Cyclic AMP-elevating agents prolong or inhibit eosinophil survival depending on prior exposure to GM-CSF.
1. Purified human eosinophils survived for up to 7 days when cultured in vitro in the presence of 1 ng ml-1 granulocyte-macrophage colony stimulating factor (GM-CSF) with a viability of 73%. In the absence of GM-CSF, eosinophil viability decreased after one day in culture, and only 4% of cells were viable by day 4. 2. Culture of eosinophils with cholera toxin produced a concentration-dependent decrease in GM-CSF-induced survival at 7 days (IC50 = 7 ng ml-1) which was associated with a 6 fold increase in the intracellular cyclic AMP concentration. This inhibition of cell survival could be prevented by the addition of the protein kinase A inhibitor, H89 (10(-6)M). 3. When eosinophils were cultured with dibutyryl cyclic AMP, there was a concentration-dependent inhibition of GM-CSF-induced survival at 7 days with an IC50 of 200 microM. The related cyclic nucleotide analogue, dibutyryl cyclic GMP did not inhibit GM-CSF-induced eosinophil survival over the same concentration range. 4. Culture of eosinophils with forskolin, or with the phosphodiesterase inhibitors, rolipram and SK&F94120, had no effect on GM-CSF-induced eosinophil survival at any concentration examined. 5. After 7 days' culture in the absence of GM-CSF, fractionation of eosinophil DNA on agarose gels demonstrated a 'ladder' pattern characteristic of apoptosis. GM-CSF prevented DNA fragmentation and this protection could be overcome by both cholera toxin and dibutyryl cyclic AMP. 6. GM-CSF did not affect intracellular cyclic AMP concentrations in unstimulated eosinophils or in cells stimulated by cholera toxin. Thus, GM-CSF does not apparently increase eosinophil survival by affecting cyclic AMP levels. 7. In the absence of GM-CSF both cholera toxin and dibutyryl cyclic AMP decreased the rate of eosinophil death, when compared to cells cultured with medium alone. The t1/2 values for cell death were 1.63 +/- 0.3, 2.46 +/- 0.3 and 4.62 +/- 1.0 days for cells cultured in the presence of medium, cholera toxin and dibutyryl cyclic AMP respectively. 8. In conclusion, cyclic AMP exerts opposing effects on eosinophil survival depending on prior exposure of the cells to GM-CSF.
Hallsworth
M P
MP
Department of Allergy and Respiratory Medicine, Guy's Hospital, London.
Giembycz
M A
MA
Barnes
P J
PJ
Lee
T H
TH
D003994:Bucladesine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004249:DNA Damage; D004804:Eosinophils / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D013997:Time Factors
D010726:Phosphodiesterase Inhibitors; D005576:Colforsin; D003994:Bucladesine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D002772:Cholera Toxin; D000242:Cyclic AMP