Vol. 27, No. 6 Prmted in Japan
Gastroenterologia Japonica Copyright 9 1992 by The Japanese Society of Gastroenterology
The effect of IL-6 on the des-gamma-carboxy prothrombin synthesis in human hepatoma cells Minoru ONO, Hironobu KOHDA, Tooru NARAKI, Hitoyoshi OHTA, Motoyuki OHHIRA, Chihiro SEKIYA, and Masayoshi NAMIKI Third Departments of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan Summary: Effects of several cytokines on des-gamma-carboxy prothrombin (PIVKA II) synthesis in human hepatoma cells were investigated to know the process of PIVKA II production during a liver allograft rejection. Human recombinant interleukin-6 (IL-6) significantly stimulated the PIVKA II synthesis without any influence on the cell proliferation. The effect was almost completely neutralized by the specific anti-IL-6 antibody. Neither tumor necrosis factor (TNF), interleukin-1 (IL-1) nor interferon-gamma (IFN-gamma) had such a stimulative effect. IL-6 appears to stimulate PIVKA II production, and would be a candidate of factors that enhance the production of PIVKA II during a liver allograft rejection. Gastroenterol Jpn 1992;27:745- 750. Key words: IL-6; des-gamma-carboxy prothrombin; hepatocellular carcinoma.
Introduction Prothrombin is a vitamin K-dependent clotting factor which is synthesized in the liver and undergoes posttranslational gamma-carboxylation on its 10 gamma-glutamic acid (Glu) residues in the amino-terminal domain, which has the calcium binding properties essential for the biological activity of prothrombin 1. This gamma-carboxylation is catalyzed by the vitamin K-dependent carboxylase (carboxylase) which is located in liver microsome 2. In the absence of vitamin K or when the carboxylation is antagonized by warfarin sodium, des-gamma-carboxy prothrombin (PIVKA II, protein induced by vitamin K absence or antagonist) is secreted into plasma. A marked increase of PIVKA II in serum of hepatocellular carcinoma (hepatoma) patients has been reported, and this abnormal prothrombin is now used as a new serological indicator for the diagnosis of
hepatoma 3,4. Very recently, a significant increase of circulating PIVKA II level was found during liver allograft rejection s. The mechanism of the increase, however, is not known at all. In the present study, we investigated the effects of several cytokines (TNF, IL-1, IFN-gamma and IL-6) on the PIVKA II synthesis in h u m a n hepatom cell line, Hu-H16, which has been shown to spontaneously produce the abnormal prothrombin 7. Materials and Methods Chemicals H u m a n recombinant TNF-alpha (TNF), specific activity 2 x 107 unit/mg protein; human recombinant IL-l-alpha (IL-1), specific activity, 8 x 106 units/mg protein; human recombinant IL-6 (IL-6), specific activity, 1 x 107 units/mg protein; human recombinant interferon-gamma (IFN-gamma), specific activity, 2.5 • 107 units/
Received April 10, 1992. Accepted June 26, 1992. Address for correspondence: Motoyuki Ohhira, M.D., The Third Department of Internal Medicine, Asahikawa Medical College, 4-5 Nishikagura, Asahikawa, Hokkaido 078, Japan. The abbreviations used are: PIVKA II, des-gamma-carboxy prothrombin; TNF, tumor necrosis factor; IL-1, interleukin-1; IL-6, interleukin-6; IFN-gamma, interferon-gamma; ELISA, enzyme-linked immunosorbent assay.
