Laisum Fung

The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis

Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin-deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin-/- mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.

Acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type 3

The acute and chronic effects of mouse hepatitis virus type 3 on the microcirculation of the liver in both semisusceptible C3HeB/FeJ and fully resistant A/J mice were studied. In the C3HeB/FeJ mice, abnormalities of microcirculatory flow were noted as early as 12 hr after infection and by 24 hr, localized avascular foci appeared. Disturbances were characterized by granular blood flow, sinusoidal microthrombi, distortion of sinusoids by edematous hepatocytes and necrotic lesions. Following the acute infection, Day 10, two patterns of chronic disease were observed. Eighty percent of the mice developed chronic granulomatous hepatitis whereas in the remaining 20% a more severe chronic aggressive hepatitis was observed which was characterized by ongoing hepatocellular necrosis and a marked mononuclear cell infiltrate. In both cases, in vivo microcirculatory abnormalities were found predominantly around visible lesions. Onset of the microcirculatory abnormalities was found to be concomitant with a rise in monocyte related procoagulant activity. Procoagulant activity rose acutely and remained elevated throughout the chronic phase but was higher in animals with severe disease. In contrast to the above, normal blood flow and histology were seen in the resistant A/J mice at all times following infection, and procoagulant activity remained at basal levels despite active viral replication as demonstrated by immunofluorescence studies and recovery of infectious virus. These observations suggest a role for monocyte procoagulant activity in the development of microcirculatory abnormalities following mouse hepatitis virus type 3 infection which may be important in the pathogenesis of the disease.

Expression of the fgl2 and Its Protein Product (Prothrombinase) in Tissues During Murine Hepatitis Virus Strain-3 (MHV-3) Infection

Murine Hepatitis Virus Strain 3 (MHV-3) produces fulminant hepatitis with 80-90% mortality in Balb/cJ mice. Previous studies in our laboratory have shown that peritoneal macrophages from MHV-3 infected mice produce a procoagulant (PCA) which has the ability to cleave prothrombin to thrombin (prothrombinase) encoded by the gene fgl2 located on chromosome 5. PCA accounts for sinusoidal thrombosis and hepatic necrosis and the necrosis and mortality can be prevented by treatment of animals with a monoclonal antibody to PCA. These present studies were designed to examine the expression of this gene (mRNA by Northern analysis and in situ hybridization) and the gene product PCA (immunochemistry) in tissues recovered from MHV-3 infected Balb/cJ mice in an attempt to explain the liver specific nature of MHV-3 disease. Fgl2 gene expression was detected as early as 8 hours after MHV-3 infection which persisted to 48 hours in the liver, spleen and lungs whereas no gene expression was seen in the brain or kidneys despite the fact that equivalent viral titers were detected in all tissues at all times. In the liver, fgl2 gene expression was confined to endothelial and Kupffer cells with no expression in hepatocytes. Immunochemistry localized the PCA protein to Kupffer cells and endothelial cells and necrotic foci within the liver. No PCA protein was detected by immunochemistry in any other tissues at any time during the course of MHV-3 infection. These results explain the liver specific nature (fulminant hepatitis) of MHV-3 infection and provides further evidence for the role of PCA in the pathogenesis of fulminant hepatitis. MHV-3 induces selective transcription of the gene fgl2 and only hepatic reticuloendothelial cells produce functional protein (PCA) which is known to account for fulminant hepatic failure produced by MHV-3.

Mechanism of Protective Effect of Prostaglandin E in Murine Hepatitis Virus Strain 3 Infection: Effects on Macrophage Production of Tumour Necrosis Factor, Procoagulant Activity and Leukotriene B4

Murine hepatitis virus strain 3 (MHV-3) produces a strain dependent spectrum of liver disease. Mice of the A strain are fully resistant whereas Balb/cJ mice die of fulminant hepatic failure (1) . The susceptibility of inbred mice to MHV-3, is dependent on host factors which are under strict genetic control. Differences in viral replication both invivo and in-vitro do not appear to account for strain-dependent differences in resistance to MHV-3 and it has been suggested that variation in susceptibility/resistance of inbred mice reflects defects in the host–s immune response (2). Experimental ablation of the immune cell populations by X irradiation (3) , antilymphocyte serum (4) , or infection with frog virus-3 (5) renders resistant A/J mice susceptible. Furthermore, reconstitution of susceptible neonatal A/J mice with adult immune cells requires both T lymphocytes and an adherent cell population (6) . More recently, it has been demonstrated that clearance of virus from the central nervous system is dependent upon the presence of both CD4+ and CD8+ cells that recognize viral antigens in the context of H-2D gene products (7).

Effect of eicosanoids on induction of procoagulant activity by murine hepatitis virus strain 3 in vitro.

