Relationship between oxidative burst activity and CD11b expression in neutrophils and monocytes from healthy individuals: effects of race and gender.

Oxidative burst activity and the expression of adhesion molecules have been used as indicators of leukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activity and the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers (n = 96). We also tested the potential role of gender and a racial background in the individual response differences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expression were determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonal antibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils and monocytes in all samples. However, the responses showed considerable variability among individuals. A positive correlation was observed between the responsiveness of neutrophils and monocytes in their basal or PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, no correlation was found between the level of adhesion molecule expression and cellular oxidant content in monocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidative burst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American males compared with cells from African American females, white females, or white males. In contrast, reactivity of monocytes was significantly elevated in white males compared with all other groups. These findings indicate that leukocytes with a relatively high degree of adhesion molecule expression may display an average or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnic background may influence the oxidative burst activity in neutrophils and monocytes. This needs consideration in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds.

Augmented TNF-alpha and IL-10 production by primed human monocytes following interaction with oxidatively modified autologous erythrocytes.

The presence of dysfunctional/damaged red blood cells (RBCs) has been associated with adverse clinical effects during the inflammatory response. The aim of this study was to elucidate whether oxidatively modified, autologous RBCs modulate monocyte cytokine responses in humans. Monocyte tumor necrosis factor alpha (TNF-alpha) and IL-10 production was measured in whole blood from healthy volunteers using ELISA and flow cytometry. Oxidatively modified RBCs (15 mM phenylhydrazine, 1 h, OX-RBC) or vehicle-treated RBCs (VT-RBC) opsonized by autologous serum were administered alone or in combination with one of three priming agents: E. coli lipopolysaccharide (LPS, 0.2 ng/ml), zymosan A (1 mg/ml), or phorbol 12-myristate 13-acetate (PMA, 50 ng/ml). OX-RBC or VT-RBC alone did not result in the release of TNF-alpha or IL-10. LPS, zymosan, and PMA caused marked and dose-dependent increases in TNF-alpha and IL-10 production. Addition of OX-RBC augmented the LPS-, zymosan-, and PMA-induced TNF-alpha release by approximately 100%. OX-RBC augmented LPS- and zymosan-induced IL-10 release by 400-600%. Flow cytometry analyses showed that monocytes were responsible for TNF-alpha and IL-10 production in whole blood. The presence of OX-RBC alone increased the complexity of CD14+ monocytes but caused no cytokine production. LPS alone induced cytokine production without altering cell complexity. After the combined (OX-RBC+LPS) treatment, monocytes of high complexity were responsible for TNF-alpha production. The presence of mannose or galactose (at 10-50 mM) did not alter the observed augmentation of cytokine production by OX-RBC, suggesting that lectin receptors are not involved in the response. These studies indicate that the interaction between damaged autologous erythrocytes and monocytes has a major impact on the cytokine responses in humans. An augmented cytokine production by the mononuclear phagocyte system may adversely affect the clinical course of injury and infections especially in genetic or acquired RBC diseases or after transfusions.

Increased incidence of sepsis and altered monocyte functions in severely injured type A- glucose-6-phosphate dehydrogenase-deficient African American trauma patients.

