Role of Kupffer cells in the pathogenesis of liver disease

Kupffer cells, the resident liver macrophages have long been considered as mostly scavenger cells responsible for removing particulate material from the portal circulation. However, evidence derived mostly from animal models, indicates that Kupffer cells may be implicated in the pathogenesis of various liver diseases including viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholostasis, activation or rejection of the liver during liver transplantation and liver fibrosis. There is accumulating evidence, reviewed in this paper, suggesting that Kupffer cells may act both as effector cells in the destruction of hepatocytes by produdng harmful soluble mediators as well as antigen presenting cells during viral infections of the liver. Moreover they may represent a significant source of chemoattractant molecules for cytotoxic CD8 and regulatory T cells. Their role in fibrosis is well established as they are one of the main sources of TGFβ1 production, which leads to the transformation of stellate cells into myofibroblasts. Whether all these variable functions in the liver are mediated by different Kupffer cell subpopulations remains to be evaluated. In this review we propose a model that demonstrates the role of Kupffer cells in the pathogenesis of liver disease.

Heme oxygenase-1 protects donor livers from ischemia/reperfusion injury:The role of Kupffer cells

AIM:To examine whether heme oxygenase (HO)-1 overexpression would exert direct or indirect effects on Kupffer cells activation, which lead to aggravation of reperfusion injury.METHODS: Donors were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4℃ for 24 h before transplantation. Kupffer cells were isolated and cultured for 6 h after liver reperfusion.RESULTS: Postoperatively, serum transaminases were significantly lower and associated with less liver injury when donors were pretreated with CoPP, as compared with the ZnPP group. Production of the cytokines tumor necrosis factor-α and interleukin-6 generated by Kupffer cells decreased in the CoPP group. The CD14 expression levels (RT-PCR/Western blots) of Kupffer cells from CoPP-pretreated liver grafts reduced.CONCLUSION: The study suggests that the potential utility of HO-1 overexpression in preventing ischemia/reperfusion injury results from inhibition of Kupffer cells activation.

Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model

AIM: To investigate Kupffer cell dynamics and phagocytic activity,using a rat nonalcoholic steatohepatitis (NASH) model. METHODS: Male F344 rats were fed either a control diet or a choline-deficient L-amino acid-defined (CDAA) diet,followed by contrast enhanced ultrasonography (CEUS) using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups,using the immunohistochemical staining technique. RESULTS: After 4 or more wk of the CDAA diet,CEUS examination revealed a decrease in the signal intensity,20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group,compared with the controls,with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group. CONCLUSION: CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase,although the Kupffer cell numbers were unchanged,indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality,which can detect NASH in fatty liver patients.

Ron receptor-dependent gene regulation of Kupffer cells during endotoxemia

BACKGROUND: Ron receptor tyrosine kinase signaling in macrophages, including Kupffer cells and alveolar macrophages,suppresses endotoxin-induced proinflammatory cytokine/chemokine production. Further, we have also identified genes from Ron replete and Ron deplete livers that were differentially expressed during the progression of liver inflammation associated with acute liver failure in mice by microarray analyses.While important genes and signaling pathways have been identified downstream of Ron signaling during progression of inflammation by this approach, the precise role that Ron receptor plays in regulating the transcriptional landscape in macrophages, and particular in isolated Kupffer cells, has still not been investigated.METHODS: Kupffer cells were isolated from wild-type(TK+/+)and Ron tyrosine kinase deficient(TK-/-) mice. Ex vivo, the cells were treated with lipopolysaccharide(LPS) in the presence or absence of the Ron ligand, hepatocyte growth factor-like protein(HGFL). Microarray and qRT-PCR analyses were utilized to identify alterations in gene expression between genotypes.RESULTS: Microarray analyses identified genes expressed differentially in TK+/+ and TK-/- Kupffer cells basally as well as after HGFL and LPS treatment. Interestingly, our studies identified Mefv, a gene that codes for the anti-inflammatory protein pyrin, as an HGFL-stimulated Ron-dependent gene.Moreover, lipocalin 2, a proinflammatory gene, which is induced by LPS, was significantly suppressed by HGFL treatment.Microarray results were validated by qRT-PCR studies on Kupffer cells treated with LPS and HGFL.CONCLUSION: The studies herein suggest a novel mechanism whereby HGFL-induced Ron receptor activation promotes the expression of anti-inflammatory genes while inhibiting genes involved in inflammation with a net effect of diminished inflammation in macrophages.

Donor denervation and elimination of Kupffer cells affect expression of P-selectin and ICAM-1 in liver graft

BACKGROUND: The non-function and dysfunction of primary liver graft likely involves dependence on Kupffer cells and hepatic innervation. The present experiment was designed to study the expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA in liver graft and to elucidate the role of Kupffer cells and the sympathetic nerve of the liver in down-regulating this expression. METHODS: Donor rats were given hexamethonium, a sympathetic ganglionic blocking agent, and/or gadolinium chloride, an inhibitor of Kupffer cells. Then the changes of graft P-selectin and ICAM-1 mRNA expression were measured after liver transplantation. RESULTS: The expressions of P-selectin and ICAM-1 mRNA were increased after liver transplantation, and down-regulated by liver denervation and elimination of Kupffer cells. CONCLUSIONS: Live donor denervation and elimination of Kupffer cells down-regulated the expressions of P-selectin and ICAM-1 mRNA in grafts. This may decrease graft ischemia/reperfusion injury.

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM:To determine the platelet-activating factor (PAF) synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis. METHODS:Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A (ETA) and endothelin B (ETB) receptors were also determined. RESULTS:A two-fold increase of PAF synthesis (1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA) and a 1.48-fold increase of membrane-bound PAF (1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA) were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor. CONCLUSION:Kupffer cells in the course of CCl4-induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.

Prokineticin 2/Bv8 is expressed in Kupffer cells in liver and is down regulated in human hepatocellular carcinoma

AIM： TO study the implication of prokineticin 1 （PKI/EGVEGF） and prokineticin 2 （PK2/13v8） in hepatocellular carcinoma angiogenesis.METHODS： The gene induction of PK1/EG-VEGF and PK2/Bv8 was investigated in 10 normal, 28 fibrotic and 28 tumoral livers by using real time PCR. Their expression was compared to the expression of VEGF （an angiogenesis marker）, vWF （an endothelial cell marker） and to CD68 （a monocyte/macrophage marker）. Furthermore, the rnRNA levels of PK1/EG-VEGF, PK2/Bv8, prokineticin receptor 1 and 2 were evaluated by real time PCR in isolated liver cell populations. Finally, PK2/Bv8 protein was detected in normal liver paraffin sections and in isolated liver cells by immunohistochernistry and immunocytochemistry.RESULTS： PK2/Bv8 mRNA but not PK1/EG-VEGF was expressed in all types of normal liver samples examined. In the context of liver tumor development, we reported that PK2/13v8 correlates only with CD68 and showed a significant decrease in expression as the pathology evolves towards cancer. Whereas, VEGF and vWF mRNA were significantly upregulated in both fibrosis and HCC,as expected. In addition, out of all isolated liver cells examined, only Kupffer cells （liver resident macrophages） express significant levels of PK2/Bv8 and its receptors, prokineticin receptor 1 and 2.CONCLUSION： In normal liver PK2/Bv8 and its receptors were specifically expressed by Kupffer cells. PK2/Bv8 expression decreased as the liver evolves towards cancer and did not correlate with HCC angiogenesis.

Kupffer cells contribute to concanavalin A-induced hepatic injury through a Th1 but not Th17 type response-dependent pathway in mice

BACKGROUND:Increasing evidence suggests that a close interaction of Kupffer cells with T cells plays a central role in concanavalin A-induced hepatic injury in mice,but the underlying mechanisms remain obscure.The present study aimed to determine the relative roles of Th1 and Th17 type responses in concanavalin A-induced hepatic injury in mice, and to investigate whether or not Kupffer cells contribute to hepatic injury via a Th1 or Th17 type response-dependent pathway. METHODS:Immune-mediated hepatic injury was induced in C57BL/6 mice by intravenous injection of concanavalin A. Kupffer cells were inactivated by pretreatment with gadolinium chloride 24 hours before the concanavalin A injection.The interferon-gamma(IFN-γ)and interleukin-17(IL-17)pathways were blocked by specific neutralizing antibodies.Hepatic injury was assessed using serum transferase activity and pathological analysis.Expression of inflammatory cytokines within the liver was detected by real-time polymerase chain reaction and immunohistochemistry. RESULTS:Neutralization of IFN-γsignificantly attenuated concanavalin A-induced hepatic injury.However,neutralization of IL-17 failed to suppress the injury.Inactivation of Kupffer cells by gadolinium chloride pretreatment protected against concanavalin A-induced injury and significantly reduced hepatic cytokine levels including TNF-α,IL-6 and IFN-γbut not IL-17.CONCLUSION:Our findings suggest that Kupffer cells contribute to concanavalin A-induced hepatic injury via a Th1 type response-dependent pathway and production of inflammatory cytokines including TNF-α,IL-6 and IFN-γ.

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperito-neally infected mice for studying the presence of chlamy-diae in Kupffer cells and hepatocytes.METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isola-tion in LLC-MK2 cells and fluorescence in situ hybridiza-tion (FISH). The releasing of TNFA-α by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay.RESULTS: C. pneumoniae isolation from liver homoge-nates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from sepa-rated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimula-tion of Kupffer cells by alive C. pneumoniae elicited high TNF-α levels. CONCLUSION: A productive infection by C. pneumo-niae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when local-ized in the liver. One could speculate that C. pneumoniaeinfection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune re-actions involving the liver, as observed in human patients with primary biliary cirrhosis.

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

Role of liver X receptors alpha agonist on expressions of LPS-induced inflammatory response associated factor IRAK-4 and NF-kappaB in Kupffer cells

Objective： To explore the role of activated liver X receptor α （LXRα） on the expressions of interleukin-1 receptor associated kinase-4 （IRAK-4） and NF-kappaB （NF-κB） in the inflammatory response which induced by LPS in the Kupffer cells and to investigate the possible mechanisms of LXRα negative regulation of inflammatory response. Methods： The Kupffer cells were isolated from male Kunming mice by collagen perfusion in situ. And these cells were divided into 4 groups： normal control group, LPS treatment group, LXRct agonist T0901317 treatment group, LPS and T0901317 combined treatment group. The LPS treatment group were treated with a final concentration of 1 μg/ml LPS in RPMI 1640 and cultured for 6 h, the T0901317 treatment group were treated with a final concentration of 5 μg/ml in RPMI 1640 and cultured for 24 h, and the combined treatment group received pre-culture for 24 h with a final concentration of 1μg/ml T0901317 in RPMI 1640 and then cultured for 6 h with a final concentration of 5 μg/ml LPS in RPMI 1640. All groups were cultured for 30 h. The expression of LXRα, IRAK-4 and NF-κB at mRNA and protein levels were detected by real-time PCR and Western blotting, and the TNF-α and IL-1β levels were detected by ELISA. Results： The levels of LXRα mRNA and protein were highest in T0901317 group, and lowest in LPS group （P〈0.05）. The level of IRAK4 and NF-κB mRNAs and proteins were evidently lower in the Combined-treated group than in LPS group （P〈0.05）. And the level of TNF-α and IL-1 were observed highest in LPS group （P〈0.05）, but no difference among the Control group, T0901317 group and Combined-treated group （P〉0.05）. Conclusion： These date suggest that the LXR agonists can effectively up-regulate the expressions of LXRα mRNA and protein and inhibit the inflammatory response. This may be via down-regulating the expressions of IRAK4 and NF-κB at mRNA and protein levels.

IN VITRO ANTI-HEPATOMA EFFECTS OF MONOCYTES AND KUPFFER CELLS ISOLATED FROM HEPATOMA PATIENTS AFTER TREATMENT WITH BIOLOGICAL IMMUNE STIMULANTS

Monocytes (MC), lymphocytes (LC) and Kupffer cells (KC) were isolated respectively from blood and surgical liver samples of patients suffering from he-patocellular carcinoma (HCC). 13 patients were given BCG, mixed bacterium vaccine (MBV) and human white blood cell interferon (IFN), the other 3 patients were not treated with any biological immune stimulants (BIS) and served as controls. The cytosta-tic and cytotoxic effects of MC and KC on human hepatoma SMMC-7721 (TC) were assayed in vitro and the numbers of T total (Tt), T helper (Th) and T suppressor (Ts) cells were counted using CD monoclonal antibody immunofluorescence. The results were as follows: (1) On the 7th day after the first administration of BIS, the cytostatic and cytotoxic effects of MC on TC showed obvious increase over pre-administration. The activity of BIS was 1 ?5 times as high as that in the controls. (2) After 3 administrations, the cytostatic effect of MC on TC increased to the normal level (84%), while the controls remained as before (45%). (3) On the 7th day after first administration, cytostatic and cytotoxic effects of KC on TC were 0.5 and 1 times higher respectively than those of the controls. (4) The numbers of Tt and Th of patients given BIS increased continuously; on the contrary Ts decreased in number. These results indicate that combined use of BCG, MBV and IFN can actively enhance the immune anti-hepatoma function of patients suffering from HCC.

A NEW METHOD OF ISOLATING KUPFFER CELLS FROM BIOPSY TISSUE OF RAT LIVER

The isolation of a high yield and purity of Kupffer cells has been reported in detail.1 This paper reports into the research about isolation Kupffer cells from biopsy tissue of liver. This method includes 5 important steps: (1) take fresh liver tissue, and mince with scissors. (2) spin at low speed to wash off red blood cells. (3) digest in collagenase for suitable time. (4) isolate Kupffer cells on a percoll density gradient. (5) cell charaterization was observed by N.S.E stain and peroxidatic activity with lumino-meter measurement and phagocytosis with latex beads.2.3

Role of Kupffer cells in acute hemorrhagic necrotizing pancreatitis-associated lung injury of rats

AIM： To investigate the role of Kupffer cells （KCs） in acute hemorrhagic necrotizing pancreatitis-associated lung injury （AHNP-U）. METHODS： Forty-two rats were allocated to four groups [sham operation, AHNP model, gadolinium chloride （GdCl3） pretreatment, GdCl3 control]. In GdCl3 pretreatment group, GdCl3 was administered by caudal vein injection 24 h before the AHNP model induction. Blood from the iliac artery, alveolar macrophages and tissues from the pancreas and lung, were collected in six animals per group 3 and 6 h after acute pancreatitis induction. TNF-α, IL-1 of Lserum, myeloperoxidase （MPO） of lung tissue, NF-κB activation of alveolar macrophages were detected. Serum AST and ALT in sham operation group and GdCl3 control group were tested. In addition, histopathological changes of the pancreas and lung were observed under light microscope. RESULTS： MPO of lung tissue and TNF-α, IL-1 levels of serum were all reduced significantly in GdCl3 pretreatment group compared to those in AHNP group （P〈0.01）. NF-KB activation of alveolar macrophages was also attenuated significantly in GdCl3 pretreatment group compared to that in AHNP group （P〈0.01）. The pathological injury of the lung was ameliorated obviously in GdCl3 pretreatment group compared to that in AHNP group. Nevertheless, the serum amylase level did not reduce and injury of the pancreas was not prevented in GdCl3 pretreatment group. CONCLUSION： Pulmonary injury induced by AHNP is mediated by KC activation and AHNP-LI can be significantly ameliorated by pretreatment with GdCh and KCs play a vital role in AHNP-LI.

Isolation of Kupffer cells and their suppressive effects on T lymphocyte growth in rat orthotopic liver transplantation

AIM： To develop a practical method for isolation, purification and culture of hepatic Kupffer cells （KCs） and to observe their suppressive effects on the proliferation of alloreactive T cells.METHODS： Perfusion in situ in vivo combined with density gradient centdfugation was applied in isolation, purification and culture of hepatic KC. The suppression by KCs on the T cell proliferation in mixed lymphocyte reaction （MLR） was observed.RESULTS：（1.1± 0.2） ×10^7 KCs per liver, （93.5% ±1.8%） viable cells, over 90% purity and positive for FD-2. After the first 24 h in culture, a great number of KCs which exhibited typical characteristics were observed. Using 3H-TdR incorporation assay, non-irradiated KCs significantly suppressed allo-MLR. The KCs recovered from accepted liver allografts in groups D and F were more effective in suppressing allo-MLR.CONCLUSION： A standardized procedure for isolation of highly purified rat KCs is proposed and KCs have suppressive effects on the proliferation of alloreactive T cells, especially those derived from accepted liver allografts.

