LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins_Journal of Biomedical Engineering

Authors：

HONG Tiantian ^1,2 , LIU Wang ¹ , HUANG Jiaqi ¹ , ZHAO Baisong ³ , FANG Ying ¹ , WU Jianhua ¹ ,  LIN Jiangguo ²

1. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P.R.China;
2. Research Department of Medical Sciences, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, P.R.China;
3. Department of Anesthesiology, Guangzhou Women and Children’s Medical Center, Guangzhou 510623, P.R.China;

Corresponding author：

LIN Jiangguo, Email: linjiangguo@gdph.org.cn

Keywords：

neutrophil extracellular traps; β2 integrin; cytoskeleton; Talin-1

DOI：

10.7507/1001-5515.202105019

Video：

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Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.

Citation： HONG Tiantian, LIU Wang, HUANG Jiaqi, ZHAO Baisong, FANG Ying, WU Jianhua, LIN Jiangguo. LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins. Journal of Biomedical Engineering, 2021, 38(5): 903-910. doi: 10.7507/1001-5515.202105019 Copy

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3.	Albrengues J, Shields M A, Ng D, et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science, 2018, 361(6409): 4227-4240.
4.	Cao W, Pham H P, Williams L A, et al. Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura. Haematologica, 2016, 101(11): 1319-1326.
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7.	Munir H, Jones J O, Janowitz T, et al. Stromal-driven and amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth. Nat Commun, 2021, 12(1): 1-16.
8.	Colón D F, Wanderley C W, Franchin M, et al. Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis. Crit Care, 2019, 23(1): 1-13.
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14.	Gorina R, Lyck R, Vestweber D, et al. β2 integrin–mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood–brain barrier. J Immunol, 2014, 192(1): 324-337.
15.	McEver R P, Zhu C. Rolling cell adhesion. Annu Rev Cell Dev Biol, 2010, 26(1): 363-396.
16.	Kawamoto E, Okamoto T, Takagi Y, et al. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin. Biochem Biophys Res Commun, 2016, 473(4): 1005-1012.
17.	Luo B-H, Carman C V, Springer T A. Structural basis of integrin regulation and signaling. Annu Rev Immunol, 2007, 25(1): 619-647.
18.	Fuchs T A, Bhandari A A, Wagner D D. Histones induce rapid and profound thrombocytopenia in mice. Blood, 2011, 118(13): 3708-3714.
19.	Obi A T, Andraska E, Kanthi Y, et al. Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia. Thromb Haemost, 2017, 117(2): 339-348.
20.	Carestia A, Kaufman T, Rivadeneyra L, et al. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets. J Leukoc Biol, 2016, 99(1): 153-162.
21.	Clark S R, Ma A C, Tavener S A, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med, 2007, 13(4): 463-469.
22.	Gloude N J, Khandelwal P, Luebbering N, et al. Circulating dsDNA, endothelial injury, and complement activation in thrombotic microangiopathy and GVHD. Blood, 2017, 130(10): 1259-1266.
23.	Li N, Long M, Mao D, et al. Distinct binding affinities of Mac-1 and LFA-1 in neutrophil activation. J Immunol, 2013, 190(8): 4371-4381.
24.	Neeli I, Dwivedi N, Radic M, et al. Regulation of extracellular chromatin release from neutrophils. J Innate Immun, 2009, 1(3): 194-201.
25.	Kanchanawong P, Shtengel G, Pasapera A M, et al. Nanoscale architecture of integrin-based cell adhesions. Nature, 2010, 468(7323): 580-584.
26.	Cui L, Chen C, Xu T, et al. c-Abl kinase is required for β2 integrin-mediated neutrophil adhesion. J Immunol, 2009, 182(5): 3233-3242.
27.	Erpenbeck L, Gruhn A L, Kudryasheva G, et al. Effect of adhesion and substrate elasticity on neutrophil extracellular trap formation. Front Immunol, 2019, 10(10): 1-12.
28.	Langereis J D. Neutrophil integrin affinity regulation in adhesion, migration, and bacterial clearance. Cell Adhes Migr, 2013, 7(6): 486-491.
29.	Sun Z, Costell M, Fässler R. Integrin activation by talin, kindlin and mechanical forces. Nat Cell Biol, 2019, 21(1): 25-31.
30.	Takagi J, Petre B M, Springer T A, et al. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell, 2002, 110(5): 599-611.

1. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol, 2018, 18(2): 134-147.
2. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science, 2004, 303(5663): 1532-1535.
3. Albrengues J, Shields M A, Ng D, et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science, 2018, 361(6409): 4227-4240.
4. Cao W, Pham H P, Williams L A, et al. Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura. Haematologica, 2016, 101(11): 1319-1326.
5. Delgado-Rizo V, Martínez-Guzmán M A, Iñiguez-Gutierrez L, et al. Neutrophil extracellular traps and its implications in inflammation: an overview. Front Immunol, 2017, 8(2): 1-20.
6. Kaplan M J, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol, 2012, 189(6): 2689-2695.
7. Munir H, Jones J O, Janowitz T, et al. Stromal-driven and amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth. Nat Commun, 2021, 12(1): 1-16.
8. Colón D F, Wanderley C W, Franchin M, et al. Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis. Crit Care, 2019, 23(1): 1-13.
9. McDonald B, Urrutia R, Kubes P, et al. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Host Microbe, 2012, 12(3): 324-333.
10. Kumar S, Gupta E, Kaushik S, et al. Quantification of NETs formation in neutrophil and its correlation with the severity of sepsis and organ dysfunction. Clin Chim Acta, 2019, 495(4): 606-610.
11. Döring Y, Soehnlein O, Weber C. Neutrophil extracellular traps in atherosclerosis and atherothrombosis. Circ Res, 2017, 120(4): 736-743.
12. Noubouossie D F, Reeves B N, Key N S, et al. Neutrophils: back in the thrombosis spotlight. Blood, 2019, 133(20): 2186-2197.
13. Fuchs T A, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A, 2010, 107(36): 15880-15885.
14. Gorina R, Lyck R, Vestweber D, et al. β2 integrin–mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood–brain barrier. J Immunol, 2014, 192(1): 324-337.
15. McEver R P, Zhu C. Rolling cell adhesion. Annu Rev Cell Dev Biol, 2010, 26(1): 363-396.
16. Kawamoto E, Okamoto T, Takagi Y, et al. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin. Biochem Biophys Res Commun, 2016, 473(4): 1005-1012.
17. Luo B-H, Carman C V, Springer T A. Structural basis of integrin regulation and signaling. Annu Rev Immunol, 2007, 25(1): 619-647.
18. Fuchs T A, Bhandari A A, Wagner D D. Histones induce rapid and profound thrombocytopenia in mice. Blood, 2011, 118(13): 3708-3714.
19. Obi A T, Andraska E, Kanthi Y, et al. Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia. Thromb Haemost, 2017, 117(2): 339-348.
20. Carestia A, Kaufman T, Rivadeneyra L, et al. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets. J Leukoc Biol, 2016, 99(1): 153-162.
21. Clark S R, Ma A C, Tavener S A, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med, 2007, 13(4): 463-469.
22. Gloude N J, Khandelwal P, Luebbering N, et al. Circulating dsDNA, endothelial injury, and complement activation in thrombotic microangiopathy and GVHD. Blood, 2017, 130(10): 1259-1266.
23. Li N, Long M, Mao D, et al. Distinct binding affinities of Mac-1 and LFA-1 in neutrophil activation. J Immunol, 2013, 190(8): 4371-4381.
24. Neeli I, Dwivedi N, Radic M, et al. Regulation of extracellular chromatin release from neutrophils. J Innate Immun, 2009, 1(3): 194-201.
25. Kanchanawong P, Shtengel G, Pasapera A M, et al. Nanoscale architecture of integrin-based cell adhesions. Nature, 2010, 468(7323): 580-584.
26. Cui L, Chen C, Xu T, et al. c-Abl kinase is required for β2 integrin-mediated neutrophil adhesion. J Immunol, 2009, 182(5): 3233-3242.
27. Erpenbeck L, Gruhn A L, Kudryasheva G, et al. Effect of adhesion and substrate elasticity on neutrophil extracellular trap formation. Front Immunol, 2019, 10(10): 1-12.
28. Langereis J D. Neutrophil integrin affinity regulation in adhesion, migration, and bacterial clearance. Cell Adhes Migr, 2013, 7(6): 486-491.
29. Sun Z, Costell M, Fässler R. Integrin activation by talin, kindlin and mechanical forces. Nat Cell Biol, 2019, 21(1): 25-31.
30. Takagi J, Petre B M, Springer T A, et al. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell, 2002, 110(5): 599-611.

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