Dataset Information

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

ABSTRACT:

Background

For 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 (Fc?RI)-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.

Methods

The nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human Fc?RI-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.

Results

Sensitization 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).

Conclusion

The 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.

SUBMITTER: Nakamura R

PROVIDER: S-EPMC3066406 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Publications

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

Nakamura Ryosuke R Uchida Y Y Higuchi M M Nakamura R R Tsuge I I Urisu A A Teshima R R

Allergy 20100330 10

<h4>Background</h4>For 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.<h4>Methods</h4>The nuclear fac ...[more]

PMID: 20374229

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Project description:Food allergy is a major health issue, affecting the lives of 8% of U.S. children and their families. There is an urgent need to identify the environmental and endogenous signals that induce and sustain allergic responses to ingested allergens. Acute reactions to foods are triggered by the activation of mast cells and basophils, both of which release inflammatory mediators that lead to a range of clinical manifestations, including gastrointestinal, cutaneous, and respiratory reactions as well as systemic anaphylaxis. Both of these innate effector cell types express the high affinity IgE receptor, Fc?RI, on their surface and are armed for adaptive antigen recognition by very-tightly bound IgE antibodies which, when cross-linked by polyvalent allergen, trigger degranulation. These cells also express inhibitory receptors, including the IgG Fc receptor, Fc?RIIb, that suppress their IgE-mediated activation. Recent studies have shown that natural resolution of food allergies is associated with increasing food-specific IgG levels. Furthermore, oral immunotherapy, the sequential administration of incrementally increasing doses of food allergen, is accompanied by the strong induction of allergen-specific IgG antibodies in both human subjects and murine models. These can deliver inhibitory signals via Fc?RIIb that block IgE-induced immediate food reactions. In addition to their role in mediating immediate hypersensitivity reactions, mast cells and basophils serve separate but critical functions as adjuvants for type 2 immunity in food allergy. Mast cells and basophils, activated by IgE, are key sources of IL-4 that tilts the immune balance away from tolerance and towards type 2 immunity by promoting the induction of Th2 cells along with the innate effectors of type 2 immunity, ILC2s, while suppressing the development of regulatory T cells and driving their subversion to a pathogenic pro-Th2 phenotype. This adjuvant effect of mast cells and basophils is suppressed when inhibitory signals are delivered by IgG antibodies signaling via Fc?RIIb. This review summarizes current understanding of the immunoregulatory effects of mast cells and basophils and how these functions are modulated by IgE and IgG antibodies. Understanding these pathways could provide important insights into innovative strategies for preventing and/or reversing food allergy in patients.

Project description:The thiol isomerase, protein disulfide isomerase (PDI), plays important intracellular roles during protein folding, maintaining cellular function and viability. Recent studies suggest novel roles for extracellular cell surface PDI in enhancing cellular activation and promoting their function. Moreover, a number of food-derived substances have been shown to regulate cellular PDI activity and alter disease progression. We hypothesized that PDI may have similar roles during mast cell-mediated allergic responses and examined its effects on IgE-induced mast cell activity during cell culture and food allergy. Mast cells were activated via IgE and antigen and the effects of PDI inhibition on mast cell activation were assessed. The effects of PDI blockade in vivo were examined by treating mice with the irreversible PDI inhibitor, PACMA-31, in an ovalbumin-induced model of food allergy. The role of dietary PDI modulators was investigated using various dietary compounds including curcumin and quercetin-3-rutinoside (rutin). PDI expression was observed on resting mast cell surfaces, intracellularly, and in the intestines of allergic mice. Furthermore, enhanced secretion of extracellular PDI was observed on mast cell membranes during IgE and antigen activation. Insulin turbidimetric assays demonstrated that curcumin is a potent PDI inhibitor and pre-treatment of mast cells with curcumin or established PDI inhibitors such as bacitracin, rutin or PACMA-31, resulted in the suppression of IgE-mediated activation and the secretion of various cytokines. This was accompanied by decreased mast cell proliferation, FcεRI expression, and mast cell degranulation. Similarly, treatment of allergic BALB/c mice with PACMA-31 attenuated the development of food allergy resulting in decreased allergic diarrhea, mast cell activation, and fewer intestinal mast cells. The production of TH2-specific cytokines was also suppressed. Our observations suggest that PDI catalytic activity is essential in the regulation of mast cell activation, and that its blockade may benefit patients with allergic inflammation.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

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