22 Hemsworth, G.R. and Kochan, 1. (1978) Infect. tmmun. 19, 170-177 23 Rook, G.A.W. (1988) Br. Med. Bull. 44, 611-623 24 Klebanoff, S.J. (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J.I., Goldstein, I.M. and Snyderman, R., eds), pp. 391-444, Raven Press 25 Green, S.J., Mellouk, S., Hoffman, S.L., Meltzer, M.S. and Nacy, C.A. (1990) Immunol. Lett. 25, 15-20 26 Stuehr, D.J. and Marietta, M.A. (1987) J. lmmunol. 139, 518-525 27 Long, E.R. (1958) The Chemistry and Chemotherapyof Tuberculosis (3rd edn), pp. 106-108 and 122-124, Williams and Wilkins 28 Kaufmann, S.H.E. (1989) Trop. Med. Parasitol.40, 251-257 29 Abou-Zeid, C., Harboe, M. and Rook, G.A.W. (1987) Infect. immun. 55, 3213-3214 30 Dannenberg, A.M., Jr (1990) Res. Microbiol. 141, 192-196 and 262-263 31 Comstock, G.W. (1988) Am. Rev. Respir. Dis. 138, 479480
32 Fine, P.E.M. (1989) Rev. Infect. Dis. 11 (Suppl. 2), $353-$359 33 Dannenberg, A.M., Jr (1968) Bact. Rev. 32, 85-102 34 Dannenberg, A.M., Jr, Meyer, O.T., Esterly, J.R. and Kambara, T. (1968) J. Immunol. 100, 931-941 35 Ando, M., Dannenberg, A.M., Jr, Sugimoto, M. and Tepper, B.S. (1977) Am. J. Pathol. 86, 623-634 36 Mackaness, G.B. (1968) Am. Rev. Respir. Dis. 97, 337-344 37 North, R.J. (1974) in Mechanisms of Cell-mediated Immunity (McCluskey, R.T. and Cohen, S., eds), pp. 185-219, John Wiley and Sons 38 Collins, F.M. and Campbell, S.G. (1982) Vet. Immunol. Immunopathol. 3, 5-66 39 Dannenberg, A.M., Jr and Sugimoto, M. (1976) Am. Rev. Respir. Dis. 113, 257-259 40 Yamamura, Y. (1958) Adv. Tuberc. Res. 9, 13-37 41 Yamamura, Y., Ogawa, Y., Maeda, H. and Yamamura, Y. (1974) Am. Rev. Respir. Dis. 109, 594--601 42 Shima, K., Dannenberg, A.M., Jr, Ando, M. et al. (1972) Am. J. Pathol. 67, 159-180
Molecular targets in pernicious anaemia Paul A. Gleeson and Ban-Hock Toh Autoimmune gastritis, leading to pernicious anaemia, is an organ-specific autoimmune disease characterized by chronic atrophic gastritis and circulating gastric parietal cell autoantibodies. The parietal cell autoantigens have recently been identified as the c~ and f3 subunit of the gastric proton pump (H+,K÷ ATPase). Here Paul Gleeson and Ban-Hock Toh discuss how the identification of these gastric parietal cell autoantigens and the development of a mouse model of autoimmune gastritis have paved the way for an understanding of the pathogenesis of the gastric lesion. Pernicious anaemia is predominantly a disease of whites of northern European origin and is considered to be the most common cause of vitamin B12 deficiency in Western populations 1. The disease is characterized by pathological lesions of type A chronic atrophic gastritis, affecting the fundus and body of the stomach, that are typified by gastric mucosal atrophy, selective loss of parietal and chief cells from the gastric mucosa and submucosal lymphocytic infiltration 2. Autoantibodies to gastric parietal cells and to intrinsic factor, itself a secretory product of parietal cells, are found in the circulation and in gastric secretions1,3. These observations have suggested that the acid-secreting, gastric parietal cell is the principal cell targeted in the disease (see Fig. 1) and since the brunt of the disease falls on the gastric mucosa, the condition can appropriately be called an 'autoimmune gastritis'. Longitudinal studies have suggested that pernicious anaemia is the end stage of type A chronic atrophic gastritis 4, the estimated prevalences of which are 0.1% and 1% respectively 1,s. The anaemic condition is a direct consequence of the lack of intrinsic factor, a product of the gastric
mucosa that is required for the dietary absorption of vitamin B12. A genetic predisposition to the disease is suggested by familial occurrence of pernicious anaemia and the presence of parietal cell autoantibodies, and of associated type A chronic atrophic gastritis, in 20-30% of relatives of patients with pernicious anaemia 1,3. In addition, these relatives have a higher than normal frequency of autoantibodies to antigens of the other organ-specific autoimmune diseases of the endocrine glands that cluster with pernicious anaemia. A number of studies have investigated the association of HLA genes with pernicious anaemia but, while an increased frequency of a number of major histocompatibility complex (MHC) alleles have been reported, the associations are generally weak and have not been observed in all studies 1,3,6. Recent studies have established that the major molecular targets recognized by the parietal cell autoantibodies in pernicious anaemia are the o~ and 13 subunits of the gastric H+,K + ATPase (gastric proton pump). These findings, together with the availability of a well
