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Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis

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Abstract

Autophagy and apoptosis cooperate to modulate cell survival. Neutrophils are short-lived cells and apoptosis is considered to be the major mechanism of their death. In the present study, we addressed whether autophagy regulates neutrophil apoptosis and investigated the effects of autophagy inhibition on apoptosis of human neutrophils. We first showed that the established autophagy inhibitors 3-methyladenine (MA) and chloroquine (CQ) markedly accelerated spontaneous neutrophil apoptosis as was evidenced by phosphatidylserine exposure, DNA fragmentation and caspase-3 activation. Apoptosis induced by the autophagy inhibitors was completely abrogated by a pan-caspase inhibitor Q-VD-OPh. Unexpectedly, both MA and CQ significantly delayed neutrophil apoptosis induced by TNF-α, although the inhibitors did attenuate late pro-survival effect of the cytokine. The effect was specific for TNF-α because it was not observed in the presence of other inflammation-associated cytokines (IL-1β or IL-8). The autophagy inhibitors did not modulate surface expression of TNF-α receptors in the absence or presence of TNF-α. Both MA and CQ induced a marked down-regulation of a key anti-apoptotic protein Mcl-1 but did not affect significantly the levels of another anti-apoptotic protein Bcl-XL. Finally, to confirm the effects of the pharmacological inhibition of autophagy by a genetic approach, we evaluated the consequences of siRNA-mediated autophagy suppression in neutrophil-like differentiated HL60 cells. Knockdown of ATG5 in the cells resulted in accelerated spontaneous apoptosis but attenuated TNF-α-induced apoptosis. Together, these data suggest that autophagy regulates neutrophil apoptosis in an inflammatory context-dependent manner and mediates the early pro-apoptotic effect of TNF-α in neutrophils.

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References

  1. Luo HR, Loison F (2008) Constitutive neutrophil apoptosis: mechanisms and regulation. Am J Hematol 83:288–295

    Article  PubMed  CAS  Google Scholar 

  2. Maggini J, Raiden S, Salamone G, Trevani A, Geffner J (2009) Regulation of neutrophil apoptosis by cytokines, pathogens and environmental stressors. Front Biosci 14:2372–2385

    Article  PubMed  CAS  Google Scholar 

  3. El Kebir D, Filep JG (2010) Role of neutrophil apoptosis in the resolution of inflammation. Sci World J 10:1731–1748

    Article  Google Scholar 

  4. Glick D, Barth S, Macleod KF (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221:3–12

    Article  PubMed  CAS  Google Scholar 

  5. Mizushima N (2009) Physiological functions of autophagy. Curr Top Microbiol Immunol 335:71–84

    Article  PubMed  CAS  Google Scholar 

  6. Komatsu M, Ichimura Y (2010) Selective autophagy regulates various cellular functions. Genes Cells 15:923–933

    Article  PubMed  CAS  Google Scholar 

  7. Harris J (2011) Autophagy and cytokines. Cytokine 56:140–144

    Article  PubMed  CAS  Google Scholar 

  8. Platini F, Pérez-Tomás R, Ambrosio S, Tessitore L (2010) Understanding autophagy in cell death control. Curr Pharm Des 16:101–113

    Article  PubMed  CAS  Google Scholar 

  9. Hedrick SM, Ch’en IL, Alves BN (2010) Intertwined pathways of programmed cell death in immunity. Immunol Rev 236:41–53

    Article  PubMed  CAS  Google Scholar 

  10. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A (2009) Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 16:966–975

    Article  PubMed  CAS  Google Scholar 

  11. Barth S, Glick D, Macleod KF (2010) Autophagy: assays and artifacts. J Pathol 221:117–124

    Article  PubMed  CAS  Google Scholar 

  12. Salamone G, Giordano M, Trevani AS, Gamberale R, Vermeulen M, Schettinni J, Geffner JR (2001) Promotion of neutrophil apoptosis by TNF-alpha. J Immunol 166:3476–3483

    PubMed  CAS  Google Scholar 

  13. Cross A, Moots RJ, Edwards SW (2008) The dual effects of TNFalpha on neutrophil apoptosis are mediated via differential effects on expression of Mcl-1 and Bfl-1. Blood 111:878–884

    Article  PubMed  CAS  Google Scholar 

  14. Murray J, Barbara JA, Dunkley SA, Lopez AF, Van Ostade X, Condliffe AM, Dransfield I, Haslett C, Chilvers ER (1997) Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro. Blood 90:2772–2783

    PubMed  CAS  Google Scholar 

  15. Pliyev BK (2009) 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

    Article  PubMed  CAS  Google Scholar 

  16. Pliyev BK, Antonova OA, Menshikov M (2011) Participation of the urokinase-type plasminogen activator receptor (uPAR) in neutrophil transendothelial migration. Mol Immunol 48:1168–1177

    Article  PubMed  CAS  Google Scholar 

  17. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461

    Article  PubMed  CAS  Google Scholar 

  18. Vázquez CL, Colombo MI (2009) Assays to assess autophagy induction and fusion of autophagic vacuoles with a degradative compartment, using monodansylcadaverine (MDC) and DQ-BSA. Methods Enzymol 452:85–95

    Article  PubMed  Google Scholar 

  19. Smith JA, Weidemann MJ (1993) Further characterization of the neutrophil oxidative burst by flow cytometry. J Immunol Methods 162:261–268

    Article  PubMed  CAS  Google Scholar 

  20. Mitroulis I, Kourtzelis I, Kambas K, Rafail S, Chrysanthopoulou A, Speletas M, Ritis K (2010) Regulation of the autophagic machinery in human neutrophils. Eur J Immunol 40:1461–1472

    Article  PubMed  CAS  Google Scholar 

  21. Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA 79:1889–1892

    Article  PubMed  CAS  Google Scholar 

  22. Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S (2007) Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy 3:154–157

    PubMed  CAS  Google Scholar 

  23. Hoang B, Benavides A, Shi Y, Frost P, Lichtenstein A (2009) Effect of autophagy on multiple myeloma cell viability. Mol Cancer Ther 8:1974–1984

    Article  PubMed  CAS  Google Scholar 

  24. Li X, Fan Z (2010) The epidermal growth factor receptor antibody cetuximab induces autophagy in cancer cells by downregulating HIF-1alpha and Bcl-2 and activating the beclin 1/hVps34 complex. Cancer Res 70:5942–5952

    Article  PubMed  CAS  Google Scholar 

  25. Chaachouay H, Ohneseit P, Toulany M, Kehlbach R, Multhoff G, Rodemann HP (2011) Autophagy contributes to resistance of tumor cells to ionizing radiation. Radiother Oncol 99:287–292

    Article  PubMed  CAS  Google Scholar 

  26. Boya P, González-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040

    Article  PubMed  CAS  Google Scholar 

  27. Tanida I, Waguri S (2010) Measurement of autophagy in cells and tissues. Methods Mol Biol 648:193–214

    Article  PubMed  CAS  Google Scholar 

  28. Ichimura Y, Kominami E, Tanaka K, Komatsu M (2008) Selective turnover of p62/A170/SQSTM1 by autophagy. Autophagy 4:1063–1066

    PubMed  CAS  Google Scholar 

  29. Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545

    PubMed  CAS  Google Scholar 

  30. Cowburn AS, Deighton J, Walmsley SR, Chilvers ER (2004) The survival effect of TNF-alpha in human neutrophils is mediated via NF-kappa B-dependent IL-8 release. Eur J Immunol 34:1733–1743

    Article  PubMed  CAS  Google Scholar 

  31. Yamashita K, Takahashi A, Kobayashi S, Hirata H, Mesner PW Jr, Kaufmann SH, Yonehara S, Yamamoto K, Uchiyama T, Sasada M (1999) Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood 93:674–685

    PubMed  CAS  Google Scholar 

  32. Weinmann P, Gaehtgens P, Walzog B (1999) Bcl-Xl- and Bax-alpha-mediated regulation of apoptosis of human neutrophils via caspase-3. Blood 93:3106–3115

    PubMed  CAS  Google Scholar 

  33. Wardle DJ, Burgon J, Sabroe I, Bingle CD, Whyte MK, Renshaw SA (2011) Effective caspase inhibition blocks neutrophil apoptosis and reveals Mcl-1 as both a regulator and a target of neutrophil caspase activation. PLoS ONE 6:e15768

