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Tumor necrosis factor alpha is not implicated in the genesis of experimental autoimmune gastritis
Journal of Autoimmunity 22 (2004) 1–11 www.elsevier.com/locate/issn/08968411
Tumor necrosis factor alpha is not implicated in the genesis of experimental autoimmune gastritis Aiden C.J. Marshall, Ban-Hock Toh, Frank Alderuccio* Department of Pathology and Immunology, Central and Eastern Clinical School, Monash University, AMREP, Commercial Road, Prahran, Victoria 3181, Australia Received 14 April 2003; revised 31 July 2003; accepted 4 September 2003
Abstract Experimental autoimmune gastritis (EAG) characterised by mononuclear cell infiltrate, parietal and zymogenic cell destruction and circulating autoantibodies to gastric H+/K+ ATPase is an animal model for human autoimmune gastritis, that leads to pernicious anaemia. We have previously shown that Fas has a role in initiating damage to target cells in EAG. Here we used three strategies to examine the role of TNF in this disease. We administered neutralising anti-TNF antibody either as a single injection or as twice weekly injections for 8 weeks to mice subjected to neonatal thymectomy-induced EAG. To address the role of apoptotic signals through TNFR1, TNFR1 deficient mice were either neonatally thymectomised or crossed to PC-GMCSF transgenic mice that spontaneously develop EAG. Neonatally thymectomised mice treated with anti-TNF antibody developed destructive gastritis and autoantibodies to gastric H+/K+ ATPase similar to control mice. Following either neonatal thymectomy or crossing to PC-GMCSF transgenic mice, TNFR1 deficient mice developed autoantibody-positive destructive gastritis at similar frequency compared with wild type and heterozygous littermates. Our observations that neutralisation of TNF and absence of TNFR1 has no discernible effect on development of EAG suggest that TNF is not required for mucosal cell damage or development of autoimmune gastritis. While blocking TNF activity has therapeutic benefit in certain autoimmune diseases, this is not the case for EAG. 2003 Elsevier Ltd. All rights reserved. Keywords: Autoimmunity; Apoptosis; Stomach; TNF
1. Introduction Autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM) [1,2], rheumatoid arthritis (RA) [3,4] and pernicious anaemia [5,6] are associated with destruction of specialised cells in the target organ. While the consequence of tissue destruction has been well documented, the mechanisms of cellular destruction remain poorly understood. * Corresponding author. Tel.: +61-(0)3-99030281; fax: +61-(0)3-99030731 E-mail address: [email protected] (F. Alderuccio). Abbreviations: bp, base pairs; EAG, experimental autoimmune gastritis; FITC, fluorescein isothiocyanate; g, gravity; H+/K+ ATPase, H+/K+ adenosine triphosphatase; mAb, monoclonal antibody; min, minutes; PE, phycoerythrin; PerCP, peridinin chlorophyll-protein; RT, room temperature; s, seconds 0896-8411/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.09.003
Autoimmune gastritis is an organ specific autoimmune disease and the underlying cause of pernicious anaemia in humans [6]. It is associated with loss of parietal and zymogenic cells within the gastric mucosa and production of autoantibodies to intrinsic factor and gastric H+/K+ ATPase [6]. Experimental autoimmune gastritis (EAG) induced in the mouse is very similar to the human disease [6]. They are both associated with a mononuclear cell infiltrate in the gastric mucosa accompanied by loss of parietal cells and zymogenic cells with circulating parietal cell specific autoantibodies reactive with the - and -subunits of gastric H+/K+ ATPase. EAG appears to be a Th1 CD4 T cell mediated disease associated with expression of IL-2, TNF, GMCSF but not IL-4 [7]. The - and -subunits of the gastric H+/K+ ATPase are the major T cell autoantigens in mouse EAG [8,9] and in human autoimmune gastritis [10]. EAG can be induced by a number of procedures
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including neonatal thymectomy, immunisation and more recently transgenic expression of GM-CSF in the gastric mucosa [11]. We have previously shown that apoptotic cell death is increased in gastric glands of mice with thymectomyinduced autoimmune gastritis [12]. Fas/FasL and TNF/ TNFR1 are two well defined apoptotic pathways [13,14] and using the neonatal thymectomy model of EAG, we have recently reported a critical role for the Fas pathway in mediating gastric mucosal damage associated with EAG [15]. Previously we found that TNF is expressed in the gastric mucosa of mice with EAG [7]. Also, TNF has been shown to have an important role in certain animal models of autoimmunity including the non-obese mouse model of IDDM [16–19], experimental autoimmune encephalomyelitis [20,21] and in animal models of RA [22–25]. Furthermore, blocking TNF function by administration of anti-TNF monoclonal antibody has had promising results in patients with RA [3,26] and Chrohn’s disease [27,28]. In view of the presence of TNF in the pathological lesion of autoimmune gastritis [7], the documented role of TNF in certain human and experimental models of autoimmunity, and the established role of TNF in mediating apoptosis [14], the role of TNF in the pathogenesis of EAG was addressed here. Administration of anti-TNF antibody either as a single dose or twice weekly for 8 weeks did not reduce the incidence of thymectomy-induced EAG. Further, we show that TNFR1o/o mice remained susceptible to neonatal thymectomy induced EAG and EAG induced by transgenic expression of GM-CSF in the gastric mucosa. These results suggest that TNF is not required for gastric pathology associated with EAG. Together with our earlier findings [15], our present observations suggest that different molecular mechanisms may account for destruction of parenchymal cells in autoimmune diseases and that it is important to define these mechanisms and therapeutic options in different model settings.
2. Methods 2.1. Mice C57/BL-6 TNF receptor 1 (TNFR1; p55) knockout (TNFR1o/o) mice were obtained from Dr J. Ruby, University of Melbourne (Melbourne, Australia) and were originally generated by Jacques Peschon [29]. Experimental autoimmune gastritis susceptible BALB/ cCrSlc and TNFR1o/o mice backcrossed six times to BALB/cCrSlc were bred and maintained under conventional conditions in the Monash Medical School Animal Facility. PC-GMCSF transgenic mice which spontaneously develop EAG have previously been described [11]. PC-GMCSFTNFR1w/o mice were generated by
crossing BALB/cCrSlc PC-GMCSF transgenic mice with BALB/cCrSlc TNFR1o/o (6th backcross) mice and intercrossing littermates to generate PC-GMCSF transgenic mice with either TNFR1o/o (homozygous knockout), TNFR1w/o (heterozygous) or TNFR1w/w (wild type) genotypes. All experiments were performed in accordance with Monash University animal ethics guidelines.
2.2. Backcrossing and PCR screening of TNFR1o/o mice C57/BL6 TNFR1o/o mice were backcrossed to the gastritis-susceptible BALB/cCrSlc strain [30]. Mice were genotyped by polymerase chain reaction (PCR). Oligonucleotides for detection of the wild type TNFR1 allele were (forward primer) 5#-GGATTGTCACGGTGCCG TTGAAG-3# and (reverse primer) 5#-CCTTTACGGCT TCCCAGAATTACC-3# while the TNFR1 mutation was identified using oligonucleotides (forward primer) 5#-GCTCCTGGCTCTGCTGATGGGGATAC-3# and (reverse primer) 5#-GTGCTGTCCATCTGCACGAG AC-3# which detected the neomycin cassette insertion. PCR was performed in 25 µl reaction volumes containing 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 µM dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech, Castle Hill, Australia), 25 pmol oligonucleotide primers, and 1.0 U of Taq DNA polymerase (Gibco BRL/Life Technologies, Rockville, Maryland). PCR conditions were 94 (C for 5 min, 34 cycles of 94 (C for 1 min, 65 (C for 30 s and 72 (C for 1 min, and a final cycle at 72 (C for 5 min. A 488 bp product is predicted from the wild type allele and a product of 307 bp is predicted from the mutant TNFR1 allele. PCR products were separated by 1.5% agarose gel electrophoresis and visualised using UV illumination. TNFR1w/o mice were intercrossed to generate TNFR1 homozygous knockouts (o/o), heterozygous (w/o) and homozygous wild type (w/w) mice. Genotypes followed the normal Mendelian frequencies of 1:2:1. Mice expressing the PC-GMCSF transgene were screened as previously described [11]. Briefly, oligonucleotides (forward primer) 5#-CCTCACACAGAGGAGACTA3# and (reverse primer) 5#-GTTAGAGACGACTTCTA CCTCTTC-3# generating a product of 350 bp were used to screen for the transgene, while DNA integrity was assessed using oligonucleotides (forward primer) 5#CGAGCTCGAGCCTGCCTATCTTTCAGG-3# and (reverse primer) 5#-CGGGATCCTAGTTGCAGTAGT TCTCCAG-3# specific for the mouse insulin gene to generate a product of 374 bp. Reaction mixtures were as above, except 50 pmol oligonucleotides were used and the PCR conditions were 95 (C for 2 min, 30 cycles of 92 (C for 30 s, 60 (C for 30 s and 72 (C for 1 min, and a final cycle at 72 (C for 5 min.
