FGL2/fibroleukin mediates hepatic reperfusion injury by induction of sinusoidal endothelial cell and hepatocyte apoptosis in mice.

BACKGROUND & AIMS: Sinusoidal endothelial cell (SEC) and hepatocyte death are early, TNF-α mediated events in ischemia and reperfusion of the liver (I/Rp). We previously reported that TNF-α induced liver injury is dependent on Fibrinogen like protein 2 (FGL2/Fibroleukin) and showed that FGL2 binding to its receptor, FcγRIIB, results in lymphocyte apoptosis. In this study we examine whether I/Rp is induced by specific binding of FGL2 to FcγRIIB expressed on SEC. METHODS: Hepatic ischemia and reperfusion was induced in wild type (WT) mice and in mice with deletion or inhibition of FGL2 and FcRIIB. Liver injury was determined by AST release, necrosis and animal death. Apoptosis was evaluated with caspase 3 and TUNEL staining. RESULTS: FGL2 deletion or inhibition resulted in decreased liver injury as determined by a marked reduction in both levels of AST and ALT and hepatocyte necrosis. Caspase 3 staining of SEC (12% vs. 75%) and hepatocytes (12% vs. 45%) as well as TUNEL staining of SEC (13% vs. 60%, p=0.02) and hepatocytes (18% vs. 70%, p=0.03), markers of apoptosis, were lower in Fgl2(-/-) compared to WT mice. In vitro incubation of SEC with FGL2 induced apoptosis of SEC from WT mice, but not FcγRIIB(-/-) mice. Deletion of FcγRIIB fully protected mice against SEC and hepatocyte death in vivo. Survival of mice deficient in either Fgl2(-/-) (80%) or FcγRIIB(-/-) (100%) was markedly increased compared to WT mice (10%) which were subjected to 75min of total hepatic ischemia (p=0.001). CONCLUSIONS: FGL2 binding to the FcγRIIB receptor expressed on SEC is a critical event in the initiation of the hepatic reperfusion injury cascade through induction of SEC and hepatocyte death.

Genetic determinants of mouse hepatitis virus strain 1 pneumovirulence.

We report here investigation into the genetic basis of mouse hepatitis virus strain 1 (MHV-1) pneumovirulence. Sequencing of the 3' one-third of the MHV-1 genome demonstrated that the genetic organization of MHV-1 was similar to that of other strains of MHV. The hemagglutinin esterase (HE) protein was truncated, and reverse transcription-PCR (RT-PCR) studies confirmed previous work that suggested that the MHV-1 HE is a pseudogene. Targeted recombination was used to select chimeric viruses containing either the MHV-1 S gene or genes encoding all of the MHV-1 structural proteins, on an MHV-A59 background. Challenge studies in mice demonstrated that expression of the MHV-1 S gene within the MHV-A59 background (rA59/S(MHV-1)) increased the pneumovirulence of MHV-A59, and mice infected with this recombinant virus developed pulmonary lesions that were similar to those observed with MHV-1, although rA59/S(MHV-1) was significantly less virulent. Chimeras containing all of the MHV-1 structural genes on an MHV-A59 background were able to reproduce the severe acute respiratory syndrome (SARS)-like pathology observed with MHV-1 and reproducibly increased pneumovirulence relative to rA59/S(MHV-1), but were still much less virulent than MHV-1. These data suggest that important determinants of pneumopathogenicity are contained within the 3' one-third of the MHV-1 genome, but additional important virulence factors must be encoded in the genome upstream of the S gene. The severity of the pulmonary lesions observed correlates better with elevated levels of inflammatory cytokines than with viral replication in the lungs, suggesting that pulmonary disease has an important immunological component.

The novel immunoregulatory molecule FGL2: a potential biomarker for severity of chronic hepatitis C virus infection.

BACKGROUND & AIMS: This report describes the use of a novel sensitive and specific ELISA for the measurement of human fibrinogen-like protein 2 (FGL2/fibroleukin), a novel effector of natural regulatory T (Treg) cells, to predict the course of chronic hepatitis C viral infection (HCV). METHODS: Plasma levels of FGL2 were measured in HCV patients and compared to healthy controls and to patients with alcoholic liver disease. RESULTS: FGL2 levels were significantly higher in HCV patients (84.3+/-89.1 ng/ml, n=80) compared to healthy controls (36.4+/-21.9 ng/ml, n=30, p<0.001), to a subset of patients who cleared HCV following anti-viral treatment (16.6+/-19.7 ng/ml, n=32, p<0.001), and to patients with inactive alcoholic liver disease (18.8+/-17.4 ng/ml, n=24, p<0.001). Among HCV patients, plasma levels of FGL2 correlated significantly with the stage of fibrosis (p=0.001) and were significantly higher in patients with cirrhosis (164.1+121.8 ng/ml, n=60) compared to non-cirrhotics (57.7+/-52.8 ng/ml, n=20, p=0.001). Genotype 1 patients had significantly higher levels of FGL2 (98.1+/-100.3 ng/ml, n=60) compared to patients with genotype 2/3 (41.5+/-38.6 ng/ml, n=20, p=0.0008). Patients with genotype 2/3 had FGL2 levels similar to healthy controls (41.5+/-38.6 vs. 36.41+/-21.9 ng/ml, p=ns). Infiltrating lymphocytes in liver biopsies of HCV patients were positive for either FGL2 or FoxP3 (a marker of Treg cells) or expressed both markers. CONCLUSIONS: This report documents the development of a sensitive ELISA for measurement of plasma levels of FGL2 an effector Treg cells, which correlates with the severity of HCV infection.

The FGL2-FcgammaRIIB pathway: a novel mechanism leading to immunosuppression.

Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcgammaR)IIB and FcgammaRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcgammaRIIB and FcgammaRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcgammaRIIB+/+ mice but not from FcgammaRIIB(-/-) mice and induced apoptosis in the FcgammaRIIB+ mouse B-cell line (A20) but not the A20IIA1.6 cell line that does not express FcgammaRIIB. Recombinant FGL2 infused into FcgammaRIIB+/+ (C57BL/6J, H-2b) mice but not FcgammaRIIB(-/-) mice inhibited rejection of fully mismatched BALB/cJ (H-2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune-mediated disease and suggests a potential for targeted FGL2 therapy.