1The Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC 3010; 2Department of Medicine, University of Melbourne, Western Hospital, Melbourne, VIC 3011; 4Monash Institute of Reproduction and Development and Australian Research Council Centre of Excellence in Biotechnology and Development, Monash University, Melbourne, VIC 3168, Australia; and 3Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, MI 481090622
Submitted 5 November 2003 ; accepted in final form 20 May 2004
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ABSTRACT |
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Reg genes; gastrin; gastric inflammation
The pathological features of autoimmune gastritis are more complex than a simple depletion of the parietal cells that are the direct cellular target of the T cell infiltrate. Rather, we have demonstrated that there is a disruption of the normal cellular homeostatic mechanisms in the gastric mucosa, resulting in the accumulation of elevated numbers of immature cells and thus pronounced epithelial hypertrophy (15). In addition, there is a block in production of parietal and zymogenic cells. At present, it is unclear what factors drive this breakdown in cellular homeostasis in gastric mucosa.
The units (glands) of the gastric mucosa contain populations of proliferating cells that are precursors of terminally differentiated cells. These self-renewing immature cells are required to generate end-stage cells during ontogenic development and to replace mature cells as they rapidly turn over in the units of adult animals (18). In addition, proliferative rates of immature cells are increased in damaged mucosa, presumably to aid in regeneration. A number of factors has been demonstrated to cause proliferation of immature gastric mucosal cells. Peptides encoded by the gastrin gene (gastrins) are potent stimulators of gastric mucosal cell proliferation (7). Gastrin levels are elevated in mice (21, 31) and humans (31) with autoimmune gastric disease and therefore may be involved in the hypertrophy observed in these diseases. Gastrins do not act by binding directly to proliferating immature cells of the gastric mucosa, because these cells do not express gastrin/CCKB receptors (7). Rather they probably induce enterochromaffin-like (ECL) cells and parietal cells to release factors that act on immature cells (7, 20). These hormones may include members of the EGF family, transforming growth factor- (TGF-
), heparin-binding EGF (HB-EGF), and amphiregulin, which are secreted by parietal cells (2, 29), and the Reg proteins (11).
Reg proteins are a recently defined family of growth factors. To date, most work has focussed on the RegI molecule. RegI is expressed by ECL and zymogenic cells in the human gastric mucosa (14) but only by ECL cells in the rat (4). Fuikui et al. (11) demonstrated that RegI induces proliferation of gastric epithelial cells and plays a role in the proliferative response mediated by gastrin. RegI is also induced in ECL cells by the neutrophil-induced chemokine CINC-2 in stress-induced gastric injury (19). Other members of the Reg family, namely the RegIII subfamily, are also expressed in the gastric mucosa (1). At present, there is no information on the roles of RegIII proteins in gastric mucosal biology. Here, we report that RegIII proteins levels are greatly induced in inflamed gastric mucosa, which suggests that they may play a role in stimulating gastric epithelial cell proliferation.
In this paper, we examined a number of factors that may be involved in causing gastric hypertrophy in mice with autoimmune gastritis. Gastrin was examined because, as indicated above, its levels are elevated in autoimmune gastritis, and we had recently shown that gastric mucosal hypertrophy in mice deficient in the gastric H+-K+-ATPase -subunit was completely dependent on elevated gastrin levels (9). Furthermore, the availability of gastrin-deficient mice allowed us to functionally determine its role in gastritis. We found that the pathological features of autoimmune gastritis in mice deficient for gastrin were identical to that normally found. We also determined whether levels of other growth factors known to induce gastric proliferation were increased. We found that expression of the Reg genes, in particular, were greatly elevated and that this increase was independent of gastrin.
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MATERIALS AND METHODS |
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Induction and analysis of autoimmune gastritis. Autoimmune gastritis was induced by thymectomy of mice 3 days after birth under cold anesthesia as described previously (3).
Autoantibodies to porcine H+-K+-ATPase present in mouse sera were detected using an ELISA as described previously (3). For histological assessment, stomachs from 12- to 17-wk-old mice were washed in PBS, fixed in 10% buffered formalin, and embedded in paraffin wax. Sections (4 µm) were cut, dewaxed, cleared, and stained in hematoxylin and eosin. Cell types of the gastric mucosa were identified by their morphological characteristics as previously described (15). For quantitative purposes, five sections from each stomach at least 30 µm apart were analyzed. From each section, the lengths of four randomly chosen gastric units were measured using Image-Pro software (Media Cybernetics). Only complete oxyntic units were analyzed.
