The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

Centre of Biosciences Slovak Academy of Sciences Bratislava Slovak Republic

Department of Biochemistry Faculty of Natural Sciences Comenius University Bratislava Slovak Republic

Institute of Experimental Endocrinology Biomedical Research Center Slovak Academy of Sciences Bratislava Slovakia

Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic Prague Czech Republic

Laboratory of Molecular Immunology Institute of Molecular Biology Slovak Academy of Sciences Bratislava Slovak Republic

Molecular Immunology Unit Institute for Hygiene and Applied Immunology Centre for Pathophysiology Infectiology and Immunology Medical University of Vienna Vienna Austria

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Dano K, Behrendt N, Hoyer‐Hansen G, et al. Plasminogen activation and cancer. Thromb Haemost. 2005;93:676–681. PubMed

Dotti CG, Galvan C, Ledesma MD. Plasmin deficiency in Alzheimer's disease brains: causal or casual?. Neurodegener Dis. 2004;1:205–212. PubMed

Gyetko MR, Aizenberg D, Mayo‐Bond L. Urokinase‐deficient and urokinase receptor‐deficient mice have impaired neutrophil antimicrobial activation in vitro. J Leukoc Biol. 2004;76:648–656. PubMed

Gyetko MR, Sud S, Chensue SW. Urokinase‐deficient mice fail to generate a type 2 immune response following schistosomal antigen challenge. Infect Immun. 2004;72:461–467. PubMed PMC

O'Mullane MJ, Baker MS. Loss of cell viability dramatically elevates cell surface plasminogen binding and activation. Exp Cell Res. 1998;242:153–164. PubMed

Rosenwald M, Koppe U, Keppeler H, et al. Serum‐derived plasminogen is activated by apoptotic cells and promotes their phagocytic clearance. J Immunol. 2012;189:5722–5728. PubMed

Das R, Ganapathy S, Settle M, Plow EF. Plasminogen promotes macrophage phagocytosis in mice. Blood. 2014;124:679–688. PubMed PMC

Leksa V, Godar S, Cebecauer M, et al. The N terminus of mannose 6‐phosphate/insulin‐like growth factor 2 receptor in regulation of fibrinolysis and cell migration. J Biol Chem. 2002;277:40575–40582. PubMed

Leksa V, Pfisterer K, Ondrovicova G, et al. Dissecting mannose 6‐phosphate‐insulin‐like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells. J Biol Chem. 2012;287:22450–22462. PubMed PMC

Ghosh P, Dahms NM, Kornfeld S. Mannose 6‐phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 2003;4:202–212. PubMed

Olson LJ, Castonguay AC, Lasanajak Y, et al. Identification of a fourth mannose 6‐phosphate binding site in the cation‐independent mannose 6‐phosphate receptor. Glycobiology. 2015. PubMed PMC

Hartman MA, Kreiling JL, Byrd JC, MacDonald RG. High‐affinity ligand binding by wild‐type/mutant heteromeric complexes of the mannose 6‐phosphate/insulin‐like growth factor II receptor. FEBS J. 2009;276:1915–1929. PubMed PMC

Olson LJ, Yammani RD, Dahms NM, Kim JJ. Structure of uPAR, plasminogen, and sugar‐binding sites of the 300 kDa mannose 6‐phosphate receptor. Embo J. 2004;23:2019–2028. PubMed PMC

Godár S, Leksa V, Cebecauer M, Hilgert I, Horejsi V, Stockinger H. CD222 (Mannose‐6 phosphate/insulin‐like growth factor II‐receptor) summary and workshop report In: Mason D, Andre P, Bensussan A, Buckley C, Civin C, Clark E, de Haas M, Goyert S, Hadam M, Hart D, Horejsi V, Jones Y, Meuer S, Morrissey J, Schwarz‐Albiez R, Shaw S, Simmons D, Turni L, Uguccioni M, van der Schoot E, Vivier E, and Zola H, eds. Leukocyte Typing VII. Oxford, UK: Oxford University Press; 2002:482–485.