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mg protein and polyclonal rabbit anti-human IL-6 antibody were obtained from Genzyme Corp., Cambridge, MA, USA. Cells and cell culture Hu-H1, a human hepatocellular carcinoma cell line, was grown in Eagle's Minimum Essential Medium (MEM) containing 10% fetal calf serum (Gibco, Grand Island, NY, USA), and 3% glutamine, 100 units/ml penicillin, and 100 ~tg/ml streptomycin. The cells were plated in culture flasks (Costar) and routinely maintained in a CO2 incubator at 37~ Cytokine treatment Hu-H1 cells (1 x 105 cells/well) were plated in 24-well plates and cultured for the indicated period with or without addition ofTNF, IL-1, IL-6 or IFN-gamma in culture medium. After incubation, amounts of PIVKA II and immunoreactive prothrombin secreted into medium were determined. Neutralization of lL-6 activity by anti-IL-6 antibody To verify the possible ability of IL-6 on PIVKA II induction, an inhibition study by specific antibody against IL-6 was carried out. Polyclonal rabbit anti-human IL-6 antibody was diluted by culture medium to various concentrations, and added to the IL-6 solution (50 U/ml) followed by incubation at 4~ over night. The cells (1 x 105/ well) plated in 24 well plates were cultured for 72 h in the pre-incubated medium containing IL-6 and anti-IL-6 antibody. After the culture, PIVKA II secreted into medium was determined. Assay of PIVKA II and immunoreactive prothrombin PIVKA II was measured with commercially available ELISA kits (Eitest Mono PII, Eisai, Tokyo, Japan) using the specific monoclonal antibody (E-1023) that has been shown to recognize an altered structure with conformational change in the Gla-domain of PIVKA 118,9, and is also known to react only with PIVKA II but not with fully carboxylated normal prothrombin ~~ Immunoreactive prothrombin was determined
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with ELISA using rabbit polycolonal antibody against human prothrombin, as we previously described1~ In our previous study, an increase of prothrombin precursor was found in human hepatoma tissues 11. To investigate the possibility that IL-6 stimulates the prothrombin synthesis, the amount of total immunoreactive prothrombin secreted into the medium was determined. Since, the polyclonal antibody recognizes PIVKA II as well as normal prothrombin 11, the amount of immunoreactive prothrombin determined by the ELISA was designated the "total immunoreactive prothrombin" in this study. Effect of IL-6 on the Hu-H1 cell proliferation IL-6 is shown to have a proliferative effect in several cell lines a214. To know whether the enhancement of PIVKA II production was due to the cell proliferative effect or out, we examined the 3H-TdR incorporation of Hu-H1 after IL-6 treatment as we previously described15. The cells were cultured for 24 h to 72 h in the presence or absence of various concentration of recombinant human IL-6 at a cell density of 1 x 104/ml in 96 wells microculture plates, then cells were pulsed for 4 h with 1 ~Ci/well 3H-TdR (New England Nuclear). After incubation, the cells were harvested and uptake of 3H-TdR was measured by liquid scintillation counter. Statistical analysis Statistical analysis was carried out by Student's t-test and P < 0.05 was considered as significant. Results
Effect of cytokines on PIVKA Il production in HuH1 With no addition of cytokine, the PIVKA II level after 24 h, 48 h and 72 h culture was 0, 55 + 4.5 and 170 _+ 38 mAU/ml (mean _+ SD; n = 3), respectively. When the cells were cultured for 72 h with an addition of IL-6 (100 U/ml), secreted PIVKA II significantly ( P < 0 . 0 1 ) increased to 325 + 3 9 m A U / m l (mean+SD; n = 3) as compared with the basal level in 72 h culture (Figure la). On the other hand, an addi-
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Figure 1. Effects of cytokines on des-gamma-carboxy prothrombin (PIVKA it) production of Hu-H1. Hu-H1 cells were cultured for 24 to 72 h with or without 11--6(1O0 U/ml; la), IFN-gamma (1 O0 U/ml; 1b), TNF (10 ng/ml; I c) and It--1 (10 U/ml; 1d). PIVKAII level was determined by the ELISA as described in the "Materials and Methods". Circles, no addition; Solid dots, cytokine treated. Each value was obtained from 3 samples. Bar indicates the standard deviation.
tion of IFN-gamma (Figure lb), TNF (Figure le) and IL-1 (Figure ld) resulted in a decrease of PIVKA II level. Then the cells were cultured for 72 h to 96 h with IL-6 in various concentrations.
As shown in Figure 2, IL-6 stimulated PIVKA II production dose-dependently in a range of 1 to 100U/ml. Maximum induction, which was almost double the basal level, was observed by
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I L - 6 (U/ml) Figure 2. Effect of IL-6 on PIVKA II production. Hu-H1 cells were cultured for 72 h to 96 h with IL-6 in various concentrations indicated. PIVKA U level was determined by ELISA as described in the "Materials and Methods". Each value was obtained from 5 samples. Bar indicates the standard deviation. Solid line, 96 h culture; Dotted line, 72 h culture.
addition of IL-6 at a concentration of 75 U/ml both in 72 h and 96 h culture. An extremely low PIVKA II level was detected in the corresponding ceil lysates (data not shown).
Effect of 1L-6 in the Hu-H1 cell proliferation As shown in Figure 3, little change in the 3HTdR incorporation was observed after 24 h culture with IL-6 at various concentrations, and similarly no significant change was found either after 48 h or 72 h culture. Neutralization of P1VKA H induction by anti-IL-6 antibody The polyclonal antibody inhibited the IL-6 induced PIVKA II production in a dose dependent manner (Figure 4). A maximum inhibition, about 80% inhibition, was achieved by an addition of the antibody to the concentration of 100 ~g/ml. An addition of the antibody alone made no significant change in the PIVKA II level (data not shown).
1
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50
75 100 5001000
I L - 6 (u/ml) Figure 3. [3H]Thymidine (3H-TdR) incorporation of Hu-H1 after IL6 treatment. Cells were cultured for 24 h with 11.-6in the indicated concentrations. After the culture, cells were pulsed with 3H-TdR. Each value was obtained from 5 samples and the bar indicates the standard deviation.
