Spontaneous autoimmune gastritis and hypochlorhydria are manifestin the ileitis-prone SAMP1/YitFcs mice

P. B. Ernst,1,2 L. D. Erickson,2 W. M. Loo,2 K. G. Scott,6 E. B. Wiznerowicz,1 C. C. Brown,7

F. J. Torres-Velez,8 M. S. Alam,9 S. G. Black,1 M. McDuffie,3 S. H. Feldman,5 J. L. Wallace,10

G. W. McKnight,10 I. T. Padol,10 R. H. Hunt,10 and K. S. Tung4

Departments of 1Medicine, 2Microbiology, 3Pediatrics, and 4Pathology and 5Center for Comparative Medicine, University ofVirginia, Charlottesville, Virginia; 6Department of Biology, University of Manitoba, Winnipeg, Manitoba; 10Department ofMedicine and The Farncombe Institute, McMaster University, Hamilton, Ontario, Canada; 7Department of Pathology,College of Veterinary Medicine, University of Georgia, Athens, Georgia; 8Infectious Disease Pathogenesis Section, NationalInstitute of Allergy and Infectious Diseases, Bethesda; and 9Immunobiology Branch, Center for Food Safety and Nutrition,US Food and Drug Administration, Laurel, Maryland

Submitted 18 May 2011; accepted in final form 8 September 2011

Ernst PB, Erickson LD, Loo WM, Scott KG, Wiznerowicz EB,Brown CC, Torres-Velez FJ, Alam MS, Black SG, McDuffie M,Feldman SH, Wallace JL, McKnight GW, Padol IT, Hunt RH,Tung KS. Spontaneous autoimmune gastritis and hypochlorhydria aremanifest in the ileitis-prone SAMP1/YitFcs mice. Am J PhysiolGastrointest Liver Physiol 302: G105–G115, 2012. First publishedSeptember 15, 2011; doi:10.1152/ajpgi.00194.2011.—SAMP1/Yit-Fcs mice serve as a model of Crohn’s disease, and we have usedthem to assess gastritis. Gastritis was compared in SAMP1/YitFcs,AKR, and C57BL/6 mice by histology, immunohistochemistry,and flow cytometry. Gastric acid secretion was measured in ligatedstomachs, while anti-parietal cell antibodies were assayed byimmunofluorescence and enzyme-linked immunosorbent spot as-say. SAMP1/YitFcs mice display a corpus-dominant, chronic gas-tritis with multifocal aggregates of mononuclear cells consisting ofT and B lymphocytes. Relatively few aggregates were observedelsewhere in the stomach. The infiltrates in the oxyntic mucosawere associated with the loss of parietal cell mass. AKR mice, thefounder strain of the SAMP1/YitFcs, also have gastritis, althoughthey do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulatingileitis had comparable effects on gastritis. The majority of the cellsin the aggregates expressed the T cell marker CD3 or the B cellmarker B220. Adoptive transfer of SAMP1/YitFcs CD4� T helpercells, with or without B cells, into immunodeficient recipientsinduced a pangastritis and duodenitis. SAMP1/YitFcs and AKRmice manifest hypochlorhydria and anti-parietal cell antibodies.These data suggest that common genetic factors controlling gas-troenteric disease in SAMP1/YitFcs mice regulate distinct patho-genic mechanisms causing inflammation in separate sites withinthe digestive tract.

parietal cell; autoantibodies; autoimmune gastritis; model of Crohn’sdisease

THE GASTROINTESTINAL TRACT faces the unique immunologicalchallenge of coping with a dynamic and vast array of dietaryand microbial antigens. Chronic inflammatory disease of theintestine, as well as the stomach, of adults and children (9,10, 38) arises in genetically susceptible individuals that failto properly regulate host responses to these luminal anti-

gens. The interrelationship of inflammation in the stomachand intestine can be appreciated in studies that examinethese tissues from subjects with inflammatory bowel disease(IBD). For example, sterile gastritis has been observed insubjects with Crohn’s disease or ulcerative colitis (7, 31).

Several animal models have been used to study IBD; how-ever, models of Crohn’s disease are limited. SAMP1/Yit micedevelop a spontaneous terminal ileitis with many features ofCrohn’s disease (17, 23). Ileitis in SAMP1/Yit mice is associ-ated with proinflammatory cytokine production: T helper (Th)type 1 (Th1) cell responses in younger mice (17), but also Th2cytokines (42) as they mature (4). It has been suggested that thelesions in SAMP1/YitFcs mice were limited to the terminalileum (17). These mice have also been shown to have skinlesions as well as hepatitis (23). The SAMP1/Yit mice wereoriginally derived from AKR mice with an unintentional back-cross to an unknown strain (18). AKR mice have not beenreported to develop spontaneous inflammation in their diges-tive tract.

