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NO-Evoked Macrophage Apoptosis Is Attenuated by cAMP-Induced Gene Expression

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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.

Materials and 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 (ΔΨ), analyzed using DiOC6(3). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c, and expression of the antiapoptotic protein Bcl-XL. 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-γ, and NG-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.

Conclusion

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.

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Acknowledgments

We thank Sabine Häckel for expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe.

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Authors and Affiliations

  1. Faculty of Medicine, Department of Medicine IV-Experimental Division, University of Erlangen-Nürnberg, Loschgestrasse 8, 91054, Erlangen, Germany

    Andreas von Knethen, Florian Brockhaus, Ingo Kleiter &  Bernhard Brüne

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  1. Andreas von Knethen
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  2. Florian Brockhaus
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  3. Ingo Kleiter
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  4. Bernhard Brüne
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Correspondence to Bernhard Brüne.

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von Knethen, A., Brockhaus, F., Kleiter, I. et al. NO-Evoked Macrophage Apoptosis Is Attenuated by cAMP-Induced Gene Expression. Mol Med 5, 672–684 (1999). https://doi.org/10.1007/BF03401986

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