© 1991, Elsevier Science Publishers Ltd, UK. 0167--4919/91/$02.00
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~Fundus Cardia ~ ? Pyl°rusi~-~ Body (corpus) ........
other tissues or cell types, but do bind parietal cells of most species. This lack of species specificity is typical of antibodies generated in most autoimmune diseases. Parietal cell autoantibodies are found in the sera of >90% of patients with pernicious anaemia, in 20-30% of relatives of patients with pernicious anaemia and in 5% of the healthy adult population 1,3.In patients, the autoantibody titre correlates with the severity of gastric atrophy.
Parietal cell autoantigens The first clue to the nature of the parietal cell autoGastric antigen was its ultrastructural localization to the secretory canaliculi of gastric parietal cells8 and to the ace mucous cells gastric 'microsomes'9. The parietal cell secretory canaliculi are channels for the passage of hydrochloric acid into the gastric lumen. These channels are absent in the resting cell but appear when cells are stimulated to secrete 0us neck cells acid by a variety of ligands including histamine, acetylcholine and gastrin. In the unstimulated cell, abundant Neck tubulovesicles are observed in the apical cytoplasm of the cell. Stimulation of parietal cells has been proposed to ~tal cells result in the translocation and fusion of these intracellular tubulovesicles to form the secretory canalicnli which are continuous with the apical surface of the cell (Ref. 10 )crine cell and Fig. 1(c)). An alternative hypothesis, which does not Base require membrane fusion, proposes that the tubulovesicular array is always confluent with the apical surf cells face, but is in a totally collapsed condition in the nonsecreting cell11. The major proteins of parietal cell canalicular and tubulovesicular membranes are the catalytic ~ subunit of the gastric proton pump 12 and a 60(c) Apical ,/, HCI 90 kDa glycoprotein13,14.The c~subunit has a molecular mass of 114kDa based on amino acid sequence, but migrates as a 92-95 kDa component on sodium dodecyl Resting ~//~oL ~ ' ~ Stimulated sulphate polyacrylamide gel electrophoresis (SDSPAGE)is. Parietal cell autoantibodies have been found to specifically immunoblot two molecules, of apparent molecular mass 60-90 kDa 16and of 92 kDa 17, from gastric membrane preparations. The same autoantibodies immunoACh H Basal precipitated molecules of similar size, together with components of 100-120kDa and >200 kDa apparent Hg. 1. (a) The stomach, (b) the gastric gland and (c) a composite model of the molecular mass 18J9, from solubilized, iodinated gastric gastric parietal cell are shown. The shaded fundus and body of the stomach (a) membrane preparations. The 60-90kDa autoantigen are the parietal-cell-containing regions that are affected by autoimmune gas- comprises a 35kDa core protein which is heavily tritis. In the resting state, parietal cells (c) contain abundant intracellular tubulo- N-glycosylated. Binding of autoantibodies from sera vesicles (TV) and after activation via recepton for a variety of ligands, such as gastrin (G), acetylcholine (ACh) and histamine (H), secretory canaliculi (SC) are from a number of patients to the 60-90 kDa molecule is formed which are channels continuous with the apical surface of the cell. The influenced by both the carbohydrate and protein moieties membranes of the tubulovesicles and canaliculi contain the H+,K + A TPase of the autoantigen since treatment with N-glycanase or reduction of disulphide bonds reduces autoantibody (gastric proton pump). binding 18. To identify the 60-90 kDa autoantigen, a number of characterized mouse model to study autoimmune gas- monospecific reagents have been obtained and two strattritis, should allow rapid progress in the understanding of egies have been adopted for its purification, namely the immunopathogenesis of the gastric lesion and of the immunoaffinity chromatography with fractionated basis for the loss of tolerance to an organ-specific extra- human autoantibodies and lectin chromatography. thymic autoantigen. Initial purification of parietal cell autoantibodies, using a column of crude gastric autoantigen, allowed the Parietal cell autoantibodies subsequent generation of an immunoaffinity support for Autoantibodies to the gastric parietal cell were initially the successful isolation of the 60-90 kDa autoantigen2°. detected by a complement fixation test and subsequently In addition, polylactosamine-specific lectins, including by immunofluorescence staining of the cytoplasm of tomato and potato lectins, were found to specifically bind gastric parietal cells7. The autoantibodies do not bind to to tubulovesicular and canalicular membranes of gastric Immunology Today
