Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor

Garry Michael Walsh, M L Williamson, F A Symon, G B Willars, A J Wardlaw

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

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. (C) 1996 by The American Society of Hematology.

Original languageEnglish
Pages (from-to)2815-2821
Number of pages7
JournalBlood
Volume87
Issue number7
Publication statusPublished - 1 Apr 1996

Keywords

  • HUMAN NEUTROPHILS
  • 3T3 FIBROBLASTS
  • ACTIVATION
  • EXPRESSION
  • SURVIVAL
  • ASTHMA
  • PROLIFERATION
  • PATHOGENESIS
  • VIABILITY
  • HYPODENSE

Cite this

Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor. / Walsh, Garry Michael; Williamson, M L ; Symon, F A ; Willars, G B ; Wardlaw, A J .

In: Blood, Vol. 87, No. 7, 01.04.1996, p. 2815-2821.

Research output: Contribution to journalArticle

Walsh, Garry Michael ; Williamson, M L ; Symon, F A ; Willars, G B ; Wardlaw, A J . / Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor. In: Blood. 1996 ; Vol. 87, No. 7. pp. 2815-2821.
@article{79f3ca488cbd4a908a04140a7c348c56,
title = "Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor",
abstract = "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. (C) 1996 by The American Society of Hematology.",
keywords = "HUMAN NEUTROPHILS, 3T3 FIBROBLASTS, ACTIVATION, EXPRESSION, SURVIVAL, ASTHMA, PROLIFERATION, PATHOGENESIS, VIABILITY, HYPODENSE",
author = "Walsh, {Garry Michael} and Williamson, {M L} and Symon, {F A} and Willars, {G B} and Wardlaw, {A J}",
year = "1996",
month = "4",
day = "1",
language = "English",
volume = "87",
pages = "2815--2821",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "7",

}

TY - JOUR

T1 - Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor

AU - Walsh, Garry Michael

AU - Williamson, M L

AU - Symon, F A

AU - Willars, G B

AU - Wardlaw, A J

PY - 1996/4/1

Y1 - 1996/4/1

N2 - 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. (C) 1996 by The American Society of Hematology.

AB - 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. (C) 1996 by The American Society of Hematology.

KW - HUMAN NEUTROPHILS

KW - 3T3 FIBROBLASTS

KW - ACTIVATION

KW - EXPRESSION

KW - SURVIVAL

KW - ASTHMA

KW - PROLIFERATION

KW - PATHOGENESIS

KW - VIABILITY

KW - HYPODENSE

M3 - Article

VL - 87

SP - 2815

EP - 2821

JO - Blood

JF - Blood

SN - 0006-4971

IS - 7

ER -