Ulm University

Institute of Orthopaedic Research and Biomechanics

Director: Prof. Dr. med. vet. Anita Ignatius

Bone as a Target for the

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. ...... 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 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 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 RANK Receptor activator of nuclear factor κB RANKL Receptor activator of nuclear factor κB Ligand RLT RNeasy™ Mini Kit lysis buffer RNA Ribonucleic acid Rox Reference dye for RT-PCR RT-PCR Reverse transcriptase polymerase chain reaction TAE Tris-acetate-EDTA TGF Transforming growth factor(s) TNF Tumor necrosis factor TRAP Tartrate resistant acid phosphatase Tris Tris(hydroxymethyl)aminomethane WNT Wingless integration-1

IV

List of Figures

Fig. 1: Bone mass regulation ...... 2 Fig. 2: OC resorption activity ...... 6 Fig. 3: Coupling of osteoblasts and osteoclasts ...... 7 Fig. 4: Shared cytokines of bone and immune cells ...... 9 Fig. 5: Blood cell development ...... 10 Fig. 6: Complement cascade ...... 11 Fig. 7: Complement activation ...... 13 Fig. 8: Complement regulators ...... 14 Fig. 9: Expression of complement regulatory proteins in MSC ...... 39 Fig. 10: Immune fluorescence staining of complement regulatory proteins in MSC ...... 40 Fig. 11: Expression of complement regulatory proteins in osteoblasts ...... 40 Fig. 12: Immune fluorescence staining of complement regulatory proteins in osteoblasts ...... 41 Fig. 13: Expression of complement regulatory proteins in OLC ...... 41 Fig. 14: Immune fluorescence staining of complement regulatory proteins in OLC ...... 42 Fig. 15: Expression of complement zymogens C3 and C5 ...... 43 Fig. 16: Expression of complement anaphylatoxin receptors in MSC ...... 44 Fig. 17: Immune fluorescence staining of complement anaphylatoxin receptors in MSC ...... 45 Fig. 18: Expression of complement anaphylatoxin receptors in osteoblasts ...... 45 Fig. 19: Immune fluorescence staining of complement anaphylatoxin receptors in osteoblasts ...... 46 Fig. 20: Expression of complement anaphylatoxin receptors in OLC ...... 46 Fig. 21: Immune fluorescence staining of complement anaphylatoxin receptors in OLC ...... 47 Fig. 22: C5 cleavage by OB and OLC ...... 48 Fig. 23: Chemotactic activity of C5a generated by osteoblasts and osteoclast-like cells ...... 49 Fig. 24: Internalisation of C3aR by OB and OLC ...... 50 Fig. 25: Internalisation of C5aR by OB and OLC ...... 51

V

Fig. 26: Internalisation of C5L2 by OB ...... 52 Fig. 27: Migration of MSC and osteoblasts towards a C5a gradient ...... 53 Fig. 28: Checkerboard analysis ...... 54 Fig. 29: IL-6 expression of OB following pro-inflammatory stimulation ...... 55 Fig. 30: Interleukin release by OB following pro-inflammatory stimulation ...... 56 Fig. 31: Interleukin expression of OLC following pro-inflammatory stimulation ..... 57 Fig. 32: Interleukin release by OLC following pro-inflammatory stimulation ...... 58 Fig. 33: Expression of osteogenic markers by MSC during differentiation in presence of complement anaphylatoxins ...... 59 Fig. 34: AP and von Kossa staining after osteogenic differentiation in presence of complement anaphylatoxins ...... 60 Fig. 35: mRNA expression of RANKL and OPG by osteoblasts (OB) following pro- inflammatory stimulation ...... 61 Fig. 36: RANKL/OPG ratio by OB following pro-inflammatory stimulation ...... 62 Fig. 37: mRNA expression of M-CSF by OB following pro-inflammatory stimulation ...... 63 Fig. 38: TRAP staining of PBMNC cultures in presence of complement anaphylatoxins ...... 63 Fig. 39: Osteoclast-like cell (OLC) formation in presence of complement anaphylatoxins ...... 64 Fig. 40: Resorption activity of osteoclast-like cells ...... 65 Fig. 41: Proposed interactions of bone cells and the complement system ...... 66

VI

List of Tables

Tab. 1: Reagents ...... 22 Tab. 2: Solutions ...... 23 Tab. 3: Consumable supplies ...... 23 Tab. 4: Primers ...... 24 Tab. 5: Antibodies ...... 25 Tab. 6: Media ...... 25 Tab. 7: Kits ...... 26 Tab. 8: Equipment ...... 26

VII 1. Introduction

1. Introduction

Fractures accompanied by severe and/or multiple injuries are significantly delayed in healing compared to single fractures [14, 64]. This correlation was recently confirmed by two studies using rat models of fracture accompanied by blunt chest trauma or soft tissue injury [26, 105]. In these studies a robust and systemic was found, shown by increased serum levels of pro-inflammatory cytokines and systemic activation of complement [26, 105]. Similar effects were observed in sera of patients with severe multiple injuries [46]. These findings raised the question if there are interactions of pro-inflammatory mediators such as interleukins or complement anaphylatoxins with bone cells. Therefore, the influences of these mediators relevant in trauma-induced inflammation on mesenchymal stem cells (MSC), osteoblasts (OB) and osteoclasts (OC) were investigated in the present study. Below, the involved cells and the underlying mechanisms of the interactions of the immune system in particular inflammation with bone are introduced.

1.1. Bone and Bone Cells

Bone is a multifunctional organ, which provides stabilisation and motility to the body, protects important organs, serves as the main calcium depot and harbours the bone marrow. Bone marrow is a complex tissue consisting of endothelial cells, fibroblasts, adipocytes, macrophages and other cell types and provides the location of haematopoiesis [3, 113]. There are at least two different types of adult stem cells in this complex environment: haematopoietic stem cells (HSC), which are precursors of various blood cell lineages [27], and MSC, which contribute to regeneration and maintenance of mesenchymal tissues including bone, cartilage, muscle and adipose tissue [103]. Although bone is one of the toughest materials in the body of vertebrates, it is not an inert material but is constantly rebuild in a process termed remodelling. Remodelling depends on a dynamic balance of bone resorption by osteoclasts and bone formation by osteoblasts [86], which is modified by various influences (Fig. 1) [52]. The process of remodelling is essential for constant repair of micro damages

1 1. Introduction and adaptation to changing mechanical requirements [21, 117]. Diseases associated with pathologically low or high bone density such as osteoporosis or osteopetrosis, respectively, are linked with an imbalance of osteoblast and osteoclast activity [52]. The different cell types relevant in bone homeostasis and therefore termed “bone cells” in this study are: mesenchymal stem cells, undifferentiated and undergoing osteogenic differentiation, osteoblasts, and osteoclasts. Osteocytes, matured osteoblasts that are completely embedded in bone matrix are bone cells as well, but were not used in this study.

Fig. 1: Bone mass regulation Bone mass depends of the balance between bone formation by osteoblast and bone resorption by osteoclasts. This balance is modified by multiple factors. Mechanical load, bone morphogenetic proteins (BMP) and LRP5/Wnt-signaling increase bone formation while oestrogen, bisphosphonates and calcium suppress bone resorption, both effects leading to higher bone mass. Sclerostin, β-adrenergic signalling and immobilisation cause a reduction of bone mass via reduced bone formation. Reduced bone mass is also an effect of increased bone resorption, caused by oestrogen deficiency, immobilisation and a calcium deficiency. [52]

1.1.1. Mesenchymal Stem Cells

In general, stem cells are undifferentiated precursor cells that can generate both other undifferentiated stem cells as well as differentiating cells with specific functions in tissues or organs [87, 146]. Stem cells are categorised by their differentiation potential. Embryonic stem cells are pluripotent which means they are able to differentiate into cells of all three embryonic germ layers [136]. Adult stem cells are multipotent cells that can differentiate into several cell types of the same germ layer. One type of adult stem cells located in the bone marrow are the

2 1. Introduction mesenchymal stem cells (MSC), which contribute to the regeneration of several mesenchymal tissues, which are bone, cartilage, muscle and adipose tissue [103]. Fate of MSC such as self-renewal or differentiation into a specific lineage is controlled by external signals in the microenvironment such as secreted factors, integrins or cell-cell interactions [122, 145]. The criteria defining MSC include plastic adherence, a multipotent differentiation potential and a defined expression pattern of specific surface [29, 57]. The expression pattern of MSC includes among others expression of transforming- growth factor (TGF)- receptor III (cluster of differentiation (CD) 105) [11], 5’- ribonucleotide phosphohydrolase (CD73) [10] and the cell surface protein STRO-1 [130]. Additionally, MSC lack the expression of CD14, CD45 and CD34 [29, 103]. Osteogenic differentiation of MSC can be induced by a defined medium composition containing supplements such as the glucocorticoid dexamethasone [12, 61]. Osteogenic differentiation of MSC is modulated by various factors. A group of factors influencing osteogenic differentiation are bone morphogenetic proteins (BMP) that were found to induce de novo bone formation in the connective tissue of animals [147]. BMP-2 induced the expression of osteogenic differentiation marker genes and up-regulated expression of BMP [24], whereas BMP-6 enhanced the expression of osteogenic marker genes as well as formation and mineralisation of extracellular matrix [43]. Wingless integration-1 (WNT) signaling also plays a crucial regulatory role in osteogenic differentiation [82]. WNT3a enhanced proliferation of MSC and suppressed osteogenic differentiation [18], whereas WNT10b promoted osteogenic differentiation and suppressed adipogenic differentiation [5]. Mice lacking low-density lipoprotein receptor-related protein 5 (LRP5) where shown to have a reduced bone mass [67] demonstrating the essential influence of WNT-signaling on bone mass regulation [96]. Osteogenic differentiation is indicated by production and mineralisation of extracellular matrix as well as by expression of osteogenic marker genes, such as bone sialoprotein (BSP), alkaline phosphatase (AP), collagen (COL) type I, osteocalcin (OCA), and osteopontin (OP). BSP binds to hydroxyapatite and extracellular matrix proteins such as collagens and integrins acting as a nucleator for the formation of hydroxyapatite crystals [44]. OP can also bind to hydroxyapatite and extracellular matrix proteins but is an inhibitor of hydroxyapatite crystal formation [68]. The function of AP in bone matrix

3 1. Introduction mineralisation is to provide free phosphates from organic substrates [13]. Osteocalcin is an osteoblast specific protein involved in matrix mineralisation. The transcription factor runt-related transcription factor 2 (RUNX2) is also used as a marker of osteogenic differentiation as it regulates the expression of osteoblast- related genes such as collagen (COL) type I, OP and OCA [83].

1.1.2. Osteoblasts

Osteoblasts derive from mesenchymal precursor cells and secrete the bone matrix mainly consisting of collagen and non-collagenous proteins, e.g. alkaline phosphatase, osteopontin, and osteocalcin that are important for e.g. the mineralisation of the matrix [66]. Mature osteoblasts, completely embedded in bone substance become osteocytes. Although metabolically barely active, they have signalling functions in bone remodelling, in phosphate and calcium metabolism as well as in mechanosensing [66]. Osteoblast differentiation and function is regulated by numerous factors among them hormones, nerve signals, and vascular agents. Moreover, paracrine factors are involved in osteoblast regulation, e.g. TGF-β, BMP, insulin-like growth factors (IGF), platelet-derived growth factors (PDGF), as well as inflammatory cytokines such as interleukin (IL)- 1β, IL-6 and tumor necrosis factor (TNF)-α [55, 73].

1.1.3. Osteoclasts

Osteoclasts are highly specialised cells deriving from hematopoietic precursor cells of the monocyte/macrophage lineage. Osteoclasts are large, multinucleated cells, that are able to degrade the bone matrix by providing an acid environment and secreting proteolytic enzymes [20]. Osteoclast development from their monocyte precursors requires the presence of osteoblasts or more precisely of osteoblast derived growth factors [135, 140]. Bone resorption by osteoclasts proceeds in sequential steps, which are recruitment and proliferation of OC precursors, differentiation and fusion, leading finally to demineralisation and degradation of bone matrix [149].