    Article  PubMed  CAS  Google Scholar 

  34. Feghali CA, Wright TM (1997) Cytokines in acute and chronic inflammation. Front Biosci 2:D12–D26

    PubMed  CAS  Google Scholar 

  35. Dri P, Gasparini C, Menegazzi R, Cramer R, Albéri L, Presani G, Garbisa S, Patriarca P (2000) TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase. J Immunol 165:2165–2172

    PubMed  CAS  Google Scholar 

  36. Porteu F, Nathan C (1990) Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 172:599–607

    Article  PubMed  CAS  Google Scholar 

  37. Sengeløv H, Kjeldsen L, Diamond MS, Springer TA, Borregaard N (1993) Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils. J Clin Invest 92:1467–1476

    Article  PubMed  Google Scholar 

  38. Alexander SR, Kishimoto TK, Walcheck B (2000) Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils. J Leukoc Biol 67:415–422

    PubMed  CAS  Google Scholar 

  39. Edwards SW, Derouet M, Howse M, Moots RJ (2004) Regulation of neutrophil apoptosis by Mcl-1. Biochem Soc Trans 32:489–492

    Article  PubMed  CAS  Google Scholar 

  40. Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW (2004) Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem 279:26915–26921

    Article  PubMed  CAS  Google Scholar 

  41. Langereis JD, Raaijmakers HA, Ulfman LH, Koenderman L (2010) Abrogation of NF-κB signaling in human neutrophils induces neutrophil survival through sustained p38-MAPK activation. J Leukoc Biol 88:655–664

    Article  PubMed  CAS  Google Scholar 

  42. Nagaoka I, Suzuki K, Murakami T, Niyonsaba F, Tamura H, Hirata M (2010) Evaluation of the effect of α-defensin human neutrophil peptides on neutrophil apoptosis. Int J Mol Med 26:925–934

    Article  PubMed  CAS  Google Scholar 

  43. Akhtar S, Li X, Kovacs EJ, Gamelli RL, Choudhry MA (2011) Interleukin-18 delays neutrophil apoptosis following alcohol intoxication and burn injury. Mol Med 17:88–94

    Article  PubMed  CAS  Google Scholar 

  44. Higo T, Duronio V, Tudan C, Burt HM, Jackson JK (2010) Calcium pyrophosphate dihydrate crystal-induced inhibition of neutrophil apoptosis: involvement of Bcl-2 family members. Inflamm Res 59:71–81

    Article  PubMed  CAS  Google Scholar 

  45. Matzner Y, Gavison R, Rachmilewitz EA, Fibach E (1987) Expression of granulocytic functions by leukemic promyelocytic HL-60 cells: differential induction by dimethylsulfoxide and retinoic acid. Cell Differ 21:261–269

    Article  PubMed  CAS  Google Scholar 

  46. Hauert AB, Martinelli S, Marone C, Niggli V (2002) Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int J Biochem Cell Biol 34:838–854

    Article  PubMed  CAS  Google Scholar 

  47. Watson RW, Rotstein OD, Parodo J, Bitar R, Hackam D, Marshall JC (1997) Granulocytic differentiation of HL-60 cells results in spontaneous apoptosis mediated by increased caspase expression. FEBS Lett 412:603–609

    Article  PubMed  CAS  Google Scholar 

  48. Cabrini M, Nahmod K, Geffner J (2010) New insights into the mechanisms controlling neutrophil survival. Curr Opin Hematol 17:31–35

    Article  PubMed  Google Scholar 

  49. Mihalache CC, Yousefi S, Conus S, Villiger PM, Schneider EM, Simon HU (2011) Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events. J Immunol 186:6532–6542

    Article  PubMed  CAS  Google Scholar 

  50. Remijsen Q, Vanden Berghe T, Wirawan E, Asselbergh B, Parthoens E, De Rycke R, Noppen S, Delforge M, Willems J, Vandenabeele P (2011) Neutrophil extracellular trap cell death requires both autophagy and superoxide generation. Cell Res 21:290–304

    Article  PubMed  CAS  Google Scholar 

  51. Carew JS, Espitia CM, Esquivel JA 2nd, Mahalingam D, Kelly KR, Reddy G, Giles FJ, Nawrocki ST (2011) Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. J Biol Chem 286:6602–6613

