Nympha D'Souza - Academia.edu (original) (raw)
Papers by Nympha D'Souza
We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to prod... more We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-a (TNF-a), superoxide anion (O,-), and nitric oxide (NO)-three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 rng/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weightlhr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 02&B6, 100 pg/lOO g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-a, 0,-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-a secmtion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-a spontaneously in culture. However, the AMs from chronic alcohor-fed group secreted 4263% less TNF-a spontaneously and in r e s m s e to LPS, interferon-y (IFN-y) or IFN-y + LPS compared with the'AMs from pair-fed group. Systemic LPS treatment inhibited in vitrPLPS-stimulated AM TNF-a secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)-and opsonized zymosan (0PZ)-induced AM 0,secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more 0,-in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)-and OPZ (66%)-induced AM 0,release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM 0,secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM 0,secretion in the pair-fed group, it enhanced the PMA-stimulated AM 0,release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-y, LPS, and IFN-y + LPS significantly.
Pulmonary infection with Pneumocystis carinii, an opportunistic pathogen, is associated with a va... more Pulmonary infection with Pneumocystis carinii, an opportunistic pathogen, is associated with a variety of immunosuppressive states, Including human immunodeficiency virus infection. We hypothesized that alcohol ingestion might compromise host defenses against this pathogen and, in an immunocompromised host, increase the sever-Hyof infection. This hypothesis was tested in both acute and chronic ethanol-treated normal and CD4+ T-cell-depleted mice challenged with f. carinii organisms.
Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-indu... more Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-a could be due, at least in part, to alterations in TNF-a cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchy-ma1 (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 pg/lOO g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K,) and capacity B, . J of binding sites, using recombinant h~rnan-['~l]TNF-cu as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K,,, in the range of 150-200 PM), low-capacity (B,,,,, in the range of 2-3 fmol/106 cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/106 cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K,, = 1 .O n M and a B,,, = 95 fmol/106 cells). Acute ETOH administration caused an increase in K , , on both Kupffer and sinusoidal endothelial cells; a decrease in K , , on hepatocytes; and an increase in Kd2, B , , , , , and Bmsx2 on endothelial cells. A second binding site on hepatocytes (Kd2 = 5.8 nM, Bmax2 = 186fmo1/106 cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K,, and B,,,, on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B,,,, on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B, , J and affinity (K,) of TNF-cu cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal From the Depaifments of Physiology (I.KD., J.J.S.) and Medicine (Pulmona yICritica1 Care) (N.B. D.), 332 endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-a.
Alcoholism-clinical and Experimental Research, 1996
We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to prod... more We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-a (TNF-a), superoxide anion (O,-), and nitric oxide (NO)-three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 rng/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weightlhr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 02&B6, 100 pg/lOO g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-a, 0,-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-a secmtion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-a spontaneously in culture. However, the AMs from chronic alcohor-fed group secreted 4263% less TNF-a spontaneously and in r e s m s e to LPS, interferon-y (IFN-y) or IFN-y + LPS compared with the'AMs from pair-fed group. Systemic LPS treatment inhibited in vitrPLPS-stimulated AM TNF-a secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)-and opsonized zymosan (0PZ)-induced AM 0,secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more 0,-in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)-and OPZ (66%)-induced AM 0,release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM 0,secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM 0,secretion in the pair-fed group, it enhanced the PMA-stimulated AM 0,release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-y, LPS, and IFN-y + LPS significantly.
Alcohol-induced hepatic sinusoidal endothelial cell dysfunction in the mouse is mediated by Kupffer cells
International Hepatology Communications, 1995
Metabolism, 1990
The increased glucose turnover seen during the hypermetabolic, hyperdynamic phase of sepsis is pa... more The increased glucose turnover seen during the hypermetabolic, hyperdynamic phase of sepsis is part of the body's defense mechanisms. In contrast, the metabolism of ethanol (ETOH] is known to compromise hepatic gluconeogenesis under certain conditions. This study tested the hypothesis that acute infusion of ETOH inhibits the elevated glucose production that is manifested during infection and thereby alters the normal responses to sepsis. In catheterized conscious rats, ETOH or saline infusion was started 24 hours before the induction of sepsis. and continued throughout the experiment.
Journal of Hepatology, 1999
Background~Aims: Functional and morphological alterations of the hepatic sinusoidal endothelial c... more Background~Aims: Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. Methods: Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. Results: At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glntathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. Conclusions: The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.
Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells
Hepatology Research, 2001
Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly chara... more Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly characterized. The purpose of this study was to determine in rats the effects of chronic alcohol consumption on the relative magnitude of apoptosis in two major targets of alcohol-induced liver injury: the hepatocyte (Hep) and sinusoidal endothelial cell (SEC). Methods: Rats were fed a liquid diet containing either alcohol or isocaloric amounts of maltose-dextrin for 14 weeks. Hep and SEC were isolated by liver perfusion with collagenase followed by centrifugal elutriation. The state of the liver was assessed on the basis of light microscopic appearance, plasma liver enzymes (alanine and aspartate:2-oxoglutarate amino transferases), and the content of malondialdehyde in Hep. Apoptosis was assessed on the basis of DNA fragmentation in the whole organ (TUNEL), and caspase-3 and -8 activity in isolated cells. A mechanistic approach was also undertaken by measuring mRNA expression and the amount of protein for Fas/CD95, Fas ligand, caspase-3, Bax, Bcl-X(L), and Bcl-2 in the isolated Hep and SEC. Results: The livers of alcohol-fed rats displayed prominent steatosis. Oxidative stress was also present as reflected by an increase in the malondialdehyde content of Hep. Alcohol consumption increased apoptosis in the whole liver assessed on the basis of TUNEL procedure and in Hep and SEC as reflected by significant increase in caspase-3 activity. Of the multiple pro- and anti-apoptotic factors determined in this study, significant changes as assessed by both mRNA expression and the amount of proteins, were observed only in the SEC compartment. Conclusions: The data presented in this study indicate that: (1) chronic alcohol consumption in rats leads to a moderate augmentation of apoptosis in the whole liver and in two liver cell types which are targets for injury in alcoholic liver disease: Hep and SEC; (2) the mechanisms recruited/activated by these two types of liver cells to initiate and execute apoptosis in response to alcohol vary with the cell type.
