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The Journal of Immunology, 2001, 166: 2090-2099.
Copyright © 2001 by The American Association of Immunologists

Local Transgenic Expression of Granulocyte Macrophage-Colony Stimulating Factor Initiates Autoimmunity1

Mark Biondo, Zeyad Nasa, Aiden Marshall, Ban Hock Toh and Frank Alderuccio2

Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mechanisms leading to breakdown of immunological tolerance and initiation of autoimmunity are poorly understood. Experimental autoimmune gastritis is a paradigm of organ-specific autoimmunity arising from a pathogenic autoimmune response to gastric H/K ATPase. The gastritis is accompanied by autoantibodies to the gastric H/K ATPase. The best characterized model of experimental autoimmune gastritis requires neonatal thymectomy. This procedure disrupts the immune repertoire, limiting its usefulness in understanding how autoimmunity arises in animals with intact immune systems. Here we tested whether local production of GM-CSF, a pro-inflammatory cytokine, is sufficient to break tolerance and initiate autoimmunity. We generated transgenic mice expressing GM-CSF in the stomach. These transgenic mice spontaneously developed gastritis with an incidence of about 80% after six backcrosses to gastritis-susceptible BALBc/CrSlc mice. The gastritis is accompanied by mucosal hypertrophy, enlargement of draining lymph nodes and autoantibodies to gastric H/K ATPase. An infiltrate of dendritic cells and macrophages preceded CD4 T cells into the gastric mucosa. T cells from draining lymph nodes specifically proliferated to the gastric H/K ATPase. CD4 but not CD8 T cells transferred gastritis to nude mouse recipients. CD4+ CD25+ T cells from the spleen retained anergic suppressive properties that were reversed by IL-2. We conclude that local expression of GM-CSF is sufficient to break tolerance and initiate autoimmunity mediated by CD4 T cells. This new mouse model should be useful for studies of organ-specific autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Autoimmune gastritis, the underlying cause of pernicious anemia in humans, is an organ-specific autoimmune disease associated with loss of parietal and zymogenic cells in the gastric mucosa (1, 2). Human gastritis is characterized by circulating autoantibodies to the {alpha}- and {beta}-subunits of the gastric H/K ATPase and to intrinsic factor. Experimental autoimmune gastritis (EAG)3 induced in susceptible mouse strains (3) is similar to human autoimmune gastritis, making it a relevant model to study organ-specific autoimmunity (1). EAG can be initiated in BALB/c mice by lymphopenia; the best characterized being that induced by neonatal thymectomy (4). EAG can also be initiated by immunization with autoantigen (5) and develops spontaneously in 30% of C3H/He mice (6). EAG is characterized by a chronic inflammatory infiltrate in the gastric mucosa with loss of parietal and zymogenic cells. It is also associated with autoantibodies to the {alpha}- and {beta}-subunits of the gastric H/K ATPase (7). Adoptive transfer and in vivo depletion studies have shown that EAG is a CD4+ T cell mediated disease (8, 9) with no role for CD8+ T cells (10). The early gastric lesion in EAG observed at 4 wk after neonatal thymectomy consists mainly of macrophages and CD4+ T cells (11). Transgenic (tg) mice expressing the gastric H/K ATPase {beta}-subunit in the thymus are resistant to the development of gastric autoimmunity. These studies suggest that an immune response to the H/K ATPase {beta}-subunit is necessary for disease induction (12, 13) although T cell responses have been shown to both the {alpha}- and {beta}-subunits of the H/K ATPase (14, 15, 16).

The mechanisms of disease induction in mouse models of EAG are unknown. A role has been suggested for regulatory CD4+CD25+ T cells in maintaining tolerance to autoantigens such as the gastric H/K ATPase (17, 18, 19, 20, 21). Sakaguchi and colleagues (18) have proposed that day 3 thymectomy prevented seeding to the periphery of thymic-derived CD4+CD25+ regulatory T cells. They have shown that normal splenocytes depleted of CD4+CD25+ T cells induce autoimmune gastritis when transferred to nude (nu/nu) mice (17, 22). Conversely, adoptive transfer of these regulatory T cells prevented autoimmunity induced by neonatal thymectomy or by adoptive transfer of pathogenic T cells (18). EAG induced by lymphopenia requires drastic manipulation of the immune system such as neonatal thymectomy. This limits their usefulness in studies aimed at understanding how tolerance in an intact immune system can be broken to initiate autoimmunity.

Our study was designed to address whether tolerance can be broken and gastric autoimmunity can be initiated in mice with an intact immune system by local expression of the pro-inflammatory cytokine GM-CSF in the stomach. We selected this cytokine for local tissue expression because we have previously identified GM-CSF in gastric lesions of mice with EAG (11) and because GM-CSF is a key cytokine required for proliferation and differentiation of not only granulocytes and macrophages but also of dendritic cells (23). Vaccination with GM-CSF strongly augments the immune response (24, 25). Too much GM-CSF can be lethal, because systemic tg expression of GM-CSF results in excessive accumulation and activation of granulocytes and macrophages (26, 27). Local GM-CSF expression has been shown to induce local inflammatory responses. For instance, mice infected with GM-CSF-expressing adenovirus have infiltrates of granulocytes and mononuclear cells in the lung (28). Tg expression of GM-CSF in GM-CSF deficient mice (29) corrects alveolar proteinoisis associated with the deficient mice (30). Mice deficient in GM-CSF have a marked reduction in incidence and pathology of collagen-induced arthritis (31), while administration of GM-CSF accelerates the onset and pathology of arthritis (32).

Here we report that local expression of GM-CSF in the stomach of gastritis-susceptible BALB/cCrSlc mice results in development of autoimmune gastritis associated with circulating parietal cell autoantibodies to the gastric H/K ATPase. These characteristics are identical with those of autoimmune gastritis induced by neonatal thymectomy, immunization, altered T cell repertoire, sublethal irradiation, and spontaneously in C3H/He mice (5, 6, 33, 34, 35).