Immunohistochemical detection of mitosis. Stomachs from mice were washed in PBS, fixed in 10% buffered formalin, and embedded in paraffin wax. Dewaxed and cleared sections (4 µm) were incubated in DAKO antigen retrieval solution (DAKO S1700) at 98°C for 25 min. Sections were allowed to cool for 20 min before incubation with an anti-PCNA specific antibody (DAKO M0879; 0.48 mg/l), followed by a streptavidin-horseradish peroxidase complex (Amersham 140169). Bound horseradish complex was detected by incubation with PBS containing 0.05% diaminobenzidine and 0.03% nickel chloride for 11 min. Sections were then counterstained for 30 s with haematoxylin.
RNA analysis.
cDNA fragments of RegI, amphiregulin, HB-EGF, EGF receptor, TGF-, and the housekeeping gene ribosomal protein L32 (rL32) were generated by RT-PCR using the following primer pairs: RegI (forward) 5'-CATCCTGCTCTCATGCCTGATC-3' (reverse) 5'-GAAGCAAGAATGTCTCTCCAGG-3', RegII (forward) 5'-ATTATTGATTTAGAATTTAAA-3', (reverse) 5'-GAGTTCTGCACATCTGTTC-3', RegIII
(forward) 5'-CAAATCCTATCATAAAGCAGT-3' (reverse) 5'-AGGGGAAGGAGATGGATGAAA-3', RegIV (forward) 5'-TCATCTCCATCGAAAGAGGAA-3' (reverse) 5'-TTCATCTCAGCGCAATGCC-3', amphiregulin (forward) 5'-ATGAGAACTCCGCTGCTACCGC-3' (reverse) 5'-ATAACGATGCCGATGCCAATAG-3', HB-EGF (forward) 5'-CATATGACCACACTACAGTCT-3' (reverse) 5'-CTTGAACACATAGCTTCTCAG-3', EGF-R (forward) 5'-GAAGTGGTCCTTGGGAACTT-3' (reverse) 5'-TTGCGGATGCCATCTTCTTTCC-3', TGF-
(forward) 5'-GCACCCTGCGCTCGGAAGAT-3' (reverse) 5'-TCTGGGATCTTCAGACCACT-3', rL32 (forward) 5'-AACCCAGAGGCATCGACAACA-3' (reverse) 5'-GAACACAAAACAGGCACACA-3'. PCR fragments were purified using Qiagen (QIAquick) PCR purification kit (catalog no. 28104). RegIII
and RegIII
probes were generated from I.M.A.G.E. consortium clones purchased from Incyte genomics. Purified cDNA (100 ng) was labeled using random primers and [
-32P]dCTP as previously described (24). Unincorporated
-32P was removed by using NucTrap (Stratagene). RNAs from pancreas and duodenum, tissues that express all Reg family members, were included in blotting experiments.
Total RNA was extracted from the gastric fundus of 12-wk-old unmanipulated and day 3-thymectomised heterozygous and gastrin-deficient mice using TRIzol (Invitrogen Life Technologies) following the manufacturer's instructions. A total of 20 µg of RNA in glyoxyl sample buffer (BioWhittaker Molecular Applications) was electrophoresed in 1% agarose/20 mM 3-(N-Morpholino)propanesulfonic acid gel and transferred to nitrocellulose membrane (Hybond) before crosslinking by ultraviolet irradiation at 150 mJ. Membranes were hybridized for at least 2 h with 32P-labeled cDNA (2 x 106 counts· min1·ml1) using RapidHyb solution (Amersham Biosciences) as per the manufacturers instructions. Blots were washed in 20.5% SSC/0.1% SDS at 65°C and exposed to X-ray film at 80°C. To correct for RNA loading and transfer, membranes were stripped and rehybridized with a complementary cDNA probe to rL32. X-ray films were scanned, and band intensities were measured using ImageMaster 1D software (Amersham-Biosciences). Relative expression units were derived by dividing the band intensities obtained from using the individual cDNAs by the band intensities for rL32.
