Immunology 1997 92 91-98

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

K. J. SCARFF, J. M. PETTITT, I. R. VAN DRIEL, P. A. GLEESON & B.-H. TOH Department ofPathology and Immunology,Monash University Medical School, Prahran, Victoria, Australia

SUMMARY

The gastric H+/K+-ATPase has been implicated as a major autoantigen in pernicious anaemia inhumans and in thymectomy-induced autoimmune gastritis in mice. Here we have shown thatautoimmune gastritis can be generated by direct immunization of non-thymectomized BALB/cmice 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+ andCD8+ T cells, and B cells and by circulating autoantibodies to the H+/K+-ATPase. Themononuclear infiltrate within the gastric mucosa was accompanied by loss of parietal andzymogenic cells and accumulation of small immature epithelial cells. Splenocytes from gastriticmice adoptively transferred gastritis to naive recipients. Cessation of immunization resulted indecrease in autoantibody titre and regeneration of parietal and zymogenic cells. The resultsdirectly confirm that the gastric H+/K+-ATPase is the causative autoantigen in the genesis ofautoimmune gastritis. Recovery of the lesion following cessation of immunization suggests thathomeostatic mechanisms can reverse a destructive autoimmune process.

INTRODUCTION

Autoimmune gastritis is the underlying pathological lesion ofpernicious anaemia in humans."2 The lesion is characterizedby infiltration of mononuclear cells into the submucosa andmucosa and loss of parietal and zymogenic cells from gastricglands. It is associated with circulating autoantibodies to theac- and fl-subunits of the gastric H+/K+ -ATPase,35- the enzymeresponsible for acidification of gastric juice. To understand thepathogenesis of autoimmune gastritis, we have adopted anddeveloped mouse models in which gastritis and circulatingautoantibodies to the gastric H+/K+-ATPase ensue afterneonatal thymectomy"8 or adult thymectomy combined withcyclophosphamide treatment.9 Thymectomy-induced murinegastritis is mediated by CD4+ T cells, not autoantibodies.47"0To ascertain whether autoreactive T cells directed to theH+/K+-ATPase are responsible for the initiation of thymec-tomy-induced gastritis we have expressed the fl-subunit of thegastric H+/K+-ATPase in the thymus to induce tolerance tothis subunit." Thymectomy-induced gastritis was preventedin these transgenic mice suggesting that the fl-subunit is amajor causative autoantigen recognized by the pathogenicT cells.9"'

There have been previous attempts to induce autoimmunegastritis in non-thymectomized animals by immunization with

Received 11 February 1997; revised 22 April 1997; accepted23 April 1997.

Correspondence: Dr B.-H. Toh, Department of Pathology andImmunology, Monash University Medical School, Commercial Road,Prahran, Victoria 3181, Australia.

crude gastric extracts. For instance, immunization of rhesusmonkeys with gastric mucosal extracts in Freund's completeadjuvant resulted in gastritis, parietal cell antibodies andcutaneous delayed-type hypersensitivity reactions to gastricantigens.'2 Immunization of dogs with gastric mucosal extractsalso resulted in gastritis and humoral and cellular reactions togastric antigens.13-'6 In a more recent study, gastritis andcirculating antibody to the H+/K+-ATPase a-subunit wasinduced by immunization of BALB/c mice with rat gastricparietal cells or gastric membranes in Freund's completeadjuvant. '7

In the present study, we have for the first time successfullyinduced autoimmune gastritis in non-thymectomized mice byimmunization with purified murine gastric H+/K+-ATPaseemulsified in complete Freund's adjuvant. Unlike thymectomy-induced autoimmune gastritis, the immunization-induced gas-tritis recovers following cessation of immunization with theATPase.

MATERIALS AND METHODS

Preparation ofmouse andpig gastric H+/K+-ATPaseGastric mucosal membranes from BALB/c mice and pig wereprepared as described.'8 Protein concentration of gastric mem-branes was determined using a micro-BCA protein assay(Pierce, Rockford, IL). Mouse and pig gastric H+/K+-ATPasewas purified from gastric membranes by tomato-lectin affinitychromatography.'8 Purity of the H +/K+-ATPase preparationswas assessed by staining with Coomassie Blue after sodiumdodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)'9 carried out under reducing conditions.

