DOCSLIB.ORG

Role of Catalase in Monocytic Differentiation of U937 Cells by TPA: Hydrogen Peroxide As a Second Messenger

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Role of Catalase in Monocytic Differentiation of U937 Cells by TPA: Hydrogen Peroxide As a Second Messenger Leukemia (2009) 23, 761–769 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger T Yamamoto1, N Sakaguchi1, M Hachiya1, F Nakayama1, M Yamakawa2 and M Akashi1 1Department of Radiation Emergency Medicine, The Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba-city, Chiba, Japan and 2Department of Pathology, Yamagata University Faculty of Medicine, Yamagata-city, Yamagata, Japan Human promonocytic cell line U937 cells can be induced to atherosclerosis.4,5 Internalization of foreign substances by differentiate into macrophages by treatment with 12-O-tetra- macrophages is mediated through distinct surface receptors that decanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and recognize their targets, such as microorganisms, tumor cells and CD36, with concomitant morphological changes. Moreover, cellular debris. Following phagocytosis, macrophages synthe- TPA enhanced reactive oxygen species (ROS) generation in size and release reactive oxygen species (ROS), a process called these cells, and phagocytic ability was also stimulated during ‘respiratory burst.’ Thus, the generation of ROS is important differentiation. The antioxidant agent N-acetyl-L-cysteine inhib- for much of the microbicidal and antitumor activity of ited the TPA-induced differentiation of U937 cells. TPA treat- macrophages. ment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H O )to Since the discovery of ROS, primary focus has been directed 2 2 at the oxidative damage to biologic macromolecules including H2O and O2. In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of proteins as well as DNA and lipids. Potential sources of ROS in superoxide into H2O2 and O2 without affecting the levels of phagocytic cells are nicotinamide adenine dinucleotide phos- copper-zinc superoxide dismutase or glutathione peroxidase 1, phate (NADPH) oxidases and the mitochondrial electron which removes H2O2 using glutathione as substrate. Treatment transport chain.6–8 NADPH oxidases are plasma membrane- of U937 cells with catalase inhibited the enhancement of ROS associated enzymes found in a variety of cells. This enzyme generation induced by TPA, and blocked the TPA-induced À differentiation of U937 cells. Human promyelocytic cell line catalyzes the production of O2 by the one-electron reduction HL60 cells were also induced to differentiate into macrophages of oxygen using NADPH as electron donor. On the other hand, by TPA. However, HP100-1 cells, its variant cell line over- it has also been shown that ROS are generated in a variety expressing catalase, were resistant to TPA-induced differentia- of cells other than phagocytes, and that ROS are involved tion. Our results suggest that catalase inhibits monocytic in normal physiologic signaling including cell proliferation or differentiation by TPA; the decrease in catalase level and the survival.6,9,10 Previously, we showed that ROS regulate cell accumulation of H2O2 are significant events for monocyte/ 11 macrophage differentiation by TPA. growth in human promyelocytes HL60. Leukemia (2009) 23, 761–769; doi:10.1038/leu.2008.353; Recently, there is a growing body of evidence regarding the published online 18 December 2008 ROS signal playing an important role in hematopoietic Keywords: catalase; hydrogen peroxide; monocytic differentiation; systems. The regulation of oxidative stress by ROS has been TPA shown to be necessary for the maintenance of the capacity of hematopoietic stem cells to self-renew.12 Activation with granulocyte-macrophage colony-stimulating factor and other growth factors, including interleukin-3, stem cell factor and thrombopoietin, have been shown to be associated with 13,14 Introduction alterations in the levels of hydrogen peroxide (H2O2). Moreover, it has been shown that differentiation of monocytes Stem cells form hematopoietic progenitor cells with a more into macrophages leads to an increase in the baseline oxidant limited differentiation potential. These progenitors yield blood tone.15,16 