Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.

We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.