In situ hybridization using digoxigenin-labeled cRNAs was used to localize RegIII mRNA in mouse stomach sections using procedures previously described (23) with hybridization and washing temperatures at 55°C. cRNAs were made by transcription of mouse RegIII
PCR products generated from a plasmid kindly provided by Prof. Hiroshi Okamoto (Tohoku University, Miyogi, Japan). Both antisense and sense cRNAs were used on each sample in every experiment for each set of conditions tested. All experiments were performed with the approval of the University of Melbourne Animal Experimentation Ethics Committee.
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RESULTS |
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These data indicate that autoimmune responses and the incidence of gastritis were not altered by the presence or absence of gastrin.
Pathological features of autoimmune gastritis do not require gastrin.
We have previously characterized the mucosal lesion of mice with autoimmune gastritis in some detail (15). In that study, we found that normal gastric mucosal cell development was disrupted and the predominant pathological feature was an amplification of immature cell types that resemble undifferentiated gastric mucosal stem cells, which resulted in a pronounced gastric mucosal hypertrophy. In addition, the numbers of parietal and zymogenic cells were severely decreased. In a minority of gastric units, large cells with cytoplasms packed with mucous granules, so called "mucus-rich" cells (15), were the most numerous cell type instead of immature cells. The presence of a mononuclear cell infiltrate is also a hallmark of this disease. Stained sections of the stomach of thymectomized wild-type and gastrin-deficient mice were examined for these features of autoimmune gastritis (Fig. 2). All of these features were also observed here in both the gastritic wild-type and gastritic gastrin-deficient mice. As previously observed, the gastric mucosa of unmanipulated wild-type and gastric-deficient mice is indistinguishable by light microscopy (Fig. 2, A and B) (9). In mice of both genotypes with autoimmune gastritis, gastric mucosal hypertrophy was observed (Fig 2, C and D), and in most gastric units, this was the result of large numbers of immature gastric epithelial cells (Fig. 2, E and F). Few parietal and zymogenic cells were observed, and there was an accompanying mononuclear infiltrate in the submucosa extending into the mucosal lamina propria (Fig. 2, C and D). In a minority of gastric units, mucus-rich cells were prevalent (Fig. 2, G and H). In some mice (17% in both genotypes), the gastric mucosa was infiltrated with mononuclear cells, but hypertrophy was not observed (Fig. 2, I and J).
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Analysis of expression of factors potentially involved in regulating gastric epithelial cell growth.
To search for other molecules that may induce proliferation of gastric mucosal cells in mice with autoimmune gastritis, we measured expression of gastric growth factors by RNA hybridization experiments. Gastrin-deficient mice were thymectomized on day 3, and at 12 wk of age, stomachs were taken. For comparison, littermates with one normal and one mutant gastrin allele (gastrin heterozygote) were examined [gastrin-heterozygote mice have normal levels of plasma gastrin (10)]. A portion of the fundus was used to isolate RNA, and the remainder was used for histological analysis. Stomachs were classified into three groups: 1) nongastritic, displayed no signs of gastric infiltrate with normal epithelial architecture; 2) gastritic/nonhypertrophic, displayed a submucosal mononuclear cell infiltrate but no mucosal cell hypertrophy (only for gastrin-heterozygote mice); and 3) gastritic/hypertrophic, had mononuclear infiltrate and significant epithelial hyperplasia. RNA was then electrophoresed, transferred to membrane, and hybridized to cDNAs corresponding to members of the Reg protein family RegI, RegII, RegIII, RegIII
, RegIII
, and RegIV and members of the EGF family TGF-
, HB-EGF, and amphiregulin and the EGF receptor.
We observed no significant variation in the levels of mRNAs encoding RegII, RegIII, RegIV, TGF-
, HB-EGF, or the EGF receptor for any of the samples (data not shown). The levels of RegII, RegIII
, RegIV mRNAs were very low in all samples.
Representative autoradiograms of the RNA blots and quantitation of hybridization that displayed significant changes between the groups are shown in Fig. 6. Consistent and very large increases were observed in the RNA samples derived from the hypertrophic stomachs of both the gastrin-deficient mice and their heterozygous littermates for the mRNAs encoding RegIII (average values increased 31- and 45-fold for gastrin heterozygotes and gastrin-deficient mice, respectively) and RegIII
(average values increased 49- and 62-fold for gastrin heterozygotes and gastrin-deficient mice, respectively). RegI levels were also increased, although not to the same extent (average values increased 16- and 4.2-fold for gastrin heterozygotes and gastrin-deficient mice, respectively).
