Jean E. Crabtree

The response of cells from low-grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobacter pylori.

An association has been shown between colonisation of gastric mucosa by Helicobacter pylori, acquisition of mucosa-associated lymphoid tissue (MALT), and occurrence of primary B-cell gastric MALT lymphoma. We investigated the immunological response of cells from 3 low-grade primary B-cell gastric MALT lymphomas to H pylori type NCTC 11637 and 12 isolates of H pylori from patients without lymphomas. After co-culture of tumour cells with bacteria, cells were examined for phenotypic evidence of activation and proliferation, and supernatant assayed to detect tumour-derived immunoglobulin and interleukin-2 (IL-2). Neoplastic B cells and non-neoplastic T cells proliferated, and IL-2-receptor expression by most cells in the cultures was increased with stimulating strains of H pylori. There were also increases in tumour immunoglobulin and IL-2 release when activation and proliferation were seen in response to stimulating bacteria. Removal of T cells from the tumour cell suspension reduced proliferation and IL-2-receptor expression. In comparison, no responses were seen in cells from high-grade gastric MALT lymphomas or low-grade B-cell MALT lymphomas of other sites. The response of low-grade B-cell gastric MALT lymphomas to stimulating strains of H pylori is dependent on H-pylori-specific T cells and their products, rather than the bacteria themselves.

Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal‐type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA+ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat‐shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL‐8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end‐points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag‐encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.

Helicobacter infection and gastric neoplasia.

Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the distal stomach, yet only a minority of people who harbour this organism ever develop cancer. H. pylori isolates possess substantial genotypic diversity, which engenders differential host inflammatory responses that influence clinical outcome. H. pylori strains that possess the cag pathogenicity island and secrete a functional cytotoxin induce more severe gastric injury and further augment the risk for developing distal gastric cancer. However, carcinogenesis is also influenced by host genetic diversity, particularly involving immune response genes such as IL‐1ß and TNF‐α. It is important to gain insight into the pathogenesis of H. pylori‐induced gastritis and adenocarcinoma, not only to develop more effective treatments for gastric cancer, but also because it might serve as a paradigm for the role of chronic inflammation in the genesis of other malignancies that arise within the gastrointestinal tract. Copyright

Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype.

AIMS--To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS--Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS--Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS--These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration.

CagA/cytotoxic strains of Helicobacter pylori and interleukin-8 in gastric epithelial cell lines.

AIMS--To investigate: (1) whether Helicobacter pylori directly induces interleukin-8 (IL-8) message expression and protein secretion in established gastric epithelial cell lines; and (2) if CagA/cytotoxin positive and negative strains of H pylori differ in their ability to induce epithelial IL-8. METHODS--Gastric epithelial cell lines were co-cultured with H pylori NCTC 11637 and 10 clinical isolates (four cytotoxic, six non-cytotoxic) and secreted IL-8 was measured by enzyme linked immunosorbent assay (ELISA). Specific induction of gastric epithelial IL-8 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR) amplification. RESULTS--H pylori (NCTC 11637) induced IL-8 secretion from three gastric epithelial cell lines (KATO-3, ST42, AGS) but not from MKN 45 (gastric) or intestinal (SW480, HT29) cell lines. H mustelae did not stimulate IL-8 secretion from KATO-3, ST42, and AGS cells. H pylori induced IL-8 secretion was reduced by heat killing, sonication, freeze thawing or formalin fixation of the bacteria. CagA/cytotoxin positive strains of H pylori induced significantly higher IL-8 secretion than CagA/cytotoxin negative strains in the three positive gastric epithelial cell lines (KATO-3, ST42: p < 0.01; AGS: p < 0.02). A significant increase (p < 0.01) in the expression of IL-8 mRNA relative to G3PDH mRNA was observed in KATO-3 cells after three hours of co-culture with CagA/cytotoxin positive strains. CONCLUSIONS--H pylori directly increases gastric epithelial IL-8 mRNA expression and IL-8 protein secretion in a strain specific manner. Induction of epithelial IL-8 by CagA/cytotoxin positive strains is likely to result in neutrophil chemotaxis and activation and thus mucosal damage. These observations on epithelial IL-8 may explain the association between CagA/cytotoxin positive strains and gastroduodenal disease.

Role of Helicobacter pylori CagA+ strains and risk of adenocarcinoma of the stomach and esophagus

Infection with Helicobacter pylori (H. pylori), especially CagA+ strains, has been associated with an increased risk of noncardia gastric adenocarcinoma. The relationship with junctional cancer (adenocarcinomas of the esophagus and gastric cardia combined) has not been adequately investigated, although some studies have reported a reduced risk associated with H. pylori and CagA seroseropositivity. We investigated this question in a subset of cases and controls from a recently completed, large population‐based case‐control study of gastric and esophageal adenocarcinomas in Los Angeles County. Using established antigen‐specific ELISAs, serum IgG antibodies to H. pylori whole‐cell antigens (Helico‐G) and CagA were measured in population controls (n = 356) and patients with incident esophageal adenocarcinoma (n = 80), gastric cardia cancer (n = 87) or distal gastric cancers (noncardia gastric adenocarcinoma) (n = 127). After controlling for demographic characteristics (age, gender, race, birthplace, education), smoking and body mass index, seropositivity for H. pylori was associated with a statistically significant increased risk of distal gastric cancer (adjusted odds ratio [OR] = 1.85, 95% confidence interval [CI] = 1.03, 3.32) but the risk of junctional cancer was not increased (adjusted OR = 1.26, 95% CI = 0.82, 1.94). The risk of junctional cancer was not changed when CagA and H. pylori were both considered, but the risk of distal gastric cancer was further increased. Subjects who were seropositive for both CagA and H. pylori compared to those who were seronegative for H. pylori showed a risk of 2.20 (95% CI = 1.13, 4.26) for distal gastric cancer and 0.86 (95% CI = 0.47, 1.59) for junctional cancer. Although tests for interaction between smoking and H. pylori were not statistically significant for junctional or distal gastric cancers, risk for both tumor types tended to be higher among current smokers who were also H. pylori seropositive. In conclusion, we find no evidence that infection with CagA+ strains of H. pylori reduces risk of esophageal and gastric cardia adenocarcinoma in this population. Our findings confirm the positive association between risk of distal gastric cancer and infection with H. pylori infection, especially CagA+ strains.

