Hepatic Phagocytes Research Articles

Age-associated disparity in phagocytic clearance affects the efficacy of cancer nanotherapeutics.

Nanomedicines have been approved to treat multiple human diseases. However, clinical adoption of nanoformulated agents is often hindered by concerns about hepatic uptake and clearance, a process that is not fully understood. Here we show that the antitumour efficacy of cancer nanomedicine exhibits an age-associated disparity. Tumour delivery and treatment outcomes are superior in old versus young mice, probably due to an age-related decline in the ability of hepatic phagocytes to take up and remove nanoparticles. Transcriptomic- and protein-level analysis at the single-cell and bulk levels reveals an age-associated decrease in the numbers of hepatic macrophages that express the scavenger receptor MARCO in mice, non-human primates and humans. Therapeutic blockade of MARCO is shown to decrease the phagocytic uptake of nanoparticles and improve the antitumour effect of clinically approved cancer nanotherapeutics in young but not aged mice. Together, these results reveal an age-associated disparity in the phagocytic clearance of nanotherapeutics that affects their antitumour response, thus providing a strong rationale for an age-appropriate approach to cancer nanomedicine.

Immune cell C/EBPβ deficiency is associated with hepatic mononuclear defects and spontaneous hepatitis but not steatohepatitis induced liver fibrosis.

BackgroundCCAAT/enhancer‐binding protein β (C/EBPβ) is a transcription factor known to be involved in macrophage differentiation and function, steatohepatitis and liver fibrosis.MethodsImmune restricted C/EBPβ deficient and control mice were investigated in steady‐state and in the CDA‐HFD steatohepatitis model. Mice were assessed for weight change, liver biochemical profile, histology and hepatic phagocytes composition.ResultsFlow cytometry analysis of hepatic nonparenchymal cells revealed reduced numbers of hepatic monocytes and Kupffer cells and an increase in hepatic MHC class II positive myeloid cells in immune cells restricted C/EBPβ deficient mice. Immune‐restricted C/EBPβ deficiency resulted in decreased weight gain and appearance of mild spontaneous liver inflammation. Nevertheless, In the CDA‐HFD steatohepatitis model, immune restricted C/EBPβ deficient and proficient mice exhibit similar grade of hepatic steatosis, liver enzymes levels and fibrosis stage.ConclusionsImmune‐restricted C/EBPβ deficiency leads to significant alteration in hepatic mononuclear phagocytes composition associated with spontaneous mild hepatitis. Steatohepatitis associated fibrosis is not dependent on C/EBPβ expression by immune cells.

Immune cells restricted C/EBPbeta deficiency is associated with distorted hepatic mononuclear phagocytes composition, weight loss and spontaneous hepatitis

Immune cells restricted C/EBPbeta deficiency is associated with distorted hepatic mononuclear phagocytes composition, weight loss and spontaneous hepatitis

Overcoming the Reticuloendothelial System Barrier to Drug Delivery with a "Don't-Eat-Us" Strategy.

Overcoming the reticuloendothelial system (RES) has long been a vital challenge to nanoparticles as drug carriers. Modification of nanoparticles with polyethylene glycol helps them avoid clearance by macrophages but also suppresses their internalization by target cells. To overcome this paradox, we developed an RES-specific blocking system utilizing a "don't-eat-us" strategy. First, a CD47-derived, enzyme-resistant peptide ligand was designed and placed on liposomes (d-self-peptide-labeled liposome, DSL). After mainline administration, DSL was quickly adsorbed onto hepatic phagocyte membranes (including those of Kupffer cells and liver sinusoidal endothelial cells), forming a long-lasting mask that enclosed the cell membranes and thus reducing interactions between phagocytes and subsequently injected nanoparticles. Compared with blank conventional liposomes (CL), DSL blocked the RES at a much lower dose, and the effect was sustained for a much longer time, highly prolonging the elimination half-life of the subsequently injected nanoparticles. This "don't-eat-us" strategy by DSL was further verified on the brain-targeted delivery against a cryptococcal meningitis model, providing dramatically enhanced brain accumulation of the targeted delivery system and superior therapeutic outcome of model drug Amphotericin B compared with CL. Our study demonstrates a strategy that blocks the RES by masking phagocyte surfaces to prolong nanoparticle circulation time without excess modification and illustrates its utility in enhancing nanoparticle delivery.

Targeting and Modulation of Liver Myeloid Immune Cells by Hard-Shell Microbubbles.

Poly n-butylcyanoacrylate (PBCA)-based hard-shell microbubbles (MB) have manifold biomedical applications, including targeted drug delivery or contrast agents for ultrasound (US)-based liver imaging. MB and their fragments accumulate in phagocytes, especially in the liver, but it is unclear if MB affect the function of these immune cells. We herein show that human primary monocytes internalize different PBCA-MB by phagocytosis, which transiently inhibits monocyte migration in vertical chemotaxis assays and renders monocytes susceptible to cytotoxic effects of MB during US-guided destruction. Conversely, human macrophage viability and function, including cytokine release and polarization, remain unaffected after MB uptake. After i.v. injection in mice, MB predominantly accumulate in liver, especially in hepatic phagocytes (monocytes and Kupffer cells). Despite efficiently targeting myeloid immune cells in liver, MB or MB after US-elicited burst do not cause overt hepatotoxicity or inflammation. Furthermore, MB application with or without US-guided burst does not aggravate the course of experimental liver injury in mice or the inflammatory response to liver injury in vivo. In conclusion, PBCA-MB have immunomodulatory effects on primary human myeloid cells in vitro, but do not provoke hepatotoxicity, inflammation or altered response to liver injury in vivo, suggesting the safety of these MB for diagnostic and therapeutic purposes.

Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice

Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be replaced by bone marrow precursors during extensive injuries, irradiation, and infections. We investigated the liver populations of myeloid immune cells and their location, as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original phagocytes by bone marrow-derived surrogates were also examined. We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt, and CX3CR1-DTR-EYFP. Liver nonparenchymal cells were immunophenotyped using mass cytometry and gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory cytokines, and dexamethasone accelerated the repopulation of liver phagocytes. In studies of hepatic phagocyte depletion in mice, we found that myeloid precursors can differentiate into liver macrophages and dendritic cells, which each localize to distinct tissue compartments. During replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.

Liver inflammation abrogates immunological tolerance induced by Kupffer cells.

The liver is essential for inducing immunological tolerance toward harmless antigens to maintain immune system homeostasis. However, the precise cellular mechanisms of tolerance induction against particle-bound antigens, the role of the local hepatic microenvironment, and implications for therapeutic targets in immune-mediated diseases are currently unclear. In order to elucidate cellular mechanisms of tolerance induction in healthy and injured liver, we developed a novel in vivo system combining the systemic delivery of low-dose peptide antigens coupled to inert particles, immunological readouts, and sophisticated intravital multiphoton microscopy-based imaging of liver in mice. We show that liver resident macrophages, Kupffer cells (KCs), but not hepatic monocyte-derived macrophages or dendritic cells (DCs), are the central cellular scavenger for circulating particle-associated antigens in homeostasis. KC-associated antigen presentation induces CD4 T-cell arrest, expansion of naturally occurring Foxp3(+) CD25(+) interleukin-10-producing antigen-specific regulatory T cells (Tregs) and tolerogenic immunity. Particle-associated tolerance induction in the liver protected mice from kidney inflammation in T-cell-mediated glomerulonephritis, indicating therapeutic potential of targeting KC for immune-mediated extrahepatic disorders. Liver inflammation in two independent experimental models of chronic liver injury and fibrosis abrogated tolerance induction and led to an immunogenic reprogramming of antigen-specific CD4 T cells. In injured liver, infiltrating monocyte-derived macrophages largely augment the hepatic phagocyte compartment, resulting in antigen redistribution between myeloid cell populations and, simultaneously, KCs lose signature markers of their tolerogenic phenotype. Hepatic induction of tissue-protective immunological tolerance against particulate antigens is dependent on KCs as well as on a noninflamed liver microenvironment, thereby providing mechanistic explanations for the clinical observation of immune dysfunction and tolerance break in patients with advanced liver diseases.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

Endogenous protease inhibitor uptake within the graft during reperfusion in human liver transplantation

In experimental liver transplantation, endogenous protease inhibitors alleviate ischemia-reperfusion (I/R) injury by inhibiting proteolysis and by direct anti-inflammatory actions. We described the kinetics of endogenous protease inhibitors and explored their anti-inflammatory potential during reperfusion and their effects on graft function in human liver transplantation. We measured circulating levels of protease inhibitors (secretory leukocyte proteinase inhibitor, SLPI; tissue inhibitor of metalloproteinases-1, TIMP-1) and proteolytic enzymes (elastase; matrix metalloproteinase-9, MMP-9) with ELISA, and neutrophil and monocyte CD11b and L-selectin expression with flow cytometry during liver transplantation in ten patients. To assess changes within the graft during reperfusion, blood samples from portal and hepatic veins were obtained simultaneously. Circulating SLPI and TIMP-1 levels decreased during surgery. During initial reperfusion, the transhepatic SLPI gradient was -27 (-35 to -22) ng/ml, P = 0.005, and TIMP-1 -510 (-636 to -362) ng/ml, P = 0.005, indicating graft protease inhibitor uptake. Concomitantly, hepatic phagocyte activation and sequestration as well as elastase and MMP-9 release into the circulation occurred. The transhepatic SLPI gradient correlated with postoperative liver enzymes (ALT R = -0.648, P = 0.043; ALP R = -0.661, P = 0.038; bilirubin R = -0.821, P = 0.004; GGT R = -0.648, P = 0.043). The results suggest a relative shortage of protease inhibitors within the liver during reperfusion, which may contribute to the development of graft injury.

Graft protein C entrapment is associated with reduced phagocyte activation during reperfusion in human liver transplantation

To explore the potential anti-inflammatory role of protein C pathway in ischemia-reperfusion injury during liver transplantation. Prospective, observational clinical study. Tertiary teaching hospital. Fifty adult patients undergoing liver transplantation for acute liver failure or chronic liver disease. To assess changes occurring across the transplanted liver, samples of blood entering and leaving the graft were obtained simultaneously from portal and hepatic veins. Plasma protein C and activated protein C levels, neutrophil and monocyte CD11b and L-selectin expression, and leukocyte differential counts were measured. Postoperative liver function and outcome of transplantation were recorded. During reperfusion, protein C became entrapped within the graft (portal vein 49% [20-96%]; graft caval effluent 25% [12-76%], p < .001), without concomitant activated protein C outflow from the graft. Simultaneously, marked neutrophil and monocyte activation occurred within the graft. Enhanced hepatic protein C entrapment was associated with reduced neutrophil and monocyte activation (R = .377, p = .011; R = .389, p = .008, respectively) during reperfusion. Protein C entrapment occurs immediately during reperfusion in the graft without concomitant activated protein C release, suggesting a shortage of activated protein C in the reperfused graft. The ongoing inflammatory response during reperfusion may lead to protein C and activated protein C utilization within the graft. Indeed, hepatic protein C entrapment is associated with reduced hepatic phagocyte activation, suggesting a regulatory role for protein C pathway in hepatic reperfusion in human liver transplantation.

Effects of a perfluorochemical emulsion on the fate of circulating Pseudomonas aeruginosa.

Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.

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 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 H2O2 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. (Hepatology 1996 Sep;24(3):691-6)

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.

Monoclonal antibody against the CD18 adhesion molecule stimulates glucose uptake by the liver and hepatic nonparenchymal cells

Neutrophils and macrophages play an important role in the body's microbicidal defense and have been implicated in the induction of tissue injury in reperfusion, endotoxemia and septic shock. Cellular host defense is accompanied by enhanced glucose use. In this study we examined the effect of monoclonal antibody 1F12 on in vivo glucose use by selected tissues and hepatic phagocytes. Monoclonal antibody 1F12 is specific for the CD18 adhesion molecule on rat neutrophils and is the only known antibody that induces profound neutropenia. The administration of monoclonal antibody 1F12 at a final dose of 2 mg/kg body weight enhanced the removal of neutrophils from the circulation within 15 min. Leukopenia was sustained for 24 hr. Large numbers of neutrophils were sequestered in the liver at 4 hr. Although the number of neutrophils in the liver at 24 hr after antibody treatment was lower than at 4 hr, these values were still higher than those in the saline controls. The rate of glucose metabolism by selected tissues was not significantly altered by the antibody, except by the liver, in which use increased 2.7-fold vs. the control value at 4 hr after treatment. Hepatocytes, endothelial cells and Kupffer cells contributed to the increase in the rate of glucose metabolism by the liver. The rate of glucose metabolism by Kupffer cells was significantly elevated at 4 and 24 hr, whereas the rate of glucose metabolism of hepatocytes and endothelial cells returned to normal at 24 hr. After antibody treatment, the glucose uptake by hepatic sequestered neutrophils was significantly reduced at 4 hr and returned to near-normal levels at 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody against the CD18 adhesion molecule stimulates glucose uptake by the liver and hepatic nonparenchymal cells