Alcohol-induced sinusoidal endothelial cell dysfunction in the mouse is associated with exacerbated liver apoptosis and can be reversed by caspase inhibition
Hepatology Research, 2001
The purpose of this study was to determine if a correlation exists between alcohol-induced liver ... more The purpose of this study was to determine if a correlation exists between alcohol-induced liver sinusoidal endothelial cell (SEC) dysfunction and alcohol-induced augmented liver apoptosis in the mouse. Mice were fed an alcohol-containing liquid diet for 7 weeks. On the last day of feeding, the animals were treated with the pan-caspase inhibitor IDN1529 (N-[(indole-2-)-alaninyl]-3-amino-4-oxo-fluoropentanoic acid), killed, and plasma amino transferase activity, plasma hyaluronan, liver caspase-3 activity, the frequency of apoptotic nuclei in the liver, liver histology and electron microscopic appearance evaluated. Alcohol feeding significantly increased (2.5-fold) plasma hyaluronan levels, frequency of apoptotic nuclei (20-fold), and caspase-3 activity (1.7-fold), but did not affect plasma amino transferase activity. Transmission electron microscopy revealed that SEC was among the cell types undergoing apoptosis. Livers of alcohol-fed mice displayed marked fat accumulation without necrosis or fibrosis. Treatment of mice with IDN1529 reversed the alcohol effects on plasma hyaluronan levels, liver caspase-3 activity, and frequency of apoptotic nuclei. However, the inhibitor did not prevent fat accumulation in the liver. These data suggest that alcohol-induced exacerbation of apoptosis in the liver, which extends to the SEC, causes functional impairment of the sinusoidal lining and can be reversed by caspase inhibition.
Hepatology Research, 2002
The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigat... more The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigated whether Fas-mediated apoptosis in the liver requires induction of apoptosis-related regulators and whether Kupffer cells play a role in this process. Mice were injected with GdCl 3 to deplete/suppress Kupffer cells, followed by treatment with an anti-Fas agonistic antibody, Jo2. Hepatic mRNA levels of several pro-and anti-apoptotic regulators were determined 0.5, 1.5 and 4.0 h after Jo2 injection. Liver histology, TUNEL response, the activity of caspases-3, -8, and -9, and reduced and oxidized glutathione, were also evaluated. Jo2 dramatically increased the number of apoptotic nuclei in the liver, up-regulated mRNA for Bcl-w, Bfl-1, and Bcl-X L, but did not affect pro-apoptotic regulator mRNA expression. Caspase-3, -8 and -9 activity increased at 1.5 h after Jo2-injection. GdCl 3 treatment was associated with an increase in the apoptotic effect of Jo2. No effect of Jo2 was recorded on redox state of the free cellular thiol system. These data suggest that: (1) the prompt apoptotic response to Fas-mediated signaling in the liver does not require induction of pro-apoptotic factors; (2) Kupffer cells may play a major role in the liver apoptotic response to Fas ligation by clearing apoptotic cells by phagocytosis; (3) oxidative stress does not seem to play an important role in Fas-mediated liver apoptosis.
Effect of chronic alcohol consumption by rats on tumor necrosis factor-α and interleukin-6 clearance in vivo and by the isolated, perfused liver
Biochemical Pharmacology, 1996
The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF... more The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF)-alpha and [125I]interleukin (IL)-6 plasma clearance and organ distribution in vivo and uptake and metabolism by the isolated, perfused liver were studied. Alcohol was administered to rats in a liquid diet for 16 weeks, and caused a decreased (48%) plasma clearance rate of IL-6 and converted the plasma clearance kinetics of the cytokine from a biphasic exponential in normal rats to a monophasic exponential decay. Alcohol feeding significantly increased (101%) plasma clearance of TNF-alpha, which followed a biphasic exponential decay and decreased the T1/2 for both the alpha (67%) and beta (76%) elimination components. The distribution of both cytokines in trichloroacetic acid precipitable and non-precipitable fractions of liver, spleen, stomach, small intestine (ileum), lung, kidney, and blood was also studied. The only effect of alcohol treatment was a significant decrease in IL-6 uptake and metabolism by the small intestine. Perfused livers, isolated from alcohol-fed rats, took up and metabolized larger amounts of IL-6 than did livers isolated from pair-fed rats. TNF-alpha uptake and metabolism by the isolated, perfused liver were not affected by chronic alcohol consumption. Regardless of the animal treatment, the isolated perfused liver took up and metabolized significantly larger (17-fold) amounts of TNF-alpha than IL-6, in spite of identical concentrations of cytokines (6 nM) in the perfusion medium. The data presented in this study along with our previous results demonstrating the effects of alcohol consumption on TNF-alpha and IL-6 receptors on various liver cells suggest that the effects of chronic alcohol treatment on cytokine clearance cannot be ascribed to changes in the receptors for the two cytokines. Also, no correlation was found between the effects of alcohol treatment on plasma cytokine clearance and uptake and metabolism of cytokines by the isolated, perfused liver. Experimental data and theoretical considerations suggest that cytokine receptor recycling may play an important role in mediating alcohol effects on cytokine clearance.