Furthermore, studies have suggested that ROS may be precursors that cause unilineage differentiation and production involved in the regulation of neuronal or cardiomyocyte of mature blood cells, including red blood cells, megakaryo- differentiation.17,18 However, the roles of ROS remain unclear, cytes, monocytes/macrophages, granulocytes and lympho- and the type of ROS involved in the differentiation of 1–3 cytes. Monocytes enter the circulation and migrate into hematopoietic cells is not yet known. various tissues, where they terminally differentiate into macro- Leukemic cell lines, such as U937 and HL60, are blocked phages in response to stimuli. Macrophages are phagocytes that at different stages of differentiation.19 These cells can differ- play important roles in the immune system, especially in the entiate to more mature cells following exposure to various host defense against infection or malignancy, and they also agents in vitro. Therefore, these leukemic cell lines provide participate in the development of a variety of diseases including not only the opportunity to study the induction of cell differentiation but also the mechanisms for cell maturation. Correspondence: Dr M Akashi, Department of Radiation Emergency Using these cell lines, we studied the roles of ROS in phagocytic Medicine, The Research Center for Radiation Emergency Medicine, differentiation induced by 12-O-tetradecanoylphorbol-13-acet- National Institute of Radiological Sciences, Chiba-city, Chiba 263- ate (TPA), a member of phorbol esters. We found that the levels 8555, Japan. E-mail: [email protected] of catalase are reduced and H2O2 acts as a second messenger Received 26 July 2008; revised 18 September 2008; accepted 14 in TPA signaling essential for the differentiation of phaogocytic October 2008; published online 18 December 2008 cells. Role of catalase in monocytic differentiation T Yamamoto et al 762 Materials and methods cells were combined and were stained with either anti-CD11b or –CD36 antibody-FITC (Immnotech, Marseille, France) at 4 1C Reagents for 30 min. Fluorescent intensities were analyzed by FACSCa- 12-O-tetradecanoylphorbol-13-acetate was purchased from libur (Becton, Dickinson and Company, Franklin Lakes, NJ, Sigma-Aldrich Co. (St Louis, MO, USA). TPA was dissolved in USA). To determine nonspecific binding, FITC-conjugated IgG1 dimethyl sulfoxide. When TPA was added to cells, the isotype antibody was used, and its intensities were subtracted concentration of dimethyl sulfoxide was less than 0.1%. from those of cells with CD11b or CD36. N-acetyl-L-cysteine (NAC) and human catalase were purchased from Calbiochem (La Jolla, CA, USA). NAC was dissolved in water at a concentration of 1 M and stored at À20 1C until use. Analysis of phagocytic activity Superoxide dismutase (SOD) (Cu/Zn type) from bovine The cells were incubated at 37 1C for 10 min with luminol- erythrocytes was purchased from Wako Pure Chemical bound microbeads (Kamakura Techno-Science Inc., Kanagawa, 20 Industries Ltd (Osaka, Japan). Anti-catalase rabbit polyclonal Japan), as directed by the manufacturer’s protocol. Chemilu- antibody was from Calbiochem. Anti-manganese SOD minescence, which was generated by the reaction between (MnSOD) rabbit polyclonal antibody was a product of Stressgen internalized microbeads-bound luminol and intracellular ROS, (Ann Arbor, MI, USA). Anti-copper-zinc SOD (CuZnSOD) rabbit was measured for 1 min using a Lumat LB 9507 Luminometer polyclonal antibody was purchased from Santa Cruz Biotech- (Berthold Technologies, Bad Wildbad, Germany). nology Inc. (Santa Cruz, CA, USA). Anti-glutathione peroxidase 1 (GSH-Px1) sheep polyclonal antibody was from Abcam plc. (Cambridge, UK). Anti-glyceraldehyde-3-phosphate dehydrogen- Intracellular levels of ROS Intracellular ROS were measured according to the methods ase (G3PDH) rabbit polyclonal antibody was purchased from 21 described previously. Briefly, cells were incubated with 5 mM Trevigen (Gaithersburg, MD, USA). Horseradish peroxidase- CM-H DCFDA (5-6-chloromethyl-20,70-dichlorodihydrofluores- conjugated anti-rabbit immunoglobulin G (IgG) antibody was a 2 cein diacetate, acetyl ester) (Molecular Probes, Eugene, OR, product of Amersham Biosciences (Buckinghamshire, UK). USA) in