Chronic infection with hepatitis B virus affects an estimated 300 million people on a global basis (24
). An estimated 1.25 million people in the United States are chronic carriers, defined as positive for HBsAg for more than 6 months (26
). Although most carriers do not develop hepatic complications from chronic hepatitis B, 15–40% will develop serious sequelae during their lifetime, which is why a greater understanding is needed of the pathogenesis of viral-induced hepatocellular injury. We chose to study chronic hepatitis B viral infection in the Asian-Pacific region, where chronic infection is especially prevalent and patients usually acquire the infection at the time of birth or early in childhood. Some of these patients, whose infection originated earlier in life, demonstrate marked chronic hepatitis in the third and fourth decades of life. This disease is characterized by recurrent flares of hepatocellular injury. Minimal chronic viral hepatitis B may reflect either clinical remission of the latter or a more benign disease. Biopsy is necessary to distinguish the two disorders and has a direct influence on therapeutic choices. Flares of marked chronic hepatitis are postulated to reflect T lymphocyte–mediated immune responses to the virus (27
). Indeed, T cell–mediated immune responses also play a prominent role in other forms of hepatocellular injury — autoimmune hepatitis, hepatotoxins, and concanavalin A hepatitis (28
). It is not clear what explains why patients present with marked chronic hepatitis versus minimal chronic viral hepatitis B or what mediates transition from one form to the other. Intrahepatic production of IFN-γ figures prominently in how viral hepatitis B–specific T cells mediate hepatocellular injury in marked chronic hepatitis (1
The current study demonstrates that expression of Fgl2/fibroleukin mRNA and protein varies markedly in patients with marked chronic hepatitis B versus those with minimal chronic viral hepatitis B. Expression of Fgl2/fibroleukin is highly correlated with fibrin expression. We have reported previously expression of Fgl2/fibroleukin in three patients with hepatitis B–mediated fulminant hepatic failure, a devastating but rare complication of hepatitis B (7
). Taken together with results reported here for marked chronic hepatitis, pharmacologic blockade of Fgl2/fibroleukin may offer an important new treatment approach in hepatitis viral B–induced disease. It is important to stress that these findings identify that an association exists between expression of Fgl2/fibroleukin, a prothrombinase, and the fibrin deposition that attends disease in marked chronic hepatitis B. Whether a cause and effect relationship exists between Fgl2/fibroleukin expression and fibrin deposition requires future studies, such as clinical trials in patients with a specific inhibitor of Fgl2/fibroleukin. Future studies will also need to address how established therapies for hepatitis viral B–induced disease modulate Fgl2/fibroleukin expression, such as the effects of IFN-α.
To begin to define a cause and effect relationship between the Fgl2/fibroleukin prothrombinase and hepatocellular injury in viral hepatitis we developed Fgl2/fibroleukin+/–
mice and examined the effect of Fgl2/fibroleukin genotype on in vitro and in vivo responses to MHV-3. Macrophages from Fgl2/fibroleukin+/+
mice responded to the MHV-3 virus with a robust procoagulant response. In contrast, macrophages from Fgl2/fibroleukin–/–
mice generated no increase in procoagulant above control levels. LPS induced a similar procoagulant response in both. LPS is known to induce TF/factor VII–dependent procoagulant activity in peritoneal macrophages. In the presence of calcium, TF/factor VIIa activates the conversion of the zymogen precursor factor X into factor Xa, the classical activator of thrombin generation. Using purified recombinant Fgl2/fibroleukin prothrombinase generated in a baculovirus system and reconstituted into phospholipid-containing vesicles and MHV-3–infected murine peritoneal macrophages, we recently assessed rates of thrombin generation using one-stage clotting assays, 125I-labeled prothrombin cleavage assays, and a chromogenic substrate of thrombin, namely H-D-hexahydrotyrosyl-L
-nitroanalide-diacetate. Like factor Xa, the Fgl2/fibroleukin prothrombinase is dependent on calcium, phospholipids, and factor Va for full activity (10
). Fgl2/fibroleukin prothrombinase differs from factor Xa in that activity on cell surfaces of MHV-3–infected peritoneal macrophages is resistant to extensive washing, consistent with the predicted transmembrane structure of the Fgl2/fibroleukin prothrombinase. Activity is not inhibited by antithrombin III and is not dependent on factor VII. The Fgl2/fibroleukin prothrombinase generates a unique thrombin cleavage fragment of approximately 24 kDa (10
). Neutralizing Ab’s directed against Fgl2/fibroleukin inhibit MHV-3–induced changes in the one-stage clotting assay (31
). Such Ab’s are specific in that they do not react with purified MHV-3– or LPS-induced procoagulants.
