CD: Celiac Disease; PBMC: Peripheral Blood Mononuclear Cells; HLA: Human Leucocyte Antigen; Ttg: Tissue Tranglutaminase; PHA: Phytohaemaglutinin A; PT: Pepsin-Trypsin; Gd: Gliadin; G33-mer, Immunogenic Peptide of α-gliadin; Z34-mer, Immunogenic Peptide of α-zein

CD is an immunologically mediated systemic disorder developed in genetically predisposal individuals, exacerbated by wheat and related cereals as barley and rye. Disease symptoms are promoted by inflammation of the intestinal mucosa, inducing gastrointestinal and/or extra-intestinal manifestations [1]. CD is a lifelong condition and gluten-free diet is the only treatment. One of the most important alternative cereals used for the gluten-free bakery products is maize; additionally, its prolamins have been used as a negative control in different studies on CD. By chance, in some of those studies maize prolamins have demonstrated adverse effects [2,3] inducing doubt about the maize use for dietary treatment of CD patients. The response to maize prolamins could be due to similarities between maize (zein) and wheat (gliadin) prolamins both with a high percentage of glutamine able to be deamidated by transglutaminase and proline residues that hinders a full digestion by gastrointestinal proteases [3].

The proposed pathogenesis of CD highlights the role of T-cells, after peptide presentation by dendritic cells to Th1 cells via the HLA-DQ2/8 context, activating them and consequently releasing cytokines, mainly IFN-γ [4]. Therefore, IFN-γ is a marker of cellular response to different gluten peptides by in vitro assays; its advantage is that promotes tissue inflammation and has no autocrine effect on other PBMC, like monocytes [5-7]. Gluten specific T-cells producing IFN-γ can be found in peripheral blood of CD patients in gluten-free diet after a short gluten challenge [5]. Isolation and subsequent in vitro stimulation of these T-cells with a wide variety of dietary peptides, generates a reliable tool to evaluate the cellular response to gluten-free foods [6]. The aim of this study was to evaluate the T-cell response in vitro to maize prolamins in comparison to wheat prolamins of peripheral blood mononuclear cells (PBMC) from CD patients and PBMC from non- CD individuals as controls, after gluten-free diet followed by a threeday gluten challenge.

Patients

Patients underwent gluten-free diet for at least one month, and a three days challenge with at least 50 g/day gluten was made and blood samples were taken at day 0 and day 6. The ethical committee of the Centro de Investigación en Alimentación y Desarrollo (CIAD A.C.) approved the study and all samples were taken under informed written consent. Whole blood was taken (14 mL) from each patient by venipuncture into Vacutainer tubes (BD Medical Systems, USA). DNA was extracted from 200 μL whole blood by the QIAamp DNA Blood Mini Kit (QIAGEN, USA) and genotyping of HLA-DQ2/DQ8 was done by real time PCR (Step One Plus, Applied Biosystems) using specific primers [8]. Isolation of peripheral blood mononuclear cells (PBMC’s) from 12 mL blood was done using Ficoll-Paque PLUS (Amersham-Biosciences, Sweden) density gradient centrifugation technique. Plasma anti-gliadin (Gd) IgG, anti-Gd IgA, anti-zein IgA and anti-transglutaminase (TG) IgA antibodies were analyzed by a direct enzyme-linked immunosorbent assay (ELISA), as previously reported [9]. IgA anti-gliadin and/or zeins and IgA anti-TG were expressed as an index value and it was calculated based on the mean of absorbance values of control individuals as reported before [9] and index values of 1.0 and above were considered as positive.

Peptide preparation

The immunogenic peptides α-gliadin 33-mer (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; MW = 3914.51 Da), later referred to as G33-mer, and α-zein 34-mer (LQQAIAASNIPLSPLLFQQSPALSLVQSLVQTIR; MW = 3646.32 Da), later referred to as Z34-mer, were supplied by United Biosystems (USA) with purities of 97.54% and 95.66%, respectively. Gliadins from wheat and zeins from maize (Sigma Chem Co, St. Louis, MO USA) were subjected to pepsin-trypsin (PT) digestion, as previously described [2]. All immunogenic peptides and digested prolamins were treated with transglutaminase (TG) from guinea pig liver (Sigma- Aldrich, St Louis, MO USA) 5 μg/500 mg of protein in CaCl₂ 2 mM for 60 min at 37°C and then placed on ice. Separation of TG was performed by ultrafiltration (UF cell, Amicon Inc. Beverly, MA. USA.), with a 30 kDa cut-off membrane and peptides were recovered in sterile water.

Cell culture and cytokine assays

Isolated PBMC were incubated at a final concentration of 2 x 10⁵ cells/mL on culture plates and cultured in Dulbecco’s Modified Medium (D-MEM) containing 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 37ºC in a 5% CO₂ atmosphere. The immunogenic peptides were used in the experiments at final concentration of 50 μg/mL and the digested prolamins at 100 μg/mL. Phytohemagglutinin A (PHA) (Sigma Aldrich, USA) was used as positive control at concentration of 25 μg/mL. After 20 h, supernatants were collected and frozen at -70ºC prior to cytokine evaluation. ELISA kits were used for IFN-γ (Mabtech, Sweden) detection according to manufacturer.

