Tolerance established in autoimmune disease by mating or bone marrow transplantation that target autoantigen to thymus
Kim Murphy1,
Mark Biondo1,
Ban-Hock Toh1 and
Frank Alderuccio1
1 Department of Pathology and Immunology, Monash University Medical School, Monash University, Commercial Road, Prahran, Victoria, 3181 Australia
The first two authors contributed equally to this work
Correspondence to: F. Alderuccio; E-mail: frank.alderuccio{at}med.monash.edu.au
Transmitting editor: D. Tarlinton
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Abstract
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Autoimmune diseases are a significant cause of death and morbidity, affecting up to 5% of the population. At present, there is no cure. Autologous bone marrow transplantation has been promoted as a treatment for achieving disease reversal and long-term remission. However, clinical trials in progress in Europe and North America report a significant risk of relapse. Here, we have addressed whether we can establish tolerance in an active autoimmune disease model by thymic expression of autoantigen. We show that tolerance and disease resistance can indeed be established in transgenic mice that spontaneously develop granulocyte macrophage colony stimulating factor-induced autoimmune gastritis, by mating them with disease-resistant transgenic mice that target autoantigen to the thymus. T cells from these double-transgenic mice are non-responsive to gastric antigen in vitro and fail to initiate disease following transfer to naive recipients. Further, we show that transplantation with bone marrow from disease-resistant transgenic mice renders recipient mice with gastritis tolerant to autoantigen as shown by a dramatic fall in autoantibody levels and T cell non-responsiveness to antigen in vitro. We suggest that genetically modified bone marrow targeting autoantigen to the thymus may be used to establish tolerance and prevent relapse of autoimmune disease following autologous bone marrow transplantation.
Keywords: autoantibody, autoimmune gastritis, autoimmunity, stomach, transgenic
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Introduction
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Autoimmune diseases are a significant cause of death and morbidity in Western countries, affecting up to 5% of the population (1). They are presently incurable. Contemporary treatment targets the effector limb of the immune response. The present status is summarized by Rose and Mackay (2):
For many years the clinical immunologist has sought the holy grail, a targeted therapy that would specifically destroy the pathogenic clone, whether of T or B cell origin, responsible for autoimmune damage. That goal still remains an unrealized ideal.
Bone marrow transplantation in autoimmunity has been hailed as a new horizon, offering the potential for reversing disease and achieving long-term remission (3). The strategy aims to remove self-reactive T lymphocytes through irradiation and toxic drugs, and replacement with allogenic or autologous lymphocytes (36). It has been used with some success in animal models of autoimmunity including diabetes, arthritis and encephalomyelitis (6,7), and in humans with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and multiple sclerosis (3,5). However, there are major drawbacks with current approaches. Allogeneic bone marrow transplantation carries the risk of graft-versus-host disease and transfer of autoimmune disease from genetically related donors (8). Autologous bone marrow transplantation currently on clinical trials for human autoimmune diseases in Europe and North America has a significant risk of relapse (2070%) because the immune system is reconstituted with the hosts own bone marrow with the generation of self-reactive T cells that re-initiate autoimmunity (9,10).
Previously, we generated transgenic mice (PC-GMCSF transgenic) that express the pro-inflammatory cytokine granulocyte macrophage colony stimulating factor (GM-CSF) in the stomach (11). In contrast to other animal models of gastritis that require perturbation of the immune system such as neonatal thymectomy (1214), the transgenic mice spontaneously develop gastritis. The gastric infiltrate in these mice comprises mainly of CD4 T cells, macrophages and dendritic cells, with macrophages and dendritic cells preceding the influx of CD4 T cells. The gastritis is accompanied by autoantibodies to gastric H/K-ATPase. It seems likely that GM-CSF expressed in the gastric mucosa has triggered the recruitment and activation of antigen-presenting cells (APC) that migrate to draining lymph nodes activating H/K-ATPase-specific T cells. Indeed the T cell response to gastric H/K-ATPase is localized to draining lymph nodes and gastritis can be transferred to naive mice by CD4+ T cells (11). These features are similar to other mouse models of gastritis (1416).
