Skip to main content
SpringerLink
Log in

Activated human neutrophils rapidly release the chemotactically active D2D3 form of the urokinase-type plasminogen activator receptor (uPAR/CD87)

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

The urokinase-type plasminogen activator receptor (uPAR/CD87) exists both in cell-bound and soluble forms. Neutrophils contain extensive intracellular pools of uPAR that are translocated to the plasma membrane upon activation. In the present study, we investigated the ability of human neutrophils to shed uPAR from cell surface following activation and addressed the possible involvement of the released receptor in the inflammatory response. We first observed that the spontaneous release of suPAR by resting neutrophils was strongly and rapidly (within minutes) enhanced by calcium ionophore ionomycin and to a lesser extent when cells were primed with TNF-α and then stimulated with fMLP or IL-8. We demonstrated that suPAR is produced by resting and activated neutrophils predominantly as a truncated form devoid of N-terminal D1 domain (D2D3 form) that lacks GPI anchor. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from activated neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. We conclude that activated neutrophils release the chemotactically active D2D3 form of suPAR that acts as a ligand of FPRL1. Interestingly, we present evidence that GPI-specific phospholipase D (GPI-PLD) that has previously been shown to shed uPAR in cancer cells is not involved in suPAR release from human neutrophils. We suggest that production of the chemotactically active D2D3 form of suPAR by activated human neutrophils in vivo could contribute to the recruitment of monocytes and other formyl peptide receptors-expressing cells to the sites of acute inflammation where neutrophil accumulation and activation occur.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

uPAR:

Urokinase-type plasminogen activator receptor

suPAR:

Soluble uPAR

fMLP:

N-formyl-methionyl-leucyl-phenylalanine

TNF-α:

Tumor necrosis factor-α

IL-8:

Interleukin-8

FPRL1:

Formyl peptide receptor-like-1

TPEN:

N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine

DTPA:

Diethylenetriaminepentaacetic acid

TAPI-1:

TNF-α protease inhibitor-1

GPI-PLD:

GPI-specific phospholipase D

References

  1. Ragno P (2006) The urokinase receptor: a ligand or a receptor? Story of a sociable molecule. Cell Mol Life Sci 63:1028–1037. doi:10.1007/s00018-005-5428-1

    Article  PubMed  CAS  Google Scholar 

  2. Blasi F, Carmeliet P (2002) uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol 3:932–943. doi:10.1038/nrm977

    Article  PubMed  CAS  Google Scholar 

  3. Sidenius N, Blasi F (2003) The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metastasis Rev 22:205–222. doi:10.1023/A:1023099415940

    Article  PubMed  CAS  Google Scholar 

  4. Llinas P, Le Du MH, Gardsvoll H et al (2005) Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide. EMBO J 24:1655–1663. doi:10.1038/sj.emboj.7600635

    Article  PubMed  CAS  Google Scholar 

  5. Behrendt N, Ploug M, Patthy L et al (1991) The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 266:7842–7847

    PubMed  CAS  Google Scholar 

  6. Ploug M, Gardsvoll H, Jorgensen TJ et al (2002) Structural analysis of the interaction between urokinase-type plasminogen activator and its receptor: a potential target for anti-invasive cancer therapy. Biochem Soc Trans 30:177–183. doi:10.1042/BST0300177

    Article  PubMed  CAS  Google Scholar 

  7. Stepanova VV, Tkachuk VA (2002) Urokinase as a multidomain protein and polyfunctional cell regulator. Biochemistry (Mosc) 67:109–118. doi:10.1023/A:1013912500373

    Article  CAS  Google Scholar 

  8. Wei Y, Waltz DA, Rao N et al (1994) Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem 269:32380–32388

    PubMed  CAS  Google Scholar 

  9. Kugler MC, Wei Y, Chapman HA (2003) Urokinase receptor and integrin interactions. Curr Pharm Des 9:1565–1574. doi:10.2174/1381612033454658

    Article  PubMed  CAS  Google Scholar 

  10. Chapman HA, Wei Y (2001) Protease crosstalk with integrins: the urokinase receptor paradigm. Thromb Haemost 86:124–129

