Abraham P. Bautista

Superoxide generation by Kupffer cells and priming of neutrophils during reperfusion after hepatic ischemia.

The objective of this study was to identify the cellular source of the vascular oxidant stress in hepatic ischemia-reperfusion injury in male Fischer rats. Nonparenchymal cells (Kupffer cells, endothelial cells) and neutrophils were isolated from postischemic liver lobes by collagenase-pronase digestion followed by centrifugal elutriation. The spontaneous and stimulated generation of superoxide by these cells were subsequently quantified in vitro. Large Kupffer cells from the postischemic lobes spontaneously generated 300% more superoxide than similar cells from control animals. No difference in spontaneous superoxide formation was found when the small Kupffer cells were compared. No other cells isolated from the postischemic lobes or control liver including neutrophils released any detectable superoxide spontaneously. In contrast, small Kupffer cells and neutrophils from the postischemic liver generated significantly more superoxide after stimulation with phorbol ester or opsonized zymosan than the controls. The considerably higher response with zymosan stimulation compared to phorbol ester indicates a particular priming for a receptor-mediated signal transduction pathway during reperfusion. These studies demonstrate that Kupffer cells are the principal source of the oxidant stress during the initial reperfusion phase after hepatic ischemia. The priming of neutrophils during this time may be an important factor for the later neutrophil-induced injury phase.

Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats.

Kupffer cells and polymorphonuclear leukocytes (PMNs) contribute to the severe reperfusion injury of the liver after ischemia at different time points. The objective of this study was to identify die cellular source(s) of reactive oxygen formation during the PMN‐ induced injury phase. Kupffer cells and PMNs were isolated from the liver after 45 min of ischemia and 5 h or 24 h of reperfusion using collagenase‐pronase digestion and a centrifugal elutriation method. Spontaneous superoxide anion (O2−) formation by large Kupffer cells (basal value 0.65 ± 0.16 nmol/h/106 cells) was increased (up to 550%) during the entire reperfusion period. No enhanced O2− generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of O2− spontaneously (0.25 ± 0.05 nmol O2−/h/106 cells), but hepatic PMNs generated significantly more superoxide: 1.90 ± 0.58 nmol O2−/h/106 cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced O2− formation during reperfusion; the priming effect was consistantly higher for stimulation with opsonized zymosan (receptor‐ mediated signal transduction pathway) compared to phor‐ bol myristate acetate (protein kinase C activation). Our data support the hypothesis that PMNs and large Kupffer cells are predominantly responsible for the postischemic oxidant stress during the later reperfusion injury phase after hepatic ischemia in viva.

Superoxide anion generation in the liver during the early stage of endotoxemia in rats.

Infiltration of polymorphonuclear neutrophils (PMN) in the rat liver 3 hr after an intravenous (IV) injection of a sublethal dose of Escherichia coli lipopolysaccharide (LPS) was observed without any significant alteration in the total number of Kupffer and endothelial cells. Since previous studies have demonstrated that phagocytic cells in the liver were in a state of metabolic activation under similar experimental conditions, we investigated the in vitro generation of superoxide anion (O2 ‐) by this cell type following the administration of LPS. Kupffer cells from normal rats did not release O2 ‐, in contrast to those obtained from LPS‐treated rats. The generation of O2 ‐ by Kupffer cells from endotoxic rats was elevated from 3.0 ± 1.9 nmol/106 cells/60 min (mean ± SD) in the absence of macrophage (Mφ) activators, to 5.0 ± 2.36, 11.33 ± 5.40, and 4.33 ± 0.90 in the presence of opsonized zymosan, phorbol myristate acetate (PMA), and the calcium ionophore A23187, respectively. Hepatocytes from normal or endotoxic rats did not produce detectable O2 ‐. Endothelial cells from LPS‐treated rats generated < 0.8 nmol/106 cells in the presence of zymosan. PMN that accumulated in the livers of endotoxic rats released O2 ‐ only in the presence of zymosan (8.12 ± 5.40), PMA (15.43 ± 5.84), or A23187 (1.70 ± 0.12). The O2 ‐ generation by blood monocytes and PMN increased significantly after endotoxin administration and in the presence of activators. These results suggest that the hypermetabolic state of phagocytic cells in the liver shortly after LPS treatment may be correlated with the increased generation of O2 ‐. The latter may subsequently contribute to the induction of hepatic injury in endotoxemia.

