Volume 119, Issue 6, Pages 1663-1671 (December 2000) Combined interleukin 6 and soluble interleukin 6 receptor accelerates murine liver regeneration  Malte Peters, *, Guido Blinn, *, Thomas Jostock, *, Peter Schirmacher, ‡, Karl–Hermann Meyer Zum Büschenfelde, *, Peter R. Galle, *, Stefan Rose–John, *  Gastroenterology  Volume 119, Issue 6, Pages 1663-1671 (December 2000) DOI: 10.1053/gast.2000.20236 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Hyper-IL-6 causes accelerated reconstitution of liver weight after partial hepatectomy. After a 50% hepatectomy, mice were either left untreated (□) or received IL-6 (20 μg/mouse; ○) or Hyper-IL-6 (2 μg/mouse; ■) IP. At the indicated time points, mice were killed, the remnant livers were removed, and the percentage of liver weight increase compared with time 0 was determined (see Materials and Methods). At least 6 mice underwent the procedure at each time point in each treatment group. The dashed line represents 50% weight gain of the liver. Results are mean ± SD. P values were calculated from Hyper-IL-6 vs. IL-6 and from Hyper-IL-6 vs. control. **P < 0.005. Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Hyper-IL-6 significantly accelerates liver proliferation after partial hepatectomy in mice. (A) After a 50% partial hepatectomy, mice were either left untreated (□) or received IL-6 (20 μg/mouse; ○) or Hyper-IL-6 (2 μg/mouse; ■) IP. One hour before the mice were killed, 50 mg/kg body wt BrdU in PBS was injected IP into the mice. After removal of the remnant livers, the organs were fixed in 4% formaldehyde and embedded in paraffin. Tissue sections were subjected to BrdU immunohistochemistry. The percentage of BrdU-positive nuclei was counted in at least 3 mice per treatment group. Values are mean ± SD. **P < 0.005. (B) Immunohistochemical detection of BrdU incorporation in S-phase liver nuclei as an indicator of liver cell proliferation. After 50% partial hepatectomy, mice were either (A and B) left untreated, (A' and B') treated with 20 μg IL-6, or (A" and B") treated with 2 μg Hyper-IL-6. Mice were killed 36 (A, A', and A") or 72 hours (B, B', and B") after surgery. One hour before the animals were killed, 50 mg/kg body wt BrdU in PBS was injected IP. Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Hyper-IL-6 significantly accelerates liver proliferation after partial hepatectomy in mice. (A) After a 50% partial hepatectomy, mice were either left untreated (□) or received IL-6 (20 μg/mouse; ○) or Hyper-IL-6 (2 μg/mouse; ■) IP. One hour before the mice were killed, 50 mg/kg body wt BrdU in PBS was injected IP into the mice. After removal of the remnant livers, the organs were fixed in 4% formaldehyde and embedded in paraffin. Tissue sections were subjected to BrdU immunohistochemistry. The percentage of BrdU-positive nuclei was counted in at least 3 mice per treatment group. Values are mean ± SD. **P < 0.005. (B) Immunohistochemical detection of BrdU incorporation in S-phase liver nuclei as an indicator of liver cell proliferation. After 50% partial hepatectomy, mice were either (A and B) left untreated, (A' and B') treated with 20 μg IL-6, or (A" and B") treated with 2 μg Hyper-IL-6. Mice were killed 36 (A, A', and A") or 72 hours (B, B', and B") after surgery. One hour before the animals were killed, 50 mg/kg body wt BrdU in PBS was injected IP. Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of Hyper-IL-6 and IL-6 on AST activity after partial hepatectomy. After partial hepatectomy, AST levels were determined in the sera of mice, which were either left untreated (□) or received 20 μg IL-6 (○) or 2 μg Hyper-IL-6 (■) at the indicated time points. Results are mean ± SD (n = 4–6). P values were calculated from Hyper-IL-6 vs. IL-6 and from Hyper-IL-6 vs. control. *P < 0.05; **P < 0.005. Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) STAT3 phosphorylation and (B) c-fos and c-myc mRNA expression after IL-6 and Hyper-IL-6 treatment in hepatectomized mice. (A) One and 2 hours after partial hepatectomy, protein extracts were obtained from remnant livers and phosphorylated. STAT3 was detected by Western blot analysis. Mice were either left untreated (control) or received 20 μg IL-6 or 2 μg Hyper-IL-6 (H-IL-6) IP immediately after the procedure. (B) Zero, 1, 4, and 8 hours after partial hepatectomy, total RNA was prepared from mice receiving either no treatment, 20 μg IL-6, or 2 μg Hyper-IL-6 IP immediately after the operation. Filters were hybridized with a 32P-labeled 1.2-kb PstI restriction fragment of human c-fos cDNA and a 1.75-kb NotI/EcoRI restriction fragment of human c-myc cDNA. Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 IL-6R expression after partial hepatectomy. (A) IL-6R and gp130 mRNA levels were analyzed by RNase protection assays. Total RNA was prepared at different time points as indicated from livers of hepatectomized mice that had received no cytokine treatment. (B) Murine soluble IL-6R was immunoprecipitated from sera of untreated animals after partial hepatectomy at time points indicated and subjected to Western blot analysis (right panel). As a control, supernatant from COS-7 cells transfected with the cDNA coding for the murine sIL-6R (transfected) and supernatant from untransfected COS-7 cells (untransfected) were subjected to immunoprecipitation (left panel). Gastroenterology 2000 119, 1663-1671DOI: (10.1053/gast.2000.20236) Copyright © 2000 American Gastroenterological Association Terms and Conditions