4 1. Introduction

Bone resorption by osteoclasts requires several cellular characteristics (Fig. 2) [84]. To fulfil its function of bone resorption in a defined area an osteoclast has to attach to the bone surface and seal the area below itself [135]. The attachment is mediated by specific integrins, using OP and BSP on the bone surface as ligands [107, 149]. Activated OC undergo cellular polarisation with formation of a ruffled border facing the bone surface [1, 135]. Sealing of the resorption lacuna involves rearrangement of the cytoskeleton resulting in the formation of an actin ring rich of filamentous actin [23], [135]. Lowering the pH to about 5 by release of hydrochloric acid (HCl) is essential for demineralisation of the bone matrix, which is a rate- limiting step in bone resorption [129, 141]. Degradation of the organic components of bone matrix is carried out by matrix metalloproteinases (MMP) [28, 120] and secreted, lytic enzymes, such as catepsin K [30], which are activated by the low pH in the resorption lacuna. Catepsin K, a papain family cysteine protease, cleaves collagens into small peptides and activates other enzymes, such as tartrate resistant acid phosphatase (TRAP) by proteolytic cleavage [30]. TRAP acts as a phosphatase in the resorption lacuna and modulates OC attachment via dephosphorylation of OC and BSP [20, 33, 78]. Although not exclusively found in OC, TRAP is, together with other characteristics, such as multiple nuclei, a common marker for osteoclast formation [20]. Matrix proteins, peptides and solubilised inorganic components are cleared from the resorption lacuna via transcytosis [95, 116].

5 1. Introduction

Fig. 2: OC resorption activity OC express several enzymes and exchange systems that are essential for their resorption activity. For instance, the calcitonin receptor (CTR), tartrate-resistant acid phosphatase (TRAP) and a vacuolar proton pump are necessary for proper OC activity [84].

1.1.4. Mutual Regulation of Osteoblasts and Osteoclasts

Bone homeostasis bases on a complex and highly regulated balance of bone resorption by osteoclasts and bone formation by osteoblasts, called remodelling (Fig. 3) [86]. The key molecules regulating osteoclastogenesis are receptor activator of nuclear factor-κB ligand (RANKL), a member of the tumor necrosis factor family, and osteoprotegerin (OPG). Both factors are expressed by osteoblasts. Binding of RANKL to its receptor RANK on osteoclast precursors induces osteoclast formation. Binding of RANKL to OPG, a soluble receptor, which acts as a decoy receptor for RANKL, inhibits osteoclast formation. The presence of macrophage-colony stimulating factor (M-CSF) is also important. M-CSF binds to the c-fms receptor of early osteoclast precursors regulating their proliferation, differentiation and survival. The RANK/RANKL/OPG and M-CSF/c-fms receptor regulatory axes strictly couple osteoblast and osteoclast activity thus controlling skeletal mass homoeostasis [19, 20, 85]. Whereas the influences of OB on formation and activity of OC are well investigated, the influences of OC on OB function are not yet completely understood. However it was shown in vitro, that cell culture supernatants from non-resorbing osteoclasts could induce bone nodule formation of a pre-osteoblastic, murine cell

6 1. Introduction line [65] as well as osteogenic differentiation of human MSC [73]. As possible mediators for the influence of osteoclasts on osteoblasts the platelet-derived growth factor subunit B dimer (PDGF-BB) [75, 86] and sphingosine 1-phosphate (S1P) were suggested [112]. Additionally, it was shown that there is bidirectional signalling of the surface molecule ephrinB2 suppressed on OC via its receptor EphB4, which is expressed on OB [150]. This bidirectional signaling suppresses osteoclastogenesis by inhibiting the c-Fos-NFATc1 cascade in osteoclast precursors and enhances osteogenic differentiation. This finely tuned regulatory mechanisms can be disturbed for example by inflammatory cytokines during systemic or local inflammatory processes in diseases such as osteoarthritis or fracture healing, especially in the case of severe multiple trauma.

Fig. 3: Coupling of osteoblasts and osteoclasts Expression of RANKL and M-CSF by MSC and osteoblasts is essential for the formation and activation of osteoclasts. On the other hand, proliferation of osteoblast precursors and osteogenic differentiation are modulated by coupling factors which can be membrane-bound on or released by osteoclasts or liberated from the bone matrix during resorption.

1.2. Interactions of Bone and the Immune System

Although the interactions of the skeletal and the immune system are manifold and complex, a key component of the mutual regulation is the RANK/RANKL/OPG system. RANKL is produced mainly by osteoblasts but also by immune cells such as T cells and neutrophils [22, 85]. Binding of RANKL to its receptor RANK on monocytes is essential for the formation of osteoclasts by fusion of monocytes. However the RANK/RANKL system also influences immune cell interactions such

7 1. Introduction as dendritic cell-T cell interactions and is necessary for the maturation of dendritic cells [101, 123]. The RANK/RANKL signalling is regulated by the expression of OPG, a decoy receptor for RANKL, which like RANKL is expressed by osteoblasts. The expression of these cytokines and the proliferation and differentiation of osteoblasts during the transition from bone resorption to bone formation is regulated by coupling factors expressed by osteoclasts. These coupling factors are secreted or membrane bound proteins expressed by osteoclasts or are liberated from the bone matrix during osteoclastic bone resorption [86, 150]. However the RANK/RANKL/OPG system is also regulated by immune cells. B cells for example can up-regulate OPG expression and down- regulate RANKL expression [150]. Besides the RANK/RANKL/OPG system there are also interactions between bone and immune system that are mediated by cytokines secreted by immune cells. Some of the most important pro-inflammatory cytokines produced by immune cells, such as IL-1β, IL-6 and TNF-α, induce osteoclastogenesis and bone resorption. Other cytokines such as the B cell mitogen IL-14, produced by Th1 and Th2 cells, have an osteoprotective effect (Fig 4) [85].

8 1. Introduction

Fig. 4: Shared cytokines of bone and immune cells Several cytokines modulating OC formation are produced by immune cells or influence osteoclast formation indirectly by affecting immune cells. A network of pro- and anti-inflammatory cytokines influencing osteoclast formation is shown. GM-CSF: granulocyte macrophage colony-stimulating factor; IFNγ: interferon-γ; IL: interleukin; RANKL: receptor activator of nuclear factor κB ligand; TNF: tumor necrosis factor. The figure is based on a review article from Takayanagi 2007 [134] and own results.

1.3. The Complement System as a Key Part of the Immune System

The immune system is classically divided into the innate and the adaptive immune system. The adaptive or specific immune system consists of highly specific cells and proteins, such as B- and T-cells and antibodies. The innate immune system is also termed unspecific immune system and consists mainly of phagocytes such as monocytes, macrophages and neutrophil granulocytes, which are supported by the complement system and the inflammatory response. The phagocytes of the immune system all derive from the haematopoietic lineage (Fig 5). The complement cascade, which is more closely introduced later in this study, has several functions including the activation and coordination of both innate and adaptive immunity by influencing different cell types of both systems [108, 152].

9 1. Introduction

Fig. 5: Blood cell development The development of important cells of the innate immune system originating from the pluripotent haematopoietic stem cell is shown. The cellular part of innate immunity as the first line of defence consists mainly of phagocytes such as monocytes, macrophages and neutrophils. Additionally, dendritic cells, important presenting cells and natural killer cells, which provide protection against intracellular threats, are a part of innate immunity. Mast cells are important for the release of mediators such as histamine and pro-inflammatory cytokines. Adapted from [90].

1.3.1. The Complement System

The complement system is a very old system for danger sensing and fighting and an essential part of the innate immunity. It defends the organism against foreign materials and pathogens directly by pathogen lysis or indirectly by recruitment of leucocytes, which perform phagocytosis. Usually complement activation appears locally at sites of danger such as injuries, cell damage and invasion of pathogens, helping to fight infections or to degrade dead or damaged cells. In contrast, severe incidents such as multiple trauma or sepsis as well as chronic inflammatory processes like in rheumatoid arthritis and other autoimmune diseases can cause systemic, excessive or enduring complement activation [108].

10 1. Introduction

Fig. 6: Complement cascade The complement system is a cascade of serine proteases activated by recognition of various pathogen associated molecular patterns (PAMP) via the classical and the lectin pathways. The alternative pathway is spontaneously activated and attacks all cells that do not express complement regulatory proteins. MBL: mannose binding lectin, CD59: MAC (membrane attack complex) inhibitory factor, DAF: decay accelerating factor, MCP: membrane cofactor of proteolysis, CR1: 1. Adapted from [137].

1.3.2. Complement Activation

Most of the complement serin proteases are present in plasma as zymogens, inactive precursors that require proteolytic cleavage for activation. Several danger or pathogen associated patterns (DAMPs, PAMPs), including DNA, ATP and pathogen surfaces can trigger complement activation via three different established pathways: the classical, the alternative and the (Fig. 6). All three pathways lead to the formation of one of two different C3 convertases and to the cleavage of C3 into and . The larger fragment, C3b can become a part of a newly formed C3 convertase or bind to a C3 convertase and form a C5 convertase that cleaves C5 to C5a and C5b. C5b promotes the acquisition of the terminal complement components C6 to C9 to the pathogen

11 1. Introduction surface and the formation of the membrane attack complex (MAC) (Fig.7). C3b and C5b bound to the cell surface of pathogens also act as and promote phagocytosis by macrophages and neutrophils [42]. In the last decade two new pathways of complement activation have been suggested. The first is a cross activation of C3 and C5 by cleavage via thrombin or factor Xa, central proteases of the other major serin protease system, the coagulation system [4, 59]. The second new pathway is a cellular activation of complement. It was shown that activated macrophages are capable of cleaving C5 via a surface bound serin protease, thereby producing chemotactically active C5a (Fig. 7) [58]. The small fragments resulting from cleavage of C3 and C5 are powerful pro- inflammatory mediators called anaphylatoxins. The anaphylatoxins cause release of histamine from basophils [74] and mast cells [35] and trigger the oxidative burst in macrophages [89] and neutrophils [36]. C5a was also shown to act as a strong chemokine for macrophages [2], neutrophils [31], mast cells [53] as well as B- and T- lymphocytes [93, 100]. Their effects are mediated by a group of highly specific G-protein coupled receptors (GPCR) the (C3aR) and the C5a receptor (C5aR). For C5a there is another receptor called C5a receptor-like 2 (C5L2), whose role is not yet completely elucidated, but may be that of a decoy receptor negatively regulating the pro-inflammatory response [45, 47].

12 1. Introduction

Fig. 7: Complement activation Complement is usually activated by recognition of antigen- complexes or pathogen associated molecular patterns (PAMP) via the pattern recognition molecules C1q, mannose binding lectin (MBL) or ficollins. Central steps of the activation are the formation of the C3 and C5 convertases. Complement activation leads to opsonisation of pathogen surfaces by C3b and C4b, to the release of the strongly pro-inflammatory anaphylatoxins and to the formation of the membrane attack complex (MAC). Additionally, complement can be activated by cell dependent cleavage of C5 or by cross-activation by other serine proteases, for instance thrombin. Complement regulatory proteins (CRegs) protect host cells against the detrimental effects of complement. Intr.: intrinsic activation. Extr.: extrinsic activation. Adapted from [32].

1.3.3. Complement Regulation

Complement is a very powerful but unspecific defence system that can have also destructive side effects on healthy host tissue. To avoid self-damage, complement activation is tightly regulated by soluble and membrane bound regulatory proteins (Fig. 8). One mechanism of the control of anaphylatoxin activity is the cleavage of the arginin residue by carboxypeptidase N resulting in the des-Arg forms of the anaphylatoxins [17]. Whereas C3a des-Arg has no more pro-inflammatory activity,

13 1. Introduction

C5a des-Arg maintains 1-10 percent of the pro-inflammatory activity of C5a [121]. Several other soluble or membrane bound proteins contribute to the control of complement system. Soluble factors such as C1-inhibitor (C1-INH), and Factor I are mainly independent from the target cells as they act in the fluid phase or bind to host-specific cell surface structures. Most of the membrane bound regulators such as membrane cofactor of proteolysis (MCP, CD46), decay- accelerating factor (DAF, CD59) and MAC (membrane attack complex) inhibitory factor (MACIF, Protectin, CD59) are expressed on most cell types with few exceptions [69]. An exeption is (CR1, CD35), which is mainly found on leukocytes and erythrocytes [69].

