IGF‐II production and IGF‐IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX‐2 expression. J. Cell. Physiol. 216: 180–188, 2008.

The Analysis of Vitamin C Concentration in Organs of Gulo-/- Mice Upon Vitamin C Withdrawal

Background Vitamin C is an essential nutrient for maintaining human life. Vitamin C insufficiency in the plasma is closely related with the development of scurvy. However, in vivo kinetics of vitamin C regarding its storage and consumption is still largely unknown. Methods We used Gulo-/- mice, which cannot synthesize vitamin C like human. Vitamin C level in plasma and organs from Gulo-/- mice was examined, and it compared with the level of wild-type mice during 5 weeks. Results The significant weight loss of Gulo-/- mice was shown at 3 weeks after vitamin C withdrawal. However, there was no differences between wild-type and vitamin C-supplemented Gulo-/- mice (3.3 g/L in drinking water). The concentration of vitamin C in plasma and organs was significantly decreased at 1 week after vitamin C withdrawal. Vitamin C is preferentially deposited in adrenal gland, lymph node, lung, and brain. There were no significant changes in the numbers and CD4/CD8 ratio of splenocytes in Gulo-/- mice with vitamin C withdrawal for 4 weeks. And the architecture of spleen in Gulo-/- mice was disrupted at 5 weeks after vitamin C withdrawal. Conclusion The vitamin C level of Gulo-/- mice was considerably decreased from 1 week after vitamin C withdrawal. Vitamin C is preferentially stored in some organs such as brain, adrenal gland and lung.

Interleukin-18 increases metastasis and immune escape of stomach cancer via the downregulation of CD70 and maintenance of CD44

Cancer cells metastasize to the other site after escaping from the immune system and CD70, CD44 and vascular endothelial growth factor (VEGF) play important roles in this process. It is recently reported that interleukin (IL)-18 is closely related with the pathogenesis of skin tumor. Therefore, we investigated the role of endogenous IL-18 from stomach cancer on the immune escape mechanism and metastasis via the regulation of CD70, CD44 and VEGF expression. IL-18 and IL-18R expressions were not only investigated on tumor tissues (n = 10), and sera (n = 20) from stomach cancer patients, but also on human stomach cancer cell lines. IL-18 and IL-18R expressions were found on stomach cancer cell lines and tumor tissues. In addition, IL-18 levels were elevated in sera from cancer patients (P < 0.05), compared with sera from normal individuals. Changes in CD70, CD44 and VEGF expression by flow cytometry, immunoblotting and enzyme-linked immunosorbent assay and immune susceptibility by (51)Cr-release assay were investigated, after silencing or neutralization of endogenous IL-18. CD70 expression was increased and it increases immune susceptibility of cancer cells. In contrast, CD44 and VEGF expression was decreased and it suppresses neovascularization and the metastasis of stomach cancer. After inoculation of IL-18 small interfering RNA (siRNA)-transfected stomach cancer cells into Balb/C (nu/nu) mice, regression of tumor mass was determined by measuring of tumor size. And the number and location of metastatic lesions were investigated by hematoxylin and eosin staining. The regression of tumor mass and the suppression of metastasis were observed in the mice, which are injected with IL-18 siRNA-transfected cell lines. Our data suggest that endogenous IL-18 might facilitate stomach cancer cell immune escape by suppressing CD70 and increasing metastatic ability by upregulating CD44 and VEGF.

Vitamin C down‐regulates VEGF production in B16F10 murine melanoma cells via the suppression of p42/44 MAPK activation

It is known that vitamin C induces apoptosis in several kinds of tumor cells, but its effect on the regulation of the angiogenic process of tumors is not completely studied. Vascular endothelial growth factor (VEGF) is the most well‐known angiogenic factor, and it has a potent function as a stimulator of endothelial survival, migration, as well as vascular permeability. Therefore, we have investigated whether vitamin C can regulate the angiogenic process through the modulation of VEGF production from B16F10 melanoma cells. VEGF mRNA expression and VEGF production at protein levels were suppressed by vitamin C. In addition, we found that vitamin C suppressed the expression of cyclooxygenase (COX)‐2 and that decreased VEGF production by vitamin C was also restored by the administration of prostaglandin E2 which is a product of COX‐2. These results suggest that vitamin C suppresses VEGF expression via the regulation of COX‐2 expression. Mitogen‐activated protein kinases are generally known as key mediators in the signaling pathway for VEGF production. In the presence of vitamin C, the activation of p42/44 MAPK was completely inhibited. Taken together, our data suggest that vitamin C can down‐regulate VEGF production via the modulation of COX‐2 expression and that p42/44 MAPK acts as an important signaling mediator in this process. J. Cell. Biochem. 112: 894–901, 2011.

Vitamin C Is an Essential Factor on the Anti-viral Immune Responses through the Production of Interferon-α/β at the Initial Stage of Influenza A Virus (H3N2) Infection

L-ascorbic acid (vitamin C) is one of the well-known anti-viral agents, especially to influenza virus. Since the in vivo anti-viral effect is still controversial, we investigated whether vitamin C could regulate influenza virus infection in vivo by using Gulo (-/-) mice, which cannot synthesize vitamin C like humans. First, we found that vitamin C-insufficient Gulo (-/-) mice expired within 1 week after intranasal inoculation of influenza virus (H3N2/Hongkong). Viral titers in the lung of vitamin C-insufficient Gulo (-/-) mice were definitely increased but production of anti-viral cytokine, interferon (IFN)-α/β, was decreased. On the contrary, the infiltration of inflammatory cells into the lung and production of pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-α/β, were increased in the lung. Taken together, vitamin C shows in vivo anti-viral immune responses at the early time of infection, especially against influenza virus, through increased production of IFN-α/β.