We have previously addressed the nature of the initiating autoantigen in gastritis by generating transgenic mice expressing the
(IE-H/K
transgenic) or ß (IE-H/Kß transgenic) subunit of the H/K-ATPase under the control of an MHC class II promoter (17,18). IE-H/Kß transgenic mice were resistant to neonatal thymectomy-induced gastritis (18), whereas IE-H/K
transgenic mice remained susceptible (17). Tolerance is established in the thymus because thymocytes from IE-H/Kß transgenic mice failed to transfer gastritis to naive mice (18). We have also been unable to induce gastritis in IE-H/Kß transgenic mice by immunization with autoantigen (17), adult thymectomy with cyclophosphamide treatment (19) or crossing to single TCR
chain TCR mice (F. Alderuccio, pers. obs.). Our findings are consistent with the notion that bone marrow-derived MHC class II+ dendritic cells are the major cells that induces irreversible tolerance to antigen in the thymus (20,21).
Here, we report that crossing PC-GMCSF x IE-H/Kß transgenic mice resulted in tolerance to antigen and prevention of gastritis in the double-transgenic mice. Further, we show that tolerance can also be established in PC-GMCSF transgenic mice by transplantation with bone marrow cells from the transgenic gastritis-resistant mice. Our observations suggest that autologous bone marrow transplantation using bone marrow stem cells that have been genetically modified to target antigen to the thymus may obviate the risk of disease relapse.
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Methods
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Mice
Generation of IE-H/Kß and PC-GMCSF has previously been described (11,18). IE-H/Kß have been backcrossed onto BALB/cCrSlc >10 times. In the present studies, PC-GMCSF transgenic mice have been backcrossed at least 4 times. Mice were maintained at Monash University Medical School animal facility and all experiments conducted according to Insti tutional animal ethics guidelines.
ELISA, indirect immunofluorescence and immunohistochemistry
Circulating H/K-ATPase autoantibodies were assayed by ELISA on 96-well plates coated with purified pig H/K-ATPase as previously described (18). Anti-parietal cell autoantibodies were detected by indirect immunofluorescence on frozen or paraffin-embedded sections of normal mouse stomach (18). mAb 1H9 and 2B6, reactive with the gastric H/K-ATPase
and ß subunit respectively, were used as controls. Immuno histochemistry was performed on frozen tissue sections using antibodies reactive with CD4 T cells (FITCanti-CD4; clone RM4-5), CD8 T cells (FITCanti-CD8; clone 53-6.7), dendritic cells (FITCanti-CD11c; clone HL3), macrophages (FITCanti-CD11b; clone M1/70), B cells (anti-B220; clone RA3.3A1) and granulocytes (FITCanti-Gr1; clone RB6-8C5). Sections were blocked with 1% normal swine serum for 15 min at room temperature and incubated with antibody for 60 min at room temperature. Sections were washed twice with PBS/0.05% Tween 20 for 5 min and mounted. To visualize parietal cells, sections were double stained together with biotinylated-DBA (Dolichos biflorus; Sigma, St Louis, MO) (22) followed by streptavidinTexas Red. Sections were viewed with a Bio-Rad (Hercules, CA) confocal microscope.
Flow cytometry
For FACS analysis, 12 x 106 cells were stained in 30 µl volumes containing allophycocyaninanti-CD4 (clone RM4-5), PerCPanti-CD8 (clone 53-6.7), phycoerythrin (PE)anti-B220 (clone RA3.3A1) and PEanti-CD25 (IL-2
chain, clone PC61) diluted in HBSS/1% FCS. Cells were analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, Sunnyvale, CA).
Histology
Tissues were fixed in 10% formalin in PBS and embedded in paraffin. Stomach sections (5 µm) were stained with hematoxylin & eosin and by modified Maxwells stain, 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 parietal and zymogenic cells within gastric glands. Other tissues were also examined for the presence of pathology.
