Renate Urbaschek

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.

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‐TNFα suggesting that TNFα is a primary mediator of these events. Nitric oxide (NO) initially appears to play an important role in these events by stabilizing the TNFα ‐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.

Serum collagen type VI and XIV and hyaluronic acid as early indicators for altered connective tissue turnover in alcoholic liver disease.

Hepatic fibrosis in alcoholic liver disease often heralds progression to cirrhosis and, therefore, noninvasive parameters are required for early diagnosis and follow-up. Collagens VI and XIV, procollagen-III-N-propeptide, hyaluronic acid, and active transforming growth factor-β1 (TGF-β1) were measured in healthy volunteers, patients with alcoholic cirrhosis, and heavy drinkers without cirrhosis. Noncirrhotic alcoholics were assigned to two groups with either normal aspartate aminotransferase or levels ≥2 normal. Collagens VI and XIV were elevated in all alcoholic patients compared to controls (P < 0.0001, all instances). Procollagen-III-N-propeptide and hyaluronic acid levels were higher in alcoholic patients with elevated liver enzymes and in cirrhotics as compared to controls. Procollagen-III-N-propeptide revealed a significant correlation with serum levels of TGF-β1(P < 0.0001). Collagens VI, and XIV, procollagen-III-N-propeptide, and hyaluronic acid appear to be sensitive markers indicating fibrotic transformation in alcoholics. The correlation between procollagen-III-N-propeptide and TGF-β1 emphasizes its role in hepatic fibrogenesis.

In vivo microscopic studies of the responses of the liver to endotoxin.

SummaryIn vivo microscopic methods concomitant with electron microscopic and histochemical procedures are being used to explore the sequelae of responses of Kupffer cells and the hepatic microvasculature to endotoxins. To gain further insight into the role of the liver in host defense and nonspecific resistance, the effects of endotoxin also are being studied in animals sensitized to endotoxin (BCG infection) or tolerant to endotoxin (pretreated with detoxified endotoxin, low doses of endotoxin, or in C3H/HeJ mice). The results to date, have demonstrated that endotoxin induces significant alterations in the hepatic microcirculation due to swelling of Kupffer and endothelial cells and the adhesion of leukocytes and platelets to the sinusoid wall. Lymphocytes frequently are associated with the Kupffer cells. Phagocytosis also is affected; following a brief period of stimulation, the rate of phagocytosis by Kupffer cells is depressed. In BCG infected animals all of these responses are exaggerated but can be minimized by pretreatment with detoxified endotoxin or minute concentrations of endotoxin 24 h prior to the challenge dose of endotoxin. The responses are not seen in the endotoxin low-responder, C3H/HeJ mouse which was found to have a deficiency in lysosomal enzymes and a paucity of functional Kupffer cells. The results provide some insight into the sequelae of cellular and microvascular events that occur in the liver during endotoxemia, endotoxin-related host defense mechanisms and non-specific resistance. In addition, support is provided for the central role of Kupffer cells in these events and that lysosomal enzymes participate in the toxic response elicited by endotoxin.

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.

Treatment of experimental sepsis-induced immunoparalysis with TNF

Following a severe septic abdominal infection induced by sublethal cecal ligation and puncture (CLP) in mice, a phase of depressed immune reactivity occurred two days after CLP characterized by a reduced capacity to produce TNF. To determine whether this reduced TNF production causes immunoparalysis as determined by increased susceptibility to bacterial infection and whether therapeutic TNF substitution can be beneficial during this phase, a super-infection with Salmonella enterica Serovar typhimurium or Listeria monocytogenes was induced two days after sublethal CLP. After CLP a state of true immunoparalysis developed during which Salmonella or Listeria super-infection led to increased lethality paralleled by increased bacterial numbers in spleens and livers. Injection of recombinant human TNF before or at the time of super-infection conferred protection to Salmonella but not to Listeria. In the latter case, the infection mortality was even enhanced. Thus, super-infection during the state of sepsis-induced immunoparalysis leads to increased lethality. TNF substitution during this state of immunoparalysis can be beneficial or deleterious, depending on the location of TNF activity in the animal, timing of TNF administration, or the type of super-infection. These results demonstrate that impaired TNF production capacity can account for some aspects of immunoparalysis, however, diagnostic parameters are required for a safe TNF substitution therapy.

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.

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.

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.

Hepatic Microcirculatory Dysfunction During Cholestatic Liver Injury in Rats

Objective: The present study was conducted to elucidate the sequential alterations in the hepatic microvascular inflammatory response to extrahepatic biliary obstruction.