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 α-and β-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 α- or β-subunits of the gastric H⁺/K⁺ ATPase, but the β-subunit appears to be the initiating autoantigen, while the α-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.     

Immunopathogenesis,loss of T cell tolerance and genetics of autoimmune gastritis

Over the past 10 years experimental autoimmune gastritis has been established as a highly defined model of organ-specific autoimmunity. Autoimmune gastritis represents one of the few autoimmune diseases in which the causative autoantigens, namely the gastric H/K ATPase alpha- and beta-subunits, are defined. Furthermore, it has been clearly established that a CD4+ T cell response to the H/K ATPase beta-subunit, in particular, is essential for the initiation of autoimmune gastritis. The immunopathology of autoimmune gastritis is due to a disruption of the normal developmental pathways of the mucosa, rather than a direct depletion of the end-stage parietal and zymogenic cells. CD4+CD25+ regulatory T cells were first described in experimental autoimmune gastritis and there has been a recent explosion of interest in the potential role of these immunoregulatory T cells in protection against a variety of autoimmune diseases. The availability of H/K ATPase deficient mice has begun to provide considerable insight into the basis for tolerance to the gastric autoantigens. Experimental autoimmune gastritis has also provided valuable insight into our understanding of the genetics of disease susceptibility and four distinct genetic regions have been identified which confer susceptibility to this organ-specific disease. The highlights of these recent advances are the subject of this review.     

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.     

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.     

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.     

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.     

The causative H+/K+ ATPase antigen in the pathogenesis of autoimmune gastritis

The gastric H+/K+ ATPase is the causative autoantigen recognized by CD4+ T cells that mediate autoimmune gastritis. Pathogenic CD4+ T cells are regulated by CD25+CD4+ T cells. Here, it is proposed that waves of activation and migration of antigen presenting cells and CD4+ T cells to and from the target organ and draining lymph node result in tissue damage.     

Animal models of human disease: experimental autoimmune gastritis--a model for autoimmune gastritis and pernicious anemia.

Human autoimmune gastritis is an organ-specific autoimmune disease of the stomach. It is characterized by the development of disease-specific autoantibodies and a pathology that specifically targets specialized cells within the gastric environment. The autoantigens associated with this disease have been defined as the gastric H+/K+ ATPase and intrinsic factor. The development of experimental disease models has been pivotal in our contemporary understanding of autoimmunity. Here we review mouse models of autoimmune gastritis and their relevance to human autoimmune gastritis associated with pernicious anemia. We appraise some historical as well as recent studies of experimental autoimmune gastritis (EAG), highlighting key findings that have formed the basis of our current understanding of the etiology and mechanism(s) associated with autoimmune gastritis. A precise understanding of the pathogenesis of autoimmune gastritis will permit the design of innovative and rational therapeutic strategies to prevent, arrest, ameliorate or reverse the disease.     

Murine experimental autoimmune gastritis models refractive to development of intrinsic factor autoantibodies, cobalamin deficiency and pernicious anemia

Researchers have developed murine lymphopenic, non-lymphopenic, transgenic, spontaneous and infectious agent based models to induce an experimental autoimmune gastritis (EAG) for the study of human organ-specific autoimmune disease. These models result in a chronic inflammatory mononuclear cell infiltrate in the gastric mucosa, destruction of parietal and zymogenic cells with autoantibodies reactive to the gastric parietal cells and the gastric H+/K+ ATPase (ATP4), arguably hallmarks of a human autoimmune gastritis (AIG). In the case of AIG, it is well documented that, in addition to parietal cell antibodies being detected in up to 90% of patients, up to 70% have intrinsic factor antibodies with the later antibodies considered highly specific to patients with pernicious anemia. This is the first report specifically investigating the occurrence of intrinsic factor antibodies, cobalamin deficiency and pernicious anemia in EAG models. We conclude, in contrast to AIG, that, in the three EAG models examined, intrinsic factor is not selected as a critical autoantigen.     

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.     

The role of natural killer cells in the induction of autoimmune gastritis

A number of experimental models of organ-specific autoimmunity involve a period of peripheral T cell lymphopenia prior to disease onset. In particular, experimental autoimmune gastritis, induced in susceptible mouse strains by neonatal thymectomy, is a CD4+ T cell mediated autoimmune disease. We have previously demonstrated that this disease displays the hallmarks of a Th1-mediated DTH inflammatory response with an essential role for IFN-gamma very early in the pathogenesis of disease. Given the interplay between the innate and adaptive immune responses, a potential source of early IFN-gamma production in these lymphopenic mice is the innate immune response. Here we have assessed the contribution of innate immunity to the induction of experimental autoimmune gastritis, in particular, the role of natural killer (NK) cells in production of IFN-gamma. Analysis of NK cells and macrophages revealed no difference in either the number or activation status between euthymic and neonatally thymectomised mice. Furthermore, in vivo depletion of NK cells immediately after neonatal thymectomy of (BALB/cCrSlcxC57BL/6) F1 mice demonstrated no reduction in disease incidence compared to control groups of neonatally thymectomised mice. Therefore, we conclude that NK cells are not the primary source of IFN-gamma required for the pathogenesis of autoimmune gastritis following neonatal thymectomy but rather the small cohort of T cells in the periphery of lymphopenic mice are likely to be responsible for the IFN-gamma production.     

Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4(+)CD25(+) T cells

Murine autoimmune gastritis, induced by neonatal thymectomy or the injection of CD25-depleted lymphocytes into nu/nu recipients, is characterized by an inflammatory infiltrate into the gastric mucosa, parietal cell destruction and circulating anti-parietal cell antibodies. Using RAG-2(-/-)mice as recipients, we determined that the induction of disease relies on CD4(+)CD25(-)effector cells and prevention relies on CD4(+)CD25(+)regulatory cells; neither requires participation of CD8 cells or B cells. The severity of gastritis was dependent on the cytokine repertoire of CD4(+)CD25(-)effector T cells. Recipients of IL-4(-/-)T cells developed more severe gastritis and recipients of INF-gamma(-/-)T cells developed milder disease than recipients of wildtype or IL-10(-/-)effector T cells. Gastritis did not develop in the absence of IL-12. Protection from gastritis does not require either IL-4 or IL-10 because CD4(+)CD25(+)cells from IL-4(-/-)or IL-10(-/-)mice completely abrogated the disease process. CD4(+)CD25(+)cells also protected RAG-2(-/-)recipients from colitis and inhibitory activity was partially dependent on IL-10 expression. These findings highlight the critical role of CD4(+)CD25(+)regulatory T cells in protection from several autoimmune syndromes and delineate the differential contribution of IL-10 to CD4(+)CD25(+)Treg activity in the settings of gastritis and colitis.     

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.     

Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ-specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase alpha chain, but only marginally to the beta chain. Two H/K ATPase-reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+, TCR alpha/beta-, and I-Ad restricted, and recognize distinct peptides from the H/K ATPase alpha chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co-transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2-day-old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well-characterized lines of autoantigen-specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.     

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.     

Gastritis in neonatal BALB/cCrSlc mice induced by immunization without adjuvant can be transferred to syngeneic nu/nu recipients

The popularly exploited murine neonatal thymectomy experimental autoimmune gastritis (nTx:EAG) model requires the animal to be in a state of lymphopenia. Here we report on a novel murine immunization (without adjuvant) model that can establish a lasting gastritis. We demonstrate that the immunization model (imm:EAG) results in a bona fide autoimmune disease and define the resulting pathology and serology observed and compare it with that reported for human autoimmune gastritis. Immune cells present in the stomachs of imm:EAG gastritic mice include CD8 T cells, CD11b and Gr1 (granulocytes/monocytes) and B cells. We detected circulating antibodies of immunoglobulin G1 (IgG1) subclass, with some IgG(2a) and IgG(2b) reactive to stomach membranes and the parietal cell proton pump. The class and subclass of autoreactive antibodies resulting from imm:EAG suggest a T helper 1 (Th1)/Th2 immune response. We establish that both self-reactive T and B cells from BALB/cCrSlc imm:EAG gastritic mice have the potential to induce a gastritis in BALB/c syngeneic nu/nu recipients. We conclude that this model is likely to be superior to the currently popularly exploited nTx:EAG and provide insight into and an understanding of the mechanisms of and remedies for autoimmunity in an intact immune system.     

Requirements for autoimmune responses to mouse gastric autoantigens

Autoimmune gastritis, in which the H+/K(+)-ATPase of parietal cells is the major antigen, is one of the most common autoimmune diseases. Here we examined if specific properties of the H+/K(+)-ATPase or parietal cells are involved in rendering them autoimmune targets. The model antigens beta-galactosidase and ovalbumin (OVA) were expressed in parietal cells of transgenic mice. On experimental induction of autoimmune gastritis by neonatal thymectomy, autoantibodies to beta-galactosidase developed in mice expressing beta-galactosidase in parietal cells, a response that was independent of either the response to the gastric H+/K(+)-ATPase or gastric inflammation. In contrast, mice that expressed OVA in parietal cells did not exhibit an antibody response to OVA after thymectomy. However, increasing the frequency of anti-OVA T lymphocytes in OVA-expressing mice resulted in autoantibodies to OVA and gastritis. These studies indicate that parietal cells can present a variety of antigens to the immune system. Factors such as the identity and expression level of the autoantigen and the frequency of autoreactive T cells play a role in determining the prevalence and outcome of the particular immune response. In addition, as not all mice of a particular genotype displayed autoimmunity, random events are involved in determining the target of autoimmune recognition.     

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.     

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.