Effect of lL-6 on total prothrombin synthesis (Figure S) When ceils were culture for 72 h to 96 h with an addition of IL-6 in various concentrations, the amount of total prothrombin was relatively increased but not statistically significant. TNF, ILl and IFN-gamma reduced the total prothrombin level (data not shown), which might be due to the cytotoxic effect of the cytokines.
Discussion The vitamin K-dependent proteins, including several clotting factors, undergo unique posttranslational gamma-carboxylation1,2. Vitamin K-dependent carboxylase is a key enzyme catalyzing the reaction2. We have previously reported that a significant decrease in the vitamin K-dependent carboxylase activity despite an increase of prothrombin precursor plays an essential role in the PIVKA II production in hepatoma16, and that no vitamin K deficiency exists in cancerous liver tissues 1~. Very recently, Nakao et al. reported a remarkable increase of plasma PIVKA II level during acute liver allograft rejection, although the patients revealed no bleeding tendency or vitamin K deficiency, and they indicated that PIVKA II is
IL-6 and des-gamma-carboxy prothrombm
December 1992
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Figure 4. Neutralization of IL-6 activity by anti-lL-6 antibody. Polyclonal anti-lL-6 antibody was diluted to the indicated concentrations, and added to IL-6 solution (50 U/ml) followed by incubation at 4~ over night. Then cells were cultured for 72 h. The PIVKA II level, which was induced by IL-6 (50 U/ml) without an addition of antibody, was expressed as 100%. Circles, with an addition of IL-6 only; Solid dots, with an addition of both IL-6 and anti-lL-6 antibody. Each value was obtained from 3 samples. Bar indicates the standard deviation.
a new sensitive serological indicator for acute liver allograft rejection5. The mechanism, however, has not yet been clarified. Several investigators reported a marked elevation of serum TNF level during acute rejection of hepatic 17 and renal allografts18. Similarly a significant elevation of serum IL-6 protein level19 and also an increased expression of IL6 mRNA 2~ are demonstrated in acute renal allograft rejection. Thus, TNF and IL-6 are thought to be the candidates that play an important role in the allograft rejection, which would be associated with the development of cytotoxic T lymphocyte2~. However, little is known about the effect of these cytokines on the posttranslational carboxylation of prothrombin. In the present study, we demonstrated that human recombinant IL-6 stimulates the PIVKA II production in vitro in human hepatoma cells with little influence on cell proliferation. The precise mechanism by which IL-6 enhances the PIVKA II production remained to be solved. One possible explanation would be that IL-6 induces prothrombin synthesis as in the case of acutephase proteins 22, which would amplify the production of PIVKA II. Indeed, IL-6 appeared to stimulate the total prothrombin synthesis to some
o o 1'
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Figure 5. Effect of IL-6 on total prothrombin synthesis. Hu-H1 cells were cultured with various concentrations of IL-6 for 72 h to 96 h. Then the total amount of immunoreactive prothrombin was determined by ELISA as described in the "Materials and Methods". Each value was obtained from 5 samples, and the bar indicates the standard deviation. Solid line, 96 h culture; Dotted line, 72 h culture.
extent as shown in Figure 5. However, the degree of the increase in the total amount ofprothrombin was not so definite as in the case of PIVKA II. Therefore, it does not seem likely that merely the increase of total amount of prothrombin in HuH1 cells can be responsible for the PIVKA II induction by IL-6. To clarify the effect of IL-6 on prothrombin synthesis more exactly, prothrombin mRNA expression should be also investigated. An alternative possibility would be a change of the carboxylation system itself. Very recently we have found similar enhancement of PIVKA II production in Hu-H 1 by cAMP analog, in which no significant increase of total prothrombin was noted 23. Although the signal transduction of IL-6 has not been understood, IL-6 might have potency to alter the carboxylation system, particularly the vitamin K-dependent carboxylase. Recently TNF is reported to inhibit the formation of bone Gla protein (osteocalcin), one of the vitamin K-dependent proteins24. However, biological effects of TNF on the carboxylation system of vitamin K-dependent proteins are unclear. In the present study, TNF did not enhance the PIVKA II
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production in human hepatoma cells, which does not seem compatible with the clinical observations described above. The discrepancy might be, in part, due to the differences between in vitro and in vivo experimental systems. Also it may be possible that TNF has a counteraction on malignant and normal hepatocytes, as in the case of hepatocyte growth factor 25, in terms of prothrombin synthesis and its carboxylation. To confirm the effect of IL-6 and TNF on PIVKA II production more exactly, further study using a primary culture of h u m a n hepatocytes should be needed as well as the in vivo model of Hu-H1 transplanted nude mouse that we recently developed26.
