A Decapeptide from Durum Wheat Prevents Celiac Peripheral Blood Lymphocytes from Activation by Gliadin Peptides

0031-3998/07/6101-0067 PEDIATRIC RESEARCH Copyright © 2006 International Pediatric Research Foundation, Inc. Vol. 61, No. 1, 2007 Printed in U.S.A. A Decapeptide from Durum Wheat Prevents Celiac Peripheral Blood Lymphocytes from Activation by Gliadin Peptides MARCO SILANO, RITA DI BENEDETTO, ANTONELLO TRECCA, GIOACCHINO ARRABITO, FABIANA LEONARDI, AND MASSIMO DE VINCENZI Division of Food Science [M.S, R.D.B, F.L., M.D.V], Human Nutrition and Health, Istituto Superiore di Sanità, Rome, Italy 00161; Division of Endoscopy and Operative Gastroenterology [A.T, GA], “Fabio Di Giovanbattista,” Rome, Italy 00100 production of interferon (IFN)-␥ by celiac PBMC incubated with the p57–73 itself (7). This therapeutic strategy could also rely upon ligand antagonists naturally occurring in some grains, such as a decapeptide (10mer) identified in the peptic-tryptic digest of the alcoholsoluble protein fraction of durum wheat (GLP) (8). This molecule, whose sequence is QQPQDAVQPF, named in previous studies “1157” after its molecular weight, has been demonstrated to protect some cellular lines from gliadin toxicity (8,9). Although in CD pathogenesis the central role is played by gluten-specific HLA DQ2/DQ8 restricted CD4⫹ intestinal T cells, recent studies indicate that the peripheral blood lymphocytes from celiac patients could be as informative as the intestinal ones. Polyclonal effector T-cell lines appear in the peripheral blood after in vivo gluten challenge and are able to secrete inflammatory cytokines after in vitro stimulation with gliadin peptides (7,10,11). In addition, the PBMC from celiac patients on a gluten-containing diet secrete inflammatory cytokines, express activation markers, and show immunologic modifications after incubation with gliadin (12–17). So, peripheral blood lymphocytes provide a system for rapid and easy assessment of the toxicity of gluten epitopes and the antagonist effects of ligand peptides. In this study, fresh PBMC from celiac patients on a glutencontaining diet are used to define the antagonist effect of 10mer against the ability of gliadin peptides to induce the activation of the immune system. As gliadin peptides, the whole peptic-tryptic digestion of alcohol-soluble protein fraction of wheat and the ␣-gliadin sequence 62–75 have been used. ABSTRACT: Identifying antagonist peptides able to inhibit the abnormal immune response triggered by gliadin peptides in celiac disease (CD) is an alternative therapeutic strategy for CD. The aim of this study was to evaluate the antagonist effect of 10mer, a decapeptide (sequence QQPQDAVQPF) from alcohol-soluble protein fraction of durum wheat, assessing its ability to prevent celiac peripheral blood lymphocytes from activation by gliadin peptides. Peripheral blood mononuclear cells (PBMC) were obtained from DQ2-positive untreated coeliac children and from healthy controls and incubated with the peptic-tryptic digest of bread wheat gliadin (GLP) and peptide 62–75 from ␣-gliadin both alone and with 10mer simultaneously. PBMC proliferation, release of pro-inflammatory Th1 cytokines interferon-␥ and tumor necrosis factor-␣, release of immunoregulatory cytokine IL-10, and analysis of CD25 expression as indexes of lymphocytes activation were carried out. Enhanced lymphocytes activation was seen after exposure to GLP and p62–75, whereas the simultaneous incubation with 10mer inhibits the lymphocytes response. These data indicate that a peptide naturally occurring in durum wheat exerts in vitro an antagonist effect against gliadin toxicity and could have a protective effect in CD disease. (Pediatr Res 61: 67–71, 2007) C D is an increasingly diagnosed auto-immune enteropathy (prevalence 1:150), driven by an abnormal T-cell response to wheat gluten– derived peptides and strongly associated with HLA-DQ2, which is present in more than 90% of CD patients (1–3). So far, the only effective treatment is the withdrawal of the gluten-containing cereals from the diet (4). Complying with a gluten-free diet is difficult because of the diffusion of the cereals-based foods and it affects patients’ quality of life; therefore, new treatments are being actively searched. An alternative therapeutic possibility may be to identify modified gliadin (a gluten fraction) peptides that are not toxic and are also able to inhibit the pathogenic immune response. In fact, some single amino acid substitutions at critical position of T-cell epitopes convert highly toxic peptides in ligand antagonists, as tested both in animal and ex vivo models (5,6). It has been reported that some peptides obtained by amino acid modifications of peptide 57–73 from ␣-gliadin abolish the MATERIALS AND METHODS Peptic-tryptic digestion. The alcohol-soluble protein fraction from whole cereal flour of bread wheat (Triticum aestivum, variety S. Pastore) was extracted and subjected to peptic-tryptic digestion, as previously described (18). Gliadin preparations were assayed for endotoxin by using the QCL-100 reagent kit (BioWhittaker, Walkersville, MD) and found to have endotoxin levels of ⬍0.5 EU/mL. Peptide 10mer and P62–75. The sequence of peptide 10mer (MW, 1157 D) was identified in the alcohol-soluble protein fraction of durum wheat (Triticum durum, variety Adamello) by De Vincenzi et al. (8). P62–75 contains one of the two overlapping 9-aminoacid sequences within the p57–73, a HLADQ2–restricted peptide able to induce an intestinal T-cell response in celiac Received February 23, 2006; accepted August 15, 2006. Correspondence: Massimo De Vincenzi, Ph.D., Division of Food Science, Human Nutrition and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy; e-mail: Abbreviations: BrdU, 5-bromo-2-deoxyuridine; CD, celiac disease; GLP, gliadin peptides; PBMC, peripheral blood mononuclear cells; tTG, tissue transglutaminase DOI: 10.1203/01.pdr.0000250173.88049.79 67 68 SILANO ET AL. Table 1. Details of peptides Abbreviation Source Sequence p62–75 A-gliadin protein sequence PQPQLPYPQPQLPY Gliadin peptides Peptic-tryptic digestion of (GLP) alcohol-soluble protein fraction of Triticum aestivum 10mer Alcohol-soluble protein QQPQDAVQPF fraction of Triticum durum patients (19). The peptides 10mer and p62–75 were synthesized (Primm Company, Milan, Italy) by the solid phase method using Applied Biosystems’ (Foster City, CA) model 431A and purified up to 99% by reverse-phase HPLC on Varian 5020 system. The details of the peptides are listed in Table 1. Transglutaminase deamidation. To deamidate GLP, p62–75, and 10mer, they were incubated for 4 h at 37 C° in a mix consisting of 100 ␮g/mL guinea pig liver tTG, 2 mM CaCl, and 400 ␮L GLP, p62–75, or 10mer. All reagents were purchased from Sigma Chemical Co., St. Louis, MO. Study population. Ten children with biopsy-proven CD were studied (7 females, 3 males; mean age, 6.2 y; range, 3–14 y). All of them showed signs and symptoms suggesting CD and underwent diagnostic gastroduodenal endosco (...truncated)