Soluble Receptor (DcR3) and Cellular Inhibitor of Apoptosis-2 (cIAP-2) Protect Human Cytotrophoblast Cells Against LIGHT-Mediated Apoptosis
2004; Elsevier BV; Volume: 165; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63298-1
ISSN1525-2191
Autores Tópico(s)Cell death mechanisms and regulation
ResumoLIGHT (tumor necrosis factor superfamily 14) is among the powerful apoptosis-inducing cytokines synthesized in human placentas. Here, we investigated mechanisms protecting cytotrophoblast (CTB) cells from LIGHT-mediated apoptosis. Viability assays and caspase-3 immunoblots using recombinant LIGHT were done to establish that CTB cells purified from term placentas resist LIGHT-induced apoptosis. Although the cells were also resistant to killing by another placental cytokine, interferon-γ (IFN-γ), a combination of the two induced apoptosis. Killing was prevented by DcR3-Fc fragment but not control human-Fc fragment, showing that apoptosis occurs via the LIGHT pathway and that soluble receptors provide protection. Next, two cellular inhibitors of apoptosis expressed in CTB cells, cellular inhibitor of apoptosis (cIAP)-1 and cIAP-2, were investigated for protection. Cellular IAP-1 was unchanged after stimulation with LIGHT whereas cIAP-2 mRNA and protein were elevated. The increase was abrogated by treating CTB cells with LIGHT + IFN-γ, implying a central role for cIAP-2 in preventing LIGHT-mediated apoptosis and an ability of IFN-γ to overcome cIAP-2 protection. Definitive evidence was provided in experiments that showed that cIAP-2 anti-sense morpholinos permit LIGHT to induce apoptosis in HT-29 cells. In summary, the data are consistent with the postulate that placental CTB cells are protected from LIGHT-mediated apoptosis by both soluble receptor, DcR3, and cIAP-2. LIGHT (tumor necrosis factor superfamily 14) is among the powerful apoptosis-inducing cytokines synthesized in human placentas. Here, we investigated mechanisms protecting cytotrophoblast (CTB) cells from LIGHT-mediated apoptosis. Viability assays and caspase-3 immunoblots using recombinant LIGHT were done to establish that CTB cells purified from term placentas resist LIGHT-induced apoptosis. Although the cells were also resistant to killing by another placental cytokine, interferon-γ (IFN-γ), a combination of the two induced apoptosis. Killing was prevented by DcR3-Fc fragment but not control human-Fc fragment, showing that apoptosis occurs via the LIGHT pathway and that soluble receptors provide protection. Next, two cellular inhibitors of apoptosis expressed in CTB cells, cellular inhibitor of apoptosis (cIAP)-1 and cIAP-2, were investigated for protection. Cellular IAP-1 was unchanged after stimulation with LIGHT whereas cIAP-2 mRNA and protein were elevated. The increase was abrogated by treating CTB cells with LIGHT + IFN-γ, implying a central role for cIAP-2 in preventing LIGHT-mediated apoptosis and an ability of IFN-γ to overcome cIAP-2 protection. Definitive evidence was provided in experiments that showed that cIAP-2 anti-sense morpholinos permit LIGHT to induce apoptosis in HT-29 cells. In summary, the data are consistent with the postulate that placental CTB cells are protected from LIGHT-mediated apoptosis by both soluble receptor, DcR3, and cIAP-2. Human placentas are sites of production of essentially all of the apoptosis-inducing tumor necrosis factor (TNF) family ligands identified to date.1Phillips TA Ni J Hunt JS Death-inducing tumour necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines.Placenta. 2001; 22: 663-672Crossref PubMed Scopus (101) Google Scholar Among these is LIGHT [homologous to lymphotoxin, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes], which is also known as TNF superfamily 14. LIGHT is a 29-kd type II transmembrane protein that is present in both the cytosol and on cell membranes, and circulates as metalloproteinase-cleaved soluble protein.2Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (334) Google Scholar, 3Morel Y Schiano de Colella J Harrop J Deen KC Holmes SD Wattam TA Khandejar SS Truneh A Sweet RW Gastaut J Olive D Costello RT Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT downregulates its own receptors.J Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (155) Google Scholar, 4Granger SW Butrovich KD Houshmand P Edwards WR Ware CF Genomic characterization of LIGHT reveals linkage to an immune response locus on chromosome 19p13.3 and distinct isoforms generated by alternate splicing or proteolysis.J Immunol. 