Statistical analysis

Experiments were performed in triplicate, results are given as mean values that were compared after ANOVA. Statistical significance among days 0 and 6 was compared by Student’s one sample T-test and statistical significance among treatments by Tukey-Kramer multiple comparison test using the statistical software NCSS, version 2001. The p-values of 0.05 or less were considered as statistically significant.

The characteristics of the three control subjects and the two celiac patients are described in Table 1. All the control individuals showed negative indexes (<1.0) of anti-Gd IgG, anti-Gd IgA and anti-TG IgA antibodies. Celiac patient 1 presented positive indexes (>1.0) for anti- Gd IgG and anti-TG IgA antibodies, while patient 2 had for anti-Gd IgG, anti-Gd IgA, anti-TG IgA and anti-Zein IgA antibodies (Table 1).

Table 1: Characteristics of control individuals and celiac disease patients; CD: celiac disease; ND: not done; HLA: human leucocyte antigen; DQA1: alpha-chain DQ alleles; DQB1: beta-chain DQ alleles; IgG: G isotype immunoglobulin; IgA: A isotype immunoglobulin; Gd: gliadins; Zn: zeins; tTG: tissue transglutaminase.

The immunogenic peptide of α-zeins (Z34-mer) induced an increased release of IFN-γ in both celiac patients, but the stimulus remained after 6 days only on patient 2. Our work team also observed cell stimulation by this proposed immunogenic peptide when duodenal bulb intestinal biopsies were challenged in vitro under cell culture conditions [2]. Cell response to Z34-mer is independent of the gluten challenge and the higher response at day 0 could be explained by the fact that maize is a common constituent food of the gluten-free diet and patients were highly exposed to larger quantities of its protein. On patient 2, the high serum IgA anti-zeins detected (Table 1), suggest a higher sensibility that is reflected on a greater response with respect to patient 1 and controls. Therefore, the response decreases significantly at day 6 (p<0.05, Figure 1) perhaps due to lower consumption of maize by consumption of the gluten challenge.

Figure 1: Release of the Th1 response related cytokine IFN-g in control individuals and CD patients. Absorbance values of IFN-γ are plotted based on log10 and represents the fold increase of cytokine in PBMC’s without stimulation. All cytokine values of controls individuals were averaged and presented as a single value. CD patients were plotted individually as patient 1 and 2. G33-mer at patient 1 in day 0 vs. day 6 (p<0.05); GFIII at patient 2, day 0 vs. day 6 (p<0.05); Z34-mer at patient 2 vs. control and patient 1 (p<0.005); Z34-mer at patient 2, day 0 vs. day 6 (p<0.05); G33-mer in both patients vs. controls (p<0.005); ZFIII at patient 2 vs. controls and patient 1 (p<0.005). IFN-γ, interferon gamma; G33- mer, α-alpha-gliadin 33-mer; GFIII, gliadin digested fraction; Z34- mer, alpha-zein 34-mer; ZFIII, zein digested fraction.

An increase of IFN-γ release was also observed by the PT-digested fraction of zeins (ZFIII) on patient 2, comparable to response to PTdigested fraction of gliadins (GFIII). Contrary to Silano et al. [6] who used smaller amounts of PT-digested wheat to obtain a T-cell response, we saw poor response to our PT-digested gliadins despite having used a larger amount of gliadin-digested fraction. It is possible that the immunogenic epitopes in this peptide fraction were insufficient to achieve cell stimulation as consequence of handling and digestion procedure. However, stimuli with gliadin peptides was clearly observed by using the α-gliadin peptide G33-mer that has been demonstrated to have a single dominant epitope that elicits an optimal IFN-γ release in gluten-sensitive T-cells [5].

Z34-mer induced cellular response in a non HLA-DQ8 patient and this was also observed in our previous work on other patients that do not have this haplotype [2], even though it had been shown in silico to have affinity to the HLA-DQ8 tetramer [3]. The amino acid sequences between Z34-mer and G33-mer peptides are quite different. However, they share prolamin features like poor digestion by mammalian proteases and their glutamine residues able to be deamidated by tTG that can increase affinity to HLA-DQ2 or DQ8 molecules in antigen presenting cells and to induce a cellular response. Isolation of PBMC and its posterior stimulation in vitro with zein peptides could be an efficient tool for finding epitopes in maize protein in some nonresponsive subjects to the gluten-free diet.

In conclusion, in PBMC of a CD patient a cellular response to maize zeins was induced and this response was even higher to that induced by wheat gliadins although independent of the gluten challenge. In vitro stimulation of PBMC with immunogenic peptide Z34-mer is comparable to that of the G33-mer with a dominant epitope that elicits an optimal IFN-γ release in gluten-sensitive T-cells.

J.P. Ortiz-Sanchez received a Ph.D. fellowship from the Mexican Conacyt (101386).