Role of tumor necrosis factor-alpha and interferon-gamma in Helicobacter pylori infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF-alpha geneknockout (TNF-alpha(-/-)) mice and IFN-gamma gene-knockout (IFN-gamma(-/-)) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild-type and knockout mice. The number of H. pylori colonized in the stomach of IFN-gamma(-/-) and TNF-alpha(-/-) mice was higher than that of wild-type mice. These findings suggest that TNF-alpha and IFN-gamma may play a protective role in H. pylori infection. Furthermore, we examined the contribution of TNF-alpha and IFN-gamma to gastric inflammation. The CPY2052-infected TNF-alpha(-/-) mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild-type mice, whereas the CPY2052-infected IFN-gamma(-/-) mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN-gamma may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF-alpha may not participate in the development of inflammatory response.     

Ghrelin attenuates plasminogen activator inhibitor-1 production induced by tumor necrosis factor-alpha in HepG2 cells via NF-kappaB pathway

Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on enterocyte shedding of syndecan-1 and associated decreases in internalization of Listeria monocytogenes and Staphylococcus aureus

Syndecan-1 is a heparan sulfate proteoglycan expressed on epithelia, and its ectodomain can be shed into the extracellular milieu, affecting a variety of cellular functions. Using two bacteria known to react with heparan sulfate, Listeria monocytogenes and Staphylococcus aureus, experiments were designed to clarify the effect of syndecan-1 shedding on bacterial internalization by human HT-29 enterocytes. Mature enterocytes were incubated with tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma for 16h prior to addition of bacteria. These cytokines acted synergistically to decrease syndecan-1 expression, assessed by visual observations of syndecan-1 expression on enterocytes using immunohistochemistry and a monoclonal antibody to the syndecan-1 core protein, by quantifying this fluorescent intensity, and by quantifying the concentration of shed syndecan-1 using an enzyme-linked immunoabsorbent assay. Neither IFN-gamma nor TNF-alpha alone had a noticeable effect on L. monocytogenes internalization, but a mixture of both cytokines resulted in decreased (P<0.01) internalization. Enterocyte preincubation with TNF-alpha alone, and with both cytokines, was associated with decreased S. aureus internalization, at P<0.05 and P<0.01, respectively. Thus, TNF-alpha and IFN-gamma acted synergistically to shed syndecan-1 ectodomains from HT-29 enterocytes, and shedding was associated with decreased internalization of two pathogenic bacteria, suggesting that syndecan-1 shedding may modulate the pathogenesis of specific microbes.     

Glycogen synthase kinase-3beta suppresses tumor necrosis factor-alpha expression in cardiomyocytes during lipopolysaccharide stimulation

This study was to investigate the role of glycogen synthase kinase-3beta (GSK-3beta) in cardiomyocyte tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). In cultured neonatal mouse cardiomyocytes, LPS induced TNF-alpha expression and increased GSK-3beta activation. Inhibition of GSK-3beta by SB216763 or by over-expression of a dominant negative mutant of GSK-3beta significantly enhanced TNF-alpha expression in LPS-stimulated cardiomyocytes, in association with an increase in p65 phosphorylation. In contrast, over-expression of GSK-3beta by adenoviral vectors containing wild-type GSK-3beta or a constitutively active GSK-3beta attenuated TNF-alpha expression induced by LPS. Further evidence to support the inhibitory role of GSK-3beta in TNF-alpha expression is that protein kinase B (Akt) signaling, an upstream inhibitor of GSK-3beta, promotes TNF-alpha expression in LPS-stimulated cardiomyocytes and this action of Akt signaling can be mimicked by GSK-3beta inactivation. Our study demonstrates that GSK-3beta plays an inhibitory role in cardiomyocyte TNF-alpha expression during LPS stimulation, and it may be a potential therapeutic target for sepsis.     

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha.

Tumour necrosis factor alpha (TNF-alpha) at 20 ng/ml induced apoptosis in human hepatoma cells in vitro. The effect of TNF-alpha-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-alpha-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-alpha elevated free Ca(2+)concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-alpha-induced apoptosis may be involved in stimulating Ca(2+)-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca(2+)regulator or antioxidant, preventing lipid peroxidation in the process.     

Long-term effects of tumor necrosis factor-alpha treatment on insulin signaling pathway in HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB

Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.     

