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• W2043981736 abstract "Apoptotic cells are rapidly engulfed by adjacent tissue cells or macrophages before they can release pro-inflammatory/proimmunogenic intracellular contents. In addition, recognition of the apoptotic cells is actively anti-inflammatory and anti-immunogenic with generation of anti-inflammatory mediators such as transforming growth factor-β (TGF-β) and anti-inflammatory eicosanoids. Here, we have investigated the role played by the induction of TGF-β in the coordinate expression of anti-inflammatory eicosanoids or peroxisome proliferator-activated receptor-γ and in the suppression of pro-inflammatory lipid mediators and nitric oxide (NO). By use of a dominant negative TGFβII receptor, TGF-β signaling was blocked, and its participation in the consequences of apoptotic cell stimulation was determined. The induction of TGF-β itself could be attributed to exposed phosphatidylserine on the apoptotic cells, which therefore appears to drive the balanced inflammatory mediator responses. Arachidonic acid release, COX-2, and prostaglandin synthase expression were shown to be significantly dependent on the TGF-β production. On the other hand, a requirement for TGF-β was also shown in the inhibition of thromboxane synthase and thromboxanes, of 5-lipoxygenase and sulfidopeptide leukotrienes, as well as of inducible nitric-oxide synthase and NO. TGF-β-dependent induction of arginase was also found and would further limit the NO generation. Finally, apoptotic cells stimulated production of 15-lipoxygenase and 15-hydroxyeicosatetraenoic acid, a potentially anti-inflammatory pathway acting through peroxisome proliferator-activated receptor-γ, and lipoxin A4 production, which were also up-regulated by a TGF-β-dependent pathway in this system. These results strongly suggest that the apoptotic cell inhibition of pro-inflammatory mediator production is pleiotropic and significantly dependent on the stimulation of TGF-β production. Apoptotic cells are rapidly engulfed by adjacent tissue cells or macrophages before they can release pro-inflammatory/proimmunogenic intracellular contents. In addition, recognition of the apoptotic cells is actively anti-inflammatory and anti-immunogenic with generation of anti-inflammatory mediators such as transforming growth factor-β (TGF-β) and anti-inflammatory eicosanoids. Here, we have investigated the role played by the induction of TGF-β in the coordinate expression of anti-inflammatory eicosanoids or peroxisome proliferator-activated receptor-γ and in the suppression of pro-inflammatory lipid mediators and nitric oxide (NO). By use of a dominant negative TGFβII receptor, TGF-β signaling was blocked, and its participation in the consequences of apoptotic cell stimulation was determined. The induction of TGF-β itself could be attributed to exposed phosphatidylserine on the apoptotic cells, which therefore appears to drive the balanced inflammatory mediator responses. Arachidonic acid release, COX-2, and prostaglandin synthase expression were shown to be significantly dependent on the TGF-β production. On the other hand, a requirement for TGF-β was also shown in the inhibition of thromboxane synthase and thromboxanes, of 5-lipoxygenase and sulfidopeptide leukotrienes, as well as of inducible nitric-oxide synthase and NO. TGF-β-dependent induction of arginase was also found and would further limit the NO generation. Finally, apoptotic cells stimulated production of 15-lipoxygenase and 15-hydroxyeicosatetraenoic acid, a potentially anti-inflammatory pathway acting through peroxisome proliferator-activated receptor-γ, and lipoxin A4 production, which were also up-regulated by a TGF-β-dependent pathway in this system. These results strongly suggest that the apoptotic cell inhibition of pro-inflammatory mediator production is pleiotropic and significantly dependent on the stimulation of TGF-β production. Recognition and clearance of apoptotic cells by phagocytes play pivotal roles in development, maintenance of tissue homeostasis, control of the immune response, and resolution of inflammation (1Fadok V.A. Bratton D.L. Henson P.M. J. Clin. Investig. 2001; 108: 957-962Crossref PubMed Scopus (401) Google Scholar, 2Serhan C.N. Savill J. Nat. Immun. 2005; 6: 1191-1197Crossref Scopus (1757) Google Scholar). Apoptotic cells are removed by professional phagocytes, members of the mononuclear phagocyte system such as macrophages and immature dendritic cells, or by nonprofessional phagocytes such as fibroblasts, endothelial, epithelial, smooth muscle, or stromal cells (3Henson P.M. Hume D.A. Trends Immunol. 2006; 27: 244-250Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Uptake of the apoptotic cell is by a specialized and highly conserved form of phagocytosis termed efferocytosis (4DeCathelineau A.M. Henson P.M. Essays Biochem. 2003; 39: 105-117Crossref PubMed Scopus (177) Google Scholar, 5Gardai S.J. McPhillips K.A. Frasch C. Janssen W.J. Starefeldt A. Murphy-Ullrich J.E. Bratton D.L. Oldenborg P.A. Michalak M. Henson P.M. Cell. 2006; 123: 321-334Abstract Full Text Full Text PDF Scopus (1016) Google Scholar). As a cell becomes apoptotic, it is generally removed in situ by near-neighbor cells or macrophages in a quiet, almost invisible fashion, i.e. the process does not induce a local tissue reaction. In fact, recognition and removal of apoptotic cells are normally both anti-inflammatory and anti-immunogenic (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar, 9Patel V.A. Longacre A. Hsiao K. Fan H. Meng F. Mitchell J.E. Rauch J. Ucker D.S. Levine J.S. J. Biol. Chem. 2006; 281: 4663-4670Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).The interaction and recognition are triggered by surface changes on the apoptotic cells. Two widely distributed surface ligands on apoptotic cells are phosphatidylserine (PS) 4The abbreviations used are: PS, phosphatidylserine; PSRS, phosphatidylserine recognition structures; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor-β; LPS, lipopolysaccharide; IFN-γ, interferon-γ; COX, cyclooxygenase; PG, prostaglandin; NO, nitric oxide; LO, lipoxygenase; PPARγ, peroxisome proliferator-activated receptor-γ; iNOS, inducible nitric-oxide synthase; LXA4, lipoxin A4; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; LRP, low density lipoprotein receptor-related protein. 4The abbreviations used are: PS, phosphatidylserine; PSRS, phosphatidylserine recognition structures; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor-β; LPS, lipopolysaccharide; IFN-γ, interferon-γ; COX, cyclooxygenase; PG, prostaglandin; NO, nitric oxide; LO, lipoxygenase; PPARγ, peroxisome proliferator-activated receptor-γ; iNOS, inducible nitric-oxide synthase; LXA4, lipoxin A4; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; LRP, low density lipoprotein receptor-related protein. (10Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 11Martin S.J. Reutelingsperger C.P. McGahon A.J. Rader J.A. van Schie R.C. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2551) Google Scholar) and calreticulin (5Gardai S.J. McPhillips K.A. Frasch C. Janssen W.J. Starefeldt A. Murphy-Ullrich J.E. Bratton D.L. Oldenborg P.A. Michalak M. Henson P.M. Cell. 2006; 123: 321-334Abstract Full Text Full Text PDF Scopus (1016) Google Scholar), which become associated in patches together on the cell surface. Indirect effects of the collectin family of molecules or direct action of calreticulin leads to stimulation of low density lipoprotein receptor-related protein (LRP) on the phagocytosing cell (5Gardai S.J. McPhillips K.A. Frasch C. Janssen W.J. Starefeldt A. Murphy-Ullrich J.E. Bratton D.L. Oldenborg P.A. Michalak M. Henson P.M. Cell. 2006; 123: 321-334Abstract Full Text Full Text PDF Scopus (1016) Google Scholar, 12Manaka J. Kuraishi T. Shiratsuchi A. Nakai Y. Higashida H. Henson P. Nakanishi Y. J. Biol. Chem. 2004; 279: 48466-48476Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, LRP activation seems to induce production of pro-inflammatory mediators (13Gardai S.J. Xiao Y.Q. Dickinson M. Nick J.A. Voelker D.R. Greene K.E. Henson P.M. Cell. 2003; 115: 13-23Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). On the other hand, there is considerable evidence to implicate PS as the main stimulus for the anti-inflammatory or anti-immunogenic effects (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar, 8Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar, 14Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 16McDonald P.P. Fadok V.A. Bratton D. Henson P.M. J. Immunol. 