Autoantibodies to Tissue Transglutaminase as Predictors of Celiac Disease

GASTROENTEROLOGY 1998;115:1317 1321 RAPID COMMUNICATIONS Autoantibodies to Tissue Transglutaminase as Predictors of Celiac Disease WALBURGA DIETERICH,*, EBERHARDT LAAG,* HEIKE SCHÖPPER,*, UMBERTO VOLTA, ANNE FERGUSON, HELEN GILLETT, ERNST OTTO RIECKEN,* and DETLEF SCHUPPAN*, *Department of Gastroenterology, Klinikum Benjamin Franklin, Free University of Berlin, Berlin, Germany; Medizinische Klinik I, University of Erlangen-Nürnberg, Erlangen, Germany; Department of Internal Medicine, Cardioangiology, and Hepatology, Policlinico S. Orsola, University of Bologna, Bologna, Italy; and Department of Medicine, Western General Hospital, University of Edinburgh, Edinburgh, Scotland See editorial on page 1584. Background & Aims: Immunoglobulin A (IgA) autoantibodies to endomysium (EMA) are highly specific and sensitive markers for celiac disease. Recently, we identified tissue transglutaminase (ttg) as the major if not sole endomysial autoantigen. Methods: An enzymelinked immunosorbent assay (ELISA) was established to measure IgA anti-ttg titers in serum samples from 106 celiac patients with partial or subtotal villous atrophy, 43 celiac patients on a gluten-free diet, and 114 diseased and healthy controls. Results were correlated with clinical and histological data and with EMA titers. Results: In patients with biopsy-proven celiac disease consuming a normal, gluten-containing diet, 98.1% of the serum samples had elevated IgA titers against ttg, whereas 94.7% of the control sera were negative. IgA anti-ttg correlated positively with semiquantitative IgA EMA titers (r 0.862; P F 0.0001). Conclusions: An ELISA based on ttg allows diagnosis of celiac disease with a high sensitivity and specificity. IgA anti-ttg and IgA EMA show an excellent correlation, further confirming the enzyme as the celiac disease autoantigen. Because the assay is quantitative, not subjected to interobserver variation, and easy to perform, it will be a useful tool for population screening of a hitherto underdiagnosed disease. Celiac disease is an enteropathy that is characterized by small intestinal lesions of variable severity. In genetically predisposed individuals, the disease is triggered by ingestion of gluten, but its manifestation seems to require additional external factors. 1,2 Celiac disease is diagnosed by small bowel biopsy, but serum immunoglobulin A (IgA) autoantibodies to endomysium (EMA), an extracellular constituent of smooth muscle, show an almost 100% sensitivity and specificity for celiac disease. 3 7 Although celiac disease is effectively treated by gluten withdrawal, the tight association of EMA with the disorder suggests the importance of autoimmunity in the pathogenesis. 8 We recently identified tissue transglutaminase (ttg) as the main if not sole endomysial autoantigen in celiac disease. 9 This enzyme is synthesized by a broad spectrum of cell types but is usually retained in intracellular compartments. Upon wounding, ttg can be released from cells, where it is thought to aid in tissue repair by cross-linking extracellular proteins, 10,11 and to activate transforming growth factor, 12,13 which enhances collagen synthesis 14 and induces intestinal epithelial cell differentiation. 15 After identifying ttg as the putative endomysial autoantigen of celiac disease, we wanted to develop and validate an enzyme-linked immunosorbent assay (ELISA) that allows a reproducible, sensitive, and specific screening of large populations, in particular because recent studies suggest a high prevalence of celiac disease that may reach 0.3% 0.4% 16,17 and because intervals from first symptoms to diagnosis may last many years. 18 Materials and Methods Patients A total of 106 serum samples from untreated celiac patients (71 female and 35 male) were examined. The patients had different clinical and histopathologic degrees of severity (malabsorption or partial or subtotal villous atrophy) and responded with clinical improvement to gluten withdrawal. The mean age was 32.6 years, ranging from 4 to 80 years. In addition, serum samples from 43 celiac patients (34 female and Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; EMA, endomysial antibody; ttg, tissue transglutaminase. 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

1318 DIETERICH ET AL. GASTROENTEROLOGY Vol. 115, No. 6 Table 1. Number, Sex, and Age of Celiac Patients on a Normal Diet, Celiacs on a Gluten-Free Diet, and Controls 9 male; age, 7 79 years; mean age, 34.3 years) consuming a gluten-free diet for 6 12 months were studied (Table 1). One hundred fourteen patients or controls (74 female and 40 male; age, 1 74 years; mean age, 36.3 years) with various unrelated diseases or no disease served as controls. They included patients with inflammatory bowel diseases, primary biliary cirrhosis, lupus erythematosus, and a variety of other inflammatory or neoplastic diseases (Table 1). IgA anti-ema titers, assessed by indirect immunofluorescence on monkey esophagal tissue slides in two reference laboratories (A.F./H.G. and U.V.), were semiquantified as follows: 0, not detectable; 1, positive at dilutions between 1/5 and 1/20; 2, positive between 1/40 and 1/80; 3, positive at 1/100; 4, positive at 1/200; and 5, positive at dilutions 1:200. ELISA for ttg No. of patients Sex (F/M) Age, range ( yr ) Age, mean ( yr ) Celiacs, biopsy-proven 106 71/35 4 80 32.6 Celiacs on a gluten-free diet 43 34/9 7 79 34.3 Controls a 114 74/40 1 74 36.3 Healthy controls 41 Inflammatory bowel disease 36 Primary biliary cirrhosis 10 Lupus erythematosus 8 Cystic fibrosis 3 Rheumatoid arthritis 2 Metastatic gastric cancer 2 Various diseases b 12 NOTE. Celiac patients on a gluten-free diet did not undergo biopsy. a In the control group, biopsy specimens were available only from 10 healthy control patients. b Included 1 patient each with infectious diarrhea, abdominal pain, anemia, unclear diarrhea, small bowel carcinoma, primary liver carcinoma, osteomyelofibrosis, pancreatic carcinoma, T-cell lymphoma, familial adenomatous polyposis coli, esophageal cancer, and hepatitis C. Microtiter plates (96-well; Greiner, Frickenhausen, Germany) were coated with 1 µg of ttg per well (single batch, 0.00167 U of ttg activity) from guinea pig liver (Sigma, Deisenhofen, Germany) in 100 µl of 50 mmol/l Tris-HCl, 150 mmol/l NaCl, and 5 mmol/l CaCl 2, ph 7.5, for 2 hours at 37 C with a coating efficiency of 23.0% 29.4% as determined by addition of a radiolabeled tracer. Wells were extensively washed with 50 mmol/l Tris-HCl, 150 mmol/l NaCl, 10 mmol/l EDTA, and 0.1% Tween 20, ph 7.4, and the plates were incubated in washing buffer for at least 10 minutes at room temperature or overnight at 4 C. Sera, diluted in 100 µl of the same buffer, were added to the wells and incubated for 1 hour at room temperature. After three washes, the wells were incubated with 100 µl of peroxidase-conjugated rabbit antihuman IgA (Dianova, Hamburg, Germany), diluted 1/1000, in the same buffer. Unbound antibodies were removed, and color was developed by addition of 200 µl 0.1 mol/l sodium citrate, 1 mg/ml o-phenylenediamine hydrochloride (Sigma), and 0.06% H 2 O 2 (Merck, Germany), ph 4.2, at room temperature for 30 minutes in the dark. Absorbances were read on an ELISA reader (MRX; Dynatech, Denkendorf, Germany) at 450 nm. All serum samples were initially tested in duplicate at a dilution of 1/25. In case of highly elevated titers (optical density [OD] values 2.5), sera were further diluted to obtain OD values in the linear range between 0.5 and 2.5 OD. After subtraction of the background (normally 0.08 OD) the OD values were multiplied with the serum dilution to obtain the ELISA titers. With the inclusion of 3 control serum samples in each assay, intra-assay and interassay variations were below 2.8% and 20% (15 independent tests), respectively. Statistics The Spearman correlation was used to compare the stochastic EMA values with the numerical ELISA titers; the Kruskal Wallis test was used to compare untreated celiacs with celiacs on a gluten-free diet or with the controls. The sensitivity of the ELISA was calculated as the frequency of positive IgA anti-ttg titers in patients with biopsy-proven, EMA-positive celiac disease consuming a normal diet, and the specificity as the frequency of negative IgA anti-ttg titers in nonceliacs without EMA, considering a titer of 15 as a cutoff value, which excluded 95% of the nonceliac patients. Results An ELISA based on serum IgA autoantibodies against ttg was performed; the results are summarized in Table 2. The ELISA procedure was improved from the protocol used in our previous study. 9 Blocking with bovine serum albumin was avoided because some serum samples from patients with celiac disease and controls showed antibodies against this food component, therefore possibly falsifying the results. Also, the addition of calcium to the coating buffer was mandatory to improve the sensitivity of the ELISA because the antibodies of celiacs reacted more strongly with the calcium-activated form of ttg. With a cutoff value of 15 (excluding 95% of nonceliacs) to define a positive reaction for circulating IgA antibodies to ttg, 4 patients with celiac disease were negative for ttg but had a weak to moderate positivity for EMA (Table 2). Ten serum samples without detectable IgA EMA (fluorescence score, 0) had elevated IgA anti-ttg (ELISA titer 15; Table 2). However, the number of false-positive IgA anti-ttg results was reduced to 6 when 1 patient with celiac small intestinal

