Regulation of IgE-Mediated Allergic Reactions by Disulfide-Linked Multimers of Histamine-Releasing Factor

Background: Stimulation with allergen and allergen-specific IgE activates mast cells, leading to allergic reactions. We studied how the recently defined alarmin, Histamine-Releasing Factor (HRF), promotes this immune response. Methods: HRF reactivity to IgE and other Ab isotypes was analyzed by in-vitro binding assays and 3D-structure modelling. Extracellular and intracellular formations of HRF multimers were studied in aqueous solution and siRNA-treated cells, respectively. Effects of HRF dimers and multimers on in-vitro mast cell activation were measured in IgE-sensitized mast cells by stimulation with high or low valency antigen. In-vivo effects of HRF in allergic reactions were studied in passive anaphylaxis and food allergy models using WT or non-multimerizable HRF mutant mice as well as humanized mice. Results: HRF binds to a substantial subset of both antigen-driven and non-antigen-driven antibodies. Extracellular HRF proteins become non-enzymatically disulfide-linked dimers and multimers, whereas intracellular HRF requires oxidoreductases for dimerization. Surprisingly, while HRF dimers can enhance antigen/IgE-induced cytokine production, stronger signals by HRF multimers are required for degranulation. HRF multimers render HRF-reactive IgE-sensitized mast cells more sensitive and responsive to suboptimal antigen doses. In-vivo anaphylactic responses induced by passive sensitization with HRF-reactive IgE and subsequent antigen challenge are drastically reduced by blockade of HRF-IgE interactions and by HRF-Cys172Ala mutation in mutant mice. Enhancement of anaphylactic responses by HRF is more prominent with low-valency antigen than with supernaturally high-valency antigen. Conclusion: The results collectively demonstrate that HRF multimers are important for IgE-mediated optimal allergic reactions.