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Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

King Abdulaziz University

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (FcɛRI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in FcɛRI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (FcɛRIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFcɛRIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction.

The research into understanding of the immunological processes is often difficult due to several factors complicating the isolation and culturing of primary degranulating cells like mast cells and basophils. The establishment of rat basophilic leukemia (RBL) cell line as an efficient and reliable experimental research tool was considered a major advance toward the understanding of the wild-type mast cell population's biology. The development of sub-clone RBL-IV (HR+) led to the isolation of histamine-secreting RBL-2H3 cell line. Since then, RBL-2H3 cells have been extensively used for studying the IgE high affinity receptor (FcɛRI) interactions with their ligand, the IgE antibody. This cell line has been employed for generating human and more recently canine and equine FcɛRIα-transfected RBL cell lines facilitating an assessment of the residues involved in the complementary interaction between the IgE molecules from these species and their cognate high affinity receptor. A proteomics-based approach to the definition of IgE-receptor-mediated signaling pathways was also carried out using this cell line. Furthermore, RBL-2H3 cells have the potential of being used to assess the potential allergenicity of antigens to humans and other animals like dogs and horses which are known to suffer from similar allergic manifestations.

Review: Diagnostic and therapeutic applications of rat basophilic leukemia cells.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcɛRI) is the central protein interaction in IgE-mediated allergic reactions. The cross-linking of the IgE/FcɛRI complex, through cognate allergens, on the surface of mast cells and basophil cells results in mediator release, and thus leads to the symptoms of type I hypersensitivity responses in mammals. To develop a baseline value for subsequent equine anti-allergy drug and vaccine research, the interaction of equine IgE with its high-affinity FcɛRI receptor was investigated following the cloning and expression of equine IgE with specificity for NIP-HSA (4-hydroxy-5-iodo-3-nitrophenylacetic acid conjugated to human serum albumin). Receptor recognition and effector functions were assessed in Rat Basophil Leukemia (RBL-2H3.1) cells transfected with the α chain of equine and canine FcɛRI. Results obtained showed that the equine FcɛRI receptor recognizes both equine and canine IgE and supports similar β-hexosaminidase release levels from RBL cells transfected with equine FcɛRI, peaking at 36.68% at 100ngml(-1) antigen and 32.00% at 100ngml(-1) antigen respectively. Furthermore, the binding kinetics of the equine IgE to the equine FcɛRI receptor and the canine IgE to the same receptor was measured to be KA=6.33×10(9)M(-1) and KA=1.84×10(9)M(-1) respectively. This research established basic reagents and vitro assay systems to underpin the development of rational therapeutic intervention strategies to combat equine allergic manifestations.

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

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (FcεRI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human Cε3 domains comprise the binding sites for FcεRIα and crystal structure determination has shown that amino acids in four sites contribute to the high affinity of the interaction. The role of homologous residues within canine IgE-Fc, i.e. amino acids located at Cε2-Cε3 interface (residues 332-337), loop BC (residues 362-365), loop DE (residues 393-396), and loop FG (residues 424-427) in canine Cε3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of β-hexosaminidase from RBL cells transfected with canine or human FcεRIα and (ii) the affinity of the mutants for the soluble extracellular domain of the α-chain expressed in Pichia pastoris were determined by Surface Plasmon Resonance (SPR). Kinetic analysis supports the observed effects of IgE mutations on stimulus secretion coupling. Potential applications of this study, leading to the generation of an IgE variant with a disabled FcεRIα binding site, are discussed.

Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcεRIα.

The Cε2 and Cε4 domains are considered as scaffolds, allowing Cε3 domains to assume an appropriate orientation to interact with FcεRI (Wurzburg and Jardetzky, 2002; Hunter et al., 2008). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of Cε2 and Cε4 domains to bind to and induce target cell degranulation via FcεRIα. Our results indicate that for (1) Cε3 domains in IgE of canine and human origin are the only necessary region for binding to FcεRIα. (2) The interaction of canine IgE with human sFcεRIα is significantly enhanced by contributions from both Cε2 and Cε4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine Cε2 and Cε4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the Cε3 domain to facilitate stable interaction with canine FcRIα. Kinetic constants for the binding of this chimera to the soluble extracellular domain of the receptor indicate an approximate 120-fold decrease in the affinity for canine sFcεRIα (ka=5.30 × 10(2)M(-1)s(-1)) and a 330-fold increase in the dissociation from canine sFcεRIα (KD=6.9 × 10(-6)M(-1)), compared to the wild type IgE kinetic constants (Ka=6.30 × 10(4)M(-1)s(-1); KD=2.1 × 10(-8)M(-1)). Although canine IgE does engage human FcεRIα, canine Cε2 and Cε4 do not contribute to the high-affinity of interaction with human FcεRIα. Upon replacement of human Cε2 and Cε4 domain by the canine homologues, human IgE Cε3 only retains a low affinity for the human receptor, which shows that Cε2 and Cε4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor.

The role of Cε2, Cε3, and Cε4 domains in human and canine IgE and their contribution to FcεRIα interaction.

To explore the relationships between the subunits (α, β and γ) of the high affinity IgE receptor (Fc&RI) and its ability to mediate transmembrane signaling.

Article	Year
Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction. Molecular immunology\| PubMed ID: 20947168	2010
Review: Diagnostic and therapeutic applications of rat basophilic leukemia cells. Molecular immunology\| PubMed ID: 22750069	2012
Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcɛRI. Veterinary immunology and immunopathology\| PubMed ID: 23485176	2013
Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcεRIα. Molecular immunology\| PubMed ID: 24084098	2014
The role of Cε2, Cε3, and Cε4 domains in human and canine IgE and their contribution to FcεRIα interaction. Molecular immunology\| PubMed ID: 24091297	2014
High affinity IgE-Fc receptor α and γ subunit interactions. Journal of the College of Physicians and Surgeons--Pakistan : JCPSP\| PubMed ID: 24491004	2014

Sari Sabban

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