OBJECTIVE: To determine whether trauma patients with the common, type A- glucose-6-phosphate dehydrogenase (G6PD) deficiency have an aggravated inflammatory response, increased incidence of septic complications, and/or more profound alterations in leukocyte functions compared with nondeficient trauma patients. SETTINGS: Intensive and surgical care units of a trauma center and flow cytometry and experimental laboratories at a teaching university hospital. DESIGN: Prospective cohort clinical study with measurements on days 2 and 5 postinjury. Monocyte and neutrophil oxidant content, apoptosis, and CD11b expression and plasma cytokine levels were compared between G6PD-deficient and nondeficient patients. PATIENTS: A total of 467 male African American trauma patients were screened for the deficiency. Forty-four type A-202/376 G6PD-deficient patients were identified and enrolled in the study; 43 nondeficient patients were also enrolled and were matched by age, clinical criteria of injury severity, and type of trauma. MAIN RESULTS: After severe injury (Injury Severity Score, > or =16), 50% of the deficient and 6.2% of nondeficient patients developed sepsis with positive bacterial blood cultures. In deficient patients, the frequency of bronchial (75%) and wound infections (25%) was also increased compared with nondeficient patients (32% and 0%). The durations of systemic inflammatory response syndrome, Sepsis Syndrome, and days on antibiotics were three times longer in deficient than in nondeficient individuals. However, adult respiratory distress syndrome occurred in 37% of both groups. Anemia was more severe in the deficient than nondeficient patients from day 10 posttrauma. On day 5, the peroxide content was doubled, apoptosis was decreased, and CD11b expression was increased in monocytes from deficient patients compared with cells from nondeficient patients. On day 5, the plasma interleukin (IL)-10 concentration was significantly lower in deficient than nondeficient patients, whereas tumor necrosis factor-alpha, IL-6, and IL-8 levels were similar. After moderate injuries (Injury Severity Score, 9-16), the deficiency was not associated with adverse clinical effects, and the trauma-induced changes in leukocyte function were similar in deficient and nondeficient patients. CONCLUSIONS: The common type A- G6PD deficiency predisposes septic complications and anemia in trauma patients after severe injuries as defined by an Injury Severity Score of > or =16. This adverse clinical course is accompanied by altered monocyte functions manifested as augmented oxidative stress, a decreased apoptotic response, increased cell adhesion properties, and a diminished IL-10 response.

Augmented resistance to oxidative stress in fatty rat livers induced by a short-term sucrose-rich diet.

Hepatic steatosis and the accompanying oxidative stress have been associated with a variety of liver diseases. It is not known if fat accumulation per se plays a direct role in the oxidative stress of the organ. This study tested if steatosis induced by a short-term carbohydrate-rich diet results in an increased hepatic sensitivity to oxidative stress. Antioxidant status was determined in a liver perfusion system and in isolated parenchymal, endothelial and Kupffer cells from rats kept on sucrose-rich diet or on regular diet for 48 h. t-Butyl hydroperoxide addition (2 mM) to the perfusion fluid resulted in a release of alanine aminotransferase (ALT) in livers from controls, whereas no ALT release was observed in fatty livers. After t-butyl hydroperoxide addition, oxidized glutathione release was 40% less in fatty than in control livers, whereas reduced glutathione (GSH) release was not different. Sinusoidal oxidant stress was mimicked by the addition of lipopolysaccharide (LPS) from Escherichia coli (10 microg/ml) followed by the addition of opsonized zymosan (8 mg/ml) to the perfusion medium. LPS plus zymosan treatments resulted in the release of ALT in control but not in fatty livers. At the end of perfusion, liver glutathione content was 3-fold elevated, and the tissue content of lipid peroxidation products was approx. 40% less in fatty livers compared to controls. GSH content was doubled and glucose-6-phosphate dehydrogenase (G6PD) expression was elevated by 3- and 10-fold in sinusoidal endothelial and parenchymal cells form fatty livers compared to cells from control animals. Following H(2)O(2) administration in vitro (0.2-1 mM), GSH remained elevated in endothelial and parenchymal cells from fatty livers compared to cells from controls. In contrast, G6PD activity and GSH content were similar in Kupffer cells isolated from fatty or control livers. The study shows that hepatic fat accumulation caused by a short-term sucrose diet is not accompanied by elevated hepatic lipid peroxidation, and an elevated hepatic antioxidant activity can be manifested in the presence of prominent steatosis. The diet-induced increase in G6PD expression and, thus, the efficient maintenance of reduced glutathione in endothelial and parenchymal cells are a supportive mechanism in the observed hepatic resistance against intracellular or sinusoidal oxidative stress.

A carbohydrate-rich diet stimulates glucose-6-phosphate dehydrogenase expression in rat hepatic sinusoidal endothelial cells.

A carbohydrate-rich diet induces glucose-6-phosphate dehydrogenase (G6PD) in liver parenchymal cells, which supports fatty acid synthesis de novo. Bacterial endotoxins stimulate G6PD expression in hepatic sinusoidal endothelial and Kupffer cells but not in parenchymal cells. This study was designed to elucidate whether G6PD expression is regulated uniformly by dietary carbohydrates in hepatic sinusoidal and parenchymal cells. Freshly isolated cells from five groups of Sprague-Dawley rats were analyzed for G6PD activity and mRNA abundance. The rats were grouped as follows: 1) food deprived for 24 h; 2) food deprived for 24 h followed by consumption of the standard diet for 48 h; 3) food deprived for 24 h followed by consumption of a carbohydrate-rich diet for 48 h; 4) fed standard diet; and 5) fed standard diet followed by consumption of a carbohydrate-rich diet for 48 h. In endothelial cells, G6PD activity was 150% greater in group 3 than in group 1 and 125% greater in group 5 than in group 4. Steady-state G6PD mRNA levels were elevated by 300% in endothelial cells from group 3 compared with those from group 1. In Kupffer cells, G6PD activity and mRNA abundance were not different among the groups. As expected, G6PD expression was 700-1200% greater in parenchymal cells from rats fed a carbohydrate diet (groups 3 and 5) than from controls. Our results indicate that short-term consumption of a carbohydrate-rich diet stimulates G6PD expression in endothelial and parenchymal cells. Because G6PD supports reactive oxygen metabolism, the response may represent a preconditioning of antioxidant pathways in the hepatic cell populations that are targets of sinusoid-born reactive oxygen species during infections.

Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid.

During the innate immune response, excessive release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver and other tissues. The consequence of oxidative stress is determined by the status and adaptive changes of antioxidant pathways. In this review, we present evidence that the synchronized response of hepatic sinusoidal endothelial cells, the primary sites of phagocyte attachment, plays an important role in defense against phagocyte-derived ROS. An essential component of the metabolic adaptation of hepatic sinusoidal cells to lipopolysaccharide (LPS)-induced oxidative stress is the stimulated expression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose cycle (hexose monophosphate shunt, HMS). All major ROS-metabolic enzymes, i.e., glutathione peroxidase, glutathione reductase, catalase, superoxide dismutases, NADPH oxidase, and nitric oxide synthase, directly or indirectly depend on NADPH, which is produced in the HMS in these cells. The functional significance of up-regulated HMS within a particular cell type depends on the accompanying adaptive changes in ROS-metabolizing enzymes. In LPS-activated Kupffer cells, the elevated expression of glucose transporter GLUT1 and G6PD mainly serves primed production of superoxide anion, hydrogen peroxide, and nitric oxide. In sinusoidal endothelial cells, the LPS-induced response pattern of glucose- and ROS-metabolizing enzymes results in elevated ROS detoxifying capacity. The described studies also suggest the existence of an intercellular oxidant balance between pro-oxidant Kupffer cells and antioxidant endothelial cells in the hepatic micro-environment. Maintenance of the intercellular oxidant/antioxidant balance between phagocytes and endothelial cells may represent an important mechanism protecting the hepatic parenchyma against exogenous oxidative stress during the inflammatory response.

Tumor necrosis factor alpha augments the expression of glucose-6-phosphate dehydrogenase in rat hepatic endothelial and Kupffer cells.

Cellular activity of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose monophosphate shunt, supports several pathways involved in the nonspecific immune response. In the present study, we investigated the in vivo effects of selected pro-inflammatory cytokines on the expression of G6PD in Kupffer and hepatic endothelial cells. Murine recombinant TNF alpha, IL-1 beta, or IL-6 (1.5 x 10(5) U/kg) was injected and cellular G6PD mRNA level determined using a quantitative reverse transcription and polymerase chain reaction method. G6PD mRNA was elevated two- to threefold seven hours after the injection of TNF alpha in Kupffer and endothelial cells as compared to cells from saline-injected animals. The elevated G6PD mRNA was accompanied by increased cellular enzyme activity in both cells. The cellular activity of 6-phosphogluconate dehydrogenase (6PGD) was also increased seven hours after TNF alpha treatment in these cells. G6PD mRNA and enzyme activity returned to control levels 22h after TNF alpha administration. In contrast to the marked effects of TNF alpha, no significant alterations were found on G6PD expression following IL-1 beta or IL-6 injections in these cells. None of these cytokines caused changes in G6PD or 6PGD expression in parenchymal cells. These data indicate that the proinflammatory cytokine TNF alpha plays an important role in the regulation of cellular G6PD expression in hepatic immune competent cells.

Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells.

The present study investigated the effect of lipopolysaccharide (LPS; from Escherichia coli, 2 mg/kg body wt ip) on selected aspects of the antioxidant status in Kupffer and sinusoidal endothelial cells. Cells were isolated 18 h after the injection of saline or LPS. In fresh suspension cultures, cellular reduced glutathione (GSH) and H2O2 were determined by monochlorobimane, and 2',7'-dichlorofluorescein diacetate, respectively, using a fluorescence plate reader. LPS injection increased GSH content two- to threefold in Kupffer cells compared with cells from control rats. Cellular GSH content was higher in endothelial than Kupffer cells. However, LPS did not increase GSH content in endothelial cells. Addition of H2O2 (40-200 microM) to Kupffer or endothelial cells caused a transient decrease in GSH, which was more pronounced in cells from control rats (approximately 45% drop) than in LPS-exposed cells (approximately 25% drop). Depleted GSH levels were accompanied by a proportional increase in cellular H2O2. After inhibition of catalase by 3-amino-1,2,4-triazole, the presence of 0.2 mM H2O2 depleted GSH content by 75% and 40% in Kupffer cells from saline- or LPS-injected rats, respectively. The same treatments caused a similar 50% decrease in both activated and control endothelial cells. LPS decreased catalase activity by 45% in Kupffer cells, whereas it had no effect on catalase in endothelial cells. Glutathione reductase activity was not altered by LPS in either cell type. These data show that in activated Kupffer cells the elevated level of cellular glutathione plays an augmented role in the protection against reactive oxygen species, whereas the contribution of catalase to H2O2 detoxification is attenuated. In LPS-stimulated endothelial and Kupffer cells, the efficient maintenance of GSH is consistent with upregulated production of reducing power through the hexose phosphate shunt observed previously.

Endotoxin stimulates hydrogen peroxide detoxifying activity in rat hepatic endothelial cells.

The study aimed to assess the effect of lipopolysaccharide (LPS) in vivo (from Escherichia coli, 2 mg/kg body weight intraperitoneally) on the production and elimination of hydrogen peroxide (H2O2) in rat hepatic endothelial and Kupffer cells. Twenty-two hours after the injection of LPS, hepatic cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation, and cell-associated H2O2 was determined by flow cytometry analysis using 2',7'-dichloroflorescin diacetate (DCF-diacetate). LPS treatment did not alter the basal or phorbol myristate acetate-stimulated levels of H2O2-related fluorescence in endothelial cells; however, it doubled phorbol myristate acetate-stimulated fluorescence in Kupffer cells. Administration of varying concentrations of H202 (range, 10(-7) - 10(-4) mol/L) in vitro caused a significantly delayed increase in fluorescence in endothelial cells from endotoxemic rats as compared with cells from saline-injected animals. The 50% effective concentration of H202 was found at 1.1 x 10(-6) and 8.1 x 10(-6) mol/L on endothelial cells after saline and LPS treatment, respectively. No differences were detected in H2O2-stimulated fluorescence between resting and LPS-stimulated Kupffer cells. Administration of varying glucose concentrations in vitro significantly decreased the H2O2-stimulated fluorescence in endothelial and Kupffer cells from LPS-injected animals. Inhibition of nitric oxide synthase by in vitro administration of NG-monomethyl-L-arginine (L-NNMMA) did not alter the H2O2- or phorbol myristate acetate-stimulated responses in endothelial and Kupffer cells. As shown earlier, LPS stimulates the gene expression of GLUT1 glucose transporter, glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutases, and glutathione peroxidase in hepatic endothelial cells. The present data indicate that the LPS-induced metabolic alterations are accompanied by an increased H2O2-detoxifying capacity in hepatic endothelial cells. This may represent a protective mechanism against exogenous oxidative stress caused by activated hepatic phagocytes during inflammation. Our observations are consistent with primed production of reactive oxygen species (ROS) in LPS-activated Kupffer cells.

