TISSUE FACTOR EXPRESSION IN HUMAN MONOCYTIC CELL LINES

Thmmbais Research, Vol. 88, No. 2, pp. 215–228, 1997 Copyright @ 1997 Elsevier Science Ltd Printed in tie USA. All rights reserved IXM9-3848/97$17.00+ .(XI

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TISSUE FACTOR EXPRESSION IN HUMAN MONOCYTIC CELL LINES Taavo Tenno’,Johan Botling2,FredrikOberg2,KennethNilsson2and Agneta Siegbahn’ ‘Laboratoryfor CoagulationResearch,Departmentof ClinicalChemistryand 2Laboratoryof Tumor Biology,Departmentof Pathology,UniversityHospital,S-75 185Uppsala,Sweden (Received 4 July 1997by Editor S. Schulman; revised/acxepted 16 September 1997)

Abstract Tissue factor (TF) is a main initiatorof the coagulationprotease cascade. Control of the expressionof this protein in monocytesis essential,since these cells are the only circulatingblood cells responsiblefor TF expression.In this report we have used two human cell lines, arrestedat differentstagesof monocyticdifferentiation,to study TF expression. The monoblastic cell line U-937 had a constitutive expression of TF surface protein and low TF mRNA levels detected by immunofluorescence or quantitativereversetranscriptasepolymerasechain reactionrespectively.The phorbol12-myristate-13-acetate (PMA) was a potent enhancer of TF expression in U-937. Lipopolysaccharide(LPS) and tumor necrosis factor (TNF) had no effect on TF expression in U-937. The Mono Mac 6 cell line, with phenotypicfeatures similar to that of mature monocytes,expressedlower basal levels of TF mRNA and surface TF antigen.However,in Mono Mac 6 cellsTF expressionwas inducedin responseto LPS and TNF. These results indicate differences in basal and induced TF expression betweenU-937 and MonoMac 6 cell lines. @1997 Elsevier Science Ltd Tissue factor is a membrane bound single chain glycoprotein(l), which on exposure to the blood activatescoagulation,ultimatelyleadingto formationof a thrombus.This protein forms a complexwith factor VII/VIIa catalyzingthe proteolyticactivationof coagulationfactors IX and X and leading to activationof the extrinsiccoagulationpathway.AlthoughTF is normally not expressedin leukocytes,the expressionof TF can be inducedin monocytes,which are the only Key words:Tissue factor,monocytes,cytokines,bloodcoagulation. Corresponding author: Agneta Siegbahn, Department of Clinical Chemistry, University Hospital,S-75 185Uppsala,Sweden,Fax:int+46-18-5525 62

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circulatingblood cells havingthis capacity.Sincethe expressionof TF on monocytesis believed to trigger pathologicalcoagulationprocesseslike disseminatedintravascularcoagulation(DIC) and unstableangina, it is of great importanceto study the mechanismcontrollingTF expression in these cells. Described inducersof monocyteTF expressionare LPS (2), PMA (3), P-selectin(4) monocyte chemotacticprotein-1and plateletderivedgrowth factor-BB(5). LPS inducedTF expressionin monocytes seems to be mediated by the LPS binding protein / CD14 pathway (6) and the couplingof adhesivereceptor CD1lb/CDl 8 has been associatedwith microphage TF response (7). In most cases the monocyteTF expressionseemsto be regulatedat the transcriptionallevel. However, in the human monocyticleukemiacell line THP-1 TF expressionis regulatedat both transcriptionaland posttranscriptionallevels(8). The human monoblastic leukemia cell line U-937 has been widely used to study monocytic differentiationand activation. This cell line was establishedfrom a histocytic Iymphoma(9). The U-937 cells are arrested at the monoblast stage of differentiationand normally do not express the monocyte associated antigen CD14 (1O).Previcmslya few reports have described some inducers of procoagulantactivity (PCA) in U-937 cells (11,12).The monocyticcell line Mono Mac 6 was establishedfrom a patientwith rnonoblasticleukemiaand has been reportedto have characteristicsroughly similarto those of mature monocytes(13). TF expressionin these cells has been a subject to one earlier report showingthat low basal levels of TF mRNA and protein can be increasedin these cells in responseto PMA (14). Studieson TF expressionhave been hampered by the difficultiesof isolatingand culturingdefinedmonocytepopulationsfrom peripheral blood. We have therefore employed established human monocytic cell lines as modelsfor normalmonocyticcells. In this paper we have studied the TF expressionduring activationof monocytic cell lines by quantitativereverse transcriptasepolymerasechain reaction RT-PCR and flow cytometry.Uninduced U-937 cells were found to express TF mRNA at a Iow level but high expression of surface protein. The levels of TF mRNA and protein expression varied during cell growth, dependingon additionof nutrients.The expressionof TF was upregulatedafter activationof U937 cells with PMA, but not by LPS or TNFa..In contrast,we show that uninducedMono Mac 6 monocyticcells expresslow basal levelsof TF, which is upregulatedin a similarmanner as in maturemonocytesafter inductionby LPS or TNFcc MATERIALSAND METHC)DS Reagents Foetal calf serum (FCS) was purchasedfrom Gibco Life Technologies(Paisley, Scotland),Lglutamine,penicillinand streptomycinfrom Biochrom(Berlin,Germany).Phorbol 12-myristate 13-acetateand endotoxin lipopolysaccharidefrom Escherichia Coli, serotype 0111:B4, were purchased from Sigma ChemicalCo. (St. Louis, MO, USA). Limulusamebocytelysate (LAL) was from Haemachem Inc. (St. Louis, MO, USA), LAL substrate (SpectrozymeLAL) from American Diagnostic (Greenwich,CT, USA) and endotoxinstandardfrom ChromogenicAB (Molndal,Sweden).T7 RNA polymerasewas from Boehringer(Mannheim,Germany),RNasin, AMV reversetranscriptaseand Taq DNA polymerasewere from Promega(Madison,WI, USA). Interleukin-1(1(IL-1P) was from R&D Systems(Abingdon,UK), interleukin-6(IL-6), TNF and

