title: Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-alpha production.
abstract: BACKGROUND
  Local tumor necrosis factor (TNF)-alpha production by resident macrophages (M phi) contributes to posttraumatic tissue injury. Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances M phi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation. Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of M phi 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 M phi. 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 M phi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO(2)) or hypoxic (95% N(2) with 5% CO(2)) condition. TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay. The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 microM) and the adenylate cyclase activator forskolin (FSK; 50 microM) 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 III-specific inhibitor milrinone (200 microM) were used to inhibit cAMP degradation. The nondegradable cAMP analogue dibutyryl cAMP (dbcAMP; 100 microM) 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 M phi. It appears that hypoxia synergizes with LPS in the induction of M phi TNF-alpha production through PDE-mediated cAMP degradation. Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of M phi TNF-alpha production by hypoxia and LPS in posttraumatic tissue.
authors.affiliation: Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. xianzhong.meng@uchsc.edu
authors.forename: X
authors.initials: X
authors.lastname: Meng
authors.forename: L
authors.initials: L
authors.lastname: Ao
authors.forename: B D
authors.initials: BD
authors.lastname: Shames
authors.forename: A H
authors.initials: AH
authors.lastname: Harken
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D000818:Animals; D000242:Cyclic AMP / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D000860:Hypoxia / Q000276:immunology*; D008070:Lipopolysaccharides / Q000633:toxicity*; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases