Trivalent Antigens for Degranulation of Mast Cells

Abstract

Degranulation of basophils and mast cells, releasing histamine, plays a central role in allergic reactions. Degranulation is a response to cell surface receptor aggregation caused by association of receptors with antibodies bound to multivalent antigens. Tools used in studying this process have included small-molecule divalent antigens, but they suffer from weak signaling due to small aggregate size. We have prepared trivalent antigens that allow formation of larger aggregates and potent responses from mast cells.Aggregation of cell surface receptors is a common mechanism involved in signal transduction across cell membranes. 1 This mechanism is used, for example, by receptors that are intrinsic protein tyrosine kinases (i. e., autophosphorylation of aggregated receptors), 2,3 such as the epidermal growth factor receptor, the platelet derived growth factor receptor, and the high affinity receptor for IgE (Fcε RI), which plays a central role in allergic reactions. 4,5 Early steps in the initiation of signaling by this class of receptors are similar: multivalent interactions with a ligand lead to aggregation of receptors and enhanced phosphorylation of tyrosines, which can be recognized by cytoplasmic regulatory molecules.FcεRI receptors are found on the surface of mast cells, basophils, and rat basophilic leukemia (RBL) cells, and these receptors bind the Fc (invariant) region of the divalent antibody IgE. When multiple IgE molecules bind a polyvalent antigen and in turn associate with FcεRI on a cell surface, aggregates of these receptors are generated (Figure 1). Formation of aggregates can in some cases trigger calcium release and degranulation (export of histamine).* paul_savage@byu.edu . Supporting Information Available:Experimental details for the degranulation experiments; a synthetic scheme for 8a and 8b; synthetic details for the preparation of the di-and tri-valent antigens (6a, 6b, 7a, 7b, 8a and 8b); and 1 H NMR spectra for 6a, 6b, 7a, 7b, 8a and 8b. For example, it has been observed that aggregation caused by IgE dimers are less effective than those caused by larger IgE oligomers at stimulating cellular responses 6 and that cellular responses are inhibited when an optimal degree of aggregation is exceeded. 9 The factors that cause an aggregate to be a robust signaling unit, an inhibitor of signal transduction, or a nonsignaling unit remain to be fully elucidated. NIH Public AccessThe importance of influences of ligand-induced receptor aggregates on cellular signals has prompted significant effort in developing means for quantitative analysis of the interactions between multivalent ligands and cell surface receptors. 10 An attractive approach to studing IgE -FcεRI aggregation involves the use of synthetic symmetric divalent ligands, which are the simplest type of ligand capable of aggregating receptors. The interaction of divalent IgE antibodies with a divalent ligand can result in an IgE -FcεRI aggregate spectrum that contains only linear chains and rings of various sizes (including one to on...

Two-Allergen Model Reveals Complex Relationship between IgE Crosslinking and Degranulation

“…This is likely a reflection of the difference in affinities of DNP for IgE DNP ( K d of 22 nM) compared to dansyl for IgE dansyl ( K d of 54 nM). In line with previous work, the bivalent allergen, HmBA [DNP 2 ] failed to stimulate a response under any condition due to insufficient valency (Figure 2) (Posner et al, 2007; Sil et al, 2007). Combined, these results validate that there is no cross-reactivity between the IgE-hapten pairs and demonstrates the suitability of the two allergen model to investigate the effect of multiple unique allergens on mast cell degranulation.…”

“…Interestingly, at the highest allergen concentrations the overall degranulation response began to increase again as observed with HmTA [dansyl 4 ] concentrations of 50 and 100 nM (Figure 3). The bell shaped dose-response has traditionally been attributed to the degree of IgE aggregation (Huber, 2013; Posner et al, 2007; Sil et al, 2007). As the allergen concentration increases, the degree of IgE cross-linking increases up to a point when monovalent allergen-IgE interactions begin to dominate due to excess allergen.…”

“…Bivalent allergens have been previously shown to form cyclic dimers with IgE and these clusters of cyclic dimers typically do not stimulate a degranulation response (Posner et al, 2007; Sil et al, 2007). We next investigated if the bivalent DNP allergen, HmBA [DNP 2 ] which was unable to stimulate mast cell degranulation, (Figure 2) was able to decrease the degranulation response caused by HmTA [dansyl 4 ].…”

Nanoparticle-Mediated IgE−Receptor Aggregation and Signaling in RBL Mast Cells

“…Although data points in the plot are obtained by subtracting the degranulation response of nonspecific interaction from unmodified AuNPs, no significant cellular degranulation is induced above baseline unstimulated levels when the nanoparticle concentration is less than 2.4 nM. The biphasic nature of this dose-response curve is typical for a polyvalent ligand interacting with a bivalent receptor such as anti-DNP IgE 14. The degree of degranulation increases with DNP-AuNP concentration up to 0.6 nM.…”

Spatio-Temporal Signaling in Mast Cells

“…It is possible for such reagents to induce immobilization in the absence of signaling, at least as measured by degranulation and Ca 2+ mobilization. 48 Bivalent ligands with flexible PEG-based spacers fail to stimulate measurable secretion 49 and can even be inhibitory. 50 In general, higher oligomers induced by higher valency antigens – or further crosslinking of antibody-bound complexes – generate better signaling from the RBL-2H3 cell line over a broad dose response range.…”