- Email: [email protected]
Celiac disease pathogenesis: The plot thickens
September 2002
EDITORIALS
30. Kaplan KB, Burds AA, Swedlow JR, Bekir SS, Sorger PK, Nathke IS. A role for the adenomatous polyposis coli protein in chromosome segregation. Nat Cell Biol 2001;3:429 – 432. 31. Fodde R, Kuipers J, Rosenberg C, Smits R, Kielman M, Gaspar C, van Es JH, Breukel C, Wiegant J, Giles RH, Clevers H. Mutations in the APC tumour suppressor gene cause chromosomal instability. Nat Cell Biol 2001;3:433– 438. 32. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 1992;256:668 – 670. 33. Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S, Hofland N, van Dijk J, White R, Edelmann W, Kucherlapati R, Khan PM, Fodde R. Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli
939
protein involved in tumorigenesis and development. Genes Dev 1999;13:1309 –1321.
Address requests for reprints to: Vincent W. Yang, M.D., Ph.D., Division of Digestive Diseases, Department of Medicine, 201 Whitehead Biomedical Research Building, Emory University School of Medicine, 615 Michael Street, Atlanta, Georgia 30322. e-mail: [email protected]: fax: (404) 727-5767. Supported by grants from the National Institutes of Health (DK52230 and CA84197). © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35773
Celiac Disease Pathogenesis: The Plot Thickens See article on page 803.
eliac disease (CD) has taken center stage recently based on advances in diagnosis and greater understanding of the mechanisms underlying its pathogenesis. There have been several exciting discoveries. First, with the advent and broader application of antibody screening tests, the most useful being the immunoglobulin (Ig)A anti-endomysial antibody and the IgA anti-tissue transglutaminase antibody, it is now clear that CD is a relatively common disorder that occurs in perhaps as many as 1:150 to 1:350 individuals in the United States and Europe.1– 4 Moreover, as increasing numbers of new patients are identified based on antibody screening and confirmation by the gold standard of small intestinal mucosal biopsy, it has became apparent that the classic textbook case of patients presenting with symptoms and signs of severe malabsorption, diarrhea, and wasting is the exception, rather than the rule. This is leading to an increased awareness of the need to include CD in the differential diagnosis of individuals presenting with symptoms and signs as diverse as unexplained iron deficiency or osteopenia, to those with mildly abnormal liver function tests, or simply what seem at first to be vague gastrointestinal symptoms.5–7 Furthermore, CD often accompanies other diseases, and its diagnosis may be overlooked if the other disease is the sole focus of attention.8 Recently, it has been recognized that CD may have its onset or become clinically apparent in individuals being treated for hepatitis C with ␣-interferon, a major activator of host innate immunity.9 –11 It is almost 3 decades since the discovery that CD is strongly associated with specific HLA class II antigens.
C
Although initially the association was thought to be with the HLA class II antigen HLA-DR3 (now termed HLADR17), it is now known the primary association is with a HLA-DQ2 molecule that is encoded at the gene level by 2 alleles, DQB1*02 and HLA DQA1*0501.12,13 Approximately 95% of CD patients have DQ2 encoded by these alleles. CD patients can inherit these alleles together on one chromosome from a parent who is DR17, because DR17 is tightly linked to both DQB1*02 and HLA DQA1*0501. Alternatively, one allele can be inherited from each parent. In this case, one parent carries HLA-DR7 that is linked to the allele DQB1*02 and the other carries HLA-DR5 that is linked to the allele DQA1*0501. These latter DQ2 CD patients, who are more prevalent in Southern Europe, are heterozygous for DR7/DR5.13–15 The remainder of celiac patients (⬃5%) mostly have a DQ8 molecule encoded by DQB1*0302 and DQA1*0301 on a DR4 haplotype.13,16,17 CD is exceedingly rare in the absence of DQ2 or DQ8, and it appears that they are necessary, although not sufficient, for developing CD. Naturally, a key question that has been addressed over the past several years is how do these particular DQ molecules play a role in the pathogenesis of CD?13 Progress in answering this question has been quite remarkable. CD has been long recognized as an ideal model in which to elucidate how HLA class II molecules such as DQ2 or DQ8 contribute to disease pathogenesis.13 The diverse group of HLA class II molecules present in the human population function to bind protein fragments (peptides) in their peptide-binding groove, and these HLA-class II- peptide complexes are recognized by specific populations of CD4⫹ T cells. In CD, the association with HLA-DQ2 and DQ8 is unequivocal, the proteins in
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EDITORIALS
