A murine model of acute liver injury induced by human monoclonal autoantibody.

We have previously reported an immunoglobulin (Ig) M autoantibody to hepatocyte-related 190-kd molecules in patients with type 1 autoimmune hepatitis (AIH). This molecule was first isolated by hepatocyte-specific human monoclonal antibody (MoAb). To elucidate the role of this IgM autoantibody in hepatocyte injury, we examined the reactivity of this MoAb to murine hepatocytes and then questioned whether acute hepatic injury could be induced in mice via injection of this MoAb. The reactivity of MoAb was examined via both FACS analysis using murine hepatocytes and immunostaining of liver tissues. We then identified the murine hepatocyte membrane molecule recognized by this MoAb. The role of this MoAb in the immunopathogenesis of AIH was assessed by testing whether its injection into mice could increase serum aminotransferase levels as well as cause changes in liver histology. The present results demonstrate that this MoAb cross-reacted with murine hepatocytes and recognized a 190-kd molecule on the murine hepatocyte membrane just as in human hepatocytes. One hour after the injection of MoAb, the deposition of both IgM and complement component 3 was found in liver tissues. At 8 hours after the injection, serum aminotransferase levels were significantly increased in MoAb-injected mice compared with controls. Histological study revealed massive hepatocyte necrosis in MoAb-injected mice. In conclusion, human MoAb recognized a 190-kd molecule of both human and murine hepatocytes, and the injection of this MoAb to mice resulted in acute liver injury, indicating that this type of autoantibody may play an important role in the immunopathogenesis of AIH.

Influence of TNF gene polymorphism in patients with acute and fulminant hepatitis.

BACKGROUND: Tumor necrosis factor (TNF) is involved in liver damage, especially in fulminant hepatitis (FH). Our previous data showed that the serum level of TNF-alpha was markedly increased in FH. To investigate the mechanism of the overproduction of TNF in FH patients, polymorphism of the TNF gene was studied. METHODS: We analyzed 120 healthy subjects (controls), 63 patients with acute hepatitis (AH), and 32 patients with FH. Of the 32 FH patients, 21 died or received liver transplantation (FH-D), and 11 survived with intensive therapy (FH-S). The TNF-alpha promoter region at -1031, -863, -857, -308, and -238, and TNF-beta Nco1 polymorphism sites were studied. RESULTS: (1) The four groups showed no differences in polymorphisms of positions -857, -308, and -238. The allelic frequencies of positions -1031C and -863A in the FH-D patients were significantly higher compared to findings in control subjects. (2) The allelic frequency of B2 in the TNF-beta gene was significantly higher in FH patients, and particularly in the FH-D patients, compared to control subjects. (3) When the patients were divided into four groups by etiology, hepatitis A virus (HAV), HBV, HCV, and non-A non-B non-C, the allelic frequencies of positions -863A and TNF-beta B2 in FH patients were increased in the non-A non-B non-C group compared to controls. CONCLUSIONS: FH patients with a poor prognosis had higher frequencies of positions -1031C and -863A in the TNF-alpha promoter region, and higher frequencies of the B2 allele of the TNF-beta gene. These data suggest that the genomic background may be associated with the prognosis of acute liver failure.

A novel IgM class autoantibody to a hepatocyte-related 190 kDa molecule in patients with type 1 autoimmune hepatitis.

It has been reported that autoantibodies to hepatocytes are frequently found in patients with autoimmune hepatitis (AIH). To elucidate the nature of these hepatocyte-specific autoantibodies, we attempted to generate a hepatocyte-specific monoclonal antibody (MoAb) from Epstein-Barr virus-transformed peripheral blood mononuclear cells obtained from a patient with AIH. We established a single clone, 2E3, that continued to produce an immunoglobulin M (IgM) antibody (lambda-type). This MoAb had the following properties: it reacted mainly with hepatocyte-derived cell lines, rather than with other cell lines, and it reacted with liver tissue but not with other tissues. By immunoblot analysis, we found that this MoAb recognized a 190 kDa molecule on hepatocytes. The MoAb was able to kill hepatocyte-derived cell lines in the presence of fresh human serum. This cytotoxic effect was completely abrogated by heat inactivation of human serum prior to its addition to cell lines. In addition, an IgM autoantibody that recognized a 190 kDa molecule was also found in patients with AIH but not in those with chronic hepatitis C; its titer correlated significantly with serum alanine aminotransferase (ALT) levels in patients with AIH. In conclusion, we generated a human MoAb that recognizes a 190 kDa molecule on hepatocytes. Because of its ability to mediate complement-dependent cytotoxicity and the presence of similar IgM autoantibody in patients with AIH, we hypothesize this autoantibody may play a role in the immunopathogenesis of AIH.

