Highly expressed cytoplasmic FcεRIß in human mast cells functions as a negative regulator of the FcRγ-mediated cell activation signal.

BACKGROUND: We recently reported that the interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIß functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIß in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIß in the cytoplasm remains unknown. METHODS: The localization of FcεRIß and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIß, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIß was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: In the subepithelial region, FcεRIß was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIß expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIß in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIß in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIß and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. CONCLUSION: Cytoplasmic FcεRIß, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell activation.

BACKGROUND: FcεRIß reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIß of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIß should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIß in human MC FcεRI expression and signaling. METHODS: FcεRIß and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIß in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs. RESULTS: The diminution of FcεRIß significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIß inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIß immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIß ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo. CONCLUSION: The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

Th1-dominant shift of T cell cytokine production, and subsequent reduction of serum immunoglobulin E response by administration in vivo of plasmid expressing Txk/Rlk, a member of Tec family tyrosine kinases, in a mouse model.

BACKGROUND: Th1 and Th2 cells, resulting from antigenic stimulation in the presence of IL-12 and IL-4, respectively, are implicated in the pathology of various diseases including allergic and autoimmune diseases. Txk/Rlk is a member of Tec family tyrosine kinases. We reported that Txk acts as a Th1-specific transcription factor in the T lymphocytes. OBJECTIVE: In this study we have asked whether administration of txk expression plasmid brings about a Th1/Th2 shift in vivo of the mice, and subsequent reduction of circulating IgE. METHODS: Mice were administered a txk expression plasmid with hemagglutinating virus of Japan (HVJ) envelope vector. Txk expressions in spleen cells were assessed by immunoblotting and immunocytochemical staining. Cytokine productions by the spleen cells and serum Ig concentrations were studied by ELISA. RESULTS: Administration of a txk expression plasmid with HVJ vector induced expression of Txk in the spleen cells. The spleen cells showed enhanced Th1-specific cytokine production; spleen cells from the txk administered mice produced more IFN-gamma as compared with those from control plasmid-administered mice in an antigen-specific manner. IL-2 and IL-4 secretions of the spleen cells were comparable between the two mouse groups. Txk administration did not reduce serum IgG concentration. It markedly reduced total IgE level and an IgG1/IgG2a ratio, reflection of Th1/Th2 balance, in sera. Furthermore, txk administration reduced ovalbumin (OVA)-specific IgE levels in sera of the OVA sensitized mice. CONCLUSION: Thus, Txk enhances IFN-gamma secretion and thus modulates Th1/Th2 cytokine balance, leading to reduction of serum IgE.