the dietary grains that activate disease are known (i.e., gluten in wheat, secalins in rye, and hordeins in barley) and, importantly, populations of potentially disease-relevant T cells from the intestinal mucosa of CD patients can be readily isolated and grown in culture.13,18 Thus, in contrast to other HLA-associated diseases (e.g., rheumatoid arthritis and insulin-dependent diabetes mellitus) where the antigens that cause disease are unknown and the T cells in the relevant patient tissues are difficult to obtain, many of the pieces necessary to understand a key aspect of CD pathogenesis are in place. However, when algorithms were developed for which type of peptides might bind in the peptide-binding groove of the DQ2 or DQ8 molecule, the gliadins and related proteins in barley and rye did not seem to fill the bill,13 raising questions about what otherwise seemed to be an obvious mechanism.19 After all, how could one envision that binding of peptides from these grain proteins to DQ2 or DQ8, and subsequent T-cell activation, was an essential step in disease pathogenesis if peptides from those grain proteins did not even seem to bind, or bound very weakly, to the relevant HLA-DQ2 or DQ8 molecules? Subsequently, it was suggested that deamidation of glutamines in gliadin peptides, possibly by gastric acid or intestinal enzymes, might generate better binding of those peptides to DQ2 and subsequent T-cell activation by converting glutamine residues to negatively charged glutamic acid.20 As so often happens, serendipity then entered the picture. In Germany, Schuppan et al.21 were attempting to identify the target antigen of anti-endomysial antibody that was being used so successfully as a diagnostic screening test for CD. They discovered that the target antigen of anti-endomysial antibody was an enzyme, tissue transglutaminase, also termed TG2.21 In Norway, Sollid et al., and in the Netherlands, Koning et al., knew that the CD-associated DQ2 and DQ8 molecules have a preference for binding peptides with a negative charge, and that negatively charged amino acids were notably lacking in the glutamine-rich proteins known to activate CD.22,23 They astutely recognized that tissue transglutaminase might be responsible for the deamidation of glutamine residues in those proteins and thereby generate negatively charged glutamic acid residues. Testing in their laboratories showed this to be the case.24,25 Moreover, some gliadin peptides in which glutamine was converted to glutamic acid by tissue transglutaminase bound more efficiently to DQ2 and DQ8 and could activate T cells grown from the small intestinal mucosa of CD patients.24,25 They had solved a dilemma and a mechanism emerged by which peptides derived from
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ingested grain proteins could activate antigen-specific T-cell responses in the intestinal mucosa of CD patients expressing the HLA-DQ2 or -DQ8 susceptibility molecules. These findings suggested novel ways for treating or preventing CD, with the ultimate hope being an alternative for the “gluten-free” diet. The search was now on to find which peptide in gluten and the other diseaseactivating dietary grains activates disease. Indeed, initial studies performed using T cells from adult CD patients indicated there might be only 1, or at most a few, peptides in wheat gluten that are responsible for T-cell activation in DQ2 or DQ8 CD patients.26,27 However, as often is the case, the plot thickens. Koning’s group studied T cells from children with CD, and found that there were multiple gliadin or glutenin peptides that could bind to DQ2 or DQ8 and activate mucosal T-cell populations from children with CD.28 Moreover, in any given individual, several different gliadin or glutenin peptides29 could activate mucosal T cells. Notably, those peptides often differed from patient to patient, deamidation of glutamines to glutamic acid was not a requirement, and one-half of the children did not respond to the previously reported gliadin peptides thought to play a dominant role in the activation of mucosal T cells from adult CD patients.28 This data led initially to the notion that perhaps there is a broad group of different gluten peptides, not deamidated as well as deamidated, that might activate CD in children, but with increasing age the T-cell repertoire responsive to gliadin in adults becomes increasingly narrow and focused on one or a few deamidated peptides. More recently, Koning et al. developed algorithms based on the spacing between glutamine and proline residues and the presence or absence of other amino acids in the peptide, to predict which peptides in the dietary grains become deamidated by transglutaminase and bind to DQ2, and are thereby candidates for activating mucosal T-cell populations from CD patients.30 Using algorithms of varying stringencies, they predicted there would be as many as 50 or more activating peptides in gluten, hordeins, and secalins. Consistent with recent reports that oats do not, or only rarely, activate CD,31,32 their algorithms found no, or at most a few, potential DQ2 or DQ8 binding peptides in avenins from oats that would be predicted to activate T cells in CD patients.30 In this issue of GASTROENTEROLOGY, Arentz-Hansen et al.33 show that DQ2 restricted mucosal T cells from adult celiac patients in fact also recognize a broader group of gliadin peptides than indicated in their previous published data,27 a finding also suggested in studies by