Effects of acute alcohol intoxication on gluconeogenesis and its hormonal responsiveness in isolated, perfused rat liver
Biochemical Pharmacology, 1992
Rats were acutely administered ethanol as a primed constant infusion in order to produce sustaine... more Rats were acutely administered ethanol as a primed constant infusion in order to produce sustained blood ethanol levels of 8-12 or 55-65 mM. At the end of ethanol infusion the livers were either freeze-clamped in vivo or isolated and perfused for metabolic studies. The rate of gluconeogenesis and its responsiveness to phenylephrine (10 microM), prostaglandin F2 alpha (5 microM) and glucagon (10 nM), as well as the redox state of the cytosolic NAD(+)-NADH system were assessed in livers isolated from acutely ethanol-treated rats, and subsequently perfused without ethanol. For liver clamped in vivo, high- but not low-ethanol treatment decreased the ATP content by 31% and slightly increased ADP and AMP content, resulting in a decreased energy charge (11%). Glutamate and aspartate content was also increased in high-dose ethanol-infused rats with no changes in malate and 2-oxoglutarate content. Gluconeogenesis with saturating concentrations of lactate (4 mM)+pyruvate (0.4 mM) was delayed in reaching a plateau in the livers of high-dose ethanol-treated rats and its response to all three stimulators was impaired. Low-dose ethanol treatment only decreased the liver response to phenylephrine. While the perfused livers of low-dose ethanol-treated rats displayed no changes in adenine nucleotide content, the livers of high-dose ethanol-treated rats had a decreased ATP (35%) and an increased AMP (77%) content, paralleled by a fall in the total adenine nucleotides (14%) and energy charge (14%). No differences were observed between the saline- and ethanol-treated rats with respect to malate-aspartate shuttle intermediate concentration in perfused livers. Also, the livers of high-, but not low-dose ethanol-treated rats had a more negative value of NAD(+)-NADH redox state as compared to the livers of control rats. The data suggest that acute ethanol intoxication produces changes in liver metabolism and its responsiveness to hormones/agonists that are demonstrable for at least 2 hr after isolation and perfusion of the liver.
Granulocyte-Macrophage colony stimulating factor (GM-CSF) priming in the treatment of elderly patients with acute myelogenous leukemia
American Journal of Hematology, 1995
Alcoholism: Clinical and Experimental Research, 1996
Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronicall... more Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronically in a complete liquid diet or in sucrosecontaining drinking water. The animals were kept on liquid diet (kalcohol) for 14 weeks or on sucrose-containing drinking water (kalcohol) for 12.5 weeks and sacrificed thereafter. To rule out possible artifact induced by fixation procedure, livers were fixed by immersion (no perfusion), immersion preceded by perfusion, and by perfusion wlth glutaraldehyde and examined with both scanning and transmission electron microscopy. Regardless of the mode of its administration, and of the fixation procedure used, alcohol induced similar changes in liver sinusoid ultrastructure. Such changes included disruption of the sieve-plate pattern of the sinusoidal endothelial cell fenestrations with the appearance of large gaps and resulting in a meshwork lining, wherein large areas of the sinusoid communicated freely with the underlying hepatocytes. Transmission electron microscopy complemented these findings. The results reported in this study demonstrate that alcohol-induced structural changes of the liver sinusoid in the rat are similar whether alcohol is fed via a liquid diet or in drinking water. Therefore, alcohol administration in drinking water may provide a simple, inexpensive, and convenient method of inducing structural changes in the rat liver sinusoid.
Acute Ethanol Intoxication Suppresses E. Coli Lipopolysaccharide Enhanced Glucose Utilization by Hepatic Nonparenchymal Cells
Alcoholism: Clinical and Experimental Research, 1991
During infection or endotoxemia, the immune system is activated and its energy needs increase. Al... more During infection or endotoxemia, the immune system is activated and its energy needs increase. Alcohol (ETOH) intoxication on the other hand suppresses the immune system and increases susceptibility to infection. Since the liver is the primary site both for metabolism of ETOH and detoxification of bacterial lipopolysaccharides (LPS), this investigation was directed at studying the effect of acute ETOH intoxication on the LPS-induced enhancement of in vivo glucose utilization in different types of hepatic cells. Rats were given an intravenous (IV) injection of ETOH followed by a constant infusion for 7 hr to maintain blood alcohol levels at about 175 mg/dl. E. coli LPS was administered IV at 4 hr and in vivo glucose utilization by the different types of liver cells was estimated 3 hr later using the 14C-2-deoxyglucose technique. Hepatocytes (HP), Kupffer (KC), and endothelial cells (EC), as well as the sequestered polymorphonuclear leukocytes (PMN), were separated from the liver by collagenase-pronase digestion followed by centrifugal elutriation and Ficoll-Hypaque density gradient centrifugation. The number of PMN in the liver was increased several-fold 3 hr after LPS administration. The presence of ETOH did not inhibit the LPS-induced neutrophil migration into the liver. ETOH depressed the LPS-induced increase in glucose uptake in both EC and KC by 50 to 80%, respectively. It also reduced the LPS-induced increase of plasma tumor necrosis factor activity by 80%. ETOH alone did not produce any significant changes in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Ethanol Alters the Metabolic Response of Isolated, Perfused Rat Liver to a Phagocytic Stimulus
Alcoholism: Clinical and Experimental Research, 1993
Intercellular communication in the liver is a potentially important mechanism for the regulation ... more Intercellular communication in the liver is a potentially important mechanism for the regulation of hepatic metabolism. Since alcohol (ethanol, ETOH) can interact with both parenchymal and nonparenchymal cells, the present study was performed to assess the possible effects of ETOH on the nonparenchymal cell-to-hepatocyte signal traffic by studying the glycogenolytic and glycolytic response of the perfused rat liver to colloidal carbon, a phagocytic stimulus for Kupffer and sinusoidal endothelial cells. Livers from fed rats were perfused with hemoglobin-free Krebs Ringer bicarbonate buffer containing ETOH (20 mM) or acetaldehyde (1 mM). Twenty minutes after initiating the infusion of ETOH or acetaldehyde, colloidal carbon was infused and the rate of carbon uptake, glucose, lactate and pyruvate output, and oxygen consumption were determined. In control livers, carbon stimulated the output of glucose (60%), lactate (25%), and pyruvate (53%), without affecting the lactate/pyruvate ratio. ETOH, but not acetaldehyde, enhanced the carbon effect on glucose output (38%), but suppressed the increased lactate and pyruvate output (48% and 91% respectively) resulting in a dramatic 10-fold increase in the lactate/pyruvate ratio. By using inhibitors of cyclooxygenase or alcohol dehydrogenase (indomethacin and 4-methylpyrazole, respectively) in the presence of carbon and/or ETOH, we determined that: (1) following carbon stimulation prostaglandins are the likely mediators secreted by nonparenchymal cells that increase carbohydrate output; and (2) the ETOH-induced enhancement of carbon-stimulated glycogenolysis is also mediated by prostaglandins and is not dependent on the oxidative metabolism of ETOH.