    Article  PubMed  CAS  Google Scholar 

  52. Espina V, Mariani BD, Gallagher RI, Tran K, Banks S, Wiedemann J, Huryk H, Mueller C, Adamo L, Deng J, Petricoin EF, Pastore L, Zaman S, Menezes G, Mize J, Johal J, Edmiston K, Liotta LA (2010) Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival. PLoS ONE 5:e10240

    Article  PubMed  Google Scholar 

  53. Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM (1997) Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol 98:673–685

    Article  PubMed  CAS  Google Scholar 

  54. Cheng Y, Qiu F, Tashiro S, Onodera S, Ikejima T (2008) ERK and JNK mediate TNFalpha-induced p53 activation in apoptotic and autophagic L929 cell death. Biochem Biophys Res Commun 376:483–488

    Article  PubMed  CAS  Google Scholar 

  55. van den Berg JM, Weyer S, Weening JJ, Roos D, Kuijpers TW (2001) Divergent effects of tumor necrosis factor alpha on apoptosis of human neutrophils. J Leukoc Biol 69:467–473

    PubMed  Google Scholar 

  56. Salamone G, Trevani A, Martínez D, Vermeulen M, Gamberale R, Fernández-Calotti P, Raiden S, Giordano M, Geffner J (2004) Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor-alpha. Immunology 113:355–362

    Article  PubMed  CAS  Google Scholar 

  57. Witko-Sarsat V, Pederzoli-Ribeil M, Hirsch E, Sozzani S, Cassatella MA (2011) Regulating neutrophil apoptosis: new players enter the game. Trends Immunol 32:117–124

    Article  PubMed  CAS  Google Scholar 

  58. Geering B, Simon HU (2011) Peculiarities of cell death mechanisms in neutrophils. Cell Death Differ 18:1457–1469

    Article  PubMed  CAS  Google Scholar 

  59. Germain M, Nguyen AP, Le Grand JN, Arbour N, Vanderluit JL, Park DS, Opferman JT, Slack RS (2011) MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner. EMBO J 30:395–407

    Article  PubMed  CAS  Google Scholar 

  60. Germain M, Slack RS (2011) MCL-1 regulates the balance between autophagy and apoptosis. Autophagy 7:549–551

    Article  PubMed  CAS  Google Scholar 

  61. Elgendy M, Sheridan C, Brumatti G, Martin SJ (2011) Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol Cell 42:23–35

    Article  PubMed  CAS  Google Scholar 

  62. Erlich S, Mizrachy L, Segev O, Lindenboim L, Zmira O, Adi-Harel S, Hirsch JA, Stein R, Pinkas-Kramarski R (2007) Differential interactions between Beclin 1 and Bcl-2 family members. Autophagy 3:561–568

    PubMed  CAS  Google Scholar 

  63. Nezis IP, Shravage BV, Sagona AP, Lamark T, Bjørkøy G, Johansen T, Rusten TE, Brech A, Baehrecke EH, Stenmark H (2010) Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J Cell Biol 190:523–531

    Article  PubMed  CAS  Google Scholar 

  64. Kovacs JR, Li C, Yang Q, Li G, Garcia IG, Ju S, Roodman DG, Windle JJ, Zhang X, Lu B (2012) Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery. Cell Death Differ 19:144–152

    Article  PubMed  CAS  Google Scholar 

  65. Choi YE, Butterworth M, Malladi S, Duckett CS, Cohen GM, Bratton SB (2009) The E3 ubiquitin ligase cIAP1 binds and ubiquitinates caspase-3 and -7 via unique mechanisms at distinct steps in their processing. J Biol Chem 284:12772–12782

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Russian Foundation for Basic Research (grants # 09-04-01664 and # 11-04-00989).

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

    Boris K. Pliyev & Mikhail Menshikov

  2. Department of Biochemistry, Institute of Experimental Cardiology, Cardiology Research Centre, 3rd Cherepkovskaya Str., 15a, Moscow, 121552, Russia

    Boris K. Pliyev & Mikhail Menshikov

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Correspondence to Boris K. Pliyev.

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Pliyev, B.K., Menshikov, M. Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis. Apoptosis 17, 1050–1065 (2012). https://doi.org/10.1007/s10495-012-0738-x

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