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Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hepatocytes

Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hepatocytes Lipopolysaccharide Binding © 1990 S. K arger AG. Basel Pathobiology 1990;58:89-94 1015 -2 0 0 8 /9 0 /0 5 8 2 -0 0 8 9 $ 2 .75/0 Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hépatocytes1 G. Ramadoria , K.-H. Meyer zum Buschenfeldea , P.S. Tobiasb, J.C. Mathisonb, R.J. Ulevitchb a Klinikum der Johannes-Gutenberg-Univcrsität, I. Medizinische Klinik und Poliklinik, Mainz, FRG; b Department o f Immunology, Research Institute o f Scripps Clinic, La Jolla, Calif., USA Key Words. Lipopolysaccharide-binding protein • Hépatocytes • Acute phase protein • LPB-biosyn thesis Abstract. Studies reported here show that the recently discovered acute-phase protein, lipopolysaccharide-binding protein (LBP), is synthesized by hepatocytes. For these studies, explanted rabbit hepatocytes were grown in the presence of 35S-methionine. Biosynthetically labelled LBP in the cells and supernatant was identified using immu­ noprécipitation with rat anti-rabbit LBP antibody. This antibody immunoprecipitates both the LBP polypeptide and the glycosylated protein. With a cell-free translation system a comparison of RNA from normal rabbit liver with that isolated from acute-phase rabbit liver indicated that a translatable LBP message is only found in the RNA from acute-phase liver. Studies with explanted rabbit hepatocytes showed that several forms of LBP distinguishable by migration in SDS-PAGE are secreted into the extracellular medium. This heterogeneity probably is a result of differences in glycosylation of LBP since pretreatment of the hepatocytes with tunicamycin results in accumulation of a single polypeptide with an apparent mass of 50 kD in SDS-PAGE. Explanted rabbit hepatocytes spontaneously synthesize and secrete LBP and express LBP mRNA as detected by cell-free translation; thus, it was not possible to upregulate the expression of LBP. Nevertheless, these studies form the basis for future investigations on the regula­ tion of LBP biosynthesis. Introduction nized; namely, the mannose-binding protein [2] and lipo­ polysaccharide-binding protein (LBP) [3, 4], An acute-phase reaction is part of the physiologic We have recently suggested that there may be a family response of an organism to disparate conditions such as of LBPs with certain structural and/or functional simi­ bacterial infections, myocardial infarction, surgery, larities [5], The first two members of this family are LBP bums or neoplastic diseases. One characteristic of the and a neutrophil granule protein called bactericidal per­ acute-phase response is changes in the rate of synthesis of meability-increasing protein (BPI) [6]. There is a striking at least 30 different plasma proteins [1], Some of these homology in the amino-terminal sequences of rabbit proteins, which are normally present in very low concen­ LBP [5], and human BPI and both proteins share at least trations prior to the acute-phase response, rise as much as one function: the ability to bind to lipopolysaccharide several hundred-fold. Examples in this group are C-reac- from a wide variety of gram-negative bacteria [7, 8], tive protein in man, serum amyloid A in man and mouse, While BPI has been proven to be a neutrophil granule and ci2-macroglobulin in rats. Recently several new mem­ protein and inducible in HL-60 cells during differentia­ bers of this class of acute-phase proteins have been recog­ tion of these cells to a neutrophil-like cell [9], the site of synthesis of LBP has not been established. The studies described in this report provide information about the 1 Supported by A l l 5136, AI25563, SFB 311 (IMMUNPATHO- site o f synthesis of LBP in rabbits. In this paper we dem­ GENESE) Project A7, Ra 362/5-1 (DFG). This is Department o f Immunology publication No. 5988-IMM. onstrate LBP gene expression in acute-phase rabbit liver 90 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch in distilled water and sacrificed 24 h after injection. The RNA and in explanted rabbit hépatocytes. LBP is synthesized extraction was performed according to Chirgwin et al. [14] as as a 50-kD protein and released from hepatocytes as a described elsewhere [13, 14]. glycosylated protein with at least two different glycosyla­ Total RNA was also extracted from hepatocyte primary cultures tion patterns. These studies form the basis for future (cells plated onto 6-well plates) using the same procedure [14]. investigations that compare and contrast the regulation Briefly guanidinium lysates were repeatedly passed through a 23- gaugc needle to shear DNA and then layered onto 2 ml o f 5.7 M of BPI and LBP biosynthesis. CsCl and centrifuged in a Beckman SW 5 0 .1 rotor (Beckman Instru­ ments, Inc., Fullerton, Calif.) for 16 -1 8 h at 35,000 rpm at 20 ° C , the R NA pellet was washed twice in ethanol, then dissolved in water, reprecipitated in ethanol and dissolved in water, RNA con­ Materials and Methods tent was determined by measurement o f absorption at 260 nm. Reagents Dulbecco's modified Eagle’s medium (DMEM) and DMEM Cell-Free Translation o f RNA Total RNA from normal and acute-phase rabbit liver as well as without methionine was purchased from Gibco (Heidelberg, FRG) from hepatocytes was translated in vitro using rabbit reticulocyte and fetal bovine serum (FBS), penicillin, streptomycin and ¿-gluta­ mine from R o w Laboratories (Bonn, FRG), crystallized bovine lysate [16] in the presence o f 35S-m ethionine according to the serum albumin (BSA) with the minimum purity o f 98% and colla­ instructions o f the manufacturer. As positive control acute-phase murine liver RNA was used together with tobacco mosaic virus génase from Clostridium histolyticum were purchased from Behring- poly-A-RNA provided with the kit. Werke (Marburg, FRG). 