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Time Dependent Production of Cytokines And 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 cytokines 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 three 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 macrophages phagocytose Some studies have investigated the regulation of inhaled particles and antigens, but as inflammatory cytokines and eicosanoids in macrophage 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 used 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 U937 can 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 leucocytes in the airway wall. 6 of proliferation 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 clusters and extend pseudopodia upon were added to the cell cultures 24h prior to phorbol ester 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 systems, USA), 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, Belgium) levels were measured using mediators was investigated. We used budesonide, enzyme- linked immunosorbent assay (ELISA). fluticasone propionate and prednisolone, because 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 with slight modifications. 21 SepPak Materials and methods C18 cartridges (Waters Ass., USA) were activated with Cell culture 10ml of methanol pre-washed with 10ml distilled water. Two hundred and fifty m l of supernatant was U937 cells (American Type Culture Collection USA, applied to the columns and rinsed with 2.5ml batch 1593.2) were cultured in RPMI 1640 medium methanol, dried with 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; Gibco, UK) in tissue culture flasks nostics, USA, tritiated antigens from Amersham, UK (Costar, UK) at 37°C humidified atmosphere with 5% and standards from Sigma, USA). LTB 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. Cell viability was assessed by 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 culture 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 cytokine 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, U937 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 PMA they became adherent by Incubation with steroids forming cell clusters, extended pseudopodia and, as To ensure that the U937 cell line in our investigations shown in Fig. 1, released IL-1 b , IL-6 and TNF- a in could be used to evaluate glucocorticoid responsive- response to LPS stimulation. However, LPS did not ness, we performed the experiment with steroids. We induce the PMA-treated cells to produce INF- g or IL-10 used three corticosteroids, namely budesonide (data not shown). After incubation with LPS the (Sigma, USA), fluticasone 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, NL). 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.
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