    PubMed  CAS  Google Scholar 

  11. Beaufort N, Leduc D, Rousselle J-C et al (2004) Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin G. J Immunol 172:540–549

    PubMed  CAS  Google Scholar 

  12. Hoyer-Hansen G, Ronne E, Solberg H et al (1992) Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem 267:18224–18229

    PubMed  CAS  Google Scholar 

  13. Andolfo A, English WR, Resnati M et al (2002) Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor. Thromb Haemost 88:298–306

    PubMed  CAS  Google Scholar 

  14. Beaufort N, Leduc D, Rousselle JC et al (2004) Plasmin cleaves the juxtamembrane domain and releases truncated species of the urokinase receptor (CD87) from human bronchial epithelial cells. FEBS Lett 574:89–94. doi:10.1016/j.febslet.2004.08.009

    Article  PubMed  CAS  Google Scholar 

  15. Montuori N, Visconte V, Rossi G et al (2005) Soluble and cleaved forms of the urokinase-receptor: degradation products or active molecules? Thromb Haemost 93:192–198

    PubMed  CAS  Google Scholar 

  16. Wilhelm OG, Wilhelm S, Escott GM et al (1999) Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J Cell Physiol 180:225–235. doi:10.1002/(SICI)1097-4652(199908)180:2<225::AID-JCP10>3.0.CO;2-2

    Article  PubMed  CAS  Google Scholar 

  17. Pyke C, Eriksen J, Solberg H et al (1993) An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator. FEBS Lett 326:69–74. doi:10.1016/0014-5793(93)81763-P

    Article  PubMed  CAS  Google Scholar 

  18. Sidenius N, Sier CFM, Blasi F (2000) Shedding and cleavage of the urokinase receptor (uPAR): identification and characterisation of uPAR fragments in vitro and in vivo. FEBS Lett 475:52–56. doi:10.1016/S0014-5793(00)01624-0

    Article  PubMed  CAS  Google Scholar 

  19. Slot O, Brunner N, Locht H et al (1999) Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis. Ann Rheum Dis 58:488–492

    Article  PubMed  CAS  Google Scholar 

  20. Sier CF, Stephens R, Bizik J et al (1998) The level of urokinase-type plasminogen activator receptor is increased in serum of ovarian cancer patients. Cancer Res 58:1843–1849

    PubMed  CAS  Google Scholar 

  21. Mustjoki S, Sidenius N, Sier CF et al (2000) Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy. Cancer Res 60:7126–7132

    PubMed  CAS  Google Scholar 

  22. Resnati M, Pallavicini I, Wang JM et al (2002) The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci USA 99:1359–1364. doi:10.1073/pnas.022652999

    Article  PubMed  CAS  Google Scholar 

  23. Selleri C, Montuori N, Ricci P et al (2005) Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood 105:2198–2205. doi:10.1182/blood-2004-06-2424

    Article  PubMed  CAS  Google Scholar 

  24. de Paulis A, Montuori N, Prevete N et al (2004) Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2. J Immunol 173:5739–5748

    PubMed  Google Scholar 

  25. Shen H, Cheng T, Olszak I et al (2001) CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells. J Immunol 166:5027–5033

    PubMed  CAS  Google Scholar 

  26. Furlan F, Orlando S, Laudanna C et al (2004) The soluble D2D3(88-274) fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion. J Cell Sci 117:2909–2916. doi:10.1242/jcs.01149

    Article  PubMed  CAS  Google Scholar 

  27. Pliyev BK (2008) Chemotactically active proteins of neutrophils. Biochemistry (Mosc) 73:970–984

    Article  CAS  Google Scholar 

  28. Scapini P, Lapinet-Vera JA, Gasperini S et al (2000) The neutrophil as a cellular source of chemokines. Immunol Rev 177:195–203. doi:10.1034/j.1600-065X.2000.17706.x

    Article  PubMed  CAS  Google Scholar 

  29. Chertov O, Ueda H, Xu LL et al (1997) Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. J Exp Med 186:739–747. doi:10.1084/jem.186.5.739

