Intracellular calcium alterations and free radical formation evaluated by flow cytometry in endotoxin-treated rat liver Kupffer and endothelial cells

Abstract

During endotoxic shock, the liver exerts an endotoxin (lipopolysaccharide, LPS) clearance function with the participation of both sinusoidal (mainly Kupffer and endothelial cells) and parenchymal cells. In order to determine the specificity and diversity of response of each liver cell type, the effect of Escherichia coli 0111:B4 endotoxin (LPS) on intracellular Ca2+ content and reactive oxygen metabolite production in rat liver Kupffer, endothelial and parenchymal cells, was evaluated by flow cytometry during short treatment times (from 0-2 min) with a low dose of LPS (10 micrograms/ml). Concerning sinusoidal cells, LPS produced a significant increase of intracellular Ca2+ in both endothelial and Kupffer cells. The LPS effect on Kupffer cells was higher than on endothelial cells. When intracellular reactive oxygen metabolite production was evaluated in LPS-treated sinusoidal cells, a fast and significant increase of Kupffer cells in activated state (cells with a high reactive oxygen intermediate production) was observed. However, endothelial cells did not show LPS-induced changes in their intracellular reactive oxygen metabolite content. All these results support a rapid activation of liver Kupffer cells by endotoxin consistent with the major role of this cellular type as active first line of defense during endotoxic shock. The liver endothelial cells are also involved in the first steps of the cell damage showing intracellular Ca2+ alterations. Liver parenchymal cells did not show any response at these experimental conditions (short treatment time and low LPS dose) indicating that longer treatment times are needed for LPS binding and action in agreement with previous studies.