Alcoholism: Clinical and Experimental Research, 1995
Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-indu... more Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-a could be due, at least in part, to alterations in TNF-a cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchy-ma1 (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 pg/lOO g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K,) and capacity B, . J of binding sites, using recombinant h~rnan-['~l]TNF-cu as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K,,, in the range of 150-200 PM), low-capacity (B,,,,, in the range of 2-3 fmol/106 cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/106 cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K,, = 1 .O n M and a B,,, = 95 fmol/106 cells). Acute ETOH administration caused an increase in K , , on both Kupffer and sinusoidal endothelial cells; a decrease in K , , on hepatocytes; and an increase in Kd2, B , , , , , and Bmsx2 on endothelial cells. A second binding site on hepatocytes (Kd2 = 5.8 nM, Bmax2 = 186fmo1/106 cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K,, and B,,,, on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B,,,, on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B, , J and affinity (K,) of TNF-cu cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal From the Depaifments of Physiology (I.KD., J.J.S.) and Medicine (Pulmona yICritica1 Care) (N.B. D.), 332 endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-a.
Modulation of F-met-leu-phe Induced Chemotactic Activity and Superoxide Production by Neutrophils during Chronic Ethanol Intoxication
Alcoholism: Clinical and Experimental Research, 1992
Chronic alcohol consumption has been associated with increased migration of neutrophils into live... more Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.
Alcoholism: Clinical and Experimental Research, 1994
The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus ... more The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus interfering in lung immune defense mechanisms. Male rats were treated with alcohol either acutely (7 hr continuous intravenous alcohol infusion at a rate of 30 mg/100 g body weight/hr after a priming dose of 175 mg/100 g body weight) or chronically (feeding an alcohol-containing liquid diet for 12-14 weeks). Three hr before killing, the rats received an intravenous injection of Gram-negative bacterial lipopolysaccharide (LPS Escherkhia cdi, 026B6, 100 pg/100 g body weight). After anesthesia with sodium pentobarbital, the trachea was cannulated, and the lungs excised and lavaged to obtain alveolar macrophages. The recovered cells were used to measure the binding of recombinant human ['ml]tumor necrosis factor-a (TNF-a) and ['wl]interleukin-6 (IL-6). Kd and B, were determined at 4OC, thus reflecting only the cell-surface binding sites and their affinity. Two binding sites were detected for both cytokines: high-affinity K d , in the range of 20-110 pM), lowcapacity (8-, in the range of 1-13 fmol/lO' cells), and low-affinity (Kdl in the range of 0.6-1.3 nM), high-capacity ( 8 , , in the range of 34-100 fmol/lO' cells). Acute alcohol treatment significantly decreased 8 , , (39%) and 8 , , (79%) for TNF-a, whereas chronic alcohol feeding abrogated the 8-, (8-, = 0), without affecting 8-*. In the acute group, LPS had an effect similar to that of alcohol.
Interleukin-6 and Tumor Necrosis Factor-alpha Clearance and Metabolism In Vivo and by the Isolated, Perfused Liver in the Rat: Effect of Acute Alcohol Administration
Alcoholism: Clinical and Experimental Research, 1996
ABSTRACT Plasma clearance and organ distribution of intravenously injected human recombinant [125... more ABSTRACT Plasma clearance and organ distribution of intravenously injected human recombinant [125I]interleukin (IL)-6 and [125I]tumor necrosis factor (TNF)-α were studied in male rats, 2 hr after intravenous alcohol (ethanol) administration (single dose, 2.2 g · kg-1 body weight). Also, the rate of uptake and degradation of the two cytokines by the isolated, perfused rat liver was studied in the absence or in the presence of ethanol (35 mM) in the perfusate. Acute ethanol administration significantly increased plasma clearance rate for both cytokines (36% and 72%, for IL-6 and TNF-α, respectively), decreased the t1/2α (30% and 11%, for IL-6 and TNF-α, respectively), abolished the slow (β)-phase component for TNF-α, and increased t1/2β for IL-6 (31%). Although alcohol did not affect organ distribution of TNF-α, it increased the IL-6 content in the liver, kidney, and blood. IL-6 uptake rate by the isolated, perfused rat liver was 2-fold higher than TNF-α uptake, whereas the rate of degradation was larger for TNF-α than for IL-6, despite the fact that both cyokines were presented to the liver at the same concentration (6 nM). Ethanol addition to the perfusate (35 mM, final concentration) significantly increased TNF-α uptake (24%), without affecting IL-6 uptake or the degradation rate of either cytokine. Also, the kinetics of degradation by the isolated, perfused rat liver was linear for TNF-α, but exponential for IL-6. Data presented in this study demonstrate that: (1) acute alcohol consumption can alter the kinetic behavior of IL-6 and TNF-α in the bloodstream, mainly by accelerating their clearance which, in turn, may counteract the outcome of cytokine secretion and delivery to the blood; and (2) short exposure of liver to ethanol levels commonly seen in humans after binge drinking may alter its capacity to take up cytokines.
