Combinations of ERK and p38 MAPK Inhibitors Ablate Tumor Necrosis Factor-α (TNF-α) mRNA Induction
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m005486200
ISSN1083-351X
AutoresKarine Rutault, Catherine A. Hazzalin, Louis C. Mahadevan,
Tópico(s)Immune Response and Inflammation
ResumoTumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-α transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-α mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-α mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 μm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-α transcripts but does not affect degradation of c-juntranscripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-α mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy. Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-α transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-α mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-α mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 μm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-α transcripts but does not affect degradation of c-juntranscripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-α mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy. interleukin tumor necrosis factor α lipopolysaccharide LPS-binding protein mitogen-activated protein MAP kinase extracellular signal-regulated kinase mitogen-activated protein ERK kinase 12-O-tetradecanoylphorbol-13-acetate 5,6-dichlorobenzimidazole riboside fetal calf serum glyceraldehyde-3-phosphate dehydrogenase enzyme-linked immunosorbent assay Monocytes and macrophages play a pivotal role in inflammation and immune regulation. Upon activation, monocytes produce and release many inflammatory mediators such as IL-1,1 IL-6, IL-8, TNF-α, or arachidonic acid metabolites, as well as the anti-inflammatory mediators IL-10, soluble TNF receptor, and IL-1 receptor antagonist. Among the different products released, TNF-α is thought to be one of the most important mediators of inflammatory disease (1Beutler B. Krochin N. Milsark I.W. Luedke C. Cerami A. Science. 1986; 232: 977-980Crossref PubMed Scopus (1014) Google Scholar), and therefore the understanding of molecular mechanisms of TNF-α gene induction is of considerable medical interest. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is a very potent inducer of TNF-α production and release in human monocytes. Two proteins, CD14 and LPS-binding protein (LBP), are implicated in mediating the cellular response to LPS (2Wright S.D. J. Immunol. 1995; 155: 6-8PubMed Google Scholar, 3Haziot A. Ferrero E. Kontgen F. Hijiya N. Yamamoto S. Silver J. Stewart C.L. Goyert S.M. Immunity. 1996; 4: 407-414Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 4Wurfel M.M. Kunitake S.T. Lichenstein H. Kane J.P. Wright S.D. J. Exp. 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Recent work indicates that Toll-like receptors, a family of human receptors related to Drosophila Toll, mediate LPS-induced signal transduction and that this response is dependent on LBP and enhanced by CD14 (6Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1100) Google Scholar, 7Kirschning C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 8Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar, 9Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar). The involvement of all three mitogen-activated protein (MAP) kinase subtypes, c-Jun N-terminal kinase (10Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar, 11Hambleton J. Weinstein S.L. Lem L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778Crossref PubMed Scopus (414) Google Scholar), p38 (12Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.R. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 13Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M.J. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar, 14Dean J.L.E. Brook M. Clark A.R. Saklatvala J. J. Biol. Chem. 1999; 274: 264-269Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar), and the extracellular signal-regulated kinase (ERK) p42/44 (13Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M.J. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar, 15Scherle P.A. Jones E.A. Favata M.F. Daulerio A.J. Covington M.B. Nurnberg S.A. Magolda R.L. Trzaskos J.M. J. Immunol. 1998; 161: 5681-5686PubMed Google Scholar), in LPS-induced cytokine production and release has been documented extensively. However, the precise role of these MAP kinase subtypes in the processes of transcriptional induction and/or translation of cytokine transcripts remains in contention. In particular, although the production of human TNF-α in response to LPS is clearly regulated at both transcriptional and post-transcriptional levels (16Raabe T. Bukrinsky M. Currie R.A. J. Biol. Chem. 1998; 273: 974-980Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17Myokai F. 