Sensitization Of Kupffer Cells Research Articles

Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-gamma, pioglitazone.

Agonists of peroxisome proliferator-activated receptor (PPAR)-gamma have been shown to reduce tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance. On the other hand, sensitization of Kupffer cells to lipopolysaccharide (LPS) and their production of TNF-alpha are critical for progression of alcoholic liver injury. This study was intended to determine whether pioglitazone, a PPAR-gamma agonist, could prevent alcohol-induced liver injury. Rats were given ethanol (5 g/kg b.wt.) and pioglitazone (500 microg/kg) once every 24 h intragastrically. Ethanol for 8 weeks caused pronounced steatosis, necrosis, and inflammation in the liver. These pathological parameters were diminished greatly by pioglitazone. Kupffer cells were sensitized to LPS after ethanol for 4 weeks as evidenced by aggravation of liver pathology induced by LPS (5 mg/kg) and enhancement of LPS (100 ng/ml)-induced intracellular Ca2+ concentration elevation in Kupffer cells. The parameters were diminished by treatment with pioglitazone. LPS-induced TNF-alpha production by Kupffer cells from the 4-week ethanol group was 3 to 4 times higher than control. This increase was blunted by 70% with pioglitazone. Gut permeability was 10-fold higher in the 4-week ethanol group, and pioglitazone treatment did not change the value. Inclusion of TNF-alpha in culture media of Kupffer cells enhanced CD14 expression, LPS-induced intracellular Ca2+ concentration response, and production of TNF-alpha. These results indicate that pioglitazone prevents alcoholic liver injury through abrogation of Kupffer cell sensitization to LPS.

Kupffer cells are a major source of increased platelet activating factor in the CCl 4-induced cirrhotic rat liver

Background/Aims: Endothelin-1 (ET-1) stimulates the synthesis of platelet-activating factor (PAF) by Kupffer cells in vitro. Hepatic concentrations of both ET-1 (a potent vasoconstrictor) and PAF (a mediator of hepatic vasoconstriction and the cirrhotic hyperdynamic state) increase in cirrhosis. The aim of this study was to determine if the responsiveness of Kupffer cells to produce PAF upon ET-1 challenge is modified by cirrhosis. Methods: Kupffer cells, isolated from the livers of control and CCl 4-induced cirrhotic rats, were placed in serum-free medium after overnight culture. PAF and ET-1 receptors, ET-1-induced PAF synthesis, and PAF- and ET-1-induced prostaglandin E 2 (PGE 2) synthesis were determined 24 h later. Results: Both basal and ET-1-stimulated PAF synthesis was increased in cirrhotic Kupffer cells as indicated by increased cell-associated and released PAF. Cirrhotic Kupffer cells also had elevated densities of functional receptors for both PAF and ET-1 (exclusively ET B), as measured by ligand binding, mRNA expression of the respective receptors, and ligand-stimulated PGE 2 synthesis. Conclusions: Cirrhosis sensitizes Kupffer cells to both ET-1 and PAF by elevating their respective receptor levels. Since both mediators individually cause portal hypertension, an increase in ET-1-stimulated PAF synthesis in Kupffer cells will exacerbate the hepatic and extrahepatic complications of cirrhosis.

