Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease.
OBJECTIVE
To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).


METHODS
Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group was fed ethanol (dose of 5g-12 g/kg/d) and control group received dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM) or the reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-alpha and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.


RESULTS
In ethanol-fed group, the percentages of FITC-CD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 % and 5.38 %), the difference was significant (P<0.05). The expressions of CD14 mRNA were 7.56+/-1.02 and 8.74+/-1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77+/-0.21 and 1.98+/-0.23) (P<0.05). Plasma endotoxin levels at 4 wk and 8 wk increased significantly in ethanol-fed group (129+/-21 ng/L and 187+/-35 ng/L) than those in control rats (48+/-9 ng/L and 53+/-11 ng/L)(P<0.05). Mean values of plasma ALT levels increased dramatically in ethanol-fed rats (112+/-15 IU/L and 147+/-22 IU/L) than those in the control animals (31+/-12 IU/L and 33+/-9 IU/L) (P<0.05). In ethanol-fed rats, the levels of TNF-alpha were 326+/-42 ng/L and 402+/-51 ng/L at 4 wk and 8 wk, respectively which were significantly higher than those in control group (86+/-12 ng/L and 97+/-13 ng/L) (P<0.05). The levels of IL-6 were 387+/-46 ng/L and 413+/-51 ng/L, which were also higher than control group (78+/-11 ng/Land 73+/-10 ng/L) (P<0.05). In liver section from ethanol-fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.


CONCLUSIONS
Ethanol administration led to a significant synthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.
Dai
Li-Li
LL
Department of Digestive Disease, the Second College of Clinical Medicine & the Second Affiliated Hospistal of Chongqing University of Medical Science, 74 Linjiang Road, Chongqing 400010, China.
Gong
Jian-Ping
JP
Zuo
Guo-Qing
GQ
Wu
Chuan-Xin
CX
Shi
Yu-Jun
YJ
Li
Xu-Hong
XH
Peng
Yong
Y
Deng
Wu
W
Li
Sheng-Wei
SW
Liu
Chang-An
CA
D000818:Animals; D005260:Female; D007728:Kupffer Cells / Q000378:metabolism* / Q000473:pathology; D008099:Liver / Q000473:pathology; D008108:Liver Diseases, Alcoholic / Q000473:pathology / Q000503:physiopathology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011971:Receptors, Immunologic; C048071:endotoxin receptor