Mechanism of tissue damage through free oxygen radicals during hepatic amoebiasis in guinea pigs

The capacity of Kupffer cells and blood monocytes to release free oxygen radicals was studied by chemiluminescence (CL) response during hepatic amebiasis in guinea pigs. Guinea pigs infected with Entamoeba histolytica intramesenterically were sacrificed on days 0, 2, 5 and 8 post-infection. Hepatic lesions were graded I-IV. A significant increase in the CL response was observed from day 2 post-infection and it increased with the progress of infection. Maximum increase was observed on the 8th post infection day. Animals with grade III or IV hepatic lesions had significantly elevated CL response. The degree of hepatic lesions correlated well with the CL response (P less than 0.01). It is postulated that tissue damage during hepatic amoebiasis may be mediated through enhanced release of free oxygen radicals by Kupffer cells and blood monocyte.     

Generation of reactive oxygen species by Kupffer cells and blood monocytes during intestinal amebiasis in guinea pigs

The reactive oxygen species generation capacity of Kupffer cells and blood monocytes was studied through chemiluminescence (CL) in guinea pigs, infected intracecally with Entameba histolytica, on days 0, 3, 7, 14 and 21 post infection. There was an elevated response up to the 14th post-infection day. The CL response of these cells was diminished on the 21st post infection day though it was significantly higher than up the controls. The CL response increased exponentially with the severity of cecal lesions. A direct correlation was observed between the CL response of phagocytic cells and cecal lesions of animals. The role of oxygen species in causation of tissue injury during intestinal amebiasis is postulated.     

Role of lysosomal enzymes in tissue damage during hepatic amoebiasis. An experimental study

Two key lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-glucuronidase of Kupffer cells and blood monocytes were studied in guinea pigs infected intramesenterically with E. histolytica. The animals were sacrificed on days 0, 2, 5 and 8 post-infection. The enzyme levels in the cell lysate and supernatants of both Kupffer cells and blood monocytes were significantly higher (P less than 0.01) in the infected animals compared with the controls. This increase was more marked on the 5th and 8th days post infection (P less than 0.001). The alteration in enzyme levels was higher in Kupffer cells than in blood monocytes (P less than 0.05). The enzyme levels increased exponentially with the severity of the hepatic lesions. A direct correlation was observed between the enzyme levels and the severity of the hepatic lesions (P less than 0.01). Hence, the role of lysosomal enzymes, released by activated mononuclear phagocytic cells, in the pathogenesis of hepatic amoebiasis is postulated.     

Kupffer cell functions during intestinal amoebiasis in guinea pigs

Kupffer cells play an important role in all alimentary tract infections. Their role in intestinal amoebiasis is not clear. Hence, the present study examined Kupffer cell functions--phagocytic capacity and levels of a key lysosomal enzyme, N-acetyl-beta-glucosaminidase -- in guinea pigs infected with Entamoeba histolytica intracaecally. Animals were sacrificed on days 0, 3, 7, 14 and 21 post-infection. During intestinal amoebiasis the phagocytic capacity in Kupffer cells was depressed, whereas lysosomal enzyme levels were highly elevated. Maximum alteration in Kupffer cell functions was observed on the 14th post-infection day (P less than 0.01). Animals which survived until 21 days post-infection did not show any significant (P greater than 0.05) change in their Kupffer cell functions compared with controls and day zero values. Phagocytic capacity was inversely correlated with severity of caecal lesions. In contrast, enzyme levels were directly correlated with degree of caecal lesions. Similar trends were observed in blood monocytes. The depressed phagocytic capacity of mononuclear phagocytes and the increased enzyme release by these cells accompanying intestinal amoebic infection may contribute to the establishment of the infection and the tissue damage that accompanies it.     