3sS-methionine (specific radioactivity ap­ proximately 800 Ci/m mol), 32P-labeled nucleotide dCTP (specific activity approximately 700 Ci/m m ol) M C-methylated protein stan­ Radiolabeling o f Hepatocyte Proteins dards (high and low molecular weight standards), Amplify®, and Hepatocyte monolayers were pulsed for a suitable interval with rabbit reticulocyte lysate were purchased from Amersham Buchler methionine-free DMEM containing 35S-m ethionine. The cells were GmbH (Braunschweig, FRG), formalin-fixed Staphylococcus aureus washed on ice with cold Hank’s balanced salt solution (HBSS) and lysed by freeze-thawing with a solution o f phosphate-buffered saline (‘Pansorbin’) later called Staph A was purchased from Calbiochem GmbH (Frankfurt, FRG), guanidinium isothyocyanate was from (pH 7.5), containing lOOmA/KCI, 1% Triton X -100, 0.5% sodium Ruka (Bern, Switzerland), CsCl and agarose from BRL (Heidelberg, deoxycholate, 2 mA/ phenylmethylsulfonyl fluoride. 10 mM EDTA. FRG). The culture supernatants and lysates were centrifuged in an Eppen- dorf minifuge for 30 min at 4 °C diluted with the lysis buffer con­ taining 1 % SDS and stored at - 8 0 °C. Total protein synthesis was Antisera measured by trichloracetic acid precipitation o f 1 pi o f cellular Rat antiserum against LBP was raised by immunizing animals with LBP purified from acute-phase rabbit serum as described else­ lysates, supernatants and cell-free translation products [ 17], where [3]. Guinea pig antiserum against rabbit serum amyloid A Im munoprécipitation an d SDS-PAGE Analysis (SAA) was obtained by im munizing anim als with SAA purified from acute-phase rabbit serum as described previously [10]. Antiserum Cellular lysates, supernatants and cell-free translation products against rabbit albumin was purchased from Cappel Laboratories. (the latter diluted to 200 pi with lysis buffer) were precleared with Staph A (50 pi o f a 10% solution/sample) and incubated with the Prim ary H epatocyte Cultures appropriate antibody overnight at 4 ° C . Immunocomplexes were precipitated by adding Staph A. Immunoprecipitates were further Hepatocytes were isolated from the livers o f young rabbits (body weight 6 0 0 -8 0 0 g) by the perfusion technique as described else­ analyzed by SDS-PAGE according to Laemmli [18] as described where [1 1-13]. Briefly, the liver was shortly perfused with a cal­ elsewhere [ 19] with reduction o f samples using p-mercaptoethanol. cium-free solution and then with a solution containing 0.05 % colla­ Incorporation o f 35S-methionine into individual immunoprecipi- génase. The liver was then teased apart and hepatocytes were sepa­ tated proteins was determined in gel slices after digestion with 15% rated from non-parenchymal cells by differential centrifugation; the hydrogen peroxide for 16 h and addition o f Scinti Verse I (Fisher contamination with non-parenchymal cells was always reduced to Scientific, Heidelberg, FRG) [20]. less than 1 % [12]. The viability assessed by the trypan blue exclu­ sion test was always above 80%. Cells were plated onto collagen- coated 24-well or 6-well Falcon plates at 2 X 10s cells/well in 500 pi or 106 cells/well in 1.5 ml culture medium, respectively. Culture Results medium (DMEM) contained 25 mA/ HEPES, 4.5 g/1 glucose, 1 pg/ml insulin, 0.2% fatty acid-free BSA. For the first 2 -4 h o f LBP Synthesis by Rabbit Hepatocyte Primary culture, cells were cultured in the presence o f 10% FBS instead o f BSA. The cells were kept in culture at 37 °C under a 5% CO2 95% Cultures air atmosphere for up to 10 days with daily change o f culture Explanted rabbit hepatocytes were found to synthe­ medium. size a biosynthetically labelled protein with a Mr of about 60,000 that is immunoprecipitated from cell ly­ Isolation o f RNA sates and supernatants by adding rat anti-rabbit LBP Total RNA was extracted from the liver o f normal rabbits and o f rabbits treated subcutaneously with 1 ml o f 3% wt/vol silver nitrate antibody. The apparent heterogeneity of LBP secreted Biosynthesis of LBP in Hepatocytes 91 Source C M c + + + Anti LBP LBP + + - MW M O ' 3) 9 2 - ,sS-methionine. To study the specificity o f the immunoprécipitation reaction, the antibody against LBP was incubated either with puri­ fied cold LBP or with PBS before use. Fig. 2. Kinetics o f LBP synthesis and secretion by rabbit hepato­ cytes. An autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP immunoprecipitated from cellular lysates or from cell super- nates. Hepatocytes were pulsed for 30 min with 250 pCi/250pl/w ell o f L-35S-methionine and then incubated for 0, 30, 60, 120 or 540 min in unlabeled L-methionine containing DMEM. Lancs 1-5 Fig. 1. Synthesis and secretion o f LBP by rabbit hepatocytes. An autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP contain the cell lysates corresponding to 0, 30, 60, 120 and 540 min immunoprecipitated from hepatocytes (C) or culture medium (M). post-chase and lanes l ' - 5 ' contain the media from the same time Hepatocyte cultures were pulsed for 2 h with medium containing points. by hepatocytes shown in figure 1 probably represents LBP and a concurrent increase of extracellular LBP. LBP with different glycosylation patterns (see below). Thus LBP is synthesized and secreted by rabbit hepato­ Importantly, the presence of LBP in the immunoprecipi- cyte primary cultures. Data from cells cultured for 2 days tates was established by inhibiting immunoprécipitation are shown in figure 2. of 35S-proteins with purified serum LBP. The identity of LBP purified from acute-phase rabbit serum is a gly­ coprotein [3], To determine whether the biosynthetic the remaining bands present in figure 1 is currently product of the explanted rabbit hepatocytes is also glyco­ unknown and may arise as a result of antibodies in the anti-LBP serum directed to other proteins synthesized in sylated, the following experiment was performed. Ex- planted hepatocytes were treated with different concen­ hepatocytes. Alternatively these may represent major trations of tunicamycin [21, 22] for 4 h and pulsed with biosynthetic products o f the rabbit hepatocytes that non- 35S-methionine for 2 h in the presence of tunicamycin. specifically appear in the immunoprecipitates. To demonstrate that LBP is a secretory protein in As shown in figure 3, tunicamycin treatment results in a rabbit hepatocytes, a ‘pulse-chase’ experiment was per­ decrease in the apparent mass of LBP reaching a 50-kD formed on days 2 and 6 after isolation of the cells. In form with 5 pg/ml. The 50-kD form of LBP accumulates both cases similar data were obtained showing a progres­ intracellularly with lesser amounts found in the extracel­ sive decrease of biosynthetically labelled intracellular lular medium. 