Nitric oxide production and iNOS mRNA expression in mice induced by repeated stimulation with live Fusobacterium nucleatum

There have been few studies on the detection of direct nitric oxide (NO) production and interferon-gamma (IFN-gamma) in vivo without using animal cell culture. We questioned whether NO and IFN-gamma could be produced at the site of infection. The peritoneal cavity of mice was used as the local infection model. NO and IFN-gamma in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram-negative rod periodontal pathogen. The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat-killed bacteria (HKB) and those untreated: normal (N). These mice were compared on the basis of cell filtration, NO and IFN-gamma production by injection of live bacteria (LFn) or heat-killed bacteria (HKFn). In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn. A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn. At the same time iNOS enzyme activity and iNOS mRNA expression were detected. IFN-gamma, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn. At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages. These findings indicate that live bacteria play important roles in NO production by macrophages. It is suggested that NO may contribute to the inflammatory response during F. nucleatum infection in periodontitis.