Oral Contraceptives Worsen Endotoxin‐Induced Liver Injury in Rats

Background Oral contraceptives are widely used; however, these drugs occasionally cause liver injury. Recently, it was reported that estriol worsens alcoholic liver injury by the mechanism involving activation of Kupffer cells as a result of gut‐derived endotoxin. However, the relationship between oral contraceptives and endotoxin‐induced liver injury has not been elucidated. Here we show that oral contraceptives sensitize Kupffer cells via a mechanism dependent on increased gut permeability to endotoxin.Methods Female Wistar rats (200–250 g) were given intraperitoneally a combination of estradiol (35 ng/kg of 17α‐Ethynylestradiol) and progesterone (2 μg/kg of Norethindrone), each dose being similar to that contained in oral contraceptives (EP treatment). After 24 hr, a sublethal dose of lipopolysaccharide (LPS; 5 mg/kg) was injected via the tail vein. In some experiments, antibiotics (150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin) were administered orally for 4 days before EP treatment. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated and cultured in RPMI 1640 + 10% fetal bovine serum for 24 hr. After addition of LPS (100 ng/ml) to the culture medium, intracellular calcium concentration ([Ca2+]i) was measured with fura‐2.Results Liver histology in rats given EP treatment intraperitoneally followed by an injection of LPS (5 mg/kg) 24 hr later revealed pronounced liver damage with massive necrosis. Whereas mean values of alanine aminotransferase (ALT) in the control, nontreated rats were 30 ± 6 IU/liter, ALT increased to 75 ± 21 IU/liter 24 hr after LPS injection. This increase was aggravated 6‐fold (483 ± 118 IU/liter;p < 0.05) by EP treatment. The EP treatment‐induced increase in ALT was completely blocked by antibiotics (82 ± 26 IU/liter;p < 0.05). Gut permeability was increased approximately 10‐fold with EP treatment. This increase in gut permeability was not altered by treatment with nonabsorbable antibiotics. In isolated Kupffer cells, LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (36 ± 8 nmol/liter) to 100 ± 8 nmol/liter. In contrast, the LPS‐induced [Ca2+]i elevation was approximately 3‐fold greater in the group given EP treatment before 24 hr (305 ± 17 nmol/liter;p < 0.05). This increase was also blocked completely by treatment with antibiotics (128 ± 13 nmol/liter). Similar results were obtained for LPS‐induced tumor necrosis factor‐α production by Kupffer cells from either control or EP treatment group. The increased tumor necrosis factor‐α production as a result of EP treatment was blocked completely by antibiotics.Conclusions These results indicate that EP treatment in vivo sensitizes Kupffer cells to LPS via a mechanism dependent on the portal increase of gut‐derived endotoxin. This event suggests that oral contraceptives exacerbate alcoholic liver injury.

Oral Contraceptives Worsen Endotoxin-Induced Liver Injury in Rats

Background Oral contraceptives are widely used; however, these drugs occasionally cause liver injury. Recently, it was reported that estriol worsens alcoholic liver injury by the mechanism involving activation of Kupffer cells as a result of gut-derived endotoxin. However, the relationship between oral contraceptives and endotoxin-induced liver injury has not been elucidated. Here we show that oral contraceptives sensitize Kupffer cells via a mechanism dependent on increased gut permeability to endotoxin. Methods Female Wistar rats (200–250 g) were given intraperitoneally a combination of estradiol (35 ng/kg of 17α-Ethynylestradiol) and progesterone (2 μg/kg of Norethindrone), each dose being similar to that contained in oral contraceptives (EP treatment). After 24 hr, a sublethal dose of lipopolysaccharide (LPS; 5 mg/kg) was injected via the tail vein. In some experiments, antibiotics (150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin) were administered orally for 4 days before EP treatment. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated and cultured in RPMI 1640 + 10% fetal bovine serum for 24 hr. After addition of LPS (100 ng/ml) to the culture medium, intracellular calcium concentration ([Ca2+]i) was measured with fura-2. Results Liver histology in rats given EP treatment intraperitoneally followed by an injection of LPS (5 mg/kg) 24 hr later revealed pronounced liver damage with massive necrosis. Whereas mean values of alanine aminotransferase (ALT) in the control, nontreated rats were 30 ± 6 IU/liter, ALT increased to 75 ± 21 IU/liter 24 hr after LPS injection. This increase was aggravated 6-fold (483 ± 118 IU/liter;p < 0.05) by EP treatment. The EP treatment-induced increase in ALT was completely blocked by antibiotics (82 ± 26 IU/liter;p < 0.05). Gut permeability was increased approximately 10-fold with EP treatment. This increase in gut permeability was not altered by treatment with nonabsorbable antibiotics. In isolated Kupffer cells, LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (36 ± 8 nmol/liter) to 100 ± 8 nmol/liter. In contrast, the LPS-induced [Ca2+]i elevation was approximately 3-fold greater in the group given EP treatment before 24 hr (305 ± 17 nmol/liter;p < 0.05). This increase was also blocked completely by treatment with antibiotics (128 ± 13 nmol/liter). Similar results were obtained for LPS-induced tumor necrosis factor-α production by Kupffer cells from either control or EP treatment group. The increased tumor necrosis factor-α production as a result of EP treatment was blocked completely by antibiotics. Conclusions These results indicate that EP treatment in vivo sensitizes Kupffer cells to LPS via a mechanism dependent on the portal increase of gut-derived endotoxin. This event suggests that oral contraceptives exacerbate alcoholic liver injury.

Estriol Enhances Lipopolysaccharide-Induced Increases in Nitric Oxide Production by Kupffer Cells via Mechanisms Dependent on Endotoxin

Background Estriol causes sensitization of Kupffer cells to lipopolysaccharide (LPS) via mechanisms dependent on gut-derived LPS. Accordingly, this study examines the effect of estriol treatment on nitric oxide (NO) production from Kupffer cells. Methods Rats were given estriol (20 mg/kg body weight) intraperitoneally, and Kupffer cells were isolated 24 hr later. Some rats were treated for 4 days with 150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract. After addition of LPS, NO production by Kupffer cell was detected using a fluorescence indicator, DAF-2. Results Twenty-four hours after estriol administration, LPS-induced NO production by Kupffer cells was enhanced as compared with control Kupffer cells. Sterilization of the gut with antibiotics blocked this enhancement. Conclusions Estriol treatment in vivo enhances LPS-induced NO production in Kupffer cells.

Estriol Enhances Lipopolysaccharide‐Induced Increases in Nitric Oxide Production by Kupffer Cells via Mechanisms Dependent on Endotoxin

Background Estriol causes sensitization of Kupffer cells to lipopolysaccharide (LPS) via mechanisms dependent on gut‐derived LPS. Accordingly, this study examines the effect of estriol treatment on nitric oxide (NO) production from Kupffer cells.Methods Rats were given estriol (20 mg/kg body weight) intraperitoneally, and Kupffer cells were isolated 24 hr later. Some rats were treated for 4 days with 150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract. After addition of LPS, NO production by Kupffer cell was detected using a fluorescence indicator, DAF‐2.Results Twenty‐four hours after estriol administration, LPS‐induced NO production by Kupffer cells was enhanced as compared with control Kupffer cells. Sterilization of the gut with antibiotics blocked this enhancement.Conclusions Estriol treatment in vivo enhances LPS‐induced NO production in Kupffer cells.

Thalidomide prevents alcoholic liver injury in rats through suppression of Kupffer cell sensitization and TNF-α production

Background & Aims: Sensitization of Kupffer cells (KCs) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF) α are critical for progression of alcoholic liver injury. Thalidomide has been shown to suppress TNF-α production from macrophages. Accordingly, the purpose of this study was to determine whether thalidomide could prevent alcohol-induced liver injury. Methods: Rats were given ethanol (5 g/kg body wt) and thalidomide (5 mg/kg) once every 24 hours intragastrically. To assess the sensitization of Kupffer cells, LPS (5 mg/kg intravenously) was administered and liver histology was evaluated 24 hours later. KCs were isolated after 4 weeks of ethanol treatment and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, whereas TNF-α was evaluated by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. CD14 was determined by Western and fluorescence staining. Results: Treatment with ethanol for 8 weeks caused marked steatosis, necrosis, and inflammation in the liver. These pathologic parameters were diminished markedly by treatment with thalidomide. In the 4-week ethanol group, the LPS-induced liver damage was aggravated and KCs were sensitized to LPS. Coadministration of thalidomide with ethanol prevented the KC sensitization completely. Furthermore, thalidomide abolished the LPS-induced increase in CD14 expression and [Ca2+]i elevation in KCs. Gut permeability was increased about 10-fold after 4 weeks of ethanol exposure, which was not affected by thalidomide. Moreover, thalidomide reduced the LPS-induced TNF-α production by KCs by decreasing TNF-α messenger RNA. Conclusions: These results collectively indicate that thalidomide prevents alcoholic liver injury through suppression of TNF-α production and abolishment of KC sensitization.GASTROENTEROLOGY 2002;123:291-300