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y-interferon (y-IFN) from Genzyme (Cambridge, MA, USA). Fluorosceine-isothiocyanatelabelled rabbit anti-mouse IgG and mouse anti-Aspergillustiger glucose oxidase monoclinal IgGl (DAK-GO1)were from Dakopatts A/S (Glostrup,Danrnark),TF monoclinal antibodies (TF9-1OH1Oand TF9-9C3) from American Diagnostic Inc., anti-Leu M5 (CD1lC) ad phycoerythrin-conjugatedmonoclinal anti-CD14 antibody (Leu-M3) from Becton Dickinson. Recombinant active-site inhibited factor VIIa (rFVIIai) was a kind gift from Novo Nordisk, Denmark. Cell cultureand media A subclone of human monoblasticcell line U-937, U-937-1 (15) and the monocytic cell line Mono Mac 6 (13) (gift of Dr. Ziegler-Heitbrock)were used. These cells were cultured in RPMI 1640supplementedwith 7.5% heat inactivatedFCS, 1%of glutamineand antibiotics(100 IU/ml of penicillinand 50 pg/ml of streptomycin).The serumfree subcloneof U-937,U-937-lSF (16) was cultured in RPMI 1640 supplementedwith glutamineand antibiotics.Cells were grown at 37°C in a 5% C02 atmosphere. Beforethe experimentsthe phenotypeof the cellswas checkedby measuringsome characteristic monocytedifferentiationmarkerswith flow cytometryas describedbelow. In all experimentsU937 cells had a slight (<1O’?4O positive cells) expressionof CD1IC and were distinctlynegative for CD14. Mono Mac 6 cells were 20-40Y0positive for CD14. The cells were taken for experimentsin the logarithmicgrowth phase: U-937 cells 24 hours and Mono Mac 6 cells 48 hours after splitting the culture. The cells were centrifuged(300g, 5 rein) and resuspended in completemediumat a densityof 2X106/ml. Endotoxindetermination All cell culture media and reagents were checked for endotoxin contamination by limulus arnebocytelysate (LAL) assay (M-695).In summary,50 pl of sampleor endotoxinstandardwas incubated for 5 min at 37°C in a 96 well microtitreplate and then 50 @ of LAL was added. After 7 min incubationat 37°C 100 @ of LAL substratewas added to the each well. Then the plate was incubatedfor 5 min at 37°C and finallythe reactionwas stoppedwith 100 pl of 20% acetic acid, and the absorbancewas measuredwith a spectrophotometerat 405 nm. All solutions containedless than 15p~ml of endotoxinat maximalexperimentalconcentrations. RNA analysis TF mRNA was identifiedby quantitativeRT-PCRas describedby P6tgenset al (17). An in vitro synthesisedinternal standard RNA was used to correct the differencesin reverse transcription and PCR reaction. The control RNA for PCR was in vitro transcribed from HindIII digested pGEM-TFayl DNA (kindlyprovidedby A. J. G. Potgens)with T7 RNA polymerase.Plasmid DNA was digested by treatment with RQ1-DNasel. Phenol extracted and ethanol precipitated RNA was then run on 1% agaroseformaldehydegel togetherwith plasmidDNA and quantitated by hybridizationto the TF probe as described below. The control RNA possessed the same primer sites for amplificationas TF mRNA, but gave differentPCR products (0.7 and 0.3 kb respectively)(17). The total RNA from 2.5x1OScells was isolated quantitativelyby the conventional method describedearlier (18). RNA concentrationwas measuredspectrophotometricallyat 260 nrn and visualizedin ethidiumbromide stainedagarosegels. This method gave quantitativelythe intact