Effects of Ca^(2+) channel blockers on store-operated Ca^(2+) channel currents of Kupffer cells after hepatic ischemia/reperfusion injury in rats

AIM： To study the effects of hepatic ischemia/ reperfusion （I/R） injury on store-operated calcium channel （SOC） currents （Isoc） in freshly isolated rat Kupffer cells, and the effects of Ca^2＋ channel blockers, 2-aminoethoxydiphenyl borate （2-APB）, SK＆F96365, econazole and miconazole, on Isoc in isolated rat Kupffer cells after hepatic I/R injury.METHODS： The model of rat hepatic I/R injury was established. Whole-cell patch-clamp techniques were performed to investigate the effects of 2-APB, SK＆F96365, econazole and miconazole on Isoc in isolated rat Kupffer cells after hepatic I/R injury.RESULTS： I/R injury significantly increased Isoc from -80.4±25.2pA to -159.5±34.5pA （^bp 〈 0.01, n = 30）. 2-APB （20, 40, 60, 80, 100 pmol/L）, SK＆F96365 （5, 10, 20, 40, 50 pmol/L）, econazole （0.1, 0.3, 1, 3, 10 μmol/L） and miconazole （0.1, 0.3, 1, 3, 10 μmol/L） inhibited Isoc in a concentration-dependent manner with IC50 of 37.41 μmol/L （n = 8）, 5.89 μmol/L （n = 11）, 0.21 μmol/L （n = 13）, and 0.28 μmol/L （n = 10）. The peak value of Isoc in the I-V relationship was decreased by the blockers in different concentrations, but the reverse potential of Isoc was not transformed. CONCLUSION： SOC is the main channel for the influx of Ca^2＋ during hepatic I/R injuries. Calcium channel blockers, 2-APB, SK＆F96365, econazole and miconazole,have obviously protective effects on I/R injury, probably by inhibiting Isoc in Kupffer cells and preventing the activation of Kupffer cells.

Augmenter of liver regeneration promotes hepatocyte proliferation induced by Kupffer cells

AIM： To observe the effects of augmenter of liver regeneration （ALR） on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells. METHODS： Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR （rrALR） were added. ^3H-thymidine, BrdU and ^3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium, rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes. RESULTS： rrALR stimulated DNA replication in Kupffer cells and protein synthesis both in cells and in medium in a non-concentration-dependent manner. The effect was significant at the concentration of 1μg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1μg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant （Kd） of 0.883 nmol/L and a maximum binding capacity （Bmax） of 126.1 pmol/g protein in the rat Kupffer cells. CONCLUSION： ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.

Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRNA and FasL mRNA in KC pretreated with IFN-γwere studied with real-time polymerase chain reaction(PCR).The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography.Allogeneic T-cell response was used to confirm the inhibition of KC in vitro.The proliferation of lymphocytes was detected using[ 3 H]thymidine incorporation.Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS:Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ,and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6,which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody.KC expressing IDO could induce allogeneic T-cell apoptosisCONCLUSION:In addition to Fas/FasL pathway,IDO may be another mechanism for KC to induce immune tolerance.

Expression of macrophage inflammatory protein-1αin Kupffer cells following liver ischemia or reperfusion injury in rats

AIM： To explore the expression of macrophage inflammatory protein-1α （MIP-1α） in Kupffer cells （KCs） following liver ischemia/reperfusion injury IRI in rats. METHODS： Forty male SD rats were divided randomly into five groups. A model of partial warm ischemia/ reperfusion injury in the rat liver was established. KCs were isolated and incubated one hour, six hours, 12 h, and 24 h after the reperfusion. Tumor necrosis factor alpha （TNF-α） and interleukin-lbeta （IL-1β） in the supernatants were measured by ELISA. MIP-1α in KCs was detected by immunocytochemical and RT-PCR. RESULTS： No or few MIP-1α protein and mRNA were expressed in the KCs of the control group. Its expression in the IRI group had a significant increase after the reperfusion （P 〈 0.05）, which was contrary to the control group. CONCLUSION： The active behavior of the MIP-1α gene in KCs following liver ischemia/reperfusion injury is assumed to be one of the major causes for the hepatic ischemia/reperfusion injury.