Fig. 8: Complement regulators Stages of complement activation where regulation occurs are shown. A: binding of C1 inhibitor (C1INH) separates C1r and C1s from the active C1 complex. B: decay accelerating factor (CD46, DAF), C4 binding protein (C4BP) and complement receptor 1 (CR1) displace C2a from the C4b2a complex. Bound C4b is cleaved by the soluble protease factor I. C: CR1 and factor H displace C3b from the complex and act as cofactors for factor I mediated cleavage. D: binding of MAC inhibitory factor (CD59, MACIF) to the C5b678 complex prevents formation of membrane pores by the membrane attack complex (MAC) Adapted from [90].

14 1. Introduction

1.4. Influence of Complement on Bone

1.4.1. Role of Complement in Bone Development

Since several years there is evidence that various complement proteins are involved in endochondral bone formation during bone development. In endochondral bone formation cartilage is formed, which is then replaced by bone. In the primary ossification centre there are three zones of proliferating, resting and hypertrophic chondrocytes. Growth of bones proceeds by proliferation of chondroblasts, which then secrete matrix proteins and become resting cells. This is followed by hypertrophy and finally apoptosis of the chondrocytes and their replacement by osteoblasts accompanied by remodelling and mineralisation of the collagen matrix. It was shown by Andrades et al. that complement proteins were present in the zones of endochondral bone development in a distinct spatial pattern [8]. In the resting zone of fetal rat tibias and femurs C3, factor B, and were expressed. Factor B and properdin were also found in the proliferating zone. In the hypertrophic zone C5 and C9 were present [8]. These findings indicate that complement might contribute to the turnover from cartilage to bone in endochondral ossification. This conclusion is supported by several other studies showing that C1s, the first component involved in the classical pathway of complement activation, is involved in cartilage degradation during ossification. C1s was found in epiphyseal cartilage and in the fracture callus of hamsters [138], in the primary ossification centre of the human femur [114] and in the secondary ossification centre of hamster tibia [115] as well as in chondrocyte cultures in vitro. In all this studies C1s was found to be located mainly in chondrocytes with an increase during differentiation and a maximum in hypertrophic chondrocytes [92, 114, 115, 138]. An immunohistochemical analysis of the secondary ossification centre of hamster tibiae using an active form-specific antibody, could show that active C1s was present mainly in degrading matrix around invading vessels, while the inactive form was found mainly inside hypertrophic chondrocytes, indicating a role of C1s in matrix degradation [115]. Indeed, C1s was found to be able to cleave type I and II collagen and gelatin by its serin protease activity [148]. Aditionally, C1s was shown to co-localize with matrix metalloproteinase-9 in the primary ossification

15 1. Introduction centre of the human femur and to be able to activate the MMP-9 zymogen [114]. In a recent review it was concluded from this findings that complement proteins could possibly stimulate turnover of cartilage to bone [111].

1.4.2. Influence of Complement on Osteoblasts and MSC

Bone cells are not only targets for activated complement but may also express and activate complement zymogens. C3 expression by mouse marrow-derived stromal cells (ST2) and primary osteoblasts was found in a dose dependent manner, following stimulation with 1α,25-dihydroxyvitamin D3 in vitro [56, 118]. This dependency on 1α,25-dihydroxyvitamin D3 turned out to be tissue-specific in stromal cells and osteoblasts whereas C3 production from hepatocytes acquired no vitamin D3 [118]. It was found that C3 production is vitamin-D dependent in bone but not in liver or serum. C3 production could be restored by supplemental administration of 1α,25-dihydroxyvitamin D3 [62]. Schraufstatter et al. found an internalisation of C3aR and C5aR on MSC following binding of the respective agonists. Internalisation of both receptors was accompanied by strong and prolonged phosphorylation of ERK1/2, which could be shown before for C5aR in various cell types. However, the strong and prolonged ERK1/2 phosphorylation following C3aR internalisation was surprising, as the effect of C3aR internalisation is transient and weak in most cell types [126].

1.4.3. Influence of Complement on Osteoclast Formation

There is evidence from several studies that complement positively influences osteoclast formation by direct and indirect effects. Osteoclasts can be derived from murine bone marrow cultures stimulated with 1α,25-dihydroxyvitamin D3. Osteoclast formation was inhibited by an anti-C3 antibody, suggesting a crucial role for C3 in osteoclastogenesis. As this inhibition appeared strongest when the anti-C3 antibody was added at early time points of culture the authors reasoned that C3 is needed for proliferation of precursor cells and in early differentiation [119]. A similar, more recent study using bone marrow cells from wild type and C3 deficient (C3-/-) mice showed that C3-/- bone marrow cultures generated fewer OCs

16 1. Introduction than wild type cells, since several features of osteoclast formation are impaired in bone marrow cultures from C3-/- mice [139]. By blocking C3aR and C5aR in human bone marrow cultures, Tu et al. could also show that the anaphylatoxin receptors are necessary for osteoclastogenesis [139]. As these studies investigated on osteoclastogenesis from mixed bone marrow cultures, they cannot predicate if osteoblasts or osteoclasts or both are influenced by complement C3. Actually, there are indications of both direct and indirect effects on osteoclast formation. Bone marrow cells from C3-/- mice produced less M-CSF and a reduced RANKL/OPG expression ratio, following stimulation with vitamin D3 [139]. Both, M-CSF and an increased RANKL/OPG ratio are essential for efficient osteoclast formation, indicating an indirect effect of complement on osteoclastogenesis. Furthermore it was shown that bone marrow cultures produce all complement components necessary for activation of C3a and C5a via the alternative pathway, and that complement activation via this pathway as well as the anaphylatoxin receptors C3aR and C5aR are required for osteoclastogenesis [139].

1.4.4. Complement induced Migration of Bone Cells

As anaphylatoxins are known to be powerful chemokines, it is most likely that migration is one of the functions of anaphylatoxins on MSC and osteoblasts. It was shown that MSC reveal a strong chemotaxis towards a C3a or C5a gradient, respectively, that could be blocked by the respective receptor antagonists, and by pertussis toxin indicating a receptor-specific Gi- activation-dependent mechanism. In contrast to other cell types, chemotaxis was accompanied by a strong and elongated ERK1/2 phosphorylation not only after C5aR activation but also following C3aR activation [126].

1.4.5. Complement triggered Inflammatory Response

Another function of anaphylatoxins in bone cells could be the induction of pro- inflammatory cytokine release as it was shown for several other cell types, mainly immune cells. A first study concentrating on complement-triggered IL-6 release

17 1. Introduction from MG-63 osteoblast-like cells found a significant increase in the IL-1β induced release of IL-6 when C5a was added. The authors conclude from this results that osteoblasts express a functional C5a receptor and that modifying critical C5a levels by C5a agonists and antagonists might be a promising therapeutically approach for inflammatory bone loss as found in periodontitis [104].

1.4.6. Complement and Bone in Disease

Due to the important role of complement in the development and homeostasis of healthy bone it is not surprising that complement proteins are involved in several diseases affecting bone and that typical disorders of the complement system also have an influence on bone. Deficiencies of different complement components lead to a number of typical diseases. In general deficiencies in early complement components of the classical and lectin activation pathways can cause immune- complex diseases. An example is the deficiency of C1q which is one of the most important risk factors for development of systemic lupus erythematosus (SLE) [131]. Other important complement disorders are deficiencies of complement regulatory proteins. A lack of the C1-inhibitor causes the hereditary angioedema (HAE), while a deficiency in factor H is accompanied by uncontrolled activation of the alternative pathway leading for instance to membranoproliferative glomerulonephritis type II. The same effect can be caused by an auto-antibody stabilizing the C3bBb complex. At the present there is no data available about bone disorders associated with HAE or membranoproliferative glomerulonephritis type II, but it is generally accepted that complement induced SLE which is accompanied by systemic inflammatory environment is a strong risk factors for bone loss [127]. Another important chronic inflammatory disease affecting bone is rheumatoid arthritis (RA), a disease of the joint, affecting cartilage as well as bone, leading to severe damage of both tissues [79]. Complement is involved in the development of RA as well as in acute inflammatory arthritis caused by trauma or cartilage degeneration. Deposits of C3c and C9 were found in the synovial vasculature and the intercellular matrix in both, patients with RA and acute arthritides, accompanied by a decreased expression of CD59, an inhibitor of MAC formation, by synovial lining cells, stromal cells, and endothelial cells. Furthermore there is

18 1. Introduction longstanding evidence that rheumatoid arthritis also induces general bone loss in dependency of disease activity, leading to osteoporosis [50]. It is most likely that the chronically and systemically high levels of pro-inflammatory cytokines, especially IL-1β and TNF-α that are pivotal in RA lead to enhanced expression of RANKL by osteoblasts and, thereby to increased osteoclast formation [97]. In contrast, no C3c and C9 deposits were found in degenerative or mechanically induced diseases of the joint such as osteoarthritis and osteochondritis dissecans or following patellar luxation and meniscus rupture [71]. Additionally the expression of CD59 was prominent in synovial lining cells, stromal cells, and epithelial cells from degenerative diseases, indicating increased protection against MAC induced cell damage in these diseases [71]. On the contrary, only recently, increased concentrations of components of the classical (C1s and ) and the alternative (factor B) complement activation pathways, the central components C3 and C5 as well as components of the MAC (C5, C7 and C9) where found in synovial fluids of individuals with osteoarthritis compared to healthy individuals [144]. Furthermore, mice deficient in C5 or C6 where protected against the development of osteoarthritis after meniscectomy, whereas mice deficient in CD59 showed an increased severity of the osteoarthritis [144]. Not only chronically but also transiently systemic inflammation interferes with bone homeostasis and fracture repair. It was shown that pro-inflammatory cytokines were significantly up regulated during systemic inflammatory response syndrome (SIRS) after severe trauma, in a rat model of blunt chest trauma. The pro- inflammatory effect of the experimental blunt chest trauma was abolished by treatment with an anti-C5a antibody, showing that the complement system is a key component in posttraumatic inflammatory response [41]. Recently Recknagel et al. from our institute could show in a rat model of femur fracture that the fracture healing was significantly impaired by a concomitant blunt chest trauma, resulting in reduced bending stiffness of the healed bone after 35 days of healing, compared to rats without blunt chest trauma [105]. Additionally it was shown in the study that the treatment with a C5aR antagonist completely restored the impaired fracture healing caused by blunt chest trauma. In the mentioned study it was concluded that complement influences fracture healing indirectly by inducing a strong inflammatory response but may also directly influence the balance of osteoblasts and osteoclasts in favour of osteoclasts [106].

19 1. Introduction

Biomaterials are often used for fracture repair and joint replacement. A possible complication of these therapeutical strategies is aseptic loosening of implants caused by complement induced excessive local inflammation. When a biomaterial is exposed to blood, plasma proteins including complement components immediately bind to the surface. Due to conformational changes of C3 during binding it is able to form an initiating C3 convertase leading to further complement activation [6]. Activation of the alternative pathway by exposure to biomaterials was demonstrated in vivo and in vitro by detection of alternative pathway activation products [25, 54]. Complement activation in C4 deficient patients during haemodialysis was slower than in normal patients demonstrating the participation of the classical and/or lectin pathways [76]. Different materials have different complement activating potential. Hydrophobic materials for instance are more potent complement activators than hydrophilic and chemical groups such as NH3, OH and COOH influence the activation of complement [34]. One chance to protect biomaterials from complement activation are complement inhibitors bound to functionalised coatings as was shown with factor H specifically bound to functionalised polyethylene oxid (PEO) coated surfaces [7, 94]. All these findings show that the interactions of the skeletal and complement system are complex and multifaceted, influencing the migration of bone cells, the osteoblast-osteoclast interaction and a modulation of the inflammatory response by osteoblasts.

1.5. Aim of the Study

The aim of the present study was to investigate specific interactions of bone cells and the complement system to achieve a better understanding of the cellular processes that may be involved in impaired fracture healing during systemic inflammation. The present study focuses mainly on direct effects of the complement anaphylatoxins C3a and C5a on osteoblasts, osteoclasts and their respective precursors, mesenchymal stem cells and peripheral blood mononuclear cells (PBMNC).

20 1. Introduction

Three hypotheses were established regarding the interactions of bone cells and the complement system

H1: Primary osteoblasts express and activate complement components. Trauma-relevant mediators such as anaphylatoxins and interleukins (IL) can induce a specific inflammatory response in osteoblasts. H2: Mesenchymal stem cells acquire immune regulatory ability during osteogenic differentiation. Trauma-relevant mediators can influence osteogenic differentiation. H3: Trauma-relevant mediators modulate the interactions of osteoblasts and osteoclasts.