Elimination of macrophages by liposome-encapsulated dichloromethylene diphosphonate suppresses the endotoxin-induced priming of Kupffer cells

This study was performed to elucidate the role of Kupffer cells during endotoxemia by assessing the consequences of macrophage depletion by liposome‐encapsulated dichloromethylene diphosphonate (L‐DMDP) following lipopolysaccharide (LPS) treatment. Results show that more than 90% of the largest Kupffer cells, arbitrarily termed KC3, were eliminated, while only 50% of the smaller Kupffer cells were depleted. The selective elimination of a subpopulation of Kupffer cells was accompanied by a significant attenuation of endotoxin (LPS)‐induced serum tumor necrosis factor activity by almost 90%. Hepatic sequestration of neutrophils into the liver after LPS injection was not altered by L‐DMDP. The priming action of LPS on superoxide release by neutrophils recovered from the liver in response to in vitro PMA or zymosan was not altered by L‐DMDP. However, the LPS‐induced priming of superoxide formation in vitro by Kupffer cells, particularly KC3, was significantly attenuated. These results indicate that selective elimination of a subpopulation of Kupffer cells by L‐DMDP down‐regulates the LPS‐induced cytokine release in vivo and endotoxin‐mediated priming of hepatic macrophages for enhanced formation of toxic oxygen metabolites. However, biological activities of neutrophils (i.e., superoxide release and hepatic sequestration) are not altered by L‐DMDP, further emphasizing the specificity of L‐DMDP action on Kupffer cells. J. Leukoc. Biol. 55: 321–327; 1994.

PLATELET ACTIVATING FACTOR STIMULATES AND PRIMES THE LIVER, KUPFFER CELLS AND NEUTROPHILS TO RELEASE SUPEROXIDE ANION

Platelet activating factor (PAF) is considered a key mediator in eliciting the immunologic and metabolic consequences of endotoxic shock and sepsis. Release of oxygen-derived radicals is one of the important and relevant actions of PAF. This study examines the direct and priming effects of PAF on superoxide anion release by perfused liver, isolated Kupffer cells and blood neutrophils. One hour after PAF infusion at a dose of 2.2 micrograms/kg body weight a significant amount of superoxide release (0.71 +/- 0.1 nmol/min/g liver) was measured in the perfused liver compared with the control livers (0.2 +/- 0.01). In the in vitro presence of either phorbol ester or opsonized zymosan, superoxide release following PAF treatment in vivo was significantly increased to 1.36 +/- 0.2 and 4.29 +/- 0.36, respectively. The administration of PAF receptor antagonist (SDZ 63-441) almost completely inhibited the release of this radical. Kupffer cells (KC1, KC2, KC3) and blood neutrophils isolated from PAF-treated rats were also primed for increased production when these cells were challenged in vitro by the activator of protein kinase C, opsonin-coated zymosan as well as the chemotactic factors, complement 5a and F-met-leu-phe. PAF added in vitro to the perfused livers, isolated Kupffer cells or neutrophils from normal animals stimulated the release of superoxide with or without the above agonists. The direct stimulatory effect of PAF on superoxide release was inhibited by the PAF receptor antagonist in vitro. The role of PAF in the LPS-induced superoxide release by the perfused liver was also examined by the administration of PAF antagonist in endotoxic rats. The antagonist inhibited the LPS-mediated superoxide release at 1 hr, but not at 3 hr post-treatment. These results indicate that PAF stimulates and primes the hepatic elements to release superoxide. PAF may be an important factor during the early phase of endotoxemia, while other bioactive substances may take over at a later phase. Therefore, PAF is a key mediator that can directly enhance the release of toxic oxygen-derived radicals which may contribute to organ failure during endotoxemia or sepsis.

Chronic alcohol intoxication primes Kupffer cells and endothelial cells for enhanced CC-chemokine production and concomitantly suppresses phagocytosis and chemotaxis.

Chemokines are involved in the pathogenesis of alcoholic hepatitis and are considered to contribute to the migration of leukocytes into the liver during chronic ethanol intoxication. This work tests the hypothesis that chronic ethanol consumption selectively enhances chemokine release by Kupffer cells and hepatic sinusoidal endothelial cells and migration of inflammatory cells into the liver. Furthermore, enhanced hepatic chemokine secretion may induce an autocrine effect on the ability of Kupffer cells and endothelial cells to chemotax and ingest microbial particles. Male Wistar rats were fed with ethanol in agar block and water for 32 weeks, and were allowed free access to solid food. Results show that after 32 weeks of feeding, leukocyte infiltration and steatosis were observed in the livers of ethanol-fed rats. The majority of the infiltrated cells were CD8+ cells. Serum ALT, endotoxin, MIP-1alpha, MCP-1 and RANTES, (but not CINC and MIP-2) were also increased in the ethanol-fed rats than in the pair-fed group. Isolated Kupffer cells from ethanol-fed rats were primed for enhanced MIP-1alpha, MCP-1, and RANTES production in vitro, while the endothelial cells were primed for enhanced MIP-1alpha release only. Chronic alcohol intoxication was also associated with increased basal H2O2 formation, enhanced nuclear translocation and binding of NF-kappaB, AP-1 and MNP-1 in Kupffer Cells. Chronic ethanol feeding significantly enhanced MNP-1 binding, but not those of NF-kappaB and AP-1 in endothelial cells. Concomitantly, chemokine-induced chemotaxis, E.coli phagocytosis and f-met-leu-phe-induced superoxide anion production by Kupffer cells were downregulated in the ethanol-fed group. Taken together these data demonstrate that prolonged alcohol consumption may compromise the host to hepatitis as a result of increased chemokine production and at the same time may suppress the innate immune function of hepatic non-parenchymal cells.

Acute ethanol intoxication regulates f-met-leu-phe-induced chemotaxis and superoxide release by neutrophils and Kupffer cells through modulation of the formyl peptide receptor in the rat.