December 1998 TRANSGLUTAMINASE ANTIBODIES IN CELIAC DISEASE 1319 Table 2. IgA Anti-tTG and Corresponding IgA EMA Titers of 106 Celiacs, 43 Celiacs on a Gluten-Free Diet, and 114 Controls No. of patients Untreated celiacs 1 6 IgA EMA IgA anti-ttg 0 1 58 12, 17 346 21 2 24 758 35 3 7, 83 1253 28 4 76 3213 15 5 176 6282 Celiacs on a gluten-free diet 20 7 0 1 3 12, 27, 39, 115 1, 12, 18 36 12 2 16 1402 2 3 83, 98 1 4 144 1 5 262 Controls 114 0 1 14, 19, 22, 31, 40, 43, 129 NOTE. IgA anti-ttg titers are averages of duplicate determinations, and EMA titers have been subdivided into 6 categories, ranging from no (0) to strong (5) reactivity. The numbers for IgA anti-ttg represent single values or the ranges of the titers. Sera that did not show congruent IgA anti-ttg and EMA titers have been highlighted: sera with negative IgA anti-ttg ( 15) and positive EMA ( 1) are marked by italic type, and sera with positive IgA anti-ttg ( 15) and negative EMA (0) are marked in bold type. For details, see text. lesions (partial villous atrophy) and 3 celiacs on a gluten-free diet were subtracted (Table 2). In patients with biopsy-proven and EMA-positive disease, the calculated sensitivity and specificity of the ELISA reached 98.1% and 94.7%, respectively. The high sensitivity of the ELISA was highlighted by the examination of sera from 6 patients with increased intraepithelial lymphocytes, an early mucosal change in celiac disease. These patients, also characterized by short stature, abdominal pain, constipation, recurrent miscarriage, or irritable bowel syndrome, had low EMA titers (score 1 in 5 patients; score 0 in 1 patient) and slightly increased IgA anti-ttg titers, with 15, 16, 19, 36, 45, and 72 (data not shown). When the IgA titers to ttg were plotted against the IgA EMA scores, a positive correlation (r 0.862; P 0.0001) was obtained (Figure 1 ). Additionally, the IgA anti-ttg titers of untreated celiacs were significantly increased compared with the titers of celiacs on a gluten-free diet and with controls (Figure 2). Figure 1. Semilogarithmic plot of the correlation between IgA anti-ttg and EMA scores of 106 patients with biopsy-proven celiac disease consuming a normal diet, 43 celiac patients after a gluten-free diet, and 114 controls. The median (25/75 percentiles) of IgA anti-ttg relative to EMA were as follows: EMA (0), 4 (2/7); EMA (1), 18 (17/23); EMA (2), 105 (43/210); EMA (3), 180 (117/363); EMA (4), 557 (191/762); and EMA (5), 637 (244/1193). r 0.862; P 0.0001. titers of IgA anti-ttg in serum samples from patients with celiac disease and from various controls. Collectively, IgA anti-ttg titers showed a good correlation with EMA titers, reaching a sensitivity of 98.1% and a specificity of 94.7%. As predicted, IgA anti-ttg were normal or significantly lower in patients on a gluten-free diet than in untreated celiacs. This is in line with EMA titers that tend to disappear after gluten withdrawal. In addition to the intriguing implications that the discovery of ttg as the celiac disease autoantigen may have for the pathophysiology of the disease, 19 21 this ELISA could replace the traditional semiquantitative, observer-dependent, and time-consuming indirect immu- Discussion We used an ELISA based on ttg, the recently discovered endomysial autoantigen in celiac disease, to determine (1) the extent to which detectable IgA antittg was predictive of a positive EMA test result by immunofluorescent examination and (2) to establish the Figure 2. Semilogarithmic plot of IgA anti-ttg titers of 106 celiac patients on a normal diet (cd), 43 celiac patients after a gluten-free diet (gfd), and of 114 controls (ctr), with the following medians (25/75 percentiles): cd, 199 (118/671); cd/gfd, 18 (5/48); and ctr, 4 (2/7). Differences between groups were significant (P 0.001).