Lactoferrin protects against development of hepatitis caused by sensitization of Kupffer cells by lipopolysaccharide.

BALB/c mice were intravenously injected with lipopolysaccharide (LPS) (0.05 microg/g of body weight) 7 days after being primed with zymosan. Recombinant human lactoferrin (250 microg/g of body weight), intravenously administered 1 day before the injection of LPS, significantly lessened the severity of hepatitis, as assessed by levels of serum alanine transaminase compared to those seen when casein was administered. The transient rise of serum tumor necrosis factor alpha (TNF-alpha) after LPS treatment was also significantly lowered by the intravenous administration of lactoferrin, suggesting that the effect of lactoferrin was due to the suppression of TNF-alpha production. The following results indicate that the sites of action of lactoferrin for the suppression of the development of this type of hepatitis are Kupffer cells. Gadolinium chloride, a substance known to eliminate Kupffer cells, administered 1 day before LPS, inhibited the transient rise of TNF-alpha and protected against the development of hepatitis. Kupffer cells isolated from mice intraperitoneally injected with recombinant human lactoferrin became refractory to LPS. The specific interaction of recombinant human lactoferrin with the Kupffer cells was shown by a binding assay, which revealed two types of binding sites on mouse Kupffer cells. Of the two dissociation constants determined in this way, the lower dissociation constant, 0.47 x 10(-6) M, was within the range of the 50% effective doses for the suppression of TNF-alpha production. These results suggest that recombinant human lactoferrin administered to mice suppresses the production of TNF-alpha by Kupffer cells by directly associating with the binding sites on these cells.

Long‐Term Alcohol Exposure Changes Sensitivity of Rat Kupffer Cells to Lipopolysaccharide

Chronic ethanol treatment enhances Kupffer cell sensitivity to lipopolysaccharide (LPS). In this model, CD14 in Kupffer cells was increased significantly 4 weeks after ethanol. Moreover, it was shown that prostaglandin E2 produced by activated Kupffer cells participated in the mechanism of ethanol-induced fatty liver. This study was designed to elucidate the temporal effect of chronic ethanol exposure on Kupffer cell sensitization to LPS. Rats were given ethanol every 24 hr intragastrically for up to 12 weeks, and Kupffer cells were isolated 24 hr after the final ethanol administration and cultured in RPMI 1640 with 10% fetal bovine serum. After addition of LPS to Kupffer cells, intracellular calcium ([Ca2+]i) was measured. CD14 in Kupffer cells was increased approximately 2-fold, and then it decreased and returned to control levels. The LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha by Kupffer cells were also increased approximately 3-fold over control values, but they also returned to control levels. Triglyceride content increased with the duration of chronic ethanol treatment. At 8 weeks, prostaglandin E2 produced by Kupffer cells increased approximately 3-fold over control values and triglycerides by approximately 4-fold before gradually decreasing to basal levels. After 12 weeks of ethanol exposure, LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha production were only approximately 50% as high as peak levels at 4 weeks. Liver triglyceride content at 12 weeks was reduced significantly compared with values at 8 weeks. Kupffer cells at the early stage of chronic ethanol exposure exhibited sensitization to LPS, but this sensitivity was blunted later. This correlated with triglyceride accumulation in the liver. These data indicate that long-term alcohol exposure changes the sensitivity of rat Kupffer cells to LPS but that the magnitude of the effect is time dependent.