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18Sand 28S ribosomalRNA bands.Preparedmonocyticcell RNA (0.1-1 pg) and control RNA (0.1-0.5 pg) were incubatedwith 0.5 pg of the TF specific reverse primer for 5 min at 65°C. Then 2 pl of 5Xreverse transcriptasebuffer (250 mM Tris-HClpH 8.3, 375 mM KC1, 15 mM MgC12,50 mM DTT), 1 pl of dNTP mix (2.5 mM each), 6 U RNasin, 6.5 U AMV reverse transcriptaseand distilled water up to 10 pl were added. Reverse transcriptionwas carried out for Ih at 42°C and enzyme was inactivatedfor 5 min at 95”C. 1/30part of reverse transcribed cDNA was used for PCR. Reaction mixture contained 50 mM Tris/HCl pH 8.3, BSA (500 ~g/ml), 2% sucrose, cresol red (0.1 mM), MgClz(4 mM), dNTP solution(0.2 mM each), 0.01 Kgof reverseprimer, 0.01 Kgof forwardprimer,0.4 U Taq DNA polymeraseand distilledwater up to 10 L1.PCR cycling was performed in 10 @ capillary tubes with an Idaho Rapidcycler. Prior to cycling the sampleswere denaturedfor 60 sec at 94°C. The sampleswere amplifiedfor 14-17 cycles (94”C, O see; 50”C, O see; 72”C, 25 see) (19). The PCR product (5 pl) was fractionated in agarose gel and DNA was transferred to a nitrocellulose filter (Hybond-C, Amersham,United Kingdom).The filterswere hybridizedwith 32PlabelledTF cDNA probe at 42°C and subsequentlywashed in 15 mM NaC1/1.5mM sodiumcitrate/O.1%SDS at 53°C for 3.30 min. The 1805 bp TF cDNA, J3amHI fragment from plasmid pJH9TF, generously provided by J. H. Morrissey,was used for hybridization.The purified fragmentswere labelled with [ctszP]dCTPby random priming (MultiprimeDNA labelling kit: Amersham). Labelled filters were exposed to luminescencescreen, and phospho-stimulatedluminescence(PSL) was measured with phosphoimager Fujix BAS 2000. The background was subtracted from the measured bands, TF mRNA signals were normalisedto control RNA bands and absolute TF mRNA quantities were calculated. Subsequentlythe filters were exposed to X-OMAT Kodak films with an intensi&ingscreenat -70”C.All mRNA quantitationexperimentswere reproduced minimumtwice. Immunojluorescencestudies The surface antigen expressionwas analysedby immunofluorescenceusing an EPICS Profile II flow cytometer(CoulterElectronics,Hialeah,FL, USA). The cells were washedtwice with PBS + O.1°/0 J3SA, incubated for 30 min on ice with primaryantibody,washedtwice, incubatedfor 30 min on ice with 10 pg/ml of fluorosceine-isothiocyanate-labelled rabbit anti-mouse IgG and washed twice before analysis. For measurementof CD14 expression the cells were labelled directly with 2.5 pg/ml of phycoerythrin-conjugatedanti-CD14 antibody (Leu-M3). The used primary monoclinal antibodieswere anti-Aspergillusniger glucose oxidase monoclinal IgGl (DAK-GO1)as negativecontrol(2.5 pg/ml),anti-LeuM5 (CD1Ic) in cone. 3 ~g/ml and anti-TF (TF9-9C3 and in the FVIIai blockingexperimentsTF9-1OH1O)in cone. 5 pg/ml. Mean channel fluorescence intensity and percentage of positive cells were determined for each sample. A calibration curve was obtained by the use of highly uniform flow cytometry standardisation microbeadswith known amounts of fluorosceine(Flow CytometryStandardsCorporation,San Juan, PR, USA). The fluorescenceintensitywas then convertedinto Molecules of Equivalent Soluble Fluorochrome (MESF) (20). All flow cytometry experiments were performed a minimumof three times.

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RESULTS QuantitativeRT-PCR methodfor tissuefactor expressionin monocyticcells We adopted the quantitative RT-PCR method with internal standard, described earlier for quantitationof TF mRNA transcriptsin endothelialcells (17). In order to approvethe reliability of the method we ran several PCR standardisations.The logarithmof TF signal dependenceof PCR cycle numberwas linearin the range 12-20cycles(datanot shown).The differentamounts of total RNA from U-937 cells, non-inducedand endotoxintreated Mono Mac 6 cells were reversetranscribedin the presenceof controlRNA and the DNA was amplifiedin PCR. The fl ng total RNR

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FIG. 1 Quantitationof TF mRNA. Differentamountsof total RNA from non-inducedU-937 cells, non-induced and LPS (1 pg/ml) induced Mono Mac 6 cells were reverse transcribed togetherwith controlRNA (0.0416pg and 0.416pg per reactionfor U-937 and Mono Mac 6 cells respectively).Samplesfor non-inducedcells were amplified 19 cycles and samples from LPS inducedcells for 17 cycles.PCR productswere Southernblotted,hybridizedwith TF cDNA probe and visualized with phosphoimager(a). Intensities of the bands were quantifiedand messenger/controlratio was plottedagainstthe inputtotal RNA amount(b).

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PCR products of these standardisationexperimentswere blotted and hybridizedand the results are shown in figure la. The intensitiesof these bands were quantifiedwith image analysisand TF messenger/controlRNA ratios were used to expressthe relativeamountof TF mRNA in the monocyticcells. These ratios were plottedagainstthe input total RNA amount (Fig. lb). These plots demonstratethe quantitativityof the RT-PCRmethod. Tissuefactor mRNA expressionin U-937cells We measured the TF mRNA in U-937 cells during cell culture. The cells were taken for experimentafter 48 hours of culture and splittedto the cell concentrationof 0.2x106/ml. Each day the total RNA was extractedand the TF mRNA contentof the cells was analysedby the c

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FIG. 2 TF expressionduring culture.U-937 cells were culturedas indicatedin materials.The cells were split to the concentrationof 0.2x106/ml. Each day 0.25xIOGcells was taken from the culture to quantitate TF mRNA by RT-PCR as described in methods. Data from a representativeexperimentis presentedas TF mRNA fold induction(a) or imageof RT-PCR gel, hybridizedwith TF cDNA probe (b). U-937 (~), U-937-1SF (0) and Mono Mac 6 (A) cells were cultured as indicated in materials and each day 0.25x106cells per sample was used to measure TF basal antigenlevelsby flow cytometry.The percentageof TF positive cells (c) is presented.A representativeexperimentout of three is shown.

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quantitativeRT-PCR method. There was a transientincreaseof TF rnRNA levels in these cells 24 hours after addition of the fresh medium (Fig. 2a, b). The TF mRNA expression then decreased,and the basal levelwas reachedon the secondday after splittingof the cells. Tissuefactor protein expressionin humanmonocyticcell lines TF antigen expressionwas measuredwith flow cytometry.The TF protein expressionin U-937 cells was dependenton the time of harvest of the cells. It peaked on the first day afler splitting and then decreased slowly. TF expressionvaried in the range of 30-90°Aof positive cells (Fig. 2c) and from 4000-14000 in MESF values (data not shown).In order to determinewhetherthe inductionof TF in U-937 cells was dependenton serum proteinsonly, we used the serum flee subcloneof U-937, U-937-1SF. These cells have earlierbeen shownto grow and differentiatein serum free media (16). A time dependentTF expressionwas also found in U-937-lSF cells with maximal levels on the second day after splitting of the cells (Fig. 2c). Furthermore, TF expressionin the serum free clonewas in the same range as in the serum dependentclone of U937. The expressionof TF was highestwhen the cells were in the logarithmicgrowth phase and declinedwhen cells reachedthe lag phase. Tissuefactor surfaceantigenexpressionin the Mono Mac 6 cells was lower and more stable as comparedto the U-937 cells. The basal TF antigen expression in Mono Mac 6 cells was less than 45°/0of positive cells in all of the experiments (Fig. 2c).

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FIG. 3 Blocking of the TF antigen with rFVIIai. U-937 and Mono Mac 6 cells taken from the culture on first and second day respectively, incubated for 15 min with different concentrations of rFVIIai at 37°C, then chilled and flow cytometry experiment was performedusing two differentTF monoclinal antibodies.Valuesare given as percentageof TF positivecells and MESF of a representativeexperimentwith duplicatesamples.

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To exclude the possibilitythat the TF surfaceexpressionon the U-937 cells was caused by the non-specific binding of antibodies to the phospholipid structures of the cell membrane we performedthe experimentshown on Fig 3. Before incubationwith the primary antibodythe TF protein was blockedwith varyingconcentrationsof recombinantactive-siteinhibitedfactor VIIa (rFVIIai). This protein binds TF with high affinity, but has no functionalactivity (21). There was a concentrationdependantdecreasein the TF flow cytometrysignal, detected with the TF monoclinal antibodyclone (TF9-9C3)againstthe epitopelocated in the factor VII binding site (22). An almost completeinhibitoryeffect was obtainedwith rFVIIai in Mono Mac 6 cells; TF expressionwas inhibited93°/0and 88°/0in the percentageof TF positivecells and MESF values respectively.As expected, the binding of the TF monoclinal antibody (TF9-1OH1O)was not inhibitedby rFVIIai since this antibodydoes not detect the epitopefor FVII binding (22) (Fig. 3). Regulationof TF mRNA in monocyticcell lines We next studied TF mRNA expressionin U-937 cells in responseto PMA (30 rig/ml),LPS (1 ~g/ml) and a numberof cytokines,involvedin the regulationof monocyticfunctions;yIFN (100 U/ml), TNF (100 U/ml), IL-I~ (20 rig/ml),IL-6 (50 rig/ml)(Fig. 4a). U-937 cells were incubated with different inducers and the mRNA was quantitated after 6 h of incubation. In our experimentalconditionsthese cytokinesand LPS did not enhancethe TF mRNA levels in U-937 cells (Fig. 4a). As shown in table 1 a 2.7 fold increaseof TF mRNA was observedin responseto PMA. The kinetics of TF expression in U-937 cells after PMA stimulation was also investigated.PMA (30 n~ml) inducedexpressionof TF mRNA was maximalafier six hours of incubation(data not shown).

