Tissue-type plasminogen activator selectively inhibits multiple toll-like receptors in CSF-1-differentiated macrophages

Autoři: Lipsa Das aff001;  Pardis Azmoon aff001;  Michael A. Banki aff001;  Elisabetta Mantuano aff001;  Steven L. Gonias aff001
Působiště autorů: Department of Pathology, University of California San Diego, La Jolla, California, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224738


Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1β, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.

Klíčová slova:

Cell differentiation – Cytokines – Flow cytometry – Gene expression – Immune receptor signaling – Macrophages – Phosphorylation – Toll-like receptors


1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124(4):783–801. Epub 2006/02/25. doi: 10.1016/j.cell.2006.02.015 16497588.

2. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140(6):805–20. Epub 2010/03/23. doi: 10.1016/j.cell.2010.01.022 20303872.

3. O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like receptors—redefining innate immunity. Nat Rev Immunol. 2013;13(6):453–60. Epub 2013/05/18. doi: 10.1038/nri3446 23681101.

4. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635–700. Epub 2002/06/05. doi: 10.1146/annurev.biochem.71.110601.135414 12045108; PubMed Central PMCID: PMC2569852.

5. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162(7):3749–52. Epub 1999/04/14. 10201887.

6. Park BS, Lee JO. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med. 2013;45:e66. Epub 2013/12/07. doi: 10.1038/emm.2013.97 24310172; PubMed Central PMCID: PMC3880462.

7. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med. 1999;189(11):1777–82. Epub 1999/06/08. doi: 10.1084/jem.189.11.1777 10359581; PubMed Central PMCID: PMC2193086.

8. Hailman E, Lichenstein HS, Wurfel MM, Miller DS, Johnson DA, Kelley M, et al. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med. 1994;179(1):269–77. Epub 1994/01/01. doi: 10.1084/jem.179.1.269 7505800; PubMed Central PMCID: PMC2191344.

9. Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost. 2005;93(4):647–54. Epub 2005/04/21. doi: 10.1160/TH04-12-0842 15841308.

10. Albers GW, Bates VE, Clark WM, Bell R, Verro P, Hamilton SA. Intravenous tissue-type plasminogen activator for treatment of acute stroke: the Standard Treatment with Alteplase to Reverse Stroke (STARS) study. JAMA. 2000;283(9):1145–50. Epub 2000/03/07. doi: 10.1001/jama.283.9.1145 10703776.

11. Mantuano E, Brifault C, Lam MS, Azmoon P, Gilder AS, Gonias SL. LDL receptor-related protein-1 regulates NFkappaB and microRNA-155 in macrophages to control the inflammatory response. Proc Natl Acad Sci U S A. 2016;113(5):1369–74. Epub 2016/01/21. doi: 10.1073/pnas.1515480113 26787872; PubMed Central PMCID: PMC4747752.

12. Mantuano E, Azmoon P, Brifault C, Banki MA, Gilder AS, Campana WM, et al. Tissue-type plasminogen activator regulates macrophage activation and innate immunity. Blood. 2017;130(11):1364–74. Epub 2017/07/08. doi: 10.1182/blood-2017-04-780205 28684538; PubMed Central PMCID: PMC5600142.

13. Zalfa C, Azmoon P, Mantuano E, Gonias SL. Tissue-type plasminogen activator neutralizes LPS but not protease-activated receptor-mediated inflammatory responses to plasmin. J Leukoc Biol. 2019;105(4):729–40. Epub 2019/01/29. doi: 10.1002/JLB.3A0818-329RRR 30690783; PubMed Central PMCID: PMC6430673.

14. Gonias SL, Campana WM. LDL receptor-related protein-1: a regulator of inflammation in atherosclerosis, cancer, and injury to the nervous system. Am J Pathol. 2014;184(1):18–27. Epub 2013/10/17. doi: 10.1016/j.ajpath.2013.08.029 24128688; PubMed Central PMCID: PMC3873482.

15. Bu G, Williams S, Strickland DK, Schwartz AL. Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1992;89(16):7427–31. Epub 1992/08/15. doi: 10.1073/pnas.89.16.7427 1502154; PubMed Central PMCID: PMC49723.

