OBJECTIVETo establish a method for simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells.

METHODSBy combining in situ perfusion, in vitro perfusion, density gradient centrifugation and differential adhesion, primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells were obtained. The purity of these cells were assessed with morphological observation, immunofluorescent staining and ink phagocytosis assay.

RESULTSWe successfully obtained the 4 primary cells simultaneously by combining in situ perfusion with in vitro perfusion, density gradient centrifugation, and differential attachment. The cell yield rate, cell viability and purity all met requirements for the subsequent cell experiment.

CONCLUSIONThe combined cell isolation and culture method is feasible to isolate primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells simultaneously.

Animals ; Cell Culture Techniques ; Cell Separation ; Endothelial Cells ; cytology ; Hepatic Stellate Cells ; cytology ; Hepatocytes ; cytology ; Kupffer Cells ; cytology ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo present an improved method to obtain pure, viable, freshly isolated hepatic stellate cells.

METHODSAdult male SD rats were used. All procedures were performed with the animals under sodium pentobarbital anesthesia. Three days after the single intravenous administration of 1 ml liposome-encapsulated CL2MDP, which has selective cytotoxicity of Kupffer cells, livers were perfused with D-Hank's solution containing 100 U/ml heparin for 10 to 15 minutes, and then with 0.05% collagenase dissolved in D-Hank's solution for 25 to 30 minutes. The liver was then gently homogenized and further incubated in 0.025% collagenase, and 0.005% DNAase I for 30 minutes at 37 degrees C under constant stirring. This suspension was filtered through stainless steel gauze and centrifuged for 2 minutes at 50 x g to remove parenchymal cells. Sinusoidal cells in the supernatant were recovered by centrifugation for 10 minutes at 300 x g. The cells were resuspended in the presence of 28.7% Nycodenz stock solution. The final concentration of Nycodenz at this stage was 11.5%. Following centrifugation for 17 minutes at 1400 x g, The cells at the top of this Nycodenz solution were collected. Cells were resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, The cells were seeded in 50 ml culture flask at a density of 500,000 cells/ml, The cell viability was determined by trypan blue exclusion staining, the purity of hepatic stellate cells was identified by the expression of Desmin using immunocytochemistry method. Endogenous peroxidase staining was used to detect Kupffer cells.

RESULTSThe yield rate of hepatic stellate cells was 3 x 10(7) per rat, the cell viability was more than 95%, the desmin positive cell rate was 90%, no endogenous peroxidase positive cells were detected.

CONCLUSIONThe method for the isolation of hepatic stellate cells was developed without Kupffer cells confusion. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.

Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Kupffer Cells ; cytology ; Liver ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Ultracentrifugation ; methods

OBJECTIVEKupffer cells (KCs) are resident macrophages in the liver. Because the densities and sizes of KCs show a significant overlap with other sinusoidal cells, it is difficult to separate and purify them. We aim to find an improved procedure that could optimize the method for isolation of highly purified rat Kupffer cells.

METHODSIn ex vivo rat liver perfusion with pronase E and collagenase IV, density gradient centrifugation by Histodenz and selective attachment of Kupffer cells were used to isolate them. Cell proliferation and morphological characterization were studied under light phase-contrast microscopes; KCs were also studied with transmission electron microscopy and scanning electron microscopy using standard techniques. Immunocytochemistry was used to detect the expression of ED2 CD163 and lysosome associated membrane protein 2 (LAMP2). Phagocytosis of latex-beads by KCs was also studied.

RESULTSThe yield rate of KCs was 5 x 10(7) and the KCs viability exceeded 98%. The purity of KCs identified by ED2 was higher than 98%, and over 99% of the collected KCs were LAMP2 positive.

CONCLUSIONIn the future this simple, stable and effective method of collecting highly purified Kupffer cells is expected to help in further studies.

Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Kupffer Cells ; cytology ; Male ; Rats ; Rats, Inbred Lew

OBJECTIVETo investigate HMGB-1 expression and its extracellular release of cultured primary hepatic parenchymal cells (HC) and Kupffer cells (KC) that were induced by lipopolysaccharides (LPS).

METHODSPrimary hepatic parenchymal cells and Kupffer cells were cultured in flasks, and some cells were treated with 500 microg/L LPS for 24 hours (induced group) and some were not treated with LPS and served as controls. All of the cells were repeatedly frozen-thawed, and the expression levels of HMGB1-mRNA and HMGB1 proteins were detected by semi-quantitative RT-PCR and Western blot respectively. Then HC and KC were subcultured in 24-well culture plates for 6 h, 12 h, 24 h and 48 h, and the HMGB1 protein in culture fluids was detected by Western blot at each time point.