References 1. Olson RE. The function and metabolism of vitamin K. Ann Rev Nutr 1984;4:284-302. 2. Suttie JW. Vitamin K-dependent carboxylase. Ann Rev Biochem 1985;54:459-477. 3. Liebman HA, Furie BC, Tong M J, et al. Des-gamma-carboxy (abnormal) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med 1984;310:1427-1431. 4. Fujiyama S, Morishita T, Hashiguchi O, et al. Plasma abnormal prothrombin (des-gamma-carboxy prothrombin) as a marker of hepatocellular carcinoma. Cancer 1988;61:1621-1628. 5. Nakao A, Iwaki Y, Iwatsuki S, et al. Significance of PIVKA II determination in human fiver transplantation. Acta Hepat Jap 1991;32(supple 1):248. (in Japanese) 6. Huh N, Utakoji T. Production of riBs-antigen by two new human hepatoma cell lines and its enhancement by dexamethasone. Jpn J Cancer Res 1981;72:178-179. 7. Ono M, Ohta H, Ohhira M, et al. Des the impairment ofgammacarboxylation have any inhibitory effect on the extracelular excretion of des-gamma-carboxy prothrombin (PIVKA II)? Igaku no Ayumi 1990;152:123-124. (in Japanese) 8. Naraki T, Watanabe K, Shimozuru Y, et al. Development and evaluation of the EIA kit for the detection of PIVKA II using double antibody sandwich system: Monoclonal antibody to PIVKA II and polyclonal antibody to prothrombin. Clin Immunol 1986;18:479-492. (in Japanese) 9. Sugo T, Watanabe K, Naraki T, et al. Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy) prothrombin. J Biochem 1990;108:382-387. 10. Motohara K, Kuroki Y, Kan H, et al. Detection of vitamin K deficiency by use of an enzyme-linked immunoadsorbent assay for circulating abnormal prothrombin. Pediatr Res 1985;19:354-357. 11. Ono M, Ohta H, Ohhira M, et al. Measurement ofimmunoreactive prothrombin, des-gamma-carboxy prothrombin, and vitamin
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K in human liver tissues: Overproduction of immunoreactive prothrombin in hepatoceilular carcinoma. Am J Gastroenterol 1990; 85:1149-1154. 12. Van Damme J, Opdenakker G, Simpson RI, et al. Identification of the human 26-kD protein, interferon beta2 (IFN-beta2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J Exp Med 1987;165:914-919. 13. Nordan RP, Pumphrey JG, Rudikoff S. Purification and NH2-terminal sequence ofa plasmacytoma growth factor derived from the murine macrophage cell line P388D1. J Immunol 1987;139:813817. 14. Tosato G, Seamon KB, Goldman ND, et al. Monocyte-derived human B-cell growth factor identified as interferon-beta2 (BSF-2, IL-6). Science 1988;239:502-504. 15. Kohda H, Uede T, Yuasa H, et al. Construction of rat-mouse T cell hybridomas that express regulatable rat interleukin 2 receptor. J Immunol 1986;137:1557-1563. 16. Ono M, Ohta H, Ohhira M, et al. Measurement ofimunoreactive prothrombin precursor and vitamin K-dependent gammacarboxylation in human hepatocellular carcinoma tissues: Decreased carboxylation of prothrombin precursor as a cause of des-gamma-carboxy prothrombin synthesis. Tumor Biol 1990;11: 319-326. 17. Imagawa DK, Millis JM, Olthoff KM, et al. The role of tumor necrosis factor in allogralt rejection. Transplantation 1990;50:219225. 18. Maury CPJ, Teppo AM. Raised serum level of cachectin/tumor necrosis factor-alpha in renal allograR rejection. J Exp Med 1987;166:1132-1137. 19. Yoshimura N, Oka T, Kahan BD: Sequential determinations of serum interleukin-6 levels as an immunodiagnostic tool to differentiate rejection from nephrotoxicity in renal allograft recipients. Transplantation 1991;51:172-176. 20. Vandenbroecke C, Caillat-Zucman S, Legendre C, et al. Differential is situ expression of cytokines in renal allogratt rejection. Transplantation 1991;51:602-609. 21. Ford HR, Hoffman RA, Tweardy D J, et al. Evidence that production of interleukin-6 within the rejecting allogralt coincides with cytotoxic T lymphocyte development. Transplantation 1991;51: 656-661. 22. Gauldie J, Richards C, Haarnish D, et al. Interferon beta2/B-cell stimulatory factor type 2 shares identify with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver ceils. Proc Natl Acad Sci USA 1987; 84:7251-7255. 23. Ohhira M, Ono M, Kohda H, et al. Effects ofdibutyryl cyclic AMP for secretion of PIVKA II from Hu-H1 cells. Jpn J Gastroenterol 1991;88:2728. (in Japanese) 24. Nanes MS, Rubin J, Titus L, et al. Tumor necrosis factor-alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism. Endocrinology 1991;128:2577-2582. 25. Shiota G, Rhoads DB, Wang TC, et al. Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc Natl Acad Sci USA 1992;89:373-377. 26. Kohda H, Ono M, Sekiya C, et al. Detection of PIVKA II produced by human hepatoma cells in nude mice. Human Cell 1991;4:63-66.