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

BALB/c and (BALB/c x C57BL/6)F1 mice used for tg mice production and BALB/cCrSlc were maintained at Monash University Medical School animal facilities (Victoria, Austrailia). PC-GM-CSF tg mice were backcrossed to BALB/cCrSlc at least four times.

Parietal cell GM-CSF (PC-GMCSF) transgene construction

The transgene directing GM-CSF expression to parietal cells of the stomach was generated as follows. The gene encoding murine GM-CSF was isolated from pUC8 (27) by BamHI and EcoRI restriction enzyme digestion to release a fragment of ~2.6 kbp. This included a 310-bp HaeIII {Phi}X174 fragment that was originally cloned into the SspI site in the 3' untranslated region (27). This fragment was subcloned into the SpeI site of p11SV between 10.9 kbp of murine gastric H/K ATPase {beta}-subunit 5' untranslated region (36) and an SV40-derived polyadenylation signal. All fragments were blunt ended before ligation and orientation of the GM-CSF gene was determined by restriction fragment mapping. The 13.95-kbp transgene was isolated by NotI and XhoI restriction enzyme digestion and purified on a nucleic acid chromotography system 52 column (Life Technologies, Gaithersburg, MD).

Generation of PC-GM-CSF tg mice

Isolated transgene was resuspended in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA, at a concentration of 2–5 ng/µl, injected into pronuclei of fertilized (BALB/c x C57BL/6) x BALB/c oocytes and transferred to oviducts of psuedopregnant BALB/c mice according to the method of Hogan et al. (37). Tg founders were identified by PCR analysis of extracted mouse-tail DNA. Oligonucelotides 5'-CCT CAC ACA GAG GAG ACT A-3' and 5'-GTT AGA GAC GAC TTC TAC CTC TTC-3' were designed to generate a 350-bp fragment spanning the H/K ATPase {beta}-subunit promoter and the GM-CSF gene. Control PCR to confirm DNA integrity contained oligonucleotides 5'-CGA GCT CGA GCC TGC CTA TCT TTC AGG-3' and 5'-CGG GAT CCT AGT TGC AGT AGT TCT CCA G-3' designed to generate a 374-bp product from the mouse insulin gene. PCR was performed in 25-µl reaction volumes containing amplification buffer; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.1% gelatin; 200 µM dATP, dCTP, dGTP and dTTP; 50 pmols oligonucelotide primers; and 1.5 U Taq DNA polymerase (Life Technologies, Melbourne, Australia). Reaction mixtures were incubated at 95°C for 2 min and 30 cycles of 92°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with a final cycle at 72°C for 5 min. Fifteen-microliter samples of PCR product were separated by agarose gel electrophoresis and visualized using UV illumination. Images were captured by digital camera and inverted for publication.

Messenger RNA analysis

Transgene expression of GM-CSF mRNA was detected by RT-PCR essentially as previously described (13). Total RNA was isolated from lung, spleen, heart, liver, and stomach using the Ultraspec II RNA isolation kit (Biotecx Laboratories, Houston, TX). Briefly, 2 µg of total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) using oligo(dT) primer in a total volume of 20 µl. Two microliters of the reaction mixture was subjected to PCR using primers designed to amplify the tg GM-CSF or actin cDNA. Actin primers were 5'-ATGGATGACGATATCGCTG-3' and 5'-ATGAGGTAGTCTGTCAGGT-3' and generated a product of 568 bp. Tg GM-CSF was detected following two rounds of PCR. The first-round primers were sense 5'-CTATAAGCCCTAGAGGACGC-3' and anti-sense 5'-CCGCATAGGTGGTAACTTGTGTTTC-3' to generate a product of 400 bp. In the second-round PCR, 2 µl of PCR product were amplified using an internal anti-sense primer 5'-GGCAGTATGTCTGGTAGTAGCTGG-3' to generate a product of 275 bp. PCR was comprised of 1 cycle at 92°C for 2 min and 30 cycles at 92°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with a final cycle at 72°C for 5 min. PCR products were subjected to agarose gel electrophoresis and visualized by UV illumination. Images were captured by digital camera and inverted for publication.

ELISA, indirect immunofluorescence, and flow cytometry

Circulating anti-H/K ATPase autoantibodies were assayed by ELISA on 96-well plates coated with purified pig H/K ATPase as previously described (13). Anti-parietal cell autoantibodies were detected by indirect immunofluorescence on frozen or paraffin-embedded sections of normal mouse stomach (13). Gastric H/K ATPase {alpha}- and {beta}-subunit reactivity was detected by immunofluorescence reactivity with Sf9 cells infected with baculovirus encoding rat ATPase {alpha}- or {beta}-subunit. Recombinant baculovirus was obtained from E. Shevach (National Institutes of Health, Bethesda, MD). mAbs 1H9 and 2B6, reactive with the gastric H/K ATPase {alpha}-subunit and {beta}-subunit, respectively, were used as controls.

Immunohistochemistry was performed on frozen tissue sections with Abs reactive with CD4 T cells (FITC-anti-CD4; clone RM4-5), CD8 T cells (FITC-anti-CD8; clone 53-6.7), dendritic cells (FITC-anti-CD11c; clone HL3), macrophages (FITC-anti-CD11b; clone M1/70), B cells (anti-B220; clone RA3.3A1) and granulocytes (FITC-anti-Gr1; clone RB6–8C5). Sections were blocked with 1% normal swine serum for 10 min at room temperature and incubated with Ab for 60 min at room temperature. Sections were washed twice with PBS/1% Tween 20 for 5 min and mounted. To visualize parietal cells, sections were double stained together with biotinylated Dolichos biflorus (Sigma, St. Louis, MO; Ref. 38) followed by streptavidin-Texas Red. Sections were viewed with a Bio-Rad (Richmond, CA) confocal microscope.

