Tissue transglutaminase the key player in celiac disease: a review

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1 Autoimmunity Reviews 3 (2004) Tissue transglutaminase the key player in celiac disease: a review a b, Shimon Reif, Aaron Lerner * a Division of Pediatric Gastroenterology, Dana Children s Hospital, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel b Department of Pediatrics, Carmel Medical Center, 7 Michal St., Rappaport Faculty of Medicine, Technion Israel Institute of Technology, Haifa 34362, Israel Received 2 April2003; accepted 13 May 2003 Abstract Gluten-sensitive enteropathy, otherwise known as celiac sprue, is characterized by an abnormal proximal small intestinal mucosa arising as a result of an inappropriate inflammatory response to ingested gluten antigens present in wheat in genetically susceptible individuals. This immune response is directed to a 33-mer peptide of the a gliadin component of gluten. The generation of an epitope for the recognition by CD4 T cells reuires deamination of the protein by tissue transglutaminase (ttg). Moreover, IgA anti ttg is highly sensitive and is specific serologic marker (95 99%) of celiac disease. They can be easily determined uantitatively, by ELISA of an accurate and relatively inexpensive techniue. Therefore, ttg can be used as the first line diagnostic test in the work-up of celiac disease, as well as for screening purposes. Finally, ttg may contribute to future strategies in treating celiac disease either by producing nontoxic wheat or by generating oral vaccination that can prevent the disease Elsevier B.V. All rights reserved. Keywords: Celiac disease; Tissue transglutaminase; Gluten 1. Introduction Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten-containing grains in genetically predisposed individuals. Elimination of gluten, the cornerstone of treatment for CD, may Abbreviations: ttg, tissue transglutaminase; CD, celiac disease; GFD, gluten-free diet. *Corresponding author. Tel.: ; fax: address: lerner_aaron@clalit.org.il (A. Lerner). dramatically influence the course of the disease and prevent complications, such as osteopenia, malignancy and miscarriage w1x. With the recent introduction of newer and more precise serologic tests, such as antibodies against tissue transglutaminase (ttg), significant progress has been achieved, not only in exploring the genetic basis, clinical course and epidemiology of the disease, but also in understanding its pathogenesis. This greater knowledge may contribute to enhanced treatment, and possibly, to the prevention of CD w2x. This review will outline ttg as a diagnostic /04/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi: /s (03)00065-x

2 S. Reif, A. Lerner / Autoimmunity Reviews 3 (2004) tool and describe its role in the immunologic basis of CD and subseuent therapeutic implications. 2. Role of serologic testing in CD Celiac patients exposed to gluten express high levels of serum antibodies to gliadin, endomysium, reticulin, jejwnum and ttg w3,4x. Elimination of gluten from their diet results in a decrease of the titer of these antibodies and to their eventual disappearance. Anti-reticulin antibodies comprise a specific, but not a very sensitive test. Antibodies of the immunoglobulin IgA and IgG class to gliadin have also been used in the past to diagnose CD and monitor response to therapy. Sensitivities range from 76 to 88% for IgG and 52 to 91% for IgA antigliadin antibodies, while the respective specificities vary from 88 to 92% and from 45 to 94% w5 7x. The main advantage of using the IgG anti-gliadin is seen in patients with IgA deficiency, a condition that is more common among CD patients than in the generalpopulation w8,9x. Anti-endomysium antibodies (EMA) of the IgA class have a reportedly much better sensitivity and specificity (97 to 100% and 98 to 99%, respectively), and a combination of antigliadin and antiendomysium antibodies can give a negative and positive predictive value approaching 100% w10x. Because these specific antibodies were found to be useful and reliable diagnostic tests for CD, the diagnostic criteria for CD, was revised by the European Society for Pediatric Gastroenterology, Hepatology and Nutrition. It has been argued that serologic tests should be used in screening and initialworkups, but a smallintestinalbiopsy with characteristic histologic abnormalities should still be the main criterion for diagnosis w11x. An intestinal biopsy to show improvement of the villous structure under Gluten free diet (GFD) could be now replaced by the measurement of serologic markers w11x. 3. Alteration in the incidence of CD due to the use of serologic tests CD was believed to affect 1:1500 individuals throughout Europe, with a higher prevalence (1:150) in Ireland w12x. Recent serologic screening Table 1 Atypicalpresentations Non-specific