Intrinsic resistance of hepatocytes to complement-mediated injury

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.     

Mechanisms of viral hepatitis induced liver injury

Among seven human hepatitis viruses (A to E, G and TT virus), hepatitis B (HBV) and C (HCV) viruses are able to persist in the host for years and principally contribute to the establishment of chronic hepatitis. During the course of persistent infection, continuous intrahepatic inflammation maintains a cycle of liver cell destruction and regeneration that often terminates in hepatocellular carcinoma (HCC). While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatistis are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. In the process of liver injury, hepatocellular death (apoptosis) induced by the proapoptotic molecules of T cells activated following antigen recognition triggers a cascade of antigen nonspecific effector systems and causes necroinflammatory disease. Accordingly, the regulation of the immune response, e.g., via the cell death pathways, in chronically infected patients should prevent the development of HCC.     

An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells

The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER^T2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K^b/OVA257-264-specific OT-I T cells to OVA_X_CreER^T2 mice or generated triple transgenic OVA_X CreER^T2_X_OT-I mice.OT-I T cells become activated in OVA_X_CreER^T2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER^T2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER^T2_X_OT-I mice indicating absence of tolerogenic mechanisms.This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.     

Modulation of granulomatous hypersensitivity. VI. T lymphocyte subsets influence mast cell density in liver granulomas of Schistosoma mansoni-infected mice

In murine schistosomiasis mansoni, a complex series of cell-cell interactions involving T cell subclasses regulates the intensity of the inflammatory granulomatous response. Recent evidence suggests that granuloma mast cells also participate in the regulatory process by the release of histamine. The current study was performed to determine factors that affect the number of granuloma mast cells. More mast cells were detected in liver granulomas from chronically (20-wk) as opposed to acutely (8-wk) infected mice. Adoptive transfer of spleen cells from 20-wk-infected donors into acutely (6-wk) infected recipients increased granuloma mast cell density. Treatment of spleen cells with anti-Thy-1.2 or anti-Lyt-1.1 antiserum and complement, but not anti-Lyt-2.1 or normal mouse serum, abrogated adoptive transfer-induced, augmentation of granuloma mast cell density. Treatment of acutely infected animals with cyclophosphamide or cimetidine (H2 antagonist enhanced granuloma mast cell density. These data suggest that granuloma mast cell density is dependent upon subsets of T lymphocytes.     

Enhanced expression of Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein induces resistance to Fas-mediated apoptosis in activated mast cells

Mast cells play a critical role in host immune responses and are implicated in the pathogenesis of allergic inflammation. Though mouse mast cell line MC/9 expresses cell surface Fas Ag and is sensitive to Fas-induced apoptosis, activated MC/9 cells are resistant to Fas-induced cell death by cross-linking of FcepsilonRI or FcgammaR. Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP), a caspase-8 inhibitor that lacks the cysteine domain, is one of the negative regulators of receptor-mediated apoptosis. In this report, we show that activation of mast cells by cross-linking of FcepsilonRI or FcgammaR can induce enhanced expression of FLIP and consequently a resistance to Fas-induced apoptosis, although the expression level of Fas Ag is not changed. Addition of antisense oligonucleotide for FLIP prevents resistance to Fas-induced apoptosis of activated mast cells, suggesting that endogenous FLIP inhibits Fas-mediated apoptosis in activated mast cells. Thus, the enhanced expression of FLIP in activated mast cells contributes to the resistance to Fas-induced apoptosis, which may result in the development and prolongation of allergic inflammation.     

Immunofluorescence studies indicate that the basic trypsin-kallikrein-inhibitor of bovine organs (Trasylol) originates from mast cells.

