Kupffer cell-derived prostaglandin E2 is involved in regulation of lipid synthesis in rat liver tissue

Our recent studies suggest that Kupffer cells play a role in the physiological regulation of lipid metabolism of the adjacent hepatocytes. In the present study, we have tested the hypothesis that inhibition of Kupffer cells decreases prostaglandin E(2) (PGE(2)) release inside liver tissue, a phenomenon contributing to lipid accumulation in hepatocytes. PGE(2) secretion as well as lipid synthesis were assessed in precision-cut liver slices (PCLS) from rats previously treated with Kupffer cell inhibitors (GdCl(3) 10 mg kg(-1) body wt, i.v. injection and glycine 5% in diet). In addition, lipid synthesis was assessed in primary rat hepatocytes cultured in the absence or presence of PGE(2) (0.01, 1 and 10 microM). Inhibition of Kupffer cell activity by GdCl(3) decreases PGE(2) secretion by PCLS and resulted in a higher lipid synthesis. Since incubation with PGE(2) over 48 h decreases lipid synthesis from acetate in cultured hepatocytes, we propose that the lower PGE(2) secretion linked to Kupffer cell inhibition, partly explains a higher rate of synthesis of lipids with a subsequent accumulation in liver tissue, as previously shown in fasted rats.     

Are Kupffer cells involved in the metabolic adaptation of the liver to dietary carbohydrates given after fasting?

In rats, a high carbohydrate fat-free (HCFF) diet, given after fasting, induces both hepatic lipogenic and glycogenic enzymes. In the present study, we evaluated the involvement of Kupffer cells in the metabolic events occurring in the liver during the fasting-refeeding transition. Male Wistar rats were fasted for 48 h and received an intravenous injection of either NaCl 0.9% (Gd-) or 10 mg/kg GdCl(3) (Gd+), an inhibitor of Kupffer cells, then fed for 12 h with a HCFF diet. The comparison of colloidal carbon uptake was similar in rats fasted and in rats fasted and then refed a HCFF diet, thus indicating that refeeding does not affect per se Kupffer cell phagocytic activity. The inhibition of Kupffer cells by GdCl(3) did not affect fatty acid synthase (FAS) induction, as shown by the analysis of both FAS mRNA and activity; refeeding a HCFF diet increased the hepatic triglyceride and glycogen content to the same extent in Gd+ and Gd- rats. Our results do not support the involvement of Kupffer cells in the metabolic events occurring in the liver tissue by feeding a HCFF diet after fasting. However, the discussion supports the involvement of Kupffer cells in the modulation of the hepatic lipid metabolism by other nutrients than carbohydrates.     

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.     

Cryopreservation of rat precision-cut liver slices by ultrarapid freezing: influence on phase I and II metabolism and on cell viability upon incubation for 24 hours

Several cryopreservation methods for precision-cut rat liver slices (PCLS) have been proposed, allowing a short-term (a few hours) maintainance of viability and functionality upon thawing. The aim of the present study was to test the metabolic capacity of PCLS cryopreserved by an ultrarapid method. The biotransformation of paracetamol to its glucuronide and sulfate conjugates and of midazolam to its hydroxylated metabolites was studied in thawed PCLS incubated for 24 hours at 37 degrees C in Williams' medium E. In addition, protein levels of the key enzymes involved in these metabolic reactions, i.e. UGT1A1, ST1A1, CYP2E1 and CYP3A2 were determinated. In addition, biological markers of cell function (ATP and glycogen levels) and toxicity (LDH leakage in the medium) were also measured. Compared to controls (non cryopreserved PCLS), CYP3A2 activity and content and CYP2E1 content were maintained at the same level all along the incubation, whereas paracetamol glucuronidation and sulfation dropped to 24 and 21% of the control value, respectively, immediately after thawing. Freezing-thawing conditions also modified cell functionality, leading to a lower intracellular ATP and glycogen content, and an increase in cell lysis, as shown by LDH released in the medium. The results of this study suggest that cryopreserved PCLS are able to maintain some phase I activities for 24 hours after thawing whereas some phase II metabolic capacities are not maintained.     

Hydrogel embedding of precision-cut liver slices in a microfluidic device improves drug metabolic activity

A microfluidic-based biochip made of poly-(dimethylsiloxane) was recently reported for the first time by us for the incubation of precision-cut liver slices (PCLS). In this system, PCLS are continuously exposed to flow, to keep the incubation environment stable over time. Slice behavior in the biochip was compared with that of slices incubated in well plates, and verified for 24 h. The goal of the present study was to extend this incubation time. The viability and metabolic activity of precision-cut rat liver slices cultured in our novel microflow system was examined for 72 h. Slices were incubated for 1, 24, 48, and 72 h, and tested for viability (enzyme leakage (lactate dehydrogenase)) and metabolic activity (7-hydroxycoumarin (phase II) and 7-ethoxycoumarin (phase I and II)). Results show that liver slices retained a higher viability in the biochip when embedded in a hydrogel (Matrigel) over 72 h. This embedding prevented the slices from attaching to the upper polycarbonate surface in the microchamber, which occurred during prolonged (>24 h) incubation in the absence of hydrogel. Phase II metabolism was completely retained in hydrogel-embedded slices when medium supplemented with dexamethasone, insulin, and calf serum was used. However, phase I metabolism was significantly decreased with respect to the initial values in gel-embedded slices with medium supplements. Slices were still able to produce phase I metabolites after 72 h, but at only about ～10% of the initial value. The same decrease in metabolic rate was observed in slices incubated in well plates, indicating that this decrease is due to the slices and medium rather than the incubation system. In conclusion, the biochip model was significantly improved by embedding slices in Matrigel and using proper medium supplements. This is important for in vitro testing of drug metabolism, drug-drug interactions, and (chronic) toxicity.     

Primary role of Kupffer cell–hepatocyte communication in the expression of oxidative stress in the post-ischaemic liver

It has been reported that hepatocyte metabolism and function can be modulated by the activated Kupffer cell through the release of different biomolecules like cytokines, eicosanoids, oxygen free radicals and enzymes. In relation to these paracrine factors involved in circuits of intercellular communication, the existence of a hepatic oxygen sensor located in the Kupffer cell has been postulated. According to this postulate the oxygen metabolism of the liver parenchymal cells could be under the control of the Kupffer cells. In order to study the role of the Kupffer cell in the reperfusion syndrome of the liver, a lobular ischaemia–reperfusion model was performed in rats with or without previous treatment with gadolinium chloride to block Kupffer cell function. Spontaneous chemiluminescence of the liver surface, oxygen uptake by tissue slices and tert-butyl hydroperoxide-initiated chemiluminescence determinations were performed to evaluate the oxygen metabolism and the oxy-radical generation by the liver. The lower basal photoemission, in parallel with a lower basal oxygen uptake registered in the hepatic lobes from the animals pretreated with gadolinium chloride clearly indicates that the gadolinium chloride-dependent functional inhibition of Kupffer cell leads to a downregulation of oxygen metabolism by the liver. Moreover, the intensity of oxidative stress exhibited by the postischaemic lobes appears to be closely linked with the Kupffer cell activity. On the basis of the data obtained we propose that a paracrine circuit between activated Kupffer cell and hepatocytes is an early key event in the induction of postischaemic oxidative stress in the liver. Furthermore the interference with the mitochondrial electron flow by some biomolecules released from the activated Kupffer cell, such as tumour necrosis factor, interleukins, eicosanoids, etc., would increase the rate of generation of reactive oxygen species by the inhibited mitochondrial respiratory chain. © 1998 John Wiley & Sons, Ltd.     

Uncoupling protein-2 induction in rat hepatocytes after acute carbon tetrachloride liver injury

This study is focused on the role of UCP-2 in hepatic oxidative metabolism following acute CCl(4) administration to rats. UCP-2 mRNA, almost undetectable in the liver of controls, was significantly increased 24 h after CCl(4) administration, peaked at 72 h and then tended to disappear. UCP-2 protein, undetectable in controls, increased 48-72 h after CCl(4) treatment. Experiments with isolated liver cells indicated that in control rats UCP-2 was expressed in non-parenchymal cells and not in hepatocytes, whereas in CCl(4)-treated rats UCP-2 expression was induced in hepatocytes and was not affected in non-parenchymal cells. Addition of CCl(4) to the culture medium of hepatocytes from control rats failed to induce UCP-2 expression. Liver mitochondria from CCl(4)-treated rats showed an increase of H(2)O(2) release at 12-24 h, followed by a rise of TBARS. Vitamin E protected liver from CCl(4) injury and reduced the expression of UCP-2. Treatment with GdCl(3) prior to CCl(4), in order to inhibit Kupffer cells, reduced TBARS and UCP-2 mRNA increase in hepatic mitochondria. Our data indicate that CCl(4) induces the expression of UCP-2 in hepatocytes with a redox-dependent mechanism involving Kupffer cells. A role of UCP-2 in moderating CCl(4)-induced oxidative stress during tissue regeneration after injury is suggested.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.     

Reoxygenation after cold hypoxic storage of cultured precision-cut rat liver slices: effects on cellular metabolism and drug biotransformation.

