Advertisement

Mast Cells pp 325-337 | Cite as

Induction of Mast Cell Apoptosis by a Novel Secretory Granule-Mediated Pathway

  • Fabio R. Melo
  • Sara Wernersson
  • Gunnar Pejler
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1220)

Abstract

Mast cells (MCs) have detrimental functions in the context of numerous pathologies, and regimens aimed at neutralizing MCs or individual MC products can thus be of therapeutic value. One way to target MCs in disease is to selectively induce MC apoptosis, but there is so far no agent available that selectively induces apoptosis in MCs. Mast cells are heavily loaded with secretory granules containing large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the secretory granules will thus lead to the release of serglycin-protease complexes into the cytosol. A potential consequence of this would be that the unleashed granular proteases cause apoptosis by proteolytic activation of proapoptotic compounds located in the cytosol. Indeed, we have recently found that MCs are highly sensitive to apoptosis induced by permeabilization of the secretory granules. In this chapter, we describe the methods used to study MC apoptosis induced by this novel, secretory granule-mediated pathway.

Key words

Mast cells Apoptosis Proteases Granules Caspases 

Abbreviations

AO

Acridine orange

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

EGTA

Ethylene glycol tetraacetic acid

FITC

Fluorescein isothiocyanate

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

LDH

Lactate dehydrogenase

LLME

H-Leu-Leu-OMe⋅HBr

NAO

Acridine orange 10-nonyl bromide

PI

Propidium iodide

PIPES

Piperazine-N,N′-bis(2-ethanesulfonic acid

R110

Rhodamine 110

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Z-DEVD-FMK

Z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone

Z-VAD-FMK

N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone

Notes

Acknowledgments

The authors of this article receive support from The Swedish Research Council, Formas, King Gustaf V 80-year Anniversary Fund, Torsten and Ragnar Söderberg Foundation, The Vårdal Foundation, The Swedish Society of Medicine, Åke Wiberg Foundation, Konsul Th C Bergh Foundation, and The Swedish Cancer Foundation.

References

  1. 1.
    Kalesnikoff J, Galli SJ (2008) New developments in mast cell biology. Nat Immunol 9: 1215–1223PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Bischoff SC (2007) Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev 7:93–104Google Scholar
  3. 3.
    Dawicki W, Marshall JS (2007) New and emerging roles for mast cells in host defence. Curr Opin Immunol 19:31–38PubMedCrossRefGoogle Scholar
  4. 4.
    Peachell P (2006) Regulation of mast cells by beta-agonists. Clin Rev Allergy Immunol 31: 131–142PubMedCrossRefGoogle Scholar
  5. 5.
    Lundequist A, Pejler G (2011) Biological implications of preformed mast cell mediators. Cell Mol Life Sci 68:965–975PubMedCrossRefGoogle Scholar
  6. 6.
    Karra L, Berent-Maoz B, Ben-Zimra M, Levi-Schaffer F (2009) Are we ready to downregulate mast cells? Curr Opin Immunol 21: 708–714PubMedCrossRefGoogle Scholar
  7. 7.
    Pejler G, Abrink M, Wernersson S (2009) Serglycin proteoglycan: regulating the storage and activities of hematopoietic proteases. Biofactors 35:61–68PubMedCrossRefGoogle Scholar
  8. 8.
    Pejler G, Åbrink M, Ringvall M, Wernersson S (2007) Mast cell proteases. Adv Immunol 95: 167–255PubMedCrossRefGoogle Scholar
  9. 9.
    Melo FR, Waern I, Ronnberg E, Abrink M, Lee DM, Schlenner SM, Feyerabend TB, Rodewald HR, Turk B, Wernersson S, Pejler G (2011) A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway. J Biol Chem 286:5423–5433PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Rönnberg E, Pejler G (2012) Serglycin- the master of the mast cell. Methods Mol Biol 836:201–217PubMedCrossRefGoogle Scholar
  11. 11.
    Ivanova S, Repnik U, Bojic L, Petelin A, Turk V, Turk B (2008) Lysosomes in apoptosis. Meth Enzymol 442:183–199PubMedCrossRefGoogle Scholar
  12. 12.
    Lutsenko GV (2010) Flow-cytometry assay for apoptosis using fluorophore 10-nonyl acridine orange. Biol Membrany 27:430–439Google Scholar
  13. 13.
    Boya P, Kroemer G (2008) Lysosomal membrane permeabilization in cell death. Oncogene 27:6434–6451PubMedCrossRefGoogle Scholar
  14. 14.
    Turk B, Turk V (2009) Lysosomes as “suicide bags” in cell death: myth or reality? J Biol Chem 284:21783–21787PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Fabio R. Melo
    • 1
  • Sara Wernersson
    • 1
  • Gunnar Pejler
    • 1
  1. 1.Department of Anatomy, Physiology and BiochemistrySwedish University of Agricultural SciencesUppsalaSweden

Personalised recommendations