Mediators of Inflammation, 8 IN Time dependent production...

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Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids Ingrid M. Garrelds 1,2,CA , Peter Th. W. van Hal 2 , Raquel C. Haakmat 1 , Henk C. Hoogsteden 2 , Pramod R. Saxena 1 and Frederik J. Zijlstra 1 1 Institute of Pharmacology, Faculty of Medicine, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam and 2 Department of Pulmonary Medicine, University Hospital Rotterdam-Dijkzigt,The Netherlands CA Corresponding Author Institute of Pharmacology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Tel: (+31) 10 4087534 Fax: (+31) 10 4089458 Email: [email protected] IN the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cyto- kines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol- 12 myristate acetate (PMA) for 24h, U937 cells were cultured in the absence or presence of lipopoly- saccharide (LPS: 1 and 5 m g ml –1 ) and glucocorticoste- roids (budesonide, fluticasone propionate and pre- dnisolone: 10 –11 , 10 –9 and 10 –7 M) for 96h. The production of interleukin- 1b (IL-1 b ), interleukin-6 (IL-6), prostaglandin E 2 (PGE 2 ) and thromboxane B 2 (Tx B 2 ) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-a ) reached its maximum between 6 and 12h. Interferon-gamma (IFN-g ), inter- leukin-10 (IL-10) and leukotriene B 4 (LTB 4 ) were not detectable. All three glucocorticosteroids (budeso- nide, fluticasone propionate and prednisolone) com- pletely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells. Key words: Glucocorticosteroids, Cytokines, Arachidonic acid metabolites, U937 Introduction Inflammation of the airways underlies a major part of the clinical symptoms in asthma and chronic obstruc- tive pulmonary diseases (COPD). A number of studies support the involvement of mononuclear phagocytes in asthma. 1–3 Alveolar macrophages phagocytose inhaled particles and antigens, but as inflammatory cells they are know n to produce potent inflammatory mediators, including proteins and lipids mediators, which are able to influence other cell types. Several features of asthma can be mimicked in vitro and in vivo by these secretory products. 4 We and others have previously shown that prostaglandin D 2 , prostaglandin F 2a and a stable analogue of thrombox- ane A 2 all constrict human airway muscle. 5 Leukotriene B 4 (LTB 4 ) increases vascular permeability and enhan- ces adherence and migration of granulocytes. Inhala- tion of LTB 4 results in peripheral neutropenia and the accumulation of leucocytes in the airway wall. 6 Leukotriene C 4 (LTC 4 ) induces bronchoconstriction, reduces the clearance of mucus and increases vascular permeability. 7 Cytokines, such as IL-1b , IL-6 and TNF- a , play an important role in immune reactions and regulate the growth and state of activity of many cells in inflammation including the airw ay inflammation present in asthma. 8 Both eicosanoids and cytokines can also be released from inflammatory cells in culture. Some studies have investigated the regulation of cytokines and eicosanoids in macrophage cell lines or macrophages stimulated with various inflammatory agents. 9–13 The human monoblastoid U937 tumour cell line is widely used as a model for the maturation of monocytic cells. 14 U937 is an established human monoblast cell line derived from the pleural exudate of a patient with diffuse histiocytic lymphoma. Maturation of U937 can be induced by incubation with different agents, including retinoic acid, 15 vita- min D derivative s, 16 cytokines 17 and phorbol esters. 18 Phorbol ester-induced maturation results in cessation of proliferation and significant alterations in the morphology of the cells. Under standard conditions, U937 cells grow in suspension and exhibit a smooth ISSN 0962-9351 print/ISSN 1466-1861 online/99/040229-07 © 1999 Taylor & Francis Ltd 229 Research Paper Mediators of Inflammation, 8, 229–235 (1999)

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Time dependent production ofcytokines and eicosanoids byhuman monocytic leukaemia U937cells; effects of glucocorticosteroids

Ingrid M. Garrelds1,2,CA, Peter Th. W. van Hal2,Raquel C. Haakmat1, Henk C. Hoogsteden2,Pramod R. Saxena1 and Frederik J. Zijlstra1

