Differential Expression of LIGHT and Its Receptors in Human Placental Villi and Amniochorion Membranes
2002; Elsevier BV; Volume: 161; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64479-3
ISSN1525-2191
AutoresRyan M. Gill, Jian Ni, Joan S. Hunt,
Tópico(s)Systemic Lupus Erythematosus Research
ResumomRNA encoding LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a member of the tumor necrosis factor superfamily of ligands, as well as mRNAs encoding LIGHT receptors [HVEM, LTβR, and TR6 (DcR3)] are present in placentas and cytotrophoblast cells at term. To establish translation of these messages and determine directions for functional studies, term placentas, amniochorion membranes, and purified cytotrophoblast cells were evaluated by immunoblotting and immunohistochemistry. Ligand and receptor proteins were identified in lysates from all three sources although the soluble receptor, TR6, was scarce in placentas and all receptors were in low abundance in cytotrophoblast cells. These results were confirmed and cell type-specific expression was documented by immunohistochemistry. Ligand and receptor proteins were differentially expressed according to cell type. For example, HVEM was identified on syncytiotrophoblast but not in villous mesenchymal cells; amnion epithelial cells were positive for all proteins whereas chorion membrane cytotrophoblasts exhibited none. Because LIGHT is a powerful cytokine that can alter gene expression and promote apoptosis, these experiments suggest that ligand-receptor interactions may critically influence structural and functional aspects of human placentas through as yet undefined autocrine/paracrine pathways. mRNA encoding LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a member of the tumor necrosis factor superfamily of ligands, as well as mRNAs encoding LIGHT receptors [HVEM, LTβR, and TR6 (DcR3)] are present in placentas and cytotrophoblast cells at term. To establish translation of these messages and determine directions for functional studies, term placentas, amniochorion membranes, and purified cytotrophoblast cells were evaluated by immunoblotting and immunohistochemistry. Ligand and receptor proteins were identified in lysates from all three sources although the soluble receptor, TR6, was scarce in placentas and all receptors were in low abundance in cytotrophoblast cells. These results were confirmed and cell type-specific expression was documented by immunohistochemistry. Ligand and receptor proteins were differentially expressed according to cell type. For example, HVEM was identified on syncytiotrophoblast but not in villous mesenchymal cells; amnion epithelial cells were positive for all proteins whereas chorion membrane cytotrophoblasts exhibited none. Because LIGHT is a powerful cytokine that can alter gene expression and promote apoptosis, these experiments suggest that ligand-receptor interactions may critically influence structural and functional aspects of human placentas through as yet undefined autocrine/paracrine pathways. The placenta and extraplacental membranes, which are derived from the implanted blastocyst, are tolerated by the mother throughout pregnancy. Mechanisms underlying maternal acceptance of these semi-allogeneic tissues are as yet unclear but are believed to include immune modulation by tumor necrosis factor α (TNFα) and its closely related superfamily members, FasL and TNF related apoptosis inducing ligand (TRAIL).1Phillips TA Ni J Pan G Ruben SM Wei Y Pace J Hunt JS TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege.J Immunol. 1999; 162: 6053-6059PubMed Google Scholar, 2Runic R Lockwood CJ Ma Y Dipasquale B Guller S Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival.J Clin Endocrinol. 1996; 81: 3119-3122Google Scholar, 3Hunt JS Vassmer D Ferguson TA Miller L Fas ligand is positioned in mouse uterus and placenta to prevent trafficking of activated leukocytes between the mother and the conceptus.J Immunol. 1997; 158: 4122-4128PubMed Google Scholar LIGHT [homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HVEM, a receptor expressed by T lymphocytes], a newly identified member of this growing family of molecules, is reportedly transcribed in human placentas.4Phillips TA Ni J Hunt JS Death-inducing tumor necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentas, cytotrophoblasts, placental macrophages, and placental cell lines.Placenta. 