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2.3. Neonatal thymectomy Neonatal thymectomy was performed on 3-day-old BALB/cCrSlc or BALB/cCrSlc TNFR1o/o mice under cold anaesthesia as described [8]. Briefly, a small incision was made over the sternum to expose the rib cage and a small notch was removed to the second intercostal joint. The exposed fascia was gently torn with fine pointed forceps to expose the thymus gland. Under gentle suction, the thymus was removed with a glass pipette. Mice were placed under a heat source and returned to their mother once they had regained activity. 2.4. In vitro testing of anti-TNF antibody TNF-sensitive WEHI 164 murine fibrosarcoma cells (obtained from Dr J. Ruby, University of Melbourne) cultured to less than 80% confluency were treated with 2 µg/ml actinomycin D (Sigma, St Louis, MO, USA) at a concentration of 7.5105 cells/ml for 1 h at 37 (C. Fifty µl aliquots of either anti-mouse TNF CV1q (Centocor, Malvern, Pennsylvania, USA) or mouse IgG2a-kappa chain isotype control (Sigma) at 1 mg/ml were mixed with 50 µl serially diluted mouse recombinant TNF (0.1 ng to 0.001 ng) (Sigma) and incubated at 37 (C 10% CO2 for 2 h. One hundred µl (7.5104) actinomycin D-treated WEHI 164 cells were added to the mixture and incubation continued at 37 (C 10% CO2 for a further 18 h. Cell suspensions were stained with propidium iodide and analysed by flow cytometry (FACScalibur and CellQuest software, Becton Dickinson, Mountain View, CA, USA) for detection of dead cells. 2.5. In vivo anti-TNF injections Day 3 neonatally thymectomised BALB/cCrSlc mice were administered with anti-mouse TNF antibody (cV1q) (Centocor), or mouse IgG2a-kappa chain (Sigma) as an isotype control. Test and control groups were weighed and injected intra-peritoneally with immunoglobulin at a dose of 5 mg/kg in 50–100 µl sterile PBS. Mice were injected either with a single dose on day 4 (1 day following neonatal thymectomy) or twice weekly from day 4 to 8 weeks of age. Mice were killed at 10 weeks of age and assessed for production of parietal cell autoantibodies, H+/K+ ATPase reactivity and histological evidence of gastritis. 2.6. ELISA Enzyme-linked immunosorbent assay was used to screen mouse sera for H+/K+ ATPase reactive autoantibodies as previously described [8]. Alternative rows of round-bottomed microtitre plates were coated with 50 µl
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purified porcine gastric H+/K+ ATPase [31] in 0.5 M carbonate–bicarbonate buffer, pH 9.6. Unbound sites were blocked with 200 µl of PBS/1% BSA at 4 (C overnight and plates stored at 20 (C. Plates were incubated with 50 µl of mouse sera at a dilution of 1/50 in PBS/0.05% Tween-20 for 1 h at room temperature. Plates were washed with PBS/0.05% Tween 20 and 50 µl biotinylated anti-mouse IgG (Amersham Pharmacia Biotech) (diluted 1:500 in PBS/0.1% BSA) added to each well for 1 h at room temperature. Plates were washed and 50 µl streptavidin-biotinylated horseradish peroxidase (HRPO) complex (Amersham Pharmacia Biotech) (1:500 in PBS/0.1% BSA) was added to each well. Bound HRPO activity was detected with 100 µl substrate containing 0.2 mg/ml O-phenylenediamine (Sigma, Castle Hill, Australia) and 0.006% H2O2 for 30 min at room temperature in the dark. The reaction was terminated by addition of 50 µl of 5 M H2SO4. Absorbance was measured at 490 nm. Background reactivity determined from wells lacking H+/K+ ATPase was subtracted from absorbance of coated wells. Controls for ELISA included monoclonal antibodies 1H9 and 2B6 specific for the - and -subunits of the gastric H+/K+ ATPase respectively, an irrelevant monoclonal antibody ET1 as an isotype control and mouse sera with known reactivity with H+/K+ ATPase. All sera and controls were tested in duplicate. For testing autoantibody reactivities at different dilutions, 50 µl of sera at a range of dilutions from 1/50 to 1/12150 (serial one-in-three dilutions) was added to the porcine gastric H+/K+ ATPase coated plates and incubated at room temperature for 1 h. The remaining steps were as described above. 2.7. Immunohistochemistry Anti-parietal cell autoantibodies were detected by indirect immunofluorescence on 4 µm sections of freshly frozen or tannic acid-processed, paraffin-embedded normal mouse stomachs. Sections were blocked with PBS/1% normal swine serum for 15 min at room temperature. Sections were rinsed in PBS/0.05% Tween-20 and incubated for 1 h at room temperature with mouse sera (diluted 1/25 in PBS/0.05% Tween-20). Slides were washed 25 min in PBS/0.05% Tween-20 and incubated with either FITC-conjugated sheep anti-mouse Ig (Silenus, Melbourne, Australia) (diluted 1/50 in PBS/ 0.05% Tween-20) or goat anti-mouse IgG Alexa 488 Fluor (Molecular Probes, Eugene, Oregon) (1/2000 in PBS/0.05% Tween-20). Following two washes in PBS/ 0.05% Tween-20, sections were mounted with mounting solution and examined by epifluorescence microscopy. 1H9 and 2B6, monoclonal antibodies specific for the and -subunits of the H+/K+ ATPase respectively, and previously tested positive and negative sera were used as controls.
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2.8. Histology Stomachs were fixed in 10% phosphate buffered formalin and processed using an automated Shandon Hypercentre 2 (Shandon Southern Instruments, Sewickley, PA, USA). Tissues were embedded in paraffin wax and 4 µm sections cut and stained with haematoxylin and eosin. Gastritis was scored as normal if no infiltrate was present in the submucosa, non-destructive gastritis if infiltrate was present within the mucosa without destruction of parietal and zymogenic cells and destructive gastritis with infiltrate and cellular damage. 2.9. Flow cytometry For flow cytometry analysis, 1–2106 cells from the thymus, spleen or paragastric lymph nodes were stained in 30 µl volumes containing FITC-anti-CD4 (clone RM4-5) (Pharmingen, San Diego, CA, USA), PerCPanti-CD8 (clone 53-6.7) (Pharmingen) and PE-antiCD25 (IL-2-chain, clone PC61) (Pharmingen) diluted in Hanks’ buffered salt solution (Gibco BRL/Life Technologies)/1% FCS/0.02% sodium azide. Cells were analysed on a FACScan using CellQuest software (Becton Dickinson). 2.10. Statistics The Fischer exact probability test (GraphPad InStat 2.00 for Win 95/NT 1997) was used to analyse gastritis incidence between two groups. Cell numbers were compared using the Kruskal–Wallis test (non-parametric ANOVA) and the Dunn post test (GraphPad InStat). Autoantibody reactivities were compared using a Mann–Whitney U test (GraphPad InStat).