For FACS analysis, 1–2 x 106 cells were stained in 30-µl volumes containing APC-anti-CD4 (clone RM4-5), PerCP-anti-CD8 (clone 53-6.7), PE-anti-B220 (clone RA3.3A1) and PE-anti-CD25 (IL-2{alpha}-chain, clone PC61) diluted in HBSS/1% FCS. Cells were analyzed on a FACScaliber using CellQuest software (Becton Dickinson, Mountain View, CA).

Histology

Tissues were fixed in 10% formalin in PBS and embedded in paraffin. Five-micrometer stomach sections were stained with hematoxylin and eosin and viewed by light microscopy. Gastritis was assessed by the presence of cellular infiltrate within the gastric mucosa. Destructive gastritis comprised the presence of cellular infiltrate within the gastric mucosa with destruction of the cells within gastric glands. Other tissues were also examined for the presence of pathology.

Gastric and liver membranes and purified gastric H/K ATPase

Purified porcine gastric H/K ATPase was prepared by tomato-lectin chromotography as previously described (39). Mouse gastric and liver membranes were prepared as follows. Tissues were homogenized in ice-cold sucrose buffer (0.25 M sucrose, 2 mM EDTA, 5 mM Tris (pH 7.5), 1 mM PMSF) with a polytron homogenizer (Kinematica, Lucerne, Switzerland). Samples were centrifuged at 360 x g for 10 min at 4°C to remove nuclei and cell debris. The supernatant was centrifuged at 5500 x g for 15 min at 4°C to pellet mitochondria. The supernatant was collected and centrifuged at 100,000 x g for 1 h at 2°C to pellet membranes. Membranes were resuspended in cold HEPES buffer (50 mM HEPES (pH 7.6), 1 mM EDTA, 1 mM PMSF) and protein concentration determined using micro BCA protein assay (Pierce, Rockford, IL). Samples were stored at -20°C. Presence of H/K ATPase in gastric membranes was confirmed by ELISA reactivity with mAbs 1H9 and 2B6 specific for gastric H/K ATPase {alpha}- and {beta}-subunits, respectively (40).

In vitro proliferation assay

Pooled single cell suspensions of lymphocytes from tg (n = 5) and non-tg (n = 6) littermates were prepared by gently grinding lymphoid tissues between frosted glass slides and used as responders in in vitro proliferation assays. Non-tg splenocytes were treated with ammonium chloride solution (0.9%) to lyse RBC and irradiated (3000 rads) for use as APCs. Cells were suspended in RPMI 1640 culture media supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 50 µM 2-ME. Proliferation assays were performed in 96-well tissue culture plates (Dynex Technologies, Chantilly, VA) in a total volume of 200 µl containing 2.5 x 105 responder cells, 2.5 x 105 irradiated APCs, and Ag. For in vitro assay for CD4+CD25+ regulatory T cells, splenocytes were sorted into CD4+CD25+ and CD4+CD2- populations, with purity of 99% and 98%, respectively. Proliferation assay was performed in a total volume of 200 µl containing 2 x 104 responders, 5 x 104 irradiated APCs, and Ag. Con A was used at a final concentration of 3 µg/ml and IL-2 was used at 100 U/ml. Cells were incubated for 48 h at 37°C/10% CO2 followed by an additional overnight incubation in the presence of 1 µCi [3H]thymidine (NEN, Boston, MA). Cells were harvested onto glass filters (Skatron, Sterling, VA) suspended in scintillant and [3H]thymidine incorporation was determined on a Wallac 1205 Betaplate liquid scintillation counter (Pharmacia, Uppsala, Sweden). Control wells were comprised of responder cells alone, APCs alone or proliferation in the absence of Ag.

Cell transfer studies

Single-cell suspensions were prepared from pooled spleens, paragastric lymph nodes, inguinal lymph nodes, and stomachs (41) in HBSS/1% FCS. Two groups of PC-GMCSF tg (n = 6 and n = 3) mice with circulating parietal cell and H/K ATPase Abs and one group of non-tg (n = 3) littermates were used in these experiments. Splenocytes were treated with ammonium chloride solution (0.9%) to lyse RBC. Cells to be injected were washed and resuspended in HBSS in a total volume of 150–200 µl. Cells were transferred to BALB/c nu/nu mice by i.v. tail vein injection. Recipient mice received 4 x 107 splenocytes, 1–2 x 107 inguinal lymph node cells, 1.3–2 x 107 paragastric lymph node cells, or 5 x 106 stomach infiltrate cells. Mice were killed at 8–12 wk following cell transfer and sera were analyzed for H/K ATPase and parietal cell autoantibodies. Stomachs and other tissues were processed for paraffin-embedded sections and examined by histology for gastritis.

For transfer of purified CD4+ and CD8+ T cells, pooled paragastric lymph node cells were isolated in two separate experiments from six and five PC-GMCSF tg mice with circulating parietal cell and H/K ATPase Abs. Cells were stained with anti-CD4-PE and anti-CD8-FITC and sorted using a FACScaliber cell sorter (Becton Dickinson). Analysis of sorted populations revealed a purity for CD4+ and CD8+ T cells of 97% and 98%, respectively. Each BALB/c nu/nu recipient received 1.5–2 x 106 cells by i.v. injection. Eight weeks following transfer, mice were killed and examined as described above.