92 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch Studies with Rabbit Liver RNA Total RNA extracted from isolated hepatocytes at dif­ ferent times after the isolation was translated in vitro in 1VIVV Cells Medium ( X 1 O'3) I------------------------- II-------------------- the presence of 35S-methionine. LBP was immunopreci- f i r m pitated from the cell-free translation products and ana­ lyzed by SDS-PAGE. As shown in figure 4, a protein with an apparent mass of about 50 kD was precipitated 9 2 - in agreement with data obtained using tunicamycin, indicating that this is the precursor of the mature full glycosylated LBP. LBP mRNA was found in all the sam­ 68- ples investigated (days 2, 4, 6, or 8 after isolation) indi­ cating that LBP is continuously synthesized by explanted hepatocytes during culture. In the present studies we have not been able to isolate rabbit hepatocytes that do not synthesize LBP. The same phenomenon was ob­ served for SAA (data not shown). The reasons for this are not clear since LBP is definitely an acute-phase reactant and the mRNA for rabbit LBP is not detectable in rabbit liver until after the induction of the acute-phase response 0 01 1.0 50 10 0 0.1 1.0 5.0 10 (fig. 5) [Schumann et al., manuscript in preparation). One possibility is that the process of adaptation to cul­ [Tunicamycin], ^g/ml ture conditions may trigger both the LBP and the SAA gene as observed in other animal species [13] for SAA. To determine whether LBP is regulated as expected Fig. 3. Effects o f tunicamycin on LBP biosynthesis by rabbit for an acute-phase protein, rabbits were treated either hepatocytes. An autoradiogram o f an SDS-PAGE gel (7.5% acryl­ with saline or a silver nitrate solution [3] and 24 h later, am ide) o f LBP immunoprecipitated from hepatocyte lysates and after sacrifice, the livers were snap frozen in liquid nitro­ from culture supemates. Cells were exposed for 4 h to different con­ gen and total RNA was extracted both from control and centrations o f tunicamycin (0, 0,1, 1,5, 10 pg/ml) and then pulsed acute-phase livers. This RNA was translated in vitro for 2 h with 250 jiCi/well 3SS-methionine in 250 pi o f ¿-m ethionine- free medium. using rabbit reticulocyte lysate and 35S-methionine. To look for proteins synthesized in this system, the radioac­ tive cell-free translation products were incubated either with antibodies to rabbit LBP, rabbit SAA or rabbit albumin and immunoprecipitates were analyzed by 60-kD protein is glycosylated and that treatment of the SDS-PAGE. As shown in figure 5, LBP was clearly de­ cells with tunicamycin reduces secretion and results in tected in the products of acute-phase RNA but not in the accumulation of an intracellular form of LBP with an those of normal RNA. The same was true of SAA. The apparent size of 50 kD. We also demonstrated that RNA quantitative difference between the two proteins reflects isolated from rabbit liver 24 h after induction of an the difference in their respective plasma levels (SAA acute-phase response would support the synthesis of plasma level is about 20 times higher than that of LBP). LBP in an in vitro translation system while RNA from Albumin, as expected, showed an opposite behavior with control rabbits yielded no detectable LBP in the same in decreased synthesis under acute-phase conditions. vitro translation system. Thus the present studies pro­ vide additional support for the concept that LBP is an acute-phase protein synthesized in hepatocytes and pro­ Discussion vides the basis for future studies that define the regula­ tion of the synthesis of this newly described acute-phase The data presented in this paper demonstrate that protein. LBP is synthesized and secreted by explanted rabbit Although the ability to synthesize LBP using mRNA hepatocytes. Using immunoprécipitation of 35S-methio- and an in vitro translation system requires initiation of nine-labelled protein it was established that the secreted an acute-phase response, the explanted rabbit hepato- Biosynthesis o f LBP in Hepatocytes MW MW ( x 10 '3) SAA LBP Albumin (x IQ 3) 68- . ■ ■ ■ - _■ ' . S > • ' * v * N A N A N A n»«* -LBP i * ’ Zj Fig. 4. LBP gene expression in rabbit hepatocytes at different S : *• & W " ' times after isolation. Autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP immunoprecipitated from cell-free translation 2 4 6 8 products o f total RNA obtained from rabbit hepatocyte primary cultures. Days After Explanting Fig. 5. Induction o f LBP and SAA gene expression in rabbit liver Cells after treatment o f the animals with silver-nitrate. Autoradiogram o f an SDS-PAGE gel (15 % acrylamide) o f SAA. LBP or albumin immu­ noprecipitated from cell-free translation product o f RNA extracted from normal control liver (N) or from acute phase liver (A). cytes constitutively express LBP at the mRNA and pro­ that correspond to LBP are completely inhibited by the tein level. The regulation of LBP synthesis has not been use of added purified LBP. Studies with tunicamycin studied but, as shown for other acute-phase reactants, demonstrate that LBP is glycosylated by the hepatocytes may be under the control of cytokines most notably IL-6 and released as a form that display slightly different [23, 24], Attempts to increase the production of LBP in mobilities on SDS gels. A similar heterogeneity was noted the explanted rabbit hepatocytes with purified rabbit for LBP in rabbit serum isolated 24 h after an acute phase TNF, partially purified rabbit IL-1 or cell-free superna­ response, the two forms noted in vivo have apparent tant from lipopolysaccharide-stimulated macrophages molecular weights of about 60 and 58 kD with the 60-kD were not successful [Ramadori, unpublished data]. Until form representing about 80% of the total LBP. The tuni­ better methods are developed to isolate explanted rabbit camycin studies also establish that the nonglycosylated hepatocytes that only express LBP after stimulation, in form of LBP has an apparent mass of about 50 kD. This vitro studies of the regulation of LBP expression in rab­ is in agreement with the result of the cell-free translation bit cannot be