Recently, it was shown that hepatocyte DNA synthesis after partial hepatectomy (PH) is impaired in interleukin-6-deficient (IL-6 ؊/؊ ) mice, which results in significantly delayed, but eventual, recovery of normal liver weight, compared with the IL-6 ؉/؉ controls. Four possible compensatory mechanisms might explain this phenomenon: 1) hepatocyte hypertrophy; 2) activation of the oval cell compartment and subsequent maturation to hepatocytes; 3) non-oval biliary epithelial cell (BEC) proliferation; and/or 4) differential rates of apoptotic cell death in the regenerating liver. These hypotheses were tested by subjecting IL-6 ؊/؊ and IL-6 ؉/؉ mice to PH and determining sequential liver weight, histology, hepatocyte and BEC 5Ј-bromo-2Ј-deoxyuridine (BrdU) labeling, liver DNA content, ␣-fetoprotein (AFP) mRNA production, and apoptosis at several time points after PH. Consistent with previous studies, we show that the absence of IL-6 significantly impairs hepatocyte DNA synthesis and delays liver weight recovery after PH, but the defect observed in this study is less severe than that previously reported, and no excess mortality, massive necrosis on histology, nor differences in liver injury test are seen. Interestingly, the IL-6 ؊/؊ mice show more hepatocyte BrdU pulse labeling than the IL-6 ؉/؉ controls at 24 hours, but less at 36, 48, and 60 hours. Continuous BrdU infusion up to 60 hours after PH showed a cumulative hepatocyte labeling index of 79.5% in IL-6 ؉/؉ mice and 70.8% in IL-6 ؊/؊ mice, respectively (P F .03). However, despite a lower labeling index and significantly delayed weight recovery, hepatic mass was equally restored in the two groups by 96 hours. There was no evidence of oval cell proliferation in the IL-6 ؊/؊ mice, as determined by routine histology and AFP mRNA analysis, and non-oval BEC proliferation was also slightly impaired in the IL-6 ؊/؊ mice compared with the IL-6 ؉/؉ mice. In addition, liver DNA content per gram of liver showed an increase compared with normal at 60 hours in both groups, but by 96 hours, there was no difference between the two groups. Thus, neither oval cell nor BEC proliferation, nor hepatocyte hypertrophy, could account for the eventual equivalent weight recovery. There was, however, a difference between the two groups in the rate of apoptosis. In normal livers of both IL-6 ؊/؊ and IL-6 ؉/؉ mice, apoptotic cells were uncommon, and even fewer such cells were detected at 24, 36, and 48 hours after PH. Between 60 and 96 hours after PH, a wave of apoptosis spread through the livers of both groups. The number of apoptotic cells was directly proportional to the magnitude of hepatocyte BrdU labeling and liver DNA content after PH, and the difference between the nadir of apoptosis at 24 hours and the peak at 96 hours was greater for the IL-6 ؉/؉ mice. In addition, a direct comparison between the two groups at 96 hours showed that hepatocyte apoptosis was significantly lower in the IL-6 ؊/؊ versus the IL-6 ؉/؉ mice (P F .02). Treatment of the IL-6 ؊/؊ mice with rIL-6 completely r...

The interleukin-6 (IL-6)/gp-80 and hepatocyte growth factor (HGF)/met ligand/receptor systems have been shown to stimulate biliary epithelial cell (BEC) DNA synthesis in vitro. The mRNA and protein production of these two in vitro mitogens were mapped in vivo during the first week after bile duct ligation (BDL) when peak BEC DNA synthesis is seen. Changes around the biliary tree were compared with those seen in the peripheral liver using a combination of Northern blotting and a unique biliary tree isolation technique, in which the bile ducts and the surrounding portal stroma and inflammatory cells are separated from the hepatocytes by perfusion digestion. Further localization was performed with in situ hybridization and immunohistochemistry. In the normal liver, there is low-level expression of HGF mRNA by periportal stellate cells, and HGF protein localizes to these cells and to neutrophils; extracellular HGF protein is present in the bile. There is no detectable IL-6 mRNA by Northern analysis or IL-6 protein expression in the normal liver, but both met and IL-6 receptor (IL-6R) mRNA are detectable; met mRNA is expressed strongly in the biliary tree, and met protein is expressed weakly on hepatocytes and strongly on BEC. IL-6R mRNA is weakly expressed in the biliary tree, and IL-6R protein is detectable on hepatocytes, with a periportal-to-perivenular gradient, but not on BEC. During the first 3 days after BDL, HGF mRNA expression is increased in both the biliary tree and in the peripheral liver, and production is localized to stellate cells, periductal neutrophils, and stromal cells, which typically accompany the proliferating ductules. IL-6 mRNA and protein were detected only near the biliary tree after BDL, and not in the peripheral liver, and the production was localized to periductal hematolymphoid cells, which had the morphological appearance of macrophages and/or dendritic cells. There is also a distinct up-regulation of met and gp-80 mRNA and protein in the biliary tree, which is stronger than that seen in the peripheral liver. Met protein expression is increased, and IL-6R(gp-80) protein is induced on the proliferating BEC, consistent with the participation of both the HGF/met and IL-6/gp-80 systems in the early phases of type I ductular reactions. These observations show that periductal hematolymphoid and stromal cells are the source of BEC growth factors, and receptors for these factors are up-regulated on BEC during active ductular proliferation. Complex interactions between the inflammatory, stromal, and BEC results in a dysmorphogenic repair response that eventually leads to cirrhosis. (HEPATOLOGY 1998;28:1260-1268.)

