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(American Journal of Pathology. 2004;165:1257-1268.)
© 2004 American Society for Investigative Pathology

CD4+CD45RBHi Interleukin-4 Defective T Cells Elicit Antral Gastritis and Duodenitis

Taeko Dohi*{dagger}, Kohtaro Fujihashi{ddagger}, Toshiya Koga{ddagger}, Yuri Etani{ddagger}, Naoto Yoshino{ddagger}, Yuki I. Kawamura* and Jerry R. McGhee{dagger}

From the Department of Gastroenterology,* Research Institute, International Medical Center of Japan, Tokyo, Japan; the Departments of Oral Biology,{ddagger} School of Dentistry, and The Immunobiology Vaccine Center, Department of Microbiology,{dagger} The University of Alabama at Birmingham, Birmingham, Alabama


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We have analyzed the gastrointestinal inflammation which develops following adoptive transfer of IL-4 gene knockout (IL-4–/–) CD4+CD45RBHi (RBHi) T cells to severe combined immunodeficient (SCID) or to T cell-deficient, T cell receptor ß and {delta} double knockout (TCR–/–) mice. Transfer of IL-4–/– RBHi T cells induced a similar type of colitis to that seen in SCID or TCR–/– recipients of wild-type (wt) RBHi T cells as reported previously. Interestingly, transfer of both wt and IL-4–/– RBHi T cells to TCR–/– but not to SCID mice induced inflammation in the gastric mucosa. Notably, TCR–/– recipients of IL-4–/– RBHi T cells developed a more severe gastritis with erosion, apoptosis of the antral epithelium, and massive infiltration of macrophages. This gastritis was partially dependent on the indigenous microflora. Recipients of both wt and IL-4–/– RBHi T cells developed duodenitis with multinuclear giant cells, expansion of mucosal macrophages, and dendritic cells. Full B cell responses were reconstituted in TCR–/– recipients of RBHi T cells; however, anti-gastric autoantibodies were not detected. We have now developed and characterized a novel model of chronic gastroduodenitis in mice, which will help in our understanding of the mechanisms involved in chronic inflammation in the upper gastrointestinal tract of humans.

A number of murine models of colitis involve either aberrant T cell or cytokine expression. For example, spontaneous colitis develops in T cell receptor (TCR)-{alpha} gene knockout mice where aberrant T cells producing Th2-type cytokines actually mediate disease.1-3 Perhaps the most useful murine model for both T cell and cytokine regulation of inflammatory bowel disease (IBD) has involved adoptive transfer of CD45RBHi T cells from normal mice to either SCID4-6 or to RAG 2 gene knockout (RAG–/–) mice.7,8 The transfer of RBHi T cells into SCID mice results in development of a severe mononuclear cell infiltration, epithelial cell hyperplasia, and tissue damage. Strong evidence has emerged provided that colitis results from enteric bacterial antigen (Ag)-driven Th1 cell induction,9 since RBHi T cell transfer into SCID mice with reduced bacterial flora failed to develop large bowel disease.10,11 Interestingly, T cell subsets producing TGF-ß12 and IL-1013 in the RBlow T cell population, when co-delivered with RBHi T cells, actually suppressed colitis. The RBlow T cell population was enriched in CD4+CD25+ T regulatory (Tr) cells.14 Despite this compelling evidence that RBHi T cells give rise to effector Th1 cells which mediate colitis, this model had not allowed study of either normal or abnormal B cell participation in the pathogenesis of CD4+ T cells. Since the majority of antibody forming cells (AFCs) in humans and higher mammals reside in the gastrointestinal (GI) tract mucosa,15,16 the impact of the presence of B cells and plasma cells in the mucosal pathology cannot be ignored. For example, we recently showed that dysregulated Th2 cells cause villus atrophy and goblet cell transformation in the small intestinal epithelium. Further, a wasting disease was seen and was mediated by excess IL-4 and the presence of B cells.17

Chronic GI tract inflammation is one of the most common types of inflammatory processes. For many years, gastric ulcers were thought to occur in susceptible individuals, especially those hypersecreting gastric hydrochloric acid.18,19 However, it has become clear that gastritis, peptic ulcer disease, and gastric cancer are the result of infection with the bacterium Helicobacter pylori.19,20 Gastritis has been reproduced in experimental animals gastrically infected with either H. pylori or related bacteria.21-24 Thus, gastritis and duodenitis are directly related to H. pylori infection; however, surprisingly little is known about what causes chronic inflammatory responses in the upper GI tract. Although many groups have shown that disease severity is related to certain virulence factors associated with H. pylori, only a minority of individuals infected with virulent strains of H. pylori develop severe disease.25 In animal models, IL-10-deficient mice infected with H. pylori exhibited enhanced gastritis, lower bacterial loads, and higher serum IgG antibody (Ab) titers when compared with control mice.26 These results have suggested the importance of the host immune system in perpetuating the gastritis and duodenitis. Indeed, a gastritis model in mice with H. pylori infection showed that CD4+ T cells were both necessary and sufficient for gastritis, and IFN-{gamma} contributed to this inflammation.27 Further, oral immunization of mice with the H. pylori urease induced protection against H. pylori infection, but was often associated with corpus gastritis, which is now recognized as post-immunization gastritis.28-30 In addition, H. pylori-infected SCID mice reconstituted with splenic T cells from H. pylori-infected, C57BL/6 mice developed severe gastritis, however, host colonization of H. pylori did not correlate to the severity of gastritis.31 Thus, cell-mediated immunity appears necessary for gastritis development, and does not necessarily relate to the bacterial load. Although still controversial, there is significant evidence that CD4+ Th1-type responses contribute to H. pylori-induced gastritis in humans.32 For example, IFN-{gamma} and TNF-{alpha} are the major cytokines produced by gastric T cells from H. pylori-infected subjects.33,34