Endotoxin stimulates gene expression of ROS-eliminating pathways in rat hepatic endothelial and Kupffer cells.

Reactive oxygen species (ROS) are mediators of cellular injury and play a putative role in the onset of hepatic damage during endotoxemia or sepsis. It has been suggested that induction of glucose-6-phosphate (G-6-P) dehydrogenase, the key enzyme of the hexose monophosphate shunt (HMS), may support ROS-producing or ROS-eliminating pathways in hepatic endothelial and Kupffer cells during endotoxemia. The aim of the study was to assess in vivo lipopolysaccharide (LPS)-induced alterations in rat gene expression of selected enzymes that are in functional relationship with the HMS. mRNA levels and activities of glucose transporter GLUT-1, Mn- and CuZn-dependent superoxide dismutases (Mn-SOD and CuZn-SOD), and Se-dependent glutathione peroxidase (Se-GPX) were determined. Cellular extracts were analyzed 7 or 22 h after injection of LPS (Escherichia coli, 2 mg/kg ip) or injection of saline. Exposure to LPS for 7 or 22 h caused a 10- to 25-fold increase in GLUT-1 mRNA levels in endothelial and Kupffer cells. In parenchymal cells, GLUT-1 mRNA expression was low, and LPS caused no marked changes. Cellular levels of Mn-SOD mRNA were 20-40 times greater in all hepatic cells from LPS-treated animals than in cells from control rats. LPS at 22 h increased Mn-SOD activity by 45% in endothelial cells but caused no significant changes in Kupffer or parenchymal cells. Message levels and enzyme activities of CuZn-SOD and Se-GPX were significantly elevated 22 h after LPS injection in endothelial cells only. Thus LPS results in marked upregulation of functionally related genes in hepatic cells. In endothelial cells, the simultaneous upregulation of GLUT-1, G-6-P dehydrogenase, Mn-SOD, CuZn-SOD, and Se-GPX may represent an important mechanism for accelerated elimination of ROS released from activated sinusoidal phagocytes. In Kupffer cells, upregulated GLUT-1 and G-6-P dehydrogenase, together with constitutively present SOD and lack of upregulated Se-GPX, suggest an elevated capacity to produce O2- and H2O2 that is consistent with primed bacterial killing.

Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin.

Lipopolysaccharide (LPS) produces a rapid and sustained reduction in the circulating concentration of insulin-like growth factor I (IGF-I), which may be responsible, in part, for the alterations in protein metabolism observed in these animals. The purpose of the present study was to determine whether this drop was due to a decreased hepatic production of IGF-I and/or an increased clearance of the peptide from the blood. Four hours after intravenous injection of LPS the plasma IGF-I concentration was decreased 50%. IGF-I release by in situ perfused livers from control rats was constant throughout the 60-min perfusion period and averaged 111 +/- 3 ng/min. In contrast, hepatic IGF-I output was decreased 46% by in vivo LPS. In contrast, livers from LPS-injected rats released more IGF binding proteins-1, -2 and -4 than did control livers. Hepatic cell isolation indicated that LPS decreased the IGF-I content in Kupffer and parenchymal cells, but not endothelial cells, by approximately 45%. Pharmacokinetic analysis of blood 125I-IGF-I decay curves indicated that the half-life for whole body clearance of 125I-IGF-I from the circulation was not altered by LPS. However, LPS increased 125I-IGF-I uptake by spleen, liver, lung, and kidney while decreasing uptake by the pancreas and gastrointestinal tract. These results indicate that the LPS-induced decrease in blood IGF-I concentration is primarily due to a reduction in hepatic production, not a change in whole body peptide clearance, and that a decreased production by both parenchymal and Kupffer cells contributes to this alteration.

Acute endotoxin tolerance is accompanied by stimulated glucose use in macrophage rich tissues.

