Nitric Oxide Acts in Conjunction with Proinflammatory Cytokines to Promote Cell Death in Osteoblasts

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Abstract
Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-1β are known modulators of bone remodeling in vitro and in vivo. The same cytokines induce the production of nitric oxide (NO) in various cell types, including osteoblasts and osteoclasts, and NO has recently been implicated in the regulation of bone resorption. We investigated the relationship between NO levels and cell viability in MC3T3-E1, a well-characterized osteoblastic cell line. NO donors at high concentrations (≥0.5 mM) produce a significant cytotoxic effect over a 48 h period. Various combinations of the three cytokines strongly promote endogenous NO production, and high NO levels are correlated with the loss of cell viability. Although TNF-α produces NO-independent cytotoxicity, NO greatly enhances this cytotoxic effect. Human and mouse TNF-α differ in their cytotoxic effects, and human TNF-α induces lower levels of NO production. In cocultures of RAW 264.7 mouse macrophages stimulated with lipopolysaccharide and IFN-γ, and untreated MC3T3-E1 osteoblasts, addition of anti–TNF-α antibody and inhibition of NO synthesis have additive, protective effects on osteoblast viability. NO cytotoxicity involves an apoptotic mechanism. Our results underline the importance of NO and TNF-α as cytotoxic mediators in the osseous microenvironment and might explain the observed deficiency of bone formation in inflammatory sites.