Free radical generation and the course of primary infection with Nippostvongylus brasiliensis in congenitally athymic (nude) rats

The course of primary infections with Nippostrongylus brasiliensis was followed in nude (CBH/R nu/nu) and heterozygote (CBH/R nu/+) rats. In both nude and heterozygote rats peak egg production by N. brasiliensis occurred on days 7 and 8 post-infection. However, whereas in heterozygote rats egg production declined rapidly thereafter and ceased completely by day 14 post-infection, in nude rats high numbers of N. brasiliensis eggs were still seen on day 27 post-infection, when the experiment was terminated. In comparison with the nude rats, heterozygote rats had a 5-fold greater loss of weight by day 9 post-infection and a 4-fold higher incidence of diarrhoea. Furthermore, infected heterozygote rats became anaemic whereas uninfected rats and infected nude rats showed no evidence of anaemia. Free radical generation was measured in infected (9 days) and uninfected rats. Leucocytes from infected heterozygote rats were able to generate copious quantities of free radicals in response to N. brasiliensis whereas leucocytes from infected nude rats produced only slightly more free radicals than uninfected rats. Thus, worm rejection, weight loss, diarrhoea, anaemia and free radical generation in response to N. brasiliensis infection are all T-cell dependent events.     

Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows

African swine fever is an important viral disease of wild and domestic pigs. To gain further knowledge of the properties of the currently circulating African swine fever virus (ASFV), experimental infections of young pigs (approximately 8 weeks of age) and pregnant sows (infected at about 100 days of gestation) with the genotype II ASFV Georgia/2007 were performed. The inoculated young pigs developed typical clinical signs of the disease and the infection was transmitted (usually within 3–4 days) to all of the “in contact” animals that shared the same pen. Furthermore, typical pathogical lesions for ASFV infection were found at necropsy. Inoculation of pregnant sows with the same virus also produced rapid onset of disease from post-infection day three; two of the three sows died suddenly on post-infection day five, while the third was euthanized on the same day for animal welfare reasons. Following necropsy, the presence of ASFV DNA was detected in tonsils, spleen and lymph nodes of some of the fetuses, but the levels of viral DNA were much lower than in these tissues from the sows. Thus, only limited transplacental transmission occurred during the course of this experiment. These studies contribute towards further understanding about the spread of this important viral disease in domestic pigs.     

Pathophysiological effect of hepatic ischemia and reperfusion after hepatectomy in dogs with obstructive jaundice,focusing on the effect of coenzyme Q10 and styrene-co-maleic acid superoxide dismutase

The purpose of the present study was to elucidate the effect of hepatic reflow following ischemia on the remnant liver after hepatectomy with occluded hepatic blood inflow in dogs with obstructive jaundice. When 40% hepatectomy was performed with 10-min occlusion of hepatic blood inflow in dogs with obstructive jaundice, the lipid peroxide content in the remnant liver increased significantly, together with a reduction in superoxide dismutase (SOD)-like activity. The levels of endotoxin and β-N-acetyl hexosaminase (NAH) in peripheral blood also increased. The phagocytic index increased transiently after 30 min, followed by a marked decrease after 3h. Histologically, degeneration and necrosis of the hepatic parenchymal cells were demonstrated, and survival rate at 7 days was only 23.1%. With the administration of coenzyme Q10 (CoQ10) or styrene-co-maleic acid SOD (SM-SOD), these phenomena were significantly inhibited, and the survival rate improved. After hepatectomy, Kupffer cells in the remnant liver were activated by increased endotoxin levels in the portal vein, inducing the production of free radicals, which, in turn, damaged the Kupffer cells by reducing endotoxin clearance. Finally, the impaired functional reserve in the remnant liver provoked liver failure. The administration of CoQ10 or SM-SOD prevented the occurrence of these phenomena triggered by the free radicals generated by Kupffer cells, stimulated by endotoxin in the portal vein. Abstracts of this study were presented at the 28th Annual Meeting of the Japan Society of Hepatology (Tokyo, 1992), at the 20th Annual Meeting of the Japanese Society for Vascular Surgery (Sapporo, 1992), and at the 41st Annual Meeting of the Japanese Society of Gastroenterological Surgery (Kobe, 1993).     

Postbinge Effects of Acute Alcohol Intoxication on Hepatic Free Radical Formation

