Research Paper

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

IN the present study the human monoblast cell line Time dependent production of U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The and eicosanoids by kinetics of the production of eicosanoids and cyto- kines, which are thought to play a role in the human monocytic leukaemia U937 pathogenesis of asthma, were studied. In addition, the cells; effects of glucocorticosteroids effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol- 1,2,CA 2 12 myristate acetate (PMA) for 24h, U937 cells were Ingrid M. Garrelds , Peter Th. W. van Hal , cultured in the absence or presence of lipopoly- Raquel C. Haakmat 1, Henk C. Hoogsteden 2, saccharide (LPS: 1 and 5 m g m l–1) and glucocorticoste- Pramod R. Saxena 1 and Frederik J. Zijlstra 1 roids (budesonide, fluticasone propionate and pre- dnisolone: 10 –11, 10–9 and 10–7 M) for 96h. The production of interleukin- 1 b (IL-1b ), interleukin-6 1Institute of Pharmacology, Faculty of Medicine, (IL-6), prostaglandin E 2 (PGE2) and thr omboxane B 2 Erasmus University Rotterdam, PO Box 1738, 3000 (Tx B2) gradually increased in time after stim ulation with LPS, whereas the transient production of tumor DR Rotterdam and 2Department of Pulmonary necrosis factor alpha (TNF- a ) reached its maximum Medicine, University Hospital Rotterdam-Dijkzigt,The betwe en 6 and 12h. Inte rferon-gamma (IFN- g ), inter- Netherlands leukin-10 (IL-10) and leukotriene B 4 (LTB4) were not detectable. All th ree glucocorticosteroids (budeso- nide, fluticasone propionate and prednisolone) com- CA pletely inhibited the production of both eicosanoids Corresponding Author and cytokines. The production of eicosanoids was Institute of Pharmacology, Erasmus University more sensitive to these glucocorticoids than the Rotterdam, PO Box 1738, 3000 DR Rotterdam, The production of cytokines. The observed differences in Netherlands the kinetics of the production of eicosanoids and Tel: (+31) 10 4087534 cytokines stress the importance of tim e course Fax: (+31) 10 4089458 experiments in studies on the effect of drugs on Email: [email protected] mononuclear cells.

Key words: Glucocorticosteroids, Cytokines, Arachidonic acid metabolites, U937

Introduction permeability. 7 Cytokines, such as IL-1 b , IL-6 and TNF- a , play an important role in immune reactions and Inflammation of the airw ays underlies a major part of regulate the growth and state of activity of many cells the clinical symptoms in asthma and chronic obstruc- in inflammation including the airw ay inflammation tive pulmonary diseases (COPD). A number of studies present in asthma. 8 Both eicosanoids and cytokines support the involvement of mononuclear phagocytes can also be released from inflammatory cells in culture. in asthma.1–3 Alveolar phagocytose Some studies have investigated th e regulation of inhaled particles and antigens, but as inflammatory cytokines and eicosanoids in cell lines or cells they are known to produce potent inflammatory macrophages stimulated with various inflammatory mediators, including proteins and lipids mediators, agents.9–13 which are able to influence other cell types. The human monoblastoid U937 tumour cell line is Several features of asthma can be mimicked in vitro widely us ed as a model for the maturation of and in vivo by these secretory products. 4 We and monocytic cells. 14 U937 is an established human others have previously shown that prostaglandin D 2, monoblast cell line derived from the pleural exudate prostaglandin F 2a and a stable analogue of thrombox- of a patient with diffuse histiocytic lymphoma. 5 ane A2 all constrict human airw ay muscle. Leukotriene Maturation of U 937 c an be induced by incubation 15 B4 (LTB4) increases vascular permeability and enhan- with different agents, including retinoic acid, vita- ces adherence and migration of granulocytes. Inhala- min D derivatives, 16 cytokines17 and phorbol esters. 18 tion of LTB 4 results in peripheral neutropenia and the Phorbol ester-induced maturation results in cessation accumulation of le ucocytes in the airway wall. 6 of p roliferation and significant alterations in the

