Auto-Oxidation and Oligomerization of Protein S on the Apoptotic Cell Surface Is Required for Mer Tyrosine Kinase-Mediated Phagocytosis of Apoptotic Cells This information is current as of October 2, 2021. Hiroshi Uehara and Emily Shacter J Immunol 2008; 180:2522-2530; ; doi: 10.4049/jimmunol.180.4.2522 http://www.jimmunol.org/content/180/4/2522 Downloaded from References This article cites 53 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/180/4/2522.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 October 2, 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 Auto-Oxidation and Oligomerization of Protein S on the Apoptotic Cell Surface Is Required for Mer Tyrosine Kinase-Mediated Phagocytosis of Apoptotic Cells Hiroshi Uehara and Emily Shacter1 Prompt phagocytosis of apoptotic cells prevents inflammatory and autoimmune responses to dying cells. We have previously shown that the blood anticoagulant factor protein S stimulates phagocytosis of apoptotic human B lymphoma cells by human monocyte-derived macrophages. In this study, we show that protein S must first undergo oxidative activation to stimulate phagocytosis. Binding of human protein S to apoptotic cells or to phosphatidylserine multilamellar vesicles promotes auto- oxidation of Cys residues in protein S, resulting in covalent, disulfide-linked dimers and oligomers that preferentially bind to and activate the human Mer tyrosine kinase (MerTK) receptor on the macrophages. The prophagocytic activity of protein S is eliminated when disulfide-mediated oligomerization is prevented, or when MerTK is blocked with neutralizing Abs. Downloaded from Protein S oligomerization is independent of phospholipid oxidation. The data suggest that membranes containing phospha- tidylserine serve as a scaffold for protein S-protein S interactions and that the resulting auto-oxidation and oligomerization is required for the prophagocytic activity of protein S. In this way, apoptotic cells facilitate their own uptake by macro- phages. The requirement for oxidative modification of protein S can explain why this abundant blood protein does not constitutively activate MerTK in circulating monocytes and tissue macrophages. The Journal of Immunology, 2008, 180: 2522–2530. http://www.jimmunol.org/ poptosis is a physiological form of cell death that plays outer leaflet of the plasma membrane (10, 11). The PS exposure is an essential role in tissue and organ development and thought to be essential for the recognition and uptake of apoptotic A homeostasis in multicellular organisms. Rapid removal cells by phagocytes (12–14). of apoptotic cells by macrophages and certain types of neighboring Several different macrophage receptors have been identified as cells before the loss of plasma membrane integrity prevents the being involved in the phagocytosis of apoptotic cells (reviewed in leakage of potentially toxic and immunogenic cellular contents and Ref. 15). These include a PS receptor, integrins, CD14, CD36, and thereby prevents inflammation and autoimmune responses to cell receptors for oxidized low-density lipoprotein. One key macro- death (1, 2). This is thought to be one of the main physiological phage receptor is Mer tyrosine kinase (MerTK), which is a mem- by guest on October 2, 2021 advantages of death through apoptosis instead of necrosis, in ber of the TAM family of receptor tyrosine kinases (16). It is which cellular macromolecules may leak out and stimulate an in- expressed on epithelial cells and monocytic cells (17), both of flammatory response before removal of the cells from the tissue (3, which have phagocytic activity. The essential role of MerTK has 4). Engulfment of apoptotic cells by macrophages also triggers been demonstrated for the phagocytosis of photoreceptor outer production of anti-inflammatory and immunosuppressive cyto- segment cells by retinal pigment epithelium cells (18, 19). kines, further limiting an immune response (5, 6). Studies using In addition to these phagocyte surface molecules, a number of gene knockout mice have shown an association between autoim- soluble molecules have been identified that may control the inter- mune disease and abnormal clearance of apoptotic cells (7, 8). action between apoptotic cells and phagocytes (2). These include The removal of apoptotic cells involves apoptotic cell surface protein S, MFG-E8, gas6, C1q, mannose-binding lectin, throm- ␤ molecules, phagocyte receptors, and soluble factors that modulate bospondin, pentraxin, 2-glycoprotein I, and surfactant proteins A cell recognition and uptake (1, 9). Upon induction of apoptosis, and D. Opsonization of target cells with bridging molecules may cells lose the phospholipid