To verify these hypotheses, the following questions were answered in this study, some of them in collaboration with our co-operation partners:

 Do bone cells express complement-related proteins, such as complement regulators, anaphylatoxin receptors and the complement components C3 and C5?  Are osteoblasts and osteoclasts able to directly activate the complement zymogens C3 and C5 by proteolytic cleavage?  Are the anaphylatoxin receptors C3aR, C5aR and C5L2 internalised by osteoblasts and osteoclasts following binding of the respective ligand?  Is there a chemotactic effect of C5a on MSC and osteoblasts?  Do osteoblasts and osteoclasts show an inflammatory response following stimulation with complement anaphylatoxins and other pro-inflammatory mediators?  Is osteogenic differentiation influenced by complement anaphylatoxins?  Are formation and resorption activity of osteoclasts directly or indirectly influenced by anaphylatoxins C3a and C5a?

21 2. Material and Methods

2. Material and Methods

2.1. Material

2.1.1. Reagents and Solutions

Tab. 1: Reagents

Reagent Company 0.5% Trypsin/0.2% EDTA (10x) Biochrom AG Accutase PAA Laboratories GmbH Agarose for DNA electrophoresis SERVA Electrophoresis Ascorbate-2-phosphate Sigma-Aldrich® Biocoll (1.077 g/ml) Biochrom AG Bisbenzimide H33342 (Hoechst 33342) AppliChem GmbH Bovine serum albumin Sigma-Aldrich® Carbon dioxide MIT IndustrieGase AG Collagenase type IV Sigma-Aldrich® Dexamethasone Sigma-Aldrich® Dimethyl sulfoxide Merck KGaA Disodium hydrogen phosphate Merck KGaA DNA ladder 100 bp AppliChem GmbH Dulbecco's MEM Biochrom AG Dulbecco's MEM calcium-free USB Corporation Dulbecco's PBS PAA Laboratories GmbH Ethanol VWR International Ethidium bromide 0.07% AppliChem GmbH Ethylenediamine tetraacetic acid VWR International FBS South American Origin CAMBREX Fetal Bovine Serum, Origin: EU-approved countries (South America) Biochrom AG Foetal Bovine Serum Origin: EU Approved (South American) Gibco® Formalin Merck KGaA Fungizone® Antimycotic, liquid Gibco® Gel loading solution, 6x Sigma-Aldrich® L-glutamine PAA Laboratories GmbH Lipopolysaccharides from Escherichia coli 055:B5 Sigma-Aldrich® Macrophage-colony stimulating factor, recombinant human CHEMICON® INT. MEM Alpha Modification PAA Laboratories GmbH Nitrogen MIT IndustrieGase AG Eurofins MWG Synthesis Oligo-desoxythymidine primers GmbH Penicillin-Streptomycin, liquid invitrogen™ Platinum® SYBR® Green qPCR SuperMix-UDG invitrogen™

22 2. Material and Methods

Primer random p(dN)6 Roche Pure water Ampuwa® Pyrogallol Merck KGaA Receptor activator of nuclear factor NF-κB ligand, recombinant human Insight Biotechnology RLT buffer QIAGEN GmbH Rnasin® RNase inhibitor Promega GmbH Rox reference dye invitrogen™ Saccharose Merck KGaA Silver nitrate AppliChem GmbH Sodium chloride Merck KGaA Sodium dihydrogen phosphate Merck KGaA Sodium thiosulfate Sigma-Aldrich® Tris(hydroxymethyl)aminomethane USB Corporation Triton X-100 Sigma-Aldrich® Trypan Blue solution Sigma-Aldrich® Türk’s solution Merck KGaA β-Glycerophosphate disodium Sigma-Aldrich® β-Mercaptoethanol Sigma-Aldrich®

Tab. 2: Solutions

Solution Composition Bleach 6% NaOCl, 0.9 M NaCl

Buffered 4% formalin solution 30 mM NaH2PO4 + 45 mM Na2HPO4 in 4% formalin, pH 7.0 TAE buffer 40 mM tris, 1 mM EDTA, 40 mM glacial acetic acid, pH 8.5

2.1.2. Consumable Supplies

Tab. 3: Consumable supplies

Consumable supplies Company BD BioCoat™ Osteologic™ Bone Cell Culture System BD Biosciences Cell culture flasks nunc™ Cell culture plates nunc™, Corning Inc. CryoTube™ Vials nunc™ Lab-TEK™ Chamber Slides (with detachable top) nunc™ MicroAmp® Fast Optical 96-Well Reaction Plate (0.1 ml) Applied Biosystems Inc. Microscope cover glasses 13 cm Ø VWR Microscope slides Menzel Glaser PCR Sealers™ Microseal® 'B' Film Bio-Rad Laboratories

23 2. Material and Methods

2.1.3. Primers

Primers were synthesised by Thermo Fisher Scientific (Germany).

Tab. 4: Primers PCR product size mRNA Primer sequence (bp) AP forward 5'-GAA CGT ATT TCT CCA GAC CCA GA-3' 224 AP reverse 5´-GTG GTC TTG GAG TGA GTG AGT GA-3´ BSP forward 5'-CGA GGG GGA GTA CGA ATA CA-3' 79 BSP reverse 5'-AGG TTC CCC GTT CTC ACT TT-3' C3 forward 5’-TGC TGC CCA GTT TCG AGG TCA -3’ 248 C3 reverse 5’-CCC GTC CAG CAG TAC CTT TCG G-3’ C5 forward 5’-TGT CGT CGC AAG CCA GCT CC-3’ 215 C5 reverse 5’-TGC CAA TGC CTT GAA TTT CCC AGG-3’ C3aR forward 5’-GCA GGT TCC TAT GCA AGC TC-3’ 221 C3aR reverse 5’-GAA GAT TTC CCG GTA CAC GA-3’ C5aR forward 5’-CTC AAC ATG TAC GCC AGC AT-3’ 168 C5aR reverse 5’-CAG GAA GGA GGG TAT GGT CA-3’ CD46 forward 5’-GTG AGG AGC CAC CAA CAT TT-3’ 177 CD46 reverse 5’-GGC GTC ATC TGA GAC AGG-3’ CD55 forward 5´-CAG CAC CAC CAC AAA TTG AC -3´ 215 CD55 reverse 5´-CTG AAC TGT TGG TGG GAC CT-3´ CD59 forward 5´-CCG CTT GAG GGA AAA TGA G -3´ 130 CD59 reverse 5´-CAG AAA TGG AGT CAC CAG CA-3´ GAPDH forward 5'-GAA GGT GAA GGT CGG AGT C-3' 224 GAPDH reverse 5'-GAA GAT GGT GAT GGG ATT TC-3' IL-6 forward 5'-AGG AGA CTT GCC TGG TGA AA-3' 180 IL-6 reverse 5'-CAG GGG TGG TTA TTG CAT CT-3' IL-8 forward 5´GTG CAG TTT GCC AAG GAG T-3’ 196 IL-8 reverse 5´- CTC TGC ACC CAG TTT TCC TT-3’ M-CSF forward 5'-GGA GAC CTC GTG CCA AAT TA-3' 223 M-CSF reverse 5'-GGC CTT GTC ATG CTC TTC-3' OCA forward 5'-GGC AGC GAG GTA GTG AAG AG-3' 52 OCA reverse 5'-CTC CCA GCC ATT GAT ACA GG-3' OP forward 5'-CTC AGG CCA GTT GCA GCC-3' 177 OP reverse 5'-GCC ACA GCA TCT GGG TAT TT-3' OPG forward 5'-AGG AAA TGC AAC ACA CGA CA-3' 168 OPG reverse 5'-TAC TTT GGT GCC AGG CAA AT-3' RANKL forward 5'-CCA GCA TCA AAA TCC CAA GT-3' 194 RANKL reverse 5'-CCC CAA AGT ATG TTG CAT CCT G-3'

24 2. Material and Methods

2.1.4. Antibodies

Tab. 5: Antibodies Target Antibody Produced in Specificity Company species

Anti-Fas (human, activating) Mouse Human Fas Millipore clone CH11

Rabbit anti-human C3aR santa cruz Rabbit Human C3aR (H300) biotechnology®

Mouse anti-human CD46 Mouse Human CD46 AbD Serotec

Mouse anti-human CD55 Mouse Human CD55 AbD Serotec

Mouse anti-human CD59 Mouse Human CD59 AbD Serotec

Mouse anti-human CD88 Mouse Human CD88 AbD Serotec

Rabbit polyclonal to GPCR Rabbit Human C5L2 abcam® C5L2 Mouse IgG1 Mouse IgG1 negative control Mouse - negative AbD Serotec control

Rabbit IgG Rabbit - IgG dianova

2.1.5. Media

Tab. 6: Media

Medium Composition

MSC expansion Dulbecco’s MEM (Biochrom AG) + 10% FCS (CAMBREX), medium 1% L-glutamine, 1% penicillin-streptomycin, 0.5% Fungizone®

MSC differentiation Osteogenic differentiation: medium MSC expansion medium + 0.1 mM dexamethasone, 10 mM β-glycerophosphate disodium, 0.2 mM ascorbate-2-phosphate

OB expansion medium Dulbecco’s MEM (Biochrom AG) + 10% FCS (Biochrom AG), 1% L-glutamine, 1% penicillin-streptomycin, 0.5% Fungizone®

OB differentiation OB expansion medium + 0.1 mM dexamethasone, 10 mM medium β-glycerophosphate disodium, 0.2 mM ascorbate-2-phosphate

OB stimulation medium Dulbecco’s MEM (Biochrom AG) + 2% FCS (Biochrom AG, heat inactivated), 1% L-glutamine, 1% penicillin-streptomycin, 0.5% Fungizone®

OLC culture medium MEM Alpha Modification (PAA Laboratories GmbH) + 10% FCS (Gibco®), 1% L-glutamine, 1% penicillin-streptomycin (after that adjustment of pH to 6.8 with HCl) + 40 ng/ml humane RANKL, 25 ng/ml humane M-CSF

25 2. Material and Methods

2.1.6. Kits

Tab. 7: Kits

Kit Company Acid Phosphatase, Leukocyte (TRAP) Kit Sigma-Aldrich® C5a ELISA Kit DRG Diagnostics HotStarTaq® Master Mix Kit QIAGEN GmbH Leukocyte Alkaline Phosphatase Kit Sigma-Aldrich® MicroVue Complement C3a EIA Kit Quidel OmniscriptTM RT Kit QIAGEN GmbH QIAshredderTM QIAGEN GmbH Quantikine® Human IL-6 Immunoassay R&D Systems Europe, Ltd. Quantikine® Human IL-8 Immunoassay R&D Systems Europe, Ltd. RNase-Free DNase Set QIAGEN GmbH RNeasyTM Mini Kit QIAGEN GmbH

2.1.7. Equipment

Tab. 8: Equipment

Equipment Type Company Camera Camedia C-5060 Wide Zoom OLYMPUS Camera (fluorescence) DFC360 FX Leica Camera DFC420C Leica Centrifuge UniCen FR Herolab GmbH Laborgeräte Clean bench HERA Safe Heraeus Holding GmbH Counting chamber Neubauer, double Jordan Gamma GmbH Gel documentation system Fusion-SL 3500.WL Vilber Lourmat National Institutes of Health, Image processing software ImageJ 1.43r USA Image processing software LAS AF Leica Image processing software MetaMorph AF, version 1.4.0 Leica Incubator BBD 6220 Heraeus Holding GmbH Microscope (fluorescence) DMI6000 B Leica Microscope Olympus IX70 OLYMPUS PCR device RoboCycler® GRADIENT 96 Stratagene PCR device Thermocycler Biometra GmbH Photometer infinite M200 Tecan StepOnePlus™ Real-Time PCR RT-PCR device Applied Biosystems Inc. System Statistical analysis software PASW Statistics 18.0 SPSS Inc. Water bath GFL®

26 2. Material and Methods

2.2. Methods

2.2.1. Isolation/Preparation of Cells

Mesenchymal stem cells (MSC) MSC were isolated from bone marrow aspirates obtained from surgical procedures on 5 male donors (20-38 years) after informed consent in accordance with the terms of the ethics committee of Ulm University, Germany. Isolation of the cells was performed by density gradient centrifugation and adhesion to tissue culture plastic [103]. Accordingly, 2-3 ml bone marrow aspirate was layered onto 5 ml Biocoll and centrifuged (400 g, 30 min, room temperature). Mononuclear cells were taken from the interphase of the gradient and washed with 2-3 volumes MSC expansion medium (250 g, 10 min, room temperature). Cells were counted in a counting chamber with Türk’s solution and seeded at 80,000-160,000 cells/cm2 in cell culture flasks. Medium was changed after 24 h and subsequently twice a week. After 7-14 days, colony forming unit-fibroblasts (CFU-F) were visible and were cultivated up to one more week. To distribute cells, CFU-F were washed twice with phosphate buffered saline (PBS) and 1 ml trypsin/ethylenediamine tetraacetic acid (EDTA) was added for 1 min at 37 °C. After that, medium was added and changed after 24 h. Cells were grown to subconfluence, trypsinised and cryo-preserved in liquid nitrogen in medium containing 20% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO).

