A mechanism for the action of the compound DA-6034 on NF-κB pathway activation in Helicobacter pylori-infected gastric epithelial cells

DA-6034 is a synthetic derivative of eupatilin, a flavonoid with anti-inflammatory effects. The aim of this study was to investigate the effects of DA-6034 on the interactions between IκB kinase (IKK) and heat shock protein 90 (Hsp90), and activation of the nuclear factor-kappaB (NF-κB) signalling pathway in human gastric epithelial cells infected with Helicobacter pylori. MKN-45 gastric epithelial cell line was treated with DA-6034 and H. pylori. DA-6034 significantly inhibited NF-κB activation and upregulated the expressions of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 in MKN-45 cells infected with H. pylori. However, DA-6034 did not influence activator protein-1 DNA binding activity in H. pylori-infected gastric epithelial cells. Pretreatment with DA-6034 attenuated the H. pylori-induced increase in IKK activity, and Hsp90 was associated with IKK-α and IKK-γ in MKN-45 cells. Treatment with DA-6034 dissociated the Hsp90 and IKK-γ complex in H. pylori-infected cells, leading to the inhibition of IL-8 expression. These results suggest that the eupatilin derivative 7-carboxymethyloxy-3',4',5-trimethoxy flavone has anti-inflammatory activity in gastric epithelial cells infected with H. pylori through the promotion of the dissociation of the IKK-γ-Hsp90 complex and suppression of NF-κB signalling.     

Conjugated linoleic acids produced by Lactobacillus dissociates IKK-gamma and Hsp90 complex in Helicobacter pylori-infected gastric epithelial cells

Although probiotics have been reported to reduce the gastric inflammatory response to Helicobacter pylori infection, little information is available regarding the molecular mechanisms behind this reduction. This study investigates the role of conjugated linoleic acids (CLA) produced by probiotics in interactions of IkappaB kinase (IKK) and heat shock protein 90 (Hsp90) to activate the nuclear factor-kappaB (NF-kappaB) signaling pathway in human gastric epithelial cells infected with H. pylori. Conditioned medium (CM) containing Lactobacillus acidophilus-producing CLA significantly inhibited the activated NF-kappaB signals and the upregulated expression of interleukin-8 (IL-8) in MKN-45 cells infected with H. pylori. Pretreatment with CM with CLA attenuated the increased IKK activity induced by H. pylori. Transfection of siRNA for IKK-beta dramatically reduced H. pylori-induced IkappaBalpha phosphorylation, but siRNA for IKK-alpha had little effect on IkappaBalpha phosphorylation, although the siRNA for IKK-alpha significantly decreased IL-8 production. Furthermore, Hsp90 was associated with IKK-alpha and IKK-gamma in H. pylori-infected cells, and CM with CLA dissociated the complex between Hsp90 and IKK-gamma. These results suggest that CLA produced by probiotics has anti-inflammatory activity in gastric epithelial cells infected with H. pylori via dissociation of the IKK-gamma and Hsp90 complex.     

Orally administered CpG oligodeoxynucleotide induces production of CXC and CC chemokines in the gastric mucosa and suppresses bacterial colonization in a mouse model of Helicobacter pylori infection

Bacterial DNA and unmethylated CpG oligodeoxynucleotides (CpG ODN) are known to be potent stimulators of the innate immune system in vitro and in vivo. We therefore investigated if oral administration of CpG ODN could enhance innate immunity in the gastric mucosa and control the extent of Helicobacter pylori infection in mice. Intragastric administration of a single dose of CpG ODN significantly increased local production of the CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES and the CXC chemokine gamma interferon-inducible protein 10 in the stomach and/or the small intestine. Importantly, intragastric administration of CpG ODN to mice with an already established H. pylori infection, in the absence of any coadministered antigen, was found to reduce the bacterial load in the stomach compared to the load in H. pylori-infected control mice, while similar administration of non-CpG ODN had no effect on the bacterial load. The reduction in the bacterial numbers in the stomachs of mice treated with CpG ODN was associated with enhanced infiltration of immune cells and increased RANTES production in the gastric mucosa compared to the infiltration of immune cells and RANTES production in H. pylori-infected control animals. These findings suggest that intragastric administration of CpG ODN without antigen codelivery may represent a valuable strategy for induction of innate immunity against H. pylori infection.     

