Differential immunotoxic effects of inorganic and organic mercury species in vitro

Abstract

Despite the fact that humans are exposed to multiple forms of mercury (elemental, inorganic, and organic), most research on mercury toxicity has focused on methylmercury (MeHg) and on neurotoxic outcomes and mechanisms. Recent work has indicated that the immunotoxic effects of mercury compounds may be significant contributors to human disease as well as mechanistically relevant to other target organ toxicities. In this study, we compared the effects of inorganic Hg (iHg) to organic Hg species (MeHg and ethylmercury, EtHg) in human peripheral blood mononuclear cells (PBMCs) in vitro at sub-cytotoxic concentrations, using methods developed to characterize response of human PBMCs to iHg in vitro. PBMCs were isolated from six volunteer blood donors (three males and three females) and cultured in the presence and absence of lipopolysaccharide (LPS) and low levels (up to 200nM of each Hg species, separately) for 24h in culture. Cell culture supernatants were analyzed for cytokine concentrations with a bead-based multiplex assay. We report that iHg and MeHg both increase pro-inflammatory cytokine release in LPS-stimulated PBMCs, while EtHg decreases IFN-gamma release as well pro-inflammatory cytokine release. IL-17 release is significantly increased only in response to iHg treatment. Levels of anti-inflammatory cytokines (IL-1Ra and IL-10) were not significantly altered by any Hg treatment. These results indicate that both organic and inorganic species of Hg can affect the human immune system, but that they may exert different effects on immune function.

Figure 1. Low concentrations of inorganic, ethyl, or methylmercury do not affect cell viability

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. Cell viability was measured by MTT assay. The optical density for cells treated with LPS only was set to be 100% viability, and each treatment condition is compared to this control. No statistically significant changes in viability were observed.

Figure 2. TNF-α release by PBMCs over time

PBMCs isolated from a male volunteer were treated with iHg (up to 200 nM) in the presence of 50 ng/ml LPS. TNF-α was measured in cell culture supernatants after 12, 24, and 48 hours in culture. Error bars represent the standard error of the mean. TNF-α signficantly increased in a concentration response at 12 (P_[Hg]<0.001), 24 (P_[Hg]<0.001), and 48 (P_[Hg]<0.001) hours, though TNF-α levels were higher at the earlier time points.

Figure 3. TNF-α release increases in a concentration-dependent manner in response to iHg, but decreases in response to EtHg treatment

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. TNF-α was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean. MeHg does not significantly affect TNF-α release, where as EtHg significantly down-regulates its release and iHg significantly up-regulates its release.

Figure 4. IL-1β release increases in a concentration-dependent manner in response to iHg and MeHg, but decreases in response to EtHg treatment

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IL-1β was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean. iHg and MeHg significantly up-regulate IL-1β release, whereas EtHg significantly down-regulates its release.

Figure 5. IL-1Ra release is not significantly affected by treatment with iHg, EtHg, or MeHg

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IL-1Ra was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean.

Figure 6. IL-10 release is not significantly affected by treatment with iHg, EtHg, or MeHg

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IL-10 was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean.

Figure 7. IL-4 release is not significantly affected by treatment with iHg, EtHg, or MeHg

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IL-4 was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean.

Figure 8. IFN-γ release decreases in response to EtHg treatment

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IFN-γ was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. EtHg significantly down-regulates release of IFN-γ. Error bars represent the standard error of the mean.

Figure 9. IL-17 release increases in a concentration-dependent manner in response to iHg treatment

PBMCs isolated from six volunteers were treated with the specified concentrations of each mercury species (up to 200 nM) in the presence of 50 ng/ml LPS. IL-17 was measured in cell culture supernatants after 24 hours in culture. Results are shown for cells treated with iHg (A), EtHg (B), and MeHg (C). P_[Hg] represents the p-value for the trend in Hg effect in the concentration-response curve. Error bars represent the standard error of the mean.