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Mast Cell Costimulation by CD226/CD112 (DNAM-1/Nectin-2)

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Mast Cell Costimulation by CD226/CD112 (DNAM-1/Nectin-2) THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 37, pp. 27190–27196, September 15, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Mast Cell Costimulation by CD226/CD112 (DNAM-1/Nectin-2) A NOVEL INTERFACE IN THE ALLERGIC PROCESS* Received for publication, March 13, 2006, and in revised form, June 13, 2006 Published, JBC Papers in Press, July 10, 2006, DOI 10.1074/jbc.M602359200 Ido Bachelet‡, Ariel Munitz‡, David Mankutad§, and Francesca Levi-Schaffer‡1 From the ‡Deparment of Pharmacology, The School of Pharmacy, The Faculty of Medicine, The Hebrew University of Jerusalem and the §Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem 91120, Israel Mast cells have critical effector functions in various immune cytes. For example, mast cells induce T lymphocyte prolifera- reactions. In allergic inflammation, mast cells interact with tis- tion and enhance their activity through OX40/OX40L engage- Downloaded from sue-infiltrating eosinophils, forming a regulatory unit in the late ment (5). Reciprocally, intracellular adhesion molecule 1 and chronic phases of the allergic process. However, the path- (ICAM-1)/LFA-12 interactions costimulate mast cells upon ways and molecules within this unit are still largely undefined. contact with T lymphocytes (6). Fc⑀RI-dependent activation of Here, we show that human mast cells and eosinophils express mast cells was shown to be enhanced by several receptors, DNAX accessory molecule 1 (DNAM-1, CD226) and its ligand including c-Kit (7), 4-1BB (8), CD28 (9), and CCR1 (10). Mast www.jbc.org Nectin-2 (CD112). CD226 synergizes with Fc⑀RI on mast cells, cell activity is also enhanced by multiple soluble factors as well. and its engagement augments degranulation through a pathway Moreover, it is likely to assume that positive signals, especially if involving Fyn, linker of activation of T-cells, phospholipase C operating via separate routes, will stack and synergize with each ␥2, and CD18. This pathway is subject to negative interference other. at Tel Aviv university-Library of Life Sciences and Medicine, on January 5, 2011 by inhibitory receptors and is completely inhibited by linking Although mast cell-eosinophil interactions were shown to IgE with IRp60 (CD300a) using a bispecific antibody. Moreover, enhance cytotoxic functions in helminthic infections, the data blocking CD112 expressed on eosinophils using neutralizing regarding mast cell-eosinophil cross-talk in allergic settings are antibodies normalized the hyperactivity resulting from IgE-de- very scarce. Early observations have demonstrated that eosino- pendent activation of mast cells co-cultured with eosinophils. phil major basic protein and eosinophil cationic protein acti- Our findings demonstrate a novel interface between these two vate mast cells and basophils to degranulate (11, 12). Eosino- effector cells, implicating relevance for in vivo allergic states. phils produce stem cell factor, potentially contributing to mast Moreover, costimulatory responses might be a critical compo- cell survival and activity (13). On the other hand, mast cells nent in allergic reactions and may therefore become novel tar- produce IL-3, IL-5, and GM-CSF, important cytokines regulat- gets for anti-allergic therapy. ing eosinophil recruitment, survival, and activity (14). Mast cell tryptase evokes eosinophil degranulation through the protease- activated receptor PAR2 (15). However, it is likely that informa- During the past decade, mast cells have been recognized as tion transfer between cells is mediated more by direct cell-cell critical immune effector cells. Solid evidence indicates their interactions, being more efficient, accurate, and liable than involvement in various settings extending far beyond their tra- paracrine communication systems. ditionally associated context of type I hypersensitivity reac- In this study, we demonstrate that human mast cells express tions, such as innate immunity (1), autoimmunity (2), athero- DNAX accessory molecule 1 (DNAM-1, CD226) and its ligands sclerosis (3), and more. Yet many aspects of their effector Nectin-2 (CD112) and the poliovirus receptor PVR (CD155). functions, even in the classical context of allergic reactions, are We show that CD226 augments Fc⑀RI-mediated activation of still unknown. mast cells by enhancing a signaling pathway dependent in part Until recently, mast cell involvement in the allergic process on Fyn, LAT, and PLC␥2 but not on Syk. Moreover, this path- was confined to its early/acute phase, of which Fc⑀RI-depend- way involves association of CD226 with integrins, and prefer- ent activation forms the central trigger (4). The late/chronic entially to