In the murine colitis models induced by adoptive transfer of RBHi T cells, the microflora is necessary for disease; however, no priming of donor T cells with pathogens was required.10,11 Furthermore, no specific colitogenic bacterial species has been identified to date. Nonetheless, regulation of colitis by Tr cells seems to occur in both innate and adaptive immunity.35 These results indicate that T cells from normal mice have the potential to induce inflammation in response to the indigenous bacteria; however, the healthy host simultaneously develops Tr cells that attenuate the disease producing T cells. Similar mechanisms for upper GI tract inflammation may be present; however, there have been no reports of a gastritis model in the absence of infection or deliberate immunization, except for autoimmune gastritis induced by neonatal thymectomy36,37 or ionizing radiation.38

In this study, we hypothesized that normal T cells prepared from pathogen-free mice would contain a subset, which potentially could cause inflammation in the upper GI tract, as well as colitis. We describe a new type of RBHi T cell transfer model in TCR–/– mice that allows assessment of RBHi T cells in the presence of responsive B cells. We discovered that transfer of RBHi T cells from IL-4 gene knockout (IL-4–/–) mice resulted in gastroduodenitis in the absence of infection by pathogenic bacteria. Our results suggest the CD4+ Th1-type T cells mediate both colitis and gastroduodenitis in TCR–/– mice reconstituted with RBHi T cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice

Normal wild-type (wt), IL-4–/–, IFN-{gamma} gene knockout (IFN-{gamma}–/–) TCR ß and {delta} chain-defective (TCR–/–) and SCID mice on the C57BL/6 background were originally obtained from the Jackson Laboratory (Bar Harbor, ME). This mouse colony was maintained under pathogen-free conditions in flexible Trexler isolators at the University of Alabama at Birmingham (UAB) Immunobiology Vaccine Center Mouse Facility. A separate colony was also maintained in the Immunocompromised Mouse Facility of the Research Institute, International Medical Center of Japan (IMCJ, Tokyo, Japan). We periodically and extensively performed health surveillance on these colonies. These tests were performed in laboratories with expertise in laboratory animal health care (Jackson Laboratories, Charles River Laboratories, Wilmington, MA and Central Laboratories for Experimental Animals, Kawasaki, Japan). This analysis included serological testing for viral infections, bacterial cultures of the nasopharynx, stomach and cecum, ecto- and endoparasitic examinations, and histology of all major organs and tissues. Feces and the stomach were also tested for Helicobacter spp. in these laboratories, including H. hepaticus, H. bilis, H. muridarum, and "H. rappini", by PCR.39-42 No lesions or pathogens including Helicobacter spp. and intestinal parasites have ever been detected in these two separate mouse colonies. We obtained essentially identical results in the mouse facilities of both UAB and the IMCJ, and these experiments are quite reproducible. Although original reports indicated that TCR–/– mice develop intestinal inflammation,3 they did not develop histologically obvious gastritis, duodenitis, or colitis in our mouse facility until they were 24 weeks of age, which is well beyond the time frame of our experiments.

Purification of T Cells

Splenic T cells for adoptive transfer were purified as described previously.17 In brief, following lysis of erythrocytes, splenic T cells were enriched by passage through a nylon wool column, and then stained with phycoerythrin (PE)-conjugated anti-CD45RB (23G2), FITC-labeled anti-B220, anti-CD11b, and anti-CD8 monoclonal antibodies (mAbs) (BD PharMingen, San Diego, CA). The CD45RBHi T cell subset was separated by flow cytometry using a FACS Vantage (Becton-Dickinson Co., Sunnyvale, CA). Sorted, CD45RBHi subsets were > 99% pure by the reanalysis using anti-CD4 Ab, anti-TCR {gamma}{delta} Ab, anti-NK1.1 Ab, and anti-Gr-1 Ab.

Adoptive Transfer of T Cells

In these studies, purified populations of RBHi T cells were adoptively transferred to 6- to 8-week-old TCR–/– mice. We routinely transferred 1.0 x 106 T cells by the intravenous (i.v.) route. Body weight was monitored weekly, and mice were taken for analysis when their weight became less than 75% of the initial weight, or at 12 weeks after adoptive transfer of RBHi T cells.

