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

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 ß- and γ-chains. 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 ß-hexosaminidase release as a readout. 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. 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.

High affinity IgE-Fc receptor α and γ subunit interactions.

OBJECTIVE: To explore the relationships between the subunits (α, ß and γ) of the high affinity IgE receptor (Fc&RI) and its ability to mediate transmembrane signaling. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. METHODOLOGY: The approach employed was to create a chimera (human αγγ) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc&RIα) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. RESULTS: The RBLs cell lines transfected with the huFc&RIα/γ/γ cDNA constructs were assessed for the cell surface expression of the huFc&RIα subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc&RIα subunit expression on the surface of transfected cells as seen by flowcytometric studies, ß-hexosaminidase assays and intracellular calcium mobilisation studies. CONCLUSION: In the present study the grounds for non-expression of huFc&RIα/γ/γ cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes.

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

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 ß-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.

Fc receptor-γ subunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells.

The high-affinity IgE receptor (FcɛRI) is formed by the IgE-binding α subunit, ß subunit and γ subunits homodimer. All three subunits are required for proper expression of the receptor on the plasma membrane of mast cells and basophils. However, the exact molecular mechanism of inter-subunit interactions required for correct expression and function of the FcɛRI complex remains to be identified. A recent study suggested that polar aspartate at position 194 within the transmembrane domain of the α subunit could interact by hydrogen bonding with polar threonine at position 22 in the transmembrane domains of the γ subunits. To verify this, we used previously isolated rat basophilic leukemia (RBL)-2H3 variant cells deficient in the expression of the FcɛRI-γ subunit (FcR-γ), and transfected them with DNA vectors coding for FcR-γ of the wild-type or mutants in which T22 was substituted for nonpolar alanine (T22A mutant) or polar serine (T22S mutant). Analysis of the transfectants showed that both T22A and T22S mutants were capable to restore surface expression of the FcɛRI similar to wild-type FcR-γ. Furthermore, cells transfected with wild-type, T22A or T22S FcR-γ showed comparably enhanced FcɛRI-mediated degranulation. Our data indicate that substitution of FcR-γ T22 with non-polar amino acid does not interfere with surface expression of the FcɛRI and its signaling capacity.

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

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.

Protein profiling of the secretome of FcεRI activated RBL-2H3.1 cells.

BACKGROUND: Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. OBJECTIVE: The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. METHODS: A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. RESULTS: Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified ~53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines (e.g. MCP-2, PF-4, CSF-1 and TGF-ß1) are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. CONCLUSION: In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes.

A novel insight to the functional role of Stathmin 1 in IgE-mediated activation of RBL-2H3 cells.

IgE-mediated cell signaling, induced by cross-linking of high affinity receptor for IgE (FcεRI) in the presence of antigen (Ag), is a well known mechanism described for mast cell activation in allergy and hypersensitivity reactions, which induces a spectrum of cellular responses such as secretion and up-regulation of cell surface FcεRI. Although for several years IgE binding to FcεRI was considered to be a passive sensitization process, the outcomes of several recent studies have revealed a variety of different cellular responses to IgE binding compared to IgE plus Antigen binding. The present study applied a functional proteomics-based approach to investigate mast cell signaling events and provided new insights to FcεRI-mediated cell signaling in RBL-2H3.1 cells, and may point to the activation of alternative signaling pathways in response to IgE or IgE plus Ag. Comparative analysis by 2-D PAGE of RBL cells activated with IgE plus Ag for three and four hours compared to non-activated cells was followed by mass spectrometric protein identification and provided evidence for the induction of Stathmin 1 (STMN1) gene expression in response to IgE plus Ag activation.Complementary SDS-PAGE analysis showed a distinct up-regulation of STMN1 induction in response to challenge with IgE plus Ag compared to sensitization with IgE only. Phosphoproteomics analysis gave evidence for significant increase at phosphorylation of STMN1 on ser16 after 1min, though a slight rise at 5 min, and on ser38 after 1 and 5min sensitization with IgE and a similar result was observed for 1min IgE plus Ag-activation. IgE plus Ag-activation was also found to induce the phosphorylation of ser38 to a greater extent than sensitization with IgE. In contrast, IgE alone was more effective than IgE plus Ag at inducing phosphorylation of ser16. Collectively this study provides further insights into the role of stathmin 1 in FcRI-mediated activation of cells of mast cell lineage and might shed light on the diverse response of these cells to IgE or IgE plus Ag.

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 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.

Proteomic identification and characterization of secreted N-glycosylated NPC2 following cross-linking of the high-affinity receptor for IgE on mast cells.

Allergen-mediated cross-linking of the high-affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL-2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol-binding glycoprotein, NPC2/RE1 (Niemann-Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE-mediated stimulus and co-localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD-2-related lipid-recognition) protein family (155 members), which includes the Toll-like receptor co-factors, MD-1 and MD-2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.

Characterization of an early signaling defect following Fc epsilonRI activation in the canine mastocytoma cell line, C2.

A comparison of IgE recognition by cognate receptors expressed on the C2 canine mastocytoma cell line with analogous events in a rat basophilic leukemia cell line transfected with the alpha-chain subunit of the canine high-affinity IgE receptor using flow cytometry show that canine IgE recognizes the alpha-chain of its cognate receptor on both cell lines. Our study confirms the expression of functional IgE receptors in both cell lines, but receptor-mediated signaling in the C2 line only supports the early stages of downstream signaling as shown by the phosphorylation of the gamma-chain and the failure to effect the phosphorylation of Syk. In contrast RBL-2H3 cells respond to sensitization with IgE and challenge with cognate antigen with tyrosine phosphorylation of the gamma-subunits of the receptor complex followed by downstream phosphorylation of Syk and Ca(2+) mobilization, culminating in beta-hexosaminidase release. We propose that the identification of the precise signaling defect in C2 cells will yield useful information regarding the pathway leading to mast cell exocytosis and facilitate the restoration of the complete signaling cascade through complementation of the missing/defective signal transducer since signaling events downstream of Ca(2+) mobilization are intact as demonstrated by beta-hexosaminidase release following non-immunologic stimulation with the calcium ionophore, A23187.