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.     

Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.     

Upregulation of CCL20 and recruitment of CCR6+ gastric infiltrating lymphocytes in Helicobacter pylori gastritis

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. There is increased T-cell infiltration at the site of infection with H. pylori. CCR6, a specific β-chemokine receptor for CCL20 (MIP-3α/LARC/exodus), has recently been reported to mediate lymphocyte homeostasis and immune responses in mucosal tissue, and it may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation. In this study, we investigated the role of CCR6 and its ligand, CCL20, in inducing an inflammatory response in the gastric mucosa during H. pylori infection. Gastric infiltrating T lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed for the expression of the CCR6 chemokine receptor. Our results demonstrated that there was significantly increased CCR6 expression in CD3⁺ T cells infiltrating the gastric mucosa, and the CCR6 ligand, the CCL20 chemokine, was selectively expressed in inflamed gastric tissues. The production of CCL20 was upregulated in response to H. pylori in gastric epithelial cells when there was stimulation by the proinflammatory cytokines interleukin-1β and tumor necrosis factor alpha. Furthermore, recombinant CCL20 induced lymphocyte chemotaxis migration in fresh gastric T cells ex vivo, indicating that the gastric T cells could migrate toward inflammatory sites via CCR6/CCL20 interaction. Our results suggest that the interaction between CCL20 and CCR6 may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation in Helicobacter infection.     

Helicobacter pylori-specific CD4+ T cells home to and accumulate in the human Helicobacter pylori-infected gastric mucosa

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.     

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.     

CD4+ and CD8+ T cell responses in Helicobacter pylori-infected individuals

In order to characterize T cell responses in human Helicobacter pylori infection, we have examined proliferative responses and cytokine production by CD4+ and CD8+ T cells isolated from duodenal ulcer patients and asymptomatic H. pylori carriers, after activation with some H. pylori antigens that may be important in disease development. For control purposes, T cells from uninfected volunteers were also examined. The different H. pylori antigens induced only modest proliferative responses in circulating CD4+ and CD8+ T cells from both H. pylori-infected and uninfected individuals. However, circulating T cells from H. pylori-infected subjects produced larger amounts of interferon-gamma (IFN-gamma) in response to the Helicobacter antigens than did T cells from uninfected volunteers. Furthermore, CD8+ T cells produced larger amounts of IFN-gamma than did CD4+ T cells, on a per cell basis. Most IFN-gamma-producing cells from both infected and uninfected volunteers appeared to be naive T cells expressing CD45RA. Increased production of IL-4 and IL-5 was, on the other hand, only seen in a few instances after stimulation of isolated CD4+ and CD8+ T cells. Stimulation of freshly isolated gastric T cells with the different H. pylori antigens did not result in increased proliferation or cytokine production. In conclusion, our results show that several different purified H. pylori antigens induce production of IFN-gamma, preferentially by CD8+ cells. Therefore, they suggest that IFN-gamma-secreting CD8+ cells contribute significantly to the cytokine response induced by H. pylori infection.     

Molecular cloning and functional characterization of porcine CCL28: possible involvement in homing of IgA antibody secreting cells into the mammary gland

Constitutive expression of chemokines by epithelial cells controls the recruitment and the localization of specialized lymphocytes. Mucosae associated-epithelial chemokine (MEC/CCL28) cloned from porcine salivary gland and colon tissues consisted of an open reading frame (ORF) of 384-bp coding for 127 amino-acids protein with 22 residues signal sequence. The resulting mature protein is composed of 105 aa with 4 conserved cysteine residues. CCL28 shows aa sequence identity with rat, mouse, macaque and human ranging from 67 to 87%. Using plasmid pQETris-CCL28 injection, a rabbit anti-serum was produced and showed a specific reactivity towards non-reduced form of CCL28 recombinant protein. Comparatively to CCL25 mRNA expression, RT-PCR analysis showed that CCL28 is expressed in various mucosal tissues, but most abundantly in nasal mucosa, colon, salivary and mammary gland (MG). Immunohistochemical analysis showed that CCL28 is produced by epithelial cells of these tissues suggesting that this chemokine can play an important role by linking homing mechanisms between the gut, nasal mucosa and MG. In addition, mRNA of CCL28 was up-regulated in the MG at late gestation and during lactation but was not found at weaning. CCL28 protein was excreted in sow's milk sustaining that this chemokine plays a key role of IgA-ASCs accumulation in this tissue and thus controls the passive transfer level of IgA antibodies from mother to infant.     