Although many immunological responses are perturbed inSAMP1/Yit mice, some evidence suggests that the primarydefect lies in the epithelial cell barrier. In the process ofstudying intestinal epithelial permeability (28), we showed adisrupted epithelial cell barrier function in the SAMP1/Yitstomach (34). While SAMP1/Yit mice develop a spontane-ous terminal ileitis and have been used as a model ofCrohn’s disease, the health of their gastric mucosa has notbeen carefully examined. The purpose of this study was tomore thoroughly investigate the gastric manifestations inSAMP1/Yit mice.

MATERIALS AND METHODS

Mice. SAMP1/YitFcs mice [the registered nomenclature for a strainthat has been referred to previously as SAMP1/YitFc (35)] werederived from the SAMP1/Yit mice originally developed at the YakultCentral Institute for Microbiological Research (Tokyo, Japan). Micewere provided initially by Drs. Pizarro and Cominelli and subse-quently by the University of Virginia. Congenic mouse strains gen-erated at the University of Virginia (M.M.) included SAMP.B6-D9Mit297-D9Mit212 (SAMP.B6C9A), SAMP.B6-D9Mit297-D9Mit12369BL (SAMP.B6C9BL), and SAMP.B6-DXMit166-DXMit156(SAMP.B6CX). Congenic strains and (AKR � SAMP1/YitFcs) F1mice were generated on site as described previously (18, 40). C57BL/6J, AKR/J, and C3Smn.CB17-Prkdcscid/J (C3H.scid) mice were ob-

Address for reprint requests and other correspondence: P. B. Ernst, Divisionof Comparative Pathology and Medicine, Dept. of Pathology, Univ. of Cali-fornia, San Diego, 9500 Gilman Dr., MC 0063, San Diego, CA 92093-0063(e-mail: pernst@ucsd.edu).

Am J Physiol Gastrointest Liver Physiol 302: G105–G115, 2012.First published September 15, 2011; doi:10.1152/ajpgi.00194.2011.

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tained from Jackson Laboratories (Bar Harbor, ME) or bred andhoused in specific pathogen-free conditions at the University ofVirginia. Male and female 8- to 58-wk-old mice were used in thestudies. Mice were not infected with any known pathogenic Helico-bacter spp. All mice were maintained specific pathogen-free withquarterly health surveillance monitoring, and protocols were per-formed with the approval of the University of Virginia InstitutionalAnimal Care and Use Committee.

Characterization of gastritis. Stomachs were collected from allmice, flushed with PBS, fixed in Bouin’s fixative, oriented symmet-rically (to allow uniform cross-sectional measurement of mucosathickness), and prepared for staining with hematoxylin and eosin (1)or processed for immunohistochemistry to detect CD45R/B220, F4/80, and CD3. For B220 (catalog no. 553086, BD Bioscience, SanDiego, CA) and F4/80 (catalog no. MCA497BB, Serotec, Raleigh,NC), tissue sections were pretreated by steaming with Diva solution(Biocare, Concord, CA) for 20 min and then incubated with theprimary antibody at a concentration of 1:1,000 and 1:200, respec-tively. Detection was achieved using a polymer detection system(Rat-on-Mouse AP kit, Biocare) and Vulcan fast red (Biocare). CD3�

cells were detected using a monoclonal antibody (Ab 16669, Abcam,Cambridge, MA) at 1:1,000 dilution, with sequential application of agoat anti-rabbit antibody (BA-1000, Vector Laboratories, Burlingame,CA), Vector Elite ABC (Vector Laboratories), and NovaRed (VectorLaboratories).

All hematoxylin-eosin-stained slides were scanned and digitallystored using ScanScope (Aperio, Vista, CA) and read using Image-Scope (Aperio) software to allow the use of a web-based file-sharing platform. Scoring was based on a modification of a com-prehensive approach described previously (1, 2, 14) developed inconsultation with a comparative pathologist (C.C.B.) and an in-vestigator with extensive experience scoring autoimmune gastritisin mice (K.S.T.). Thickness of the corpus tissue was measuredusing the “ruler function” of ImageScope, which allows the startand end of the measurement to be precisely placed from the edgeof the lumen to the outside of the serosa �5 mm from the junctionof the forestomach and corpus. Multiple criteria for the three majorregions of the murine stomach were used to grade the histopathol-ogy (Table 1). All photomicrographs were captured using Image-Scope.