Statistical analysis

Cell numbers and populations were compared using a two-tailed t test. Cell transfer results were compared using a Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of PC-GMCSF tg mice

PC-GMCSF tg mice were generated by injecting into fertilized mouse embryos, a transgene composed of murine gastric H/K ATPase {beta}-subunit promoter and mouse GM-CSF gene (Fig. 1GoA). We have previously shown that expression of OVA or {beta}-galactosidase under the control of the same murine H/K ATPase {beta}-subunit promoter does not induce spontaneous autoimmune gastritis in mice (K. Scarff, unpublished observations). Tg mice were identified by PCR using primers spanning the H/K ATPase {beta}-subunit promoter and GM-CSF gene junction (Fig. 1Go, A and B). DNA integrity was assessed by PCR amplification of mouse insulin gene (Fig. 1GoB). Demonstration of GM-CSF expression was attempted by immunohistochemistry with no success (data not shown). RT-PCR was then used to demonstrate tg expression of GM-CSF in stomachs of PC-GMCSF tg mice. RNA isolated from tg and non-tg lung, spleen, heart, liver, and stomach was subjected to RT-PCR. The quality and integrity of the cDNA generated was assessed by PCR amplification of mouse {beta}-actin gene (Fig. 1GoC). Expression of tg GM-CSF was observed only in tg stomachs and not in other tg or non-tg tissues (Fig. 1GoC). Two rounds of PCR were required to visualize mRNA expression suggesting low levels of transgene mRNA or as previously observed, a short biological half-life of GM-CSF mRNA (30, 42).



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  		FIGURE 1. Generation and analysis of PC-GMCSF tg mice. A, The parietal cell GM-CSF transgene was constructed by ligating the murine GM-CSF gene to the 10.9-kbp H/K ATPase {beta}-subunit 5' nontranslated region. The black and open boxes indicate the GM-CSF exons and introns, respectively. The {phi}X174 fragment previously introduced into the gene (27 ) is indicated by the speckled box and the SV40-derived poly(A) signal by a striped box. NotI and XhoI sites were used to excise the transgene from the cloning vector and position of oligonucleotides used for PCR screening and RT-PCR analysis are indicated. B, PCR was used to discriminate between tg and non-tg (N-tg) mice. Oligonucleotides A and A' were designed to span the H/K ATPase {beta}-subunit and GM-CSF gene to amplify a product of 350 bp. DNA integrity was confirmed by PCR analysis of the mouse insulin gene and inclusion of positive (+ve) and negative (-ve) DNA controls. M, DNA markers. C, RT-PCR analysis was used to confirm expression of tg GM-CSF (Tg-GM-CSF) in the stomachs of tg mice. Total RNA was isolated from lung (L), spleen (Sp), liver (Liv), heart (H), and stomach (St) of tg and non-tg mice. mRNA was reversed transcribed and used in two rounds of PCR. Oligonucleotides B/B' were used in the first round followed by oligonucleotides B/B'' in the second reaction to produce a product of 275 bp. PCR of actin mRNA was used to confirm integrity of RNA.

 
PC-GMCSF tg mice develop spontaneous gastritis and H/K ATPase autoantibodies

Tg and non-tg mice backcrossed four times to gastritis-susceptible BALB/cCrSlc mice were examined at 12 wk of age for gastritis and autoantibodies to gastric parietal cells H/K ATPase. Six of 16 (38%, p = 0.02) PC-GMCSF tg mice developed gastric H/K ATPase reactive Abs assessed by ELISA, compared with 0/16 non-tg littermates (Fig. 2Go). ELISA-positive sera reacted by immunofluorescence with baculoviral {alpha}- and {beta}-subunits of the gastric H/K ATPase expressed in insect Sf9 cells. Reactivity of anti-H/K ATPase reactive sera with parietal cells was confirmed by indirect immunofluorescence (Fig. 3GoB) in which the staining pattern was identical with that observed with mAbs to gastric H/K ATPase (Fig. 3GoC). We noted that three sera that reacted with baculoviral Ags did not react with parietal cells or H/K ATPase by ELISA. This may reflect differences in sensitivity of the assays or in the Ags used. It is difficult to draw any conclusions because it is not known whether the mice in question may have gone on to develop gastritis if left for a longer period of time. Mice that displayed parietal cell and H/K ATPase autoantibodies had morphological and histological evidence of destructive gastritis. The gastritis was characterized macroscopically by mucosal hypertrophy (Fig. 3GoE) accompanied by dramatic enlargement of draining para-gastric lymph nodes (not shown) and microscopically by submucosal mononuclear cell infiltrate that extended into the lamina propria with destruction of parietal and zymogenic cells (Figs. 2Go and 3GoE). With further backcrossing of PC-GMCSF tg mice to BALB/cCrSlc mice, the incidence of gastritis increased to 57% (8/14) and 82% (9/11), respectively, in mice backcrossed five and six times. Non-tg mice did not develop circulating parietal cell autoantibodies or gastritis (Fig. 3Go, A and D). Histological examination of nongastric tissues including, heart, kidney, liver, and pancreas indicated that the inflammatory cellular infiltrate was confined to the gastric mucosa of PC-GMCSF tg mice (data not shown).



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  		FIGURE 2. Incidence of autoimmune gastritis in PC-GMCSF tg mice. Twelve-week-old tg and non-tg mice were analyzed for evidence of autoimmune gastritis by the following: 1) ELISA to detect the presence of H/K ATPase Abs on gastric H/K ATPase coated wells; 2) indirect immunofluorescence (IF) on frozen or paraffin-embedded normal mouse stomach sections to detect anti-parietal cell autoantibodies; 3) immunofluorescence staining of Spodoptera frugiperda cells infected with recombinant H/K ATPase {alpha}- or {beta}-subunit expressing baculovirus to detect reactivity with individual subunits. A filled box indicates reactivity. Gastritis was determined on paraffin-embedded stomach sections stained with hemotoxylin and eosin and assessed by the presence of mononuclear cell infiltrate and tissue destruction within the gastric mucosa and indicated by a filled box.