Neutrophils and macrophages play an important role in the body's microbicidal defense and have been implicated in the induction of tissue injury in reperfusion, endotoxemia and septic shock. Cellular host defense is accompanied by enhanced glucose use. In this study we examined the effect of monoclonal antibody 1F12 on in vivo glucose use by selected tissues and hepatic phagocytes. Monoclonal antibody 1F12 is specific for the CD18 adhesion molecule on rat neutrophils and is the only known antibody that induces profound neutropenia. The administration of monoclonal antibody 1F12 at a final dose of 2 mg/kg body weight enhanced the removal of neutrophils from the circulation within 15 min. Leukopenia was sustained for 24 hr. Large numbers of neutrophils were sequestered in the liver at 4 hr. Although the number of neutrophils in the liver at 24 hr after antibody treatment was lower than at 4 hr, these values were still higher than those in the saline controls. The rate of glucose metabolism by selected tissues was not significantly altered by the antibody, except by the liver, in which use increased 2.7-fold vs. the control value at 4 hr after treatment. Hepatocytes, endothelial cells and Kupffer cells contributed to the increase in the rate of glucose metabolism by the liver. The rate of glucose metabolism by Kupffer cells was significantly elevated at 4 and 24 hr, whereas the rate of glucose metabolism of hepatocytes and endothelial cells returned to normal at 24 hr. After antibody treatment, the glucose uptake by hepatic sequestered neutrophils was significantly reduced at 4 hr and returned to near-normal levels at 24 hr. Another CD18 monoclonal antibody (monoclonal antibody WT.3), which does not cause neutropenia, did not alter the glucose uptake by tissues and hepatic cells, although it induced slight sequestration of neutrophils in the liver. The N-formylmet-leu-phe–induced superoxide release by neutrophils was attenuated by monoclonal antibody 1F12 in vitro and in vivo. These data indicate that as a consequence of monoclonal antibody 1F12 treatment, a major portion of the neutrophil pool is found in the liver and that Kupffer cells and endothelial cells become activated. It is postulated that this is part of the mechanism for increased clearance of effete leukocytes from the circulation. (HEPATOLOGY 1993;17:924–931.)

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/10(6) 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.

Alcohol-induced downregulation of superoxide anion release by hepatic phagocytes in endotoxemic rats

Bacterial endotoxins [lipopolysaccharide (LPS)] are potent immunomodulators, and ethanol is known to depress certain immune defense mechanisms. Thus the combined impact of these two agents on the generation of superoxide anion (O2(-).) by isolated hepatic phagocytic cells was investigated. Ethanol was infused intravenously into rats for 7 h, and Escherichia coli LPS was injected intravenously at 4 h after ethanol administration. Control groups received an equal volume of saline or ethanol alone. Nonparenchymal cells that were composed of endothelial and Kupffer cells and few polymorphonuclear neutrophils (PMN; less than 1%) were obtained after collagenase-pronase digestion. In the LPS-treated rats, the total number of PMN per liver increased significantly. Histological sections of the liver showed PMN infiltration and areas of necrosis after LPS treatment with or without ethanol. In the presence of either phorbol 12-myristate 13-acetate or opsonized zymosan in vitro, Kupffer cells and hepatic PMN from LPS-treated rats generated large amounts of O2(-).. Ethanol intoxication in vitro by these cells to 50%. Ethanol alone (without LPS) had no effect on the production of O2(-).. These studies demonstrate that ethanol intoxication was associated with the downregulation of the LPS-enhanced in vivo priming of hepatic phagocytes to generate O2(-). in vitro and may thus contribute to the enhanced susceptibility of alcoholic subjects to develop an infection.

Heinz body anemia and methemoglobinemia in ponies given red maple (acer rubrum L.) leaves.

Ponies given dried red maple (Acer rubrum L.) leaves at a dose of 3.0 gm/kg body weight became ill and died one to five days after administration of the leaves. Two clinical patterns of disease were seen. Ponies given dried leaves collected after September 15 died by 18 hours, while ponies given dried leaves collected before September 15 became ill with a hemolytic syndrome and died by three to five days. Freshly harvested leaves administered immediately after collection did not produce disease in ponies, but when dried, they became toxic and remained so for at least 30 days. Overnight freezing did not alter the toxicity of the leaves. Leaves were toxic when administered at doses of 1.5 gm/kg of body weight. The clinical signs of ponies with the hemolytic syndrome included polypnea, tachycardia, icterus, cyanosis, scleral petechiation, and brownish discoloration of the urine and blood. Blood changes of ponies with the hemolytic syndrome included anemia, hemoglobinemia, Heinz bodies, depletion of erythrocyte reduced glutathione, increased erythrocyte fragility, and increased serum levels of aspartate amino transferase, sorbitol dehydrogenase, plasma protein, and bilirubin. Lesions of ponies that died from the hemolytic syndrome included icterus, centrilobular hepatic degeneration, hemoglobinemic nephrosis, and erythrophagocytosis by splenic, adrenal, and hepatic phagocytes. Only brownish discoloration of the blood and mild centrilobular hepatic degeneration were observed in the four ponies that died peracutely.