Skip to main content
SpringerLink
Log in

Granzyme B is recovered by natural killer cells via clathrin-dependent endocytosis

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

When they recognize a target cell, natural killer (NK) cells mount an attack to kill the target by exerting their cytotoxicity via the exocytosis of cytotoxic granules. Although the details of this process (which includes the movement of cytotoxic granules in the immune synapse and their fusion with the plasma membrane, releasing granzymes and perforin into the synaptic cleft) are relatively better understood, the post-exocytosis regulation of the process is still largely unknown. Here we show that a clathrin-dependent endocytosis stimulated by target cell occurs in NK92 cell line, which is closely correlated with granzyme B recovery. Inhibition of the endocytosis significantly attenuates the cytotoxicity of NK92 cells. The NK cell recovery of its released effector molecules, in turn, suggests that endocytosis may well play a key role in the post exocytosis regulation of immune cells.

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
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Smyth MJ, Cretney E, Kelly JM, Westwood JA, Street SE, Yagita H, Takeda K, van Dommelen SL, Degli-Esposti MA, Hayakawa Y (2005) Activation of NK cell cytotoxicity. Mol Immunol 42:501–510

    Article  CAS  PubMed  Google Scholar 

  2. Blott EJ, Griffiths GM (2002) Secretory lysosomes. Nat Rev Mol Cell Biol 3:122–131

    Article  CAS  PubMed  Google Scholar 

  3. Liu D, Xu L, Yang F, Li D, Gong F, Xu T (2005) Rapid biogenesis and sensitization of secretory lysosomes in NK cells mediated by target-cell recognition. Proc Natl Acad Sci USA 102:123–127

    Article  CAS  PubMed  Google Scholar 

  4. Clark R, Griffiths GM (2003) Lytic granules, secretory lysosomes and disease. Curr Opin Immunol 15:516–521

    Article  CAS  PubMed  Google Scholar 

  5. Bossi G, Griffiths GM (2005) CTL secretory lysosomes: biogenesis and secretion of a harmful organelle. Semin Immunol 17:87–94

    Article  CAS  PubMed  Google Scholar 

  6. Bossi G, Trambas C, Booth S, Clark R, Stinchcombe J, Griffiths GM (2002) The secretory synapse: the secrets of a serial killer. Immunol Rev 189:152–160

    Article  CAS  PubMed  Google Scholar 

  7. Fischer A, Latour S, de Saint Basile G (2007) Genetic defects affecting lymphocyte cytotoxicity. Curr Opin Immunol 19:348–353

    Article  CAS  PubMed  Google Scholar 

  8. Krzewski K, Strominger JL (2008) The killer’s kiss: the many functions of NK cell immunological synapses. Curr Opin Cell Biol 20:597–605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Orange JS (2008) Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol 8:713–725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk T, Holmes CF, Gauldie J, Bleackley RC (2000) Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 103:491–500

    Article  CAS  PubMed  Google Scholar 

  11. Keefe D, Shi L, Feske S, Massol R, Navarro F, Kirchhausen T, Lieberman J (2005) Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 23:249–262

    Article  CAS  PubMed  Google Scholar 

  12. Chowdhury D, Lieberman J (2008) Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol 26:389–420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA (2002) Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation. J Exp Med 196:493–503

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA, Trapani JA, Bird PI (1996) A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J Biol Chem 271:27802–27809

    Article  CAS  PubMed  Google Scholar 

  15. Xu T, Naraghi M, Kang H, Neher E (1997) Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J 73:532–545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    Article  CAS  PubMed  Google Scholar 

  17. Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, Yee H, Zender L, Lowe SW (2008) Senescence of activated stellate cells limits liver fibrosis. Cell 134:657–667

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Piriou L, Chilmonczyk S, Genetet N, Albina E (2000) Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species. Cytometry 41:289–297

    Article  CAS  PubMed  Google Scholar 

  19. Heidelberger R, Heinemann C, Neher E, Matthews G (1994) Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371:513–515

    Article  CAS  PubMed  Google Scholar 

  20. Forte P, Baumann BC, Schneider MK, Seebach JD (2009) HLA-Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a + human NK cells. Xenotransplantation 16:19–26