 


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  		FIGURE 3. Parietal cell autoantibodies and gastritis in PC-GMCSF tg mice. Parietal cell reactivity was assessed by indirect immunofluorescence on normal stomach sections. Non-tg mice (A) do not develop anti-parietal cell Abs, and in contrast to PC-GMCSF tg mice (B), display a strong parietal cell reactivity that is identical with the staining produced with mAbs 1H9 and 2B6 (C), which are specific for the gastric H/K ATPase (40 ). Hematoxylin- and eosin-stained stomach sections from non-tg mice (D) display normal gastric histology while PC-GMCSF tg mice reactive with gastric H/K ATPase develop mononuclear cell infiltrates (E) within the gastric mucosa (arrows) and cellular destruction within the gastric glands (arrow-heads). Bar, 100 µm.

 
Influx of dendritic cells and macrophages precedes CD4 T cells in the gastric lesion of PC-GMCSF tg mice

Frozen stomach sections from 4- and 8-wk-old PC-GMCSF tg mice with parietal and H/K ATPase autoantibodies and non-tg littermates were examined by confocal microscopy with Abs specific for CD4 T cells, CD8 T cells, B cells, macrophages, dendritic cells, and granulocytes (Fig. 4Go). Representative sections stained with hemotoxylin and eosin confirm hypertrophy and presence of mononuclear infiltrates within the gastric mucosa of PC-GMCSF tg mice (Fig. 4GoB) compared with non-tg littermates (Fig. 4GoA). Sections were double stained to visualize parietal cells and various cell surface markers specific for CD4+ T cells, CD8+ T cells, B cells (B220), dendritic cells (CD11c), macrophages (CD11b,) and granulocytes (Gr1). Firstly, we found that stomach sections from 8-wk-old non-tg littermates were not completely void of leukocytes (Fig. 4Go) with occasional staining of cells observed in some sections. This is consistent with our earlier findings in nonthymectomized mice in which occasional macrophages and lymphocytes were also observed in the gastric mucosa (11). In contrast, there was a profound difference in the staining pattern observed with 8-wk-old PC-GMCSF tg mice. There was a dramatic influx of CD4 T cells (Fig. 4GoE), dendritic cells (Fig. 4GoK), macrophages (Fig. 4GoN), and granulocytes (Fig. 4GoQ). However, it should be noted that CD11b can also be found on dendritic cells and granulocytes, and thus the staining observed in Fig. 4GoN may not be entirely associated with macrophages. In some sections, B cells were observed in follicle-like aggregates (Fig. 4GoT) similar to that observed in the neonatal thymectomy model of EAG. CD8 T cells were not present (Fig. 4GoH), which is also similar to the neonatal thymectomy model (11, 41) in which CD8 T cells do not appear to be implicated in the pathogenesis of EAG (10). To identify early cellular events associated with the initiation of autoimmunity, we examined stomachs of 4- (with H/K ATPase reactivity) and 2-wk-old tg mice. Immunohistochemical analysis of the stomachs of 4-wk-old PC-GMCSF tg mice showed the presence of a heterogeneous cellular infiltrate similar to that observed in the 8-wk-old group except that B cell follicles were not observed (data not shown). This indicates that autoimmune gastritis in this model is well established by 4 wk of age and, therefore, accelerated compared with the neonatal thymectomy model in which only a minority of mice display evidence of disease at 4 wk of age (11). However, examination of 2-wk-old tg mice did reveal a difference in the composition of the cellular infiltrate. Of the leukocyte markers examined, staining was observed only with CD11c and CD11b (Fig. 4Go, L and O) markers. No staining was observed for CD4 or CD8 T cells, B cells or granulocytes (Fig. 4Go, F, I, U, and R). Taken together, these observations support our hypothesis that in PC-GMCSF tg mice the first stage of autoimmune gastritis is associated with proliferation and activation of APCs, which then leads to activation and migration of pathogenic CD4 T cells into the gastric mucosa.



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  		FIGURE 4. Immunohistochemistry of stomachs from PC-GMCSF tg mice. Five-micrometer frozen stomach sections were prepared from 8-wk-old non-tg (A, D, G, J, M, P, S) and PC-GMCSF tg mice (B, E, H, K, N, Q, T) and 2-wk-old PC-GMCSF tg mice (C, F, I, L, O, R, U). Sections were stained with hemotoxylin and eosin (A–C) to visualize stomach morphology and confirm the presence of gastritis in tg mice. Parietal cells (stained red) were identified using the lectin, Dolichos biflorus, which specifically binds to carbohydrates on parietal cells (D–R) or human sera with parietal cell reactivity (S–U). Fewer parietal cell staining in the 2-wk-old Tg mice is due to incomplete development of the gastric mucosa in young mice (36 ). FITC-conjugated Abs were used to identify cell surface markers: CD4 (D–F), CD8 (G–I), CD11c (J–L), CD11b (M–O) and Gr1 (P–R). B220 surface Ag was identified using a biotinylated-anti-B220 mAb followed by streptavidin-conjugated Texas Red (S–U). Fluorescence images were captured on Bio-Rad confocal microscope with identical exposure times. Sections from 8-wk-old mice were captured with a x10 objective and 2-wk-old mice with a x20 objective.