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However, in the study of Leeet al., it was claimed that inhibition of p38 primarily affected the translation of the TNF-α transcripts but not the level of TNF-α mRNA in these cells. By contrast, we have shown that induction of several immediate-early genes is clearly inhibited at the transcriptional level by these compounds (18Hazzalin C.A. Cano E. Cuenda A. Barratt M.J. Cohen P. Mahadevan L.C. Curr. Biol. 1996; 6: 1028-1031Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 19Hazzalin C.A. Cuenda A. Cano E. Cohen P. Mahadevan L.C. Oncogene. 1997; 15: 2321-2331Crossref PubMed Scopus (89) Google Scholar). In addition, pyridinyl imidazoles have now been reported to affect the LPS-stimulated transcription of several cytokine genes including IL-1β (20Manthey C.L. Wang S.-W. Kinney S.D. Yao Z. J. Leukocyte Biol. 1998; 64: 409-417Crossref PubMed Scopus (156) Google Scholar, 21Baldassare J.J. Bi Y. Bellone C.J. J. Immunol. 1999; 162: 5367-5373PubMed Google Scholar). Manthey et al. (20Manthey C.L. Wang S.-W. Kinney S.D. Yao Z. J. Leukocyte Biol. 1998; 64: 409-417Crossref PubMed Scopus (156) Google Scholar) reproduced earlier findings (12Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.R. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 22Pritchett W. Hand A. Sheilds J. Dunnington D. J. Inflamm. 1995; 45: 97-105PubMed Google Scholar, 23Young P. McDonnell P. Dunnington D. Hand A. Laydon J. Lee J. Agents & Actions. 1993; 39: C67-C69Crossref PubMed Scopus (126) Google Scholar) that p38 inhibitors appeared to suppress TNF-α protein levels more than TNF-α mRNA. By contrast, Dean et al. (14Dean J.L.E. Brook M. Clark A.R. Saklatvala J. J. Biol. Chem. 1999; 274: 264-269Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar) using these inhibitors to study LPS-stimulated cyclooxygenase-2 and TNF-α production, came to the very opposite conclusion and found no discrepancy between the suppression of TNF-α protein and mRNA levels, suggesting that inhibition of mRNA accumulation might account completely for the effect of this compound on TNF-α secretion in human blood monocytes. A further complication is the very recent indication of a novel mode of control of TNF-α transcript levels, namely through modulation of their sensitivity to degradation (24Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (636) Google Scholar, 25Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 26Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar). The differences outlined above may arise from the use of different cell types or different experimental methods to measure gene induction. The low numbers of primary monocytes obtainable by purification from human blood is a restrictive factor in studying the role of MAP kinases in TNF-α gene regulation and necessitates the use of model systems. However, cell lines such as U937 or THP-1, commonly used as models of human monocytes to overcome this problem, represent quite immature stages of monocyte differentiation and may not accurately reflect the situation in mature monocytes. Here, we show first that although THP-1 cells are a poor model for studying LPS-stimulated TNF-α mRNA induction, they become more sensitive to LPS stimulation after differentiation with TPA and compare well with mature human monocytes purified from human blood. Moreover, we show in TPA-differentiated THP-1 cells and in purified blood monocytes that individual blockade of p38 or ERK pathways does certainly affect TNF-α transcript levels, although neither inhibitor is capable of completely blocking TNF-α mRNA induction. Most importantly however, using a combination of these inhibitors it is possible to ablate TNF-α induction totally at the mRNA level. Finally, we show that the inhibition of p38 MAP kinase selectively destabilizes TNF-α transcripts without affecting other inducible transcripts such as c-jun. These results are discussed in relation to the controversy in the literature outlined above and to the possible use of combinations of MAP kinase inhibitors at reduced concentrations as a therapeutic strategy. Lymphoprep was from Nycomed Pharma (Oslo, Norway). RPMI 1640 medium and Hanks' balanced salt solution were purchased from Life Technologies, Inc. FCS was obtained from Globepharm (Guildford, UK). LPS (Escherichia coliserotype 055:B5) and TPA were from Sigma. SB203580 was a gift from J. Souness (Aventis Pharma Ltd., Dagenham, UK). PD98059 was obtained from Alexis (Nottingham, UK). U0126 was purchased from Promega(Southampton, UK). The following monoclonal antibodies were used: fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulins and CD54 were from Dako (Glostrup, Denmark); CD14 (Hb246) and CD3 (UCHT-1) were from ATCC; CD19 (BU12) was a gift of D. Hardie (University of Birmingham, UK), CD58 was from Roche Molecular Biochemicals. Rabbit anti-phospho-p38 antibody was purchased from New England Biolabs (Hitchin, UK). Dynabeads M-450 sheep anti-mouse IgG were from Dynal (Oslo, Norway). [α-32P]CTP was purchased from ICN Pharmaceuticals (Irvine, CA). The random primed DNA labeling kit was obtained from Roche Molecular Biochemicals. THP-1 