| Summary: | Thesis (Ph.D.)--Memorial University of Newfoundland, 1999. Biochemistry Bibliography: leaves 227-298 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-y. 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-kB. 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-kB. 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-kB 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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