OBJECTIVETo observe the expression of CD(14) protein and CD(14) gene in liver sinusoidal endothelial cells (LSECs) of rats during endotoxemia and the role of CD(14) protein in the activation of lipopolysaccharide (LPS)-induced LSECs.

METHODSWistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein of the rats, then sacrificed immediately, at 3, 6, 12, and 24 h, respectively. LSECs were isolated from normal and LPS-injected rats by the in situ collagenase perfusion technique. The isolated LSECs were incubated with anti CD(14) polyclonal antibody, then followed by staining with goat anti-rabbit IgG conjugated fluorescein isothiocyanate (FITC). The percentage and mean fluorescence intensity (MFI) of CD(14)-positive cells were detected by the flow cytometric analysis (FCM). LSECs were collected to measure the expression of CD(14) mRNA by the in situ hybridization analysis. The isolated LSECs from normal rats were divided into two groups. Group of LPS: LSECs were induced with different concentration of LPS (0, 0.01 microg/ml, 1 microg/ml, 10 microg/ml, and 100 microg/ml). Group of anti-CD(14) blockade: LSECs were pre-incubated for 30 min with CD(14) antibody before different concentrations of LPS were added. The supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)- alpha and interleukin (IL)-6.

RESULTSIn rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. The number of FITC-CD(14) positive LSECs was 54.32%, 65.83%, 85.61%, and 45.65% at 3, 6, 12, and 24 h, respectively, which increased markedly when compared to control rats (4.45%, P<0.01). The expression of CD(14) mRNA in LSECs was stronger than that in control rats. The levels of TNF-alpha were significantly increased in group of LPS (54.49 +/- 6.02 pg/ml, 84.65 +/- 10.16 pg/ml, 206.54 +/- 23.55 pg/ml, 349.87 +/- 39.47 pg/ml, and 365.76 +/- 40.31 pg/ml) than those in group of anti-CD(14) blockade (55.93 +/- 6.95 pg/ml, 63.32 +/- 7.81 pg/ml, 85.34 +/- 9.72 pg/ml, 112.75 +/- 13.54 pg/ml, and 198.66 +/- 21.54 pg/ml) (P<0.01). The levels of IL-6 also increased significantly in group of LPS (103.34 +/- 12.52 pg/ml, 187.39 +/- 20.31 pg/ml, 243.87 +/- 27.83 pg/ml, 289.51 +/- 30.15 pg/ml, and 298.53 +/- 31.94 pg/ml) than those in group of anti-CD(14) blockade (104.37 +/- 11.49 pg/ml, 125.02 +/- 13.58 pg/ml, 164.59 +/- 19.47 pg/ml, 183.47 +/- 20.17 pg/ml, and 221.76 +/- 26.43pg/ml) (P<0.01).

CONCLUSIONSLSEC can synthesize CD(14) protein and express CD(14) gene during endotoxemia. Anti CD(14) antibody can inhibit the production of TNF-alpha and IL-6 in LSECs induced by LPS. The expression of CD(14) protein may take an important part in the activation of LSECs induced by LPS.