Sistema FMUSP-HC: Faculdade de Medicina da Universidade de São Paulo (FMUSP) e Hospital das Clínicas da FMUSPADNAN, AtherACHARYA, ShreeALENAZY, Leila A.VECILLAS, Leticia de lasBIANCHI, Pedro GiavinaPICARD, MatthieuCALBACHE-GIL, LuciaROMERO-PINEDO, SalvadorABADIA-MOLINA, Ana ClaraKERR, WilliamPEDICONE, ChiaraNAGAI, JunHOLLERS, EleanorDWYER, DanielCASTELLS, Mariana2023-08-162023-08-162023JOURNAL OF IMMUNOLOGY, v.210, n.6, 20230022-1767https://observatorio.fm.usp.br/handle/OPI/54592Multistep mast cell desensitization blocks the release of mediators following IgE crosslinking with increasing doses of Ag. Although its in vivo application has led to the safe reintroduction of drugs and foods in IgE-sensitized patients at risk for anaphylaxis, the mechanisms of the inhibitory process have remained elusive. We sought to investigate the kinetics, membrane, and cytoskeletal changes and to identify molecular targets. IgE-sensitized wild-type murine (WT) and FcERIa humanized (h) bone marrow mast cells were activated and desensitized with DNP, nitrophenyl, dust mites, and peanut Ags. The movements of membrane receptors, FcERI/ IgE/Ag, actin, and tubulin and the phosphorylation of Syk, Lyn, P38-MAPK, and SHIP-1 were assessed. Silencing SHIP-1 protein was used to dissect the SHIP-1 role. Multistep IgE desensitization of WT and transgenic human bone marrow mast cells blocked the release of 13-hexosaminidase in an Ag-specific fashion and prevented actin and tubulin movements. Desensitization was regulated by the initial Ag dose, number of doses, and time between doses. FcERI, IgE, Ags, and surface receptors were not internalized during desensitization. Phosphorylation of Syk, Lyn, p38 MAPK, and SHIP-1 increased in a dose -response manner during activation; in contrast, only SHIP-1 phosphorylation increased in early desensitization. SHIP-1 phosphatase function had no impact on desensitization, but silencing SHIP-1 increased 13-hexoxaminidase release, preventing desensitization. Multistep IgE mast cell desensitization is a dose-and time-regulated process that blocks 13-hexosaminidase, impacting membrane and cytoskeletal movements. Signal transduction is uncoupled, favoring early phosphorylation of SHIP-1. Silencing SHIP-1 impairs desensitization without implicating its phosphatase function.engrestrictedAccessarticleCopyright AMER ASSOC IMMUNOLOGISTS10.4049/jimmunol.2100485Immunology1550-6606