Essential Roles of PKA, iNOS, CD95/CD95L, and Terminal Caspases in Suppression of Eosinopoiesis by PGE2 and Other cAMP‐Elevating Agents

Abstract

Up- and downregulation of eosinopoiesis control pulmonary eosinophilia in human asthma. In mice, eosinopoiesis is suppressed in vitro by prostaglandin E2 (PGE2) and in vivo by diethylcarbamazine, through a proapoptotic mechanism sequentially requiring inducible NO synthase (iNOS) and the ligand for death receptor CD95 (CD95L). We examined the roles of iNOS, cAMP-mediated signaling, caspases, and CD95L/CD95 in suppression of eosinopoiesis by PGE2 and other agents signaling through cAMP. Bone-marrow collected from BALB/c mice, or from iNOS-, CD95-, or CD95L-deficient mutants (and wild-type controls), was cultured with interleukin-5 (IL-5), alone or associated with PGE2, cAMP-inducing/mimetic agents, caspase inhibitor zVAD-fmk, iNOS inhibitor aminoguanidine, or combinations thereof, and eosinopoiesis was evaluated at various times. PGE2, added up to 24 hours of culture, dose-dependently suppressed eosinopoiesis, by inducing apoptosis. This effect was (a) paralleled by induction of iNOS in eosinophils; (b) duplicated by sodium nitroprusside, isoproterenol, and cAMP-inducing/mimetic agents; (c) prevented by protein kinase A inhibition. NO was produced through iNOS by dibutyryl-cAMP-stimulated bone-marrow. Overall, PGE2 and isoproterenol shared a requirement for four effector elements (iNOS, CD95L, CD95, and terminal caspases), which together define a pathway targeted by several soluble up- and downmodulators of eosinopoiesis, including drugs, mediators of inflammation, and cytokines.

Roles of 5-Lipoxygenase and Cysteinyl-Leukotriene Type 1 Receptors in the Hematological Response to Allergen Challenge and Its Prevention by Diethylcarbamazine in a Murine Model of Asthma

“…Previous studies [24, 25] had shown DEC effects to be dependent on inducible NO synthase, which is an essential part of a proapoptotic pathway activated by a variety of soluble ligands [30]. The present study makes it unlikely that DEC induces or activates iNOS directly, as no effect of DEC was observed in 5-LO-deficient mice.…”

“…Eosinopoiesis in liquid culture was strictly dependent on IL-5, and culture conditions were adequate for demonstrating both enhancing and suppressive effects [9, 29, 30]. Cells present in 7-day culture were resuspended, collected, counted, cytocentrifuged, and stained for eosinophil peroxidase (EPO; cyanide-resistant peroxidase), a murine eosinophil lineage-specific marker, present from the earliest precursors to terminally differentiated eosinophils [27, 28], and scored as detailed in [9].…”

α‐Galactosylceramide suppresses murine eosinophil production through interferon‐γ‐dependent induction ofNOsynthase andCD95

“…Eosinopoiesis and its modulation by various agents were strictly dependent upon IL‐5 (Gaspar‐Elsas et al ., 1997; 2000a; 2009). Where indicated, cultures received α‐GalCer (10 −6 –10 −12 M), together with indomethacin (10 −7 M), aspirin (10 −7 M) (Xavier‐Elsas et al ., ; Queto et al ., ), the iNOS inhibitor aminoguanidine (10 −4 M; Queto et al ., ) or the caspase inhibitor zVAD‐fmk (20 μM; de Luca et al ., ). Each agonist or inhibitor was added only once, immediately after IL‐5, at the beginning of the culture period.…”

“…We had previously characterized (Jones et al ., ; Gaspar‐Elsas et al ., ; Queto et al ., ) a proapoptotic mechanism for regulation of eosinophil production, dependent upon iNOS and the ligand for CD95/Fas (CD95L/FasL), which operates in vivo during airway allergen challenge of sensitized mice, and accounts for the therapeutic effect of diethylcarbamazine (DEC), a leukotriene synthesis inhibitor (Matthews and Murphy, ), in experimental asthma (Queto et al ., ). Although activation of this mechanism by DEC and other drugs (de Luca et al ., ) prevents the induction of eosinophilia by allergen (Masid‐de‐Brito et al ., ), its blockade by LTD 4 and non‐steroidal anti‐inflammatory drugs (NSAIDs) (Xavier‐Elsas et al ., ) protects developing eosinophils from apoptosis. Furthermore, dexamethasone protects developing eosinophils from apoptosis by preventing expression of iNOS (de Luca et al ., ).…”

“…Although activation of this mechanism by DEC and other drugs (de Luca et al ., ) prevents the induction of eosinophilia by allergen (Masid‐de‐Brito et al ., ), its blockade by LTD 4 and non‐steroidal anti‐inflammatory drugs (NSAIDs) (Xavier‐Elsas et al ., ) protects developing eosinophils from apoptosis. Furthermore, dexamethasone protects developing eosinophils from apoptosis by preventing expression of iNOS (de Luca et al ., ).…”