Immunology 1996 89 606-612

MUTZ-3, a monocytic model cell line for interleukin-4 andlipopolysaccharide studies

H. QUENTMEIER,* A. DUSCHLt Z.-B. HU,* B. SCHNARR,t M. ZABORSKI* & H. G. DREXLER*

*DSMZ, German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany and tTheodor-Boveri-Institut,

Physiologische Chemie II, Wiirburg, Germany

SUMMARY

The human monocytic cell lines MUTZ-3 and MONO-MAC-6 express the lipopolysaccharide (LPS)receptor CD14. Paralleling the situation in peripheral blood monocytes (PBMo), recombinant human

interleukin-4 (IL-4) down-regulated the expression of CD14 on the cell surface of MUTZ-3, but not thatofMONO-MAC-6 cells. In addition, preincubation with IL-4 prevented the LPS-induced up-regulationof IL-1p mRNA levels in MUTZ-3, but not in MONO-MAC-6 cells. We examined whether thedifferential responsiveness of the cell lines was due to the missing expression of the IL-4 receptor

(IL-4R) a or Yc chain in MONO-MAC-6 cells. Flow cytometric and immunoprecipitation analysisrevealed expression of both IL-4R chains in both cell lines. In addition, short-term stimulation with 11L-4induced tyrosine-phosphorylation of the Yc chain. As both cell lines also expressed signal transducer andactivator of transcription 6 (STAT 6), our data suggested that the differential reaction patterns ofMUTZ-3 and MONO-MAC-6 cells were not due to a generally defective IL-4R complex. Interestingly,long-term (48 hr) treatment with LPS rendered MONO-MAC-6 cells sensitive to IL-4. LPS up-regulatedexpression of monocyte-specific esterase (MSE) mRNA as well as CD14 protein in MONO-MAC-6cells; both effects were inhibited by IL-4. This stimulation was not paralleled by an increase of IL-4RmRNA or protein expression supporting the above hypothesis of a constitutively present and active IL-4R. We discuss possible causes for the differential reaction patterns of MUTZ-3 and MONO-MAC-6cells to IL-4.

INTRODUCTION

Lipopolysaccharide (LPS), a membrane glycolipid of Gram-negative bacteria, induces the release of inflammatory cytokinesfrom mononuclear phagocytes by interaction with the specificreceptor CD14, eventually contributing to the pathogenesis ofseptic shock.' Interleukin-4 (IL-4) down-regulates CD142'3'4 andinhibits the LPS-induced expression of tumour necrosis factor-a(TNF-a), IL-10( and IL-6 mRNA as well as protein secretionin peripheral blood monocytes (PBMo).5'6 In addition, 1L-4diminishes the LPS-induced binding activity of the transcriptionfactor AP-17 and suppresses the activation of NFB.8 Both

transcription factors bind to regulating target sequences in the

promoters of the monocyte-related cytokine genes mentionedabove. Thus IL-4 may inhibit the LPS-stimulated cytokine releasefrom monocytes by down-regulating the LPS receptor and, more

directly, by down-regulating cytokine gene expression. The

signalling events following stimulation of some cytokine receptors

Received 21 May 1996; revised 28 July 1996; accepted 12 August 1996.

Abbreviations: MSE, monocyte-specific esterase; PBMo, peripheralblood monocytes.

Correspondence: Dr H. Quentmeier, German Collection of Micro-

organisms and Cell Cultures, Mascheroder Weg 1 B, D-38124 Braunsch-

weig, Germany.

involve the Janus kinase-signal transducer and activator of tran-scription (JAK-STAT) pathway. Stimulation of the IL-4R inducestyrosine-phosphorylation of the receptor chains as well as of theassociated JAKs.9 l0 1 A substrate of the IL-4R-associated kinase,signal transducer and activator of transcription 6 (STAT 6),12 inturn is phosphorylated and supposedly acts on the expression ofresponsive genes after dimerization and association with therespective promoter sequences. Unlike other cytokines, IL-4 doesnot activate the ras pathway. However, the IL-4 response isassociated with the phosphorylation of IRS-2, a protein related tothe major substrate of the insulin receptor (IRS-1). Myeloid cellsthat lack this protein do not respond to IL-4, and the ability torespond can be conferred on the cells by introduction of IRS-1.13

Thus, stimulation of monocytic cells with LPS serves as modelfor studying inflammatory events, and cytokines like IL-4 thatmodulate the cellular responses to this bacterial stimulus are ofmajor clinical interest. Central scientific questions relate to thesignalling pathways triggered by stimulating monocytes with LPSand IL-4 or other effectors influencing the inflammatory response.However, monocytes are a minor fraction of peripheral bloodmononuclear cells. In in vitro studies, it is nearly impossible toavoid contamination with other residual cell populations, leadingto the presence of additional, undefined cytokines in the experi-mental setting. As cell lines have the advantage of monoclonality,we compared the effects of IL-4 and LPS on monocytic cell lines.

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IL-4 down-regulates CDJ4 expression

The novel acute myeloid leukaemia (AML)-derived cell lineMUTZ-3 represents a monocytic model system for both stimuli.MONO-MAC-6 cells, showing monocyte-specific responses toLPS, react to IL-4 in some experimental settings, but areunresponsive in others. Here, we tried to elucidate whether thedifferential response patterns of MUTZ-3 and MONO-MAC-6cells to IL-4 were caused by differences in the expression of theIL-4R complex.

MATERIALS AND METHODS

Cell cultureThe cell line MUTZ-3 (DSM ACC 295) was maintained ina-minimal essential medium (a-MEM) (Gibco BRL, Eggenstein,Germany) with 20% fetal bovine serum (FBS) (Sigma, Deisen-hofen, Germany) and 10% conditioned medium (CM) of the cellline 5637 (DSM ACC 35) containing several cytokines includinggranulocyte-macrophage colony-stimulating factor, granulocytecolony-stimulating factor and - to a lesser extent - stem-cell factorand macrophage colony-stimulating factor.14 MONO-MAC-6(DSM ACC 124) was grown in RPMI-1640 medium (GibcoBRL) with 10% FBS, 2mM L-glutamine, 1 x nonessential aminoacids, 1 mm Na-pyruvic acid and 9 jig/ml bovine insulin (GibcoBRL). MOLT-3 (DSM ACC 84) cells were kept in RPMI-1640with 10% FBS. All cell lines were taken from the cell bank of theDSMZ (German Collection of Microorganisms and Cell Cultures,Braunschweig, Germany),'5 were free of contamination withmycoplasmas and were harvested in the logarithmic growth phasewith a viability exceeding 90% as determined by trypan blue dyeexclusion.

Growth factors, antibodies and LPSRecombinant human 1L-4, a generous gift of Professor W. Sebald(Wiirzburg, Germany), was applied in final concentrations of60ng/ml. VIM-13 (CD14) and M57 (anti-hIL-4R at chain)monoclonal antibodies (mAb) were kindly provided by ProfessorW. Knapp (Vienna, Austria) and Dr H. J. Gruss (Immunex, Seattle,WA). Chicken anti-,yc chain Ab were obtained from Serva(Heidelberg, Germany), mouse anti-STAT 6 mAb from Dianova(Hamburg, Germany). The anti-h 'yc immunoglobulin G (IgG)TUGh4 was generously provided by Dr K. Sugamura (Sendai,Japan). Rabbit anti--yc chain antiserum N-20 was obtained fromSanta Cruz (Heidelberg, Germany) and anti-phosphotyrosineantibody from Zymed, WAK-Chemie (Bad Homburg, Germany).LPS from Salmonella typhimurium was obtained from Sigma.

[3HJThymidine uptakeFor [3H]Thymidine incorporation, 5 x 103 (100 td) MONO-MAC-6 or 5 x 104 (1001 1) MUTZ-3 cells were seeded in 96-well flat-bottom microtitre plates after washing and resuspending in growthfactor-free medium. Effectors were added as 2 x concentratedsolutions in a 100 11 volume. At different times, cells were pulsedfor 4 hr with 1 14Ci (10o l) [3H]Thymidine (Amersham-Buchler,Braunschweig, Germany). Cells were harvested on glass fibrefilters with a multiple automatic sample harvester, and radio-activity was determined in a liquid scintillation counter.

