1. Volume 126, Issue 5, Pages 1387-1399 (May 2004)
Activated natural killer T cells induce liver injury by Fas and tumor necrosis factor-α during alcohol consumption 
Masahiro Minagawa, Qinggao Deng, Zhang-xu Liu, Hidekazu Tsukamoto, Gunther Dennert 
Gastroenterology 
Volume 126, Issue 5, Pages (May 2004)
DOI: /j.gastro

2. Figure 1
Increase in body weights, liver weights, and serum ALT values in mice fed control and alcohol diets. The alcohol diet was initiated at time 0. (A ) Increase in body weights over time. (B) Serum ALT values in control- and alcohol-fed mice. (C ) Liver weights of control- and alcohol-fed mice after complete bleeding.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Changes in liver histology induced by alcohol feeding and injection of α-GalCer. (A ) Liver of a B6 mouse fed the control glucose diet for 4 weeks. (B) Liver of a B6 mouse fed the alcohol diet for 4 weeks. (C ) Liver of a mouse fed the control glucose diet for 1 week. (D) Liver of a B6 mouse fed the alcohol diet for 1 week. (E ) Liver of a B6 mouse fed the control glucose diet for 1 week as in (C ) and then injected with α-GalCer. The liver was harvested 1 day later. (F ) Liver of a B6 mouse fed the alcohol diet for 1 week as in (C ) and then injected with α-GalCer. The liver was harvested 1 day later. (G) Liver of an NKT cell-deficient CD1d−/− mouse fed the alcohol diet for 1 week and then injected with α-GalCer. The liver was harvested 1 day later (original magnification, 200×).
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Increase of NKT cells in the liver of alcohol- and glucose-fed control mice. Mice were fed control glucose or alcohol diets for 1 or 4 weeks, at which time liver mononuclear cells were isolated and analyzed for the expression of cell-surface markers NK1.1 and CD3. Numbers in the panels indicate percentages of each cell subset.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Alcohol sensitizes animals to liver injury and hepatocyte lysis. (A and B) Animals were fed control glucose or alcohol diets for 1 week, at which time they were injected with α-GalCer. (A ) ALT values are shown for the 24-hour time point after α-GalCer injection (glucose group, 138 ± 31 U/L; ethanol group, 2819 ± 849 U/L). (B) Survival 2 days after α-GalCer injection (n = 7 for each group). (C and D) α-GalCer was injected on the day the alcohol diet was started (day 0) or 3 or 5 days thereafter, as indicated. (C ) Serum ALT values for the 24-hour time point after α-GalCer injection. (D) Survival 2 days after α-GalCer injection (n = 4 for each time point). (E ) Hepatocytes were isolated from B6 mice fed glucose control or alcohol diets for 1 week. They were tested for sensitivity to hepatic mononuclear effector cells isolated from 2 mice 2 hours after injection of α-GalCer. After a 4-hour incubation, the release of 51Cr from hepatocytes was assayed at an effector/target ratio (E:T) of 20:1, which was determined in pilot studies to yield optimal results. The plotted values represent net increases over spontaneous release as described in the Materials and Methods section. Shown is 1 of 3 similar experiments.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
NKT cells are effectors in liver injury. (A ) Liver mononuclear cells were isolated from normal mice or mice that had been injected with anti-AsGM1 antibody or rabbit serum 24 and 48 hours before harvesting of the liver. The cells were analyzed by flow cytometry for expression of NK1.1 and CD3, as indicated. (B) Mice were fed alcohol for 1 week and injected with anti-AsGM1 or rabbit serum on days 5 and 6 after initiation of alcohol feeding. Serum ALT values were determined on day 8. (C ) Mice were fed alcohol and injected with anti-AsGM1 or rabbit serum as in (B). Mice were injected with α-GalCer on day 7, and serum ALT values were determined on day 8. (D) Normal B6 mice were injected with anti-AsGM1 or rabbit serum 24 and 48 hours before injection with α-GalCer. Two hours after injection with α-GalCer, liver mononuclear cells were isolated and assayed on L1210-Fas+ or L1210-Fas− target cells in an 18-hour 51Cr-release assay. The effector/target ratio (E:T) of 10:1 was determined to be optimal from pilot experiments. Where indicated, effector cells were treated with anti-NK1.1 and complement before use in the cytotoxicity assay. GC, alpha galactosyl ceramide.