cells were grown in RPMI 1640 medium with 10% fetal calf serum and 20 μm β-mercaptoethanol. Differentiated THP-1 cells were obtained by treatment with 5 nm TPA for 2 days and then starved overnight in 0.5% FCS-RPMI in the presence of 5 nm TPA before stimulation. Human peripheral blood monocytes were freshly prepared from the buffy coat fraction of 1 unit of donor blood. Mononuclear cells were separated on Ficoll Lymphoprep (400 g, 30 min) and the remaining red blood cells were lysed for 15 min in 0.01 m Tris-HCl containing 8.3 g/liter ammonium chloride. Mononuclear cells were then allowed to adhere (5 × 106 cells/ml) in 10% FCS-RPMI. After 90 min at 37 °C, 6% CO2, nonadherent cells were removed, and adherent cells were recovered by a 30-min treatment in 0.02% EDTA. Monocytes were then depleted of contaminating T and B cells using CD3 and CD19 monoclonal antibodies followed by sheep anti-mouse IgG-coated Dynabeads. Monocyte purity was assessed by fluorescence-activated cell sorting in a Becton Dickinson FACSscan. Cells were 90% CD14. Cells were rendered quiescent by an overnight incubation in 0.5% FCS-RPMI before stimulation for MAPK analysis or Northern blots. THP-1 or TPA-differentiated THP-1 cells were lysed in ice-cold lysis buffer consisting of 20 mm Hepes, pH 8, 5 mm EDTA, 10 mmEGTA, 5 mm NaF, 10% glycerol, 1 mmdithiothreitol, 400 mm KCl, 0.4% Triton X-100, 20 mm sodium β-glycerophosphate, 1 μmmicrocystin, 0.2 μm okadaic acid, 0.1 mmsodium orthovanadate, and proteases inhibitors. Lysates were incubated on ice for 10 min and cleared by centrifugation at 13,000 ×g for 10 min. Protein concentrations were measured in the supernatants by the Bradford method (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), and equal amounts of proteins were loaded onto electrophoresis gels. Analysis of p38 activation was performed using specific phospho-p38 antibodies. Cell extracts were loaded on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore). Membranes were blocked overnight at 4 °C in 5% skimmed milk in TBST (20 mm Tris-HCl, pH 8, 137 mm NaCl containing 0.1% Tween 20). After several washes, membranes were probed with phospho-p38 antibodies diluted in 5% skimmed milk in TBST for 1 h at room temperature. After washes, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h, and blots were then developed using a chemiluminescent detection. Total cellular RNA in THP-1 cells was isolated according to the method of Chomczynski and Sacchi (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). RNA from human blood monocytes was purified using the Purescript RNA isolation kit (Gentra system, Minneapolis, MN). Aliquots containing 5 μg of RNA were resolved on 1% agarose gels containing 0.41m formaldehyde. RNA was transferred onto nylon membranes (Hybond N+, Amersham Pharmacia Biotech) by capillary transfer in 50 mm NaOH. cDNA probes for TNF-α,c-jun, and GAPDH were labeled with 50 μCi of [α-32P]CTP by random priming. GAPDH mRNA was detected using a 1-kilobase. PstI fragment of murine cDNA in pBluescript KS (Stratagene) kindly provided by D. R. Edwards (University of East Anglia, Norwich, UK). TNF-α mRNA was detected using a 738-base pair EcoRI fragment of human TNF-α cDNA clone in pBluescript SK+, kindly provided by D. R. Katz (University College London). c-junmRNA was detected using a 749-base pairEcoRI/SacII fragment derived from the mouse c-jun pAH119 plasmid (generously provided by R. Bravo, European Molecular Biology Laboratory, Heidelberg, Germany). TNF-α and c-jun mRNA were visualized by autoradiography and quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and corrected by reference to the corresponding GAPDH reading to compensate for slight variation in loading. THP-1, TPA-differentiated THP-1, or freshly purified blood monocytes (105 cells/well) were incubated for 16–18 h with 10 ng/ml of LPS, unless differently noted, in RPMI containing 1% FCS. TNF-α levels were detected using the Duoset kit (Genzyme Diagnostic, Cambridge, MA) according to the manufacturer's instructions. Cells (106 cells/ml) in RPMI containing 10% rabbit serum and 0.1% NaN3 were incubated for 30 min at 4 °C with murine monoclonal antibodies followed by a 30-min incubation with fluorescein isothiocyanate rabbit anti-mouse IgG. After washing, cells were fixed in 3.7% formaldehyde in Hanks' balanced salt solution and analyzed using a FACScan flow cytometer (Becton Dickinson). The human monocytic cell line THP-1 corresponds to an immature stage of monocyte differentiation and thus does not represent an ideal model of human monocytes for studying cytokine regulation. Several studies have shown that TPA treatment of THP-1 cells resulted in a more differentiated phenotype in terms of adherence, loss of proliferation, or expression of surface markers (29Hoff T. Spencker T. Emmendoerffer A. Goppelt-Struebe M. J. Leukocyte Biol. 1992; 52: 173-182Crossref PubMed Scopus (32) Google Scholar, 30Shwende H. Fitzke E. Ambs P. Dieter P. J. Leukocyte Biol. 1996; 59: 