A single injection of E. coli LPS at a dose of 10 mg/kg b.w. ("high dose") is lethal in Sprague Dawley rats. However, animals given a sublethal dose of LPS (0.5 mg/kg bw; "low dose") at time zero, followed by a second high dose injection at 48 h, display endotoxin tolerance with 100% survival. The aim of the present study was to assess the relationship between this observed endotoxin tolerance and the endotoxin-induced glucose metabolic response in selected tissues and nonparenchymal hepatic cells. In each experimental group two injections, the first at time zero, the second at 48 h were given in vivo. Four experimental groups constituted these studies: A) saline followed by saline, B) low dose LPS followed by saline, C) saline followed by high dose LPS, and D) low dose LPS followed by high dose LPS. In vivo glucose use in tissues and cells was measured 3h after the last treatments employing the 2-deoxy-glucose tracer technique. Glucose use by liver, lung, spleen and intestine was not different between saline/saline (group A) and low dose LPS/saline injected (group B) animals. Saline/high dose LPS injection (group C) doubled glucose uptake, while the sequential LPS injections (group D) caused an additional, 2-3 fold increase in the glucose use by these tissues. Hepatic endothelial cells showed a similarly elevated glucose use in vivo in both group C and D. Kupffer cells from group D animals, however, displayed markedly elevated glucose use in vivo as compared to cells from group C. Our data indicate that high dose LPS in endotoxin tolerant animals is accompanied by a more markedly stimulated tissue glucose use than found following lethal LPS treatment alone. This increased peripheral glucose use may support cellular functions responsible for the protection of the host.

Acute alcohol administration attenuates insulin-mediated glucose use by skeletal muscle.

The aim of the present work was to test the effect of acute in vivo alcohol administration (180-190 mg/dl plasma for 3 h) on glucose utilization by tissues under basal conditions or after a hyperinsulinemic (100-130 microU/ml) euglycemic clamp in fasted rats. In vivo glucose use by individual tissues was assessed by the tracer 2-deoxy-D-glucose technique. Alcohol administration to saline-infused rats markedly inhibited glucose use by skeletal muscles, including the soleus, white and red quadriceps, and gastrocnemius, as well as by the heart. Ethanol infusion, however, had no effect on glucose use by the diaphragm, lung, liver, skin, ileum, brain, and adipose tissue. The insulin-stimulated glucose use was also inhibited by alcohol selectively in the muscles, with no effect on other tissues tested, except a moderate inhibition in the brain. Ethanol inhibited muscle glucose use by an average of approximately 50% under both basal and insulin-stimulated conditions. However, because insulin treatment more than doubled basal glucose use by these muscles, the 50% inhibition by ethanol treatment represents a greater inhibition of absolute glucose use under insulin-stimulated rather than under basal conditions. Our data demonstrate that acute alcohol intake attenuates basal and hormone-induced glucose utilization in a tissue-specific fashion. The inhibitory effect of alcohol on skeletal muscle glucose use could contribute to the previously observed decreased glucose recycling in humans after acute alcohol intake.

Endotoxin stimulates the expression of glucose-6-phosphate dehydrogenase in Kupffer and hepatic endothelial cells.

The aim of the study was to elucidate the effect of lipopolysaccharide (LPS) administration in vivo (Escherichia coli endotoxin, 1 mg/kg body weight) on the expression and cellular activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the rate-limiting enzyme of the hexose monophosphate shunt in hepatic cells. Under basal conditions, Kupffer cells displayed higher activity of G6PDH than endothelial or parenchymal cells. In vivo LPS treatments for 7 and 22 h resulted in 40 and 60% increases, respectively, in the cellular activity of G6PDH in Kupffer cells. G6PDH activity was increased by 140 and 90% after 7- and 22-h LPS treatments in endothelial cells. G6PDH activity in parenchymal cells prepared from animals after 22 h of LPS treatment was decreased by approximately 60% compared with that in cells from saline-injected animals. Total cellular RNA or protein extracts from these cells were analyzed by Northern or Western blots. Under basal conditions, G6PDH mRNA levels relative to total cellular RNA were higher in Kupffer than in endothelial cells and were not detectable in parenchyma cells. LPS injection caused a time-dependent increase in G6PDH mRNA expression in Kupffer and endothelial cells. Western blot analysis of Kupffer cell extracts also showed that LPS treatments caused markedly elevated expression of protein in these cells. These results show that endotoxemia results in marked induction of G6PDH in Kupffer and hepatic endothelial cells but has no such effect in the parenchymal cells. These findings also suggest that the elevated cellular expression of G6PDH is an important regulatory event in the adaptive responses of hepatic nonparenchymal cells to infections. The elevated expression of G6PDH may be important for support of the upregulated NADPH-dependent pathways, such as superoxide anion and nitric oxide production, macromolecular synthesis, or the maintenance of cellular glutathione status.

