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 ap optosis. Objective: We have investigated the effects on eosinophil apoptosis of 3 me diators that are likely to be produced by the airway epithelium, namely PGE (2), TNF-alpha, and nitric oxide. Methods: Peripheral blood eosinophils from healthy adult volunteers were pu rified by density gradient centrifugation and negative immunomagnetic selec tion. Eosinophils were cultured for 16 or 40 hours with PGE(2) (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 mu mol/L), TNF-alpha (5 00 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 mu mol/L), and 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dib utyryl cyclic guanosine monophosphate (100 mu mol/L). Control cultures cons isted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL ) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of a nnexin V-FITC binding to externalized phosphatidylserine, by electrophoresi s of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assess ment of hypodiploid DNA with propidium iodide, Results: PGE(2) and cyclic AMP inhibited spontaneous eosinophil apoptosis a t both 16 and 40 hours as did the PGEP(2) receptor agonist, 11-deoxy PGE(1) , at 40 hours, but these effects were not inhibited by a protein kinase A a ntagonist. 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 red uced the inhibitory effects of PGE(2), cyclic AMP, and TNF-alpha. Assessmen ts of apoptosis by DNA fragmentation gave similar but quantitatively less s ensitive results. Conclusion: Inhibition of spontaneous eosinophil apoptosis by PGE(2) appear s to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE(2) may increase the proinflammatory pot ential of these cells in chronic asthma.