Kupffer cells become activated in response to elevated levels of LPS during
ethanol feeding, but the role of
ethanol in the molecular processes of activation remains unclear. Because
cytochrome P4502E1 (
CYP2E1) is upregulated in Kupffer cells after
ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase
TNF-alpha production in response to LPS. However, cultured Kupffer cells rapidly lose their
CYP2E1. This difficulty was overcome by transfecting
CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased
CYP2E1 expression (E2) displayed increased levels of CD14/
Toll-like receptor 4,
NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and
NF-kappaB. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased
TNF-alpha production.
Diphenyleneiodonium, a
NADPH oxidase inhibitor, and
diallyl sulfide, a
CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that
NADPH oxidase and
CYP2E1 contribute equally to H2O2 generation. Because
CYP2E1 expression also enhanced the levels of the membrane localized
NADPH oxidase subunits p47phox and
p67phox, thereby contributing to the
oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from
NADPH and
CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated
TNF-alpha production via activation of
NF-kappaB, whereas p38 promoted
TNF-alpha production by stabilizing
TNF-alpha mRNA.
Oxidant generation after
CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly,
CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by
ethanol, a critical early step in
alcoholic liver disease.