Primary osteoblasts (OB) OB were isolated according to Robey & Termine [109] from bone samples (femur or tibia) of 4 male and 1 female healthy patients (23-77 years) undergoing surgery for fracture repair after informed consent in accordance with the terms of the ethics committee of Ulm University, Germany. Bone pieces were subjected to collagenase digestion and cell isolation was performed in calcium-free Dulbecco’s MEM to inhibit fibroblast growth. OB were stored in liquid nitrogen in medium containing 20% FCS and 10% DMSO.

27 2. Material and Methods

Peripheral blood mononuclear cells (PBMNC) For the generation of osteoclast-like cells (OLC, defined as tartrate resistant acid phosphatase (TRAP) positive, multinucleated cells), PBMNC were isolated from blood donations of voluntary donors (male, 30 to 40 years) in accordance with the terms of the ethics committee of Ulm University, Germany, via density gradient centrifugation. Blood donations were mixed with PBS at a ratio of 1:2 and 20 ml of the mixture was layered onto 20 ml Biocoll and centrifuged (400 g, 30 min, room temperature). PBMNC were taken from the interphase of the gradient and were washed twice with PBS (250 g, 10 min, room temperature). Cells were counted in a Neubauer chamber with Türk’s solution and used directly for experiments or cryo-preserved in liquid nitrogen in medium containing 20% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO).

2.2.2. Cultivation of Cells

Mesenchymal stem cells MSC stored in liquid nitrogen were thawed in a water bath (37 °C) and added to 9 ml medium to dilute DMSO. Cells were centrifuged (250 g, 10 min, room temperature), counted in a counting chamber with trypan blue and used for cell expansion. Cells were seeded in cell culture flasks at 2,000-3,000 cells/cm2 and cultured in MSC expansion medium at 37 °C, 8.5% CO2 and saturated humidity. Medium was changed twice a week. At subconfluence, MSC were trypsinised and reseeded for further expansion or used for experiments (cells of passage 1-4).

Induction of osteogenic differentiation of mesenchymal stem cells Osteogenic differentiation of MSC was induced to compare the complement expression and the influence of complement anaphylatoxins on undifferentiated and osteogenicly differentiated MSC. Cell lysates or fixed cells from day 0 were used as a control for the state of differentiation. For the induction of osteogenic MSC differentiation in all differentiation experiments, MSC were seeded at 10,000 cells/cm2 in cell culture plastic wells in osteogenic differentiation medium at 37 °C, 8.5% CO2 and saturated humidity. Medium was changed twice a week and cells were cultured for 14, 21,or 28 days.

28 2. Material and Methods

Cell differentiation was analysed via alkaline phosphatase (AP)- and von Kossa staining and expression analysis of specific marker genes. Additionally the expression of complement components at different time points of osteogenic differentiation was investigated. For immune fluorescence experiments MSC of passage 2-4 were seeded at 10,000 cells/cm2 on 13 mm Ø microscope cover glasses or on eight-well Lab-Tek chamber slides in OB expansion Medium for 3-4 days. As dexamethasone is known to suppress immune reactions, cells used for any experiments involving pro-inflammatory stimulation, were cultured in MSC differentiation medium without dexamethasone for the last 4 days prior to the experiments.

Primary osteoblasts OB stored in liquid nitrogen were prepared for cell expansion as described for MSC. Cells were seeded at 4,000-5,000 cells/cm2 in OB expansion medium and were otherwise treated as MSC. For stimulation experiments, OB of passage 4-6 were seeded at 10,000 cells/cm2 in OB differentiation medium in cell culture plastic wells and were cultured for

14 days at 37 °C, 8.5% CO2 and saturated humidity. For immune fluorescence experiments OB of passage 4-6 were seeded at 10,000 cells/cm2 on 13 mm Ø microscope cover glasses or on eight-well Lab-Tek chamber slides in OB expansion Medium for 3-4 days. OB used for any experiments involving pro- inflammatory stimulation, were cultured in OB differentiation medium without dexamethasone for the last 4 days prior to the experiments.

Osteoclast-like cell generation from PBMNC After isolation, PBMNC were seeded at 500,000 cells/cm2 in OLC culture medium containing 40 ng/ml RANKL and 20 ng/ml recombinant human (rh) M-CSF in cell culture plastic wells and were cultured at 37 °C, 5% CO2 and saturated humidity. Osteoclast formation was analysed by TRAP staining and counting of multinucleated cells. For immune fluorescence cells were seeded on eight-well Lab-Tek chamber slides. The medium was changed twice a week and cells were cultured up to 21 days.

29 2. Material and Methods

2.2.3. mRNA Expression

The mRNA expression of different groups of marker genes in MSC, osteoblasts and osteoclasts was analysed to give information about osteogenic differentiation, complement expression, inflammatory response and RANKL/OPG/M-CSF expression.

RNA isolation Cell lysates were collected from MSC at day 0 (500,000 cells) and at the end of the respective MSC, OLC, OB experiment and stored at -80 °C in RLT buffer containing 10 µl/ml -mercaptoethanol. Lysates were homogenised with QIAshredder™ and RNA was isolated using the RNeasy™ Mini Kit and RNase- Free DNase Set, all according to the manufacturer instructions. RNA concentration was measured photometrically. cDNA synthesis 1 µg RNA was transcribed into cDNA using the Omniscript™ RT Kit completed with oligo-desoxythymidine primers (5 µM), random hexamer primers (50 µM) and RNase inhibitor (10 units) in a total volume of 20 µl according to the manufacturer instructions. The obtained cDNA was diluted at a ratio of 1:3 and used for polymerase chain reaction (PCR).

Real-time reverse transcriptase polymerase chain reaction Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed in a total volume of 25 µl in 96 well plates sealed with tape. Samples contained 12.5 µl Platinum® SYBR® Green qPCR SuperMix-UDG, 0.5 µl Rox reference dye, 0.4 µM of each primer and 2 µl cDNA. Specific primer pairs (Tab. 4) were designed using published gene sequences (PubMed, NCBI Entrez Nucleotide Database). For the analysis of the mRNA expression in human cells, cloned amplification products were provided and used as standards for real-time RT-PCR (50 °C for 2 min; 95 °C for 2 min; 40 cycles with 95 °C for 15 sec, 60 °C for 1 min and 95 °C for 15 sec; 60 °C for 1 min; heating in 0.3 °C steps to 95 °C; 95 °C for 15 sec). The amount of each respective amplification product was determined relative to the house-keeping gene glyceraldehyde-3-phosphate

30 2. Material and Methods dehydrogenase (GAPDH). Normalised values of samples collected at the end of the experiments were compared to the control at day 0 for osteogenic differentiation marker genes of MSC. For some of the examined genes, no cloned standards where available. In this cases real-time RT-PCR was performed analogously, however without using standards. Instead, the amount of each respective amplification product was determined via the Ct (cycle threshold) method. The average Ct value of the respective gene of interest of each sample measured (in duplicate, resulting in n = 2-4) was normalised to the average Ct value of Gapdh of the respective sample (Ct=Ct(gene of interest)-Ct(Gapdh)). Ct values were calculated by referring Ct values to the respective control (Ct=Ct(specimen)-Ct(control)).

The relative mRNA expression was then calculated by the term 2-Ct.

Semi-quantitative polymerase chain reaction Semi-quantitative PCR was used to analyze expression of the complement zymogens C3 and C5. Samples (total volume 20 µl) containing 10 µl HotStarTaq® Master Mix, 0.5 µM of each primer (for specific primer pairs see Tab. 4) and 1 µl cDNA were prepared and used for PCR (95 °C for 14 min; 32 cycles with 95 °C for 60 sec, 60 °C for 45 sec and 72 °C for 60 sec; 72 °C for 20 min; 4 °C for cooling samples).

Agarose gel electrophoresis The PCR amplification products (mixed with gel loading solution at a ratio of 6:1) and the DNA ladder were separated on a 2% agarose gel in tris-acetate-EDTA (TAE) buffer, visualised by ethidium bromide staining (0.9 µM) and documented with a gel documentation system.

2.2.4. ELISAs

Interleukin release was analysed in supernatants of OB and OLC cultures after 24 hours of stimulation with trauma-relevant mediators. In the complement activation experiment specific ELISAs for C3a and C5a were used to determine the amount of C3 or C5 cleavage. Supernatants were stored at -80 °C until use. The analysis

31 2. Material and Methods was performed using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer instructions. Briefly, interleukin concentrations in the samples are determined via the binding of the molecules to an immobilised monoclonal antibody. An enzyme-linked polyclonal antibody specific for IL-6 is added. After the subsequent addition of the substrate solution, colour develops in proportion to the amount of the interleukin that was initially bound.

2.2.5. Immune Fluorescence Staining

Indirect immune fluorescence staining The presence of complement regulators and receptors in bone cells was investigated by indirect immune fluorescence staining. MSC and OB were seeded 3 - 4 days before staining with 10.000 cells per cm2 on 13 mm Ø or on eight-well Lab-Tek chamber slides in MSC expansion medium (hMSC) or in MSC (o-hMSC) or OB (OB) osteogenic differentiation medium. PBMNC were cultured on eight-well Lab-Tek chamber slides for 21 days in OLC culture medium to promote OLC formation. Cells were fixed with 4% phosphate-buffered formalin for 5 min and permeabilised with 0.1% Triton X-100 in PBS for 10 min. Unspecific binding was blocked with BSA 2% in PBS for 30 min at 37° C. Visualisation of the regulators and receptors was performed by detection of specific primary antibodies (see Tab. 5) against CD46, CD55, CD59, C3aR, C5aR and C5L2 by the binding of a suitable, fluorescence labeled secondary antibody. Cell nuclei were counterstained with Hoechst 33342. For negative controls the primary antibody was substituted with mouse or rabbit immune globulin G (IgG).

Double immune fluorescence staining for receptor localisation Osteoblasts were seeded on 13 mm Ø microscope cover glasses in 24-well plates or on 8-well LabTek chamber slides with 10.000 cells/cm2 and were cultured in osteoblast differentiation medium for 3-4 days before the experiment. PBMNC were seeded with 500,000 cells/cm2 in 8-well LabTek chamber slides and cultured for 21 days in OLC culture medium to promote formation of osteoclast-like cells. To investigate the internalisation of the complement anaphylatoxin receptors C3aR and C5aR and C5L2 following binding of the respective ligand, OB or OLC were incubated with 1 µg/ml C3a or 0,1 µg/ml C5a in OB stimulation medium for 0, 15 or

32 2. Material and Methods

45 min. Cells were fixed with 4% phosphate-buffered formalin and unspecific binding was blocked with BSA 2% in PBS for 30 min at 37 °C. To detect the internalisation of complement receptors, a two-step protocol for immune fluorescence staining was performed. In the first step, the cells were incubated with a specific primary antibody against the respective receptor for 60 min at RT followed by 60 min incubation with a green fluorescence-labelled secondary antibody. In the second step cells were permeabilised with 0.1% Triton X-100 in PBS for 10 min and blocking with BSA was repeated. Cells were incubated with the same primary antibody followed by a red fluorescence-labelled secondary antibody. Cell nuclei were counterstained with Hoechst 33342. Images were acquired separately for each fluorescent dye. In the resulting overlay images receptors at the cell surface appear green or yellow whereas receptors inside the cell appear red.

2.2.6. Osteogenic Differentiation of Mesenchymal Stem Cells

Stainings regarding the qualitative analysis of MSC differentiation were documented via bright field microscopy.

