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

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 cytokines, 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 24 h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 microg ml(-1)) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10(-11), 10(-9) and 10(-7) M) for 96 h. The production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-alpha) reached its maximum between 6 and 12 h. Interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely 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.


Introduction
Inflammation of the airw ays unde rlie s a major part of the clinic al symptoms in asthma and chronic obstructive p ulmonary dise ase s (COPD). A number of studie s support the involve me nt of mononuclear phagocyte s in asthma. [1][2][3] Alve olar mac rophages phagoc ytose inhale d partic le s and antige ns, but as inflammatory ce lls the y are know n to produc e pote nt inflammatory me diato rs, inc luding prote ins and lipids me diato rs, w hich are able to influe nce other c ell types.
Several feature s of as thma can be mimicke d in vitro and in v ivo by these se cre tory p roduc ts . 4 We and othe rs have pre viously show n that prostaglandin D 2 , prostaglandin F 2a and a stable analog ue of thromboxane A 2 all constric t human airw ay muscle . 5 Le ukotrie ne B 4 (LTB 4 ) inc re ases vascular pe rmeability and e nhance s adhere nc e and migratio n of granuloc yte s. Inhalation of LTB 4 re sults in p eriphe ral ne utrop e nia and the accumulation of le ucocyte s in the airw ay w all. 6 Le ukotrie ne C 4 (LTC 4 ) induc es bronchoc ons tric tion, re duces the clearanc e of muc us and inc re ases vasc ular permeability. 7 Cytokine s, such as IL-1b , IL-6 and TNF-a , play an important role in immune re ac tions and re gulate the grow th and state of activ ity of many ce lls in inflammatio n inc luding the airw ay inflammation pre sent in as thma. 8 Both e ic osanoids and cytokine s can also be re leased from inflammatory c ells in c ulture . Some studie s have inve stigate d the re gulation of cytokine s and eic osanoids in macrophage c ell line s or macrophage s stimulate d w ith vario us inflammatory agents. [9][10][11][12][13] The human monoblastoid U937 tumour ce ll line is w ide ly use d as a model for the maturatio n of monocytic c ells. 14 U937 is an e stablishe d human monoblast c ell line de rive d from the ple ural ex udate of a patie nt w ith diffuse histioc ytic lymphoma. Maturation of U937 c an be induc ed by inc ubatio n w ith diffe re nt agents, inc luding re tino ic acid, 15 vitamin D de rivative s, 16 cytokine s 17 and phorbol este rs. 18 Phorbol e ste r-induc ed maturatio n re sults in c essatio n of prolife ratio n and signific ant alte rations in the morphology of the c ells. Unde r standard c onditio ns, U937 c ells grow in suspe nsion and ex hibit a smooth and round surfac e, but the y become adhe re nt, start to form cell cluste rs and ex te nd pse udopodia up on phorbol e ste r tre atme nt. [18][19][20] The morphologic al change s observe d during this maturatio n proce ss suggest func tional change s of the U937 c ells.
We studie d the kine tic s of the produc tion of se veral cytokine s, IL-1b , IL-6, IL-10, IFN-g and TNF-a and eic osanoids PGE 2 , Tx B 2 and LTB 4 in U937 to clarify w hether mediators are se que ntially re lease d by activate d mononuc le ar ce lls. These me diato rs w e re studie d, bec ause the y are thought to be of importanc e in the pathogene sis of the airw ay inflammatio n in lung diseases. Furthermore , the e ffec tive ne ss of glucoc orticoste roids to suppre ss the formation of inflammatory me diato rs w as inve stigate d. We used budesonide , flutic asone propionate and pre dnisolone , be cause the y are the most imp ortant drugs c urre ntly use d in the tre atment of asthma.
Maturatio n of U937 c ells w as induc e d w ith 250 ng ml -1 PMA (Sigma, USA). Ce lls w e re c ulture d for 24 h w ith PMA and furthe r grow n for 48 h in fre sh comple te medium. Ce ll viability w as assessed by trypan-blue ex clusion in the non-adhere nt untre ate d U937 as w e ll as in the PMA tre ate d U937 ce lls. The viability of the ce lls w as > 95%.
The adhe re nt c ells w ere scrap ed from the tis sue culture flasks w ith a rubber police man and aliquots of 0.5 3 10 6 c ells in 1 ml w ere put in 24 w ells plastic multiw e ll culture plate s (Cos tar, UK) and inc ubate d w ith 1 m g ml -1 or 5 m g ml -1 or w ithout LPS from Esc h e rich ia c o li (Serotype 0111:B4; Sigma, USA). The ce lls w ere inc ubate d for 1, 3, 6, 8, 10, 12, 24, 48 and 96 h in se parate w e lls at 37°C. Ce ll-fre e supe rnatants w ere c ollec te d and store d at -80°C until measureme nts of lipid mediato rs and c ytokine s.