pursued. experiment (fig. 5) and expected size of the LBP polypep­ Data with anti-LBP reveal a number of proteins that tide deduced from the cDNA sequence of the LBP gene may be nonspecifically associated with the immunopreci- [Schumann et al., manuscript in preparation]. pitate since they are not blocked by the addition of puri­ Recently we suggested that LBP is part of a gene fied LBP to the antibody (fig. 1). In contrast the bands family of proteins that have evolved to interact with 94 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch 11 Ramadori G, Lenzi M, Dienes HP, et al: Binding proper ties o f lipopolysaccharide [5] and that LBP has a specific bind­ mechanically and enzymatically isolated hepatocytes for IgG ing site for the lipid A region of LPS [8], The biological and C. Liver 1983;3:358-368. role of LBP is just now beginning to be understood; 12 Ramadori G, Rasokat G, Burger H, et al: Quantitative determi­ recently it has been shown that LBP can enhance the nation o f complement components produced by purified hepa­ adherence of lipopolysaccharide-coated particles and tocytes. Clin Exp Immunol 1984;55:189-195. 13 Ramadori G, Sipe JD, Dinarello CA, et al: Pretranslational mod­ gram-negative bacteria to macrophages [Wright et al., ulation o f acute phase hepatic protein synthesis by murine manuscript submitted]. It is our contention that LBP recombinant interleukin-1 (IL-1) and purified human IL-1. J plays an important role in the host response to gram­ Exp Med 1985;162:930-942. negative bacteria and to lipopolysaccharide. The present 14 Chirgwin JM, Przybyla AE, McDonald RJ, et al: Isolation o f studies provide the basis for the future work aimed at biologically active ribonucleic acid from sources enriched in ribonucléase. Biochemistry 1979;18:5284-5287. elucidating the host mechanisms that control LBP bio­ 15 Ramadori G, Sipe JD, Colten HR: Expression and regulation o f synthesis and secretion that is essential for a full under­ the murine serum amyloid A (SAA) gene in extrahepatic sites. J standing of the fraction of this protein. Immunol 1985;135:3645-3647. 16 Pelham HRB, Jackson RJ: An efficient m RNA dependent trans­ lation system from reticulocyte lysates. Eur J Biochem 1976;67: Acknowledgments 2 4 7 -2 5 1 . 17 Roberts BE, Patterson BM: Efficient translation o f tobacco mo­ We wish to thank Velda Comstock for her assistance in the prep­ saic virus RNA and rabbit globin GS RNA in a cell free system aration o f the manuscript and Christine Waldmann for technical from commercial wheat germ. Proc Natl Acad Sei USA 1973;70: assistance. 2 3 3 0 -2 3 3 2 . 18 Laemmli UR: Cleavage o f structural proteins during the assem­ bly o f the head o f bacteriophage T4. Nature (Lond) 1970;227: 6 8 0 -6 8 2 . References 19 Strunk RC, Cole FS, Perlmutter DH, et al: y-Interferon increases 1 Koj A, et al: The acute-phase response to injury and infection: expression o f class III complement genes C2 and factor B in The roles o f interleukin one and other mediators; in Gordon AH human monocytes and in murine fibroblasts transfected with (ed): Research Monographs in Cell Tissue Physiology. Amster­ human C2 and factor B genes. J Biol Chem 1985,260:15280— dam, Elsevier, 1985, vol 10, pp 5 3 4 -1 2 0 1 . 15285. 2 Kuhlman M, Joiner K, Ezekowitz, RAB: The human mannose­ 20 Sipe JD, Colten HR, Goldberger G, et al: Human serum amyloid binding protein functions as an opsonin. J Exp Med 1989; 169: A (SAA): Biosynthesis and post-synthetic processing o f pre-SAA 1733-1745. and structural variants defined by complementary DNA. Bio­ 3 Tobias PS, Soldau K, Ulevitch RJ: Isolation o f a lipopolysaccha- chemistry 1985;24:2931-2935. ride-binding acute-phase reactant from rabbit serum. J Exp Med 21 Elbein AD: The tunicamycin-useful tools for studies on glyco­ 1986;164:777-793. proteins. Trends Biochem Sei 1981;6:219-221. 4 Tobias PS, McAdam K.PWJ, Soldau K, et al: Control o f lipo- 22 Ramadori G, Rieder H, Knittel TH, et al: Fat storing cells (FSC) polysaccharide-high density lipoprotein interactions by an acute o f rat liver synthesize and secrete fibronectin. Comparison with phase reactant in human serum. Infect Immun 1985;50:73-76. hepatocytes. J Hepatol 1987;2:208-217. 5 Tobias PS, Mathison JC, Ulevitch RJ: A family o f lipopolysac­ 23 Ramadori G, Van Dam m e J. Rieder H, et al: Interleukin-6, the charide binding proteins involved in responses to gram-negative third mediator o f acute-phase reaction, modulates hepatic pro­ sepsis. J Biol Chem 1988;263:13479-13488. tein synthesis in human and mouse. Comparison with interleu­ 6 Gray PW, Flaggs G, Leong SR, et al: Cloning o f the cDNA o f a kin lß and tumor necrosis factor-a. Eur J Immunol 1988; 18: human neutrophil bactericidal protein. J Biol Chem 1989;264: 1 2 5 9 -1 2 6 4 . 9 5 0 5 -9 5 0 9 . 24 Ramadori G, Mitsch A, Rieder H, et al: Alpha- and gamma- 7 Weiss J, Muello K, Victor M, et al: The role o f lipopolysaccha­ interferon (IFNa, IFNy) but not interleukin-1 (IL-1) modulate rides in the action o f the bactericidal/permeability-increasing synthesis and secretion o f ßi-microglobulin by hepatocytes. Eur neutrophil protein on the bacterial envelope. J Immunol 1984; J Clin Invest 1988;18:343-351. 132:3109-3115. 8 Tobias PS, Soldau K. Ulevitch RJ: Identification o f a lipid A binding site in the acute phase reactant lipopolysaccharide bind­ Received: June 29, 1989 ing protein. J Biol Chem 1989;264:10867-10871. Accepted: August 5, 1989 9 W eiss J, Olssen I: Cellular and subcellular localization o f the bactericidal/permeability-increasing protein o f the neutrophils. Giuliano Ramadori, MD Blood 1987;69:652-659. Klinikum der Johannes-Gutenberg-Universität 10 Tobias PS, McAdam KPWJ, U levitch RJ: Interactions o f bacte­ I. M edizinische Klinik und Poliklinik rial lipopolysaccharide with acute-phase rabbit serum and isola­ Postfach 3960 tion o f two forms o f rabbit serum amyloid A. J Immunol 1982; Langenbeckstrasse 1 128:1420-1427. D -6 5 0 0 Mainz (FRG) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pathobiology Karger

Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hepatocytes

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Karger
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© 1990 S. Karger AG, Basel