1999; 163: 6164-6172PubMed Google Scholar). We suspect that these two stimuli, acting through different signaling pathways, are balanced, with a normal bias toward the anti-inflammatory. Unfortunately, the receptor(s) that recognizes PS (PS recognition structures, PSRS) that is responsible for this effect is unknown, although it does seem to distinguish between stereoisomeric forms of the phosphoserine head group (10Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 14Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar) and does seem to react with an activating IgM antibody mAb217 (17Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekewitz R.A. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar) whose binding is blocked by PS. The antibody binds to and activates cells and mimics exactly the effects of PS on apoptotic cells in contributing to uptake and on the generation of anti-inflammatory mediators (7Hoffmann P.R. Kench J.A. Vondracek A. Kruk E. Daleke D.L. Jordan M. Marrack P. Henson P.M. Fadok V.A. J. Immunol. 2005; 174: 1393-1404Crossref PubMed Scopus (169) Google Scholar, 18Hoffmann P.R. deCathelineau A.M. Ogden C.A. Leverrier Y. Bratton D.L. Daleke D.L. Ridley A.J. Fadok V.A. Henson P.M. J. Cell Biol. 2001; 155: 649-659Crossref PubMed Scopus (44) Google Scholar), and it has been used here along with apoptotic cells to stimulate macrophages for production or suppression of eicosanoids.A major anti-inflammatory mediator induced in response to apoptotic cells, mAb217, or PS liposomes is TGF-β (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar, 8Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar, 16McDonald P.P. Fadok V.A. Bratton D. Henson P.M. J. Immunol. 1999; 163: 6164-6172PubMed Google Scholar). Blockade of TGF-β has been shown to reverse the suppressive effects of apoptotic cells or PS in vivo on either inflammation or adaptive immunity (7Hoffmann P.R. Kench J.A. Vondracek A. Kruk E. Daleke D.L. Jordan M. Marrack P. Henson P.M. Fadok V.A. J. Immunol. 2005; 174: 1393-1404Crossref PubMed Scopus (169) Google Scholar, 8Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar). On the other hand, earlier studies also suggested induction of other candidates such as IL-10 (19Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1494) Google Scholar), PGE2 (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar), and even platelet-activating factor (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar), although the last two can have both pro- or anti-inflammatory effects. The ability of apoptotic cell recognition to alter the production of eicosanoids had first been noted for thromboxane (20Meagher L.C. Savill J.S. Baker A. Fuller R.W. Haslett C. J. Leukocyte Biol. 1992; 52: 269-273Crossref PubMed Scopus (247) Google Scholar, 21Stern M. Savill J. Haslett C. Am. J. Pathol. 1996; 149: 911-921PubMed Google Scholar) and exemplified by roles for TGF-β and prostaglandins in the apoptotic cell enhancement of Trypanosoma cruzi growth in macrophages (22Freire-de-Lima C.G. Nascimento D.O. Soares M.B. Bozza P.T. Castro-Faria-Neto H.C. de Mello F.G. DosReis G.A. Lopes M.F. Nature. 