Differential expression and tumor necrosis factor‐mediated regulation of TNFRSF11b/osteoprotegerin production by human melanomas

Tumors escape host immune responses, in part, through the release of immunomodulatory factors and decoy receptors into their microenvironment. Several cancers express surface‐bound and soluble members of the tumor necrosis factor (TNF) receptor superfamily, including TNFRSF11b/osteoprotegerin (OPG). In its physiologic role, OPG regulates bone remodeling through competition for osteoclast‐activating cytokines and protects newly forming bone from T cell‐mediated apoptosis. In multiple tumor types, OPG production is associated with an aggressive phenotype and increased metastasis to bone, but no study has examined OPG production in human metastatic melanoma. We demonstrate that a significant proportion of human metastatic melanomas constitutively produces OPG through a mechanism governed by membrane‐bound TNF‐α signaling through TNF receptor 1 (TNFR1). These observations both define a specific mechanism that regulates melanoma production of OPG and establish a new molecular target for the therapeutic regulation of OPG.     

Genetic regulation of MUC1 expression by Helicobacter pylori in gastric cancer cells

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.     

Effect of lactoferrin on the production of tumor necrosis factor‐α and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor‐α and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor‐α production or nitric oxide production, but lipopolysaccharide‐stimulated tumor necrosis factor‐α production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin. J. Cell. Biochem. 76:30–36, 1999. © 1999 Wiley‐Liss, Inc.     

Prolactin induced expression of interleukin-1 alpha,tumor necrosis factor-alpha,and transforming growth factor-alpha in cultured astrocytes

Prolactin (PRL) is a potent mitogen in cultured astrocytes. Because one of the major effects of astrocyte proliferation is the expression of inflammatory cytokines, we examined the effect of PRL-induced mitogenesis on the expression of interleukin-1 (IL-1α), tumor necrosis factor-α (TNF-α), and transforming growth factor-α (TGF-α) in cultured astrocytes. Astrocytes were stimulated with PRL or growth hormone (GH), and the expression of cytokines was determined by immunohistochemistry and Western blot analysis. Following incubation of astrocytes with 1 nM PRL for 6 h, strong positive staining of IL-1α and TNF-α, but not TGF-α, was found. No detectable staining for the above cytokines was found in vehicle, or GH treated astrocytes. When astrocytes were incubated in the presence of 1 nM PRL for 18 h, strong positive staining for IL-1α and TGF-α was found. Immunocytochemical analysis of the expression of TNF-α and IL-1α in PRL stimulated astrocytes suggested that the expression of IL-1α preceded the expression of TNF-α. To confirm this observation, Western blot analyses were performed on extracts from astrocytes incubated with 1 nM PRL. In unstimulated astrocytes, IL-1α levels were not detectable. In astrocytes stimulated with 1 nM PRL, expression of IL-1α was clearly detected after 1 h of incubation, and IL-1α levels continued to increase during the course of the experiment (6 h). In contrast, in astrocytes stimulated with 1 nM PRL, an increase in the expression of TNF-α was first apparent after 2 h of incubation. TNF-α levels peaked 3 to 4 h after the addition of PRL, and returned to near control levels after 6 h. Finally, injection of PRL into a wound site in female rats increased the expression of glial fibrillary acid protein (GFAP), an astrocyte specific protein. These data suggest that PRL can stimulate astrogliosis at the wound site in vivo. These data clearly indicate that PRL can stimulate the expression of TNF-α and IL-1α in cultured astrocytes and suggest that PRL may play a role in the regulation of the neuroimmune response in vivo.     

Ginsenoside Rg1 inhibits tumor necrosis factor-alpha (TNF-alpha)-induced human arterial smooth muscle cells (HASMCs) proliferation

In China, the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The aim of this study was to determine the effects of ginsenoside Rg1 on the proliferation and molecular mechanism in cultured human arterial vascular smooth muscle cell (HASMC) induced by tumor necrosis factor-alpha (TNF-alpha). It was shown that ginsenoside Rg1 significantly inhibited TNF-alpha-induced HASMC proliferation in a dose-dependent manner. Treatment with ginsenoside Rg1, which blocked the cell cycle in the G1-phase, induced a downregulation of cyclin D1 and an upregulation in the expression of p53, p21(WAF/CIP1), and p27(KIP1). MEK inhibitors PD98059, U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not p38-inhibitor SB203580 or JNK-inhibitor SP600125 significantly aggravated ginsenoside Rg1-inhibited HASMC proliferation. Ginsenoside Rg1 markedly inactivated the extracellular signal-regulated kinases (ERK1/2) and protein kinase B (PKB), indicating that the inhibition of ginsenoside Rg1 on HASMC proliferation was associated with ERK and PI3K/PKB pathways. The inactivation of ERK and PI3K/PKB pathways and modulation of cell-cycle proteins by ginsenoside Rg1 may be of importance in inhibition of HASMCs proliferation.