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-α, 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 μmol/L), TNF-α (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 μmol/L), and 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 μmol/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-α 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-α. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. Conclusion: 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.
- Annexin V