Abstract
To investigate the expression of CD14 on Kupffer cells during the course of carbon tetrachloride (CCl(4))-mediated liver injury and its role in the activation of Kupffer cells. Rats were administered CCl(4) twice weekly for up to 8 weeks. Kupffer cells were isolated from normal and CCl(4)-treated rats by the combined 'collagenase-pronase' perfusion method, discontinuous density gradient centrifugation. On the day after isolation, the cells were incubated with RPMI-1640 containing varying doses of lipopolysaccharide (LPS) for 6 h. Supernatants were then collected for measuring the concentration of tumor necrosis factor-alpha (TNF-alpha) by enzyme-linked immunosorbent assay (ELISA). The expression of CD14 mRNA on Kupffer cells were determined by RT-PCR. The plasma concentrations of endotoxin were determined by chromogenic substrate Limulus amebocyte lysate assay. Basic TNF-alpha production of Kupffer cells isolated from CCl(4)-treated rats at 4 and 6 weeks was significantly higher than that of normal (P < 0.05). Following LPS stimulation the production of TNF-alpha was markedly increased in Kupffer cells from the 2-, 4- and 6-week treatment groups (P < 0.05). Moreover, LPS-induced TNF-alpha production was dose-dependent. CD14 mRNA expression on Kupffer cells isolated from CCl(4)-treated rats was elevated following 2 weeks of CCl(4) administration and the maximum elevation occurred at 6 weeks. Gene expression was decreased in Kupffer cells after 8 weeks of CCl(4) treatment. CCl(4) administration elicited extensive changes in liver morphology, including steatosis, inflammation and necrosis. The plasma concentrations of endotoxin of CCl(4)-treated rats were increased during the time of liver injury. Up-regulation of CD14 expression in Kupffer cells during CCl(4)-mediated chronic liver injury indicates cell activation and that they are more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.
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