Activation of Peroxisome Proliferator-Activated Receptor γ by Nitric Oxide in Monocytes/Macrophages Down-Regulates p47phox and Attenuates the Respiratory Burst

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Abstract
NO appears as an important determinant in auto and paracrine macrophage function. We hypothesized that NO switches monocyte/macrophage function from a pro- to an anti-inflammatory phenotype by activating anti-inflammatory properties of the peroxisome proliferator-activated receptor (PPAR)γ. NO-releasing compounds (100 μM S-nitrosoglutathione or 50 μM spermine-NONOate) as well as inducible NO synthase induction provoked activation of PPARγ. This was proven by EMSAs, with the notion that supershift analysis pointed to the involvement of PPARγ. PCR analysis ruled out induction of PPARγ mRNA as a result of NO supplementation. Reporter assays, with a construct containing a triple PPAR response element in front of a thymidine kinase minimal promoter driving the luciferase gene, were positive in response to NO delivery. DNA binding capacity as well as the transactivating capability of PPARγ were attenuated by addition of the antioxidant N-acetyl-cysteine or in the presence of the NO scavenger 2-phenyl-4,4,5,6-tetramethyl-imidazoline-1-oxyl 3-oxide. Having established that NO but not lipophilic cyclic GMP analogs activated PPARγ, we verified potential anti-inflammatory consequences. The oxidative burst of macrophages, evoked by phorbol ester, was attenuated in association with NO-elicited PPARγ activation. A cause-effect relationship was demonstrated when PPAR response element decoy oligonucleotides, supplied in front of NO delivery, allowed to regain an oxidative response. PPARγ-mediated down-regulation of p47 phagocyte oxidase, a component of the NAD(P)H oxidase system, was identified as one molecular mechanism causing inhibition of superoxide radical formation. We conclude that NO participates in controlling the pro- vs anti-inflammatory phenotype of macrophages by modulating PPARγ.