Gastroenterologia Japonica Copyright 9 1992 by The Japanese Society of Gastroenterology
The effect of IL-6 on the des-gamma-carboxy prothrombin synthesis in human hepatoma cells Minoru ONO, Hironobu KOHDA, Tooru NARAKI, Hitoyoshi OHTA, Motoyuki OHHIRA, Chihiro SEKIYA, and Masayoshi NAMIKI Third Departments of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan Summary: Effects of several cytokines on des-gamma-carboxy prothrombin (PIVKA II) synthesis in human hepatoma cells were investigated to know the process of PIVKA II production during a liver allograft rejection. Human recombinant interleukin-6 (IL-6) significantly stimulated the PIVKA II synthesis without any influence on the cell proliferation. The effect was almost completely neutralized by the specific anti-IL-6 antibody. Neither tumor necrosis factor (TNF), interleukin-1 (IL-1) nor interferon-gamma (IFN-gamma) had such a stimulative effect. IL-6 appears to stimulate PIVKA II production, and would be a candidate of factors that enhance the production of PIVKA II during a liver allograft rejection. Gastroenterol Jpn 1992;27:745- 750. Key words: IL-6; des-gamma-carboxy prothrombin; hepatocellular carcinoma.
Introduction Prothrombin is a vitamin K-dependent clotting factor which is synthesized in the liver and undergoes posttranslational gamma-carboxylation on its 10 gamma-glutamic acid (Glu) residues in the amino-terminal domain, which has the calcium binding properties essential for the biological activity of prothrombin 1. This gamma-carboxylation is catalyzed by the vitamin K-dependent carboxylase (carboxylase) which is located in liver microsome 2. In the absence of vitamin K or when the carboxylation is antagonized by warfarin sodium, des-gamma-carboxy prothrombin (PIVKA II, protein induced by vitamin K absence or antagonist) is secreted into plasma. A marked increase of PIVKA II in serum of hepatocellular carcinoma (hepatoma) patients has been reported, and this abnormal prothrombin is now used as a new serological indicator for the diagnosis of
hepatoma 3,4. Very recently, a significant increase of circulating PIVKA II level was found during liver allograft rejection s. The mechanism of the increase, however, is not known at all. In the present study, we investigated the effects of several cytokines (TNF, IL-1, IFN-gamma and IL-6) on the PIVKA II synthesis in h u m a n hepatom cell line, Hu-H16, which has been shown to spontaneously produce the abnormal prothrombin 7. Materials and Methods Chemicals H u m a n recombinant TNF-alpha (TNF), specific activity 2 x 107 unit/mg protein; human recombinant IL-l-alpha (IL-1), specific activity, 8 x 106 units/mg protein; human recombinant IL-6 (IL-6), specific activity, 1 x 107 units/mg protein; human recombinant interferon-gamma (IFN-gamma), specific activity, 2.5 • 107 units/
Received April 10, 1992. Accepted June 26, 1992. Address for correspondence: Motoyuki Ohhira, M.D., The Third Department of Internal Medicine, Asahikawa Medical College, 4-5 Nishikagura, Asahikawa, Hokkaido 078, Japan. The abbreviations used are: PIVKA II, des-gamma-carboxy prothrombin; TNF, tumor necrosis factor; IL-1, interleukin-1; IL-6, interleukin-6; IFN-gamma, interferon-gamma; ELISA, enzyme-linked immunosorbent assay.
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mg protein and polyclonal rabbit anti-human IL-6 antibody were obtained from Genzyme Corp., Cambridge, MA, USA. Cells and cell culture Hu-H1, a human hepatocellular carcinoma cell line, was grown in Eagle's Minimum Essential Medium (MEM) containing 10% fetal calf serum (Gibco, Grand Island, NY, USA), and 3% glutamine, 100 units/ml penicillin, and 100 ~tg/ml streptomycin. The cells were plated in culture flasks (Costar) and routinely maintained in a CO2 incubator at 37~ Cytokine treatment Hu-H1 cells (1 x 105 cells/well) were plated in 24-well plates and cultured for the indicated period with or without addition ofTNF, IL-1, IL-6 or IFN-gamma in culture medium. After incubation, amounts of PIVKA II and immunoreactive prothrombin secreted into medium were determined. Neutralization of lL-6 activity by anti-IL-6 antibody To verify the possible ability of IL-6 on PIVKA II induction, an inhibition study by specific antibody against IL-6 was carried out. Polyclonal rabbit anti-human IL-6 antibody was diluted by culture medium to various concentrations, and added to the IL-6 solution (50 U/ml) followed by incubation at 4~ over night. The cells (1 x 105/ well) plated in 24 well plates were cultured for 72 h in the pre-incubated medium containing IL-6 and anti-IL-6 antibody. After the culture, PIVKA II secreted into medium was determined. Assay of PIVKA II and immunoreactive prothrombin PIVKA II was measured with commercially available ELISA kits (Eitest Mono PII, Eisai, Tokyo, Japan) using the specific monoclonal antibody (E-1023) that has been shown to recognize an altered structure with conformational change in the Gla-domain of PIVKA 118,9, and is also known to react only with PIVKA II but not with fully carboxylated normal prothrombin ~~ Immunoreactive prothrombin was determined
