Dose- and time-dependent changes in viability and IL-6, CXCL8 and CCL2 production by HaCaT-cells exposed to cobalt. Effects of high and low calcium growth conditions

Background Sensitization requires exposure to an allergen with subsequent production of a danger signal. In the skin, keratinocytes are the main producers of these signals. Objective To compare dose- and time-effects of cobalt on the viability of and cytokine release from HaCaT cells cultured at low or high calcium. Method To model two separate states of differentiation of keratinocytes, HaCaT cells were cultured under low or high calcium conditions. HaCaT were exposed to different concentrations of cobalt chloride (10 mu m to 5 mM) over time (30 minutes- 48 hours). Cell viability was measured with the Cell-Titer Blue Viability assay. Cytokine production was measured using a bead-based immunoassay and flow cytometry. Gene expression was quantified using qPCR. Data was analyzed by ANOVA and linear mixed model. Results Viability of the cells was dose- and time-dependent. A linear mixed statistical model showed that cobalt exposure induces increase in IL-6, CXCL8 and CCL2 production over time and whereas increase of IL-6 and a decrease of CCL2 was associated with increasing cobalt chloride concentrations. When comparing the cells incubated under high and low calcium conditions, the more differentiated cells in the high concentration were found to exert a stronger response in terms of IL-6 release. Conclusions Our data suggest that cobalt chloride triggered an alarm system in HaCaT cells, and proinflammatory cytokines/chemokines were secreted in a dose- and time-dependent manner. When high and low calcium incubations were compared, the difference was seen only for IL-6. These findings indicate that the effect of cobalt chloride on cell toxicity occurs throughout the living epidermis.

Automated summary

The study compared the dose- and time-effects of cobalt on the viability and cytokine release of HaCaT cells cultured at low or high calcium conditions. Results showed that cobalt exposure triggered the production of proinflammatory cytokines and chemokines in a dose- and time-dependent manner, with more differentiated cells in high calcium conditions exhibiting a stronger response in terms of IL-6 release. This suggests that the effect of cobalt chloride on cell toxicity occurs throughout the living epidermis.