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Immunization with mouse gastric H+/K+-ATPaseFigure (a) shows the immunization schedule. Six-week-oldmice were injected subcutaneously (s.c.) over four sites with50 ug H+/K+-ATPase prepared from C57BL/6 and BALB/cmice and emulsified in 50 yl of Freund's complete adjuvant(Difco Laboratories Inc, Detroit, MI). Three weeks after eachimmunization, two or three mice were killed and examined forgastritis and autoantibodies to gastric H+/K+-ATPase. Afterfour immunizations, the mice were divided into two groups.

Immunizations were continued in the first group to a total ofseven immunizations. The second group was not immunizedfurther. In this group, three mice were analysed every 2 weeksfor 10 weeks to assess the development and recovery of thegastritis once immunization ceased. A separate group of micewas immunized four times with pig gastric H+/K+-ATPase.Four mice received four injections of Freund's complete adju-vant emulsified in phosphate-buffered saline (PBS) at 3-weekintervals and were killed 9 weeks after the final injection.

Processing and staining ofmouse stomachsStomachs were removed from mice immediately after theywere killed. The body portion of stomachs was opened alongthe lesser curvature, washed in PBS, fixed in 10% phosphate-

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buffered formalin for 6-8 hr at room temperature, dehydratedin ethanol and embedded in paraffin wax. Sections were

dewaxed and stained by a modification of Maxwell'smethod20'21 and with haematoxylin and eosin.

Assessment ofgastritisTwo to five mice were killed after each immunization andtheir stomachs were processed as described above. Eleven to25 stomach sections from each mouse were examined forgastritis after staining with a modification of Maxwell's methodand haematoxylin and eosin.

Detection ofgastric H+/K+-ATPase autoantibodiesSera were screened for parietal cell autoantibodies by immuno-fluorescence reactivity with paraffin-embedded sections ofmouse stomach' and for H+/K+-ATPase autoantibodies byenzyme-linked immunosorbent assays (ELISA) as described.22Specificity of these autoantibodies to the cx- or the fl-subunitof the mouse H +/K+-ATPase was determined by immunoblot-ting.23 Monoclonal antibodies 1H9 and 2B6, directed respect-ively to the a- and fl-subunits of the mouse gastric H+/K+-ATPase,24 were used as positive controls and anti-a-lactal-bumin monoclonal antibody, ET-1,25 was used as an isotype-negative control.

ImmunocytochemistrySamples of stomach tissue from immunized mice were snap-frozen in isopentane/liquid nitrogen. Frozen sections were cut

Mouse PigMW x 103

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Figure 1. (a) Immunization schedule. Mice received between one andseven immunizations with H+/K+-ATPase and were killed either: 3weeks after one to three immunizations; 3, 5, 7, 9, 11 or 13 weeksafter four immunizations; or 3 weeks after seven immunizations(arrows represent time of injection, crosses represent time-points atwhich mice were killed). (b) Coomassie blue staining and immunoblotanalysis of mouse and pig H+/K+-ATPase. Purified H+/K+-ATPasefrom mouse (lanes 1-3) and pig (lanes 4-6) was subjected to 10%/oSDS-PAGE under reducing conditions. Proteins (15 jg/well) in lanes1 and 4 were stained with Coomassie blue. Proteins (10 jg/well) inlanes 2, 3, 5 and 6 were immunoblotted with anti-a-subunit monoclonalantibody 1H9 (lanes 2 and 5), or anti-fl-subunit monoclonal antibody2B6 (lanes 3 and 6).

Figure 2. Circulating parietal cell autoantibodies demonstrated byindirect immunofluorescence. Sections of paraffin-embedded normalmouse stomach were reacted with serum from a BALB/c mouse givenfour immunizations with mouse H+/K+-ATPase (a), or adjuvantalone (b); magnification, x 220.