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1015-2008
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1423-0291
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10.1159/000163569
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Abstract

Lipopolysaccharide Binding © 1990 S. K arger AG. Basel Pathobiology 1990;58:89-94 1015 -2 0 0 8 /9 0 /0 5 8 2 -0 0 8 9 $ 2 .75/0 Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hépatocytes1 G. Ramadoria , K.-H. Meyer zum Buschenfeldea , P.S. Tobiasb, J.C. Mathisonb, R.J. Ulevitchb a Klinikum der Johannes-Gutenberg-Univcrsität, I. Medizinische Klinik und Poliklinik, Mainz, FRG; b Department o f Immunology, Research Institute o f Scripps Clinic, La Jolla, Calif., USA Key Words. Lipopolysaccharide-binding protein • Hépatocytes • Acute phase protein • LPB-biosyn thesis Abstract. Studies reported here show that the recently discovered acute-phase protein, lipopolysaccharide-binding protein (LBP), is synthesized by hepatocytes. For these studies, explanted rabbit hepatocytes were grown in the presence of 35S-methionine. Biosynthetically labelled LBP in the cells and supernatant was identified using immu­ noprécipitation with rat anti-rabbit LBP antibody. This antibody immunoprecipitates both the LBP polypeptide and the glycosylated protein. With a cell-free translation system a comparison of RNA from normal rabbit liver with that isolated from acute-phase rabbit liver indicated that a translatable LBP message is only found in the RNA from acute-phase liver. Studies with explanted rabbit hepatocytes showed that several forms of LBP distinguishable by migration in SDS-PAGE are secreted into the extracellular medium. This heterogeneity probably is a result of differences in glycosylation of LBP since pretreatment of the hepatocytes with tunicamycin results in accumulation of a single polypeptide with an apparent mass of 50 kD in SDS-PAGE. Explanted rabbit hepatocytes spontaneously synthesize and secrete LBP and express LBP mRNA as detected by cell-free translation; thus, it was not possible to upregulate the expression of LBP. Nevertheless, these studies form the basis for future investigations on the regula­ tion of LBP biosynthesis. Introduction nized; namely, the mannose-binding protein [2] and lipo­ polysaccharide-binding protein (LBP) [3, 4], An acute-phase reaction is part of the physiologic We have recently suggested that there may be a family response of an organism to disparate conditions such as of LBPs with certain structural and/or functional simi­ bacterial infections, myocardial infarction, surgery, larities [5], The first two members of this family are LBP bums or neoplastic diseases. One characteristic of the and a neutrophil granule protein called bactericidal per­ acute-phase response is changes in the rate of synthesis of meability-increasing protein (BPI) [6]. There is a striking at least 30 different plasma proteins [1], Some of these homology in the amino-terminal sequences of rabbit proteins, which are normally present in very low concen­ LBP [5], and human BPI and both proteins share at least trations prior to the acute-phase response, rise as much as one function: the ability to bind to lipopolysaccharide several hundred-fold. Examples in this group are C-reac- from a wide variety of gram-negative bacteria [7, 8], tive protein in man, serum amyloid A in man and mouse, While BPI has been proven to be a neutrophil granule and ci2-macroglobulin in rats. Recently several new mem­ protein and inducible in HL-60 cells during differentia­ bers of this class of acute-phase proteins have been recog­ tion of these cells to a neutrophil-like cell [9], the site of synthesis of LBP has not been established. The studies described in this report provide information about the 1 Supported by A l l 5136, AI25563, SFB 311 (IMMUNPATHO- site o f synthesis of LBP in rabbits. In this paper we dem­ GENESE) Project A7, Ra 362/5-1 (DFG). This is Department o f Immunology publication No. 5988-IMM. onstrate LBP gene expression in acute-phase rabbit liver 90 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch in distilled water and sacrificed 24 h after injection. The RNA and in explanted rabbit hépatocytes. LBP is synthesized extraction was performed according to Chirgwin et al. [14] as as a 50-kD protein and released from hepatocytes as a described elsewhere [13, 14]. glycosylated protein with at least two different glycosyla­ Total RNA was also extracted from hepatocyte primary cultures tion patterns. These studies form the basis for future (cells plated onto 6-well plates) using the same procedure [14]. investigations that compare and contrast the regulation Briefly guanidinium lysates were repeatedly passed through a 23- gaugc needle to shear DNA and then layered onto 2 ml o f 5.7 M of BPI and LBP biosynthesis. CsCl and centrifuged in a Beckman SW 5 0 .1 rotor (Beckman Instru­ ments, Inc., Fullerton, Calif.) for 16 -1 8 h at 35,000 rpm at 20 ° C , the R NA pellet was washed twice in ethanol, then dissolved in water, reprecipitated in ethanol and dissolved in water, RNA con­ Materials and Methods tent was determined by measurement o f absorption at 260 nm. Reagents Dulbecco's modified Eagle’s medium (DMEM) and DMEM Cell-Free Translation o f RNA Total RNA from normal and acute-phase rabbit liver as well as without methionine was purchased from Gibco (Heidelberg, FRG) from hepatocytes was translated in vitro using rabbit reticulocyte and fetal bovine serum (FBS), penicillin, streptomycin and ¿-gluta­ mine from R o w Laboratories (Bonn, FRG), crystallized bovine lysate [16] in the presence o f 35S-m ethionine according to the serum albumin (BSA) with the minimum purity o f 98% and colla­ instructions o f the manufacturer. As positive control acute-phase murine liver RNA was used together with tobacco mosaic virus génase from Clostridium histolyticum were purchased from Behring- poly-A-RNA provided with the kit. Werke (Marburg, FRG). 