Regulation of nitric oxide synthesis and superoxide generation by C-reactive protein

Ratnam, Shobhitha (1999) Regulation of nitric oxide synthesis and superoxide generation by C-reactive protein. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (40MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Activated macrophages utilize both nitric oxide (NO) and superoxide (O2˙￣) for defense against microbes. During periods of inflammation, cells of the immune system are activated by microbial products and/or cytokines, triggering the induction of immune/inflammatory nitric oxide synthase (iNOS) and the assembling of NADPH oxidase complex resulting in the generation of NO and O2˙￣. However, simultaneous generation of NO and O2˙￣ could be harmful to host cells since this may lead to the production potentially toxic molecules. Therefore, the regulation of the production of NO and O2˙￣ is critical to host survival. Biological response to tissue injury or infection is characterized by an acute phase response that includes the amplified synthesis of C-reactive protein (CRP). Previous reports have indicated that CRP modulates O2˙￣ generation. Therefore, this thesis has examined the role of CRP in the regulation of NO synthesis and O2˙￣ generation in a macrophage model. -- CRP stimulated NO synthesis in rat peritoneal macrophages and in the murine 264.7 cell line alone or in synergy with interferon-γ. The CRP-induced increase in NO production was due to an increased synthesis of the inducible isoform of iNOS preceded by an increase in steady state iNOS mRNA level. -- CRP decreased the production of O2˙￣ by stimulated macrophages, and activated phosphatidylcholine-directed phospholipase C (PC-PLC). Decreased production of O2˙￣ corresponded with the activation of PC-PLC. In contrast, PC- PLC activation was found to be necessary in CRP-mediated signaling leading to iNOS transcription. Increased PC hydrolysis may be one mechanism cells use to avoid simultaneous generation of NO and O2˙￣. -- CRP also activated extracellular signal-regulated kinase (ERK) signaling cascade and increased the phosphorylation of I-κB. Studies with various cell- permeable inhibitors of PC hydrolysis and tyrosine kinases suggested that PC- PLC activation and tyrosine phosphorylation are essential for CRP-mediated phosphorylation of ERK and I-κB. Furthermore, the induction of iNOS by CRP was shown to be at least partly mediated by the activation of ERK signaling pathway resulting in I-κB phosphorylation and the ensuing iNOS transcription. -- The specific role of CRP in host defense was examined by using Chlamydia trachomatis, an intracellular pathogen. CRP decreased the viability of C. trachomatis , and this effect was reversed by blocking NO production with NG- monomethyl L- arginine (NMMA), a competitive inhibitor of iNOS. This suggested that the CRP-induced increase in NO production during inflammatory periods plays an important role in the protection against microbial pathogens.

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