1Institute of Pharmacology, Faculty of Medicine,Erasmus University Rotterdam, PO Box 1738, 3000DR Rotterdam and 2Department of PulmonaryMedicine, University Hospital Rotterdam-Dijkzigt,TheNetherlands

CACorresponding AuthorInstitute of Pharmacology, Erasmus UniversityRotterdam, PO Box 1738, 3000 DR Rotterdam, TheNetherlandsTe l: (+31) 10 4087534Fax : (+31) 10 4089458Email: [email protected]

IN the present s tudy th e hum an m onoblast ce ll lineU937 has been used as a m ode l to s tudy the fun ctionof hum an m ononuclear phagocyte s in asthm a. Thekin etic s of th e production of e icos anoids and cyto-kin es , w hich are though t to play a role in thepath ogenes is of asthm a, w ere s tudied. In addition, theeffe cts of glucocor ticosteroids were in ves tiga ted, asthe se drugs are of gr eat im portance for the treatm entof asth m atic patie nts . Afte r s tim ulation w ith phorbol-12 m yris tate acetate (PMA) for 24 h, U937 cells werecultured in the absence or pre sence of lipopoly-saccharide (LPS: 1 and 5 m g m l–1) and glucocorticoste -roids (bude son ide , fluticasone propionate and pre-dn isolone : 10–11, 10–9 and 10–7 M) for 96 h . Th eproduction of in te rleukin- 1 b (IL-1 b ), in te rleukin -6(IL-6), prostaglandin E2 (PGE2) and th rom box ane B2

(Tx B2) gradually in creased in tim e after s tim ulationw ith LPS, w hereas th e tran sien t production of tum ornecrosis factor alpha (TNF-a ) r eached its m ax im umbe tw een 6 and 12 h. In ter feron -gam m a (IFN-g ), inter -leukin -10 (IL-10) and leukotrie ne B4 (LTB4) were notde tectable. All three glucocorticos te roids (budeso-n ide , fluticasone propionate and prednisolone) com -pletely inh ibited th e production of both e icosanoidsand cytokine s . Th e production of eicosanoids wasm ore sensitive to th ese glucocorticoids th an theproduction of cytokin es . Th e observed diffe rence s inthe kin etics of the production of eicosanoids andcytokin es s tr ess th e im portance of tim e courseex perim ents in s tudie s on th e e ffe ct of drugs onm ononuclear cells.

Key words: Glucocorticoste roids, Cytokine s, Arachidonicacid metabolite s, U937

Introduction

Inflammation of the airw ays unde rlie s a major part ofthe clinic al symptoms in asthma and chronic obstruc-tive pulmonary dise ase s (COPD). A number of studie ssupport the involve ment of mononuclear phagocytesin asthma.1–3 Alveolar mac rophages phagocytoseinhaled partic le s and antige ns, but as inf lammatoryce lls they are know n to produce potent inf lammatorymediators, inc luding prote ins and lipids mediators,w hich are able to inf luence other cell types.

Several feature s of as thma can be mimicked in vitroand in v ivo by these se cre tory products .4 We andothe rs have pre viously show n that prostaglandin D2,prostaglandin F2 a and a stable analogue of thrombox -ane A2 all constric t human airw ay muscle .5 Leukotrie neB4 (LTB4) inc reases vascular pe rmeability and enhan-ce s adherence and migration of granulocytes. Inhala-tion of LTB4 re sults in peripheral ne utropenia and theaccumulation of leucocyte s in the airw ay w all.6

Leukotrie ne C4 (LTC4) induces bronchoconstric tion,reduces the clearanc e of mucus and inc reases vascular

permeability.7 Cytokine s, such as IL-1 b , IL-6 and TNF-a ,play an important role in immune re ac tions andregulate the grow th and state of activ ity of many ce llsin inf lammation inc luding the airw ay inf lammationpresent in as thma.8 Both e icosanoids and cytokine scan also be re leased from inf lammatory cells in culture .Some studie s have inve stigated the re gulation ofcytokine s and eicosanoids in macrophage cell line s ormacrophage s stimulate d w ith various inf lammatoryagents.9–13

