Article | Published:

Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation

Nature Immunologyvolume 4pages138144 (2003) | Download Citation

Subjects

Abstract

Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD−/− thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II−/−CAD−/− embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-β gene was strongly up-regulated in the thymus of DNase II−/−CAD−/− embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Kerr, J.F., Wyllie, A.H. & Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972).

  2. 2

    Jacobson, M.D., Weil, M. & Raff, M.C. Programmed cell death in animal development. Cell 88, 347–354 (1997).

  3. 3

    Vaux, D.L. & Korsmeyer, S.J. Cell death in development. Cell 96, 245–254 (1999).

  4. 4

    Raff, M. Cell suicide for beginners. Nature 396, 119–122 (1998).

  5. 5

    Nagata, S. Apoptosis by death factor. Cell 88, 355–365 (1997).

  6. 6

    Ashkenazi, A. & Dixit, V.M. Death receptors: signaling and modulation. Science 281, 1305–1308 (1998).

  7. 7

    Green, D.R. & Reed, J.C. Mitochondria and apoptosis. Science 281, 1309–1312 (1998).

  8. 8

    Thornberry, N.A. & Lazebnik, Y. Caspases: enemies within. Science 281, 1312–1316 (1998).

  9. 9

    Earnshaw, W.C., Martins, L.M. & Kaufmann, S.H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424 (1999).

  10. 10

    Los, M., Wesselborg, S. & Schulze-Osthoff, K. The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10, 629–639 (1999).

  11. 11

    Savill, J. & Fadok, V. Corpse clearance defines the meaning of cell death. Nature 407, 784–788 (2000).

  12. 12

    Wyllie, A.H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556 (1980).

  13. 13

    Nagata, S., Nagase, H., Kawane, K., Mukae, N. & Fukuyama, H. Degradation of chromosomal DNA during apoptosis. Cell Death Differ. (in the press).

  14. 14

    Enari, M. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50 (1998).

  15. 15

    Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184 (1997).

  16. 16

    Sakahira, H., Enari, M. & Nagata, S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96–99 (1998).

  17. 17

    McCarty, J.S., Toh, S.Y. & Li, P. Multiple domains of DFF45 bind synergistically to DFF40: roles of caspase cleavage and sequestration of activator domain of DFF40. Biochem. Biophys. Res. Commun. 264, 181–185 (1999).

  18. 18

    Sakahira, H. & Nagata, S. Co-translational folding of caspase-activated DNase with Hsp70, Hsp40 and inhibitor of caspase-activated DNase. J. Biol. Chem. 277, 3364–3370 (2002).

  19. 19

    Zhang, J. et al. Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45. Proc. Natl. Acad. Sci. USA 95, 12480–12485 (1998).

  20. 20

    McIlroy, D. et al. An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes. Genes Dev. 14, 549–558 (2000).

  21. 21

    Li, L.Y., Luo, X. & Wang, X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412, 95–99 (2001).

  22. 22

    Parrish, J. et al. Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature 412, 90–94 (2001).

  23. 23

    van Loo, G. et al. Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation. Cell Death Differ. 8, 1136–1142 (2001).

  24. 24

    Hanayama, R. et al. Identification of a factor that links apoptotic cells to phagocytes. Nature 417, 182–187 (2002).

  25. 25

    Bernardi, G. in The Enzymes (ed. Boyer, P.D.) 271–287 (Academic Press, New York and London, 1971).

  26. 26

    Kawane, K. et al. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science 292, 1546–1549 (2001).

  27. 27

    Krieser, R.J. et al. Deoxyribonuclease IIa is required during the phagocytic phase of apoptosis and its loss causes lethality. Cell Death Differ. 9, 956–962 (2002).

  28. 28

    Kawane, K. et al. Structure and promoter analysis of murine CAD and ICAD genes. Cell Death Differ. 6, 745–752 (1999).

  29. 29

    Oberhammer, F. et al. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684 (1993).

  30. 30

    Godfrey, D.I., Kennedy, J., Suda, T. & Zlotnik, A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3CD4CD8 triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150, 4244–4252 (1993).