Mortality in MHV-3–infected Fgl2/fibroleukin–/–
mice was significantly reduced compared with Fgl2/fibroleukin+/+
littermates; therefore, infection of Fgl2/fibroleukin–/–
mice tended to recapitulate the resistant and susceptible phenotypes reported previously for C57Bl/6 and A/J inbred strains, respectively (16
). Also, targeted ablation of the Fgl2/fibroleukin gene and neutralizing Ab’s against the Fgl2/fibroleukin prothrombinase resulted in comparable effects on MHV-3 pathogenesis (23
). The response to LPS was not modified by Fgl2/fibroleukin genotype and contrasted with the effect in the LPS-hyporesponsive C3H/HeJ strain. When we assessed fibrin deposition in the liver and quantified hepatocellular necrosis, we observed effects of MHV-3 infection that were consistent with the effect of the Fgl2/fibroleukin genotype on overall mortality, namely, markedly decreased fibrin deposition and hepatocellular injury in Fgl2/fibroleukin–/–
mice. Previous studies have suggested the existence of multiple genetic modifiers for the MHV-3–induced fulminant hepatitis phenotype (19
), including the CEACAM1 MHV viral receptor glycoprotein (32
). MHV-3 viral loads in the livers did not vary with the Fgl2/fibroleukin genotype. This contrasts with the effects of targeted disruption of the CEACAM1 MHV viral receptor, which modifies hepatotrophic host-viral interactions and decreases viral loads in the livers of MHV-3–infected mice (33
). We did not detect an important effect of ablating the Fgl2/fibroleukin gene on MHV-3 viral loads in the liver. To the extent that MHV-3 viral hepatitis can be used to model viral hepatitis B in humans, these cumulative findings can be taken to indicate that hepatocellular injury induced by MHV-3 is critically dependent on the Fgl2/fibroleukin prothrombinase.
What are the other biological roles for Fgl2/fibroleukin? When generating the Fgl2/fibroleukin–deficient murine model, we identified loss of Fgl2/fibroleukin–/–
mice on both a mixed 129Sv/J × C57Bl/6 genetic background and C57Bl/6 inbred background. Coagulation and procoagulant factor deficiencies are known to result in early or “occult” pregnancy failure due to perturbations at the fetal-maternal interface and can be maternal (34
) or fetal in origin (36
). We have reported previously constitutive Fgl2/fibroleukin prothrombinase mRNA and protein expression on murine trophectoderm (17
). Future studies will be necessary to determine whether reconstitution of Fgl2/fibroleukin expression in extraembryonic tissues rescues Fgl2/fibroleukin–/–
mice from early lethality or whether loss of Fgl2/fibroleukin–/–
mice reflects other important functional contributions of Fgl2/fibroleukin during development. When studying adult Fgl2/fibroleukin–deficient mice, we failed to identify baseline alterations in standard hematological and coagulation profiles or bleeding times. Although adult Fgl2/fibroleukin–/–
mice were unremarkable compared with littermates with respect to bleeding episodes or longevity, we anticipate more detailed future analysis of the coagulation cascade.
This Fgl2/fibroleukin prothrombinase bypasses the TF/factor VII extrinsic pathway and directly converts prothrombin to thrombin. Stimuli known to induce expression of the Fgl2/fibroleukin mRNA and protein (e.g, IFN-γ treatment [see ref. 13
]), MHV-3 viral infection, xenotransplantation) are distinct from those that elicit TF/factor VII–dependent fibrin generation (e.g., LPS). Future studies will need to define the disease settings in which Fgl2/fibroleukin expression directs local fibrin generation. It is of interest that a coronavirus can induce tissue–specific expression of the Fgl2/fibroleukin prothrombinase. Using a set of parental and recombinant MHV-3 strains, we mapped the viral genetic determinants that induce fulminant hepatitis in susceptible murine strains (40
). These studies identified a critical role for the MHV-3 coronavirus nucleocapsid gene product in the transcriptional activation of the gene. Whether coronavirus infection in other species also induces Fgl2/fibroleukin prothrombinase expression remains to be determined. In this respect it will be important to define whether expression of the Fgl2/fibroleukin prothrombinase contributes to pulmonary parenchymal injury in human patients with severe acute respiratory syndrome (SARS), given that coronavirus infection contributes to this newly recognized illness (41
Our results provide compelling evidence for a role of the Fgl2/fibroleukin prothrombinase in viral hepatitis. Collectively, these data argue that the Fgl2/fibroleukin prothrombinase is a logical target for molecular manipulation and offers the hope for the development of new treatment approaches for patients with fulminant and marked chronic viral hepatitis.