3. Results 3.1. Administration of anti-TNF antibody does not abrogate thymectomy-induced experimental autoimmune gastritis To examine the potential benefit of anti-TNF administration in the treatment of autoimmune gastritis, we examined the development of thymectomy induced EAG following a single dose or prolonged exposure to neutralising anti-TNF antibody. The biological activity of the anti-TNF CV1q antibody was confirmed on TNF sensitive WEHI 164 cells (data not shown). Following a single injection of anti-TNF at day 4 of age, one day after thymectomy, 11/14 (79%) mice developed gastritis at 10 weeks of age, comprising a mononuclear infiltrate within the gastric mucosa (Fig. 1A and D), with the majority (10/14) showing evidence of parietal and zymogenic cell loss (Fig. 1A and E). All mice with gastritis also developed parietal cell autoanti-
bodies reactive with gastric H+/K+ ATPase (Fig. 1A). In comparison, 4/6 (67%) mice injected with IgG2a-kappa chain control antibody developed gastritis and autoantibodies (P>0.05, anti-TNF compared to isotype control) (Fig. 1A). Interestingly, the incidence of destructive gastritis appeared to be increased in anti-TNF treated mice compared to control mice (P<0.05). However, the incidence of destructive gastritis following neonatal thymectomy is consistent with the observed frequency from previous findings. To examine the influence of the anti-TNF antibody over an extended period in our model system, we injected a group of mice twice a week for 8 weeks following neonatal thymectomy. Three of 6 (50%) mice developed gastritis and autoantibodies, with two mice developing destructive gastritis (Fig. 1B). In comparison, 3/4 (75%) IgG2a-kappa chain injected mice developed gastritis with parietal cell specific autoantibodies (Fig. 1B) (P>0.05, anti-TNF compared to isotype control). There was no statistical difference between incidence of destructive gastritis in anti-TNF treated mice compared to control mice (P>0.05). Likewise, there was also no statistical difference in the incidence of autoantibodies or destructive gastritis between the single and long-term injected groups. 3.2. BALB/cCrSlc mice deficient in TNFR1 develop experimental autoimmune gastritis following neonatal thymectomy To examine the role that TNFR1-mediated signaling may play in the pathogenesis of EAG, we assessed the incidence of EAG in susceptible BALB/cCrSlc mice deficient in TNFR1. TNFR1 gene targeted mice were backcrossed six times to BALB/cCrSlc mice and heterozygous TNFR1w/o mice intercrossed and litters thymectomised at day 3 of age. At 14 weeks of age, mice were killed and assessed for production of parietal and H+/K+ ATPase reactive autoantibodies and histological evidence of gastritis. Overall, we found no statistical difference in the incidence of autoantibody reactivity or gastritis between the various genotypes (Fig. 2). As expected, the majority of TNFR1w/w mice (4/5) developed characteristics of EAG including parietal cell associated H+/K+ ATPase reactivity (Figs. 2 and 3A) and gastritis with associated tissue damage (Fig. 3E). We found 12/16 (75%) BALB/cCrSlc TNFR1w/o mice developed gastritis with the majority (10/16) displaying mucosal damage (Figs. 2 and 3F) and parietal cell specific autoantibodies (Figs. 2 and 3B). Finally, 11/14 (79%) BALB/cCrSlc TNFR1o/o mice developed gastritis characterised by a prominent gastric infiltrate extending into the lamina propria of the mucosa, with destruction of zymogenic and parietal cells apparent in the majority of these mice (8/14) (Figs. 2 and 3G). All of the TNFR1o/o mice with gastritis developed antibodies to parietal cells
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Fig. 1. Mice were thymectomised at day 3 and injected either at (A) day 4 or (B) twice weekly for 8 weeks with either anti-TNF antibody or IgG2a as an isotype control. Mice were assessed at 10 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes. Gastritis was graded as either normal (C); non-destructive gastritis if cellular infiltrate was present within the gastric mucosa (arrow) (D); or destructive gastritis if infiltrate was present in addition to parietal and zymogenic cell destruction (arrow-head) (E). Bar: 100 µm.
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Fig. 2. Incidence of autoimmune gastritis in neonatally thymectomised BALB/cCrSlc TNFR1 deficient mice. C57/BL-6 TNFR1o/o mice were backcrossed six times to BALB/cCrSlc and intercrossed to generate wild type (w/w), heterozygous (w/o) and TNFR1 deficient (o/o) mice. Mice were thymectomised at day 3 and assessed at 14 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes.
and the gastric H+/K+ ATPase (Figs. 2 and 3C). In addition, we quantified the total number of cells obtained from the spleens and paragastric lymph nodes of these mice and found no difference in the total number of cells and CD4+ and CD8+ T cell populations within these organs between genotypes (data not shown). 3.3. PC-GMCSF transgenic mice which spontaneously develop experimental autoimmune gastritis develop disease in the absence of TNFR1 We have recently described a transgenic mouse model that spontaneously develops EAG due to the transgenic expression of the pro-inflammatory cytokine GM-CSF in gastric parietal cells [11]. Unlike many other models of EAG (such as neonatal thymectomy) that are associated with an induced state of lymphopenia [32], PCGMCSF mice are not lymphopenic and the immune repertoire has not been targeted. PC-GMCSF transgenic mice develop EAG without further manipulation such as neonatal thymectomy and thus represent an alternative system to examine pathogenic mechanisms associated with EAG [11]. Therefore we examined the induction of EAG in TNFR1o/o mice that were also PC-GMCSF transgenic. All PC-GMCSF transgenic (Tg) TNFR1w/w
mice (13/13) developed autoantibodies to gastric H+/K+ ATPase detected by ELISA, compared to 14/17 (82%) Tg-TNFR1w/o (P>0.05) and 6/11 (55%) Tg-TNFR1o/o mice (P<0.05 compared to Tg-TNFR1w/w mice, P>0.05 compared to Tg-TNFR1w/o mice) (Fig. 4). Similarly, all Tg-TNFR1w/w mice (13/13) developed parietal cellspecific autoantibodies, compared to 15/17 (88%) TgTNFR1w/o mice (P>0.05) and 7/11 (64%) Tg-TNFR1o/o mice (P<0.05 compared to Tg-TNFR1w/w mice) (Fig. 4). The observed difference in antibody reactivity of sera diluted 1/50 between Tg-TNFR1o/o and Tg-TNFR1w/w mice was reproduced over a range of serum dilutions and found that the reactivity of autoantibody positive sera was consistently lower in Tg-TNFR1o/o mice compared to Tg-TNFR1w/w mice (P=0.0073) (data not shown). Histological evidence of gastritis was found in all PC-GMCSF transgenic mice regardless of TNFR1 genotype. To some extent, this may be due to the pro-inflammatory properties of the transgenic PCGMCSF mice. In these groups of mice, destructive gastritis was evident in 5/13 (38%) PC-GMCSF transgenic TNFR1w/w mice, compared to 3/17 (18%) transgenic TNFR1w/o mice and 2/11 transgenic TNFR1o/o mice (18%) (P>0.05 between all groups) (Fig. 4). While
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Fig. 3. Parietal cell autoantibodies and gastritis in neonatally thymectomised mice. Sera from neonatally thymectomised (A) TNFR1w/w, (B) TNFR1w/o and (C) TNFR1o/o mice all developed parietal cell autoantibodies in contrast to normal mice (D). Haematoxylin and eosin stained stomach sections (E–H) of 14 week old thymectomised TNFR1w/w (E), TNFR1w/o (F) and TNFR1o/o (G) mice displayed prominent mononuclear cell infiltrate within the gastric mucosa (arrow), accompanied by cellular destruction (arrow-head) and tissue hypertrophy. These mice lacked zymogenic (Z) and parietal cell (P) regions, which are indicated in the stomach of a normal mouse (H). Bar: 100 µm.
there was no statistical difference within these groups, the incidence of destructive gastritis in the TNFR1 genotypes was lower in PC-GMCSF transgenic mice compared to neonatally thymectomised mice (Table 1). These differences may relate to the different methods of disease induction of the two groups. The total number of cells obtained from the spleens and paragastric lymph nodes of these mice were similar, with no differences in cell proportions within these organs between genotypes (data not shown).