Gastric mononuclear cells (MNC) from the corpus dissected freeof the forestomach and antrum, gastric lymph nodes, or mesenen-teric lymph nodes were prepared as reported previously (1, 2, 37),labeled for CD3, CD45, and B220 (reagents described in Table 2)immediately after isolation, and assayed by flow cytometry, aspreviously described (3, 25, 45). After gating on CD45� cellsexcluding aggregates, samples were assayed using a flow cytom-eter (Fortessa, Becton Dickinson, San Jose, CA). Data were ana-lyzed using Flowjo software (Treestar, Ashland, OR). Controlsincluded beads coated with each antibody for compensation, andgates were set using gastric MNC labeled with combinations of allantibodies minus one (36), such that �97.5% of all nonspecificfluorescence was excluded.

Adoptive transfer studies. To investigate the pathogenicity oflymphocytes in the gastritis, adoptive transfer studies were per-formed as previously described for studying the role of these cellsin ileitis (27). Briefly, mesenteric lymph node cells from SAMP1/YitFcs or C3H mice were prepared using standard techniques (15)and used as a source of lymphocytes that were enriched into CD4�

or B220� subsets by magnetic beads; then 5 � 105 T cells, B cells,or both were adoptively transferred into the major histocompati-bility complex-compatible C3H.scid mice by intraperitoneal injec-tion. After 6 wk, tissues were collected and scored for histology.

Evaluation of gastric acid secretion. Mice were fasted in metaboliccages overnight, weighed, and anesthetized. The pylorus was isolated,and a snug ligature was applied at the junction of the antrum andpylorus. After 3 h of recovery, the mice were anesthetized, theproximal end of the stomach was ligated at the junction of theesophagus, and the entire stomach was removed. The gastric contentswere collected, weighed to estimate the volume, and assayed for pH.In some cases, pentagastrin (50 �g/kg ip) was administered in a bolus,but no differences were observed between this treatment and the 3-hunstimulated gastric acid secretion (data not shown).

Autoantibody detection. Sera were screened for autoantibodies byimmunofluorescence. Briefly, frozen sections of normal mousestomach were incubated with a 1:50 dilution of the serum to bescreened from SAMP1/YitFcs, AKR, or C57BL/6 mice and thenwith a FITC-conjugated anti-murine antibody (IgM, IgG1, IgG2a,IgG2b, or IgA) and detected by fluorescent microscopy (43). Theintensity of fluorescence was graded (assigned a score of 0 –3)

Table 1. Summary of scoring criteria

Parameter

Score

0 1 2 3

Thickness �750 �m �750 �mForestomach

Granulocytes Absent PresentMononuclear cells Absent Focal, 1–5 cells in a �40 field AggregatesHyperkeratosis �10 layers �10 layers

CorpusGranulocytes Absent or rare Focal, 2–5 cells in a �40 field �1 field with 5 cells LuminalMononuclear cells Rare Focal Diffuse, �30 cells in a �40 field AggregatesParietal cell loss None Irregular staining and morphology Moderate loss of parietal cell mass Marked loss of parietal cell mass

AntrumGranulocytes Absent or rare Focal, 2–5 cells in a �40 field �1 field with 5 cells LuminalMononuclear cells Rare Focal Diffuse Aggregates

Table 2. Reagents used for flow cytometry

Description Fluorochrome Channel Source Stock No. Isotype

Anti-CD3 FITC FL1 eBioscience 11003182 Hamster IgGAnti-B220 PE-TXred D yellow/green BD 551489 Rat IgG2a, IgG2kAnti-CD45 PerCP B blue BD 557235 Rat IgG2b, IgG2k

PE, phycoerythrin; TX red, Texas red; PerCP, peridinin-chlorophyll-protein complex.

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against negative and positive control sections. In addition, gastricMNC isolated as described above were compared with MNCisolated from bone marrow and spleen (25, 45) by enzyme-linkedimmunosorbent spot (ELISpot) assay for total IgM-, IgG-, andIgA-producing cells (16), as well as antigen-specific spots, usingplates coated with 50 �g/ml of a lysate of enriched parietal cellsisolated from C57BL/6 mouse stomachs prepared as describedpreviously (29) or porcine H�-K�-ATPase (Arotec Diagnostics).Plates coated with AGS cell lysates served as a negative control.