2000; 403: 199-203Crossref PubMed Scopus (380) Google Scholar). Therefore, a key issue is whether apoptotic cell-induced TGF-β, acting in an autocrine/paracrine fashion, mediates the alterations in eicosanoid generation. By use of a dominant negative TGF-β receptor construct, we have been able to show that apoptotic cells stimulate via their induction of active TGF-β, a coordinate production of generally anti-inflammatory and simultaneous inhibition of generally pro-inflammatory, eicosanoids. The effect is mediated by effects on the synthases for these mediators. Additional coordinate effects were seen on related proteins, including iNOS, which was down-regulated, and PPARγ or arginase, which were induced, i.e. combining to reduce NO production and also potentially in keeping with the anti-inflammatory balance.EXPERIMENTAL PROCEDURESAntibodies and Reagents—TGF-β was purchased from R&D Systems. Lipopolysaccharide (LPS, Escherichia coli 0111:B4) was from List Biological Laboratories, Inc. Recombinant murine interferon-γ was from BD Biosciences. Human factor Va was from Hematologic Technologies Inc. Cyclosporin A and protease inhibitor mixture set I were from Calbiochem. Anti-arginase 1 antibody was from Santa Cruz Biotechnology. Antibodies against COX-2, human PGD-2 synthase, murine PGE-1 synthase 1, PGI synthase, thromboxane synthase, 5-LO, 15-LO II, PPARγ1, iNOS, and indomethacin were from Cayman Chemical. Anti-β-actin antibody was from Cell Signaling. mAb217 is an IgM monoclonal antibody that was originally raised against PS-recognizing macrophages. It was obtained from concentrated hybridoma supernatants. Unfortunately, attempts to label, fragment, or convert the antibody to an IgG isotype have universally resulted in loss of activity. [5,6,8,9,11,12,14,15-3H]Arachidonate acid (200 Ci/mol) was from American Radiolabeled Chemical. Lipofectamine Plus reagent was from Invitrogen.Induction of Apoptotic Cells—Jurkat T cells were exposed to UV irradiation at 254 nm for 10 min. Jurkat T cells were cultured in RPMI 1640 with 10% fetal calf serum (Gemini Bio-Products) for 3 h at 37°C in 5% CO2. The cells were generally ≥80% apoptotic by nuclear morphology and maintained intact cell membranes that excluded trypan blue (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar).Cell Culture, Stimulation and Measurement of Pro-inflammatory Mediators by ELISA—Murine peritoneal macrophages were obtained from BALB/c mice 4 days after intraperitoneal injection of 1 ml of thioglycollate. Murine RAW 264 cells were obtained from the American Type Culture Collection. RAW-V and RAW-TβRII were made by stable transfection of empty vector and truncated TGF-β receptor II constructs, respectively. The cells (1 × 106 cells/well) were plated in each well of a 24-well tissue culture plate and were cultured in DMEM supplemented with 10% heat-inactivated endotoxin-free fetal bovine serum, 2 mml-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin under a humidified 5% CO2 atmosphere at 37 °C for 24 h. Then the cells were cultured in serum-free DMEM in the absence or presence of LPS, IFN-γ, factor Va, or cyclosporin A for 18 h with 3 × 106 apoptotic Jurkat cells (apoJ), viable Jurkat cells (ViableJ), 50 μg/ml mAb217, 50 μg/ml control isotype IgM, or 100 μm liposomes (containing 30:70 molar ratios of PS:PC or PC alone). The supernatants were collected and measured for TGF-β, TNF-α, PGE2, PGF1α, 15-HETE, LXA4, and leukotrienes by ELISA according to the manufacturer's instructions (ELISA TECH, Aurora, CO). In some experiments, to inhibit pro-inflammatory mediators, the macrophages were treated with stimuli cited before and 100 ng/ml LPS or LPS plus 40 units/ml IFN-γ.NO Production Assay—NO levels produced by RAW 264 cells, RAW-TβRII cells, and murine peritoneal macrophages were measured by reducing the nitrate accumulated over 18 h to nitrite with nitrate reductase (23Schmidt H.H. Wilke P. Evers B. Bohme E. Biochem. Biophys. Res. Commun. 1989; 165: 284-291Crossref PubMed Scopus (142) Google Scholar) and measuring the nitrite concentration by the method of Green et al. (24Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10674) Google Scholar). The nitrite concentrations were quantified by using a double three-point standard curve of NaNO2 concentrations (in a linear range between 1 and 80 μm).Measurement of Arachidonate Release—Arachidonic acid release was measured as described (25Evans J.H. Fergus D.J. Leslie C.C. BMC Biochem. 