Isolation of gastric and liver membranes and purified H/K-ATPase
Purified porcine gastric H/K-ATPase was prepared by tomato lectin chromatography as previously described (23). 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 and 1 mM PMSF) with a polytron homogenizer (Kinematica, Switzerland). Samples were centrifuged at 360 g for 10 min at 4°C to remove nuclei and cell debris. The supernatant was centrifuged at 5500 g for 15 min at 4°C to pellet mitochondria. The supernatant was collected and centrifuged at 100,000 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 and 1 mM PMSF) and protein concentration determined using the BCA protein assay (Pierce, Rockford, IL). Samples were stored at 20 °C. The presence of H/K-ATPase in gastric membranes was confirmed by ELISA reactivity with mAb 1H9 and 2B6 specific for gastric H/K-ATPase
and ß subunits respectively (24).
In vitro T cell proliferation assay
Pooled single-cell suspensions of lymphocytes from 10-week-old PC-GMCSF (n = 3) and PC-GMCSF x IE-H/Kß (n = 4) littermates were prepared from paragastric lymph nodes by gently grinding between frosted glass slides and used as responders in in vitro proliferation assays. Splenocytes from normal BALB/cCrSlc mice were treated with ammonium chloride solution (0.9%) to lyse red blood cells and irradiated (3000 rad) for use as APC. 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-mercaptoethanol. Proliferation assays were performed in 96-well tissue culture plates in a total volume of 200 µl containing 45 x 105 responder cells 1 x 106 irradiated APC and antigen.
For T cell proliferation assays involving bone marrow transfers, pooled single-cell suspensions were from paragastric lymph nodes of PC-GMCSF transgenic mice transferred with bone marrow from non-transgenic (n = 3) or IE-H/Kß transgenic (n = 3) mice. Proliferation assay was performed in a total volume of 200 µl containing 1 x 105 responders, 2 x 105 irradiated splenocytes as APC and antigen. 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). Cell were harvested onto glass filters (Skatron, Sterling, VA) suspended in scintillant and [3H]thymidine incorporation determined on a LKB Rackbeta scintillation counter. Control wells comprised responder cells alone, APC alone or proliferation in the absence of antigen.
Cell transfer study
Single-cell suspensions were prepared from pooled paragastric lymph nodes in HBSS/1% FCS. PC-GMCSF transgenic (n = 6) mice with circulating parietal cell and H/K-ATPase antibodies and PC-GMCSF x IE-H/Kß transgenic (n = 4) littermates were used in these experiments. Cells to be injected were washed and resuspended in HBSS in a total volume of 150200 µl, and transferred to BALB/c nu/nu mice by i.v. tail vein injection. Recipient mice received 1 x 107 paragastric lymph node cells. Mice were killed at 12 weeks following cell transfer, and sera 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.
Bone marrow transplantation
Donor IE-H/Kß transgenic mice were killed by CO2 asphyxiation, and bone marrow was harvested by flushing isolated femurs and tibia with sterile HBSS/1% FCS. Cells were collected, counted and resuspended at a concentration of 4 x 107 viable cells/150 µl in HBSS. Recipient PC-GMCSF mice were selected on the basis of autoantibody reactivity with the gastric H/K-ATPase by ELISA. On day 0, mice received a single dose of 600700 rad (GammaCell 1000 irradiator) and were i.v. injected with 4 x 107 cells via the tail vein. Following irradiation and bone marrow transfer, mice received three weekly i.v. injection of 0.25 mg depleting anti-CD4 (clone GK1.5) mAb. CD4 depletion was assessed on peripheral blood lymphocytes. Mice were bled at intervals following bone marrow transfer for autoantibody analysis. At the completion of the experiment, mice were killed and stomachs analyzed histologically for evidence of gastritis. Paragastric lymph node cells were isolated and used in in vitro T cell proliferation assays as described above.
Statistical analysis
Cell transfer results were compared using Fishers exact test.
