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1997 90: 1501-1507 Franca Citarella, Angelina Felici, Mieke Brouwer, John Wagstaff, Antonio Fantoni and C. Erik Hack Cell Line (HepG2) Interleukin-6 Downregulates Factor XII Production by Human Hepatoma http://bloodjournal.hematologylibrary.org/content/90/4/1501.full.html Updated information and services can be found at: (2334 articles)Hemostasis, Thrombosis, and Vascular Biology • Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Copyright 2011 by The American Society of Hematology; all rights reserved. 20036. the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom Interleukin-6 Downregulates Factor XII Production by Human Hepatoma Cell Line (HepG2) By Franca Citarella, Angelina Felici, Mieke Brouwer, John Wagstaff, Antonio Fantoni, and C. Erik Hack Involvement of the contact system of coagulation in the the human hepatoma cell line HepG2 by up to 75%. The decrease in protein secretion correlated with an equivalentpathogenesis of various inflammatory diseases is suggested by reduced plasma levels of factor XII (Hageman factor) and decrease of factor XII mRNA likely indicating a pretransla- tional control of factor XII gene expression by IL-6. Downreg-prekallikrein generally considered to result from activation of the contact system. However, in many of these diseases ulation of factor XII production by IL-6 in vitro parallelled that of transthyretin, a known negative acute-phase protein.patients develop an acute-phase response and, therefore, an alternative explanation for the decreased levels of factor XII Moreover, we show that, in patients developing an acute- phase response after immunotherapy with IL-2, plasma lev-could be the downregulation of factor XII gene expression in the liver as described for negative acute-phase proteins. els of factor XII correlate (r ! .76, P Ú .0001) with those of transthyretin. Taken together, these results suggest thatWe report here that interleukin-6 (IL-6), the principal cyto- kine mediating the synthesis of most acute-phase proteins factor XII behaves as a negative acute-phase protein. q 1997 by The American Society of Hematology.in the liver, downregulates the production of factor XII by (TTR; prealbumin), a well-known negative acute-phase pro-T tein.HE PLASMA CONTACT system is involved in themaintenance of homeostasis of the organism; indeed, activated contact system proteins trigger the activation of MATERIALS AND METHODSthe intrinsic pathways of coagulation and fibrinolysis, the activation of the complement system, and the production of Cell culture. HepG2 cells were cultured in Dulbecco’s modified Eagles medium supplemented with 10% (vol/vol) fetal calf serumkinins.1-5 (FCS), penicillin-streptomycin, and glutamine (all purchased fromThe activation of the contact system is initiated by factor GIBCO BRL, Paisley, UK). For the stimulation experiments, cellsXII (FXII), a serine protease synthesized by the liver6 as a were plated at a density of 1 1 105 cells/cm2 in 25-cm2 flasks; aftersingle-chain inactive zymogen. Upon binding to a negatively 24 hours, the medium was replaced with fresh medium with or charged surface, FXII is converted to activated FXII (FXIIa), without 10% (vol/vol) FCS and with or without added cytokines (0 which, in turn, activates prekallikrein to kallikrein and FXI time). After 24 hours, culture media were harvested and replaced to activated FXI. Kallikrein activates additional FXII and with fresh medium containing the appropriate cytokines and cells cleaves high molecular weight kininogen generating the va- cultured for an additional 24 hours. This procedure was repeated on soactive peptide bradykinin. 3 successive days. Cells and culture fluids were harvested at 24- hour intervals for RNA analysis by Northern blot and for proteinFXII and prekallikrein plasma levels are low in sepsis, (FXII, TTR, and fibrinogen) quantification by enzyme-linked immu-which has been ascribed to increased consumption resulting noassorbent assays (ELISAs), respectively. Cell supernatants werefrom activation of the contact system. However, evidence centrifuged at 4,000g for 20 minutes at 47C to remove detached cellsfor such an activation process is often lacking. For example, and stored at 0207C until analysis. All stimulation experiments were circulating levels of FXIIa- and kallikrein-C1-inhibitor com- repeated at least three times, with duplicate incubations and duplicateplexes, which reflect activation of the contact system in vivo, determination of the secreted proteins. are also low in most patients with sepsis.7 Cytokines. Human recombinant IL-6 (rIL-6) produced in Esche- Mammalian liver responds to acute systemic injury by a richia coli was purified to homogeneity by gel filtration and ion