Xu-Hong Li1, Jian-Ping Gong, Yu-Jun Shi, Chang-An Liu, Yong Peng. 1. Department of General Surgery, Second College of Clinical Medicine & Second Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400010, China.
Abstract
OBJECTIVE: To observe expression of CD14 protein and its gene in Kupffer cells induced by lipopolysaccharide (LPS) and explore the role of CD14 in LPS-induced Kupffer cell activation. METHODS: Kupffer cells were isolated from Wistar rat livers by in situ collagenase digestion, followed by culture and incubation with 100 microg/ml LPS for 0, 30, 60 and 120 min, respectively. CD14 protein expressed on the membrane of Kupffer cells was examined using confocal microscopy and Western blotting analysis. Expression of CD14 mRNA in Kupffer cells was determined by reverse transcription polymerase chain reaction (RT-PCR). Tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: Expression of CD14 mRNA and CD14 protein in isolated Kupffer cells was upregulated quickly after LPS stimulation and increased with time. Likewise, there was a time-dependent increase of TNF-alpha and IL-6 in the supernatant with upregulation of CD14 expression. There were significant differences between normal and LPS-stimulated Kupffer cells (P<0.05). CONCLUSIONS: LPS can upregulate expression of CD14 protein and its gene in isolated Kupffer cells. CD14 may play an important role in the activation of LPS-induced Kupffer cells.
OBJECTIVE: To observe expression of CD14 protein and its gene in Kupffer cells induced by lipopolysaccharide (LPS) and explore the role of CD14 in LPS-induced Kupffer cell activation. METHODS: Kupffer cells were isolated from Wistar rat livers by in situ collagenase digestion, followed by culture and incubation with 100 microg/ml LPS for 0, 30, 60 and 120 min, respectively. CD14 protein expressed on the membrane of Kupffer cells was examined using confocal microscopy and Western blotting analysis. Expression of CD14 mRNA in Kupffer cells was determined by reverse transcription polymerase chain reaction (RT-PCR). Tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: Expression of CD14 mRNA and CD14 protein in isolated Kupffer cells was upregulated quickly after LPS stimulation and increased with time. Likewise, there was a time-dependent increase of TNF-alpha and IL-6 in the supernatant with upregulation of CD14 expression. There were significant differences between normal and LPS-stimulated Kupffer cells (P<0.05). CONCLUSIONS:LPS can upregulate expression of CD14 protein and its gene in isolated Kupffer cells. CD14 may play an important role in the activation of LPS-induced Kupffer cells.