phosphate-buffered saline for 15 min at 37 1C in the Horseradish peroxidase-conjugated anti-sheep/goat IgGs dark. During incubation, the acetate groups on CM-H DCFDA antibody was purchased from Binding Site LTD (Birmingham, 2 are cleaved by intracellular esterase, trapping the probe inside UK). cells. CM-H2DCFDA was chosen because it showed better retention in cells than other derivatives. Endogenous ROS levels Cells and cell culture were analyzed by FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with excitation and U937 cells, derived from a diffuse human histiocytic lymphoma emission settings of 488 and 530 nm, respectively. (American Type Tissue Culture Collection, Manassas, VA, USA), were cultured in minimum essential medium-a (Gibco Invitro- gen, Carlsbad, CA, USA) supplemented with 10% fetal calf Cytotoxicity assay serum (Biological Industries, Kibbutz Beit Haemek, Israel) at The cytotoxicity of cells was determined
Load more

Recommended publications
  • Stimulation of Tumor Necrosis Factor Release from Monocytic Cells by the A375 Human Melanoma Via Granulocyte-Macrophage Colony-Stimulating Factor1
    [CANCER RESEARCH 50, 2673-2678. May 1, 1990] Stimulation of Tumor Necrosis Factor Release from Monocytic Cells by the A375 Human Melanoma via Granulocyte-Macrophage Colony-stimulating Factor1 Massimo Sabatini,2 Jeffery Chavez, Gregory R. Mundy, and Lynda F. Bonewald Division of Endocrinology and Metabolism, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284- 7877 ABSTRACT cell line A375, the target cell line used to show that GM-CSF induced monocyte-mediated cytoxicity (3), we noted that con It has long been known that complex interactions occur between tumors ditioned medium harvested from A375 tumor cell cultures and normal host immune cells. The human melanoma cell line A375 has been used previously as an indicator cell for tumor cell cytotoxicity induced TNF production in human blood monocytes and the mediated by monocytes. During other studies on this tumor cell line, we human monocytoid cell line U937 by the secretion of a soluble noted that the conditioned media harvested from A375 cultures induced factor. By multiple criteria, we have identified this soluble factor both the human monocytoid cell line U937 and human blood monocytes which causes TNF production as GM-CSF. These results sug to release the cytokine tumor necrosis factor (TNF). We characterized gest that in this human tumor, production of GM-CSF by the this tumor factor which induced TNF release by monocytic cells. Purifi tumor may retard tumor growth by causing release of cytotoxic cation was performed using ammonium sulfate precipitation, ion exchange cytokines of host cell origin. (DEAE) chromaiography, gel filtration, and reversed-phase high per formance liquid chromatography.
    [Show full text]
  • 81943049.Pdf
    Cytokine 43 (2008) 181–186 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/issn/10434666 The role of the chemokines MCP-1, GRO-a, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques Charikleia Papadopoulou a, Valerie Corrigall b, Peter R. Taylor c, Robin N. Poston a,* a Centre for Cardiovascular Biology and Medicine, King’s College London, Guy’s Campus, London SE1 1UL, UK b Department of Rheumatology, King’s College London, London, UK c Academic Department of Surgery, King’s College London, London, UK article info abstract Article history: Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central Received 14 October 2007 events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemo- Received in revised form 17 March 2008 kines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses Accepted 7 May 2008 the role of chemokines IL-8, MCP-1 and GRO-a, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic Keywords: U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhe- Atherosclerosis Chemokine sion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, che- Monocyte mokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all Leukocyte–endothelial adhesion chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Cellular adhesion assay Immunohistochemistry demonstrated the expression of MCP-1, GRO-a and their receptors in the endo- thelial cells and intima of all atherosclerotic lesions.
