Epithelial Transport of Immunogenic and Toxic Gliadin Peptides In Vitro

Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, Spain Received: March Epithelial Transport of Immunogenic and Toxic Gliadin Peptides In Vitro Christian Zimmermann 0 Silvia Rudloff 0 1 G unter Lochnit 2 Sevgi Arampatzi 3 Wolfgang Maison 4 Klaus-Peter Zimmer 0 0 Department of Pediatrics, Justus Liebig University Giessen , Feulgenstr. 12, D-35392, Giessen, Germany, 1 Institute of Nutritional Science, Justus Liebig University Giessen , Wilhelmstr. 20, D-35392, Giessen, Germany, 2 Institute of Biochemistry, Medical Faculty, Justus Liebig University Giessen , Friedrichstr. 24, D-35392, Giessen, Germany, 3 Institute of Organic Chemistry, Justus Liebig University Giessen , Heinrich-Buff-Ring 58, D-35392, Giessen, Germany, 4 Pharmaceutical and Medicinal Chemistry, Universita t Hamburg , Bundesstr. 45, D-20146, Hamburg , Germany Scope: Celiac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31-43 and the immunogenic P56-68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length. Methods and Results: Fluorescence labeled P31-43 and P56-68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOFMS. According to fluorescence intensities, significantly more P31-43 compared to P56-68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56-68, but we observed for the first time the degradation of the toxic peptide P31-43 at the apical side of epithelial cells. Conclusion: Considering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the - integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response. With a prevalence of 1% celiac disease is one of the most common inflammatory disorders in Western populations; it is mainly triggered and maintained by gluten, storage proteins in wheat, rye, and barley [1, 2]. In addition to gluten as environmental factor, a genetic predisposition is required to develop celiac disease. The vast majority of celiac patients express human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8 by which immunogenic gluten-derived gliadin peptides (GP) are presented and thereby induce the proliferation of gluten sensitive T-cells [1]. The oral intake of gluten may then lead to an initiation of innate and adaptive immune responses which result in cell damage and villus atrophy in the small intestine with subsequent malabsorption and diarrhoea. Up to now the only treatment is a life-long gluten-free diet not only to enable mucosal recovery but also to minimize the risk for the development of enteropathyassociated T-cell lymphoma which has been seen to be markedly increased in patients with untreated celiac disease [2, 3]. In this context, gliadins as a fraction of gluten proteins [4] were identified as important inflammatory stimuli in celiac patients [5]. Currently, two groups of GP are considered to contribute to the disease process in a different manner. Therefore, a distinction is made between immunogenic and toxic GP [6]. Immunogenic GP contain sequences which stimulate HLA-DQ2 or HLA-DQ8 restricted T-cell clones derived from celiac patients thereby inducing an adaptive immune response [711]. For example, P5668 is considered to be a sequence representative of immunogenic peptides located at amino acid position 5668 of a-gliadin [11]. In contrast, toxic GP were shown to induce non T-cell mediated mucosal damage in biopsies from celiac small intestine [1214]. P3143, a peptide sequence located at position 3143 of a-gliadin represents a GP from the group of toxic peptides [12]. As a prerequisite for these immune reactions, GP have to resist proteolytic digestion and to pass the intestinal epithelial barrier and gain access to the lamina propria. Dietary proteins are digested by soluble gastric and pancreatic proteases and are subsequently hydrolysed by peptidases located in the brush border membrane of enterocytes [15]. Furthermore, antigens can be degraded in the endocytotic pathway of enterocytes [10]. However, proline-rich peptides are inaccessible for most proteases leading to the hypothesis that GP which are rich in proline are resistant to proteolytic breakdown [16]. These antigens may then cross the epithelial barrier via paracellular pathways regulated by tight junctions which are thought to be accessible for small antigens with a maximal molecular weight of about 5,500 Da [17]. Alternatively, antigens may pass the monolayer transcellular by endocytosis [18]. Up to now, underlying mechanisms of translocation and processing of the GP P3143 and P5668 in enterocytes are not clear. Matysiak-Budnik et al. [19] reported that the immunogenic P5768 was partially degraded by brush-border peptidases in biopsies from control but not in those from celiac patients. During the transport process across the epithelial barrier, however, it was completely degraded in biopsies from both, control and celiac patients. In contrast, the toxic P3149 was shown to be resistant against degradation by brush-border peptidases in celiac and control patients. Still, the intact peptide was only able to pass the epithelial barrier in biopsies from celiac patients but not in those from controls. Thus, the specific ways of epithelial translocation of immunogenic and toxic GP are considered to be a crucial step in the pathogenesis of celiac disease. Using fluorescence labeled immunogenic and toxic GP in enterocytes and investigating their intracellular transport, it has been suggested that the immunogenic P5668 was transported into late endosomes whereas the toxic P3143 and P3149 remained in early endosomes. Due to the transport of P5668 into HLA-class-II positive late endosomes a degradation and/or a presentation by enterocytes was hypothesized. In contrast, it is supposed that toxic GP stay intact on the endocytotic route and ar (...truncated)