The gastric inflammatory response induced by H. pylori consists of neutrophils, lymphocytes (T and B cells), plasma cells, and macrophages, along with varying degrees of epithelial cell degeneration and injury.81 Invasion of the gastric mucosa is rarely if at all identified in vivo, and therefore other potential mechanisms for induction of inflammation must be postulated. One possibility is that H. pylori secretes substances that stimulate mucosal inflammation from afar. For example, urease has been detected within the lamina propria and the urease complex of H. pylori stimulates chemotaxis by both monocytes and neutrophils and activates mononuclear cells as well.82 Similarly, H. pylori porins and low-molecular-weight molecules, such as Paf-acether, possess chemotactic properties.83, 84 Finally, H. pylori water extracts promote neutrophil–endothelial cell interactions in vitro and increase leucocyte adherance via CD11a/CD18 and CD11b/CD18 interactions with intercellular adhesion molecule type-1 (ICAM-1).85
Contact-mediated induction of cytokine release and development of acute inflammation
Another mechanism by which H. pylori may induce inflammation is through direct contact with gastric epithelial cells and stimulation of cytokine release. Gastric epithelium from H. pylori-infected persons demonstrates enhanced levels of interleukin-1β (IL-1β), IL-2, IL-6, IL-8 and TNF-α.86–90 This model is consistent with previous data demonstrating that interaction of Salmonella typhimurium with epithelial cells results in chemotactic cytokine (chemokine)-mediated signalling that directs polymorphonuclear cell migration towards the epithelial cell surface.91, 92 Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine and expression of this peptide is localized to gastric epithelial cells in vivo.93 As might be predicted, levels of IL-8 are also directly related to the severity of gastritis.89In vitro, H. pylori stimulates IL-8 release from gastric epithelial cells and these events require an active interplay between live bacteria and epithelial cells.94–96 Thus, a paradigm for the acute component of H. pylori-induced gastric inflammation is that contact between bacteria and epithelial cells stimulates IL-8 secretion, which then regulates neutrophilic infiltration into the gastric mucosa.
The human IL-8 gene contains several binding sites within its promoter region, including a NF-κB binding motif and a binding site for c-fos and c-jun, which together comprise the transcription factor AP-1.97 NF-κB is a transcription factor sequestered in the cytoplasm, whose activation and regulation is tightly controlled by a class of inhibitory proteins termed IκB’s that mask the nuclear localization signals of NF-κB, thereby preventing movement of NF-κB to the nucleus. Upon stimulation, phosphorylation of IκB leads to its ubiquitination and proteosome-mediated degradation, thereby liberating NF-κB to enter the nucleus where it regulates transcription of a variety of genes.98 Stimulation and activation of NF-κB does not require protein synthesis, thereby allowing efficient activation of target genes, such as IL-8. This system is particularly utilized in immune and inflammatory responses where rapid activation of defense genes following exposure to pathogens such as bacteria is critical for survival of an organism. Several studies have demonstrated that contact between H. pylori and gastric epithelial cells results in brisk activation of NF-κB, which is followed by increased IL-8 expression.99–101 The ability of H. pylori to activate NF-κB in vitro has also been corroborated in vivo as activated NF-κB is present within gastric epithelial cells of infected, but not uninfected patients, which mirrors the location of increased IL-8 protein within colonized mucosa.93, 99