555-561Crossref PubMed Scopus (446) Google Scholar). To study the levels at which TNF-α production is regulated, we first compared LPS-induced TNF-α mRNA and protein in TPA-differentiated and nondifferentiated THP-1 cells. Normal THP-1 cells or THP-1 cells differentiated for 2 days with 5 nm TPA were rendered quiescent and stimulated with 100 ng/ml of LPS, and TNF-α gene induction was evaluated by Northern blot analysis (Fig. 1 A). In THP-1 cells, LPS only weakly induces TNF-α transcripts, whereas after differentiation with TPA, much higher levels of TNF-α mRNA are seen in response to LPS (Fig. 1 A). In the differentiated cells, TNF-α mRNA was detectable 30 min after LPS treatment. Maximal induction was observed between 1 and 2 h, and transcript levels declined thereafter, with barely detectable levels remaining after 6 h. We have shown further that this response is detectable in the presence of translational inhibitors (data not shown), and this finding, together with the transient response, characterizes the TNF-α gene as an immediate-early gene in response to LPS stimulation. We next analyzed levels of TNF-α protein released in the culture medium of these two cell systems in response to LPS. Dose-response analysis showed that normal THP-1 cells secreted only low levels of TNF-α protein when stimulated with up to 1 μg/ml of LPS, and we could only detect significant amounts of TNF-α in these supernatants using 5 μg/ml of LPS (Fig. 1 B). By contrast, the TPA-differentiated cells secreted large quantities of TNF-α protein following treatment with concentrations of LPS as low as 1 ng/ml, the lowest concentration tested (Fig. 1 B); this was approaching the maximal level and showed no significant further increase at higher concentrations. Time-course analysis showed that normal THP-1 cells produce no detectable TNF-α protein over a 48-h incubation period using 100 ng/ml LPS (Fig. 1 C), the same stimulus as used for analysis of transcript levels shown above. By contrast, after TPA differentiation, these cells release detectable quantities of TNF-α cytokine as early as 2 h after LPS-stimulation. The level of secreted protein peaks at around 6 h and diminishes to a quite stable but submaximal level over 48 h. Dose-response and time course analyses were also performed in freshly isolated monocytes from human blood (discussed further below). TNF-α mRNA and protein levels compared well with those seen in TPA-differentiated THP-1 cells (data not shown). These studies revealed a very marked enhancement of TNF-α response in the TPA-differentiated cells, especially when stimulated with lower concentrations of LPS. Thus, TPA-differentiated THP-1 cells are a better model for events in purified human monocytes than the nondifferentiated THP-1 cells, in particular for the study of TNF-α mRNA and protein. To confirm that differentiated THP-1 cells mimic human monocytes more closely than undifferentiated cells, we next determined the levels of expression of two monocyte surface markers, CD14 and CD54, in undifferentiated and TPA-differentiated cells by FACScan and compared it with expression levels in purified human blood monocytes. This showed that TPA treatment enhanced the expression of monocyte markers in THP-1 cells (Fig. 2). Undifferentiated THP-1 cells very weakly express the LPS receptor CD14; only 13.1% of the cells are positive. By contrast, 42.2% of differentiated THP-1 cells express the CD14 marker compared with 94.7% in purified blood monocytes. The same observation was made in analyses of the adhesion molecule CD54 (ICAM-1). CD54 is not detectably expressed in normal THP-1 cells but is expressed in 53.9% of the TPA-differentiated cells compared with 61.3% in purified blood monocytes. This confirms indications from the TNF-α expression studies above that TPA-differentiated THP-1 cells are a better model for human monocytes than the undifferentiated cells. Because CD14 is part of the LPS receptor, the increased levels of CD14 seen here may explain why the differentiated cells mount a stronger response to LPS than undifferentiated cells. Because TPA differentiation increases the expression of the LPS receptor CD14, we then compared the ability of LPS to activate p38 MAP kinase in THP-1 and TPA-differentiated THP-1 cells to that in purified blood monocytes. For positive controls, stimuli such as anisomycin, UV radiation, and hyperosmotic stress, which are known to strongly activate p38, were used. As expected, anisomycin, UV radiation, or osmotic stress using sodium chloride or sorbitol strongly induced p38 activation in normal THP-1 cells (Fig.3) showing that the p38 cascade is present and activated normally in these cells. However, LPS stimulation only produced very weak and barely detectable activation of p38 in these cells. By contrast, in TPA-differentiated THP-1 cells, as in purified blood monocytes, all of the five stimuli tested, including LPS, proved to be very efficient in activating the p38 pathway. This finding reiterates at the signal transduction level all previous indications that the differentiated cells more closely resemble mature monocytes than the undifferentiated cells. Lee et al.