Activation Phenotype Stem Cell Factor Programs the Mast Cell
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Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 is online at: average * The Journal of Immunology , 27 of which you can access for free at: 2012; 188:5428-5437; Prepublished online 23 from submission to initial decision 4 weeks from acceptance to publication April 2012; doi: 10.4049/jimmunol.1103366 http://www.jimmunol.org/content/188/11/5428 Stem Cell Factor Programs the Mast Cell Activation Phenotype Tomonobu Ito, Daniel Smrz, Mi-Yeon Jung, Geethani Bandara, Avanti Desai, Sárka Smrzová, Hye Sun Kuehn, Michael A. Beaven, Dean D. Metcalfe and Alasdair M. Gilfillan J Immunol cites 52 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2012/04/24/jimmunol.110336 6.DC1 This article http://www.jimmunol.org/content/188/11/5428.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Stem Cell Factor Programs the Mast Cell Activation Phenotype Tomonobu Ito,*,1 Daniel Smrzˇ,*,1 Mi-Yeon Jung,* Geethani Bandara,* Avanti Desai,* Sˇa´rka Smrzˇova´,* Hye Sun Kuehn,* Michael A. Beaven,† Dean D. Metcalfe,* and Alasdair M. Gilfillan* Mast cells, activated by Ag via Fc«RI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of Fc«RI-mediated degranulation and cytokine production. The hyporesponsive phenotype Downloaded from was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders. The Journal of Immunology, 2012, 188: 5428–5437. http://www.jimmunol.org/ ntigen-mediated mast cell (MC) activation, via FcεRI, Little is known about how SCF and other extrinsic factors, or the results in the release of an array of inflammatory combination thereof, may dictate the MC phenotype with regard to A mediators that underlies allergic reactions in atopic responsiveness to Ag and other stimulants. Under acute experi- disease (1, 2). MCs are derived from bone marrow progenitors that mental conditions, SCF is one of several endogenous agents known migrate into the circulation and peripheral tissues where they to potentiate Ag-mediated MC degranulation and cytokine pro- expand and mature under the influence of cytokines contained duction (11–13). Nevertheless, in contrast to its acute effects on within the surrounding milieu (3). KIT activation, as a conse- MC activation in vivo (14), it is reported that repetitive s.c. in- quence of binding of its ligand stem cell factor (SCF) produced jection of SCF over a period of 21 d into mice may actually protect within tissues by stromal cells, is critical for the development of against fatal anaphylactic reactions (15). Indeed, at the sites of by guest on September 25, 2021 MCs from bone marrow progenitor cells and their subsequent injection, the MCs exhibited little morphological evidence of de- accumulation, maturation, and survival in tissues (4). Unlike hu- granulation after induction of anaphylaxis via IgE in these mice man MCs (5), mouse MC development and survival in culture are (figure 2 in Ref. 15), suggesting that chronic exposure to SCF may supported by IL-3 in the absence of SCF (6). However, the sup- have a profoundly different impact on MC activation than does plementary presence of SCF markedly enhances the rate of growth short-term exposure. Thus, we investigated the hypothesis that of these cells and is described to skew the development of the cells prolonged exposure of MCs to SCF, as likely occurs in vivo to to that of a serosal/connective tissue phenotype. In contrast, cells maintain MC homeostasis, may lead to transcriptional modifi- grown in IL-3 alone are considered to more resemble the mucosal cations that alter the underlying activation properties of the cells. phenotype based on the types of proteases expressed (7–10). As reported in this article, these studies led us to identify a novel mechanism for the regulation of the extent of MC activation *Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Dis- through SCF-dependent induction of a hyporesponsive phenotype eases, National Institutes of Health, Bethesda, MD 20892; and †Laboratory of Mo- with respect to both cytokine production and degranulation. This lecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of