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FIG. 4 Induced TF mRNA in cell lines. U-937 (a) and Mono Mac 6 (b) cells at density 2X106/ml were stimulatedin completemedium for 6 and 2 hours respectivelywith various inducers: yIFN (100 U/ml), TNF (100 U/ml), IL-1P (20 rig/ml),IL-6 (50 rig/ml), LPS (1 pg/ml), PMA (30 rig/ml). TF mRNA was quantitated with described RT-PCR method. Data is presentedas TF mRNA fold inductionof the representativeexperiments.

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TABLEI Inductionof TF mRNA and SurfaceAntigenin MonocyticCell Lines. u-937 fold ind. *SD

p

Mono Mac 6 fold ind.*SD p 0/0 ofpos. cells*SD p

Noninduced 1 1 PMA 2.7 +0.75 (n=5)<0.05 ND LPS ND 7.5 +5.3 (n=6)<0.05 TNF ND 1.8+0.1 (n=3)<0.05

8.3 +4.2 (n=5) ND 31.2+9.1 (n=5) <0.01 17.1*5.1 (n=5) <0.01

The experimental conditions for mRNA experimentsare shown by figure 4 and fold induction of TF mRNA is shown. Protein expressionin Mono Mac 6 cells quantitated with flow cytometer after 5 hours culture in the absence or presence of inducer and percentage of TF positive cells is shown. Values are presented as mean *SD. Statistics not determinedfor ND. The same set of inducersas for U-937 cells was used to examinethe regulationof TF mRNA in Mono Mac 6 cells (Fig. 4b). LPS was found to be a potent inducerof TF rnRNA in these cells. The kinetics of LPS (1 pg/ml) induction of TF in Mono Mac 6 cells showed a rapid accumulation of TF rnRNA peaking around 1.5-2 hours after start of stimulation (data not shown). We found also a statistically significant increase in TF mRNA levels after TNF induction (Table 1 and Fig. 4b). In contrast to the circulatingblood monocytes,Mono Mac 6 cells did not respond to PMA (3). Other well-knownregulatorsof inflammatory responses in monocyticcells, such as IL-11$IL-6 and y-IFNdid not induceTF mRNA in Mono Mac 6 cells. Regulationof TFprotein expressionin humanmonocyticcell lines The expressionof surface TF antigen in U-937 cells was high during the cell culture (Fig. 2b) and the culture of these cells in the presenceof PMA (30 rig/ml)could not induce a significant elevation of the surface receptor over a prolongedtime period. Also the LPS or inflammatory mediators(@N, TNF, IL-1~, IL-6) did not give a significantresponseof TF. Culture of Mono Mac 6 cells with 1 pg/ml of LPS resultedin a rapid inductionof TF antigen, with maximal expression from 4 to 8 hours after stimulation(data not shown). Mono Mac 6 cells were induced with the same pattern of inducers as shown for mRNA experiments. Corroboratingwith mRNA experiments,a statisticallysignificantinductionof TF antigenon the surfaceof Mono Mac 6 cells was obtainedonly by LPS and TNF (Table 1). Other inducersdid not induce the expressionof TF (data not shown). We also found that other concentrationsof PMA, in the range 0.1-600rig/ml,did not inducesurtlaceTF (datanot shown). DISCUSSION The regulationof TF expressionin the cells of the monocytoidlineageis of major importancein inflammationand woundhealing.High exposureof TF on the surfaceof these cells is associated with disorderslikcDICTandarteriosclerosis.It is also known that leukemicpatients often have problems with bleedings and alterations in coagulation (23). The constitutively high