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Koning et al.28 Thus, in addition to the 2 major ␣-gliadin sequences and a ␥-gliadin sequence that are deamidated by TG2, bind to DQ2 and activate T cells from adult celiac patients, they describe several additional ␥-gliadin epitopes and one new ␣-gliadin sequence that can bind DQ2 and activate CD patient T cells.33 Although deamidation of glutamine residues by transglutaminase clearly favors the generation of peptides that activate DQ2 restricted T cells, they further report, as Koning noted in studies of children,28 that deamidation is not an absolute requirement for T-cell activation.33 In contrast to the prior findings of this group,34 their new findings render more plausible the notion that deamidation of peptides occurs as disease develops, but may not be required for the initial activation of mucosal T cells in the very early stages of disease. Notably, all of the sequences they detected are located in regions of gliadin with high proline content, and not all of those sequences would have been predicted by the Koning algorhithm.30 It is now evident that a relatively large number of peptides in dietary grains are capable of activating DQ2 and DQ8 T cells from CD patients, although this number is certainly not unlimited. This finding may have significant implications when considering alternative therapeutic approaches, at least to the extent those approaches are based on altering T-cell activation. One way to view CD pathogenesis is to consider the events that occur before T-cell activation, the T-cell activation events themselves as discussed previously, and the tissue-damaging mechanisms and events that ensue following T-cell activation. When viewed in that context, several interesting questions arise, most notably relevant to the events that precede T-cell activation. First, do the same peptides that activate T cells in in vitro studies, as presented herein, activate CD in vivo? Do the proteins that activate CD come in several flavors, i.e., those that activate early events leading to innate immune and inflammatory responses in those susceptible to CD, and those that ultimately active DQ2 and DQ8 restricted T cells. At least a few early studies suggest this may be the case.13,35–36 Moreover, this would not be surprising given that proteins in these dietary grains initially undergo changes in the intestinal tract related to their encounter with gastric acid and digestive enzymes, followed by additional changes after encounter with host antigen-presenting cells in the mucosa and mucosal enzymes such as tissue transglutaminase. In this regard, it is interesting that a recent study found that the prolineglutamine-rich epitopes present in the peptides that activate CD are normally resistant to enzymatic processing in the human intestinal tract and dipeptidyl pepti-
EDITORIALS
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dase IV and dipeptidyl carboxypeptidase I appear to be key rate-limiting enzymes in the digestive breakdown of those peptides.37 Is the unique amino acid composition of these proteins a clue as to why myriads of other dietary proteins do not seem to generate peptides that cause similar diseases, including in people with other HLA class II genes? Do these glutamine and proline-rich proteins or their partially digested peptides also have effects on innate responses of the intestinal mucosa and, if so, are those unique to patients susceptible to CD? What cells produce the TG2 that deamidates these peptides, and how is it, or is it, differentially released or active in CD patients? It seems elucidation of these and other early events represents a new critical frontier to conquer as we increase our knowledge of CD pathogenesis. Finally, it is clear that HLA-DQ2 genes are the predominant known genetic factor in CD. Nonetheless, given the prevalence of CD in the United States and Europe and the frequency of DQ2 and DQ8 in these populations, only approximately 1%–2% of individuals with DQ2 or DQ8 would be predicted to develop CD. Studies to detect other CD susceptibility genes, based on linkage analysis, have revealed additional potential genetic loci that may contain genes contributory to disease, but these associations are weak relative to the HLA association and, with a few exceptions, have not been confirmed when additional populations were studied.38 – 43 It is possible that different genes may play a role in disease susceptibility in different groups of patients, and the mechanisms by which those genes contribute to the pathogenesis of CD may be diverse. MARTIN F. KAGNOFF Department of Medicine University of California at San Diego La Jolla, California
References 1. Catassi C, Fabiani E, Ratsch IM, Coppa GV, Giorgi PL, Pierdomenico R, Alessandrini S, Iwanejko G, Domenici R, Mei E, Miano A, Marani M, Bottaro G, Spina M, Dotti M, Montanelli A, Barbato M, Viola F, Lazzari R, Vallini M, Guariso G, Plebani M, Cataldo F, Traverso G, Ventura A, et al. The coeliac iceberg in Italy. A multicentre antigliadin antibodies screening for coeliac disease in school-age subjects. Acta Paediatr Suppl 1996;412:29 –35. 2. Not T, Horvath K, Hill ID, Partanen J, Hammed A, Magazzu G, Fasano A. Celiac disease risk in the USA: high prevalence of antiendomysium antibodies in healthy blood donors. Scand J Gastroenterol 1998;33:494 – 498. 3. Weile I, Grodzinsky E, Skogh T, Jordal R, Cavell B, Krasilnikoff PA. High prevalence rates of adult silent coeliac disease, as seen in Sweden, must be expected in Denmark. APMIS 2001;109:745– 750. 4. Farrell RJ, Kelly CP. Diagnosis of celiac sprue. Am J Gastroenterol 2001;96:3237–3246.
