Role of Oxidative Stress and NF-κB in Metal Ion-induced Interleukin-1β Release by Murine Bone Marrow-Derived Macrophages

Ferko, Maxime-Alexandre (Department of Mechanical Engineering; University of Ottawa)
Catelas, Isabelle (Department of Mechanical Engineering, Department of Surgery, Department of BMI; University of Ottawa)


Metal ions released from implantable alloys (e.g., CoCrMo and stainless steel) have been associated with adverse tissue reactions that can limit implant longevity (1). Previous studies have shown that Co2+, Cr3+, and Ni2+ can induce oxidative stress in macrophages (2), as well as activate the NLRP3 inflammasome (3), a multiprotein complex responsible for the activation of caspase-1, which in turn activates pro-interleukin(IL)-1β, a potent pro-inflammatory cytokine. In addition, activation of the NRLP3 inflammasome is known to require an earlier priming step involving the NF-κB transcription factor (4). However, the mechanisms by which metal ions activate the NLRP3 inflammasome in macrophages remain largely unknown. The objectives of this study were to: 1. determine if metal ion-induced IL-1β release by macrophages is oxidative stress-dependent; and 2. determine if metal ion-induced IL-1β release is NF-κB-dependent.

Materials and Methods

This research has been approved by the local animal care committee. Bone marrow-derived macrophages (BMDM) prepared from 4- to 16-week old female wild-type C57BL/6J mice (The Jackson Laboratory) were resuspended at 300,000 cells/well in 24-well plates.

The BMDM were primed for 6h with 500 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich) in a humidified environment at 370C and 5% CO2. The cells were then exposed to 18 ppm Co2+ (Fisher Scientific), 300 ppm Cr3+ (Sigma-Aldrich), or 48 ppm Ni2+ (Sigma-Aldrich) for 18-24h. These ion concentrations have been shown to induce the highest IL-1β release (5). Cells incubated without metal ions and with 5 μM nigericin (Cayman Chemical) were used as a negative and positive control, respectively. To analyze the effects of oxidative stress, 2 mM L-ascorbic acid (L-AA; Sigma-Aldrich), an oxidative stress inhibitor, was added during the incubation with the ions. To analyze the effects of NF-κB, 60 μM JSH-23 (Cayman Chemical), an inhibitor of NF-κB, was added during either priming (with LPS), activation (with the ions), or both priming and activation incubations. In all cases, supernatants were snap-frozen for storage at -800C at the end of the incubation with the ions, and later analyzed by western blot for active caspase-1 detection and/or by ELISA (Thermo Fisher Scientific) for IL-1β release.

Statistical analysis was performed using two-way analysis of variance (ANOVA) and the Tukey-Kramer post-hoc test. p<0.05 was considered significant.


Immunoblotting results revealed the presence of the cleaved p20 subunit of caspase-1 in supernatants of BMDM incubated with Cr3+, but not with Ni2+ or Co2+ (Fig. 1). Importantly, the cleaved subunit, present with 300 ppm Cr3+, was undetectable when 2 mM L-AA was also present.

ELISA results showed that the presence of 2 mM L-AA induced a decrease in IL-1β release with both 300 ppm Cr3+ (Fig. 2A) and 48 ppm Ni2+ (Fig. 2B), down to or below the level of the negative control (p<0.001 in both cases). Co2+ did not induce a significant IL-1β increase (Fig. 2C).

ELISA results also revealed that when present during both the 6h-LPS priming incubation and the 18-24h incubation with metal ions, 60 μM JSH-23 induced a decrease of 89% in IL-1β release (p<0.001), down to the level of the negative control (Fig. 3). When present during either the 6h-LPS priming incubation or the 18-24h-ion incubation, 60 μM JSH-23 induced only a partial decrease of 67% and 65% in IL-1β release (p<0.001 in both cases), respectively, and the levels remained higher than those in the negative control.

Discussion and Conclusion

Results showed oxidative stress-dependent activation of caspase-1 and release of IL-1β in BMDM exposed to Cr3+, suggesting the activation of the NLRP3 inflammasome and pointing to oxidative stress as a mediator. The absence of active caspase-1 by western blotting with Ni2+ (possibly because of signal below detection limit), coupled with the much lower IL-1β release  suggests that Cr3+ is a more potent activator of the NLRP3 inflammasome than Ni2+, in agreement with our previous study (5). Interestingly, active caspase-1 was detected in the lysates of THP-1 monocytes exposed to 90 ppm Ni2+ (6). This apparent discrepancy may be due to differences in cell types and experimental design. Finally, together with our previous study showing a marginal increase in IL-1β release with Co2+ (5), the absence of IL-1β release with Co2+ in the present study suggests that this ion has either a limited or no effect on the activation of the NLRP3 inflammasome pathway.

The inhibition of Cr3+-induced IL-1β release in the presence of JSH-23 during both the priming and activation incubations suggests that Cr3+-induced IL-1β release (a probable indicator of inflammasome assembly) is dependent on the NF-κB pathway. The relative involvement of this pathway during the priming and activation incubations remains, however, to be further investigated. It is possible that the partial, as opposed to complete, inhibition of IL-1β in the presence of JSH-23 during only the priming or the activation incubation may have been due to residual LPS on cell membranes and culture substrate surfaces during the activation incubation.

Overall, this study shows that Cr3+-induced IL-1β release by macrophages, a likely consequence of inflammasome activation, is dependent on oxidative stress and on the NF-κB pathway.

Fig. 1: Caspase-1 activation in BMDM after 18-24h with no ions, Cr3+, Ni2+, or Co2+, with and without L-AA. Representative immunoblot of 3 experiments.

Fig. 2: IL-1β release by BMDM after 18-24h with (A) Cr3+, (B) Ni2+, or (C) Co2+, with and without L-AA. * and †: significant difference (p<0.05) between a given condition and the neg. control (no ions, no L-AA) and a given condition with and without L-AA, respectively. Means ± SEM of 3 experiments.

Fig. 3: IL-1β release by BMDM after 18-24h with Cr3+ with and without JSH-23. * and †: significant difference (p<0.05) between a given condition and the neg. control (no ions, no JSH-23) and a given condition with and without JSH-23, respectively. Means ± SEM of 3 experiments.


This study was supported by the Canadian Institutes of Health Research (CIHR) and the Canada Research Chairs (CRC) program (I.C.). The authors thank Hallie Arnott for data collection, as well as Dr. Eric Lehoux and Kriti Kumar for technical assistance.


1. Magone K, et al. Arch Orthop Trauma Surg 2015;135(5):683–695; 2. Niki Y, et al. Biomaterials 2003;24(8):1447–1457; 3. Caicedo MS, et al. J Orthop Res 2009;27(7):847–854; 4. Bauernfeind FG, et al. J Immunol 2009;183(2):787–791; 5. Ferko M-A, et al. 33rd Annual Meeting of the Canadian Biomaterials Society, Winnipeg, MB, 2017; 6. Li X, et al. Inflammation 2014;37(2):457–466.

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