We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to prod... more We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-a (TNF-a), superoxide anion (O,-), and nitric oxide (NO)-three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 rng/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weightlhr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 02&B6, 100 pg/lOO g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-a, 0,-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-a secmtion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-a spontaneously in culture. However, the AMs from chronic alcohor-fed group secreted 4263% less TNF-a spontaneously and in r e s m s e to LPS, interferon-y (IFN-y) or IFN-y + LPS compared with the'AMs from pair-fed group. Systemic LPS treatment inhibited in vitrPLPS-stimulated AM TNF-a secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)-and opsonized zymosan (0PZ)-induced AM 0,secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more 0,-in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)-and OPZ (66%)-induced AM 0,release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM 0,secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM 0,secretion in the pair-fed group, it enhanced the PMA-stimulated AM 0,release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-y, LPS, and IFN-y + LPS significantly.
Pulmonary infection with Pneumocystis carinii, an opportunistic pathogen, is associated with a va... more Pulmonary infection with Pneumocystis carinii, an opportunistic pathogen, is associated with a variety of immunosuppressive states, Including human immunodeficiency virus infection. We hypothesized that alcohol ingestion might compromise host defenses against this pathogen and, in an immunocompromised host, increase the sever-Hyof infection. This hypothesis was tested in both acute and chronic ethanol-treated normal and CD4+ T-cell-depleted mice challenged with f. carinii organisms.
Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-indu... more Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-a could be due, at least in part, to alterations in TNF-a cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchy-ma1 (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 pg/lOO g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K,) and capacity B, . J of binding sites, using recombinant h~rnan-['~l]TNF-cu as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K,,, in the range of 150-200 PM), low-capacity (B,,,,, in the range of 2-3 fmol/106 cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/106 cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K,, = 1 .O n M and a B,,, = 95 fmol/106 cells). Acute ETOH administration caused an increase in K , , on both Kupffer and sinusoidal endothelial cells; a decrease in K , , on hepatocytes; and an increase in Kd2, B , , , , , and Bmsx2 on endothelial cells. A second binding site on hepatocytes (Kd2 = 5.8 nM, Bmax2 = 186fmo1/106 cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K,, and B,,,, on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B,,,, on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B, , J and affinity (K,) of TNF-cu cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal From the Depaifments of Physiology (I.KD., J.J.S.) and Medicine (Pulmona yICritica1 Care) (N.B. D.), 332 endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-a.
Alcoholism-clinical and Experimental Research, 1996
We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to prod... more We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-a (TNF-a), superoxide anion (O,-), and nitric oxide (NO)-three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 rng/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weightlhr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 02&B6, 100 pg/lOO g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-a, 0,-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-a secmtion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-a spontaneously in culture. However, the AMs from chronic alcohor-fed group secreted 4263% less TNF-a spontaneously and in r e s m s e to LPS, interferon-y (IFN-y) or IFN-y + LPS compared with the'AMs from pair-fed group. Systemic LPS treatment inhibited in vitrPLPS-stimulated AM TNF-a secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)-and opsonized zymosan (0PZ)-induced AM 0,secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more 0,-in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)-and OPZ (66%)-induced AM 0,release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM 0,secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM 0,secretion in the pair-fed group, it enhanced the PMA-stimulated AM 0,release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-y, LPS, and IFN-y + LPS significantly.
Alcohol-induced hepatic sinusoidal endothelial cell dysfunction in the mouse is mediated by Kupffer cells
International Hepatology Communications, 1995
Metabolism, 1990
The increased glucose turnover seen during the hypermetabolic, hyperdynamic phase of sepsis is pa... more The increased glucose turnover seen during the hypermetabolic, hyperdynamic phase of sepsis is part of the body's defense mechanisms. In contrast, the metabolism of ethanol (ETOH] is known to compromise hepatic gluconeogenesis under certain conditions. This study tested the hypothesis that acute infusion of ETOH inhibits the elevated glucose production that is manifested during infection and thereby alters the normal responses to sepsis. In catheterized conscious rats, ETOH or saline infusion was started 24 hours before the induction of sepsis. and continued throughout the experiment.
Journal of Hepatology, 1999
Background~Aims: Functional and morphological alterations of the hepatic sinusoidal endothelial c... more Background~Aims: Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. Methods: Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. Results: At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glntathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. Conclusions: The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.
Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells
Hepatology Research, 2001
Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly chara... more Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly characterized. The purpose of this study was to determine in rats the effects of chronic alcohol consumption on the relative magnitude of apoptosis in two major targets of alcohol-induced liver injury: the hepatocyte (Hep) and sinusoidal endothelial cell (SEC). Methods: Rats were fed a liquid diet containing either alcohol or isocaloric amounts of maltose-dextrin for 14 weeks. Hep and SEC were isolated by liver perfusion with collagenase followed by centrifugal elutriation. The state of the liver was assessed on the basis of light microscopic appearance, plasma liver enzymes (alanine and aspartate:2-oxoglutarate amino transferases), and the content of malondialdehyde in Hep. Apoptosis was assessed on the basis of DNA fragmentation in the whole organ (TUNEL), and caspase-3 and -8 activity in isolated cells. A mechanistic approach was also undertaken by measuring mRNA expression and the amount of protein for Fas/CD95, Fas ligand, caspase-3, Bax, Bcl-X(L), and Bcl-2 in the isolated Hep and SEC. Results: The livers of alcohol-fed rats displayed prominent steatosis. Oxidative stress was also present as reflected by an increase in the malondialdehyde content of Hep. Alcohol consumption increased apoptosis in the whole liver assessed on the basis of TUNEL procedure and in Hep and SEC as reflected by significant increase in caspase-3 activity. Of the multiple pro- and anti-apoptotic factors determined in this study, significant changes as assessed by both mRNA expression and the amount of proteins, were observed only in the SEC compartment. Conclusions: The data presented in this study indicate that: (1) chronic alcohol consumption in rats leads to a moderate augmentation of apoptosis in the whole liver and in two liver cell types which are targets for injury in alcoholic liver disease: Hep and SEC; (2) the mechanisms recruited/activated by these two types of liver cells to initiate and execute apoptosis in response to alcohol vary with the cell type.