The human monoblastoid U937 tumour ce ll line isw ide ly used as a model for the maturatio n ofmonocytic cells.14 U937 is an e stablished humanmonoblast c ell line de rived from the pleural ex udateof a patie nt w ith diffuse histiocytic lymphoma.Maturation of U937 can be induc ed by inc ubationw ith diffe rent agents, inc luding re tino ic ac id,15 vita-min D de rivative s ,16 cytokine s17 and phorbol esters.18

Phorbol e ste r-induc ed maturatio n results in cessationof prolife ration and s ignific ant alte rations in themorphology of the cells. Under standard conditio ns,U937 cells grow in suspension and ex hibit a smooth

ISSN 0962-9351 print/ISSN 1466-1861 online/99/040229-07 © 1999 Taylor & Francis Ltd 229

Research Paper

Mediators of Inflammation, 8, 229–235 (1999)

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and round surfac e, but they become adhe re nt, start toform cell c luste rs and ex tend pseudopodia uponphorbol e ste r tre atment.18–20 The morphologic alchanges observed during this maturatio n proce sssuggest func tional changes of the U937 cells.

We studied the kine tic s of the production of severalcytokine s, IL-1 b , IL-6, IL-10, IFN-g and TNF-a andeicosanoids PGE2, Tx B2 and LTB4 in U937 to clarifyw hether mediators are sequentially re leased by acti-vated mononuclear ce lls . These mediators w e re stud-ied, because they are thought to be of importanc e inthe pathogene sis of the airw ay inf lammation in lungdiseases . Furthermore , the e ffec tivene ss of glucocorti-costeroids to suppress the formation of inf lammatorymediators w as inve stigated. We used budesonide ,fluticasone propionate and prednisolone , becausethey are the most important drugs curre ntly used inthe treatment of asthma.

Materials and methods

Cell culture

U937 cells (American Type Culture Collection USA,batch 1593.2) w ere cultured in RPMI 1640 medium(Gibco, UK) supplemented w ith penic illin (ICN, USA;100 m g ml–1), streptomycin (ICN, USA; 100 m g ml–1),glutamax -I (Gibco, UK; 10 mM) and 10% foetal bovinese rum (FBS; Gibco, UK) in tissue culture flasks(Costar, UK) at 37°C humidifie d atmosphere w ith 5%carbon diox ide .

Maturatio n of U937 cells w as induced w ith 250 ngml–1 PMA (Sigma, USA). Ce lls we re cultured for 24 hw ith PMA and furthe r grow n for 48 h in fre shcomple te medium. Ce ll viability w as assessed bytrypan-blue ex clusion in the non-adherent untre ate dU937 as we ll as in the PMA treate d U937 ce lls . Theviability of the ce lls w as >95%.

The adhe rent cells w ere scraped from the tis sueculture flasks w ith a rubber policeman and aliquots of0.5 3 106 cells in 1 ml w ere put in 24 w ells plas ticmultiw e ll culture plate s (Costar, UK) and inc ubate dw ith 1 m g ml–1 or 5 m g ml–1 or w ithout LPS fromEsche rich ia co li (Serotype 0111:B4; Sigma, USA). Thece lls w ere incubated for 1, 3, 6, 8, 10, 12, 24, 48 and96 h in separate we lls at 37°C. Cell-free supe rnatantsw ere collec te d and stored at –80°C until measure-ments of lipid mediators and cytokine s.

Incubation with steroids

To ensure that the U937 cell line in our inve stigationscould be used to evaluate glucocorticoid re sponsive -ne ss, w e performed the ex perime nt w ith ste roids . Weused three cortic osteroids , namely budesonide(Sigma, USA), flutic asone propionate (gift of Glax oWellcome, UK) and prednisolone sodium phosphate(Genfarma, NL). The drugs we re used in the finalconcentratio ns: 10–11, 10–9 and 10–7 M.

The ce lls we re pre-tre ated w ith PMA, the drugsw ere added to the cell culture s 24 h prior tostimulation w ith LPS. From this set point [t = 0] thece lls w ere incubated for 1, 3, 6, 8, 10, 12, 24, 48 and96 h in the pre sence or absence of the drug and/orLPS. The ce ll-free supe rnatants we re stored at –80°Cuntil measurements .

Ce ll viability w as not alte re d afte r drug treatme nt.