3sS-methionine (specific radioactivity ap­ proximately 800 Ci/m mol), 32P-labeled nucleotide dCTP (specific activity approximately 700 Ci/m m ol) M C-methylated protein stan­ Radiolabeling o f Hepatocyte Proteins dards (high and low molecular weight standards), Amplify®, and Hepatocyte monolayers were pulsed for a suitable interval with rabbit reticulocyte lysate were purchased from Amersham Buchler methionine-free DMEM containing 35S-m ethionine. The cells were GmbH (Braunschweig, FRG), formalin-fixed Staphylococcus aureus washed on ice with cold Hank’s balanced salt solution (HBSS) and lysed by freeze-thawing with a solution o f phosphate-buffered saline (‘Pansorbin’) later called Staph A was purchased from Calbiochem GmbH (Frankfurt, FRG), guanidinium isothyocyanate was from (pH 7.5), containing lOOmA/KCI, 1% Triton X -100, 0.5% sodium Ruka (Bern, Switzerland), CsCl and agarose from BRL (Heidelberg, deoxycholate, 2 mA/ phenylmethylsulfonyl fluoride. 10 mM EDTA. FRG). The culture supernatants and lysates were centrifuged in an Eppen- dorf minifuge for 30 min at 4 °C diluted with the lysis buffer con­ taining 1 % SDS and stored at - 8 0 °C. Total protein synthesis was Antisera measured by trichloracetic acid precipitation o f 1 pi o f cellular Rat antiserum against LBP was raised by immunizing animals with LBP purified from acute-phase rabbit serum as described else­ lysates, supernatants and cell-free translation products [ 17], where [3]. Guinea pig antiserum against rabbit serum amyloid A Im munoprécipitation an d SDS-PAGE Analysis (SAA) was obtained by im munizing anim als with SAA purified from acute-phase rabbit serum as described previously [10]. Antiserum Cellular lysates, supernatants and cell-free translation products against rabbit albumin was purchased from Cappel Laboratories. (the latter diluted to 200 pi with lysis buffer) were precleared with Staph A (50 pi o f a 10% solution/sample) and incubated with the Prim ary H epatocyte Cultures appropriate antibody overnight at 4 ° C . Immunocomplexes were precipitated by adding Staph A. Immunoprecipitates were further Hepatocytes were isolated from the livers o f young rabbits (body weight 6 0 0 -8 0 0 g) by the perfusion technique as described else­ analyzed by SDS-PAGE according to Laemmli [18] as described where [1 1-13]. Briefly, the liver was shortly perfused with a cal­ elsewhere [ 19] with reduction o f samples using p-mercaptoethanol. cium-free solution and then with a solution containing 0.05 % colla­ Incorporation o f 35S-methionine into individual immunoprecipi- génase. The liver was then teased apart and hepatocytes were sepa­ tated proteins was determined in gel slices after digestion with 15% rated from non-parenchymal cells by differential centrifugation; the hydrogen peroxide for 16 h and addition o f Scinti Verse I (Fisher contamination with non-parenchymal cells was always reduced to Scientific, Heidelberg, FRG) [20]. less than 1 % [12]. The viability assessed by the trypan blue exclu­ sion test was always above 80%. Cells were plated onto collagen- coated 24-well or 6-well Falcon plates at 2 X 10s cells/well in 500 pi or 106 cells/well in 1.5 ml culture medium, respectively. Culture Results medium (DMEM) contained 25 mA/ HEPES, 4.5 g/1 glucose, 1 pg/ml insulin, 0.2% fatty acid-free BSA. For the first 2 -4 h o f LBP Synthesis by Rabbit Hepatocyte Primary culture, cells were cultured in the presence o f 10% FBS instead o f BSA. The cells were kept in culture at 37 °C under a 5% CO2 95% Cultures air atmosphere for up to 10 days with daily change o f culture Explanted rabbit hepatocytes were found to synthe­ medium. size a biosynthetically labelled protein with a Mr of about 60,000 that is immunoprecipitated from cell ly­ Isolation o f RNA sates and supernatants by adding rat anti-rabbit LBP Total RNA was extracted from the liver o f normal rabbits and o f rabbits treated subcutaneously with 1 ml o f 3% wt/vol silver nitrate antibody. The apparent heterogeneity of LBP secreted Biosynthesis of LBP in Hepatocytes 91 Source C M c + + + Anti LBP LBP + + - MW M O ' 3) 9 2 - ,sS-methionine. To study the specificity o f the immunoprécipitation reaction, the antibody against LBP was incubated either with puri­ fied cold LBP or with PBS before use. Fig. 2. Kinetics o f LBP synthesis and secretion by rabbit hepato­ cytes. An autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP immunoprecipitated from cellular lysates or from cell super- nates. Hepatocytes were pulsed for 30 min with 250 pCi/250pl/w ell o f L-35S-methionine and then incubated for 0, 30, 60, 120 or 540 min in unlabeled L-methionine containing DMEM. Lancs 1-5 Fig. 1. Synthesis and secretion o f LBP by rabbit hepatocytes. An autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP contain the cell lysates corresponding to 0, 30, 60, 120 and 540 min immunoprecipitated from hepatocytes (C) or culture medium (M). post-chase and lanes l ' - 5 ' contain the media from the same time Hepatocyte cultures were pulsed for 2 h with medium containing points. by hepatocytes shown in figure 1 probably represents LBP and a concurrent increase of extracellular LBP. LBP with different glycosylation patterns (see below). Thus LBP is synthesized and secreted by rabbit hepato­ Importantly, the presence of LBP in the immunoprecipi- cyte primary cultures. Data from cells cultured for 2 days tates was established by inhibiting immunoprécipitation are shown in figure 2. of 35S-proteins with purified serum LBP. The identity of LBP purified from acute-phase rabbit serum is a gly­ coprotein [3], To determine whether the biosynthetic the remaining bands present in figure 1 is currently product of the explanted rabbit hepatocytes is also glyco­ unknown and may arise as a result of antibodies in the anti-LBP serum directed to other proteins synthesized in sylated, the following experiment was performed. Ex- planted hepatocytes were treated with different concen­ hepatocytes. Alternatively these may represent major trations of tunicamycin [21, 22] for 4 h and pulsed with biosynthetic products o f the rabbit hepatocytes that non- 35S-methionine for 2 h in the presence of tunicamycin. specifically appear in the immunoprecipitates. To demonstrate that LBP is a secretory protein in As shown in figure 3, tunicamycin treatment results in a rabbit hepatocytes, a ‘pulse-chase’ experiment was per­ decrease in the apparent mass of LBP reaching a 50-kD formed on days 2 and 6 after isolation of the cells. In form with 5 pg/ml. The 50-kD form of LBP accumulates both cases similar data were obtained showing a progres­ intracellularly with lesser amounts found in the extracel­ sive decrease of biosynthetically labelled intracellular lular medium. 