IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes

We explored the effect of extracellular matrix (ECM) produced by fetal and adult hepatocytes on tissue-specific gene expression and proliferation of fetal and adult hepatocytes. Adult hepatocytes ECM strongly induced expression of both albumin and HNF-4 in adult hepatocytes. In contrast, fibroblast ECM reduced the expression of mRNAs for albumin and alpha-fetoprotein in fetal hepatocytes. Adult hepatocytes ECM also increased the activity of liver-specific enzymes of adult hepatocytes (DPP IV and glucose-6-phosphatase) in both fetal and adult hepatocytes, while fetal hepatocyte-derived ECM increased activity of the fetal hepatocyte enzyme GGT in fetal hepatocytes. Fibroblast ECM was inhibitory for the activity of all enzymes assayed. Removal of heparin chains from the various matrices by pretreatment of the ECM with heparinase resulted in reduction of glucose-6-phosphatase and DPP IV in adult hepatocytes. Removal of chondroitin sulfate chains from fetal hepatocyte-derived ECM resulted in loss of induction of GGT in the fetal cells. Fetal hepatocytes proliferated best on adult hepatocyte-derived ECM. Adult hepatocytes showed only modest proliferation on both fetal and adult hepatocytes ECM and their growth was inhibited by fibroblast ECM. In conclusion, adult hepatocyte ECM better supports the expression of adult genes, whereas fetal hepatocyte ECM induced expression of fetal genes. Fibroblast derived-ECM was inhibitory for both proliferation and tissue-specific gene expression in fetal and adult hepatocytes. The data support a role for heparan sulfate being the active element in adult ECM, and chondroitin sulfate being the active element in fetal ECM.     

Norepinephrine stimulates interleukin-6 mRNA expression in primary cultured rat hepatocytes

Non-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of alpha- and beta-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1beta stimulated IL-6 mRNA expression in hepatocytes. IL-1beta was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1beta production from non-parenchymal liver cells.     

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained for 6 d in the hierarchical co-culture. When a rat hepatoma cell line H4-II-E-C3, which retains the induction of tyrosine aminotransferase (TAT), was co-cultured in a monolayer with NPLCs, dose-dependent stimulation of TAT induction was observed. In a hierarchical co-culture, NPLCs further stimulated TAT induction in H4-II-E-C3 cells. Since peritoneal macrophages could stimulate TAT induction in hepatocytes in both monolayer and hierarchical co-cultures, bone marrow cells, which can proliferate and differentiate into macrophages in vitro, were investigated as a possible substitute for NPLCs. Bone marrow cells isolated from rat femurs were cultivated in the presence of IL-3 and macrophage colony-stimulating factor (M-CSF), and co-cultured with hepatocytes. Urea synthesis and TAT induction of hepatocytes were stimulated in the co-culture. The co-culture of bone marrow and H4-II-E-C3 cells, both of which have proliferation ability in vitro, was also shown to be effective in stimulating liver functions. The hierarchical configuration, in which two cell types can communicate with the soluble factor(s) through a collagen layer, was found to be more effective than a monolayer in long-term co-culture.     

Tyrosine aminotransferase and gamma-glutamyl transferase activity in human fetal hepatocyte primary cultures under proliferative conditions

