Anti-acids lead to immunological and morphological changes in the intestine of BALB/c mice similar to human food allergy

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Abstract

We have shown that anti-acid medication for treating dyspeptic disorders can block protein digestion and induce a higher risk for food sensitization. This mechanism was confirmed in human and animal studies on the humoral as well as the cellular level. Here we aimed to investigate the outcome of the treatment with the anti-acid drug sucralfate on the intestine in our murine model, assuming that morphological and immunological changes will occur. BALB/c mice were fed codfish extract plus sucralfate. Antibodies were examined in ELISA, RBL assay and Western blot. Quantitative morphological analysis of the intestine was performed by design-based stereology, focussing on epithelium, lamina propria, smooth muscle, eosinophils and CD3+ cells. Histological analyses were performed after H&E-, PAS- and Congo red-staining, while immune histochemistry was done for detection of CD3+ cells. Codfish-specific IgE and its activity in RBL assay confirmed the Th2-response after treatment with sucralfate. The reactivity pattern of murine IgE in Western blot was similar to allergic patients’ IgE. Histological examination showed more slender villi in the duodenum, and increased goblet cell mucus in the cecum after sucralfate treatment. Stereological analyses of the intestine revealed higher eosinophil/CD3+ ratios, decreased mean thickness of the epithelium of duodenum and cecum, and thinner smooth muscle cell layer in the colon of food allergic mice. Anti-acid treatment with sucralfate induces changes in the structure of epithelium and villi, and an increase in eosinophils and mucus-producing cells in the intestine. Therefore, this medication leads to sensitization against food with changes typical for food allergy also in the intestine.

Introduction

The increase of allergies, especially asthma and hay fever, may have reached a plateau lately (Braun-Fahrländer et al., 2004; Grize et al., 2006). Regarding food allergies, an average of 2% of the adult population (Sicherer et al., 2004) and about 6% of the pediatric population are affected (Venter et al., 2006). A novel factor responsible for the induction of food allergy was detected in our working group. We could show that anti-acid drugs inhibit the gastric protein breakdown and increase the risk for sensitization against persisting food proteins (Schöll et al., 2005; Untersmayr et al., 2004, Untersmayr et al., 2003). With the anti-acid drug sucralfate, this effect could be due to the elevation of the gastric pH and the adjuvant activity of the contained aluminum component (Brunner et al., 2007). This Th2-inducing influence of anti-acids was confirmed in human patients (Schöll et al., 2005; Untersmayr et al., 2005) and in murine models (Schöll et al., 2007; Untersmayr et al., 2003). Furthermore, the established allergic milieu of BALB/c mouse mothers fed with anti-acids and fish protein was stable enough to impact a subsequent immune response in their offspring (Schöll et al., 2007). The allergic immune status in these mice was confirmed by high levels of allergen-specific IgE- and IgG1-antibodies, positive skin tests and oral provocation test reactions (Schöll et al., 2005; Untersmayr et al., 2003), as well as increased numbers of mast cells and eosinophils in the gastric mucosa (Untersmayr et al., 2004). A detailed characterization of changes at the presumed induction site of food allergy, the intestine, has not been performed in our mouse studies yet.

Several papers show that food allergy in mice is characterized by villous atrophy and goblet cell hyperplasia, as well as infiltration of IgE-positive mast cells performing degranulation in the jejunum (Nakajima-Adachi et al., 2006). Elevated levels of IL-4 and histamine were also found in the intestinal tissue. Furthermore, a loss of body weight, jejunal edema, eosinophil infiltration, mast cell hyperplasia and increased mucus production were observed in food allergic mice (Saldanha et al., 2004). However, it is important to note that these studies either used transgenic mouse strains (Nakajima-Adachi et al., 2006) or the parenteral route in combination with adjuvants for sensitization (Saldanha et al., 2004). The aim of our current work was to characterize the effect of sucralfate on the intestine in a murine model. Due to the effective Th2-response induced in our earlier murine and human studies, we suggest that sucralfate may induce changes in morphology and immunology. In contrast to previous work, sensitization in our study is achieved by the oral route, comparable to the human situation.

Section snippets

Preparation of codfish extract

Commercially available frozen codfish was used to prepare an extract as described previously (Untersmayr et al., 2003). The protein content was determined according to the method of Bradford (1976), using a Bio-Rad Protein Assay (Bio-Rad, Munich, Germany).

Protocol of sensitization of mice

Female BALB/c mice (8 weeks) were purchased from Harlan Winkelmann (Borchen, Germany) and treated according to the European Community rules of animal care (Waldegrave, 1986) with the permission of the Austrian Ministry of Science (GZ

Sucralfate treatment of BALB/c mice induces allergen-specific antibodies

Female mice were fed intragastrically with PBS or with codfish+sucralfate. Mice treated with sucralfate showed significantly higher levels of codfish-specific IgE, IgG1, IgG2b, IgG3 and IgA compared to the PBS-treated group (Fig. 1). Antibody levels of IgE and IgG1 after sucralfate treatment were not significantly different from the codfish+alum treatment (IgE 0.8–1.0 vs. 0.8–1.1 μg/mL, IgG1 3–116 vs. 37–186 μg/mL). The biological relevance of codfish-specific IgE was confirmed in an RBL assay,

Discussion

In earlier studies, we could show that anti-acids induce food allergy and a Th2-bias at the humoral and cellular level. To characterize the effect of these drugs on the presumed induction and effector site of food allergy, we investigated the intestine in the present study.

Using anti-acid treatment with sucralfate and codfish as model allergens, we were able to induce significantly elevated allergen-specific IgE and IgG1 in BALB/c mice. This humoral immune response confirmed again the ability

Acknowledgments

We thank Anja Spies, Tanja Rausch and Mag. Julia Wallmann for their excellent technical assistance, and Mag. Krisztina Szalai for helpful scientific discussion for this manuscript. Funding was provided by a Hertha-Firnberg stipend T283-B13 of the Austrian Science Fund FWF, and by the Deutsche Forschungsgemeinschaft (SFB/TR 22).

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