Incubation with steroids
To ensure that the U937 c ell line in our inve stigatio ns could be used to e valuate glucocortic oid re sponsivene ss, w e performed the ex perime nt w ith ste roids. We used thre e cortic oste roids , name ly budesonide (Sigma, USA), flutic as one propionate (gift of Glax o Wellcome , UK) and pre dnisolone sodium phosphate (Ge nfarm a, NL). The drugs w e re used in the final conc entratio ns: 10 -11 , 10 -9 and 10 -7 M.
The ce lls w e re pre -tre ate d w ith PMA, the drugs w ere adde d to the c ell c ulture s 24 h prior to stimulatio n w ith LPS. From this set point [t = 0] the ce lls w ere inc ubate d for 1, 3, 6, 8, 10, 12, 24, 48 and 96 h in the p re se nce or abse nce of the drug and/or LPS. The ce ll-fre e supe rnatants w e re store d at -80°C until measure me nts .
Ce ll viability w as not alte re d afte r drug tre atme nt.

Measurements of eicosanoids
The supe rnatants w ere proce sse d as de scribe d in detail pre viously w ith slight modific atio ns. 21

LPS-induced cytokine production after pre-treatment with PMA
In standard me dium w ithout PMA, U937 c ells had a smooth and round surfac e and did not produce the cytokine s and eic osanoids studie d (data not show n). Afte r addition of PMA the y became adhe re nt by forming ce ll cluste rs, ex te nde d pseudopodia and, as show n in Fig. 1, re lease d IL-1b , IL-6 and TNF-a in re sponse to LPS stimulatio n. How ever, LPS did not induc e the PMA-tre ate d cells to produce INF-g or IL-10 (data not show n). Afte r inc ubatio n w ith LPS the amounts of IL-1b inc re ased signific antly at 48 and 96 h, w hich se emed to be concentration depende nt. IL-6 leve ls alre ady inc re ased signific antly afte r 6 h, afte r w hich the levels re ached a plate au. TNF-a production show ed a diffe re nt patte rn compare d w ith IL-1b and IL-6; its levels inc re as ed afte r 6 h of inc ubation w ith LPS, but starte d to de cre ase afte r 24 h.

LPS-induced eicosanoid production after pre-treatment with PMA
We dete rmine d the effe cts of LPS on PGE 2 , Tx B 2 and LTB 4 produc tion by U937 cells w hich w e re pre -tre ate d w ith PMA. Without PMA, U937 ce lls did not produc e any of the e icosanoids studie d (data not show n). The ce lls primed w ith PMA re lease d PGE 2 and Tx B 2 , but not LTB 4 , in re sponse to LPS (Fig. 2). This e ffec t w as timere late d. The le ve ls of Tx B 2 alre ady re ached a plate au phase afte r 6 h, w hilst the leve ls of PGE 2 inc re ase d slow ly w ith time.
Effect of glucocorticosteroids on the LPS-induced production of inflammatory mediators by PMA pre-treated U937 cells Glucocortic oste roids dow nre gulate the cytokine ex pre ssion of human alve olar macrophage s. 22 To inve stigate w he the r the U937 ce lls c ould be used as a model to study gluc oc ortic oid re sponsive ne ss in monocyte s/macrophage s, w e inc ubate d the cells w ith budesonide , flutic asone propionate or prednisolone . w ith bude sonide , flutic asone proprionate and prednisolone re spec tive ly. The production of IL-1b and IL-6 w as dose-depe nde ntly inhibite d by all thre e ste roids. At 10 -7 M conc entratio n, all thre e ste roids comple te ly abolished the produc tion of IL-1b , IL-6 and TNF-a induc e d by LPS. In the abse nce of LPS no production could be found in the supernatant (data not show n). Similarly, the produc tion of the eic osanoids (PGE 2 and Tx B 2 ) w as alre ady inhibite d by all thre e gluc oc ortic oste roids at c onc entrations of 10 -11 M.