Influence of TNF gene polymorphism and HLA-DRB1 haplotype in Japanese patients with chronic liver disease caused by HCV.

OBJECTIVE: To clarify the host genomic role in chronic liver disease associated with hepatitis C virus (HCV), we investigated the relationship between the severity of hepatitis and the polymorphisms of the tumor necrosis factor (TNF) gene or the human leukocyte antigen (HLA)-DRB1 haplotypes. METHODS: We analyzed 40 healthy subjects, 50 patients with chronic inactive hepatitis caused by HCV with mean serum ALT concentrations under 40 IU/ml (group A), and 50 patients with chronic active liver disease caused by HCV and mean ALT concentrations over 50 IU/ml (group B). RESULTS: There were no significant differences in the frequencies of TNF promoter gene variants at positions -238 and -308 between the groups. Regarding polymorphisms at the TNF-beta NcoI site, the frequency of B1B1 homozygotes in group A was significantly increased, compared with the healthy subjects and those in group B (controls 7.5%, group A 34%, group B 10%). Regarding the analysis of HLA-DRB1, DRB1*0901 was significantly more frequent in group A than in group B (group A 19%, group B 5%). TNF B1B1 homozygotes were associated with HLA-DRB1*0901 and *1302, and negatively associated with DRB1*0405. Combination analysis revealed that HCV was inactive in the majority of patients who were both DRB1*0901 and B1B1 homozygotes. CONCLUSION: Our data suggest that TNF gene polymorphisms and HLA-DRB1 haplotype may influence the activity of HCV in chronic liver disease.

In vitro binding of hepatitis C virus to CD81-positive and -negative human cell lines.

AIM: The aim of the present study is to study the mechanism of entry of hepatitis C virus (HCV) into human cells. We examined the in vitro binding of HCV to various human cell lines with or without CD81 expression. METHODS: We first used three cell lines, two hepatocyte-derived, huH-7 and HepG2, and one colon cancer cell line, Cw2. Among them, HepG2 did not express TAPA-1 (CD81) on their surface but two others did. They were incubated with HCV + serum for 1 h and HCV-RNA in extracted RNA obtained from these cells was examined by using both a quantitative test and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We found that a significant amount of HCV-RNA was detected in huH-7 and HepG2 but not in Cw2. In addition, the titer of HCV-RNA in serum-incubated CD81-transfected HepG2 was similar to that of the non-transfected titer, and the binding between HCV and huH-7 was not inhibited by anti-CD81. Under the same condition, HCV-RNA tended to be detectable in serum-pulsed hepatocyte-derived cell lines, but not in the others. CONCLUSION: These results suggest that while CD81, as reported, specifically binds to HCV-E2 protein, the entry of HCV into human hepatocytes might be regulated by CD81-unrelated molecule(s).

Analysis of natural killer cells and interleukin-15 in patients with acute and fulminant hepatitis.

To clarify the role of natural killer (NK) cells in acute hepatitis (AH) and fulminant hepatitis (FH), we measured the prevalence of CD16+ and CD57+ NK cells and the serum levels of IL-15, an inducer of NK cell activity. We analyzed 14 healthy subjects, 30 AH patients, and 13 FH patients for the percentages of CD16+ and CD57+ cells in peripheral blood lymphocytes and the serum IL-15 levels. In the AH patients with HAV, the percentage of CD16+ cells was significantly increased compared to healthy subjects, but that of CD57+ cells did not differ between the groups. The mean values of IL-15 levels of AH with HAV and unknown etiology, as well as of FH were higher than those of healthy subjects. Four subacute type FH (S-FH) patients had an increase in serum IL-15 levels, as well as the percentage of CD16+ cells throughout the course of their illness. However, in four acute type FH patients, this phenomenon was not observed. Our data suggest that NK cell immunity appears to be important in patients with AH secondary to HAV infection. In addition, the number of IL-15 and NK cells increased in the later stages of the illness, which may have contributed to the pathogenesis of S-FH.