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Autoimmune gastritis induced by gastric H+/K+-ATPase

and fixed in 2% paraformaldehyde/30 mm lysine/lO mm sodiumperiodate,26 blocked with 10% normal swine serum (NSS) andexamined for reactivity with rat anti-mouse CD4 (GK1 5); ratanti-mouse CD8 (53-6-7); anti-Mac-i (MI-70); and anti-B220(RA3-3A1), as described.26

Adoptive transfer experimentsAdoptive transfer experiments were performed using a modifi-cation of a method described previously.'0 Briefly, BALB/cmice which had received seven immunizations of H+/K+-ATPase were killed 3 weeks after the last immunization. Spleencell suspensions were resuspended in Hank's balanced saltsolution (ICN Pharmaceuticals Inc., Costa Mesa, CA). Redcells were lysed in 17 mM Tris-HCl pH 7-2 containing 0-144 MNH4Cl at 370 for 7 min and the cells were pelleted in Hank'sbalanced salt solution. The cells, resuspended in Dulbecco'smodified Eagle's essential medium (Gibco BRL, Grand Island,NY) containing 10% fetal calf serum (Gibco BRL), 50 mM2-mercaptoethanol (Sigma Chemical Co. St Louis, MO), 2 mML-glutamine (Gibco BRL), 100 U/ml penicillin and 100 mg/mlstreptomycin (ICN Pharmaceuticals Inc., Costa Mesa, CA)were incubated with 3 pg/ml concanavalin A (ICNPharmaceuticals Inc.) for 48 h at 370 in 5% CO2. Cells wereisolated on a Hypaque-Ficoll gradient and 2 x 107 cells injectedintraperitoneally (i.p.) into 8-day-old BALB/c mice. Sixteenweeks after transfer, sera were examined for parietal cellautoantibodies and stomachs for gastritis. Adoptive transferexperiments were also carried out with concanavalinA-stimulated splenocytes from mice injected with adjuvantalone.

RESULTS

Parietal cell H+/K+-ATPase

To examine the capacity of syngeneic compared to xenogeneicantigen in the development of autoimmune gastritis, H+/K+-

ATPase was purified from gastric mucosal membrane extractsof BALB/c mice and pigs by tomato-lectin affinity chromatog-raphy."8 Analysis of these preparations by SDS-PAGErevealed a major 92000 MW component and 70000-90000and 60000-90000 MW components in the mouse and pigpreparations, respectively. The 92 000 MW component con-forms to the size of the a-subunit and the other components,to the glycosylated #-subunit of the H+/K+-ATPase.Components of more than 100 000 MW were also seen in bothpreparations. Immunoblotting with gastric H+/K+-ATPasesubunit-specific monoclonal antibodies confirmed the presenceof the a- and fl-subunits (Fig. lb). The a-subunit-specificmonoclonal antibody also showed reactivity with componentswith MW of 120 000 and more than 150000, consistent withthe known behaviour of the a-subunit on SDS gels.27 Thedifference in size between the mouse and pig fl-subunit prob-ably reflects differences in N-glycosylation. Therefore, theseanalyses indicate that the H+/K+-ATPase heterodimer is themajor component of the mouse and pig preparations.

Parietal cell autoantibodies to the H+/K +-ATPase

After the second immunization with mouse gastric H+/K+-ATPase, 64% (14/22) of mice developed circulating autoanti-bodies to parietal cells demonstrated by immunofluorescence(Fig. 2). All mice (20/20) had these autoantibodies after thethird immunization. The titre of parietal cell autoantibodyincreased with booster immunizations, peaking between 3 and5 weeks after the fourth immunization. Thereafter, the anti-body titre declined in the group that received four immuniza-tions (Fig. 3a). However, in the group where immunizationwas continued after the fourth immunization, parietal cellantibody titre remained high throughout the course of theexperiment (data not shown).