In addition to NF-κB, mitogen-activated protein kinases (MAPK) have been implicated as mediators of H. pylori-induced IL-8 expression. Mitogen-activated protein kinases cascades are signal transduction networks that target transcription factors and thus participate in a diverse array of cellular functions including cytokine production.102–104 The cascades are organized in three-kinase tiers consisting of a mitogen-activated protein kinases, a MAPK kinase (MKK), and a MKK kinase (MKKK). Transmission of signals occurs by sequential phosphorylation and activation of the components specific to a respective cascade. In mammalian systems, five mitogen-activated protein kinases modules have been identified and characterized to date; these include extracellular signal-regulated kinase 1 and 2 (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK).102–104 In addition to regulating NF-κB, mitogen-activated protein kinases can activate other transcription factors, such as AP-1, that regulate cytokine gene expression. H. pylori rapidly induces activation of ERK, p38, and JNK mitogen-activated protein kinases in cell culture systems, and similar to NF-κB activation, these effects are dependent upon the presence of live bacteria, raising the question as to whether H. pylori-induced IL-8 production is dependent upon activation of NF-κB, mitogen-activated protein kinases, or both.105–107In vitro studies utilizing IL-8 reporter constructs have now revealed that H. pylori-induced IL-8 gene expression is dependent upon activation of both NF-κB and AP-1 (via mitogen-activated protein kinases activation), indicating that synergistic interactions between AP-1 and NF-κB within the IL-8 promoter are required for maximal H. pylori-induced IL-8 production.97
Another mechanism through which H. pylori may regulate phagocyte activation is via production of H. pylori neutrophil-activating protein. H. pylori neutrophil-activating protein is a 150-kDa dodecameric iron-binding protein originally identified as a component of the H. pylori outer membrane that promotes adhesion of neutrophils to endothelial cells.108, 109H. pylori neutrophil-activating protein is immunogenic in humans and mice, and stimulates phagocyte chemotaxis, NADPH oxidase assembly, and production of reactive oxygen species in vitro.110 All H. pylori strains analysed to date possess the gene encoding H. pylori neutrophil-activating protein (napA), although protein expression levels are variable.108 However, the relationship between H. pylori neutrophil-activating protein expression and clinical outcome, as well as the effects of isogenic disruption of napA on pathogenic responses, remain to be determined.
H. pylori colonization induces an exuberant systemic and mucosal humoral response directed at multiple antigens.111–116 However, antibody production does not result in eradication even though H. pylori is susceptible in vitro to antibody-dependent complement-mediated phagocytosis and killing.117, 118 These observations suggest that gastric mucus may be a protective niche in which H. pylori exist and are relatively inaccessible to specific antibodies or their effector functions. The ineffective humoral response generated towards H. pylori and its components (i.e. Lewis antigens) may instead actually contribute to pathogenesis. Monoclonal antibodies directed against H. pylori cross-react with gastric epithelium of both mice and humans and delivery of these antibodies alone to mice can induce gastritis.70 In colonized human patients, H. pylori induces the formation of antibodies that recognize the H+,-K+ ATPase epitope on the luminal surface of acid-secreting parietal cells.70, 71 IgM antibodies generated by immortalized B cells obtained from H. pylori-colonized gastric mucosa also recognize gastric epithelium.119 These findings indicate that the mucosal humoral response to H. pylori, in addition to bacterial factors, may contribute to the development of distinct histological lesions. For example, an autoimmune reaction against parietal cells may lead to gastric atrophy with a concomitant reduction in gastric acidity; conversely, immunoglobulin-mediated destruction of epithelial cells may initiate and/or maintain mucosal inflammation and epithelial cell injury.