Histological Analysis

The stomach was removed by excising the esophagus and the anal side of the gastro-duodenal junction, opened along the greater curvature, washed and extended. Next the stomach was cut longitudinally in the middle first, and a 3- to 4-mm-wide strip was prepared. The lateral side of this strip included the fundus, antrum, and gastro-duodenal junction. This side was cut for preparation of sections for histological examination. At least one such section from each mouse prepared in this manner was examined. Duodenal tissues were obtained from 0.5 cm to 3 cm from the gastro-duodenal junction. Colonic tissue was taken from the middle and distal parts of the large intestine. The tissues were opened, fixed in 5% glacial acetic acid in ethanol, and paraffin-embedded. Tissue sections (4 µm) were prepared and stained with hematoxylin and eosin (H&E). In some experiments, 6 µm-frozen sections were prepared, dried and fixed with cold acetone for 10 minutes, and subjected to histological analysis. The immunohistochemical staining was performed using FITC- or biotin-labeled anti-CD3, anti-CD4, anti-B220, anti-CD11b, and anti-CD11c mAbs, respectively (all from BD PharMingen, San Diego, CA), followed by FITC- or TRITC-labeled streptavidin (BD PharMingen). For the detection of IgA+, IgG+ or IgM+ plasma cells, sections were reacted with biotin-labeled anti-mouse IgA Ab, TRITC-labeled anti-mouse IgG Ab and FITC-labeled anti-mouse IgM Ab (BD PharMingen) followed by aminocoumarin-labeled streptavidin. Apoptotic cells were detected by TdT-mediated dUTP nick end labeling (TUNEL) using the Apoptosis in Situ Detection Kit Wako (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) on paraffin-embedded sections, according to the vender’s protocol. To detect myeloperoxidase in granulocytes, sections were directly incubated with a substrate solution, 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) in the presence of H2O2. Histological scores for gastritis were based on a summation of scores for cell infiltration (0, none; 1, moderate; 2, severe), epithelial hypertrophy (0, none; 1, moderate; or 2, severe), erosion (0, none; 1, focal; or 2, diffuse), giant cells (0, none; 1, less than 2; or 2, more than 3 per section), and deformity of pits (0, none; 1, moderate; or 2, severe). Histological scores for duodenitis and colitis were determined by severity of the inflammation (0, none; 1, mild; 2, moderate; or 3, severe). For histological examination, two observers performed all of the scoring. One was a pathologist who was not involved in this research, and for this examiner, the samples were provided in blinded fashion.

Purification of Lamina Propria Lymphocytes

Peyer’s patches were excised from the intestinal wall, and small intestinal lamina propria lymphocytes (LPLs) were prepared as described previously.43 Briefly, the intestinal tissue was treated with 1 mmol/L EDTA in phosphate-buffered saline (PBS) for 20 minutes to remove the epithelium. The tissue was then digested with type V collagenase (Sigma) for 20 minutes, and this step was repeated once more. The mononuclear cells were further purified by using a discontinuous Percoll gradient of 75% and 40%.

ELISPOT Assay

Enumeration of antibody forming cells (AFCs) was performed as described previously.44 In brief, 96-well nitrocellulose plates (Millititer HA, Millipore, Bedford, MA) were coated with goat anti-mouse Ig (H+L) [Southern Biotechnology Associates (SBA), Birmingham, AL]. After blocking with 1% bovine serum albumin (BSA) in PBS, the LPLs were incubated for 4 hours at 37°C in moisturized atmosphere of 5% CO2 in an incubator. After washing the plates, the captured Ig was visualized using peroxidase-labeled anti-mouse IgG, IgA or IgM (SBA). The spots in the individual wells were counted with the aid of a stereomicroscope.

Quantification of Plasma Immunoglobulin Levels

Plasma immunoglobulin (Ig) levels were determined by a sandwich ELISA using the combination of anti-mouse Ig (H+L) (SBA) and peroxidase-labeled anti-mouse IgG, IgA, or IgM (SBA) as described previously.17

RT-PCR for mRNA Analysis

Total RNA was prepared from gastric tissue or from separated cells using an RNA-Bee RNA isolation reagent (Tel-Test, Inc., Friendswood, TX). Complementary DNA was synthesized from RNA by reverse transcription (RT). The PCR primers for murine GAPDH used were 5'-AGCCAAACGGGTCATCATCTC and 5'-TGCCTGCTTCACCACCTTCTT; for TNF-{alpha}, 5'-TTCTGTCTACTGAACTTCGGGGTCATCGGTCC-3' and 5'-GTATGAGATAGCAAATCGGCTGACGTGTGCC. The step-cycle program was set for denaturing at 95°C for 45 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 45 seconds for a total of 40 cycles. Expression of mRNA was assessed by quantitative PCR using a SYBR Green PCR Master Mix (Applied Biosystems, Warrington, United Kingdom) and ABI PRISM 7700 Sequence Detector (Applied Biosystems). Quantification of mRNA for IFN-{gamma} and IL-10 was performed using ABI Taqman probes (Applied Biosystems). The PCR primers for IFN-{gamma} were 5'-TGATCCTTTGGACCCTCTGA and 5'-GCAAAGCCAGATGCAGTGT, for IL-10, 5'-GCTCTTGACTACCAAAGCCAC and 5'-CATGCCAGTCAGTAAGAGCAGG. The Taqman probes used for IFN-{gamma} were 5'-CCTCCTGCGGCCTAGCTCTGAGAC and for IL-10, AAGAGAGCTCCATCATGCCTGGCTCA. Threshold cycle numbers (CT) were determined with Sequence Detector Software (version 1.7; Applied Biosystems) and transformed using the {Delta}CT/{Delta}{Delta}CT method as described by the manufacturer, with GAPDH used as the calibrator gene.