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The levels of mRNAs encoding the factors were very similar in the nongastritic mice and in the mice with gastric mononuclear cell infiltrate but no hypertrophy (gastrin heterozygote gastritic/nonhypertrophic). Hence, it appears that elevated levels of Reg and amphiregulin are associated with hypertrophy and not gastric inflammation per se.
Analysis of cells expressing RegIII in inflamed gastric mucosa.
To discover which cells in the gastric mucosa of gastritic mice express RegIII, we hybridized tissue sections with cRNAs corresponding to the sequence of RegIII
(Fig. 7). Hybridization with sense cRNA resulted in negligible staining (Fig. 7A). Antisense cRNA hybridized to a large number of epithelial cells (Fig. 7B). The cells expressing the RegIII
mRNA appeared to be small immature cells, as judged by their morphology and position in the gastric units (Fig. 7C). The location of cells binding the RegIII
cRNA was similar to the location of cells that stained with PCNA (Fig. 5), supporting their assignment as immature cells. Another prominent cell population in the units of mice with autoimmune gastritis is mucus-rich cells (15). These cells did not appear to express RegIII
mRNA (Fig. 7D).
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DISCUSSION |
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Previous investigations (21) had indicated that gastrin levels were elevated in mice with autoimmune gastritis. Hence, we examined the contribution of this hormone to the pathological features of autoimmune gastritis by using gastrin-deficient mice. When gastrin-deficient mice were thymectomized on day 3, we found that both the incidence of mononuclear cell infiltrate and anti-H+-K+-ATPase autoantibodies, two hallmarks of autoimmune gastritis, were very similar to those of wild-type mice of a similar genetic background (Fig. 1). Hence, gastrin is not required for the development of an autoimmune response to gastric autoantigens and development of chronic disease.
Gastrin was also not required for the pathological features observed in mice with autoimmune gastritis. Gastrin-deficient mice had the same degree of gastric mucosal hypertrophy, as measured by an increase in gastric mucosal unit length, as wild-type animals (Fig. 3). Anti-H+-K+-ATPase antibody levels were also quantitated (Fig. 4), and no differences were found between mice of the two genotypes. Gastric mucosal hypertrophy in wild-type mice with autoimmune gastritis is primarily the result of the accumulation of abnormally high levels of immature epithelial cells (15) that accumulate due to increased levels of cellular proliferation. The gastric mucosae of gastrin-deficient gastritic mice also had increased numbers of small immature cells (Fig. 2), and an elevated number of cells in mitosis was detected (Fig. 5). In a minority of gastric units from mice with autoimmune gastritis, large mucus-rich cells accumulate (15) that resemble the recently described spasmolytic polypeptide-expressing metaplasia lineage (32). These cells also appeared in gastrin-deficient mice (Fig. 2). Finally, gastrin-deficient mice also displayed depletion of parietal and zymogenic cells, another feature of autoimmune gastritis (Fig. 2). From these analyses, we conclude that gastrin does not contribute to the pathological features of autoimmune gastritis. This contrasts with our recent data (9) in another system of gastric mucosal hypertrophy, the H+-K+-ATPase -subunit-deficient mouse, in which the severe hypertrophy was entirely gastrin dependent. Clearly, various pathways involving different mitogenic stimuli may lead to disruption of gastric epithelial cell homeostasis.
Ligands of the EGF receptor have been implicated in inducing gastric cell proliferation and function. We found that the EGF receptor ligands TGF- and HB-EGF mRNA levels were not significantly elevated in mice with autoimmune gastritis. Hence, we believe it is unlikely that these molecules are responsible for the observed hypertrophy. Levels of another EGF receptor ligand amphiregulin were increased by 3.7- and 2.5-fold in two of three gastrin-heterozygote hypertrophic gastritic mice but were not elevated in one mouse. This relatively small and variable increase suggests that this hormone may play only a minor role in hypertrophy observed in this system. The increase in amphiregulin levels in gastrin-deficient gastritic/hypertrophic mice was greater than in the gastrin-heterozygote gastritic/hypertrophic mice (average values increased 2.1-fold in gastrin heterozygotes and 5.5-fold in gastrin-deficient mice). This suggests that gastrin may negatively regulate release of this hormone in inflammatory situations or that amphiregulin levels are increased to compensate for a lack of gastrin. It has been suggested that parietal cells are primarily responsible for the production of amphiregulin (2). Because parietal cells are severely depleted from mice with severe autoimmune gastritis, it would appear likely that other cell types are also responsible for its production in this inflammatory situation.