血管紧张素Ⅱ对人内皮细胞转录因子NF－kB的激活机制及其对血小板源生长因子B链基因转录的影响

目的：研究血管紧张素Ⅱ对ECV304细胞中转录因子NF－kB的作用和对血小板源生长因子B链（PDGF－B）基因表达的影响。方法：采用电泳迁移率移动分析（EMSA）,免疫组织化学方法,共聚焦显微镜及金颗粒标记免疫电镜技术;荧光素酶报告基因与变异型激酶质粒共转染的方法及Northern印迹法研究血管紧张素Ⅱ激活ECV304细胞NK－kB的信号传递路径和检测了血管紧张素Ⅱ刺激前后ECV304细胞PGF－BmRNA的表达水平。结果：血管紧张素Ⅱ刺激后,在ECV304细胞内有NK－kB的激活及核易位过程,应用免疫荧光聚集显微镜,免疫镜及Northern印迹等方法均可观察到PDGF－B或其基因表达增高。变异型激酶质粒IKKa-KM,IKKβ－KM及NIK－Km可抑制经血管紧张素Ⅱ刺激的转染细胞内与NF－kB启动相连的荧光素酶的表达。结论：血管紧张素Ⅱ可激活胞质内NK－kB并出现核易位,激酶NIK、IKKα和IKKβ参与了此信号传递路径,血管紧张素Ⅱ刺激后PDGF－B链mRNA水平增高。     

T cell receptor-induced lipid raft recruitment of the I kappa B kinase complex is necessary and sufficient for NF-kappa B activation occurring in the cytosol

TCR-induced NF-kappa B activation is necessary for the innate immune response and involves induced lipid raft recruitment of the I kappa B kinase (IKK) complex. In this study, we systematically investigated lipid raft recruitment of members of the NF-kappa B activation pathway in human T cells. All upstream components leading to IKK activation were found constitutively or inducibly in lipid rafts, while the NF-kappa B/I kappa B complex and phosphorylated forms of IKK alpha/beta, I kappa B alpha and p65 are exclusively found in the cytosolic fraction. Disruption of raft organization precluded NF-kappaB activation induced by T cell costimulation, but IL-1-triggered NF-kappa B activation remained unaffected. Targeting of the IKK complex to lipid rafts caused constitutive IKK activation and NF-kappa B DNA binding, which was further triggered upon T cell costimulation. Various experimental approaches revealed that costimulation-induced IKK alpha/beta activation loop phosphorylation is independent from IKK beta-mediated transautophosphorylation, but rather involves phosphorylation by the IKK-interacting protein NIK and its upstream activator COT.     

Helicobacter pylori infection produces expression of a secretory component in gastric mucous cells

Helicobacter pylori infection induces the expression of a secretory component (SC) in gastric epithelial cells. We investigated the cell lineage of the SC- and immunoglobulin (Ig) A-expressing epithelial cells in H. pylori-infected gastric mucosa. Materials were obtained by means of gastric biopsy from H. pylori-infected patients (24 cases) before and after the eradication of H. pylori, from five normal uninfected volunteers, and from three gastrectomy cases. Acetic acid-ethanol-fixed and paraffin-embedded specimens were examined using histochemical staining for gastric mucins (periodic acid oxidation-thionine Schiff reaction-concanavalin A-horse radish peroxidase staining) by means of immunostaining for gastric mucins (45M1 and HIK1083), intestinal cells (MUC2 and CD10), Ki67, H. pylori, SC, and IgA. The SC and IgA were not found in normal gastric mucosa. The expressions of the SC and IgA in gastric surface mucous cells and mucous neck cells in the generating zone of the gastric mucosa of H. pylori-infected patients were significantly higher before eradication of H. pylori than after the eradication. These mucous cells have the potential for SC-mediated translocation of IgA into the gastric lumen, and this may act as part of the antibacterial defense system against H. pylori infection in the gastric generating zone.     

Activation of intercellular adhesion molecule 1 expression by Helicobacter pylori is regulated by NF-kappaB in gastric epithelial cancer cells

Interactions between leukocytes and epithelial cells may play a key role in Helicobacter pylori-associated gastric mucosal inflammation. This process is mediated by various cell adhesion molecules. The present study examined the molecular mechanisms leading to H. pylori-induced epithelial cell intercellular adhesion molecule-1 (ICAM-1; also called CD54) expression. Coculture of epithelial cells with cytotoxin-associated gene pathogenicity island-positive (cag PAI(+)) H. pylori strains, but not with a cag PAI(-) strain or H. pylori culture supernatants, resulted in upregulation of steady-state mRNA levels and cell surface expression of ICAM-1. Coculture with H. pylori induced an increase in luciferase activity in cells which were transfected with a luciferase reporter gene linked to the 5'-flanking region of the ICAM-1 gene. H. pylori activated the ICAM-1 promoter via the NF-kappaB binding site. An inducible nuclear protein complex bound to the ICAM-1 NF-kappaB site and was identified as the NF-kappaB p50-p65 heterodimer. H. pylori induced the degradation of IkappaB-alpha, a major cytoplasmic inhibitor of NF-kappaB, and stimulated the expression of IkappaB-alpha mRNA. Pretreatment of epithelial cells with pyrrolidine dithiocarbamate, which blocks NF-kappaB activation, inhibited H. pylori-induced ICAM-1 expression. THP-1 macrophagic cells, peripheral blood mononuclear cells, and purified neutrophils adhered to H. pylori-infected epithelial cells to a greater extent than to uninfected cells. These results show that H. pylori directly induces expression of ICAM-1 on gastric epithelial cells in an NF-kappaB-dependent manner that may support leukocyte attachment during inflammation.     

