Effects of metal ions on caspase-1 activation and interleukin-1β release in murine bone marrow-derived macrophages

Ions released from metal implants have been associated with adverse tissue reactions and are therefore a major concern. Studies with macrophages have shown that cobalt, chromium, and nickel ions can activate the NLRP3 inflammasome, a multiprotein complex responsible for the activation of caspase-1 (a proteolytic enzyme converting pro-interleukin [IL]-1β to mature IL-1β). However, the mechanism(s) of inflammasome activation by metal ions remain largely unknown. The objectives of the present study were to determine if, in macrophages: 1. caspase-1 activation and IL-1β release induced by metal ions are oxidative stress-dependent; and 2. IL-1β release induced by metal ions is NF-κB signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone marrow-derived macrophages (BMDM) were exposed to Co(2+) (6–48 ppm), Cr(3+) (100–500 ppm), or Ni(2+) (12–96 ppm), in the presence or absence of a caspase-1 inhibitor (Z-WEHD-FMK), an antioxidant (L-ascorbic acid [L-AA]), or an NF-κB inhibitor (JSH-23). Culture supernatants were analyzed for caspase-1 by western blotting and/or IL-1β release by ELISA. Immunoblotting revealed the presence of caspase-1 (p20 subunit) in supernatants of BMDM incubated with Cr(3+), but not with Ni(2+) or Co(2+). When L-AA (2 mM) was present with Cr(3+), the caspase-1 p20 subunit was undetectable and IL-1β release decreased down to the level of the negative control, thereby demonstrating that caspase-1 activation and IL-1β release induced by Cr(3+) was oxidative stress-dependent. ELISA demonstrated that Cr(3+) induced the highest release of IL-1β, while Co(2+) had no or limited effects. In the presence of Ni(2+), the addition of L-AA (2 mM) also decreased IL-1β release, below the level of the negative control, suggesting that IL-1β release induced by Ni(2+) was also oxidative stress-dependent. Finally, when present during both priming with LPS and activation with Cr(3+), JSH-23 blocked IL-1β release, demonstrating NF-κB involvement. Overall, this study showed that while both Cr(3+) and Ni(2+) may be inducing inflammasome activation, Cr(3+) is likely a more potent activator, acting through oxidative stress and the NF-κB signaling pathway.