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fMLP-induced in vitro nitric oxide production and its regulation in murine peritoneal macrophages
1School of Biotechnology, Banaras Hindu University, Varanasi, India. ajit.sodhi@lycos.com
Abstract:
Bacterial N-formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) are important mediators of monocyte/macrophage recruitment and activation at the sites of inflammation. In the current study, the role of nitric oxide (NO) in the activation of murine peritoneal macrophages to tumoricidal state in response to in vitro fMLP treatment has been investigated. Murine peritoneal macrophages on treatment with fMLP showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. L-NMMA, a specific inhibitor of L-arginine pathway, inhibited the fMLP-induced NO secretion and macrophage-mediated tumoricidal activity against P815 cells. These results indicate the L-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of fMLP-treated macrophages. The expression of iNOS protein and iNOS mRNA is also observed. The pharmacological inhibitors genistein, wortmannin, H7, PD98059, TPCK, and pertussis toxin (PTX) blocked the fMLP-induced NO production, suggesting the involvement of tyrosine kinases, PI3K, PKC, p42/44 MAPkinase, NF-kappa B, and G-proteins. The expression of phospho-p42/44 MAPK and phospho-I kappa B was also observed. The role of protein phosphatases in the above pathway has been suggested using the specific inhibitors of these phosphatases, i.e., okadaic acid and sodium orthovanadate.
Insights
Bacterial N-formyl peptides activate macrophages to kill tumors via nitric oxide (NO) production. This NO generation is dependent on L-arginine and involves multiple signaling pathways, including kinases and G-proteins.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Bacterial N-formyl peptides, like N-formyl-methionyl-leucyl-phenylalanine (fMLP), are key inflammatory mediators.
- Monocyte and macrophage activation are crucial for immune responses at inflammation sites.
Purpose of the Study:
- To investigate the role of nitric oxide (NO) in activating murine peritoneal macrophages to a tumoricidal state following fMLP treatment.
Main Methods:
- Murine peritoneal macrophages were treated with fMLP in vitro.
- Nitric oxide (NO) production and tumoricidal activity against P815 mastocytoma cells were measured.
- Inhibitors of the L-arginine pathway (L-NMMA) and various signaling molecules were used.
- Expression of inducible nitric oxide synthase (iNOS) and phosphorylated signaling proteins was analyzed.
Main Results:
- fMLP treatment induced dose- and time-dependent NO production and enhanced macrophage tumoricidal activity.
- L-NMMA inhibited fMLP-induced NO secretion and tumoricidal effects, indicating L-arginine dependence.
- Pharmacological inhibitors implicated tyrosine kinases, PI3K, PKC, p42/44 MAPK, NF-kappa B, and G-proteins in fMLP-induced NO production.
- Expression of iNOS, phospho-p42/44 MAPK, and phospho-I kappa B was observed.
Conclusions:
- L-arginine-dependent NO production is a key mechanism for fMLP-induced macrophage tumoricidal activity.
- Multiple signaling pathways, including tyrosine kinases, PI3K, PKC, p42/44 MAPK, NF-kappa B, and G-proteins, are involved in fMLP-mediated NO production.
- Protein phosphatases may also play a role in regulating this pathway.