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

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 interleukin-13 ^{8, 9} as do particles in cigarette smoke or diesel exhaust particles which enhance IgE synthesis ¹⁰.

The rise in allergic manifestations in industrialized countries in the last 50 years has been attributed to a combination of the effect of environmental pollutants and a trend to a more sanitized environment, which combine to shift the immune response towards a profile predominated by TH₂ cytokines, as proposed by the ‘Hygiene Hypothesis’ ¹¹.

As mentioned above, humans are not the only mammals afflicted by allergy. Notably horses and dogs can also develop classic allergic responses and a study by ¹² has shown that, as in humans, equine allergy is attributed to genetic and environmental factors. As a consequence, these animals present good models for studying the interplay between genetic and environmental causes of allergy, its progression from sensitization to disease, and possible intervention strategies once clinical manifestations have set in

In 1887, Stömmer was the first person to describe the similarity between human and equine asthma ¹³, the effect of histamine on the equine cardiovascular system is very similar to that of humans ¹⁴. Horses are also the cornerstone of the horse racing industry, which is worth US$72 billion with a betting turnover of US$115 billion annually ¹⁵.

Most contemporary racehorses are descendants of the small number of Arabian horses bred by Lady Anne Blunt from 1878 onwards. Modern racehorses are commonly inbred to select for performance abilities. They are prone to genetic disorders, one of which is their susceptibility to mount allergic responses. They also have 1000 times higher serum IgE levels than even the most severely allergic humans ¹⁶. Horse allergic responses are usually manifested as insect bite hypersensitivity (IBH) ^{17, 18}. IBH results in dermatitis due to bites form insects in the genus Culicoides. Another form of equine allergic disease is recurrent airway obstructions (RAO), this is manifested in the lungs and airways. It is characterized by wheezing and labored breathing. RAO commonly occurs in response to mould spores, and high allergen-specific IgE levels have been recorded in horses suffering from RAO in one study ¹⁹ although another investigation has not confirmed this ²⁰.

Studies on equine allergy revolved around the attempt at monitoring and neutralizing equine IgE by developing anti-equine IgE monoclonal antibodies (mAbs) ^{21, 22}. Furthermore the study by ²³ discusses the production of the extracellular domains of the equine high-affinity Fc receptor’s α chain (FcεRIα) receptor in an attempt to detect and quantitate equine serum IgE. A related study by Ledin²⁴ discusses a new approach aimed at neutralizing serum IgE by priming the immune system using a self/non-self immunogen. All these studies, however, lacked an effective assay to test the safety and efficacy of their protocols. In this article, we now present such an assay system applicable to the study of diagnostic and therapeutic strategies relevant to the equine system, where β-hexosaminidase release, as an indicator of cell mediator degranulation, was assessed on RBL-2H3.1 cells expressing equine FcεRIα. This protocol is based on previous publications ^{25, 4, 5, 2, 3} describing the engineering of RBL cells transfected with the gene encoding the IgE binding domain of the high-affinity receptor for IgE from different species. The protocol explains how to perform a β-hexosaminidase release assay, the results of which are presented as the mean ± standard deviation of triplicate experiments.

The release assay was first developed by Siraganian and Hook ²⁵ to study human allergy. The lab group led by Dr. Reuben Siraganian also developed the RBL cell line. These RBL cells were developed to express the human FcεRIα and the protocol was published by ⁴. The final piece of the assay came with the development of the pSV plasmid in the paper by Neuberger ²⁶ which described the production of an IgE antibodies by cloning its heavy chain gene downstream of a mouse gene for an IgE variable region that targets the hapten 4-hydroxy-3-nitro-phenacetyl (NP), the resulting chimeric antibody was fully functional. The ability to develop any IgE targeting the same hapten, while also cloning its receptor on the surface of RBL cells resulted in the standardization of the assay making it a useful protocol to measure the degranulation of basophil cells.

The assay does have pros and cons. The pros of the assay is its adaptability to be used in any mammalian system, our lab has thus used it to test for the degranulation in the human, canine and equine systems, and this is achievable simply by synthesizing the organism's IgE and cloning its receptor onto the surface of the RBL cells.

On the other hand, the cons of the assay is that the RBL cells are very sensitive to thermal, mechanical and PH changes, making them give a variation of degranulation levels within the same assay. It is thus strongly advised that the assays are always repeated in triplicates and then an average is taken from them. Furthermore, the RBL cells tend to shift toward a non-releasing phenotype if they are left in tissue culture for an extended times (>10 weeks) ²⁷, making their maintenance cumbersome. They are also prone to infections by mycoplasma bacteria, which are not visible from a light microscope and do not change the cell's morphology, but would drastically change their degranulation levels. Thus regular mycoplasma tests are needed.