Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis.

BACKGROUND & AIMS: Murine autoimmune gastritis, induced by day-3 thymectomy, is characterized by cellular infiltrates and circulating autoantibodies to gastric hydrogen/potassium adenosine triphosphatase. The aim of this study was to analyze the cellular infiltrates and cytokines in autoimmune gastritis. METHODS: Stomachs and blood samples from day-3 thymectomized BALB/c mice were obtained from 2 to 12 weeks after thymectomy for analysis. RESULTS: At 4 weeks, the gastritic infiltrates were composed of macrophages and CD4+ T cells, accompanied by major histocompatibility complex class II expression on gastric epithelial cells. Mucosal B cells, scant at 4 weeks, were abundant at 8 weeks, coincident with the peaking of autoantibodies to gastric hydrogen/potassium adenosine triphosphatase. CD8+ T cells increased marginally during the 12 weeks. Mononuclear cells from diseased stomachs transferred gastritis to nu/nu recipients. At 4 weeks, interleukins 2, 3, 5, 6, and 10; interferon gamma; tumor necrosis factor alpha; and granulocyte-macrophage colony-stimulating factor were detected in gastritic mucosa, but interleukin 4 was not. CONCLUSIONS: The early lesion of autoimmune gastritis is composed of macrophages and CD4+ T cells with major histocompatibility complex class II expression in gastric epithelial cells. Autoantibody production is a late event. Our results are consistent with a lesion mediated by CD4+ T cells producing a mix of Th1- and Th2-type cytokines.

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.

The 60- to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a beta subunit of the gastric H+/K(+)-ATPase (proton pump).

Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the alpha subunit of the gastric H+/(+)-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the alpha subunit of the gastric H+/K(+)-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the alpha subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the beta subunit of the pig kidney Na+/K(+)-ATPase. We therefore propose that the 60- to 90-kDa glycoprotein autoantigen is the beta subunit of the gastric H+/K(+)-ATPase and that the alpha and beta subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.