 
T cells from PC-GMCSF tg mice proliferate to gastric membranes and H/K ATPase

Cell suspensions from paragastric lymph nodes were pooled from five H/K ATPase-reactive tg mice and from six nonreactive non-tg littermates. Cells were challenged in vitro with preparations of gastric or liver membranes or purified gastric H/K ATPase. T cells from tg mice responded specifically to gastric membranes and to H/K ATPase and not to liver membranes (Fig. 5GoA). Non-tg T cells did not respond to gastric or liver membranes or to gastric H/K ATPase (Fig. 5GoA). These observations suggest that the response to gastric membranes is likely to be directed to the gastric H/K ATPase. We next determined whether the T cell response was confined to cells isolated from the paragastric lymph node, or whether T cells isolated from other lymphoid organs can respond to gastric membranes. Cells were isolated from the spleen and from the paragastric, mesenteric, and inguinal lymph nodes. A proliferative response was observed only with T cells isolated from the paragastric lymph node (Fig. 5GoB). None of the cell preparations responded to liver membranes, included as controls (Fig. 5GoB). From these results it appears that PC-GMCSF T cells proliferate specifically to the H/K ATPase of gastric membranes and that the response is confined to cells from the paragastric lymph node.



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  		FIGURE 5. In vitro proliferation of PC-GMCSF T cells to gastric membranes and H/K ATPase. A, Paragastric lymph node cells were pooled from four PC-GMCSF tg and from six non-tg mice and proliferation was determined in response to gastric or liver membranes or purified H/K ATPase. In each well, 2.5 x 105 responder cells were incubated with 2.5 x 105 irradiated normal splenocytes as APCs and Ag at various concentrations. Cells were incubated for 48 h followed by overnight incubation with 1 µCi of [3H]thymidine. Each point represents the mean of duplicate wells. Control wells included responder cells alone (1400 cpm), APCs alone 141 cpm), and responders plus APCs in the absence of Ag (1269 cpm), with mean cpm indicated. B, Single-cell suspensions from spleen, paragastric, inguinal, and mesenteric lymph nodes were assessed for in vitro proliferation to gastric and liver membranes. Assay conditions were as described above. Controls included responder cells alone (600–966 cpm), APCs alone (369 cpm), and responders plus APCs in the absence of Ag (500–800 cpm) with mean cpm indicated.

 
CD4+ T cells from PC-GMCSF tg mice transfer gastritis

To determine whether PC-GMCSF tg mice can adoptively transfer gastritis, cells isolated from pooled tg and non-tg stomach, spleen, or paragastric or inguinal lymph nodes were transferred to BALB/c nu/nu mice in two separate experiments. Eight to 12 wk following transfer, recipient mice were killed, sera were assessed for H/K ATPase, and parietal cell autoantibodies and stomachs were examined for gastritis (Fig. 6Go). A destructive gastritis was observed in mice that received cells from tg spleen (1/2) (Fig. 6GoA), inguinal lymph nodes (3/5), paragastric lymph nodes (2/5), and stomach (1/1); the gastritis was associated with circulating autoantibodies to parietal cells demonstrated by indirect immunofluorescence (Fig. 6GoB). These observations indicate that pathogenic lymphocytes are not confined to the paragastric lymph node or stomach, but were present in all lymphoid sources examined. Cells from non-tg spleen (n = 1), inguinal lymph node (n = 1), and paragastric lymph node (n = 1) did not induce gastritis in nu/nu mice (data not shown). This is consistent with previous observations that cells from normal mice do not transfer gastritis (9, 20, 21). To determine whether the pathogenic cells were CD4+ or CD8+ T cells, CD4+ and CD8+ cells from pooled paragastric lymph nodes were sorted by flow cytometry and transferred to syngeneic nu/nu mice. Pooled data from two experiments indicate that mice transferred with CD4+ T cells develop gastritis with circulating autoantibodies to parietal cells (5/6) while mice transferred with CD8+ T cells remained disease free (0/4, p = 0.048) (data not shown). The finding that CD4+ T cells transferred autoimmune gastritis is similar to that observed in EAG induced by neonatal thymectomy (10), in which CD4+ T cells have been shown to be the pathogenic T cells responsible for disease.



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  		FIGURE 6. Transfer of autoimmune gastritis from PC-GMCSF tg mice to BALB/c nu/nu mice. Splenocytes from PC-GMCSF tg mice with parietal cell reactive autoantibodies were transferred i.v. to recipient BALB/c nu/nu mice. Eight-wk following transfer, mice were killed and assessed for parietal cell autoantibodies and histological evidence of gastritis. Nu/nu mice transferred with splenocytes from tg mice developed gastritis (A) with characteristic mononuclear cell infiltrate within gastric mucosa (arrow-heads) and cellular destruction within gastric glands (arrows) and autoantibodies (B) reactive with parietal cells by immunofluorescence and to H/K ATPase by ELISA (not shown).

 
Lymphocyte and CD4+CD25+ regulatory T cell populations are not perturbed in PC-GMCSF tg mice

The majority of mouse models of EAG result from an induced state of lymphopenia; be it neonatal thymectomy (43), repeated lymphoid irradiation (35), or tg skewing of the T cell repertoire (34). Therefore, total cell numbers and lymphocyte populations were analyzed in 6- to 8-wk-old PC-GMCSF tg to determine whether there were any differences in these parameters compared with non-tg mice. We found no difference in total number of cells recovered from the thymus (tg, n = 4, 1.48 ± 0.5 x 108; non-tg, n = 4, 1.62 ± 1.83 x 108; p = 0.89), spleen (tg, 1.79 ± 0.4 x 108; non-tg, 1.85 ± 0.31 x 108; p = 0.81), and inguinal lymph nodes (tg, 3.90 ± 2.74 x 106; non-tg, 2.86 ± 1.59 x 106; p = 0.54). Not unexpectedly, given the dramatic enlargement of the draining paragastric lymph node of tg mice, we observed a corresponding 10-fold increase in the total number of cells recovered from this site (tg, 5.53 ± 2.08 x 106; non-tg, 0.58 ± 0.19 x 106; p = 0.017). FACS analysis of thymocyte and splenocyte populations using CD4, CD8, and B220 markers revealed no difference in these populations (data not shown). These data indicate that the induction of autoimmune gastritis was not associated with alteration or skewing of T or B lymphocyte populations.