2001; 22: 663-672Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 5Mauri 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 (645) Google Scholar, 6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar It is not known whether these messages are translated and no potential functions have been suggested. Much is known of the involvement of LIGHT in the human immune system. LIGHT, as with other members in this superfamily, forms a homotrimer.6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar, 7Rooney 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: 14307-14315Crossref PubMed Scopus (196) Google Scholar LIGHT has both cytosolic and membrane-bound forms.8Granger 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: 5122-5128PubMed Google Scholar The membrane-bound form may be cleaved by matrix metalloproteinases to act as a soluble protein.8Granger 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: 5122-5128PubMed Google Scholar, 9Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, co-stimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110PubMed Google Scholar, 10Morel 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 down-regulates its own receptors.J Immunol. 2000; 165: 4397-4404PubMed Google Scholar Soluble LIGHT enhances mixed lymphocyte reactions (MLR)6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar and is a major mediator of graft-versus-host disease.11Tamada 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 (272) Google Scholar LIGHT is reportedly required for dendritic cell-mediated primary allogeneic T cell responses,9Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, co-stimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110PubMed Google Scholar is known to induce T lymphocyte proliferation and secretion of Th-1 cytokines,9Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, co-stimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110PubMed Google Scholar, 11Tamada 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 (272) Google Scholar and participates in the induction of cell-mediated immunity.9Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, co-stimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110PubMed Google Scholar, 11Tamada 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 (272) Google Scholar, 12Morel Y Truneh A Sweet RW Olive D Costello RT The TNF superfamily members LIGHT and CD154 (CD40 ligand) co-stimulate induction of dendritic cell maturation and elicit specific CTL activity.J Immunol. 2001; 167: 2479-2486PubMed Google Scholar LIGHT, as with most members of the TNF superfamily, has the ability to trigger apoptosis in some tumor cells in culture and in vivo.11Tamada 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 (272) Google Scholar, 13Chen 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: 38794-38801Crossref PubMed Scopus (43) Google Scholar The increasingly varied roles postulated for LIGHT indicate that function will depend on receptor expression and cytokine environment. LIGHT binds to three receptors, TR6 (DcR3), HVEM, and LTβR5Mauri 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 (645) Google Scholar, 6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar, 7Rooney 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: 14307-14315Crossref PubMed Scopus (196) Google Scholar, 14Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 15Yu K Kwon B Ni J Zhai Y Ebner R Kwon BS A newly identified member of the tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.J Biol Chem. 1999; 274: 13733-13736Crossref PubMed Scopus (343) Google Scholar (Figure 1). TR6 is a soluble receptor that competes with HVEM for LIGHT binding,15Yu K Kwon B Ni J Zhai Y Ebner R Kwon BS A newly identified member of the tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.J Biol Chem. 1999; 274: 13733-13736Crossref PubMed Scopus (343) Google Scholar, 16Zhang J Salcedo TW Wan X Ullrich S Hu B Gregorio T Feng P Qi S Chen H Cho YH Li Y Moore PA Wu J Modulation of T-cell responses to alloantigens by TR6/DcR3.J Clin Invest. 2001; 107: 1459-1468Crossref PubMed Scopus (108) Google Scholar abrogates LIGHT-mediated apoptosis,15Yu K Kwon B Ni J Zhai Y Ebner R Kwon BS A newly identified member of the tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.J Biol Chem. 1999; 274: 13733-13736Crossref PubMed Scopus (343) Google Scholar, 16Zhang J Salcedo TW Wan X Ullrich S Hu B Gregorio T Feng P Qi S Chen H Cho YH Li Y Moore PA Wu J Modulation of T-cell responses to alloantigens by TR6/DcR3.J Clin Invest. 