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parietal cells; tomato lectin specifically interacts with the 60-90 kDa membrane autoantigen 13,zl. Two murine monoclonal antibodies have been generated, using isolated dog tubulovesicles as antigen, which also react with a 60-90 kDa gastric membrane glycoprotein 22. Both monoclonal antibodies react with the purified 60-90kDa autoantigen, and, in contrast to,the human autoantibodies, with the 35 kDa protein core =. As the monoclonal antibodies recognize the polypeptide backbone and bind all the 60-90 kDa autoantigen, the protein of the 60-90 kDa antigen must be a single molecular species.
60-90 kDa autoantigen: the [3 subunit of the gastric proton pump After a single-step chromatographic purification of the 60-90 kDa autoantigen from pig stomach using tomato lectin, trypdc peptide sequences were obtained from both the 60-90 kDa glycoprotein and the 35 kDa core protein 21. These sequences, together with the deduced full length amino acid sequence from cDNA clones encoding the autoantigen, showed a 33% sequence identity with the [3 subunit of the pig kidney Na+,K + ATPase 21, itself a 55 kDa glycoprotein with a 35 kDa core protein. A physical association between the autoantigen and the c~ subunit of the proton pump was shown by immunoprecipitation studies with the monoclonal antibodies to the autoantigen and with a rabbit antibody specific for the ~ subunit of the proton pump 21,22. Furthermore, immunoaffinity chromatography using one of the monoclonal antibodies to the 60-90kDa autoantigen showed that most of the ~ subunit copurified with the 60-90 kDa glycoprotein from detergent extracts of gastric membranes 21,=. The 60-90 kDa autoantigen was therefore identified as a [3 subunit of the gastric H+,K + ATPase (proton pump) (Fig. 2). The gastric H+,K + ATPase together with the Na+,K + ATPase and the Ca + ATPase belong to a family of ATPases characterized by a catalytic ~ subunit of approximately 100kDa. These c~ subunits have a high degree of amino acid sequence identity and are phosphorylated during their reaction cycles24. The Na+,K + ATPase was thought to be unique in possessing a 13 subunit. However, it is now established that the gastric proton pump also consists of two subunits 14,2L2s,26. In the case of the Na+,K + ATPase 27, the [3 subunit appears to be critical for the assembly and surface expression of a functional Na+,K + ATPase. Studies to determine if the same is true of the gastric proton pump 13 subunit are currently under way in a number of laboratories. The proton pump subunits as major antibody targets in autoimmune gastritis Karlsson et al,17 suggested that the 92 kDa parietal cell autoantigen detected by immunoblotting is the catalytic (~) subunit of the gastric proton pump, based on the mass of this component and on autoantibody-mediated reduction of enzyme activity from gastric membrane detergent extracts. This group also demonstrated autoantibody-mediated inhibition of ATPase activity associated with gastric tubulovesicular membranes 28. Components of 100-120 kDa and >200 kDa were also observed in immunoprecipitates with human parietal cell autoImmunology Today
Fig. 2. The model of the gastric H+,K + A TPase shows the (~subunit with eight transmembrane domains, the aspartic acid phosphorylation site (*P) and the FITC-binding site (#), which may be a component of the ATP-binding sitd 2. These sites are localized on the hydrophilic domain between transmembrane domains four and five, which is generally considered to be intracellular. However, a recent report indicates that the single potential N-glycosylation site found on this hydrophilic domain is indeed glycosylated23; this model is therefore likely to be an oversimplification. The pig ~ subunit contains a single hydrophobic domain and six potential N-glycosylation sites on the large extraceIlular carboxy-terminal domain. The molar ratio of the c~and ~ subunits is approximately 1:1; however, the arrangement of the two subunits shown is purely hypothetical.