4. Discussion The mechanisms of targeted cell death in many autoimmune diseases are poorly understood. In the case of autoimmune gastritis, we have recently reported that the Fas pathway plays a major role in target cell loss in this disease [15]. In the present study we have examined for a role for TNF in EAG. The effect of neutralising the activity of TNF was assessed in two studies of EAG induced by neonatal
thymectomy in which the neutralising antibody was administered either as a single dose or as repeated doses over 8 weeks. We found that administration of neutralising anti-TNF antibody as a single dose did not reduce the incidence of autoimmune gastritis. It is possible that this may have been due to the short period of anti-TNF antibody action and to address this, a second group of neonatally thymectomised mice were injected twice weekly over an 8 week period. This second group of mice also developed autoantibodies and destructive gastritis, suggesting that TNF may not be required for disease induction or gastric parenchymal cell death. However, it is possible that the injected anti-TNF antibody, although now present in higher amounts than the single injection, and for a prolonged period of time, may not have efficiently accessed the gastric mucosa. Using a more definitive approach, we assessed the induction of EAG in TNFR1 (p55) deficient mice, since the majority of, if not all, of the apoptotic potential of TNF is mediated through TNFR1 [14,29]. TNFR1 deficient mice were backcrossed to the gastritissusceptible BALB/cCrSlc mouse strain and subjected to
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Fig. 4. Incidence of autoimmune gastritis in PC-GMCSFTNFR1o/o mice. C57/BL-6 TNFR1o/o mice were backcrossed six times to BALB/cCrSlc, then crossed with BALB/cCrSlc PC-GMCSF transgenic mice. Mice were intercrossed to generate PC-GMCSF transgenic mice that were either TNFR1o/o, TNFR1w/o or TNFR1w/w. Mice were assessed at approximately 20 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes. Table 1 Incidence of destructive gastritis in TNFR1 genotypes in neonatally thymectomised and PC-GMCSF transgenic mice EAG induction method
Neonatal thymectomy PC-GMCSF Tg P valuea a
TNFR1 genotype o/o
w/o
w/w
8/14 (57%) 2/11 (18%) 0.099
10/16 (63%) 3/17 (18%) 0.013
2/5 (40%) 5/13 (38%) 1.000
Fischer exact statistical test used.
neonatal thymectomy or crossed with PC-GMCSF transgenic mice. Following neonatal thymectomy, all TNFR1 genotypes developed characteristics of EAG, including autoantibody production, mononuclear cell infiltrate and cellular destruction within the gastric mucosa. A similar finding was observed in TNFR1 gene targeted mice crossed on to PC-GMCSF transgenic mice. PC-GMCSF transgenic mice have recently been described as a model for EAG in which the proinflammatory cytokine GM-CSF is transgenically expressed in gastric parietal cells [11]. In both models, it was evident that the absence of TNFR1 did not affect the induction of EAG compared to wild type littermates. These results suggest that TNFR1 is not required on the parietal cell
surface to receive an apoptotic signal from infiltrating cells following neonatal thymectomy and that TNFR1 does not have a pro-inflammatory role in EAG. However, a difference that did become apparent between these two models was the reduced incidence of destructive gastritis in the PC-GMCSF model compared to the thymectomy model (Table 1). While these were two experiments performed on different groups of animals, the outcome probably relates to the different methods and mechanisms associated with each model. Neonatal thymectomy is thought to deplete CD4+CD25+ regulatory cells in the periphery [33,34], resulting in activation and expansion of pathogenic CD4+ T cells. In contrast, the PC-GMCSF transgenic model has not targeted the lymphoid compartment but instead is likely to have activated the immune response through the local pro-inflammatory action of GM-CSF in the gastric mucosa. Indeed we have shown that PC-GMCSF transgenic mice maintain a functional CD4+CD25+ T cell population [11]. It is possible that in PC-GMCSF transgenic mice, immune regulation has not been totally perturbed and still has some influence on the pathogenesis of EAG. While an immune response is clearly generated, evident by the induction of parietal cell and H+/K+ ATPase specific autoantibodies, cellular
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destruction may be limited by the presence of the regulatory T cell population. This observation may be similar to the non-pathogenic insulitis observed around pancreatic islets in mouse models of diabetes prior to development of clinical diabetes associated with cellular infiltrates in the pancreas and -cell destruction [35,36]. The apparent absence of a role for TNF in the genesis of autoimmune gastritis should be viewed in the context of a role for this molecule in other human and experimental autoimmune diseases. For instance in murine collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis, neutralising anti-TNF antibody decreased production of IL-1 and other proinflammatory cytokines [22]. Combined with anti-CD4 antibody [23] or anti-IL-12 antibody [24], anti-TNF therapy reduced swelling and histological severity of arthritis and joint erosion. However, TNF is not an absolute requirement for development of CIA [37,38]. Although some TNFR1o/o mice had reduced clinical parameters of CIA, severe disease, in addition to lymphadenopathy, was observed in many mice [37]. The antibody neutralisation experiments suggested that antiTNF monoclonal antibody may be used to treat rheumatoid arthritis. Indeed anti-TNF mAb trials in rheumatoid arthritis reduced disease severity [26]. Clinical trials of infliximab, a chimeric anti-TNF mAb [28,39,40] showed that TNF regulates local chemokine and cytokine production, recruitment of inflammatory cells into joints, angiogenesis, and blood levels of matrix metalloproteinases-1 and -3 [3]. Infliximab also gave promising results in Chrohn’s disease refractory to conventional drugs [27,28,41–44], active ankylosing spondylitis, psoriatic arthritis [40] and Sjogren’s syndrome [45]. Infliximab induced apoptosis in monocytes from patients with Chrohn’s disease suggesting that the antibody reduced the number of activated cells at the inflamed site [43]. However, the results of our present study and previous observations suggest that anti-TNF antibody treatment may not be suitable for all autoimmune diseases. For instance, whereas a single administration of anti-TNF mAb in neonatal NOD mice completely prevented diabetes [46], administration at later time points increased disease incidence [47]. The former result is consistent with the observation that mice lacking TNFR1 were protected from diabetes [16]. Transgenic TNF expression within -islet cells of the pancreas reduced T cell responses [48] and protected transgenic NOD mice from diabetes and -cell destruction [17,18]. However, in a transgenic model of virally induced diabetes (rat insulin promoter-lymphocytic choriomeningitis virus), early islet-specific TNF expression enhanced disease incidence, while late TNF expression abrogated disease [19]. TNF has also been implicated in multiple sclerosis [49] and neutralisation of TNF in experimental autoimmune encephalomyelitis (EAE)
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[20,21] gave promising results. However, mice in which TNF and LT genes were inactivated were still susceptible to EAE initiated by immunisation with mouse spinal cord homogenate suggesting that these molecules are not essential for EAE development [50]. Furthermore, in a large clinical study of multiple sclerosis, systemic blockade of TNF with Lenercept resulted in detrimental clinical effects [51]. The situation is further complicated because TNFR1 has the capacity to bind ligands other than TNF including lymphotoxin (LT), that can induce cytotoxic effects on T cells [52]. In summary, our studies suggest that TNFR1 mediated apoptosis does not have a role in experimental autoimmune gastritis. Using neutralising antibody and gene targeted approaches, we found EAG induction was not compromised with the neutralisation of TNF or in the absence of TNFR1. Indeed our previous study [15] suggests that the main pathway of cellular destruction in EAG is associated with the Fas pathway of cell death. In addition and as a general point, it also needs to be emphasised that not all autoimmune diseases may respond favorably to anti-TNF treatment and that even when beneficial effects are recorded in animal models, caution should be taken in extrapolating these to the human disease.
Acknowledgements We thank Centocor (Malvern, Pennsylvania) for providing the CV1q anti-TNF antibody and Dr Janet Ruby (University of Melbourne) for the C57/BL-6 TNFR1 knockout mice and WEHI164 cells. This work is supported by grants from the National Health and Medical Research Council (NHMRC) of Australia and The Alfred Hospital. A.C.J.M is a recipient of an Australian post-graduate scholarship.
