Immunization with gastric H+/K(+)-ATPase induces a reversible autoimmune gastritis.

The gastric H+/K(+)-ATPase has been implicated as a major autoantigen in pernicious anaemia in humans and in thymectomy-induced autoimmune gastritis in mice. Here we have shown that autoimmune gastritis can be generated by direct immunization of non-thymectomized BALB/c mice with mouse gastric H+/K(+)-ATPase in complete Freund's adjuvant. The gastritis was characterized by infiltration of the gastric submucosa and mucosa with macrophages, CD4+ and CD8+ T cells, and B cells and by circulating autoantibodies to the H+/K(+)-ATPase. The mononuclear infiltrate within the gastric mucosa was accompanied by loss of parietal and zymogenic cells and accumulation of small immature epithelial cells. Splenocytes from gastritic mice adoptively transferred gastritis to naive recipients. Cessation of immunization resulted in decrease in autoantibody titre and regeneration of parietal and zymogenic cells. The results directly confirm that the gastric H+/K(+)-ATPase is the causative autoantigen in the genesis of autoimmune gastritis. Recovery of the lesion following cessation of immunization suggests that homeostatic mechanisms can reverse a destructive autoimmune process.     

The role of O-antigen polysaccharide in the activation of neutrophils by lipopolysaccharides of Salmonella species.

Autoimmune gastritis develops spontaneously in approximately 60% of BALB/c mice thymectomized neonatally. Histologically and clinically it is similar to the atrophic gastritis associated with pernicious anaemia in humans. Here we identified antigenic protein relating to the pathogenesis of autoimmune gastritis in these mice. All sera from 32 thymectomized mice with gastritis contained autoantibodies to the vesicular fraction prepared from rat gastric parietal cells. Immunoblot analysis revealed all of these to react with a 94-kD protein corresponding in molecular mass with the H+/K(+)-ATPase alpha subunit. Some sera were also reactive with 65-85-kD and/or 60-kD proteins, whose sizes correspond to the H+/K(+)-ATPase beta subunit and intrinsic factor, respectively. The finding that immuno-adsorption with these sera resulted in reduction of H+/K(+)-ATPase activity in the vesicular fraction, supported a conclusion of H+/K(+)-ATPase alpha and/or beta subunits as the antigenic proteins. After immunization of normal syngeneic mice with various doses of gastric parietal cells or their vesicular fraction, all sera from animals demonstrating atrophic gastric mucosa with lymphocyte infiltration reacted with the H+/K(+)-ATPase alpha subunit. No antibodies to other proteins were induced even in mice immunized with higher doses of antigen. We therefore conclude that H+/K(+)-ATPase alpha subunit is important as the target antigen in pathogenesis of autoimmune gastritis in neonatally thymectomized mice, probably due to a high affinity for the MHC molecule.     

Autoimmune gastritis: tolerance and autoimmunity to the gastric H+/K+ ATPase (proton pump).

The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.     

Expression of a gastric autoantigen in pancreatic islets results in non-destructive insulitis after neonatal thymectomy

Autoimmune gastritis, induced by day-3 thymectomy of BALB/c mice, is a destructive CD4⁺ T cell-mediated disease characterized by leukocyte infiltrates in the gastric mucosa, loss of parietal and chief cells and anti-gastric H/K ATPase autoantibodies. Our previous studies have indicated that a T cell response to the H/K ATPase β subunit is required for the onset of autoimmune gastritis (Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R., J. Exp. Med. 1993. 178: 419). To determine whether a response to the β subunit autoantigen is alone sufficient to induce autoimmunity, or whether other tissue-specific factors are required, we have generated transgenic mice expressing the gastric H/K ATPase β subunit in β islet cells of the pancreas (RIP-H/Kβ). RIP-H/Kβ mice developed autoimmune gastritis and insulitis after day-3 thymectomy. Significantly, insulitis, observed as a peri-islet infiltrate, was only detected in thymectomized mice with autoimmune gastritis. There was no apparent immune destruction of the pancreas as insulitis did not progress to invasion of the islets or diabetes. Double transgenic mice, expressing the gastric H/K ATPase β subunit in the thymus and in the pancreas, were protected from both gastritis and insulitis after day-3 thymectomy. Therefore, insulitis in the RIP-H/Kβ mice appears to be dependent on a T cell response to the H/K ATPase β subunit. This is the first example where an organ-specific initiating autoantigen has been expressed in another peripheral tissue. Autoimmune destruction in the stomach, but not the pancreas, indicates that tissue-specific factors play a fundamental role in the development of organ-specific autoimmunity.     