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Results
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PC-GMCSF transgenic mice crossed to H/K-ATPase ß subunit transgenic mice do not develop autoantibodies or a destructive gastritis
We recently reported that PC-GMCSF transgenic mice backcrossed onto the gastritis-susceptible BALB/cCrSlc strain of mice develop spontaneous gastritis (11). We have also reported that ectopic expression of the gastric H/K-ATPase ß subunit in the thymus driven by an MHC class II promoter renders transgenic mice tolerant and resistant to gastritis (18). Here, we crossed gastritis-susceptible PC-GMCSF transgenic mice with gastritis-resistant IE-H/Kß transgenic mice and assessed the double-transgenic mice for development of gastritis. An important feature of PC-GMCSF transgenic mice and one that would allow for rigorous testing of our hypothesis is that the local environmental trigger for autoimmunity (GM-CSF expression in the stomach) would remain throughout the experiment. As expected, PC-GMCSF single-transgenic mice (seven of 16) developed gastritis with circulating antibodies reactive with gastric H/K-ATPase and gastric parietal cells (Fig. 1). The gastritis in PC-GMCSF transgenic mice was characterized by marked mononuclear cell infiltrate in the gastric mucosa accompanied by parietal and zymogenic cell destruction (Fig. 2A and C). Non-transgenic and single IE-H/Kß transgenic littermates did not develop autoantibodies or gastritis (not shown).

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Fig. 1. Experimental autoimmune gastritis in PC-GMCSG x IE-H/Kß double-transgenic mice. PC-GMCSF and IE-H/Kß single-transgenic mice were crossed to generate non-, single- and double-transgenic mice. At 12 weeks of age, mice were killed, genotyped, and assessed for circulating autoantibodies and gastritis. ELISA was used to determine autoantibody reactivity with purified gastric H/K-ATPase, while indirect immunofluorescence (IIF) on normal mouse stomach was used to detect parietal cell reactivity (filled box). Sections of paraffin-embedded stomachs (5 µm) were stained with hematoxylin & eosin and assessed for the presence of gastritis. Gastritis was graded as non-destructive (diagonal stripped box) or destructive (filled box). Open boxes indicate the absence of parietal cell autoantibodies or gastritis.
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Fig. 2. Gastric pathology in transgenic mice. Sections (5 µm) from paraffin-embedded stomachs from PC-GMCSF (A and C) and PC-GMCSF x IE-H/Kß (B and D) transgenic mice were stained with hematoxylin & eosin, and assessed for the presence of mononuclear cell infiltrate (arrowheads) and evidence of cellular destruction of parietal and zymogenic cells within the gastric glands (arrows). (C and D) Higher magnification images to illustrate examples of destructive (C) and non-destructive (D) gastritis. Bar: 100 µm.
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Interestingly, PC-GMCSF x IE-H/Kß double-transgenic mice did not develop autoantibodies reactive with parietal cell-associated H/K-ATPase (P = 0.022; PC-GMCSF compared to PC-GMCSF x IE-H/Kß transgenic mice) (Fig. 1). However, these mice had a mild gastritis that was not associated with tissue destruction (Figs 1, and 2B and D). The gastric infiltrate comprised mononuclear cells localized mainly to the submucosa with little evidence of migration into the lamina propria between gastric glands (Fig. 2B and D). We have previously reported that the infiltrate in the gastric mucosa of PC-GMCSF transgenic mice comprised CD4+ T cells, macrophages, dendritic cells, B cells and granulocytes (11), with the influx of macrophages and dendritic cells preceding that of CD4 T cells. We observed few CD8+ T cells in the gastric mucosa, consistent with observations in other models of experimental autoimmune gastritis. These findings were reproduced in the present study (Fig. 3A, D, G and J). As expected, IE-H/Kß transgenic mice did not display any significant cellular infiltrate (Fig. 3B, E, H and K) apart from the occasional cell observed also in normal non-transgenic mice. The cellular infiltrate in PC-GMCSF x IE-H/Kß double-transgenic mice comprised mainly CD11c+ and CD11b (Mac-1)+ with few CD4+ T cells, and virtually no CD8+ T cells (Fig. 3C, F, I and L). The data are consistent with the pro-inflammatory activity of GM-CSF on dendritic cells and macrophages (25).