Quantification of Cytokine Production and Intracellular Cytokine Analysis

For quantification of cytokines, CD4+ T cells were purified from freshly prepared LPLs by staining with FITC-labeled anti-CD4 mAb (BD PharMingen) followed by flow cytometry with a FACS Vantage system. The purified CD4+ T cells were added to wells of plates coated with anti-CD3 mAb (10 µg/ml, clone 145–2C11; BD PharMingen) and cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), sodium pyruvate, L-glutamine, HEPES, and 50 µmol/L 2-mercaptoethanol (complete medium). Cultures were incubated for 48 hours at 37°C in 5% CO2 in a moist air incubator. Cells in culture were removed, separated by centrifugation, and the supernatants were then subjected to a cytokine-specific ELISA, as described previously.43 Briefly, microtiter plates were coated with mAbs to individual cytokines and blocked with 3% BSA in PBS at 37°C for 2 hours, and then diluted samples were added to well and incubated overnight at 4°C. Captured cytokines were detected using biotinylated detection mAbs and peroxidase-labeled anti-biotin mAb (Vector Laboratories Inc., Burlingame, CA). The following mAbs were used for coating and detection, respectively: anti-IFN-{gamma}, R4–6A2 and XMG 1.2; anti-IL-2, JES6–1A12 and JES6–5H4 mAbs; anti-IL-4, BVD4-1D11 and BVD6–23G2. For intracellular cytokine analysis, LPLs were cultured in plates coated with anti-CD3 mAb in complete medium with anti-CD28 mAb. After 48 hours of culture, the cells were subjected to intracellular cytokine staining as described previously.45 In brief, cells were stained with FITC-labeled anti-CD4 mAb (BD PharMingen), fixed, permealized, and then stained with PE-labeled anti-IFN-{gamma} mAb (BD PharMingen) for analysis using flow cytometry.

Treatment with Antibiotics and Bacterial Culture

In some experiments, recipient mice were given a combination of antibiotics in their drinking water. Metronidazole (0.6 g/L, Wako Pure Chemical Industries Ltd.), neomycin (0.35 g/L, Wako), streptomycin (0.2 g/L, Wako), and bacitracin (0.35 g/L, Wako) were added to the drinking water, and the mice were continuously given this water ad libitum until the histological and cytological analyses were performed. To determine total colony forming units (CFU), mice were treated with antibiotics as above for 6 weeks, fasted for 6 hours and then the entire stomach was removed, washed three times with PBS, minced and homogenized with 2 ml of PBS in a Teflon-glass homogenizer. This homogenate was then subjected to serial dilution and spread over culture plates. Anaerobic bacteria were cultured on GAM agar plates (Nissui Pharmaceuticals, Tokyo, Japan) in culture jars (GasPack System BBL, Becton Dickinson) with AneroPac (Mitsubishi Gas Chemical Co, Inc., Tokyo, Japan). For aerobic cultures, brain-heart infusion agar (Difco, Becton Dickinson), chocolate agar, and sheep blood agar plates (both from Nissui) were used. To examine for the presence of indigenous microflora in the oral cavity, the mucosal surfaces of oral cavity were swabbed, and the swabs were placed in 300 µl of PBS. Then 100 µl of this suspension was spread over a culture plate. Anaerobic cultures were initiated within 20 minutes after taking samples. After 24 hours of incubation at 37°C, numbers of CFU were enumerated.

Statistics

The results were compared by the Mann-Whitney test using the Statview II statistical program (Abacus Concepts, Berkeley, CA) adapted for Macintosh computers. The results were considered to be statistically significant if P values were less than 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Inflammation of the GI Tract in TCR–/– and SCID Mouse Recipients

Previous studies have shown that the transfer of RBHi T cells resulted in severe colitis in either SCID or RAG–/– mouse recipients, and the colitis was mediated by CD4+ Th1-type cells.4-6,9,46 A similar type of colitis was induced in TCR–/– recipients of wt RBHi T cells. Recipients of IL-4–/– RBHi T cells also developed a severe colitis (Figure 1) . We noted that TCR–/– recipients of either wt or IL-4–/– RBHi T cells developed a duodenitis with a heavy cell infiltration, which was rarely described in SCID recipients previously. These changes were limited to the duodenum within 5 cm of the pylorus, and both the jejunum and ileum remained normal. We also found that these mouse recipients developed an inflammation of the stomach, especially in the antral region. When recipients of wt, IL-4–/–, or IFN-{gamma}–/– RBHi T cells were compared, recipients of IL-4–/– T cells developed the most severe gastritis and those mice receiving IFN-{gamma}–/– T cells showed only minimal changes (Figure 1) . Interestingly, SCID recipients of either wt or IL-4–/– RBHi T cells showed mild lesions in the stomach, with only slight cell infiltration. Adoptive transfer of wt or IL-4–/– CD45RBlow T cells did not result in gastritis, duodenitis, or colitis (data not shown).



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  		Figure 1. Histological scores of gastritis, duodenitis and colitis. TCR–/– or SCID mouse recipients were given RBHi T cells prepared from splenocytes of wt (blank column, n = 8), IL-4–/– (solid column, n = 10) or IFN-{gamma}–/– (hatched column, n = 7) mice. The results shown are the mean and SEM. *, Difference between wt and IFN-{gamma}–/– mice was statistically significant.