Human IgA‐secreting cells induced by intestinal,but not systemic,immunization respond to CCL25 (TECK) and CCL28 (MEC)

Organ‐specific homing of lymphoid cells depends on the expression of tissue‐specific adhesion molecules and production of specific chemokines. CCL25 (TECK) and CCL28 (MEC) have been reported to direct circulating memory/effector B cells to mucosal tissues. Here, we examined if differential responsiveness to mucosal and systemic chemokines could explain the differential migration pattern of circulating human antibody‐secreting cells (ASC), induced by mucosal and systemic immunization. There was a robust migration of specific IgA‐ and IgM‐ASC induced by Salmonella vaccination toward the mucosal chemokines CCL25 and CCL28. In contrast, tetanus‐specific ASC migrated to the systemic chemokine CXCL12 (SDF‐1α) and showed no response to CCL25 or CCL28, not even tetanus‐specific IgA‐ASC. Cell sorting experiments demonstrated that Salmonella‐specific ASC co‐expressed CCR9 and CCR10. Our results show that induction site, rather than isotype commitment, determines the chemokine responsiveness and migration pattern of human effector B cells.     

The local and systemic T-cell response to Helicobacter pylori in gastric cancer patients is characterised by production of interleukin-10

Helicobacter pylori causes a life-long infection that may lead to development of gastric adenocarcinoma (GC) and thereby cause major worldwide health problems. The present study was designed to study whether those that develop GC have an altered immune response to H. pylori compared to individuals that remain asymptomatic. When stimulated with H. pylori antigens, T cells from both peripheral blood and gastric mucosa of H. pylori-infected GC patients produced high amounts of IL-10, while the IL-10 production from blood T cells of H. pylori-infected asymptomatic subjects was low. Furthermore, mRNA levels of IL-10 were increased in the gastric mucosa of GC patients. In addition, the frequency of activated CD8(+) T cells was markedly reduced in stomach mucosa of patients with GC compared to asymptomatic individuals. We propose that the increased production of the suppressive cytokine IL-10 in H. pylori-infected GC patients leads to a diminished cytotoxic anti-tumour T-cell response in the stomach, which may contribute to tumour progression in subjects suffering from GC.     

Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients

Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4(+) CD25(high) T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4(+) CD25(low) and CD4(+) CD25(-) cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4(+) CD25(high) T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4(+) CD25(high) cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.     

CCL25和CCL28及其相应受体CCR9和CCR10

CCL25和CCL28是CC趋化因子家族成员,主要表达于胸腺树突状细胞和黏膜上皮细胞,CCL25和CCL28相应的受体分别是CCR9和CCR10,表达于T淋巴细胞和B淋巴细胞。CCL25和CCL28及其相应受体对循环IgA浆母细胞和T淋巴细胞的归巢起重要作用,而且CCL28还具有广谱的抗微生物活性。     