RegI was initially described as an inducer of pancreatic islet cell proliferation. Subsequently, several members of the Reg family have been identified in humans, rats, and mice with 4070% identity between family members (1, 25). Analysis of the mouse genome has identified four Reg subfamilies (1, 13). RegI (pancreatic stone protein) and RegII are the only members of their subfamilies. The RegIII subfamily has four members: RegIII, RegIII
, RegIII
, and RegIII
. RegIII
, RegIII
, and RegIII
appear to have similar tissue distributions and are expressed at significant levels in the gastrointestinal tract. RegIII
, on the other hand, appears to be expressed only in the pancreas (1). RegIV is also expressed throughout the gastrointestinal tract (13). Recently, a Reg-like protein with
45% identity to Reg proteins has also been identified, which is also expressed in the gastrointestinal mucosa (17).
To date, little emphasis has been placed on examining the role of members of the Reg family apart from RegI in the gastric mucosa. We examined expression of RegI, RegII, RegIII, RegIII
, RegIII
, and mRNA. RegII, RegIII
, and RegIV mRNAs were very low to undetectable in stomachs from all mouse groups. In contrast to this were the levels of RegIII
and RegIII
mRNAs, which were increased 30- to 60-fold in all of the gastritic/hypertrophic mice. RegI mRNA levels were more modestly increased (4- to 16-fold). Importantly, we also examined mice that had a mononuclear cell infiltrate but had not developed hypertrophy. In these mice, the level of the Reg mRNAs were not significantly different from mice without gastritis, indicating a strong correlation with hypertrophy and increased Reg levels rather than just with the presence of a mononuclear infiltrate. These data were the first demonstration of the induction of RegIII
and RegIII
mRNAs in inflamed gastric mucosa, and they suggest that these genes may play a significant role in the pathological manifestations of autoimmune gastritis, whereas other Reg family members, namely RegII, RegIII
, and RegIV are unlikely to be involved. It is also noteworthy that the genes for the RegIIIs can be regulated independently despite their close proximity.
In situ hybridization with a RegIII cRNA demonstrated that an abundant population of epithelial cells in the gastritic mucosa expressed the RegIII mRNA. These cells were small and were positioned immediately below the pit cell region in the area containing cells that stained with the mitosis associated-antigen PCNA. Thus these cells are likely to be immature cells. Immature cells are the most abundant cell type in the mucosa of gastritic mice; hence, elevated expression in this cell population is consistent with the abundance of the RegIII transcripts. Expression of the RegIII genes in the rapidly proliferating immature cells of the mucosa raises the possibility that proliferation is driven by an autocrine loop.
This work has demonstrated that gastrin is not involved in the pathological features of autoimmune gastritis and that members of the Reg family are strong candidates to stimulate the proliferation of immature cells. Importantly, this study has shown that gastrin is not essential for the production of Reg proteins. Recently, it was demonstrated that cytokines associated with inflammation, such as CINC-2 (19), TGF-
, and interferon-
are able to induce production of RegI and RegIII (pancreatitis-associated protein I) (8) in rats. Previously, it was shown (22) that the inflammatory infiltrate in stomachs of mice with autoimmune gastritis produces a number of cytokines including interferon-
. It has also been shown (5) that interferon-
neutralization is able to prevent the development of autoimmune gastritis. Hence, it is likely that in a chronic inflammatory situation such as autoimmune disease or chronic bacterial infection (e.g., Helicobacter pylori), the continuous stimulation of production of Reg family members leads to hyperproliferation and hypertrophy of the gastric mucosa. It will now be important to precisely define the role of Reg genes in normal development of the gastric mucosa and pathological conditions such as inflammatory conditions and gastric carcinoma (16).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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