Western blot analysis and immunoprecipitationFor Western blot analysis, 2 x 106 cells were pelleted, resuspendedin 25 Id protease inhibitor buffer containing 0O1 M NaCl, 1OmmTris-HCl pH 8-0, 1 mm ethylene diamine tetra-acetic acid (EDTA),

1 ,g/ml aprotinin and 100Ig/ml phenylmethylsulphonyl fluoride,and lysed by boiling in 25,l SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) sample buffer con-

taining 15% glycerol, 125 mm Tris-HCl (pH 6-8), 5 mM EDTA, 2%SDS, 0-1% bromophenol blue and 1% (3-mercaptoethanol. Proteinswere separated in 8% SDS-PAGE using a Protean II chamber(BioRad, Munchen, Germany) and were blotted onto nitrocellulosemembranes (Trans-Blot Transfer Medium, BioRad) by elec-troblotting (Sartoblot II-S; Sartorius, Gottingen, Germany). ForCD14 detection, the membranes were labelled with VIM-13 mAb(1: 100) and specific bands were visualized with an alkalinephosphatase-coupled anti-mouse immunoglobulin (Amersham)with the dyes 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and4-nitroblue tetrazolium chloride (NBT) (BioRad, Munchen,Germany). For STAT 6 detection, the membranes were labelledwith the anti-STAT 6 mAb (1:500) and specific bands were

visualized applying a biotin/streptavidin-horseradish peroxidasesystem (Amersham) in combination with the Renaissance WesternBlot Chemoluminescence Reagent protocol (Du Pont, NEN, BadHomburg, Germany).

For immunoprecipitation, 3 x 107 cells were pelleted, washedwith ice-cold phophate-buffered saline (PBS) and resuspended in250 l protease inhibitor buffer (50mm HEPES pH7-1, 150mMNaCl, 1 mm MgCl2, 2 jig/ml aprotinin, 5,ug/ml leupeptin, 20,tMantipain, 20 $M pepstatin). After adding 250 41 lysis buffer (50mmHEPES pH7-1, 150mM NaCl, mM MgCl2, 20% glycerol, 3%Triton-X100 cells were rotated for 30 min at 4°. Cell lysates werecentrifuged at 100000g for 10 min. Cleared supernatants wereincubated for 4 hr at 40 with the anti-'yc Ab TUGh4 and the immunecomplexes were collected on anti-rat agarose for 1 hr at 4°. Theimmunoprecipitates were washed twice with ice-cold lysis buffer,twice with 0-5 M LiCl in 100mM Tris-HCL, pH 8-0, and boiled for3 min in SDS sample buffer. The samples were subjected to SDS-gel electrophoresis on 7 5% gels followed by Western blot analysisusing the enhanced chemoluminescence detection system.

ImmunophenotypingIL-4R a and CD14 expressions were determined with therespective antibodies described above. For immunophenotyping,5x 105 cells were stained with 501.I of 1/50 diluted VIM-13 and400 ng ofM57 mAb. Non-reactive isotype-specific Ab were appliedas controls. Binding of mAb was assessed by an immunofluores-cence technique using fluorescein isothiocyanate-conjugated goatanti-mouse (Dunn, Asbach, Germany) immunoglobulin heavychain-specific antisera. Distribution of antigens was analysedby flow cytometry (FACScan; Becton Dickinson, Heidelberg,Germany). Addition of propidium iodide (10 14g/ml) (Sigma) to thecells prior to analysis allowed gating out of dead cells.

RNA extraction and analysisTotal cellular RNA was isolated by the guanidine isothiocyanate-CsCl method. Total RNA (0Ijg; equal amounts per lane in eachexperiment) was electrophoresed in a 1% formaldehyde-agarosegel, transferred to a nylon membrane (Gene Screen; DuPont, BadHomburg, Germany) and cross-linked with ultraviolet light (UVStratalinker 1800; Stratagene, Heidelberg, Germany). After pre-

hybridization, filters were hybridized overnight at 620 withrandom-primed 32P-labelled probes (Amersham). Filters were

then stringently washed and exposed at -80° to X-ray films (FujiRX; INTAS, Gottingen, Germany) with intensifying screens forautoradiography.