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Fig. 3. Immunohistochemical analysis of stomachs from PC-GMCSF x IE-H/Kß transgenic mice. Frozen stomach sections (5 µm) were prepared from 10-week-old PC-GMCSF (A, D, G and J) and IE-H/Kß (B, E, H and K) single-transgenic mice and PC-GMCSF x IE-H/Kß (C, F, I and L) double-transgenic mice. Parietal cells (stained red) were identified using the lectin, D. biflorus, which binds specifically carbohydrates on parietal cells. Note the fewer number of parietal cells in PC-GMCSF transgenic mice which is a result of the cellular destruction associated with these mice. FITC-conjugated antibodies were used to identify the cell-surface markers CD4 (AC), CD8 (DF), CD11c (GI) and CD11b (JL). Fluorescence images were captured on a Bio-Rad confocal microscope with identical exposure times. Images were captured with a x10 objective.
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PC-GMCSF x IE-H/Kß double-transgenic mice are tolerant to gastric autoantigen
We have previously shown that the autoimmune response to the gastric autoantigen in PC-GMCSF transgenic mice was confined to draining paragastric lymph nodes, with no reactivity associated with non-draining lymphoid organs (11). In addition, lymphocytes from the paragastric lymph node can transfer gastritis to nu/nu mice (11). To demonstrate the lack of gastritis in PC-GMCSF x IE-H/Kß double-transgenic mice was associated with tolerance to gastric autoantigen, we performed T cell proliferation and transfer studies. As reported previously, paragastric lymph node cells from PC-GMCSF transgenic mice responded in vitro to gastric membranes and H/K-ATPase (Fig. 4). In contrast, paragastric lymph node cells from PC-GMCSF x IE-H/Kß double-transgenic mice failed to respond to gastric H/K-ATPase or gastric membranes (Fig. 4). Furthermore, pooled paragastric lymphocytes from PC-GMCSF x IE-H/Kß double-transgenic mice (n = 4) failed to transfer gastritis to nu/nu mice (none of four), whereas pooled paragastric cells from PC-GMCSF transgenic mice (n = 6) readily transferred gastritis (four of four) complete with anti-H/K-ATPase autoantibody reactivity and gastritis (P = 0.029) (Fig. 5). These data indicate that PC-GMCSF x IE-H/Kß double-transgenic mice are tolerant to gastric autoantigen and lack pathogenic T cells capable of transferring gastritis to naive recipients.

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Fig. 4. PC-GMCSF x IE-H/Kß double-transgenic mice are tolerant to gastric antigens. Single-cell suspensions of pooled paragastric lymph nodes from 10-week-old PC-GMCSF single-transgenic (n = 3) and PC-GMCSF x IE-H/Kß double-transgenic (n = 4) mice were prepared and used in in vitro proliferation assays with gastric or liver membranes or with purified gastric H/K-ATPase. In each well, 45 x 105 responder cells were incubated with 1 x 106 irradiated normal splenocytes as APC and antigen (in parenthesis) at the indicated concentration. Cells were incubated for 48 h followed by overnight incubation with 1 µCi [3H]thymidine. Each time point represents the mean of duplicate wells. Controls included responder cells alone (PC-GMCSF, 8833 c.p.m.; PC-GMCSF x IE-H/Kß, 7487 c.p.m.), APC alone (472 c.p.m.) and responders plus APC in the absence of antigen (PC-GMCSF, 8963 c.p.m.; PC-GMCSF x IE-H/Kß, 6192 c.p.m.).
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Fig. 5. PC-GMCSF x IE-HKß double-transgenic mice fail to transfer gastritis to naive recipients. Pooled paragastric lymphocytes (1 x 107) from PC-GMCSF (n = 6) and PC-GMCSF x IE-H/Kß (n = 4) were injected into BALB/c nu/nu mice and examined at 12 weeks for autoantibody production and gastritis. Autoantibody reactivity with purified gastric H/K-ATPase was determined by ELISA and parietal cell reactivity by indirect immunofluorescence (IIF, filled box). Gastritis was assessed by histological examination of hematoxylin & eosin-stained stomach sections. Gastritis was graded as non-destructive (diagonal stripped box) or destructive (filled box). Open boxes indicate the absence of parietal cell autoantibodies or gastritis. Controls included mAb reactive with gastric H/K-ATPase (1H9/2B6), isotype control (ET1), and mouse sera with known positive (+) and negative () reactivity with gastric H/K-ATPase.