RESULTSCompared with the cells in the control group, the expression levels of HMGB1-mRNA in the induced group were significantly increased in both HC and KC at 24 h (t=31.32 and 45.90, P<0.05) and the protein levels of HMGB1 showed the same results (t=46.19 and 38.44, P<0.05). There was a small quantity of HMGB1 protein in the culture fluids of two control groups and the induced group of HC. However the HMGB1 protein in the induced group of KC were obviously increased with prolonged culture time (F=42.74, P<0.05). Compared with the control group, the level of HMGB1 protein in the induced group of KC was not increased at 6 h (t=9.57, P>0.05) but was significantly increased at 12 h, 24 h and 48 h (t=21.95, 32.39, 44.16, respectively P<0.05).

CONCLUSIONLPS could increase HMGB1 expression of HC and KC and HMGB1 release from KC, but not from HC. The results suggest that KC play an important role in triggering inflammation and liver injury.

Animals ; Cells, Cultured ; Female ; HMGB1 Protein ; metabolism ; Hepatocytes ; metabolism ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; cytology ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of angiotensin II type-1 (AT-1) alpha receptor gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic Kupffer cells.

METHODSThe expression of AT-1 alpha receptors in primary isolated cultured hepatic Kupffer cells was detected by immunohistochemistry. pSilencer/AT-1 alpha receptor siRNA plasmids were transfected into Kupffer cells, which were subsequently exposed to 10(-6) mol/L angiotensin II (Ang II) for 60 min. The changes in the DNA binding activity of NF-kappaB in the cells was assessed using electrophoretic gel mobility shift assay (EMSA).

RESULTSAT-1 alpha receptor expression was detected in Kupffer cells. NF-kappaB DNA binding activity was markedly increased in Kupffer cells after Ang II stimulation, and obviously inhibited by transfectiom with pSilencer/AT-1 alpha receptor siRNA plasmid.

CONCLUSIONAng II stimulation of Kupffer cell results in increased activation of NF-kappaB via AT-1 alpha receptor.

Cells, Cultured ; Humans ; Kupffer Cells ; cytology ; NF-kappa B ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism

OBJECTIVETo explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).

METHODSIsolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.

RESULTSThe ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).

CONCLUSIONSPretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.

Animals ; Cells, Cultured ; Endotoxins ; immunology ; Immune Tolerance ; Interleukin-1 Receptor-Associated Kinases ; biosynthesis ; genetics ; Kupffer Cells ; cytology ; immunology ; metabolism ; Lipopolysaccharides ; immunology ; Male ; Mice ; Mice, Inbred BALB C

The effects of estriol on oxygen uptake, glucose release, lactate and pyruvate production, beta-hydroxybutyrate and acetoacetate production in perfused rat liver as well as, carbon uptake in rat liver and intracellular calcium in isolated Kupffer cells were investigated. Basal oxygen consumption of perfused liver increased significantly in estriol or ethanol-treated rats. But these increased effects were blocked by gadolinium chloride pretreatment. In a metabolic study, pretreatment with estriol resulted in a decrease in glucose production and in glycolysis while an increase in ketogenesis. A more oxidized redox state of the mitochondria was indicated by increased ratios of perfusate [lactate]/[pyruvate] and decreased ratios of perfusate [beta-hydroxybutyrate]/[acetoacetate]. Carbon uptake of Kupffer-cell increased significantly in estriol-treated rats. But these increased uptake were not shown in rats pre-treated by gadolinium chloride blocking phagocytosis. In isolated Kupffer cells from estriol-treated rats, intracellular calcium was more significantly increased after addition of lipopolysaccharide (LPS) than in controls. These findings suggest that the metabolic effects of estriol (two mg per 100 mg body wt) can be summarized to be highly toxic in rat liver, and these findings suggest that oral administration of estrogens may induce hepatic dysfunctions and play a role in the development of liver disease.
3-Hydroxybutyric Acid/metabolism ; Acetoacetates/metabolism ; Animal ; Calcium/metabolism ; Carbohydrates/metabolism ; Carbon/metabolism ; Cells, Cultured ; Colloids/metabolism ; Estriol/pharmacology* ; Estriol/metabolism ; Ethanol/pharmacology ; Female ; Gadolinium/pharmacology ; Glucose/biosynthesis ; Intracellular Fluid/metabolism ; Kupffer Cells/metabolism ; Kupffer Cells/cytology ; Lactates/metabolism ; Lipids/metabolism ; Liver/metabolism ; Liver/drug effects* ; Oxygen Consumption ; Phagocytosis ; Pyruvic Acid/metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the possible mechanism of the inhibitory effect of liver X receptor alpha (LXRalpha) on lipopolysaccharide (LPS)-induced inflammation in mouse Kupffer cells (KCs).