Gastric mucosal inflammatory responses to Helicobacter pylori.

Within the gastroduodenal mucosa Helicobacter pylori infection stimulates local production of a range of proinflammatory and immunoregulatory cytokines, neutrophil infiltration, specific T‐ and B‐ cell responses and the development of gastric lymphoid follicles. Following bacterial eradication this mucosal inflammatory response resolves. Infiltrating neutrophils are likely to be one of the major mediators of mucosal damage. Neutrophil activation, including reactive oxygen metabolite production and the release of myeloperoxidase, will be induced directly by bacterial factors and indirectly through products of complement activation, bioactive lipids and host‐derived cytokines. Interleukin‐8, and related peptides of the chemokine family secreted by gastric epithelial cells, are likely to be important host mediators inducing neutrophil migration to sites of infection. Epithelial IL‐8 is upregulated by TNF‐alpha and IL‐1 and directly by H. pylori strains expressing the CagA phenotype. The extent of mucosal injury may reflect bacterial density, the variability of different strains of H. pylori to induce chemokine expression in epithelial cells and the oxidative burst in neutrophils. Recent evidence from in vivo and in vitro studies shows that CagA+ VacA+ strains of H. pylori are associated with enhanced inflammatory responses and mucosal damage. Defining the specific bacterial mediators of mucosal inflammation will be important in elucidating the role of H. pylori in the pathogenesis of gastroduodenal disease.

Immune and Inflammatory Responses to Helicobacter pylori Infection

The gastroduodenal response to chronic Helicobacter pylori infection is characterized by the infiltration of plasma cells, lymphocytes, neutrophils and monocytes into the mucosa. Eradication studies have shown that this inflammatory response represents a specific reaction to the presence of H. pylori. As well as stimulating specific local T and B cell responses and a systemic antibody response, H. pylori infection also induces a local pro-inflammatory cytokine response. Interleukin-8 (IL-8), which is expressed and secreted by gastric epithelial cells, may be an important host mediator inducing neutrophil migration and activation. IL-8 mRNA and protein secretion in gastric epithelial cell lines can be up-regulated by the cytokines tumour necrosis factor-alpha and IL-1 and also by type I strains of H. pylori (expressing the vacuolating toxin and cytotoxin-associated protein, CagA). The gastric epithelium thus plays an active role in mucosal defence. Neutrophil activation and the production of reactive oxygen metabolites will be induced directly by bacterial factors and indirectly via host-derived cytokines, products of complement activation and bioactive lipids. Strain variation in the induction of both IL-8 from epithelial cells and the oxidative burst in neutrophils may be an important factor determining the extent of mucosal injury. There is now increasing evidence from both in vivo and in vitro studies that type I strains induce an enhanced inflammatory response and mucosal damage. An understanding of the bacterial mediators of mucosal inflammation is important in elucidating the role of chronic H. pylori infection in the pathogenesis of gastroduodenal disease.

Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.

Chemokine mRNA expression in gastric mucosa is associated with Helicobacter pylori cagA positivity and severity of gastritis.

AIM: To investigate the association between the quantity of gastric chemokine mRNA expression, severity of gastritis, and cagA positivity in Helicobacter pylori associated gastritis. METHODS: In 83 dyspeptic patients, antral and corpus biopsies were taken for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and histological grading of gastritis. Gastritis was evaluated by visual analogue scales. Quantities of chemokine (IL-8, GRO alpha, ENA-78, RANTES, MCP-1) RT-PCR products were compared with G3PDH products. Each sample was also evaluated for the presence of cagA and ureA mRNA by RT-PCR. RESULTS: mRNA expression of all five chemokines was significantly greater in H pylori positive than in H pylori negative mucosa. In H pylori positive patients, in the antrum C-X-C chemokine mRNA expression was significantly greater in cagA positive patients than in cagA negative patients, but there were no significant differences in C-C chemokine mRNA expression. In H pylori positive patients, chemokine mRNA expression in the corpus was less than in the antrum. In contrast to the antrum, only GRO alpha mRNA expression was significantly greater in cagA positive infection. Polymorphonuclear cell infiltration was correlated with C-X-C chemokine mRNA expression. Significant correlations were also found between bacterial density and C-X-C chemokine mRNA expression. CONCLUSIONS: In H pylori infection, C-X-C chemokines may play a primary role in active gastritis. Infection with cagA positive H pylori induces greater gastric chemokine mRNA expression in the antral mucosa, which may be relevant to the increased mucosal damage associated with cagA positive H pylori infection.