Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease.

Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alcoholic liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hydroxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alcoholic cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alcoholic as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead observed in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition experiments showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidation contribute to the development of immune responses associated with alcoholic liver disease.

Anti-phospholipid antibodies associated with alcoholic liver disease target oxidized phosphatidylserine on apoptotic cell plasma membranes.

BACKGROUND/AIMS: Circulating anti-phospholipid antibodies (aPL) are often present in patients with alcoholic liver disease (ALD). The observations that defects in the disposal of apoptotic corpses leads to the development of aPL prompted us to investigate whether ALD-associated aPL might recognize antigens in apoptotic cells. METHODS: Apoptosis was induced in HuT-78 human T-lymphoma and HepG2 hepatoma cells by, respectively, FAS ligation with CH11 monoclonal antibodies or the incubation with ethanol (400 mmol/L). RESULTS: Flow cytometry revealed that IgG from ALD patients with high aPL titers selectively bind to the surface of apoptotic, but not to viable cells. No binding was instead evident using either control or aPL-negative ALD sera. ELISA assays using different oxidized phospholipids as antigens showed that anti-phospholipid reactivity of ALD sera was mainly directed towards oxidized cardiolipin and phosphatidylserine. The pre-adsorption of aPL-positive sera with oxidized phosphatidylserine, but not with oxidized cardiolipin, lowered aPL binding to apoptotic HuT-78 cells by about 50%. No effect was instead observed by pre-adsorption with oxidation-protected phospholipids or with human serum albumin adducted with different lipid peroxidation products. CONCLUSIONS: aPL associated with ALD target apoptotic cells by specifically recognizing oxidized phosphatidylserine, suggesting a possible link between hepatocyte apoptosis and anti-phospholipid auto-reactivity in ALD.

Valine-alanine manganese superoxide dismutase polymorphism is not associated with alcohol-induced oxidative stress or liver fibrosis.

The role of genetic factors in the pathogenesis of alcohol-induced liver disease (ALD) is receiving increasing attention. Recently, it has been reported that homozygosity for a valine to alanine substitution in the mitochondrial targeting sequence of manganese superoxide dismutase (Mn-SOD) represents a risk factor for severe ALD. Because this mutation is postulated to modify enzyme transport into mitochondria, we have sought confirmatory evidence of this association in a larger group of patients and investigated whether this polymorphism might influence alcohol-induced oxidative stress. Genotyping for the valine-alanine (Val-Ala) polymorphism of the Mn-SOD gene in 281 patients with advanced ALD (cirrhosis/fibrosis) and 218 drinkers without liver disease showed no differences in either the heterozygote (55% vs. 50%) or the homozygote (19% vs. 23%) frequency for the alanine allele. By measuring the titers of circulating antibodies against oxidized cardiolipin (OX-CL) and malondialdehyde (MDA) or hydroxy-ethyl radical (HER) adducts as markers of oxidative stress, we found a significant increase in ALD patients compared with healthy controls. However, the carriers of the alanine Mn-SOD allele had titers of anti-MDA, anti-HER, and anti-OX-CL IgG comparable with heterozygotes and patients homozygous for the valine allele. Similarly, the frequency of subjects with antibody titers above the 95th percentile of controls was not increased among homozygotes for the alanine Mn-SOD allele. In conclusion, in our population Val-Ala polymorphism in Mn-SOD influences neither susceptibility to alcohol-induced liver fibrosis nor alcohol-induced oxidative stress.

Moderate alcohol consumption increases oxidative stress in patients with chronic hepatitis C.

The mechanisms by which alcohol consumption worsens the evolution of chronic hepatitis C (CHC) are poorly understood. We have investigated the possible interaction between hepatitis C virus (HCV) and ethanol in promoting oxidative stress. Circulating IgG against human serum albumin (HSA) adducted with malondialdehyde (MDA-HSA), 4-hydroxynonenal (HNE-HSA), or arachidonic acid hydroperoxide (AAHP-HSA) and against oxidized cardiolipin (Ox-CL) were evaluated as markers of oxidative stress in 145 CHC patients with different alcohol consumption, 20 HCV-free heavy drinkers (HD) without liver disease, and 50 healthy controls. Anti-MDA IgG was increased in CHC patients irrespective of alcohol intake as well as in the HD group. CHC patients with moderate alcohol intake (<50 g ethanol/d), but not HD, also had significantly higher values of anti-AAHP-HSA, anti-HNE-HSA, and anti-Ox-CL IgG (P <.05) than controls. A further elevation (P <.001) of these antibodies was evident in CHC patients with heavy alcohol intake (>50 g ethanol/d). Anti-AAHP and anti-Ox-CL IgG above the 95th percentile in the controls were observed in 24% to 26% of moderate and 58% to 63% of heavy drinkers but only in 6% to 9% of the abstainers. The risk of developing oxidative stress during CHC was increased 3-fold by moderate and 13- to 24-fold by heavy alcohol consumption. Heavy drinking CHC patients had significantly more piecemeal necrosis and fibrosis than abstainers. Diffuse piecemeal necrosis was 4-fold more frequent among alcohol-consuming patients with lipid peroxidation-related antibodies than among those without these antibodies. In conclusion, even moderate alcohol consumption promotes oxidative stress in CHC patients, suggesting a role for oxidative injury in the worsening of CHC evolution by alcohol.

Use of molecular simulation for mapping conformational CYP2E1 epitopes.

The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature. In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1. A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of alpha helices and beta sheets. Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease. Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects. Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively. The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies.

Genetic and epigenetic factors in autoimmune reactions toward cytochrome P4502E1 in alcoholic liver disease.

Autoimmune reactions are often associated with alcoholic liver disease; however, the mechanisms responsible are largely unknown. This study investigates the potential role of the immune response against hydroxyethyl free radical (HER)-derived antigens and of polymorphisms in immunoregulatory genes in the development of anti-cytochrome P4502E1 (CYP2E1) autoantibodies in alcohol abusers. Immunoglobulin G (IgG) recognizing human CYP2E1 and HER-derived epitopes were measured by microplate immunosorbent assay in the sera of 90 patients with alcoholic fibrosis/cirrhosis (ALD), 37 heavy drinkers without liver disease or steatosis only (HD), and 59 healthy subjects. Single nucleotide polymorphisms in the interleukin 10 (IL-10) promoter and in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. The titers and frequency of anti-CYP2E1 autoantibodies were significantly higher in ALD than in HD subjects or controls. ALD patients with anti-HER IgG had higher titers and a 4-fold increased risk (OR: 4.4 [1.8-10.9]) of developing anti-CYP2E1 autoantibodies than subjects without anti-HER antibodies. The mutant CTLA-4 G allele, but not the IL-10 polymorphism, was associated with an enhanced risk of developing anti-CYP2E1 IgG (OR: 3.8 [1.4-10.3]). CTLA-4 polymorphism did not influence antibody formation toward HER-antigens. ALD patients with concomitant anti-HER IgG and the CTLA-4 G allele had a 22-fold higher (OR: 22.9 [4.2-125.6]) risk of developing anti-CYP2E1 autoreactivity than subjects negative for these factors. In conclusion, antigenic stimulation by HER-modified CYP2E1 combined with an impaired control of T-cell proliferation by CTLA-4 mutation promotes the development of anti-CYP2E1 autoantibodies that might contribute to alcohol-induced liver injury.