Statistical analysis. The inflammation scores were compared byStudent’s t-test with Bonferroni’s correction for multiple comparisonswhen indicated. Multivariate analysis was used to examine the effectsof age and sex on inflammation. The correlation of corpus inflamma-tion and anti-parietal cell antibody titers was also assessed. The pH ofgastric secretions was compared using Wilcoxon’s rank sum test. Allanalyses were done using the statistical package in Sigmaplot (Graph-Pad Software, San Diego, CA).

RESULTS

SAMP1/YitFcs mice develop a corpus-dominant gastritis.Pathological changes in gastric tissue were assessed in theforestomach, corpus, and antrum. Figure 1 shows no signif-icant evidence of gastritis in C57BL/6 mice. In contrast,tissue from the SAMP1/YitFcs mice was thickened (mean of10 animals: 905.3 and 479 �m as measured at the junctionof the forestomach and corpus for SAMP1/YitFcs andC57BL/6 respectively, P � 0.05) and had a marked chronic,mononuclear cell infiltrate, including defined multifocalaggregates (Fig. 1A vs. Fig. 1, B and C) and occasionalaccumulations in the lumen of the gastric glands that ap-peared to include dead mononuclear cells but rarely identi-fiable polymorphonuclear cells, in the corpus. Infiltrationwith polymorphonuclear cells was remarkably modest. Ep-ithelial glands in the corpus were often distended or tortu-ous, with evidence of epithelial cell proliferation. In areas ofinflammation, there was loss of parietal cell mass. Inflam-mation was more modest in the antrum, and when inflam-matory cells were increased, they were focal and located atthe base of the glands close to or on either side of themuscularis mucosa. Histological scoring is summarized inFig. 1D.

Ileitis and gastritis in SAMP1/YitFcs mice share some of thesame genetic control. The SAMP1/YitFcs mice were origi-nally derived from AKR mice (40), so gastritis was assessedin this strain as well. AKR mice displayed a corpus-domi-nant, spontaneous, chronic gastritis. As neither strain hadbeen reported previously to have gastritis, these observa-tions represent two new murine models of gastritis.

When SAMP1/YitFcs mice are crossed to AKR mice, theresultant F1 mice lack ileitis (40). When F1 mice from thiscross were examined, the gastritis was markedly attenuated(Fig. 1E), approaching the degree of inflammation inC57BL/6 mice. In other reports (18), congenic mice gener-ated by backcrosses onto C57BL/6 mice yielded offspringwith decreased inflammation in the terminal ileum. Thecongenic strains with attenuated ileitis (SAMP.B6C9A andSAMP.B6CX) also had decreased gastritis, while theSAMP.B6C9/BL congenic strain had no effect on gastritis(Fig. 1F), which, again, mimicked the effect of this cross onileitis (18).

A mixed T and B cell infiltrate constitutes the majority of thechronic gastritis. The mononuclear cells within the aggregatesexpressed B220 (Fig. 2, A and C), as well as CD3 (Fig. 2, B andD). Both of these markers identified the majority of the mono-nuclear cells in the aggregates in the corpus, as well as thosethat accumulated diffusely throughout the tissue. Cells express-ing F4/80 were not detected in the aggregates and were sparsethroughout the mucosa (data not shown).

Gastric mononuclear cells were isolated from the corpus orfrom the draining gastric lymph nodes. These cells expressedCD3 (60%) and B220 (30%) in preparations from the mucosa(Fig. 2E), as well as the draining gastric lymph nodes (data notshown), approximating the results observed by immunohisto-chemistry. Interestingly, a small percentage (4–7%) of CD3�

cells expressed B220, which would be consistent with T cellsbeing highly activated (44), as recognized previously in theliver (26) and gut (6, 12).

Adoptive transfer of CD4� Th cells induces a pangastritisand duodenitis distinct from the spontaneous disease inSAMP1/YitFcs mice. As the ileitis in SAMP1/YitFcs micecan be adoptively transferred into immunodeficient mice(27), we determined if the same was true for the gastritis.CD4� Th cells purified from the mesenteric lymph nodes ofSAMP1/YitFcs mice were transferred into severe combinedimmunodeficiency (SCID) recipient mice (Fig. 3). Whilegastritis emerged, it shifted from a corpus-predominantgastritis found in the SAMP1/YitFcs donors to a pangastritisand lacked the characteristic mononuclear cell aggregatesobserved in the donor mice. Cotransfer of B cells fromSAMP1/YitFcs donors did not significantly alter the lesionsthat developed in the recipients after 6 wk. Moreover, asevere duodenitis with total ablation of the villi was detectedin virtually all recipients given T cells alone or in combi-nation with B cells (Fig. 3B). The latter was surprising, asduodenitis was a relatively unusual finding in SAMP1/YitFcs mice and has not been investigated previously in thetransfer model. These findings suggest that the adoptivetransfer of Th cells was proinflammatory but failed todirectly mimic the characteristic gastritis that developsspontaneously in these mice.

