Inhibition of Lipopolysaccharide Activation of Kupffer Cells by Transition Metals

Background

Bacterial endotoxins are the principal agents causing sepsis and septic shock. Cytokine cascades produced by cellular interactions to endotoxins can cause cardiovascular failure followed by multi-organ failure and death. Endotoxin intravenously administered to mice can have fatal consequences. Previous studies have shown that the transition metals Mn²⁺ and Cr³⁺ can be protective.

Methods

The effects of Mn²⁺, Cr³⁺, Zn²⁺, and Cu²⁺ on lipopolysaccharide (LPS) binding to rat Kupffer cell extracts were analyzed using dot-blots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western transfer. Kupffer cells were isolated from rat livers by collagenase perfusion, differential centrifugation, and adhesion to plastic.

Results

Five millimolar of Mn²⁺, Zn²⁺, Cr³⁺, and Cu²⁺ completely inhibited LPS binding. Isolated Kupffer cells were also exposed to Mn²⁺ and to LPS and tumor necrosis factor-alpha release measured. The presence of Mn²⁺ significantly (P < 0.05) reduced tumor necrosis factor-alpha production by Kupffer cells in response to LPS. Experiments to determine if these effects were mediated by binding to LPS-binding proteins showed this was not the case. More likely a complex occurs between the metal and LPS. We also showed significantly enhanced uptake of LPS into Kupffer cells in the presence of Mn^2+.

Conclusions

The data are consistent with the metals binding to LPS via its two phosphate groups and neutralizing their charge. These data also support the hypothesis that there is enhanced cellular uptake by non-receptor-mediated methods such as absorptive pinocytocis. At the same time receptor binding and activation of the cells is inhibited. This can explain the effects of transition metals on LPS toxicity.

Key Words: endotoxin, Kupffer cells, cytokines, transition metals