2001; 167: 5122-5128Crossref PubMed Scopus (75) Google Scholar LIGHT is not only a powerful mediator of T-cell activation2Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (334) Google Scholar, 3Morel Y Schiano de Colella J Harrop J Deen KC Holmes SD Wattam TA Khandejar SS Truneh A Sweet RW Gastaut J Olive D Costello RT Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT downregulates its own receptors.J Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (155) Google Scholar, 5Harrop JA McDonnell PC Brigham-Burke M Lyn SD Minton J Tan KB Dede K Spampanato J Silverman C Hensley P DiPrinzio R Emery JG Deen K Eichman C Chabot-Fletcher M Truneh A Young P Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T-cells and inhibits HT29 cell growth.J Biol Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 6Tamada K Shimozaki K Chapoval AI Zhu G Sica G Flies D Boone T Hsu H Fu Y Nagata S Ni J Chen L Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway.Nat Med. 2000; 6: 283-289Crossref PubMed Scopus (278) Google Scholar but also induces apoptosis in some tumor cells.3Morel Y Schiano de Colella J Harrop J Deen KC Holmes SD Wattam TA Khandejar SS Truneh A Sweet RW Gastaut J Olive D Costello RT Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT downregulates its own receptors.J Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (155) Google Scholar, 5Harrop JA McDonnell PC Brigham-Burke M Lyn SD Minton J Tan KB Dede K Spampanato J Silverman C Hensley P DiPrinzio R Emery JG Deen K Eichman C Chabot-Fletcher M Truneh A Young P Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T-cells and inhibits HT29 cell growth.J Biol Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 6Tamada K Shimozaki K Chapoval AI Zhu G Sica G Flies D Boone T Hsu H Fu Y Nagata S Ni J Chen L Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway.Nat Med. 2000; 6: 283-289Crossref PubMed Scopus (278) Google Scholar, 7Zhai Y Guo R Hsu T Yu G Ni J Kwon BS Jiang G Lu J Tan J Ugustus M Carter K Rojas L Zhu F Lincoln C Endress G Xing L Wang S Oh K Gentz R Ruben S Lippman ME Hsieh S Yang D LIGHT, a novel ligand for lymphotoxin β receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer.J Clin Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (236) Google Scholar, 8Wu MY Wang PY Han SH Hsieh SL The cytoplasmic domain of the lymphotoxin-beta receptor mediates cell death in HeLa cells.J Biol Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Rooney IA Butrovich KD Glass AA Borboroglu S Benedict CA Whitbeck JC Cohen GH Eisenberg RJ Ware CF The lymphotoxin β receptor is necessary and sufficient for LIGHT mediated apoptosis of tumor cells.J Biol Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 10Chen M Hsu T Luh T Hseih S Overexpression of Bcl-2 enhances LIGHT and interferon-γ-mediated apoptosis in Hep3BT2 cells.J Biol Chem. 2000; 275: 38794-38801Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar LIGHT is biologically active as a homotrimer5Harrop JA McDonnell PC Brigham-Burke M Lyn SD Minton J Tan KB Dede K Spampanato J Silverman C Hensley P DiPrinzio R Emery JG Deen K Eichman C Chabot-Fletcher M Truneh A Young P Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T-cells and inhibits HT29 cell growth.J Biol Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 9Rooney IA Butrovich KD Glass AA Borboroglu S Benedict CA Whitbeck JC Cohen GH Eisenberg RJ Ware CF The lymphotoxin β receptor is necessary and sufficient for LIGHT mediated apoptosis of tumor cells.J Biol Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar and has three receptors. Two of these are membrane-bound and transduce intracellular signals [HVEM, lymphotoxin-β receptor (LTβ-R)], and one is a soluble receptor (DcR3, also known as TR6). Although it was originally suggested that coincident expression of the two membrane-bound receptors is required for apoptotic signaling,7Zhai Y Guo R Hsu T Yu G Ni J Kwon BS Jiang G Lu J Tan J Ugustus M Carter K Rojas L Zhu F Lincoln C Endress G Xing L Wang S Oh K Gentz R Ruben S Lippman ME Hsieh S Yang D LIGHT, a novel ligand for lymphotoxin β receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer.J Clin Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (236) Google Scholar more recent studies have demonstrated that LTβ-R alone is capable of transducing an apoptotic signal.8Wu MY Wang PY Han SH Hsieh SL The cytoplasmic domain of the lymphotoxin-beta receptor mediates cell death in HeLa cells.J Biol Chem. 1999; 274: 11868-11873Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Rooney IA Butrovich KD Glass AA Borboroglu S Benedict CA Whitbeck JC Cohen GH Eisenberg RJ Ware CF The lymphotoxin β receptor is necessary and sufficient for LIGHT mediated apoptosis of tumor cells.J Biol Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar DcR3, the soluble receptor, can prevent LIGHT from binding to either HVEM or LTβ-R.11Pitti RM Marsters SA Lawrence DA Roy M Kischkel FC Dowd P Huang A Donahue CJ Sherwood SW Baldwin DT Godowski PJ Wood WI Gurney AL Hillan KJ Cohen RL Goddard AD Botstein D Ashkenazi A Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer.Nature. 