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We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.

Interleukin-11 is a hematopoietic cytokine that signals via the signal transducer gp130. Although gp130 is ubiquitously expressed, interleukine-11 responsiveness is restricted to cells that express the interleukine-11 receptor alpha-subunit. The interleukine-11 receptor alpha-subunit can be functionally replaced by its soluble form indicating that the transmembrane and cytoplasmic parts are not required for signal transduction. Here, we show that a recombinant fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 acts as a superagonist on cells expressing gp130 but lacking the membrane-bound interleukine-11 receptor alpha-subunit. It induces acute phase protein synthesis in hepatoma cells and efficiently promotes proliferation of Ba/F3 cells stably, transfected with gp130. In these bioassays, the fusion protein of a fragment of the human interleukine-11 receptor alpha-subunit ectodomain linked to human interleukine-11 is 50 times more potent than the combination of interleukine-11 and the soluble interleukine-11 receptor alpha-subunit. Thus, our findings support the concept that covalent fusion of two soluble proteins required for receptor activation dramatically increases their bioactivity.

Interleukin-6 mediates its pleiotropic effects by interacting with its membrane bound receptor (gp80) or the soluble counterpart gp54, resulting in activation of a complex that includes the transducer protein gp130. We have generated a polyclonal antibody against the rat soluble IL-6 receptor (anti-rat sIL-6R) in rabbits. By Western blot analysis we show that purified anti-rat sIL-6R IgG antibody reacts specifically with recombinant rat sIL-6R generated from E. coli, baculovirus or adenovirus expression systems. Anti-rat sIL-6R inhibited IL-6-induced acute phase protein synthesis in rat (H35) but not human (HepG2) hepatoma cells, and did not affect stimulation of those cells by Oncostatin-M. Conversely, on the mouse hybridoma B9 cell line, IgG anti-rat sIL-6R showed a dose-dependent stimulation of proliferation. Fab fragments of this antibody did not stimulate, but abrogated IL-6-mediated hepatoma cell stimulation and B9 cell proliferation. Gel shift analysis of STAT nuclear factors showed activation of STAT DNA binding in nuclei of B9 cells treated with IgG anti-rat sIL-6R, whereas in H35, NIH-3T3 and M1 cells, only IL-6 could trigger a similar STAT activation. Our data suggest that mechanisms of IL-6 receptor activation and signalling in mouse B9 hybridoma cells show subtle but important differences from other IL-6-responsive cells.

Recently, a novel cytokine, cardiotrophin-1 (CT-1), was cloned and found to induce cardiac myocyte hypertrophy in vitro. Amino acid sequence similarity showed CT-1 to be a member of the IL-6/LIF/CNTF/OSM/IL-11 cytokine family. Since all known members of the IL-6 cytokine family induce an hepatic acute phase protein (APP) gene expression, we investigated the ability of CT-1 to induce a liver acute phase response. Upon stimulation of rat hepatoma cells, CT-1 and LIF induced the strongest rat fibrinogen mRNA expression, OSM and IL-6 induced a less pronounced response. When human hepatoma cells and primary rat hepatocytes were stimulated with CT-1, the expression of human haptoglobin and rat alpha 2-macroglobulin mRNA was induced. The induction of the acute phase response was dose- and time-dependent. In this study we demonstrate that CT-1, a novel cytokine belonging to the IL-6 cytokine family, is a hepatocyte stimulating factor.

Interleukin 4 (IL4) has been shown to exhibit anti-inflammatory effects by inhibiting the secretion by monocytes of proinflammatory cytokines such as interleukin 1 (IL1), interleukin 6 (IL6), and tumor necrosis factor (TNF) and by inducing the secretion of the IL1 receptor antagonist. We investigated the role of this cytokine on the production of acute-phase proteins in primary human hepatocyte cultures. Cells were exposed to either IL4 and/or IL6, the most potent mediator of hepatic acute phase proteins. IL4 led to decreased production of haptoglobin, C-reactive protein and albumin while alpha 1-antitrypsin and fibrinogen remained unaffected. These inhibitory effects of IL4 were also observed at the mRNA level. In addition, IL4 inhibited the IL6-induced production of haptoglobin although it had no effect on the induced C-reactive protein and fibrinogen. Our results demonstrate that IL4 can affect the production of a subset of acute-phase proteins by human hepatocytes and can antagonize some of the effects of IL6. These observations reinforce the notion that IL4 can be considered as an anti-inflammatory cytokine.

During inflammatory states, hepatocytes are induced to synthesize and secrete a group of proteins called acute-phase proteins. It has recently been shown that besides interleukin-6 (IL-6), related cytokines such as leukemia inhibitory factor, oncostation M and interleukin-11 are also mediators of the hepatic acute-phase response. All these mediators belong to the hematopoietic family of alpha-helical cytokines. Here we show that an additional member of this cytokine family, ciliary neurotrophic factor (CNTF), induces the hepatic acute-phase protein genes haptoglobin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and beta-fibrinogen in human hepatoma cells (HepG2) and in primary rat hepatocytes with a time course and dose-response comparable with that of IL-6. Our next aim was to define the receptor components used by CNTF on hepatic cells. Using a cell-free binding assay we exclude that CNTF binds to the 80 kDa IL-6 receptor, a protein with significant homology to the CNTF receptor which has recently been cloned from neuroblastoma cells. In human hepatoma cells (Hep3B) which lack the leukemia inhibitory factor receptor, CNTF was not able to induce acute-phase protein synthesis, indicating that this receptor protein may be part of the functional CNTF receptor on hepatic cells.

The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6-mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin.

Rat hepatocytes cultured for 2 days with interleukin-6 show increased synthesis of acute phase proteins and enhanced accumulation of 14C-labelled alpha-aminoisobutyric acid. Transforming growth factor-beta 1 (0.1-10 ng/ml) inhibits whereas epidermal growth factor (1-100 ng/ml) enhances both basal and interleukin-6-induced amino acid uptake by rat hepatocytes with only a slight alteration of acute phase protein synthesis.

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.

Synthesis of the growth hormone-regulated anti-protease GHR-P63 by rat hepatocytes was strongly reduced during acute inflammation. This decrease was detected 8 h after the onset of inflammation and reached a maximum after 24 h. A decrease in the GHR-P63 mRNA level measured by in vitro translation and by hybridization mainly accounted for the alteration of GHR-P63 synthesis. Besides this major pretranslational mechanism, inflammation also interfered with GHR-P63 synthesis at a posttranslational level. This was indicated by the production of abnormal immunoprecipitable species at early stages of the acute-phase response.