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5. Bardella MT, Fraquelli M, Quatrini M, Molteni N, Bianchi P, Conte D. Prevalence of hypertransaminasemia in adult celiac patients and effect of gluten-free diet. Hepatology 1995;22:833– 836. 6. Kaukinen K, Halme L, Collin P, Farkkila M, Maki M, Vehmanen P, Partanen J, Hockerstedt K. Celiac disease in patients with severe liver disease: gluten-free diet may reverse hepatic failure. Gastroenterology 2002;122:881– 888. 7. O’Leary C, Wieneke P, Buckley S, O’Regan P, Cronin CC, Quigley CM, Shanahan F. Celiac disease and irritable bowel-type symptoms. Am J Gastroenterol 2002;97:1463–1467. 8. Barera G, Bonfanti R, Viscardi M, Bazzigaluppi E, Calori G, Meschi F, Bianchi C, Chiumello G. Occurrence of celiac disease after onset of type 1 diabetes: a 6-year prospective longitudinal study. Pediatrics 2002;109:833– 838. 9. Bardella MT, Marino R, Meroni PL. Celiac disease during interferon treatment. Ann Intern Med 1999;131:157–158. 10. Cammarota G, Cuoco L, Cianci R, Pandolfi F, Gasbarrini G. Onset of coeliac disease during treatment with interferon for chronic hepatitis C. Lancet 2000;356:1494 –1485. 11. Adinolfi LE, Mangoni ED, Andreana A. Interferon and ribavirin treatment for chronic hepatitis C may activate celiac disease. Am J Gastroenterol 2001;96:607– 608. 12. Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. Evidence for a primary association of celiac disease to a particular HLA-DQ ␣/ heterodimer. J Exp Med 1989;169:345–350. 13. Kagnoff MF. HLA genes in coeliac disease. In: Auricchio S, Greco L, Maiuri L, Troncone R, eds. Coeliac disease. Italy: JGC Editions, 2000:5–14. 14. Lundin KE, Sollid LM, Qvigstad E, Markussen G, Gjertsen HA, Ek J, Thorsby E. T lymphocyte recognition of a celiac disease-associated cis- or trans- encoded HLA-DQ ␣/-heterodimer. J Immunol 1990;145:136 –139. 15. Sollid LM, Thorsby E. The primary association of celiac disease to a given HLA-DQ ␣/ heterodimer explains the divergent HLA-DR associations observed in various Caucasian populations. Tissue Antigens 1990;36:136 –137. 16. Lundin KE, Scott H, Fausa O, Thorsby E, Sollid LM. T cells from the small intestinal mucosa of a DR4, DQ7/DR4, DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8. Hum Immunol 1994;41:285–291. 17. Lundin KE, Gjertsen HA, Scott H, Sollid LM, Thorsby E. Function of DQ2 and DQ8 as HLA susceptibility molecules in celiac disease. Hum Immunol 1994;41:24 –27. 18. Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. Gliadin-specific, HLA-DQ(␣ 1*0501, 1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med 1993;178:187– 196. 19. Johansen BH, Gjertsen HA, Vartdal F, Buus S, Thorsby E, Lundin KE, Sollid LM. Binding of peptides from the N-terminal region of ␣-gliadin to the celiac disease-associated HLA-DQ2 molecule assessed in biochemical and T cell assays. Clin Immunol Immunopathol 1996;79:288 –293. 20. Sjostrom H, Lundin KE, Molberg O, Korner R, McAdam SN, Anthonsen D, Quarsten H, Noren O, Roepstorff P, Thorsby E, Sollid LM. Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition. Scand J Immunol 1998;48:111–115. 21. Schuppan D, Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO. Identification of the autoantigen of celiac disease. Ann N Y Acad Sci 1998;859:121–126. 22. Johansen BH, Jensen T, Thorpe CJ, Vartdal F, Thorsby E, Sollid LM. Both ␣ and  chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules, with  chain residues being most influential. Immunogenetics 1996;45: 142–150. 23. van de Wal Y, Kooy YM, Drijfhout JW, Amons R, Koning F. Peptide
