Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated state in advanced liver cirrhosis.

Advanced cirrhosis is associated with generalized vasodilation of unknown origin, which contributes to mortality. Cirrhotic patients are endotoxemic, and activation of vascular cannabinoid CB1 receptors has been implicated in endotoxin-induced hypotension. Here we show that rats with biliary cirrhosis have low blood pressure, which is elevated by the CB1 receptor antagonist SR141716A. The low blood pressure of rats with CCl4-induced cirrhosis was similarly reversed by SR141716A, which also reduced the elevated mesenteric blood flow and portal pressure. Monocytes from cirrhotic but not control patients or rats elicited SR141716A-sensitive hypotension in normal recipient rats and showed significantly elevated levels of anandamide. Compared with non-cirrhotic controls, in cirrhotic human livers there was a three-fold increase in CB1 receptors on isolated vascular endothelial cells. These results implicate anandamide and vascular CB1 receptors in the vasodilated state in advanced cirrhosis and indicate a novel approach for its management.

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.

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.

Biliary obstruction exacerbates the hepatic microvascular inflammatory response to endotoxin.

Gram-negative sepsis is a serious complication for patients with obstructive jaundice. The present study was conducted to elucidate the response of hepatic microcirculation to endotoxin 2 weeks after bile duct ligation (BDL) or sham-operation in rats. Two hours after lipopolysaccharide (LPS) injection (1, 10, or 100 microg/kg, iv.), the hepatic microvasculature was examined using in vivo microscopy. BDL elicited increases in leukocytes adhering to the sinusoidal wall, swelling of sinusoidal endothelial cells as well as phagocytic activity of hepatic macrophages and a decrease in the numbers of perfused sinusoids. LPS (1, 10, 100 microg/kg) further increased leukocyte adhesion and reduced the numbers of perfused sinusoids in a dose-dependent manner. Leukocyte adhesion in response to LPS (1, 10, 100 microg/kg) in BDL rats was increased 6.1-fold, 5.9-fold, and 3.3-fold, respectively when compared with sham-operated rats. The numbers of perfused sinusoids in response to LPS (1, 10, 100 microg/kg) in BDL rats were decreased by 42%, 36%, and 45%. While 1 and 10 microg/kg LPS also elicited an increase in phagocytic activity in BDL rats when compared with sham-operated rats, the response to 100 microg/kg LPS was suppressed. LPS did not affect the numbers of swollen endothelial cell in BDL rats. The present study demonstrated that chronic biliary obstruction enhanced the hepatic microvascular response to low doses of endotoxin. This observation suggests that exaggerated hepatic microcirculatory dysfunction during sepsis contributes to the development of liver injury and a high incidence of morbidity and mortality in biliary obstruction.

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.

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.

Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes.

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however, seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation.

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.

Effects of bacterial products on granulopoiesis.

Methylated endotoxin and Freeman-type polysaccharide each stimulate granulopoiesis and the production of CSF in mice. These same preparations also protect pretreated mice from lethal X-irradiation. The role of CSF in stimulating granulopoiesis in vivo was shown by the ability of anti-CSF to reduce the number of CFUc in endotoxin-treated mice. C3H/HeJ low responder mice cannot be protected against lethal X-irradiation by pretreatment with endotoxin and they fail to produce CSF in response to phenol water extracted endotoxin and the Freeman-type polysaccharide, but do respond to trichloroacetic acid endotoxin with elevated serum CF levels.

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.

Aspects of beneficial endotoxin-mediated effects.

The status of hyperreactivity and hyporeactivity following the administration of endotoxin in a susceptible host represents phenomena which are of interest in an attempt to understand the role of endotoxins in pathophysiological events in general. Two experimental approaches designed to examine these events are reported herein; i.v. injection with minute concentrations of endotoxin (10 ng of a BOIVIN endotoxin from E. coli 0111) induces tolerance against lethal doses of endotoxin (0.5 microgram or 5.0 microgram) within 24 h in hyperreactive NMRI mice that were infected 14 days before with BCG. Transfer of post-endotoxin serum from BCG infected mice, which contains a myriad of macrophage mediators and which induces nonspecific resistance to X-irradiation, renders a strain of mice (C3H/HeJ) that is hyporeactive to endotoxins, susceptible to the lethal effect of endotoxin. Studies of the role of the macrophage and its mediators in the experimental models described here may contribute to a further understanding of the mechanisms underlying endotoxin-induced biological activities.

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.

The inflammatory response to endotoxins.

1. Endotoxins are very potent and widely spread inflammation-inducing substances. 2. In the course of local infections endotoxins represent one of the main principles of the pathogenicity of gram-negative bacteria by inducing acute nonspecific inflammation. 3. The pharmacological activities of endotoxins consist primarily in generating and liberating the classic mediators of acute nonspecific inflammation. 4. Endotoxins are able to enter into the circulation through their capicity to activate pharmacological mediators. 5. The endotoxic mediators which increase the permeability of the microcirculation of the intestinum enable endotoxins as components of the physiological intestinal flora to enter into the circulation; these induce systemic disease or shock depending on their concentration in the circulation. 6. In the course of chronic inflammation recidivism or recrudescence as trasient acute inflammatory outburst can be caused by local effects of endotoxins. 7. According to some recent observations the inflammation inducing capacity of endotoxins may promote the entry of aerobic bacteria into the blood stream which can result in mixed septicemia.

[Detection of endotoxin in plasma: specificity and value for development and prognosis of infection]. / Endotoxinnachweis im Plasma: Spezifität und Aussagekraft für Entwicklung und Prognose einer Sepsis.

A number of problems may be involved in the detection of endotoxin in plasma of patients using LAL (Limulus amebocyte lysate). When collecting blood or processing samples, contamination with endotoxin or its adsorption to material must be avoided. In our laboratory a kinetic LAL microtiter assay was developed that takes into account plasma-related interferences with the LAL endotoxin reaction by performing an internal standardization in each sample. Negative results do not absolutely exclude the involvement of endotoxins in the underlying disease. High levels of endotoxins do not necessarily reflect the severeness of the clinical status of the patient. Due to nonendotoxin-specific reactions with some complete lysates, false-positive levels may result, e.g., following immunoglobulin therapy. In spite of these limitations, the LAL test remains a valuable tool in the evaluation of gram-negative infections.

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.

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.

Potential transfer of endotoxin across high-flux polysulfone membranes.

It has been postulated that synthetic membranes, such as polysulfone membranes, are rather impermeable for endotoxin or endotoxin fragments and can be used for sterile filtration of dialysate. It has never been investigated, however, whether endotoxin permeability may be different in commercially available polysulfone membranes. In vitro, we found a significantly different permeability for endotoxin in two standard dialyzers and one test dialyzer with high-flux polysulfone membranes. In contrast to the F-60 dialyzer with a very low permeability for endotoxin, a stepwise increasing load of endotoxin concentration in the dialysate compartment of the PN 1913 test dialyzer and Primus 1350 polysulfone dialyzer was followed by a stepwise increase of endotoxin in the blood compartment. A significant transfer across the membranes was found when the endotoxin concentration in the dialysate compartment was > 10 ng/mL in the PN 1913 and > 0.5 ng/mL in the Primus 1350. In the latter, about 0.5% of the endotoxin concentration of the dialysate compartment was found in the blood compartment. The data suggest that manufacturers have to evaluate the performance and other properties of their synthetic membranes in detail.