Nonsteroidal Anti-inflammatory Drugs Suppress T-cell Activation by Inhibiting p38 MAPK Induction

• DOI: 10.1074/jbc.m110676200
• Publication Date: 2002-07-26T15:03:26Z
• Authors: Silvia Rossi Paccani; Marianna Boncrist...; Cristina Ulivieri; Mario Milco D'Elios; Gianfranco Del Prete; Cosima T. Baldari
• Keywords: 0301 basic medicine; MAP Kinase Signaling System; Recombinant Fusion Proteins; T-Lymphocytes; Lymphocyte Activation; Biochemistry; Cell Line; 03 medical and health sciences; Genes, Reporter; Humans; nonsteroidal anti-inflammatory drugs (NSAID); Signal trasduction; Molecular Biology; nonsteroidal anti-inflammatory drugs (NSAID); T cell receptor; MAPK; Signal trasduction; NFATC Transcription Factors; Anti-Inflammatory Agents, Non-Steroidal; Membrane Proteins; Nuclear Proteins; Cell Biology; MAPK; 3. Good health; DNA-Binding Proteins; Enzyme Activation; Isoenzymes; Cyclooxygenase 2; Prostaglandin-Endoperoxide Synthases; Enzyme Induction; Cyclooxygenase 1; T cell receptor; Mitogen-Activated Protein Kinases; Transcription Factors

In addition to antagonizing inflammation by inhibiting the activity of cyclooxygenases (COX), nonsteroidal anti-inflammatory drugs (NSAID) block T-cell activation. The immunosuppressant NSAID correlates with their ability transcription factors required for expression inducible response genes triggered antigen receptor (TCR) engagement. Whereas inhibition nuclear factor-κB aspirin and sodium salicylate can be partly accounted binding IκB kinase-β, broad range transcriptional targets suggests that products COX might affect one or more among early steps in TCR-signaling cascade. Here we show NF-AT activation a selective p38 MAP kinase induction. suppression TCR-dependent fully overcome prostaglandin E2, underlining requirement Furthermore, COX-1 results defective induction theCOX-2 gene, which behaves as an TCR responsive gene. data identify COX-2 integral sequential components signaling contribute elucidate molecular basis immunosuppression NSAID. cyclooxygenase mitogen-activated protein reactive oxygen species extracellular signal-regulated monoclonal antibody prostaglandins E2 factor activated T-cells peripheral blood lymphocytes lipopolysaccharide glyceraldehyde-3-phosphate dehydrogenase kinase/extracellular cluster differentiation Nonsteroidal (NSAID)1 catalyze first step biosynthesis (PG) related eicosanoids. Two isoforms have been identified eucaryotic cells. Although closely enzymatic activity, are implicated different facets interaction cells internal external environments. is constitutively expressed most thought play important role tissue homeostasis. contrast, inducibly limited array cell types proinflammatory mitogenic stimuli considered crucial mediator (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Google Scholar). agreement dominant inflammation, COX-2-selective but unwanted side effects such gastric damage hemorrhage, typical nonselective NSAID, likely (2DeWitt Mol. Pharmacol. 1999; 55: 625-631Google harbor additional immunomodulatory properties correlated cytokines result, at least part, from well characterized on exemplified NF-κB cyclopentenone PG through covalent modification β (3Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 403: 103-108Google However, COX-independent emerging, including itself (4Yin M.-J. 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Chem. 272: 9962-9970Google these not only inhibit distal individual pathways also cascade leading activation, potentially implicating this process. support potential T-cells, whereas gene encoding T-cell-activating (9Iñiguez assessed effect Using read-out specifically kinase, reversed PGE2 therefore dependent activity. engagement, highlighting p38. Cell lines included stably transfected Jurkat line expressing luciferase under control trimer site human interleukin-2 promoter (11Di Baldari C.T. Somma M.M. Majolini M.B. Ulivieri Milia Telford J.L. Biologicals. 