Long-Term Alcohol Exposure Changes Sensitivity of Rat Kupffer Cells to Lipopolysaccharide

Background: Chronic ethanol treatment enhances Kupffer cell sensitivity to lipopolysaccharide (LPS). In this model, CD14 in Kupffer cells was increased significantly 4 weeks after ethanol. Moreover, it was shown that prostaglandin E2 produced by activated Kupffer cells participated in the mechanism of ethanol-induced fatty liver. This study was designed to elucidate the temporal effect of chronic ethanol exposure on Kupffer cell sensitization to LPS. Methods: Rats were given ethanol every 24 hr intragastrically for up to 12 weeks, and Kupffer cells were isolated 24 hr after the final ethanol administration and cultured in RPMI 1640 with 10% fetal bovine serum. After addition of LPS to Kupffer cells, intracellular calcium ([Ca2+]i) was measured. Results: CD14 in Kupffer cells was increased approximately 2-fold, and then it decreased and returned to control levels. The LPS-induced increases in [Ca2+]i and tumor necrosis factor-α by Kupffer cells were also increased approximately 3-fold over control values, but they also returned to control levels. Triglyceride content increased with the duration of chronic ethanol treatment. At 8 weeks, prostaglandin E2 produced by Kupffer cells increased approximately 3-fold over control values and triglycerides by approximately 4-fold before gradually decreasing to basal levels. After 12 weeks of ethanol exposure, LPS-induced increases in [Ca2+]i and tumor necrosis factor-α production were only approximately 50% as high as peak levels at 4 weeks. Liver triglyceride content at 12 weeks was reduced significantly compared with values at 8 weeks. Conclusions: Kupffer cells at the early stage of chronic ethanol exposure exhibited sensitization to LPS, but this sensitivity was blunted later. This correlated with triglyceride accumulation in the liver. These data indicate that long-term alcohol exposure changes the sensitivity of rat Kupffer cells to LPS but that the magnitude of the effect is time dependent.

Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin.

Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol-induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS). Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western analysis. Ethanol levels exhibited a cyclic pattern in ethanol-treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3-fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS-induced intracellular Ca2+, and TNF-alpha all were increased. These phenomena were blocked by antibiotics. Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

Kupffer Cell Sensitization by Alcohol Involves Increased Permeability to Gut‐Derived Endotoxin

Background: Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol‐induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS).Methods: Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura‐2, and tumor necrosis factor (TNF)‐α was measured by enzyme‐linked immunosorbent assay. CD14 was evaluated by Western analysis.Results: Ethanol levels exhibited a cyclic pattern in ethanol‐treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3‐fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (&lt;20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS‐induced intracellular Ca2+, and TNF‐α all were increased. These phenomena were blocked by antibiotics.Conclusions: Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

Tolerance and Sensitization to Endotoxin in Kupffer Cells Caused by Acute Ethanol Involve Interleukin-1 Receptor-Associated Kinase

Ethanol changes sensitivity of Kupffer cells to endotoxin. Here, the hypothesis that interleukin-1 receptor-associated kinase (IRAK), a downstream signaling molecule of toll-like receptors, regulates the response to LPS in Kupffer cells after ethanol treatment was evaluated. C57BL/6 mice were given ethanol intragastrically, and LPS was injected 1 or 21 h later. One hour after ethanol treatment, serum transaminases after LPS were 60% of control, while ethanol increased these parameters about 3-fold 21 h after ethanol. Pretreatment with antibiotics blocked these effects of ethanol. In Kupffer cells from mice treated with ethanol 1 h earlier, LPS-induced TNFα production, and IRAK expression and activity and NFκB were decreased 50–60% of control. In contrast, in Kupffer cells from mice treated with ethanol 21 h earlier, LPS-induced TNFα production, expression and activity of IRAK were increased 1.5-fold over controls, while NFκB was elevated 3-fold. These data indicate that ethanol-induced tolerance and sensitization of Kupffer cells to endotoxin in mice involve IRAK.