Tumor necrosis factor alpha is not implicated in the genesis of experimental autoimmune gastritis

Experimental autoimmune gastritis (EAG) characterised by mononuclear cell infiltrate, parietal and zymogenic cell destruction and circulating autoantibodies to gastric H(+)/K(+)ATPase is an animal model for human autoimmune gastritis, that leads to pernicious anaemia. We have previously shown that Fas has a role in initiating damage to target cells in EAG. Here we used three strategies to examine the role of TNFalpha in this disease. We administered neutralising anti-TNFalpha antibody either as a single injection or as twice weekly injections for 8 weeks to mice subjected to neonatal thymectomy-induced EAG. To address the role of apoptotic signals through TNFR1, TNFR1 deficient mice were either neonatally thymectomised or crossed to PC-GMCSF transgenic mice that spontaneously develop EAG. Neonatally thymectomised mice treated with anti-TNFalpha antibody developed destructive gastritis and autoantibodies to gastric H(+)/K(+)ATPase similar to control mice. Following either neonatal thymectomy or crossing to PC-GMCSF transgenic mice, TNFR1 deficient mice developed autoantibody-positive destructive gastritis at similar frequency compared with wild type and heterozygous littermates. Our observations that neutralisation of TNFalpha and absence of TNFR1 has no discernible effect on development of EAG suggest that TNFalpha is not required for mucosal cell damage or development of autoimmune gastritis. While blocking TNFalpha activity has therapeutic benefit in certain autoimmune diseases, this is not the case for EAG.     

Characterisation of proton pump antibodies and stomach pathology in gastritis induced by neonatal immunisation without adjuvant

It has previously been reported that neonatal BALB.D2 mice injected with native proton pump antigens without adjuvant develop an irreversible gastritis (Claeys et al, 1997). The ease of inititating gastritis in the neonate stands in contrast with the difficulty in initiating gastritis in adult mice that require repeated immunisation in adjuvant that is reversible following cessation of immunisation (Scarff et al, 1997). In view of these contrasting observations, we set out to ascertain whether we could confirm the observations in neonatal mice as well as further characterise the pathology and the autoantibody response. We found that neonatal gastritis-susceptible BALB/c mice (n=12), immunised with either pig or mouse gastric membranes in the absence of adjuvant, develop gastritis without circulating antibody to parietal cells detected by immunofluorescence, a hallmark of murine and human gastritis (Toh et al, 1997). However, mice immunized with pig gastric membranes (n=6) had circulating antibodies reactive by immunofluorescence to recombinant alpha and/or beta subunit of gastric H+/K+-ATPase expressed by insect cells (Sfalpha and Sfbeta). Four mice from this cohort with antibodies to Sfbeta also had reactivity to gastric H+/K+-ATPase by ELISA, and 3 immunoblotted the beta but not the alpha subunit of the ATPase. In the cohort of mice immunised with mouse gastric membranes (n=6), four produced antibodies reactive by immunofluorescence to Sfalpha, two of which were also reactive to Sfbeta and one developed antibodies detected by ELISA to gastric H+/K+-ATPase. However, no members of this cohort had antibodies reactive by immunoblotting to either the beta or alpha subunit of the ATPase. In all cases gastritic stomachs were characterised by areas deficient in ribosome-rich zymogenic cells and marked reductions in H+/K+-ATPase-positive parietal cells. Metaplasia detected by Maxwell stain, as clusters of mucus-producing cells throughout gastric units, were non-reactive to stomach mucin autoantibody suggesting the mucins comprise other and/or aberrant form(s). Compared to our previous observations in adult mice, our present data confirms that gastric autoimmunity is more readily induced in the neonate than the adult. Our data also affirms that while the neonatal immune system can mount a damaging inflammatory cellular immune response to gastric antigens, it develops an altered antibody response.     