Weight loss, lethargy, chronic fatigue Hematologic complaints Bruising Anemia: isolated vitamin deficiency or a combination of iron, folate, and B12 deficiency Hyposplenism Neurologic disease Cerebellar ataxia Peripheralneuropathy Posterior and lateral column abnormalities Neuromyopathies Endocrine/Metabolic Short stature Pubertaldelay Osteoporosisyosteomalacia Gynecologic Primary or secondary amenorrhea Primary or secondary infertility Gastrointestinal Constipation Unexplained transaminitis Psychiatric Depression Psychosis, including schizophrenia of normal populations, however, has yielded prevalence between 1:100 to 1:300 w13,14x. In the past, CD had been considered a disease of childhood that was usually diagnosed before the age of 2 years, and presented with symptoms of diarrhea, malabsorption and weight loss following the introduction of cereals into the infant diet. Adults and adolescents often present atypically, with vague and non-specific or problems that at first glance appear unconnected to an intestinalpathology. Such atypical symptoms are listed in Table 1. Indeed, the variety of symptoms, signs, and severity of CD led to the concept of there being a celiac iceberg, with the patients who had complaints associated with the gastrointestinal tract being the ones above the surface, and many more as yet undiagnosed patients with minimalor no symptoms or with atypicalones making up a silent, unseen majority hidden below the surface w15x. Testing for CD is, therefore, recommended even when the clinical symptoms are indicative however, subtle for the disease, or for individuals who are at increased risk either because of heredity,

3 42 S. Reif, A. Lerner / Autoimmunity Reviews 3 (2004) extra-intestinalsymptoms, or an associated disorder (e.g. IgA deficiency, Diabetes Mellitus w16x). 4. ttg as a serologic marker for CD After it has been shown that ttg is the substrate recognized by Antiendomysium antibodies (EMA) w17x, an Enzyme-linked immunosorbent assay (ELISA) was developed and validated as an alternative screening test for CD w18x. The currently used method employs a commercially available guinea pig ttg as a substrate for the ELISA. The recent cloning of human ttg w19x, however, led to a human ttg base assay, which has higher specificity and sensitivity w20x. Furthermore, in certain circumstances, such as the evaluation of a possible CD among patients with elevated liver transaminases and obscure chronic liver disease, there was a high freuency of false positive anti ttg tested on guinea pig as the antigen due to the presence of hepatic proteins in the commercial ttg obtained from guinea pig liver, which disappeared when human ttg was used as the Ag in the ELISA system w21x. Initialseries uoted a sensitivity of 95 98% and a specificity of 94 95% for the IgA anti-ttg assay, respectively, in untreated celiac patients w18,22,23x. The results of ELISA anti-ttg tests correlated well with the traditionaliga-ema tests and the titer of ttg antibodies fluctuated with the dietary gluten exposure in a manner consistent with previous observation of EMA antibody monitoring w24x. Furthermore, EMA assays are based on indirect immunofluorescence using monkey esophagus or umbilical cord, which is expensive, labor intensive and subjective. In contrast to EMA, ttg autoantibodies can easily be measured in immunoassays with diagnostic sensitivity and specificity that are comparable to those of EMA w18,23x. Therefore, ttg emerged as the recommended standard initial diagnostic study w19 22x. The presence of a positive anti-ttg test should prompt a small intestinal biopsy: if the patient is IgA deficient a condition with a 10-fold risk of developing CD IgG antigliadin should be used or a direct duodenal biopsy should be performed w25x. In their recent study, Agardh et al. w24x suggested that since IgA ttg is a more specific and sensitive test than IgG1 ttg and IgM ttg, it should, therefore, be implemented as screening test for CD. Nevertheless, IgG1 ttg is more commonly used for children diagnosed at a young age (-2 years) and may be used as sufficiently sensitive substitute for IgA ttg in IgA-deficient children; nevertheless, it has not been practically implemented. Why do CD patients produce ttg antibodies? The most plausible explanation was put forward by Sollid et al. w26x where they reasoned that ttg can crosslink itself to gluten and this gluten ttg complex will be taken up by B-cells that express ttg-specific immunoglobulin on their membrane. As a result of this uptake, the gluten ttg complex will be degraded intracellularly and gluten peptides will bind to HLA-DQ and be expressed on the cell surface. In CD patients, gluten-specific T cells will recognize this HLA-DQ peptide complex and this results in T cell help for the production of ttgspecific antibodies by the B cells. The fact that