    [Show full text]
  • Cellular Models and Assays to Study NLRP3 Inflammasome Biology
    International Journal of Molecular Sciences Review Cellular Models and Assays to Study NLRP3 Inflammasome Biology 1 1, 1, 2 2,3 Giovanni Zito , Marco Buscetta y, Maura Cimino y, Paola Dino , Fabio Bucchieri and Chiara Cipollina 1,3,* 1 Fondazione Ri.MED, via Bandiera 11, 90133 Palermo, Italy; [email protected] (G.Z.); [email protected] (M.B.); [email protected] (M.C.) 2 Dipartimento di Biomedicina Sperimentale, Neuroscenze e Diagnostica Avanzata (Bi.N.D.), University of Palermo, via del Vespro 129, 90127 Palermo, Italy; [email protected] (P.D.); [email protected] (F.B.) 3 Istituto per la Ricerca e l’Innovazione Biomedica-Consiglio Nazionale delle Ricerche, via Ugo la Malfa 153, 90146 Palermo, Italy * Correspondence: [email protected]; Tel.: +39-091-6809191; Fax: +39-091-6809122 These authors contributed equally to this work. y Received: 19 May 2020; Accepted: 12 June 2020; Published: 16 June 2020 Abstract: The NLRP3 inflammasome is a multi-protein complex that initiates innate immunity responses when exposed to a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Inflammasome activation leads to the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and to pyroptotic cell death. Over-activation of NLRP3 inflammasome has been associated with several chronic inflammatory diseases. A deep knowledge of NLRP3 inflammasome biology is required to better exploit its potential as therapeutic target and for the development of new selective drugs. To this purpose, in the past few years, several tools have been developed for the biological characterization of the multimeric inflammasome complex, the identification of the upstream signaling cascade leading to inflammasome activation, and the downstream effects triggered by NLRP3 activation.
    [Show full text]
  • Dharmacon™ Accell™ Sirna References
    RECOMMENDED READING Dharmacon™ Accell™ siRNA References Dharmacon™ Accell™ siRNA reagents are specially modified for use in T47D (ductal breast epithelial tumor cell line) - 23 difficult-to-transfect cells without the need for transfection reagents, virus, T98 glioma cells - 13 or electroporation. The following selected peer-reviewed publications have THP-1 monocytes - 11, 26, 45, 50, 62 cited their successful use in a variety of experimental systems. U266 (peripheral blood B lymphocyte myeloma) - 43 U937 (leukemic monocyte lymphoma) - 53 *For more references that use our siRNA for in vivo applications, please see our in vivo siRNA reading list. Primary cells & in vivo β-islet cells - 15 Established cell lines Bone marrow cells - 10, 17 ARPE-19 (human retinal epithelial cells) - 38 Bronchial smooth muscle cells (BSMC) - 29, 30 BxPC3 (pancreatic tumor cell lines) - 9 Cardiomyocytes - 5 C1 tumor derived cells - 51 Cerebellar granule neurons (CGN) - 8, 69 CD4+ primary human T cells - 4, 70 Colon stem/progenitor cells - 75 CD14+ primary monocytes - 21, 35 Corneal endothelial cells (adult human CECs), and ex vivo human corneal DG-75 human B lymphocytes – 77 endothelium - 74 GH3 (rat somatolactotrophs pituitary cell line) - 61 Cortical neurons - 1, 8, 44, 58, 68 H9 stem cell lines - 48 Endometrial cells - 16 HCT-116 (colorectal carcinoma) - 27 Endothelial cells - 7, 36 HUVEC - 28 Extravillous trophoblasts (EVT) - 31 JJN3(plasma cell leukemia) - 43 Fibroblasts (primary) - 72 KG1 (human acute myelogenous leukemia (AML) macrophage cell line) -
    [Show full text]
  • Differentiation of U-937 Monocytes to Macrophage-Like Cells
    DIFFERENTIATION OF U-937 MONOCYTES TO MACROPHAGE-LIKE CELLS POLARIZED INTO M1 OR M2 PHENOTYPES ACCORDING TO THEIR SPECIFIC ENVIRONMENT: A STUDY OF MORPHOLOGY, CELL VIABILITY, AND CD MARKERS OF AN IN VITRO MODEL OF HUMAN MACROPHAGES A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science. By FATMA ABDULHADI B.S., Seventh of April University, 2007 2014 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL April 25, 2014 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Fatma Abdulhadi ENTITLED Differentiation of U-937 Monocytes to Macrophage- like Cells Polarized to M1 and M2 Phenotypes According to Their Specific Environment: A Study of Morphology, Cell Viability, and CD Markers of An In Vitro Model of Human Macrophages BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. Nancy J. Bigley, Ph.D. Thesis Director Committee on Final Examination Barbara E. Hull, Ph.D. Nancy J. Bigley, Ph.D. Director of Microbiology Professor of Microbiology and and Immunology Program, Immunology College of Science and Mathematics Barbara E. Hull, Ph.D. Professor of Biological Sciences Gerald M. Alter, Ph.D. Professor, Department of Biochemistry & Molecular Biology Robert E.W. Fyffe, Ph.D. Vice President for Research and Dean of the Graduate School ABSTRACT Abdulhadi, Fatma. M.S. Microbiology and Immunology Graduate Program, Wright State University, 2014. Differentiation of U-937 Monocytes to Macrophage- like Cells Polarized to M1or M2 Phenotypes According to Their Specific Environment: A Study of Morphology, Viability, and CD Markers of An In Vitro Model of Human Macrophages.