Thymic-derived CD4+CD25+ T cells have been implicated in the regulation of pathogenic CD4 T cells in mouse models of autoimmune gastritis (18, 19, 20, 21). Therefore, we examined for the presence of this regulatory population of cells in our tg mice. We found no difference in the proportions of thymic or peripheral populations of CD4+CD25+ cells of tg and non-tg mice (data not shown). To address whether there was any functional alteration in this population, we tested the in vitro proliferative and regulatory property of these cells (20, 44). CD4+CD25+ and CD4+CD25- cells were purified from pooled spleens of three gastritic PC-GMCSF tg mice. Although CD4+CD25- cells readily proliferated to Con A, CD4+CD25+ regulatory cells were resistant to Con A stimulation (Fig. 7Go). Furthermore, CD4+CD25+ regulatory cells inhibited proliferation of CD4+CD25- cells to Con A stimulation and this inhibition was abrogated by IL-2 (Fig. 7Go). These characteristics are similar to those previously described for this regulatory population (20, 44) and indicate that autoimmune gastritis in PC-GMCSF tg mice is not due to global perturbation of the CD4+CD25+ regulatory population.



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  		FIGURE 7. In vitro CD4+CD25+ regulatory T cell assay. CD4+CD25+ and CD4+CD2- cells from pooled splenocytes of three gastric PC-GMCSF tg mice were sorted by flow cytometry to 99% and 98% purity respectively (not shown). Each population was assayed in vitro for proliferative response to 3 µg/ml Con A in the absence or presence of 100 U/ml IL-2. The ability of CD4+CD25+ cells to prevent proliferation of CD4+CD2- cells to Con A stimulation was assessed in mixed culture assays with reducing proportion of CD4+CD25+ cells. Abrogation of CD4+CD25+-induced regulation was shown by addition of IL-2. Cells were incubated for 48 h followed by overnight incubation with 1 µCi of [3H]thymidine. Each point represents the mean of duplicate wells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EAG in mice is an animal model of human autoimmune gastritis, sharing the same target autoantigens, gastric pathology, and circulating autoantibodies to the {alpha}- and {beta}-subunits of the gastric H/K ATPase (1). The best characterized EAG models are those induced by lymphopenia (4). These models are of limited use for studies directed toward addressing how tolerance can be broken and autoimmunity initiated in animals with an intact immune system.

In this study, we generated tg mice that locally expressed GM-CSF in the stomach under the control of the gastric H/K ATPase {beta}-subunit promoter (36). PC-GMCSF tg mice spontaneously developed characteristics of autoimmune gastritis with an incidence increasing from 40% in mice backcrossed four times to ~80% in mice backcrossed six times to gastritis-susceptible BALB/c/CrSlc mice. Circulating autoantibodies to gastric parietal cells were generated that reacted with the {alpha}- and {beta}-subunits of the gastric H/K ATPase. Stomachs from these PC-GMCSF tg mice with parietal cell Abs displayed an inflammatory infiltrate in the gastric mucosa. The infiltrates extended into the lamina propria with accompanying destruction of mucosal parietal and zymogenic cells. The gastric hypertropy observed in PC-GMCSF tg mice is similar to that described in other models of EAG (8, 35, 43) and has been attributed to the replacement of parietal and zymogenic cells with proliferating epithelial stem cells (7). Lymphoid cells recovered from the draining paragastric lymph nodes of PC-GMCSF tg mice specifically proliferated in response to stimulation with gastric Ags and purified H/K ATPase. CD4, but not CD8, T cells transferred gastritis to syngeneic nu/nu mice, These observations confirm the immunological nature of the gastritis. These characteristics are identical with the autoimmune gastritis observed in mice following neonatal thymectomy (13, 33, 43, 45), suggesting that the immunopathology in both models are similar.

GM-CSF is a pleiotrophic cytokine that stimulates proliferation and maturation of macrophages and dendritic cells (23, 46, 47). In previous studies we have shown that GM-CSF is expressed in the gastric lesion of mice with EAG induced by neonatal thymectomy (11). A role for GM-CSF in autoimmune pathology has previously been suggested in collagen-induced arthritis in mice (31, 32). Although the exact mechanism by which GM-CSF expression induces autoimmune gastritis is not known, we suggest that local expression of GM-CSF transgene in the gastric mucosa has initiated an autoimmune response through activation of local APCs, most likely dendritic cells. In normal mice, immature APCs are implicated in the removal of apoptotic cells resulting from cellular turnover in the stomach (7); and in itself, this process would not be expected to result in activation of these APCs (48). In the presence of a pro-inflammatory cytokine such as GM-CSF, gastric APCs may be activated directly (49) or through the action of other induced cytokines such as TNF-{alpha} (50) and migrate to the local draining lymph node where they activate naive CD4+ T cells specific for the gastric H/K ATPase. The proposed effect of GM-CSF in enhancing dendritic cell activation and presentation has previously been exploited in designing vaccines against pathogens and tumors (24, 51, 52). The earlier influx of CD11c and CD11b reactive cells within the gastric mucosa of PC-GMCSF tg mice observed at 2 wk after birth preceding the influx of CD4 T cells observed at 4 wk supports a role for these APCs in the initiation of gastric autoimmunity. It is not known whether these cells are recruited to the gastric mucosa, arise from local proliferation or both. Similar findings have been observed following intramuscular injections with plasmids encoding GM-CSF (53). In these studies, expression of GM-CSF in muscle resulted in local accumulation of macrophages, dendritic cells and granulocytes but not CD4 T cells. The lack of CD4 T cells in the DNA vaccination study compared with our findings may reflect differences in the time span of the two experiments. The finding that an in vitro T cell proliferative response was only observed with T cells isolated from the local draining paragastric lymph node and not from other lymphoid organs supports the suggestion that activated APCs migrate to the draining lymph node to activate naive T cells homing to the lymph node. As with other models of autoimmune gastritis (8, 9, 10), the transfer and histochemical studies in this report suggest that CD4+ T cells are the pathogenic cells in EAG. Ag presentation and activation of T cells may also occur in the gastric mucosa, as has been suggested in animal models of diabetes (54). Certainly, we have observed organized lymphoid structures similar to those described by Ludewig and colleagues (54) in the gastric mucosa of mice with thymectomy-induced EAG (11) and also in the PC-GMCSF mice in the present study.