only CD patients have measurable numbers of gluten-specific T cells explains why only CD patients make ttg-specific antibodies and also, importantly, why the antibody titer drops after gluten withdrawal w27,28x. 5. ttg: the master regulator of CD In 1997, Dieterich et al. w17x described ttg as being the target of the endomysium-specific antibodies that are characteristic for CD w29x. As a result of that observation, a series of studies have revealed that screening for the presence of ttgspecific antibodies is a very specific indicator for CD w18 24x. ttg is expressed in many different tissue and organs, and is found intracellularly as well as extracellularly. In the extracellular environment, ttg was shown to play a role in extracellular matrix assembly, cell adhesion and wound healing w30,31x. The calcium-dependent ttg catalyzes selective crosslinking or deamination of proteinbound glutamine residues w32x. Binding of calcium ions induces conformationalalterations in ttg that result in the acuisition of transglutaminase activity w33x. Recent evidence has indicated that ttg is not only a diagnostic marker of CD, but that it is also directly involved in the pathogenesis of the dis-

4 S. Reif, A. Lerner / Autoimmunity Reviews 3 (2004) ease. Antibodies against ttg in CD patients interfere with fibroblast-induced differentiation of epithelial cells, possibly by inhibiting the crosslinking activity of ttg. In addition, ttg is necessary for activation of transforming growth factor b (TGF-b) w34x, which is involved in the differentiation of intestinalepithelium w35,36x. Reduced formation of TGF-b could also lead to increased activation of mucosal Th1 cells that produce inflammatory cytokines, because TGF-b is a suppressor factor for T cells w37,38x. Moreover, there is now compelling evidence that ttg is linked to the humoral response that is also involved in generating gluten peptides, which stimulates the T cells in the small intestine of CD patients w39x. The currently accepted theory is that susceptible people, e.g. HLA type DQA*0501 and DQB1*0201, exhibit an aberrant response to dietary gluten, and that the resulting small intestinal damage is caused by locally activated CD4 T- lymphocytes. Gluten-sensitive T lymphocytes recognize gluten-derived peptides epitopes when presented in association with DQ2. Activation of these normally silent CD4 T-lymphocytes trig- gers a T-helper type 1 pattern of cytokine production, including the release of IFN-g and leading to mucosaldamage w3x. Glutamine is the most abundant amino acid in gliadin, making up 35% of its composition, and it may be centralto the toxic effect of these proteins in celiac patients. The ttg enzyme selectively deamidates gluten protein glutamine to glutamic acid. This introduction of negative charges results in peptides that bind with relatively high affinity to the disease-associated HLA-DQ2 or HLA-DQ8 molecules w28,40x. In addition to this process, ttg increases their stimulatory effect on gluten-sensitive intestinal T lymphocytes and exposes neoepitopes in wheat proteins. One of severaltoxic epitopes may reside in the N-terminalregion of a gliadin, a constituent of gluten, corresponding to amino acid residues This peptide causes the histologic characteristic of CD in both organ culture and in-vivo challenge studies. In addition, it can bind DQ2 molecules, and when presented in association with DQ2, it can stimulate peripheral blood T-lymphocytes. In an attempt to find the precise structure of the toxic element of gluten, a single epitope of a gliadin was recently recognized as the dominant epitope recognized by T-lymphocytes in patients with CD w41x. This 33-mer peptide is stable invivo and in-vitro to breakdown by all gastric, pancreatic and intestinalbrush-border membrane peptidase, and it reacts with ttg by deamination of specific glutamine residues within this seuence, which is essentialfor HLA-DQ2 binding and subseuent T lymphocyte stimulation w28,40x. The importance of the modification of gluten by ttg has been underscored by the observation that the enzyme specificity correlates with the toxicity of cereals for CD patients w42x. Moreover, ttg specificity can be combined with the reuirements for peptide binding to HLA-DQ2, a finding that resulted in a search that identified T-cell stimulatory peptides in wheat, barely and rye but not in oats, the latter being considered safe for CD patients. 6. ttg as a main therapeutic target in treating CD ttg can be considered a master regulator of CD. It is involved in both the humoral and cellular response and is tightly linked to cereal toxicity w43x. The enzyme ttg is considered the ideal assay for screening CD patients in making the initialdiagnosis. An intestinalbiopsy is then carried out for finalconfirmation and ttg levelfor monitoring clinical response to gluten-free diets w44x. Finally, the discovery of the immune regulation and the specific epitope in gluten-sensitive enteropathy opens the way for severalpotential interventionalstrategies for treating CD: 1. The discovery of the specific epitope of gluten which is rich with proline is a crucial factor in the resistance of the 33-mer gliadin peptide to gastrointestinalbreakdown. This will enable the use of a bacterial prolyl endopeptide that catalyzes this peptid, and diminishes its toxic effect. 2. The induction of tolerogenic T cells or promotion of T cells anergy. 3. ttgase-mediated endocytosis might be an effective mechanism for oralvaccination with immunogenic peptide epitopes as long as they are

