Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol.
OBJECTIVE
We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity.


METHODS
Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used.


RESULTS
Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes.


CONCLUSIONS
Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.
Clot
P
P
Department of Medical Sciences, University of Torino, Novara, Italy.
Parola
M
M
Bellomo
G
G
Dianzani
U
U
Carini
R
R
Tabone
M
M
Aricò
S
S
Ingelman-Sundberg
M
M
Albano
E
E
D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects*; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D005609:Free Radicals; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000276:immunology*; D008108:Liver Diseases, Alcoholic / Q000276:immunology*; D008297:Male; D018613:Microscopy, Confocal; D008862:Microsomes, Liver / Q000187:drug effects / Q000276:immunology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D005609:Free Radicals; C065424:1-hydroxyethyl radical; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1