1320 DIETERICH ET AL. GASTROENTEROLOGY Vol. 115, No. 6 nofluorescence test on sections of monkey esophagus and may allow the screening of large populations for celiac disease. Four serum samples with positive IgA EMA (2 at a dilution of 1/5 and 1 each at dilutions of 1/10 and 1/100) showed no detectable IgA anti-ttg even on repeated testing. The endomysial antigen used for these immunofluorescence tests was of monkey origin, whereas the ttg used for the ELISA is derived from guinea pig liver. The occasional but unequivocal discrepancies between the results of the two techniques may be explained either by (minor) interspecies differences or by the existence of other minor autoantigens in the extracellular matrix. In addition, sera may preferentially react with antigenic neoepitopes generated by cross-linking of ttg with gliadin or by potentiation of gliadin antigenicity by deamidation. 21,22 We have shown a preferred crosslinking of gliadin by ttg and an incorporation of ttg itself in these complexes, as well as IgA antibodies that are directed to these cross-links rather than to ttg or gliadin alone (Dieterich et al., unpublished observations). Ten serum samples had no detectable IgA EMA but an elevated IgA anti-ttg titer (Table 2). Three of these samples were from celiac patients on a gluten-free diet and 1 from a patient with partial villous atrophy, a finding consistent with celiac disease. This suggests that the sensitivity of the ttg-elisa to detect celiac disease may be greater than that of the indirect immunofluorescence method, without significant loss of specificity. Because small intestinal biopsy specimens had not been obtained from the other 6 persons in this group (3 with systemic lupus erythematosus, 1 with primary biliary cirrhosis, 1 with Crohn s disease, and 1 without known disease), prospective studies must clarify if such patients have silent or latent celiac disease rather than representing false-positive results. Preliminary data on patients with IgA deficiency, a condition with an at least 10-fold increased incidence of celiac disease, indicate either elevated IgA anti-ttg titers despite low IgA levels or high levels of IgG anti-ttg (Dieterich et al., unpublished results). Because the IgG autoantibodies may also be elevated in other inflammatory or autoimmune diseases, 9 the predictive value of the IgG anti-ttg test in these patients has to be confirmed in a larger study. In summary, our data on a spectrum of patients confirm that ttg is the major if not sole endomysial autoantigen of celiac disease 9 and suggest that an ELISA based on this protein could be a useful tool to screen large populations for the prevalence of this fairly common disorder, which might otherwise remain undetected for many years. References 1. Trier JS. Celiac sprue. N Engl J Med 1991;325:1709 1719. 2. Marsh MN. Gluten, major histocompatibility complex, and the small intestine: a molecular and immunobiologic approach to the spectrum of gluten sensitivity ( celiac sprue ). Gastroenterology 1992;102:330 354. 3. Walker-Smith JA, Guandalini S, Schmitz J, Shmerling DH, Visakorpi JK. Revised criteria for diagnosis of coeliac disease. Arch Dis Child 1990;65:909 911. 4. Bürgin-Wolff A, Gaze H, Hadziselimovic F, Huber H, Lentze MJ, Nusslé D, Reymond-Berthet C. Antigliadin and antiendomysium antibody determination for coeliac disease. Arch Dis Child 1991; 66:941 947. 5. Volta U, Molinaro N, Fusconi M, Cassani F, Bianchi FB. IgA antiendomysial antibody test. A step forward in celiac disease screening. Dig Dis Sci 1991;36:752 756. 6. Corrao G, Corazza GR, Andreani ML, Torchio P, Valentini RA, Galatola G, Quaglino D, Gasbarrini G, di Orio F. Serological screening of coeliac disease: choosing the optimal procedure according to various prevalence values. Gut 1994;35:771 775. 