Allergen-induced histamine release in rat mast cells transfected with the α subunits of FcεRI

Background: Antigenic profiles obtained by ELISA with IgE from patients with wheat food allergy (WFA) established that major allergens are albumins/globulins (AG) for children suffering from atopic eczema/dermatitis syndrome (AEDS), ω5-gliadins for adults suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA), anaphylaxis or urticaria and low-molecular-weight (LMW) glutenin subunits for patients with anaphylaxis. We aimed to characterize a new mast cell transfectant for its ability to degranulate with wheat proteins and patient sera and compare these results to those obtained by ELISA. Methods: Thirty sera from patients with WFA were tested: 14 with AEDS (group 1) and 16 with WDEIA, anaphylaxis or urticaria (group 2). An IgE Fc receptor (FcΕRI) humanized rat RBL-2H3 line was established by transfection with cDNAs encoding α-, β- and γ-subunits for the human IgE receptor. Results: A humanized RBL clone was selected for its capacity to express mRNA α-, β- and γ-subunits of FcΕRI, to bind allergen-specific human IgE and to degranulate. In group 1, sera induced enhanced degranulation with AG extract, but rarely reacted with gliadins and glutenins. In group 2, half of the sera showed degranulation with LMW glutenins whereas the AG fraction and lipid transfer proteins were rarely positive. ω5-Gliadins did not appear as a major allergen in degranulation assays, although functional allergen-specific IgE was measurable in appreciable amounts. Conclusion: Our data demonstrate that in wheat food allergen evaluation, correlation exists between mast cell degranulation and IgE measurements, depending on the type of allergen. Therefore, the biological activity of some allergen types may also be affected by other parameters.

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“…2). IgE-sensitized cells were stimulated with anti-human IgE in the absence or presence of D 2 O [38]. …”

Section: Resultsmentioning

confidence: 99%

Evaluation of an in vitro Mast Cell Degranulation Test in the Context of Food Allergy to Wheat

Bodinier

Brossard

Triballeau

et al. 2008

Int Arch Allergy Immunol

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“…It is conceivable that basophil cell lines, such as the KU812 [52], or animal cell lines transfected with the human IgE high-affinity receptor (FcεRI) [53] may also be used as recipient cells for this purpose, though these experimental systems are, at present, generally less sensitive than using primary basophils.…”

Section: Additional Applications Of Batmentioning

confidence: 99%

Diagnostic Tests Based on Human Basophils: Potentials, Pitfalls and Perspectives

Kleine-Tebbe¹,

Erdmann

Knol

et al. 2006

Int Arch Allergy Immunol

168

164

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Human basophils are important tools for studying immediate-type hypersensitivity reactions since they release a variety of mediators (e.g., histamine, leukotriene C₄, IL-4 and IL-13) following allergen triggering. Several diagnostic tools have been introduced that measure either leukotriene production or the upregulation of surface markers (CD63 and CD203c) from these cells after antigen stimulation. However, a broad variability in basophil activity exists between different basophil donors and different antigens within one donor. This manifests itself in terms of their reactivity (maximum secretory response), based on the intracellular signaling of the basophils studied, and in terms of their sensitivity. The latter is governed by the number of IgE receptors per basophil, the ratio of antigen-specific IgE to total IgE, and by the number of cell surface antigen-specific IgE molecules for half-maximal responses, termed ‘intrinsic sensitivity’. These variables give rise to shifts in the dose-response curves which, in a diagnostic setting where only a single antigen concentration is employed, may produce false-negative data. Thus, in order to meaningfully utilize the current basophil activation tests for diagnostic purposes, each allergen should be pre-evaluated separately in order to determine a suitable stimulation range. Additionally, anti-IgE or anti-FcΕRIα antibodies should serve as positive controls, bearing in mind that 10–20% of basophil donors are not responsive to IgE-mediated stimulation. Diagnostic studies using CD63 or CD203c in hymenoptera, food and drug allergy are critically discussed. Basophil-based tests are indicated for allergy testing in selected cases but should only be performed by experienced laboratories.

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“…Moreover, the level of degranulation was relatively low after such treatments, so artificial ‘accelerators’ of degranulation, such as an adenosine analogue (15) or deuterium oxide (D 2 O; 12–14), were required to measure meaningful responses. These compounds have been reported to potentiate the degranulation of mast cells (19–22), but the addition of high concentrations of D 2 O increased spontaneous mediator release from these cells (18, 20, 21). …”

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confidence: 99%

A convenient and sensitive allergy test: IgE crosslinking‐induced luciferase expression in cultured mast cells

Uchida

Higuchi

Nakamura

et al. 2010

Allergy

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BackgroundFor the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible.MethodsThe nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human FcεRI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1 : 100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen.ResultsSensitization with 15 pg/ml IgE was sufficient to detect IgE crosslinking–induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1 fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (P = 0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well (R = 0.9127, Spearman's test).ConclusionThe EXiLE test using RS-ATL8 cells is a promising in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.

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Allergen-induced histamine release in rat mast cells transfected with the α subunits of FcεRI

Cited by 20 publications

References 24 publications

Evaluation of an in vitro Mast Cell Degranulation Test in the Context of Food Allergy to Wheat

Evaluation of an in vitro Mast Cell Degranulation Test in the Context of Food Allergy to Wheat

Diagnostic Tests Based on Human Basophils: Potentials, Pitfalls and Perspectives

A convenient and sensitive allergy test: IgE crosslinking‐induced luciferase expression in cultured mast cells

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