Alcohol Enhances Lipopolysaccharide‐Induced Increases in Nitric Oxide Production by Kupffer Cells Via Mechanisms Dependent on Endotoxin

Ethanol causes both tolerance and sensitization of Kupffer cells. Accordingly, this study examines the effect of acute ethanol consumption on nitric oxide (NO) production from Kupffer cells with or without lipopolysaccharide (LPS) treatment. Rats were given ethanol (4 g/kg body weight) intragastrically, and Kupffer cells were isolated 2 and 24 hr later. Some rats were treated for 4 days with 150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract. After addition of LPS, NO was measured by the Griess reaction. Two hours after ethanol administration, LPS-induced NO production by Kupffer cells was diminished by 50% but was enhanced 2-fold at 24 hr. Sterilization of the gut with antibiotics blocked this enhancement. Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. It is likely that sensitization to Kupffer cell is caused by gut-derived endotoxin.

Estriol sensitizes rat Kupffer cells via gut-derived endotoxin.

The relationship between gender and alcohol-induced liver disease is complex; however, endotoxin is most likely involved. Recently, it was reported that estriol activated Kupffer cells by upregulation of the endotoxin receptor CD14. Therefore, the purpose of this work was to study how estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg ip), and Kupffer cells were isolated 24 h later. After addition of lipopolysaccharide (LPS), intracellular Ca2+ concentration was measured using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-alpha was measured by ELISA. CD14 was evaluated by Western analysis. One-half of the rats given estriol intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by sterilization of the gut with antibiotics. A similar pattern was obtained with liver histology and serum transaminases. Translocation of horseradish peroxidase was increased about threefold in gut segments by treatment with estriol. This increase was not altered by treatment with nonabsorbable antibiotics. On the other hand, endotoxin levels were increased to 60-70 pg/ml in plasma of rats treated with estriol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular Ca2+ concentration, tumor necrosis factor-alpha production, and CD14 were increased, as previously reported. All these phenomena were blocked by antibiotics. Therefore, it is concluded that estriol treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14. This is most likely due to elevated portal blood endotoxin caused by increased gut permeability.

Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells.

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, while tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 +/- 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In addition, TNF-alpha production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin.

II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin.

It is well known that females show a greater susceptibility to alcohol-induced liver injury than males. Additionally, females who consume alcohol regularly and have been obese for 10 years or more are at greater risk for both hepatitis and cirrhosis. Female rats on an enteral alcohol protocol exhibit injury more quickly than males, with widespread fatty changes over a larger portion of the liver lobule. Levels of plasma endotoxin, intercellular adhesion molecule-1, free radical adducts, infiltrating neutrophils, and nuclear factor-kappaB are increased about twofold more in livers from female than male rats after enteral alcohol treatment. Estrogen treatment in vivo increases the sensitivity of Kupffer cells to endotoxin. Evidence has been presented that Kupffer cells are pivotal in the development of alcohol-induced liver injury. Destruction of Kupffer cells with gadolinium chloride (GdCl3) or reduction of bacterial endotoxin by sterilization of the gut with antibiotics blocks early inflammation due to alcohol. Similar results have been obtained with anti-tumor necrosis factor-alpha antibody. These findings led to the hypothesis that alcohol-induced liver injury involves increases in circulating endotoxin, leading to activation of Kupffer cells, which causes a hypoxia-reoxygenation injury. This idea has been tested using pimonidazole, a nitroimidazole marker, to quantitate hypoxia in downstream pericentral regions of the liver lobule. After chronic enteral alcohol, pimonidazole binding increases twofold. Enteral alcohol also increases free radicals detected with electron spin resonance. Importantly, hepatic hypoxia and radical production detected in bile are decreased by destruction of Kupffer cells with GdCl3. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by alcohol.

The role of gut-derived bacterial toxins and free radicals in alcohol-induced liver injury.