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Abrogation of autoantibody response with persistent pathology after transplantation with bone marrow from gastritis-resistant transgenic mice
Given our success with establishing tolerance and disease resistance by crossing gastritis-resistant IE-H/Kß transgenic mice with gastritis-susceptible PC-GMCSF, we set out to ascertain whether transplantation with bone marrow cells from gastritis-resistant IE-H/Kß transgenic mice can establish tolerance and disease resistance in the PC-GMCSF transgenic mice. PC-GMCSF mice with established disease were selected for transplantation by the presence of autoantibody to gastric H/K-ATPase because these autoantibodies strongly correlated with the presence of gastritis (11,18).
We used total-body irradiation to deplete both the myeloid and lymphoid compartments, a 3-week course of CD4-depleting antibody to remove residual CD4 T cells remaining in the periphery or that might emigrate from the thymus and transfer of whole bone marrow cells from IE-H/Kß transgenic or non-transgenic mice. Ten autoantibody-positive PC-GMCSF mice were irradiated and treated with anti-CD4 antibody. Six mice received whole bone marrow from non-transgenic mice and served as controls, and four mice received bone marrow from IE-H/Kß transgenic mice. Amalgamated data from two separate experiments is presented. PCR analysis of thymus and other lymphoid tissues demonstrated the presence of donor cells in recipient mice (not shown). Using donor bone marrow cells from enhanced green fluorescent protein transgenic mice (26), we found that the pre-conditioning protocol we used achieved 6080% chimerism in recipient mice including expression in CD11c+ thymus dendritic cells (data not shown). In the control group that received non-transgenic bone marrow (six of six), antibody reactivity with gastric H/K-ATPase remained similar to pretreatment levels when tested 5 and 10 weeks following transplantation (Fig. 6). In contrast, PC-GMCSF transgenic mice (four of four) that received bone marrow from gastritis resistant IE-H/Kß transgenic mice displayed a marked drop in H/K-ATPase reactivity that was maintained for the length of the experiment (Fig. 6). The segregation of these two groups based on the reduction in antibody reactivity was highly significant (P = 0.0048). Histological examination of stomachs revealed that cellular infiltration and destruction was still present in both groups. There was no obvious difference in the level of cellular infiltrate within the glands or of destruction (not shown). In experimental autoimmune gastritis, areas of cellular destruction are characterized by replacement of parietal cells with mucous-secreting immature cells (27) that can be easily identified using the modified Maxwell stain that stains these areas yellow. The level of destruction within the gastric glands was quantified by comparing the percentage areas of the stomach that stained yellow. These comparisons revealed no difference between the two groups of mice (data not shown).

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Fig. 6. Reversal of autoimmune response in PC-GMCSF mice transferred with IE-H/Kß transgenic bone marrow cells. PC-GMCSF transgenic mice with reactive autoantibodies to the gastric H/K-ATPase were subjected to 600700 rad of total body irradiation and transferred with 4 x 107 whole bone marrow cells from non-transgenic mice (Group A) or IE-H/Kß transgenic mice (Group B). In addition, mice received weekly i.v. anti-CD4 antibody (0.25 mg) for 3 weeks following bone marrow transfer. At 5 and 10 weeks following transfer, mice were bled and the presence of anti-H/K-ATPase reactivity was determined by ELISA. Ten weeks following transfer, mice were killed and organs removed for histological examination.
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PC-GMCSF transgenic mice are tolerant to gastric antigen following transplantation with bone marrow from gastritis-resistant transgenic mice
The reversal of autoantibody production in PC-GMCSF transgenic mice receiving bone marrow from experimental autoimmune gastritis-resistant IE-H/Kß transgenic mice suggests that these mice are no longer mounting an immune response to the gastric autoantigen and have become tolerant. To further address this, we performed in vitro T cell proliferation assays using pooled lymphocytes from the stomach draining paragastric lymph node. Pooled paragastric lymphocytes from PC-GMCSF transgenic mice that received normal bone marrow and maintained their H/K-ATPase autoantibody production (Fig. 6) responded specifically to gastric membranes, and not with control liver membranes (Fig. 7). In contrast, pooled paragastric lymphocytes from PC-GMCSF transgenic mice that received IE-H/Kß transgenic bone marrow and demonstrated a reduction in H/K-ATPase autoantibody reactivity did not respond to liver or gastric membranes (Fig. 7). These results suggest that T cells capable of responding to the gastric autoantigen have been rendered tolerant in these mice.