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Tumor necrosis factor alpha is not implicated in the genesis of experimental autoimmune gastritis Aiden C.J. Marshall, Ban-Hock Toh, Frank Alderuccio* Department of Pathology and Immunology, Central and Eastern Clinical School, Monash University, AMREP, Commercial Road, Prahran, Victoria 3181, Australia Received 14 April 2003; revised 31 July 2003; accepted 4 September 2003
Abstract Experimental autoimmune gastritis (EAG) characterised by mononuclear cell infiltrate, parietal and zymogenic cell destruction and circulating autoantibodies to gastric H+/K+ ATPase is an animal model for human autoimmune gastritis, that leads to pernicious anaemia. We have previously shown that Fas has a role in initiating damage to target cells in EAG. Here we used three strategies to examine the role of TNF in this disease. We administered neutralising anti-TNF antibody either as a single injection or as twice weekly injections for 8 weeks to mice subjected to neonatal thymectomy-induced EAG. To address the role of apoptotic signals through TNFR1, TNFR1 deficient mice were either neonatally thymectomised or crossed to PC-GMCSF transgenic mice that spontaneously develop EAG. Neonatally thymectomised mice treated with anti-TNF antibody developed destructive gastritis and autoantibodies to gastric H+/K+ ATPase similar to control mice. Following either neonatal thymectomy or crossing to PC-GMCSF transgenic mice, TNFR1 deficient mice developed autoantibody-positive destructive gastritis at similar frequency compared with wild type and heterozygous littermates. Our observations that neutralisation of TNF and absence of TNFR1 has no discernible effect on development of EAG suggest that TNF is not required for mucosal cell damage or development of autoimmune gastritis. While blocking TNF activity has therapeutic benefit in certain autoimmune diseases, this is not the case for EAG. 2003 Elsevier Ltd. All rights reserved. Keywords: Autoimmunity; Apoptosis; Stomach; TNF
1. Introduction Autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM) [1,2], rheumatoid arthritis (RA) [3,4] and pernicious anaemia [5,6] are associated with destruction of specialised cells in the target organ. While the consequence of tissue destruction has been well documented, the mechanisms of cellular destruction remain poorly understood. * Corresponding author. Tel.: +61-(0)3-99030281; fax: +61-(0)3-99030731 E-mail address: [email protected] (F. Alderuccio). Abbreviations: bp, base pairs; EAG, experimental autoimmune gastritis; FITC, fluorescein isothiocyanate; g, gravity; H+/K+ ATPase, H+/K+ adenosine triphosphatase; mAb, monoclonal antibody; min, minutes; PE, phycoerythrin; PerCP, peridinin chlorophyll-protein; RT, room temperature; s, seconds 0896-8411/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.09.003
Autoimmune gastritis is an organ specific autoimmune disease and the underlying cause of pernicious anaemia in humans [6]. It is associated with loss of parietal and zymogenic cells within the gastric mucosa and production of autoantibodies to intrinsic factor and gastric H+/K+ ATPase [6]. Experimental autoimmune gastritis (EAG) induced in the mouse is very similar to the human disease [6]. They are both associated with a mononuclear cell infiltrate in the gastric mucosa accompanied by loss of parietal cells and zymogenic cells with circulating parietal cell specific autoantibodies reactive with the - and -subunits of gastric H+/K+ ATPase. EAG appears to be a Th1 CD4 T cell mediated disease associated with expression of IL-2, TNF, GMCSF but not IL-4 [7]. The - and -subunits of the gastric H+/K+ ATPase are the major T cell autoantigens in mouse EAG [8,9] and in human autoimmune gastritis [10]. EAG can be induced by a number of procedures
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including neonatal thymectomy, immunisation and more recently transgenic expression of GM-CSF in the gastric mucosa [11]. We have previously shown that apoptotic cell death is increased in gastric glands of mice with thymectomyinduced autoimmune gastritis [12]. Fas/FasL and TNF/ TNFR1 are two well defined apoptotic pathways [13,14] and using the neonatal thymectomy model of EAG, we have recently reported a critical role for the Fas pathway in mediating gastric mucosal damage associated with EAG [15]. Previously we found that TNF is expressed in the gastric mucosa of mice with EAG [7]. Also, TNF has been shown to have an important role in certain animal models of autoimmunity including the non-obese mouse model of IDDM [16–19], experimental autoimmune encephalomyelitis [20,21] and in animal models of RA [22–25]. Furthermore, blocking TNF function by administration of anti-TNF monoclonal antibody has had promising results in patients with RA [3,26] and Chrohn’s disease [27,28]. In view of the presence of TNF in the pathological lesion of autoimmune gastritis [7], the documented role of TNF in certain human and experimental models of autoimmunity, and the established role of TNF in mediating apoptosis [14], the role of TNF in the pathogenesis of EAG was addressed here. Administration of anti-TNF antibody either as a single dose or twice weekly for 8 weeks did not reduce the incidence of thymectomy-induced EAG. Further, we show that TNFR1o/o mice remained susceptible to neonatal thymectomy induced EAG and EAG induced by transgenic expression of GM-CSF in the gastric mucosa. These results suggest that TNF is not required for gastric pathology associated with EAG. Together with our earlier findings [15], our present observations suggest that different molecular mechanisms may account for destruction of parenchymal cells in autoimmune diseases and that it is important to define these mechanisms and therapeutic options in different model settings.
2. Methods 2.1. Mice C57/BL-6 TNF receptor 1 (TNFR1; p55) knockout (TNFR1o/o) mice were obtained from Dr J. Ruby, University of Melbourne (Melbourne, Australia) and were originally generated by Jacques Peschon [29]. Experimental autoimmune gastritis susceptible BALB/ cCrSlc and TNFR1o/o mice backcrossed six times to BALB/cCrSlc were bred and maintained under conventional conditions in the Monash Medical School Animal Facility. PC-GMCSF transgenic mice which spontaneously develop EAG have previously been described [11]. PC-GMCSFTNFR1w/o mice were generated by
crossing BALB/cCrSlc PC-GMCSF transgenic mice with BALB/cCrSlc TNFR1o/o (6th backcross) mice and intercrossing littermates to generate PC-GMCSF transgenic mice with either TNFR1o/o (homozygous knockout), TNFR1w/o (heterozygous) or TNFR1w/w (wild type) genotypes. All experiments were performed in accordance with Monash University animal ethics guidelines.
2.2. Backcrossing and PCR screening of TNFR1o/o mice C57/BL6 TNFR1o/o mice were backcrossed to the gastritis-susceptible BALB/cCrSlc strain [30]. Mice were genotyped by polymerase chain reaction (PCR). Oligonucleotides for detection of the wild type TNFR1 allele were (forward primer) 5#-GGATTGTCACGGTGCCG TTGAAG-3# and (reverse primer) 5#-CCTTTACGGCT TCCCAGAATTACC-3# while the TNFR1 mutation was identified using oligonucleotides (forward primer) 5#-GCTCCTGGCTCTGCTGATGGGGATAC-3# and (reverse primer) 5#-GTGCTGTCCATCTGCACGAG AC-3# which detected the neomycin cassette insertion. PCR was performed in 25 µl reaction volumes containing 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 µM dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech, Castle Hill, Australia), 25 pmol oligonucleotide primers, and 1.0 U of Taq DNA polymerase (Gibco BRL/Life Technologies, Rockville, Maryland). PCR conditions were 94 (C for 5 min, 34 cycles of 94 (C for 1 min, 65 (C for 30 s and 72 (C for 1 min, and a final cycle at 72 (C for 5 min. A 488 bp product is predicted from the wild type allele and a product of 307 bp is predicted from the mutant TNFR1 allele. PCR products were separated by 1.5% agarose gel electrophoresis and visualised using UV illumination. TNFR1w/o mice were intercrossed to generate TNFR1 homozygous knockouts (o/o), heterozygous (w/o) and homozygous wild type (w/w) mice. Genotypes followed the normal Mendelian frequencies of 1:2:1. Mice expressing the PC-GMCSF transgene were screened as previously described [11]. Briefly, oligonucleotides (forward primer) 5#-CCTCACACAGAGGAGACTA3# and (reverse primer) 5#-GTTAGAGACGACTTCTA CCTCTTC-3# generating a product of 350 bp were used to screen for the transgene, while DNA integrity was assessed using oligonucleotides (forward primer) 5#CGAGCTCGAGCCTGCCTATCTTTCAGG-3# and (reverse primer) 5#-CGGGATCCTAGTTGCAGTAGT TCTCCAG-3# specific for the mouse insulin gene to generate a product of 374 bp. Reaction mixtures were as above, except 50 pmol oligonucleotides were used and the PCR conditions were 95 (C for 2 min, 30 cycles of 92 (C for 30 s, 60 (C for 30 s and 72 (C for 1 min, and a final cycle at 72 (C for 5 min.