Sixty-five per cent (13/20) of mice developed fl-subunit-specific autoantibodies after the third immunization and 100%

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Figure 3. Autoantibodies in immunized mice. (a) Circulating parietal cell autoantibodies detected by immunofluorescence. Micewere given four immunizations with mouse H+/K+-ATPase (arrows). Filled circles represent 24 mice, open circles, one mouse.(b) Anti-H+/K+-ATPase antibodies by ELISA and immunoblotting. Time interval after the first immunization is represented onthe same scale as (a). Results are shown directly below the corresponding immunofluorescence titre readings.

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(15/15) of mice had these autoantibodies after the fourthimmunization (Figs. 3b and 4). Autoantibodies to the H+/K+-ATPase a-subunit were first detected after the fourth immuniz-ation, corresponding to the peak antibody response, with 20%(3/15) of the mouse sera containing a-subunit autoantibodies.Injections with adjuvant alone did not result in the generationof antibodies to parietal cells or to the gastric H+/K+-ATPase.

Immunization of mice with pig H+/K+-ATPase resultedin an antibody response to the pig antigen detected by ELISA(not shown). However, the antibody response was predomi-nantly pig-specific with only low-titre anti-mouse H+/K+-ATPase antibodies, even after four immunizations with pigantigen.

Development of gastritis in immunized mice

Figure 5 compares the histology of stomachs from mice withand without gastritis. Mononuclear infiltration into the submu-cosa was detected after two immunizations with mouseH+/K+-ATPase. After four immunizations, areas of themucosa showed advanced gastritis where large pockets ofmononuclear cells had penetrated the lamina propria betweengastric glands and extended to the mucosal surface (Fig. 6c).Mononuclear cells were not detected in gastric mucosa ofnormal mice (Fig. 6a) nor in the mucosa of mice injected withadjuvant alone. The stomachs of mice immunized with pigH+/K+-ATPase showed only scant mononuclear cell infil-tration into the gastric mucosa, consistent with the absence ofautoantibodies in these mice. Mice treated with adjuvant aloneshowed no gastric infiltrate.

Phenotype of gastric mononuclear infiltrates

The phenotype of the infiltrating mononuclear cells in thegastric mucosa of a mouse with advanced gastritis after four

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Figure 5. Histology of stomachs from normal and H+/K+-ATPase-immunized mice: modified Maxwell's staining. (a) Section through thenormal stomach showing distribution of gastric glands; (b) sectionthrough stomach of an immunized mouse killed 3 weeks after sevenimmunizations with H+/K+-ATPase. The majority of the gastricglands in the arc of the mucosa contain large deposits of neutralpolysaccharides, revealed by Alcian yellow. Note that the flank of themucosa at left of the section is less affected. Bar= 450 gm for panel a;900 jim for panel b.

immunizations was characterized immunocytochemically(Fig. 7). CD4+ and CD8+ T cells were present in the submu-cosa and throughout the lamina propria (Fig. 7a and b). Smallclusters of B220+ cells (B cells) were detected in the submucosaassociated with advanced gastritis. The majority of the infiltrating cells appeared to be Mac-i-positive cells concentrate(at the bases of the glands and throughout the lamina propri;(Fig. 7c). Since the infiltrate is mononuclear, these Mac1-positive cells are probably macrophages. No staining waobserved with conjugate alone (Fig. 7d).

-49.5

Figure 4. Immunoblot reactivity of mouse H+/K+-ATPase with serafrom mice immunized with mouse gastric H+/K+-ATPase. MouseH+/K+-ATPase (4 pg/lane) was subjected to 7 5% SDS-PAGE underreducing conditions. Immunoblots show reactivity to either (lane 1)a- and fl-subunits; (lane 2) fl-subunit alone; (lane 3) anti-a-subunitmonoclonal antibody 1H9; (lane 4) anti-f-subunit monoclonal anti-body 2B6; (lane 5) anti-a-lactalbumin monoclonal antibody ET-l(isotype negative control); or (lane 6) normal mouse serum.