Discussion
In the pre se nt study w e have show n that PMA induc ed morphological diffe re ntiatio n of U937 cells.
We also found that PMA pre -tre ate d U937 ce lls ne e de d LPS for the production of both c ytokine s (IL-1b , IL-6 and TNF-a and e icosanoids (PGE 2 and Tx B 2 ). IL-6 w as alre ady dete c table afte r 6 h of inc ubation, w here as IL-1b could be de te c te d afte r 48 h. The production of both c ytokine s re ached a plate au, in contras t to the trans ie nt induc tion of TNF-a w hich w as max imal betw e en 6 and 24 h. Furthermore , w e show ed that the produc tion of e icosanoids c ould be inhibite d by low er c onc entratio ns of glucocortic oids as c ompare d w ith the produc tion of c ytokine s. Unstimulate d U937 ce lls ex hibite d a smooth and round surfac e, but afte r PMA additio n the y be came adhere nt by forming ce ll cluste rs and ex te nde d pse udopodia, w hich is in agre e ment w ith the finding s of Hosoya & Maruno uchi. 20 Unlike the se change s in morphology, the produc tion of c ytokine s and eic osanoids c ould be induc ed by LPS and PMA together only. How ever, Hass e t a l. 23 found that U937 c ells re lease d IL-1b and TNF-a afte r TPA tre atme nt w ithout LPS. IL-1a and IL-6 c ould not be dete c te d in their syste m. The basis of the se diffe re nce s is unc lear. A I. M. Ga rr e lds e t al.