Gastritis in SAMP1/YitFcs mice is associated with an auto-immune gastritis and hypochlorhydria. Since the adaptivetransfer of Th cells failed to recreate the corpus-dominantgastritis in SAMP1/YitFcs mice, the role of other cells wasexplored. Approximately 50% of the SAMP1/YitFcs micedisplayed hyperkeratosis at the junction of the forestomachand corpus (Fig. 4, A–C), often associated with an over-growth of rod-shaped bacteria (Fig. 4D). As the number ofbacteria on the surface of the forestomach epithelium ap-peared increased, gastric acid secretion was assessed. Mea-surement of the 3-h unstimulated gastric acid secretionyielded an average of 0.765 ml of gastric juice from all micesampled and showed that approximately half of the SAMP1/YitFcs and AKR mice displayed hypo- or achlorhydria (Fig.4E). As parietal cell damage and loss of acid production areusually associated with autoimmune gastritis, the serum wasassayed for anti-parietal cell antibodies. As shown in Fig. 5,serum from SAMP1/YitFcs mice had significant titers ofautoantibodies that recognized parietal cells specifically, butnot adjacent cells. These antibodies were not detected in theC57BL/6 (Fig. 5B) and (AKR � SAMP1/YitFcs) F1 mice

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(data not shown) that lacked gastritis. Moreover, the titer ofthe anti-parietal cell antibody correlated to the intensityof the corpus gastritis (Fig. 5C). Examination of the isotypeof these anti-parietal cell antibodies revealed multiple iso-

types, including IgG1, IgG2a, IgG2b, and IgA (data notshown). ELISpot assays revealed that antibodies recogniz-ing parietal cell lysates and H�-K�-ATPase were producedpredominantly by IgA-secreting B cells isolated from the

Fig. 1. SAMP1/YitFcs mice develop a corpus-dominant gastritis. Gastric tissue was collected from control C57BL/6 mice (A) and SAMP1/YitFcs mice (B), andinflammation was assessed by histology. A: normal, uninflamed gastric mucosa. Higher power (A2) shows a typical uniform population of parietal cells in thecorpus; representative parietal cells are indicated by yellow arrows in A3. Gastric mucosa of SAMP1/YitFcs mice is typically thickened (B; scale bars, �500�m in A and B). At higher magnification, mononuclear cell aggregates are apparent in the corpus (green arrows, B2–B4); dilated glands containing accumulationsof dead cells are indicated by white arrows (B2–B4). Images captured at a higher power show intact parietal cells in control mouse (A4) and inflammation withassociated loss of parietal cell mass in SAMP1/YitFcs mouse (B4). Aggregates (encircled) were observed in the forestomach of some mice (C; scale bar, �100�m). D: scoring for forestomach (FS), corpus, and antrum from 5 SAMP1/YitFcs (SAMP1) mice and 6 C57BL/6 (BL/6) age-matched mice, representing a sampleof �60 mice. Values are means � SE. *P � 0.05. As several genetic elements regulating the onset of ileitis in SAMP1/YitFcs mice have been identified, gastritiswas assessed in these crosses. E: scoring for the forestomach, corpus, and antrum from 10 SAMP1/YitFcs, 9 AKR, and 9 (AKR � SAMP1/YitFcs) F1 mice.F: gastritis scores for 10 C57BL/6 mice and 13 SAMP1/YitFcs mice compared with scores for SAMP1/Yit/Fcs-C57BL/6 congenic mice, including 8SAMP.B6C9A (B6C9A), 3 SAMP.B6C9BL (B6C9BL), and 3 SAMP.B6CX (B6CX) mice. Values are means � SE. *P � 0.05 vs. SAMP1.

Fig. 2. Characterization of mononuclear cellswithin the aggregates. Photomicrographsrepresent 2 magnifications of sectionsstained for B220 (A and C) and CD3 (B andD). Number of B220� cells within theseaggregates exceeded the number of CD3�

cells. Images are from 1 mouse and arerepresentative of 5 separate SAMP1/YitFcsmice. E: flow cytometric analysis of mono-nuclear cells isolated from the gastric corpuslamina propria and stained for B220 andCD3. Results from representative experi-ment show that almost all the freshly isolatedgastric mononuclear cells expressed CD3 orB220, while a significant subset expressedboth markers. PE, phycoerythrin.