  31. 31

    Yaegashi, Y., Nielsen, P., Sing, A., Galanos, C. & Freudenberg, M.A. Interferon β, a cofactor in the interferon γ production induced by gram-negative bacteria in mice. J. Exp. Med. 181, 953–960 (1995).

  32. 32

    Su, D.M., Wang, J., Lin, Q., Cooper, M.D. & Watanabe, T. Interferons α/β inhibit IL-7-induced proliferation of CD4 CD8 CD3 CD44+ CD25+ thymocytes, but do not inhibit that of CD4 CD8 CD3 CD44 CD25 thymocytes. Immunology 90, 543–549 (1997).

  33. 33

    Lin, Q., Dong, C. & Cooper, M.D. Impairment of T and B cell development by treatment with a type I interferon. J. Exp. Med. 187, 79–87 (1998).

  34. 34

    Montgomery, R.A. & Dallman, M.J. Semi-quantitative polymerase chain reaction analysis of cytokine and cytokine receptor gene expression during thymic ontogeny. Cytokine 9, 717–726 (1997).

  35. 35

    Arends, M.J., Morris, R.G. & Wyllie, A.H. The role of the endonuclease. Am. J. Pathol. 136, 593–608 (1993).

  36. 36

    Gavrieli, Y., Sherman, Y. & Ben-Sasson, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501 (1992).

  37. 37

    Staley, K., Blaschke, A. & Chun, J. Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ. 4, 66–75 (1997).

  38. 38

    Peitsch, M.C. et al. Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J. 12, 371–377 (1993).

  39. 39

    Susin, S.A. et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446 (1999).

  40. 40

    Wu, Y.C., Stanfield, G.M. & Horvitz, H.R. NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis. Genes Dev. 14, 536–548 (2000).

  41. 41

    Mukae, N., Yokoyama, H., Yokokura, T., Sakoyama, Y. & Nagata, S. Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation. Genes Dev. 16, 2662–2671 (2002).

  42. 42

    Durrieu, F. et al. Caspase activation is an early event in anthracycline-induced apoptosis and allows detection of apoptotic cells before they are ingested by phagocytes. Exp. Cell Res. 240, 165–175 (1998).

  43. 43

    Binder, D., Fehr, J., Hengartner, H. & Zinkernagel, R.M. Virus-induced transient bone marrow aplasia: major role of interferon-α/β during acute infection with the noncytopathic lymphocytic choriomeningitis virus. J. Exp. Med. 185, 517–530 (1997).

  44. 44

    Doly, J., Civas, A., Navarro, S. & Uze, G. Type I interferons: expression and signalization. Cell Mol. Life Sci. 54, 1109–1121 (1998).

  45. 45

    Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760 (2002).

  46. 46

    Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

  47. 47

    Bird, A.P. CpG-rich islands and the function of DNA methylation. Nature 321, 209–213 (1986).

  48. 48

    Leadbetter, E.A. et al. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603–607 (2002).

  49. 49

    Kondoh, G. et al. Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method. J. Biochem. Biophys. Methods 39, 137–142 (1999).

  50. 50

    Platt, N., Suzuki, H., Kurihara, Y., Kodama, T. & Gordon, S. Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro. Proc. Natl. Acad. Sci. USA 93, 12456–12460 (1996).

  51. 51

    Takeshita, S., Kaji, K. & Kudo, A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J. Bone Miner. Res. 15, 1477–1488 (2000).

  52. 52

    Laird, P.W. et al. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293 (1991).

  53. 53

    Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2001).

Download references

Acknowledgements

We thank M. Adachi for help in the initial stage of this study; K. Miwa for PCR; A. Kudo for CMG-12 cells; K. Ishihara for the advice on the fetal thymus organ culture; and S. Aoyama and M. Harayama for secretarial assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture in Japan. K.K. is supported by a research fellowship from the Japan Society for the Promotion of Science.