antibodies18,19; an antibody specific for the (~ subunit of the proton pump (raised against a fusion protein expressed in bacteria) has been used to show that these higher molecular mass components are derived from the 92 kDa proton pump a subunit 22. This behaviour of the c~ subunit appears to be typical of highly hydrophobic proteins 29. Thus, all the evidence suggests that the c~and [3 subunits of the pump are major molecular targets for antibodies in autoimmune gastritis. The nature of the autoantigen detected by serum immunofluorescence with the cell surface membranes of gastric parietal cells3° and by cytotoxic autoantibody reactivity to parietal cells in vitro 31 remains unresolved since, to date, the only membrane molecules identified by immunoblotting or immunoprecipitation with parietal cell autoantibodies are the ~ and the [3 subunits of the gastric proton pump. In addition, the basis for the observed autoantibody inhibition of the binding of radiolabelled gastrin to isolated parietal cells 32, an observation disputed by Burman et al. zS, is not clear at this stage. The gastrin receptor is considered to be restricted to the basolateral surface, although the nature of the receptor has not yet been elucidated. As parietal cells are isolated from the gastric mucosa by enzyme digestion, it is possible that considerable membrane damage may occur during their preparation, resulting in loss of membrane polarity. It is possible, therefore, that these cell surface reactions with autoantibodies are due to interactions with the proton pump present on the membranes of the isolated parietal cells. If so, then the inhibition of gastrin binding could be attributed to steric hindrance after the binding of the autoantibodies to the proton Vol. 12 No. 7 1991
pump. These interactions observed in vitro may, therefore, not be relevant in vivo. The role of the autoantibodies in the pathogenesis of the disease is currently unresolved. A direct role for the circulating autoantibodies in parietal cell depletion from the gastric mucosa was suggested by an early study showing that passive transfer of human autoantibodies to rodents is accompanied by gastric atrophy 33 and also by the observation that sera containing parietal cell autoantibodies are cytotoxic to gastric parietal cells in vitro 31. Recent studies have shown that the [3 subunit is localized only to the intracellular membranes and the apical surface of parietal cells and is absent from the basolateral cell surface (J. Pettitt, unpublished). Further, the autoepitopes of the ot subunit may be localized on the cytoplasmic domains of this membrane protein 34. Since access of circulating autoantibodies to the proton pump of intact parietal cell in vivo may be problematic, a direct role for the anti-proton pump antibodies in mediating the disease must remain in question. However, it is possible that there are other autoantigens on the basolateral membrane of parietal cells that have not been detected by the biochemical techniques used, and that autoantibodies to these components may have a role in the genesis of the gastric lesion of the disease. Alternatively, autoantibodies to the proton pump in gastric secretions could have direct access to the apical surface of parietal cells: parietal cell autoantibodies have been detected in gastric secretions of patients with pernicious anaemia 3. However, the specificity of these autoantibodies has not been characterized and their potential pathogenic role in the acidic gastric environment has not been assessed. Recent data suggest that the titre of autoantibodies declines when the parietal cells are lost, indicating that the immune response is antigen driven 3s. Irrespective of the precise role of the parietal cell autoantibodies in the pathogenesis of the gastric lesion, it is therefore likely that an autoreactive T-cell response is also elicited in this disease. It will be important to determine the nature and specificity of this T-cell response. The parietal cells and also the pepsinogen-secreting chief cells are lost in the gastric lesion, whereas there is no decrease in the number of endocrine cells. The basis for the selective loss of chief cells is not known. Circulating autoantibodies do not react with the membranes of chief cells, although they may react with the secreted protein, pepsinogen 34. Again, it will be imperative to investigate the autoreactive T-cell response in patients with this disease, including reactivity to chief cells.