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procoagulant activity in leukemic cells caused by TF has been suspected to trigger DIC in patients (24). Moreover, the expressionof TF by monocyteprogenitorcells has been reported (25). These reports emphasisethe requirementfor better understandingof TF regulationin the monocyte-microphagedifferentiationprocess. Most of the earlier studies on tissue factor expressionin monocyticcells have been performed by using diverse procoagulantactivity assays. There are variationsin experimentaltechniques and usually it is difficultto interpretatethe resultsobtainedon PCA. The procoagulantactivity, equated with the activity of TF is multifactorialand can be modulated by the phospholipid constitutionand structureof cell membrane.Moreover,the interpretationof results obtainedby cell lysate PCA is difficult. However, the role of TF in the cells of early monocytoid differentiationis unclear,and there have been reportsaboutthe role of TF independentof blood coagulation(26), In our study we have used two methods,which reflect the different stages of TF receptor expression.The quantitativeRT-PCR with reliable controlsprovides a method to detect low levels of TF mRNA from smallnumbersof cells. The flow cytometrymethoddetects the cell-surfaceexposed TF protein, but can not be equatedwith the PCA assays. Competition experiments with rFVIIai confirmed the specificity of the immunofluorescencemethod and showed,that detectedTF proteinpossessedthe FVIIabindingactivity. We have used two monocytoidcell lines to study the activation-associatedTF expression of monocytic cells arrested in different stages of differentiation.It is known, that U-937 cells presentthe immaturephenotypeof monoblastcells (27), whereasMono Mac 6 cells are arrested in a late stage of the monocytic differentiation.Thus, Mono Mac 6 cells express the main monocytic differentiationmarker, CD14 (13,28). The monoblastcell line, U-937 expressed a low basal level of tissue factor mRNA. About the same amount of mRNA was found in the monocyticcell line Mono Mac 6. The TF mRNA in thesecell lineswas not detectedby northern blotting (data not shown).TF surfaceantigenexpressionduring conventionalcell culture in the serum dependent clone of U-937 peaked on the first day after splitting of the cells. Accumulationof TF mRNA transcriptsby addition of fresh FCS supplementedmedium was also significant. Presumably this TF response after splitting of the cells was mediated via inadvertentactivationof the cells by serumproteinsand/orthe release from density dependent inhibitorysignals. We thereforeused the serum free clone of U-937 (U-937-1SF) to determine the part of the serum in the activation of the cells during culture. There was also a similar expressionof TF peakingon the secondday after splitting.Thus,the TF inductionin these early monocytoidcells is obviouslyrelatedto the growthof the cells,the consumptionof nutrientsor the inhibitionby cell-densitydependentmechanisms. In our study LPS did not significantlyaffect TF mRNA inductionin U-937 monoblastcells in any experiment.This is in accordancewith the negligibleexpressionof the LPS receptor,CD14, in U-937 cells. In contrastthe U-937 cells have been previouslyreportedto respondto LPS and express TF antigen and PCA (29). The signaling mechanismof LPS induced surface TF in CD14 negativeU-937 cells remainsto be explained. We found that the stimulationof Mono Mac 6 cells with LPS enhancedthe TF mRNA content 7.5 times. However, this cell line does not possess all phenotypicand functionalproperties of mature monocytes (30), and thus the sensitivitywas still lower compared to that of human

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circulatingmonocytes(31).An increasein responsivenessto LPS, measuredby PCA assays,has also been observedduringthe differentiationof humanmonocytesto macrophages(32). Phorbol esters are known to be potent activatorsof procoagulantactivity (3) and protein kinase C in monocytes. A PMA concentration of 30 rig/ml induces PKC to become membrane associatedin monocytesbut did not induceTF activityin lysed cells (33). In other experiments this high concentrationof PMA inducedTF mRNA (34) as well as half of the maximalPCA in monocytes(35). The same authorsalso show the participationof PKC in the LPS inductionof TF in monocytes. In human hematopoieticcell lines PMA often induces differentiationand activation(10). PMA inducedTF mRNA and surfaceproteinaccumulationin the monoblastcell line U-937, but no detectableincreaseof TF in the monocytelike cell line Mono Mac 6. Thus we suppose, that PMA induces TF in earlier stages of monocytic differentiationand has no influencein mature monocytes.One might also speculatethat the contradictoryresults of PMA inductionof monocyteTF expressioncan relateto their differentiationstage. In U-937 monoblaststhe main inducerof TF mRNA and antigen was PMA. Activation of TF transcriptionby PMA is mediatedthroughthe serumresponseregion in the promotersite of the gene (36). LPS did not induceTF in these cells.In contrast,TF inductionin Mono Mac 6 cells is very similar to the pattern in mature monocytes,involvingthe mediators like TNF and LPS. Both inducers are the representativesof TF signaltransductionpathway through LPS response region, which includes the activation of transcription factors NF-IcB and AP-1 (37). The differentpatternsof inducersand differencesin time of achievementof maximalinductionof TF in U-937 and Mono Mac 6 cell lines suggest that TF in different monocytoiddifferentiation stagesis mediatedthroughdistinctsignaltransductionpathways. Our studies on these two cell lines may indicate that TF expression is regulated during the processof monocyte/microphagedifferentiation.The monoblasticU-937 cellshad a constitutive surface TF expression while Mono Mac 6 cells with phenotypicproperties similar to that of more mature monocytic cells had significantlylower surface TF expression.Supportingthese findings our unpublished observationsshow, that induction of differentiationin U-937 cells leads to downregulationof TF (manuscriptin preparation).Consideringthe leukemic origin of these cell lines the direct translationof the results to the normal differentiatingmonocyticcells can be done only with due reservation. However, studies on bone marrow derived normal monocyticprogenitorcells supportour findings(25).Thesecellshad a constitutiveprocoagulant activityand expressedthe TF mRNA after inductionwith LPS. Acknowledgements This study was supportedby grants from the SwedishMedicalResearch Council,Project B9613X-11568-OIA, Swedish Cancer Society and the Swedish Heart and Lung Foundation. We thank Prof. U. Hedner and Dr. M. Ezban for the generousgifi of the rFVIIai, Dr. R. M. W. de Waal for the plasmid pGEM-TFayl and Dr. J. H. Morrissey for providing us the plasmid pJH9TF.

