Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes.

Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF-beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response.     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

肝细胞生长因子刺激大鼠离体肝细胞DNA合成的剂量—与时间—效应

本工作采用３ＨＴｄＲ掺入ＤＮＡ法观察重组人肝细胞生长因子（ｒｈＨＧＦ）刺激大鼠离体肝细胞ＤＮＡ合成的剂量与时间效应。实验结果表明：ｒｈＨＧＦ是最强的促肝细胞分裂剂,在一定剂量范围内,ｒｈＨＧＦ与肝细胞ＤＮＡ合成有明显的量效关系。１ｎｇ／ｍｌｒｈＨＧＦ即可引起３ＨＴｄＲ掺入显著增加（Ｐ＜０．０１）,随剂量增加,刺激ＤＮＡ合成的效应也随之增强;１０ｎｇ／ｍｌ时３ＨＴｄＲ掺入量最大,较对照组高７倍（Ｐ＜０．００１）,剂量再增加即出现抑制效应;ｒｈＨＧＦ刺激肝细胞ＤＮＡ合成存在时间效应关系,表现为ｒｈＨＧＦ作用２４ｈ,ＤＮＡ合成量明显高于对照组（Ｐ＜０．０１）,４８ｈ作用达最高（Ｐ＜０．００１）,随后开始下降,至９６ｈ下降到相当于２４ｈ的水平。     

Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression.

Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.     

Limited effects of recombinant human and murine interleukin 1 and tumour necrosis factor on production of acute phase proteins by cultured rat hepatocytes

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.     

IL-1beta and IL-6 modulate apolipoprotein E gene expression in rat hepatocyte primary culture

Incubation of rat hepatocytes in primary culture with IL-1beta at a concentration of 2.5 units/ml resulted in an increase (+80%) in the amount of apoE mRNA without any effect upon apoE synthesis. IL-6 at a low concentration (10 units/ml) induced a decrease (-35%) in the amount of apoE mRNA, but increased apoE synthesis (+28%). No effect was observed with higher concentrations of IL-1beta (10 units/ml) or IL-6 (100 units/ml). These results suggest that inflammatory cytokines IL-1beta and IL-6 modulate the expression of apoE gene in cultured rat hepatocytes, at a concentration that does not induce the acute phase response.     

Glutamic acid potentiates hepatocyte response to mitogens in primary culture

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1–20 μM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [¹²⁵I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley-Liss, Inc.     

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

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.     

Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines.

Hepatocyte growth factor (HGF) has potent mitogenic activity for mature hepatocytes and various normal epithelial cells. We now have evidence that HGF at 1-10 ng/ml, strongly inhibits the growth of HepG2 hepatocellular carcinoma cells, B6/F1 melanoma cells and KB squamous carcinoma cells. These tumor cells express high affinity receptors for HGF with a Kd of 25-28 pM, similar to findings with hepatocytes. HGF at 1-100 ng/ml had no significant cytolytic effect on tumor cells. Therefore, the anti-proliferative effect of HGF on tumor cells seems to be cytostatic, not cytolytic. As HGF apparently has bidirectional effects on cell growth, the possibility that it can serve as an anti-tumor agent merits attention.     

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).     

Acute effects of interleukin 1 alpha and 6 on intermediary metabolism in freshly isolated rat hepatocytes

Using hepatocytes in suspension, freshly isolated from adult male fed rats, we studied the acute influence of recombinant human interleukins 1 alpha, 2 and 6 on glycogen and fatty acid metabolism. By far the largest effects were observed with interleukin-1 alpha: short incubations (up to 60 min) sufficed to depress glycogen synthesis in a dose-dependent manner, while the rates of glycogenolysis and glycolysis were increased as indicated by the release of glucose and lactate. Interleukin-6 acted similarly, though being much less effective on a molar basis, whereas interleukin-2 only caused a small increase in lactate production. In hepatocytes from 24h-starved rats interleukin-1 alpha caused a minor stimulation of gluconeogenesis. Although neither fatty acid synthesis nor oxidation of fatty acids in quiescent hepatocytes from fed rats was significantly affected by interleukins, interleukin-1 alpha was able to cause appreciable inhibition of fatty acid synthesis in hepatocytes from regenerating liver (isolated 22h after partial hepatectomy). It is concluded (i) that interleukins, in particular interleukin-1 alpha, acutely promote hepatic glucose release, and (ii) that transition of adult hepatocytes from a quiescent into a proliferatory state allows the occurrence of rapid effects of interleukin-1 alpha on fatty acid metabolism.     

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.     