(12Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.R. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar) showed that pyridinyl imidazole compounds that inhibit p38 MAPK are very potent inhibitors of LPS-induced TNF-α secretion in human monocytes. Further, PD98059, a MEK inhibitor that blocks ERK activation, also causes a marked inhibition of LPS-stimulated TNF-α release in human blood monocytes (13Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M.J. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar). To investigate the mechanism of action of these two inhibitors, we first carried out dose-response analyses to assess the level of inhibition of TNF-α protein released by purified blood monocytes. Human monocytes were purified from buffy coat preparations as described (see "Experimental Procedures") and pretreated for 30 min with the indicated concentrations of the p38 inhibitor SB203580 (Fig.4 A) or the MEK1/2 inhibitor PD98059 (Fig. 4 B), followed by a 16-h incubation with 10 ng/ml LPS. Cell supernatants were then harvested and assayed for the presence of TNF-α protein. These data confirmed that both inhibitors inhibit LPS-induced TNF-α release from human monocytes in a dose-dependent manner. PD98059 only partially blocked TNF-α production (47% inhibition using 50 μm PD98059). In agreement with previous data, the p38 inhibitor SB203580 was shown to be a very powerful inhibitor of TNF-α release, since it blocked more than 77% of the TNF-α production at a concentration of 120 nm (Fig. 4 A). No obvious loss of viability of human monocytes cells was detected at the concentrations of inhibitors used. The inhibition seen with these compounds, particularly the p38 inhibitor, has previously been ascribed principally to translational inhibition of TNF-α transcripts, with minimal effect on the levels of transcripts (12Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.R. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 22Pritchett W. Hand A. Sheilds J. Dunnington D. J. Inflamm. 1995; 45: 97-105PubMed Google Scholar,23Young P. McDonnell P. Dunnington D. Hand A. Laydon J. Lee J. Agents & Actions. 1993; 39: C67-C69Crossref PubMed Scopus (126) Google Scholar). We next asked whether the presence of SB203580 or PD98059 would have any effect on the level of TNF-α mRNA in purified human monocytes. These experiments were done in triplicate, but because of the difficulty of obtaining purified monocytes in sufficient numbers for mRNA analysis, only a subset of data points corresponding to the protein data from Fig. 4, A and B, was chosen for mRNA analysis (Fig. 5,A and B). This showed that over the concentration range selected, SB203580 produced increasing and pronounced inhibition of TNF-α transcript levels, contradicting the proposal that SB203580 acts primarily at the level of translation. It is important to note that a reduction in secreted TNF-α protein (Fig.4 A) was always more substantial than its effects on transcript levels (Fig. 5 A). This finding is in agreement with published data indicating TNF-α translation as another point of action of this inhibitor. However, contrary to the prevailing view, the data showed that SB203580 also has a clear concentration-dependent effect on TNF-α transcript levels, and this must also contribute to the overall loss of TNF-α production. By contrast, the milder reduction of TNF-α protein seen with varying concentrations of PD98059 (Fig. 4 B) corresponds well with a reduction in transcripts (Fig. 5 B) and conceivably could account for most of the reduced protein levels seen under these conditions. Although strict comparisons of protein and transcripts levels are not safe, the data suggest that as opposed to SB203580, which affects TNF-α production at both mRNA and translational levels, the less marked effects of PD98059 may occur principally at the mRNA level. We next investigated whether combinations of the two inhibitors described above might produce more pronounced inhibition of TNF-α transcript levels. Because of the difficulty of obtaining sufficient quantities of purified monocytes for these studies, we first analyzed these phenomena in TPA-differentiated THP-1 cells, shown above to be a good model of human monocytes. Cells were pretreated with different concentrations of SB203580, PD98059, or a combination of both compounds as indicated (Fig. 6 A) for 60 min before LPS stimulation (100 ng/ml, 60 min). Used on its own, each inhibitor at the highest concentration tested blocked slightly more than 50% of LPS-stimulated TNF-α mRNA accumulation. Note that SB203580 was less efficient in inhibiting TNF-α mRNA expression in differentiated THP-1 cells than in purified human monocytes, (50% inhibition compared with >75% inhibition using 10 μmSB203580). More interestingly, we observed t
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