Degranulation and Anaphylaxis Efficient Ige-Mediated Mast Cell
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The Tetraspanin CD63 Is Required for Efficient IgE-Mediated Mast Cell Degranulation and Anaphylaxis This information is current as Stefan Kraft, Marie-Hélène Jouvin, Nitin Kulkarni, Sandra of September 26, 2021. Kissing, Ellen S. Morgan, Ann M. Dvorak, Bernd Schröder, Paul Saftig and Jean-Pierre Kinet J Immunol 2013; 191:2871-2878; Prepublished online 14 August 2013; doi: 10.4049/jimmunol.1202323 Downloaded from http://www.jimmunol.org/content/191/6/2871 Supplementary http://www.jimmunol.org/content/suppl/2013/08/14/jimmunol.120232 Material 3.DC1 http://www.jimmunol.org/ References This article cites 48 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/191/6/2871.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Tetraspanin CD63 Is Required for Efficient IgE-Mediated Mast Cell Degranulation and Anaphylaxis Stefan Kraft,*,† Marie-He´le`ne Jouvin,* Nitin Kulkarni,* Sandra Kissing,‡ Ellen S. Morgan,x Ann M. Dvorak,x Bernd Schro¨der,‡ Paul Saftig,‡ and Jean-Pierre Kinet* Mast cell (MC) activation through the high-affinity IgE receptor Fc«RI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit Fc«RI-induced MC degranulation, the intrinsic role of these molecules in Fc«RI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow–derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow–derived MC showed a significant decrease in Fc«RI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by Downloaded from b-hexosaminidase release assays. The secretion of TNF-a, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, Fc«RI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kitw/w-v mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly atten- uated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of http://www.jimmunol.org/ MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation. The Journal of Immunology, 2013, 191: 2871–2878. ast cells (MC) originate in the bone marrow and mi- serotonin, proteases, IL-4, and also a significant proportion of grate to most connective tissues and epithelia in pro- TNF-a, are preformed and stored in granules from which they are M cesses that involve various integrins and extracellular released upon MC activation in a process called degranulation or matrix proteins (1). Within those peripheral tissues, MC mature regulated exocytosis. Others, such as numerous cytokines, leu- and can be long-lived, constitutional tissue residents, or recruited kotrienes, and PGs, are synthesized de novo and released upon to sites of inflammation. Their best-established role is in IgE- MC activation (4–8). by guest on September 26, 2021 mediated allergic reactions, but they also positively and nega- MC can be activated by various mechanisms, such as Igs, tively regulate multiple aspects of the inflammatory reaction in cytokines, physical factors, and microbial products. MC activation autoimmunity, contact hypersensitivity, cancer response, and host takes place in acute IgE-mediated allergic reactions such as ana- defense directly and indirectly by influencing dendritic cells, Th1, phylaxis. In this case, Ag-specific IgE bound to its high-affinity Th2, regulatory T cells, Th17, endothelial cells, neurons, and receptor FcεRI expressed at the surface of MC is cross linked possibly B cells (2, 3). These effects are mediated by the nu- by multivalent Ags, leading to the activation of multiple signaling merous mediators that MC release upon activation. Two main cascades. Key features are the activation of protein tyrosine kinases release mechanisms operate in MC. Mediators such as histamine, of the src and spleen tyrosine kinase family, multiple downstream adapter molecules, GTPases and serine-threonine kinases, and calcium mobilization, which ultimately results in the release of *Laboratory of Allergy and Immunology, Department of Pathology, Beth Israel † the different mediators (7, 9, 10). Deaconess Medical Center, Boston, MA 02215; Department of Pathology, Massa- ε chusetts General Hospital, Boston, MA 02114; ‡Institute of Biochemistry, University Fc RI-induced MC activation can be positively or negatively of Kiel, 24098 Kiel, Germany; and xCenter for Vascular Biology Research, Beth affected by a variety of mechanisms (11). One involves tetraspa- Israel Deaconess Medical Center, Boston, MA 02215 nins, proteins that are expressed in various membrane compart- Received for publication August 31, 2012. Accepted for publication July 5, 2013. ments and regulate cell morphology, motility, invasion, fusion, and This work was supported by National Institutes of Health Grant RO1 AI41087 (to signaling (12–14). Using mAb that recognize Ags expressed at the J.-P.K.) and grants from the Deutsche Forschungsgemeinschaft (DFG-GRK1459 and surface of MC and inhibit FcεRI-induced MC degranulation, we SPP1580) and the Center of Excellence Inflammation at Interfaces (to B.S. and P.S.). previously identified the tetraspanins CD63 and CD81 as regu- Address correspondence and reprint requests to Dr. Jean-Pierre Kinet or Dr. Paul ε Saftig, Laboratory of Allergy and Immunology, Department of Pathology, Beth Israel lators of Fc RI-induced MC activation (15, 16). Anti-CD63 Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215 (J.-P.K.) or inhibits MC degranulation without affecting early signals, such Institute of Biochemistry, University of Kiel, Otto-Hahn Platz 9, 24098 Kiel, Germany as protein tyrosine phosphorylation and calcium mobilization, (P.S.). E-mail addresses: [email protected] (J.-P.K.) or [email protected] kiel.de (P.S.) suggesting that it interferes with exocytosis (16). CD63 is known The online version of this article contains supplemental material. to be expressed in intracellular membranes, such as secretory Abbreviations used in this article: BMMC, bone marrow–derived mast cell; IKK2, lysosomes (SL) including serotonin-containing granules (17–20). IkB kinase 2; LTC4, leukotriene C4; MC, mast cell; PCA, passive cutaneous anaphy- Upon basophil degranulation, CD63 expression at the plasma laxis; PM, plasma membrane; SL, secretory lysosome; SNARE, soluble N-ethyl- membrane (PM) increases and is classically used as a marker of maleimide–sensitive factor-attachment protein receptor; WT, wild-type. basophil activation (21). Recently, an isoform of CD63 has been Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 identified as specific form of CD63 expressed at the PM in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202323 2872 CD63 IS ESSENTIAL FOR MAST CELL DEGRANULATION IN ALLERGY degranulated MC (22). Anti-CD63 also inhibits adhesion of the immunoblotting were performed using 10% polyacrylamide gels. Anti- MC model cell line RBL-2H3 to extracellular matrix proteins, mouse CD63 rabbit antiserum (25) was used at a 1:400 dilution and ε b which could be explained by the known interaction of CD63 with control anti-Fc RI mAb supernatant [JRK1 (32)] pure, followed by in- cubation with HRP-conjugated anti-rabbit and anti-mouse Abs (Sigma- integrins in PM microdomains or by inhibition of signaling mech- Aldrich). Anti-phosphotyrosine (4G10; EMD Millipore), anti–phospho- anisms common to FcεRI and integrins (16, 23, 24). Akt (anti–phospho-Ser473 Akt; Cell Signaling Technology, Beverly, MA) Recently, CD63-deficient mice were generated. Despite the and anti-Akt blots (Cell Signaling Technology) of cells triggered as de- broad tissue and cell distribution of CD63, these mice display no scribed for degranulation assays were performed as described (in Refs. 16, 33). obvious morphologic or functional abnormalities of their lyso- somes (25). However, they exhibit morphologic changes in the Degranulation assays collecting ducts in