Sphingomyelinase mediates macrophage activation by titanium particles independent of phagocytosis: A role for free radicals, NFκB, and TNFα

The manner in which wear debris initiates intracellular signaling and macrophage activation remains poorly understood. While particle phagocytosis has been implicated in this process, recent studies have shown that phagocytosis is not required for macrophage activation. We examined the hypothesis that titanium particles stimulate macrophages through membrane associated signaling events involving free radicals, sphingomyelinase, NFκB, and TNFα. Titanium particles stimulated peroxidation of linoleic acid, producing malondialdehyde, while neither lipopolysaccharide nor PBS pre-incubated with particles did, suggesting that the increased peroxidation is related to the presence of the particles themselves. Furthermore, particles stimulated sphingomyelin metabolism in a neutral sphingomyelinase (NSmase) containing cell free system; this effect was inhibited by glutathione, indicating that NSmase activation was due to titanium induced free radicals. Titanium particles also stimulated NSmase activity in cultures of ANA-1 murine macrophages. Addition of purified NSmase to ANA-1 cell cultures stimulated NFκB binding, increased transcriptional activity in cells transfected with NFκB responsive promoters, and induced TNFα expression. These effects were also inhibited by addition of glutathione. Similarly, glutathione inhibited the ability of titanium particles to induce NFκB signaling and TNFα expression in ANA-1 cells. The findings demonstrate that titanium particles generate free radicals and induce plasma membrane peroxidation and NSmase activation. NSmase, in turn, hydrolyzes sphingomyelin, with activation of the NFκB signaling pathway and induction of responsive genes, including TNFα. This study demonstrates a mechanism for phagocytosis-independent macrophage activation and defines the sphingomyelin cycle as a potential therapeutic target for the prevention of wear debris induced osteolysis.