Measurements of cytokines

IL-1 b (R&D syste ms, USA), IL-6 (CLB, NL), IL-10(Pharming, USA), TNF-a (Pharming, USA) and IFN-g(Medgenix , Belgium) leve ls we re measure d usingenzyme- linked immunosorbent as say (ELISA).

Measurements of eicosanoids

The supe rnatants w ere proce ssed as de scribed indetail previously w ith slight modific ations.21 SepPakC18 cartridge s (Wate rs Ass ., USA) w ere activate d w ith10 ml of methanol pre -w ashed w ith 10 ml distille dw ate r. Tw o hundred and fifty m l of supernatant w asapplied to the columns and rins ed w ith 2.5 mlmethanol, drie d w ith a Savant Speed-Vac concen-trato r, and dissolved in 1 ml radio immunoassay (RIA)buffer. PGE2 and Tx B2 production were measured byRIA (antibodie s w ere obtaine d from Pe rSeptive Diag-nostic s, USA, tritiate d antige ns from Amersham, UKand standards from Sigma, USA). LTB4 le ve ls we remeasured by enzyme immuno as say (EIA) kits , w hichw ere obtaine d from Amersham, USA.

Statistical analysis

Each drug w as studie d in four separate ex perime ntsand the values are ex pre ssed as mean ± s.e . mean.Statis tical comparisons betw een contro l and drug-tre ate d cell culture s w ere made by ANOVA follow ed bypaire d t-te st. A p<0.05 w as cons ide re d signific ant.

Results

LPS-induced cytokine production afterpre-treatment with PMA

In standard medium w ithout PMA, U937 cells had asmooth and round surfac e and did not produce thecytokine s and eicosanoids studied (data not show n).Afte r addition of PMA they became adherent byforming ce ll c lusters, ex tended pseudopodia and, asshow n in Fig. 1, re leased IL-1 b , IL-6 and TNF-a inresponse to LPS stimulation. However, LPS did notinduce the PMA-treated cells to produce INF-g or IL-10(data not show n). Afte r inc ubation w ith LPS theamounts of IL-1 b inc reased signific antly at 48 and 96 h,w hich se emed to be concentration dependent. IL-6leve ls already inc re ased signific antly afte r 6 h, afte rw hich the leve ls reached a plateau. TNF-a production

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show ed a diffe re nt patte rn compared w ith IL-1 b and IL-6; its leve ls inc reased afte r 6 h of incubation w ith LPS,but starte d to decrease afte r 24 h.

LPS-induced eicosanoid production afterpre-treatment with PMA

We determine d the effe cts of LPS on PGE2, Tx B2 andLTB4 production by U937 cells w hich were pre -treatedw ith PMA. Without PMA, U937 ce lls did not produceany of the e icosanoids studied (data not show n). Thece lls primed w ith PMA re leased PGE2 and Tx B2, but notLTB4, in re sponse to LPS (Fig. 2). This e ffec t w as time-re lated. The leve ls of Tx B2 already reached a plate au

phase afte r 6 h, w hilst the leve ls of PGE2 inc reasedslow ly w ith time.

Effect of glucocorticosteroids on theLPS-induced production of inflammatorymediators by PMA pre-treated U937 cells

Glucocortic osteroids dow nregulate the cytokineex pre ssion of human alveolar macrophage s.22 Toinve stigate w he the r the U937 ce lls could be used asa model to study glucocortic oid re sponsivene ss inmonocyte s/macrophage s, w e inc ubate d the cellsw ith budesonide , fluticasone propionate or pre-dnisolone . Figures 3, 4 and 5 show the data obtaine d

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FIG. 1. Kinetics of IL-1b (top panel), IL-6 (middle panel) andTNF-a (lower panel) production in PMA-treated U937 cellswhich were stimulated with LPS. Data represent mean ± s.e.mean obtained from four separate experiments. *p<0.05versus response without LPS. When no error bar is visible,error falls within the limit of the symbol.

FIG. 2. Kinetics of LTB4 (upper panel), PGE2 (middle panel)and TxB2 (lower panel) production in PMA-treated U937 cellswhich were stimulated with LPS. Data represent mean ± s.e.mean obtained from four separate experiments. *p<0.05versus response without LPS. When no error bar is visible,error falls within the limit of the symbol.

