Z Gastroenterol 2012; 50 - V07
DOI: 10.1055/s-0032-1323857

Purifikation, Charakterisierung und biologische Wirkungen von alpha-Amylase-/Trypsininhibitoren aus Pflanzen, Effektoren von intestinalen TLR4 bei Zöliakie

V Zevallos 1, Y Junker 2, M Laparra 3, B Hebich 1, N Rüssel 1, D Schuppan 1
  • 1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany
  • 2Dept. of Paediatrics, Univ. of Kiel Medical School, Kiel, Germany
  • 3Agrochemistry and Food Technology Institute, National Research Council (CSIC), Valencia, Spain

Aims: The family of cereal alpha-amylase/trypsin inhibitors (ATIs), pest resistance molecules of grains, has been implicated in the development of allergic responses. We have identified wheat ATIs (CM3 and 0.19) as potent activators of innate immunity in celiac disease by engaging the toll like receptor 4 (TLR4)-MD2-CD14 complex in monocytes, macrophages and dendritic cells. ATIs are also present in other plants but have not been evaluated in relation to innate immune activation.

Objective: To extract, purify and characterize ATIs from cereals, grains and legumes and to evaluate their ability to stimulate innate immunity using a cellular bioassay.

Methods: ATIs were exhaustively extracted from milled and defatted samples using salt solutions and comparatively dialyzed against acidic and neutral buffers, sterile filtrated and lyophilized. Total protein content, ATIs, CM3 and 0.19 were analyzed by Western-blotting and quantified by reverse phase (RP) HPLC. Extracts were cultured with monocytic cell lines and concentrations of inflammatory cytokines measured in cell culture supernatants after overnight incubation.

Results: We identified four groups of plants based on the relative potency to induce innate immune reactions expressed in units of IL-8 released (100 units per gram of sample). Group 1, >100 units (wheat, barley and rye), Group 2, between 100 and 50 units (soya, quinoa, buckwheat, peas and spelt) and Group 3 <50 units (rice, white corn, millet, amaranth, popcorn) and <10 units (oats and rice). In addition, we observed up to 5 fold differences between different wheat grains.

Conclusions:

  • Our method permits quantitative extraction of ATIs from different plants and their testing as stimulators of innate immunity using a bioassay based on cytokine secretion by monocyte cell lines;

  • Using this methodology we can define 4 classes of grains and their substitutes according to stimulatory activity;

  • Since we implicate ATIs both in the pathogenesis of celiac disease and of other autoimmune diseases, these results will form the basis for further experimental and human studies.

Zöliakie: Volkskrankheit mit Tarnkappe?
Donnerstag, 20. September 2012/10:30–12:00/Saal 8