5 44 S. Reif, A. Lerner / Autoimmunity Reviews 3 (2004) resistant to the action of gastric and intestinal digestive enzymes. In addition, by blocking ttg activity, it would prevent the generation of the majority of T cells stimulatory gluten peptide. Further knowledge of the substrate specificity of ttg will facilitate the design of specific blockers, bearing in mind, however, that long-term blocking of ttg might have undesirable conseuences. Acknowledgments Esther Eshkolis thanked for editorialassistance. Take-home messages Celiac Disease is an enteropathy caused by inflammatory response to ingestion of gluten in genetically susceptible individuals. The immune response is directed to a 33-mer peptide of the a gliadin component of gluten. Deamination of glutamin to glutamic acid by tissue transglutaminase (ttg) generate an epitope reuired for recognition by CD4 T cells. TTG is the target of the endomysium specific antibodies that are characterized in Celiac disease. IgA anti ttg is highly sensitive and specific marker of celiac disease, except in IgA deficient patients. IgA against ttg can be easily and non expensively determined uantitatively by ELISA, and is now considered as the first line in the diagnosis of Celiac disease. Finally, ttg may be used in the future as a therapeutic target of celiac disease. References w1x Mowat AMcI. Coeliac disease: a future for peptide therapy? Lancet 2000;356: w2x Maki M, Collin P. Coeliac disease. Lancet 1997;349: w3x Sollid LM, Scott H. New tool to predict celiac disease on its way to the clinics. Gastroenterology 1998;115: w4x Lerner A, Kumar V, Iancu TC. Immunological diagnosis of childhood coeliac disease: comparison between antigliadin, antireticulin and antiendomysial antibodies. Clin Exp Immunol1994;95: w5x Russo PA, Charstand LJ, Siedeman E. Comparative analysis of serologic screening tests for the initial diagnosis of celiac disease. Pediatrics 1999;104:75 8. w6x Vitoria JC, Arrieta A, Arranz C, Ayesta A, Sojo A, Maruri N, et al. Antibodies to gliadin, endomysium, and tissue transglutaminase. J Pediatr Gastroenterol Nutr 1999;29: w7x Lerner A. The controversy of the use of anti-gluten antibody (AGA) as a diagnostic toolin celiac disease. J Pediatr GastroenterolNutr 1991;12: w8x Cataldo F, Marino V, Bottaro G, Ventura A. Celiac disease and selective IgA deficiency. J Pediatr 1997;131: w9x Cataldo E, Marino V, Ventura A, Bottaro G, Coraza GR. Prevalence and clinical feature of selective immunoglobulin A deficiency in celiac disease: an Italian multicenter study. Gut 1998;42: w10x Ladinser B, RossipelE, Pitschieler K. Endomysium antibodies in celiac disease: an improved method. Gut 1994;35: w11x Walker Smith J. Revised criteria for diagnosis of celiac disease: report of working group of European Society of Pediatric Gastroentretology and Nutrition (ESPAGN). Arch Dis Child 1990;65: w12x Ciclitira PJ. AGA technical review on celiac sprue. Gastroenterology 2001;120: w13x Not T, Horvath K, Hill ID, Partanen J, Hammed A, Magazzu G, et al. Celiac disease risk in the USA: high prevalence of antiendomysium antibodies in healthy blood donors. Scand J Gastroenterol 1998;33: w14x Shamir R, Lerner A, Shinar E, Lahat N, SobelE, Baror R, et al. The use of single serologic marker underestimates the prevalence of celiac disease in Israel: a study of blood donors. Am J Gastroenterol 2002;97: w15x LebenthalE, Branski D. Celiac disease: an emerging global problem. J Pediatr Gastroenterol Nutr 2002;35: w16x Branski D, Troncone R, Maurano F, Rossi M, Micillo M, Greco L, et al. Celiac disease: a reappraisal. J Pediatr 1998;133: w17x Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3: w18x Dieterich W, Laag E, Schopper E, Volta U, Ferguson A, Gillet H, et al. Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology 1998;115: w19x Troncone R, Maurano F, Rossi M, Micillo M, Greco L, Auricchio R, et al. IgA antibodies to tissue transglutaminase: an effective diagnostic test for celiac disease. J Pediatr 1999;134: w20x Sblattero D, Berti I, Trevisiol C, Marzari R, Tommasini A, Bradbury A, et al. Human recombinant tissue trans-

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