ABSTRACT: 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]-1? to mature IL-1?). 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-1? release induced by metal ions are oxidative stress-dependent; and 2. IL-1? release induced by metal ions is NF-?B signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone marrow-derived macrophages (BMDM) were exposed to Co2+ (6-48 ppm), Cr3+ (100-500 ppm), or Ni2+ (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-?B inhibitor (JSH-23). Culture supernatants were analyzed for caspase-1 by western blotting and/or IL-1? release by ELISA. Immunoblotting revealed the presence of caspase-1 (p20 subunit) in supernatants of BMDM incubated with Cr3+, but not with Ni2+ or Co2+. When L-AA (2 mM) was present with Cr3+, the caspase-1 p20 subunit was undetectable and IL-1? release decreased down to the level of the negative control, thereby demonstrating that caspase-1 activation and IL-1? release induced by Cr3+ was oxidative stress-dependent. ELISA demonstrated that Cr3+ induced the highest release of IL-1?, while Co2+ had no or limited effects. In the presence of Ni2+, the addition of L-AA (2 mM) also decreased IL-1? release, below the level of the negative control, suggesting that IL-1? release induced by Ni2+ was also oxidative stress-dependent. Finally, when present during both priming with LPS and activation with Cr3+, JSH-23 blocked IL-1? release, demonstrating NF-?B involvement. Overall, this study showed that while both Cr3+ and Ni2+ may be inducing inflammasome activation, Cr3+ is likely a more potent activator, acting through oxidative stress and the NF-?B signaling pathway.