2002; 3: 30Crossref PubMed Scopus (42) Google Scholar). Briefly, the cells were cultured in 24-well plates to incorporate arachidonate by incubation for 24 h in 1 ml of DMEM containing 10% fetal bovine serum and [3H]arachidonate (1 μCi/ml) for 24 h. Then the cells were washed and incubated in serum-free DMEM containing 0.1% human serum albumin and stimulated with LPS or apoJ or ViableJ or mAb217 or isotype control IgM. The medium was removed 2 h after stimulation and centrifuged at 500 × g for 10 min, and the amount of radioactivity (arachidonate and arachidonate products) in the supernatant was determined.Transient Cell Transfection and Reporter Gene Assay—The p3TP-luc (26Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1366) Google Scholar) luciferase reporter gene construct was transfected into RAW-TβRII and RAW-V cells using Lipofectamine Plus reagent according to the manufacturer's instructions. pSV-β-galactosidase vector (Promega) was co-transfected as an internal control to measure differences in transfection efficiency. Luciferase and β-galactosidase activities were measured 18 h after TGF-β stimulation using the luciferase assay system (Promega) and Galacto-Light (Tropix), respectively.Immunoblotting Analysis—Immunoblotting analysis was carried out as described previously with some modification (27Frasch S.C. Nick J.A. Fadok V.A. Bratton D.L. Worthen G.S. Henson P.M. J. Biol. Chem. 1998; 273: 8389-8397Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Briefly, cells (3.0 × 105 cells/well) were plated in each well of a 12-well tissue culture plate and incubated overnight. Following stimulation, the cells were lysed in lysis buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm dithiothreitol, 0.5% Triton X-100 and 1× protease inhibitor mixture set I), resolved on 10% SDS-PAGE, and blotted onto nitrocellulose membranes. The membranes were probed with primary antibodies at 4 °C overnight and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions. Equal loading of proteins in each lane was confirmed by Ponceau S staining or re-probed with corresponding antibodies against the native proteins (28Xiao Y.Q. Malcolm K. Worthen G.S. Gardai S. Schiemann W.P. Fadok V.A. Bratton D.L. Henson P.M. J. Biol. Chem. 2002; 277: 14884-14893Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) or β-actin. The results shown are representative of at least three separate experiments.Statistical Analysis—All data are presented as means ± S.E. from three or more separate experiments. The means were analyzed using analysis of variance for multiple comparisons. When analysis of variance indicated significance, the Tukey-Kramer honestly significant difference test for all pairs was used to compare groups. All data were analyzed using JMP statistical software (version 5; SAS Institute) for the Macintosh computer.RESULTSApoptotic Cells or Antibody to PSRS on Murine Macrophages Stimulate Production of TGF-β and Concomitant Blockade of LPS-induced TNF-α, NO, and iNOS—Murine macrophages (peritoneal or RAW 264, 1.0 × 106 cells/ml) were stimulated with LPS (100 ng/ml) as positive control or mAb217 (50 μg/ml) or apoptotic Jurkat T cells (3 × 106 cells/ml) for 18 h. These stimuli each induced TGF-β production in both types of macrophages (Fig. 1A). Isotype control IgM or viable Jurkat T cells were inactive. These data are in accordance with previous reports (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar, 16McDonald P.P. Fadok V.A. Bratton D. Henson P.M. J. Immunol. 1999; 163: 6164-6172PubMed Google Scholar). The stimulation of TGF-β production by apoptotic cells in this system was blocked by preincubation of the targets with the PS-binding protein factor Va (Fig. 1B) as had been shown earlier with annexin V (29Seabra S.H. de Souza W. Damatta R.A. Biochem. Biophys. Res. Commun. 