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Dietary Wheat Germ Agglutinin Modulates Ovalbumin-Induced Immune Responses in Brown Norway Rats

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Dietary Wheat Germ Agglutinin Modulates Ovalbumin-Induced Immune Responses in Brown Norway Rats British Journal of Nutrition (2001), 85, 483±490 DOI: 10.1079/BJN2000287 q The Authors 2001 Dietary wheat germ agglutinin modulates ovalbumin-induced immune responses in Brown Norway rats Bernhard Watzl1*, Christian Neudecker2, Gertrud M. HaÈnsch3, Gerhard Rechkemmer1 and Beatrice L. Pool-Zobel3² 1Institute of Nutritional Physiology, 2Institute of Hygiene and Toxicology, Federal Research Centre for Nutrition, Haid-und-Neu-Str, 9, 76131 Karlsruhe, Germany 3Institute of Immunology, Ruprecht Karls University, Heidelberg, Germany (Received 7 July 2000 ± Revised 23 October 2000 ± Accepted 1 November 2000) The trend towards an increased consumption of minimally processed plant food results in a higher intake of non-nutritive compounds such as lectins. Lectins are typically globular proteins that are resistant to digestion in the gastrointestinal tract. They affect the integrity of the intestinal epithelium and the absorption of dietary antigens, and induce the release of allergic mediators from mast cells in vitro. Based on this information we have studied whether dietary wheat germ agglutinin (WGA) could be involved in triggering food allergies. Brown Norway rats were immunized intraperitoneally using ovalbumin (OVA; 10 mg/rat) and 10 d later treated for five consecutive days with WGA (10 mg/rat per d) administered intragastrically. Rats were then orally challenged with OVA (100 mg/rat) 1 h after the last WGA application, and blood was collected 4 h later. Immunological responses (anti-OVA immunoglobulins E and G, rat mast cell protease II, interferon-g and lymphocyte proliferation) were measured and lymphocyte subpopulations were determined. In immunized rats WGA treatment resulted in increased serum rat mast cell protease II concentrations (pre-challenge 0´26 (SE 0´08) mg/ml, post-challenge 0´49 (SE 0´09) mg/ml; P , 0´01) 4 h after the OVA challenge. After 5 d serum concentrations of anti- OVA immunoglobulin E were significantly increased only in the immunized controls (absor- bance at 405 nm on days 14 and 19 was 0´09 (SE 0´008) and 0´24 (SE 0´046) respectively; P 0´02), while in WGA-treated rats no significant increase was seen (0´08 (SE 0´004) and 0´15 (SE 0´037 respectively; P 0´14). CD4+ : CD8+ T lymphocytes in the spleen was significantly increased at this time (OVA 1´1 (SD 0´2), OVA WGA 1´4 (SD 0´1), P , 0´05). The treatment did not impair the proliferation and interferon-g production of mesenteric lymphocytes. In conclusion, these data suggest that high dietary intake of lectins such as WGA may affect the allergic response towards oral antigens in the gut-associated lymphoid tissue. Wheat germ agglutinin: Immune response: Allergy Recent dietary recommendations have emphasized the Cordain et al. 2000). Lectins occur in many crop plants and increased consumption of whole-wheat products (World are part of the normal diet (Nachbar & Oppenheim, 1980). Cancer Research Fund and American Institute for Cancer They are typically globular proteins that are resistant to Research, 1997). This may result in an elevated dietary digestion by mammalian and bacterial enzymes and may intake of lectins such as wheat-germ agglutinin (WGA). pass intact through the gastrointestinal tract. Lectins may The health effects of dietary lectins have recently been further become internalized and circulate intact in the addressed by several authors who have pointed out that peripheral blood (Pusztai et al. 1993; Wang et al. 1998). currently only limited information is available on the They provoke diverse biological consequences in mammals immunotoxicity of lectins (Freed, 1999; Kilpatrick, 1999; such as hyperplasia and hypertrophy of the small intestine Abbreviations: BN, Brown Norway; GALT, gut-associated lymphoid tissue; Ig, immunoglobulin; IFNg, interferon-g; IL, interleukin; OVA, ovalbumin; RMCPII, rat mast cell protease II; WGA, wheat-germ agglutinin. * Corresponding author: Dr B. Watzl, fax +49 721 6625 404, email [email protected] ² Present address: Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University, Jena, Germany. 484 B. Watzl et al. and could increase the permeability of the intestinal wall, Materials and methods resulting in enhanced absorption of dietary antigens Materials (Pusztai, 1991; Freed, 1999; Kilpatrick, 1999; Cordain et al. 2000). WGA was purchased from Sigma (Deisenhofen, Germany). There are no data available on the average dietary Fetal bovine serum, glutamine, penicillin and streptomycin intake of WGA in human subjects. In cereals, WGA were obtained from Boehringer (Mannheim, Germany). concentrations ranged from 13 to 53 mg/kg (Ortega- Reagents for immunofluorescence staining were all from Barria et al. 1994). The highest WGA concentrations are Pharmingen (Hamburg, Germany). Antibodies were pur- found in wheat germ (#0´5 g/kg; Peumans & van chased from Sera Lab, Crawley Down, West Sussex, UK Damme, 1996). The estimated quantity of total dietary (anti-rat IgE), from The Binding