16. Bu G, Maksymovitch EA, Schwartz AL. Receptor-mediated endocytosis of tissue-type plasminogen activator by low density lipoprotein receptor-related protein on human hepatoma HepG2 cells. J Biol Chem. 1993;268(17):13002–9. Epub 1993/06/15. 8389767.

17. Bacskai BJ, Xia MQ, Strickland DK, Rebeck GW, Hyman BT. The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-D-aspartate receptors. Proc Natl Acad Sci USA. 2000;97(21):11551–6. Epub 2000/10/04. doi: 10.1073/pnas.200238297 11016955; PubMed Central PMCID: PMC17238.

18. Mantuano E, Lam MS, Gonias SL. LRP1 assembles unique co-receptor systems to initiate cell signaling in response to tissue-type plasminogen activator and myelin-associated glycoprotein. J Biol Chem. 2013;288(47):34009–18. Epub 2013/10/17. doi: 10.1074/jbc.M113.509133 24129569; PubMed Central PMCID: PMC3837140.

19. Mantuano E, Lam MS, Shibayama M, Campana WM, Gonias SL. The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration. J Cell Sci. 2015;128(18):3478–88. Epub 2015/08/15. doi: 10.1242/jcs.173765 26272917; PubMed Central PMCID: PMC4594737.

20. Sheng Z, Prorok M, Brown BE, Castellino FJ. N-methyl-D-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein. Neuropharmacology. 2008;55(2):204–14. Epub 2008/07/08. doi: 10.1016/j.neuropharm.2008.05.016 18602124; PubMed Central PMCID: PMC2610414.

21. Imber MJ, Pizzo SV. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem. 1981;256(15):8134–9. Epub 1981/08/10. 6167573.

22. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem. 1999;274(25):17406–9. Epub 1999/06/11. doi: 10.1074/jbc.274.25.17406 10364168.

23. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity. 1999;11(4):443–51. Epub 1999/11/05. doi: 10.1016/s1074-7613(00)80119-3 10549626.

24. Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M, et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34(1):251–62. Epub 2004/02/19. doi: 10.1002/eji.200324032 14971051.

25. Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol. 2003;4(7):702–7. Epub 2003/06/11. doi: 10.1038/ni945 12796777.

26. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278(11):8869–72. Epub 2003/01/16. doi: 10.1074/jbc.C200651200 12527755.

27. Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008;Chapter 14:Unit 14 1. Epub 2008/11/20. doi: 10.1002/0471142735.im1401s83 19016445; PubMed Central PMCID: PMC2834554.

28. Assouvie A, Daley-Bauer LP, Rousselet G. Growing Murine Bone Marrow-Derived Macrophages. Methods Mol Biol. 2018;1784:29–33. Epub 2018/05/16. doi: 10.1007/978-1-4939-7837-3_3 29761385.

29. Schneewind O, Missiakas D. Lipoteichoic acids, phosphate-containing polymers in the envelope of gram-positive bacteria. J Bacteriol. 2014;196(6):1133–42. Epub 2014/01/15. doi: 10.1128/JB.01155-13 24415723; PubMed Central PMCID: PMC3957714.

30. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol. 2000;18:621–63. Epub 2000/06/03. doi: 10.1146/annurev.immunol.18.1.621 10837071.

31. Jacobs MD, Harrison SC. Structure of the IkappaBalpha/NF-kappaB complex. Cell. 1998;95(6);749–58. doi: 10.1016/s0092-8674(00)81698-0 9865693.

32. Sun S-C, Ganchi PA, Ballard DW, Greene WC. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science, 1993; 259(5103):1912–5. doi: 10.1126/science.8096091 8096091.

33. Yasuda K, Yu P, Kirschning CJ, Schlatter B, Schmitz F, Heit A, et al. Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways. J Immunol. 2005;174(10):6129–36. Epub 2005/05/10. doi: 10.4049/jimmunol.174.10.6129 15879108.

34. Strober W, Murray PJ, Kitani A, Watanabe T. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006;6(1):9–20. Epub 2006/02/24. doi: 10.1038/nri1747 16493424.

35. Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity. 2008;28(2):246–57. Epub 2008/02/12. doi: 10.1016/j.immuni.2007.12.012 18261938.