© 1996 Blackwell Science Ltd, Immunology, 89, 606-612

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H. Quentmeier et al.

DNA probesThe hIL-2R yYc probe was a 880 bp EcoRI-HindIH fragment from a

pGEM plasmid, the hIL-1 probe a 1369 bp EcoRI fragmentpurified from a pBS plasmid, the monocyte-specific esterase(MSE) probe was a 1700bp EcoRI fragment from a pUC19plasmid, and the hamster (-actin probe a 1250bp PstI fragmentfrom a pBR322 plasmid. Plasmids or inserts were generouslyprovided by Dr P. M. Sondel (Madison, WI) (IL-2R Yc chain),Dr M. Brach (Berlin, Germany) (IL-1 j3), and Dr F. Zschunke(Gottingen, Germany) (MSE).

Reverse transcriptase-polymerase chain reaction (RT-PCR)First-strand cDNA was synthesized using a reverse transcriptasepreamplification system kit (Super Script; Gibco BRL) followingthe manufacturer's instructions. Five micrograms of total RNA as a

template were incubated with 500 ng of oligo dT in a final volumeof 16 H20. After heating the mixture at 700 for 10 min, 2 ,l lOx

synthesis buffer, 1 pl of 10mm 2'-deoxynucleoside 5 '-triphosphate(dNTP) mix, and 1 pl (200 U) of Moloney murine leukaemia virusreverse transcriptase were added to the reaction system. Thereaction mixture was then incubated at 420 for 50 min, at 900 forS min and was then quickly chilled on ice. Four microlitres of thereverse transcriptase reaction mixture containing the first-strandcDNA were diluted with PCR buffer containing 50 pmol of eachupstream and downstream primer, 10 nmol of dNTP mix, and1-25 U of Taq DNA polymerase (polymerase and lOx buffer:Amersham). The following oligonucleotide 5' and 3' primersequences were used: IL-2R -yc chain, 5'-GAAGACACCAC-AGCTGAT'T'C-3' (99-119 from ATG) and 5'-ACTCTCA-GCCAGTCCCTTAGA-3' (959-939 from ATG); ,B-actin, 5'-ATGGATGATGATATCGCCGCG-3' (1-21 from ATG) and 5'-CTAGAAGCATTTGCGGTGGAC-3' (1128-1108 from ATG).Oligonucleotide primers were prepared on an automated DNAsynthesizer (Cyclone Plus; Millipore, Eschborn, Germany). ThePCR was then performed with aDNA thermal cycler (Perkin ElmerCetus, Heidelberg, Germany) for the number of cycles indicated inFig. 4 under the following conditions: 30 seconds at 950 fordenaturation, 30 seconds at 600 for annealing, and 2 min at 720 forextension. The amplified PCR products were electrophoresed in1-1% agarose gels, were stained with ethidium bromide, and were

observed under UV light. The PCR products were transferred to a

nylon membrane by Southern blotting and hybridized with therespective random primed 32P-labelled DNA probes.

RESULTS

[3H]Thymidine incorporation of MUTZ-3 cells is increasedby IL-4

MUTZ-3 cells were routinely kept in medium containing 5637CM. For continuous proliferation, the supernatant could bereplaced by several single cytokines.16 Especially in combinationwith insulin, IL-4 increased short-term [3H]Thymidine incorpora-tion of MUTZ-3 cells (Fig. 1) demonstrating that the cell lineexpresses an active IL-4R. [3H]Thymidine incorporation of thegrowth factor-independent cell line MONO-MAC-6 was notaffected by IL-4 or insulin (data not shown).