 
Pathological Features of Gastritis

In all 10 TCR–/– recipients of IL-4–/– RBHi T cells, the gastric antral and duodenal mucosa were increased in thickness and exhibited an overall turbid appearance. In five recipients of IL-4–/– RBHi T cells, the gastric fundic area was also edematous, and erosions with petechiae were seen (Figure 2, A and B) . Histological examination revealed that the inflammation was accompanied by hypertrophy of glands and elongation of pits, and these changes were most evident at the gastro-duodenal junction (Figure 2, C to F) . Most typically, the antral glands were elongated more than twofold longer than their normal length with pit dilatation in some parts, and mononuclear cell infiltration in the recipients of wt and IL-4–/– RBHi T cells was seen (Figure 2G) . In addition to these findings, recipients of IL-4–/– RBHi T cells developed surface erosions with multinuclear giant cell infiltration (Figure 2H) . Neutrophil infiltration was not frequent in the stomach or the duodenum as shown in the histochemical reaction of myeloperoxidase (Figure 2, K and L) . In 50% of IL-4–/– T cell recipients, a fundic inflammation with mononuclear cell infiltration and deformity of glandular pits with cells migrating into the dilated pits were seen (Figure 2J) , while the other 50% of mice showed only mild changes (Figure 2I) . In both cases, destruction specific for parietal cells was not seen. In the inflamed stomach, a massive infiltration of CD4+ T cells occurred (Figure 3B) , an event scarcely seen in SCID recipients (Figure 3A) . Furthermore, B cell aggregates were also detected by staining with anti-B220 mAb (Figure 3C) . We also assessed the presence of plasma cells in the inflamed areas; however, no IgG-, IgA- or IgM-containing cells were seen in the gastric mucosa by immunostaining (data not shown). The infiltrating cells were mostly macrophage-like cells as shown by staining with anti-CD11b Ab (Figure 3D) . On the other hand, there was no expansion of CD11c+ dendritic cells in the inflamed stomach (data not shown). Since surface erosion was enhanced in the gastric mucosa of recipients of IL-4–/– RBHi T cells, we also assessed apoptosis. In recipients of wt RBHi T cells, although there was elongation of glands and a cell infiltration, apoptotic cells were rare in the epithelium (Figure 3E) . In contrast, in recipients of IL-4–/– RBHi T cells, the numbers of apoptotic cells were clearly increased. Apoptosis was readily detected in the infiltrating cells in all layers of the mucosa and especially in the surface epithelium (Figure 3, F and G) .



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  		Figure 2. Macroscopic and histological features of murine gastritis. A: Normal stomach tissue taken from naïve TCR–/– mice. B: Inflamed stomach from TCR–/– mice after adoptive transfer of IL-4–/– RBHi T cells. C: The gastroduodenal junction of naïve TCR–/– mice. D: The gastroduodenal junction of the SCID recipients given IL-4–/– RBHi T cells. E: The gastroduodenal junction of the TCR–/– recipients given IL-4–/– RBHi T cells. F: The inflamed gastroduodenal junction with erosion. G and H: Antral gastritis with surface erosion and elongation of pits of TCR–/– recipients of IL-4–/– RBHi T cells. Arrows indicate a multinuclear giant cell. I: The fundic lesion with mild changes was taken from the same specimen as shown in G. J: Cell infiltration and dilated pits including migrating cells in the surface of the gastric fundic region in TCR–/– recipients of IL-4–/– RBHi T cells. K: Histochemistry for peroxidase of the antral region TCR–/– recipients of IL-4–/– RBHi T cells. As positive control, a section from a mouse with colitis induced by dextran sulfate with neutrophil infiltration was used (L). Sections were counterstained with methylgreen. Images were captured using a x4 objective lens (C, D and E), x20 lens (G, I, K, and L) or x40 lens (F, H, and J).

 


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  		Figure 3. Immunohistochemistry and detection of apoptosis. CD4+ T cells in the antrum of a SCID recipient of IL-4–/– RBHi T cells (A). CD4+ T cells (B), B220+ cells (C), and CD11b+ cells (D) in the inflamed antrum of TCR–/– mouse recipients of IL-4–/– RBHi T cells. Apoptotic cells were detected by TUNEL in TCR–/– recipients of wt RBHi T cells (E) or IL-4–/– RBHi T cells (F and G). Images were captured using a x20 objective lens (A-F) or x100 lens (G).

 
Pathological Features of Duodenitis

Duodenal inflammation was seen in TCR–/– mouse recipients of both wt and IL-4–/– RBHi T cells. The villi and crypts were both remarkably elongated, and the villi were dilated due to the cell infiltration (Figure 4, B and C) . In the lamina propria, multinuclear giant cells were frequently seen; however, granulomas were absent (Figure 4D) . Immunohistological analysis revealed an infiltration of CD4+ cells (Figure 4E) , expansion of dendritic cells and an infiltration of macrophages (Figure 4F) , which were not seen in naive TCR–/– mice.



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  		Figure 4. Histological features of murine duodenitis. A: H&E staining of duodenum of TCR–/– mice without cell transfer. B: Inflamed duodenum of TCR–/– recipients of wt RBHi T cells. C and D: Inflamed duodenum of TCR–/– recipients of IL-4–/– RBHi T cells. Dilated villus of duodenal tissue was stained with anti-CD4 (green, E), and anti-CD11b (green) and anti-CD11c (red) mAbs (F). Some CD11c+ cells also express CD11b. G: IgA AFCs in the duodenum of recipients of wt RBHi T cells. H: Peyer’s patches from recipients of wt RBHi T cells stained with anti-B220 (green) and anti-CD3 (red) mAbs. I: Peyer’s patches of TCR–/– mice without T cell transfer. Images were captured using a x10 objective lens (A-C), x20 (E), or x40 lens (D, F–I).

