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.     

The chemokine receptor CCR7 is expressed on epithelium of non-inflamed gastric mucosa, Helicobacter pylori gastritis, gastric carcinoma and its precursor lesions and up-regulated by H. pylori

CCR7 chemokine-receptor expression on tumour cells of gastric carcinoma has been associated with lymph-node metastasis and is thought to play an important role in metastasis. However, so far it is unknown whether CCR7 is newly up-regulated on gastric carcinoma or already expressed in non-neoplastic gastric epithelium. Therefore, epithelial CCR7 expression was investigated in the process of gastric carcinogenesis: non-inflamed mucosa --Helicobacter pylori gastritis -- intestinal metaplasia/dysplasia -- gastric carcinoma. CCR7 was expressed by gastric epithelium in non-inflamed gastric mucosa (n = 5), H. pylori gastritis (n = 17), intestinal metaplasia (n = 10), dysplasia (n = 3) and on tumour cells in 20 of 24 patients with gastric carcinoma (13/14 intestinal-type; 7/10 diffuse-type) as tested by immunohistochemistry. As CCR7 expression by gastric epithelium was significantly stronger in H. pylori gastritis than in non-infected mucosa, the influence of H. pylori on CCR7 receptor expression of gastric epithelial cells was investigated by fluorescence activated cell sorter analysis. H. pylori strains up-regulated the CCR7 chemokine-receptor in CCR7-positive cell lines. No difference in CCR7 up-regulation between cag(+) and cag(-)H. pylori strains was found. Epithelial CCR7 up-regulation by H. pylori may alter the metastatic fate of gastric carcinoma. Additionally, CCR7 expression not only on gastric carcinoma, but also on non-neoplastic gastric epithelium, suggests a novel biological function.     

Functional Analysis of the Helicobacter pylori cag Pathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms

The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering ("hummingbird") phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.     

Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens

The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H. pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains. Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR. The levels of anti-H. pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island. For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE. H. pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H. pylori, and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H. pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H. pylori infections.     

Epithelial Intestinal Cell Apoptosis Induced by Helicobacter pylori Depends on Expression of the cag Pathogenicity Island Phenotype

Helicobacter pylori has been shown to induce chronic active gastritis and peptic ulcer and may contribute to the development of duodenal ulcer. Previous studies have shown that H. pylori mediates apoptosis of gastric epithelial cells via a Fas-dependent pathway. However, evidence for the induction of such a mechanism in intestinal epithelial cells (IEC) by H. pylori infection has not been demonstrated yet. This study was performed (i) to ascertain that H. pylori can induce IEC apoptosis; (ii) to delineate the role of the cag pathogenicity island (PAI), cagE, and vacA gene products in this process; and (iii) to verify whether the Fas-dependent pathway is involved in this phenomenon. When T84 cells were exposed to VacA(+)/cag PAI(+) H. pylori strains (CCUG 17874 and 60190), they exhibited apoptosis hallmarks as assessed by morphological studies, as well as annexin V and 3,3'-dihexyloxacarbocyanine iodide staining. In contrast, few or no apoptotic features could be detected after incubation with an isogenic mutant of strain 60190 in which the cagE gene was disrupted (60190:C(-) strain) or with a VacA(-)/cag PAI(-) H. pylori strain (G21). In addition, activation of caspase-3 during infection with VacA(+)/cag PAI(+) H. pylori strains was inhibited by pretreatment of IEC with an antagonistic anti-Fas antibody (ZB4). Taken together, these findings indicate that H. pylori triggers apoptosis in IEC via a Fas-dependent pathway following a process that depends on the expression of the cag PAI.     

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.     

The Helicobacter pylori cag pathogenicity island protein CagN is a bacterial membrane-associated protein that is processed at its C terminus

Helicobacter pylori infects nearly half the world's population and is associated with a spectrum of gastric maladies. Infections with cytotoxin-associated gene pathogenicity island (cag PAI)-containing strains are associated with an increased risk for gastric cancer. The cag PAI contains genes encoding a type IV secretion system (T4SS) and a delivered effector, CagA, that becomes tyrosine phosphorylated upon delivery into host cells and initiates changes in cell signaling. Although some cag PAI genes have been shown to be required for CagA delivery, a subset of which are homologues of T4SS genes from Agrobacterium tumefaciens, the majority have no known function or homologues. We have performed a detailed investigation of one such cag PAI protein, CagN, which is encoded by the gene HP0538. Our results show that CagN is not delivered into host cells and instead is associated with the bacterial membrane. We demonstrate that CagN is cleaved at its C terminus by a mechanism that is independent of other cag PAI proteins. Finally, we show that a delta cagN mutant is not impaired in its ability to deliver CagA to gastric epithelial cells and initiate cell elongation.     

Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells

Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis.     

IFN-gamma synergizes with TNF-alpha but not with viable H. pylori in up-regulating CXC chemokine secretion in gastric epithelial cells.

Helicobacter pylori colonizes the gastric epithelial surface and induces epithelial cells to increase production of the neutrophil attractant IL-8. Little is known about the role of the gastric epithelium in regulating mucosal T cell trafficking. We therefore characterized constitutive and regulated epithelial expression of the CXC chemokines IP-10, I-TAC and Mig, which specifically attract CXCR3 expressing CD4(+) T cells. Human gastric epithelial cell lines (AGS, Kato III, NCI) were used to characterize the constitutive and regulated expression of three CXC chemokines in response to IFN-gamma, TNF-alpha and different H. pylori preparations. Chemokine mRNA and protein production were measured by RT-PCR and ELISA. Gastric epithelial cells constitutively expressed mRNA for IP-10, Mig and I-TAC. IFN-gamma in combination with TNF-alpha strongly induced secretion of those chemokines. Soluble or membranous fractions of H. pylori significantly inhibited IFN-gamma/TNF-alpha induced epithelial cell IP-10 and Mig production. Gastric epithelial cells may contribute to mucosal T cell trafficking. The capacity of H. pylori products to inhibit IP-10 and Mig secretion may explain, at least in part, the failure to induce protective immunity against this bacterium and the ability of H. pylori to affect the presentation of the local inflammation.     

Expression of Helicobacter pylori virulence factors and associated expression profiles of inflammatory genes in the human gastric mucosa

Helicobacter pylori virulence factors have been suggested to be important in determining the outcome of infection. The H. pylori adhesion protein BabA2 is thought to play a crucial role in bacterial colonization and in induction of severe gastric inflammation, particularly in combination with expression of CagA and VacA. However, the influence of these virulence factors on the pathogenesis of H. pylori infection is still poorly understood. To address this question, the inflammatory gene expression profiles for two groups of patients infected with triple-negative strains (lacking expression of cagA, babA2, and vacAs1 but expressing vacAs2) and triple-positive strains (expressing cagA, vacAs1, and babA2 but lacking expression of vacAs2) were investigated. The gene expression patterns in the antrum gastric mucosa from patients infected with different H. pylori strains were very similar, and no differentially expressed genes could be identified by pairwise comparisons. Our data thus suggest that there is a lack of correlation between the host inflammatory responses in the gastric mucosa and expression of the babA2, cagA, and vacAs1 genes.