dramatic change in the synthesis of various plasma proteins. exchange high-performance liquid chromatography as described.14,15 This phenomenon is known as hepatic acute-phase re- sponse.8,9 The rate and amplitude of changes in plasma levels of the acute-phase proteins reflect the induction of their syn- From the Central Laboratory of The Netherlands Red Cross Blood thesis by various cytokines, such as interleukin-1b (IL-1b), Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Nether-tumor necrosis factor a (TNFa), and IL-6. In particular, the lands; the Dipartimento di Biopatologia Umana, Sezione di Biologialatter cytokine has been shown to regulate the entire set of Cellulare, Universita` di Roma ‘‘La Sapienza’’, Roma, Italy; andacute-phase proteins. IL-6 increases the expression of genes the Department of Medical Oncology, Free University Hospital,coding for the positive acute-phase proteins and downregu- Amsterdam, The Netherlands.lates that of the so-called negative acute-phase proteins.10-13 Submitted December 3, 1996; accepted April 8, 1997. Decreased plasma levels of a given protein in human diseases Supported by Human Capital and Mobility Award Contract No. may therefore be due to a negative acute-phase behavior of ERBCHBGCT920109, by Fondazione Istituto Pasteur-Fondazione that protein. Cenci Bolognetti, and by Ministero dell’Universita` e Ricerca Scien- Thus far, there are no data on the regulation of FXII tifica. Address reprint requests to Franca Citarella, Central Laboratorysynthesis by liver cells during the acute-phase reactions. We, of the Netherlands Red Cross Transfusion Service, Department oftherefore, investigated the effect of cytokines on the synthe- Pathophysiology of Plasma Proteins, Plesmanlaan 125, 1066 CXsis of FXII by the human hepatoma cell line HepG2, which Amsterdam, The Netherlands.is generally considered to be a suitable model for studies of The publication costs of this article were defrayed in part by page acute-phase protein regulation. Our results indicate that, in charge payment. This article must therefore be hereby marked HepG2 cells, FXII synthesis is downregulated by IL-6. Fur- ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to thermore, we show that, in patients who develop an acute- indicate this fact. phase response during IL-2 therapy, decreased plasma levels q 1997 by The American Society of Hematology. 0006-4971/97/9004-0007$3.00/0of FXII correlated with decreased levels of transthyretin 1501Blood, Vol 90, No 4 (August 15), 1997: pp 1501-1507 AID Blood 0024 / 5h3b$$$461 07-15-97 13:18:45 bldal WBS: Blood For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom CITARELLA ET AL1502 The specific activity of rIL-6 was 109 U per milligram as measured in the B9 bioassay.16 Endotoxin content was less than 3.5 pg/mg as measured in the Limulus assay. A second preparation of rIL-6 was kindly provided by G. Ciliberto (IRBM, Pomezia, Rome, Italy). rIL- 1b was kindly provided by Dr P.T. Lomedico (Hoffmann-La Roche, Nutley, NJ) and had a specific activity of 51 106 U/mg, as measured in D10 bioassay, and an endotoxin content of 6 pg/mg.16 rTNFa, produced in E coli, was generously provided by W. Haas (Hoffmann- La Roche, Basel, Switzerland). The specific activity was 5.9 1 107 U/mg as measured in the WEHI 164 bioassay.17 Immunoassays. Proteins secreted in the culture media were quantified by sandwich-type ELISA. The ELISAs used to quantify fibrinogen and TTR were performed using a first polyclonal antibody for coating and a second polyclonal antibody labeled with biotin. The rabbit polyclonal antibodies used were separated from antiserum (Rabbit-antihuman fibrinogen and rabbit-anti-TTR; both from the Fig 1. Effect of rIL-1b, rIL-6, and rTNFa on FXII synthesis by HepG2Department of Immune Reagents, Central Laboratory of The Nether- cells. HepG2 cells were grown to 80% confluence in 24-well plateslands Red Cross Transfusion Service, Amsterdam, The Netherlands) and incubated under serum-free conditions with 50 ng/mL of rIL-1b,by affinity chromatography on protein-G-Sepharose (Pharmacia Fine rIL-6, rTNFa, and a combination of rIL-1b and rIL-6 for 72 hours. Chemicals, Uppsala, Sweden) according to the manufacturer’s in- Culture medium was refreshed every 24 hours. FXII concentration in structions. FXII was measured using two monoclonal antibodies, culture media obtained after the third 24-hour period was measured both directed against the catalytic region of FXII, as previously by ELISA as described in the Materials and Methods. Results repre- sent the mean Ô SD of three independent experiments. *P Ú .05 asdescribed.18 The amount of proteins present in the culture media compared with unstimulated cells.was calculated by reference to a standard curve of pooled normal