The present studies were performed to test the hypothesis that Kupffer and endothelial cells are activated after recovery from an acute alcohol binge, which is accompanied by formation of oxygen-derived radicals. These radicals have been implicated in the pathogenesis of alcohol-mediated tissue injury in a number of organs. Male Sprague-Dawley rats received an intravenous injection of 20% ethanol in saline (1.75 g/kg), followed by an intravenous infusion (250 to 300 mg/kg/hr) for 12 hr. At the end of 12-hr infusion, ethanol was replaced by saline, and the infusion was continued for a further 6 hr. This was referred to as the recovery period. The 6-hr recovery period was selected because superoxide anion generation by the perfused liver peaked at this time point. Superoxide anion formation by the perfused liver was measured by the superoxide dismutase-inhibit-able reduction of ferricytochrome c. Kupffer and endothelial cells were isolated for the determination of in vivo glucose uptake and in vitro superoxide anion release. Results show that a significant ( p < 0.05) amount of superoxide (1.54 nmol/min/g) was generated by the perfused liver at 6 hr recovery after 12 hr of ethanol infusion. Serum ALT activity was also elevated in this treatment group. Time-matched control-saline infused animals or ethanol-treated animals without a recovery period released <0.2 nmol/min/g of superoxide. The postrecovery superoxide production and an accompanying increase in the in vivo glucose uptake were also observed in isolated Kupffer and endothelial cells. Depletion of Kupffer cells by gadolinium chloride before ethanol treatment and recovery was associated with significant attenuation of free radical formation by the perfused liver and reduction of serum ALT. These studies demonstrate that recovery from an acute alcohol binge has a stimulating effect on hepatic sinusoidal superoxide production, and it may also affect liver function.     

Corynebacterium parvum-elicited hepatic macrophages demonstrate enhanced respiratory burst activity compared with resident Kupffer cells in the rat

We have recently demonstrated that release of oxygen-derived free radicals by activated hepatic macrophages may be involved in the pathogenesis of a rat model of liver injury induced by Corynebacterium parvum and endotoxin. In the present study we have compared the respiratory burst activity of isolated normal rat Kupffer cells with that of hepatic macrophages elicited by C. parvum. Superoxide production (O2-.) and glucose oxidation via the hexose monophosphate shunt (HMPS) were low in normal Kupffer cells, but were significantly increased (O2-. 2.1-fold, HMPS 1.7-fold) by phorbol myristate acetate, a stimulant of the respiratory burst. Corynebacterium parvum-elicited hepatic macrophages demonstrated significantly enhanced superoxide production and HMPS activity compared with normal Kupffer cells, both in the absence of specific stimuli (O2-. 3.3-fold, HMPS 5.3-fold) and after exposure to phorbol myristate acetate (O2-. 4.5-fold, HMPS 5.3-fold). These results demonstrate that normal Kupffer cells are capable of exhibiting respiratory burst activity, but this is markedly increased for hepatic macrophages elicited by an inflammatory stimulus.     

Modulation of ischemia-reperfusion-induced hepatic injury by kupffer cells

To elucidate the role of Kupffer cells in ischemia-reperfusion-induced hepatic injury, hepatic injury induced by ischemia-reperfusion was analyzed after modulation of Kupffer cell function. Ischemia of the liver was performed by occlusion of both the portal vein and hepatic artery, which enter into the left lateral and median lobes of the liver. Blood flow in the ischemic lobe was reduced, in contrast to an increased blood flow in the nonischemic lobe during occlusion of the veins. Although hepatocyte damage was not demonstrated by ischemia for <60 min, hepatic injury was found after reperfusion of the liver, and activation of Kupffer cells was morphologically demonstrated by electron microscopies. Suppression of Kupffer cells, induced by previous administration of gadolinium chloride or latex particles, reduced the grade of hepatic injury induced by ischemia-reperfusion. On the other hand, stimulation of Kupffer cell phagocytosis, induced by administration of latex particles at the time of reperfusion, aggravated the ischemia-reperfusion-induced hepatotoxicity, which was then reduced by simultaneous administration of superoxide dismutase. Kupffer cells, isolated from the rats treated with the ischemia-reperfusion procedure, have been found to release increased amounts of oxygen radical intermediates. These results suggest that hepatic injury induced by ischemia-reperfusion is modulated by the function of Kupffer cells and that superoxide anion released from Kupffer cells cound play an important role in ischemia-reperfusion hepatic injury.     

