PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro.
BACKGROUND
Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis.


OBJECTIVE
We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE2, TNF-alpha, and nitric oxide.


METHODS
Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 micromol/L), TNF-alpha (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 micromol/L), and 2,2;-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 micromol/L). Control cultures consisted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide.


RESULTS
PGE2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP2 receptor agonist, 11-deoxy PGE1, at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-alpha delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2, 2;-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE2, cyclic AMP, and TNF-alpha. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results.


CONCLUSIONS
Inhibition of spontaneous eosinophil apoptosis by PGE2 appears to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE2 may increase the proinflammatory potential of these cells in chronic asthma.
Asthma and Allergy Research Institute and Department of Medicine, University of Western Australia, Perth, Australia.
C D
CD
Peacock
N L
NL
Misso
D N
DN
Watkins
P J
PJ
Thompson
D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D053938:DNA Fragmentation; D015232:Dinoprostone / Q000494:pharmacology*; D004804:Eosinophils / Q000166:cytology*; D053222:Fas Ligand Protein; D005260:Female; D006801:Humans; D015848:Interleukin-5 / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology; D008563:Membrane Lipids / Q000097:blood; D008875:Middle Aged; D009589:Nitrogen Oxides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000097:blood; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017304:Annexin A5; D000911:Antibodies, Monoclonal; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D015848:Interleukin-5; D008562:Membrane Glycoproteins; D008563:Membrane Lipids; D009589:Nitrogen Oxides; D010718:Phosphatidylserines; D014409:Tumor Necrosis Factor-alpha; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone