Nitric oxide production by high molecular weight water-soluble chitosan via nuclear factor-kappaB activation.

High molecular weight water-soluble chitosan (WSC), having an average molecular weight of 300000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the effect of WSC on the production of nitric oxide (NO) in RAW 264.7 macrophages was evaluated. Water-insoluble chitosan alone has been previously shown to exhibit in vitro stimulatory effect on macrophages NO production. However, WSC had no effect on NO production by itself. When WSC was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of WSC on NO synthesis was shown 24 h after treatment with rIFN-gamma. The increased production of NO from rIFN-gamma plus WSC-stimulated RAW 264.7 macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine (N(G)MMA). The increase in NO synthesis was reflected, as an increased amounts of inducible NO synthase protein. In addition, synergy between rIFN-gamma and WSC was mainly dependent on WSC-induced tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) activation. The present results indicate that the capacity of WSC to increase NO production from rIFN-gamma-primed RAW 264.7 macrophages is the result of WSC-induced TNF-alpha secretion via the signal transduction pathway of NF-kappaB activation.