Electron microscopic observations on the accumulation of large granular lymphocytes (pit cells) and Kupffer cells in the liver of rats treated with continuous infusion of interleukin-2

Treatment schedules were investigated for in vivo induction of lymphokine-activated killer cells in the rat liver. Treatment of rats with continuous systemic or regional infusion of recombinant human interleukin-2 with a dose of 4 to 8 x 10(4) U/day during 7 days, resulted in an increase in number of large granular lymphocytes or pit cells in the liver up to 43 times normal. Kupffer cells, nongranular lymphocytes, monocytes and neutrophils also increased in number, but with a maximal fivefold increase this was much less pronounced than for large granular lymphocytes. Kupffer cells showed morphological signs of activation and were frequently seen in mitosis. Frequent mitoses were also observed for large granular lymphocytes, but not for other leukocytes. This indicates that the effect of interleukin-2 treatment on hepatic (sinusoidal) cells was primarily directed to large granular lymphocytes and Kupffer cells. The large granular lymphocyte accumulation occurred mainly intrasinusoidally, but they were also frequently observed in the space of Disse where they are not found in control rats. This may be explained partly by the observed damage or gaps in the endothelial lining. The intrasinusoidal large granular lymphocytes adhered to the endothelium and to Kupffer cells. Higher responses, for all cell types, were found when interleukin-2 was administered regionally, that is, through the hepatic artery rather than through the systemic route (jugular vein), although the differences were not statistically significant. Doses below 4 x 10(4) U/day did not result in significant increases of large granular lymphocytes in the liver.     

T cell subset involvement in immune responses to Fasciola hepatica infection in cattle

The lymphocyte response to F. hepatica during a primary infection in cattle was analysed to define the role of T cell subsets in the immune response. Blood lymphocytes were isolated from eight cattle infected with F. hepatica via trickle infection over a ten-day period and from two non-infected controls. CD4+, CD8+ and gamma delta + T cells were depleted from whole lymphocyte populations by magnetic bead depletion. Lymphocytes from infected animals demonstrated a transient, but marked elevation in responsiveness to F. hepatica antigen between weeks 3 and 8 post-infection. Responses were attenuated by depletion of CD4+ and CD8+ T cells during this period. Depletion of gamma delta + T cells attenuated antigen responses at one time point only, and at an earlier stage post-infection than when alpha beta + T cells were depleted. Responses to antigen correlated positively with both hepatic fluke burden and with the degree of hepatic damage. This suggests that the cellular immune response was not protective. Antigen responses in gamma delta + T cell-depleted populations were also associated with post-mortem fluke burden and with hepatic damage. This suggests that gamma delta + T cells are involved in down regulating alpha beta + lymphocytes which may have a role in a non-protective or immunopathological immune responses.     

Superoxide produced by Kupffer cells is an essential effector in concanavalin A-induced hepatitis in mice

Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl(3)) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-gamma levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, alpha-galactosylceramide. Furthermore, GdCl(3) pretreatment changed neither the activation-induced down-regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68(+) Kupffer cells greatly increased in proportion in the early phase after Con-A injection; this increase was abrogated by GdCl(3) pretreatment. Anti-TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68(+) Kupffer cells and Con-A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con-A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con-A hepatitis. Conclusion: Superoxide produced by Kupffer cells may be the essential effector in Con-A hepatitis, and TNF and NKT cells support their activation and superoxide production.     

Kupffer cell exacerbation of hepatocyte hypoxia/reoxygenation injury.

In order to examine the possible contribution of the Kupffer cell to the generation of hypoxia/reoxygenation injury in the liver, primary cultures of hepatocytes, either alone or in coculture with Kupffer cells, were exposed to 90 min of sublethal hypoxia followed by 120 min of reoxygenation. Prolonged incubation of cocultured hepatocytes and Kupffer cells resulted in increased release of lactic dehydrogenase (LDH) indicating cell injury even under normoxic conditions. LDH release was further increased by the presence of Kupffer cells during hypoxia/reoxygenation. To determine whether or not this effect of Kupffer cells might be the result of oxygen-derived free radical production, we assessed the efficacy of the enzymatic scavengers superoxide dismutase (SOD) + catalase in ameliorating the Kupffer cell mediated injury. SOD + catalase was effective in preventing free radical injury generated by hypoxanthine + xanthine oxidase. However, SOD + catalase did not ameliorate hepatocyte injury caused by Kupffer cells. Thus, activation of Kupffer cells may be an important factor in the genesis of liver injury, but the mediator of Kupffer cell exacerbation of hepatocyte injury appears to be a mechanism other than free radicals released into the medium. These results indicate that chemical substances from the activated Kupffer cells may cause hepatocyte damage, which cannot be blocked by SOD + catalase, and suggest that these substances at reflow may be important for the genesis of reperfusion injury in vivo.     

