LIGATION OF CD69 INDUCES APOPTOSIS AND CELL-DEATH IN HUMAN EOSINOPHILS CULTURED WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR

Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell d eath in vitro unless cultured in the presence of cytokines such as gra nulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane an tigen expressed by cytokine-activated, but not freshly isolated, PB hu man eosinophils. We have examined the effect of ligation of CD69 by sp ecific monoclonal antibody (MoAb) on the viability of human eosinophil s cultured with recombinant human (rh)GM-CSF. Eosinophils were purifie d 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 c ontrols (anti-CR3 or isotype-matched control MoAb) were added, Viabili ty 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 apopt osis 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 liga tion. More apoptotic eosinophils were observed at later time-points an d this was associated with loss of viability. At 120 hours post-additi on of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable c ompared with 84% +/- 3.4% for the CR3 control (P <.001). A F(ab)(2) fr agment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosi nophils. A more rapid induction of eosinophil apoptosis was obtained w ith CD69 MoAb immobilized via their Fc portions on protein-A coated pl astic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-lig ation. 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 apopt osis induction nor were there detectable quantities of TGF beta 1 in s upernatants from GM-CSF-cultured eosinophils ligated with CD69 or cont rol MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling med iated by perturbation of CD69. This may represent an important physiol ogic mechanism for eosinophil removal in vivo. (C) 1996 by The America n Society of Hematology.