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.