    [Show full text]
  • Emerging Roles of Chemokines in Prostate Cancer
    Endocrine-Related Cancer (2009) 16 663–673 REVIEW Emerging roles of chemokines in prostate cancer David Vindrieux1,2, Pauline Escobar1,2 and Gwendal Lazennec1,2 1INSERM, U844, Site Saint Eloi, Baˆtiment INM, 80 rue Augustin Fliche, Montpellier F-34091, France 2University of Montpellier I, Montpellier F-34090, France (Correspondence should be addressed to G Lazennec, INSERM, U844, Site Saint Eloi, 80 rue Augustin Fliche, 34295 Montpellier, France; Email: [email protected]) Abstract Prostate cancer (PCa) represents the second leading cause of death among all cancer types in men in Europe and North America. Among the factors suspected to control PCa, incidence and progression, chemokines, and their receptors are now intensively studied. Chemokines are produced by tumor cells and also by the stromal microenvironment, both in the primary tumor site and in distant metastatic locations. The wide and differential distribution of chemokines and their receptors account for the pleiotropic actions of chemokines in PCa, including the modulation of growth, angiogenesis, invasion, metastasis, and hormone escape. This review will focus on the roles and the mechanisms of action and regulation of chemokines in the different steps of PCa development and will discuss the novel strategies that are currently envisioned to target chemokines in PCa. Endocrine-Related Cancer (2009) 16 663–673 Introduction hyperplasia (BPH) and the putative precursor of cancer, prostatic intraepithelial neoplasia. All three stages of Prostate cancer and chemokines prostate disease increase in prevalence with age and Prostate cancer (PCa) is the most commonly diagnosed require androgens for growth and development. cancer in males and the second leading cause of death So far, the factors responsible for PCa progression from cancer in men.
    [Show full text]
  • IL-33 Promotes IL-10 Production in Macrophages: a Role for IL-33 in Macrophage Foam Cell Formation
    OPEN Experimental & Molecular Medicine (2017) 49, e388; doi:10.1038/emm.2017.183 Official journal of the Korean Society for Biochemistry and Molecular Biology www.nature.com/emm ORIGINAL ARTICLE IL-33 promotes IL-10 production in macrophages: a role for IL-33 in macrophage foam cell formation Hai-Feng Zhang1,2,4, Mao-Xiong Wu1,2,4, Yong-Qing Lin1,2, Shuang-Lun Xie1,2, Tu-Cheng Huang1,2, Pin-Ming Liu1,2, Ru-Qiong Nie1,2, Qin-Qi Meng3, Nian-Sang Luo1,2, Yang-Xin Chen1,2 and Jing-Feng Wang1,2 We evaluated the role of IL-10- in IL-33-mediated cholesterol reduction in macrophage-derived foam cells (MFCs) and the mechanism by which IL-33 upregulates IL-10. Serum IL-33 and IL-10 levels in coronary artery disease patients were measured. The effects of IL-33 on intra-MFC cholesterol level, IL-10, ABCA1 and CD36 expression, ERK 1/2, Sp1, STAT3 and STAT4 activation, and IL-10 promoter activity were determined. Core sequences were identified using bioinformatic analysis and site- specific mutagenesis. The serum IL-33 levels positively correlated with those of IL-10. IL-33 decreased cellular cholesterol level and upregulated IL-10 and ABCA1 but had no effect on CD36 expression. siRNA-IL-10 partially abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse the decreased CD36 levels. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased the luciferase expression driven by the IL-10 promoter, with the highest extent within the − 2000 to − 1752 bp segment of the 5′-flank of the transcription start site; these effects were counteracted by U0126.