Leukotriene C 4 (LTC4) induces bronchoconstriction, morphology of the cells. Under standard conditions, reduces the clearance of mucus and increases vascular 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 I. M. Garrelds et al. and round surface, but they become adherent, start to The cells were pre-treated with PMA, the drugs form cell c lusters and extend pseudopodia upon were added to the cell c ultures 24h p rior to phorbol e ster treatment. 18–20 The morphological stimulation with LPS. From this set point [t = 0] the changes observed during this maturation process cells were incubated for 1, 3, 6, 8, 10, 12, 24, 48 and suggest functional changes of the U937 cells. 96h in the presence or absence of the drug and/or We studied the kinetics of the production of several LPS. The cell-free supernatants were stored at –80°C cytokines, IL-1 b , IL-6, IL -10, IFN - g and TNF-a and until measurements. eicosanoids PGE 2, TxB2 and LTB4 in U937 to clarify Cell viability was not altered after drug treatment. whether mediators are sequentially released by acti- vated mononuclear cells. These mediators were stud- Measurements of cytokines ied, because they are thought to be of importance in the pathogenesis of the airw ay inflammation in lung IL-1b (R&D s ystems, U SA), IL -6 (CLB, NL), IL -10 diseases. Furthermore, the effectiveness of glucocorti- (Pharming, USA), TNF- a (Pharming, USA) and IFN- g costeroids to suppress the formation of inflammatory (Medgenix, Be lgium) le vels were measured us ing mediators was investigated. W e used b udesonide, enzyme- linked immunosorbent assay (ELISA). fluticasone propionate and prednisolone, b ecause they are the most important drugs currently used in Measurements of eicosanoids the treatment of asthma. The supernatants were processed as described in detail previously w ith slight modifications. 21 SepPak Materials and methods C18 cartridges (Waters Ass., USA) were activated with 10ml o f methanol pre-washed with 10ml dis tilled water. Two hundred and fifty m l of s upernatant was U937 cells (American Type Culture Collection USA, applied to the columns and rinsed w ith 2.5ml batch 1593.2) were cultured in RPMI 1640 medium methanol, drie d w ith a Savant Speed-Vac concen- (Gibco, UK) supplemented with penicillin (ICN, USA; trator, and dissolved in 1ml radioimmunoassay (RIA) 100 m g ml–1), streptomycin (ICN, USA; 100 m g ml–1), buffer. PGE and TxB production were measured by glutamax-I (Gibco, UK; 10mM) and 10% foetal bovine 2 2 RIA (antibodies were obtained from PerSeptive Diag- serum (FBS; Gib co, U K) in tissue culture flasks nostics, USA, tritiate d antigens from Amersham, UK (Costar, UK) at 37°C humidified atmosphere with 5% and standards from Sigma, U SA). L TB levels were carbon dioxide. 4 measured by enzyme immuno assay (EIA) kits, which Maturation of U937 cells was induced with 250ng were obtained from Amersham, USA. ml–1 PMA (Sigma, USA). Cells were cultured for 24h with PMA and further grown for 48h in fresh complete medium. C ell v iability was assessed b y Statistical analysis trypan-blue exclusion in the non-adherent untreated Each drug was studied in four separate experiments U937 as well as in the PMA treated U937 cells. The and the values are expressed as mean ± s.e. mean. viability of the cells was >95%. Statistical comparisons between control and drug- The adherent cells were scraped from the tissue treated cell cultures were made by ANOVA follow ed by culture flasks with a rubber policeman and aliquots of paire d t-test. A p<0.05 was considered significant. 0.5 3 106 cells in 1ml were put in 24 wells plastic multiwell c ulture plates (Costar, UK) and incubated w ith 1 m g ml–1 or 5 m g ml–1 or without LPS from Results Escherichia coli (Serotype 0111:B4; Sigma, USA). The LPS-induced production after cells were incubated for 1, 3, 6, 8, 10, 12, 24, 48 and pre-treatment with PMA 96h in separate wells at 37°C . Cell-free supernatants were collected and stored at –80°C until measure- In standard medium without PMA, U 937 cells had a ments of lipid mediators and cytokines. smooth and round surface and did not produce the cytokines and eicosanoids studied (data not shown). After addition of PM A they b ecame adherent by Incubation with steroids forming cell c lusters, extended pseudopodia and, as To ensure that the U937 cell line in our investigations shown in Fig. 1, re leased IL-1 b , IL-6 and TNF- a in could be used to evaluate glucocorticoid responsive- response to LPS stimulation. However, L PS did not ness, we performed the experiment with steroids. We induce the PMA-treated cells to produce INF- g or IL-10 used thre e corticosteroids, name ly b udesonide (data not shown). Afte r incubation with LPS the (Sigma, U SA), f luticasone propionate (gift of Glaxo amounts of IL-1 b increased significantly at 48 and 96h, Wellcome, UK) and prednisolone sodium phosphate which seemed to be concentration dependent. IL-6 (Genfarma, N L). The drugs were used in the final levels already increased significantly after 6h, after concentrations: 10 –11, 10–9 and 10–7 M. which the levels reached a plateau. TNF- a production

230 Mediators of Inflammation · Vol 8 · 1999 Inflam matory mediators by U937

phase after 6h, whilst the levels of PGE 2 increased slowly with time.

