The interaction between Lyn and FcεRIβ is indispensable for FcεRI-mediated human mast cell activation.
ABSTRACT FcεRIβ 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 FcεRIβ 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 FcεRIβ should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIβ in human MC FcεRI expression and signaling.
FcεRIβ and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIβ in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs.
The diminution of FcεRIβ 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 FcεRIβ 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 FcεRIβ immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIβ ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo.
The interaction between Lyn and FcεRIβ 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.
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ABSTRACT: Although the existence of Fc(epsilon)RI-alphabetagamma(2) and Fc(epsilon)RI-alphagamma(2) receptor subtypes was reported, there has been no direct evidence of these two subtypes of Fc(epsilon)RI in vivo. To investigate the existence of these two subtypes of Fc(epsilon)RI in vivo, the authors evaluated the expression of Fc(epsilon)RI-beta in the giant papillae of chronic allergic conjunctivitis and compared the expression level of Fc(epsilon)RI-beta with control conjunctivae using the anti-human Fc(epsilon)RI-beta antibody. Fc(epsilon)RI-beta expression in giant papillae obtained from patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis in control conjunctivae was evaluated by immunohistochemistry using anti-Fc(epsilon)RI-beta, -alpha, -gamma, and anti-human mast cell tryptase, anti-chymase, anti-basophil, and anti-CD1a antibodies. Statistical analyses revealed that the densities of Fc(epsilon)RI-beta(+) cells, Fc(epsilon)RI-alpha(+) cells, tryptase(+) cells, and Fc(epsilon)RI-beta(+)/tryptase(+) cells were significantly increased in giant papillae compared with controls. There were two types of Fc(epsilon)RI (alphabetagamma(2) and alphagamma(2)) on the mast cells of the giant papillae. The ratio of the Fc(epsilon)RI-beta(+) cell number/Fc(epsilon)RI-alpha(+) cell number in the giant papillae (0.69 +/- 0.08 [mean +/- SD]) was significantly higher than that of the controls (0.07 +/- 0.16). Fc(epsilon)RI-beta/tryptase double immunostaining revealed that 81% +/- 13% of tryptase(+) cells expressed Fc(epsilon)RI-beta. Fc(epsilon)RI-beta(+) cells were preferentially localized within and around epithelial tissue. The authors also found that Fc(epsilon)RI-beta was expressed by basophils but not by Fc(epsilon)RI-alphagamma(2)-positive Langerhans cells in the giant papillae samples. Preferential Fc(epsilon)RI-beta expression observed in the mast cells and basophils of giant papillae suggests important roles of Fc(epsilon)RI-beta in the pathophysiology of atopic keratoconjunctivitis and vernal keratoconjunctivitis.Investigative ophthalmology & visual science 02/2009; 50(6):2871-7. · 3.43 Impact Factor
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ABSTRACT: To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 3365-3369) to allow cloning of intracellular antigens. A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes. Individual cells expressing intracellular CD63 were identified by autoradiography. cDNA was extracted from positive spots and reintroduced into Escherichia coli. After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis. The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites. The CD63 gene was expressed in a wide variety of cells. Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955-2962). By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein. CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.Journal of Biological Chemistry 03/1991; 266(5):3239-45. · 4.65 Impact Factor
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ABSTRACT: The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.Molecular and Cellular Biology 01/2002; 21(24):8318-28. · 5.37 Impact Factor