Alcohol-induced sinusoidal endothelial cell dysfunction in the mouse is associated with exacerbated liver apoptosis and can be reversed by caspase inhibition
Hepatology Research, 2001
The purpose of this study was to determine if a correlation exists between alcohol-induced liver ... more The purpose of this study was to determine if a correlation exists between alcohol-induced liver sinusoidal endothelial cell (SEC) dysfunction and alcohol-induced augmented liver apoptosis in the mouse. Mice were fed an alcohol-containing liquid diet for 7 weeks. On the last day of feeding, the animals were treated with the pan-caspase inhibitor IDN1529 (N-[(indole-2-)-alaninyl]-3-amino-4-oxo-fluoropentanoic acid), killed, and plasma amino transferase activity, plasma hyaluronan, liver caspase-3 activity, the frequency of apoptotic nuclei in the liver, liver histology and electron microscopic appearance evaluated. Alcohol feeding significantly increased (2.5-fold) plasma hyaluronan levels, frequency of apoptotic nuclei (20-fold), and caspase-3 activity (1.7-fold), but did not affect plasma amino transferase activity. Transmission electron microscopy revealed that SEC was among the cell types undergoing apoptosis. Livers of alcohol-fed mice displayed marked fat accumulation without necrosis or fibrosis. Treatment of mice with IDN1529 reversed the alcohol effects on plasma hyaluronan levels, liver caspase-3 activity, and frequency of apoptotic nuclei. However, the inhibitor did not prevent fat accumulation in the liver. These data suggest that alcohol-induced exacerbation of apoptosis in the liver, which extends to the SEC, causes functional impairment of the sinusoidal lining and can be reversed by caspase inhibition.
Hepatology Research, 2002
The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigat... more The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigated whether Fas-mediated apoptosis in the liver requires induction of apoptosis-related regulators and whether Kupffer cells play a role in this process. Mice were injected with GdCl 3 to deplete/suppress Kupffer cells, followed by treatment with an anti-Fas agonistic antibody, Jo2. Hepatic mRNA levels of several pro-and anti-apoptotic regulators were determined 0.5, 1.5 and 4.0 h after Jo2 injection. Liver histology, TUNEL response, the activity of caspases-3, -8, and -9, and reduced and oxidized glutathione, were also evaluated. Jo2 dramatically increased the number of apoptotic nuclei in the liver, up-regulated mRNA for Bcl-w, Bfl-1, and Bcl-X L, but did not affect pro-apoptotic regulator mRNA expression. Caspase-3, -8 and -9 activity increased at 1.5 h after Jo2-injection. GdCl 3 treatment was associated with an increase in the apoptotic effect of Jo2. No effect of Jo2 was recorded on redox state of the free cellular thiol system. These data suggest that: (1) the prompt apoptotic response to Fas-mediated signaling in the liver does not require induction of pro-apoptotic factors; (2) Kupffer cells may play a major role in the liver apoptotic response to Fas ligation by clearing apoptotic cells by phagocytosis; (3) oxidative stress does not seem to play an important role in Fas-mediated liver apoptosis.
Effect of chronic alcohol consumption by rats on tumor necrosis factor-α and interleukin-6 clearance in vivo and by the isolated, perfused liver
Biochemical Pharmacology, 1996
The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF... more The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF)-alpha and [125I]interleukin (IL)-6 plasma clearance and organ distribution in vivo and uptake and metabolism by the isolated, perfused liver were studied. Alcohol was administered to rats in a liquid diet for 16 weeks, and caused a decreased (48%) plasma clearance rate of IL-6 and converted the plasma clearance kinetics of the cytokine from a biphasic exponential in normal rats to a monophasic exponential decay. Alcohol feeding significantly increased (101%) plasma clearance of TNF-alpha, which followed a biphasic exponential decay and decreased the T1/2 for both the alpha (67%) and beta (76%) elimination components. The distribution of both cytokines in trichloroacetic acid precipitable and non-precipitable fractions of liver, spleen, stomach, small intestine (ileum), lung, kidney, and blood was also studied. The only effect of alcohol treatment was a significant decrease in IL-6 uptake and metabolism by the small intestine. Perfused livers, isolated from alcohol-fed rats, took up and metabolized larger amounts of IL-6 than did livers isolated from pair-fed rats. TNF-alpha uptake and metabolism by the isolated, perfused liver were not affected by chronic alcohol consumption. Regardless of the animal treatment, the isolated perfused liver took up and metabolized significantly larger (17-fold) amounts of TNF-alpha than IL-6, in spite of identical concentrations of cytokines (6 nM) in the perfusion medium. The data presented in this study along with our previous results demonstrating the effects of alcohol consumption on TNF-alpha and IL-6 receptors on various liver cells suggest that the effects of chronic alcohol treatment on cytokine clearance cannot be ascribed to changes in the receptors for the two cytokines. Also, no correlation was found between the effects of alcohol treatment on plasma cytokine clearance and uptake and metabolism of cytokines by the isolated, perfused liver. Experimental data and theoretical considerations suggest that cytokine receptor recycling may play an important role in mediating alcohol effects on cytokine clearance.