Cytokine expression in pediatric Helicobacter pylori infection

Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide and almost invariably causes chronic gastritis in the infected host. A predominant Th1 profile has been demonstrated in H. pylori-infected mucosa from adults, but no previous study has evaluated in situ cytokine expression in children. We therefore examined expression of proinflammatory, anti-inflammatory, and regulatory cytokines by immunohistochemistry in cryopreserved antral biopsy specimens from 10 H. pylori-infected and 10 uninfected children and correlated expression of cytokines with histology scores. Concomitant expression of interleukin-8 (IL-8), gamma interferon (IFN-gamma), IL-4, transforming growth factor beta, and tumor necrosis factor alpha was seen in 8/10 H. pylori-infected cases and in 5/10 noninfected cases; all H. pylori-infected subjects showed staining for at least two of the cytokines. The proportion of epithelial cytokine-specific staining did not differ significantly between the groups, either in surface or glandular epithelium. Furthermore, no significant differences were noticed between intraepithelial or lamina propria lymphocyte staining in the groups. There was, however, a tendency of higher numbers of IFN-gamma- and IL-8-positive cells in the H. pylori-infected group. IFN-gamma and IL-8 lamina propria lymphocyte expression correlated significantly with antrum chronic inflammation, but there was no correlation between histology scores and epithelial cytokine expression. When the same techniques were used, the cytokine response appeared to be smaller in H. pylori-infected children than in adults, and there was no clear Th1 dominance. These results therefore suggest a different mucosal immunopathology in children. It remains to be determined whether the gastric immune response is downregulated in children with H. pylori infection and whether this is relevant to the outcome of infection.     

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.     

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.     

Chemokine receptor 5 expression in gastric mucosa of Helicobacter pylori-infected and noninfected children

Experimental data from human adults or animal models indicate that the Helicobacter pylori-specific immune response is dominated by inflammatory T cells of the Th1 type. To investigate whether a Th1 immune response is established in early H. pylori infection, gastric biopsy samples from 70 children were subjected to immunohistochemical analysis. To this end, T cells, B cells, monocytes, neutrophils, and chemokine receptor 5 (CCR5)-expressing (CCR5(+)) cells, which are associated with Th1 immune responses, were quantified. Children were classified according to H. pylori status and clinical, laboratory, and macroscopic (during endoscopy) findings, without knowledge of histological findings. Group 1 included 31 H. pylori-infected children, group 2 contained 24 children with other conditions possibly affecting the stomach, and group 3 contained 15 children without verifiable pathological findings in the stomach. Lymphoid follicles were present in 90% of biopsy samples from group 1 and 48% of those from group 2 but absent in group 3 biopsy samples. Intraepithelial T cells and CCR5(+) cells were regularly detected in all groups without significant differences. B cells, monocytes, and neutrophils were not found. In contrast, the numbers of lamina propria T cells (P < 0.003) and CCR5(+) cells (P < 0.001) were increased significantly in H. pylori-infected children. B cells (in 13 of 66 children) were detected in children with active (n = 11) or previously cleared (n = 2) H. pylori infections but were absent in healthy children. The numbers of monocytes (in 10 of 67 children) did not differ among the groups. Calculations indicated that the majority of gastric T cells express CCR5; this finding is in contrast to the low percentage of CCR5(+) T cells in the peripheral circulation. Thus, an increase in the numbers of CCR5(+) cells in H. pylori-infected stomach mucosa suggests that this molecule may play an important role in gastric immune responses.     

Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells

During Helicobacter pylori infection, T cells are recruited to the gastric mucosa, but the host T-cell response is not sufficient to clear the infection. Some of the recruited T cells respond in a polarized manner to a Th1 response, while others become anergic. We have previously shown that T-cell anergy may be induced during infection by the interaction of T cells with B7-H1, which is up-regulated on the gastric epithelium during H. pylori infection. Recently, regulatory T (Treg) cells with a CD4(+) CD25(high) FoxP3(+) phenotype were found at an increased frequency in the gastric mucosa of biopsy specimens from H. pylori-infected patients. While Treg cells are important in maintaining tolerance, they can also suppress immune responses during infection. In this study, we examined the induction of the Treg phenotype when na?ve T cells were incubated with gastric epithelial cells exposed to H. pylori. The frequency of this phenotype was markedly decreased when B7-H1 was blocked with monoclonal antibodies or its expression was blocked with small interfering RNA. The functional role of these Treg cells was assessed in proliferation assays when the cells were cocultured with activated T cells, which effectively decreased proliferation of the cells.