human plasma. By comparing dose-response curves of the latter with those of purified FXII, the pooled plasma was estimated to contain 35.3 { 2.9 mg of FXII per milliliter. The interassay coefficient of samples. A difference was considered significant, using a two-tailed variation of all assays was less than 10%. P, at P › .05.Results were corrected for cell number by assessing the total amount of RNA extracted from the flasks and were expressed as the RESULTS amount of protein secreted into the culture medium during 24 hours by 106 HepG2 cells. Data are presented as the mean { SD from FXII production by HepG2 cells. FXII was constitu- four to six replicate cultures. tively synthesized and secreted by HepG2 cells in the ab- Preparation of RNA and Northern blot analysis. Cells were sence of any stimulus. In initial experiments, we studied the washed with ice-cold phosphate-buffered saline and lysed in guanidi- effects of rIL-1b, rIL-6, and rTNFa on the production of nium thiocyanate solution (4 mol/L guanidinium thiocyanate, 25 FXII. Cells were cultured under serum-free conditions in the mmol/L sodium citrate, and 0.5% sodium lauryl sarcosylate, pH 7). presence of rIL-1b, rIL-6, rTNFa, and combinations of rIL-Total RNA was isolated by phenol-chloroform extraction.19 Twenty- 1b and rIL-6. As shown in Fig 1, the amount of FXII presentfive micrograms of total RNA, as determined spectrophotometrically, in the culture media of HepG2 cells stimulated for 72 hourswas fractionated on 1% agarose-formaldehyde gel and transferred with rIL-6 was three times lower than in the controls (P onto nylon membranes (Hybond-N; Amersham, Buckinghamshire, UK). Hybridization, washing, and rehybridization were performed .022), whereas changes induced by rIL-1b and rTNFa were according to the manufacturer’s instructions. Filters were hybridized not statistically significant. Moreover, rIL-1b did not sig- with 32P-labeled 0.5-kb FXII cDNA probe. The FXII probe was the nificantly modify the effect of rIL-6 on FXII production. HindIII-Pst I fragment of the pBFXII plasmid20 containing nucleo- Based on the initial results, we focused our attention on the tides 1-543 of hFXII cDNA. Each filter was rehybridized with a 0.3- effect of IL-6 on FXII production by HepG2 cells cultured in kb human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the presence or absence of FCS. Dose-response experiments cDNA probe to perform a normalization for mRNA loading. Filters using rIL-6 concentrations ranging from 0.1 to 50 ng/mL were washed and exposed to Kodak X-Omat film (Eastman Kodak, indicated that rIL-6 was already effective at a concentrationRochester, NY) at 0707C with intensifying screens. The abundance of 0.5 ng/mL (data not shown) and reached a plateau atof specific FXII transcripts in each lane was directly quantified on concentrations less than 5 ng/mL. Therefore, all subsequentfilters by a Phosphoimager Analyser (Canberra Packard, Meriden, experiments were performed using 5 ng of rIL-6 per milliliterCT). Analysis of FXII and TTR levels in IL-2–treated patients. Four of culture medium. Under these conditions, rIL-6 signifi- patients who received high doses of recombinant IL-2 as a treatment cantly downregulated the production of FXII by HepG2 cells for metastatic melanoma or renal cell carcinoma malignant diseases cultured both in the presence and absence of FCS (Fig 2A). were studied with regard to the changes occurring in the contact The amount of FXII in culture media after 24 hours of expo- system proteins.21 Further details regarding the patients, the IL-2 sure to rIL-6 was 75% of that in medium from cells cultured treatment, and the blood sampling are given elsewhere.22 FXII levels in absence of rIL-6 and decreased to 25% at 48 and 72 hours.in plasma samples were determined by radioimmunoassay as de- The amount of FXII in culture media from unstimulated scribed.7,21 TTR levels were measured by ELISA, as described above. cells cultured with or without FCS was comparable at 24Data analysis. All statistical analyses were performed by and 48 hours, whereas it was twofold increased in HepG2Graphpad Instat (San Diego, CA). Intergroups and intragroups dif- cells maintained without FCS for 72 hours.ferences were assessed by the Mann-Whitney U test and the Wil- Synthesis of fibrinogen, studied as positive control ofcoxon test, respectively. The Spearman rank correlation analysis was used to assess the correlation between protein levels in plasma acute-phase reaction, was threefold to fourfold increased in AID Blood 0024 / 5h3b$$$461 07-15-97 13:18:45 bldal WBS: Blood For personal use only. by guest on November 16, 2011. bloodjournal.hematologylibrary.orgFrom IL-6 DOWNREGULATES FXII PRODUCTION 1503 decrease resembled that of TTR. These