CD14 expression on MUTZ-3 and MONO-MAC-6 cells ismodulated by IL-4 and LPS

Western blot analysis demonstrated that both cell lines expressed

600

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100

A -Ins - IL-4_l -Ins +IL-4A +Ins -IL-4

+Ins +IL-4

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Figure 1. Proliferative response of MUTZ-3 on IL-4 and insulin.[3H]Thymidine uptake of MUTZ-3 cells treated with IL-4 (60ng/ml),insulin (100 ng/ml) or IL-4+ insulin. The data are expressed as percentageincorporation of medium control at the respective day. Standard deviationsare always less than 10%. The data represent means of triplicates.

the 53 000MW CD14 glycoprotein (Fig. 2). In MUTZ-3, but not inMONO-MAC-6 cells, IL4 on its own reduced the constitutiveCD14 expression (Table 1). LPS treatment induced the upregula-tion of this protein on the cell surface of both cell lines (Table 2),with maximal expression after 48 hr and at concentrations as low as

50 pg/ml. Costimulation with IL-4 (60 ng/ml) reduced the LPSeffect (Table 2, Fig. 3) in both cell lines.

IL-4 prevents the LPS-induced upregulation of IL-1,B mRNAexpression in MUTZ-3, but not in MONO-MAC-6 cells

A 75-min-stimulation of MUTZ-3 and MONO-MAC-6 cells withLPS (50pg/ml) induced highly elevated IL-113 mRNA levels. Pre-incubation with LL-4 (60 ng/ml) for 19 hr to 48 hr prevented thiseffect in MUTZ-3, but not in MONO-MAC-6 cells (Fig. 4).

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CD 14 STAT 6

Figure 2. Western blot analysis of CD14 and STAT 6 molecules fromMUTZ-3 and MONO-MAC-6 cells. Proteins from cell lysates were

separated by PAGE and blotted onto nitrocellulose membranes. Mwestimates were based on Mw markers (left).

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IL-4 down-regulates CDJ4 expression

Table 1. Effects of IL-4 on CD14 surface expression of MUTZ-3 andMONO-MAC-6

MUTZ-3 (%) MONO-MAC-6 (%)

Experiment Medium IL-4 Medium IL-4

#1 19* 6 16 18#2 1 1 4 10 12#3 25 7 15 16

*Percentage of VIM-13 reactive MUTZ-3 and MONO-MAC-6 cellsafter 72 hr incubation with or without IL-4 (60 ng/ml).

IL-4 inhibits the LPS-induced upregulation of MSE mRNA

A 48-hr stimulation with LPS (3 ng/ml) induced high expression ofMSE mRNA exclusively in MONO-MAC-6 cells. Coincubationwith IL-4 (60 ng/ml) suppressed this effect (Fig. 4).

MUTZ-3 and MONO-MAC-6 cells express an active IL-4Rcomplex

The IL-4R consists of an a and a Yc chain, the latter being part alsoof several other cytokine receptors, thus being called the commonchain. Though Northern blot and RT-PCR analysis showed adramatically lower expression of PYc chain mRNA in MONO-MAC-6 than in MUTZ-3 cells (Fig. 4), protein studies revealed thatboth cell lines showed comparable expression of the -yc chain at the

100-

CD

co66z0

102

102

Table 2. Effects of LPS and IL-4 on CD14 expression of MUTZ-3 andMONO-MAC-6

MUTZ-3 (%) MONO-MAC-6 (%)

Experiment Medium LPS LPS +IL-4 Medium LPS LPS + IL-4

# 1 12* 21 9 13 63 49# 2 10 22 n.d. 16 81 53#3 21 31 18 16 62 49#4 23 40 n.d. 13 78 57

*Percentage of VIM-13 reactive MUTZ-3 and MONO-MAC-6 cellsafter 48 hr incubation with or without IL-4 (60 ng/ml) and LPS (MUTZ-3 #1, 2: 1,ug/ml; #3: 100ng/ml; # 4: 1 ng/ml. MONO-MAC-6 # 1, 2: 3 ng/ml;#3: 1 ng/ml; #4: 50 pg/ml).

n.d., not done.

protein level, as also found for the IL4Ra chain (Fig. 3, Fig. 5,upper panel). Incubation with LPS for 48 hr did not influence the zYcexpression (Fig. 5, upper panel). Short-term stimulation with 11L-4increased tyrosine phosphorylation of the -Yc chain, when comparedto unstimulated or long-term 1L4 plus LPS-stimulated cells (Fig.5, lower panel). The yc chain was identified applying Molt-3 cellsstrongly expressing the yc chain (data not shown). Coprecipitatedproteins, associated with the -Yc chain explain bands observable inthe anti- phosphotyrosine blot (Fig. 5, lower panel). In addition tothe IL4R, both cell lines expressed STAT 6, the 100000MWsubstrate of the IL-4R-associated JAKs (Fig. 2). We were not able