 
Production of Cytokines

To assess cytokine production by T cells that infiltrated the mucosa of adoptive hosts, CD4+ T cells were isolated from LPLs of the small intestine. The CD4+ T cells recovered from recipients of wt or IL-4–/– RBHi T cells released both IFN-{gamma} and IL-2 (Figure 5A) . It should be noted that T cells from the recipients of wt RBHi T cells also produced high levels of IL-4 (Figure 5A) . We also noted that T cells infiltrating the gastric mucosa produced IFN-{gamma} in the TCR–/– recipients of IL-4–/– T cells (Figure 5B) , although T cells from recipients of IL-4–/– T cells tended to contain slightly more IFN-{gamma} producing cells than recipients of wt RBHi T cells, as determined by flow cytometry (Figure 5B) . Thus, IFN-{gamma} release by T cells recovered from recipients of wt or IL-4–/– RBHi T cells were, for the most part, comparable. However, the lack of IL-4 secretion resulted in a remarkable phenotype, which contributed to the distinct and severe gastritis in TCR–/– recipients of IL-4–/– RBHi T cells. Further, to prove that the milder gastritis in SCID than in TCR–/– recipients of IL-4–/– RBHi T cells, we quantitatively assessed TNF-{alpha}, IFN-{gamma}, and IL-10 by RT-PCR from total RNA extracts taken from the gastric antrum. Expression of IFN-{gamma} and TNF-{alpha} in TCR–/– recipients was higher than those seen in SCID recipients (Figure 5C) .



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  		Figure 5. Production of cytokines by CD4+ T cells infiltrating the GI tract. A: CD4+ T cells were isolated from the small intestine of TCR–/– recipients of wt (blank column) or IL-4–/– (solid column) RBHi T cells and stimulated with anti-CD3 mAb for 48 hours. Culture supernatants were subjected to a cytokine ELISA. Values shown are the mean of three experiments obtained from pooled cells from three mice in each group. The results shown are the mean and SD. The difference was statistically significant. B: Production of IFN-{gamma} by infiltrating cells. LPLs were prepared from the inflamed gastric mucosa of TCR–/– recipients of IL-4–/– (left) or wt (right) RBHi T cells, stimulated with anti-CD3 and anti-CD28 mAbs for 48 hours, and subjected to intracellular cytokine analysis. C: Relative expression of mRNA for cytokines in the stomach in TCR–/– and SCID recipients of IL-4–/– RBHi T cells determined by quantitative RT-PCR. Total RNA was extracted from the antral mucosa, and mRNA for individual cytokines and GAPDH were analyzed. Based on the average {Delta}CT value of four SCID recipients, data from individual mice were shown as relative expression to SCID mice. Results from four mice are shown and the results indicated as (+) are the average of relative expression for each group. *, Statistically significant difference from SCID recipients.

 
Mucosal and Systemic B Cell Responses in TCR–/– Recipients of RBHi T Cells

It should be noted that TCR–/– recipients of either wt or IL-4–/– RBHi T cells exhibited IgA-positive cells in the small intestine, including the duodenum (Figure 4G) . Peyer’s patches were also reconstituted with distinct T and B cell zones (Figure 3H) , which were filled with B220+ cells in TCR–/– mice before T cell transfer (Figure 3I) . The plasma Ig levels were also elevated following adoptive transfer of either wt or IL-4–/– RBHi T cells. The IgG levels were comparable in both groups; however, the IgA levels were lower in recipients of IL-4–/– RBHi T cells than recipients of wt RBHi T cells (Figure 6A) . The numbers of AFCs in the small intestine in TCR–/– recipients were also reconstituted by adoptive transfer of RBHi T cells. Numbers of IgG and IgA secreting cells were much less frequent in TCR–/– recipients of IL-4–/– T cells than those mice given wt T cells (Figure 6B) . Thus, systemic IgG responses were fully reconstituted in both groups of mice; however, recipients of IL-4–/– T cells exhibited little class switching to the IgG or IgA isotypes. Plasma from these mice did not contain autoreactive Abs when assessed by the binding capacity to sections prepared from the stomach of naïve mice.



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  		Figure 6. Reconstitution of systemic and mucosal Ig production. A: Plasma Ig levels in TCR–/– recipients of wt RBHi T cells (blank column), IL-4–/– RBHi T cells (solid column) or naïve TCR–/– mice (shaded column). B: Numbers of IgG, IgA, or IgM secreting cells in the small intestinal LPLs isolated from TCR–/– recipients of wt (blank column), IL-4–/– RBHi (solid column) or naïve TCR–/– mice (hatched column) was determined by ELISPOT assay. Values shown are the mean and a SD of each group containing 4 to 7 mice.

 
Gastritis Was Dependent on the Presence of a Microflora

To this point, we found that gastritis was induced in the TCR–/– recipients of RBHi T cells without infection of pathogenic bacterial strains. However, the indigenous microflora in the upper GI tract or bacteria in ingested food may play a role in the induction of gastritis. To clarify this, we gave mice neomycin, streptomycin, bacitracin, and metronidazole in their drinking water following transfer of IL4–/– RBHi T cells. This treatment essentially eliminated the indigenous flora in the oral cavity and the stomach (Table 1) , and efficiently suppressed the gastritis. Interestingly, the effect on gastritis was efficient but only partial, whereas colitis was completely blocked in these mice (Figure 7) . These results indicated that induction of gastritis was partially dependent on the indigenous microflora, while colitis essentially required its presence.


