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Folate Receptor Β Regulates Integrin Cd11b/CD18 Adhesion of a Macrophage Subset to Collagen

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Folate Receptor Β Regulates Integrin Cd11b/CD18 Adhesion of a Macrophage Subset to Collagen Folate Receptor β Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen This information is current as Christian Machacek, Verena Supper, Vladimir Leksa, Goran of September 24, 2021. Mitulovic, Andreas Spittler, Karel Drbal, Miloslav Suchanek, Anna Ohradanova-Repic and Hannes Stockinger J Immunol 2016; 197:2229-2238; Prepublished online 17 August 2016; doi: 10.4049/jimmunol.1501878 Downloaded from http://www.jimmunol.org/content/197/6/2229 Supplementary http://www.jimmunol.org/content/suppl/2016/08/17/jimmunol.150187 Material 8.DCSupplemental http://www.jimmunol.org/ References This article cites 49 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/197/6/2229.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Folate Receptor b Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen Christian Machacek,* Verena Supper,* Vladimir Leksa,*,† Goran Mitulovic,‡ Andreas Spittler,x Karel Drbal,{,1 Miloslav Suchanek,{ Anna Ohradanova-Repic,* and Hannes Stockinger* Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) b, a GPI-anchored protein belonging to the folate receptor family. As FRb shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRb, it represents a promising target for future thera- peutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenviron- ment of FRb in the plasma membrane of human FRb+ macrophages and FRb-transduced monocytic THP-1 cells. In this manner, Downloaded from we identified a novel role of FRb: that is, we report functional interactions of FRb with receptors mediating cellular adhesion, in particular the CD11b/CD18 b2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRb+ human primary macrophages and THP-1 cells to collagen in comparison with their FRb2 counterparts. We further show that FRb is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRb as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen. The Journal of Immunology, 2016, 197: 2229–2238. http://www.jimmunol.org/ olate receptor (FR) b, a GPI-anchored protein belonging to Unlike the broadly expressed reduced folate carrier (SLC19A1) the folate receptor family (1), constitutes one of the pathways and proton-coupled folate transporter (SLC46A1) (6), FRb shows F providing folic acid necessary for essential cellular functions a restricted expression to cells of the myeloid origin, and only a such as DNA synthesis, repair, and methylation (2, 3). Contrary to the subset of activated macrophages and placental cells has been embryonically lethal FRa knockout mice (4), genomic ablation of shown to express functional FRb (7). Macrophages show remark- FRb has no reported phenotype except for an increased risk of em- able plasticity, and their phenotype can vary greatly depending on bryonic lethality through arsenate or valproic acid exposure (5). the stimulus. On the one hand, classically activated (e.g., LPS plus by guest on September 24, 2021 However, a comprehensive functional analysis of this receptor, as IFN-g) macrophages (M1 type) drive Th1 responses and mediate well as an identification of its interacting proteins, is still missing. host defense from bacteria as well as viruses and propagate anti- tumor functions. On the other hand, alternatively activated (e.g., by *Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infecti- Th2 cytokine IL-4) macrophages (M2 type) induce a Th2-mediated ology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; immune response against parasites and fungi and display an anti- † Laboratory of Molecular Immunology, Institute of Molecular Biology, Slovak Acad- inflammatory as well as wound healing phenotype (8). Reports that emy of Sciences, 845 51 Bratislava, Slovakia; ‡Department of Clinical Chemistry and Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; xDepart- FRb is expressed on subpopulations of monocytes and macro- ment of Surgery and Core Facility Flow Cytometry, Medical University of Vienna, { phages in rheumatoid arthritis (9, 10), on M2 macrophages in 1090 Vienna, Austria; and EXBIO Praha, 252 42 Vestec, Czech Republic cancer (11, 12) as well as on myeloid leukemia cells (13, 14), have 1 Current address: Department of Cell Biology, Faculty of Science, Charles University placed attention on FRb as a tool for targeting pathogenic and in Prague, Prague, Czech Republic. malignant myeloid cells. ORCIDs: 0000-0003-2831-600X (V.S.); 0000-0003-1964-3965 (G.M.); 0000-0003- 2657-6836 (A.S.); 0000-0003-4450-4625 (K.D.); 0000-0002-8005-8522 (A.O.