    Article  PubMed  Google Scholar 

  21. Xu J, McNeil B, Wu W, Nees D, Bai L, Wu LG (2008) GTP-independent rapid and slow endocytosis at a central synapse. Nat Neurosci 11:45–53

    Article  CAS  PubMed  Google Scholar 

  22. Gaffield MA, Betz WJ (2007) Synaptic vesicle mobility in mouse motor nerve terminals with and without synapsin. J Neurosci 27:13691–13700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B (2003) Cell-penetrating peptides A reevaluation of the mechanism of cellular uptake. J Biol Chem 278:585–590

    Article  CAS  PubMed  Google Scholar 

  24. Mousavi SA, Malerod L, Berg T, Kjeken R (2004) Clathrin-dependent endocytosis. Biochem J 377:1–16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nishi K, Saigo K (2007) Cellular internalization of green fluorescent protein fused with herpes simplex virus protein VP22 via a lipid raft-mediated endocytic pathway independent of Caveolae and Rho Family GTPases but dependent on Dynamin and Arf6. J Biol Chem 282:27503–27517

    Article  CAS  PubMed  Google Scholar 

  26. Mayor S, Pagano RE (2007) Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8:603–612

    Article  CAS  PubMed  Google Scholar 

  27. Masilamani M, Narayanan S, Prieto M, Borrego F, Coligan JE (2008) Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor. Traffic 9:1019–1034

    Article  CAS  PubMed  Google Scholar 

  28. Kummer JA, Kamp AM, Tadema TM, Vos W, Meijer CJ, Hack CE (1995) Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood. Clin Exp Immunol 100:164–172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cullen SP, Martin SJ (2008) Mechanisms of granule-dependent killing. Cell Death Differ 15:251–262

    Article  CAS  PubMed  Google Scholar 

  30. Baran K, Ciccone A, Peters C, Yagita H, Bird PI, Villadangos JA, Trapani JA (2006) Cytotoxic T lymphocytes from cathepsin B-deficient mice survive normally in vitro and in vivo after encountering and killing target cells. J Biol Chem 281:30485–30491

    Article  CAS  PubMed  Google Scholar 

  31. Rajagopalan S, Bryceson YT, Kuppusamy SP, Geraghty DE, van der Meer A, Joosten I, Long EO (2006) Activation of NK cells by an endocytosed receptor for soluble HLA-G. PLoS Biol 4:e9

    Article  PubMed  Google Scholar 

  32. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227

    Article  CAS  PubMed  Google Scholar 

  33. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82–86

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Phil (Oxon) Foad Katirai for his professional English editing and thank Prof. Tao Xu, Ms. Jingze Lu, and Mr. Yinong Zhang for their help with the confocal work. We thank Dr. Zhigang Tian, Dr. Ming Zhu, and Dr. Shi Chen for their help in the work of cell cultures. This work was supported by grants from the National Natural Sciences Foundation of China (No. 30400390) and Major State Basic Research Development Program of China (973 Program) (No. 2007CB512402) and the Ministry of Science and Technology (No. 2009DFA31940).

Author information

Authors and Affiliations

  1. Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Pan Li, Yan Yang, Ping Xiong, Yong Xu, Min Fang, Zheng Tan, Fang Zheng & Feili Gong

  2. Key Laboratory of Occupational Health and Safety, Department of Prevention Medicine, North China Coal Medical University, Tangshan, Hebei, 063000, China

    Guoying Zheng

  3. Institute of Biophysics and Biochemistry, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China

    Chunguang Zhang

Authors
  1. Pan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guoying Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chunguang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ping Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Min Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zheng Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fang Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Feili Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Fang Zheng or Feili Gong.

Electronic supplementary material

The files are unfortunately not in the Publisher's archive anymore.