2001; 107: 1459-1468Crossref PubMed Scopus (108) Google Scholar, 17Roth W Isenmann S Nakamura M Platten M Wick W Kleihues P Bahr M Ohgaki H Ashkenazi A Weller M Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis.Cancer Res. 2001; 61: 2759-2765PubMed Google Scholar prolongs the survival time of mice receiving heart allografts,16Zhang J Salcedo TW Wan X Ullrich S Hu B Gregorio T Feng P Qi S Chen H Cho YH Li Y Moore PA Wu J Modulation of T-cell responses to alloantigens by TR6/DcR3.J Clin Invest. 2001; 107: 1459-1468Crossref PubMed Scopus (108) Google Scholar and favors Th-2 lymphokine production in a MLR.16Zhang J Salcedo TW Wan X Ullrich S Hu B Gregorio T Feng P Qi S Chen H Cho YH Li Y Moore PA Wu J Modulation of T-cell responses to alloantigens by TR6/DcR3.J Clin Invest. 2001; 107: 1459-1468Crossref PubMed Scopus (108) Google Scholar HVEM is present on T cells and is important in LIGHT-mediated T cell costimulation.5Mauri 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 (645) Google Scholar, 6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar, 9Tamada K Shimozaki K Chapoval AI Zhai Y Su J Chen S Hsieh S Nagata S Ni J Chen L LIGHT, a TNF-like molecule, co-stimulates T cell proliferation and is required for dendritic cell-mediated allogenic T cell response.J Immunol. 2000; 164: 4105-4110PubMed Google Scholar, 11Tamada 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 (272) Google Scholar, 18Harrop JA Reddy M Dede K Brigham-Burke M Lyn S Tan KB Silverman C Eichman C DiPrinzio R Spampanato J Porter T Holmes S Young PR Truneh A Antibodies to TR2 (herpesvirus entry mediator), a new member of the TNF receptor superfamily, block T cell proliferation, expression of activation markers, and production of cytokines.J Immunol. 1998; 161: 1786-1794PubMed Google Scholar, 19Kwon BS Tan KB Ni J Oh-OK-Kwi Lee ZH Kim KK Kim Y-J Wang S Gentz R Yu G-L Harrop J Lyn SD Silverman C Porter TG Truneh A Young PR A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation.J Biol Chem. 1997; 272: 14272-14276Crossref PubMed Scopus (252) Google Scholar LTβR is not found on T or B cells but is found on stromal cells and in some tumor cells, where it transduces apoptotic signals.5Mauri 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 (645) Google Scholar, 6Harrop 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: 27548-27556Crossref PubMed Scopus (201) Google Scholar, 7Rooney 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: 14307-14315Crossref PubMed Scopus (196) Google Scholar, 20Browning JL Sizing ID Lawton P Bourdon PR Rennert PD Majeau GR Ambrose CM Hession C Miatkowski K Griffiths DA Ngam-ek A Meier W Benjamin CD Hochman PS Characterization of lymphotoxin-αβ complexes on the surface of mouse lymphocytes.J Immunol. 1997; 159: 3288-3298PubMed Google Scholar The messages for all three receptors have been detected in the human placenta, but translation and cellular localization remain unknown.4Phillips TA Ni J Hunt JS Death-inducing tumor necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentas, cytotrophoblasts, placental macrophages, and placental cell lines.Placenta. 2001; 22: 663-672Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 14Montgomery RI Warner MS Lum BJ Spear PG Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 21Pitti 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 (678) Google Scholar, 22Degli-Esposti MA Davis-Smith T Din W Smolak PJ Goodwin RG Smith CA Activation of the lymphotoxin β receptor by cross-linking induces chemokine production and growth arrest in A375 melanoma cells.J Immunol. 1997; 158: 1756-1762PubMed Google Scholar In this study, our goal was to identify translated proteins in the LIGHT ligand/receptor system and establish their cellular localization in term placentas, amniochorion, and purified cytotrophoblast cells. We have uncovered potential autocrine, juxtacrine, and paracrine signaling pathways that may be important in placental structure and function. All reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. The human trophoblast-derived choriocarcinoma cell line, JAR, was purchased from the American Type Culture Collection (ATCC no. HTB-144) (Manassas, VA). Human placentas were obtained from normal cesarean section delivery at term, in accordance with a protocol approved by the Human Subjects Committee of the University of Kansas Medical Center. Samples were taken randomly from the floating villi and reflected amniochorion for further analysis. Underlying pathology was not evident on histological examination of the samples. Cytotrophoblasts were isolated from term placenta by enzymatic digestion and gradient centrifugation as described.23Kliman 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 (1399) Google Scholar, 24Douglas GC King BF Isolation of pure villous cytotrophoblasts from term human placenta using immunomagnetic microspheres.J Immunol Methods. 