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Expression of Fas ligand (FasL) on NKT and NK cells and of Fas on hepatocytes. (A ) Animals were injected with α-GalCer 7 days after initiation of alcohol feeding. Two hours later, liver mononuclear cells were isolated and stained for FasL, CD3, and NK1.1. Expression of FasL on NKT cells (CD3+ NK1.1+ cells) and NK cells (CD3− NK1.1+cells) is shown. Gates were set to exclude 99% of cells not expressing the respective markers. Numbers in panels indicate percentages of FasL-staining cells. (B) Livers were harvested on day 7 of alcohol feeding, and hepatocytes were isolated. Expression of Fas on freshly isolated hepatocytes was assayed. Thick lines on the left constitute unstained controls, and thin lines on the right indicate Fas-staining cells. Numbers in panels indicate mean fluorescence channel values. Shown is 1 of 2 similar experiments. (C ) Fas-deficient lpr or normal mice were fed alcohol or control diet for 1 week. On day 7 after initiation of alcohol feeding, mice were injected with α-GalCer. One day later, ALT values were assayed (glucose-fed B6, 376 ± 149 U/L; ethanol-fed B6, 2494 ± 585 U/L; glucose-fed lpr, 78 ± 37 U/L; ethanol-fed lpr, 146 ± 40 U/L). All alcohol-fed lpr mice injected with α-GalCer survived.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Time course of serum ALT values in alcohol-fed B6, lpr, TNFR1-deficient, and Jα281 gene-deleted mice. Mice were fed alcohol for 4 weeks. At the times indicated, the average ALT values determined from groups of 3–4 mice are plotted. KO, knockout.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Functional Fas and TNF pathways are required for α-GalCer-induced liver injury in alcohol-fed mice. (A ) B6, lpr, TNFR1-deficient, and Jα281 gene-deleted mice were fed glucose control or alcohol diets for 4 weeks. At the end of the fourth week, they were injected with α-GalCer. Serum ALT values were determined 1 day after α-GalCer injection (glucose-fed B6 mice, 438 ± 155 U/L; ethanol-fed B6 mice, 3299 ± 1120 U/L; other groups, <200 U/L). All alcohol-fed B6 mice died within 48 hours of injection, whereas all other mice survived. (B) By using the same mice as in (A ), serum TNF-α levels were determined 3 hours after α-GalCer injection. There was no statistically significant difference in TNF-α levels between the control and ethanol-fed groups after injection of α-GalCer. (C ) Intracellular TNF-α staining in NKT cells from normal B6 mice. NKT cells were isolated from livers 2 hours after injection of α-GalCer or vehicle. Gray shaded areas represent isotype antibody controls. Gates were set to exclude 99% of all cells stained with isotype control antibody. Numbers in panels indicate percentages of TNF-α–staining NKT cells. Glu, glucose; EtOH, alcohol.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Hepatocytes from alcoholic livers display increased sensitivity to Fas-mediated apoptosis. TNF-α provides synergistic effects to Fas-mediated cell death. (A ) B6 mice were fed alcohol or glucose control diet for 1 week, at which time livers were harvested and hepatocytes prepared. Hepatocytes labeled with 51Cr were incubated with the indicated concentrations of anti-Fas antibody only or with antibody and protein A (100 ng/mL) and were assayed for release of radioactivity in a 4-hour assay. Shown is 1 of 3 similar experiments. (B) Hepatocytes were isolated from mice fed glucose control or alcohol diet for 1 week, labeled with 51Cr, and incubated with various concentrations of recombinant TNF-α and with anti-Fas antibody 0.1 μg/mL and protein A 100 ng/mL, as indicated. The 51Cr release was determined after a 4-hour incubation.
Gastroenterology  , DOI: ( /j.gastro )