Cultured rat precision-cut liver slices (PCLS) were used to study the influence of hypothermic preservation and reoxygenation at 37 degrees C on cellular metabolism and drug biotransformation. Cold hypoxic storage caused a depressed metabolism in rat liver slices, but reoxygenation for 8 h at 37 degrees C partially restored the levels of both ATP and GSH and totally restored the capacity to synthesize proteins. Metabolism of midazolam (CYP3A-dependent oxidation) by cold preserved liver slices was decreased by 30% but no further affected by reoxygenation, showing the same profile as freshly cut slices. Such a reoxygenation at 37 degrees C is accompanied by a dramatic loss of CYP3A2 protein while CYP3A1 protein was unaffected. These results suggest that CYP3A2 did not play a major role in midazolam oxidation. Such results are not consistent with a putative reoxygenation injury but rather with cold hypoxic damage. Since cold preserved liver slices did not respond to bacterial endotoxin stimulation (lipopolysaccharides), a minor role of non-parenchymal cells is suggested as mediators for deleterious effects developed during the cold storage.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3 h at 37 °C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Iron activates NF-kappaB in Kupffer cells

Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury.     

Reoxygenation after cold hypoxic storage of cultured precision-cut rat liver slices: effects on cellular metabolism and drug biotransformation

Cultured rat precision-cut liver slices (PCLS) were used to study the influence of hypothermic preservation and reoxygenation at 37°C on cellular metabolism and drug biotransformation. Cold hypoxic storage caused a depressed metabolism in rat liver slices, but reoxygenation for 8 h at 37°C partially restored the levels of both ATP and GSH and totally restored the capacity to synthesize proteins. Metabolism of midazolam (CYP3A-dependent oxidation) by cold preserved liver slices was decreased by 30% but no further affected by reoxygenation, showing the same profile as freshly cut slices. Such a reoxygenation at 37°C is accompanied by a dramatic loss of CYP3A2 protein while CYP3A1 protein was unaffected. These results suggest that CYP3A2 did not play a major role in midazolam oxidation. Such results are not consistent with a putative reoxygenation injury but rather with cold hypoxic damage. Since cold preserved liver slices did not respond to bacterial endotoxin stimulation (lipopolysaccharides), a minor role of non-parenchymal cells is suggested as mediators for deleterious effects developed during the cold storage.     

Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O(2) uptake (105 +/- 5 micromol. g(-1). h(-1)) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 +/- 8 micromol. g(-1). h(-1) and binding of pimonidazole, a hypoxia marker, increased about threefold (P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-alpha and PGE(2), whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE(2) from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE(2) from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.     

Release of peptide leukotrienes from rat Kupffer cells

Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, and the levels of peptide leukotrienes (LTC4, LTD4, and LTE4) contained in the culture supernatant were determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. In response to A23187, Kupffer cells released LTC4, LTD4, and LTE4. After 10 min-preincubation of Kupffer cells with AA861, a 5-lipoxygenase inhibitor, the generation of LTC4, LTD4, and LTE4 from A23187-stimulated Kupffer cells was significantly suppressed. Platelet activating factor (PAF), a phospholipid mediator, significantly enhanced the release of LTC4, LTD4, and LTE4 from Kupffer cells stimulated with A23187. These results suggested that Kupffer cells may participate in inflammatory and immunologic events in the liver tissue by the release of peptide leukotrienes.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

Expression of osteopontin in Kupffer cells and hepatic macrophages and Stellate cells in rat liver after carbon tetrachloride intoxication: a possible factor for macrophage migration into hepatic necrotic areas

Activated Kupffer cells and macrophages accumulate in necrotic areas in the liver. Osteopontin, an extracellular matrix with RGD sequence, has been shown to act as a chemokine that can induce monocyte migration. The possibility that osteopontin can play a role in infiltration of both cells into hepatic necrotic areas was investigated in rats. Northern blot analysis revealed that osteopontin mRNA expression was minimal in Kupffer cells and hepatocytes immediately after isolation from normal rats, but slight in hepatic stellate cells assumed nearly quiescent in function after 3 days of culture on plastic dishes. When rat received carbon tetrachloride, liver necrosis developed between 1 and 3 days following the intoxication. In these rats, osteopontin mRNA expression assessed by quantitative competitive RT-PCR was increased in the liver later than 1 day with its peak at 2 days following the intoxication. Kupffer cells and hepatic macrophages and hepatic stellate cells isolated from such liver showed marked expression of osteopontin mRNA on Northern blotting. Immunohistochemical examination disclosed that osteopontin was stained in macrophages including Kupffer cells and stellate cells in the necrotic areas. On electron microscopy, osteopontin stains were present in the Golgi apparatus in these cells. Recombinant human osteopontin promoted migration of Kupffer cells isolated from normal rats and cultured in a Transwell cell culture chamber in a dose-related manner. We conclude that activated Kupffer cells and hepatic macrophages and stellate cells express osteopontin. These cells might contribute to the infiltration of Kupffer cells and macrophages into hepatic necrotic areas by expressing osteopontin.