Medium IL-4Ra

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I , . , ,(vf)3 1 04

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3 104Medium IL-4Ra

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104LPS CD14

104

LPS + IL-4 CD14

104

Figure 3. Flow cytometric analysis of 1L-4Ra and CD14 expression of MUTZ-3 and MONO-MAC-6 cells. Cells were incubated for 48 hrwith LPS (100 ng/ml for MUTZ-3; 3 ng/ml for MONO-MAC-6) with or without IL-4 (60 ng/ml). Isotype controls are included as open graphs.

© 1996 Blackwell Science Ltd, Immunology, 89, 606-612

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Figure 4. Effects of LPS and IL-4 on levels of IL-1j0, MSE and IL-4R 'yC mRNA in MUTZ-3 and MONO-MAC-6 cells. Cells were treated as

follows: A, 48 hr medium; B, 48 hr IL-4 (60 ng/ml); C, 48 hr medium followed by 1-25 hrLPS (5Opg/ml); D, 48 hr IL-4 (60 ng/ml) followed by1-25 hr LPS (5Opg/ml); E, 48hr LPS (3ng/ml); F, 48hr IL-4 (60ng/ml) and LPS (3ng/ml). NB: Northern blot analysis: total RNA (IOg/lane) was hybridized with 32P-labelled probes. Rehybridization of the filters with j3-actin cDNA confirmed identical RNA loading in singlelanes. PCR: semiquantitative RT-PCR was carried out with primers specific for -Yc chain and fl-actin for the indicated number of cycles.Southern blots were hybridized with the respective 32P-labelled probes.

to detect tyrosine-phosphorylated STAT 6 molecules after a 20 minLPS stimulation (data not shown) suggesting a very rapidphosphorylation process.

DISCUSSION

1L4, like other cytokines, induces a complex array of effects on

cells expressing the respective receptor. These processes are

accompanied by tyrosine-phosphorylation of the receptor and otherintracellular substrates, eventually leading to activation of thecells.17 In PBMo, LL-4 exerts inhibitory effects on the cellularresponses to inflammatory stimuli like LPS. For instance, IL-4down-regulates the LPS-R CD142,3'4 and inhibits the LPS-inducedexpression of TNF-a, IL-1,8 and IL-6 mRNA as well as proteinsecretion. 5,6 Both signalling pathways, following stimulation ofthe LPS-R and the LL-4R, are currently major points of scientificinterest.

The search of the role of individual members of thesesignalling chains is pursued mostly in experiments applyingheterologous expression systems. However, signal transductionstudies which are designed not only to explain early events likephosphorylation of the receptor itself but aimed to elucidateactivation and differentiation processes lastly must include studiesof cells with the correct tissue-specific background. Therefore,the understanding of the IL-4-induced events, preceding down-regulation of CD14 for example, will have to include studies on

monocytic cells. However, elucidation of these regulatory eventsin PBMo is hampered by the probable contamination of the primarycells with other residual cell populations or by unwarrantedstimulation during the purification procedure, both certainlyleading to the presence of additional, undefined cytokines in theexperimental setting. As cell lines have the advantages of mono-clonality and nearly unlimited availability, we looked for a cell linereacting to LPS and to LL-4 in a monocyte-specific manner.

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MUTZ3

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IL-1p

MSE

YOchan

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No. of cycdesF

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NfM cean

No. of cycles

IL-4 down-regulates CDJ4 expression 611

MONO-MAC-6 MUTZ-3A B C A B C Blot: anti-y.

A B C A B C Bo~niP

90-80-70-60-

MONO-MAC-6 MUTZ-3A B C A B C Blot: anti-PY

60- at;S

70-... ..

BI i 1- 60 -gm) C, 48 hr1L- 6 ml) plu _P 3ngm

antiserum N-20 (upper panel) or an anti-phosphotyrosine antiserum (lowerpanel). Mw of marker proteins is indicated to the left in kDa.