Using the indirect immunofluorescence technique, the basic kallikrein-trypsin inhibitor of bovine organs, Trasylol, could be localized in tissue mast cells of bovine lung, liver, pancreas and parotid gland. Identification of cells exhibiting specific fluorescence as tissue mast cells was achieved by combined light and electron microscopic diagnosis of bovine liver tissue sections. The presence of Trasylol in mast cells explains the widespread distribution of this inhibitor in functionally totally different organs or tissues of the bovine organism, as determined earlier by biochemical means. Identification of Trasylol as a mast cell constituent will facilitate the search for the biological function of this inhibitory protein in connection with a unique and highly specialized cell population.     

A role for CD21/CD35 and CD19 in responses to acute septic peritonitis: a potential mechanism for mast cell activation

Although it is now appreciated that mast cell-mediated release of TNF-alpha is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2(null)) were examined in the present study. Along with CD19-deficient (CD19(null)) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2(null) mice and only partially improves survival of CD19(null) mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-alpha release is also impaired in Cr2(null) and CD19(null) animals, suggesting that these molecules directly affect mast cell activation. Cr2(null) and CD19(null) mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.     

Cytokine-induced inflammatory liver injuries

IL-18 is a pleiotropic cytokine and is produced by various types of cells including activated macrophages, particularly Kupffer cells. IL-18 has potential to activate inflammatory responses through induction of IFN-gamma production in collaboration with IL-12. Somewhat paradoxically, IL-18 also has the capacity to induce allergic responses via induction of IL-4 production by T helper cells and to activate mast cells and basophils to release atopic effector molecules such as histamine. Indeed, IL-18 is involved in inflammatory tissue injuries, such as Crohn's disease and atherosclerosis, and also in hyper IgE and atopic dermatitis. IL-18 is particularly important for induction of experimental liver diseases. Endotoxin-induced liver injury or Fas ligand-induced hepatitis is caused by endogenous IL-18 in mice. Moreover, patients with liver diseases such as fulminant hepatitis, liver cirrhosis due to hepatitis virus infection and primary biliary cirrhosis show elevation of serum levels of IL-18, that correlates with the corresponding disease severity. Therefore, endogenous IL-18 plays a major role in induction of some types of liver injuries in mice and human. NKT cells that express both T cell receptor and NK cell marker are abundant in the liver of mice and human. Recent studies have revealed that NKT cells participate in some types of liver injuries, such as concanavalin A-induced T cell-mediated hepatitis and malaria hepatitis. In this review article, we focus on IL-18-involving liver damages and NKT-cell-mediated liver injuries.     

A mapping study of caspase-3 activation following acute spinal cord contusion in rats.

Spinal cord injury (SCI) initiates a cascade of biochemical changes that results in necrotic and apoptotic cell death. There is evidence that caspase-3 activation and apoptotic cell death occur within hours after SCI. However, the time course and cellular localization of activated caspase-3 has not been examined. Such information is essential because caspase-3-independent apoptotic pathways do exist. In this experiment, we describe the distribution of and cell types containing activated caspase-3 at 4 hr, 1 day, 2 days, 4 days, and 8 days following SCI in rats. Numerous caspase-3-positive cells were observed at 4 hr and 1 day postinjury and colocalized most often with CC1, a marker for oligodendroglia. Both markers disappeared near the injury epicenter over the next several days. Activated caspase-3 was again present in the injured spinal cord on postoperative day 8, which coincided with a reemergence of CC1-positive cells. Many of these CC1-positive cells again colocalized activated caspase-3. NeuN-positive neurons of the dorsal horn were occasionally immunopositive for activated caspase-3 at early time points. OX42-positive microglia/macrophages rarely contained activated caspase-3. The results indicate a biphasic pattern of caspase-3 activation during the first 8 days postinjury, suggesting that at least two mechanisms activate caspase-3 following SCI. This time-course study provides a framework for investigating and understanding the different signaling events contributing to this biphasic pattern of caspase-3 activation.     

The appearance and degradation of specific hepatocellular cytoplasmic inclusion bodies in rat liver due to D-galactosamine. I. The relation between the amount of liver glycogen and the appearance of the atypical dense bodies in the liver cell.

