Eosinophils isolated with two different methods show different characteristics of activation.
Eosinophils can be isolated from a mixed suspension of granulocytes by different procedures. We compared functional responses of human eosinophils purified according to two different principles: (1) an fMLP-induced difference in specific gravity between eosinophils and neutrophils and (2) selective removal of neutrophils by means of immunomagnetic beads coated with CD16 mAb. The results showed that eosinophils isolated with the CD16 beads method have a higher capacity to synthesize platelet activating factor (PAF) after stimulation with serum-treated zymosan (STZ) than eosinophils purified with the fMLP method. Binding of STZ and subsequent activation of the respiratory burst were also increased in CD16-isolated eosinophils. Furthermore, eosinophils isolated with the CD16 beads showed stronger chemotactic responses towards C5a and PAF. The difference in activity of these eosinophil preparations might be explained by a loss of the more active cells during the isolation with the fMLP method: only 30-60% of the eosinophils were recovered with this method, in contrast to a recovery of more than 95% with the CD16 beads method. Indeed, this 'lost' population of eosinophils, subsequently purified with CD16-coated beads, had a higher respiratory burst activity. The alternative explanation, i.e., an enhancement of eosinophil function by the beads method, appeared not to be valid, because repurification of fMLP-isolated eosinophils in the presence of fresh neutrophils and CD16-coated beads did not change the reactivity of the eosinophils. We conclude that the fMLP method leads to selective purification of eosinophils with a resting (or 'unprimed') phenotype.
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
M
M
Blom
A T
AT
Tool
F P
FP
Mul
E F
EF
Knol
D
D
Roos
A J
AJ
Verhoeven
D015703:Antigens, CD / Q000096:biosynthesis; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000502:physiology; D004804:Eosinophils / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000096:biosynthesis; D017452:Receptors, IgG / Q000276:immunology; D016897:Respiratory Burst / Q000502:physiology; D015054:Zymosan / Q000276:immunology
D015703:Antigens, CD; D010972:Platelet Activating Factor; D017452:Receptors, IgG; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015054:Zymosan