The ontogeny of gamma-glutamyl transferase (GGTase; E.C.2.3.2.2) and tyrosine aminotransferase (TAT; E.C.2.6.1.5) activities in 14 to 36 weeks gestational and neonatal hepatocytes during development of human fetal liver was studied. Subsequently, 20-24 weeks gestational hepatocytes were cultured in media supplemented with epidermal growth factor (EGF) and insulin with or without glucagon and dexamethasone to investigate the proliferation and differentiation of fetal hepatocyte in vitro using GGTase and TAT as biochemical markers. During the development of the liver, the activity of GGTase increased continuously from the first trimester through the third trimester and decreased (p < 0.001) in neonates. A low basal level of TAT activity was seen only during the third trimester, which then increased significantly (p < 0.001) in neonates. Fetal hepatocytes, in the presence of EGF and insulin, undergo proliferation from the fourth to 10th day with an increase in cell number (p < 0.001) and concomitant increase (p < 0.001) in GGTase activity. As the cells attain confluence, enzyme activity decreased significantly (p < 0.001) from the 10th to 16th day. Maximal TAT activity (p < 0.001) was observed at 48 h of culture, which decreased, but not significantly, during cell proliferation and the enzyme activity was regained as the cultures attained confluence. Furthermore, TAT activity was induced synergistically (p<0.001) in the presence of glucagon and dexamethasone, while GGTase was inhibited (p<0.001). These results indicate that GGTase increases with proliferation, whereas TAT, once it has been expressed, is not suppressed during cell proliferation. In conclusion, human fetal hepatocytes undergo enzymic differentiation by 48 h of culture, and proliferate with an increase in GGTase in the presence of growth factors with maintenance of differentiated status up to the studied 16 days of culture.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2)

The effect of rIL-6 on the growth and differentiation of highly purified human peripheral blood B cells was examined. IL-6 alone induced minimal incorporation of [3H]thymidine by unstimulated or Staphylococcus aureus (SA)-stimulated B cells and did not augment proliferation induced by SA and IL-2. Similarly, IL-6 alone did not support the generation of Ig-secreting cells (ISC) or induce the secretion of Ig by unstimulated or SA-stimulated B cells. However, IL-6 did augment the generation of ISC and the secretion of all isotypes of Ig induced by SA and IL-2. Maximal enhancement of B cell responsiveness by IL-6 required its presence from the initiation of culture. Delaying the addition of IL-6 to B cells stimulated with SA and IL-2 beyond 24 h diminished its effect on ISC generation. However, increased Ig production but not ISC generation was observed when IL-6 was added to B cells that had been preactivated for 48 h with SA and IL-2. This effect was most marked when the activated B cells were also stimulated with IL-2. IL-6 in combination with other cytokines such as IL-1 and IL-4 did not induce the secretion of Ig or generation of ISC in the absence of IL-2. Moreover, antibody to IL-6 did not inhibit the effect of IL-2 on the growth and differentiation of B cells stimulated with SA, but did inhibit the IL-6-induced augmentation of Ig secretion by B cells stimulated with SA and IL-2. IL-6 alone enhanced T cell dependent induction of B cell differentiation stimulated by PWM. Part of this enhancement was related to its capacity to increase the production of IL-2 in these cultures. These results indicate that IL-6 has several direct enhancing effects on the differentiation of B cells, all of which are at least in part dependent on the presence of IL-2. In addition, IL-6 can indirectly increase B cell differentiation by increasing IL-2 production by T cells.     

Estrogen and progesterone regulate the IL-6 signal transduction pathway in antibody secreting cells

Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and traslocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

Differential effects of LPS and TGF-beta on the production of IL-6 and IL-12 by Langerhans cells, splenic dendritic cells, and macrophages

We examined modulatory effects of lipopolysaccharide (LPS) on IL-6 and IL-12 production by mouse Langerhans cells (LC), spleen-derived CD11c+ dendritic cells (DC), and macrophages (Mphi). Low dose LPS (1 ng/ml) increased IL-6 and IL-12 p40 production by Mphi. LPS slightly augmented IL-6 production but showed no effect on IL-12 p40 production by DC. In contrast, only high dose LPS (1 microg/ml) induced IL-6 but not IL-12 p40 production by LC. CD14 expression was the highest on Mphi and then on DC, but not on LC, which may explain the difference in responsiveness to LPS. We also found that TGF-beta inhibited IL-6 and IL-12 p40 production by LPS-stimulated Mphi. However, TGF-beta did not inhibit IL-6 production and even enhanced IL-12 p40 production by anti-CD40/IFN-gamma-stimulated Mphi. Concerning LC, TGF-beta enhanced IL-6 and IL-12 p40 production when stimulated with anti-CD40/IFN-gamma alone or with anti-CD40/IFN-gamma and LPS. Taken together, these findings indicate diverse effects of LPS and TGF-beta on these antigen presenting cells, which probably represents their differential roles in the innate immunity.     

Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.