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gastric corpus (Fig. 5D) compared with plates coated withirrelevant proteins, including IgG or IgM or cell lysatesfrom AGS cells.

DISCUSSION

This report describes two new models of autoimmune gas-tritis in the SAMP1/YitFcs and AKR mice. As SAMP1/YitFcsmice are derived from AKR mice (40), the presence of gastritisin both strains may not be unexpected, although only theformer manifest terminal ileitis (17). F1 mice derived from across of SAMP1/YitFcs and AKR lose their ileitis (40). Sur-prisingly, they also had markedly attenuated gastritis, whichmay reflect an interaction between distinct alleles of a givengene or chromosomal segment that produces a phenotypedistinct from either parental phenotype. Examination of con-genic strains derived from crosses of C57BL/6 with SAMP1/YitFcs mice (18) showed that ileitis and gastritis fell under thesame genetic control. Although the pathogenesis of the twolesions may differ, these studies suggest that control of disease

in the ileum and stomach of the SAMP1/YitFcs mice sharessome of the same genetic elements.

The major histological changes reflected a chronic inflam-mation with multifocal aggregates of mononuclear cells com-posed predominantly of B220� B cells and CD3� T cells.These aggregates were primarily in the oxyntic mucosa, withoccasional lesions in the forestomach but substantially fewerinflammatory cells infiltrating the antral mucosa. The presenceof T cells was associated with mRNA for IFN-� and TNF- inthe gastric mucosa from the AKR and SAMP1/YitFcs mice,with little evidence of IL-4 or IL-17A (data not shown). Toaddress the pathogenesis of the gastritis more completely, weassessed the role of B cells in the production of autoantibodies.The morphology of the oxyntic mucosa in SAMP1/YitFcs micewas often very perturbed, including loss of parietal cell mass.In approximately half of the SAMP1/YitFcs and AKR mice,pH was significantly increased. Parietal cell-specific antibody-producing cells were detected in the mucosa of the SAMP1/YitFcs mice, while anti-parietal cell antibodies were detected

Fig. 3. Adoptive transfer of CD4� T helper(Th) cells induces a pangastritis and duodeni-tis distinct from the spontaneous disease inSAMP1/YitFcs mice. To investigate the roleof Th cells in the pathogenesis of gastritis,severe combined immunodeficiency (SCID)recipient mice were given 5 � 105 CD4� Thcells with or without an equal number of Bcells, both prepared from the mesentericlymph node of SAMP1/YitFcs mice. After 6wk, tissues were collected. A: control SCIDtissue had little inflammation and a com-pletely healthy duodenum (black arrow).SCID recipients given Th cells and B cellsfrom SAMP1/YitFcs mice (same magnifica-tion as SCID control) had a slightly thickenedcorpus and evidence of a generalized pangas-tritis, with more obvious lesions in the antrumin the recipients than the donor SAMP1/Yit-Fcs mice. In addition, their duodenal architec-ture was virtually obliterated (red arrow andhigher power in B; scale bar, �100 �m).Parietal cells are obvious and intact in theserecipients. C: histological scoring. Data arefrom 2 SCID and 4 reconstituted mice pergroup.

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in the majority of the SAMP1/YitFcs mice and �50% of theAKR mice. Interestingly, IgA-producing cells were the pre-dominant isotype in the stomach, which is unlike the pattern ofisotype expression observed in the MLN of the SAMP1/YitFcsmice (27) or in the mucosa of human subjects with IBD (20) orgastritis (8, 10). Direct examination of the antisera for their

ability to react to host cells in the ileum failed to identify anobvious autoantibody reaction (data not shown).

The adoptive transfer of ileitis by CD4� Th cells is exacer-bated by the coadministration of B cells (11, 27). It has beensuggested that B cells from SAMP1/YitFcs mice express aglucocorticoid-induced TNF receptor-related protein (GITR)

Fig. 4. SAMP1/YitFcs mice have evidence ofbacterial overgrowth and achlorhydria. Hy-perkeratosis was observed in the forestomachin �50% of the SAMP1/YitFcs mice (B andC) but was not evident in the C57BL/6 mice(A). In some cases, a marked accumulation ofrod-shaped bacteria was evident (D). Thesefindings suggest that there may be bacterialovergrowth due to altered gastric acid pro-duction. Examination of the gastric pH over 3h in unstimulated, ligated stomachs revealedabnormal gastric acid production that reachedpH 7 in some cases in SAMP1/YitFcs andAKR mice, suggestive of achlorhydria (E).(AKR � SAMP1/YitFcs) F1 mice, whichlacked gastritis, had essentially normal gastricacid production. Data are from 6–9 mice pergroup. Values are median scores. *P � 0.05 vs.C57BL/6 mice (Wilcoxon’s rank sum test).