Pfisterer K, Forster F, Paster W, et al. The late endosomal transporter CD222 directs the spatial distribution and activity of Lck. J Immunol. 2014;193:2718–2732. PubMed

Leksa V, Loewe R, Binder B, et al. Soluble M6P/IGF2R released by TACE controls angiogenesis via blocking plasminogen activation. Circ Res. 2011;108:676–685. PubMed

Leksa V, Godar S, Schiller HB, et al. TGF‐{beta}‐induced apoptosis in endothelial cells mediated by M6P/IGFII‐R and mini‐plasminogen. J Cell Sci. 2005;118:4577–4586. PubMed

Machacek C, Supper V, Leksa V, et al. Folate receptor beta regulates integrin CD11b/CD18 adhesion of a macrophage subset to collagen. J Immunol. 2016;197:2229–2238. PubMed

Ohradanova‐Repic A, Machacek C, Fischer MB, Stockinger H. Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel. Clin Transl Immunol. 2016;5:e55. PubMed PMC

Cossarizza A, Chang HD, Radbruch A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 2017;47:1584. PubMed PMC

Schiller HB, Szekeres A, Binder BR, Stockinger H, Leksa V. Mannose 6‐phosphate/insulin‐like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase‐type plasminogen activator receptor. Mol Biol Cell. 2009;20:745–756. PubMed PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real‐time quantitative PCR and the 2(–Delta Delta C(T)) Method. Methods. 2001;25:402–408. PubMed

Ohradanova‐Repic A, Machacek C, Charvet C, et al. Extracellular purine metabolism is the switchboard of immunosuppressive macrophages and a novel target to treat diseases with macrophage imbalances. Front Immunol. 2018;9:852. PubMed PMC

Hattey E, Wojta J, Binder BR. Monoclonal antibodies against four different epitopes of plasminogen exhibit different effects on plasminogen activation kinetics. Thromb Haemost. 1987;58.

Baqui AA, Meiller TF, Turng BF, Kelley JI. Functional changes in THP‐1 human monocytic cells after stimulation with lipopolysaccharide of oral microorganisms and granulocyte macrophage colony stimulating factor. Immunopharmacol Immunotoxicol. 1998;20:493–518. PubMed

Elliott MR, Koster KM, Murphy PS. Efferocytosis signaling in the regulation of macrophage inflammatory responses. J Immunol. 2017;198:1387–1394. PubMed PMC

Birge RB, Boeltz S, Kumar S, et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 2016;23:962–978. PubMed PMC

Gordon S, Pluddemann A. Macrophage clearance of apoptotic cells: a critical assessment. Front Immunol. 2018;9. PubMed PMC

Schwende H, Fitzke E, Ambs P, Dieter P. Differences in the state of differentiation of THP‐1 cells induced by phorbol ester and 1,25‐dihydroxyvitamin D3. J Leukoc Biol. 1996;59:555–561. PubMed

Hall SE, Savill JS, Henson PM, Haslett C. Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose‐specific lectin. J Immunol. 1994;153:3218–3227. PubMed

Ouyang L, Shi Z, Zhao S, et al. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 2012;45:487–498. PubMed PMC

Mattson MP. Neuronal life‐and‐death signaling, apoptosis, and neurodegenerative disorders. Antioxid Redox Signal. 2006;8:1997–2006. PubMed

Ravichandran KS. Beginnings of a good apoptotic meal: the find‐me and eat‐me signaling pathways. Immunity. 2011;35:445–455. PubMed PMC

Kobayashi N, Karisola P, Pena‐Cruz V, et al. TIM‐1 and TIM‐4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity. 2007;27:927–940. PubMed PMC

DeKruyff RH, Bu X, Ballesteros A, et al. T cell/transmembrane, Ig, and mucin‐3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells. J Immunol. 2010;184:1918–1930. PubMed PMC

Park D, Tosello‐Trampont AC, Elliott MR, et al. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature. 2007;450:430–434. PubMed

Voss OH, Tian LJ, Murakami Y, Coligan JE, Krzewski K. Emerging role of CD300 receptors in regulating myeloid cell efferocytosis. Mol Cell Oncol. 2015;2. PubMed PMC

Park SY, Jung MY, Lee SJ, et al. Stabilin‐1 mediates phosphatidylserine‐dependent clearance of cell corpses in alternatively activated macrophages. J Cell Sci. 2009;122:3365–3373. PubMed

Park SY, Jung MY, Kim HJ, et al. Rapid cell corpse clearance by stabilin‐2, a membrane phosphatidylserine receptor. Cell Death Differ. 2008;15:192–201. PubMed

Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature. 2002;417:182–187. PubMed

Jun JI, Kim KH, Lau LF. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing. Nat Commun. 2015;6. PubMed PMC

Stepanek O, Brdicka T, Angelisova P, et al. Interaction of late apoptotic and necrotic cells with vitronectin. PLoS One. 2011;6:e19243. PubMed PMC

Seitz HM, Camenisch TD, Lemke G, Earp HS, Matsushima GK. Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells. J Immunol. 2007;178:5635–5642. PubMed

D'Mello V, Singh S, Wu Y, Birge RB. The urokinase plasminogen activator receptor promotes efferocytosis of apoptotic cells. J Biol Chem. 2009;284:17030–17038. PubMed PMC

Greenberg ME, Sun M, Zhang R, Febbraio M, Silverstein R, Hazen SL. Oxidized phosphatidylserine‐CD36 interactions play an essential role in macrophage‐dependent phagocytosis of apoptotic cells. J Exp Med. 2006;203:2613–2625. PubMed PMC

Ogden CA, deCathehneau A, Hoffmann PR, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med. 2001;194:781–795. PubMed PMC

Gregory CD, Devitt A, Moffatt O. Roles of ICAM‐3 and CD14 in the recognition and phagocytosis of apoptotic cells by macrophages. Biochem Soc Trans. 1998;26:644–649. PubMed

Hawkes C, Kabogo D, Amritraj A, Kar S. Up‐regulation of cation‐independent mannose 6‐phosphate receptor and endosomal‐lysosomal markers in surviving neurons after 192‐IgG‐saporin administrations into the adult rat brain. Am J Pathol. 2006;169:1140–1154. PubMed PMC

Nykjaer A, Christensen EI, Vorum H, et al. Mannose 6‐phosphate/insulin‐like growth factor‐II receptor targets the urokinase receptor to lysosomes via a novel binding interaction. J Cell Biol. 1998;141:815–828. PubMed PMC

Amritraj A, Posse de Chaves EI, Hawkes C, Macdonald RG, Kar S. Single‐transmembrane domain IGF‐II/M6P receptor: potential interaction with G protein and its association with cholesterol‐rich membrane domains. Endocrinology. 2012;153:4784–4798. PubMed

Guzik K, Bzowska M, Smagur J, et al. A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages. Cell Death Differ. 2007;14:171–182. PubMed

Martin M, Leffler J, Blom AM. Annexin A2 and A5 serve as new ligands for C1q on apoptotic cells. J Biol Chem. 2012;287:33733–33744. PubMed PMC

Herren T, Swaisgood C, Plow EF. Regulation of plasminogen receptors. Front Biosci. 2003;8:d1–8. PubMed

Miles LA, Hawley SB, Baik N, Andronicos NM, Castellino FJ, Parmer RJ. Plasminogen receptors: the sine qua non of cell surface plasminogen activation. Front Biosci. 2005;10:1754–1762. PubMed

Stillfried GE, Saunders DN, Ranson M. Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity. Breast Cancer Res. 2007;9:R14. PubMed PMC

Andronicos NM, Chen EI, Baik N, et al. Proteomics‐based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg‐RKT, a major regulator of cell surface plasminogen activation. Blood;115:1319–1330. PubMed PMC