    [Show full text]
  • Immune Complexes Induce TNF-Α and BAFF Production from U937 Cells by HMGB1 and RAGE Tory Mediators8,9
    European Review for Medical and Pharmacological Sciences 2017; 21: 1810-1819 Immune complexes induce TNF-a and BAFF production from U937 cells by HMGB1 and RAGE X.-J. GAO1,2, Y.-Y. QU2, X.-W. LIU2, M. ZHU2, C.-Y. MA2, Y.-L. JIAO2, B. CUI2, Z.-J. CHEN3, Y.-R. ZHAO2 1Center of Laboratory Medicine, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China 2Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan, China 3Research Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan, China Abstract. – OBJECTIVE: This study investi- Key Words: gated the effects of immune complexes (ICs) on Immune complexes, U937 cells, High mobility group tumor necrosis factor α (TNF-α) and B cell-ac- box protein 1, Receptor for advanced glycation end tivating factor (BAFF) production from U937 products, Systemic lupus erythematosus. cells and further explored the mechanism. MATERIALS AND METHODS: U937 cells were incubated with necrosis supernatant or system- ic lupus erythematosus (SLE) sera alone, or Introduction their combination. The expression of TNF-α and BAFF was determined by Real-time poly- Systemic lupus erythematosus (SLE) is a mul- merase chain reaction and enzyme-linked im- ti-systemic involvement and chronic progressive munosorbent assay. High mobility group box protein 1(HMGB1) A-box was produced by gene autoimmune disorder characterized by production recombination. HMGB1 A-box and anti-receptor of autoantibodies against nucleic components such for advanced glycation end products (RAGE) as DNA and nucleosomes and formation of immu- antibody were adopted in the blocking experi- ne complexes (ICs) due to polyclonal B cell acti- ments.
    [Show full text]
  • Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures
    Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jacqueline D. Corry, B.A. Graduate Program in Integrated Biomedical Science Program The Ohio State University 2015 Dissertation Committee: Mark E. Peeples, Ph.D.—Advisor Douglas M. McCarty, Ph.D. Ian Davis, DVM, Ph.D. Stefan Niewiesk, DVM, Ph.D. Copyright by Jacqueline D. Corry 2015 Abstract Live attenuated respiratory syncytial virus (RSV) vaccine candidates are produced in Vero cells, a cell line that cleaves the attachment (G) glycoprotein. As a result, Vero- derived virus is 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cultures than virus grown in HeLa. HAE cultures are isolated directly from the human airways, so it is likely that Vero-grown vaccine virus would be similarly inefficient at initiating infection of the nasal epithelium following vaccination, requiring a larger inoculum, thereby raising the cost per dose. Using protease inhibitors with increasing specificity, we identified cathepsin L as the responsible protease and confirmed that virus grown in the presence of protease inhibitors was more infectious for HAE cultures. Our evidence suggests that the G protein interacts with cathepsin L in the late endosome or lysosome via endocytic recycling. While essential for Nipah virus F protein cleavage, endocytic recycling is detrimental to the production of infectious RSV from Vero cells. We found that cathepsin L is able to cleave the G protein in Vero-grown, but not in HeLa-grown virions suggesting a difference in G protein posttranslational modification.
    [Show full text]
  • Differentiation of U-937 Histiocytic Lymphoma Cells Towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3 ',5 '-Monophosphate1
    (CANCER RESEARCH 50. 20-25, January I, I9TO] Differentiation of U-937 Histiocytic Lymphoma Cells towards Mature Neutrophilic Granulocytes by Dibutyryl Cyclic Adenosine-3 ',5 '-monophosphate1 Debra 1 . Laskin,2 Andrew J. Beavis, Andrea A. Sirak, Sean M. ()'( 'onncl 1,and Jeffrey D. Laskin Departments of Pharmacology and Toxicology. Rutgers University [D. L. L., A. J. B..A. A. S.J, Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School [J. I). L.], and (.'enter for Advanced Biotechnology and Medicine [D. L. L., S. M. O.J, Piscataway, New Jersey 08854 ABSTRACT of mature phagocytic cells and are not spécifietothe monocytic or granulocytic lineage. Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl Treatment of U-937 cells with agents that elevate intracellu- cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to lar cAMP such as dBcAMP, prostaglandin E2, or cholera toxin differentiate towards granulocytes. dBcAMP produced a dose- and time- has been reported to potentiate the differentiating effects of dependent inhibition of U-937 cell growth reaching a maximum after 48- both retinoic acid and vitamin D, (4, 13, 14). This suggests that h treatment with 500 /JM. At this concentration, dBcAMP had no effect cAMP-dependent phosphorylation reactions may modulate dif on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a ferentiation of myeloid cells (4). If cAMP is involved in differ concomitant increase in cells in the (.„<(.,phase.dBcAMP also induced entiation, then one would predict that agents that increase the appearance of f-met-leu-phe receptors on U-937 cells as well as the intracellular levels of this mediator would, by themselves, in ability to produce hydrogen peroxide and Superoxide anión.These data duce differentiation.