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binding characteristics of the coeliac disease-associated DQ(␣1*0501, 1*0201) molecule. Immunogenetics 1996;44: 246 –253. van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 1998;161:1585–1588. Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjostrom H, Sollid LM. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4:713–717. Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 2000;6:337–342. Arentz-Hansen H, Korner R, Molberg O, Quarsten H, Vader W, Kooy YM, Lundin KE, Koning F, Roepstorff P, Sollid LM, McAdam SN. The intestinal T cell response to ␣-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 2000;191:603– 612. Vader W, Kooy Y, Van Veelen P, De Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002;122:1729 –1737. van de Wal Y, Kooy YM, van Veelen P, Vader W, August SA, Drijfhout JW, Pena SA, Koning F. Glutenin is involved in the gluten-driven mucosal T cell response. Eur J Immunol 1999;29: 3133–3139. Vader LW, de Ru A, van der Wal Y, Kooy YM, Benckhuijsen W, Mearin ML, Drijfhout JW, van Veelen P, Koning F. Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 2002;195:643– 649. Janatuinen EK, Pikkarainen PH, Kemppainen, Kosma VM, Jarvinen RM, Uusitupa MI, Julkunen RJ. A comparison of diets with and without oats in adults with celiac disease. N Engl J Med 1995;333:1033–1037. Janatuinen EK, Kemppainen TA, Julkunen RJ, Kosma VM, Maki M, Heikkinen M, Uusitupa MI. No harm from five year ingestion of oats in coeliac disease. Gut 2002;50:332–335. Arentz-Hansen H, McAdam SN, Molberg Ø, Fleckenstein B, Lundin KEA, Jørgensen TJD, Jung G, Roepstorff P, Sollid LM. Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology 2002;123: 803– 809. Molberg O, McAdam S, Lundin KE, Kristiansen C, Arentz-Hansen H, Kett K, Sollid LM. T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol 2001;31:1317–1323. Maiuri L, Picarelli A, Boirivant M, Coletta S, Mazzilli MC, De Vincenzi M, Londei M, Auricchio S. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients. Gastroenterology 1996;110:1368 – 1378. Chowers Y, Marsh MN, De Grandpre L, Nyberg A, Theofilopoulos AN, Kagnoff MF. Increased proinflammatory cytokine gene expression in the colonic mucosa of coeliac disease patients in the early period after gluten challenge. Clin Exp Immunol 1997;107: 141–147. Hausch F, Shan L, Santiago NA, Gray GM, Khosla C. Intestinal digestive resistance of immunodominant gliadin peptides. Am J Physiol Gastrointestinal Liver Physiol 2002;10.1152/ajpgi.00136. Houlston RS, Tomlinson IP, Ford D, Seal S, Marossy AM, Ferguson A, Holmes GK, Hosie KB, Howdle PD, Jewell DP, Godkin A, Kerr GD, Kumar P, Logan RF, Love AH, Johnston S, Marsh MN, Mitton S, O’Donoghue D, Roberts A, Walker-Smith JA, Stratton
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MF. Linkage analysis of candidate regions for coeliac disease genes. Hum Mol Genet 1997;6:1335–1339. Greco L, Corazza G, Babron MC, Clot F, Fulchignoni-Lataud MF, Percopo S, Zavattari P, Bouguerra F, Dib C, Tosi R, Troncone R, Ventura A, Mantavoni W, Magazzu G, Gatti R, Lazzari R, Giunta A, Perri F, Iacono G, Cardi E, de Virgiliis S, Cataldo F, De Angelis G, Musumeci S, Clerget-Darpoux F, et al. Genome search in celiac disease. Am J Hum Genet 1998;62:669 – 675. King AL, Yiannakou JY, Brett PM, Curtis D, Morris MA, Dearlove AM, Rhodes M, Rosen-Bronson S, Mathew C, Ellis HJ, Ciclitira PJ. A genome-wide family-based linkage study of coeliac disease. Ann Hum Genet 2000;64:479 – 490. Naluai AT, Nilsson S, Gudjonsdottir AH, Louka AS, Ascher H, Ek J, Hallberg B, Samuelsson L, Kristiansson B, Martinsson T, Nerman O, Sollid LM, Wahlstrom J. Genome-wide linkage analysis of Scandinavian affected sib-pairs supports presence of susceptibility loci for celiac disease on chromosomes 5 and 11. Eur J Hum Genet 2001;9:938 –944. Liu J, Juo SH, Holopainen P, Terwilliger J, Tong X, Grunn A, Brito
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M, Green P, Mustalahti K, Maki M, Gilliam TC, Partanen J. Genomewide linkage analysis of celiac disease in Finnish families. Am J Hum Genet 2002;70:51–59. 43. Greco L, Babron MC, Corazza GR, Percopo S, Sica R, Clot F, Fulchignoni-Lataud MC, Zavattari P, Momigliano-Richiardi P, Casari G, Gasparini P, Tosi R, Mantovani V, De Virgiliis S, Iacono G, D’Alfonso A, Selinger-Leneman H, Lemainque A, Serre JL, Clerget-Darpoux F. Existence of a genetic risk factor on chromosome 5q in Italian coeliac disease families. Ann Hum Genet 2001;65:35– 41.