One of the most sensitive and specific signs of the galactosamine effect upon the rat liver cell is the appearance of PAS-positive and diastase-resistant granules within the cytoplasm of hepatocytes. Light-microscopic, histochemical, biochemical, and electron-microscopic findings reveal that the appearance of these ADB (= atypical dense bodies) depends upon a working glycogen metabolism at the time of GalN treatment. The ADB are composed of particles resembling, due to shape and size, ribosomes and beta particles of glycogen. Most of them are surrounded by the rER, but they are never enclosed by a limiting membrane. Due to sequential changes they can be generally classified into three types; the early, the intermediate, and the late type. In seven experiments it can be shown, that the appearance of the ADB depends upon the time and dosage after GalN treatment. They occur even if an additional treatment with galactose or uridine prevents the liver from the features of a hepatitis, as also shown in the livers of newborn animals up to 3 weeks of age. The histochemical response against various glucosidases, hexosaminidases, pronase, and RNAse as well as against various fixatives indicates that ADB are composed of, at least, two different constituents, the former RNAse-sensitive and visible with routine light-microscopic staining procedures, the latter RNA-resistant, PAS-positive, and invisible after staining with H & E or toluidine blue. The latter is diastase-resistant, suggesting that this portion of ADB does not represent the usual glycoproteins but some abnormal metabolite of glycogen. The ADB can be detected with maximal accumulation in the cytoplasm of hepatocytes at that time when the glycogen content determined in the liver homogenate by biochemical methods is greatly reduced.     

Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death.

Programmed cell death (pcd) is activated during the hypersensitive response (HR) of plants to avirulent pathogens. We have recently shown that, similar to pcd in animal cells, nuclei of cells undergoing HR cell death contain fragmented nuclear DNA (nDNA). Here, we report that cell death occurring during the HR is accompanied by an increase in the activity of several deoxyribonucleases. Induction of nuclease activities was coordinated with cell death and may account for the degradation of nDNA during the HR. HR-associated nuclease activities were not induced during senescence, following necrotic cell death resulting from abiotic stress, or in response to induction of plant defense mechanisms by salicylic acid. HR-associated nuclease activities were stimulated by Ca2+ and inhibited by EGTA, EDTA, and Zn2+. At least one of the HR-associated nuclease activities was detected in nuclei purified from leaves undergoing pcd. A nuclease with an electrophoretic mobility similar to that of the nuclease activity found in nuclei isolated from leaves undergoing HR cell death was purified. Our findings are in accordance with some of the biochemical events that occur during pcd in animal cells. However, further analysis of the pattern of nDNA fragmentation and the corresponding structural changes that occur in the nuclei of tobacco cells undergoing HR cell death revealed that these features may have differences from those that take place during apoptosis in animal cells.     

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.     

STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury

Interferon-gamma (IFN-gamma) has been implicated in liver damage in animal models and chronic hepatitis C infection; however, the underlying mechanism is not clear. Here we examined the role of STAT1, a key signaling molecule for IFN-gamma, in a model of murine hepatitis induced by the injection of LPS/D-galactosamine and in human hepatoma Hep3B cells. STAT1 is rapidly activated and highly induced after injection of LPS/D-galactosamine. Both overexpression of STAT1 and hepatocellular damage are located in the same pericentral region. Disruption of the STAT1 gene abolishes LPS/D-galactosamine-induced liver injury. Studies from IFN-gamma-deficient mice indicate that IFN-gamma is the major cytokine responsible for activation and hyperexpression of STAT1 in LPS/D-galactosamine-induced hepatitis. Hep3B cells overexpressing dominant negative STAT1 are resistant to IFN-gamma and IFN-gamma + TNF-alpha-induced cell death, whereas Hep3B cells overexpressing wild-type STAT1 are more susceptible to cell death. Taken together, these findings suggest that STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury.     

Antigen-specific primary activation of CD8+ T cells within the liver

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8(+) T cells specific for H-2K(b) were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-K(b) mice expressing H-2K(b) in the liver. Activated CD8(+) cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8(+) cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8(+) T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8(+) T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.     

