Baiba J. GrubeSg, Charles G. CochaneS, Richard D. Yes, Carol E. Greenn, Mary E. McPhailfl, ..... RNA Type XI R-6750, Sigma) (38), DNA by the diphenylamine reaction ... and autoradiographed (HyperfilmTM-MP, Amersham, Sweden).
Vol. 269, No. 11, Issue of March 18, pp. 8477-8482, 1994 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Lipopolysaccharide Binding Protein Expressionin Primary Human Hepatocytes andHepG2 Hepatoma Cells* (Received for publication, October 11, 1993)
Baiba J. GrubeSg, Charles G. CochaneS, Richard D. Yes, Carol E. Greenn, Mary E. McPhailfl, Richard J. UlevitchS, and Peter S. Tobias$ From the Department of Immunology, The Scripps Research Institute, L a Jolla, California 92037 and ISRI International, Menlo Park, California94025
Lipopolysaccharide (LPS)-binding protein (LBP)is a (1).A variety of cytokines are released for host defense from cells of monocytic origin in response to challenge by LPS (2). In normal plasma protein and an acute phase reactant important for host responses to Gram-negative bacteria overwhelming sepsis, the inflammatory cascade may no longer a n escalation of and LPS. LBP forms high affinity complexes with LPS be held in check, but may actually lead to by t h e very mediators initially released to which bindto CD14, a monocyte surface protein,to ini- injury to host tissues has emerged as one of the key tiate the release of inflammatory mediators. We found preserve host function (3). TNF-a that human primary hepatocytes synthesize LBP and mediators of shock (4). It is released from cells of monocytic that the synthesis is up-regulatedby interleukin (1L)-6. origin in response to picomolar concentrations of circulating To examine this phenomenon in more detail, we evalu- LPS (5). ated the capacity of IL-6, IL-1, and tumor necrosis factor The lipidA structure of LPS is involved in initiating a signal to induce LBP synthesis in HepG2 cells in the presence in the macrophage (Me)) (6) through a novel receptor-dependor absence of dexamethasone.IL-6 induced LBP synthe- ent mechanism(7, 8). The lipidA moiety is bound to the circusis. Dexamethasone, IL-1, and tumor necrosis factor hadlating binding protein, lipopolysaccharide-binding protein a synergistic effect when combined withIL-6, but dem- (LBP) (9, 10). LBP delivers LPS to the MO receptor, CD14 (8). onstrated minimal effect independently. LBP biosynthe- Although other mechanisms for LPS activation of monocytes sis was evaluatedby immunoprecipitation of 35S-labeled are postulated, only LBP-dependent LPS binding to CD14 reLBP from HepG2supernatants, measurementof steadystate LBP mRNA levels, and analysisof LBP-dependent sults in a lo3 greater production of TNF-a (11). Depletion of LPS binding to CD14 positive cells. An 35S-labeled, 60- LBP from serum suppresses TNF-a production (7). LBP also kDa protein was immunoprecipitated with anti-LBP an- serves as a n opsonin for Gram-negative bacteria and reduces tibody from IL-6-stimulated HepG2 cell supernatants. the threshold concentrationfor LPS to induce cytokine release Northern blot analysis of cellular RNA revealed an in- from the MO (12). LBP and CD14 have emerged as the two central molecules crease in LBP mRNA in IL-6-stimulatedcells. CD14 expressing cells bound fluoresceinated LPS in the pres- for LPS activationof monocytes. The work herein isfocused on ence of supernatants from HepG2cells treated withIL-6. the regulationof LBP expression. Elucidationof the regulatory These data provide the first information about specific mechanism for LBP synthesis is integral to understanding the cytokine and dexamethasone regulation of LBP expres- physiologic changes of shock initiated by LPS. LBP is a circusion in HepG2 cells. LBP behaves like a Type 1 acute lating plasma protein present in normal