    [Show full text]
  • (U937) Cell Line
    Microbial Pathogenesis 1988; 5: 87-95 Growth of Legionella pneumophila i n a human macrophage-like (U937) cell line Eric Pearlman,' Asmina H . Jiwa, 1 N . Cary Engleberg' •2.3 and Barry I . Eisenstein' •2 * 'Departments of Microbiology and Immunology and 'Internal Medicine, University of Michigan, Ann Arbor, M/ 48109-0620, U.S .A. and 3Ann Arbor Veterans Administration Hospital (Received March 16,1988; accepted April 18, 1988) Pearlman E . (Dept . of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109-0620, U .S.A.), A. H . Jiwa, N . C. Engleberg and B . I . Eisenstein . Growth of Legionella pneumophila in a human macrophage-like (U937) cell line . Microbial Pathogenesis 1988; 5: 87-95 . We established a model of the bacteria-macrophage interaction to study the cellular basis of Legionella pneumophila pathogenesis and to characterize avirulent L. pneumophila. We found that U937 cells, which are derived from a human histiocytic lymphoma cell line, support intracellular growth of L. pneumophila with a doubling time of 6 h, and that sustained intracellular growth is associated with a cytopathic effect (CPE) that can be detected by microscopic examination and quantified with the vital stain 3-(4,5-dimethyl thiazol-2-yl)-2,5,- diphenyl tetrazolium bromide (MTT) . An L. pneumophila isolate obtained directly from infected guinea-pig spleens can grow and produce CPE in these cells, destroying most of the cell layer after 72 h of growth . Only 106 organisms of this strain are required to kill 50% of guinea-pigs inoculated by the intraperitoneal route. In contrast, an avirulent isolate derived by 203 successive plate passages of the same strain can neither kill guinea-pigs at an intraperitoneal inoculum of 10' nor grow or produce CPE in U937 cells .
    [Show full text]
  • Conversion of the U937 Monocyte Into "Macrophage-Like" Populations Exhibiting M1 Or M2 Characteristics
    Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2013 Conversion of the U937 Monocyte into "Macrophage-Like" Populations Exhibiting M1 or M2 Characteristics Bradley M. Sharp Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons Repository Citation Sharp, Bradley M., "Conversion of the U937 Monocyte into "Macrophage-Like" Populations Exhibiting M1 or M2 Characteristics" (2013). Browse all Theses and Dissertations. 782. https://corescholar.libraries.wright.edu/etd_all/782 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. CONVERSION OF THE U937 MONOCYTE INTO “MACROPHAGE-LIKE” POPULATIONS EXHIBITING M1 OR M2 CHARACTERISTICS. A thesis submitted in partial fulfillment Of the requirement fOr the degree Of Master of Science By BRADLEY M. SHARP B.S., OhiO State University, 2009 2013 Wright State UniVersity WRIGHT STATE UNIVERSITY GRADUATE SCHOOL May 6, 2013 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Bradley M. Sharp ENTITLED Conversion of the U937 Monocyte into “MacrOphage-like” POpulatiOns Exhibiting M1 or M2 Characteristics BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. Nancy J. Bigley, Ph.D. Thesis Director Barbara Hull, Ph.D., Director MicrObiOlOgy and ImmunOlOgy Graduate PrOgram COmmittee On Final ExaminatiOn Barbara Hull, Ph.D. Gerald Alter, Ph.D.
    [Show full text]