1998; 396: 699-702Crossref PubMed Scopus (693) Google Scholar, 12Yu K Kwon B Ni J Zhai Y Ebner R Kwon B A newly identified member of the tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.J Biol Chem. 1999; 274: 13733-13736Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 13Zhang M Guo R Zhai Y Fu XY Yang D LIGHT stimulates IFNγ-mediated intercellular adhesion molecule-1 upregulation of cancer cells.Hum Immunol. 2003; 64: 416-426Crossref PubMed Scopus (12) Google Scholar A second cytokine, interferon (IFN)-γ has been shown to facilitate LIGHT-mediated apoptosis in tumor cells using an as yet unidentified pathway.7Zhai Y Guo R Hsu T Yu G Ni J Kwon BS Jiang G Lu J Tan J Ugustus M Carter K Rojas L Zhu F Lincoln C Endress G Xing L Wang S Oh K Gentz R Ruben S Lippman ME Hsieh S Yang D LIGHT, a novel ligand for lymphotoxin β receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer.J Clin Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (236) Google Scholar, 13Zhang M Guo R Zhai Y Fu XY Yang D LIGHT stimulates IFNγ-mediated intercellular adhesion molecule-1 upregulation of cancer cells.Hum Immunol. 2003; 64: 416-426Crossref PubMed Scopus (12) Google Scholar LIGHT and its three receptors are abundant in human placentas. Specific mRNAs as well as proteins have been reported.1Phillips TA Ni J Hunt JS Death-inducing tumour necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines.Placenta. 2001; 22: 663-672Crossref PubMed Scopus (101) Google Scholar, 5Harrop JA McDonnell PC Brigham-Burke M Lyn SD Minton J Tan KB Dede K Spampanato J Silverman C Hensley P DiPrinzio R Emery JG Deen K Eichman C Chabot-Fletcher M Truneh A Young P Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T-cells and inhibits HT29 cell growth.J Biol Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 14Mauri DN Ebner R Montgomery RI Kochel KD Cheung TC Yu GL Ruben S Murphy M Eisenberg RJ Cohen GH Spear P Ware C LIGHT, a new member of the TNF superfamily, and lymphotoxic α are ligands for herpesvirus entry mediator.Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 15Gill RM Ni J Hunt JS Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes.Am J Pathol. 2002; 161: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Immunohistochemical studies have identified LIGHT signals in both the syncytiotrophoblast layer and in the villous mesenchymal cells of term placentas, and immunoblots have detected abundant LIGHT protein in placental lysates. Although light microscopic identification of cytotrophoblast (CTB) cells is difficult in term placentas because of the rarity of this subpopulation, lysates of CTB cells purified from term placentas contain LIGHT.15Gill RM Ni J Hunt JS Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes.Am J Pathol. 2002; 161: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar LIGHT is also found in the amnion membrane, in the fetal mesenchymal cells located between the amnion and chorion membranes, and in the decidua.15Gill RM Ni J Hunt JS Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes.Am J Pathol. 2002; 161: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar All three LIGHT receptors have been localized to specific cells in human term placentas and the extraplacental membranes by immunohistochemistry, and isolated CTB cells have been shown to contain all three receptor mRNAs1Phillips TA Ni J Hunt JS Death-inducing tumour necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines.Placenta. 2001; 22: 663-672Crossref PubMed Scopus (101) Google Scholar and specific proteins.15Gill RM Ni J Hunt JS Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes.Am J Pathol. 2002; 161: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Neither LIGHT nor any of its receptors is present in the extravillous CTB cells that comprise the chorion membrane, indicating that signaling through the LIGHT/LIGHT-R system is not an invariable function of all trophectoderm-derived trophoblast cell subpopulations. The human placenta is also a site of production of IFN-γ and its receptors,16Peyman JA Hammond GL Localization of IFN-gamma receptor in first trimester placenta to trophoblasts but lack of stimulation of HLA-DRA, -DRB, or invariant chain mRNA expression by IFN-gamma.J Immunol. 