Local tg expression of other pro-inflammatory cytokines such as TNF-{alpha}, IFN-{gamma}, and IL-2 have previously been attempted to induce other models of organ-specific autoimmunity. For instance, expression of IL-2 or TNF-{alpha} in pancreatic islets of BL/6 mice resulted in insulitis without diabetes (55, 56), while in the NOD mouse, islet expression of TNF-{alpha} accelerated diabetes onset (50). These studies indicate that TNF-{alpha} expression in islets can promote local inflammation or aggravate diabetes in diabetes-prone mice. However, whether TNF-{alpha} can promote by itself a destructive autoimmune lesion is not clear. Although IFN-{gamma} expression in pancreatic islets resulted in diabetes, it is unclear whether this is a consequence of autoimmunity or the result of local destruction induced by IFN-{gamma} (57). In the present study, we show for the first time that expression of the pro-inflammatory cytokine, GM-CSF, in the gastric environment of gastritis-susceptible mice has induced autoimmune gastritis. This was confirmed by production of autoantibodies to the gastric H/K ATPase, a specific T cell response to the ATPase and transfer of disease by CD4 T lymphocytes from gastritic to nu/nu mice. These observations suggest that expression of GM-CSF in the local environment of the stomach is sufficient to break tolerance and initiate autoimmunity.

A defining feature of our study is that tolerance to gastric H/K ATPase has been broken without a major perturbation of the immune system, a feature associated with lymphopenic models of EAG (4, 58). EAG is not observed in normal BALB/c or BALB/cCrSlc mice. Experimental evidence suggests a role for regulatory CD4+CD25+ T cells in maintaining tolerance and that removal of this population from the normal repertoire renders the remaining lymphocytes pathogenic (17, 18). This is not the case in PC-GMCSF tg mice because we found CD4+CD25+ cells in the thymus and in the periphery of these mice. In vitro, we found that CD4+CD25+ T cells from the spleens of tg mice were anergic, could prohibit proliferation of CD4+CD25- cells and their anergy could be reversed by exogenous IL-2. These properties are identical with those previously described for CD4+CD25+ regulatory cells (20, 44). Therefore, in PC-GMCSF tg mice, it appears that local production of GM-CSF in the stomach has initiated a pathogenic autoimmune response, and overcome suppressor activity of CD4+CD25+ regulatory T cells. Our observation that cells from the draining paragastric lymph node can be specifically stimulated to proliferate in the presence of gastric Ags supports this suggestion. The observation that autoimmune gastritis can be transferred from lymphoid sites other than the draining lymph node seems to contradict this. This apparent discrepancy may reflect the ability of circulating activated autoreactive T cells to expand following transfer to an "empty" periphery in nu/nu mice. In vitro, CD4+CD25+ regulatory cells are naturally anergic and do not proliferate in response to stimulation through the TCR. However, the anergy and suppressive activity of CD4+CD25+ T cells can be abrogated by IL-2 coupled with TCR stimulation (20, 44). Therefore, it is possible that in PC-GMCSF tg mice, local IL-2 generated by activated self-reactive CD4 T cells in the paragastric lymph node may have rendered CD4+CD25+ regulatory cells nonfunctional. The presence of this regulatory population may also explain why not all PC-GMCSF tg mice develop autoimmunity. However, with the incidence of gastritis approaching 100% with increasing numbers of backcrosses to gastritis-susceptible BALB/c/CrSlc mice, local expression of GM-CSF alone may be sufficient to break tolerance and initiate autoimmunity in this mouse strain.

The PC-GMCSF tg mice generated in this study will permit us to address several important questions pertaining to the development of organ-specific autoimmunity. We have shown for the first time that local expression of a proinflammatory cytokine in the stomach of genetically susceptible mice can induce a damaging autoimmune response. Our observations support the recent findings of Sarvetnick and colleges (59) that bystander damage resulting from responses to "danger" signals (60, 61) may be sufficient to initiate autoimmunity. Taken together, these observations support our proposed genesis of the gastric lesion in autoimmune gastritis initiated by the gastric H/K ATPase (62). The induction of damaging autoimmunity in the presence of an intact immune system will make this model useful for understanding mechanisms associated with the breakdown of immune regulation in the local lymph nodes leading to autoimmunity. Understanding this process can be expected to lead to strategies designed to restore tolerance and/or arrest progression of damaging autoimmunity.


   Acknowledgments
 
We thank Dr. R. Lang for providing pUC8 plasmid encoding the mouse GM-CSF gene and Dr. E. Randle-Barrett and D. Pellicci for cell sorting.


   Footnotes
 
1 This work was funded by the National Health and Medical Research Council of Australia and the Alfred Hospital Healthcare Group. Back

2 Address correspondence and reprint requests to Dr. Frank Alderuccio, Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria, 3181 Australia. Back

3 Abbreviations used in this paper: EAG, experimental autoimmune gastritis; tg, transgenic; PC-GMCSF tg, parietal cell-GM-CSF tg; nu/nu, nude. Back

Received for publication May 4, 2000. Accepted for publication November 9, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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