Gastritis is associated with depletion of parietal and zymogeniccells and accumulation of small columnar cells

In normal mouse stomachs, parietal cells, located mainly inthe neck of the glands, stained blue-green, due to Luxol fastblue binding to phospholipids28 in the extensive secretorymembranes and the mitochondria (Figs 5a and 6a). Zymogeniccells at the bases of glands stained red-purple due to theaffinity of pyronin for RNA29 in the abundant ribosomes(Figs. 5a and 6a).

Mononuclear cell infiltration into the lamina propria was

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Autoimmune gastritis induced by gastric H+/K+-ATPase

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Figure 6. Histology of stomachs from normal and H+/K+-ATPase-immunized mice; modified Maxwell's staining. Gastric mucosaof: (a) normal mouse showing parietal cells (p; blue-green) in the neck of the gland extending to the zone of zymogenic cells (z;red-purple); (b) mouse 3 weeks after two immunizations with H+/K+-ATPase showing neutral polysaccharides (yellow) in thecytoplasm of parietal cells (p) and in the gland lumen and degenerating zymogenic cells (z); (c) mouse 3 weeks after fourimmunizations with H+/K+-ATPase showing columns of immature pyronin-staining columnar cells (large arrowhead), parietalcells (small arrows) interspersed with the columnar cells in the neck, luminal neutral polysaccharides and mononuclear cells in thelamina propria; (d) mouse 13 weeks after four immunizations with H+/K+-ATPase showing differentiating and fully differentiatedparietal cells (small arrows), zymogenic cells (large arrowhead), fewer columnar cells, gland lumina with neutral polysaccharidesand mononuclear cells in the lamina propria. Bar= 30 glm for panels a, c, d; 15 gm for panel b.

associated with degeneration of parietal and zymogenic cells.While the severity of the epithelial lesion increased withcontinued immunization with the gastric H+/K+-ATPase, itsdevelopment was localized to areas of mononuclear cell infil-tration. Gastric mucosa which had not been infiltrated withmononuclear cells showed normal histology. After two immun-izations with H+/K+-ATPase, some parietal cells had cavitiescontaining neutral polysaccharides, zymogenic cells were

reduced in number and neutral polysaccharides were presentin gland lumina (Fig. 6b). After four immunizations, someareas of the mucosa showed advanced gastritis (Fig. 5b). Inthese areas, parietal cells had cytoplasmic cavities filled withneutral polysaccharides (Fig. 6c) and were reduced in numberor completely absent. A few degenerating zymogenic cells weredetected and massive deposits of neutral polysaccharides filledthe gland lumina (Figs. 5b and 6c). There was an increase in

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Figure 7. Immunoperoxidase characterization of mononuclear cell infiltrate in cryosections of stomach of mouse killed 5 weeksafter four immunizations with H+/K+-ATPase. Nuclei are stained with haematoxylin. CD4+ cells (a), CD8+ cells (b) andmacrophages (c) are found in the submucosa and throughout the lamina propria between gastric glands. (d) No primary antibody.Bar= 60 gm for panels a-d.

number of small immature columnar cells staining withpyronin, extending almost to the base of many glands, replac-ing parietal and zymogenic cells (Fig. 6c). Mice immunizedwith pig H+/K+-ATPase showed very little damage to thegastric mucosa.

Differentiation of parietal cells and zymogenic cells and recoveryof the lesion

Thirteen weeks after the fourth immunization, there wassubstantial restoration of both parietal cell and zymogenic cellpopulations (Fig. 6d). The glands were obviously in a recoveryphase since there was still a larger than normal population of

immature cells and a mononuclear infiltrate was still presentin the submucosa. Therefore, cessation of immunization withthe gastric H+/K+-ATPase resulted in restoration of parietaland zymogenic cell populations.

Adoptive transfer of splenocytes from gastric mice

To determine whether gastritis induced by immunization withgastric H+/K+-ATPase can be transferred to naive recipients,concanavalin A-stimulated splenocytes (2 x 107) from micewhich had received seven immunizations of H+/K+-ATPasewere transferred to 8-day-old BALB/c mice. At 16 weeks aftertransfer, four of five recipients had gastritis with infiltration

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Autoimmune gastritis induced by gastric H+/K+-ATPase 97

of the gastric mucosa with a large number of mononuclearcells and depletion of parietal and zymogenic cells frominfiltrated glands. However, none of the recipient mice hadcirculating parietal cell autoantibodies (data not shown). Asin the H +/K+-ATPase-immunized mice, the infiltrate graduallyreceded and parietal and zymogenic cells redeveloped in theglands. The two recipients of concanavalin A-stimulated splen-ocytes from adjuvant alone-injected mice did not show anygastritis nor circulating parietal cell autoantibodies.