    Article  PubMed  CAS  Google Scholar 

  30. De Yang , Chen Q, Schmidt AP et al (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192:1069–1074. doi:10.1084/jem.192.7.1069

    Article  PubMed  CAS  Google Scholar 

  31. Chertov O, Michiel DF, Xu L et al (1996) Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 271:2935–2940. doi:10.1074/jbc.271.6.2935

    Article  PubMed  CAS  Google Scholar 

  32. Cassatella MA (1999) Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol 73:369–509. doi:10.1016/S0065-2776(08)60791-9

    Article  PubMed  CAS  Google Scholar 

  33. Plesner T, Ploug M, Ellis V et al (1994) The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils. Blood 83:808–815

    PubMed  CAS  Google Scholar 

  34. Pedersen TL, Plesner T, Horn T et al (2000) Subcellular distribution of urokinase and urokinase receptor in human neutrophils determined by immunoelectron microscopy. Ultrastruct Pathol 24:175–182. doi:10.1080/01913120050132912

    Article  PubMed  CAS  Google Scholar 

  35. Owen CA, Campbell MA, Sannes PL et al (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol 131:775–789. doi:10.1083/jcb.131.3.775

    Article  PubMed  CAS  Google Scholar 

  36. Heiple JM, Ossowski L (1986) Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis. J Exp Med 164:826–840. doi:10.1084/jem.164.3.826

    Article  PubMed  CAS  Google Scholar 

  37. Böyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest 97:77–89

    Google Scholar 

  38. Bordier C (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256:1604–1607

    PubMed  CAS  Google Scholar 

  39. Cham BP, Gerrard JM, Bainton DF (1994) Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation. Am J Pathol 144:1369–1380

    PubMed  CAS  Google Scholar 

  40. Lacal P, Pulido R, Sanchez-Madrid F et al (1988) Intracellular location of T200 and Mo1 glycoproteins in human neutrophils. J Biol Chem 263:9946–9951

    PubMed  CAS  Google Scholar 

  41. Sengelov H, Kjeldsen L, Borregaard N (1993) Control of exocytosis in early neutrophil activation. J Immunol 150:1535–1543

    PubMed  CAS  Google Scholar 

  42. Dahlgren C, Johansson A, Lundqvist H et al (1992) Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin). Biochim Biophys Acta 1137:182–188. doi:10.1016/0167-4889(92)90200-U

    Article  PubMed  CAS  Google Scholar 

  43. Bajaj MS, Kew RR, Webster RO et al (1992) Priming of human neutrophil functions by tumor necrosis factor: enhancement of superoxide anion generation, degranulation, and chemotaxis to chemoattractants C5a and F-Met-Leu-Phe. Inflammation 16:241–250. doi:10.1007/BF00918813

    Article  PubMed  CAS  Google Scholar 

  44. Behrendt N, Ronne E, Ploug M et al (1990) The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J Biol Chem 265:6453–6460

    PubMed  CAS  Google Scholar 

  45. Bae YS, Lee HY, Jo EJ et al (2004) Identification of peptides that antagonize formyl peptide receptor-like 1-mediated signaling. J Immunol 173:607–614

    PubMed  CAS  Google Scholar 

  46. Matityahu E, Feniger-Barish R, Meshel T et al (2002) Intracellular trafficking of human CXCR1 and CXCR2: regulation by receptor domains and actin-related kinases. Eur J Immunol 32:3525–3535. doi:10.1002/1521-4141(200212)32:12<3525::AID-IMMU3525>3.0.CO;2-1

    Article  PubMed  CAS  Google Scholar 

  47. McFarlane SM, Pashmi G, Connell MC et al (2002) Differential activation of nuclear factor-kappaB by tumour necrosis factor receptor subtypes. TNFR1 predominates whereas TNFR2 activates transcription poorly. FEBS Lett 515:119–126. doi:10.1016/S0014-5793(02)02450-X