Murid herpesvirus-4 induces chronic inflammation of intrahepatic bile ducts in mice deficient in gamma-interferon signalling

Aim:  Infection of gamma interferon receptor defective mice with murid herpesvirus-4 also known as murine gammaherpesvirus-68 results in multi-organ fibrosis. In this paper we characterise the pathological changes occurring in the liver in this model.
Methods:  Standard immunohistochemistry and in situ hybridisation techniques were used to identify the cellular changes and the presence of virus at different times post infection.
Results:  In liver sections from infected gamma interferon receptor defective mice sampled on day 16 to at least day 120, 79% showed proliferating intrahepatic bile ducts associated with a chronic mononuclear cell inflammation. Only 8% of wild type mice showed similar lesions. Coincident with the inflammatory response bile duct epithelial cells were positive for arginase 1. Around day 50 post infection onwards focal fibrotic lesions appeared in approximately 30% of gamma interferon receptor defective mice resulting in destruction of intrahepatic bile ducts. In contrast to the chronic persisting inflammatory response the presence of virus infected cells were only observed between day 12–20 post-infection.
Conclusion:  Infection of gamma interferon receptor defective mice with a murine gammaherpesvirus initiates a chronic persisting inflammatory response with a pathological profile similar to the human fibrotic liver disorder Primary Sclerosing Cholangitis.     

Inflammation and platelet-activating factor production during hepatic ischemia/reperfusion.

The role of platelet-activating factor as a potential mediator of hepatic inflammatory injury associated with liver ischemia/reperfusion was investigated using a partial no-flow model in rats in vivo. Platelet-activating factor levels of livers from sham-operated rats and from animals experiencing hepatic reperfusion for less than 6 hr were very low. They were observed to increase significantly after 12 hr of reperfusion and reached peak levels after a 24-hr reperfusion period, a time when maximal hepatic injury and inflammation occurred. Treatment of experimental rats with WEB2170, a platelet-activating factor receptor antagonist, attenuated the hepatic injury and inflammation, as evidenced by decreases in plasma ALT and in hepatocyte necrosis and neutrophil infiltration. Both inactivation of Kupffer cells with gadolinium chloride and inhibition of the formation of reactive oxygen species with allopurinol reduced platelet-activating factor production in the liver, whereas induction of neutropenia had no effect, suggesting that interaction of Kupffer cells with oxygen-derived free radicals may be a plausible mechanism for hepatic platelet-activating factor accumulation. It is concluded that platelet-activating factor contributes to the inflammatory consequences of ischemia/reperfusion underlying late-phase hepatic injury.     

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.     

Superoxide Anion Release Into the Hepatic Sinusoid After an Acute Ethanol Challenge and Its Attenuation by Kupffer Cell Depletion

Superoxide anion release into the hepatic sinusoids and subsequent damage to the endothelial cells of the hepatic sinusoids after ethanol challenge was examined. A 250 mg/kg body weight/hr dose of ethanol was given to rats for 3 hr, and superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome c method. Ethanol treatment resulted in superoxide anion release into the hepatic sinusoids (0.20 ± 0.01 vs. 0.12 ± 0.02 o.d., p < 0.05) and an increase in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate, a marker of damage to the endothelial cells of the hepatic sinusoids (0.003 ± 0.002 vs. 0.008 ± 0.002; p < 0.05). Tumor necrosis factor-alpha was not detectable in either group, and there were no significant differences in the population of hepatic macrophages, leukocytes, or Kupffer cells between the two groups. To clarify the role of Kupffer cells in the mechanism, 10 mg/kg of body weight of gadolinium chloride was given to rats twice, 24 hr apart, resulting in depletion of ED2-positive cells from the hepatic lobules. The superoxide anion release after the ethanol challenge was significantly attenuated in the Kupffer cell-depleted rats, compared with the controls (0.14 ± 0.02;p <0.05, compared with ethanol alone). The change was associated with a significant decrease in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate (0.004 ± 0.002; p < 0.05, compared with ethanol alone). Ethanol causes superoxide anion release into the hepatic sinusoid and subsequent damage to the sinusoidal endothelial cells. These changes were reduced by Kupffer cell depletion. This supports the view that Kupffer cell depletion has a protective effect on ethanol-induced liver injury.