Effects of acute alcohol intoxication on gluconeogenesis and its hormonal responsiveness in isolated, perfused rat liver
Biochemical Pharmacology, 1992
Rats were acutely administered ethanol as a primed constant infusion in order to produce sustaine... more Rats were acutely administered ethanol as a primed constant infusion in order to produce sustained blood ethanol levels of 8-12 or 55-65 mM. At the end of ethanol infusion the livers were either freeze-clamped in vivo or isolated and perfused for metabolic studies. The rate of gluconeogenesis and its responsiveness to phenylephrine (10 microM), prostaglandin F2 alpha (5 microM) and glucagon (10 nM), as well as the redox state of the cytosolic NAD(+)-NADH system were assessed in livers isolated from acutely ethanol-treated rats, and subsequently perfused without ethanol. For liver clamped in vivo, high- but not low-ethanol treatment decreased the ATP content by 31% and slightly increased ADP and AMP content, resulting in a decreased energy charge (11%). Glutamate and aspartate content was also increased in high-dose ethanol-infused rats with no changes in malate and 2-oxoglutarate content. Gluconeogenesis with saturating concentrations of lactate (4 mM)+pyruvate (0.4 mM) was delayed in reaching a plateau in the livers of high-dose ethanol-treated rats and its response to all three stimulators was impaired. Low-dose ethanol treatment only decreased the liver response to phenylephrine. While the perfused livers of low-dose ethanol-treated rats displayed no changes in adenine nucleotide content, the livers of high-dose ethanol-treated rats had a decreased ATP (35%) and an increased AMP (77%) content, paralleled by a fall in the total adenine nucleotides (14%) and energy charge (14%). No differences were observed between the saline- and ethanol-treated rats with respect to malate-aspartate shuttle intermediate concentration in perfused livers. Also, the livers of high-, but not low-dose ethanol-treated rats had a more negative value of NAD(+)-NADH redox state as compared to the livers of control rats. The data suggest that acute ethanol intoxication produces changes in liver metabolism and its responsiveness to hormones/agonists that are demonstrable for at least 2 hr after isolation and perfusion of the liver.
Granulocyte-Macrophage colony stimulating factor (GM-CSF) priming in the treatment of elderly patients with acute myelogenous leukemia
American Journal of Hematology, 1995
Alcoholism: Clinical and Experimental Research, 1996
Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronicall... more Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronically in a complete liquid diet or in sucrosecontaining drinking water. The animals were kept on liquid diet (kalcohol) for 14 weeks or on sucrose-containing drinking water (kalcohol) for 12.5 weeks and sacrificed thereafter. To rule out possible artifact induced by fixation procedure, livers were fixed by immersion (no perfusion), immersion preceded by perfusion, and by perfusion wlth glutaraldehyde and examined with both scanning and transmission electron microscopy. Regardless of the mode of its administration, and of the fixation procedure used, alcohol induced similar changes in liver sinusoid ultrastructure. Such changes included disruption of the sieve-plate pattern of the sinusoidal endothelial cell fenestrations with the appearance of large gaps and resulting in a meshwork lining, wherein large areas of the sinusoid communicated freely with the underlying hepatocytes. Transmission electron microscopy complemented these findings. The results reported in this study demonstrate that alcohol-induced structural changes of the liver sinusoid in the rat are similar whether alcohol is fed via a liquid diet or in drinking water. Therefore, alcohol administration in drinking water may provide a simple, inexpensive, and convenient method of inducing structural changes in the rat liver sinusoid.
Acute Ethanol Intoxication Suppresses E. Coli Lipopolysaccharide Enhanced Glucose Utilization by Hepatic Nonparenchymal Cells
Alcoholism: Clinical and Experimental Research, 1991
During infection or endotoxemia, the immune system is activated and its energy needs increase. Al... more During infection or endotoxemia, the immune system is activated and its energy needs increase. Alcohol (ETOH) intoxication on the other hand suppresses the immune system and increases susceptibility to infection. Since the liver is the primary site both for metabolism of ETOH and detoxification of bacterial lipopolysaccharides (LPS), this investigation was directed at studying the effect of acute ETOH intoxication on the LPS-induced enhancement of in vivo glucose utilization in different types of hepatic cells. Rats were given an intravenous (IV) injection of ETOH followed by a constant infusion for 7 hr to maintain blood alcohol levels at about 175 mg/dl. E. coli LPS was administered IV at 4 hr and in vivo glucose utilization by the different types of liver cells was estimated 3 hr later using the 14C-2-deoxyglucose technique. Hepatocytes (HP), Kupffer (KC), and endothelial cells (EC), as well as the sequestered polymorphonuclear leukocytes (PMN), were separated from the liver by collagenase-pronase digestion followed by centrifugal elutriation and Ficoll-Hypaque density gradient centrifugation. The number of PMN in the liver was increased several-fold 3 hr after LPS administration. The presence of ETOH did not inhibit the LPS-induced neutrophil migration into the liver. ETOH depressed the LPS-induced increase in glucose uptake in both EC and KC by 50 to 80%, respectively. It also reduced the LPS-induced increase of plasma tumor necrosis factor activity by 80%. ETOH alone did not produce any significant changes in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Ethanol Alters the Metabolic Response of Isolated, Perfused Rat Liver to a Phagocytic Stimulus
Alcoholism: Clinical and Experimental Research, 1993
Intercellular communication in the liver is a potentially important mechanism for the regulation ... more Intercellular communication in the liver is a potentially important mechanism for the regulation of hepatic metabolism. Since alcohol (ethanol, ETOH) can interact with both parenchymal and nonparenchymal cells, the present study was performed to assess the possible effects of ETOH on the nonparenchymal cell-to-hepatocyte signal traffic by studying the glycogenolytic and glycolytic response of the perfused rat liver to colloidal carbon, a phagocytic stimulus for Kupffer and sinusoidal endothelial cells. Livers from fed rats were perfused with hemoglobin-free Krebs Ringer bicarbonate buffer containing ETOH (20 mM) or acetaldehyde (1 mM). Twenty minutes after initiating the infusion of ETOH or acetaldehyde, colloidal carbon was infused and the rate of carbon uptake, glucose, lactate and pyruvate output, and oxygen consumption were determined. In control livers, carbon stimulated the output of glucose (60%), lactate (25%), and pyruvate (53%), without affecting the lactate/pyruvate ratio. ETOH, but not acetaldehyde, enhanced the carbon effect on glucose output (38%), but suppressed the increased lactate and pyruvate output (48% and 91% respectively) resulting in a dramatic 10-fold increase in the lactate/pyruvate ratio. By using inhibitors of cyclooxygenase or alcohol dehydrogenase (indomethacin and 4-methylpyrazole, respectively) in the presence of carbon and/or ETOH, we determined that: (1) following carbon stimulation prostaglandins are the likely mediators secreted by nonparenchymal cells that increase carbohydrate output; and (2) the ETOH-induced enhancement of carbon-stimulated glycogenolysis is also mediated by prostaglandins and is not dependent on the oxidative metabolism of ETOH.