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w ith bude sonide , fluticasone proprionate and pre-dnisolone respec tive ly. The production of IL-1 b andIL-6 w as dose-depende ntly inhibited by all thre esteroids. At 10–7 M concentratio n, all thre e steroidscomple te ly abolished the production of IL- 1 b , IL-6and TNF-a induced by LPS. In the absence of LPS noproduction could be found in the supernatant (datanot show n). Similarly, the production of the eicosa-noids (PGE2 and Tx B2) w as already inhibited by allthree glucocortic osteroids at concentrations of10–11 M.

Discussion

In the present study we have show n that PMAinduc ed morphological diffe re ntiatio n of U937 cells.We also found that PMA pre -treated U937 ce lls neededLPS for the production of both cytokine s (IL-1 b , IL-6and TNF-a and e icosanoids (PGE2 and Tx B2). IL-6 w as

alre ady detec table afte r 6 h of incubation, w hereas IL-1 b could be de tec ted afte r 48 h. The production ofboth cytokine s re ached a plateau, in contras t to thetrans ient induc tion of TNF-a w hich w as max imalbetw een 6 and 24 h. Furthermore , w e show ed thatthe produc tion of e icosanoids could be inhibited bylow er conc entratio ns of glucocortic oids as compare dw ith the production of cytokine s.

Unstimulate d U937 ce lls ex hibited a smooth andround surfac e, but afte r PMA additio n they becameadherent by forming ce ll c lusters and ex te nde dpseudopodia, w hich is in agreement w ith the finding sof Hosoya & Marunouchi.20 Unlike the se changes inmorphology, the production of cytokine s and eicosa-noids could be induc ed by LPS and PMA togetheronly. How ever, Hass e t a l.23 found that U937 cellsre leased IL-1 b and TNF-a afte r TPA treatme nt w ithoutLPS. IL-1 a and IL-6 could not be detec ted in theirsystem. The basis of these diffe re nce s is unclear. A

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FIG. 3. Effects of budesonide on IL-1b , IL-6, TNF-a , PGE2 and TxB2 production by U937 cells pre-treated and stimulated withPMA and LPS respectively. Data represent mean ± s.e. mean obtained from four separate experiments. *p<0.05 compared withcontrol cultures. When no error bar is visible, error falls within the limit of the symbol.

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possible ex planatio n may be that a subclone of U937w as used diffe re nt from the one used in our ex peri-ments. How ever, Hass e t a l.23 also show ed in theirsystem that TNF-a w as ex pre ssed earlie r (alre ady afte r2–4 h) than IL-1 b (afte r 24–48 h), and this diffe rencein kine tic s is in agreement w ith our re sults.

In this inve stigation w e show ed an inc re ase in TNF-a production until 12 h of inc ubation w ith LPS. TNF-ais know n as a mac rophage activato r.12 Therefore , itcan be ex pected that afte r 12 h all the U937 cellspresent in the w ell w ould be activate d and thus theproduction of TNF-a w ill be te rminate d. Taimi e t a l.24

observed that IL-6 is produced by PMA-diffe rentiate dU937 cells afte r stimulation w ith LPS. We show ed thatthe produc tion inc reased s ignific antly afte r 6 h ofinc ubation, afte r w hich the leve ls re ached a plate au.This may indic ate that at this point the cells havestopped producing IL-6 and that the amount of IL-6 isthe ac tual amount that the cells have produced

w ithout any bre akdow n of IL-6. This plateau couldalso have been reached because the rate of break-dow n of IL-6 equalled the production of IL-6. Wedemonstrate d that the amount of IL-1 b secre ted byPMA pre-tre ated U937 gradually inc reased afte r incu-bation w ith LPS even afte r 48 and 96 h. Previousstudie s have show n that IL-1 generation is depende nton the stage of diffe rentiatio n.25 The bas is of thediffe rential and sequential ex pre ssion of the thre ecytokine s studie d he re may be caused by sequentialse cretion or gene ex press ion during PMA/LPS-induc ed diffe rentiatio n.