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REFERENCES 1. EDGINGTON, T.S., MACKMAN,N., BRAND, K. AND RUF, W. The structuralbiology of expressionand functionof tissuefactor.ThrombHaemost66, 67-79, 1991. 2. RIVERS, R.P., HATHAWAY, W.E. AND WESTON, W.L. The endotoxin-induced coagulantactivityof human monocytes.Br J Haematol30, 311-316,1975. 3. LYBERG, T., PRYDZ, H. Phorbol esters induce synthesis of thromboplastinactivity in human monocytes.BiochemJ 194,699-706,1981. 4. CELI, A., PELLEGRINI, G., LORENZET, R., DE BLASI, A., READY, N., FURIE, B.C. AND FURIE, B. P-selectin induces the expression of tissue factor on monocytes. Proc Natl Acad Sci U S A 91, 8767-8771,1994. 5. ERNOFSSON, M., SIEGBAHN, A. Platelet-derived growth factor-BB and monocyte chemotactic protein-1 induce human peripheral blood monocytes to express tissue factor. Thromb Res 83, 307-320,1996. 6. STEINEMANN,S., ULEVITCH,R.J. AND MACKMAN,N. Role of the lipopolysaccharide (LPS)-bindingprotein/CD14pathway in LPS inductionof tissuefactor expressionin monocytic cells. ArteriosclerThromb 14, 1202-1209,1994. 7. FAN, S.T., EDGINGTON, T.S. Coupling of the adhesive receptor CD1lb/CD18 to fictional enhancementof effecter microphage tissue factor response.J Clin Invest 87, 50-57, 1991. 8. BRAND, K., FOWLER, B.J., EDGINGTON, T.S. AND MACKMAN, N. Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptionaland posttranscriptional levelsin responseto lipopolysaccharide.Mol Cell Biol 21, 4732-4738,1991. 9. SUNDSTROM,C., NILSSON, K. Establishmentand characterizationof a human histocytic lymphomaceil line (U-937).Int J Cancer17, 565-577,1976. 10. NILSSON, K., IHVED, I. AND FORSBECK, K. Induced differentiation in human malignant hematopoietic cell lines. In: Gene expression during normal and malignant a?~erentiation.Andersson,L.C. (Ed.),pp. 57-71,AcademicPress,Inc. Ltd. London 1985. 11.LYBERG, T., NILSSON, K. AND PRYDZ,H. Synthesisof thromboplastinby U-937 cells. Br J Haematol51, 631-641,1982. 12. CONKLING, P.R., GREENGERG, C.S. AND WEINBERG,J.B. Tumor necrosis factor induces tissue factor-like activity in human leukemia cell line U-937 and peripheral blood monocytes.Blood 72, 128-133,1988. 13.ZIEGLER HEITBROCK,H.W., THIEL, E., FYTTERER,A., HERZOG, V. AND WIRTZ, A. Establishmentof a human cell line (Mono Mac 6) with characteristicsof mature monocytes. Int J Cancer 41, 456-461, 1988. 14. DICKINSON, J.L., ANTALIS, T.M. Tissue factor and plasminogen activator inhibitor expressionin the differentiationof myeloidleukemiccells.Leukemia7, 864-871,1993. 15. ~SJO, B., IHVED, I., GIDLUND, M., FUERSTENBERG,S., FENYO, E.M., NILSSON, K. AND WIGZELL, H. Susceptibilityto infectionby the human immunodeficiencyvirus (HIV) correlates with T4 expression in a parential monocytoidcell line and its subclones. Virology 157, 359-365, 1987. 16. OBERG, F., HULT, N., BJARE, u., IVHED, I., KIVI, s., BERGH, J., LARSSON, L.G., SUNDSTROM, C. AND NILSSON, K. Characterizationof a U-937 subline which can be inducedto differentiatein serum-freemedium.Int J Cancer50, 153-160,1992.

