The development and compensation of biliary cirrhosis in interleukin-6-deficient mice

Abstract

In an effort to understand the role of IL-6/gp130 signaling in chronic liver injury, IL-6 deficient (IL-6(-/-)) and wild-type control (IL-6(+/+)) mice were subjected to bile duct ligation (BDL) for 12 weeks. This maneuver causes chronic biomechanical stress and liver injury, fueling sustained biliary epithelial and hepatocyte proliferation. By 12 weeks after BDL, IL-6(-/-) mice develop significantly higher total serum bilirubin levels (23.2 +/- 2.3 versus 14.9 +/- 2.1 mg/dl, P < 0.0001; delta bilirubin subfraction 16.7 +/- 4.0% versus 9.2 +/- 1.8%; P < 0.002), and the majority (15/18) show "black" gallbladder bile, compared to IL-6(+/+) mice (5/16; P < 0.003). The IL-6(-/-) mice also cannot sustain the compensatory liver mass increase commonly seen with chronic obstructive cholangiopathy, because of less hepatocyte proliferation, despite a rate of hepatocyte apoptosis similar to that of IL-6(+/+) mice. Moreover, IL-6(-/-) mice show a more advanced stage of biliary fibrosis and a higher mortality rate than the IL-6(+/+) controls (51% versus 23%; P < 0.02). These phenotypic changes in the IL-6(-/-) mice are associated with decreased expression and phosphorylation of gp130 and the transcription factor STAT3, compared to IL-6(+/+) mice. Daily treatment with exogenous recombinant IL-6 for 3-6 weeks starting at 6 weeks after BDL significantly lowers the serum total bilirubin in both groups. In the IL-6(-/-) mice, exogenous IL-6 treatment also increases the level of gp130 protein expression and completely reverses the loss of liver mass by increasing the hepatocyte proliferation. In conclusion, IL-6 appears to contribute to biliary tree integrity and maintenance of hepatocyte mass during chronic injury.

Figure 1.

Measures of morbidity, liver injury, and function and mortality after bile duct ligation (BDL). a: BDL results in a 15–20% decrease in total body weight. The percentage of weight loss was the same in the IL-6^+/+ and the IL-6^−/− mice (□, IL-6^−/− sham; ▪, IL-6^+/+ sham; ○, IL-6^−/− BDL; •, IL-6^+/+ BDL). b: Serum alanine aminotransferase (ALT) levels indicate similar hepatocellular injury in the two groups of mice after BDL, except for 3 weeks (*P < 0.05), when ALT levels were higher in the IL-6^−/− mice (○, IL-6^−/−; •, IL-6^+/+). c: Total serum bilirubin (TB) increased similarly in the two groups during the first 3 weeks after BDL. In the IL-6^+/+ mice, the TB stabilized at values near 15 mg/dl for the duration of the study. In contrast, TB in the IL-6^−/− mice continued to increase throughout the study (•, IL-6^+/+; ○, IL-6^−/−; *P < 0.004; **P < 0.0001). d: Although survival was similar for the first 7–8 weeks, after that time the IL-6^−/− mice experienced an increased mortality (•, IL-6^+/+ (n = 30); ○, IL-6^−/− (n = 45); P < 0.02).

Figure 2.

Gross appearance of bile in the distended biliary tracts. Note the presence of relatively clear or “white” bile in the IL-6^+/+ mice (left), in contrast to the dark green or “black” bile in the distended gallbladder of IL-6^−/− mice (right).

Figure 3.

Assessment of liver weight/body weight ratio (liver mass), actual liver weights, BrdU, and TUNEL+ labeling indexes after bile duct ligation (BDL). a: The initial increase in liver mass during the first week after BDL was independent of the IL-6 status, but the IL-6^−/− mice were unable to sustain the increased liver mass during longer follow-up (•, IL-6^+/+; ○, IL-6^−/−; *P < 0.0005). b: Similar results were seen for the actual liver weights (*P < 0.02). c: BrdU labeling indexes showed fewer hepatocytes but more BEC labeling in the IL-6^−/− mice at both 6 and 12 weeks after BDL, but the difference in BEC labeling was statistically significant only at 12 weeks (*P < 0.04; **P < 0.02; ***P < 0.0001; □, IL-6^+/+; ▧, IL-6^−/−). d: There was no significant difference between IL-6^+/+ and IL-6^−/− mice in the number of TUNEL+ cells/50 high power fields (HPFs) (1000×) at either 6 or 12 weeks after BDL (□, IL-6^+/+; ▧, IL-6^−/−).