CD18. We also demonstrate that this pathway is phase, characterized by tissue inflammation and remodeling, prone to inhibition by inhibitory receptors. Finally, we show was attributed solely to infiltrating leukocytes, notably to the that in the presence of eosinophils, mast cell Fc⑀RI-mediated eosinophils. However, several lines of evidence indicate that activation is augmented and that this effect is mediated at least mast cells participate in modulation of the late/chronic phase partially by a CD226/CD112 interaction. In conclusion, these mainly by their interactions with eosinophils and T lympho- findings demonstrate a novel interface between mast cells and eosinophils and suggest that CD226 and CD112 participate in * This work was supported by grants from the Aimwell Charitable Trust (UK), modulation of the allergic response. the Israeli Science Foundation (Israel) and a grant from the Italian Ministry of Foreign Affairs for joint research with Israel (Italy). The costs of publica- 2 The abbreviations used are: LFA-1, leukocyte function-associated antigen 1; tion of this article were defrayed in part by the payment of page charges. DNAM-1, DNAX accessory molecule 1; PVR, poliovirus receptor; ICAM-1, This article must therefore be hereby marked “advertisement” in accord- intracellular adhesion molecule 1; LAT, linker for activation of T cells; ERK, ance with 18 U.S.C. Section 1734 solely to indicate this fact. extracellular-signal regulated kinase; MAPK, mitogen-activated protein 1 Affiliated with the David R. Bloom Center of Pharmacy at the Hebrew Uni- kinase; PLC, phospholipase C; HMC-1, human mast cell leukemia 1; FACS, versity of Jerusalem. To whom correspondence should be addressed. Tel.: fluorescence-activatedcellsorter;IL,interleukin;GM-CSF,granulocyte-mac- 972-2-6757512; Fax: 972-2-6758144; E-mail: [email protected]. rophage colony-stimulating factor. 27190 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 37•SEPTEMBER 15, 2006 Mast Cell Costimulation by CD226/CD112 EXPERIMENTAL PROCEDURES Activation System and Mediator Assays—For mediator Antibodies and Reagents—All the cell culture media, assays, cells were activated in Immunolon-2HB 96-well plates reagents, and buffers were purchased from Biological Indus- (ThermoLabsystems, MA). The plates were coated with sheep Ј ␮ tries, Beit Haemek, Israel. Stem cell factor is a kind gift from anti-mouse F(ab )2 (25 g/ml) followed by anti-CD226 (clone ␮ Amgen, Inc. (Thousand Oaks, CA). IL-6 was purchased from F5), anti-CD112, anti-CD155, or isotype control (10 g/ml, 2 h Peptrotech (Rocky Hill, NJ). The following mouse monoclonal at 37 °C). Mast cells were sensitized with human myeloma IgE ␮ antibodies were prepared as described (16, 17) and used: F22 (5 g/ml, 2 h at 37 °C) 5 days prior to activation. On activation, (anti-DNAM-1/CD226, IgG1), F5 (anti-DNAM-1/CD226, cell were washed with Tyrode’s buffer (137 mM NaCl, 12 mM IgM), L14 (anti-Nectin-2/CD112, IgG2a), L95 (anti-PVR/ NaHCO3, 5.5 mML-glucose, 2 mM KCl, 0.3 mM Na2HPO4, 1.8 CD155, IgG1), E59 (anti-CD300a, IgG1), P192 (anti-CD300a, mM CaCl2, 0.9 mM MgCl2), added to the plate on ice simulta- ␮ IgG2a). The following antibodies and reagents were purchased neously with mouse anti-human IgE antibody (5 g/ml), and and used: rat anti-mouse CD300a (clones (NKRL1-172206.111, the plate was incubated at 37 °C for 30 min. Activation was Downloaded from stopped by removing supernatants from cells by centrifugation -172219.111, -172224.111, -172238.111)) from R&D Systems and freezing of both fractions for analysis. Tryptase was ana- (Minneapolis, MN); mouse anti-human IgE (clone GE-1) from lyzed by a chromogenic assay as described (16). IL-4 was ana- Sigma; anti-human tryptase (clone AA1) and isotype control lyzed by a commercial Duoset enzyme-linked immunosorbent (IgG1 and IgG2A) antibodies from Dako (Glostrup, Denmark); Ј assay kit (Diaclone, Besanc¸on, France). Prostaglandin D2 was sheep anti-mouse F(ab ) from ICN Biomedicals (Aurora, OH); www.jbc.org 2 analyzed by a commercial enzymatic immunoassay kit (Cay- human myeloma IgE/␬ from Calbiochem-Merck (Schwalbach, man, Ann Arbor, MI). Germany); polyclonal anti-human phosphotyrosine, phospho- For intracellular flow cytometry, antibody incubations (spe- serine, Fyn, CD18 and CD29 from Santa Cruz Biotechnology cific and sheep anti-mouse antibody) and activation were per- (Santa Cruz, CA); rabbit anti-human phospho-syk, phospho- at Tel Aviv university-Library of Life Sciences and Medicine, on January 5, 2011 formed with cells in suspension (30 min on ice for each anti- LAT, phospho-ERK, phospho-PLC␥1, and phospho-PLC␥2 body incubation). Cells were fixed at the stated time points (see from Cell Signaling (Beverly, MA); horseradish peroxidase- below). For calcium mobilization, antibody incubations (spe- conjugated anti-rabbit
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