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with ELISA using rabbit polycolonal antibody against human prothrombin, as we previously described1~ In our previous study, an increase of prothrombin precursor was found in human hepatoma tissues 11. To investigate the possibility that IL-6 stimulates the prothrombin synthesis, the amount of total immunoreactive prothrombin secreted into the medium was determined. Since, the polyclonal antibody recognizes PIVKA II as well as normal prothrombin 11, the amount of immunoreactive prothrombin determined by the ELISA was designated the "total immunoreactive prothrombin" in this study. Effect of IL-6 on the Hu-H1 cell proliferation IL-6 is shown to have a proliferative effect in several cell lines a214. To know whether the enhancement of PIVKA II production was due to the cell proliferative effect or out, we examined the 3H-TdR incorporation of Hu-H1 after IL-6 treatment as we previously described15. The cells were cultured for 24 h to 72 h in the presence or absence of various concentration of recombinant human IL-6 at a cell density of 1 x 104/ml in 96 wells microculture plates, then cells were pulsed for 4 h with 1 ~Ci/well 3H-TdR (New England Nuclear). After incubation, the cells were harvested and uptake of 3H-TdR was measured by liquid scintillation counter. Statistical analysis Statistical analysis was carried out by Student's t-test and P < 0.05 was considered as significant. Results
Effect of cytokines on PIVKA Il production in HuH1 With no addition of cytokine, the PIVKA II level after 24 h, 48 h and 72 h culture was 0, 55 + 4.5 and 170 _+ 38 mAU/ml (mean _+ SD; n = 3), respectively. When the cells were cultured for 72 h with an addition of IL-6 (100 U/ml), secreted PIVKA II significantly ( P < 0 . 0 1 ) increased to 325 + 3 9 m A U / m l (mean+SD; n = 3) as compared with the basal level in 72 h culture (Figure la). On the other hand, an addi-
IL-6 and des-gamma-carboxy prothrombm
December 1992
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(b)
500
500
400
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Figure 1. Effects of cytokines on des-gamma-carboxy prothrombin (PIVKA it) production of Hu-H1. Hu-H1 cells were cultured for 24 to 72 h with or without 11--6(1O0 U/ml; la), IFN-gamma (1 O0 U/ml; 1b), TNF (10 ng/ml; I c) and It--1 (10 U/ml; 1d). PIVKAII level was determined by the ELISA as described in the "Materials and Methods". Circles, no addition; Solid dots, cytokine treated. Each value was obtained from 3 samples. Bar indicates the standard deviation.
tion of IFN-gamma (Figure lb), TNF (Figure le) and IL-1 (Figure ld) resulted in a decrease of PIVKA II level. Then the cells were cultured for 72 h to 96 h with IL-6 in various concentrations.
As shown in Figure 2, IL-6 stimulated PIVKA II production dose-dependently in a range of 1 to 100U/ml. Maximum induction, which was almost double the basal level, was observed by
748
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I L - 6 (U/ml) Figure 2. Effect of IL-6 on PIVKA II production. Hu-H1 cells were cultured for 72 h to 96 h with IL-6 in various concentrations indicated. PIVKA U level was determined by ELISA as described in the "Materials and Methods". Each value was obtained from 5 samples. Bar indicates the standard deviation. Solid line, 96 h culture; Dotted line, 72 h culture.
addition of IL-6 at a concentration of 75 U/ml both in 72 h and 96 h culture. An extremely low PIVKA II level was detected in the corresponding ceil lysates (data not shown).
Effect of 1L-6 in the Hu-H1 cell proliferation As shown in Figure 3, little change in the 3HTdR incorporation was observed after 24 h culture with IL-6 at various concentrations, and similarly no significant change was found either after 48 h or 72 h culture. Neutralization of P1VKA H induction by anti-IL-6 antibody The polyclonal antibody inhibited the IL-6 induced PIVKA II production in a dose dependent manner (Figure 4). A maximum inhibition, about 80% inhibition, was achieved by an addition of the antibody to the concentration of 100 ~g/ml. An addition of the antibody alone made no significant change in the PIVKA II level (data not shown).
1
5
i 10
50
75 100 5001000
I L - 6 (u/ml) Figure 3. [3H]Thymidine (3H-TdR) incorporation of Hu-H1 after IL6 treatment. Cells were cultured for 24 h with 11.-6in the indicated concentrations. After the culture, cells were pulsed with 3H-TdR. Each value was obtained from 5 samples and the bar indicates the standard deviation.