Von Kossa staining As osteogenic differentiation comprises the mineralisation as the last step after proliferation of cells and matrix maturation [132], the differentiation was verified by demonstrating mineralised matrix via von Kossa staining. Incubation with silver nitrate leads to the replacement of calcium or other positively charged ions. After the reduction of silver ions, stained sections appear dark [15]. At day 23, cells were washed with PBS, fixed with buffered 4% formalin solution, washed with pure water and incubated with silver nitrate (0.05 g/ml) for 60 min. After washing with pure water, cells were incubated with pyrogallol (0.01 g/ml) for 10 min, fixed with sodium thiosulfate (0.05 g/ml in pure water) for 5 min and air- dried.

Alkaline phosphatase staining The activity of AP, an enzyme crucial for the initiation of mineralisation, however not for the progression of bone nodule mineralisation [13], was analysed with

33 2. Material and Methods

Leukocyte Alkaline Phosphatase Kit following the manufacturer instructions after fixation of the cells with buffered 4% formalin solution at day 23. Briefly, incubation in the dark with a solution containing an AP substrate results in a red dye deposit, indicating sites of AP activity in cells differentiated into the osteogenic lineage.

2.2.7. Formation and Activity of Osteoclast-like Cells

Non-fluorescent stainings regarding OLC formation and activity were documented via bright field microscopy.

Tartrate-resistant acid phosphatase assay TRAP assay was performed to confirm the expression of this enzyme, which is present in cells of the macrophage lineage, influences the attachment of OC to the bone surface [33] and presumably acts as a phosphatase in the resorption lacuna [20, 33]. TRAP assay was performed with Acid Phosphatase, Leukocyte (TRAP) Kit according to the manufacturer instructions at day 21. Briefly, after fixing cells, incubation with a TRAP substrate results in a red staining, indicating TRAP- positive cells in the culture. In order to quantitatively assess the formation of multi-nucleated cells, TRAP- positive cells with at least three nuclei were counted as OLC. For each donor and condition, cells in 3 wells of a 96-well plate were counted at 400x magnification.

Calcium phosphate resorption Calcium phosphate resorption was quantitatively assessed in BD BioCoat™ Osteologic™ Bone Cell Culture System plates coated with synthetic calcium phosphate. To detach the cells after 28 days of culture in OLC culture medium, wells were filled with pure water for 24 h or bleach for 5-10 min, respectively. Subsequently, still mineralised surface was visualised via von Kossa staining as described above (chapter 2.2.6.) beginning with the step of the incubation with silver nitrate. White and grey sections indicated released calcium by the cells. The partially resorbed calcium phosphate surfaces were documented and the percentaged resorption area was quantified using image analysis software (MetaMorph AF, version 1.4.0).

34 2. Material and Methods

2.2.8. Migration of Bone Cells

The cell migration assays were performed in cooperation with the group of Prof. Brenner from the Department of Orthopaedics at the University Hospital Ulm. The chemotactic response of MSC and OB was investigated using a 48-well microchemotaxis chamber and polycarbonate filters with 8 μm pores. The lower wells of the chemotaxis chamber were filled with DMEM without or with 10, 100 or 1000 ng/ml human recombinant C5a and covered by the migration filter. Cells were seeded in the upper wells with 1 x 104 cells in 50 µl/well. The filter was removed after 4 h of incubation. The filter was rinsed with cold water and non- migrated cells on the upper side of the filter were scraped of with a rubber wiper. The migrated cells on the lower side were fixed with 4% formaldehyde, Giemsa stained and counted. Additionally, migration of osteoblasts was tested in response to 100 ng/ml C5a with and without pre-incubation with a specific C5aR antagonist (C5aRA) at a concentration of 1 mg/ml for 1 h (kindly provided by Prof. John D. Lambris, Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA). A checkerboard analysis with 100 ng/ml C5a added to both the upper and lower wells or only to the lower well was performed with osteoblasts in order to distinguish between chemotaxis and random migration [38].

2.2.9. Stimulation of Bone Cells with Trauma Mediators

Inflammatory response of osteoblasts To assess the inflammatory response of osteoblasts treated with pro-inflammatory mediators relevant in severe trauma, cells of passage 3-5 where cultivated in OB differentiation medium until confluence. Osteoblasts where detached with Accutase™ and seeded with 100.000 cells/cm2 in osteoblast differentiation medium in 24-well plates at least 12 h before treatment with pro-inflammatory mediators. Cells were treated for 24 h with 1 µg/ml C3a, 0.1 µg/ml C5a or 0.1 ng/ml IL-1β or were co-stimulated with IL-1β and C3a or Il-1β and C5a in osteoblast stimulation medium to investigate their ability to respond to inflammatory stimuli. The concentrations of the trauma-relevant mediators used in the experiments were within the range of the patho-physiological concentrations

35 2. Material and Methods measured in the sera of patients with polytrauma [46]. 0.1 µg/ml lipopolysaccharide (LPS) was used as a positive control. After 24 h of incubation, cell culture supernatants were collected, centrifuged at 800 g for 5 min and stored at -80 °C until use. Release of the pro-inflammatory cytokines IL-6 and IL-8 into the medium was assessed using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. Cell lysates were harvested to determine mRNA expression of IL-6, RANKL, M-CSF and osteoprotegerin (OPG).

Inflammatory response of osteoclast-like cells PBMNC were cultured for 21 days in 24-well plates in OLC culture medium to promote the formation of osteoclast-like cells. After 21 days, the cells were treated with single or combined trauma-relevant pro-inflammatory mediators as described for osteoblasts. After 24 h of incubation, cell culture supernatants were collected, centrifuged at 800 g for 5 min and stored at -80 °C until use. Release of the pro- inflammatory cytokines IL-6 and IL-8 into the medium was assessed using ELISA kits according to the manufacturer’s instructions. Cell lysates were harvested to determine mRNA expression of IL-6, RANKL, M-CSF and osteoprotegerin (OPG).

2.2.10. Cleavage of Complement Zymogens by Bone Cells

The C3a and C5a ELISAs and the chemotaxis assay were performed in collaboration with the laboratory of Prof. Huber-Lang from the Department of Traumatology, Hand-, Plastic-, and Reconstructive Surgery at the University Hospital Ulm. Osteoblasts were seeded with 10.000 cells/cm2 in 24-well plates and cultured in OB differentiation medium for 14 days. After 14 days, cells were incubated for 4 h with 5 g C3 or C5 in 250 l FCS-free medium per well. PBMNC were seeded with 500,000 cells/cm2 in 96-well plates and cultured for 14 days in modified OLC culture medium with reduced concentrations of 20 ng/ml RANKL and 10 ng/ml rh M-CSF to promote formation of OLC and were then incubated for 4 h with 2.5 g of purified human complement protein C5 or C3 in 50 l FCS-free medium per well. Parallel cultures were treated with C3 or C5 in the presence of 25 ng/ml phorbol 12-myristate 13-acetate (PMA). PMA, an activator of protein kinase C (PKC) is often used as an activator for neutrophils and macrophages. Human neutrophils and rat alveolar macrophages PMA activated by PMA where

36 2. Material and Methods able to effectively cleave C5 to C5a [58]. Following incubation, the cell culture supernatants were centrifuged at 800 g for 5 min and stored at -80 °C until use. C3 and C5 cleavage was analysed using C3a and C5a ELISA kits, according to the manufacturer’s instructions. To test the functional activity of OLC- and OB-derived cleavage products, a neutrophil chemotaxis assay was performed with the cell culture supernatants as described previously[58].

2.2.11. Statistical Analysis

Experiments were performed independently 3-7 times in triplicate or quadruplicate cultures. The expression of complement components during osteogenic differentiation was evaluated using a non-parametric Wilcoxon signed rank test for paired samples. A non-parametric Mann-Whitney U-test for non-paired samples was performed to evaluate differences between control and cells treated with anaphylatoxins and IL-1β (SPSS Version 18.0.0, SPSS Inc., Chicago, Illinois, USA). To evaluate C3 and C5 cleavage activity of osteoclasts as well as the inflammatory response of osteoclasts a Student’s t-test was performed. Statistical differences of p<0.05 were considered significant. Boxplots depict median, lower and upper quartiles as well as minimum and maximum; outlier values are indicated as single data points.

37 3. Results

3. Results

3.1. Expression of Complement Components by Bone Cells Many cell types express zymogens of the complement cascade, complement regulators or complement receptors. Therefore, the expression of important complement regulatory proteins, anaphylatoxin receptors and complement zymogens in MSC, osteoblasts and osteoclasts was examined via qRT-PCR and immune fluorescence staining. Differences in complement expression by undifferentiated and differentiated MSC were also investigated.

3.1.1. Complement Regulatory Proteins CD46, CD55 and CD59

Expression of complement regulators in mesenchymal stem cells The cell surface bound complement regulatory proteins membrane cofactor of proteolysis (MCP, CD46), decay accelerating factor (DAF, CD55) and MAC inhibitory factor (MACIF, CD59) were expressed by undifferentiated and osteogenicly differentiated MSC as shown by qRT-PCR (Fig. 9). Additionally CD46 and CD59 expression was significantly increased after 14, 21 and 28 days of osteogenic differentiation. CD55 expression was very low compared to the house- keeping gene GAPDH and did not increase during osteogenic differentiation. The expression of another complement regulatory protein, complement receptor 1 (CR1, CD35), which is mainly expressed in immune cells, was also examined but CD35 was not expressed in MSC.

38 3. Results

Fig. 9: Expression of complement regulatory proteins in MSC mRNA expression of complement regulatory proteins membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in undifferentiated MSC and after 14, 21 and 28 days of osteogenic differentiation. Outliers are shown as circles, asterisks indicate significant increase vs. control (p < 0.05).

The expression of CD46, CD55 and CD59 was also shown via immune fluorescence staining of the receptors on undifferentiated MSC and after 28 days of osteogenic differentiation (Fig. 10). All three regulatory proteins were present in undifferentiated and differentiated MSC as it was shown on the mRNA level. In contrast to the mRNA expression the immune fluorescence staining showed an increase not only of CD46 but also of CD55 after 28 days of osteogenic differentiation. There was a very strong staining of CD59 in undifferentiated as well as in differentiated MSC; however, there was no visible increase during differentiation.

39 3. Results

Fig. 10: Immune fluorescence staining of complement regulatory proteins in MSC Immune fluorescence staining of membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in undifferentiated and differentiated (28 days) MSC.

Expression of complement regulators in osteoblasts The complement regulatory proteins CD46, CD55 and CD59 were expressed by primary osteoblasts as shown by qRT-PCR (Fig. 11). CD55 was expressed at very low levels compared to the house-keeping gene GAPDH. CD35 was not expressed in osteoblasts.

Fig. 11: Expression of complement regulatory proteins in osteoblasts mRNA expression of complement regulatory proteins membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in primary osteoblasts. Outliers are shown as circles.

40 3. Results

The expression of CD46, CD55 and CD59 by osteoblasts was also shown via immune fluorescence staining (Fig. 12). Immune fluorescence images reveal strong expression of all three regulators.

Fig. 12: Immune fluorescence staining of complement regulatory proteins in osteoblasts Immune fluorescence staining of membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in osteoblasts.

Expression of complement regulators in osteoclast-like cells Consistent with the findings in MSC and primary osteoblasts the complement regulatory proteins CD46, CD55 and CD59 were expressed by osteoclast-like cells (Fig. 13). The expression of the complement regulators compared to the house- keeping gene GAPDH was higher than in MSC and osteoblasts. CD35 was not expressed in osteoclast-like cells.

Fig. 13: Expression of complement regulatory proteins in OLC mRNA expression of complement regulatory proteins membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in OLC after 21 days of osteoclastogenesis.

41 3. Results

The immune fluorescence staining confirmed the expression of CD46, CD55 and CD59 by osteoclast-like cells (Fig. 14). However, the staining was only weak compared to MSC and primary osteoblasts.

Fig. 14: Immune fluorescence staining of complement regulatory proteins in OLC Immune fluorescence staining of membrane cofactor of proteolysis (CD46), decay accelerating factor (CD55) and MAC inhibitory factor (CD59) in osteoclast-like cells (OLC) after 21 days of culture.

