Chronic ethanol feeding increases activation of NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role of increased reactive oxygen in LPS-stimulated ERK1/2 activation and TNF-alpha production

Abstract

Reactive oxygen species (ROS) contribute to the development of chronic ethanol-induced liver injury. Although ROS modulate the activity of many signal transduction pathways, the molecular targets of ROS during ethanol exposure are not well understood. Here, we investigated whether specific ROS-sensitive signal transduction pathways contribute to increased tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells after chronic ethanol feeding to rats. Lipopolysaccharide (LPS) rapidly increased ROS production, measured by dihydrorhodamine fluorescence, in Kupffer cells from ethanol- and pair-fed rats, and ROS production was 2.5-fold greater in ethanol-fed compared with pair-fed. Pretreatment with diphenyleneiodonium (DPI), which inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalized ROS production in Kupffer cells from ethanol-fed rats. LPS rapidly increased Rac1-guanosinetriphosphatase (GTPase) activity and p67(phox) translocation to the plasma membrane in Kupffer cells from pair-fed rats. After ethanol feeding, Rac1-GTPase activity was already increased over pair-fed at baseline and remained elevated over pair-fed after LPS stimulation. Further, LPS-stimulated p67(phox) translocation to the plasma membrane was enhanced after chronic ethanol feeding. LPS-stimulated extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, two signaling pathways regulated by ROS, were increased twofold in Kupffer cells from ethanol-fed rats compared with pair-fed controls. However, only LPS-stimulated ERK1/2 phosphorylation was inhibited by DPI, which also reduced LPS-stimulated TNF-alpha production in Kupffer cells from pair- and ethanol-fed rats. These results demonstrate that chronic ethanol feeding increases LPS-stimulated NADPH oxidase-dependent production of ROS in Kupffer cells. Further, ERK1/2 is an important target of NADPH oxidase-derived ROS in Kupffer cells, contributing to enhanced LPS-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding.

Fig. 1

Chronic ethanol feeding increases LPS-stimulated ERK1/2 phosphorylation in Kupffer cells. (A) LPS-stimulated ERK1/2 phosphorylation (p-ERK1/2) was measured in Kupffer cells from pair- and ethanol (EtOH)-fed rats after culture for 16–18 h. Cells were then treated with or without 100 ng/ml LPS for 0–60 min, and ERK1/2 phosphorylation was assessed by Western blot. (B) Dephosphorylation of ERK1/2 after LPS treatment was measured in Kupffer cells from pair- and ethanol-fed rats. After culture for 16–18 h, cells were stimulated with 100 ng/ml LPS for 30 min and then treated or not with 10 mM 2-deoxy glucose and 5 μM rotenone to deplete adenosine 5′-triphosphate (ATP) and inhibit further phosphorylation. Phospho-ERK1/2 relative to total ERK1/2 was measured by Western blotting. Values represent mean ± sem, n = 4 (A) and n = 7 (B); *, P < 0.05, compared with pair-fed at the same time-point.

Fig. 2

Chronic ethanol feeding increases LPS-stimulated DHR fluorescence in isolated Kupffer cells, which, isolated from pair- and ethanol-fed rats, were cultured 16–18 h and then stimulated with or without 100 ng/ml LPS for 0–15 min (A). Media were then replaced with 10 μM DHR, and cells were incubated for 15 min. Fluorescence was measured at an excitation wavelength of 505 nm and emission detection wavelength of 530 nm. Values represent mean ± sem, corrected for DHR fluorescence at zero time; n = 5; *, P < 0.05, compared with pair-fed. DHR fluorescence at zero time did not differ between pair-fed (2185± 307 arbitrary units of fluorescence) and ethanol-fed (2135± 290). (B) Pretreatment with DPI chloride decreased LPS-stimulated ROS production. Kupffer cells were treated with or without 10 μM DPI for 2 h and then stimulated with LPS for 5 min, followed by incubation with DHR as described above; n = 13, values with different superscripts; ^a, P < 0.05 compared with pair-fed cells not treated with LPS; ^b, P < 0.05, compared with pair-fed cells treated with LPS; ^c, P < 0.05 compared with cells treated with LPS but not treated with DPI. DHR fluorescence is expressed as percent of pair-fed LPS treated (177±87), as several different lots of DHR were used in these experiments, and each lot had a different relative fluorescence.

