Mast cell activation by supra-optimal antigen concentrations - a promising condition to identify novel regulators of FcεRI signaling

• Mastzellaktivierung mit supraoptimalen Antigenkonzentrationen - eine vielversprechende Bedingung zur Identifikation neuer Regulatoren der FcεRI-Signaltransduktion

Gast, Mathias; Huber, Michael (Thesis advisor); Lüscher, Bernhard (Thesis advisor)

Aachen (2019, 2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

Activation of the high-affinity receptor for IgE (FcεRI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5’-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen stimulation. The low-affinity IgG receptor, FcγRIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, antigen/IgE-dependent and progressive co-localization of FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon Ag stimulation. Absence of FcγRIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca2+ mobilization, which indicates a contribution of this receptor to SHIP1-dependent negative regulation of mast cell activation. Though, degranulation and IL-6 production were not or only weakly enhanced, respectively. Altogether our data establish the LYN/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal antigen concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control. Initiation of mast cell responses through FcεRI and the receptor tyrosine kinase KIT involves similar signaling pathways and mechanisms such as the PI3K pathway, the MAPK pathway, as well as Ca2+ mobilization. While separate stimulation of both receptors results in distinct cellular responses, co-stimulation with SF and Ag causes KIT-dependent enhancement of FcεRI-driven degranulation and cytokine production. To initiate this synergistic effect, signaling processes that are induced by both receptors must be integrated. Considering the high level of structural synergism between FcεRI and KIT signaling, we describe Ag-driven tyrosine phosphorylation and internalization of KIT in the absence of SF. We observed that KIT was phosphorylated at Y719 by LYN upon Ag stimulation in a concentration-dependent manner. As this process did not require the intrinsic tyrosine-protein kinase activity of the receptor it must be assumed that Ag-driven Y719 phosphorylation is most likely independent of KIT dimerization. This is triggered by SF-based KIT activation and accompanied by its autophosphorylation at Y719. Our data further indicate that Ag-based KIT recruitment could possibly exert a negative regulatory influence on early Ag-driven MC-responses. Comparison of KIT-deficient and WT BMMCs revealed that in the absence of KIT β-hexosaminidase release in response to Ag stimulation was significantly enhanced. In contrast, Ag-triggered cytokine production remained unaffected in KIT-deficient BMMCs.