Spontaneous Autoimmune Gastritis in C3H/He Mice : A New Mouse Model for Gastric Autoimmunity

Autoimmune gastritis is the underlying pathological lesion of pernicious anemia in humans. The lesion is characterized by a chronic inflammatory infiltrate in the gastric mucosa with loss of parietal and zymogenic cells. It is associated with circulating autoantibodies to the gastric H/K-ATPase, the enzyme responsible for acidification of gastric juice. Experimental models of autoimmune gastritis have previously been produced in mice after a variety of manipulations, including thymectomy. Here we report for the first time a spontaneous mouse model of autoimmune gastritis in C3H/He mice. The spontaneous gastritis is also accompanied by circulating autoantibodies to the gastric H/K-ATPase. The spontaneous mouse model should be useful for studies directed toward the immunopathogenesis and treatment of autoimmune gastritis.     

Helicobacter pylori und antigastrale Autoimmunität

Recent studies report a significant association between Helicobacter pylori gastritis and autoimmune reaction. Antigastric autoantibodies are detectable in about 30% of H. pylori infected patients. Two major in situ binding sites have been found: first, at the luminal membrane of the foveolar epithelium in antrum and corpus mucosa and, second, at canalicular membranes within parietal cells in the corpus mucosa. The presence of latter type of autoantibodies is correlated with histological and clinical parameters of corpus mucosa atrophy. The gastric H+/K(+)-ATPase, which is already known as an autoantigen in classic autoimmune gastritis, also represents a major target in atrophic H. pylori gastritis. According to recent data molecular mimicry between H. pylori and the host does not play a pathogenic role in the formation these autoantibodies. In conclusion, antigastric autoimmunity represents a relevant host factor which contributes to the final outcome of H. pylori gastritis.     

CD4+ T cells, but not CD8+ T cells, are required for the development of experimental autoimmune gastritis.

Murine autoimmune gastritis, induced by neonatal thymectomy, is characterized by a mononuclear infiltrate within the gastric mucosa, loss of parietal and zymogenic cells and circulating autoantibodies to the gastric H/K ATPase. The infiltrate contains both CD4+ and CD8+ T cells. Here we have investigated the roles of CD4+ and CD8+ T cells in the development of gastritis by in vivo treatment with depleting rat anti-CD4 and anti-CD8 monoclonal antibodies. Depletion of CD4+ T cells decreased the incidence of gastric mononuclear infiltrates from 63% (5/8), observed in normal rat immunoglobulin G (IgG)-injected mice, to 8% (1/12) and also abolished the production of antigastric autoantibodies. In contrast, depletion of CD8+ T cells did not reduce the incidence of gastritis. The absence of CD8+ T cells in the infiltrate of the stomach of anti-CD8(+)-treated mice was confirmed by immunocytochemistry. These results argue that neonatal thymectomy-induced autoimmune gastritis is mediated by CD4+ T cells and that CD8+ T cells do not play a significant role in the development of the gastric lesion.     

Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease

Experimental autoimmune gastritis (EAG) is an excellent model of human autoimmune gastritis, the underlying cause of pernicious anaemia. Murine autoimmune gastritis replicates human gastritis in being characterized by a chronic inflammatory mononuclear cell infiltrate in the gastric mucosa, destruction of parietal and zymogenic cells, and autoantibodies to the alpha-and beta-subunits of the gastric H+/K+ ATPase. Disease is induced strain specifically in gastritis-susceptible BALB/c mice by methods with a greater variety than those for most other experimental autoimmune diseases. The disease is induced in the regional gastric lymph node in which pathogenic CD4+ T cells are recruited. The model provides an excellent illustration of regulation by CD4+CD25+T cells, and, indeed, the removal of such regulatory cells, e.g., by neonatal thymectomy, is thought to be a major mechanism by which disease can develop. The culprit T helper type 1 (Th1) CD4+ T cells recognize either the alpha- or beta-subunits of the gastric H+/K+ ATPase, but the beta-subunit appears to be the initiating autoantigen, while the alpha-subunit may have a role in perpetuating disease. Since no specific environmental modifiers are identifiable, the origins of the disease are intrinsic; this is illustrated by the capacity of a cytokine (GM-CSF)-dependent inflammatory stimulus in the stomach to initiate EAG, according to a transgenic model in which thymectomy is dispensible. Thus, EAG is an exquisite model for a reductionist analysis of the multiple elements that in combination induce autoimmunity in humans.     