asymmetry normally found in the expand the repertoire of potential phagocytic targets by allowing plasma membrane and expose phosphatidylserine (PS)2 on the classical phagocyte receptors that are customarily associated with infection and immunity to mediate recognition and uptake of ap- optotic cells and thereby limit the undesirable side effects from Laboratory of Biochemistry, Division of Therapeutic Proteins, Center for Drug Eval- uation and Research, Food and Drug Administration, Bethesda, MD 20892 exposure to dead cell debris (20, 21). We and others showed that the anticoagulant factor protein S is Received for publication June 28, 2007. Accepted for publication November 30, 2007. required for the efficient uptake of apoptotic lymphoma cells by The costs of publication of this article were defrayed in part by the payment of page macrophages in vitro (22, 23). Protein S, originally defined as an charges. This article must therefore be hereby marked advertisement in accordance anticoagulant protein that is a nonenzymatic cofactor for activated with 18 U.S.C. Section 1734 solely to indicate this fact. protein C, is a vitamin K-dependent, 68-kDa monomeric protein 1 Address correspondence and reprint requests to Dr. Emily Shacter, Center for Drug present at a concentration of ϳ25 ␮g/ml in the blood (24, 25). It Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Building 29A, Room 2A-11, HFD-121, Bethesda, MD 20892-4555. E-mail address: [email protected] 2 Abbreviations used in this paper: PS, phosphatidylserine; MerTK, Mer tyrosine 2-methyl-propaimidaamide; (Ϯ)9-HODE, (Ϯ)-9-hydroxy-10E,12Z-octadecadienoic kinase; CFDA, carboxyfluorescein diacetate, succinimidyl ester mixed isomer; PI, acid; IAA, iodoacetamide; NEM, N-ethylmaleimide; TnCl, taurine chloramine; sMer, propidium iodide; PC, phosphatidylcholine; DOPS, dioleoyl PS; DOPC, dioleoyl PC; soluble Mer; LMV, large multilamellar vesicle; oxPLPS, oxidized PLPS; LDS, lith- PLPS, 1-palmitoyl-2-linoleoyl-sn-glycero-3-[phospho-L-serine]; AAPH, 2.2Ј-azobis- ium dodecyl sulfate. www.jimmunol.org The Journal of Immunology 2523 ϩ is known to bind to PS on cell surfaces in a Ca2 -dependent man- 30–50% apoptosis as assessed by two different techniques: Hoechst 33342 ner using a Gla domain at the N terminus of the protein. Aberrantly and PI staining followed by nuclear morphology assessment using fluo- low levels of protein S may lead to inefficient uptake of early rescence microscopy (4) or by two-color flow cytometry using a FACScan (BD Biosciences) following labeling of cells with FITC-annexin V in apoptotic cells and expose immune cells to potentially immuno- binding buffer (10 mM HEPES, 0.14 M NaCl, 5 mM KCl, 2.5 mM genic cellular contents and thus trigger an autoimmune response CaCl2, 1 mM MgCl2 (pH 7.4)) followed by addition of PI as described (26). Deficiencies in protein S, either hereditary or acquired previously (13). through autoantibody formation, lead to excess thrombosis (27) Modification of protein S with iodoacetamide (IAA), and are associated with autoimmune diseases such as systemic N-ethylmaleimide (NEM), or taurine chloramine (TnCl) lupus erythematosus (28, 29). The newly discovered role of protein S in stimulating the phagocytosis of apoptotic cells re- Protein S was covalently modified by incubating with 100 mM IAA or 10 mM NEM in PBS for 30 min at room temperature. The reaction was veals a novel functional link between the coagulation system stopped by passing the solution through a Sephadex G-25 desalting col- and autoimmunity (26). umn. TnCl was prepared by mixing 10 mM taurine with 2.5 mM HOCl, In this report, we investigated the biochemical and molecular which results in immediate and quantitative consumption of all of the mechanisms for stimulation of phagocytosis by protein S. We HOCl into TnCl (30). This solution was diluted to 0.5 mM TnCl in PBS containing 1.5 mM CaCl and incubated with protein S alone (0.25 mg/ml) or demonstrate that in order for protein S to serve as a prophagocytic 2 protein S bound to PS vesicles at 25°C for 1 h. The sulfhydryl-specific cross- molecule, it must bind to membranous PS, either on the surface of linking of protein S was performed in PBS containing 0.5 mM BM(PEO)2 apoptotic cells or in artificial phospholipids vesicles. Protein S then under the same condition as described for oxidation with TnCl. The reactions undergoes oligomerization through oxidation of cysteine residues were stopped by desalting with Sephadex G-25. and formation of intermolecular disulfide bonds. This oxidative Preparation of phospholipid vesicles Downloaded from activation of the protein is required for binding to and activation the macrophage MerTK.