Site, Heidelberg, Germany lectins is in the range 0±200 mg/person per d (calcula- (anti-rat IgG), and from Southern Biotechnology, Birming- tions based on data from Peumans & van Damme, 1996). ham, AL, USA (anti-mouse IgG1). All the other reagents Such a dose range is expected to be too low to induce were obtained from Sigma. non-nutritive or toxic effects in the gastrointestinal tract (Peumans & van Damme, 1996). In volunteers consum- ing wheat germ with the equivalent of 200 mg active Animals and treatment schedule agglutinin, no side effects were reported (Brady et al. Adult male BN rats were obtained from the breeding 1978). In another group of volunteers the consumption of colony of the Federal Research Centre for Nutrition, whole wheat resulted in the generation of serum anti- Karlsruhe, Germany. Permission for the animal studies WGA antibodies (Tchernychev & Wilchek, 1996), was obtained from the State Veterinary Office and indicating that intact WGA passes through the intestinal experiments were performed in compliance with their wall and reaches cells of the gut-associated lymphoid guidelines for the care and use of laboratory animals. BN tissue (GALT) located in the lamina propria. Only few rats were fed ad libitum with a WGA- and OVA-free diet data are currently available on the immunotoxicity of (C1000; Altromin, Lage, Germany); 12 h before substance orally administered WGA on the GALT (Pusztai et al. application and before termination of the experiment, 1993), although this is probably the immune tissue with feeding was stopped. All test substances were administered the highest WGA exposure. The immunological con- intragastrically by tube. OVA and WGA were dissolved in sequences of the trans-epithelial transport of intact WGA 1 ml saline (9 g NaCl/l). from the diet are unknown. BN rats (six to eight rats per treatment) were immunized In the rat WGA binds to surface glycans on epithelial intraperitoneally on day 0 using 1 ml OVA (10 mg/ml) cells causing damage to the base of the villi. The resulting dissolved in saline without use of an adjuvant. Control structural changes include increased intestinal permeability animals were treated intraperitoneally with 1 ml saline. for different permeability markers (Pusztai et al. 1993). In During days 10±14, rats were treated for five consecutive tissue culture experiments WGA blocked as well as days: group A, non-immunized control animals were treated enhanced the epithelial permeability of various perme- with 1 ml saline/d; group B, immunized rats were ability markers, depending on their molecular size treated with 1 ml saline/d; group C, immunized rats were (Lorenzsonn & Olsen, 1982; SjoÈlander et al. 1984). In treated with 10 mg WGA (27 mg/kg body weight)/d. At vitro WGA interacts with the interleukin (IL) 2 receptor on 1 h after the last treatment with WGA, the animals of all T and B lymphocytes, and thereby impairs lymphocyte three groups were challenged orally with 100 mg OVA/rat function (Pusztai, 1991). WGA binds to immunoglobulin dissolved in 1 ml saline. Doses of OVA were based on (Ig) on leucocytes from allergic patients and induces previous experiments (Watzl et al. 1999a,b). The dose of histamine secretion (Shibasaki et al. 1992), it stimulates WGA was chosen to be ten times higher than the average histamine release by murine peritoneal non-sensitized mast total dietary intake of lectins in human subjects consuming cells (Lansman & Cochrane, 1980) as well as IL-4 and IL- vegetarian diets (calculations based on data from Peumans 13 secretion by human basophils (Haas et al. 1999). These & van Damme, 1996). cytokines are key promotors of both T-helper 2 lymphocyte responses and IgE synthesis. Such in vitro activities suggest a potential of WGA-rich food to stimulate allergic Blood collection responses to dietary antigens. The hypothesis of the present study was that WGA could Blood was obtained from the tail vein at different time impair the intestinal epithelial integrity, thereby facilitating points during the experiment, and at the end of the the penetration of antigens and impairing the tolerance of experiments from the axillary plexus of rats anaesthetized the gut towards these antigens. In addition, WGA might with diethyl ether. Serum was obtained and stored at further directly interact with mucosal mast cells and non- 2208C until further processing. specifically induce mediator release. The objective of the present study was to investigate whether toxic effects of Cell isolation and culture WGA in the intestinal tissue could be involved in triggering food allergies. An established animal model (Watzl et al. Lymphocytes were isolated from spleen and mesenteric 1999a,b) was used to study whether dietary WGA affected lymph nodes and were suspended in RPMI 1640 the immune response to intragastrically applied ovalbumin medium, supplemented with 10 mM-HEPES buffer solu- (OVA) in immunized Brown Norway (BN) rats. tion, and 5 % (v/v) heat-inactivated fetal bovin serum, Wheat germ agglutinin and immune response 485 2-mercaptoethanol (50 mM), L-glutamine (2 mmol/l), peni- (IFNg) was quantified by ELISA as described earlier cillin (100 U/ml), and streptomycin (100 mg/ml).
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