7. Ladinser B, Rossipal E, Pittschieler K. Endomysium antibodies in coeliac disease: an improved method. Gut 1994;35:776 778. 8. Picarelli A, Maiuri L, Frate A, Greco M, Auricchio S, Londei M. Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease. Lancet 1996;348:1065 1067. 9. Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797 801. 10. Upchurch HF, Conway E, Patterson MK, Maxwell MD. Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme. J Cell Physiol 1991;149:375 382. 11. Bowness JM, Folk JE, Timpl R. Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen. J Biol Chem 1987;262:1022 1024. 12. Kojima S, Nara K, Rifkin DB. Requirement for transglutaminase in the activation of latent transforming growth factor- in bovine endothelial cells. J Cell Biol 1993;121:439 448. 13. Nunes I, Gleize, PE, Metz CN, Rifkin DB. Latent transforming growth factor- binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-. J Cell Biol 1997;136:1151 1163. 14. Lyons RM, Moses HL. Review. Transforming growth factors and the regulation of cell proliferation. Eur J Biochem 1990;187:467 473. 15. Halttunen T, Marttinen A, Rantala I, Kainulainen H, Mäki M. Fibroblasts and transforming growth factor induce organization and differentiation of T84 human epithelial cells. Gastroenterology 1996;111:1252 1262. 16. Catassi C, Rätsch IM, Fabiani E, Rossini M, Bordicchia F, Candela F, Coppa GV, Giorgi PL. Coeliac disease in the year 2000: exploring the iceberg. Lancet 1994;343:200 203. 17. Not T, Horvath K, Hill ID, Fasano A, Hammed A, Magazzu G. Endomysium antibodies in blood donors predicts a high prevalence of celiac disease in the USA (abstr). Gastroenterology 1996;110:A351. 18. Lankisch PG, Martinez Schramm A, Petersen F, Dröge M, Lehnick D, Lembcke B. Diagnostic intervals for recognizing celiac disease. Z Gastroenterol 1996;34:473 477.

December 1998 TRANSGLUTAMINASE ANTIBODIES IN CELIAC DISEASE 1321 19. Sollid LM, Molberg O, McAdam S, Lundin KEA. Autoantibodies in coeliac disease: tissue transglutaminase guilt by association. Gut 1997;41:851 852. 20. Cronin CC, Shanahan F. A significant step in the celiac puzzle. Gastroenterology 1998;114:1339 1341. 21. Schuppan D, Dieterich W, Riecken EO. Exposing gliadin as a tasty food for lymphocytes. Nat Med 1998;4:666 667. 22. Molberg Ø, McAdam SN, Körner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KEA, Sjöström 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. Received June 16, 1998. Accepted July 31, 1998. Address requests for reprints to: Detlef Schuppan, M.D., Ph.D., Medizinische Klinik I, University of Erlangen-Nürnberg, Krankenhausstrasse 12, 91054 Erlangen, Germany. e-mail: detlef. schuppan@med1.med.uni-erlangen.de; fax: (49) 9131-8536003. Supported by grants SFB 366 C5 and Schu 646/4-1 from the Deutsche Forschungsgesellschaft and by a grant from the German Celiac Society. Laënnec of Laënnec s cirrhosis Réné Théophile Hyacinthe Laënnec (1781 1826) was born at Quimper in Lower Brittany. The early death of his mother and abandonment by his father led to his being placed in the care of an uncle who was a professor in the medical school at Nantes. After provincial tutelage, he completed his education at the Charité hospital where he became a protégé of the famed Baron Corvisart. Though of a delicate constitution (he later succumbed to tuberculosis, the disease that had taken his mother), he applied himself with remarkable diligence to correlation of clinical observations and pathological findings. Publication of numerous papers on a variety of topics established his reputation. In 1816 he conceived design of the stethoscope and defined auscultation. In 1819, impressed by the color of a chronically diseased micronodular liver, he coined the term cirrhosis, taken from the Greek word for tawny. Contributed by WILLIAM S. HAUBRICH, M.D. Scripps Clinic and Research Foundation, La Jolla, California