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2.3. Neonatal thymectomy Neonatal thymectomy was performed on 3-day-old BALB/cCrSlc or BALB/cCrSlc TNFR1o/o mice under cold anaesthesia as described [8]. Briefly, a small incision was made over the sternum to expose the rib cage and a small notch was removed to the second intercostal joint. The exposed fascia was gently torn with fine pointed forceps to expose the thymus gland. Under gentle suction, the thymus was removed with a glass pipette. Mice were placed under a heat source and returned to their mother once they had regained activity. 2.4. In vitro testing of anti-TNF antibody TNF-sensitive WEHI 164 murine fibrosarcoma cells (obtained from Dr J. Ruby, University of Melbourne) cultured to less than 80% confluency were treated with 2 µg/ml actinomycin D (Sigma, St Louis, MO, USA) at a concentration of 7.5105 cells/ml for 1 h at 37 (C. Fifty µl aliquots of either anti-mouse TNF CV1q (Centocor, Malvern, Pennsylvania, USA) or mouse IgG2a-kappa chain isotype control (Sigma) at 1 mg/ml were mixed with 50 µl serially diluted mouse recombinant TNF (0.1 ng to 0.001 ng) (Sigma) and incubated at 37 (C 10% CO2 for 2 h. One hundred µl (7.5104) actinomycin D-treated WEHI 164 cells were added to the mixture and incubation continued at 37 (C 10% CO2 for a further 18 h. Cell suspensions were stained with propidium iodide and analysed by flow cytometry (FACScalibur and CellQuest software, Becton Dickinson, Mountain View, CA, USA) for detection of dead cells. 2.5. In vivo anti-TNF injections Day 3 neonatally thymectomised BALB/cCrSlc mice were administered with anti-mouse TNF antibody (cV1q) (Centocor), or mouse IgG2a-kappa chain (Sigma) as an isotype control. Test and control groups were weighed and injected intra-peritoneally with immunoglobulin at a dose of 5 mg/kg in 50–100 µl sterile PBS. Mice were injected either with a single dose on day 4 (1 day following neonatal thymectomy) or twice weekly from day 4 to 8 weeks of age. Mice were killed at 10 weeks of age and assessed for production of parietal cell autoantibodies, H+/K+ ATPase reactivity and histological evidence of gastritis. 2.6. ELISA Enzyme-linked immunosorbent assay was used to screen mouse sera for H+/K+ ATPase reactive autoantibodies as previously described [8]. Alternative rows of round-bottomed microtitre plates were coated with 50 µl
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purified porcine gastric H+/K+ ATPase [31] in 0.5 M carbonate–bicarbonate buffer, pH 9.6. Unbound sites were blocked with 200 µl of PBS/1% BSA at 4 (C overnight and plates stored at 20 (C. Plates were incubated with 50 µl of mouse sera at a dilution of 1/50 in PBS/0.05% Tween-20 for 1 h at room temperature. Plates were washed with PBS/0.05% Tween 20 and 50 µl biotinylated anti-mouse IgG (Amersham Pharmacia Biotech) (diluted 1:500 in PBS/0.1% BSA) added to each well for 1 h at room temperature. Plates were washed and 50 µl streptavidin-biotinylated horseradish peroxidase (HRPO) complex (Amersham Pharmacia Biotech) (1:500 in PBS/0.1% BSA) was added to each well. Bound HRPO activity was detected with 100 µl substrate containing 0.2 mg/ml O-phenylenediamine (Sigma, Castle Hill, Australia) and 0.006% H2O2 for 30 min at room temperature in the dark. The reaction was terminated by addition of 50 µl of 5 M H2SO4. Absorbance was measured at 490 nm. Background reactivity determined from wells lacking H+/K+ ATPase was subtracted from absorbance of coated wells. Controls for ELISA included monoclonal antibodies 1H9 and 2B6 specific for the - and -subunits of the gastric H+/K+ ATPase respectively, an irrelevant monoclonal antibody ET1 as an isotype control and mouse sera with known reactivity with H+/K+ ATPase. All sera and controls were tested in duplicate. For testing autoantibody reactivities at different dilutions, 50 µl of sera at a range of dilutions from 1/50 to 1/12150 (serial one-in-three dilutions) was added to the porcine gastric H+/K+ ATPase coated plates and incubated at room temperature for 1 h. The remaining steps were as described above. 2.7. Immunohistochemistry Anti-parietal cell autoantibodies were detected by indirect immunofluorescence on 4 µm sections of freshly frozen or tannic acid-processed, paraffin-embedded normal mouse stomachs. Sections were blocked with PBS/1% normal swine serum for 15 min at room temperature. Sections were rinsed in PBS/0.05% Tween-20 and incubated for 1 h at room temperature with mouse sera (diluted 1/25 in PBS/0.05% Tween-20). Slides were washed 25 min in PBS/0.05% Tween-20 and incubated with either FITC-conjugated sheep anti-mouse Ig (Silenus, Melbourne, Australia) (diluted 1/50 in PBS/ 0.05% Tween-20) or goat anti-mouse IgG Alexa 488 Fluor (Molecular Probes, Eugene, Oregon) (1/2000 in PBS/0.05% Tween-20). Following two washes in PBS/ 0.05% Tween-20, sections were mounted with mounting solution and examined by epifluorescence microscopy. 1H9 and 2B6, monoclonal antibodies specific for the and -subunits of the H+/K+ ATPase respectively, and previously tested positive and negative sera were used as controls.
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2.8. Histology Stomachs were fixed in 10% phosphate buffered formalin and processed using an automated Shandon Hypercentre 2 (Shandon Southern Instruments, Sewickley, PA, USA). Tissues were embedded in paraffin wax and 4 µm sections cut and stained with haematoxylin and eosin. Gastritis was scored as normal if no infiltrate was present in the submucosa, non-destructive gastritis if infiltrate was present within the mucosa without destruction of parietal and zymogenic cells and destructive gastritis with infiltrate and cellular damage. 2.9. Flow cytometry For flow cytometry analysis, 1–2106 cells from the thymus, spleen or paragastric lymph nodes were stained in 30 µl volumes containing FITC-anti-CD4 (clone RM4-5) (Pharmingen, San Diego, CA, USA), PerCPanti-CD8 (clone 53-6.7) (Pharmingen) and PE-antiCD25 (IL-2-chain, clone PC61) (Pharmingen) diluted in Hanks’ buffered salt solution (Gibco BRL/Life Technologies)/1% FCS/0.02% sodium azide. Cells were analysed on a FACScan using CellQuest software (Becton Dickinson). 2.10. Statistics The Fischer exact probability test (GraphPad InStat 2.00 for Win 95/NT 1997) was used to analyse gastritis incidence between two groups. Cell numbers were compared using the Kruskal–Wallis test (non-parametric ANOVA) and the Dunn post test (GraphPad InStat). Autoantibody reactivities were compared using a Mann–Whitney U test (GraphPad InStat).