Author information

Author notes

    • Hidehiro Fukuyama

    Present address: Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY, 10021, USA

Affiliations

  1. Department of Genetics, Osaka University Medical School, Osaka, 565-0871, Japan

    • Kohki Kawane
    • , Hidehiro Fukuyama
    • , Hideyuki Yoshida
    • , Hiroko Nagase
    •  & Shigekazu Nagata
  2. Departments of Cell Biology and Neuroscience, Osaka University Medical School, Osaka, 565-0871, Japan

    • Yoshiyuki Ohsawa
    •  & Yasuo Uchiyama
  3. Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan

    • Kazuhisa Okada
    •  & Tetsuya Iida
  4. Laboratory of Genetics, Integrated Biology Laboratories, Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan

    • Shigekazu Nagata
  5. Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka, 565-0871, Japan

    • Shigekazu Nagata

Authors

  1. Search for Kohki Kawane in:

  2. Search for Hidehiro Fukuyama in:

  3. Search for Hideyuki Yoshida in:

  4. Search for Hiroko Nagase in:

  5. Search for Yoshiyuki Ohsawa in:

  6. Search for Yasuo Uchiyama in:

  7. Search for Kazuhisa Okada in:

  8. Search for Tetsuya Iida in:

  9. Search for Shigekazu Nagata in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Shigekazu Nagata.

About this article

Publication history

Received

Accepted

Published

Issue Date

DOI

https://doi.org/10.1038/ni881

Further reading

  • Delicate regulation of the cGAS–MITA-mediated innate immune response

    • Wei-Wei Luo
    •  & Hong-Bing Shu

    Cellular & Molecular Immunology (2018)

  • Apoptosis regulation by subcellular relocation of caspases

    • Evgeniia A. Prokhorova
    • , Gelina S. Kopeina
    • , Inna N. Lavrik
    •  & Boris Zhivotovsky

    Scientific Reports (2018)

  • Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018

    • Lorenzo Galluzzi
    • , Ilio Vitale
    • , Stuart A. Aaronson
    • , John M. Abrams
    • , Dieter Adam
    • , Patrizia Agostinis
    • , Emad S. Alnemri
    • , Lucia Altucci
    • , Ivano Amelio
    • , David W. Andrews
    • , Margherita Annicchiarico-Petruzzelli
    • , Alexey V. Antonov
    • , Eli Arama
    • , Eric H. Baehrecke
    • , Nickolai A. Barlev
    • , Nicolas G. Bazan
    • , Francesca Bernassola
    • , Mathieu J. M. Bertrand
    • , Katiuscia Bianchi
    • , Mikhail V. Blagosklonny
    • , Klas Blomgren
    • , Christoph Borner
    • , Patricia Boya
    • , Catherine Brenner
    • , Michelangelo Campanella
    • , Eleonora Candi
    • , Didac Carmona-Gutierrez
    • , Francesco Cecconi
    • , Francis K.-M. Chan
    • , Navdeep S. Chandel
    • , Emily H. Cheng
    • , Jerry E. Chipuk
    • , John A. Cidlowski
    • , Aaron Ciechanover
    • , Gerald M. Cohen
    • , Marcus Conrad
    • , Juan R. Cubillos-Ruiz
    • , Peter E. Czabotar
    • , Vincenzo D’Angiolella
    • , Ted M. Dawson
    • , Valina L. Dawson
    • , Vincenzo De Laurenzi
    • , Ruggero De Maria
    • , Klaus-Michael Debatin
    • , Ralph J. DeBerardinis
    • , Mohanish Deshmukh
    • , Nicola Di Daniele
    • , Francesco Di Virgilio
    • , Vishva M. Dixit
    • , Scott J. Dixon
    • , Colin S. Duckett
    • , Brian D. Dynlacht
    • , Wafik S. El-Deiry
    • , John W. Elrod
    • , Gian Maria Fimia
    • , Simone Fulda
    • , Ana J. García-Sáez
    • , Abhishek D. Garg
    • , Carmen Garrido
    • , Evripidis Gavathiotis
    • , Pierre Golstein
    • , Eyal Gottlieb
    • , Douglas R. Green
    • , Lloyd A. Greene
    • , Hinrich Gronemeyer
    • , Atan Gross
    • , Gyorgy Hajnoczky
    • , J. Marie Hardwick
    • , Isaac S. Harris
    • , Michael O. Hengartner
    • , Claudio Hetz
    • , Hidenori Ichijo
    • , Marja Jäättelä
    • , Bertrand Joseph
    • , Philipp J. Jost
    • , Philippe P. Juin
    • , William J. Kaiser
    • , Michael Karin
    • , Thomas Kaufmann
    • , Oliver Kepp
    • , Adi Kimchi
    • , Richard N. Kitsis
    • , Daniel J. Klionsky
    • , Richard A. Knight
    • , Sharad Kumar
    • , Sam W. Lee
    • , John J. Lemasters
    • , Beth Levine
    • , Andreas Linkermann
    • , Stuart A. Lipton
    • , Richard A. Lockshin
    • , Carlos López-Otín
    • , Scott W. Lowe
    • , Tom Luedde
    • , Enrico Lugli
    • , Marion MacFarlane
    • , Frank Madeo
    • , Michal Malewicz
    • , Walter Malorni
    • , Gwenola Manic
    • , Jean-Christophe Marine
    • , Seamus J. Martin
    • , Jean-Claude Martinou
    • , Jan Paul Medema
    • , Patrick Mehlen
    • , Pascal Meier
    • , Sonia Melino
    • , Edward A. Miao
    • , Jeffery D. Molkentin
    • , Ute M. Moll
    • , Cristina Muñoz-Pinedo
    • , Shigekazu Nagata
    • , Gabriel Nuñez
    • , Andrew Oberst
    • , Moshe Oren
    • , Michael Overholtzer
    • , Michele Pagano
    • , Theocharis Panaretakis
    • , Manolis Pasparakis
    • , Josef M. Penninger
    • , David M. Pereira
    • , Shazib Pervaiz
    • , Marcus E. Peter
    • , Mauro Piacentini
    • , Paolo Pinton
    • , Jochen H.M. Prehn
    • , Hamsa Puthalakath
    • , Gabriel A. Rabinovich
    • , Markus Rehm
    • , Rosario Rizzuto
    • , Cecilia M.P. Rodrigues
    • , David C. Rubinsztein
    • , Thomas Rudel
    • , Kevin M. Ryan
    • , Emre Sayan
    • , Luca Scorrano
    • , Feng Shao
    • , Yufang Shi
    • , John Silke
    • , Hans-Uwe Simon
    • , Antonella Sistigu
    • , Brent R. Stockwell
    • , Andreas Strasser
    • , Gyorgy Szabadkai
    • , Stephen W.G. Tait
    • , Daolin Tang
    • , Nektarios Tavernarakis
    • , Andrew Thorburn
    • , Yoshihide Tsujimoto
    • , Boris Turk
    • , Tom Vanden Berghe
    • , Peter Vandenabeele
    • , Matthew G. Vander Heiden
    • , Andreas Villunger
    • , Herbert W. Virgin
    • , Karen H. Vousden
    • , Domagoj Vucic
    • , Erwin F. Wagner
    • , Henning Walczak
    • , David Wallach
    • , Ying Wang
    • , James A. Wells
    • , Will Wood
    • , Junying Yuan
    • , Zahra Zakeri
    • , Boris Zhivotovsky
    • , Laurence Zitvogel
    • , Gerry Melino
    •  & Guido Kroemer

    Cell Death & Differentiation (2018)

  • ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation

    • Michael A Schumacher
    • , Matija Hedl
    • , Clara Abraham
    • , Jessica K Bernard
    • , Patricia R Lozano
    • , Jonathan J Hsieh
    • , Dana Almohazey
    • , Edie B Bucar
    • , Shivesh Punit
    • , Peter J Dempsey
    •  & Mark R Frey

    Cell Death & Disease (2017)

  • Programmed cell death and the immune system

    • Shigekazu Nagata
    •  & Masato Tanaka

    Nature Reviews Immunology (2017)