BAFF-induced NEMO-independent processing of NF-kappa B2 in maturing B cells

NF-kappa B is usually activated by signal-induced, ubiquitin-mediated degradation of its inhibitor, I kappa B. This process is initiated by phosphorylation of I kappa B by the I kappa B kinase (IKK) complex, predominantly by the IKK beta catalytic subunit, and requires the regulatory subunit IKK gamma (NEMO). Another activation pathway, with no known physiological inducers, involves ubiquitin-mediated processing of the NF-kappa B2 inhibitory protein p100 and is dependent on phosphorylation of p100 by IKK alpha. We show here that B cell-activating factor (BAFF) activates this second pathway and that this requires the BAFF receptor (BAFF-R), the NF-kappa B-inducing kinase (NIK) and protein synthesis, but not NEMO. This NEMO-independent cascade is physiologically relevant for the survival and, hence, progression of maturing splenic B cells.     

Survival signaling in resting B cells

The survival of mature resting B cells in the periphery depends on signaling from the B-cell receptor (BCR) and the B-cell activating factor of the TNF family receptor (BAFF-R). Engagement of both receptors promotes NF-kappa B activity, which contributes to B-cell survival through different pathways. BCR signaling leads to activation of the inhibitor of NF-kappa B kinase (IKK) complex via Carma1, Bcl10 and MALT1, whereas BAFF-R engagement promotes processing of NF-kappa B2 protein p100, which is dependent on NF-kappa B-inducing kinase (NIK) and IKK alpha. Proximal signaling intermediates are potentially common to both pathways. We suggest that BCR and BAFF-R survival signaling are mutually dependent. In addition, we propose that BAFF-R signaling enhances the expression of survival genes through direct chromatin modifications in NF-kappa B target gene promoters.     

Helicobacter pylori infection stimulates plasminogen activator inhibitor 1 production by gastric epithelial cells

Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.     

CCL28 is increased in human Helicobacter pylori-induced gastritis and mediates recruitment of gastric immunoglobulin A-secreting cells

Human Helicobacter pylori infection gives rise to an active chronic gastritis and is a major risk factor for the development of duodenal ulcer disease and gastric adenocarcinoma. The infection is accompanied by a large accumulation of immunoglobulin A (IgA)-secreting cells in the gastric mucosa, and following mucosal immunization only H. pylori-infected volunteers mounted a B-cell response in the gastric mucosa. To identify the signals for recruitment of gastric IgA-secreting cells, we investigated the gastric production of CCL28 (mucosa-associated epithelial chemokine) and CCL25 (thymus-expressed chemokine) in H. pylori-infected and uninfected individuals and the potential of gastric B-cell populations to migrate toward these chemokines. Gastric tissue from H. pylori-infected individuals contained significantly more CCL28 protein and mRNA than that from uninfected individuals, while CCL25 levels remained unchanged. Chemokine-induced migration of gastric lamina propria lymphocytes isolated from patients undergoing gastric resection was then assessed using the Transwell system. IgA-secreting cells and IgA(+) memory B cells from H. pylori-infected tissues migrated toward CCL28 but not CCL25, while the corresponding cells from uninfected patients did not. Furthermore, IgG-secreting cells from H. pylori-infected patients did not migrate to CCL28 but instead to CXCL12 (SDF-1alpha). However, chemokine receptor expression did not correlate to the migratory pattern of the different B-cell populations. These studies are the first to show increased CCL28 production during gastrointestinal infection in humans and provide an explanation for the large influx of IgA-secreting cells to the gastric mucosa in H. pylori-infected individuals.     

B cells pulsed with Helicobacter pylori antigen efficiently activate memory CD8+ T cells from H. pylori-infected individuals

Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects.     

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer

Recently, we identified increased cathepsin X expression in H. pylori-infected gastric mucosa. Here, we describe further up-regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up-regulation in H. pylori-infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori-infected and non-infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non-contact co-cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5-fold) and protein (1.6-fold) expression than H. pylori-negative patients. Cathepsin X was also up-regulated in gastric cancer (3-12-fold) compared to non-neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)-positive H. pylori-infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up-regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer.     

Helicobacter pylori infection induces interleukin-8 receptor expression in the human gastric epithelium

The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.