Effect of lL-6 on total prothrombin synthesis (Figure S) When ceils were culture for 72 h to 96 h with an addition of IL-6 in various concentrations, the amount of total prothrombin was relatively increased but not statistically significant. TNF, ILl and IFN-gamma reduced the total prothrombin level (data not shown), which might be due to the cytotoxic effect of the cytokines.
Discussion The vitamin K-dependent proteins, including several clotting factors, undergo unique posttranslational gamma-carboxylation1,2. Vitamin K-dependent carboxylase is a key enzyme catalyzing the reaction2. We have previously reported that a significant decrease in the vitamin K-dependent carboxylase activity despite an increase of prothrombin precursor plays an essential role in the PIVKA II production in hepatoma16, and that no vitamin K deficiency exists in cancerous liver tissues 1~. Very recently, Nakao et al. reported a remarkable increase of plasma PIVKA II level during acute liver allograft rejection, although the patients revealed no bleeding tendency or vitamin K deficiency, and they indicated that PIVKA II is
IL-6 and des-gamma-carboxy prothrombm
December 1992
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Figure 4. Neutralization of IL-6 activity by anti-lL-6 antibody. Polyclonal anti-lL-6 antibody was diluted to the indicated concentrations, and added to IL-6 solution (50 U/ml) followed by incubation at 4~ over night. Then cells were cultured for 72 h. The PIVKA II level, which was induced by IL-6 (50 U/ml) without an addition of antibody, was expressed as 100%. Circles, with an addition of IL-6 only; Solid dots, with an addition of both IL-6 and anti-lL-6 antibody. Each value was obtained from 3 samples. Bar indicates the standard deviation.
a new sensitive serological indicator for acute liver allograft rejection5. The mechanism, however, has not yet been clarified. Several investigators reported a marked elevation of serum TNF level during acute rejection of hepatic 17 and renal allografts18. Similarly a significant elevation of serum IL-6 protein level19 and also an increased expression of IL6 mRNA 2~ are demonstrated in acute renal allograft rejection. Thus, TNF and IL-6 are thought to be the candidates that play an important role in the allograft rejection, which would be associated with the development of cytotoxic T lymphocyte2~. However, little is known about the effect of these cytokines on the posttranslational carboxylation of prothrombin. In the present study, we demonstrated that human recombinant IL-6 stimulates the PIVKA II production in vitro in human hepatoma cells with little influence on cell proliferation. The precise mechanism by which IL-6 enhances the PIVKA II production remained to be solved. One possible explanation would be that IL-6 induces prothrombin synthesis as in the case of acutephase proteins 22, which would amplify the production of PIVKA II. Indeed, IL-6 appeared to stimulate the total prothrombin synthesis to some
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Figure 5. Effect of IL-6 on total prothrombin synthesis. Hu-H1 cells were cultured with various concentrations of IL-6 for 72 h to 96 h. Then the total amount of immunoreactive prothrombin was determined by ELISA as described in the "Materials and Methods". Each value was obtained from 5 samples, and the bar indicates the standard deviation. Solid line, 96 h culture; Dotted line, 72 h culture.
extent as shown in Figure 5. However, the degree of the increase in the total amount ofprothrombin was not so definite as in the case of PIVKA II. Therefore, it does not seem likely that merely the increase of total amount of prothrombin in HuH1 cells can be responsible for the PIVKA II induction by IL-6. To clarify the effect of IL-6 on prothrombin synthesis more exactly, prothrombin mRNA expression should be also investigated. An alternative possibility would be a change of the carboxylation system itself. Very recently we have found similar enhancement of PIVKA II production in Hu-H 1 by cAMP analog, in which no significant increase of total prothrombin was noted 23. Although the signal transduction of IL-6 has not been understood, IL-6 might have potency to alter the carboxylation system, particularly the vitamin K-dependent carboxylase. Recently TNF is reported to inhibit the formation of bone Gla protein (osteocalcin), one of the vitamin K-dependent proteins24. However, biological effects of TNF on the carboxylation system of vitamin K-dependent proteins are unclear. In the present study, TNF did not enhance the PIVKA II
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production in human hepatoma cells, which does not seem compatible with the clinical observations described above. The discrepancy might be, in part, due to the differences between in vitro and in vivo experimental systems. Also it may be possible that TNF has a counteraction on malignant and normal hepatocytes, as in the case of hepatocyte growth factor 25, in terms of prothrombin synthesis and its carboxylation. To confirm the effect of IL-6 and TNF on PIVKA II production more exactly, further study using a primary culture of h u m a n hepatocytes should be needed as well as the in vivo model of Hu-H1 transplanted nude mouse that we recently developed26.
