Inhibition of cyclic-3′,5′-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-α production

Background. Local tumor necrosis factor (TNF)-alpha production by resident macrophages (Mphi) contributes to posttraumatic tissue injury. Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances Mphi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation. Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of Mphi TNF-alpha production through an influence on cAMP generation or degradation. It is unclear whether elevation of cellular cAMP inhibits LPS-stimulated TNF-alpha production by hypoxic Mphi. Moreover, it is unknown whether the synergism of hypoxia with LPS can be abrogated by promotion of cAMP generation or inhibition of cAMP degradation. Methods. Rat peritoneal Mphi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO2) or hypoxic (95% N-2 with 5% CO2) condition. TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay. The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 muM) and the adenylate cyclase activator forskolin (FSK; 50 muM) were applied to promote cAMP generation. The nonselective cyclic-3',5'-nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM) and the PDE Ill-specific inhibitor milrinone (200 muM) were used to inhibit cAMP degradation. The nondegradable cAMP analogue dibutyryl cAMP (db-cAMP; 100 muM) was applied to further determine the role of PDE. Results. Although hypoxia alone had a minimal effect on TNF-alpha production, it dramatically enhanced LPS-stimulated TNF-alpha production (4.08 +/- 0.28 ng/10(6) cells in hypoxia plus LPS vs 1.63 +/- 0.26 ng/10(6) cells in LPS, 2.5-fold, P < 0.01). Promotion of cAMP generation by either ISP or FSK reduced TNF-alpha production by hypoxic cells. However, neither of these two agents abolished the synergism of hypoxia with LPS (1.68 +/- 0.13 ng/10(6) cells in ISP plus hypoxia plus LPS vs 0.55 +/- 0.04 ng/10(6) cells in ISP plus LPS, threefold; 1.17 +/- 0.03 ng/10(6) cells in FSK plus hypoxia plus LPS vs 0.33 +/- 0.02 ng/10(6) cells in FSK plus LPS, 3.5-fold; both P < 0.01). Inhibition of cAMP degradation with IBMX reduced TNF-alpha production in hypoxic cells and abrogated the synergism (0.31 +/- 0.11 ng/10(6) cells in IBMX plus hypoxia plus LPS vs 0.27 +/- 0.04 ng/10(6) cells in IBMX plus LPS, P > 0.05), and the PDE III inhibitor milrinone had a comparable effect. Moreover, dbcAMP also attenuated TNF-alpha production with abrogation of the synergistic effect of hypoxia (0.56 +/- 0.08 ng/10(6) cells in dbcAMP plus hypoxia plus LPS vs 0.46 +/- 0.04 ng/10(6) cells in dbcAMP plus LPS, P > 0.05). Conclusions. The results show that elevation of cellular cAMP, either by promotion of generation or by inhibition of degradation, suppresses LPS-stimulated TNF-alpha production in hypoxic Mphi. It appears that hypoxia synergizes with LPS in the induction of Mphi TNF-alpha production through PDE-mediated cAMP degradation. Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of Mphi TNF-alpha production by hypoxia and LPS in posttraumatic tissue. (C) 2001 Elsevier Science.