Address requests for reprints to: Martin F. Kagnoff, M.D., University of California at San Diego, Department of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0623. e-mail: [email protected]; fax: (858) 534-5691. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35840
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30. Kaplan KB, Burds AA, Swedlow JR, Bekir SS, Sorger PK, Nathke IS. A role for the adenomatous polyposis coli protein in chromosome segregation. Nat Cell Biol 2001;3:429 – 432. 31. Fodde R, Kuipers J, Rosenberg C, Smits R, Kielman M, Gaspar C, van Es JH, Breukel C, Wiegant J, Giles RH, Clevers H. Mutations in the APC tumour suppressor gene cause chromosomal instability. Nat Cell Biol 2001;3:433– 438. 32. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 1992;256:668 – 670. 33. Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S, Hofland N, van Dijk J, White R, Edelmann W, Kucherlapati R, Khan PM, Fodde R. Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli
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protein involved in tumorigenesis and development. Genes Dev 1999;13:1309 –1321.
Address requests for reprints to: Vincent W. Yang, M.D., Ph.D., Division of Digestive Diseases, Department of Medicine, 201 Whitehead Biomedical Research Building, Emory University School of Medicine, 615 Michael Street, Atlanta, Georgia 30322. e-mail: [email protected]: fax: (404) 727-5767. Supported by grants from the National Institutes of Health (DK52230 and CA84197). © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35773
Celiac Disease Pathogenesis: The Plot Thickens See article on page 803.
eliac disease (CD) has taken center stage recently based on advances in diagnosis and greater understanding of the mechanisms underlying its pathogenesis. There have been several exciting discoveries. First, with the advent and broader application of antibody screening tests, the most useful being the immunoglobulin (Ig)A anti-endomysial antibody and the IgA anti-tissue transglutaminase antibody, it is now clear that CD is a relatively common disorder that occurs in perhaps as many as 1:150 to 1:350 individuals in the United States and Europe.1– 4 Moreover, as increasing numbers of new patients are identified based on antibody screening and confirmation by the gold standard of small intestinal mucosal biopsy, it has became apparent that the classic textbook case of patients presenting with symptoms and signs of severe malabsorption, diarrhea, and wasting is the exception, rather than the rule. This is leading to an increased awareness of the need to include CD in the differential diagnosis of individuals presenting with symptoms and signs as diverse as unexplained iron deficiency or osteopenia, to those with mildly abnormal liver function tests, or simply what seem at first to be vague gastrointestinal symptoms.5–7 Furthermore, CD often accompanies other diseases, and its diagnosis may be overlooked if the other disease is the sole focus of attention.8 Recently, it has been recognized that CD may have its onset or become clinically apparent in individuals being treated for hepatitis C with ␣-interferon, a major activator of host innate immunity.9 –11 It is almost 3 decades since the discovery that CD is strongly associated with specific HLA class II antigens.