2004; 324: 744-752Crossref PubMed Scopus (64) Google Scholar). PS liposomes also stimulated the production of TGF-β but less efficiently. This is probably because the presentation to the PSRS from PS exposed on the apoptotic cell is from a quite different environment compared with a liposome. However, PS liposomes increased the production of TGF-β in the presence of LPS or cyclosporin A (Fig. 1C). These findings suggest that PS liposomes themselves may up-regulate TGF-β translation when TGF-β message has been induced by other stimuli (17Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekewitz R.A. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar, 30Waiser J. Dell K. Bohler T. Dogu E. Gaedeke J. Budde K. Neumayer H.H. Nephrol. Dial. Transplant. 2002; 17: 1568-1577Crossref PubMed Google Scholar). Classically activated macrophages (LPS and IFN-γ stimulation) exhibit release of TNF-α and NO as well as up-regulation of iNOS. As shown in Fig. 1, D and E, these three responses to stimulation with LPS and IFN-γ were inhibited by exposure of the macrophages to apoptotic cells or mAb217. Previous studies implicated the TGF-β produced, in the suppression of TNF-α induction, and might be expected to serve the same role for suppression of iNOS and NO.To demonstrate this presumed requirement for TGF-β in the suppression, a dominant negative form of the TGFβRII was employed. Transfection of RAW 264 cells with this construct was shown to block the ability of TGF-β to signal for 3xPT-luc reporter (which contains three consecutive 12-O-tetradecanoylphorbol-13-acetate-response elements and a portion of the plasminogen activator inhibitor (PAI-1) promoter region) gene activation (Fig. 2A). Because TGF-β can induce its own synthesis, the effect of the dominant negative receptor was also examined on the production of TGF-β itself after stimulation with apoptotic cells, mAb217, or LPS. As shown in Fig. 2B, this treatment blocked 60–70% of the TGF-β produced by each of the stimuli, i.e. supporting an additional autocrine/paracrine effect of TGF-β on its own induction in these systems.FIGURE 2Transfection of truncated TGFβRII into RAW 264 cells reverses the suppression of TNF-α, NO, iNOS, and arginase 1 up-regulation induced by apoptotic cells or PSRS stimulation. A, RAW macrophages transfected with truncated TGF-β receptor II (RAW-TβRII) and empty vector (RAW-V) were transiently co-transfected with 3xPT-luc and pSV-β-galactosidase constructs. After 48 h, the cells were incubated in the presence of TGF-β (10 ng/ml) for 18 h. The luciferase assays, which were normalized to β-galactosidase, are expressed as relative luciferase unites. #, significantly different from controls. *, significantly different from RAW-V cells stimulated with TGF-β. B, RAW-V or RAW-TβRII cells were incubated in the presence of LPS, mAb217, isotype IgM, apoJ, or ViableJ for 18 h. #, significantly different from controls. *, significantly different from RAW-V cells. C and D, RAW-V or RAW-TβRII cells were incubated with the stimuli for 30 min and then with LPS plus IFN-γ for 18 h. TNF-α or NO were measured in the conditioned medium. #, significantly different from LPS/IFN-γ.*, significantly different from RAW-V cells. E and F, iNOS or arginase protein levels in the total cell lysate were analyzed by Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transfection of the truncated TGFβRII was found to completely reverse the suppression of TNF-α and NO production caused by apoptotic cells or PSRS stimulation and also restored the up-regulation of the iNOS protein (Fig. 2, C–E). In keeping with the suppression of NO production by blocking up-regulation of iNOS, exposure of macrophages to apoptotic cells or PSRS stimuli also led to increases in intracellular levels of arginase 1 (Fig. 2F), which could further reduce the production of nitric oxide.Macrophages with Truncated TGFβRII Are Defective in Prostaglandin Production and Prostaglandin Synthase Expression in Response to Stimulation