Chronic plus binge ethanol feeding synergistically induces neutrophil infiltration and liver injury in mice: a critical role for E-selectin

Abstract

Chronic plus binge ethanol feeding acts synergistically to induce liver injury in mice, but the mechanisms underlying this phenomenon remain unclear. Here, we show that chronic plus binge ethanol feeding synergistically up-regulated the hepatic expression of interleukin-1β and tumor necrosis factor alpha and induced neutrophil accumulation in the liver, compared with chronic or binge feeding alone. In vivo depletion of neutrophils through administration of an anti-Ly6G antibody markedly reduced chronic-binge ethanol feeding-induced liver injury. Real-time polymerase chain reaction analyses revealed that hepatic E-selectin expression was up-regulated 10-fold, whereas expression of other neutrophil infiltration-related adhesion molecules (e.g., P-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1) was slightly up- or down-regulated in this chronic-binge model. The genetic deletion of E-selectin prevented chronic-binge ethanol-induced hepatic neutrophil infiltration as well as elevation of serum transaminases without affecting ethanol-induced steatosis. In addition, E-selectin-deficient mice showed reduced hepatic expression of several proinflammatory cytokines, chemokines, and adhesion molecules, compared to wild-type mice, after chronic-binge ethanol feeding. Finally, the expression of E-selectin was highly up-regulated in human alcoholic fatty livers, but not in alcoholic cirrhosis.

Conclusions: Chronic-binge ethanol feeding up-regulates expression of proinflammatory cytokines, followed by the induction of E-selectin. Elevated E-selectin plays an important role in hepatic neutrophil infiltration and injury induced by chronic-binge feeding in mice and may also contribute to the pathogenesis of early stages of human alcoholic liver disease.

Fig. 1

Comparison of liver injury and inflammation induced by chronic, binge and chronic-binge feeding. C57BL6/J mice were subjected to chronic, binge or chronic-binge feeding. (A) Liver injury was assessed by measuring serum ALT and AST levels. (B, C) Liver mRNA levels of cytokines (B) and chemokines (C) were analyzed by real-time PCR. *P<0.05, **P<0.01, ***P<0.001.

Fig. 2

Comparison of the hepatic inflammatory cell recruitment induced by chronic, binge, and chronic-binge feeding. C57BL6/J mice were subjected to chronic, binge or chronic-binge feeding. (A, B) Liver mRNA levels of monocyte/macrophage (A, F4/80 and CD68) and neutrophil (B, Ly6G) markers were analyzed by real-time PCR. (C) Liver sections were stained for MPO and the number of MPO⁺ cells per ×100 field was counted. (D, E) Hepatic leukocytes were isolated and analyzed by flow cytometry. Panel D: Representative flow cytometry data for hepatic CD11b⁺Gr-1^high neutrophil infiltration; Panel E: Mean fluorescence intensity of the cell surface levels of CD11b and CD62L on neutrophils. Neutrophil activation is associated with the upregulation of CD11b and downregulation of CD62L expression. *P<0.05, **P<0.01, ***P<0.001.

Fig. 3

The effect of neutrophil depletion on chronic-binge feeding-induced liver injury. C57BL6/J mice were subjected to chronic-binge feeding and were injected intravenously with 200μg and 100μg of anti-Ly6G or isotype control antibodies 24h and 4h before ethanol gavage, respectively. Mice were sacrificed 9 hours post gavage. (A) Liver mRNA levels of neutrophil (Ly6G) and macrophage (F4/80) markers were analyzed by real-time PCR. (B) Liver sections were stained for MPO and the number of MPO⁺ cells per ×100 field was counted. (C) Liver injury was assessed by measuring serum ALT and AST levels. (D) Steatosis was quantified by measuring hepatic triglyceride levels. *P<0.05, *** P<0.001.

Fig. 4

Hepatic E-selectin expression is upregulated by chronic-binge feeding and positively correlates with neutrophil infiltration. (A) Real-time PCR analyses of hepatic mRNA expression of adhesion molecules from chronic-binge-fed or pair-fed mice. (B) Real-time PCR analyses of hepatic E-selectin mRNA from chronic-, binge-, or chronic-binge-fed mice. *P<0.05, **P<0.01, ***P<0.001. (C) The correlation between the hepatic mRNA levels of E-selectin and Ly6G from pair-fed and chronic-binge-fed mice was analyzed using the Spearman rank correlation test.

Fig. 5

Chronic-binge feeding-induced hepatic neutrophil recruitment is decreased in SELE^−/− mice. WT and SELE^−/− mice were subjected to chronic-binge ethanol or pair feeding. (A, B) Real-time PCR analyses of hepatic mRNA levels of monocyte/macrophage (A, F4/80 and CD68) and neutrophil (B, Ly6G) markers. (C) Liver sections were stained for MPO and the number of MPO⁺ cells per ×100 field was counted. ***P<0.001 for EtOH-fed vs. corresponding pair-fed; ^•••P<0.001 for EtOH-fed SELE^−/− vs. EtOH-fed WT. (D, E) Hepatic leukocytes were isolated and analyzed by flow cytometry. Panel D: Representative flow cytometry data of hepatic neutrophil infiltration; Panel E: Mean fluorescence intensity of cell surface levels of CD11b and CD62L. *P<0.05 and ***P<0.001.

Fig. 6

Chronic-binge feeding-induced liver injury and inflammation are attenuated in SELE^−/− mice. WT and SELE^−/− mice were subjected to chronic-binge ethanol or pair feeding. (A, B) Liver injury was assessed by measuring serum ALT and AST levels (A) and liver-to-body weight ratios (B). (C) Steatosis was quantified by measuring hepatic triglyceride levels. (D–F) Liver mRNA levels of cytokines (D), chemokines (E), and adhesion molecules (F) were analyzed by real-time PCR. *P<0.05, **P<0.01, ***P<0.001 for EtOH-fed vs corresponding pair-fed; P<0.05, P<0.01 for EtOH-fed SELE^−/− vs. EtOH-fed WT.

Fig. 7

Hepatic expression of E-selectin is upregulated in patients with early stages of ALD. (A–C) Real-time PCR analyses of hepatic mRNA levels of adhesion molecules (A), Il-1β (B), and TGF-β (C) in normal human livers (n=10) and livers from patients with alcoholic fatty liver (AFL, n=10) and alcoholic cirrhosis (n=11). *P<0.05, **P<0.01 compared to normal livers, ^•P<0.05 compared to AFL (Kruskal-Wallis test followed by Dunn’s multiple comparison test). (D) Correlation between the E-selectin and MPO mRNA levels in normal, AFL, and cirrhotic livers (Spearman rank correlation test).

Fig. 8

A model depicting the key role of E-selectin in the pathogenesis of chronic-binge- induced liver injury. Chronic-binge ethanol consumption increases gut permeability and subsequently elevates portal LPS levels. LPS stimulates Kupffer cells to produce TNF-α and IL-1β which, together with LPS, have been shown to upregulate E-selectin on endothelial cells and may contribute to hepatic E-selectin upregulation in this chronic-binge model. E-selectin then binds to neutrophils, inducing neutrophil activation, sequestration, and transmigration. Activated neutrophils kill steatotic hepatocytes and induce Kupffer cell activation via the interaction of ICAM-1 and CD11b, thereby promoting hepatocellular necrosis and inflammation.