C
Although initially the association was thought to be with the HLA class II antigen HLA-DR3 (now termed HLADR17), it is now known the primary association is with a HLA-DQ2 molecule that is encoded at the gene level by 2 alleles, DQB1*02 and HLA DQA1*0501.12,13 Approximately 95% of CD patients have DQ2 encoded by these alleles. CD patients can inherit these alleles together on one chromosome from a parent who is DR17, because DR17 is tightly linked to both DQB1*02 and HLA DQA1*0501. Alternatively, one allele can be inherited from each parent. In this case, one parent carries HLA-DR7 that is linked to the allele DQB1*02 and the other carries HLA-DR5 that is linked to the allele DQA1*0501. These latter DQ2 CD patients, who are more prevalent in Southern Europe, are heterozygous for DR7/DR5.13–15 The remainder of celiac patients (⬃5%) mostly have a DQ8 molecule encoded by DQB1*0302 and DQA1*0301 on a DR4 haplotype.13,16,17 CD is exceedingly rare in the absence of DQ2 or DQ8, and it appears that they are necessary, although not sufficient, for developing CD. Naturally, a key question that has been addressed over the past several years is how do these particular DQ molecules play a role in the pathogenesis of CD?13 Progress in answering this question has been quite remarkable. CD has been long recognized as an ideal model in which to elucidate how HLA class II molecules such as DQ2 or DQ8 contribute to disease pathogenesis.13 The diverse group of HLA class II molecules present in the human population function to bind protein fragments (peptides) in their peptide-binding groove, and these HLA-class II- peptide complexes are recognized by specific populations of CD4⫹ T cells. In CD, the association with HLA-DQ2 and DQ8 is unequivocal, the proteins in
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the dietary grains that activate disease are known (i.e., gluten in wheat, secalins in rye, and hordeins in barley) and, importantly, populations of potentially disease-relevant T cells from the intestinal mucosa of CD patients can be readily isolated and grown in culture.13,18 Thus, in contrast to other HLA-associated diseases (e.g., rheumatoid arthritis and insulin-dependent diabetes mellitus) where the antigens that cause disease are unknown and the T cells in the relevant patient tissues are difficult to obtain, many of the pieces necessary to understand a key aspect of CD pathogenesis are in place. However, when algorithms were developed for which type of peptides might bind in the peptide-binding groove of the DQ2 or DQ8 molecule, the gliadins and related proteins in barley and rye did not seem to fill the bill,13 raising questions about what otherwise seemed to be an obvious mechanism.19 After all, how could one envision that binding of peptides from these grain proteins to DQ2 or DQ8, and subsequent T-cell activation, was an essential step in disease pathogenesis if peptides from those grain proteins did not even seem to bind, or bound very weakly, to the relevant HLA-DQ2 or DQ8 molecules? Subsequently, it was suggested that deamidation of glutamines in gliadin peptides, possibly by gastric acid or intestinal enzymes, might generate better binding of those peptides to DQ2 and subsequent T-cell activation by converting glutamine residues to negatively charged glutamic acid.20 As so often happens, serendipity then entered the picture. In Germany, Schuppan et al.21 were attempting to identify the target antigen of anti-endomysial antibody that was being used so successfully as a diagnostic screening test for CD. They discovered that the target antigen of anti-endomysial antibody was an enzyme, tissue transglutaminase, also termed TG2.21 In Norway, Sollid et al., and in the Netherlands, Koning et al., knew that the CD-associated DQ2 and DQ8 molecules have a preference for binding peptides with a negative charge, and that negatively charged amino acids were notably lacking in the glutamine-rich proteins known to activate CD.22,23 They astutely recognized that tissue transglutaminase might be responsible for the deamidation of glutamine residues in those proteins and thereby generate negatively charged glutamic acid residues. Testing in their laboratories showed this to be the case.24,25 Moreover, some gliadin peptides in which glutamine was converted to glutamic acid by tissue transglutaminase bound more efficiently to DQ2 and DQ8 and could activate T cells grown from the small intestinal mucosa of CD patients.24,25 They had solved a dilemma and a mechanism emerged by which peptides derived from
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ingested grain proteins could activate antigen-specific T-cell responses in the intestinal mucosa of CD patients expressing the HLA-DQ2 or -DQ8 susceptibility molecules. These findings suggested novel ways for treating or preventing CD, with the ultimate hope being an alternative for the “gluten-free” diet. The search was now on to find which peptide in gluten and the other diseaseactivating dietary grains activates disease. Indeed, initial studies performed using T cells from adult CD patients indicated there might be only 1, or at most a few, peptides in wheat gluten that are responsible for T-cell activation in DQ2 or DQ8 CD patients.26,27 However, as often is the case, the plot thickens. Koning’s group studied T cells from children with CD, and found that there were multiple gliadin or glutenin peptides that could bind to DQ2 or DQ8 and activate mucosal T-cell populations from children with CD.28 Moreover, in any given individual, several different gliadin or glutenin peptides29 could activate mucosal T cells. Notably, those peptides often differed from patient to patient, deamidation of glutamines to glutamic acid was not a requirement, and one-half of the children did not respond to the previously reported gliadin peptides thought to play a dominant role in the activation of mucosal T cells from adult CD patients.28 This data led initially to the notion that perhaps there is a broad group of different gluten peptides, not deamidated as well as deamidated, that might activate CD in children, but with increasing age the T-cell