The development of hepatitis secondary to murine hepatitis virus strain 3 (MHV-3) infection correlates with the induction of macrophage procoagulant activity (PCA). 16,16 dimethyl prostaglandin E2 (dmPGE2) has previously been shown to inhibit the development of disease in this model and in parallel, inhibit induction of PCA, a macrophage effector molecule which has previously been shown to correlate with resistance/susceptibility to MHV-3 infection. These studies were undertaken to determine if inhibition of PCA was a specific property of dmPGE2 or if this property was shared by other eicosanoids including prostacyclin (PGI2), PGF2a and leukotriene B4 (LTB4). Furthermore, using the recently developed anti-PCA monoclonal antibody 3D4.3 (IgG2ak) which reacted with and inhibited functional PCA, studies were then undertaken to determine the mechanism by which PCA was inhibited by dmPGE2 (transcriptional, post-transcriptional or post-translational). Treatment with dmPGE2 resulted in inhibition of PCA induction compared with vehicle control over a range of 10(-12) to 10(-6) M. Utilizing the monoclonal antibody 3D4.3, it was demonstrated by Western immunoblot and immunofluorescence studies that although PCA was functionally inhibited by dmPGE2, it was still antigenically expressed as proteins of molecular weights 74 and 70 kd. Treatment of macrophages with PGI2, PGF2a or LTB4 failed to inhibit or augment PCA induction to MHV-3 stimulation at all concentrations tested (10(-12) to 10(-6) M). These results suggest that inhibition of PCA by dmPGE2 is a specific property of this eicosanoid and that its actions occur at a post-translational level.

A sensitive radioimmunoassay for the determination of antibodies to mouse hepatitis virus.

Abstract A solid-phase radioimmunoassay is described for the detection of antibodies to mouse hepatitis virus. Viruses were purified by velocity and isopycnic gradient centrifugation and 96-well plastic plates were coated with viral antigens. To allow the detection of most serotypes of low titered antisera, a pool of antigens from several viral serotypes were employed. The second antibody, an affinity-purified goat antimouse immunoglobulin, detects IgG, IgM and IgA antibodies. This assay is more sensitive than either the plaque reduction assay or the commercially available enzyme-linked immunosorbant assay and proved to be useful for screening mouse colonies for the presence of mouse hepatitis virus, following seroconversion in experimental animals and in the production of monoclonal antibodies to both structural and nonstructural proteins.

MHV-3 Induced Prothrombinase is Encoded by MUSFIBLP

Previously, we demonstrated induction of a unique macrophage prothrombinase, PCA, in MHV-3 infected BALB/cJ mice. By immunologic screening, a clone representing PCA was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp, but when infected with MHV-3, synthesized musfiblp-specific mRNA. Musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrate when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.

The Effects of Mouse Hepatitis Virus Type 3 on the Microcirculation of the Liver in Inbred Strains of Mice

Mouse Hepatitis Virus Type 3 (MHV-3) infection results in strain dependant liver disease. The acute effects of MHV-3 on the in vivo microcirculation of the liver in fully susceptible (Balb/cJ), fully resistant (A/J) mice and the chronic effects on C3H mice were studied. In Balb/cJ mice by 6 to 12 hours following infection, granular flow and sinusoidal microthrombi were present predominantly in periportal areas. By 24 to 48 hours, liver cell edema and small focal necrotic lesions were prominent. After 48 hours, thrombi and hepatocellular necrosis were widespread. The animals succumbed to the infection within 5 days.

Role of Macrophage Procoagulant Activity in Mouse Hepatitis Virus (MHV) Infection: Studies Using T Cell MHV-3 Clones and Monoclonal Antibody 3D4.3

Murine hepatitis virus strain 3 (MHV) infection produces a strain dependent spectrum of disease1,2. Mice of the A and SJL strain are fully resistant to the effects of viral infection, whereas mice of semisusceptible strains (C3H/HeJ) develop acute hepatitis which progesses to varying degrees of chronic hepatitis. Mice of fully susceptible strains (Balb/cJ, C57BL/6J) die of fulminant hepatic failure. The resistance of the A strain mice cannot be explained by a lack of a cellular receptor for MHV as viral binding occurs on cells from these resistant mice3,4. Furthermore, restriction of viral replication does not explain resistance, as resistance occurs despite the presence of active viral replication in the resistant A strain. Bang and Warwick previously reported that differences in viral replication in cultures of macrophages reflects the relative susceptibility/resistance (S/R) to viral infections5. However, several laboratories have shown that MHV replicates in cultures of macrophages, endothelial cells and hepatocytes from both susceptible and resistant animals4,6–8although viral replication occurs to a lesser degree in cells derived from resistant animals6,9. Thus, absolute differences in viral replication do not account for the strain dependent S/R pattern seen in MHV infection.