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  		Table 1. Total Viable Aerobic and Anaerobic Counts of the Whole Stomach and the Oral Mucosa of Antibiotic-Treated and Nontreated TCR–/– Mice (or mean CFU ± SD of 3 mice)

 


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  		Figure 7. Treatment of recipient mice with antibiotics. A preparation of IL-4–/– RBHi T cells was transferred to eight TCR–/– mice (including two sets of male littermates in each experimental group). Four of these mice were given a combination of antibiotics in their drinking water (blank squares), and after 10 weeks histological scores were compared with control mice without treatment (solid squares).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have established a new model for gastritis that is induced by adoptive transfer of RBHi T cells incapable of production of IL-4 (Th1-prone T cells). The gastritis and duodenitis develops in the absence of a particular bacterial pathogen, such as Helicobacter spp. and is also distinct from autoimmune models of gastritis. The first major finding was that this pathogen-free gastritis developed after adoptive transfer of IL-4–/– RBHi T cells into TCR–/– mice. Gastritis did not occur after transfer of IL-4–/– RBHi T cells into SCID mice, clearly suggesting a requirement for B cell responses for full-blown gastric inflammation. Finally, adoptive transfer of IL-4–/– RBHi T cells yielded greater mucosal damage when compared with TCR–/– recipients of wt RBHi T cells. Each of these significant new findings is discussed in more detail below.

Numerous studies have attempted to establish in vivo models for gastric inflammation following infection with H. pylori. In our model, we successfully induced gastritis without bacterial infection or specific immunization. Of course, several models for autoimmune gastritis are induced in the absence of pathogens. Autoimmune disease induced by thymectomy36,37 or ionizing radiation38 have both resulted in gastritis. These types of gastritis were associated with damage of parietal cells or loss of parietal and chief cells by autoantibodies to the gastric H+/K+-ATPase.47,48 A similar type of autoimmune gastritis occurs spontaneously in C3H/He mice.49 "Autoimmune gastritis" and "colitis induced in SCID/Rag2–/– recipients of RBHi T cell transfer" are both caused by the absence of CD4+CD25+ regulatory T cells, because gastritis was induced in nu/nu mice recipients of CD25– T cells prepared from CD25+ cell-depleted mice.50 However, these two models have not been compared with each other very often. Importantly, autoimmune gastritis induced by neonatal thymectomy was not dependent on a microflora.51 Autoimmune gastritis was seen in germ-free mice with similar severity of inflammation, and autoantibody levels were comparable to those seen in conventional mice. In contrast, as shown in Figure 7 , our gastritis model was dependent, in a significant way, on the microflora. Further, no anti-parietal cell autoantibody could be detected. Besides a requirement for a microflora and the absence of autoantibody, our model is different from autoimmune gastritis in several important ways. For example, although neonatal thymectomy caused gastritis as well as autoimmune oophoritis, orchitis, thyroiditis, pancreatitis, and prostatitis,37,52 colitis has not been described previously. In contrast, the colon was a major organ affected by RBHi T cell transfer in SCID/RAG2–/–/TCR–/– recipients, which requires the presence of a microflora. Further, in autoimmune gastritis, lesions are limited to the gastric corpus, and parietal cell destruction was the main histological feature. On the other hand, our model showed more severe inflammatory changes in the antral mucosa. Since other autoimmune disease models such as spontaneous gastritis in MRL-lpr mice were also independent of a microflora or infection,37,52 it seems that different subsets of T regulatory cells are affected in autoimmune models and RBHi T cell-induced colitis/gastritis models. Thus, our model is quite distinct from autoimmune gastritis reported previously.

TCR–/– recipients of wt or IL-4–/– T cells developed gastroduodenitis as well as colitis; however, no obvious changes in the jejunum or ileum were seen. This anatomical localization suggests the possible involvement of lumenal foreign antigens in the development of this type of inflammation. Colitis induced in SCID or RAG2–/– mice by adoptive transfer of RBHi T cells does not occur in the absence of an indigenous flora.10,11 Through extensive testing for Helicobacter spp., no pathogenic strains (including Helicobacter spp.) were detected in mice, which developed gastritis and duodenitis. In addition, neutrophil infiltration, which generally indicates bacterial infection, was not seen in the stomach or the duodenum in our model. On the other hand, TCR–/– recipients of IL-4–/– RBHi T cells developed a milder form of gastritis when they were treated with antibiotics. Of note, colitis was totally blocked in this group of mice. These results indicate that gastritis was partially dependent on an indigenous microflora, while colitis essentially required its presence. We speculate that orally ingested microbes or indigenous microflora in upper GI tract, in addition to food antigens, which have not been fully degradated in the stomach or duodenum, may play a similar role in this type of inflammation.

