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Survival Pathways That Regulate Macrophage Tec and Btk in M-CSF

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Survival Pathways That Regulate Macrophage Tec and Btk in M-CSF Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival This information is current as of September 26, 2021. Martin Melcher, Bernd Unger, Uwe Schmidt, Iiro A. Rajantie, Kari Alitalo and Wilfried Ellmeier J Immunol 2008; 180:8048-8056; ; doi: 10.4049/jimmunol.180.12.8048 http://www.jimmunol.org/content/180/12/8048 Downloaded from References This article cites 36 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/180/12/8048.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival1 Martin Melcher,2* Bernd Unger,2† Uwe Schmidt,3* Iiro A. Rajantie,‡ Kari Alitalo,‡ and Wilfried Ellmeier4* Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec؊/؊Btk؊/؊ BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways Downloaded from revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec؊/؊Btk؊/؊ macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor ␣ (GM-CSFR␣) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage- specific regulation of GM-CSFR␣ expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling- induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM- CSFR␣ expression. The Journal of Immunology, 2008, 180: 8048–8056. http://www.jimmunol.org/ acrophages are large phagocytic mononuclear cells review see Ref. 4 and references therein). Thus, a better under- that play important roles in innate and adaptive im- standing of M-CSFR signaling may also be of medical relevance. munity. Their progenitors, the monocytes, enter the Members of the Tec kinase family (Bmx, Btk, Itk, Rlk, and Tec) M 5 blood stream from the bone marrow (BM) and migrate to tissues constitute the second largest family of nonreceptor tyrosine ki- where they mature into resident tissue macrophages (1). The dif- nases and are preferentially expressed in the hematopoietic system. ferentiation, proliferation, and survival of macrophages are regu- A large number of studies have shown important roles for these lated by the M-CSF. In fact, M-CSF receptor (M-CSFR)-deficient kinases in the lymphoid system. Furthermore, mice with combi- by guest on September 26, 2021 mice or mice with an inactivating mutation of M-CSF have pleio- natorial deletions of Tec family kinases revealed both unique and tropic phenotypes including decreased macrophage numbers in redundant functions in B cells (Tec, Btk) and T cells (Rlk, Itk). vivo (2, 3). In mice, impaired M-CSF signaling has also been Although the Tec family kinase members Tec, Btk, and Bmx are implicated in the pathogenesis of several disorders (for a detailed expressed in monocytes/macrophages (5–7), little is known about their function in this lineage. Several studies implicated Tec family kinases in the LPS-induced signaling in macrophages leading to the induction of TNF-␣ production. Btk-defective X-linked immu- *Institute of Immunology, Center for Physiology, Pathophysiology and Immunology, † nodeficient (xid) macrophages have impaired secretion of the Medical University of Vienna, Vienna, Austria; Competence Center for Biomolec- ␣ ␤ ular Therapeutics, Vienna, Austria; and ‡Molecular/Cancer Biology Laboratory, Bio- proinflammatory cytokines TNF- and IL-1 after stimulation medicum Helsinki, University of Helsinki, Helsinki, Finland with LPS (8) and are also incapable of producing efficient bursts of Received for publication August 7, 2007. Accepted for publication April 10, 2008. reactive oxygen intermediates (9). In line with this, xid macro- The costs of publication of this article were defrayed in part by the payment of page phages show impaired p65 phosphorylation and transactivation charges. This article must therefore be hereby marked advertisement in accordance upon LPS stimulation, whereas IkB␣-degradation is normal (10). with 18 U.S.C. Section 1734 solely to indicate this fact. Meanwhile, another study could not find any differences in TNF-␣ 1 This work was supported by the START program (Grant Y-163) of the Fonds zur Fo¨rderung der Wissenschaftlichen Forschung and the Austrian Ministry of Education, expression between control and xid macrophages after LPS stim- Science and Culture (to W.E.), by the K-Plus Competence Center for Biomolecular ulation (5), which may reflect differences in the genetic back- Therapeutics (to W.E.), by the Sonderforschungsbereich project F2305-B13 of the Austrian Research Fund (to W.E.), and by a postdoctoral fellowship (to U.S.) from the grounds or different macrophage populations used in these studies. Deutsche Forschungsgemeinschaft (Schm 2128/1-1). However, the importance of Tec family kinases for monocyte 2 M.M. and B.U. contributed equally to this work. function has been confirmed through the analysis of Btk-deficient 3 Current address: Nabriva Therapeutics, Brunnerstrasse 59, 1235 Vienna, Austria. human monocytes. Blood monocytes isolated from X-linked 4 Address correspondence and reprint requests to Dr. Wilfried Ellmeier, Institute of agammaglobulinemia (XLA) patients who lack a functional Btk Immunology, Medical University Vienna, Center for Physiology, Pathophysiology gene have impaired phagocytic functions and altered chemotactic and Immunology, Lazarettgasse 19, A-1090 Vienna, Austria. E-mail address: ␣ [email protected] responses (11) and are impaired in the production of TNF- and IL-1␤ upon stimulation of TLR 2 or 4 (12), although another study 5 Abbreviations used in this paper: BM, bone marrow; BMDC, BM-derived dendritic cell; BMM, BM-derived macrophage; HPRT, hypoxanthine phosphoribosyltrans- reports that Btk is not essential for LPS/TLR4 signaling (13). ferase; LCM, L929 cell-conditioned medium; M-CSFR, M-CSF receptor; PI, pro- Overexpression of Btk in wild-type human monocytes leads to the pidium; xid, X-linked immunodeficient (gene); XLA, X-linked agammaglobulinemia. stabilization of TNF-␣ mRNA and therefore to an increase in Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 TNF-␣ production (5, 12). Interestingly, incubation of XLA www.jimmunol.org The Journal of Immunology 8049 monocytes with M-CSF leads to an increase in the expression of containing M-CSF (10 and 30 ng/ml), GM-CSF (500 and 1500 U/ml), or Tec and restores their ability to produce TNF-␣ upon LPS stimu- LCM was exchanged daily until the end of the culture. For the supernatant 7 Ϫ/Ϫ Ϫ/Ϫ lation (5). This suggests a compensatory role for Tec similar to the exchange experiments, 10 wild-type and Tec Btk cells were re- seeded onto 10-cm dishes on day 6, and the corresponding supernatants situation observed in murine B cells (14), which may also explain were added daily to the culture. For the M-CSF titration experiments, cells why XLA patients show normal innate immune responses (5). were reseeded in 6-well plates at 0.8 ϫ 106 cells per well on day 5 and In this study, we aimed to further dissect the role of Tec family different concentrations of M-CSF (15, 30, 60, and 90 ng/ml) were added. kinases in monocytes/macrophages. We used a genetic approach to Generation of BM-derived dendritic cells (BMDCs) study macrophages lacking various members of the Tec kinase family and generated combinatorial Tec family kinase knockout BM cells were isolated and RBCs were lysed as described above for the 6 mice. We could show that Tec and Btk regulate the survival of generation of BMM. To generate BMDCs, 2 ϫ 10 BM cells were cultured on 10-cm dishes in RPMI 1640 medium with 10% FCS, 100 U/ml peni- BM-derived macrophages (BMM) by controlling M-CSFR signal- ␮ L Ϫ/Ϫ Ϫ/Ϫ cillin, 10 g/ml streptomycin, 2 mM -glutamine, 10 mM HEPES (Sigma- ing. A severe drop in cell numbers in Tec Btk macrophage Aldrich), 50 ␮M 2-ME (Invitrogen) and 700 U/ml recombinant murine cultures was observed, and this correlated with increased cell death
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