Distinct effects of transforming growth factor-beta on EGF receptors and EGF-induced DNA synthesis in primary cultured rat hepatocytes

Previous studies showed that transforming growth factor-beta (TGF-beta, 1 ng/ml) strongly inhibited DNA synthesis induced by epidermal growth factor (EGF) in primary cultures of adult rat hepatocytes. This paper reports that TGF-beta (4 ng/ml) caused marked increase of EGF-binding to cultured rat hepatocytes. The binding increased biphasically with time to a maximum after treatment with TGF-beta for 12 h. Scatchard analysis showed that adult rat hepatocytes had a single class of non-cooperative binding sites with a Kd of 1.5 nM, that there were 1.4 X 10(5) binding sites/cell, and that TGF-beta increased the number of binding sites without changing the Kd value. The increase in EGF binding sites by TGF-beta was dose-dependent and the dose that elicited the maximum increase was about 10 times that which inhibited DNA synthesis stimulated by EGF. These findings suggest that the effect of TGF-beta in modulating the EGF receptor is not directly related to that in inhibiting DNA synthesis in adult rat hepatocytes.     

Appearance of hybridoma growth factor/interleukin-6 in the serum of mice bearing a methylcholanthrene-induced sarcoma

Serum concentrations of hybridoma growth factor/interleukin-6 progressively increased in mice bearing a transplantable methylcholanthrene-induced sarcoma with tumor growth. Elevated HGF/interleukin-6 concentrations were also positively correlated with increased serum concentrations of the hepatic acute phase reactant protein, amyloid P. Daily Indomethacin treatment of sarcoma-bearing mice prolonged survival and reduced the magnitude of the serum amyloid P response, but failed to attenuate either tumor growth or serum HGF/interleukin-6 responses. Since previous studies have demonstrated that neither interleukin-1 nor tumor necrosis factor-alpha can be detected in the serum of these sarcoma-bearing mice, and that HGF/interleukin-6 is a principal mediator of the hepatic acute phase response, we conclude that circulating HGF/interleukin-6 may contribute significantly to the host responses which accompany experimentally-introduced cancer. Furthermore, prostanoid inhibition does not appear to regulate the synthesis and release of HGF/interleukin-6 during tumor growth.     

Plasma levels of growth hormone, corticosterone and insulin, during induced hepatocyte synchronization in young rats

A wave of synchronous hepatocytes entering the cell cycle can be obtained in vivo after a subcutaneous injection (e.g. of casein) in rats at around Post-natal Day 10, when plasma growth hormone (GH) levels reach a low plateau (40 +/- 2 ng/ml) and liver cell proliferation rate is high. The present work reports the following changes in plasma hormone concentrations after synchronization of 20% of the hepatocyte population: (1) during the G1 phase (i.e. 6-12 hr after the mitogenic trigger), plasma GH concentration has dropped further (25 +/- 1.5 ng/ml). It was back to 90% of control levels during the S phase, mitosis and the following response including a transitory decrease in labelling index below control values. Injected together with the irritating mitotic trigger, a single dose of rat GH reduced the cell synchronization and post-synchronization effects by 50%. (2) Plasma corticosterone levels varied inversely to those of GH, increasing to twice the control values during G1 and were back to physiological levels when synchronized hepatocytes entered the S phase. (3) Variations in insulin levels were similar to that of corticosterone, with narrower ranges and reduced amplitudes. Our data suggest a possible correlation between the observed variations in plasma hormone levels and the induced synchronous hepatocyte response.     

Biological effects of biosynthetic human EGF on the growth of mammalian cells in vitro

The effects were examined of biosynthetic human epidermal growth factor (Bh-EGF) produced from cloned E. coli on DNA synthesis and all divisions of 13 different kinds of primary and established cell lines. Primary cultures of mammary epithelia, hepatocytes and stomach cells were strongly stimulated by EGF to undergo DNA synthesis in serum-free culture medium with concentrations of Bh-EGF as low as 0.1-10 ng/ml. In sharp contrast, 0.1-100 ng/ml of Bh-EGF failed to enhance thymidine incorporation into DNA when applied to established cell lines using the serum-free medium. Higher concentrations of Bh-EGF (30-100 ng/ml) promoted morphological changes only in hepatocytes, e.g., elongation, enlargement and projection of their cytoplasm. The above results were also obtained in mouse EGF (m-EGF). In our binding assay, Bh-EGF competed against [125I]-m-EGF with a one-fourth to one-fifth efficacy when compared with m-EGF. It was concluded that the in vitro biological activity of Bh-EGF was similar to that of m-EGF.     

A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide.

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.