Alcoholism: Clinical and Experimental Research, 1995
Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-indu... more Tumor necrosis factor-a (TNF-a) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-a could be due, at least in part, to alterations in TNF-a cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchy-ma1 (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 pg/lOO g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K,) and capacity B, . J of binding sites, using recombinant h~rnan-['~l]TNF-cu as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K,,, in the range of 150-200 PM), low-capacity (B,,,,, in the range of 2-3 fmol/106 cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/106 cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K,, = 1 .O n M and a B,,, = 95 fmol/106 cells). Acute ETOH administration caused an increase in K , , on both Kupffer and sinusoidal endothelial cells; a decrease in K , , on hepatocytes; and an increase in Kd2, B , , , , , and Bmsx2 on endothelial cells. A second binding site on hepatocytes (Kd2 = 5.8 nM, Bmax2 = 186fmo1/106 cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K,, and B,,,, on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B,,,, on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B, , J and affinity (K,) of TNF-cu cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal From the Depaifments of Physiology (I.KD., J.J.S.) and Medicine (Pulmona yICritica1 Care) (N.B. D.), 332 endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-a.
Modulation of F-met-leu-phe Induced Chemotactic Activity and Superoxide Production by Neutrophils during Chronic Ethanol Intoxication
Alcoholism: Clinical and Experimental Research, 1992
Chronic alcohol consumption has been associated with increased migration of neutrophils into live... more Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.
Alcoholism: Clinical and Experimental Research, 1994
The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus ... more The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus interfering in lung immune defense mechanisms. Male rats were treated with alcohol either acutely (7 hr continuous intravenous alcohol infusion at a rate of 30 mg/100 g body weight/hr after a priming dose of 175 mg/100 g body weight) or chronically (feeding an alcohol-containing liquid diet for 12-14 weeks). Three hr before killing, the rats received an intravenous injection of Gram-negative bacterial lipopolysaccharide (LPS Escherkhia cdi, 026B6, 100 pg/100 g body weight). After anesthesia with sodium pentobarbital, the trachea was cannulated, and the lungs excised and lavaged to obtain alveolar macrophages. The recovered cells were used to measure the binding of recombinant human ['ml]tumor necrosis factor-a (TNF-a) and ['wl]interleukin-6 (IL-6). Kd and B, were determined at 4OC, thus reflecting only the cell-surface binding sites and their affinity. Two binding sites were detected for both cytokines: high-affinity K d , in the range of 20-110 pM), lowcapacity (8-, in the range of 1-13 fmol/lO' cells), and low-affinity (Kdl in the range of 0.6-1.3 nM), high-capacity ( 8 , , in the range of 34-100 fmol/lO' cells). Acute alcohol treatment significantly decreased 8 , , (39%) and 8 , , (79%) for TNF-a, whereas chronic alcohol feeding abrogated the 8-, (8-, = 0), without affecting 8-*. In the acute group, LPS had an effect similar to that of alcohol.
Interleukin-6 and Tumor Necrosis Factor-alpha Clearance and Metabolism In Vivo and by the Isolated, Perfused Liver in the Rat: Effect of Acute Alcohol Administration
Alcoholism: Clinical and Experimental Research, 1996
ABSTRACT Plasma clearance and organ distribution of intravenously injected human recombinant [125... more ABSTRACT Plasma clearance and organ distribution of intravenously injected human recombinant [125I]interleukin (IL)-6 and [125I]tumor necrosis factor (TNF)-α were studied in male rats, 2 hr after intravenous alcohol (ethanol) administration (single dose, 2.2 g · kg-1 body weight). Also, the rate of uptake and degradation of the two cytokines by the isolated, perfused rat liver was studied in the absence or in the presence of ethanol (35 mM) in the perfusate. Acute ethanol administration significantly increased plasma clearance rate for both cytokines (36% and 72%, for IL-6 and TNF-α, respectively), decreased the t1/2α (30% and 11%, for IL-6 and TNF-α, respectively), abolished the slow (β)-phase component for TNF-α, and increased t1/2β for IL-6 (31%). Although alcohol did not affect organ distribution of TNF-α, it increased the IL-6 content in the liver, kidney, and blood. IL-6 uptake rate by the isolated, perfused rat liver was 2-fold higher than TNF-α uptake, whereas the rate of degradation was larger for TNF-α than for IL-6, despite the fact that both cyokines were presented to the liver at the same concentration (6 nM). Ethanol addition to the perfusate (35 mM, final concentration) significantly increased TNF-α uptake (24%), without affecting IL-6 uptake or the degradation rate of either cytokine. Also, the kinetics of degradation by the isolated, perfused rat liver was linear for TNF-α, but exponential for IL-6. Data presented in this study demonstrate that: (1) acute alcohol consumption can alter the kinetic behavior of IL-6 and TNF-α in the bloodstream, mainly by accelerating their clearance which, in turn, may counteract the outcome of cytokine secretion and delivery to the blood; and (2) short exposure of liver to ethanol levels commonly seen in humans after binge drinking may alter its capacity to take up cytokines.