-R.); As GPI-anchored proteins are inserted into the outer leaflet of the 0000-0001-6404-4430 (H.S.). cell membrane, they require interaction partners in the form of Received for publication August 20, 2015. Accepted for publication July 14, 2016. transmembrane proteins to transmit outside–in signals. Therefore, This work was supported by the European Union Seventh Framework Programme characterization of the protein microenvironment of FRb is one (FP7/2007-2013) under Grant Agreement NMP4-LA-2009-228827 NANOFOL. V.L. possible route to investigate its function. Recently, a functional was supported by Austrian Science Fund Grant P22908 and Slovak Scientific Grant interaction between FRa and gp130 was reported (15), explaining Agency Grant 2/0063/14. how FRa is able to convey a proliferative advantage to cancer Address correspondence and reprint requests to Prof. Hannes Stockinger and Dr. Anna Ohradanova-Repic, Institute for Hygiene and Applied Immunology, cells. As FRb exhibits restricted expression to cells of myeloid Center for Pathophysiology, Infectiology and Immunology, Medical University origin, we investigated whether FRb is involved in the regulation of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria. E-mail addresses: hannes. of immune cell function. [email protected] (H.S.) and [email protected] (A.O.-R.) One central function is the ability of immune cells to locate in The online version of this article contains supplemental material. areas with active inflammation. This, however, can drive disease Abbreviations used in this article: AF, Alexa Fluor; ATRA, all-trans retinoic acid; CR3, complement receptor type 3; ECM, extracellular matrix; FR, folate receptor; progression in instances of chronic inflammation (e.g., rheumatoid HA, hemagglutinin; IP, immunoprecipitation; LC-MS/MS, liquid chromatography– arthritis) (16). The integrin family of proteins constitutes a major MS/MS; MFI, mean fluorescence intensity; MS, mass spectrometry; MS/MS, tandem factor in the regulation of leukocyte trafficking (17), and its MS; TFA, trifluoroacetic acid; vD , 1,25-dihydroxyvitamin D . 3 3 members were shown to associate with various GPI-anchored Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 proteins that modulate their activity (18–20). In this study, we www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501878 2230 CD11b/CD18 ADHESION TO COLLAGEN REGULATED BY FRb report a functional interaction of FRb with the CD11b/CD18 (A-11070) and AF 680–conjugated goat anti-mouse IgG were purchased integrin heterodimer Mac-1 resulting in modulated adhesion to from Invitrogen. Anti-hemagglutinin (HA) mAb (clone 12CA5) and the extracellular matrix (ECM) component collagen. peroxidase-conjugated anti-mouse IgG (A9044) were purchased from Sigma-Aldrich. IRDye 800CW–conjugated donkey anti-rabbit IgG was purchased from LI-COR Biosciences (Lincoln, NE). AF 488–conjugated Materials and Methods goat-anti-mouse IgG (H+L) was purchased from Thermo Fischer Sci- Cell isolation and cultivation entific (Waltham, MA). Human PBMCs were isolated from leukocyte reduction chambers of Flow cytometry healthy adult blood donors by density gradient centrifugation using Cells were detached using 1.5 mM EDTA in HBSS. Cells were washed with Lymphoprep (Axis-Shield, Oslo, Norway). CD14+ monocytes were purified staining buffer (PBS supplemented with 1% BSA [Carl Roth, Karlsruhe, from PBMCs by MACS using CD14 microbeads (Miltenyi Biotec, Ber- Germany] and 0.02% NaN ), and nonspecific binding of the mAbs to Fc gisch Gladbach, Germany) with a minimal purity of 98%. THP-1 cells 3 receptors was prevented by blocking
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