Antineutrophil monoclonal antibody (1F12) alters superoxide anion release by neutrophils and Kupffer cells.

The formation of oxygen-derived radicals by phagocytes is regulated by chemotactic agents, cytokines, and adhesion molecules, such as CD11b/CD18 (Mac-1). In the rat system, we investigated the effect of monoclonal antibody 1F12 against rat neutrophils on hepatic sequestration of neutrophils and superoxide release by hepatic phagocytes. Within 15 min after 1F12 injection, there was profound neutropenia, which persisted for 24 h. The majority of the "lost" neutrophils were sequestered in the liver 4 h after treatment. Zymosan-induced superoxide release in vitro by isolated hepatic neutrophils from 1F12-treated rats was significantly attenuated at 4 and 24 h. The phorbol myristate acetate mediated superoxide release was inhibited 24 h after treatment. Superoxide anion release by normal adherent neutrophils in the presence of agonists was also inhibited by 1F12 in vitro. The in vivo administration of 1F12 primed the Kupffer cells to release superoxide. In vitro treatment of Kupffer cells with 1F12 also stimulated superoxide release. Monoclonal antibody WT.3 (also directed against rat neutrophils), which does not cause neutropenia, did not alter superoxide generation by neutrophils and Kupffer cells. These results indicate that 1F12 may be useful in attenuating inflammation and tissue injury associated with neutrophil activation. However, the activation of Kupffer cells to release toxic oxygen-derived metabolites may predispose the liver to injury in certain pathological conditions.

Alcohol administration attenuates LPS-induced expression of inducible nitric oxide synthase in Kupffer and hepatic endothelial cells.

This study investigates the effects of in vivo ethanol (primed infusion, causing 170-190 mg% plasma alcohol for 12 hours) and/or LPS (12 hours after injection of E. coli LPS 1 mg/kg bw.) on the mRNA expression of inducible nitric oxide synthase (NOS II) in hepatic cells measured by competitive PCR technique, and on hepatic release of reactive nitrogen intermediates (RNI, NO2- + NO3-). Perfused livers from alcohol- or saline-infused animals did not release measurable amounts of RNI. Under these conditions small amounts of NOS II mRNA were expressed in Kupffer and endothelial cells, while it was not detectable in parenchymal cells. LPS treatment along with markedly elevating hepatic RNI release increased NOS II mRNA levels by 35- and 200-fold, in endothelial and Kupffer cells, respectively. LPS injection and alcohol infusion to the same animal decreased hepatic RNI release by about 70% and almost completely inhibited the LPS-induced, elevated NOS II mRNA in Kupffer or endothelial cells. No similar changes were observed in the parenchymal cells. These data suggest that the primary target of in vivo LPS in upregulating hepatic NO release are the nonparenchymal cells. Furthermore, alcohol inhibits the LPS-induced response which may influence immune-related hepatic function.

Primed pentose cycle activity supports production and elimination of superoxide anion in Kupffer cells from rats treated with endotoxin in vivo.