A well characterized human cholangiocarcinoma (CC) cell line, SG231, was compared with primary cultures of normal human biliary epithelial cells (BECs) for alterations in interleukin 6 (IL-6) and hepatocyte growth factor (HGF)-mediated stimulation and transforming growth factor ␤1 (TGF-␤1) and activin A-mediated inhibition of growth. Results were compared with immunolabeling of the original tumor and after injection of SG231 into the liver of BALB/cByJ-scid mice. In vitro, both BECs and CCs expressed met, gp80, and gp130 messenger RNA (mRNA) and protein, but the levels of expression were higher in the CCs than in the BECs. In both the CCs and BECs, exogenous HGF or IL-6 induced phosphorylation of met or gp130, respectively, and a concentration-dependent increase in DNA synthesis. However, the CCs but not BECs, continued to grow in basal serum-free medium (SFM) and spontaneously produced both IL-6 and HGF under these conditions, which resulted in auto-phosphorylation of gp130 and met, respectively; and neutralizing anti-HGF or anti-IL-6 alone inhibited CC growth, indicative of autocrine growth control circuits. Conversely, activin A inhibits the growth of both BECs and CCs, but does not significantly increase apoptosis. Activin-A-induced growth inhibition of both CCs and BECs can be reversed by 100 ng/mL exogenous IL-6, but not by 10 to 100 ng/mL HGF. TGF-␤1 inhibited the growth of BECs but had no mitoinhibitory or proapoptotic effects on CCs. Immunolabeling of the original tumor and after inoculation into scid mice showed positive staining for met, gp130, gp80, and IL-6. This study contributes to a further understanding of BEC growth control and derangements that can occur during cholangiocarcinogenesis. (HEPATOL-OGY 2000;32:26-35.)

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. Interleukin-6 (IL-6)/gp-130 signaling is involved acutely in hepatocyte 1,2 and biliary epithelial cell (BEC) growth control and pathobiology. For example, liver regeneration after partial hepatectomy (PH) is delayed in IL-6 Ϫ/Ϫ mice, 1,2 whereas IL-6/sIL-6R double transgenic mice develop hepatocellular hyperplasia and adenomas. 3 Although data on the in vitro effects of IL-6 on hepatocyte proliferation are conflicting, 4,5 there are at least two mechanisms by which IL-6/gp-130 promotes BEC growth in vitro. IL-6 is able to directly stimulate BEC DNA synthesis 6 and inhibit apoptosis, via an increase of the bcl-2/ bax ratio. 7 When stimulated by other proinflammatory cytokines or phorbol esters, nonneoplastic BEC can also produce and secrete IL-6, which can then act as an autocrine growth factor under conditions of BEC stress. 6,8,9 Serum and liver IL-6 levels are also elevated in patients with chronic inflammatory liver diseases. 10 -13 In this setting, IL-6 has traditionally been considered to exert a profibrogenic and mitoinhibitory influence on the development of cirrhosis. 10,14 -17 However, in other "chronic inflammatory proliferative disorders," such as psoriasis and rheumatoid arthritis, IL-6 has been linked with keratinocyte and synovial cell proliferation, respectively. 18 In addition, recent studies have shown that gp130 sign...

The mild phenotypical differences between IL-6+/+ and IL-6-/- mice in the acute response to BDL is most likely attributable to the redundancy of the gp-130 signaling system. However, the long-term response to BDL results in a distinct phenotype in the IL-6-/- mice, marked by a relentless rise in serum total bilirubin and an inability to maintain compensatory increase in liver mass.