1989; 119: 259-268Crossref PubMed Scopus (181) Google Scholar Cytotrophoblasts were further purified from this cell suspension by removal of HLA-A,B,C-positive cells using the monoclonal antibody W6/32 (ATCC no. HB95) and goat anti-mouse Ig-conjugated magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA) according to the protocol recommended by the manufacturer. To assess purity of cytotrophoblasts, Cytospin (Shandon, Pittsburgh, PA) preparations of cells were analyzed by immunohistochemical staining using mouse anti-pan cytokeratin (Lu-5) (Bio Genex, San Ramon, CA), which detects all trophoblast cells, and mouse anti-CD14 (Zymed, San Francisco, CA), which detects contaminating macrophages. Less than one percent of the cells was immunoreactive for CD14. We further qualified the purity of our samples by immunoblotting and immunohistochemical staining using mouse anti-βhCG (clone CG05) (Neomarkers, Fremont, CA) to detect any contaminating syncytial fragments.23Kliman 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 (1399) Google Scholar Less than 4% of the cytospin-prepared cells demonstrated immunoreactivity for βhCG, suggesting very few contaminating syncytial fragments. βhCG protein was not detectable by immunoblot in these samples indicating that highly pure populations of cytotrophoblasts were isolated. Purified cells were immediately lysed for protein and RNA preparations. Whole placenta (n = 1), cytotrophoblasts purified from a different placenta (n = 1), and JAR cells were analyzed. RNA was isolated from 8 × 106 cells, or 100 mg tissue, using 1 ml TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. Total RNA was treated with DNase I (AMP-D1) according to the manufacturer's instructions. First-strand cDNA synthesis was then performed using this treated total RNA and Moloney murine leukemia virus reverse transcriptase (Life Technologies) according to the enzyme manufacturer's protocol. Ten μl of each 1:5 diluted cDNA sample were used in the subsequent 50 μl PCR reaction for the LIGHT primer pair. Four μl (50 μl reaction) of cDNA were used for the β-actin primer pair. Primers were derived from human LIGHT cDNA (forward: 5′-CAAGAGCGAAGGTCTCACGAGGTC-3′ and reverse 5′-TCACACCATGAAAGCCCCGAAGTAAG-3′) and human β-actin cDNA (forward 5′-CACCCCGTGCTGCTGACCGAGGCC-3′ and reverse 5′-CCACACGGAGTACTTGCGCTCAGG-3′) sequences, respectively (National Center for Biotechnology Information databases), using the PrimerSelect program (DNASTAR Inc., Madison, WI), and were synthesized by Gemini Biotech (Alachua, FL). The amplification schedule for LIGHT was: 94°C for 5 minutes; 38 cycles of 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 2 minutes; and 72°C for 7 minutes. The amplification schedule for β-actin was: 94°C for 45 seconds; 30 cycles of 94°C for 45 seconds; 60°C for 30 seconds; 72°C for 2 minutes; and 72°C for 7 minutes. All reactions were conducted in a GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, CA). Products were analyzed by electrophoresis of 10 μl of each reaction through 2% agarose (Amresco 3:1, Solon, OH), 0.05 μg/ml ethidium bromide, followed by UV transillumination. The correct sequence of product derived from LIGHT primers was confirmed by dRhodamine terminator cycle sequencing using the ABI 310 DNA sequencer (Applied Biosystems) in the Center for Reproductive Sciences at the University of Kansas Medical Center. Protein samples were prepared from homogenized placental tissue (n = 3) and lysed primary cells purified from the same placentas (n = 3). Protein quantification was performed as per the manufacturer's protein assay protocol (Bio-Rad Laboratories, Richmond, CA). Fifty μg of total protein were separated by electrophoresis on 15% polyacrylamide SDS-PAGE gels. Proteins were electrophoretically transferred to 0.2 μm-supported nitrocellulose (Schleicher & Schuell, Keene, NH), for 75 minutes at 100 volts (27°C) in Na2CO3/NaHCO3 buffer (pH 9.9). For detection of specific proteins, primary antibodies were prepared in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T) and 3% nonfat milk (Bio-Rad Laboratories) and incubated on the membranes for approximately 15 hours at 4°C (Table 1). Membranes were washed in TBS-T and incubated