Intestinal Mucosa of Celiacs in Remission Is Unable To Abolish Toxicity of Gliadin Peptides on in Vitro Developing Fetal Rat Intestine And Cultured Atrophic Celiac Mucosa

003 I-3998/88/2402-0233$02.00/0 PEDIATRIC RESEARCH Copyright 0 1988 International Pediatric Research Foundation, Inc. ~ 1 Vol. 24, No. 2, 1988 Printed in U.S.A. Intestinal Mucosa of Celiacs in Remission Is Unable To Abolish Toxicity of Gliadin Peptides on in Vitro Developing Fetal Rat Intestine And Cultured Atrophic Celiac Mucosa H. J. CORNELL, R. S. AURICCHIO, G. DE RITIS, M. DE VINCENZI, L. MAIURI, V. RAIA, AND V. SILANO Department of Applied Chemistry, Royal Melbourne Institute of Technology, Melbourne, Australia [H.J.C.]; Clinica Pediatrica 11 Facolta' di Medicina e Chirurgia, Naples, Gruppo di Ricerca del Consiglio Nazionale delle Richerche sulla Fisiologia e Patologia dell'Apparato Digerente [S.A., G.d.R., L.M., V.R.]; and Department of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanita: Roma, Programme of Preventive Medicine, Project of Perinatal Pathologies and Their Consequences, Consiglio Nazionale delle Richerche, Rome, Italy [M.D. V., Y S . ] ABSTRACT. Subfraction 2R of fraction 9 from a peptictryptic-pancreatic digest of wheat gliadin is known to be toxic in vivo to celiac patients. We have found that fractions 9 and 2R inhibit the in vitro development of fetal rat intestine and the increase of enterocyte height occurring in organ culture of atrophic celiac mucosa (0.1-0.5 mg/ml medium). Other peptide fractions of the gliadin digest are devoid of such in vitro effects. Subfraction ZR, after incubation with morphologically normal small intestinal mucosa of celiacs in remission and ultrafiltration, was still very active in both culture systems at low concentration (0.1 mg/ml); on the contrary, subfraction 2R was inactivated after incubation with normal mucosa. These results are compatible with the hypothesis that there is a mucosal defect in handling gliadin peptides in celiac disease, and suggest that there is either a primary (or secondary) enzyme deficiency or some other mechanism operating in the intestinal mucosa of celiac patients in remission. (Pediatr Res 24: 233-237,1988) Abbreviation PTC, peptic-tryptic-cotazym The toxicity of wheat in celiac disease results from the gliadin protein fraction (1-6). The ingestion of peptide mixtures obtained from wheat gluten after in vitro sequential digestion with proteolytic enzymes also induced the typical symptoms in patients affected by celiac disease (7, 8). Numerous studies have been done using in vitro systems which could identify the toxic peptide(s) and the mechanism(s) of their toxic activity. The organ culture of human small intestinal biopsies has been proposed as an in vitro model of celiac disease (9, 10). Jejunal specimens obtained from patients with active enteropathy show morphological and biochemical improvement when cultured in a medium free from gliadin peptides. No improvement occurs when the tissue is cultured in the presence of gliadin peptides (1 1-21). The in vitro developing fetal rat intestine also has been demonstrated to be a suitable model for the identification of peptides Received August 10, 1987; accepted April 13, 1988. Correspondence Prof. Salvatore Auricchio, Via S. Pansini, 5, 80131-Naples, Italy. that are toxic in celiac disease. Gliadin and prolamin peptides from cereals that are toxic in celiac disease (wheat, rye, barley, and oats) are very active in inhibiting in vitro development and morphogenesis of small intestine from 17-day-old rat fetuses (22, 23). Cornell and Townley (24) fractionated a peptic-tryptic-pancreatic digest of gliadin into 10 primary fractions by chromatography on S.P. Sephadex. Fraction 9 of this digest is the most active in causing significant reduction in D-xylose absorption in celiac patients in remission (25), preventing the morphological recovery of the epithelium of the atrophic intestinal mucosa of celiac patients (26) and inhibiting the in vitro development and differentiation of the fetal rat intestine (22). Subfractions 1 and 2, obtained by QAE Sephadex chromatography of fraction 9, and the purified forms 9-1B and 9-2B also have been shown to decrease the urinary excretion of xylose when fed to celiac patients in remission (27). Fraction 9 and subfractions 1 and 2 also were the only ones that were incompletely digested by histologically normal celiac mucosa and by mucosa of first degree relatives of celiac patients when compared to digestion by mucosa of normal controls (24,27,28), suggesting a primary mucosal defect in celiac disease (28). The aim of our study was to evaluate the capability of the peptide fractions 9 and (subfraction) 2R to inhibit the in vitro development of the fetal rat intestine and the morphological recovery of cultured atrophic celiac mucosa before and after normal digestion of the fractions with recovered celiac mucosa and normal mucosa. MATERIALS AND METHODS Preparation of gliadin peptides. Gliadin (BDH, U.K.), prepared from the wheat variety "Capella Desprez," was used for the preparation of the digests. PTC digests were prepared by treatment of the gliadin with each of the enzymes in succession and the final digests adjusted to pH 3.1 and centrifuged as described previously by Cornell and Townley (24). The PTC digests were fractionated on S.P. Sephadex (C-25) into 10 major peptide fractions as previously described (24). Subfractions of fraction 9 were prepared on QAE Sephadex A-25 as described previously (24, 28). Briefly, this involved the application of fraction 9 (400 mg) to a column (23 x 1.4 cm) of QAE Sephadex equilibrated in 0.02 M Tris-citrate buffer, pH 9.2. Subfraction 1 was eluted in the starting buffer and subfraction 2 was then 234 CORNELL ET AL. eluted on application of a 0.02 M Tris-citrate buffer of pH 6.0. The lyophilized subfraction 2 was desalted on Biogel P-2 (BioRad Laboratories, Richmond, CA) and again lyophilized. The major components of these subfractions have an apparent mol. ~ t of. 1400- 1500 (28). Subfraction 2 of fraction 9 was incubated with remission celiac mucosa at 37" C and pH 7.5 for 2 h after which it was heated to 60" C for 5 min and ultrafiltered using "Centriflo" CF 50 membrane cones (Amicon Corp., Redwood City, CA) as reported previously (24). The concentration of the peptides and amino acids remaining after digestion were measured by the microKjeldahl method. In vitro culture of fetal rat intestine. Pregnant Wistar rats were anesthetized with ether and 17-day-old fetuses were removed at laparotomy. Fetal jejunum segments were isolated and cultured in vitro for 48 h in a serum-free medium according to the method described by de Ritis et al. (29). Jejunal segments from the same fetus were cultured in the absence and presence of the peptides. All the peptide mixtures were sterilized before addition to incubation medium by filtration through 0.22 mp Millipore filters. Differentiation of the fetal rat jejunuin was followed morphologically by light microscopy as reported by de Ritis et al. (29) without knowledge of the culture conditions. A morphol (...truncated)