DISCUSSION

Previous studies have implicated the fl-subunit of the gastricH+/K+-ATPase in the initiation of thymectomy-induced gas-tritis.9 " However, the development of gastritis by active directimmunization of normal non-thymectomized mice with gastricH+/K+-ATPase has not previously been reported. Whileprevious studies used crude gastric antigens to induce auto-immune gastritis,12-17 our study is the first report of anautoimmune gastritis following immunization with a purifiedgastric autoantigen. Our findings suggest that the gastricH+/K+-ATPase is the causative autoantigen responsible forthe genesis of the gastritis. Furthermore, we have recentlyshown that transgenic mice expressing the fl-subunit of thegastric H+/K+-ATPase in the thymus failed to develop auto-immune gastritis following the same schedule of immunizationwith the gastric H+/K+-ATPase as used here.30 Takentogether, the observations suggest that the f-subunit is theinitiating autoantigen not only in thymectomy-induced gastritisbut also in immunization-induced gastritis. Nishio et al. haveshown that a T-cell clone reactive with the a-subunit derivedfrom thymectomy-induced gastritis can induce gastritis.3' Thelatter observation suggests that a-subunit-reactive T cells cancontribute to the gastritis after it has been initiated by f,-subunit-reactive T cells.A critical factor in the development of gastritis by immuniz-

ation is the species of origin of the H+/K+-ATPase.Immunization with pig H+/K+-ATPase resulted in a strongantibody response to the immunogen but little, if any, gastritisnor autoantibody production. However, immunization withmouse H+/K+ -ATPase resulted in a severe destructive gastritisand autoantibodies to the H+/K+-ATPase. These observationssuggest that autoepitopes in murine H+/K+-ATPase are moreeffective than porcine epitopes in the initiation of murinegastric autoimmunity.

The destruction of parietal and zymogenic cells is probablycaused by invasion of the gastric mucosa by mononuclear cellssince degenerative changes in these cells were localized toregions with cellular infiltrates and splenocytes from gastriticmice can adoptively transfer a destructive gastritis to naiverecipients. The epithelial lesion is similar to that seen in thegastritis accompanying pernicious anaemia and in thymec-tomy-induced gastritis.1 A convincing demonstration of anautoimmune response to zymogenic cells in pernicious anae-mia1 and in murine gastritis has not been demonstrated.Zymogenic cell loss may be due to 'by-stander' cytotoxicity.Alternatively, it may be due to perturbation of the normaldevelopmental pathway of gastric epithelial cells, a suggestionsupported by the increased number of immature cells seen inthe gastritic glands. However, it is possible that the zymogeniccell loss observed here may be the result of an immune

response to a contaminant in the gastric H+/K+-ATPasesample.

The recovery of gastritis following cessation of immuniz-ation is in striking contrast to the persistent thymectomy-induced gastritis.7'9 The basis for this difference is not known.None the less, the findings are consistent with the suggestionthat inadequate autoantigen presentation6 and/or homeostaticmechanisms32 can prevent expansion of potentially pathogenicT cells in normal individuals. Recent studies have supported arole for a CD25+ subset of CD4' T cells in T-cell homeost-asis.3335 Whatever the nature of these mechanisms, the presentresults suggest that they can reverse a destructive autoimmunelesion after cessation of immunization with the autoantigen.A better understanding of these mechanisms may lead to newstrategies for reversal of destructive autoimmune lesions.

ACKNOWLEDGMENTS

This study was supported by grants from the National Health andMedical Research Council of Australia.