Figure S1. CPZ concentration titration. a,b The effect of chlorpromazine (CPZ) on the vitality of NK92 cells. 1 × 106 NK92 cells were pretreated with different concentrations of CPZ for 30 min at 37°C, respectively. The cells were then washed twice and stained with 10 μg/ml propidium iodide (PI) for 30 min. The death rate of NK92 cells was immediately analyzed by flow cytometry. c,d Effect of CPZ on transferrin (Tfr) internalization. 1 × 106 NK92 cells were pretreated with different concentrations of CPZ for 30 min at 37°C, respectively, and then washed and incubated with Alexa488-transferrin (10 µg/ml) for 30 min on ice for binding, washed, and transferred to 37°C for 30 min for internalization. After that, cells were washed with acid buffer (50 mM glycine, 150 mM NaCl, pH 2.5) to remove uninternalized transferrin, followed by neutralization with RPMI (pH 10). The endocytosis of Alexa488-transferrin is calculated as the fold change in mean fluorescence intensities (MFI). MFI of bound Alexa488-transferrin on the cell surface on ice was named MFI(maximum). MFI of internalized Alexa488-transferrin was named MFI(experiment). The percentage internalization is calculated by the following formula: percentage internalization = MFI(experiment)/MFI(maximum) × 100. Error bar indicates SD. Data are representative of three independent experiments (18_2010_377_MOESM1_ESM.tiff 779 kb)

Figure S2. Internalization of FM1-43FX and transferrin assays. A Effect of CPZ treatment on uptake of FM1-43FX by NK cells with PEC stimulation. a NK92 cells that were labeled with 5 μg/ml FM1-43FX for 5 min and kept always at 4℃ for maximum fluorescence. b NK92 cells that internalized FM1-43FX after stimulation with PEC for 3 h and washed with PBS to remove surface FM1-43FX. c NK92 cells that were pretreated with 10 μg/ml CPZ for 30 min and internalized FM1-43FX after stimulation with PEC. d NK92 cells that were continuously treated with CPZ for 3 h and internalized FM1-43FX with PEC stimulation; e No treatment of NK92 cells were as a control. B: Effect of CPZ treatment on endocytosis of Alexa488-transferrin. a NK92 cells that were labeled with 10 μg/ml Alexa488-transferrin for 30 min and kept always on ice for maximum transferrin fluorescence. b NK92 cells that were labeled with Alexa488-transferrin then transferred to 37°C to allow internalization for 3 h, then washed with acid wash buffer to remove surface ligands. c NK92 cells that were pretreated with 10 ug/ml CPZ for 30 min, and internalized Alexa488-transferrin. d NK92 cells that were continuously treated with CPZ for 3 h and internalized Alexa488-transferrin; e: No treatment of NK92 cells were as a control. Data are representative of three independent experiments. (18_2010_377_MOESM2_ESM.tiff 467 kb)

Figure S3. Cytotoxicity of NK cells is attenuated by pretreatment with sucrose. a, b The cytotoxicity of NK92 cells against K562 cells. After incubation of 0.45 M sucrose pretreated or non-pretreated NK cells and CFSE pre-labeled K562 cells at indicated ratio in the graft for 3 h, all cells were stained with PI and analyzed by FCM. a: Upper right quadrant shows the K562 death rate. b The cytotoxicity was calculated with formula described in the Materials and methods section. c The cytotoxicity of NK92 cells to PEC. After NK cells with or without sucrose pretreatment and PEC cells were cocultured at indicated ratio in the graft for 3 h, NK cells were discarded and PEC survivals were stained with crystal violet. The absorbance value was measured to calculate the cytotoxicity. *: p < 0.05. Data are representative of three independent experiments. (18_2010_377_MOESM3_ESM.tiff 1173 kb)

Figure S4. Colocalization of CD63 and granzyme B. a NK cells with CPZ pretreatment (+CPZ) or non-treatment (−CPZ) were stimulated by PEC for 0 min or 60 min, then fixed, permeabilized, and non-specific binding sites blocked and incubated with anti-CD63 (1:200), anti-granzyme B (1:200) and FITC- or TRITC-conjugated secondary antibody (1:200), respectively. b After NK cells with CHC-specific RNAi pretreatment (CHC) or scrambled RNAi (scrambled) pretreatment were stimulated by PEC for 0 min or 60 min, colocalization of LAMP-1 and granzyme B was observed by immunostaining as described above. CD63 is shown in green. Granzyme B (GrmB) is shown in red. Nucleus is shown in blue. Scale bar is 10 μm. Data are representative of three independent experiments. (18_2010_377_MOESM4_ESM.tiff 721 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, P., Zheng, G., Yang, Y. et al. Granzyme B is recovered by natural killer cells via clathrin-dependent endocytosis. Cell. Mol. Life Sci. 67, 3197–3208 (2010). https://doi.org/10.1007/s00018-010-0377-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-010-0377-8

Keywords

Search

Navigation