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ligand, which, when engaged into its receptor GITR, decreasesthe function of regulatory Th cells (27). B cells in SAMP1/YitFcs mice also produce less IL-10 and transforming growthfactor- (24). Thus it is possible that the B cell defect(s) inSAMP1/YitFcs mice affect(s) immune regulatory function,which is one mechanism known to control gastritis (2, 13, 32,37). However, reconstitution with T and B cells isolated fromSAMP1/YitFcs mice failed to recapitulate the corpus-predom-inant gastritis observed in SAMP1/YitFcs mice. These obser-vations support the notion that the B cells are producing anautoantibody that is necessary for the corpus-dominant gastritisto emerge and that the accumulation of this antibody and itsdamage takes �6 wk. The role of B cells in this process isbeing addressed through long-term reconstitutions, which mayprovide the time required for the development of autoantibod-ies and parietal cell damage.

Reconstitution of the recipients with donor cells fromSAMP1/YitFcs mice induced a novel duodenitis. The presenceof gastritis in the recipients is in agreement with the recentfindings of Reuter et al. (33); however, because they did notreport examining the duodenum, a direct comparison of theirstudy with the present study is difficult. Both studies used thesame H2k-matched recipients, as described previously (27),which should prevent a major contribution of graft vs. hostdisease. However, the presence of duodenitis may reflect aminor mismatch in the major histocompatibility complex or thetendency of donor lymphocytes from these mice to promotedisease easily in multiple tissues of recipients.

It has been reported that, in addition to ileitis, SAMP1/YitFcs mice develop skin lesions and display hepatic mono-nuclear cell aggregates (23). However, in another report, “nosignificant extraintestinal inflammation” was detected in thestomach, liver, spleen, thymus, and various lymph nodes (17).There are several possible explanations, besides reader error,for the varying ability to detect gastritis. 1) The morphology ofthe SAMP1/YitFcs mice was compared with that of thefounder AKR strain (33), which, based on the evidence in thepresent study, also develops autoimmune gastritis. Such acomparison lacking a negative control would complicate aninterpretation, while contrasting the morphology to a strainwithout gastritis, such as the C57BL/6 strain, reveals the lesioneasily. 2) There could also be genetic drift between lines.While possible, this seems unlikely, since by genetic analysisat our institution, founding and current strains appear identical(Drs. Jesus Rivera-Nieves and M. McDuffie, personal commu-nication). Moreover, SAMP1/YitFcs mice were originally de-rived from AKR mice (41); thus the tendency to developgastritis may lie in the AKR background, which is embedded inthe SAMP1/YitFcs strain. 3) Epigenetic factors may also affectthe outcome, as could age and sex. However, lesions arepresent in males and females and in some mice as young as3–4 wk old (unpublished observations; 33). 4) The inability to

detect gastritis could be due to differences in the microbiomein the mice. This is quite possible, as bacteria, while notnecessary, exacerbate the lesions in the SAMP1/Yit mice (5,33). In other cases, the presence of certain bacterial speciesattenuates ileitis (21, 22, 30). Together, these observations leadone to conclude that the immunological abnormalities inSAMP1/YitFcs mice facilitate heightened responses in multi-ple tissues that are modulated by microbial communities.

The presence of gastritis in association with defects in epithelialpermeability has been described in SAMP1/YitFcs mice (34).Gastritis in SAMP1/YitFcs mice has been proposed as a model ofCrohn’s gastritis (33), although these investigators did not reportany investigations of autoimmune gastritis and, hence, could notdiscount it. As the data in the present study have shown unequiv-ocally that B cells are present in the corpus and include autore-active B cells likely recognizing H�-K�-ATPase from parietalcells, it is our conclusion that gastritis in these mice is moreevident in the corpus in association with the antigen that drives theB cell expansion. It is possible that the epithelial permeabilitydefect favors the translocation of microbes that modifies the hostresponses in these mice.