3 2
Mediators of Inflammation · Vol 8 · 1999 FIG. 3. Effects of budesonide on IL-1b , IL-6, TNF-a , PGE 2 and TxB 2 production by U937 cells pre-treated and stimulated with PMA and LPS respectively. Data represent mean ± s.e. mean obtained from four separate experiments. *p<0.05 compared with control cultures. When no error bar is visible, error falls within the limit of the symbol.
possible ex planatio n may be that a subclone of U937 w as use d diffe re nt from the one used in our ex perime nts. How ever, Hass e t a l. 23 also show ed in their syste m that TNF-a w as ex pre sse d earlie r (alre ady afte r 2-4 h) than IL-1b (afte r 24-48 h), and this diffe re nc e in kine tic s is in agre e ment w ith our re sults. In this inve stigatio n w e show ed an inc re as e in TNFa production until 12 h of inc ubatio n w ith LPS. TNF-a is know n as a mac rophage activato r. 12 There fore , it can be ex p ecte d that afte r 12 h all the U937 c ells pre sent in the w ell w ould be activate d and thus the production of TNF-a w ill be te rminate d. Taimi e t a l. 24 observe d that IL-6 is produc e d by PMA-diffe re ntiate d U937 cells afte r stimulatio n w ith LPS. We show ed that the produc tion inc re ase d signific antly afte r 6 h of inc ubatio n, afte r w hich the le vels re ache d a plate au. This may indic ate that at this point the c ells have stoppe d producing IL-6 and that the amount of IL-6 is the ac tual amount that the c ells have produc e d w ithout any bre akdow n of IL-6. This plate au c ould also have bee n re ached bec ause the rate of bre akdow n of IL-6 e qualle d the production of IL-6. We demonstrate d that the amount of IL-1b secre te d by PMA pre -tre ate d U937 gradually inc re ase d afte r inc ubation w ith LPS e ve n afte r 48 and 96 h. Pre vious studie s have show n that IL-1 ge ne ratio n is depe nde nt on the stage of diffe re ntiatio n. 25 The bas is of the diffe re ntial and sequential ex pre ssion of the thre e cytokine s studie d he re may be c ause d by se que ntial se cre tion or ge ne ex pre ssion during PMA/LPSinduc ed diffe re ntiatio n.
We obse rve d that PMA-tre ate d U937 produc e d the eic osanoids PGE 2 and Tx B 2 , but no LTB 4 , afte r inc ubatio n w ith LPS. Sinc e LTB 4 w as not dete ctable, the gene ratio n of leukotrie ne s appe ars to re pre se nt a property of ce lls at a late r state in the diffe re ntiatio n along the monocyte /macrophage line age . Köhler found that U937 sec re te d PGE 2 and Tx B 2 , but no LTB 4 afte r inc ubatio n w ith TPA and arachidonic acid for 1-24 h. 26 It is know n that human pe ritone al mac rophage s do gene rate lipox ygenase produc ts afte r inc ubatio n w ith Ca-ionop hore A23187. 13 How ever, in this study w e found no effect of A23187 (1 and 5 m M for 15 min) on U937 (data not show n).
Pre vious studie s have re porte d the pre senc e of gluc ocortic oid re ce ptors in U937 ce lls. 27 In a re ce nt study w e have demonstrate d unde r our culture conditio ns, using the unadap te d re c eptor assay, specific binding of 3 H-labelled dex ame thasone to the se ce lls. U937 c ells appe are d to have 17.1 + /-5.6 3 10 3 site s per c ell and a K d of 5.3 + /-1.0 nM. 28 These finding s suggeste d that U937 ce lls are gluc oc ortic oid re spons ive . Knuds en has de monstrate d that glucocortic oste roids dow nre gulate the IL-1b ge ne ex pre ssion by U937, 29 w hich has been confirme d in human alve olar mac rophages re c ently. 22 In this study w e confirme d the gluc oc ortic oid-induc e d dow nre gulatio n of IL-1b in U937 cells, and additio nally w e show e d that also the induc tion of IL-6 and TNF-a is inhibite d by diffe re nt classes of glucocortic oids. Inte re stingly, glucocortic oids w ere able to inhibit the induc tion of both PGE 2 and Tx B 2 at a much low er conc entratio n (10 -11 M) as c ompare d w ith the inhibitio n of the cytokine s studie d (10 -7 M). This diffe re nc e may be ex plaine d by diffe re nc es in the unde rlying w orking mechanism of glucocortic oids. Glucoc ortic oids are thought to inte rfe re w ith the produc tion of eic osanoids via the induc tion of lipocortins . Lipocortin-1 can be induc e d in diffe rentiate d U937 c ells by glucoc ortic oids. 30 How ever, the dow nre gulatio n of c ytokine ex pre ssion by gluc ocortic oids is thought to oc cur at the le vel of either trans c ription or trans latio n; glucocortic oids may inte rac t w ith ne gative gluc oc ortic oid re sponse eleme nts in the gene of some cytokine s, may inte rac t w ith other trans c ription fac tors or may de cre ase the I. M. Ga rr e lds e t al. stability of mRNA. 31 Future studie s in U937 should foc us on the que stion, w hich of these thre e mechanisms, or c ombination of mechanisms, unde rlie s the gluc ocortic oid e ffec ts on the cytokine ex pre ssion observe d he re .
In conclusion, the re sults of this study show that U937 c ells c an be used as a model to study the production of selec te d inflammatory mediato rs that are be lie ved to be imp ortant in the pathogene sis of asthma. Additio nally, U937 ce lls can be used to study the effe cts of glucocortic oids on these mediato rs. The observe d diffe re nce s in the kine tic s of the produc tion of eicosanoids and c ytokine s stre ss the importanc e of time c ourse ex perime nts in studie s on the e ffec ts of drugs on mononuc lear phagoc yte s.