individuals at a conphase protein. centration of 0.1-5 pg/ml (131, which rise to 2 5 0 pg/ml in response to injury (14). LBP is known to be synthesized in the liver of rabbits constitutively (15) a n d i n rat liver in several The release of lipopolysaccharide (LPS)’ from the outer cell inflammatory models (161, but its synthesis in human hepatowall of Gram-negative bacteria produces the acute systemic cytes has not been demonstrated. Moreover, nothing is known inflammatoryresponsesyndromewhichinthemostsevere about the humoral mediators that regulate LBP synthesis. clinical scenario evolves into multiple organ failure and death Synthesis of acute phase proteins (APP) in the liver is regulated by IL-1 (17, 181, TNF-a (191, IL-6 (17, 201, IL-11 (211, in* This research was supported in part by Grants GM-37696, HL- terferon-? (22), leukemia inhibitory factor (23), oncostatin M 23584, AI-15136, AI-25563, and UM-28485 from the National Institutes (241, transforming growth factor+ (251, and dexamethasone of Health and Grant NO0014 fromthe Office of Naval Research. This is (26). Regulation of APP synthesis encompasses a spectrum of publication7913 IMM from the Department of Immunology,The Scripps Research Institute, La Jolla, CA92037. The costsof publication stimulatory, synergistic, and inhibitory responses (26, 27). We of this article were defrayed in part by the payment of page charges. establish herein that primary human hepatocytes secrete LBP This article must therefore be hereby marked “aduertisement” in ac- and that expressionof LBP in the presence of IL-6 is elevated. cordance with 18 U.S.C. Section 1734 solely to indicate this fact. The investigation of LBP synthesis in response to four huI Recipient of National Research ServiceAward Trainee Grant 2 T32 GM07037-17.On leave of absence from the Department of Surgery, moral mediators, IL-1, TNF-a, IL-6 and dexamethasone, repUniversity of Washington, Seattle, WA 98104. To whom correspondence resentative of the two principal APP regulatory classes (20) and reprint requests should be addressed: Dept. of Immunology, The was undertaken in the HepG2 model utilized for elucidating Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA the regulation of other cytokine-modulated hepatic APPs (28, 92037. Tel.: 619-554-4549;Fax: 619-554-6123. The abbreviations used are: LPS, lipopolysaccharide/endotoxin; 29). Technical problems such as possible contaminating nonLBP, lipopolysaccharide binding protein; IL, interleukin; TNF, tumor parenchymal cells in primary hepatocyte cultures (30) or the necrosis factor; DEX, dexamethasone; M 0 , monocyte; APP, acute phase effect of primary hepatocyte culture techniques on their funcprotein; MEM, minimal essential medium; FCS, fetal calf serum; CHO, tion (31) are circumvented by investigating APP regulation in Chinese hamster ovary; r, recombinant; h, human; ELISA,enzymelinked immunosorbent assay; FIB, fibrinogen; FITC, fluorescein iso- hepatocyte cell lines derived from human hepatomas, such as thiocyanate; AGP, al-acid glycoprotein. the HepG2 cell line. LBP synthesis in HepG2 cells is stimu-
8477
Human LBP Expression
8478
TABLEI Comparison of LBP and a,-acidglycoprotein secretion by primary human hepatocytes Primary human hepatocytes were cultured as described under "Experimental Procedures." Cells were treated a t time 0,12 h, and 39 h with fresh stimulatory media. Data are expressed as mean 2 S.D. APP suuernatant collected at 12 h
Treatment
LBP
LBP
nglh
Control" IL-lb IL-6"
52 2 9 34282 4 49 * 13
84 h
39 h
AGP
AGP
LBP
49 252 23 2 8 40 13
17
29 22 2 8 45
AGP nglh
nglh
35 23f 13 1714ir 2 55 f 31
2
13
*6
59 2 28
34 * 20 14 * 7 61 2 30
"n=3. b n = 2.
lated by IL-6, and this effect is further enhanced by dexamethasone, IL-1, and TNF categorizing it as a Type 1APP.
