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Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells

Nature Immunology volume 4, pages 87–91 (2003)Cite this article

Abstract

Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.

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Figure 1: FCS stimulates the ability of human macrophages to phagocytose apoptotic cells.
Figure 2: Protein S stimulates phagocytosis of apoptotic BL-41 cells by macrophages.
Figure 3: Immunodepletion of protein S from serum removes prophagocytic activity from serum.
Figure 4: Protein S binds to apoptotic cells.
Figure 5: Protein S binds to phosphatidylserine on the surface of apoptotic cells.

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References

  1. Savill, J. & Fadok, V. Corpse clearance defines the meaning of cell death. Nature 407, 784–788 (2000).

    Article  CAS  Google Scholar 

  2. Shacter, E., Williams, J.A., Hinson, R.M., Senturker, S. & Lee, Y.J. Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell apoptosis and phagocytosis. Blood 96, 307–313 (2000).

    CAS  PubMed  Google Scholar 

  3. Scaffidi, P., Misteli, T. & Bianchi, M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195 (2002).

    Article  CAS  Google Scholar 

  4. Scott, R.S. et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411, 207–211 (2001).

    Article  CAS  Google Scholar 

  5. Cohen, P.L. et al. Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J. Exp. Med. 196, 135–140 (2002).

    Article  CAS  Google Scholar 

  6. Fadok, V.A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216 (1992).

    CAS  Google Scholar 

  7. Verhoven, B., Schlegel, R.A. & Williamson, P. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J. Exp. Med. 182, 1597–1601 (1995).

    Article  CAS  Google Scholar 

  8. Martin, S.J. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–1556 (1995).

    Article  CAS  Google Scholar 

  9. Moffatt, O.D., Devitt, A., Bell, E.D., Simmons, D.L. & Gregory, C.D. Macrophage recognition of ICAM-3 on apoptotic leukocytes. J. Immunol. 162, 6800–6810 (1999).

    CAS  Google Scholar 

  10. Anderson, H.A., Englert, R., Gursel, I. & Shacter, E. Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine. Cell. Death Differ. 9, 616–625 (2002).

    Article  CAS  Google Scholar 

  11. Fadok, V.A. et al. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405, 85–90 (2000).

    Article  CAS  Google Scholar 

  12. Devitt, A. et al. Human CD14 mediates recognition and phagocytosis of apoptotic cells. Nature 392, 505–509 (1998).

    Article  CAS  Google Scholar 

  13. Ogden, C.A. et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J. Exp. Med. 194, 781–796 (2001).

    Article  CAS  Google Scholar 

  14. Platt, N., Suzuki, H., Kurihara, Y., Kodama, T. & Gordon, S. Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro. Proc. Natl. Acad. Sci. USA 93, 12456–12460 (1996).

    Article  CAS  Google Scholar 

  15. Savill, J., Dransfield, I., Hogg, N. & Haslett, C. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343, 170–173 (1990).

    Article  CAS  Google Scholar 

  16. Finnemann, S.C. & Rodriguez-Boulan, E. Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for αvβ3 and αvβ5 integrins, and protein kinase C regulates αvβ5 binding and cytoskeletal linkage. J. Exp. Med. 190, 861–874 (1999).

    Article  CAS  Google Scholar 

  17. Albert, M.L., Kim, J.I. & Birge, R.B. αvβ5 integrin recruits the CrkII-Dock 180-rac1 complex for phagocytosis of apoptotic cells. Nat. Cell Biol. 2, 899–905 (2000).

    Article  CAS  Google Scholar 

  18. Esmon, C.T. Regulation of blood coagulation. Biochim. Biophys. Acta. 1477, 349–360 (2000).

    Article  CAS  Google Scholar 

  19. Dahlback, B. Blood coagulation. Lancet 355, 1627–1632 (2000).

    Article  CAS  Google Scholar 

  20. Thomas, R.H. Hypercoagulability syndromes. Arch. Intern. Med. 161, 2433–2439 (2001).

    Article  CAS  Google Scholar 

  21. Merrill, J.T. What causes the antiphospholipid syndrome? Curr. Rheumatol. Rep. 3, 293–300 (2001).

    Article  CAS  Google Scholar 

  22. Rand, J.H. Molecular pathogenesis of the antiphospholipid syndrome. Circ. Res. 90, 29–37 (2002).

    Article  CAS  Google Scholar 

  23. Mevorach, D., Mascarenhas, J.O., Gershov, D. & Elkon, K.B. Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188, 2313–2320 (1998).

    Article  CAS  Google Scholar 

  24. Esmon, C.T. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J. Biol. Chem. 264, 4743–4746 (1989).

    CAS  PubMed  Google Scholar 

  25. Nelsestuen, G.L., Kisiel, W. & Di Scipio, R.G. Interaction of vitamin K dependent proteins with membranes. Biochem. 17, 2134–2138 (1978).

    Article  CAS  Google Scholar 

  26. Walker, F.J. Regulation of activated protein C by a new protein. A possible function for bovine protein S. J. Biol. Chem. 255, 5521–5524 (1980).