Effect of glucocorticosteroids on the LPS-induced production of inflammatory mediators by PMA pre-treated U937 cells Glucocorticosteroids downregulate the cytokine expression of human alveolar macrophages. 22 To investigate whether the U937 cells could be used as a model to study glucocorticoid responsiveness in /macrophages, w e incubated the cells with budesonide, f luticasone propionate or pre- dnisolone. Figures 3, 4 and 5 show the data obtained

FIG. 1. Kinetics of IL-1 b (top panel), IL-6 (middle panel ) and TNF-a (lower panel ) production in PMA-treated U937 cells which were stimulated with LPS. Data represent mean ± s.e. mean obtained from four separate experiments. * p<0.05 versus response without LPS. When no error bar is visible, error falls within the limit of the symbol. showed a different pattern compared with IL-1 b and IL- 6; its levels increased after 6h of incubation with LPS, but started to decrease after 24h.

LPS-induced eicosanoid production after pre-treatment with PMA

We determined the effects of LPS on PGE 2, TxB2 and LTB4 production by U937 cells which were pre-treated with PMA. Without PMA, U937 cells did not produce FIG. 2. Kinetics of LTB 4 (upper panel ), PGE2 (middle panel ) any of the eicosanoids studied (data not shown). The and TxB2 (lower panel ) production in PMA-treated U937 cells cells primed with PMA released PGE and TxB , but not which were stimulated with LPS. Data represent mean ± s.e. 2 2 mean obtained from four separate experiments. * p<0.05 LTB4, in response to LPS (Fig. 2). This effect was time- versus response without LPS. When no error bar is visible, related. The levels of TxB 2 already reached a plateau error falls within the limit of the symbol.

Mediators of Inflammation · Vol 8 · 1999 231 I. M. Garr elds et al. with budesonide, fluticasone proprionate and pre- already detectable after 6h of incubation, whereas IL- dnisolone respectively. The production of IL-1 b and 1b could b e detected after 48h. The production of IL-6 w as dose-dependently inhibited b y all th ree both cytokines reached a plateau, in contrast to the steroids. At 10 –7 M concentration, all three steroids transient induction of T NF- a which w as maximal completely ab olished the production of IL- 1 b , IL-6 between 6 and 24h. Furthermore, we showed that and TNF-a induced by LPS. In the absence of LPS no the production of eicosanoids could be inhibited by production could b e found in the supernatant (data lower concentrations of glucocorticoids as compared not shown). Similarly, the production of the eicosa- with the production of cytokines. noids (PGE2 and TxB2) was already inhibited by all Unstimulated U937 cells exhibited a smooth and three glucocorticosteroids at concentrations of round surface, but after PMA addition they became 10–11 M. adherent by fo rming cell c lusters and extended pseudopodia, which is in agreement with the findings 20 Discussion of H osoya & Marunouchi. Unlike these changes in morphology, the production of cytokines and eicosa- In the present study we have shown that PMA noids could b e induced by LPS and PMA together induced morphological differentiation of U937 cells. only. H owever, H ass et a l.23 found that U937 cells We also found that PMA pre-treated U937 cells needed released IL-1 b and TNF-a after TPA treatment without LPS for the production of both cytokines (IL-1 b , IL-6 LPS. IL-1a and IL-6 could no t be detected in their and TNF-a and eicosanoids (PGE 2 and TxB2). IL-6 was system. The basis of these differences is unclear. A

FIG. 3. Effects of budesonide on IL-1 b , IL-6, TNF- a , PGE2 and TxB2 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.

232 Mediators of Inflammation · Vol 8 · 1999 Inflam matory mediators by U937

FIG. 4. Effects of fluticasone propionate on IL-1 b , IL-6, TNF- a , PGE2 and TxB2 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 explanation may be that a subclone of U937 without any breakdown of IL-6. This plateau could was used different from the one used in our experi- also have been reached because the rate of break- ments. However, H ass et a l.23 also showed in their down of IL-6 equalled the production of IL-6. We system that TNF- a was expressed earlier (already after demonstrated that the amount of IL-1 b secreted by 2–4h) than IL-1 b (after 24–48h), and this difference PMA pre-treated U937 gradually increased after incu- in kinetics is in agreement with our results. bation with LPS even after 48 and 96h. Pre vious In this investigation we showed an increase in TNF- studies have shown that IL-1 generation is dependent a production until 12h of incubation with LPS. TNF- a on the stage of differentiation. 25 The basis of th e is known as a macrophage activator. 12 Therefore, it differential and sequential expression of the three can be expected that after 12h all the U937 c ells cytokines studied here may be caused by sequential present in the well would be activated and thus the secretion or gene expression during PMA/LPS- production of TNF- a will be terminated. Taimi et a l.24 induced differentiation. observed that IL-6 is produced by PMA-differentiated We observed that PMA-treated U937 produced the