REFERENCES

1. TOH B.-H., VAN DRIEL I.R. & GLEESON P.A. (1997) Autoimmunegastritis and pernicious anemia. In: The Autoimmune Diseases.(eds. N.R. Rose & I.R. Mackay) Academic Press, London (inpress).

2. STRICKLAND R. & MACKAY I.R. (1973) A reappraisal of thenature and significance of chronic atrophic gastritis. Am J DigDis 18, 426.

3. TOH B.-H., GLEESON P.A., SIMPSON R.J. et al. (1990) The60-90 kDa parietal cell autoantigen associated with autoimmunegastritis is a novel f3-subunit of the gastric H+/K+-ATPase. ProcNatl Acad Sci USA 87, 6418.

4. GLEESON P.A. & TOH B.-H. (1991) Molecular targets in perniciousanaemia. Immunol Today 12, 233.

5. CALLAGHAN J.M., KHAN M.A., ALDERUCCIO F., VAN DRIEL I.R.,GLEESON P.A. & TOH B.-H. (1993) The ot and 3 subunits of thegastric H+/K+-ATPase are concordantly targeted by parietal cellautoantibodies associated with autoimmune gastritis.Autoimmunity 16, 289.

6. GLEESON P.A., TOH B.-H. & VAN DRIEL I.R. (1996) Organ-specificautoimmunity induced by lymphopenia. Immun Rev 149, 97.

7. SAKAGUCHI S., FUKUMA K., KURIBAYASHI K. & MASUDA T. (1985)Organ-specific autoimmune diseases induced in mice by elimin-ation of T cell subset. J Exp Med 161, 72.

8. JONES C.M., GLEESON P.A., CALLAGHAN J.M., MORI Y.,MASUDA Y. & TOH B.-H. (1991) The parietal cell autoantigensrecognised in neonatal thymectomy-induced murine gastritis arethe a and 1-subunits of the gastric proton pump. Gastroenterology101, 287.

9. BARRETT S., TOH B. -H., ALDERUCCIO F., VAN DRIEL I.R. &GLEESON P.A. (1995) Organ-specific autoimmunity induced byadult thymectomy and cyclophosphamide-induced lymphopenia.Eur J Immunol 25, 238.

10. SMITH H., SAKAMOTO Y., KAsm K. & TUNG K.S.K. (1991) Effectorand regulatory cells in autoimmune oophoritis elicited by neonatalthymectomy. J Immunol 147, 2928.

11. ALDERUCCIO F., TOH B.-H., TAN S.-S., GLEESON P.A. & VAN DRIELI.R. (1993) An autoimmune disease with multiple moleculartargets abrogated by the transgenic expression of a single autoan-tigen in the thymus. J Exp Med 178, 419.

12. ANDRDA J.A., ROSE N.R. & ANDRADA E.C. (1969) Experimentalautoimmune gastritis in the Rhesus monkey. Clin Exp Immunol4, 293.

© 1997 Blackwell Science Ltd, Immunology, 92, 91-98

98 K J. Scarifet al.

13. HENNES A.R., SEVELIUS H., LEWELLEN T., JOEL W., WOODS A.H.& WOLF S. (1962) Atrophic gastritis in dogs. Production ofintradermal injection of gastric juice in Freund's adjuvant. ArchPathol 73, 281.

14. FIXA B., VEJBORA O., KOMARKOVA O., LANR F. & PARIZEK J.(1964) On immunologically induced gastric atrophy in dogs.Gastroenterologia 102, 331.

15. FIXA B., THIELE H.G., KOMARKOVS 0. & NozICK A.Z. (1972)Gastric autoantibodies and cell-mediated immunity in perniciousanemia - a comparative study. Scand J Gastroenterol 7, 237-40.

16. KROHN K.J.E. & FINDLAYSON N.D.C. (1973) Interactions ofhumoral and cellular immune responses in experimental caninegastritis. Clin Exp Immunol 14, 237-45.

17. KONTANI K., TAGUCHI 0. & TAKAHASHI T. (1992) Involvementof the H+/K+-ATPase a subunit as a major antigenic protein inautoimmune gastritis induced by neonatal thymectomy in mice.Clin Exp Immunol 89, 63.