with the respective secondary antibody-enzyme conjugates for 1 hour at 27°C (Table 1). Membranes were washed in TBS-T and subjected to chemiluminescent detection (Pierce, Rockford, IL).Table 1Summary of Antibodies UtilizedHuman antigenSpeciesIsotypeConjugationImmunoblot concentrationImmunohistochemistry concentrationSource*J. Ni: Dr. J. Ni, Human-Human Hybridoma, Inc., Bethesda, MD; SCB: Santa Cruz Biotechnology, Santa Cruz CA; BDP: BD Pharmingen, San Diego CA; Vector: Vector, Burlingame CA; JL: Jackson Immunoresearch Laboratories, West Grove, PA.LIGHTMouseIgG1 (monoclonal)–2.00 μg/ml–J. NiLIGHT (C-20)GoatIgG (polyclonal)––10.0 μg/mlSCBTR6/DcR3RabbitIgG (polyclonal)–0.10 μg/ml10.0 μg/mlJ. NiHVEM (N-19)GoatIgG (polyclonal)–0.40 μg/ml10.0 μg/mlSCBLTβR (N-15)GoatIgG (polyclonal)–0.20 μg/ml10.0 μg/mlSCBControlMouseIgG1 (monoclonal)–2.00 μg/ml10.0 μg/mlBDPControlGoatIgG (polyclonal)–0.40 μg/ml10.0 μg/mlVectorControlRabbitIgG (polyclonal)–0.10 μg/ml10.0 μg/mlVectorMouse IgGGoatIgG (polyclonal)HRP†Horseradish peroxidase.0.08 μg/ml–BDPGoat IgGRabbitIgG (polyclonal)HRP0.08 μg/ml–JLRabbit IgGGoatIgG (polyclonal)HRP0.08 μg/ml–JLMouse IgGHorseIgG (polyclonal)Biotin–5.0 μg/mlVectorGoat IgGHorseIgG (polyclonal)Biotin–5.0 μg/mlVectorRabbit IgGGoatIgG (polyclonal)Biotin–5.0 μg/mlVector* J. Ni: Dr. J. Ni, Human-Human Hybridoma, Inc., Bethesda, MD; SCB: Santa Cruz Biotechnology, Santa Cruz CA; BDP: BD Pharmingen, San Diego CA; Vector: Vector, Burlingame CA; JL: Jackson Immunoresearch Laboratories, West Grove, PA.† Horseradish peroxidase. Open table in a new tab One cm3 samples were surgically excised, at random from 4 placentas, from both cotyledons embedded in basal plate and amniochorion membranes, fixed in 4% paraformaldehyde, and then embedded in paraffin blocks. Paraffin sections were deparaffinized in Histoclear (National Diagnostics, Atlanta, GA) and rehydrated in an ethanol gradient. Tissue sections were blocked with 10% normal horse or goat serum for 1 hour at 27°C. The blocker was removed and primary antibody was added (Table 1). The tissue sections were incubated for 15 hours at 4°C. Washes were performed in phosphate-buffered saline (PBS) with 0.3% Tween-20 (PBS-T) and the peroxidase block was performed with 0.5% H2O2 in methanol for 30 minutes. The respective biotinylated secondary antibody was added and incubated for 1 hour at 27°C (Table 1). Following washes with PBS-T, the streptavidin peroxidase label (Zymed) was incubated for 10 minutes at 27°C. The sections were washed with PBS and the 3-amino-9-ethylcarbozole in N,N-dimethylformamide (AEC) color development substrate (Zymed) was incubated for 10 minutes at 27°C. Sections were washed in water and counterstained with Mayer's hematoxylin. We used RT-PCR to test for LIGHT message in placenta, purified cytotrophoblasts, and JAR cells (a choriocarcinoma-derived cell line). We detected LIGHT message in all three samples (Figure 2). Our findings agree with previous reports that placenta and JAR cells contain message for LIGHT4Phillips TA Ni J Hunt JS Death-inducing tumor necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentas, cytotrophoblasts, placental macrophages, and placental cell lines.Placenta. 2001; 22: 663-672Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 5Mauri 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 (645) Google Scholar, 7Rooney 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: 14307-14315Crossref PubMed Scopus (196) Google Scholar and we demonstrate, for the first time, LIGHT message in purified cytotrophoblast cells. Immunoblotting was used to establish the presence of LIGHT protein in human term placentas. LIGHT protein was detected in lysates from three placentas as well as in their matching amniochorion membranes (Figure 3). LIGHT protein was also detected in lysates of purified cytotrophoblast cells (Figure 3). LIGHT protein was localized in sections of placentas and fetal membranes using immunohistochemistry. LIGHT protein was observed in placental mesenchymal cells (Figure 4A) and in the syncytiotrophoblast layer (Figure 4A). We noted that a small proportion of placental syncytiotrophoblast demonstrated patchy immunoreactivity with anti-LIGHT (< 1 to 2%, data not shown). In the amniochorion, LIGHT protein was strongly expressed by the amnion and fetal mesenchymal cells and was detectable in the decidua capsularis (Figure 4B). LIGHT protein was not detected in the chorion (Figure 4B). The negative cont
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