The studies presented here WLre canried out with MUTZ-3'16and MONO-MAC-6 cells.18 Both are CD14-positive cell linesestablished from patients with acute myelomonocytic leukaemia(AML FAB M4 and AML FAB M5, respectively). Especially,MUT'Z-3 represents a reliable model system for both stimuli, LPSand IL-4.

11L-4 induced [3H]Thymidine uptake of MUTZ-3 cells, provid-ing evidence that these cells expressed an active LL-4R. In MONO-MAC-6 cells, we did not observe a proliferative response to 11L-4.To look at a more monocyte-specific parameter than [3H]Thymi-dine uptake, we then analysed the influence of 11L-4 on thepercentage of CD14 positive cells. Analogous to PBMo,2.319 .1-4down-regulated the expression of CD14 in MUTZ-3 cells. Inaddition and again paralleling the situation in the primary cells,5'611.4 abrogated the rise of 11_-1f3 mRNA levels in M-UTZ-3 cellsafter short-term stimulation (75 m.) with LPS. Both 11-4 effects,down-regulation of CD14 and inhibition of LPS-triggered 11-113mRNA upregulation, were not seen in MONO-MIAC-6 cells.

Consequently, we analysed whether the lack of response ofMONO-MAC-6 cells to 11-4 might be due to the absence of one ofthe components of the 11-4R. However, both cell lines expressedthei1-4R a and -yc chain. In addition, short-term stimulation with11is4 induced tyrosine-phosphorylation of the yc chain. Both celllines expressed STAT 6, the substrate of JAKs associated with11L-4-induced signalling events. Taken together, our results showedthat the missing responsiveness of MONO-MAC-6 was not theconsequence of the absence of 11-4R components. On the otherhand, our data could not exclude that the receptor had lost itsfunction by a locally restricted change, for example by a pointmutation. However, this change would not completely abolish theresponsiveness to 114. This is, because up-regulation of CD14 bya two-day stimulation with LPS was reduced by 1L-4 in MONO-MAC-6 as well as in MUTZ-3. In addition, 114 inhibited theLPS-triggered MSE mRNA upregulation in MONO-MAC-6 cells.

addi1996 BackelScaiencprlelnLtd eImmuntoloy 89, 606-612 ycels

These events were not paralleled by an increase of IL-4R a or 'Ycchain supporting the hypothesis of a constitutively present and (atleast in some experimental settings) active IL-4R. The partialunresponsiveness of MONO-MAC-6 cells to IL-4 might be due tolocal defects in the receptor complex, for example due to a pointmutation, exerting influence only on some parameters (like IL-1Bexpression) but not on others (like MSE expression).

Alternatively, the responsiveness of MONO-MAC-6 afterLPS-treatment might be caused by induction of proteins otherthan the IL-4R complex itself but still obligatory for IL-4responses. One candidate would be IRS-2, an intracellularmolecule obligatory for LL-4 signalling. Myeloid cells that lackthis protein which is closely related to the substrate of the insulinreceptor IRS-1 fail to respond to JIL4.13

In conclusion, the reactions of the novel monocytic cell lineMUTZ-3 to LPS and IL-4 closely resemble those ofPBMo makingit a promising model for signal transduction studies. Resultsobtained from MONO-MAC-6 cells differ in several aspects fromthose of PBMo and MUTZ-3. Revealing the causes of thisdifferential responsiveness might help to elucidate the complexstructure of the LL-4R signalling pathway.

REFERENCES

1. LANDMANN R., ZmmEmu W., SANsANo S. et al. (1995) Increasedcirculating soluble CD14 is associated with high mortality in gram-negative septic shock. J Infect Dis 171, 639.

2. RuPPERT J., FRmDRicHs D., Xu H. & PErERs J.H. (1991) IL-4 decreasesthe expression of the monocyte differentiation marker CD14,paralleled by an increasing accessory potency. Immunobiology 182,449.

3. LAUENER R.P., GOYERT S.M., GERA R.S. & VERcELiw D. (1990)Interleukin 4 down-regulates the expression of CD14 in normalhuman monocytes. Eur J Immunol 20, 2375.