    Article  PubMed  CAS  Google Scholar 

  48. Todd I, Radford PM, Draper-Morgan KA et al (2004) Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour. Immunology 113:65–79. doi:10.1111/j.1365-2567.2004.01942.x

    Article  PubMed  CAS  Google Scholar 

  49. Mann KJ, Hepworth MR, Raikwar NS et al (2004) Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism. Biochem J 378:641–648. doi:10.1042/BJ20031326

    Article  PubMed  CAS  Google Scholar 

  50. Metz CN, Brunner G, Choi-Muira NH et al (1994) Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D. EMBO J 13:1741–1751

    PubMed  CAS  Google Scholar 

  51. Arribas J, Borroto A (2002) Protein ectodomain shedding. Chem Rev 102:4627–4638. doi:10.1021/cr010202t

    Article  PubMed  CAS  Google Scholar 

  52. Gasser O, Hess C, Miot S et al (2003) Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils. Exp Cell Res 285:243–257. doi:10.1016/S0014-4827(03)00055-7

    Article  PubMed  CAS  Google Scholar 

  53. Vittorelli ML (2003) Shed membrane vesicles and clustering of membrane-bound proteolytic enzymes. Curr Top Dev Biol 54:411–432. doi:10.1016/S0070-2153(03)54017-0

    Article  PubMed  CAS  Google Scholar 

  54. Doherty DE, Downey GP, Worthen GS et al (1988) Monocyte retention and migration in pulmonary inflammation. Requirement for neutrophils. Lab Invest 59:200–213

    PubMed  CAS  Google Scholar 

  55. Page AR, Good RA (1958) A clinical and experimental study of the function of neutrophils in the inflammatory response. Am J Pathol 34:645–669

    PubMed  CAS  Google Scholar 

  56. Taub DD, Anver M, Oppenheim JJ et al (1996) T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 97:1931–1941. doi:10.1172/JCI118625

    Article  PubMed  CAS  Google Scholar 

  57. Zhou J, Stohlman SA, Hinton DR et al (2003) Neutrophils promote mononuclear cell infiltration during viral-induced encephalitis. J Immunol 170:3331–3336

    PubMed  CAS  Google Scholar 

  58. Chavakis T, Willuweit AK, Lupu F et al (2001) Release of soluble urokinase receptor from vascular cells. Thromb Haemost 86:686–693

    PubMed  CAS  Google Scholar 

  59. Rieu P, Porteu F, Bessou G et al (1992) Human neutrophils release their major membrane sialoprotein, leukosialin (CD43), during cell activation. Eur J Immunol 22:3021–3026. doi:10.1002/eji.1830221138

    Article  PubMed  CAS  Google Scholar 

  60. Sun R, Iribarren P, Zhang N et al (2004) Identification of neutrophil granule protein cathepsin G as a novel chemotactic agonist for the G protein-coupled formyl peptide receptor. J Immunol 173:428–436

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. J. M. Wang (National Cancer Institute at Frederick, Frederick, USA) and Dr. P. M. Murphy (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, USA) for providing FPRL1/293 cells. We also wish to thank Dr. V. Stepanova (University of Pennsylvania, Philadelphia, USA) for sharing recombinant D1 and D2D3 fragments of human suPAR and Dr. T. Arefyeva (Institute of Experimental Cardiology, Cardiology Research Center, Moscow, Russia) for her help in flow cytometry analysis. This work was supported by Welcome Trust grant No. 75154 and CRDF grant No. RB1-2454-MO-02.

Author information

Authors and Affiliations

  1. Department of Biological and Medical Chemistry, Faculty of Fundamental Medicine, Moscow State University, Lomonosovsky pr., 31-5, Moscow, 119192, Russia

    Boris K. Pliyev

Authors
  1. Boris K. Pliyev
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Boris K. Pliyev.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pliyev, B.K. Activated human neutrophils rapidly release the chemotactically active D2D3 form of the urokinase-type plasminogen activator receptor (uPAR/CD87). Mol Cell Biochem 321, 111–122 (2009). https://doi.org/10.1007/s11010-008-9925-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-008-9925-z

Keywords

Search

Navigation