ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fc« receptor I-mediated activation of Syk (immunoreceptor tyrosine-based activation motifysecretion)

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), Fc«RI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the Fc«RI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-g1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor a were also inhibited. ER-27319 did not inhibit the antiCD3-induced tyrosine phosphorylation of phospholipase C-g1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of Fc«RI g subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igb ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the Fc«RI g phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in Fc«RI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease. Allergen-induced activation of the high-affinity receptor for IgE (Fc«RI) in mast cells and basophils plays a pivotal role in the initiation of allergic and inflammatory reactions. As a result of receptor engagement, mast cells and basophils release a variety of inflammatory mediators, such as histamine, arachidonic acid metabolites, and cytokines (1, 2). The activation of protein tyrosine kinases (PTKs) is one of the early and critical signaling events following Fc«RI engagement (3). Since Fc«RI belongs to the family of multisubunit antigen receptors that do not possess intrinsic PTK activity in their structure (1), the recruitment and activation of non-receptor PTKs is crucial for Fc«RI-mediated signal transduction (4). Fc«RI is a tetrameric receptor composed of an a subunit, a b subunit, and a homodimer of disulfide-linked g subunits (5). The cytoplasmic domains of b and g subunits contain sequences known as the immunoreceptor tyrosine-based activation motifs (ITAMs) (6, 7). The phosphorylation of the tyrosines of the ITAM allows the interaction of ITAMcontaining receptors with signaling proteins that have phosphotyrosine-recognizing Src homology 2 (SH2) domains (8, 9). Syk kinase is activated in Fc«RI-mediated signaling in mast cells (10, 11). Syk possesses two SH2 domains toward the amino terminus of the protein that mediate high-affinity interactions with the tyrosine-phosphorylated ITAM of the b and g subunits of Fc«RI (9, 12). The association of Syk with the ITAM of the g subunit induces the phosphorylation and activation of Syk (11–13) and stimulates subsequent signaling events, including activation of phospholipase C-g1 (PLC-g1) and protein kinase C (PKC). Thus it is possible that selective inhibitors of Syk or specific blockers of the association of Syk with ITAMs may prevent signaling by Fc«RI. Here, we report that a synthetic acridone-related compound, ER-27319, inhibits mast cell responses by inhibiting the phosphorylation and activation of Syk in these cells. MATERIALS AND METHODS Reagents and Animals. Minimal essential medium (MEM), macrophage-SFM medium, and Dulbecco’s phosphate-buffered saline (D-PBS) were from GIBCOyBRL. 5-Hydroxy[214C]tryptamine binoxalate, 5-hydroxy[1,2-3H(N)]tryptamine binoxalate, [1-14C]arachidonic acid, and myo-[2-3H(N)]inositol were obtained from DuPont NEN. Histamine EIA (enzyme immunoassay) Kit was from Immunotech (Marseille, France). Peptide-leukotriene (pLT) EIA Kit and prostaglandin D2-MOX EIA Kit were from Cayman Chemicals (Ann Arbor, MI). Mouse tumor necrosis factor a (TNFa) EIA Kit was obtained from Genzyme. Recombinant human stem cell factor (SCF) was from PeproTech (Rocky Hill, NJ). Mouse anti-human mast cell tryptase antibody and anti-human mast cell chymase antibody were from Chemicon. Antibody to phosphotyrosine (PY20) coupled to horseradish peroxidase was from ICN Immunologicals (Cleveland, OH). Agarose-conjugated PY20 and a mouse monoclonal antibody to protein kinase C d were from Transduction Laboratories (Lexington, KY). Antibodies to PLC-g1 and Lyn were from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antibody to Syk was prepared by the method of Minoguchi et al. (13). Goat antibody to glutathione S-transferase (GST) was from Pharmacia. o-Dinitrophenyl (DNP)25-BSA and DNP-specific monoclonal mouse IgE were kindly supplied by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9412539-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ER-27319, 3,4-dimethyl-10-(3-aminopropyl)-9acridone oxalate; Fc«RI, the high-affinity receptor for IgE; SH2, src homology domain 2, ITAM, immunoreceptor tyrosine-based activation motif; PLC-g1, phospholipase C-g1; PKC, protein kinase C; TNFa, tumor necrosis factor a; GST, glutathione S-transferase; DNP, o-dinitrophenyl; pLTs, peptide leukotrienes. ‡To whom reprint requests should be addressed. e-mail: k1-yamada@ eisai.co.jp.