results raised the question as to whether FXII might behave as a negative acute-phase protein during the acute-phase reaction in vivo. We therefore evaluated a possible correlation between plasma levels of FXII and TTR in patients treated with high doses of rIL-2 who develop an acute-phase reaction after high plasma levels of IL-6. Similarly to FXII,21 TTR also significantly decreased during the treatment with rIL-2 (P › .0001), and this decrease correlated with that of FXII (r  .76, P › .0001; Fig 4). DISCUSSION The results presented here show that the synthesis of FXII in the human hepatoma cell line HepG2 is downregulated by IL-6. The hepatoma cell line HepG2 is very similar to hepatocytes in terms of biologic responsiveness23-25 and is widely used as model system for studying the regulation of acute-phase protein synthesis in human liver.26-29 The decrease in FXII production by HepG2 cells was significant after 24 hours of rIL-6 stimulation and reached a nadir at 48 hours. Neither rIL1-b nor rTNFa had a significant effect on FXII levels. In fact, IL1-b and TNFa directly control the synthesis of only a limited subset of acute-phase proteins,27,30-35 and their effect appears to be mediated, at least in part, by the induction of IL-6 synthesis.10,11,36-38 In vitro studies using hepatocyte cultures and correlations of serum IL-6 concentrations with acute-phase protein levels in various inflammatory states in vivo indicate that IL-6Fig 2. FXII and TTR production in control and rIL-6–treated HepG2 cells cultured in the presence or in the absence of 10% FCS. HepG2 may be the principal regulator for most acute-phase protein cells were plated at a density of 1 Ì 105 cells/cm2 in 25-cm2 flasks. genes.11,26,33,34,38 After 24 hours, the medium was replaced with fresh medium with Hepatocyte activation with IL-6 can result in both positiveor without 10% (vol/vol) FCS with or without rIL-6 (0 time). After and negative regulation of acute-phase proteins. Positive24 hours of culture, media were harvested and replaced with fresh medium (ÔrIL-6) and the cells were cultured for an additional 24 acute-phase proteins are represented by a large number of hours. FXII (A) and TTR (B) concentrations in the culture media were proteins, including various protease inhibitors, clotting fac- determined by ELISA. Results were corrected for cell number by as- tors, and transport plasma proteins, whose plasma levelssessing the total amount of RNA extracted from the flasks and are may increase from twofold to 1,000-fold. Concomitant toexpressed as the amount of protein secreted in the culture medium in 24 hours by 106 HepG2 cells. Results (mean Ô SD) are the average the increase of positive acute-phase protein concentrations, of three independent experiments in duplicate. * PÚ .05 as compared a decrease in plasma levels of negative acute-phase proteins, with corresponding control. which include albumin, a2-HS-glycoprotein, TTR, trans- ferrin, and retinol-binding protein, has been observed. De- creased levels have generally been thought to be due to transfer to the extravascular space or to increased metabo-the presence of rIL-6 (data not shown). As control for nega- lism; nevertheless, recent studies indicate that IL-1, TNF,tive acute-phase protein, we evaluated the production of and IL-6 are all capable of decreasing transcription of genesTTR, whose levels decreased by 37% after 24 hours and by for albumin29,39-41 and that IL-6 decreases transcription of the70% after 72 hours after rIL-6 stimulation of HepG2 cells TTR gene in HepG2 cells.29(Fig 2B). Although TTR levels were generally higher in FXII and TTR levels were decreased in the culture mediaculture media from cells maintained without FCS, the extent of rIL-6–stimulated HepG2 cells cultured either in the pres-of the decrease induced by rIL-6 was comparable with that ence or in the absence of FCS, thus ruling out the possibilityobserved in cells cultured in the presence of 10% FCS (Fig that the observed decreases were due to negative regulators2B). present in the FCS. However, higher levels of FXII proteinFXII mRNA levels in HepG2 cells. Northern blot analy- and specific mRNA as well as of TTR were found in thesis showed that FXII mRNA levels were decreased by 23% medium from cells cultured without FCS especially after 72after 24 hours and by 66.5% after 48 and 72 hours of rIL- hours. This my have been due to the presence of IL-6 in the6 stimulation (Fig 3A and B). An increase of FXII mRNA FCS, but the finding that this FCS was negative in a bioassaywas observed at 72 hours in unstimulated HepG2 cells cul- for IL-6, ie, the B9 assay,16 allowed us to exclude this possi-tured without FCS. bility. Possibly, the higher levels of proteins and mRNAFXII and TTR levels in IL-2–tr