title: NO-Evoked macrophage apoptosis is attenuated by cAMP-induced gene expression.
abstract: BACKGROUND
  Previous work has suggested that an increase in expression of cyclooxygenase-2, concomitant formation of E-type prostanoids, and in turn intracellular cAMP conveys macrophage resistance against apoptosis.
  
  
  METHODS
  We analyzed the effects of lipophilic cAMP analogs on nitric oxide (NO)-induced apoptosis in RAW 264.7 macrophages and human primary monocyte-derived macrophages. Parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarization (DeltaPsi), analyzed using DiOC(6)(3). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c, and expression of the antiapoptotic protein Bcl-X(L). A cAMP response-element decoy approach confirmed cAMP-dependent gene induction.
  
  
  RESULTS
  We verified resistance of murine and human macrophages against NO donors such as S-nitrosoglutathione or spermine-NO by pre-exposing cells to lipophilic cAMP analogs or by pretreatment with lipopolysaccaride, interferon-gamma, and N(G)-nitroarginine-methylester for 15 hr. Cellular prestimulation decreased NO-evoked apoptosis, as apoptotic parameters were basically absent. Macrophage protection was not achieved during a short period of preexposure, i.e., 1 hr. To verify gene induction as the underlying protective principle, we treated RAW cells with oligonucleotides containing a cAMP-responsive element in order to scavenge cAMP response element-binding protein prior to its promoter-activating ability. Decoy oligonucleotides, but not an unrelated control oligonucleotide, weakened cAMP-evoked protection and re-established a p53 response following NO addition.
  
  
  CONCLUSIONS
  Gene induction by cAMP protects macrophages against apoptosis that occurs as a result of excessive NO formation. Decreasing programmed cell death of macrophages may perpetuate inflammatory conditions in humans when macrophages become activated in close association with innate immune responses.
authors.affiliation: Faculty of Medicine, Department of Medicine IV-Experimental Division, University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: von Knethen
authors.forename: F
authors.initials: F
authors.lastname: Brockhaus
authors.forename: I
authors.initials: I
authors.lastname: Kleiter
authors.forename: B
authors.initials: B
authors.lastname: Brüne
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D051546:Cyclooxygenase 2; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D008264:Macrophages / Q000166:cytology*; D008565:Membrane Proteins; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009841:Oligonucleotides / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000502:physiology; D015533:Transcriptional Activation
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D008565:Membrane Proteins; D020030:Nitric Oxide Donors; D009841:Oligonucleotides; D009569:Nitric Oxide; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases