Endotoxin, endotoxin-neutralizing-capacity, sCD14, sICAM-1, and cytokines in patients with various degrees of alcoholic liver disease.

BACKGROUND: Chronic alcohol ingestion leads to endotoxemia which is believed to play an important role in the pathogenesis of alcoholic liver disease (ALD). The purpose of this study was to determine if chronic ethanol consumption, in addition to affecting plasma endotoxin and cytokines, also affects the endotoxin-neutralizing capacity (ENC), sCD14, and sICAM-1, in patients with ALD. A second aim was to identify correlations between these latter parameters, endotoxin, and cytokines, especially IL-10. METHODS: Hospitalized patients with various degrees of ALD (n = 59), and 20 healthy volunteers were studied. Plasma endotoxin and ENC were determined using our kinetic Limulus amebocyte lysate test. Cytokines, sCD14, and sICAM-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Patients with ALD exhibited a mild endotoxemia (p < 0.01) and a marked decrease in ENC (p < 0.0002). TNF-alpha (p < 0.05), IL-6 (p < 0.0001), sICAM (p < 0.005), and sCD14 (p < 0.0005) were significantly elevated in all patients with ALD, and IL-10 (p < 0.05) in patients with cirrhotic ALD. With the exception of IL-10, the cytokines correlated with each other and with sICAM-1. No correlations occurred between endotoxin, ENC, and sCD14, and between these and the cytokines and sICAM-1. Elevated levels of endotoxin correlate with acute excessive alcohol ingestion. No gender differences were observed. CONCLUSIONS: Acute alcohol intoxication rather than severe ALD results in significant endotoxemia. The limited capacity of plasma to neutralize endotoxin in liver injury seems to be an important factor in ALD which may be responsible for the release of endotoxin-induced mediators, such as cytokines, as well as s-ICAM-1, that are relevant in the pathogenesis of ALD.

The microcirculation during endotoxemia.

The initial responses to endotoxemia are detectable in the microcirculation as a microvascular inflammatory response characterized by activation of the endothelium stimulating these cells from their normal anticoagulant state to a procoagulant state with increased adhesiveness for leukocytes and platelets. Concomitantly, arteriolar tone is lost and reactivity to a variety of agonists is modified. Tissue damage subsequently results not only from reduced perfusion of the exchange vessels, but also from injurious substances released from activated, sequestered leukocytes as well as activated endothelial cells, macrophages, and platelets. This is the result of endotoxins inducing activation and interaction of a number of effector cells, cascades, and acute-phase responses, such as the complement, coagulation, bradykinin/kinin, and hematopoietic systems accompanied by the release of a myriad of mediators. These include eicosanoids, cytokines, chemokines, adhesion molecules, reactive free radicals, platelet-activating factor, and nitric oxide. This paper briefly reviews the microvascular responses to endotoxemia and discusses some of the mechanisms involved.

Effect of immunoglobulin G on the hepatic microvascular inflammatory response during sepsis.

The effects of intravenous immunoglobulin G (ivIG) on the hepatic microvascular inflammatory response to sepsis were studied in rats by in vivo microscopy. High doses of ivIG (300 mg/kg bw) (Sandoglobulin or rat IgG) significantly improved the 48 h survival of septic rats from 25-66% when ivIG was given before or immediately after cecal ligation and puncture. Circulating endotoxin also was significantly reduced. Eight hours after inducing sepsis, the average number of leukocytes adhering to the sinusoidal endothelium increased 15-fold and the average decrease in the number of perfused sinusoids was 22%. IvIG administration minimized these responses. In both septic and nonseptic animals, ivIG also reduced the phagocytic activity of Kupffer cells. The results suggest that high doses of ivIG not only reduce lethality but also limit hepatic microcirculatory dysfunction during sepsis by minimizing leukocyte-endothelial interactions that may be a result of reducing circulating endotoxin and modifying Kupffer cell function.

Role of endotoxin in the hepatic microvascular inflammatory response to ethanol.

Kupffer cells (KC) and gut-derived bacterial endotoxin have been implicated in the aetiology of alcoholic liver disease. Using in vivo microscopic methods, we have shown that ethanol ingestion in mice causes a dose dependent increase in leucocyte adhesion and endothelial cell swelling in hepatic sinusoids. Activation of KC is elicited at low doses while depression occurs at high doses and with chronic exposure. The responses are exacerbated in the presence of endotoxaemia or sepsis and are not seen in endotoxin-resistant animals, implicating a role for endotoxin in the ethanol-induced inflammatory response. In addition, the responses are abolished with anti-TNF alpha suggesting that TNF alpha is a primary mediator of these events. Nitric oxide (NO) initially appears to play an important role in these events by stabilizing the TNF alpha-mediated hepatic microvascular inflammatory response to acute ethanol ingestion, thereby helping to protect the liver from ischaemia and leucocyte induced oxidative injury. Finally, an ongoing clinical study has confirmed a mild systemic endotoxaemia in patients hospitalized for alcoholic liver disease. All of these results support important roles for endotoxin, cytokines, nitric oxide and sinusoidal lining cells in the pathophysiology of liver injury resulting from ethanol alone or in combination with infection.

Ethanol exacerbates hepatic microvascular dysfunction, endotoxemia, and lethality in septic mice.

The effect of acute ethanol administration on the hepatic microvascular responses to sepsis was studied. Polymicrobial sepsis was induced 30 min after mice had received ethanol (1 g/kg b.w.) or isocaloric maltose-dextrin by gastric gavage. Lethality within 24 h was 91.7% in the ethanol-treated animals and 40.0% in septic controls. Endotoxin levels in ethanol treated animals were 107 pg/ml at 6 hr and 1205 pg/ml at 12 h, compared with 32 pg/ml and 104 pg/ml, respectively in the controls. In vivo microscopy revealed that at 3 h in the ethanol treated septic animals, Kupffer cell phagocytic activity was increased by 41%, whereas the number of sinusoids containing blood flow were reduced by 34% concomitant with a 144% increase in the adherence of leukocytes to the sinusoidal walls when compared with the septic controls. By 6 h, however, Kupffer cell phagocytic activity was reduced by 48% in the ethanol treated animals; this was accompanied by a further deterioration in sinusoidal blood flow. Thus, a small, acute dose of ethanol causes significant impairment of the hepatic microcirculation followed by suppression of Kupffer cell activity. This results in exacerbation of endotoxemia and lethality during polymicrobial sepsis.

[Endotoxin contents of phytopharmaceuticals: correlation with clinically observed side effects]. / Untersuchungen zum Endotoxingehalt von Phytopharmaka. Korrelation zu klinisch beobachteten Nebenwirkungen.

Four phytopharmaceutics (Carnivora, Pascotox forte-Injectopas, Esberitox N, Iascador M), which sometimes cause side effects after parenteral administration (fever, rigor, nausea), were examined for their endotoxin content by the kinetic turbidometric Limulus-amebocyte-lysate (LAL) microtitre test. Contaminations of over 10(5) EU/ml (endotoxin units; 1 EU = 0.1 ng of the FDA standard EC-5) were found in correlation with the clinical picture. In one preparation (Carnivora) very high endotoxin levels were always found; contaminations were only occasionally found in the others. These endotoxin measurements are supported by tests of endotoxin-dependent parameters in in-vivo experiments (reduction in leukocytes, acute death in hyperreactive mice). These findings underline the urgent need for a widening of the regulations on testing for pyrogens to include those parenteral preparations which now do not have to be tested because of their small volume (less than 15 ml).

Tumor necrosis factor and interleukin 1 as mediators of endotoxin-induced beneficial effects.