References 1. Olson RE. The function and metabolism of vitamin K. Ann Rev Nutr 1984;4:284-302. 2. Suttie JW. Vitamin K-dependent carboxylase. Ann Rev Biochem 1985;54:459-477. 3. Liebman HA, Furie BC, Tong M J, et al. Des-gamma-carboxy (abnormal) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med 1984;310:1427-1431. 4. Fujiyama S, Morishita T, Hashiguchi O, et al. Plasma abnormal prothrombin (des-gamma-carboxy prothrombin) as a marker of hepatocellular carcinoma. Cancer 1988;61:1621-1628. 5. Nakao A, Iwaki Y, Iwatsuki S, et al. Significance of PIVKA II determination in human fiver transplantation. Acta Hepat Jap 1991;32(supple 1):248. (in Japanese) 6. Huh N, Utakoji T. Production of riBs-antigen by two new human hepatoma cell lines and its enhancement by dexamethasone. Jpn J Cancer Res 1981;72:178-179. 7. Ono M, Ohta H, Ohhira M, et al. Des the impairment ofgammacarboxylation have any inhibitory effect on the extracelular excretion of des-gamma-carboxy prothrombin (PIVKA II)? Igaku no Ayumi 1990;152:123-124. (in Japanese) 8. Naraki T, Watanabe K, Shimozuru Y, et al. Development and evaluation of the EIA kit for the detection of PIVKA II using double antibody sandwich system: Monoclonal antibody to PIVKA II and polyclonal antibody to prothrombin. Clin Immunol 1986;18:479-492. (in Japanese) 9. Sugo T, Watanabe K, Naraki T, et al. Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy) prothrombin. J Biochem 1990;108:382-387. 10. Motohara K, Kuroki Y, Kan H, et al. Detection of vitamin K deficiency by use of an enzyme-linked immunoadsorbent assay for circulating abnormal prothrombin. Pediatr Res 1985;19:354-357. 11. Ono M, Ohta H, Ohhira M, et al. Measurement ofimmunoreactive prothrombin, des-gamma-carboxy prothrombin, and vitamin
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K in human liver tissues: Overproduction of immunoreactive prothrombin in hepatoceilular carcinoma. Am J Gastroenterol 1990; 85:1149-1154. 12. Van Damme J, Opdenakker G, Simpson RI, et al. Identification of the human 26-kD protein, interferon beta2 (IFN-beta2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J Exp Med 1987;165:914-919. 13. Nordan RP, Pumphrey JG, Rudikoff S. Purification and NH2-terminal sequence ofa plasmacytoma growth factor derived from the murine macrophage cell line P388D1. J Immunol 1987;139:813817. 14. Tosato G, Seamon KB, Goldman ND, et al. Monocyte-derived human B-cell growth factor identified as interferon-beta2 (BSF-2, IL-6). Science 1988;239:502-504. 15. Kohda H, Uede T, Yuasa H, et al. Construction of rat-mouse T cell hybridomas that express regulatable rat interleukin 2 receptor. J Immunol 1986;137:1557-1563. 16. Ono M, Ohta H, Ohhira M, et al. Measurement ofimunoreactive prothrombin precursor and vitamin K-dependent gammacarboxylation in human hepatocellular carcinoma tissues: Decreased carboxylation of prothrombin precursor as a cause of des-gamma-carboxy prothrombin synthesis. Tumor Biol 1990;11: 319-326. 17. Imagawa DK, Millis JM, Olthoff KM, et al. The role of tumor necrosis factor in allogralt rejection. Transplantation 1990;50:219225. 18. Maury CPJ, Teppo AM. Raised serum level of cachectin/tumor necrosis factor-alpha in renal allograR rejection. J Exp Med 1987;166:1132-1137. 19. Yoshimura N, Oka T, Kahan BD: Sequential determinations of serum interleukin-6 levels as an immunodiagnostic tool to differentiate rejection from nephrotoxicity in renal allograft recipients. Transplantation 1991;51:172-176. 20. Vandenbroecke C, Caillat-Zucman S, Legendre C, et al. Differential is situ expression of cytokines in renal allogratt rejection. Transplantation 1991;51:602-609. 21. Ford HR, Hoffman RA, Tweardy D J, et al. Evidence that production of interleukin-6 within the rejecting allogralt coincides with cytotoxic T lymphocyte development. Transplantation 1991;51: 656-661. 22. Gauldie J, Richards C, Haarnish D, et al. Interferon beta2/B-cell stimulatory factor type 2 shares identify with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver ceils. Proc Natl Acad Sci USA 1987; 84:7251-7255. 23. Ohhira M, Ono M, Kohda H, et al. Effects ofdibutyryl cyclic AMP for secretion of PIVKA II from Hu-H1 cells. Jpn J Gastroenterol 1991;88:2728. (in Japanese) 24. Nanes MS, Rubin J, Titus L, et al. Tumor necrosis factor-alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism. Endocrinology 1991;128:2577-2582. 25. Shiota G, Rhoads DB, Wang TC, et al. Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc Natl Acad Sci USA 1992;89:373-377. 26. Kohda H, Ono M, Sekiya C, et al. Detection of PIVKA II produced by human hepatoma cells in nude mice. Human Cell 1991;4:63-66.
