Sensitization and Elicitation of an Allergic Reaction to Wheat Gliadins in Mice (1)
Wheat flour proteins are divided in albumins and globulins (AG) that are soluble in water and saline buffers and in gliadins and glutenins that are insoluble. All were shown to be involved in food allergy to wheat (WFA). Relevant animal models are of interest for improving our understanding of the mechanisms of allergic sensitization and elicitation of symptoms. Mouse has been widely used to study the induction and polarization of the immune response toward a Th1, Th2, or Th17 phenotype. The Balb/cJ strain is a Th2-biaised high-IgE responder strain. It has been widely used for sensitization to food allergens, e.g., cow’s milk proteins (including b-lactoglobulin), peanut proteins, or ovalbumin. C3H/HeJ mice can also be sensitized to milk and peanut because they are deprived of “Toll- like” receptor 4 (TLR 4).
Acid-PAGE confirmed the presence of the four gliadins, i.e., a-, b-, g-, and ω-gliadins. A slight contamination by LMW glutenins was evidenced by SDS-PAGE but represented only a small percentage of the total protein amount. Gliadins in solution in 50% ethanol (1 mg/mL) were slowly diluted in 50 mM carbonate buffer (pH 9.6) at 5 μg/mL and then used to coat 96-well microtiter plates. Children demonstrated an intense IgE response against a- and b-gliadins, a moderate response against γ-gliadin, and a weak response against ω5-gliadin. Inversely, in adults the strongest IgE response was toward ω5-gliadin.
Freeze-dried GE was solubilized in 70% ethanol at 5 mg/mL and then slowly diluted at 0.2 mg/mL in sterile phosphate- buffered saline. Intraperitoneal immunizations with 10 or 20 μg of GE adsorbed onto alum were performed on days 0, 10, 20, and 30 (0.2 mL per mouse).
In the three strains, no significant difference was observed between the two doses, i.e., 10 and 20 μg, after three and four i.p. administrations. In Balb/ cJ mice, specific IgE and IgG1 production was significantly higher than in the two other strains, whatever the dose, except for IgE production in C3H/ HeJ mice at the 10 μg dose.
In Balb/cJ mice, high concentrations of Th2 cytokines (i.e., IL-4, IL-5, IL- 10, and GM-CSF) but moderate concentrations of Th1 cytokine (IFNγ) were produced. More than 10-fold lower levels of Th2 cytokines were secreted by reactivated splenocytes from C3H/HeJ and B10.A mice.
Intense IgE response against a-, b-, and γ-gliadins and a moderate one against ω-gliadin. Balb/cJ mice produced the significantly highest concentration of specific IgE against all gliadins, but the pattern of IgE specificity was similar in the three strains.
Wheat is part of the normal diet of mice, to minimize the induction of oral tolerance by feeding young weaned mice, a semisynthetic diet deprived of plant proteins were served. Different adjuvants can be used for i.p. immunization. Mineral oil-based aluminum hydroxide that preferentially stimulates Th2 cells was used, while complete Freund’ s adjuvant (CFA) preferentially stimulates a Th1-type response. The use of Freund’s adjuvant may modify, partially denature, the structure of the allergen. Several linear epitopes of ω5-gliadin were identified in patients suffering from WDEIA, including a consensus QQX1PX2QQ motif, where X1 represents leucine, phenylalanine, serine, or isoleucine and X2 represents glutamine, glutamic acid, or glycine.