Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

Summary

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Abstract

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains ¹. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout ^{2, 3}. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml^-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses ^{4, 5}. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease ^{6, 2, 3}.

Introduction

Allergy has been known for millennia. An asthma treatment was described in the Ancient Egyptian medical text known as the Ebers Papyrus (~1550 BCE) and discussed herbal remedies to treat it ⁷.

Today allergy is classified as a type I hypersensitivity response, where the T helper cell type 2 (TH₂) arm of the immune system steers the production of immunoglobulin E (IgE) antibodies in response to environmental antigens called allergens. These are diverse substances that commonly interact with cells in the immune system and stimulate the synthesis and secretion of pro-inflammatory cytokines, including interleukin-4 and inte....

Protocol

1) Preparation of Cell Line:

1. Developing the RBL-2H3.1 cell line expressing equine FcεRIα:
   1. Using basic tissue culture techniques for monolayer cell lines, transfect parental RBL-2H3.1 cells using the pEE6 plasmid, carrying the equine FcεRIα gene (GenBank: Y18204.1) ²⁸. Add 2 μg μl^-1 of the plasmid DNA to 0.8 ml of cells at a density of 1.2 x10⁷ cells ml^-1. Electroporate the cells at 250 V 960 μF using a 0.4 cm electrocuvette then immediately incubate on ice for 10 min.
   2. Select the transformed cells using media containing 0.4 g of geneticin G418 sulphate, then sort the remain....

Results

The parental RBL-2H3.1 cells and those transfected with the equine FcεRIα receptor gene were first sensitized with mouse IgE anti DNP-HSA and challenged with the DNP-HSA antigen. Mouse IgE binds to the endogenous rat receptor in both cell lines and thus acts as a control to test the release viability of both cell lines to release mediators (Figure 1 A). This is an important check and should be carried out routinely since upon extended (> 10 weeks) passage in cell culture, RBL-2H3.1 cells drift towards.......

Discussion

In summary the results of this investigation showed that when RBL-2H3.1 cells expressing equine FcεRIα are sensitized with equine IgE and challenged by an antigen, they give a peak mediator release of 36.68% ± 4.88% of the total amount of mediator inside the cells, compared to the RBL-2H3.1 parental cells not expressing equine FcεRIα.

Thus this assay provides a useful tool for investigating and studying equine allergic responses in vitro. Its allows the determ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RBL-2H3.1 Expressing Equine FcεRIα	-	-	Produced in the lab
Equine IgE anti NIP-HSA	-	-	Produced in the lab
96 Well Plate	Sigma	CLS3595	-
Multi Channel Pipette	Anachem	-	-
Incubator	Galaxy R	-	-
4Hydroxy-5-iodo-3-nitrophenylacetic acid	Cambridge Research Biochemicals	N-1070-1	NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab
Dinitrophenyl Conjugated to Human Serum Albumin	Sigma	A6661	Abbreviated DNP-HSA
Plate Spectrophotometer	Anthos Labtec HT2	-	-
Pipes	Sigma	P1851	-
Sodium Chloride	Sigma	S7653	-
Potassium Chloride	Sigma	P9333	-
Magnesium Chloride	Sigma	M2670	-
Calcium Chloride	Sigma	C1016	-
Triton x100	Sigma	X100	-
4-nitrophenyl N-acetyl-β-D-glucosaminide	Sigma	N9376	Stock solution called β-hexosaminidase substrate was 50mM prepared in DMSO
Dimethyl Sulfoxide	Sigma	D2650	-
Citric Acid	Sigma	251275	-
Sodium Acetate	Sigma	S7670	-
Tris	Sigma	T5941	-