Essential Roles of PKA, iNOS, CD95/CD95L, and Terminal Caspases in Suppression of Eosinopoiesis by PGE2 and Other cAMP-Elevating Agents
Figure 6
NO is generated through iNOS in response to dibutyryl cAMP in the presence of IL-5. Bone-marrow cells from wild-type BALB/c ((a)–(c)) or C57BL/6 mice ((d) and (f)) or iNOS-deficient (iNOS KO) mutant C57BL/6 mice ((e) and insert in (f)) were preincubated with DAF-FM in HBSS/PhR- containing 100 M L-arginine and further incubated for 8 h at 37°C in this medium, in the presence of IL-5, alone or associated with 10−6 M dibutyryl cAMP (dbcAMP), aminoguanidine (AmGua), 10−4 M, or combinations of these agents. Cells were harvested and incubated with DAF-FM and NO-specific fluorescence was monitored by flow cytometry with a gate in the granulocyte region. (a)–(c): dot-plot of NO production as a function of FSC for cells in the gated region, in cultures exposed to IL-5 alone (a) or associated with dbcAMP (b) or with dbcAMP plus aminoguanidine (c). Numbers indicate the % of the gated cells in the positive zone. (d)-(e): histogram of fluorescence intensities (-axis) as a function of cell numbers (-axis), from one representative experiment out of 3. Gray profile: IL-5 alone. Continuous line: IL-5 plus dbcAMP. Broken line: IL-5 plus dbcAMP and AmGua (wild-type controls only). (f) and insert: mean fluorescence intensities (mean + SEM; ) from the same experiments as in (d)-(e), showing NO-specific fluorescence in wild-type control (f) and iNOS-deficient cultures (insert); (f), cultures exposed to IL-5 alone (black bar on the left) or associated with AmGua alone (white bar on the left, wild-type controls only), with dbcAMP (black bar on the right) or dbcAMP plus AmGua (white bar on the right, wild-type controls only); insert, cultures exposed to IL-5 alone (black bar) or associated with dbcAMP (white bar). (f): for the indicated difference.