U937 cells after stimulation with LPS. We showed that eicosanoids PGE 2 and TxB2, b ut no LTB 4, after the production increased s ignificantly after 6h of incubation with LPS. Since LTB 4 was not detectable, incubation, after which the levels reached a plateau. the generation of leukotrienes appears to represent a This may indicate that at this point the cells have property of cells at a later state in the differentiation stopped producing IL-6 and that the amount of IL-6 is along the /macrophage lineage. K¨ ohler the actual amount that the cells have produced found that U937 secreted PGE 2 and TxB2, but no LTB 4

Mediators of Inflammation · Vol 8 · 1999 233 I. M. Garr elds et al. after incubation with TPA and arachidonic acid for downregulation of IL-1 b in U937 cells, and addition- 1–24 h.26 It is known that human peritoneal macro- ally we showed that also the induction of IL -6 and phages do generate lipoxygenase products after TNF-a is inhibited b y diffe rent classes of gluc o- incubation with Ca-ionophore A23187. 13 However, in corticoids. Inte restingly, g lucocorticoids were able this study we found no effect of A23187 (1 and 5 m M to inhibit the induction of b oth PGE 2 and TxB2 at a for 15min) on U937 (data not shown). much lo wer concentration (10 –11M) as compared Previous studies have reported the presence of with the inhibition of the cytokines studied (10 –7 M). glucocorticoid receptors in U937 cells. 27 In a recent This difference may be explained by differences in study we have demonstrated unde r our culture the underlying working mechanism of g lucocor- conditions, using the unadapted receptor assay, spe- ticoids. Gluc ocorticoids are thought to interfere cific binding of 3H-labelled dexamethasone to these with the production of eicosanoids via the induction cells. U937 cells appeared to have 17.1 +/– 5.6 3 of lip ocortins. Lipocortin-1 can be induced in differ- 3 28 30 10 sites per cell and a K d of 5.3 +/– 1.0nM. These entiated U937 c ells by glucocorticoids. How ever, findings suggested that U937 cells are glucocorticoid the downregulation of cytokine expression by gluco- responsive. Knudsen has demonstrated that gluco- corticoids is thought to occur at the level of e ither corticosteroids downregulate the IL-1 b gene expres- transcription or translation; glucocorticoids may sion by U 937, 29 which h as been confirmed interact with negative glucocorticoid response ele- in human alveolar macrophages recently. 22 In this ments in the gene of s ome cytokines, may interact study we confirmed the glucocorticoid-induced with other transcription factors or may decrease the

FIG. 5. Effects of prednisolone on IL-1 b , IL-6, TNF- a , PGE2 and TxB2 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.

234 Mediators of Inflammation · Vol 8 · 1999 Inflam matory mediators by U937

31 14.Sunds tr¨om C, Nilsson K. Establishment and characterization of a human stability of mRN A. Future studies in U937 should histiocytic lymphoma cell line (U-937). Int J Ca ncer 1976; 17: focus on the question, which of these three mecha- 565–577. 15.Ols son IL, Bre itman TR. Induction of diffe re ntiation of the human nisms, or combination of mechanisms, underlies the histiocytic lymphoma cell line U937 b y retinoic ac id and cyclic glucocorticoid effects on the cytokine expression adenosine 3 9 :59 -monophosphate-inducing agents. Ca ncer Res 1982; 42: 3924–3927. observed here. 16.Dodd RC, Cohe n MS, Ne wman, SL, Gray TK. V itamin D metabolites In conclusion, the results of this study show that change the phenotype of monoblastic U 937 cells. Proc Natl Acad Sci USA 1983; 80: 7538–7541. U937 c ells can be used as a model to study the 17.H arris PE, Ralph P, Litcofsky P , M oore MAS. Dis tinct ac tivities of production of selected inflammatory mediators that interferon- g , lymphokine and cytokine differentiation-inducing factors acting on the human monoblastic leukae mia cell line. Cance r Res 1985; are believed to be important in the pathogenesis of 45: 9–13. asthma. Additionally, U937 cells can be used to study 18.Wie derholt MD, Anderson KM , H arris JE. Labe lling of lipids and phospholipids w ith [ 3H]arachidonic acid and the biosynthesis of the effects of glucocorticoids on these mediators. The eicosanoids in U937 ce lls differentiated by phorbol e ster. Bio chem observed differences in the kinetics of the production Biophys Acta 1988; 959: 296–304. 19.H ass R, Barthe ls H, Topley N, Hadam M, Kohle r L, Goppe lt-Struebe M, of eicosanoids and cytokines stress the importance of Resch K. Regulation of TNF- a , IL-1b and IL-6 synthesis in differentiating time course experiments in studies on the effects of human monoblastoid le ukaemic U937 cells. 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