A mouse model of autoimmune gastritis In mice, neonatal thymectomy 2-4 days after birth induces organ-specific autoimmune diseases affecting a variety of endocrine organs and the stomach 36,37. The autoimmune response in one of these diseases, namely oophoritis, has been shown to be antigen driven, as removal of the ovaries prior to thymectomy prevents the development of an autoimmune response 38. The frequency of the particular organ-specific autoimmune disease depends on the genetic background of the mouse, with BALB/c strain showing the highest frequency of autoimmune gastritis36: 50-60% of BALB/c nu/+ mice develop autoimmune gastritis three months after neoImmunology Today
natal thymectomy 37. The gastric lesions of these mice are similar to those of human autoimmune gastritis in that there is selective loss of parietal and chief cells from the gastric mucosa coupled with a significant lymphocytic infiltration into this site. However, unlike the human disease, there is compensatory hyperplasia of the mucous cells with resultant thickening of the gastric mucosa. These mice also have parietal-cell-specific autoantibodies in their sera that show a similar pattern of reactivity by immunoblotting and by immunoprecipitation to that obtained with human parietal cell autoantibodies 19. Furthermore, sera from mice with induced gastritis have been shown to contain autoantibodies directed to the cx/[3 subunit of the proton pump 39. A number of monoclonal autoantibodies have been derived from these mice and two of these, representative of two sets of monoclonal autoantibodies, react with either the cxor the [3 subunit of the proton pump 39. This murine disease therefore appears to be an ideal model for the study of the human disease. Organ-specific autoimmunity can also be induced by administration of cyclosporin A (CsA) in the first week of neonatal life4°. While the molecular targets of the CsAinduced gastric autoimmune disease have not been defined, it is probable that they will also include the proton pump, given the similarity of the gastric lesion with that induced by neonatal thymectomy.
Immunopathogenesis of experimental autoimmune gastritis The immunopathogenesis of the mouse disease appears to be cell mediated, as autoimmune gastritis induced by neonatal thymectomy can be transferred by spleen cells but not by circulating autoantibodies (Ref. 3 7 and Fig. 3). The effector cells that cause autoimmune disease by adoptive transfer in this system are CD4+Lyt-l(CD5)hig h (Ref. 41), indicating a central role for autoreactive T cells in disease induction. Precursors of these pathogenic autoreactive T cells are present in the periphery of normal BALB/c mice; the phenotype of these precursor, or dormant, cells is CD4+Lyt-I(CD5) l°w (Ref. 42 and S. Sakaguchi, pers. commun.). The specificity of the T cells that can transfer autoimmune gastritis is not known; however, a T-cell-mediated response to an enriched parietal cell population has been demonstrated 37. Clearly, it will be important to establish whether these autoreactive T cells also recognize the proton pump subunits and, if so, whether T cells with this specificity can induce disease. Peptides derived from the intracellular proton pump may be expressed at the cell surface in the cleft of MHC class I molecules and thereby be recognized by pathogenic autoreactive T cells. While it is possible that the pathogenesis of the murine disease is different from that of the human condition, the murine model is consistent with the proposal that the human disease may also be mediated by T cells. There is now considerable evidence to indicate that one mechanism for maintainiffg tolerance of autoreactive T cells is clonal deletion during T-cell development in the thymus. It is likely, however, that there are additional mechanisms to maintain T-cell tolerance, especially to extrathymic antigens. It has been proposed that tolerance to extrathymic antigens, known as peripheral tolerance,
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Neonate . ~
2 Thy-1 + Adult
or cyclosporin A treatment
Z$ Serum /Sp'een
I Spleen cells
Autoimmune No Autoimmune No Autoimmune No gastritis disease gastritis disease gastritis disease
Fig. 3. Experimental autoimmune gastritis induced in mice by neonatal thymectomy or neonatal cyclosporin A treatment. Splenocytes from mice with autoimmune gastritis can transfer the disease to mice with a T-cell-deficient periphery, for example nu/nu, while splenocytes from the neonatally-treated mice cannot transfer the disease to normal mice. Transplantation of neonatal thymuses into nude mice can result in autoimmune gastritis, the autoreactive T cells therefore emigrate from the thymus and can expand in a T-cell-deficient periphery. In addition, T-cell-enriched populations from normal mice can prevent autoimmune gastritis in neonatally-treated mice, indicating that the autoreactive T cells are subjected to immunoregulation by other T cells present in the normal adult peripheral environment.