with Apoptotic Cells or LPS—In the original studies of anti-inflammatory effects of apoptotic cells, PGE2 was also shown to be generated, and it too seemed to play a role in suppression of inflammatory mediators (27Frasch S.C. Nick J.A. Fadok V.A. Bratton D.L. Worthen G.S. Henson P.M. J. Biol. Chem. 1998; 273: 8389-8397Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Accordingly, we next examined the effect of apoptotic cells and stimulation with mAb217 on induction of potentially anti-inflammatory prostaglandins as well as the role of TGF-β in their regulation. The original studies did not address the probable induction of PGI2 (detected as PGF1α) along with PGE2, and accordingly, this was included in the analysis. Supernatants from the cell culture were collected 18 h after stimulation and analyzed for PGE2 and PGF1α. The cell lysates were collected, and the levels of synthases for PGE2 (PGES1), PGD2, and PGI2 were determined by Western blotting. As expected, the two stimuli induced production of PGE2 and PGF1α starting at 2 h or earlier and extending out to 18 h of incubation (Fig. 3, A–C). Importantly, they also increased the intracellular levels of the prostaglandin synthases (Fig. 3D).FIGURE 3Prostaglandin production and prostaglandin synthase expression in response to apoptotic cells or mAb217 stimulation is TGF-β-dependent. A and B, RAW-V or RAW-TβRII cells were incubated in the presence of LPS, mAb217, isotype IgM, apoJ, or ViableJ for 18 h. PGE2 or PGF1α concentrations in the conditioned medium were analyzed by ELISA. *, significantly different from RAW-V. C, PGE2 and PGF1α time course. RAW cells were incubated in the presence of the stimuli, and the supernatant was collected after 30 min and 2, 4, 8, and 18 h. D, PGE-1, human PGD and PGI synthase protein levels in the total cell lysate were analyzed by Western blot. E, RAW cells were stimulated with mAb217, isotype IgM, apoJ, or ViableJ in the presence or absence of indomethacin (Indo) for 18 h. TGF-β concentration was analyzed by ELISA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been reported previously that TGF-β can induce prostaglandin production (22Freire-de-Lima C.G. Nascimento D.O. Soares M.B. Bozza P.T. Castro-Faria-Neto H.C. de Mello F.G. DosReis G.A. Lopes M.F. Nature. 2000; 403: 199-203Crossref PubMed Scopus (380) Google Scholar, 31Diaz A. Varga J. Jimenez S.A. J. Biol. Chem. 1989; 264: 11554-11557Abstract Full Text PDF PubMed Google Scholar, 32Ristimaki A. Ylikorkala O. Viinikka L. Arteriosclerosis. 1990; 10: 653-657Crossref PubMed Google Scholar), and in data not shown, the direct addition of active TGF-β to the macrophage cultures did stimulate production of PGE2 and PGF1α. When the macrophage response to TGF-β was blocked with the dominant negative receptor, induction of PGE2 and PGF1α by either apoptotic cells or mAb217 was prevented (Fig. 3, A and B). Interestingly, LPS-induced PGE2 and PGF1α were also reduced by about 80%. We suspect that the lack of complete blockade with this stimulus reflects the possible use of alternative pathways not involving TGF-β. In keeping with the data on the prostaglandins themselves, the truncated receptor also reduced up-regulation of the synthases (Fig. 3D), although not to as great an extent as seen for the secreted prostaglandins.These prostaglandins are lipid mediators that like TGF-β have been reported to have pro- or anti-inflammatory properties in different circumstances. For example, we earlier showed PGE2 to decrease TNF-α production from macrophages (6Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y." @default.
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• W2043981736 date "2006-12-01" @default.
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• W2043981736 title "Apoptotic Cells, through Transforming Growth Factor-β, Coordinately Induce Anti-inflammatory and Suppress Pro-inflammatory Eicosanoid and NO Synthesis in Murine Macrophages" @default.
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