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17.POTGENS, A.J., LUBSEN,N.H., VAN ALTENA,G., SCHOENMAKERS,J.G., RUITER, D.J. AND DE WAAL, R.M. Measurementof tissue factor messenger RNA levels in human endothelialcells by a quantitativeRT-PCRassay.ThrombHaemost71,208-213,1994. 18. SAMBROOK, J., FRITSCH, E.F. AND MANIATIS, T. Molecular cloning. 2nd, Cold SpringHarbor LaboratoryPress, 1989. 19. WITTWER, C.T., REED, G.B. AND RIRIE, K.M. Rapid cycle DNA amplification.In: The polymerase chain reaction. Mullis, K.B., Ferre, F. and Gibbs, R.A. (Eds.), pp. 174-181, Birkhauser,Boston 1994. 20. SCHWARTZ, A., FERNANDEZ-REPOLLET,E. Developmentof clinical standards for flow cytometry.Clin Flow Cytometry677,28-39, 1993. 21. SORENSEN, B.B., PERSSON, E., FRESKG~RD, P.O., KJALKE, M., EZBAN, M., WILLIAMS, T. AND RAO, L.V. Incorporationof an active site inhibitorin factor VIIa alters the affinityfor tissuefactor.J Biol Chem272, 11863-11868,1997. 22. MORRISSEY,J.H., FAIR, D.S. AND EDGINGTON,T.S. Monoclinal antibodyanalysisof purifiedand cell-associatedtissuefactor.ThrombRes 52, 247-261,1988. 23. BRATT, B., BLOMBACK, M., PAUL, C., SCHULMAN, S., TORNEBOHM, E. AND LOCKNER, D. Factors and inhibitors of blood coagulation and fibrinolysis in acute nonlymphoblasticleukaemia.ScandJ Haematol34, 332-339,1985. 24. TANAKA, M., YAMANISHI,H. The expressionof tissuefactorantigenand activityon the surfaceof leukemiccells. Leuk Res 17, 103-111,1993. 25. STEPHENS, A.C., ZHENG, R.Q., RUSSELL, A.R., LEVIN, M. AND RIVERS, R.P. Productionof tissue factorby monocyteprogenitorcells.ThrombRes 76,33-45, 1994. 26. BROMBERG, M.E,, KOENIGSBERG,W.H., MADISON, J.F., PAWASHE, A. AND GAREN, A. Tissue factor promotesmelanomametastasisby a pathway independentof blood coagulation.Proc Natl Acad Sci U S A 92, 8205-8209,1995. 27. OBERG, F., BOTLING,J. AND NILSSON,K. FunctionalantagonismbetweenvitaminD3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiationof U-937 cells.J Imrnunol150,3487-3495,1993. 28. TODD, R.F., NADLER, L.M. AND SCHLOSSMAN,S.F. Antigenson human monocytes identifiedby monoclinal antibodies.J Imrnunol126, 1435-1442,1981. 29. CONSONNI, R., BERTINA, R.M. Antigenicand functionalexpressionof tissue factor in endotoxin stimulated U937 cells: regulation of activity by calcium and ionophore A23187. ThrombHaemost 74,904-909,1995. 30. ~BRINK, M., GOBL, A.E., HUANG,R., NILSSON,K. AND HELLMAN,L. Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocytemacrophagecell lineage.Leukemia8, 1579-1584,1994. 31. GREGORY, S.A., MORRISSEY, J.H. AND EDGINGTON, T.S. Regulation of Tissue Factor Gene Expressionin the MonocyteProcoagulantResponseto Endotoxin.Molecular and CellularBiology9, 2752-2755,1989. 32. JUNGI, T.W., MISEREZ, R., BRCIC, M. AND PFISTER, H. Change in sensitivityto lipopolysaccharideduring the differentiationof human monocytes to macrophages in vitro. Experiential50, 110-114,1994. 33. BROZNA, J.P., CARSON, S.D. Monocyte- associated tissue factor is suppressed by phorbolmyristateacetate.Blood 73,456-462,1988. 34. VAN DER LOGT, C.P., DIRVEN, R.J., REITSMA, P.H. AND BERTINA, R.M. Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacteriallipopolysaccharideand phorbolester.BloodCoagulFibrinolysis5,211-220,1994.

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35. TERNISIEN, C., RAMANI,M., OLLIVIER,V., KHECHAI,F., VU, T., HAKIM, J. AND DE PROST, D. Endotoxin-inducedtissuefactor in humanmonocytesis dependentupon protein kinaseC activation.ScandJ Immunol70,800-806,1993. 36. CUI, M.Z., PARRY, G.C., EDGINGTON,T.S. AND MACKMAN,N. Regulationof tissue factor gene expression in epithelial cells. Induction by serum and phorbol 12-myristate 13acetate.ArteriosclerThromb 14, 807-814,1994. 37. MACKMAN, N., BRAND, K. AND EDGINGTON, T.S. Lipopolysaccharide-mediated transcriptionalactivation of the human tissue factor gene in THP-1 monocytic cells requires both activatorprotein 1 and nuclear factor kappa B binding sites. J Exp Med 174, 1517-1526, 1991.