Abstract
Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.
Highlights
Mast cells (MCs) are found at the internal and external interfaces of the body, in particular at mucosal surfaces, surrounding blood vessels, and directly interacting with peripheral nerves
To interrogate MC activation and inhibition in vitro, we developed a system using MCs derived from the bone marrow of SignalingSialic-acid-binding immunoglobulin-like lectin (Siglec)-8 transgenic mice (S8-BMMCs) [16]
To confirm natively expressed Siglec-8 functioned we evaluated Siglec-8 mAbmediated inhibition in human primary MCs derived from peripheral blood CD34+ cells activated with agonistic antihuman FceRI antibody CRA-1
Summary
Mast cells (MCs) are found at the internal and external interfaces of the body, in particular at mucosal surfaces, surrounding blood vessels, and directly interacting with peripheral nerves They respond to a broad array of both IgE-dependent and -independent activating signals, including allergens, cytokines, complement proteins, Toll-like receptor (TLR) ligands, and neuropeptides [1]. Consistent with Siglec-8 functioning as an inhibitory receptor, the membrane proximal ITIM was shown to be required for Siglec-8 mAbmediated inhibition of FceRI-dependent calcium flux and secretory responses in MCs [9] Despite these studies identifying specific components of Siglec-8 signaling in MCs, the intracellular mechanisms that contribute to Siglec-8-mediated inhibition of MCs have not been comprehensively evaluated
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