Rat hepatic sinusoidal endothelial cells in monolayer culture. Biochemical and ultrastructural characteristics.

Sinusoidal endothelial cells were isolated by collagenase-pronase digestion of rat livers followed by centrifugal elutriation. The main endothelial cell fraction consisted of more than 85% endothelial cells as shown by electron microscopy and enzyme histochemistry. Contamination by Kupffer cells was less than 5%. The endothelial cells formed a coherent stable monolayer on dishes coated with collagen type IV in the presence of an RPMI 1640 medium supplemented with 4% Ultroser. Fc receptors were undetectable immediately after elutriation but reappeared after 12 h in culture. Von Willebrand factor (formerly factor VIII-related antigen) could not be detected unequivocally by immunofluorescence. Unchallenged endothelial cells did not produce eicosanoids. In the presence of free arachidonate, however, prostaglandins D2 and E2 as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha were detected by radioimmunoassay and by high-performance liquid chromatography analysis of [3H]arachidonate-exposed cells. Cells treated with the Ca2+ ionophore A23187 produced the same spectrum of immunologically measured prostanoids. In contrast to Kupffer cells in primary culture, eicosanoid formation by endothelial cells was neither triggered by phagocytotic stimuli nor suppressed by pretreatment with dexamethasone.

Beta-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture.

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.