may be maintained by functional inactivation of autoreactive T cells (clonal anergy) 43 or by T cells with suppressor activity44. Clonal anergy of T cells has been demonstrated towards an MHC class I gene product expressed in the pancreatic islets of transgenic mice4s, whereas studies using inactivated, autoreactive T-cell clones as vaccines to suppress autoimmune disease suggest that peripheral T-cell tolerance can be maintained by clonal suppression46. The induction of autoimmune gastritis by neonatal thymectomy suggests that the responsible autoreactive lymphocytes (irrespective of whether they are directed towards the proton pump or some other parietal cell autoantigen) are not deleted from the lymphocyte repertoire during thymic development but seed the periphery prior to neonatal thymectomy at day 2-4. Autoimmune gastritis in susceptible strains of mice after neonatal thymectomy may be due to the removal of an immunoregulatory or 'suppressor' activity (Fig. 3). This suggestion is supported by the observation that spleen cells from thymectomized mice can transfer the disease to nude, but not to normal, mice47. In addition, nonfractionated splenic cells or T-cell-enriched (Thy-l+) spleen cells from adult mice have been shown to prevent the induction of autoimmune gastritis in CsA-treated animals, suggesting that an immunoregulatory or 'suppressor' T cell inhibits the activity of the autoreactive T cells4°. The phenotype of the 'suppressor' T-cell population naturally present in the periphery of the normal immune system is CD4+Lyt-l(CD5)hig h, (Ref. 48 and S. Sakaguchi, pers. commun.). The recent demonstration of the induction of autoimmune gastritis by the transplantation of neonatal thymuses into nude or irradiated mice also indicates that the autoreactive T cells can spontaneously expand and cause autoimmune disease when released into a T-celldeficient periphery 49.
An alternative explanation is that neonatal thymectomy prevents the normal deletion of the offending autoimmune T cells. It has been demonstrated that neonatal thymectomy of (C57BL/6 x AJ)F 1 (B6AF1) mice results in the enrichment of V~ 11 + T cells (I-E-reactive) in adult lymph nodes and spleen, compared with normal adult mice where the Vf311+ T cells would normally be deleted in the thymus s°. The enrichment of the same population of V~311+ cellssl and V~17a + cellss2 has also been observed after CsA treatment. It is therefore possible that tolerance to parietal cells may be attributed to the clonal deletion of T cells in susceptible strains and that the prevention of deletion of these cells by neonatal thymectomy or by CsA may have a role in the genesis of the gastric autoimmune disease. However, the capacity of neonatal thymuses to transfer the disease 49argues against this possibility. Furthermore, the V¢11 + T cells in the periphery of neonatally thymectomized mice have been shown to be nonfunctional in vitro s3. Studies of recombinant inbred strains of mice show that the susceptibility to gastritis is not associated with the H-2 haplotype but appears to be influenced by a minor histocompatibility locus36. Recent studies have indicated a close relationship between Mls-1 b and susceptibility to diseaseS4; however, Mls-1 a strains are not susceptible. T cells, expressing. V ~3.6and V ¢8.1 are known to be deleted m Mls-1 a strata mice, whereas these VB chains are present in Mls-I b mice55 . Clearly, it will be important to establish if there is a direct link between autoimmune gastritis and V~ gene use. Conclusion The cx and [3 subunits of the proton pump have been shown to be major targets recognized by the parietal cell autoantibodies in autoimmune gastritis and pernicious anaemia. The experimentally-induced mouse disease