1992; 149: 2675-2680Crossref PubMed Google Scholar, 17Paulesu L Romagnoli R Cintorino M Ricci MG Garotta G First trimester human trophoblast expresses both interferon-gamma and interferon-gamma-receptor.J Reprod Immunol. 1994; 27: 37-48Crossref PubMed Scopus (67) Google Scholar, 18Paulesu L Romagnoli R Fortino V Cintorino M Bischof P Distribution of type-I interferon-receptors in human first trimester and term placental tissues and on isolated trophoblast cells.Am J Reprod Immunol. 1997; 37: 443-448Crossref PubMed Scopus (16) Google Scholar, 19Veith GL Rice GE Interferon gamma expression during human pregnancy and in association with labour.Gynecol Obstet Invest. 1999; 48: 163-167Crossref PubMed Scopus (44) Google Scholar which is relevant to potential LIGHT-mediated apoptosis because of its known ability to enhance LIGHT-mediated apoptosis.7Zhai Y Guo R Hsu T Yu G Ni J Kwon BS Jiang G Lu J Tan J Ugustus M Carter K Rojas L Zhu F Lincoln C Endress G Xing L Wang S Oh K Gentz R Ruben S Lippman ME Hsieh S Yang D LIGHT, a novel ligand for lymphotoxin β receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer.J Clin Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (236) Google Scholar, 13Zhang M Guo R Zhai Y Fu XY Yang D LIGHT stimulates IFNγ-mediated intercellular adhesion molecule-1 upregulation of cancer cells.Hum Immunol. 2003; 64: 416-426Crossref PubMed Scopus (12) Google Scholar The binding of IFN-γ to its receptor activates two Janus family kinases, Jak1 and Jak2, which phosphorylate the IFN-γ receptor subunit, IFNGR-1, on specific tyrosine residues. These then provide docking sites for the STAT-1 transcriptional activator. The importance of this series of interactions to mobilization of cell death pathways is revealed in studies on cells lacking STAT-1, which show reduced cell death in response to apoptotic stimuli.20Janjua S Stephanou A Latchman DS The C-terminal activation domain of the STAT-1 transcription factor is necessary and sufficient for stress-induced apoptosis.Cell Death Differ. 2002; 9: 1140-1146Crossref PubMed Scopus (27) Google Scholar Local production of LIGHT and its receptors as well as the facilitator cytokine, IFN-γ, raises a critical question about how might placental cells be protected from LIGHT-mediated apoptosis. Although one pathway could be through interference by the soluble receptor, DcR3, other mechanisms are possible. For example, human CTB cells contain a number of proteins in the inhibitor of apoptosis (IAP) family.21Ka H Hunt JS Temporal and spatial patterns of expression of inhibitors of apoptosis in human placentas.Am J Pathol. 2003; 163: 413-422Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Two members of this family, cIAP-1 and cIAP-2 (also known as HIAP-2 and HIAP-1, respectively), interfere with TNF-α-mediated apoptosis by blocking activation of caspase-3.22Rothe M Sarma V Dixit VM Goeddel DV TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40.Science. 1995; 269: 1424-1427Crossref PubMed Scopus (987) Google Scholar, 23Huang H Joazeiro CA Bonfoco E Kamada S Leverson JD Hunter T The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.J Biol Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar, 24Salvesen GS Duckett CS IAP proteins: blocking the road to death's door.Nat Rev Mol Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1591) Google Scholar Apoptosis-inducing ligands in the TNF superfamily are highly homologous, with 27 to 34% identity, suggesting that cIAP-1 and cIAP-2 might also interfere with LIGHT-mediated apoptosis. To address this critical biological question, we investigated LIGHT-mediated apoptosis in purified CTB cells harvested from term placentas, and explored the possibility that DcR3, cIAP-1, and/or cIAP-2 protect these cells. The results indicate that LIGHT is prevented from killing CTB cells by both soluble receptor and cIAP-2. Simultaneous exposure to IFN-γ overcomes this latter protective effect, suggesting that in cases of infection, placental cells may become more susceptible to LIGHT-mediated damage. Recombinant human LIGHT (rhLIGHT), rhDcR3/TR6-Fc, and rhIFN-γ were purchased from R&D systems (Minneapolis, MN). The characteristics of these proteins have been reported.5Harrop JA McDonnell PC Brigham-Burke M Lyn SD Minton J Tan KB Dede K Spampanato J Silverman C Hensley P DiPrinzio R Emery JG Deen K Eichman C Chabot-Fletcher M Truneh A Young P Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T-cells and inhibits HT29 cell growth.J Biol Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Pitti RM Marsters SA Lawrence DA Roy M Kischkel FC Dowd P Huang A Donahue CJ Sherwood SW Baldwin DT Godowski PJ Wood WI Gurney AL Hillan KJ Cohen RL Goddard AD Botstein D Ashkenazi A Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer.Nature. 