T lymphocyte-mediated responses
The gastrointestinal tract represents an important barrier between human hosts and microbial populations; the ability to distinguish pathogenic bacteria from commensuals is regulated through T-cell-dependent responses. CD4+ T-cells can be divided into two functional subsets: type 1 (Th1) and type 2 (Th2) T-helper cells, which are defined by distinct patterns of cytokine secretion. Th1 cells produce IL-2 and IFN-γ and promote cell-mediated immune responses, while Th2 cells secrete IL-4, IL-5, IL-6, and IL-10 and induce B-cell activation and differentiation.120 The type of immune response to a particular microbial agent is governed by preferential expansion of one T helper cell subset accompanied by a corresponding down-regulation of the other.120 Most intracellular bacteria induce Th1 responses, while extracellular pathogens stimulate Th2 type responses. Based on the fact that H. pylori is non-invasive and that infection is accompanied by an exuberant humoral response, one might predict that a Th2 response would be predominant within H. pylori-colonized gastric mucosa. Paradoxically, the majority of H. pylori antigen-specific T-cell clones isolated from infected gastric mucosa produce higher levels of IFN-γ than IL-4, which is reflective of a Th1 type response.121H. pylori also stimulates production of IL-12 in vitro, a cytokine that promotes Th1 differentiation.122 These findings raise the hypothesis that an aberrant host response (Th1) to an organism predicted to induce secretory immune responses (Th2) may influence and perpetuate gastric inflammation (Figure 3); animal models of H. pylori-induced gastritis have supported these conclusions. H. pylori infection of IFN-γ-deficient mice that cannot mount an appropriate Th1 response leads to decreased levels of gastric inflammation compared to wild-type mice, and in vivo neutralization of IFN-γ in mice infected with a related Helicobacter species (H. felis) similarly reduces the severity of gastritis.123–125 Certain strains of mice (C57/BL6) infected with H. felis that develop a Th1-type response exhibit extensive gastric inflammation, while other genetically distinct strains (BALB/c) that respond to infection with a Th2-like response develop only minimal gastritis.126 Adoptive transfer of Th2 lymphocytes from infected mice into infected recipients reduces bacterial colonization density, while transfer of Th1 cells increases the severity of gastritis.127 Recently, elegant studies have extended these observations and demonstrated that preceding challenge with a helminth (Heligmosomoides polygyrus) that induces a Th2-type mucosal reaction significantly attenuates the development of Th1-mediated gastritis and atrophy in response to H. felis infection.128 These data are consistent with a model in which an inappropriate host T-cell response towards H. pylori facilitates the development of gastric inflammation and injury (Figure 3).
Figure 3. Working model for pathogenesis of H. pylori-induced diseases. Acquisition of H. pylori leads to the development of a Th1-mediated response, which facilitates induction and maintenance of gastric inflammation. Persons harbouring strains containing particular disease-related alleles, such as cagA, vacA s1 and iceA1, develop more severe mucosal inflammation, injury and atrophy.
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Epithelial cell injury
H. pylori-induced inflammation may also lead to other forms of epithelial cell injury and damage. Activated neutrophils generate reactive oxygen or nitrogen species that can induce oxidative DNA damage via formation of DNA adducts. Upon contact with H. pylori in vitro, an oxidative burst occurs within polymorphonuclear cells.129, 130 The specific types of cellular damage resulting from generation of reactive oxygen species include lipid peroxidation, protein oxidation, and oxidation of DNA. H. pylori increases oxidative damage of DNA in gastric epithelial cells.131, 132 Another consequence of persistent inflammation may be alterations in cellular turnover. Gastric epithelial cell proliferation rates within colonized mucosa are significantly increased compared to those in uninfected controls.133–135 In chronic H. pylori infection, there is a notable lack of epithelial cell necrosis, suggesting that other forms of cellular demise, such as apoptosis, may be induced.136 Apoptosis is a form of programmed cell death that consists of a tightly regulated series of energy-dependent molecular events, and the ability of H. pylori to alter apoptosis may influence clinical manifestations of infection.137 For example, enhanced rates of apoptosis could potentially accelerate the progression to atrophic gastritis, with a concomitant increase in the risk for distal gastric adenocarcinoma. In contrast, reduced rates of cell loss, especially when accompanied by hyperproliferation, could lead to a heightened retention of mutagenized cells, which may also predispose certain colonized individuals towards gastric carcinogenesis.