repertoire responsive to gliadin in adults becomes increasingly narrow and focused on one or a few deamidated peptides. More recently, Koning et al. developed algorithms based on the spacing between glutamine and proline residues and the presence or absence of other amino acids in the peptide, to predict which peptides in the dietary grains become deamidated by transglutaminase and bind to DQ2, and are thereby candidates for activating mucosal T-cell populations from CD patients.30 Using algorithms of varying stringencies, they predicted there would be as many as 50 or more activating peptides in gluten, hordeins, and secalins. Consistent with recent reports that oats do not, or only rarely, activate CD,31,32 their algorithms found no, or at most a few, potential DQ2 or DQ8 binding peptides in avenins from oats that would be predicted to activate T cells in CD patients.30 In this issue of GASTROENTEROLOGY, Arentz-Hansen et al.33 show that DQ2 restricted mucosal T cells from adult celiac patients in fact also recognize a broader group of gliadin peptides than indicated in their previous published data,27 a finding also suggested in studies by
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Koning et al.28 Thus, in addition to the 2 major ␣-gliadin sequences and a ␥-gliadin sequence that are deamidated by TG2, bind to DQ2 and activate T cells from adult celiac patients, they describe several additional ␥-gliadin epitopes and one new ␣-gliadin sequence that can bind DQ2 and activate CD patient T cells.33 Although deamidation of glutamine residues by transglutaminase clearly favors the generation of peptides that activate DQ2 restricted T cells, they further report, as Koning noted in studies of children,28 that deamidation is not an absolute requirement for T-cell activation.33 In contrast to the prior findings of this group,34 their new findings render more plausible the notion that deamidation of peptides occurs as disease develops, but may not be required for the initial activation of mucosal T cells in the very early stages of disease. Notably, all of the sequences they detected are located in regions of gliadin with high proline content, and not all of those sequences would have been predicted by the Koning algorhithm.30 It is now evident that a relatively large number of peptides in dietary grains are capable of activating DQ2 and DQ8 T cells from CD patients, although this number is certainly not unlimited. This finding may have significant implications when considering alternative therapeutic approaches, at least to the extent those approaches are based on altering T-cell activation. One way to view CD pathogenesis is to consider the events that occur before T-cell activation, the T-cell activation events themselves as discussed previously, and the tissue-damaging mechanisms and events that ensue following T-cell activation. When viewed in that context, several interesting questions arise, most notably relevant to the events that precede T-cell activation. First, do the same peptides that activate T cells in in vitro studies, as presented herein, activate CD in vivo? Do the proteins that activate CD come in several flavors, i.e., those that activate early events leading to innate immune and inflammatory responses in those susceptible to CD, and those that ultimately active DQ2 and DQ8 restricted T cells. At least a few early studies suggest this may be the case.13,35–36 Moreover, this would not be surprising given that proteins in these dietary grains initially undergo changes in the intestinal tract related to their encounter with gastric acid and digestive enzymes, followed by additional changes after encounter with host antigen-presenting cells in the mucosa and mucosal enzymes such as tissue transglutaminase. In this regard, it is interesting that a recent study found that the prolineglutamine-rich epitopes present in the peptides that activate CD are normally resistant to enzymatic processing in the human intestinal tract and dipeptidyl pepti-
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dase IV and dipeptidyl carboxypeptidase I appear to be key rate-limiting enzymes in the digestive breakdown of those peptides.37 Is the unique amino acid composition of these proteins a clue as to why myriads of other dietary proteins do not seem to generate peptides that cause similar diseases, including in people with other HLA class II genes? Do these glutamine and proline-rich proteins or their partially digested peptides also have effects on innate responses of the intestinal mucosa and, if so, are those unique to patients susceptible to CD? What cells produce the TG2 that deamidates these peptides, and how is it, or is it, differentially released or active in CD patients? It seems elucidation of these and other early events represents a new critical frontier to conquer as we increase our knowledge of CD pathogenesis. Finally, it is clear that HLA-DQ2 genes are the predominant known genetic factor in CD. Nonetheless, given the prevalence of CD in the United States and Europe and the frequency of DQ2 and DQ8 in these populations, only approximately 1%–2% of individuals with DQ2 or DQ8 would be predicted to develop CD. Studies to detect other CD susceptibility genes, based on linkage analysis, have revealed additional potential genetic loci that may contain genes contributory to disease, but these associations are weak relative to the HLA association and, with a few exceptions, have not been confirmed when additional populations were studied.38 – 43 It is possible that different genes may play a role in disease susceptibility in different groups of patients, and the mechanisms by which those genes contribute to the pathogenesis of CD may be diverse. MARTIN F. KAGNOFF Department of Medicine University of California at San Diego La Jolla, California
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Address requests for reprints to: Martin F. Kagnoff, M.D., University of California at San Diego, Department of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0623. e-mail: [email protected]; fax: (858) 534-5691. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35840