phenotype was not due to downregulation of the expression of Health, Bethesda, MD 20892 either FcεRI or KIT, but it could be explained by an inability of the 1T.I. and D.S. are co-first authors. cells to undergo the cytoskeletal reorganization required for me- Received for publication December 2, 2011. Accepted for publication March 28, 2012. diator release, potentially as a consequence of decreased expres- sion of the Src kinase Hck. These findings reveal that the This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases and the National Heart, Lung, and Blood sensitivity of MCs to IgE/Ag stimulation is highly regulated by Institute within the National Institutes of Health. SCF and presumably other cytokines in the surrounding tissue The array data presented in this article have been submitted to Gene Expression milieu. This may have important implications for understanding Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE35332. how the activation capacity of tissue MCs may be phenotypically Address correspondence and reprint requests to Dr. Alasdair M. Gilfillan, Laboratory modified in health and in disease. of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11C206, 10 Center Drive MSC 1881, Be- thesda, MD 20892-1881. E-mail address: agilfi[email protected] Materials and Methods The online version of this article contains supplemental material. Cell culture and coculture Abbreviations used in this article: BMMC, bone marrow-derived mast cell; cSCF, chronic SCF; GPCR, G protein-coupled receptor; b-hex, b-hexosaminidase; HSA, Experiments on mice were conducted under a protocol approved by the human serum albumin; MC, mast cell; NIH, National Institutes of Health; PKC, Animal Care and Use Committee at National Institutes of Health (NIH). protein kinase C; PL, phospholipase; RT, room temperature; SCF, stem cell factor. Bone marrow-derived MCs (BMMCs) were developed from bone marrow www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103366 The Journal of Immunology 5429 obtained from femurs of C57BL/6 mice (The Jackson Laboratory, Bar Retroviral transduction Harbor, ME), as described (16). Essentially, the cells were cultured for 4–6 wk in media containing mouse rIL-3 (30 ng/ml; PeproTech, Rocky Hill, Retroviral vector pMX-puro with/without the mouse hck cDNA (a kind gift NJ) or a combination of mouse rIL-3 (30 ng/ml) and mouse recombinant from Dr. Toshiaki Kawakami, La Jolla Institute for Allergy and Immu- SCF (unless otherwise indicated: 100 ng/ml) (PeproTech). The cells were nology, San Diego, CA) was transfected into PLAT-E cells (Cell Biolabs, maintained at 37˚C in a humidified incubator gassed with 95% air and 5% San Diego, CA) with FUGENE 6 transfection reagent (Roche Applied Science) in Opti-MEM (Invitrogen). Transfected cells were grown in CO2. The purity of the cultures, as assessed by toluidine blue staining (17) and FcεRIa and KIT expression, was .99%. The NIH 3T3 mouse fibro- DMEM containing FBS (10%) and L-glutamine (4 mM), and the media blast cell line (American Type Culture Collection, Manassas, VA) was were replaced 16–20 h posttransfection with fresh media containing grown or cocultured (18) with BMMCs in the same media as for BMMCs penicillin (100 U/ml) and streptomycin (100 mg/ml). After 24 h, virus but in the absence of IL-3 and SCF. particles in the media were collected by centrifugation (8000 rpm, over- night, 4˚C), and the pellet was resuspended in complete BMMC medium. Cell sensitization, activation, degranulation, and cytokine/ Viral titer was determined using a QuickTiter retrovirus quantitation kit 7 chemokine release (Cell Biolabs). A total of 0.5–1 3 10 BMMCs (2–3 wk old) was trans- duced with 4.5 3 1010 virus particles in hexadimerthrine bromide (10 mg/ BMMCs were sensitized overnight in cytokine-containing or cytokine-free ml). After 72 h, the medium was replaced with fresh BMMC medium media (as indicated) with mouse anti–DNP-IgE (100 ng/ml; clone SPE-7; with/without SCF, and transduced cells were selected in the presence of Sigma). After sensitization, the cells were processed and activated as de- 1.2 mg/ml puromycin for 2 wk.