3.1.2. Complement Zymogens C3 and C5

To investigate if bone cells can produce central proteins of the complement cascade, the expression of the complement zymogens C3 and C5 was examined via semi quantitative RT-PCR and agarose gel elctrophoresis.

Expression of C3 and C5 in mesenchymal stem cells The semi quantitative analysis of the mRNA expression of C3 and C5 in undifferentiated MSC and after 14, 21 and 28 days of osteogenic differentiation revealed the expression of C3 and C5 by MSC (Fig 16). There was no visible change in the expression of C3 or C5 during differentiation.

Expression of C3 and C5 in osteoblasts Semi quantitative RT-PCR revealed the expression of C3 and C5 by primary osteoblasts (Fig. 16). The expression of C5 by osteoblasts was slightly less than in MSC.

42 3. Results

Expression of C3 and C5 in osteoclast-like cells In contrast to MSC and osteoblasts, osteoclast-like cells express only C3 but no C5 (Fig. 15). Additionally there is an increase in the C3 expression during osteoclastogenesis from PBMNC, which only express small amounts of C3.

Fig. 15: Expression of complement zymogens C3 and C5 mRNA expression of the complement zymogens C3 and C5 and the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in MSC during osteogenic differentiation, in osteoblasts (OB) and during formation of osteoclast-like cells (OLC) from peripheral blood mononuclear cells (PBMNC).

3.1.3. Complement Anaphylatoxin Receptors C3aR, C5aR and C5L2

For a specific cellular answer to activated complement anaphylatoxins, it is necessary that the cells express receptors for activated complement components. Therefore the expression of the receptors for the most important anaphylatoxins C3a and C5a was analysed in bone cells to determine whether these cells are possible targets for activated complement.

Expression of anaphylatoxin receptors in mesenchymal stem cells Quantitative mRNA analysis of C3aR and C5aR mRNA revealed expression of both receptors in MSC (Fig. 16). Moreover there was a strong and significant increase in C5aR expression during osteogenic differentiation. C3aR expression also increased during differentiation but the difference was not significant.

43 3. Results

Fig. 16: Expression of complement anaphylatoxin receptors in MSC mRNA expression of the anaphylatoxin receptors C3aR and C5aR in undifferentiated MSC (hMSC) and after 14, 21 and 28 days of osteogenic differentiation(o-hMSC). Outliers are shown as circles, asterisks indicate significant increase vs. control (p < 0.05).

Consistent with the mRNA expression, the expression of the anaphylatoxin receptors C3aR and C5aR was also detected via immune fluorescence staining in undifferentiated and differentiated MSC (Fig. 17). Additionally the second receptor for C5a, the C5L2 was expressed by both, differentiated and undifferentiated MSC.

44 3. Results

Fig. 17: Immune fluorescence staining of complement anaphylatoxin receptors in MSC Immune fluorescence staining of the anaphylatoxin receptors C3aR, C5aR and C5L2 in undifferentiated and differentiated (21 days) MSC.

Expression of anaphylatoxin receptors in osteoblasts The quantitative mRNA analysis revealed the expression of both the C3aR and the C5aR in primary osteoblasts (Fig. 18).

Fig. 18: Expression of complement anaphylatoxin receptors in osteoblasts mRNA expression of the anaphylatoxin receptors C3aR and C5aR in primary osteoblasts. Outliers are shown as circles.

45 3. Results

The expression of the anaphylatoxins C3aR and C5aR by primary osteoblasts was confirmed via immune fluorescence staining (Fig. 19). C5L2 was also expressed by primary osteoblasts.

Fig. 19: Immune fluorescence staining of complement anaphylatoxin receptors in osteoblasts Expression of the anaphylatoxin receptors C3aR, C5aR and C5L2 in osteoblasts detected by immune fluorescence staining.

Expression of anaphylatoxin receptors in osteoclasts Osteoclast-like cells, as well as MSC and primary osteoblasts, express C3aR and C5aR, as was shown by qRT-PCR in PBMNC cultures after 21 days under osteoclastogenic conditions (Fig. 20).

Fig. 20: Expression of complement anaphylatoxin receptors in OLC mRNA expression of the anaphylatoxin receptors C3aR, C5aR and C5L2 in OLC after 21 days of osteoclastogenesis.

46 3. Results

Immune fluorescence staining revealed the expression of C3aR, C5aR and C5L2, by osteoclast-like cells derived from PBMNC cultures (Fig. 21).

Fig. 21: Immune fluorescence staining of complement anaphylatoxin receptors in OLC Expression of the anaphylatoxin receptors C3aR, C5aR and C5L2 in osteoclast-like cells (OLC) after 21 days of culture detected by immune fluorescence staining.

3.2. Complement Activation by Bone Cells In cooperation with the group of Prof. Huber-Lang the ability of bone cells to independently activate complement via direct cleavage of C3 or C5 by osteoblasts or osteoclast-like cells. After 4h incubation of osteoblasts and osteoclast-like cells with complement component C5, the concentration of C5a in the cell culture supernatants was measured using a C5a specific ELISA kit to assess the amount of activated complement generated by the cells (Fig. 22). Osteoblasts and osteoclast-like cells both were able to cleave C5. While osteoblasts only generated small amounts of C5a osteoclast-like cells cleaved C5 far more efficient. In presence of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) the ability of OLC to cleave C5 was further increased. PMA had no influence on C5 cleavage by osteoblasts. Respective experiments were performed with complement component C3 to investigate C3 cleavage, but no C3a was found in the cell culture supernatants.

47 3. Results

Fig. 22: C5 cleavage by OB and OLC C5a concentrations in cell culture supernatants of OB and OLC were determined via ELISA after 4h of incubation with C5 in absence and presence of phorbol 12-myristate 13-acetate (PMA). The amount of C5a in the cell culture supernatants is proportional to the amount of C5 cleaved by the cells. The asterisk indicates a significant difference (p < 0.05).

To test the functional activity of C5 generated by osteoblasts and osteoclasts, a chemotaxis assay with human neutrophils was performed using the cell culture supernatants from the complement activation experiments (Fig. 23). Controls with medium containing C5 or different concentrations of C5a revealed a dose dependent chemotactic activity of C5a on neutrophils whereas medium containing C5 without cell contact had only a weak chemotactic effect. Osteoblast culture supernatants had only weak chemotactic effects, which did not increase in presence of PMA. The supernatants of osteoclast-like cell cultures showed a strong chemotactic effect on neutrophil granulocytes, that however, was not significantly increased compared to the control medium without C5 or C5a. The chemotactic effect of OLC culture supernatants significantly increased in presence of PMA.

48 3. Results

Fig. 23: Chemotactic activity of C5a generated by osteoblasts and osteoclast-like cells Chemotactic activity of neutrophil granulocytes induced by media containing specific C5a concentrations and by cell culture supernatants of osteoblasts (OB) and osteoclast-like cells (OLC). The asterisk indicates a significant difference (p < 0.05).

3.3. Functional Studies on Anaphylatoxin Receptors

Several studies were performed on possible functions of the anaphylatoxin receptors on bone cells, including chemotaxis and inflammation.

3.3.1. Internalisation of Anaphylatoxin Receptors

The ability of osteoblasts and osteoclasts to internalize the anaphylatoxin receptors was investigated.

Internalisation of C3aR A two-step immune fluorescence staining experiment was used to show the location of the C3aR inside or outside the cell (Fig. 24). A green dye was used before permeabilisation of the cells and red dye was used afterwards. In the overlay of the phase contrast and the two fluorescent images, receptors on the cell surface appear green or yellow whereas receptors inside the cell appear red. In absence of C3a most of the receptor is located on the surface of osteoblasts.

49 3. Results

Following 45 minutes incubation of osteoblasts with C3a most of the C3aR receptors are located inside the cells. In contrast, C3aR on osteoclast-like cells was not internalised after 45 min incubation with C3a.

Fig. 24: Internalisation of C3aR by OB and OLC Internalisation of C3a receptor (C3aR) in osteoblasts (OB) and osteoclast-like cells (OLC) following stimulation with C3a was analysed by double fluorescence staining after 45 min incubation with C3a and in non-stimulated controls. C3aR on OB and OLC was stained with a green fluorochrome before permebilisation and with a red fluorochrome following permeabilisation. This procedure results in a green or yellow staining for receptors on the cell surface and in a red staining for receptors located inside the cell.

50 3. Results

Internalisation of C5aR Consistent with the internalisation of C3aR after incubation with C3a, it was shown that C5a also is internalised by osteoblasts but not by osteoclasts (Fig. 25).

Fig. 25: Internalisation of C5aR by OB and OLC Internalisation of C5a receptor (C5aR) in osteoblasts (OB) and osteoclast-like cells (OLC) following stimulation with C5a was analysed by double fluorescence staining after 45 min incubation with C5a and in non-stimulated controls. C5aR on OB and OLC was stained with a green fluorochrome before permebilisation and with a red fluorochrome following permeabilisation. This procedure results in a green or yellow staining for receptors on the cell surface and in a red staining for receptors located inside the cell.

51 3. Results

Internalisation of C5L2 In contrast with C3aR and C5aR, in absence of C5a, the second receptor for C5a, the C5L2 was located mainly inside the cells (Fig. 26). After 45 min incubation with C5a most of the C5L2 was transported to the cell surface.

Fig. 26: Internalisation of C5L2 by OB Internalisation of C5a receptor-like 2 (C5L2) in OB following stimulation with C5a was analysed by double fluorescence staining after 45 min incubation with C5a and in non-stimulated controls. C5L2 on OB was stained with a green fluorochrome before permeabilisation and with a red fluorochrome following permeabilisation. This procedure results in a green or yellow staining for receptors on the cell surface and in a red staining for receptors located inside the cell.

3.3.2. Chemotactical Response of Bone Cells to C5a

It is well known, that C5a is a powerful chemokine especially for immune cells such as neutrophil granulocytes. In cooperation with the group of Prof. Brenner it was investigated if C5a could also induce chemotaxis of bone cells. Chemotaxis of osteoblasts was examined with a modified Boyden chamber assay against different concentrations of C5a (Fig. 27). MSC showed a significantly increased number of migrated cells only at the highest C5a concentration of 1000 ng/ml. In contrast, osteoblasts revealed a dose dependent, significantly increased migration at concentrations from 10 to 1000 ng/ml. The chemotactic effect of C5a was stronger in osteoblasts compared to MSC for each C5a concentration.

52 3. Results

Fig. 27: Migration of MSC and osteoblasts towards a C5a gradient The number of cells migrated towards a C5a gradient was analysed via a modified boyden chamber assay. Different concentrations of C5a were used. Medium without C5a served as control. Asterisks indicate significant differences (p < 0.05).

A checkerboard analysis was performed with primary osteoblasts at a concentration of 100 ng/ml C5a to ascertain that the observed migration was a directed chemotaxis and not an undirected chemokinesis (Fig. 28). The assay showed that the migration was strictly dependent of a positive concentration gradient. To confirm the direct involvement of the C5aR receptor a specific C5aR antagonist was used (Fig 29). Pre-incubation with this antagonist completely abolished the chemotactic effect of C5a on primary osteoblasts.

53 3. Results

Fig. 28: Checkerboard analysis A checkerboard analysis with C5a in the lower chamber or in the upper and lower chamber was performed to test whether the observed cell movement was a directed chemotaxis. The receptor specificity of the migration was analysed using a specific C5a receptor antagonist.

3.3.3. Inflammatory Response of Bone Cells

Another typical function of complement anaphylatoxins is triggering the inflammatory response in cells of the innate immunity. Therefore it was investigated if osteoblasts and osteoclast-like cells are able to show a specific response to anaphylatoxins and pro-inflammatory cytokines.

Inflammatory response of osteoblasts Expression of IL-6 mRNA was assessed via qRT-PCR after 24h incubation with different single or combined trauma mediators (Fig. 29). These trauma mediators where used in concentrations as they are found in patients with severe multiple trauma. Lipopolysaccharide (LPS) from E. coli was used as a positive control for an inflammatory reaction. LPS caused a significant increase in IL-6 mRNA expression by primary osteoblasts. A similar result was found for the co- stimulatory conditions with IL-1β and C3a or IL1β and C5a. Both co-stimulatory conditions caused a strong, significant increase of IL-6 expression. In contrast, the single stimulation with IL-1β, C3a or C5a did not significantly increase the IL-6 expression relative to non-stimulated control.