26: 1-5Google Scholar), monocyte U937, Raji B-leukemia line. A was generated described previously Peripheral mononuclear were isolated whole density centrifugation Ficoll-Paque (Amersham Biosciences, Inc.) subsequently depleted adherence. SB203580, PD098059, purchased Calbiochem Sigma. Phosphospecific antibodies recognizing phosphorylated active forms Erk1/Erk2 Signaling Technology (Beverly, MA). Anti-p38 anti-Erk2 Santa Cruz Biotechnology (Santa Cruz, CA), anti-actin mAb Amersham Pharmacia, anti-COX-2 Transduction Laboratories (Lexington, UK). IgG OKT3 hybridoma supernatants purified Mabtrap titrated flow cytometry. Anti-IgM Cappel (Durham, NC). All highest concentration used tested lack toxicity trypan blue exclusion. RAV-2 reverse transcriptase Taqpolymerase Takara Shutzo Co. (Shiga, Japan) Roche Diagnostics SpA (Milan, Italy). To assay phosphorylation, 2–5 × 106 PBL cross-linking min 37 °C using anti-CD3 (12Plyte Pacini Scarpini F. Bianchini Lanfrancone Pelicci P.G. Oncogene. 19: 1529-1537Google Macrophages U937 10 μg/ml LPS (Sigma) °C. Ig anti-IgM antibodies. analyze expression, either TCR/CD3 secondary antibody-coated plates (PBL) (macrophages U937) combination phorbol 12-myristate 13-acetate (50 ng/ml) A32187 (100 16–24 h. carrier added culture medium h prior 30 lysates, obtained separated SDS-PAGE, transferred nitrocellulose filters, subjected immunoblot peroxidase-labeled Biosciences) chemoluminescence detection kit (Pierce). reporter CD3 plate A23187 (500 With exception time course experiments, collected 5 after processed assays (10Dong samples duplicate, each experiment repeated 3–5 times. 105 cells/sample resuspended RPMI 1640 supplemented insulin, 5.5 transferrin, 6.5 ng/ml selenite (Life Technologies, Inc.), 20 μm 2′,7′dichlorofluorescein diacetate preincubated dark room temperature. Carrier added, further incubated ice. Cells spun, same medium, saturating concentrations OKT3, After two washes phosphate-buffered saline/1% serum, 500 μl °C, washed analyzed cytometry Becton Dickinson FACScan (San Jose, CA). Total RNA extracted 2, 4, 6, 8, 16 above WIZ reagent (Ambion Inc, Austin, Texas). Reverse transcription-PCR carried out strand oligo(dT), pairs primers specific andGAPDH cDNA amplification. cDNAs amplified Perkin-Elmer 2400 Thermal Cycler (Norwalk, CT). PCR agarose gel electrophoresis, intensity ethidium bromide-stained bands quantitated laser densitometry. identity theCOX-2-specific product confirmed automatic sequencing. Confocal microscopy transiently DEAE/dextran procedure plasmid pEGFP/NF-AT1-D (13Plyte Boncristiano Fattori Galvagni Rossi Paccani Oliviero Ciliberto 2001; 276: 14350-14358Google treatment presence absence cyclosporin (Sandoz, East Hanover, NJ) 600 ibuprofen, Leica Microsystems confocal microscope (Heidelberg, Germany). Scanning COX-2gene (GenBankTM accession number AF276953) sites matrix search program TRANSFAC MatInspector (14Wingender X. Hehl Karas H. Liebich I. Matys Meinhardt Prüβ Reuter Schacherer Nucleic Acids Res. 28: 316-319Google assess impact cascade, assayed capacity firefly inhibitors, COX-1-selective inhibitor resveratrol (15Jang Cai Udeani G.O. Slowing K.V. Thomas C.F. Beecher C.W. Fong H.H. Farnsworth N.R. Kinghorn A.D. Mehta R.G. Moon R.C. Pezzuto J.M. 275: 218-220Google NS-398 (16Futaki N. Arai Hamasaka Higuchi Otomo Pharm. 