Gastritis in humans has many causes (39) and includes ulcer-ative colitis and Crohn’s disease (31). While the SAMP1/YitFcsmice have been used as a model of Crohn’s disease, it is not clearthat this gastritis resembles the “sterile” Helicobacter-free gastritisassociated with Crohn’s disease or ulcerative colitis (19). Sincemice are coprophagic, it is likely that gastric microbiota play asignificant role in modifying the inflammation, making this modelvery distinct from sterile gastritis associated with Crohn’s disease.Crohn’s gastritis is characterized as “focally enhanced gastritis”(31), usually limited to superficial regions in the antrum (7).However, in SAMP1/YitFcs mice, lesions included multifocalmononuclear cell infiltrates predominantly in the corpus, usuallyadjacent to the muscularis mucosae, which is more typical ofautoimmune gastritis. While autoimmune gastritis in Crohn’sdisease has been described in a case report (19), it is not generallyrecognized as being typical.

In summary, the SAMP1/YitFcs mouse continues to providean interesting model for studying the pathogenesis of inflam-mation. However, disease in these mice is not limited to theileum but is also manifest in the skin and liver and, inparticular, the stomach. How these lesions relate to each otherremains unclear, although they share genetic control of theinflammation in the stomach and ileum. It is likely that theydiffer significantly in their tissue-specific effector mechanismsthat escape regulation due to one or more abnormalities in theSAMP1/YitFcs mice.

ACKNOWLEDGMENTS

We thank Joanne Lannigan and Michael Solga (University of Virginia FlowCytometry Core Facility) for expert technical assistance in flow cytometry, aswell as Sheri VanHoose (University of Virginia Research Histology Core). The

Fig. 5. SAMP1/YitFcs and AKR mice have an autoimmune gastritis and produce anti-parietal cell antibodies. Given the effect on gastric pH, SAMP1/YitFcs(n � 6), AKR (n � 3), and (AKR � SAMP1/YitFcs) F1 mice (n � 5) were assayed for anti-parietal cell antibodies and compared with C57BL/6 mice (n �6). A: representative immunofluorescence image in which serum from SAMP1/YitFcs mice (left) binds selectively to the parietal cells, in contrast to the samplefrom C57BL/6 mice (right). B: percentage of each strain with titer scores of 0 (negative), 1 (�), 2 (��), or 3 (���). C: analysis of the inflammation scorein the corpus of all 20 samples and the titers of anti-parietal cell antibodies showed a significant correlation between the 2 variables (r � 0.9274, P � 0.05).D: number of nonspecific (IgM, IgG, or IgA) and antigen-specific antibody-producing cells in gastric mononuclear cells (gMNC), spleen (SPL), and bone marrow(BM) isolated from SAMP1/YitFcs mice detected by enzyme-linked immunosorbent spot assay. Values are means � SE for 3 observations.

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assistance of Robert Sampson in the immunofluorescence was greatly appre-ciated.

GRANTS

This research was supported by National Institute of Diabetes and Digestiveand Kidney Diseases Grants DK-051677 and DK-84063 (to P. B. Ernst), aResearch Fellowship from the Canadian Association of Gastroenterology (toK. G. Scott), National Institute of Allergy and Infectious Diseases AI-41236and AI-51420 (to K. S. Tung), and DK 57880 (M. McDuffie).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.B.E., L.D.E., K.G.S., C.C.B., M.S.A., M.M., J.L.W., R.H.H., and K.S.T.are responsible for conception and design of the research; P.B.E., W.M.L.,K.G.S., E.B.W., F.J.T.-V., M.S.A., S.G.B., M.M., S.H.F., G.W.M., I.T.P.,R.H.H., and K.S.T. performed the experiments; P.B.E., L.D.E., K.G.S., C.C.B.,F.J.T.-V., M.M., J.L.W., G.W.M., and K.S.T. analyzed the data; P.B.E.,L.D.E., W.M.L., K.G.S., C.C.B., F.J.T.-V., M.S.A., M.M., J.L.W., and K.S.T.interpreted the results of the experiments; P.B.E., E.B.W., and K.S.T. preparedthe figures; P.B.E. and M.M. drafted the manuscript; P.B.E., L.D.E., E.B.W.,C.C.B., F.J.T.-V., M.S.A., M.M., J.L.W., R.H.H., and K.S.T. edited andrevised the manuscript; P.B.E., L.D.E., W.M.L., K.G.S., E.B.W., C.C.B.,F.J.T.-V., M.S.A., S.G.B., M.M., S.H.F., J.L.W., G.W.M., I.T.P., R.H.H., andK.S.T. approved the final version of the manuscript.

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