Several studies have reported increased levels of gastric epithelial cell apoptosis among H. pylori-infected persons.136, 138–140 However, substantial variation exists among apoptosis scores between infected and uninfected persons within these clinical populations. An important question raised by these observations is whether differing levels of apoptosis are a result of particular bacterial products or the induced host response. Recent data have suggested that epithelial cell apoptosis in vivo is likely to be regulated by host mediators present within inflamed mucosa. For example, IFN-γ, a Th1 lymphocyte-derived cytokine that is increased within colonized mucosa, is synergistic with H. pylori in inducing Fas–Fas ligand (FasL) regulated apoptosis of gastric epithelial cells in vitro.141, 142 Fas, a member of the TNF/nerve growth factor receptor superfamily, mediates apoptosis following activation by its cognate oligomerizing ligand, FasL. FasL is expressed by a variety of cells, including epithelial cells and activated T lymphocytes, and H. pylori can induce apoptosis in certain gastric epithelial and T-cell lines by activating Fas.140, 141, 143–146 Gastric T-cell lines that display a Th1 phenotype specifically recognize H. pylori antigens, and these same cells express FasL and produce TNF-α and IFN-γ, cytokines that are synergistic with H. pylori in inducing apoptosis and enhancing Fas expression on gastric epithelial cells in vitro141, 142, 147H. felis infection of Fas-deficient mice is also associated with reduced levels of inflammation, and mucosal apoptosis scores are decreased in parallel compared to infected wild-type mice.148 A paradigm invoked by these findings is that cytokines released by Th1 lymphocytes within H. pylori-inflamed mucosa may increase apoptosis by regulating Fas–FasL interactions between T-cells and gastric epithelial cells.121, 142
However, there are additional host genetic factors that may also contribute to the ability of H. pylori to alter apoptosis in vivo. Mice lacking secretory phospholipase A2 that are infected by H. felis, develop increased epithelial cell apoptosis.149 Apoptosis in response to H. pylori may reflect heterogeneity of class II MHC host genotypes. Gastric epithelial cells express class II MHC antigens on their surfaces and expression is higher in H. pylori-infected compared to uninfected tissue.150 Although class II MHC molecules are well-recognized for their role in regulating the immune response through binding and presentation of foreign antigens to CD4+ T-cells, ligation of class II MHC molecules also results in apoptosis, and binding of H. pylori to these IFN-γ-inducible molecules can directly stimulate apoptosis in vitro.151–153 El-Omar et al. have recently identified particular polymorphisms of the human interleukin-1β gene promoter that are genetic risk factors for both precursor lesions of gastric cancer (atrophic gastritis) as well as gastric adenocarcinoma among H. pylori-infected persons.154 IL–1β can stimulate multiple intracellular signalling pathways involved in apoptosis, including Fas, NF-κB, and MAP kinases, and thus differing levels of IL-1β expression within gastric mucosa may alter the ability of H. pylori to induce apoptosis.99, 105, 141, 142
In addition to differences in host genotype, particular bacterial constituents may influence the ability of H. pylori to alter cellular turnover. Two independent studies have demonstrated that persons infected with H. pylori isolates containing the strain-specific gene cagA (see below) have significantly higher proliferation rates, but lower apoptotic indices, than either cagA– or uninfected persons.135, 155 Increases in proliferation that are not balanced by concordant increases in apoptosis over years of colonization may therefore contribute to the ability of cagA+ strains to augment the risk for gastric cancer in certain populations. This model does not, however, explain the enhanced risk of duodenal ulcer disease associated with cagA+ strains, as duodenal ulceration and distal gastric cancer appear to be mutually exclusively outcomes of chronic H. pylori infection. Further, a recent study in an ethnically distinct population has demonstrated increased apoptotic indices within cagA+-colonized mucosa.156 Therefore, the precise relationship between H. pylori cagA+ strains and cellular turnover in vivo remains to be clarified.
In vitro, urease can induce apoptosis by binding to class II MHC molecules expressed on epithelial cell surfaces.153 VacA has also been shown to induce apoptosis in cell culture systems by inserting into the mitochondrial membrane, inducing the release of cytochrome c, and activating caspase-3 (see below).140, 157, 158 Collectively, these data suggest that heterogeneity in degrees of epithelial cell apoptosis among infected clinical populations is probably dependent upon the tremendous genetic diversity that exists both between isolates of H. pylori and between human hosts.