3. Results 3.1. Administration of anti-TNF antibody does not abrogate thymectomy-induced experimental autoimmune gastritis To examine the potential benefit of anti-TNF administration in the treatment of autoimmune gastritis, we examined the development of thymectomy induced EAG following a single dose or prolonged exposure to neutralising anti-TNF antibody. The biological activity of the anti-TNF CV1q antibody was confirmed on TNF sensitive WEHI 164 cells (data not shown). Following a single injection of anti-TNF at day 4 of age, one day after thymectomy, 11/14 (79%) mice developed gastritis at 10 weeks of age, comprising a mononuclear infiltrate within the gastric mucosa (Fig. 1A and D), with the majority (10/14) showing evidence of parietal and zymogenic cell loss (Fig. 1A and E). All mice with gastritis also developed parietal cell autoanti-
bodies reactive with gastric H+/K+ ATPase (Fig. 1A). In comparison, 4/6 (67%) mice injected with IgG2a-kappa chain control antibody developed gastritis and autoantibodies (P>0.05, anti-TNF compared to isotype control) (Fig. 1A). Interestingly, the incidence of destructive gastritis appeared to be increased in anti-TNF treated mice compared to control mice (P<0.05). However, the incidence of destructive gastritis following neonatal thymectomy is consistent with the observed frequency from previous findings. To examine the influence of the anti-TNF antibody over an extended period in our model system, we injected a group of mice twice a week for 8 weeks following neonatal thymectomy. Three of 6 (50%) mice developed gastritis and autoantibodies, with two mice developing destructive gastritis (Fig. 1B). In comparison, 3/4 (75%) IgG2a-kappa chain injected mice developed gastritis with parietal cell specific autoantibodies (Fig. 1B) (P>0.05, anti-TNF compared to isotype control). There was no statistical difference between incidence of destructive gastritis in anti-TNF treated mice compared to control mice (P>0.05). Likewise, there was also no statistical difference in the incidence of autoantibodies or destructive gastritis between the single and long-term injected groups. 3.2. BALB/cCrSlc mice deficient in TNFR1 develop experimental autoimmune gastritis following neonatal thymectomy To examine the role that TNFR1-mediated signaling may play in the pathogenesis of EAG, we assessed the incidence of EAG in susceptible BALB/cCrSlc mice deficient in TNFR1. TNFR1 gene targeted mice were backcrossed six times to BALB/cCrSlc mice and heterozygous TNFR1w/o mice intercrossed and litters thymectomised at day 3 of age. At 14 weeks of age, mice were killed and assessed for production of parietal and H+/K+ ATPase reactive autoantibodies and histological evidence of gastritis. Overall, we found no statistical difference in the incidence of autoantibody reactivity or gastritis between the various genotypes (Fig. 2). As expected, the majority of TNFR1w/w mice (4/5) developed characteristics of EAG including parietal cell associated H+/K+ ATPase reactivity (Figs. 2 and 3A) and gastritis with associated tissue damage (Fig. 3E). We found 12/16 (75%) BALB/cCrSlc TNFR1w/o mice developed gastritis with the majority (10/16) displaying mucosal damage (Figs. 2 and 3F) and parietal cell specific autoantibodies (Figs. 2 and 3B). Finally, 11/14 (79%) BALB/cCrSlc TNFR1o/o mice developed gastritis characterised by a prominent gastric infiltrate extending into the lamina propria of the mucosa, with destruction of zymogenic and parietal cells apparent in the majority of these mice (8/14) (Figs. 2 and 3G). All of the TNFR1o/o mice with gastritis developed antibodies to parietal cells
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Fig. 1. Mice were thymectomised at day 3 and injected either at (A) day 4 or (B) twice weekly for 8 weeks with either anti-TNF antibody or IgG2a as an isotype control. Mice were assessed at 10 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes. Gastritis was graded as either normal (C); non-destructive gastritis if cellular infiltrate was present within the gastric mucosa (arrow) (D); or destructive gastritis if infiltrate was present in addition to parietal and zymogenic cell destruction (arrow-head) (E). Bar: 100 µm.
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Fig. 2. Incidence of autoimmune gastritis in neonatally thymectomised BALB/cCrSlc TNFR1 deficient mice. C57/BL-6 TNFR1o/o mice were backcrossed six times to BALB/cCrSlc and intercrossed to generate wild type (w/w), heterozygous (w/o) and TNFR1 deficient (o/o) mice. Mice were thymectomised at day 3 and assessed at 14 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes.
and the gastric H+/K+ ATPase (Figs. 2 and 3C). In addition, we quantified the total number of cells obtained from the spleens and paragastric lymph nodes of these mice and found no difference in the total number of cells and CD4+ and CD8+ T cell populations within these organs between genotypes (data not shown). 3.3. PC-GMCSF transgenic mice which spontaneously develop experimental autoimmune gastritis develop disease in the absence of TNFR1 We have recently described a transgenic mouse model that spontaneously develops EAG due to the transgenic expression of the pro-inflammatory cytokine GM-CSF in gastric parietal cells [11]. Unlike many other models of EAG (such as neonatal thymectomy) that are associated with an induced state of lymphopenia [32], PCGMCSF mice are not lymphopenic and the immune repertoire has not been targeted. PC-GMCSF transgenic mice develop EAG without further manipulation such as neonatal thymectomy and thus represent an alternative system to examine pathogenic mechanisms associated with EAG [11]. Therefore we examined the induction of EAG in TNFR1o/o mice that were also PC-GMCSF transgenic. All PC-GMCSF transgenic (Tg) TNFR1w/w
mice (13/13) developed autoantibodies to gastric H+/K+ ATPase detected by ELISA, compared to 14/17 (82%) Tg-TNFR1w/o (P>0.05) and 6/11 (55%) Tg-TNFR1o/o mice (P<0.05 compared to Tg-TNFR1w/w mice, P>0.05 compared to Tg-TNFR1w/o mice) (Fig. 4). Similarly, all Tg-TNFR1w/w mice (13/13) developed parietal cellspecific autoantibodies, compared to 15/17 (88%) TgTNFR1w/o mice (P>0.05) and 7/11 (64%) Tg-TNFR1o/o mice (P<0.05 compared to Tg-TNFR1w/w mice) (Fig. 4). The observed difference in antibody reactivity of sera diluted 1/50 between Tg-TNFR1o/o and Tg-TNFR1w/w mice was reproduced over a range of serum dilutions and found that the reactivity of autoantibody positive sera was consistently lower in Tg-TNFR1o/o mice compared to Tg-TNFR1w/w mice (P=0.0073) (data not shown). Histological evidence of gastritis was found in all PC-GMCSF transgenic mice regardless of TNFR1 genotype. To some extent, this may be due to the pro-inflammatory properties of the transgenic PCGMCSF mice. In these groups of mice, destructive gastritis was evident in 5/13 (38%) PC-GMCSF transgenic TNFR1w/w mice, compared to 3/17 (18%) transgenic TNFR1w/o mice and 2/11 transgenic TNFR1o/o mice (18%) (P>0.05 between all groups) (Fig. 4). While
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Fig. 3. Parietal cell autoantibodies and gastritis in neonatally thymectomised mice. Sera from neonatally thymectomised (A) TNFR1w/w, (B) TNFR1w/o and (C) TNFR1o/o mice all developed parietal cell autoantibodies in contrast to normal mice (D). Haematoxylin and eosin stained stomach sections (E–H) of 14 week old thymectomised TNFR1w/w (E), TNFR1w/o (F) and TNFR1o/o (G) mice displayed prominent mononuclear cell infiltrate within the gastric mucosa (arrow), accompanied by cellular destruction (arrow-head) and tissue hypertrophy. These mice lacked zymogenic (Z) and parietal cell (P) regions, which are indicated in the stomach of a normal mouse (H). Bar: 100 µm.
there was no statistical difference within these groups, the incidence of destructive gastritis in the TNFR1 genotypes was lower in PC-GMCSF transgenic mice compared to neonatally thymectomised mice (Table 1). These differences may relate to the different methods of disease induction of the two groups. The total number of cells obtained from the spleens and paragastric lymph nodes of these mice were similar, with no differences in cell proportions within these organs between genotypes (data not shown).
