Expression of CD14 in Kupffer′s cells induced by Lipopolysaccharide / 第三军医大学学报

ABSTRACT

Objective　To investigate the expression of LPS receptor-CD14(CD14) on the membrane of Kupffer′s cells (KCs) induced by Lipopolysaccharide (LPS )and its role in activation of KCs and production of cytokines. Methods　KCs were isolated by collagenase perfused Wistar rats and routinely cultured in 24-well dishes for 12 h. Cells were harvested and adjusted to a concentration of 1×106/ml/well and were devided into two groups. Group of LPS KCs were induced with different concentration of LPS (0, 100 ng/ml, 1 μg/ml and 100 μg/ml). Group of PI-PLC KCs were pre-incubated for 30 min with one unit of phophatidy linositol specific phospholipase C (PI-PLC) before different concentrations of LPS were added. KCs were cultured for 30 and 60 min respectively. Supernatants were then collected for measuring the level of TNFα and IL-6. Cells were stained by indirect immunofluorescent method ( rabbit anti-CD14 antibody and goat anti-rabbit IgG conjugated with FITC ) and analyzed with flow cytometer (FCM). The percentage and mean fluorescence intensity (FI) of CD14-positive cells were taken as the indexes. Results　In LPS group, after incubation of cells with increasing concentration of LPS, a significant increase in the percentage of CD14 positive KCs were found and the mean FI was stronger when compared with the control points or the group of PI-PLC. The levels of TNFα and IL-6 in supernatant also increased (P<0.01). In group of PI-PLC, decreased percentage of CD14 positive KCs and weakened mean FI were found when compared with group of LPS. The increasing production of TNFα and IL-6 slowed down in the group of PI-PLC. Conclusion　CD14 expression of KCs might be up-regulated by LPS with increase of some cytokines. The production of cytokines in KCs induced by LPS is partially inhibited by PI-PLC.