A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils

G Walsh, G Dewson, A J Wardlaw, F Levi-Schaffer, R Moqbel

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Abstract

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells, Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS. (C) 1998 Elsevier Science B.V. All rights reserved.

Original languageEnglish
Pages (from-to)153-163
Number of pages11
JournalJournal of Immunological Methods
Volume217
Issue number1-2
Publication statusPublished - 1 Aug 1998

Keywords

  • eosinophil
  • apoptosis
  • necrosis
  • annexin V
  • kimura stain
  • flow cytometric detection
  • cell-death
  • annexin-v
  • phosphatidylserine expression
  • phagocytosis
  • recognition
  • inhibition
  • macrophages
  • exposure
  • undergo

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