Lábrea hepatitis in Salvador, Bahia? (Presentation of a possible case)

A case of a four-year-old boy from Salvador, Bahia, Brazil, with a probable Lábrea hepatitis is reported. He ran a rapid course of fever, jaundice, abdominal pain, agitation, coma and death. Histologically, the liver presented widespread hepatic cell lytic and coagulative necrosis, acute fatty changes, mononuclear cell infiltration and cholestasis. The study of the case raises the question whether Lábrea hepatitis can occur outside the Amazonian region, or else its clinico-pathological features lack specificity.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

Metabolic response of macrophages to injury promoted by the activated complement system

In nucleated cells, the swelling promoted by a complement system (CS) attack is not enough to promote cell death, because unlike erythrocytes these cells are able to eliminate cytolytic complement channels from the plasma membrane, by processes that include endocytosis. Several studies have demonstrated that the resistance of nucleated cells to the injury promoted by the CS is related to the cellular metabolism. Despite this, to the present day, no study has clearly related cell survival capacity to injury by the CS to its energetic metabolic status. In macrophages, the challenge imposed by the CS provoked an increase in the total amount of glucose incorporated into fatty acids, including phospholipids and cholesterol; substrates for membrane synthesis. The inhibition of cholesterol synthesis promoted an increase of the cell death rate. These data support the importance of cholesterol metabolism for macrophage resistance to necrosis induced by the activated complement system. Copyright © 1999 John Wiley & Sons, Ltd.     

Mast cells chemotax to laminin with enhancement after IgE-mediated activation

The increase of mast cells at sites of tissue inflammation suggests the production of local factors chemotactic for mast cells. In this report, we demonstrate that the murine mast cell line PT18 and primary mouse bone marrow-derived mast cells chemotax to the basement membrane glycoprotein laminin, and that the synthetic laminin A chain-derived peptide, PA22-2, represents a region of laminin that contains a major chemoattractant site. Mast cell chemotaxis to laminin is enhanced after activation of mast cells by the calcium ionophore, A23187, or PMA and by sensitization of the cells with IgE followed by exposure to antigen. Chemotaxis is not increased in the presence of IL-3 and is independent of mast cell degranulation, as histamine release did not occur when cells were activated with PMA. Mast cell chemotaxis to laminin and its enhancement by IgE-dependent mast cell activation provides a mechanism by which these cells may be attracted to sites of tissue injury. Such activity may be particularly relevant in the response of host tissues to inflammation accompanying parasitic infestations, allergic reactions, and wound healing.     

Essential role of the adhesion receptor LFA-1 for T cell-dependent fulminant hepatitis

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1(-/-) (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1(+/+) mice following ConA injection. By contrast, LFA-1(-/-) mice were completely resistant to ConA-induced hepatitis and none of the LFA-1(-/-) mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1(+/+) mice, activated T cells were rapidly cleared from the livers of LFA-1(-/-) mice. Mechanistically, T cells from LFA-1(-/-) mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1(+/+) mice, but not from LFA-1(-/-) mice, sensitized LFA-1(-/-) mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.     

Influence of D-galactosamine upon the NAD-metabolism in rat liver

After application of D-galactosamine a hepatitis develops in the rat liver. This can be prevented by different agents, including tryptophan. Yet it has not been possible to give definitive conclusions about the mechanism of galactosamine hepatitis. In this paper we report about the influence of galactosamine on the NAD metabolism. D-galactosamine inhibits the NAD synthesis initiated by nicotinamide in normal and adrenalectomized animals. The NAD synthesis from tryptophan is prevented in normal animals, in adrenalectomized ones however there is an increase of NAD in the presence of D-galactosamine reduces the activity of the ADPR transferase. Inhibitors of the ADPR transferase prevent the galactosamine hepatitis. From the results presented we conclude that the ADPR transferase plays an important role in the development of the galactosamine hepatitis.