We obse rved that PMA-treate d U937 produced theeicosanoids PGE2 and Tx B2, but no LTB4, afte rinc ubation w ith LPS. Sinc e LTB4 w as not dete ctable,the generation of leukotriene s appears to re present aproperty of ce lls at a late r state in the diffe rentiatio nalong the monocyte /macrophage lineage . Kohlerfound that U937 sec re te d PGE2 and Tx B2, but no LTB4

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FIG. 4. Effects of fluticasone propionate on IL-1b , IL-6, TNF-a , PGE2 and TxB2 production by U937 cells pre-treated andstimulated with PMA and LPS respectively. Data represent mean ± s.e. mean obtained from four separate experiments. *p<0.05compared with control cultures. When no error bar is visible, error falls within the limit of the symbol.

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afte r incubation w ith TPA and arachidonic ac id for1–24 h.26 It is know n that human pe ritone al mac ro-phage s do gene rate lipox ygenase produc ts afte rinc ubation w ith Ca-ionophore A23187.13 How ever, inthis study we found no effect of A23187 (1 and 5 m Mfor 15 min) on U937 (data not show n).

Pre vious studie s have reported the pre senc e ofglucocorticoid receptors in U937 ce lls.27 In a re centstudy w e have demonstrate d under our cultureconditions, using the unadap te d receptor assay, spe-cific binding of 3H-labelled dex ame thasone to the sece lls . U937 cells appeared to have 17.1 +/– 5.6 3103 s ite s per cell and a Kd of 5.3 +/– 1.0 nM.28 Thesefinding s suggested that U937 ce lls are glucocortic oidrespons ive . Knudsen has demonstrate d that gluco-cortic osteroids dow nregulate the IL-1 b gene ex pres-sion by U937,29 w hich has been confirme din human alveolar mac rophages recently.22 In thisstudy w e confirme d the glucocorticoid-induced

dow nre gulation of IL-1 b in U937 cells, and additio n-ally w e showed that also the induc tion of IL-6 andTNF-a is inhibite d by diffe re nt clas ses of gluco-cortic oids. Inte re stingly, glucocorticoids w ere ableto inhibit the induc tion of both PGE2 and Tx B2 at amuch low er concentratio n (10–11M) as compare dw ith the inhibition of the cytokine s studied (10–7 M).This diffe renc e may be ex plained by diffe renc es inthe underlying w orking mechanism of glucocor-tic oids. Glucocortic oids are thought to inte rfe rew ith the production of e icosanoids via the induc tionof lipocortins . Lipocortin-1 can be induced in diffe r-entiate d U937 cells by glucocortic oids.30 How ever,the dow nregulation of cytokine ex pression by gluco-cortic oids is thought to occur at the leve l of e ithertransc ription or trans lation; glucocorticoids mayinte rac t w ith ne gative glucocortic oid response e le-ments in the gene of some cytokine s, may inte rac tw ith other transc ription factors or may decrease the

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FIG. 5. Effects of prednisolone on IL-1b , IL-6, TNF-a , PGE2 and TxB2 production by U937 cells pre-treated and stimulated withPMA and LPS respectively. Data represent mean ± s.e. mean obtained from four separate experiments. *p<0.05 compared withcontrol cultures. When no error bar is visible, error falls within the limit of the symbol.

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stability of mRNA.31 Future studie s in U937 shouldfocus on the question, w hich of these thre e mecha-nisms, or combination of mechanisms, unde rlie s theglucocorticoid e ffec ts on the cytokine ex press ionobserved he re .

In conclusion, the results of this study show thatU937 cells c an be used as a model to study theproduction of se lec ted inf lammatory mediators thatare be lie ved to be important in the pathogenesis ofasthma. Additio nally, U937 ce lls can be used to studythe effe cts of glucocorticoids on these mediators. Theobserved diffe re nce s in the kine tic s of the productionof eicosanoids and cytokine s stre ss the importanc e oftime course ex periments in studie s on the e ffec ts ofdrugs on mononuc lear phagocytes .

ACKNOWLEDGEMENTS. This investigation w as supported by the Ne the r-lands Asthma Foundation (Grant 93.71)

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Received 16 September 1999;accepted 8 October 1999

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