RBL cells expressing human FcεRI are a sensitive tool for exploring functional IgE–allergen interactions: studies with sera from peanut-sensitive patients

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Abstract

Rat basophilic leukemia cells (RBL SX-38) express the α, β, and γ chains of human FcεRI. Following sensitization with IgE from a subset of allergic human donors, these cells can be triggered by exposure to anti-IgE or to very low concentrations of specific allergens. We examined 18 sera from patients who were highly sensitive to peanuts by history and had anti-peanut IgE by in vitro testing. The ability of these sera to sensitize the RBL SX-38 cells for degranulation with peanut allergens correlates very well with the absolute amount of anti-peanut IgE (r=0.95; p<0.001). The most effective sera contained at least 50 kU/l of total IgE and at least 15 kU/l of peanut-specific IgE. RBL SX-38 cells sensitized with these sera degranulated optimally upon exposure to anti-IgE (net degranulation of 40±8%, means±S.D.; n=8) and to a 105–106 dilution of crude peanut extract (CPE) (37±7% net degranulation; 93±13% of that seen with anti-IgE). This assay is quite sensitive. Cells sensitized with selected sera are activated by exposure to a 1:107 dilution of the CPE containing picogram amounts of peanut allergens. This assay is also quite specific. Cells sensitized with sera from patients with anti-peanut IgE and no detectable IgE against soybean, walnut or grass pollen did not degranulate following exposure to these latter antigens. The converse was also true; cells sensitized with sera from patients without anti-peanut IgE did not react to peanut. These data demonstrate that RBL cells expressing human FcεRI form the basis of a useful model system for the detection of allergens and for the study of IgE–allergen interactions.

Introduction

Type I immediate hypersensitivity reactions are mediated through the effects of antigen-specific IgE. IgE binds to high-affinity receptors for IgE (FcεRI) on human mast cells and, when cross-linked by antigen, causes release of preformed mediators, release of newly formed lipid-derived mediators, and later production of proinflammatory cytokines (Schwartz and Huff, 1998). Allergic individuals express a very wide range of concentrations of total and antigen-specific IgE and this range correlates with the broad spectrum of severity of allergic reactions Homburger, 1998, Yunginger et al., 2000.

There are an estimated 4 million Americans with well-substantiated food allergies (Sampson, 1997). Reactions to foods are thought to be the most common cause of anaphylaxis when it occurs outside of the hospital and are estimated to cause 125 deaths per year in the U.S. (Burks et al., 1999). A recent analysis of 32 fatalities thought to be due to food-induced anaphylaxis revealed that peanut was the likely responsible food in 62% of the cases (Bock et al., 2001). In placebo-controlled food challenges, peanut-sensitive patients can react to as little as 100 μg of peanut protein (Hourihane et al., 1997). Thus, anaphylactic reactions and other allergic reactions to peanuts are significant medical problems Hourihane, 1998, Burks et al., 1999, Warner, 1999.

The peanut, Arachis hypogaea, is a member of the legume family and is related to beans and peas, but not to tree nuts (Sampson, 1998). Immediate hypersensitivity reactions to peanuts are unusual in that they are much more prevalent and severe than for other legumes and tend to persist into adulthood (Sampson, 1997). For this reason, substantial effort has been made to identify the major peanut allergens in hopes of desensitization of patients or production of peanuts without allergenic activity Burks et al., 1998, Moneret-Vautrin, 1998, Nelson et al., 1997. Seven potentially important allergens of peanut have been identified (Ara h 1–Ara h 7) based on binding of IgE from allergic sera on Western blots Burks et al., 1995, Stanley et al., 1997, Kleber-Janke et al., 1999, Rabjohn et al., 1999.

RBL SX-38 cells are rat basophilic leukemia cells that stably express the α, β, and γ chains of the human high-affinity receptor for IgE, FcεRI (Wiegand et al., 1996). This receptor gives these cells the important property that they can bind IgE from the sera of allergic individuals and can be activated in an allergen-specific manner (Wiegand et al., 1996). We have studied the ability of sera from 18 subjects with documented sensitivity to peanuts to sensitize these cells for subsequent degranulation following exposure to crude peanut extract (CPE). In this report, we characterize the parameters of those sera that are successful in sensitizing the RBL SX-38 cells to subsequent degranulation upon exposure to the appropriate antigen. An unexpected finding is that, with selected sera, these cells can be activated by dilute extracts containing less than 1 ng/ml of major allergen. RBL cells expressing human FcεRI should be a useful model for the detection of very small amounts of functional allergen and for the analysis of functional interactions between allergen-specific IgE and allergens.

Section snippets

Patients and preparation of sera

This protocol was approved by the Colorado Multiple Institutional Review Board. All subjects gave informed consent. We studied sera from a total of 18 patients with a history of systemic reactions to peanuts. Most of these had documented positive skin tests to peanuts. Nine patients were identified by colleagues and referred to SCD. These sera are identified by the letter “D” followed by a number. For these nine patients, blood was drawn into plain (“red top”) tubes (Becton-Dickinson), allowed

Binding of human IgE to RBL SX-38 cells

Binding of IgE to FcεRI on these cells was assessed by adding dilutions of serum D15 containing IgE. As shown in Fig. 1, with sera containing greater than 500 ng/ml of IgE/ml (210 IU/ml), when added at a 1:10 dilution (50 ng/ml), binding of IgE to FcεRI reaches a plateau.

Sensitivity and specificity of the assay

The RBL SX-38 cells, when sensitized with sera from patients with large amounts of anti-peanut IgE, degranulate when exposed to very dilute preparations of CPE. In Fig. 2a, cells were sensitized with serum D17 (44 IU/ml of

Discussion

RBL cells expressing the human α, β and γ chains of the FcεRI receptor provide a convenient cell culture system for analysis of functional IgE–allergen interactions. These cells have distinct advantages over primary cultures of human mast cells and human basophils. The former are inconvenient to culture whereas the latter must be harvested immediately before using and are nonfunctional in 8–20% of subjects Saito et al., 1996, Kepley et al., 1999.

We sensitized RBL SX-38 cells with sera from

Acknowledgements

This work was supported by a Jaffe Food Allergy Foundation Fellowship to Dr. Dibbern and by Divisional funds. We thank J.P. Kinet for providing the RBL SX-38 cells and Gary Bannon for assaying the Ara h 1 and Ara h 2 content of our crude peanut extract. We also thank Gary Bannon, Wesley Burks and Patsy Giclas for helpful discussions. Finally, we thank Sara Dale for excellent technical assistance.

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