    CAS  PubMed  Google Scholar 

  27. May, W.S. &. Cuatrecasas, P. Transferrin receptor: its biological significance. J. Membr. Biol. 88, 205–215 (1985).

    Article  CAS  Google Scholar 

  28. Stenflo, J. Contributions of Gla and EGF-like domains to the function of vitamin K- dependent coagulation factors. Crit. Rev. Eukary. Gene Expr. 9, 59–88 (1999).

    Article  CAS  Google Scholar 

  29. Carreras, L.O. & Forastiero, R.R. Pathogenic role of antiprotein-phospholipid antibodies. Haemostasis 26 (Suppl. 4) 340–357 (1996).

    CAS  PubMed  Google Scholar 

  30. Gal, A. et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat. Genet. 26, 270–271 (2000).

    Article  CAS  Google Scholar 

  31. Krahling, S., Callahan, M.K., Williamson, P. & Schlegel, R.A. Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages. Cell Death Differ. 6, 183–189 (1999).

    Article  CAS  Google Scholar 

  32. Stitt, T.N. et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 80, 661–670 (1995).

    Article  CAS  Google Scholar 

  33. Esmon, C.T. Inflammation. They're not just for clots anymore. Curr. Biol. 5, 743–746 (1995).

    Article  CAS  Google Scholar 

  34. Godowski, P.J. et al. Reevaluation of the roles of protein S and Gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3. Cell 82, 355–368 (1995).

    Article  CAS  Google Scholar 

  35. Ishimoto, Y., Ohashi, K., Mizuno, K. & Nakano, T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. J. Biochem. (Tokyo) 127, 411–717 (2000).

    Article  CAS  Google Scholar 

  36. Hall, M.O. et al. Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6. Exp. Eye Res. 73, 509–520 (2001).

    Article  CAS  Google Scholar 

  37. Lu, Q. et al. Tyro-3 family receptors are essential regulators of mammalian spermatogenesis. Nature 398, 723–728 (1999).

    Article  CAS  Google Scholar 

  38. Balasubramanian, K., Chandra, J. & Schroit, A.J. Immune clearance of phosphatidylserine-expressing cells by phagocytes. The role of β2-glycoprotein I in macrophage recognition. J. Biol. Chem. 272, 31113–31117 (1997).

    CAS  Google Scholar 

  39. Savill, J., Hogg, N., Ren, Y. & Haslett, C. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J. Clin. Invest. 90, 1513–1522 (1992).

    Article  CAS  Google Scholar 

  40. Hanayama, R. et al. Identification of a factor that links apoptotic cells to phagocytes. Nature 417, 182–187 (2002).

    Article  CAS  Google Scholar 

  41. Lee, Y.J. & Shacter, E. Oxidative stress inhibits apoptosis in human lymphoma cells. J. Biol. Chem. 274, 19792–19798 (1999).

    Article  CAS  Google Scholar 

  42. Chang, M.K. et al. Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes mediate macrophage recognition. Proc. Natl. Acad. Sci. USA 96, 6353–6358 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank V. Calvert for supplying human monocytes; E. Shores and G. Tosato for critical reading of the manuscript; and E. Petricoin and Y. M. Zhao for expert technical advice.

Author information

Author notes

  1. Howard A. Anderson and Caroline A. Maylock: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Therapeutic Proteins, Laboratory of Biochemistry, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, 20892, MD, USA

    Howard A. Anderson & Emily Shacter

  2. Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, 20815, MD, USA

    Caroline A. Maylock

  3. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, MD, USA

    Joy A. Williams

  4. Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, MD, USA

    Cloud P. Paweletz

  5. Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, 75390, TX, USA

    Hongjun Shu

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Correspondence to Emily Shacter.

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Supplementary information

Web Fig. 1.

Identification of protein S in the prophagocytic serum fraction. (a) Two-dimensional gel of the purified fraction stained with Coomassie blue. Proteins that were identified by tandem mass spectrometry (MS) are indicated (Pr.S, protein S; Kin., kininogen, BSA, bovine serum albumin). (b) MS spectra of tryptic digests of the Coomassie blue-stained protein S spot. CID represents collision-induced dissociation of peptide analyzed in c. (c) MS/MS profile analysis of ion 908.50 identifying protein S from the unique peptide sequence VYFAGVPR. Peptides from kininogen (YSIVFIAR) and BSA (KVPQVSTPTLVEVSR) were analyzed similarly (data not shown). (PDF 162 kb)

Web Fig. 2.

Immunoanalysis of protein S in enriched phagocytosis fraction. Proteins from serum (25 μg), the purified prophagocytic fraction from serum (5 μg) and human protein S (5 μg) were resolved by SDS-PAGE and stained for protein with Sypro Ruby Protein Gel Stain (Molecular Probes). Immunoblotting of proteins transferred to PVDF membranes was performed using a rabbit anti-protein S IgG primary antibody followed by HRP-conjugated goat anti-rabbit IgG. The three polypeptides seen in the commercial human protein S preparation and the differences in molecular weight observed between bovine and human protein S are most likely attributed to the numerous post-translational modifications that are known to occur to this molecule (for example, proteolysis, glycosylation, γ-glutamyl carboxylation). (PDF 195 kb)

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Anderson, H., Maylock, C., Williams, J. et al. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat Immunol 4, 87–91 (2003). https://doi.org/10.1038/ni871

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