Bacterial lipopolysaccharides or endotoxins are known to induce tumor necrosis; enhanced nonspecific resistance to bacterial, viral, and parasitic infections and to radiation sickness; and tolerance to lethal doses of endotoxin. These beneficial effects are achieved by pretreatment with minute amounts of endotoxin. Recombinant tumor necrosis factor (TNF) and interleukin 1 (IL-1) are among the mediators capable of invoking radioprotection or resistance to the consequences of cecal ligation and puncture. Both cytokines are potent inducers of serum colony-stimulating factor (CSF) in C3H/HeJ mice (low responders to endotoxin). The number of splenic granulocyte-macrophage precursors was found to increase 5 days after injection of TNF in these mice. Although with IL-1 no increase in the number of granulocyte-macrophage colonies occurred in culture in the presence of serum CSF, a marked stimulation was observed when TNF was added. This stimulation of myelopoiesis observed in vivo and in vitro may be related to the radioprotective effect of TNF. The data presented suggest that TNF and IL-1 released after injection of endotoxin participate in the mediation of endotoxin-induced enhancement of nonspecific resistance and stimulation of hematopoiesis.

Kupffer cell function in host defense.

High-resolution in vivo microscopic methods have been used to explore the responses to endotoxin of Kupffer cells in the livers of anesthetized mice, rats, hamsters, and guinea pigs under a variety of experimental conditions. These include studies of normal animals as well as of animals sensitized or tolerant to endotoxin, C3H/HeJ mice with a low response to endotoxin, mice rendered septic by cecal ligation and puncture, mice with Kupffer cells selectively destroyed by frog virus 3, and rats with portacaval shunts. The functional state of Kupffer cells was evaluated by measuring both the number of these cells per microscopic field that phagocytosed 1.0-micron latex particles and the rate at which individual Kupffer cells phagocytosed single latex particles. The intrahepatic density and level of activation of Kupffer cells were found to play a role in determining endotoxin sensitivity and to be involved, in conjunction with endotoxin, in the development of tolerance. All of these studies support the concept of a central role for Kupffer cells in host defense mechanisms and of the possible modulation and of this role by gut-derived endotoxins contained in the portal blood.

No evidence for endotoxin transfer across high flux polysulfone membranes.

Recently, there has been some concern that high-flux membranes may expose dialysis patients to the risk of endotoxin transfer secondary to backfiltration within the dialyzer. To evaluate the safety of high-flux polysulfone dialyzers, we examined in an in vitro recirculation system whether lipopolysaccharides (LPS) and lipid A respectively penetrate from the dialysate to the blood compartment and vice versa using a F-60 hemofilter (Fresenius AG). For the detection of endotoxin, a sensitive, kinetic limulus amebocyte lysate (LAL) microtiter test was used. It can be concluded that LPS and lipid A do not pass from either side through the filter system used when saline was recirculated for more than 10 h on both sides of the membrane.

Tumor necrosis factor induced stimulation of granulopoiesis and radioprotection.

Human recombinant tumor necrosis factor, TNF, was used to assess its ability to stimulate granulopoiesis and to protect mice against lethal irradiation, effects known to be inducable with TNF-rich postendotoxin serum from BCG infected mice (BCG/ET serum). Although the endotoxin contamination of this TNF preparation is extremely low its effects were compared in endotoxin low responder C3H/HeJ mice and susceptible NMRI mice. TNF is a potent inducer of serum colony stimulating activity, CSA, in both mouse strains. In peripheral blood a marked granulocytosis with a concomitant decrease in lymphocytes and monocytopenia occurs at 2 hours after injection of TNF. Moreover, TNF induces an increase in the number of splenic myelopoietic committed stem cells (GM-CFC, granulocyte-macrophage colony forming cells) determined five days after injection. The lethality rate, registered over 30 days after exposure to 660 cGy whole body X-irradiation is reduced to 40% in C3H/HeJ mice as compared to 75% in control animals. The reduction in lethality is observed both, when TNF was injected 24 hours before or after irradiation. In vitro, TNF significantly increases the number of colonies in the presence of CSA in bone marrow cultures. TNF per se does not effect colony growth. The studies reported here demonstrate that TNF is a myelopoiesis stimulating factor in mice which may be related to the reduction in lethality following whole body irradiation.

Deficient Kupffer cell phagocytosis and lysosomal enzymes in the endotoxin-low-responsive C3H/HeJ mouse.

Various substances, including lysosomal enzymes, are produced by Kupffer cells and other macrophages; their release has been implicated in the toxic response to endotoxins. C3H/HeJ mice exhibit little or no response to doses of endotoxin that are lethal in syngeneic C3HeB/FeJ mice. To explore the nature of this deficient response, the Kupffer cells of these mice were studied using in vivo microscopic as well as histochemical and electron microscopical methods. In vivo, the rate of phagocytosis of single 0.8 micron latex particles was measured in individual Kupffer cells as was the number of phagocytic cells per microscopic field. Frozen sections of livers were stained for a variety of lysosomal enzymes and liver specimens also were processed for electron microscopy. In comparison to the endotoxin-sensitive C3HeB/FeJ mice, the livers of the C3H/HeJ mice contained 60% fewer Kupffer cells that phagocytosed latex. However, the rate of phagocytosis by these cells was not statistically different and ranged from 19-26 sec. The volume density of acid-phosphatase-positive Kupffer cells was 40% less in the C3H/HeJ mice. Similar differences were observed with other lysosomal enzymes including cathepsins B and H and dipeptidyl peptidases I and II. However, light and electron microscopy revealed a relatively normal number of Kupffer cells in livers stained for peroxidase, a nonlysosomal enzyme. The results suggest that the insensitivity of C3H/HeJ mice to endotoxin may be related in part to a lysosomal enzyme deficiency and a paucity of phagocytic Kupffer cells in these animals.

Species differences in Kupffer cells and endotoxin sensitivity.

The relative species sensitivity to Escherichia coli O111:B4 endotoxin was found to be guinea pig greater than hamster greater than mouse greater than rat. The 50% lethal dose of this endotoxin correlated with both the rate at which single latex particles were phagocytosed by individual Kupffer cells and the number of Kupffer cells in hepatic lobules that phagocytosed latex. The results suggests that the intrahepatic density and the level of activation of Kupffer cells participate in determining endotoxin sensitivity.

Protective effects and role of endotoxin in experimental septicemia.

An experimental model was used in mice in which septicemia develops following invasion of the animals' own intestinal flora after cecal ligation and puncture. Pretreatment with 1 microgram of endotoxin administered 24 hours before surgery significantly reduced the rate of lethality. Bacteria were counted and differentiated in cardiac blood at various times throughout a 48-hour period after induction of septicemia in mice, with and without pretreatment. Endotoxin levels and plasma-related interference of the Limulus-amebocyte-lysate (LAL)-endotoxin reaction also were determined as were hematologic and metabolic parameters. A shift from mixed gram-positive and gram-negative to predominantly gram-negative bacteria occurred in both groups. In pretreated mice, a reduction in aerobic bacterial counts and reduced hyperglycemia were seen in the initial phase; and a decrease in anaerobic and aerobic bacteria and in endotoxin levels were observed at the end of the experiment. This appears to be related to endotoxin-induced increased resistance against the consequences of intraabdominal sepsis. These studies also indicate that the measured amount of circulating endotoxin does not necessarily correlate to the severeness of infection. Individual differences in plasma-related interference with the LAL-endotoxin reaction also emphasize the requirement for sample-internal standardization in order to reliably quantify endotoxin in plasma.

In vivo microscopic observations of the responses of Kupffer cells and the hepatic microcirculation to Mycobacterium bovis BCG alone and in combination with endotoxin.