4. JANSEN J.H., FIBBE W.E., WIENTJENs G.J.H.M. et al. (1991) Interleukin 4down-regulates the expression of CD14 and the production ofinterleukin 6 in acute myeloid leukemia cells. Lymphokine CytokineRes 10, 457.

5. EssNER R., RHoADEs K., McBRIuE W.H., MORTON D.L. & ECONOMOUJ.S. (1989) IL-4 down-regulates IL-1 and TNF gene expression inhuman monocytes. J Immunol 142, 3857.

6. TE VELDE A.A., HuuBENs R.J.F., HEuE K., DE VRiEs J.E. & FIGDOR C.G.(1990) Interleukin-4 (IL-4) inhibits secretion of IL--1j, tumor necrosisfactor at, and IL-6 by human monocytes. Blood 76, 1392.

7. DoKTER W.H.A., ESsELINK M.T., HAuIE M.R. & VELLENGA E. (1993)Interleukin-4 inhibits the lipopolysaccharide-induced expression ofc-jun and c-fos messenger RNA and activator protein-i bindingactivity in human monocytes. Blood 81, 337.

8. DoNELLY R.P., CRoFFoRD L.J., FREMAN S.L. et al. (1993) Tissue-specific regulation of 11L-6 production by IL-4. J Immunol 151, 5603.

9. iLuE J.N., Wn-muN B.A., QuELLE F.W., YAMAMoTo K. & SiLvsNoWIEN0. (1995) Signaling through the hematopoietic cytokine receptors.Annu Rev Immunol 13, 369.

10. WnnHuHN B.A., SnvENNomIEN O., MiruRA 0. et al. (1994) Involvementof the Jak-3 Janus kinase in signalling by interleukins 2 and 4 inlymphoid and myeloid cells. Nature 370, 153.

11. Musso T., JoHNsToN J.A., LINNEiN D. et al. (1995) Regulation ofJAK3expression in human monocytes: phosphorylation in response tointerleukins 2, 4, and 7. J Exp Med 181, 1425.

12. Hou J., ScHimmLER U., HENEzL W.J., Ho T.C., BRAsswUR M. &McKGHTrr S.L. (1994) An interleukin-4-induced transcription factor:IL-4 STAT. Science 265, 1701.

13. WANG L.M., Min~s M.G., SUN X.J., AARONSON S.A., WinE M. &PERCE J.H. (1993) IRS-1: Essential for insulin- and 1L-4-stimulatedmitogenesis in hematopoietic cells. Science 261, 1591.

612 H. Quentmeier et al.

14. QuENamoEIR H., ZABoRSKi M. & DRExLER H.G. The human bladdercarcinoma cell line 5637 constitutively secretes functional cytokines. JImmunol Methods (submitted).

15. DREXLER H.G., DIRKs W., MACLEOD R.A.F., QuEN~mmR H. & STEUBEK. (eds). (1995) DSM Catalogue ofHuman & Animal Cell Lines, 5thedn. Braunschweig, Germany.

16. Hu Z.B., MA W., ZABORSKI M., MACLEOD R.A.F., QuENTmEImR H. &DREXLER H.G. (1996) Establishment and characterization of two novelcytokine-responsive acute myeloid and monocytic leukemia cell lines,MUTZ-2 and MUTZ-3. Leukemia 10, 1025.

17. WANG L.M., KEEGAN A., FRANKEL M., PAUL W.E. & PIERCE J.H. (1995)Signal transduction through the IL-4 and insulin receptor families.Stem Cells 13, 360.

18. ZIELGER-HElTEROCK H.W.L., THmIL E., ForrumR A., HauzoG V., WntTzA. & Ri nnouER G. (1988) Establishment of a human cell line(MONO MAC 6) with characteristics of mature monocytes. Int JCancer 41, 456.

19. LANDmANN R., LuDwiG C., OBRIST R. & OBREctrr J.P. (1991) Effect ofcytokines and lipopolysaccharide on CD14 antigen expression inhuman monocytes and macrophages. J Cell Biochem 47, 317.

X 1996 Blackwell Science Ltd, Immunology, 89, 606-612