METHODSThe KCs isolated from the liver of male KM mice and cultured in RPMI 1640 containing 20% FBS for 24 h were divided into control, LPS, T0901317, and LPS+T0901317 groups with corresponding treatments. The expressions of LXRalpha, interferon regulatory factor 3 (IRF3) and glucocorticoid receptor interacting protein 1 (GRIP1) in the KCs were detected by Western blotting. The levels of interferon beta (IFNbeta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe level of LXRalpha protein was highest in T0901317 group and lowest in LPS group, and was significantly higher in LPS+T0901317 group than in LPS group but lower than in T0901317 group (P<0.05). The levels of IRF3 and GRIP1 protein were the highest in LPS group, and significantly lowered by T0901317 treatment (P<0.05). The expression of IRF3 and GRIP1 proteins in LPS group and LPS+ T0901317 group were significantly higher than those in the control and T0901317 groups (P<0.05). The concentration of IFN-beta was significantly higher in LPS group than in the control and T0901317 group (P<0.05), and decreased in LPS+T0901317 group in comparison with that in LPS group (P<0.05). IFN-beta was the lowest in T0901317 group. The levels of TNF-alpha and IL-1beta were the highest in LPS group (P<0.05), and comparable between the other 3 groups (P>0.05).

CONCLUSIONPre-treatment with T0901317 before LPS stimulation can suppress the expressions of IRF3 and GRIP1 to inhibit the inflammation and hence Kupffer cell activation.

Animals ; Cells, Cultured ; Hydrocarbons, Fluorinated ; pharmacology ; Inflammation ; chemically induced ; Interferon Regulatory Factor-3 ; metabolism ; Kupffer Cells ; cytology ; metabolism ; Lipopolysaccharides ; pharmacology ; Liver X Receptors ; Male ; Mice ; Nuclear Receptor Coactivator 2 ; metabolism ; Orphan Nuclear Receptors ; physiology ; Sulfonamides ; pharmacology

Animals ; Colonic Neoplasms ; pathology ; Disease Models, Animal ; Humans ; Kupffer Cells ; cytology ; metabolism ; physiology ; Liver Neoplasms ; prevention & control ; secondary ; Mice ; Mice, Nude ; Neoplasm Metastasis ; Phagocytosis ; physiology ; Reactive Oxygen Species ; metabolism ; Splenectomy ; methods

OBJECTIVETo investigate the effect of the metabolite of leflunomide, A771726,on proliferation and collagen synthesis of hepatic stellate cell (HSC).

METHODSHSC and Kupffer cells were isolated from the rat liver by collagenase IV and pronase perfusion, and purified by density gradient separation. The effects of A771726 on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. The TGF-beta, TNF-alpha and IL-1 levels in Kupffer cell conditioned medium (KCCM) were determined by enzyme-linked immunosorbent assay (ELISA).

RESULTSHSC and Kupffer cells in rat liver were well separated. The KCCM of CCl4-injured rats had significant stimulation effect on proliferation and collagen synthesis of HSC in primary culture. Addition of A771726 (0.001-10 micromol/Lein HSC culture stimulated by KCCM significantly inhibited proliferation and collagen synthesis of HSC. Furthermore, the elevated TGF-beta, TNF-alpha and IL-1 levels in KCCM of CCl4-injured rats were significantly reduced in A771726 treatment groups.

CONCLUSIONA771726 has markedly inhibitory effect on proliferation and collagen synthesis of HSC and secretion of TGF-beta,TNF-alpha and IL-1 from Kupffer cells.

Aniline Compounds ; pharmacology ; Animals ; Carbon Tetrachloride ; Cell Division ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Hepatocytes ; cytology ; Hydroxybutyrates ; pharmacology ; Immunosuppressive Agents ; pharmacology ; Interleukin-1 ; metabolism ; Isoxazoles ; metabolism ; pharmacology ; Kupffer Cells ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; metabolism