This study was performed to assess alcohol-induced alterations in superoxide release and chemotaxis by Kupffer cells and blood neutrophils. Male Sprague-Dawley rats received a bolus injection of alcohol (1.75 g/Kg) followed by an intravenous infusion (250-300 mg/Kg/hr). Three or 24 hr after alcohol infusion, blood neutrophils and Kupffer cells were isolated and assayed for f-met-leu-phe-induced chemotaxis and superoxide release, and formyl peptide receptor expression. At 3 hr post-ethanol, f-met-leu-phe-induced-chemotaxis and superoxide release by blood neutrophils were increased 2 and 3-fold, compared to saline-treated group, and were further increased at 24 hr. The expression of formyl peptide receptors was also increased from 65,000 +/- 8,000 sites per cell to 120,000 +/- 13,000 and 200,000 +/- 16,400 sites at 3 and 24 hr post-ethanol, respectively. The equilibrium dissociation constant (KD) of these receptors on neutrophils was increased at the same time interval. In contrast, alcohol infusion for 3 hr attenuated f-met-leu-phe-induced superoxide release by Kupffer cells (0.8 +/- 0.25 nmol/10(6) cells), compared to saline-treated rats (3.7 +/- 0.3). Chemotaxis by Kupffer cells in response to f-met-leu-phe was also blunted by ethanol at 3 and 24 post-treatment. At 3 hr post-ethanol, the total number of binding sites and KD for f-met-leu-phe on these cells were reduced by almost 30%. The concentration and KD of high affinity binding sites and chemotactic activity of Kupffer cells were not significantly altered by ethanol at 3 hr. However, by 24 hr these were profoundly depressed.

In vivo latex phagocytosis primes the Kupffer cells and hepatic neutrophils to generate superoxide anion.

Activation of liver macrophages during clearance of endotoxins, bacteria, or other particulate materials may be accompanied by the migration of polymorphonuclear neutrophils (PMNs) into the liver and priming of the hepatic phagocytes to release toxic oxygen metabolites. In the present study we investigated the effect of in vivo administration of latex particles on the hepatic sequestration of PMNs and the release of superoxide anion (O2 ‐) by the in situ perfused rat liver and isolated hepatic phagocytes. One hour after an intravenous injection of latex beads, a significant amount of O2 ‐ (0.7 nmol/min/g) was produced by the in situ perfused liver. Administration of latex particles into the perfused liver also elicited O2 ‐ production. Hepatic phagocytes from latex‐treated rats generated large amounts of O2 ‐ (2–14 nmol/60 min/106 cells) when these cells were stimulated in vitro with opsonized zymosan or phorbol myristate acetate (PMA), whereas phagocytes from saline‐treated rats released less than 0.8 nmol O2 ‐. Intravenous infusion of superoxide dismutase or ibuprofen did not prevent the immigration of PMNs to the liver. However, ibuprofen inhibited the production of O2 ‐ by the perfused liver. Also, after addition of ibuprofen in vitro to isolated cells, there was more than 50% inhibition of O2 ‐ generation by Kupffer cells and hepatic PMNs treated with either zymosan or PMA. These observations suggest that arachidonic acid metabolites play a role in O2 ‐ release under these conditions. Thus, activation of the reticuloendothelial system by latex phagocytosis induces the migration of PMNs into the liver and enhances the production of toxic oxygen‐derived radicals by these cells and the resident Kupffer cells. The toxic oxygen radicals may also contribute to hepatic injury.

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.

Chronic ethanol intoxication enhances the production of cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein-2 by hepatocytes after human immunodeficiency virus-1 glycoprotein 120 vaccination

Chemokines are implicated in the pathogenesis of alcoholic liver disease and human immunodeficiency virus-1 (HIV-1) infection. Thus, this work examined the regulation of chemokines --i.e., cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2)--produced by hepatocytes after HIV-1 glycoprotein 120 (gp120) vaccination in Wistar rats fed with ethanol for 30 weeks. HIV-1 gp120 in complete Freunds adjuvant was given by intrainguinal route at a dose of 5 g/kg, followed by two booster shots in incomplete Freunds adjuvant at a weekly interval. Samples were taken 1 week after the last injection was given. Results show that anti-HIV-1 gp120 antibody titer was suppressed by 40% in the ethanol-fed rats, compared with findings in the parallel controls. However, serum CINC and MIP-2 levels were more elevated in the ethanol-fed rats than in the pair-fed group. The likely sources of these chemokines are the hepatocytes. After HIV-1 gp120 treatment, isolated hepatocytes obtained from the ethanol-fed group produced more CINC and MIP-2 than did those of pair-fed rats. Concomitantly, mRNA expression for these two chemokines and hepatic sequestration of neutrophils were upregulated. Ethanol feeding alone suppressed chemokine release, but it did not alter mRNA expression in isolated hepatocytes. Administration of Freunds adjuvant (without HIV-1 gp120) did not induce chemokine release in vivo and did not prime isolated hepatocytes for enhanced chemokine production in vitro. These results show that chronic ethanol intoxication affects the ability of the host to respond to HIV-1 gp120 vaccination.