Interferon-γ is required during the initiation of an organ-specific autoimmune disease

Autoimmune gastritis induced by neonatal thymectomy of mice is a CD4⁺ T cell-mediated organ-specific autoimmune disease. The characteristic features of autoimmune gastritis, which include a mononuclear infiltrate within the gastric mucosa, loss of parietal and chief cells and circulating autoantibodies to the gastric H⁺/K⁺ ATPase, appear 6–10 weeks after thymectomy. Here we have assessed the role of interferon-γ (IFN-γ) in the pathogenesis of the gastric lesion. Splenic T cells derived from mice with gastritis produced three- to tenfold more IFN-γ than T cells from normal animals after stimulation with anti-CD3 antibodies. Treatment of neonatally thymectomized mice at weekly intervals for 6 or 12 weeks with a neutralizing rat monoclonal antibody to mouse IFN-γ abolished the production of anti-gastric autoantibodies and decreased the incidence of gastric mononuclear infiltrates from the 69% observed in normal rat immunoglobulin (Ig)-injected mice to 16%. Further, in mice treated with only a single dose of anti-IFN-γ immediately after thymectomy at 3 days after birth, the incidence of autoimmune gastritis was 1/19 compared to 8/19 in normal rat Ig-injected mice. Prevention of autoimmunity by neutralization of IFN-γ several weeks prior to the detection of a pathological lesion strongly suggests that IFN-γ plays an essential role in the initiation of the gastric autoimmune response.     

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.     

Shaping the T cell repertoire to a bona fide autoantigen: lessons from autoimmune gastritis

Murine autoimmune gastritis is one of the most well-defined organ-specific autoimmune diseases. CD4(+) T cells, which mediate the disease, recognize the highly abundant gastric H(+)/K(+) ATPase heterodimer. The H(+)/K(+) ATPase alpha subunit is also expressed in the thymus, in an aire-independent manner, whereas the H(+)/K(+) ATPase beta subunit is absent from the thymus. Analysis of both H(+)/K(+) ATPase-specific T cell receptor transgenic mice with different affinities for the gastric antigen and mice deficient in the H(+)/K(+) ATPase subunits has provided information on thymic and peripheral selection events. The H(+)/K(+) ATPase antigens play an important role in purging the repertoire of gastritogenic T cells, and recent data have suggested that this tolerance induction occurs primarily in the periphery. The gastritis system provides a powerful approach to determine the impact of peripheral antigen presentation in the target organ draining lymph node on tolerance and autoimmune disease.     

Prednisolone promotes remission and gastric mucosal regeneration in experimental autoimmune gastritis

A cardinal feature of organ-specific autoimmunity is destructive pathology in the target organ. In human and experimental models of autoimmune gastritis, mononuclear cell infiltration and cellular destruction in the gastric mucosa are disease hallmarks. Strategies to cure autoimmune disease must not only establish immunological tolerance to autoantigen, but also rid the organ of pathogenic autoreactive cells. The present study has assessed the effect of prednisolone treatment in clearing the inflammatory infiltrate in experimental autoimmune gastritis and in preventing disease relapse in athymic compared with euthymic mice. Experimental autoimmune gastritis was induced by neonatal thymectomy or by transgenic expression of GM-CSF (PC-GMCSF mice). Groups of mice were treated with prednisolone (10 mg/kg per day) for 10 weeks or with prednisolone for 10 weeks followed by 10 weeks without prednisolone. Stomachs were examined for gross morphological changes, and by histology and immunohistochemistry for composition of inflammatory infiltrate and gastric mucosal integrity. Autoantibody to gastric H+/K+ ATPase was determined by ELISA. Prednisolone promoted remission of gastritis in both mouse models of experimental autoimmune gastritis, evident by reduction in stomach size, clearing of gastric inflammatory infiltrate, and regeneration of the gastric mucosa. Prednisolone withdrawal resulted in disease relapse in all PC-GMCSF mice, whereas approximately 40% of neonatal thymectomy mice retained normal stomach morphology and remained free of gastric pathology. It is concluded that prednisolone promotes remission and gastric mucosal regeneration in experimental autoimmune gastritis. Prolonged remission of autoimmune gastritis in some athymic mice suggests a role for the thymus in disease relapse.     