Figure 4.

Assessment of liver architecture and cell composition of the liver after bile duct ligation. a: Routine histology, using Van Gieson’s picric acid-acid fuschin-stained slide at 12 weeks, showed that the IL-6^−/− mice had progressed to stage III disease (right), with the formation of portal-to-portal bridging, whereas the IL-6^+/+ mice showed mostly portal expansion, or stage II disease (left). b: Determination of the volume proportions of hepatocytes (▧), mesenchymal cells (□), and BEC (▩) showed that by 12 weeks, there was a greater percentage of mesenchymal (29.0 ± 2.9% versus 18.9 ± 0.7%; P < 0.005) and BEC (3.5 ± 0.6% versus 1.5 ± 0.2%; P < 0.006) in the IL-6^−/− mice.

Figure 5.

Analysis of the IL-6/gp130 signaling system before and after bile duct ligation (BDL). a: RT-PCR for IL-6 and LIF showed an increase in mRNA for both of these cytokines at 12 weeks after BDL (12W) in the IL-6^+/+ mice, compared to normal mice (−) without BDL. The IL-6^−/− mice also showed increased LIF compared to normals (−), but IL-6 mRNA was not detected, as expected. b: Immunoprecipitation (IP) followed by Western blotting (WB) showed no difference between IL-6^−/− and IL-6^+/+ mice in the level of IL-6Rα protein expression either before BDL(−) or 12 weeks after BDL(12W) (top row). The level of gp130 protein expression was similar between normal IL-6^+/+ and IL-6^−/− mice before BDL(−), but 12 weeks after BDL(12W) the IL-6^+/+ clearly showed more gp130 protein expression (center row) and higher levels of gp130 phosphorylation (botttom row; PY, anti-phosphotyrosine) than the IL-6^−/− mice. c: Nuclear STAT3 protein levels were slightly higher in normal IL-6^+/+ mice before BDL. Nuclear phospho-STAT3 was weakly expressed in normal mice from both groups before BDL, although an occasional IL-6^+/+ mouse showed slightly higher levels of expression. By 12 weeks after BDL, both groups of mice showed a small increase in nuclear STAT3 and a clear increase in nuclear phospho-STAT3. However, the level of expression of both STAT3 and phospho-STAT3 was greater in the IL-6^+/+ mice. HeLa cells were used as a positive control for STAT3 and a negative control for phosph-STAT3. HeLa cells treated with interferon-α (IFN-α) were used as a positive control for STAT3 and phospho-STAT3.

Figure 6.

Effect of exogenous rhIL-6 treatment. a: TB values at 6 weeks (pre-, □) and 9 weeks (post-, ▧) for both IL-6^+/+ and IL-6^−/− mice showed that treatment with rhIL-6 but not normal saline significantly lowered the serum TB levels in both the IL-6^−/− mice and in the IL-6^+/+ mice, although the decrease was more dramatic in the IL-6^−/− mice (*P < 0.03; **P < 0.0005; n = 6 in each group). b: Treatment with exogenous rhIL-6 for 3 weeks also significantly lowered the ratio of delta bilirubin to total bilirubin in the IL-6^−/− mice, indicative of increased biliary tree integrity and decreased exposure of conjugated bilirubin to the blood after therapy (see text). ▧, conjugated bilirubin; □, delta bilirubin; ▩, unconjugated bilirubin). c: Immunoprecipitation followed by Western blotting (IP/WB) showed that treatment with exogenous rhIL-6 for 6 weeks also increased the level of gp130 protein expression in the IL-6^−/− mice after BDL, consistent with previous studies. d: Treatment with exogenous rhIL-6 for 6 weeks also increased the number of portal plasma cells in the IL-6^−/− mice (inset), which were uncommonly detected in the IL-6^−/− mice treated with normal saline.