Inhibition of lytic reactivation of Kaposi's sarcoma― associated herpesvirus by alloferon

BACKGROUND Alloferon, an immunomodulatory peptide, has antiviral capability against herpesvirus. In this research, we aimed to investigate the effect of alloferon on the regulation of the life cycle of Kaposis sarcoma-associated herpesvirus (KSHV), and its mechanisms. We also assessed the antiviral activity of alloferon on natural killer (NK) cells as an early antiviral immune responder. METHODS We first examined the change in cell proliferation and the expression of the viral genes in a KSHV-infected cell line, body-cavity-based B lymphoma (BCBL)-1, under the lytic cycle by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment. To elucidate the antiviral mechanism of alloferon, we tested calcium influx and the activation of the extracellular signal-regulated kinase (ERK) pathway. Furthermore, we evaluated the cytotoxicity of NK cells against BCBL-1 by alloferon. RESULTS Alloferon effectively recovered the suppressed proliferation of BCBL-1 by TPA, which was achieved by the down-regulation of lytic-cycle-related viral genes, RTA, K8 and vIRF2. To clarify the signal transduction pathways related to the regulation of the viral genes by alloferon, we confirmed that the calcium influx into BCBL-1 was apparently inhibited by alloferon, which preceded the suppression of the phosphorylation of ERK and the activation of AP-1 by TPA. Moreover, when NK cells were exposed to alloferon, their cytolytic activity was improved, and this was mediated by the enhancement of perforin/granzyme secretion. CONCLUSIONS The results of this study suggest that alloferon can be used as an effective antiviral agent for the regulation of the KSHV life cycle by the down-regulation of AP-1 activity and for the the enhancement of antiviral immunity by up-regulation of NK cell cytotoxicity.

The anti-inflammatory effect of alloferon on UVB-induced skin inflammation through the down-regulation of pro-inflammatory cytokines.

UVB irradiation can induce biological changes in the skin, modulate immune responses and activate inflammatory reactions leading to skin damage. Alloferon, which is isolated from the blood of an experimentally infected insect, the blow fly Calliphora vicina, is known for its anti-viral and anti-tumor activities in mice model. However, the effect of alloferon against UVB irradiation and its specific mechanism are still unknown. In this study, we investigated the effect of alloferon on UVB-induced cutaneous inflammation in a human keratinocyte cell line, HaCaT. RPA and ELISA data showed that alloferon decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6 and IL-18, both on the mRNA and protein level. Western blot analysis was done to determine if alloferon regulates the MAPK signaling pathway since the MAPK signaling pathway is activated by numerous inflammatory mediators and environmental stresses including UVB irradiation. Alloferon inhibited the activation of p38 mitogen-activated protein kinase (MAPK) induced by UVB irradiation. Furthermore, the topical application of alloferon on the UVB exposed skin of hairless mice showed that alloferon treatment significantly inhibited an increase in epithelial thickness in chronic UVB-irradiated mouse skin. These findings suggest that alloferon has significant anti-inflammatory effects not only on UVB-induced inflammation in the human keratinocyte cell line, HaCaT, but also on mouse skin.

Vitamin C prevents stress-induced damage on the heart caused by the death of cardiomyocytes, through down-regulation of the excessive production of catecholamine, TNF-α, and ROS production in Gulo(−/−)Vit C-Insufficient mice

It is thought that vitamin C has protective roles on stress-induced heart damage and the development of cardiovascular diseases, but its precise role and mechanisms are unclear. In the present study, we investigated the specific mechanisms by which vitamin C leads to protecting the heart from stress-induced damage in the Gulo(-/-) mice which cannot synthesize vitamin C like humans. By exposure to stress (1h/day), the heartbeat and cardiac output in vitamin C-insufficient Gulo(-/-) mice were definitely decreased, despite a significant increase of adrenaline (ADR) and noradrenaline (NA) production. A change of cardiac structure caused by the death of cardiomyocytes and an increased expression of matrix metalloprotease (MMP)-2 and -9 were also found. Moreover, lipid peroxidation and the production of tumor necrosis factor-alpha (TNF-α) in the heart were increased. Finally, all vitamin C-insufficient Gulo(-/-) mice were expired within 2 weeks. Interestingly, all of the findings in vitamin C-insufficient Gulo(-/-) mice were completely prevented by the supplementation of a sufficient amount of vitamin C. Taken together, vitamin C insufficiency increases the risk of stress-induced cardiac damage with structural and functional changes arising from the apoptosis of cardiomyocytes.

Vitamin C Up-regulates Expression of CD80, CD86 and MHC Class II on Dendritic Cell Line, DC-1 Via the Activation of p38 MAPK

Vitamin C is an essential water-soluble nutrient which primarily exerts its effect on host defense mechanisms and immune homeostasis, but the mechanism related to immune-potentiation is poorly understood. Since dendritic cells (DCs) are known as a potent antigen presenting cell (APC) that could enhance the antigen specific immune responses, we investigate the effects of vitamin C on activation of DCs and its related mechanism by using dendritic cell lines, DC-1. First, we found that there was no damage on DC-1 by 2.5 mM of vitamin C. In the presence of vitamin C, the expression of CD80, CD86, and MHC molecules was increased, but it was decreased by the pre-treatment of SB203580, p38 MAPK-specific inhibitor. We confirmed the phosphorylation of p38 MAPK was increased by the treatment of vitamin C. Taken together, these results suggest that vitamin C could enhance the activity of dendritic cells via the up-regulation of the expression of CD80, CD86, and MHC molecules and the activation of p38 MAPK is related to this process.