Expression of CD]4 protein and its gene in liver sinusoidal endothelial cells during endotoxemia

AIM：To observe expression of CD14 protein and CD14 gene in rat liver sinsoidal endothelial cells(LSECs) during endotoxemia,and the role of CD14 protein in the activation of lipopolysaccharide(LPS)-induced LSECs.METHODS:Wistar rat endotoxemia model was established first by injection of a dose of LPS(5mg/kg,Escherichia coli O111:B4)via the tail vein,then sacrificed after 0h,3h,6h,12h,and 24h,respectively,LSECs were isolated from normal and LPS-injected rats by an in situ colagenase perfusion technique.The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody,then stained with goat anti rabbit IgG conjgated fluorescein isothiocyanate(FITC) and flow cytometric analysis(FCM) was performed.The percentage and mean fluorescence intensity(MFI) of CD14-positive cells were taken as the indexes.LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis.The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody,then stimulated with different concentrations of LPS,and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and lnterleukin(IL)-6 with ELISA.RESULTS:In rats with endotoxemia,LSECs displayed a strong MFI distinct from that of control rats .CD14 positive cells in rats with endotoxemia were 54.32%,65.83%,85.64%,and 45.65%,at 3h,6h,12h,and 24h respectively there was significant difference when compared to normal group of animals(4.45%)(P<0.01).The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats,In LPS group,the levels of TNF-αand IL-6were 54±6ng.L^-1,85±9ng.L^-1,206±22ng.L^-1,350±41ng.L^-1,366±42ng.L^-1and103±11ng.L^-1,187±20ng.L^-1,244±26ng.L^-1,290±31ng.L^-1,and 299±34ng.L^-1,respectively at different concentration points,In anti-CD14 group,the levels of TNF-αand IL-6 were 56±5ng.L^-1,67±8ng.L^-1,85±10ng.L^-1,113±12ng.L^-1, ,199±22ng.L^-1and104±12ng.L^-1,125±12ng.L^-1,165±19ng.L^-1,185±21ng.L^-1,and222±23ng.L^-1，respectively at different concentration poirts.THere was significant difference between the two groups(P<0.01).CONCLUSION:LSECs can synthesize CD14 protein and espress CD14 gene during endotoxemia .CD14 protein plays an important role in the activation of LPS-induced LSECs.This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.

AIM:To observe expression of CD14 protein and CD14 gene in rat liver sinusoidal endothelial cells(LSECs)during endotoxemia,and the role of CD14 protein in the activation of lipopolysaccharide(LPS)-induced LSECs. METHODS:Wistar rat endotoxemia model was established first by injection of a dose of LPS(5 mg/kg,Escherichia coil O111:B4)via the tail vein,then sacrificed after 0h,3h, 6h,12h,and 24h,respectively.LSECs were isolated from normal and LPS-injected rats by an in situ collagenase perfusion technique.The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody,then stained with goat anti rabbit IgG conjugated fluorescein isothiecyanate(FITC)and flow cytometric analysis(FCM) was performed.The percentage and mean fluorescence intensity(MFI)of CD14-positive cells were taken as the indexes.LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis.The isolated LSECs from normal rats were incubated firstly with anti- CD14 antibody,then stimulated with different concentrations of LPS,and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and Interleukin(IL)-6 with ELISA. RESULTS:In rats with endotoxemia,LSECs displayed a strong MFI distinct from that of control rats.CD14 positive cells in rats with endotoxemia were 54.32%,65.83%,85. %,and 45.65% at 3 h,6 h,12h,and 24 h respectively, there was significant difference when compared to normal group of animals(4.45%)(P<0.01).The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats.In LPS group,the levels of TNF-α and IL-6 were 54±6 ng.L^(-1),85±9 ng.L^(-1),206±22 ng.L^(-1),350±41 ng. L^(-1),366±42ng.L^(-1)and 103±11 ng.L^(-1),187±20ng.L^(-1),244± 26 ng.L^(-1),290±31 ng.L^(-1),and 299±34 ng.L^(-1),respectively at different concentration points.In anti-CD14 group,the levels of TNF-α and IL-6 were 56±5 ng.L^(-1),67±8 ng.L^(-1),85 ±10 ng.L^(-1),113±12 ng.L^(-1),199±22 ng.L^(-1)and 104±12 ng.L^(-1), 125±12 ng.L^(-1),165±19 ng.L^(-1),185±21 ng.L^(-1),and 222±23 ng. L^(-1),respectively at different concentration points.There was significant difference between the two groups(P<0.01). CONCLUSION:LSECs can synthesize CD14 protein and express CD14 gene during endotoxemia.CD14 protein plays an important role in the activation of LPS-induced LSECs. This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.