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Effects of inhibitors of ADP-ribosylation on macrophage activation
C Le Page1, C Pellat-Deceunynck, J C Drapier
1Unité 365 INSERM Interférons et Cytokines, Institut Curie, Section de Recherche, Paris, France.
Abstract:
Nitric oxide (NO) is a potent mediator involved in many biological functions including macrophage cytotoxicity and non-specific immunity against parasites, bacteria and viruses. Murine macrophages possess the capacity to express the inducible NO synthase (iNOS) which is not constitutively expressed but induced at the transcriptional level by interferon gamma (IFN-gamma) alone or synergistically with LPS. We have investigated the possible role of ADP-ribosylation reactions in the signaling pathway involved in NO synthase induction, since ADP-ribosylation has been reported to be involved in the expression of certain IFN-gamma and LPS-inducible genes. We found that inhibitors of ADP-ribosylation inhibited nitrite synthesis in RAW 264.7 macrophages after stimulation by IFN-gamma and LPS. These ADP-ribosylation inhibitors acted by preventing NO synthase mRNA induction, without inhibiting NO synthase enzyme activity. IRF-1, a transcription factor induced and activated by IFN-gamma was recently shown to be involved in iNOS induction. We showed that inhibitors of ADP-ribosylation had no effect on IFN-gamma-mediated mRNA induction of IRF-1 nor on its activation and binding to its target sequence in the iNOS gene. In addition, the inhibitors failed to impair the IFN-gamma-mediated antiviral activity against VSV virus. Since induction by IFN-gamma of IRF-1 and induction of the antiviral state proceed through the JAK/STAT signalling pathway, our results imply that ADP-ribosylation reactions are not involved in triggering this pathway. Although the precise mechanism requires further investigation, our results indicate that ADP-ribosylation is a crucial step restricted to the signalling pathway which leads to iNOS mRNA induction, as well as TNF and MHC class II induction during macrophage activation.
Insights
ADP-ribosylation inhibitors block nitric oxide synthase (NOS) mRNA induction in macrophages, revealing a key role in immune signaling pathways. This process is distinct from interferon-gamma-induced pathways.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Nitric oxide (NO) is vital for macrophage functions like cytotoxicity and immunity.
- Inducible nitric oxide synthase (iNOS) is upregulated by interferon-gamma (IFN-γ) and LPS.
- ADP-ribosylation's role in IFN-γ and LPS-inducible gene expression is under investigation.
Purpose of the Study:
- To investigate the role of ADP-ribosylation in the signaling pathway for nitric oxide synthase induction in macrophages.
- To determine if ADP-ribosylation is involved in IFN-γ-mediated signaling pathways.
Main Methods:
- RAW 264.7 macrophages were stimulated with IFN-γ and LPS.
- Inhibitors of ADP-ribosylation were used to assess their impact on nitrite synthesis and iNOS mRNA induction.
- The effect of inhibitors on IRF-1 induction, activation, and DNA binding was evaluated.
- IFN-γ-mediated antiviral activity against VSV was assessed.
Main Results:
- ADP-ribosylation inhibitors significantly reduced nitrite synthesis by preventing iNOS mRNA induction, not enzyme activity.
- These inhibitors did not affect IFN-γ-induced IRF-1 mRNA levels, activation, or DNA binding.
- IFN-γ-mediated antiviral activity remained unaffected by ADP-ribosylation inhibitors.
- Results suggest ADP-ribosylation is not involved in the JAK/STAT pathway triggered by IFN-γ.
Conclusions:
- ADP-ribosylation is a critical step specifically in the signaling pathway leading to iNOS mRNA induction in activated macrophages.
- This finding differentiates the iNOS induction pathway from the JAK/STAT pathway involved in IFN-γ responses.
- ADP-ribosylation also appears crucial for TNF and MHC class II induction during macrophage activation.