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Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells.

ABSTRACT: BACKGROUND:DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown. OBJECTIVE:We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation. METHODS:DJ-1 was silenced in human CD34+-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of ?-hexosaminidase, prostaglandin D2, and GM-CSF and changes in reactive oxygen species levels were measured after Fc?RI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies. RESULTS:DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following Fc?RI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling. CONCLUSION:We demonstrate a novel role for DJ-1 in the early activation of Lyn by Fc?RI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

SUBMITTER: Kim DK

PROVIDER: S-EPMC5899056 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells.

Kim Do-Kyun DK Beaven Michael A MA Metcalfe Dean D DD Olivera Ana A

The Journal of allergy and clinical immunology 20171013 1

<h4>Background</h4>DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown.<h4>Objective</h4>We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation.<h4>Methods</h4>DJ-1 was silenced in human CD34<sup>+</sup>-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constr ...[more]

PMID: 29031599

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Project description:Mast cells and basophils have long been implicated in the pathogenesis of IgE-mediated hypersensitivity reactions. They express the high-affinity IgE receptor, FcϵRI, on their surface. Antigen-induced crosslinking of IgE antibodies bound to that receptor triggers a signaling cascade that results in activation, leading to the release of an array of preformed vasoactive mediators and rapidly synthesized lipids, as well as the de novo production of inflammatory cytokines. In addition to bearing activating receptors like FcεRI, these effector cells of allergy express inhibitory ones including FcγR2b, an IgG Fc receptor with a cytosolic inhibitory motif that activates protein tyrosine phosphatases that suppress IgE-mediated activation. We and others have shown that food allergen-specific IgG antibodies strongly induced during the course of oral immunotherapy (OIT), signal via FcγR2b to suppress IgE-mediated mast cell and basophil activation triggered by food allergen challenge. However, the potential inhibitory effects of IgA antibodies, which are also produced in response to OIT and are present at high levels at mucosal sites, including the intestine where food allergens are encountered, have not been well studied. Here we uncover an inhibitory function for IgA. We observe that IgA binds mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells. Binding to BMMCs is dependent on calcium and sialic acid. We also found that IgA antibodies inhibit IgE-mediated mast cell degranulation in an allergen-specific fashion. Antigen-specific IgA inhibits IgE-mediated mast cell activation early in the signaling cascade, suppressing the phosphorylation of Syk, the proximal protein kinase mediating FcεRI signaling, and suppresses mast cell production of cytokines. Furthermore, using basophils from a peanut allergic donor we found that IgA binds to basophils and that activation by exposure to peanuts is effectively suppressed by IgA. We conclude that IgA serves as a regulator of mast cell and basophil degranulation, suggesting a physiologic role for IgA in the maintenance of immune homeostasis at mucosal sites.

Dataset Information

Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells.

Publications

Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells.

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