Organ-specific autoimmunity induced by adult thymectomy and cyclophosphamide-induced lymphopenia

Autoimmune gastritis, a CD4⁺ T cell-mediated organ-specific autoimmune disease, can be induced by thymectomy of neonatal, but not of older, BALB/c mice. Here we have shown that autoimmune gastritis can also be induced in 6–8-week-old BALB/c mice by thymectomy combined with a single dose of cyclophosphamide (300 mg/kg). This treatment reduced the numbers of splenic T and B cells approximately 25-fold. However, by 8 days after treatment, the number of splenic lymphocytes had returned to normal adult levels. Approximately 50% of treated mice developed autoimmune gastritis after 10–12 weeks. These mice had mononuclear cellular infiltrates within the gastric mucosa and serum autoantibodies to the α and β subunits of the gastric H⁺/K⁺ ATPase. Transgenic mice, expressing the gastric H⁺/K⁺ ATPase β-subunit in the thymus (Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R., J. Exp. Med. 1993. 178: 419), did not develop autoimmune gastritis after the adult thymectomy/cyclophosphamide treatment. Thus a T cell response to the H⁺/K⁺ ATPase β-subunit is likely to be required for the onset of gastritis. These observations suggest that pathogenic autoreactive T cells exist in the periphery of normal adult mice and that autoimmunity can be induced by the activation of these autoreactive T cells following transient lymphopenia. Cyclophosphamide-treatment of adult mice without thymectomy did not induce autoimmune gastritis, suggesting thymic regulation of these pathogenic T cells.     

Reciprocal changes in trefoil 1 and 2 expression in stomachs of mice with gastric unit hypertrophy and inflammation

H+/K+-ATPase beta-subunit-deficient mice (129/Sv background) display numerous pathologies in the stomach. Expression of the mutation in BALB/cCrSlc mice results in the development of an aberrant 'mucus-rich' cell population. 'Mucus-rich' cells have been described in stomachs of mice with autoimmune gastritis, a disease mediated by CD4+ T cells. Other pathological features of autoimmune gastritis are similar to those in H+/K+ beta-deficient mice and include a mononuclear cell infiltrate in the gastric mucosa, non-functional or absent parietal cells, depletion of zymogenic cells, hypergastrinaemia, and gastric unit hypertrophy caused by immature cell hyperplasia. The present study investigates further the aberrant gastric 'mucus-rich' cell lineage and analyses the mRNA expression of mucus cell products TFF1 and TFF2. 'Mucus-rich' cells stained for both acidic and neutral mucins, and with a TFF2-specific antibody. Stomachs from both models expressed decreased TFF1 mRNA and reciprocally increased TFF2 mRNA. The involvement of gastrin in regulating trefoil mRNA expression was also investigated using gastrin-deficient mice. In contrast to previous findings, gastrin did not positively regulate TFF1 mRNA expression, but there was possible augmentation of TFF2. Additionally, a clear role for inflammation was established involving both polymorphonuclear and mononuclear cells in these models, and a link was found between mucosal hypertrophy and increased interleukin-11 (IL-11) expression.     

A T cell receptor transgenic model of severe, spontaneous organ-specific autoimmunity.

The development of mouse models of human organ-specific autoimmune diseases has been hampered by the need to immunize mice with autoantigens in potent adjuvants. Even autoantigen-specific T cell receptor transgenic models of autoimmunity have proven to be complex as the transgenic mice frequently fail to develop disease spontaneously. We have isolated a CD4(+) T cell clone (TxA23)that recognizes the gastric parietal cell antigen, H/K ATPase alpha-chain(630-641), from a mouse with autoimmune gastritis that developed after thymectomy on day 3 of life. The T cell receptor alpha and beta genes from this clone were used to generate A23 transgenic mice. All A23 transgenic animals spontaneously developed severe autoimmune gastritis, and evidence of disease was detected as early as day 10 of life. Gastritis could be transferred to immunocompromised mice with a limited number of transgenic thymocytes (10(3)), but as many as 10(7) induced only mild disease in wild-type animals. Due to the complete penetrance of spontaneous disease, identity of the auto-antigen, susceptibility to immunoregulation, and close relation to autoimmune gastritis in man, A23 transgenic mice represent a unique CD4(+) T cell-mediated disease model for understanding the multiple factors regulating organ-specific autoimmunity.     

Molecular targets in pernicious anaemia.

Autoimmune gastritis, leading to pernicious anaemia, is an organ-specific autoimmune disease characterized by chronic atrophic gastritis and circulating gastric parietal cell autoantibodies. The parietal cell autoantigens have recently been identified as the alpha and beta subunit of the gastric proton pump (H+, K+ ATPase). Here Paul Gleeson and Ban-Hock Toh discuss how the identification of these gastric parietal cell autoantigens and the development of a mouse model of autoimmune gastritis have paved the way for an understanding of the pathogenesis of the gastric lesion.