was terminated with 4 M H2S0, and read a t 490 nm. Human fibrinogen(hFIB)anda,-acid glycoprotein (hAGP)were quantitated as above using the p-nitrophenylphosphate chromogen in diethanolamine buffer (Sigma 104-105) (36). Plates were coated with EXPERIMENTAL PROCEDURES gt-a-hFIB(Cappel 55123, Durham,NC)orgt-a-hAGP (Calbiochem Cell Lines and Media-HepG2 cells (generously provided by Dr. G. 122163, La Jolla, CAI, incubated with hFIB (Sigma 850-80) or hAGP Fey, The Scripps Research Institute (TSRI), La CAI Jolla, were cultured (Calbiochem 112251) and HepG2 supernatants, followed by rb-a-hFIB to confluence in minimal essential media (MEM, Flow, 11-10022, Ir- (Calbiochem 341552) or rb-a-hAGP (Accurate Chemical & Scientific vine, CA) with 10% FCS heat-inactivated at 56 "C for 30 min. The Corp. 2357096, Westbury, N Y ) and detected with alkaline phosphataseculture medium was changed to2% FCS plus MEM stimulatory media conjugated gt-a-rb-IgG (Tag0 8500, Burlingame, CAI. (SM) plus mediators for assessment of APP regulation (26). SWya"et~ Total Cellular RNA, DNA, and Protein-Total RNA, DNA, and pro(cystine and methionine-freeMEM, Select-amine Kit 300-9050AV, Life tein were precipitatedfrom cytokine-stimulated HepG2 cells disrupted Technologies, Inc.)plus 2% dialyzedFCSand[35Slmethionineand in 5 ml of phosphate-buffered saline for 30 s at 4 "C (Microson Ultra[35Slcysteine ( T ~ - a n ~ ~ S - l a b1089 e l ~Ci/nmol, ~, ICN, 51006, Costa Mesa, sonic Cell Disruptor X L ) according to the modified Ogur-%sen ProceCA) was used for metabolic labeling. Dexamethasone (DEX, D 4902, dure (37). RNA was assayedby the orcinol reaction (Orcinol01875 and Sigma) was added at 1p where indicated. RNA Type XI R-6750, Sigma) (38),DNA by the diphenylamine reaction Primary human hepatocytes were isolated by biopsy perfusion (32)of (diphenylamine D-2385 and DNA D-3664, Sigma) (39) and protein by a non-transplanted liver from a donor who died from an intracranial the BCA assay (Pierce ChemicalCo., 23225). hemorrhage (SRI International Human Subjects Use Committee apImmunoprecipitation of 35S-LabeledLBP-The HepG2 supernatants proval case 616). Cells were plated24 h after liver harvest. Initial cell were immunoprecipitated with polyclonal gt-a-rhLBP or preimmune viability was85% by trypan blueexclusion, and purity was greater thanrb-IgG a t 50 pg/ml and precipitated withtype-specific second antibody 90% by microscopy. Liver cells (1 x lo6 celld35-mm collagen-coated (9). Immunoprecipitates weresolubilized in Laemmli's (40) buffer with plate) were cultured in5% FCS in modified Waymouths 752Aiter (Life 5% 2-ME at 100 "C for 5 min, analyzed by 10% SDS-polyacrylamidegel Technologies, Inc.) (33). Medium was aspirated after 2 h to remove electrophoresis, fixed in acetic acid/H,O/isopropyl alcohol (10:65:25), nonviable, unattached cells and replacedwithfresh modified Way- and autoradiographed (HyperfilmTM-MP,Amersham, Sweden). mouth's 752Aiter without FCS. Cells were cultured between a "collagen Analysis of Steady StateTotal RNA from Cytokine-stimulatedHepG2 sandwich for 21 h prior t o cytokine stimulation (34). Cells for LBP mRNA-Total RNA from cytokine-stimulated HepG2 cells CHO-K1 cells stably transfected with human CD14 (CHO-hCD14) or wasextracted by themethod of Chomczynskiand Sacchi (41), vector (CHO-RSV) were generously supplied by J . D. Lee (TSRI, La formamidelformaldehyde denatured, electrophoresed, and transferred Jolla, CA) and cultured as described (35). to Hybond-N+ (Amersham) (42). Theblot was stained with methylene Cytokines-Human rIL-6 (1 x lo6 unit/ml) was obtained from Bioblue, hybridized with [a-32PldCTP (Amersham PB 10205)-labeled hLBP source International (Camarillo,CA, CY-02-025). Human rTNF-a (6 x cDNA (a generous gift of J. Han, TSRI), and autoradiographed (Hyperlo6 unit/ml) and rIL-1-P(1x lo' unit/ml) were generouslyprovided by filmTM-MP) (42). J . Mathison (TSRI, La Jolla,CAI. FITC-Re595LPS Bindingto CHO-hCD14 and CHO-RSVCells-LPS Mediator Stimulation of Primary Human Hepatocytes and HepG2 was isolated from lyophilized Salmonella minnesota Re595 (431, and Hepatoma Cells-Primary hepatocytes were changed to Waymouth's FITC-Re595 LPSwasprepared(44).The 48-h supernatants from medium alone or with IL-1 (lo3 unit/ml) or IL-6 (lo3 unit/ml). Superof dexamethasone plus natants werecollected and fresh stimulatory media applied a t 12 and 39 HepG2 cells cultured in the presence or absence cytokines were utilized. Cells (150 pl of either CHO-hCD14 or CHOh after onsetof stimulation; final supernatants werecollected at 84h. RSV at 2.8 x lo5 celldml in Hanks' balanced salt solution plus 0.1% HepG2 cells (2.5 x lo5 celldml), cultured in T25 flasks t o 80-100% 0.05%NaN3) were added the to concentrated confluence, were stimulated for two sequential 24-h time periods (24). bovine serum albumin and (5 pl) diluted in buffer (100pl The effect of single or combined mediators on LBP secretion i n HepG2 cytokine-stimulated HepG2 supernatants cells was evaluated inSM -c DEX plus 1)no cytokine, 2) IL-1,3)TNF, 4) of Hanks' as for cells), and incubated withFITC-Re595 LPS (final conIL-6, 5) IL-1 + TNF, 6) IL-6 + IL-1, 7) IL-6+ TNF, and 8) IL-6 + IL-1 + centration 5 ng/ml) for 30 min at 24 "C. In some instances theHepG2 supernatants diluted in buffer were incubated with either polyclonal TNF. For metabolic labeling, the medium was changedt o SMCYS-metat 48 h for 15 min andfollowed by fresh SMc~s""'t-* mediators and 0.625 gt-a-rhLBP or preimmune gt-IgGat 4 mg/ml for 30 minat 37 "C before mCi of P5S]methionine and[35Slcysteine for 21 h. Cytolune concentra- addition of the cells. FITC-Re595 LPS binding was determinedby fluotions are reported in the figure and table legends. Supernatants were rescence-activated cell sorter analysis (FACS, Becton Dickinson). filtered (0.22 nm Millipore@) and concentrated to 300 pl (Centriconm RESULTS 10, Amicon@,Beverly, MA). ELISA for LBP, Fibrinogen, and a,-Acid Glycoprotein4oat antiLBP Expression in Primary HumanHepatocytes and HepG2 recombinant human LBP (gt-a-rhLBP), biotinylated-gt-a-rhLBP and hLBP were generouslyprovided by L. Hatlen (TSRI). Microtiter plates Hepatoma Cells-In order to determine if LBP is synthesized by human liver cells, the supernatants of primary cultured were coated with gt-a-rhLBP (75 pg/ml) in carbonate-bicarbonate buffer overnight a t 4 "C, washed with phosphate-buffered saline + 0.1% Tween human hepatocytes were assayed for the secretion of LBP as a (all washes), blocked for 1 h at 37 "C with 10% milk powder in phos- function of time after stimulation and compared with the sephate-buffered saline. Plates were incubated with hLBP, primary hu- cretion of the control hepatic acute phase protein, AGP. The man hepatocyte, and HepG2 supernatants for 2 h at 37 "C followed by results are shown in Table I. LBP is expressed by primary biotinylated-gt-a-rhLBP (75 pg/ml) for 1h at 37 "C, horseradish peroxidasestreptavidin(HRP-Streptavidin43-4323, Zymed Laboratories human hepatocytes. Primary hepatocytes in control medium Inc., San Francisco, CA.) for 30 min a t 37 "C and o-phenylenediamine alone roughly halvedtheir rateof LBP and AGP output by 39 h, (DAKO 52000, Denmark)for 20 min a t room temperature. The reaction and a similar effect was seen when the hepatocytes were cul-
Human LBP Expression
8479
TABLEI1 Comparison of LBP, fibrinogen and a,-acid glycoproteinsecretion by HepG2 cells as a function of cytokine and dexamethasone stimulation APP supernatants were collected from HepG2 cells stimulated with the indicated cytokines 2 1p~ dexamethasone for 48 h a s described under "Experimental Procedures." AF'P supernatant (-)-Dexamethasone
Treatment
IO1unitslml No cytokines IL-1 TNF IL-1 + TNF IL-6 IL-6 + IL-1 IL-6 + TNF 134.5 IL-6 + IL-1 + TNF
(+)-Dexametha
ng LBPI pg DNA".h
n Fib/ pg %NA'.h
ng AGP/ pg DNA",b
ng LEPI pg DNAT-"
0.2 0.4 0.1 0.3 52.0 92.0 74.2 122.6
0.7 0.1 0.2 0.1 477.5 104.7
19.3 23.5 8.5 14.9 23.0 143.4 25.1 181.7
0.2 0.9 0.4 1.0 282.5 640.2 409.9 1212.0
Conversion to (ng/pgRNA) x 0.28. Conversion to (ng/pgcell protein) x 0.06. e Conversion to (ng/pg RNA) x 0.31. Conversion to (ng/pgcell protein) x 0.06.
51.0
n Fib/ pg %NAc.d
0.5 CO.1
0.3