Another novel aspect of our model is the use of TCR–/– mice, as opposed to SCID or RAG–/– mice. Thus, TCR–/–mice lack T cells but have a fully responsive B cell repertoire. Indeed, after adoptive transfer of RBHi T cells, TCR–/– mice exhibit increased plasma IgG levels, and AFCs were seen in the mucosal tissues. The fact that transfer of RBHi T cells resulted in colitis and duodenitis but not gastritis in SCID mice clearly suggests the involvement of B cells in the pathogenesis of gastritis. In this regard, a different colitis model has also shown that B cells play protective roles from inflammation.53 However, no AFCs were seen in the inflamed stomach tissues themselves, although small B cell aggregates were detected. Further, mucosal IgA production and IgA AFCs were actually lower in TCR–/– recipients of IL-4–/– RBHi T cells when compared with recipients of wt RBHi T cells, despite the more significant gastritis which characterize IL-4–/– RBHi T cell recipients. On the other hand, plasma IgG levels in IL-4–/– RBHi T cell recipients were comparable to recipients of wt RBHi T cells. The role of B cells and Ab production in our model certainly needs to be further investigated. We speculate that the presence of B cells and antibody production increases the sensitivity of T cells to be triggered into an inflammatory expansion as well as the activation of macrophage-type cells. Our results suggest that TCR–/– recipients were more sensitive than SCID recipients and the former mice would be able to fully respond to the foreign antigens in the upper GI tract. To increase this sensitivity, T/B cell interactions are likely involved, although this interaction may not necessarily occur in the local mucosa but could occur in any lymphoid tissue, such as spleen or mesenteric lymph nodes. In the case of H. pylori infection, it is known that the presence of anti-H. pylori Abs are not required for the exclusion of bacteria,53 but rather the Abs are actually involved in the pathogenesis of gastritis in humans54 and mice due to antigenic mimicry.55 Although we could not detect autoantibodies against the gastric parietal cells or H+/K+-ATPase, the presence of elevated Ig levels of particular idiotypes could play a role in the gastritis. In this regard, for our model of gastritis in the absence of infection or immunization, one could implicate molecular mimicry between any bacterial LPS and host blood group determinants56-59 as an attractive hypothesis, although there is no direct evidence that this phenomenon actually occurs.

There is now clear evidence for a central role of T cell-mediated immunity in gastric inflammation. In the RBHi T cell transfer model described here, Th1-type cytokine production was required for the induction of colitis and gastroduodenitis, since transfer of IFN-{gamma}–/– RBHi T cells resulted in much milder gastroduodenal inflammation (Figure 1) and colitis.17 On the other hand, it is well known that IL-4 suppresses cytokine gene expression induced by IFN-{gamma} and IL-2 in murine peritoneal macrophages.60,61 The severe tissue damage in recipients of IL-4–/– RBHi T cells was likely caused by enhanced IFN-{gamma} and IL-2 production by IL-4–/– T cells due to their predisposition toward a Th1-phenotype. However, the levels of IFN-{gamma} release by isolated T cells were comparable in these mice. These quantitative differences in Th1 cytokines may not fully explain the fact that the epithelial cell apoptosis and surface erosion was much more frequent in IL-4–/– than in wt RBHi T cell recipients. Since IL-4 is a cytokine that has pleiotropic effects on a variety of cell types, including epithelial cells and other non-hematopoietic cells, a lack of IL-4 production by infiltrating T cells may have an impact on tissue repair in addition to a cytokine imbalance. Gastritis induced by infection with pathogenic H. pylori in IL-4–/– mice was more severe than that in IFN-{gamma}–/– mice.62 In a rat acute gastric ulcer model, healing was accompanied by a rapid rise in tissue IL-4 levels.63 IL-4–/– mice were more susceptible to the colitis induced by administration of trinitrobenzene sulfonic acid, and tended to develop focal but penetrating ulcers, which were not frequently seen in IFN-{gamma}–/– mice.64 It is also known that fibroblasts express the IL-4 receptor, and Th2-type cells activate lung fibroblasts with resultant increase in deposition of collagen and fibronectin.65,66 In the airway or ileal epithelium, IL-4 induces mucin gene expression67 and goblet cell metaplasia.17,67 Thus, IL-4 may be significantly involved in the epithelial cell turnover and tissue protection required for the maintenance of the gastrointestinal tract architecture, in addition to its role as a mediator for allowing immunological homeostasis in the gut.

In summary, we have established a novel murine model for the upper gastrointestinal tract, which does not require pathogen infection or deliberate immunization. The inflammation was mediated by Th1-type immune responses restricted to a particular subset of T cells isolated from normal mice. This model also points to the significance of the host immune system in gastric lesions and should be of importance to help better understand the pathophysiology of chronic gastroduodenitis seen in H. pylori infection of humans.


   Acknowledgements
 
We thank Dr. Teruo Kirikae, Research Institute, International Medical Center of Japan, for his expert advice for bacterial culture.


   Footnotes
 
Address reprint requests to Taeko Dohi, M.D., Ph.D., Department of Gastroenterology, Research Institute, International Medical Center of Japan, 1–21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: dohi{at}ri.imcj.go.jp

Supported by U.S. Public Health Service National Institutes of Health Grants DK 44240, AI 18958, AI 43197, DE 12242, AI 35932, and DC 04976, and by grants and contracts from International Health Cooperation Research, the Ministry of Health, Labor, and Welfare, the Ministry of Education, Culture, Sports, Science, and Technology; the Japan Health Sciences Foundation and Organization; and The Organization for Pharmaceutical Safety and Research.

Accepted for publication June 21, 2004.


   References
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 Results
 Discussion
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