1998; 396: 699-702Crossref PubMed Scopus (693) Google Scholar, 14Mauri DN Ebner R Montgomery RI Kochel KD Cheung TC Yu GL Ruben S Murphy M Eisenberg RJ Cohen GH Spear P Ware C LIGHT, a new member of the TNF superfamily, and lymphotoxic α are ligands for herpesvirus entry mediator.Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 25Gray PW Leung DW Pennica D Yelverton E Najarian R Simonsen CC Derynck R Sherwood PJ Wallace DM Berger SL Levinson AD Goeddel DV Expression of human immune interferon cDNA in E. coli and monkey cells.Nature. 1982; 295: 503-508Crossref PubMed Scopus (552) Google Scholar Chrompure human IgG1 Fc fragment (Hu-Fc) was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Human placentas from cesarean section deliveries performed at term for benign conditions or to relieve fetal distress were obtained under a protocol approved by the Human Subjects Committee of the University of Kansas Medical Center. Samples were taken randomly from floating villi after dissecting and discarding the fibrous plate. Underlying pathology was not evident on either gross or histological examination of samples. CTB cells were purified from term placenta by enzymatic digestion, gradient centrifugation, and immunomagnetic purification using a monoclonal antibody against HLA-A,B,C (W6/32, no. HB95; American Type Culture Collection, Manassas, VA), as described.16Peyman JA Hammond GL Localization of IFN-gamma receptor in first trimester placenta to trophoblasts but lack of stimulation of HLA-DRA, -DRB, or invariant chain mRNA expression by IFN-gamma.J Immunol. 1992; 149: 2675-2680Crossref PubMed Google Scholar, 26Kliman HJ Nestler JE Sermasi E Sanger JM Strauss III, JM Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.Endocrinology. 1986; 118: 1567-1582Crossref PubMed Scopus (1451) Google Scholar, 27Douglas GC King BF Isolation of pure villous cytotrophoblasts from term human placenta using immunomagnetic microspheres.J Immunol Methods. 1989; 119: 259-268Crossref PubMed Scopus (187) Google Scholar To assess purity of CTBs, Cytospin (Shandon, Pittsburgh, PA) preparations of cells were analyzed by immunohistochemical staining using mouse anti-pan cytokeratin (Lu-5; Biogenex, San Ramon, CA), which detects all trophoblast cells, and mouse anti-CD14 (Zymed, San Francisco, CA), which detects contaminating macrophages. Less than 1% of cells were immunoreactive for CD14. We further qualified the purity of our samples by immunoblotting and immunohistochemical staining using mouse anti-β-hCG (clone CG05; Neomarkers, Fremont, CA) to detect any contaminating syncytial fragments.26Kliman HJ Nestler JE Sermasi E Sanger JM Strauss III, JM Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.Endocrinology. 1986; 118: 1567-1582Crossref PubMed Scopus (1451) Google Scholar Less than 4% of the cytospin-prepared cells demonstrated immunoreactivity for β-hCG suggesting very few contaminating syncytial fragments. β-hCG protein was not detectable by immunoblot in these samples indicating that highly pure populations of CTBs were isolated. The human colon adenocarcinoma cell line, HT-29, was purchased from American Type Culture Collection. HT-29 cells and CTB cells were grown in their respective complete growth media.12Yu K Kwon B Ni J Zhai Y Ebner R Kwon B A newly identified member of the tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.J Biol Chem. 1999; 274: 13733-13736Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 28Yui J Garcia-Lloret M Wegmann TG Guilbert LJ Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts.Placenta. 