Glucose use and pentose cycle activity were determined in freshly isolated rat Kupffer cells 3 h after an i.v. injection of Escherichia coli endotoxin (0.1 mg/kg body weight), by using [1-14C], [6-14C] and [2-3H]glucose. Endotoxin treatment in vivo caused a 5-fold increase in the basal glucose uptake in Kupffer cells. Pentose cycle activity was elevated from 8.7 to 13.6 nmol/h per 10(7) cells after endotoxin. In vitro treatment of the cells from saline- and endotoxin-treated animals with phorbol ester (10(-6) M) increased pentose cycle activity 2-fold and 8-fold, respectively. Phorbol ester caused a 50% increase in glucose uptake in both groups. t-Butyl hydroperoxide (0.5 mM) caused a similar increase in pentose cycle activity as phorbol ester. Glucose oxidation in the Krebs cycle was also doubled after endotoxin. KC from endotoxin-treated animals produced O2- spontaneously, and were primed to produce additional large amounts of O2- upon phorbol ester treatment. Addition of t-butyl hydroperoxide inhibited O2- production by Kupffer cells. Depletion of glutathione by N-ethylmaleimide (0.1 mM), or inhibition of NADPH oxidase by diphenyliodonium (0.1 mM) inhibited both the pentose cycle activity and the O2- production. Increasing the concentration of exogenous glucose in the cell medium elevated the glycolytic rate, while pentose cycle flux was not affected either under basal conditions or following subsequent challenges by phorbol ester or t-butyl hydroperoxide. Our data suggest that the endotoxin-induced elevated glucose use in Kupffer cells is accompanied by a primed state of the pentose cycle. This condition supports superoxide and macromolecule synthesis and could also represent a potentiated protective mechanism against oxidative cellular injury during bacterial infections.

Effect of high-dose endotoxin on glucose production and utilization.

The purpose of the present study was to determine how a high dose of endotoxin (lipopolysaccharide [LPS]), which produces hypoglycemia, alters in vivo glucose uptake by individual tissues. Catheterized conscious fasted rats were injected intravenously (i.v.) with either saline, LPS (1 mg/100 g body weight [BW], lethal dose [LD] 100), or 3-mercaptopicolinic acid (3-MP), an inhibitor of gluconeogenesis. In the latter two groups, blood glucose levels were clamped at either 6 mmol/L (euglycemia) or 3 mmol/L (hypoglycemia). In the first series of experiments, whole-body glucose flux was determined using [3-3H]glucose, and in the second study in vivo glucose uptake (Rg) by individual tissues was estimated by the tracer [U-14C]-2-deoxyglucose technique. The relative contribution of hypoglycemia per se to the LPS effect was determined by comparing the values from LPS- versus 3-MP-treated animals. There was no difference in the rate of whole-body glucose utilization (Rd) between saline-infused control rats and LPS-treated animals that were hypoglycemic. However, Rg by diaphragm, spleen, liver, and lung was increased in hypoglycemic LPS-treated rats. The increased Rg in these tissues was not observed in 3-MP-treated rats with a comparable hypoglycemia. Only the gastrocnemius muscle showed a reduction in Rg under hypoglycemic conditions, and the decrease was similar in both LPS- and 3-MP-treated animals. When sufficient glucose was infused into LPS-injected rats to maintain euglycemia, whole-body glucose Rd was increased compared with that in hypoglycemic LPS-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Brief endotoxemia markedly increases expression of GLUT1 glucose transporter in Kupffer, hepatic endothelial and parenchymal cells.

Expression of various glucose transporter isoforms was studied in hepatic cells from fasted rats 3h after an injection of E. coli LPS (1mg/kg bw., i.v.). Glucose transporter isoform content of plasma membranes from hepatic cells was determined by western blot analysis using polyclonal antibodies. The predominant glucose transporter isoform expressed in parenchymal cells was GLUT2. GLUTS-1 and -4 were also observed to be present; however, GLUT4 protein was expressed to a relatively minor extent. GLUT3 was not detectable. LPS injection resulted in a 50% decrease in GLUT2 while GLUT1 protein doubled. GLUT4 was not altered after LPS. In the plasma membranes of Kupffer and endothelial cells only the GLUT1 isoform was detected. LPS treatment resulted in a 7- and 4-fold increase in the GLUT1 protein content in these cells. These data indicate that the predominant glucose transporter of hepatic nonparenchymal cells is the GLUT1 isoform and its synthesis and/or membrane translocation is augmented in response to LPS. The observed alterations in the contents of glucose transporters indicate adaptive changes to endotoxemia in the glucose consuming and glucose producing populations of hepatic cells.