Vol. 12 No. 7 1991
model will allow us (1) to identify the mechanisms which (in press) maintain T- and B-cell tolerance to the gastric proton 21 Toh, B.H., Gleeson, P.A., Simpson, R.J. et al. (1990) pump subunits, (2) to precisely identify the key molecules Proc. Natl Acad. Sci. USA 87, 6418-6422 in the induction of the autoimmune response, that is the 22 Jones, C.M., Toh, B.H., Pettitt, J.M. et al. Eur. J. T-cell and B-cell autoepitopes, the particular MHC mol- Biochem. (in press) 23 Tai, M.M., Im, W.B., Davis, J.P. et al. (1989) ecules that present these autoepitopes to lymphocytes Biochemistry 28, 3183-3187 and the TCR and Ig genes used for the recognition of the 24 Pedersen, P.L. and Carafoli, E. (1987) Trends Biochem. autoepitopes and (3) to explore the role of the auto- Sci. 12, 146-150 immune response to these subunits in the immuno- 25 Shull, G. (1990)J. Biol. Chem. 265, 12123-12126 pathogenesis of the disease. 26 Reuben, M.A., Lasater, L.S. and Sachs, G. (1990) Proc. NatI Acad. Sci. USA 87, 6767-6771 27 Horowitz, B., Eakle, K.A., Scheiner-Bobis, G. et al. The authors are indebted to Ian van Driel for stimulating (1990) J. Biol. Chem. 265, 4189-4192 discussions and critical appraisal of the manuscript, and gradu- 28 Burman, P., Mardh, S., Norberg, L. and Karlsson, F.A. ate students for many fruitful discussions. This work was (1989) Gastroenterology 96, 1434-1438 supported by the National Health and Medical Research Coun- 29 Hennessey, J.P. and Scarborough, G.A. (1989) Anal. Biochem. 176, 284-289 cil of Australia. 30 De Aizpurua, H.J., Ungar, B. and Toh, B.H. (1982) Clin. Exp. Immunol. 52, 341-349 31 De Aizpurua, H.J., Cosgrove, L., Ungar, B. and Toh, B.H. Paul Gleeson and Ban-Hock Toh are at the Dept of Pathol- (1983) New Engl. J. Med. 309, 625-629 ogy and Immunology, Monash University Medical School, 32 De Aizpurua, H.J., Ungar, B. and Toh, B.H. (1985) New Prahran, Victoria 3181, Australia. Engl. J. Med. 313,479-483 33 Tanaka, N. and Glass, G.B.J. (1970) Gastroenterology 58,482-494 References 34 Mardh, S. and Song, T-H. (1989) Acta Physiol. Scand. 1 Strickland, R. (1990) in Immunology and 136, 581-587 lmmunopathology of the Liver and Gastrointestinal Tract 35 Burman, P., Karlsson, F.A., Loof, L., Szeschi, P.B. and (Targan, S.R. and Shanahan, F., eds), pp. 535-546, IgakuBorch, K. (1991) Scand. J. Gastroenterol. 26, 207-214 Shoin 36 Kojima, A. and Prehn, R.T. (1981) Immunogenetics 14, 2 Strickland, R. and Mackay, I.R. (1973) Am. J. Dig. Dis. 15-27 18,426-440 37 Fukuma, K., Sakaguchi, S., Kuribayashi, K. et al. (1988) 3 Whittingham, S. and Mackay, I.R. (1985) in The Gastroenterology 94, 274-283 Autoimmune Diseases (Rose, N.R. and Mackay, I.R., eds), 38 Taguchi, O. and Nishizuka, Y. (1980) Clin. Exp. pp. 243-266, Academic Press Immunol. 42, 324-331 4 Irvine, W.J., Cullen, D.R. and Mawhinney, H. (1974) 39 Jones, C.M., Callaghan, J.M., Gleeson, P.A. et al. 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