A COMPARATIVE-STUDY OF DIFFERENT METHODS FOR THE ASSESSMENT OF APOPTOSIS AND NECROSIS IN HUMAN EOSINOPHILS

Eosinophils, prominent cells in asthmatic inflammation, undergo apopto sis or programmed cell death following deprivation of contact with sur vival-promoting cytokines such as IL-5 and GM-CSF. The aim of this stu dy was to assess a number of techniques for the quantification of apop tosis 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' i n vitro for 48 h exhibited endonuclease DNA degradation, apoptotic mor phology, increased red autofluorescence and externalisation of phospha tidylserine (PS) as assessed by binding of FITC-labelled annexin V. An nexin 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 pr eparations 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 apopto sis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidi um iodide (PI) staining and flow cytometry revealed that, by 24 h, app roximately 75% of cells had compromised membrane integrity. Eosinophil s maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology an d levels of annexin V-FITC binding and PI uptake similar to that of fr eshly isolated cells, Staurosporine (10(-5) M) treatment of eosinophil s maintained in IL-5 or GM-CSF resulted in significant levels of apopt otic 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 morphol ogy. Exclusion of PI demonstrated membrane integrity at all time point s up to 6 h. Thus, eosinophils aged in vitro in the absence of viabili ty-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibi ted both membrane integrity and rapid apoptosis-associated morphologic al changes detected by single step Kimura staining which preceded exte rnalisation of PS. (C) 1998 Elsevier Science B.V. All rights reserved.