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Fig. 7. Diseased PC-GMCSF transgenic mice reconstituted with IE-H/Kß transgenic bone marrow are tolerant to gastric autoantigen. Single-cell suspensions of pooled paragastric lymph nodes from PC-GMCSF single-transgenic mice which received bone marrow cells from non-transgenic BALB/cCrSlc (n = 3) or IE-H/Kß (n = 3) transgenic donors were used in in vitro proliferation assays using gastric or liver membranes. In each well, 1 x 105 responder cells were incubated with 2 x 105 irradiated normal splenocytes as APC and antigen (in parenthesis) at the indicated concentration. Cells were incubated for 48 h followed by overnight incubation with 1µCi [3H]thymidine. Each time point represents the mean of duplicate wells. Controls included responder cells alone (non-transgenic to PC-GMCSF, 1369 c.p.m.; IE-H/Kß to PC-GMCSF, 1607 c.p.m.), APC alone (2420 c.p.m.) and responders plus APC in the absence of antigen (non-transgenic to PC-GMCSF, 1587 c.p.m.; IE-H/Kß to PC-GMCSF, 1393 c.p.m.).
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Discussion
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Autologous bone marrow transplantation as a means of treating human autoimmune diseases is currently undergoing clinical trials in Europe and North America (9). However, this procedure carries the risk of significant relapse. This is not surprising since the process essentially re-establishes the immune repertoire of the patient, including the generation of autoreactive lymphocytes. The fact that the relapse rate is not 100% is interesting. Relapse may depend on the presence of an environmental trigger.
We were the first to show that transgenic mice that directed expression of autoantigen to the thymus under control of an MHC class II promoter led to tolerance and resistance to development of gastritis initiated by a variety of methods (1719,28). Our transgenic strategy has been successfully adopted for NOD diabetes (29) and experimental autoimmune uveitis (30), in which pro-insulin II and part of the retinal interphotoreceptor retinoid-binding protein respectively were expressed in the thymus. These transgenic mice were also tolerant to antigen and resistant to disease. In all three transgenic models targeting antigen to MHC class II+ cells, autoantigen in the thymus was detectable only by RT-PCR, suggesting that low antigen level may have rendered high-affinity CD4 T cells tolerant. Reports of an increasing number of peripheral autoantigens expressed naturally in the thymus (3133) have prompted suggestions that this may have a role in establishing tolerance (34,35).
Here, we have found that tolerance can also be established and the gastritis that develops spontaneously in PC-GMCSF transgenic mice prevented by crossing these gastritis-susceptible mice to gastritis-resistant IE-HKß transgenic mice that expresses antigen in the thymus driven by an MHC class II promoter. This was demonstrated by the absence of parietal and H/K-ATPase-specific autoantibodies, destructive gastric pathology and T cell responses to gastric antigen in the PC-GMCSF x IE-H/Kß double-transgenic mice, and by failure of T cells from double-transgenic mice to transfer disease to naive recipients. The cellular infiltrate in the gastric mucosa of double-transgenic mice comprised mainly of dendritic cells and macrophages restricted to the submucosa. The data is consistent with our previous report that these cells are the earliest migrants to the gastric mucosa in PC-GMCSF transgenic mice and that their influx precedes CD4 T cells (11). It seems likely that these cells have been recruited to the gastric mucosa by local expression of GM-CSF, a cytokine known to act on these cells (25). It indicates that the pro-inflammatory stimulus of GM-CSF that induces autoimmune gastritis is maintained in the double-transgenic mice, and that tolerance and disease resistance has been established despite this potent local pro-inflammatory stimulus. The absence of destructive gastric pathology in the double-transgenic mice is consistent with the notion that self-reactive H/K-ATPase ß-subunit-reactive T cells have been rendered tolerant in the thymus of these mice and have therefore failed to migrate to the gastric mucosa to initiate destructive tissue pathology. The present findings are consistent with our suggestion that an autoimmune response to gastric H/K-ATPase ß subunit is essential to initiate a destructive gastritis (14,17,18) even though both
and ß subunits of the H/K-ATPase are T cell targets in gastritis (36,37).