Previous research from this laboratory using a continuous enteral ethanol (EtOH) administration model demonstrated that Kupffer cells are pivotal in the development of EtOH-induced liver injury. When Kupffer cells were destroyed using gadolinium chloride (GdCl3 ) or the gut was sterilized with polymyxin B and neomycin, early inflammation due to EtOH was blocked. Anti-tumour necrosis factor (TNF)-α antibody markedly decreased EtOH-induced liver injury and increased TNF-mRNA. These findings led to the hypothesis that EtOH-induced liver injury involves increases in circulating endotoxin leading to activation of Kupffer cells. Pimonidazole, a nitro-imidazole marker, was used to detect hypoxia in downstream pericentral regions of the lobule. Following one large dose of EtOH or chronic enteral EtOH for 1 month, pimonidazole binding was increased significantly in pericentral regions of the liver lobule, which was diminished with GdCl3 . Enteral EtOH increased free radical generation detected with electron spin resonance (ESR). These radical species had coupling constants matching α-hydroxyethyl radical and were shown conclusively to arise from EtOH based on a doubling of the ESR lines when 13 C-EtOH was given. α-Hydroxyethyl radical production was also blocked by the destruction of Kupffer cells with GdCl3 . It is known that females develop more severe EtOH-induced liver injury more rapidly and with less EtOH than males. Female rats on the enteral protocol exhibited more rapid injury and more widespread fatty changes over a larger portion of the liver lobule than males. Plasma endotoxin, ICAM-1, free radical adducts, infiltrating neutrophils and transcription factor NFκB were approximately two-fold greater in livers from females than males after 4 weeks of enteral EtOH treatment. Furthermore, oestrogen treatment increased the sensitivity of Kupffer cells to endotoxin. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by ethanol.

Alcohol causes both tolerance and sensitization of rat Kupffer cells via mechanisms dependent on endotoxin

Background & Aims: Ethanol causes both tolerance and sensitization of Kupffer cells. This study was designed to evaluate temporal effects of ethanol in an attempt to understand this paradox. Methods: Rats were given ethanol (4 g/kg body wt) intragastrically, and Kupffer cells were isolated 0–48 hours later. After addition of lipopolysaccharide (LPS), intracellular calcium concentration ([Ca 2+] i) was measured using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor α (TNF-α) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Results: Two hours after ethanol administration, the LPS-induced increase in [Ca 2+] i and TNF-α release by Kupffer cells was diminished by 50%, and these parameters were reciprocally enhanced twofold at 24 hours. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca 2+] i and TNF-α release completely. Twenty-four hours after ethanol, CD14 in Kupffer cells was elevated about fivefold. Conclusions: Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. It is likely that both of these phenomena are caused by gut-derived endotoxin and that sensitization in Kupffer cells is caused by increases in CD14. GASTROENTEROLOGY 1998;115:443-451

Estrogen increases sensitivity of hepatic Kupffer cells to endotoxin.

The relationship among gender, lipopolysaccharide (LPS), and liver disease is complex. Accordingly, the effect of estrogen on activation of Kupffer cells by endotoxin was studied. All rats given estrogen intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by pretreatment with gadolinium chloride, a Kupffer cell toxicant. Peak serum tumor necrosis factor-alpha (TNF-alpha) values as well as TNF-alpha mRNA in the liver after LPS were twice as high in the estrogen-treated group as in the untreated controls. Plasma nitrite levels and inducible nitric oxide synthase in the liver were also elevated significantly in estrogen-treated rats 6 h after LPS. Furthermore, Kupffer cells isolated from estrogen-treated rats produced about twice as much TNF-alpha and nitrite as controls did in response to LPS. In addition, Kupffer cells from estrogen-treated rats required 15-fold lower amounts of LPS to increase intracellular Ca2+ than controls did, and Kupffer cells from estrogen-treated animals expressed more CD14, the receptor for LPS/LPS binding protein, than controls. Moreover, estrogen treatment increased LPS binding protein mRNA dramatically in liver in 6-24 h. It is concluded that estrogen treatment in vivo sensitizes Kupffer cells to LPS, leading to increased toxic mediator production by the liver.