18. CALLAGHAN, J.M., TOH B.-H., SIMPSON R.J., BALDWIN G.S. &GLEESON P.A. (1992) Rapid purification of the gastric H+/K+-ATPase complex by tomato-lectin affinity chromatography.Biochem J 283, 63.

19. LAEMMLI U.K. (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227, 680.

20. MAXWELL A. (1963) The alcian dyes applied to gastric mucosa.Stain Technol 38, 286.

21. BEINBORN M., GIEBEL J., LINCK M., CETIN Y., SCHWENK M. &SEWING K.-F. (1993) Isolation, identification and quantitativeevaluation of specific cell types from the mammalian gastricmucosa. Cell Tissue Res 274, 229.

22. CHUANG J.S., CALLAGHAN J.M., GLEESON P.A. & TOH B.-H.(1992) Diagnostic ELISA for parietal cell autoantibody usingtomato lectin-purified gastric H+/K+-ATPase (proton pump).Autoimmunity 12, 1.

23. JONES C.M., TOH, B.-H., PETTITT J.M. et al. (1991) Monoclonalantibodies specific for the core protein of the n-subunit of thegastric proton pump (H+/K+-ATPase). Eur J Biochem 197, 49.

24. MORI Y., FUKUMA K., ADACHI Y. et al. (1989) Characterisationof parietal cell autoantigens involved in neonatal thymectomy-

induced murine autoimmune gastritis using monoclonal autoanti-bodies. Gastroenterology 97, 364.

25. THEAN E.T. & TOH B.-H. (1989) Production and characterisationof murine monoclonal antibody to human ot-lactalbumin. ImmunolCell Biol 67, 41.

26. MARTINELLI T.M., VAN DRIEL I.R., ALDERUCCIO F., GLEESON P.A.& TOH B.-H. (1996) Analysis of mononuclear cell infiltrate andcytokine production in murine autoimmune gastritis.Gastroenterology 110, 1791.

27. GOLDKORN I., GLEESON P.A. & TOH B.-H. (1989) Gastric parietalcell antigens of 60-90, 92, and 100-120 kDa associated withautoimmune gastritis and pernicious anaemia. J Biol Chem 264,18 768.

28. SALTHOUSE T.N. (1962) A quantitative histochemical method forestimating phospholipids. Nature 195, 187.

29. BRACHET J. (1953) The use of basic dyes and ribonuclease for thecytochemical detection of ribonucleic acid. Quart J Microbiol Sci94, 1.

30. ALDERUCCIO F., GLEESON P.A., BERZINS S.P. et al. (1997)Expression of the gastric H/K ATPase a subunit in the thymusmay explain the dominant role of the ,B subunit in the pathogenesisof autoimmune gastritis. Autoimmunity in press.

31. NISHIo A., HOSONO M., WATANABE Y., SAKAI M., OKUMA M. &MASUDA T. (1994) A conserved epitope on H+,K+-adenosinetriphosphastase of parietal cells discerned by a murine gastritog-enic T cell clone. Gastroenterology 107, 1408.

32. BONOMo A., KEHN P.J. & SHEVACH E.M. (1995) Post-thymectomyautoimmunity: abnormal T cell homeostasis. Immunol Today 16,61.

33. SAKAGUCHI S., SAKAGUCHI N., ASANO M., ITOH M. & TODA M.(1995) Immunologic self-tolerance maintained by activated T cellsexpressing IL-2 receptor a-chains (CD25). J Immunol 155, 1151.

34. ASANO M., TODA M., SAKAGUCHI N. & SAKAGUCHI S. (1996)Autoimmune disease as a consequence of developmental abnor-mality of a T cell population. J Exp Med 184, 387.

35. TAGUCHI 0. & TAKAHASHI T. Administration of anti-interleukin-2receptor a antibody in vivo induces localised autoimmune disease.Eur J Immunol 26, 1608.

C) 1997 Blackwell Science Ltd, Immunology, 92, 91-98