The potential of inducing tolerance to gastric H/K-ATPase and resistance to disease was further explored by transplantation of bone marrow cells from disease-resistant IE-H/Kß transgenic mice to irradiated, diseased PC-GMCSF transgenic mice that had been given depleting anti-CD4 antibody. Failure of in vitro T cell proliferation and the dramatic fall-off of antibody reactivity to gastric H/K-ATPase suggest that we have established tolerance to gastric autoantigen in the PC-GMCSF transgenic mice. Loss of anti-H/K-ATPase production suggests that antigen-specific helper CD4 T cells required for antibody production have been made tolerant. Therefore, like the PC-GMCSF x IE-H/Kß double-transgenic mice, even in the continued presence of the initiating stimulus of GM-CSF in the stomach, evidence of a specific autoimmune response is lacking in transgenic mice transplanted with bone marrow cells from gastritis-resistant transgenic mice that direct expression of antigen to thymus by an MHC class II promoter. The significance of our observation is that it has been obtained in mice with established disease, typically associated with autoantibodies and T cell reactivity to gastric antigen.
However, gastric pathology was not diminished in PC-GMCSF transgenic mice that were transplanted with IE-H/Kß transgenic bone marrow despite the apparent successful establishment of tolerance in these mice. It is unlikely that we have not allowed sufficient time for recovery of the stomach epithelium to become evident, given the half-life of 54 days for the regeneration of gastric parietal from stem cell precursors (38). The assumption that the gastric mucosa can recover from an autoimmune pathology rests on the findings of our earlier studies of immunization-induced gastritis (39) and the parietal cell ablation transgenic model of Canfield et al. (40).
We suggest that the most likely explanation for the persistent gastritis lies with the highly pro-inflammatory transgenic model we have adopted. Our data suggest that it is difficult if not impossible to reverse gastritis after the disease has been established because GM-SCF is continuously present in the stomach and can recruit inflammatory cells to the site (41,42). The suggestion is supported by our observation that even in double-transgenic mice in which we observed tolerance to autoantigen, a non-destructive gastritis was evident, comprised mainly of macrophages and dendritic cells recruited by GM-CSF. The transgenic situation of the PC-GMCSF transgenic mice is clearly very different from that of the spontaneous autoimmune diseases characterized by remissions and relapses that probably depend on whether the trigger for autoimmunity is present in the external or internal environment. Our suggestion that PC-GMCSF transgenic mice are particularly recalcitrant to disease remission is further supported by our observation that remissions were not observed in any of our PC-GMCSF transgenic mice transplanted with normal bone marrow. These findings contrast with the reported remissions in experimental and human autoimmune diseases following transplantation with autologous bone marrow (9). Notwithstanding the limitations of our transgenic experimental system, our findings lend persuasive support to the notion that bone marrow transplantation with hematopoietic stem cells that have been genetically modified to drive expression of antigen in the thymus can induce tolerance and prevent relapse of naturally occurring spontaneous autoimmune diseases, and therefore provide a significant advance to current autologous bone marrow transplantation protocols. An obvious limitation to this approach is that it requires the causative autoantigen to be identified. However, significant advances continue to be made towards the identification of antigens that drive the damaging autoimmune response (1).
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Acknowledgements
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This work is supported by funding from the Australian National Health and Medical Research Council. M. B. is a recipient of a Monash University post-graduate scholarship.
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Abbreviations
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APCantigen-presenting cell
GM-CSFgranulocyte macrophage colony stimulating factor
PC-GMCSFparietal cell GM-CSF
PEphycoerythrin
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References
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