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Bone As a Target for the Complement System

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Bone As a Target for the Complement System Ulm University Institute of Orthopaedic Research and Biomechanics Director: Prof. Dr. med. vet. Anita Ignatius Bone as a Target for the Complement System Dissertation for the Doctoral Degree in Human Biology (Dr. biol. hum.) from the Medical Faculty, Ulm University Philipp Schoengraf Born in Cologne Ulm 2011 Dean: Prof. Dr. Thomas Wirth 1st reviewer: Prof. Dr. med. vet. Anita Ignatius 2nd reviewer: Prof. Dr. med. Hubert Schrezenmeier Day of defense: 4th May 2012 How blest in whom the fond desire From error’s sea to rise, hope still renews! What a man knows not he to use requires And what he knows, he cannot use for good. Johann Wolfgang von Goethe Faust, The First Part Of The Tragedy, lines 727-730 Table of Contents 1. Introduction ......................................................................................................... 1 1.1. Bone and Bone Cells ....................................................................................... 1 1.1.1. Mesenchymal Stem Cells ............................................................................. 2 1.1.2. Osteoblasts .................................................................................................. 4 1.1.3. Osteoclasts ................................................................................................... 4 1.1.4. Mutual Regulation of Osteoblasts and Osteoclasts ...................................... 6 1.2. Interactions of Bone and the Immune System ................................................. 7 1.3. The Complement System as a Key Part of the Immune System ..................... 9 1.3.1. The Complement System ........................................................................... 10 1.3.2. Complement Activation ............................................................................... 11 1.3.3. Complement Regulation ............................................................................. 13 1.4. Influence of Complement on Bone ................................................................ 15 1.4.1. Role of Complement in Bone Development ................................................ 15 1.4.2. Influence of Complement on Osteoblasts and MSC ................................... 16 1.4.3. Influence of Complement on Osteoclast Formation .................................... 16 1.4.4. Complement induced Migration of Bone Cells ............................................ 17 1.4.5. Complement triggered Inflammatory Response ......................................... 17 1.4.6. Complement and Bone in Disease ............................................................. 18 1.5. Aim of the Study ............................................................................................ 20 2. Material and Methods ....................................................................................... 22 2.1. Material .......................................................................................................... 22 2.1.1. Reagents and Solutions ............................................................................. 22 2.1.2. Consumable Supplies................................................................................. 23 2.1.3. Primers ....................................................................................................... 24 2.1.4. Antibodies ................................................................................................... 25 2.1.5. Media .......................................................................................................... 25 2.1.6. Kits ............................................................................................................. 26 2.1.7. Equipment .................................................................................................. 26 2.2. Methods ......................................................................................................... 27 2.2.1. Isolation/Preparation of Cells ...................................................................... 27 2.2.2. Cultivation of Cells ...................................................................................... 28 2.2.3. mRNA Expression ...................................................................................... 30 I 2.2.4. ELISAs ....................................................................................................... 31 2.2.5. Immune Fluorescence Staining .................................................................. 32 2.2.6. Osteogenic Differentiation of Mesenchymal Stem Cells ............................. 33 2.2.7. Formation and Activity of Osteoclast-like Cells ........................................... 34 2.2.8. Migration of Bone Cells .............................................................................. 35 2.2.9. Stimulation of Bone Cells with Trauma Mediators ...................................... 35 2.2.10. Cleavage of Complement Zymogens by Bone Cells ................................ 36 2.2.11. Statistical Analysis .................................................................................... 37 3. Results ............................................................................................................. 38 3.1. Expression of Complement Components by Bone Cells ............................... 38 3.1.1. Complement Regulatory Proteins CD46, CD55 and CD59 ........................ 38 3.1.2. Complement Zymogens C3 and C5 ........................................................... 42 3.1.3. Complement Anaphylatoxin Receptors C3aR, C5aR and C5L2 ................. 43 3.2. Complement Activation by Bone Cells .......................................................... 47 3.3. Functional Studies on Anaphylatoxin Receptors ........................................... 49 3.3.1. Internalisation of Anaphylatoxin Receptors ................................................ 49 3.3.2. Chemotactical Response of Bone Cells to C5a .......................................... 52 3.3.3. Inflammatory Response of Bone Cells ....................................................... 54 3.4. Influence of Anaphylatoxins on Maturation and Function of Bone Cells ........ 58 3.4.1. Osteogenic Differentiation of MSC ............................................................. 58 3.4.2. Expression of M-CSF/RANKL/OPG by Stimulated Osteoblasts ................. 60 3.4.3. Formation of TRAP+ Multinucleated Cells .................................................. 63 3.4.4. Resorption Activity of Osteoclasts .............................................................. 64 4. Discussion ........................................................................................................ 66 4.1. Expression of Complement Components by Bone Cells ............................... 66 4.2. Complement Activation by Bone Cells .......................................................... 68 4.3. Functional Studies on Anaphylatoxin Receptors ........................................... 69 4.4. Influence of Anaphylatoxins on Maturation and Function of Bone Cells ........ 72 5. Summary .......................................................................................................... 74 6. References ....................................................................................................... 76 Acknowledgements .............................................................................................. 85 Curriculum vitae ................................................................................................... 86 II List of Abbreviations AM Alveolar Macrophage AP Alkaline phosphatase ATP Adenosine triphosphate BMP Bone morphogenetic protein(s) BSA Bovine serum albumin BSP Bone sialo protein C1INH C1 Inhibitor C5 Complement component C5 C5a Anaphylatoxin C5a C5aR C5a receptor C5aRA C5a receptor antagonist C5L2 C5a receptor-like 2 CD Cluster of differentiation cDNA Copy desoxyribonucleic acid CFU-F Colony forming unit-fibroblast(s) CO2 Carbon dioxide COL Collagen CR1 Complement receptor1 Ct Cycle threshold DAF Decay accelerating factor DAMP Danger associated molecular pattern DAPI 4’,6-Diamidino-2-phenylindole dihydrochloride DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid EDTA Ethylenediamine tetraacetic acid ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FCS Fetal calf serum FU Fluorescence units GAPDH, Gapdh Glyceraldehyde-3-phosphate dehydrogenase GPCR G-protein coupled receptor(s) HCl Hydrochloric acid hMSC Human mesenchymal stem cell HSC Haematopoietic stem cell IGF Insulin-like growth factor IgG Immunglobulin G IL Interleukin LPS Lipopolysaccharide LRP5 Low-density lipoprotein receptor-related protein 5 MAC Membrane attack complex MACIF MAC inhibitory factor MCP Membrane cofactor of proteolysis M-CSF Macrophage-colony stimulating factor MMP Matrix metalloproteinase(s) mRNA Messenger ribonucleic acid MSC Mesenchymal stem cell(s) Na2HPO4 Disodium hydrogen phosphate NaCl Sodium chloride NaH2PO4 Sodium dihydrogen phosphate NaOCl Sodium hypochlorite OB Osteoblast(s) OC Osteoclast(s) OCA Osteocalcin o-hMSC Osteogenicly differentiated mesenchymal stem cell(s) OLC Osteoclast-like cell(s) III OP Osteopontin OPG Osteoprotegerin PAMP Pathogen associated molecular pattern PBMNC Peripheral blood mononuclear cell(s) PDGF Platelet derived growth factor PDGF-BB Platelet-derived growth factor subunit B dimer PBS Phosphate buffered saline PCR Polymerase chain reaction PKC Protein kinase C PMA Phorbol 12-myristate 13-acetate qRT-PCR Quantitative RT-PCR RA Reumathoid arthritis
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