4. Discussion The mechanisms of targeted cell death in many autoimmune diseases are poorly understood. In the case of autoimmune gastritis, we have recently reported that the Fas pathway plays a major role in target cell loss in this disease [15]. In the present study we have examined for a role for TNF in EAG. The effect of neutralising the activity of TNF was assessed in two studies of EAG induced by neonatal
thymectomy in which the neutralising antibody was administered either as a single dose or as repeated doses over 8 weeks. We found that administration of neutralising anti-TNF antibody as a single dose did not reduce the incidence of autoimmune gastritis. It is possible that this may have been due to the short period of anti-TNF antibody action and to address this, a second group of neonatally thymectomised mice were injected twice weekly over an 8 week period. This second group of mice also developed autoantibodies and destructive gastritis, suggesting that TNF may not be required for disease induction or gastric parenchymal cell death. However, it is possible that the injected anti-TNF antibody, although now present in higher amounts than the single injection, and for a prolonged period of time, may not have efficiently accessed the gastric mucosa. Using a more definitive approach, we assessed the induction of EAG in TNFR1 (p55) deficient mice, since the majority of, if not all, of the apoptotic potential of TNF is mediated through TNFR1 [14,29]. TNFR1 deficient mice were backcrossed to the gastritissusceptible BALB/cCrSlc mouse strain and subjected to
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Fig. 4. Incidence of autoimmune gastritis in PC-GMCSFTNFR1o/o mice. C57/BL-6 TNFR1o/o mice were backcrossed six times to BALB/cCrSlc, then crossed with BALB/cCrSlc PC-GMCSF transgenic mice. Mice were intercrossed to generate PC-GMCSF transgenic mice that were either TNFR1o/o, TNFR1w/o or TNFR1w/w. Mice were assessed at approximately 20 weeks for H+/K+ ATPase antibodies by ELISA, parietal cell autoantibodies by indirect immunofluorescence (IF) and gastritis by haematoxylin and eosin staining of paraffin embedded stomach sections. ELISA reactivity is indicated by absorbance at 490 nm. Controls included positive (+) and negative () sera. Parietal cell reactivity is indicated by filled boxes. Gastritis was scored as destructive (filled box) or non-destructive (striped box). Lack of parietal cell reactivity or gastritis is indicated by open boxes. Table 1 Incidence of destructive gastritis in TNFR1 genotypes in neonatally thymectomised and PC-GMCSF transgenic mice EAG induction method
Neonatal thymectomy PC-GMCSF Tg P valuea a
TNFR1 genotype o/o
w/o
w/w
8/14 (57%) 2/11 (18%) 0.099
10/16 (63%) 3/17 (18%) 0.013
2/5 (40%) 5/13 (38%) 1.000
Fischer exact statistical test used.
neonatal thymectomy or crossed with PC-GMCSF transgenic mice. Following neonatal thymectomy, all TNFR1 genotypes developed characteristics of EAG, including autoantibody production, mononuclear cell infiltrate and cellular destruction within the gastric mucosa. A similar finding was observed in TNFR1 gene targeted mice crossed on to PC-GMCSF transgenic mice. PC-GMCSF transgenic mice have recently been described as a model for EAG in which the proinflammatory cytokine GM-CSF is transgenically expressed in gastric parietal cells [11]. In both models, it was evident that the absence of TNFR1 did not affect the induction of EAG compared to wild type littermates. These results suggest that TNFR1 is not required on the parietal cell
surface to receive an apoptotic signal from infiltrating cells following neonatal thymectomy and that TNFR1 does not have a pro-inflammatory role in EAG. However, a difference that did become apparent between these two models was the reduced incidence of destructive gastritis in the PC-GMCSF model compared to the thymectomy model (Table 1). While these were two experiments performed on different groups of animals, the outcome probably relates to the different methods and mechanisms associated with each model. Neonatal thymectomy is thought to deplete CD4+CD25+ regulatory cells in the periphery [33,34], resulting in activation and expansion of pathogenic CD4+ T cells. In contrast, the PC-GMCSF transgenic model has not targeted the lymphoid compartment but instead is likely to have activated the immune response through the local pro-inflammatory action of GM-CSF in the gastric mucosa. Indeed we have shown that PC-GMCSF transgenic mice maintain a functional CD4+CD25+ T cell population [11]. It is possible that in PC-GMCSF transgenic mice, immune regulation has not been totally perturbed and still has some influence on the pathogenesis of EAG. While an immune response is clearly generated, evident by the induction of parietal cell and H+/K+ ATPase specific autoantibodies, cellular
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destruction may be limited by the presence of the regulatory T cell population. This observation may be similar to the non-pathogenic insulitis observed around pancreatic islets in mouse models of diabetes prior to development of clinical diabetes associated with cellular infiltrates in the pancreas and -cell destruction [35,36]. The apparent absence of a role for TNF in the genesis of autoimmune gastritis should be viewed in the context of a role for this molecule in other human and experimental autoimmune diseases. For instance in murine collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis, neutralising anti-TNF antibody decreased production of IL-1 and other proinflammatory cytokines [22]. Combined with anti-CD4 antibody [23] or anti-IL-12 antibody [24], anti-TNF therapy reduced swelling and histological severity of arthritis and joint erosion. However, TNF is not an absolute requirement for development of CIA [37,38]. Although some TNFR1o/o mice had reduced clinical parameters of CIA, severe disease, in addition to lymphadenopathy, was observed in many mice [37]. The antibody neutralisation experiments suggested that antiTNF monoclonal antibody may be used to treat rheumatoid arthritis. Indeed anti-TNF mAb trials in rheumatoid arthritis reduced disease severity [26]. Clinical trials of infliximab, a chimeric anti-TNF mAb [28,39,40] showed that TNF regulates local chemokine and cytokine production, recruitment of inflammatory cells into joints, angiogenesis, and blood levels of matrix metalloproteinases-1 and -3 [3]. Infliximab also gave promising results in Chrohn’s disease refractory to conventional drugs [27,28,41–44], active ankylosing spondylitis, psoriatic arthritis [40] and Sjogren’s syndrome [45]. Infliximab induced apoptosis in monocytes from patients with Chrohn’s disease suggesting that the antibody reduced the number of activated cells at the inflamed site [43]. However, the results of our present study and previous observations suggest that anti-TNF antibody treatment may not be suitable for all autoimmune diseases. For instance, whereas a single administration of anti-TNF mAb in neonatal NOD mice completely prevented diabetes [46], administration at later time points increased disease incidence [47]. The former result is consistent with the observation that mice lacking TNFR1 were protected from diabetes [16]. Transgenic TNF expression within -islet cells of the pancreas reduced T cell responses [48] and protected transgenic NOD mice from diabetes and -cell destruction [17,18]. However, in a transgenic model of virally induced diabetes (rat insulin promoter-lymphocytic choriomeningitis virus), early islet-specific TNF expression enhanced disease incidence, while late TNF expression abrogated disease [19]. TNF has also been implicated in multiple sclerosis [49] and neutralisation of TNF in experimental autoimmune encephalomyelitis (EAE)
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[20,21] gave promising results. However, mice in which TNF and LT genes were inactivated were still susceptible to EAE initiated by immunisation with mouse spinal cord homogenate suggesting that these molecules are not essential for EAE development [50]. Furthermore, in a large clinical study of multiple sclerosis, systemic blockade of TNF with Lenercept resulted in detrimental clinical effects [51]. The situation is further complicated because TNFR1 has the capacity to bind ligands other than TNF including lymphotoxin (LT), that can induce cytotoxic effects on T cells [52]. In summary, our studies suggest that TNFR1 mediated apoptosis does not have a role in experimental autoimmune gastritis. Using neutralising antibody and gene targeted approaches, we found EAG induction was not compromised with the neutralisation of TNF or in the absence of TNFR1. Indeed our previous study [15] suggests that the main pathway of cellular destruction in EAG is associated with the Fas pathway of cell death. In addition and as a general point, it also needs to be emphasised that not all autoimmune diseases may respond favorably to anti-TNF treatment and that even when beneficial effects are recorded in animal models, caution should be taken in extrapolating these to the human disease.
Acknowledgements We thank Centocor (Malvern, Pennsylvania) for providing the CV1q anti-TNF antibody and Dr Janet Ruby (University of Melbourne) for the C57/BL-6 TNFR1 knockout mice and WEHI164 cells. This work is supported by grants from the National Health and Medical Research Council (NHMRC) of Australia and The Alfred Hospital. A.C.J.M is a recipient of an Australian post-graduate scholarship.
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