Kupffer cell function and hepatic microvascular hemodynamics were studied by in vivo microscopy in Mycobacterium bovis BCG-infected NMRI mice before and after treatment with minute (0.01 mu mg) tolerance-producing doses and doses causing 70% lethality (0.5 micrograms) of Escherichia coli 0111:B5 endotoxin alone and in combination. BCG-induced granulomas distorted the hepatic microvasculature and impeded blood flow in many sinusoids; flow also was altered further by leukocytes adhering to the sinusoidal walls and by enlarged Kupffer cells that bulged into the lumen. Nevertheless, in BCG-infected mice, the ratio of Kupffer cells which phagocytosed latex to sinusoids containing blood flow and capable of delivering these particulates to Kupffer cells was significantly greater than that in uninfected mice. The phagocytosis of single latex particles by individual Kupffer cells also was more rapid. This indicated an expansion of the numbers and activation of Kupffer cells. In this hyperreactive state, the tolerance-inducing dose of endotoxin produced no change in the rate of phagocytosis after 2 h. In contrast, the 70% lethal dose reduced the rate by 123%, unless tolerance was induced, in which case there was no reduction in the rate of phagocytosis. Twenty-four hours after injection of the tolerance-inducing dose, however, the rate of phagocytosis was accelerated slightly (17%). This suggested that the Kupffer cells had been activated and perhaps were more effective in clearing subsequent endotoxin from the blood but without sufficient release of toxic substances to be lethal. That some mediators were released, however, was suggested by the microvascular alterations that accompanied the above phagocytic responses. These results further support the concept of a central role for Kupffer cells in endotoxin-mediated, nonspecific host defense mechanisms.

Ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxemia in mice.

The efficacy of various adsorbents for endotoxin was tested in vitro and in vivo using a murine experimental model of gut-derived endotoxemia. A quantitative limulus amebocyte lysate microtiter test and the limulus amebocyte lysate tube test were used to determine intestinal and circulating levels of endotoxin. Kaopectate, kaolin/pectin mixture, kaolin, pectin, bentonite, charcoal particles, and lactulose were tested for their ability to bind endotoxins both in vitro and in vivo. The most effective material in the prevention of endotoxemia provided to be bentonite followed by Kaopectate and charcoal particles. Kaolin least effectively bound endotoxin at similar concentrations, while lactulose and pectin had minimal effects. Good correlation was shown between the ability of these drugs to bind endotoxin in vitro as compared with in vivo action.

Ability of Post-endotoxin serum from BCG-infected mice to induce nonspecific resistance and stimulation of granulopoiesis.

Serum from BCG-infected mice obtained 2 h after injection with endotoxin induced elevated levels of colony-stimulating factor and an increase in splenic granulocyte-macrophage progenitor cells in C3H/HeJ mice. The capacity of such serum to stimulate granulopoiesis may be related to its ability to increase nonspecific resistance to lethal irradiation.

[Quantitative endotoxin determination. Automated kinetic Limulus amebocyte lysate microtiter test with measurement of sample-related interferences]. / Quantitativer Endotoxin-Nachweis. Automatisierter, kinetischer Limulus-Amöbozyten-Lysat-Mikrotiter-Test mit Messung probenabhängiger Interferenzen.

A turbidometric, automated limulus amoebocyte lysate (LAL) microtiter test has been developed based on the evaluation of the LAL-endotoxin reaction kinetics. The maximal increase in optical density of each reaction mixture within 1 min is recorded. With this method an endotoxin standard curve is achieved which is linear over a concentration range of six decades. With presently available LAL methods sample-related inhibition or enhancement of the LAL endotoxin reaction may be overlooked and lead to false results. The quality of interfering factors can be characterized with our methods by spiking serial dilutions of the sample with constant endotoxin concentrations. The additional introduction of an internal standardization in our system allows the determination of endotoxin with simultaneous detection of quality and quantity of sample-induced interference. This procedure is based on a mathematic model which describes interference-caused alterations of the reaction revealed by addition of endotoxin in increasing concentrations. In comparison to the LAL tube test and the turbidometric determination at a given time the advantages of the developed method are demonstrated using three different samples (gelatin solution, adenine-HCl solution and a concentrate of coagulation factors (PPSB)). These are paradigmaticly selected because and enhancement of the LAL endotoxin reaction.