1994; 15: 819-835Crossref PubMed Scopus (339) Google Scholar After a 12-hour incubation at 37°C in 5% CO2, cells were washed once with media to remove nonadherent cells, and were cultured with or without cytokines for 2 or 4 days as indicated in the Results section and in the figure legends. Purified CTB cells (1 × 105/well) were treated with serial dilutions of rhLIGHT alone or together with rhIFN-γ (100 U/ml), rhTR6-Fc (4 μg/ml), Hu-Fc (4 μg/ml), or control medium in a total volume of 100 μl of serum-freecomplete Iscove's-modified Dulbecco's (IMDM) medium(Cellgro, Herndon, VA) in 96-well flat-bottom microplates. The CellTiter 96 nonradioactive assay (Promega, Madison, WI) was used to assess cell viability. In brief, the cells were incubated with modulators or control medium for 48 or 96 hours then the indicator dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] was added and cultures were continued for 6 hours at 37°C, 5% CO2. Subsequently, 100 μl of stop solution was added and, after incubating the microplates overnight in a humidified chamber to facilitate solubilization of formazan crystals, the A570 was measured by spectrophotometer (Elx-808; Bio-Tek Instruments Inc., Winooski, VT). HT-29 cells (5 × 103/well) grown in medium containing 10 U/ml rhIFN-γ served as a positive control. In some experiments these cells were incubated with serial dilutions of rhLIGHT in complete Dulbecco's modified Eagle's medium (Cellgro), as above for CTB cells. Protein samples were prepared from lysed cells by standard methods as previously reported.15Gill RM Ni J Hunt JS Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes.Am J Pathol. 2002; 161: 2011-2017Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Protein quantification was performed using the manufacturer's protocol (Bio-Rad Laboratories, Richmond, CA). Twenty-five μg of total protein were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, then were electrophoretically transferred to 0.2-μm-supported nitrocellulose (Schleicher & Schuell, Keene, NH), for 75 minutes at 100 V (27°C) in Tris/glycine buffer (Bio-Rad Laboratories). For detection of cIAP-2, caspase-3, and actin, 0.75 μg/ml of rabbit anti-human cIAP-2 antibody (H-85; Santa Cruz Biotechnology, Santa Cruz, CA), 1 μg/ml of rabbit anti-human caspase-3 antibody (H-277; Santa Cruz Biotechnology), or 1:5000 rabbit anti-human actin (Sigma Chemical Co., St. Louis, MO), respectively, was prepared in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T) and 2% nonfat milk (Bio-Rad Laboratories) and incubated with the membranes for ∼15 hours at 4°C. Membranes were washed in TBS-T and incubated with 0.08 μg/ml of goat anti-rabbit Ig-horseradish peroxidase conjugate (Jackson Immunoresearch Laboratories) for 1 hour at 27°C. Membranes were washed in TBS-T and subjected to chemiluminescent detection (Pierce, Rockford, IL). For these experiments, HT-29 cells were used as a model system because in preliminary experiments, solutions containing a morpholino delivery agent reduced the viability of normal CTB cells. The Gene Tools LLC (Philomath, OR) special delivery protocol was used after optimization of morpholino delivery. Briefly, a 25-mer, fluoresceinated morpholino oligomer with the sequence, 5′-TGCTGTTTTCTACTATGTTCATAAT-3′ and a fluoresceinated standard control oligomer with the sequence, 5′-CCTCTTACCTCAGTTACAATTTATA-3′ were generated by Gene Tools LLC. Oligomers were incubated with the delivery agent, ethoxylated polyethylenimine, for 20 minutes and then mixed with serum-free media. HT-29 cells (5 × 103 cells/well in 96-well plates and 4 × 105 cells/well in 6-well plates) were incubated for 12 hours in complete Dulbecco's modified Eagle's medium at 37°C, 5% CO2. Cells were washed with D-PBS (Sigma Chemical Co.) and the morpholino/ethoxylated polyethylenimine/Dulbecco's modified Eagle's medium mixture was added. After incubation for 3 hours at 37°C, 5%CO2, the delivery mixture was aspirated and fresh complete Dulbecco's modified Eagle's medium (in some cases with cytokines) was added. After 72 hours cells were photographed using a fluorescent microscope. After 96 hours the cells were lysed to acquire protein or the MTT assay was conducted on cells in wells using the protocol described above. All quantitative data were subjected to analysis of variance or paired t-test as appropriate, using statistical analysis systems (SAS Institute, Inc., Cary, NC). Preplanned comparisons were conducted to determine differences between controls and treatments. Data are presented as means ± SEM. To investigate the ability of rhLIGHT to initiate cell death in CTB cells, purified preparations of these cells obtained from term placentas were treated with medium alone or with increasing concentrations of the cytokine. Viability was measured using the MTT assay. Because CTB cells do not proliferate in vitro, MTT values closely approximate the number of viable cells.26Kliman HJ Nestler JE Sermasi E Sanger JM Strauss III, JM Purification, characterization, and in vitro differen
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