Volume 125, Issue 1, Pages 202-215 (July 2003) In vitro interleukin-6 treatment prevents mortality associated with fatty liver transplants in rats1   Zhaoli Sun, Andrew S Klein, Svetlana Radaeva, Feng Hong, Osama El-Assal, Hong-na Pan, Barbara Jaruga, Sandor Batkai, Sumito Hoshino, Zhigang Tian, George Kunos, Anna mae Diehl, Bin Gao  Gastroenterology  Volume 125, Issue 1, Pages 202-215 (July 2003) DOI: 10.1016/S0016-5085(03)00696-6

Figure 4 IL-6 treatment protects against hepatocyte and sinusoidal lining cell death in steatotic Zucker rat liver isografts. (A ) Representative photomicrographs of trypan blue-stained IL-6-pretreated and untreated steatotic Zucker rat liver isografts 1 hour and 3 hours posttransplantation (400 × magnification). The enlarged insets are shown in the middle lane. Isografts were perfused with trypan blue solution and fixed as described in Materials and Methods. (B) Representative photomicrographs of TUNEL-stained sections from IL-6-pretreated and untreated steatotic liver isografts obtained at 1, 3, and 6 hours posttransplantation (× 400). Slides were treated and developed as described in Methods. Positive apoptotic cells are stained with brown color. NC, nonparenchymal cells; H, hepatocytes. (C ) Caspase-3 activity was measured as described in Materials and Methods. Bars represent means ± SE (n = 3). (D) Nonparenchymal cell killing by necrosis or apoptosis was quantified as the percentage of trypan blue-positive cells (left) or TUNEL positive cells/total RECA-1-stained endothelial cells (right). Hepatocyte cell death was quantified as the percentage of trypan blue-positive cells (left) or TUNEL positive cells/total hepatocytes (right). Bars represent means ± SE from 4 rats in each group. ∗P < 0.05, ∗∗P < 0.01 vs. nontreated group. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 5 IL-6 prevents hypothermia/rewarming-induced necrapoptosis in cultured hepatic endothelial cells. Cultured hepatic endothelial cells were exposed to hypothermia/rewarming as described in Materials and Methods, cell death was determined by trypan blue staining (A ) and apoptosis was assayed by FACS (B), or caspase-3 activity (C ). Values are shown as means ± SEM (n = 4). ∗P < 0.001, significant difference from control group. ∗∗P < 0.01, significant difference from hypothermia/rewarming groups (HR). Ctr, normal control cultured endothelial cells; HR, hypothermia/rewarming-treated endothelial cells, endothelial cells were cultured at 4°C for 12 hours, then at 37°C for 2 hours; HR+IL-6: endothelial cells were incubated with IL-6 for 4 hours, followed by culturing at 4°C for 12 hours, then at 37°C for 2 hours. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 1 IL-6 treatment increases the survival of steatotic Zucker rat liver isografts. Lean recipient rats were transplanted with lean rat livers (L to L), obese Zucker rat livers (Fa to L), and IL-6-pretreated Zucker rat livers (IL-6+Fa to L), which were perfused and cold preserved in (A ) UW solution for 6 hours; or (B) saline solution (S) for 1 hour. For the IL-6 treatment group, IL-6 (2 μg/mL) was included in the UW or saline solution. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 2 IL-6 pretreatment protects against steatotic Zucker rat liver isograft injury. (A ) Photomicrographs of representative, H & E-stained liver sections from IL-6-treated and untreated steatotic liver isografts at 1, 3, and 12 hours following transplantation (original magnification × 100). Note the significant necrosis in untreated but not in IL-6-treated steatotic liver isografts 12 hours after reperfusion. (B) Serum transaminase levels (ALT and AST) at various time points following liver transplantation. Values are means ± SEM from 3 independent experiments. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 3 IL-6 treatment protects against sinusoidal endothelial cell loss in steatotic Zucker rat liver isografts. Liver sections from steatotic liver isografts and IL-6-treated steatotic liver isografts obtained at 1 hours and 3 hours following transplantation were immunostained with an anti-RECA-1 antibody (blue color, membranous and cytoplasmic staining) and shown at ×100 (A ) or ×400 magnification (B). Marked sinusoidal congestion and sinusoidal endothelial cell apoptosis is visible in untreated but not in IL-6-pretreated steatotic isografts. (C ) Immunostained RECA-1 positive endothelial cells were counted at 200-fold magnification from ten randomly selected fields. Values are means ± SEM (n = 3). Significant difference from corresponding nontreated steatotic isografts is indicated by ∗P < 0.001. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 6 IL-6 treatment activates STAT3 in steatotic Zucker rat liver isografts. (A ) Western blot analysis of activated STAT3 in liver isografts. Protein extracts from lean liver isografts, steatotic liver isografts, and IL-6-treated steatotic liver isografts at various time points after transplantation were immunoblotted with anti-phospho-STAT3, anti-STAT3, and anti-Bcl-XL antibodies. L to L: lean donor to lean recipient; F to L: fatty donor to lean recipient; IL-6+F to L: fatty donor treated with IL-6 to lean recipient. (B) Western blot analysis of activated STAT3 in steatotic liver isografts treated with various concentrations of IL-6 1 hour after transplantation. (C ) Immunohistochemistry of activated STAT3 in liver isografts. Liver sections from steatotic liver isografts and IL-6-treated steatotic liver isografts 1 hour following transplantation were immunstained with an anti-phospho-STAT3 antibody (brown color, nuclear staining). Red arrows: activated STAT3 in hepatocytes; Black arrows: activated STAT3 in endothelial cells in central vein; Yellow arrows: activated STAT3 in sinusoidal lining endothelial cells. (D) Liver sections from IL-6-treated steatotic liver isografts 1 hour following transplantation were doubly immunostained with an anti-RECA-1 antibody (brown color, membranous and cytoplasmic staining) and an anti-phospho-STAT3 antibody (blue color, nuclear staining). (E ) Sinusoidal endothelial cells and Kupffer cells were isolated and stimulated with IL-6 (50 ng/mL) for various time points. Cell extracts were prepared and immunoblotted with anti-phospho-STAT3 and anti-STAT3 antibodies. (F ) Zucker rat livers and lean rat livers were perfused and stored in saline solution containing IL-6 (2 μg/mL) for 1 hour and 3 hours. Liver extracts were then prepared and immunoblotted with anti-phospho-STAT3 and anti-STAT3 antibodies. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 7 IL-6 improves microcirculation in steatotic liver isografts following transplantation. (A ) Lean liver isografts, steatotic liver isografts, and IL-6-treated steatotic liver isografts at 5 minutes following transplantation. Note the heterogeneous mottled appearance of steatotic isografts and the homogenous, normal appearance of lean and IL-6-treated steatotic isografts. (B) Laser Doppler imaging of the microcirculation at before and at 10 and 60 minutes after transplantation (in panel g the image shown is before the in vitro treatment with IL-6). (C ) The density of microcirculation in panel B was quantified. Values are shown as means ± SEM from 3 rats at each time point. ∗P < 0.05, ∗∗P < 0.01 vs. corresponding value in F to L group. (D) Fluorescent microsphere analysis of microcirculation was performed as described in Materials and Methods. Values are shown as means ± SEM from 3 rats at each time point. ∗P < 0,001, P < 0.01, significant differences from corresponding nontreated groups. L to L: lean to lean; F to L: fatty to lean; and IL-6+F to L: IL-6 treated fatty liver to lean transplantation. Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)

Figure 8 In vitro IL-6 treatment markedly reduces mortality associated with fatty liver transplants from alcohol-fed rats. Donor livers from alcohol-fed rats were perfused and cold preserved in saline solution with or without IL-6 (2 μg/mL) for 1 hour, followed by transplantation into normal control rats. IL-6-treated group had a 40% survival rate at day 20 (4 of 10), whereas, untreated group had a 100% death rate at day 5 (10 of 10). Gastroenterology 2003 125, 202-215DOI: (10.1016/S0016-5085(03)00696-6)