36. Hedl M, Li J, Cho JH, Abraham C. Chronic stimulation of Nod2 mediates tolerance to bacterial products. Proc Natl Acad Sci U S A. 2007;104(49):19440–5. Epub 2007/11/23. doi: 10.1073/pnas.0706097104 18032608; PubMed Central PMCID: PMC2148308.

37. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411(6837):603–6. Epub 2001/06/01. doi: 10.1038/35079114 11385577.

38. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature. 2001;411(6837):599–603. Epub 2001/06/01. doi: 10.1038/35079107 11385576.

39. McIlhinney RA, Le Bourdelles B, Molnar E, Tricaud N, Streit P, Whiting PJ. Assembly intracellular targeting and cell surface expression of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A in transfected cells. Neuropharmacology. 1998;37(10–11):1355–67. Epub 1998/12/16. doi: 10.1016/s0028-3908(98)00121-x 9849671.

40. Fukaya M, Kato A, Lovett C, Tonegawa S, Watanabe M. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice. Proc Natl Acad Sci U S A. 2003;100(8):4855–60. Epub 2003/04/05. doi: 10.1073/pnas.0830996100 12676993; PubMed Central PMCID: PMC153645.

41. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol. 2002;169(1):10–4. Epub 2002/06/22. doi: 10.4049/jimmunol.169.1.10 12077222.

42. Latz E, Visintin A, Espevik T, Golenbock DT. Mechanisms of TLR9 activation. J Endotoxin Res. 2004;10(6):406–12. Epub 2004/12/14. doi: 10.1179/096805104225006525 15588423.

43. Shi Y, Mantuano E, Inoue G, Campana WM, Gonias SL. Ligand binding to LRP1 transactivates Trk receptors by a Src family kinase-dependent pathway. Sci Signal. 2009;2(68):ra18. Epub 2009/04/30. doi: 10.1126/scisignal.2000188 19401592; PubMed Central PMCID: PMC2696635.

44. Keck S, Freudenberg M, Huber M. Activation of murine macrophages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and promoted by SHIP1. J Immunol. 2010;184(10):5809–18. Epub 2010/04/14. doi: 10.4049/jimmunol.0901423 20385881.

45. Pierik M, Joossens S, Van Steen K, Van Schuerbeek N, Vlietinck R, Rutgeerts P, et al. Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases. Inflamm Bowel Dis. 2006;12(1):1–8. Epub 2005/12/24. doi: 10.1097/01.mib.0000195389.11645.ab 16374251.

46. Netea MG, Ferwerda G, de Jong DJ, Jansen T, Jacobs L, Kramer M, et al. Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release. J Immunol. 2005;174(10):6518–23. Epub 2005/05/10. doi: 10.4049/jimmunol.174.10.6518 15879155.

47. Carter AB, Monick MM, Hunninghake GW. Both Erk and p38 kinases are necessary for cytokine gene transcription. Am J Respir Cell Mol Biol. 1999;20(4):751–8. doi: 10.1165/ajrcmb.20.4.3420 10101008.

48. Shang LH, Luo ZQ, Deng XD, Wang MJ, Huang FR, Feng DD, et al. Expression of N-methyl-D-aspartate receptor and its effect on nitric oxide production of rat alveolar macrophages. Nitric Oxide. 2010;23(4):327–31. Epub 2010/10/05. doi: 10.1016/j.niox.2010.09.004 20884369.

49. Lee YS, Lee SJ, Seo KW, Bae JU, Park SY, Kim CD. Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways. Free Radic Res. 2013;47(5):422–31. Epub 2013/03/15. doi: 10.3109/10715762.2013.784965 23485152.

50. Metcalf D. Acute antigen-induced elevation of serum colony stimulating factor (CFS) levels. Immunology. 1971;21(3):427–36. Epub 1971/09/01. 4936184; PubMed Central PMCID: PMC1408143.

51. Jones CV, Ricardo SD. Macrophages and CSF-1: implications for development and beyond. Organogenesis. 2013;9(4):249–60. Epub 2013/08/27. doi: 10.4161/org.25676 23974218; PubMed Central PMCID: PMC3903694.

52. Hussaini IM, Srikumar K, Quesenberry PJ, Gonias SL. Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages. J Biol Chem. 1990;265(32):19441–6. Epub 1990/11/15. 1700978.

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