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Ligation of CD69 induces apoptosis and cell death in human eosinophils
cultured with granulocyte-macrophage colony-stimulating factor
GM Walsh, ML Williamson, FA Symon, GB Willars and AJ Wardlaw
Department of Respiratory Medicine, Leicester University School of
Medicine, Glenfield Hospital, UK.
Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death
in vitro unless cultured in the presence of cytokines such as
granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their
survival is prolonged for up to 10 days. CD69 is a type II membrane antigen
expressed by cytokine-activated, but not freshly isolated, PB human
eosinophils. We have examined the effect of ligation of CD69 by specific
monoclonal antibody (MoAb) on the viability of human eosinophils cultured
with recombinant human (rh)GM-CSF. Eosinophils were purified by
immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen
hours after the start of culture, a panel of CD69 MoAb or controls
(anti-CR3 or isotype-matched control MoAb) were added. Viability was
assessed by trypan blue exclusion and apoptosis by morphologic assessment,
DNA laddering, and flow cytometric analysis of eosinophil red
autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48
hours after addition of anti- CD69 MoAb compared with less than 10%
apoptosis for CR3 or the isotype matched control. The majority of apoptotic
eosinophils excluded trypan blue at 48 hours post CD69 ligation. More
apoptotic eosinophils were observed at later time-points and this was
associated with loss of viability. At 120 hours post-addition of the
anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with
84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69
MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more
rapid induction of eosinophil apoptosis was obtained with CD69 MoAb
immobilized via their Fc portions on protein-A coated plastic 96 well
plates. Ligation of CD69 or CR3 resulted in the release of comparable
quantities of eosinophil peroxidase at 48 hours post-ligation. These levels
of EPO were consistent with the viability of these cells at 48 hours as
assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF
beta 1 had no effect on CD69-dependent apoptosis induction nor were there
detectable quantities of TGF beta 1 in supernatants from GM-CSF-- cultured
eosinophils ligated with CD69 or control MoAb. These results suggest that
eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a
result of cell signalling mediated by perturbation of CD69. This may
represent an important physiologic mechanism for eosinophil removal in
vivo.
Volume 87,
Issue 7,
pp. 2815-2821,
04/01/1996
Copyright © 1996 by The American Society of Hematology
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