1993; 45: 753-755Google all inhibited dose-dependent fashion induced triggering complex agonistic (Fig. 1) pharmacologically ester calcium ionophore (data shown). similar profile observed NF-κB/luciferase pharmacological agonists Hence, both activities T-cells. COX-2-specific mRNA detectable 2 stimulation 2A), partially blocked shown) suggesting costitutively process.Figure 2COX-1 COX-2.A, analysis G6PDH (lower panel) control. B, lysates PBL, macrophages, nonactivated (0) 24 50 100 (PI) LPS, indicated.C, (3) (res). filter reprobed loading migration mass shown.View Large Image Figure ViewerDownload (PPT) initiates tyrosine kinase-based within minutes main pathways, Ras/MAPK Ca2+/calcineurin synergistically activate (17Cantrell D. 1996; 14: 259-274Google We pathways. inhibitors Erk MAPKs reduced engagement 1). However TCR-induced dramatically phosphospecific 3A). Consistent before 2A Ref. no NS-398. Similar selectively B-cells stimulated oxidative stress induce (18Ono K. Han Signalling. 12: 1-13Google normal LPS-activated H2O2-treated 3B). coupling B-cell receptors increase ROS, contributes (19Finkel FEBS Lett. 476: 52-54Google (20Tatla Woodhead Foreman J.C. Chain B.M. Free Radical Med. 14-24Google any up-regulation intracellular ROS resveratrol, has anti-oxidant upstream rule molecules target responsible inhibitory did pathway. As shown Fig. 3D, localization completely cytosolic construct green fluorescent protein-tagged NF-AT1 stimulation, massive translocation nucleus repressible ionophore. Ibuprofen 3D). achieved MAPK pathway Of note, although heterologous cells, dampen promoting its phosphorylation-dependent export (21Del Arco Mattinez-Martinez Maldonado Ortega-Perez Redondo 13871-13878Google SB203580 (22Matsuda Moriguchi Koyasu Nishida 273: 12378-12382Google opposing roles subsequent deactivation expression. signaling. indeed increased low basal level resulted Erk1/Erk2, 3C). could ibuprofen reduction synthesis account failed distinct opposed H2O2 3, B) PGE2, produced stimuli, does directly component receptor-signaling Specifically, because kinase-dependent believed diverge small GTPases Ras Rac, respectively (23Waskiewicz A.J. Cooper J.A. Curr. Opin. 7: 798-805Google our indicate serine-threonine initiated Rac. post-transcriptional stabilization (24Pouliot Baillargeon Lee Cleland L.G. James M.J. 158: 4930-4937Google 25Jang B.C. Sanchez Schaefers H.J. Trifan O.C. Liu C.H. Creminon C.K. Hla 39507-39515Google (Fig.3A) COX-1, initial possibility, PBL. COX-2, albeit levels significantly lower than 2B). high (Fig.2B), parallelled constitutive 3B) key pretreatment 2C). participates probably contributing LPS-dependent 3B), impaired COX-1−/− (26Langenbach Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. 83: 483-492Google Interestingly, A, potently blocks inhibits scanning ∼500-bp region containing essential elements regulation (27Yamamoto K Arakawa Ueda 270: 31315-31320Google revealed four positions −308/−297, −292/−281, −109/−98 −80/−69. underlines cytokine during process lymphocyte genetic evidence supports mediated CD4−CD8−to CD4+CD8+ transition thymocyte development (28Rocca Spain L.M. Pure Langenbach Patronom FitzGerald G.A. Clin. Invest. 103: 1469-1477Google COX-2-mediated thymic stromal maturation CD4+thymocytes Our strategic it promotes regulated MAPK. Among COX-2. scenario, burst rapid arachidonic acid due phospholipase Cγ A2 (cPLA2), would result PG, thereby This turn lead delayed phase synthesis. context, partial COX-2-dependent sustaining later stages mast cPLA2−/− mice fail express cytokines, however restores (29Fujishima Mejia R.O. Bingham C.O. Lam B.K. Sapirstein Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. 96: 4803-4807Google supporting proposed COX-1-dependent ofCOX-2 transcription. Collectively, provide novel insight into mechanisms underlying suggest valid alternative classical immunosuppressants tool disease. thank Sonia Grassini technical assistance, Giancarlo Benocci secretarial Luisa useful suggestions, John Telford, Antonio Lanzavecchia, Gioacchino productive discussions critical reading manuscript.