Sugimoto MA, Ribeiro ALC, Costa BRC, et al. Plasmin and plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via annexin A1. Blood. 2017;129:2896–2907. PubMed PMC

Miles LA, Lighvani S, Baik N, et al. New insights into the role of Plg‐RKT in macrophage recruitment. Int Rev Cell Mol Biol. 2014;309:259–302. PubMed PMC

Wu XW, Molinaro C, Johnson N, Casiano CA. Secondary necrosis is a source of proteolytically modified forms of specific intracellular autoantigens ‐ Implications for systemic autoimmunity. Arthritis Rheum. 2001;44:2642–2652. PubMed

Zwirzitz A, Reiter M, Skrabana R, et al. Lactoferrin is a natural inhibitor of plasminogen activation. J Biol Chem. 2018;293:8600–8613. PubMed PMC

Lin SX, Mallet WG, Huang AY, Maxfield FR. Endocytosed cation‐independent mannose 6‐phosphate receptor traffics via the endocytic recycling compartment en route to the trans‐Golgi network and a subpopulation of late endosomes. Mol Biol Cell. 2004;15:721–733. PubMed PMC

MacDonald RG, Pfeffer SR, Coussens L, et al. A single receptor binds both insulin‐like growth factor II and mannose‐6‐phosphate. Science. 1988;239:1134–1137. PubMed

Tong PY, Tollefsen SE, Kornfeld S. The cation‐independent mannose 6‐phosphate receptor binds insulin‐like growth factor II. J Biol Chem. 1988;263:2585–2588. PubMed

Braulke T, Causin C, Waheed A, et al. Mannose 6‐phosphate/insulin‐like growth factor II receptor: distinct binding sites for mannose 6‐phosphate and insulin‐like growth factor II. Biochem Biophys Res Commun. 1988;150:1287–1293. PubMed

Vreys V, Delande N, Zhang Z, et al. Cellular uptake of mammalian heparanase precursor involves low density lipoprotein receptor‐related proteins, mannose 6‐phosphate receptors, and heparan sulfate proteoglycans. J Biol Chem. 2005;280:33141–33148. PubMed

Wood RJ, Hulett MD. Cell surface‐expressed cation‐independent mannose 6‐phosphate receptor (CD222) binds enzymatically active heparanase independently of mannose 6‐phosphate to promote extracellular matrix degradation. J Biol Chem. 2008;283:4165–4176. PubMed

Blanchard F, Raher S, Duplomb L, et al. The mannose 6‐phosphate/insulin‐like growth factor II receptor is a nanomolar affinity receptor for glycosylated human leukemia inhibitory factor. J Biol Chem. 1998;273:20886–20893. PubMed

Lee SJ, Nathans D. Proliferin secreted by cultured cells binds to mannose 6‐phosphate receptors. J Biol Chem. 1988;263:3521–3527. PubMed

Volpert O, Jackson D, Bouck N, Linzer DI. The insulin‐like growth factor II/mannose 6‐phosphate receptor is required for proliferin‐induced angiogenesis. Endocrinology. 1996;137:3871–3876. PubMed

Kovacina KS, Steele‐Perkins G, Purchio AF, et al. Interactions of recombinant and platelet transforming growth factor‐beta 1 precursor with the insulin‐like growth factor II/mannose 6‐phosphate receptor. Biochem Biophys Res Commun. 1989;160:393–403. PubMed

Godar S, Horejsi V, Weidle UH, Binder BR, Hansmann C, Stockinger H. M6P/IGFII‐receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor‐beta1. Eur J Immunol. 1999;29:1004–1013. PubMed

Braulke T, Tippmer S, Neher E, von Figura K. Regulation of the mannose 6‐phosphate/IGF II receptor expression at the cell surface by mannose 6‐phosphate, insulin like growth factors and epidermal growth factor. EMBO J. 1989;8:681–686. PubMed PMC

van Meel E, Klumperman J. TGN exit of the cation‐independent mannose 6‐phosphate receptor does not require acid hydrolase binding. Cell Logist. 2014;4:e954441. PubMed PMC