Fig. 3

(A) Chronic ethanol feeding increases LPS-stimulated p67^phox translocation. Kupffer cells isolated from pair (P)- and ethanol (E)-fed rats were cultured 16–18 h and then stimulated with or without 100 ng/ml LPS for 10 min. Kupffer cells were lysed, and plasma membrane-enriched fractions and homogenates were probed for p67^phox by Western blot. NaK-ATPase was used as a control for equal loading of plasma membrane fractions. Values represent means ± sem; n = 5; +, P < 0.05, compared with pair-fed at each time-point; *, P < 0.05, compared with cells not treated with LPS. (B) Chronic ethanol feeding increases guanosine 5′-triphosphate (GTP)-bound Rac1. Kupffer cells isolated from pair- and ethanol-fed rats were cultured 16–18 h and then stimulated with or without 100 ng/ml LPS for up to 2.5 min. Kupffer cells were lysed, and GTP-bound Rac1 was measured using a Rac1 pull-down assay. Values represent means ± sem; n = 6; +, P < 0.05, compared with pair-fed at each time-point; *, P < 0.05, compared with cells not treated with LPS.

Fig. 4

DPI chloride suppresses LPS-stimulated ERK1/2 phosphorylation. Kupffer cells from pair- and ethanol-fed rats were cultured 16–18 h. Cells were then preincubated with or without 1 μM DPI for 2 h and then treated with 100 ng/ml LPS for 0–60 min. Phosphorylation of ERK1/2 was assessed by Western blot. Values represent means ± sem; n = 8; *, P < 0.05, compared with pair-fed; #, P < 0.05, compared with cells not treated with DPI (within a diet group).

Fig. 5

(A) Chronic ethanol feeding increases CYP2E1 protein expression in Kupffer cells, which were isolated from pair- and ethanol-fed rats and cultured for 16–18 h. Homogenates were prepared, and CYP2E1 protein was measured by Western blot. Values represent means ± sem; n = 9; *, P < 0.05, compared with pair-fed. (B) LPS-stimulated ERK1/2 phosphorylation in Kupffer cells treated with inhibitors of CYP2E1 or xanthine oxidase. Kupffer cells from pair- and ethanol-fed rats were cultured 16–18 h. Cells were then preincubated with or without 5 mM 4-methylpyrazole (4-MP), 5 μM chlor-methiazole (CMZ), or allopurinol (Allo) for 2 h and then treated with 100 ng/ml LPS for 0–60 min. Phosphorylation of ERK1/2 was assessed by Western blot. Blots are representative of three (for chlormethiazole and allopurinol) or seven (for 4-methylpyrazole) independent experiments.

Fig. 6

DPI chloride does not suppress LPS-stimulated p38 phosphorylation (A) or IκBα degradation (B) in Kupffer cells from ethanol-fed rats. Kupffer cells from pair- and ethanol-fed rats were cultured 16–18 h. Cells were then preincubated with or without 1 μM DPI for 2 h. (A) Phosphorylation of p38 was measured in response to treatment with 100 ng/ml LPS for 0–60 min. (B) Quantity of IκB-α was assessed by Western blot after 30 min stimulation with or without 100 ng/ml LPS. Values represent means ± sem; n = 7 for p38, and n = 6 for IκB-α. (A) *, P < 0.05, compared with pair-fed; #, P < 0.05, compared with cells not treated with DPI (within a diet group). (B) *, P < 0.05, compared with basal values within each diet group.

Fig. 7

DPI chloride inhibits LPS-stimulated TNF-α production after chronic ethanol feeding. Kupffer cells from pair- and ethanol-fed rats were cultured 16–18 h. Cells were then preincubated with or without 1 μM DPI for 2 h and then treated with 100 ng/ml LPS. (A) After 60 min LPS treatment, total RNA was isolated, and TNF-α mRNA accumulation was measured by real-time PCR and normalized to β-actin mRNA. Values represent means ± sem; n = 3; *, P < 0.05, compared with pair-fed; +, P < 0.05, compared with LPS-treated cells not treated with DPI. (B) After 4 h, cell culture media were removed, and TNF-α concentrations were measured by ELISA. Values represent means ± sem; n = 7–8; *, P < 0.05 compared with pair-fed; +, P < 0.05 compared with LPS-treated cells not treated with DPI.