92 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch Studies with Rabbit Liver RNA Total RNA extracted from isolated hepatocytes at dif­ ferent times after the isolation was translated in vitro in 1VIVV Cells Medium ( X 1 O'3) I------------------------- II-------------------- the presence of 35S-methionine. LBP was immunopreci- f i r m pitated from the cell-free translation products and ana­ lyzed by SDS-PAGE. As shown in figure 4, a protein with an apparent mass of about 50 kD was precipitated 9 2 - in agreement with data obtained using tunicamycin, indicating that this is the precursor of the mature full glycosylated LBP. LBP mRNA was found in all the sam­ 68- ples investigated (days 2, 4, 6, or 8 after isolation) indi­ cating that LBP is continuously synthesized by explanted hepatocytes during culture. In the present studies we have not been able to isolate rabbit hepatocytes that do not synthesize LBP. The same phenomenon was ob­ served for SAA (data not shown). The reasons for this are not clear since LBP is definitely an acute-phase reactant and the mRNA for rabbit LBP is not detectable in rabbit liver until after the induction of the acute-phase response 0 01 1.0 50 10 0 0.1 1.0 5.0 10 (fig. 5) [Schumann et al., manuscript in preparation). One possibility is that the process of adaptation to cul­ [Tunicamycin], ^g/ml ture conditions may trigger both the LBP and the SAA gene as observed in other animal species [13] for SAA. To determine whether LBP is regulated as expected Fig. 3. Effects o f tunicamycin on LBP biosynthesis by rabbit for an acute-phase protein, rabbits were treated either hepatocytes. An autoradiogram o f an SDS-PAGE gel (7.5% acryl­ with saline or a silver nitrate solution [3] and 24 h later, am ide) o f LBP immunoprecipitated from hepatocyte lysates and after sacrifice, the livers were snap frozen in liquid nitro­ from culture supemates. Cells were exposed for 4 h to different con­ gen and total RNA was extracted both from control and centrations o f tunicamycin (0, 0,1, 1,5, 10 pg/ml) and then pulsed acute-phase livers. This RNA was translated in vitro for 2 h with 250 jiCi/well 3SS-methionine in 250 pi o f ¿-m ethionine- free medium. using rabbit reticulocyte lysate and 35S-methionine. To look for proteins synthesized in this system, the radioac­ tive cell-free translation products were incubated either with antibodies to rabbit LBP, rabbit SAA or rabbit albumin and immunoprecipitates were analyzed by 60-kD protein is glycosylated and that treatment of the SDS-PAGE. As shown in figure 5, LBP was clearly de­ cells with tunicamycin reduces secretion and results in tected in the products of acute-phase RNA but not in the accumulation of an intracellular form of LBP with an those of normal RNA. The same was true of SAA. The apparent size of 50 kD. We also demonstrated that RNA quantitative difference between the two proteins reflects isolated from rabbit liver 24 h after induction of an the difference in their respective plasma levels (SAA acute-phase response would support the synthesis of plasma level is about 20 times higher than that of LBP). LBP in an in vitro translation system while RNA from Albumin, as expected, showed an opposite behavior with control rabbits yielded no detectable LBP in the same in decreased synthesis under acute-phase conditions. vitro translation system. Thus the present studies pro­ vide additional support for the concept that LBP is an acute-phase protein synthesized in hepatocytes and pro­ Discussion vides the basis for future studies that define the regula­ tion of the synthesis of this newly described acute-phase The data presented in this paper demonstrate that protein. LBP is synthesized and secreted by explanted rabbit Although the ability to synthesize LBP using mRNA hepatocytes. Using immunoprécipitation of 35S-methio- and an in vitro translation system requires initiation of nine-labelled protein it was established that the secreted an acute-phase response, the explanted rabbit hepato- Biosynthesis o f LBP in Hepatocytes MW MW ( x 10 '3) SAA LBP Albumin (x IQ 3) 68- . ■ ■ ■ - _■ ' . S > • ' * v * N A N A N A n»«* -LBP i * ’ Zj Fig. 4. LBP gene expression in rabbit hepatocytes at different S : *• & W " ' times after isolation. Autoradiogram o f an SDS-PAGE gel (7.5% acrylamide) o f LBP immunoprecipitated from cell-free translation 2 4 6 8 products o f total RNA obtained from rabbit hepatocyte primary cultures. Days After Explanting Fig. 5. Induction o f LBP and SAA gene expression in rabbit liver Cells after treatment o f the animals with silver-nitrate. Autoradiogram o f an SDS-PAGE gel (15 % acrylamide) o f SAA. LBP or albumin immu­ noprecipitated from cell-free translation product o f RNA extracted from normal control liver (N) or from acute phase liver (A). cytes constitutively express LBP at the mRNA and pro­ that correspond to LBP are completely inhibited by the tein level. The regulation of LBP synthesis has not been use of added purified LBP. Studies with tunicamycin studied but, as shown for other acute-phase reactants, demonstrate that LBP is glycosylated by the hepatocytes may be under the control of cytokines most notably IL-6 and released as a form that display slightly different [23, 24], Attempts to increase the production of LBP in mobilities on SDS gels. A similar heterogeneity was noted the explanted rabbit hepatocytes with purified rabbit for LBP in rabbit serum isolated 24 h after an acute phase TNF, partially purified rabbit IL-1 or cell-free superna­ response, the two forms noted in vivo have apparent tant from lipopolysaccharide-stimulated macrophages molecular weights of about 60 and 58 kD with the 60-kD were not successful [Ramadori, unpublished data]. Until form representing about 80% of the total LBP. The tuni­ better methods are developed to isolate explanted rabbit camycin studies also establish that the nonglycosylated hepatocytes that only express LBP after stimulation, in form of LBP has an apparent mass of about 50 kD. This vitro studies of the regulation of LBP expression in rab­ is in agreement with the result of the cell-free translation bit cannot be pursued. experiment (fig. 5) and expected size of the LBP polypep­ Data with anti-LBP reveal a number of proteins that tide deduced from the cDNA sequence of the LBP gene may be nonspecifically associated with the immunopreci- [Schumann et al., manuscript in preparation]. pitate since they are not blocked by the addition of puri­ Recently we suggested that LBP is part of a gene fied LBP to the antibody (fig. 1). In contrast the bands family of proteins that have evolved to interact with 94 Ramadori/Meyer zum Buschenfelde/Tobias/Mathison/Ulevitch 11 Ramadori G, Lenzi M, Dienes HP, et al: Binding proper ties o f lipopolysaccharide [5] and that LBP has a specific bind­ mechanically and enzymatically isolated hepatocytes for IgG ing site for the lipid A region of LPS [8], The biological and C. Liver 1983;3:358-368. role of LBP is just now beginning to be understood; 12 Ramadori G, Rasokat G, Burger H, et al: Quantitative determi­ recently it has been shown that LBP can enhance the nation o f complement components produced by purified hepa­ adherence of lipopolysaccharide-coated particles and tocytes. Clin Exp Immunol 1984;55:189-195. 13 Ramadori G, Sipe JD, Dinarello CA, et al: Pretranslational mod­ gram-negative bacteria to macrophages [Wright et al., ulation o f acute phase hepatic protein synthesis by murine manuscript submitted]. It is our contention that LBP recombinant interleukin-1 (IL-1) and purified human IL-1. J plays an important role in the host response to gram­ Exp Med 1985;162:930-942. negative bacteria and to lipopolysaccharide. The present 14 Chirgwin JM, Przybyla AE, McDonald RJ, et al: Isolation o f studies provide the basis for the future work aimed at biologically active ribonucleic acid from sources enriched in ribonucléase. Biochemistry 1979;18:5284-5287. elucidating the host mechanisms that control LBP bio­ 15 Ramadori G, Sipe JD, Colten HR: Expression and regulation o f synthesis and secretion that is essential for a full under­ the murine serum amyloid A (SAA) gene in extrahepatic sites. J standing of the fraction of this protein. Immunol 1985;135:3645-3647. 16 Pelham HRB, Jackson RJ: An efficient m RNA dependent trans­ lation system from reticulocyte lysates. Eur J Biochem 1976;67: Acknowledgments 2 4 7 -2 5 1 . 17 Roberts BE, Patterson BM: Efficient translation o f tobacco mo­ We wish to thank Velda Comstock for her assistance in the prep­ saic virus RNA and rabbit globin GS RNA in a cell free system aration o f the manuscript and Christine Waldmann for technical from commercial wheat germ. Proc Natl Acad Sei USA 1973;70: assistance. 2 3 3 0 -2 3 3 2 . 18 Laemmli UR: Cleavage o f structural proteins during the assem­ bly o f the head o f bacteriophage T4. Nature (Lond) 1970;227: 6 8 0 -6 8 2 . References 19 Strunk RC, Cole FS, Perlmutter DH, et al: y-Interferon increases 1 Koj A, et al: The acute-phase response to injury and infection: expression o f class III complement genes C2 and factor B in The roles o f interleukin one and other mediators; in Gordon AH human monocytes and in murine fibroblasts transfected with (ed): Research Monographs in Cell Tissue Physiology. Amster­ human C2 and factor B genes. J Biol Chem 1985,260:15280— dam, Elsevier, 1985, vol 10, pp 5 3 4 -1 2 0 1 . 15285. 2 Kuhlman M, Joiner K, Ezekowitz, RAB: The human mannose­ 20 Sipe JD, Colten HR, Goldberger G, et al: Human serum amyloid binding protein functions as an opsonin. J Exp Med 1989; 169: A (SAA): Biosynthesis and post-synthetic processing o f pre-SAA 1733-1745. and structural variants defined by complementary DNA. Bio­ 3 Tobias PS, Soldau K, Ulevitch RJ: Isolation o f a lipopolysaccha- chemistry 1985;24:2931-2935. ride-binding acute-phase reactant from rabbit serum. J Exp Med 21 Elbein AD: The tunicamycin-useful tools for studies on glyco­ 1986;164:777-793. proteins. Trends Biochem Sei 1981;6:219-221. 4 Tobias PS, McAdam K.PWJ, Soldau K, et al: Control o f lipo- 22 Ramadori G, Rieder H, Knittel TH, et al: Fat storing cells (FSC) polysaccharide-high density lipoprotein interactions by an acute o f rat liver synthesize and secrete fibronectin. Comparison with phase reactant in human serum. Infect Immun 1985;50:73-76. hepatocytes. J Hepatol 1987;2:208-217. 5 Tobias PS, Mathison JC, Ulevitch RJ: A family o f lipopolysac­ 23 Ramadori G, Van Dam m e J. Rieder H, et al: Interleukin-6, the charide binding proteins involved in responses to gram-negative third mediator o f acute-phase reaction, modulates hepatic pro­ sepsis. J Biol Chem 1988;263:13479-13488. tein synthesis in human and mouse. Comparison with interleu­ 6 Gray PW, Flaggs G, Leong SR, et al: Cloning o f the cDNA o f a kin lß and tumor necrosis factor-a. Eur J Immunol 1988; 18: human neutrophil bactericidal protein. J Biol Chem 1989;264: 1 2 5 9 -1 2 6 4 . 9 5 0 5 -9 5 0 9 . 24 Ramadori G, Mitsch A, Rieder H, et al: Alpha- and gamma- 7 Weiss J, Muello K, Victor M, et al: The role o f lipopolysaccha­ interferon (IFNa, IFNy) but not interleukin-1 (IL-1) modulate rides in the action o f the bactericidal/permeability-increasing synthesis and secretion o f ßi-microglobulin by hepatocytes. Eur neutrophil protein on the bacterial envelope. J Immunol 1984; J Clin Invest 1988;18:343-351. 132:3109-3115. 8 Tobias PS, Soldau K. Ulevitch RJ: Identification o f a lipid A binding site in the acute phase reactant lipopolysaccharide bind­ Received: June 29, 1989 ing protein. J Biol Chem 1989;264:10867-10871. Accepted: August 5, 1989 9 W eiss J, Olssen I: Cellular and subcellular localization o f the bactericidal/permeability-increasing protein o f the neutrophils. Giuliano Ramadori, MD Blood 1987;69:652-659. Klinikum der Johannes-Gutenberg-Universität 10 Tobias PS, McAdam KPWJ, U levitch RJ: Interactions o f bacte­ I. M edizinische Klinik und Poliklinik rial lipopolysaccharide with acute-phase rabbit serum and isola­ Postfach 3960 tion o f two forms o f rabbit serum amyloid A. J Immunol 1982; Langenbeckstrasse 1 128:1420-1427. D -6 5 0 0 Mainz (FRG)

Journal

Pathobiology – Karger

Published: Jan 1, 1990

Keywords: Lipopolysaccharide-binding protein; Hepatocytes; Acute phase protein; LPB-biosynthesis

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