54 3. Results

Fig. 29: IL-6 expression of OB following pro-inflammatory stimulation The expression of the pro-inflammatory cytokine IL-6 by OB stimulated with trauma mediators was quantitatively assessed by qRT-PCR. Outliers are shown as circles, asterisks indicate significant increase vs. control (p < 0.05).

Additionally to the mRNA expression of IL-6, the release of the pro-inflammatory interleukins IL-6 and IL-8 into the medium was measured using specific ELISA kits (Fig. 30). In consistence with the mRNA expression of IL-6 the release of IL-6 was significantly increased after 24h incubation with the positive control LPS as well as under both co-stimulatory conditions but not with single IL-1β, C3a or C5a. The same results were found for the release of IL-8.

55 3. Results

Fig. 30: Interleukin release by OB following pro-inflammatory stimulation The release of the pro-inflammatory cytokines IL-6 and IL-8 into the medium by OB stimulated with trauma mediators was measured by ELISA. Outliers are shown as circles, asterisks indicate significant increase vs. control (p < 0.05).

Inflammatory response of osteoclast-like cells The relative expression of IL-6 and IL8 mRNA was assessed via qRT-PCR following 24h incubation with LPS, IL-1β, C3a, C5a or IL-1β and C5a (Fig. 31). In contrast to the inflammatory response of osteoblasts only the positive control (LPS) showed a strong increase of IL-6 and IL-8 expression whereas the co- stimulation with IL-1β and C5a only caused a little increase in IL-6 expression. However IL-6 and IL-8 expression was not significantly increased.

56 3. Results

Fig. 31: Interleukin expression of OLC following pro-inflammatory stimulation The expression of the pro-inflammatory cytokines IL-6 and IL-8 by OLC stimulated with trauma mediators was quantitatively assessed by qRT-PCR.

The release of the pro-inflammatory interleukins IL-6 and IL-8 was measured using specific ELISA kits (Fig. 32). LPS strongly increased the IL-6 and IL-8 release from osteoclast-like cells whereas the co-stimulation with IL-1β and C5a only caused an increase in IL-6 release and IL-1β single stimulation increased IL-8 release However, the increase in IL-6 and IL-8 expression was not significant.

57 3. Results

Fig. 32: Interleukin release by OLC following pro-inflammatory stimulation The release of the pro-inflammatory cytokines IL-6 and IL-8 into the medium by OLC stimulated with trauma mediators was measured by ELISA.

3.4. Influence of Anaphylatoxins on Maturation and Function of Bone Cells

A possible reason for impaired fracture healing during chronic or systemic complement activation for example after severe multiple trauma is a shifted balance of osteoblast and osteoclast activity in favour of osteoclasts. This may be caused by impaired osteoblast maturation or by increased osteoclast formation and activity.

3.4.1. Osteogenic Differentiation of MSC

The influence of C3a and C5a on osteogenic differentiation of MSC was examined after 21 days of osteogenic differentiation in absence (control) or in presence of C3a or C5a in relevant concentrations as they are found in the serum of patients systemic inflammation (Fig. 33).

58 3. Results

Fig. 33: Expression of osteogenic markers by MSC during differentiation in presence of complement anaphylatoxins The expression of the osteogenic markers bone sialo protein (BSP), alkaline phosphatase (AP) and osteopontin (OP) by MSC was quantitatively measured after 21 days of osteogenic differentiation to investigate an influence of complement anaphylatoxins on the osteogenic differentiation of MSC.

After 21 days of osteogenic differentiation AP and von Kossa staining were performed to examine the osteogenic differentiation of MSC (Fig. 34). Images of the cells stained for AP showed a robust staining in nearly all cells independent from the presence or absence of C3a and C5a. The von Kossa staining also revealed a strong mineralisation in cell cultures with and without C3a or C5a.

59 3. Results

Fig. 34: AP and von Kossa staining after osteogenic differentiation in presence of complement anaphylatoxins AP and von Kossa staining was performed to investigate an influence of complement anaphylatoxins on the osteogenic differentiation of MSC.

3.4.2. Expression of M-CSF/RANKL/OPG by Stimulated Osteoblasts

The cytokines RANKL and OPG are the most important regulators of osteoclast formation. Therefore the mRNA expression of these cytokines was examined following 24 h incubation of osteoblast with trauma-relevant mediators (Fig. 35). LPS that was used as positive control for inflammatory response caused a significant increase of both RANKL and its adversary OPG. Co-stimulation with IL- 1β and C3a or IL-1β and C5a also significantly increased the expression of both RANKL and OPG. In contrast, the single factors did not increase the RANKL or OPG expression.

60 3. Results

Fig. 35: mRNA expression of RANKL and OPG by osteoblasts (OB) following pro- inflammatory stimulation mRNA expression of the OC-regulating genes osteoprotegerin (OPG) and receptor activator of nuclear factor B ligand (RANKL) in OB cultures stimulated with IL-1β, C3a and C5a single and in different combinations. Lipopolysaccharide (LPS) served as a positive control. Levels of mRNA were normalised to non-stimulated control. Outliers are shown as circles, asterisks indicate significant increase vs. control (p < 0.05).

Even more important for osteoclastogenesis than the expression of RANKL and OPG is the RANKL/OPG ratio. The ratio was calculated from the mRNA expression relative to GAPDH and related to the non-stimulated control (Fig. 36). There was no significant change in the RANKL/OPG ratio following stimulation with trauma-relevant mediators.

61 3. Results

Fig. 36: RANKL/OPG ratio by OB following pro-inflammatory stimulation Ratio of RANKL/OPG mRNA expression in OB cultures stimulated with IL-1β, C3a and C5a singly and in different combinations. Lipopolysaccharide (LPS) served as a positive control. RANKL/OPG ratio was normalised to non-stimulated control. Outliers are shown as circles.

Besides RANKL and OPG, the growth factor M-CSF is important for osteoclast formation. The expression of M-CSF mRNA was analysed after 4h incubation with trauma-relevant mediators (Fig. 37). M-CSF expression was increased following stimulation with LPS, IL-1β and C3a or IL-1β and C5a. However, the increase was not significant.

62 3. Results

Fig. 37: mRNA expression of M-CSF by OB following pro-inflammatory stimulation mRNA expression of the OC-regulating gene macrophage-colony stimulating factor (M-CSF) in OB cultures stimulated with IL-1β and C3a or IL-1β and C5a, respectively. Lipopolysaccharide (LPS) served as a positive control. Levels of mRNA were normalised to non-stimulated control.

3.4.3. Formation of TRAP+ Multinucleated Cells

It was also examined if there is a direct influence of complement anaphylatoxins C3a and C5a on osteoclast formation. PBMNC were cultivated in OLC culture medium without RANKL and M-CSF for 21 days in presence or absence of 1 µg/ml C3a or 0.1 µg/ml C5a (Fig. 38). Regular OLC culture medium containing RANKL and M-CSF was used as positive control. Cells were stained for tartrate resistant acid phosphatase (TRAP) and TRAP+ cells with three or more nuclei were considered as osteoclast-like cells. PBMNC cultures supplemented with RANKL/M-CSF, C3a or C5a contained notably more osteoclast-like cells than non- supplemented cultures.

Fig. 38: TRAP staining of PBMNC cultures in presence of complement anaphylatoxins TRAP staining of PBMNC cultures after 21 days of culture in presence of FCS (negative control), RANKL/M-CSF (positive control), C3a and C5a was performed to determine the number of osteoclast-like cells (OLC). TRAP-positive stained cells (red) with at least 3 nuclei were considered as osteoclast-like cells.

63 3. Results

The numbers of OLC in PBMNC cultures after 21 days of cultivation in presence of RANKL/M-CSF, C3a or C5a were counted and related to OLC numbers in non- supplemented control medium (Fig. 39). As expected, medium supplemented with RANKL and M-CSF significantly increased the OLC formation compared to non- supplemented control-medium. Medium containing C3a or C5a also significantly increased the OLC formation, respectively. OLC numbers found in PBMNC cultures containing RANKL/M-CSF, C3a or C5a were similar.

Fig. 39: Osteoclast-like cell (OLC) formation in presence of complement anaphylatoxins Number of TRAP-positive multinucleated (≥ 3 nuclei) cells generated in PBMNC cultures supplemented with RANKL/M-CSF, C3a or C5a relative to non-supplemented control cultures. Outliers are shown as circles, asterisks indicate a significant increase vs. control (p < 0.05).

3.4.4. Resorption Activity of Osteoclasts

The ability of C3a and C5a to induce formation of functional OLC was also examined by a resorption assay using special, calcium phosphate coated cell culture plates. Following 28 days of cultivation in medium containing RANKL/M-CSF, C3a or C5a, remaining calcium phosphate was von Kossa stained to facilitate the detection of resorbed areas (Fig. 40). As expected the calcium phosphate coatings in wells containing PBMNC cultures in medium with RANKL/M-CSF showed strong resorption. In contrast OLC cultured with C3a or C5a failed to resorb the calcium phosphate coating.

64 3. Results

Fig. 40: Resorption activity of osteoclast-like cells Resorption of calcium phosphate coating on cell culture plates by OLC in presence of RANKL/M-CSF (positive control), C3a or C5a. The remaining calcium phosphate was von Kossa stained (black) to make the resorbed areas visible (white and grey).

65 4. Discussion

4. Discussion

There is evidence for a multifaceted role of complement in formation and regeneration of bone but the underlying mechanisms are still poorly understood. This work has confirmed some findings of complement-bone interactions and added new insights in the complex network of mutual regulation of bone and the complement system (Fig. 41).

Fig. 41: Proposed interactions of bone cells and the complement system There are multiple interactions between the different types of bone cells and the complement system.

4.1. Expression of Complement Components by Bone Cells

Many cell types express complement regulatory proteins or anaphylatoxin receptors and several cell types express inactive precursors of complement- related serin proteases called zymogens. The complement regulatory proteins CD46, CD55 and CD59 are expressed on nearly all cell types investigated, with the exception of erythrocytes [69]. The present study confirms the expression of these complement regulatory proteins on MSC, OB and OLC. Other complement regulators such as the complement 66 4. Discussion receptor 1 (CR1, CD35) are only expressed on leukocytes and erythrocytes [69]. As expected, CD35 expression was not found in MSC, OB and OLC. An interesting new finding was that the expression of complement regulators increases during osteogenic differentiation. Complement regulatory proteins protect host cells against complement activated spontaneously via the alternative pathway and against damage from complement activated by nearby pathogens. The increased expression of complement leads to the conclusion that osteogenicly differentiated MSC and osteoblasts may be better protected against complement proteins than undifferentiated MSC. Additionally, many cell types express receptors for complement anaphylatoxins, which induce different effects, including migration of phagocytes, induction of phagocytosis and expression of pro-inflammatory cytokines by immune cells. The expression of C5aR on osteoblasts was already confirmed in a recent in vivo study from our group by immuno-histo staining [60]. Even before there was evidence for the expression of functional C5aR receptors on human osteosarcoma cell line MG- 63 as these cells showed a specific pro-inflammatory response after stimulation with C5a [104]. In the present study the anaphylatoxin receptors C3aR, C5aR and C5L2 were shown to be expressed by MSC, OB and OLC. There was also an increase in the expression of the anaphylatoxin receptors C3aR and C5aR during osteogenic differentiation of MSC. This may result in a higher sensitivity of osteoblasts for complement anaphylatoxins. Indeed, the chemotactic effect of C5a was significantly stronger on osteoblasts compared to MSC (3.3.2). Complement proteins are generally expressed by liver cells but also, in significant amounts by monocytes or macrophages [143]. Therefore it is a very interesting finding, that MSC and OB express C3 and C5, which are the precursor proteins of the two most important anaphylatoxins and key components in all activation pathways. OLC, which derive from the same origin as macrophages, however were found to express only C3. By expressing and releasing these central complement components bone cells may modify the local complement activation. However, the release of C3 and C5 by bone cells may differ from the mRNA expression and was not investigated in the present study. Therefore it is not possible to deduce a real influence of complement components produced by bone cells on local or systemic complement concentrations from the present results.

67 4. Discussion

4.2. Complement Activation by Bone Cells

The three well-known pathways of complement activation completely depend on soluble plasma proteins. Complement activation is usually triggered by binding of pattern recognition molecules to specific molecular patterns on target cells or spontaneously via the alternative pathway [108]. Later an additional, cell dependent activation mechanism via direct cleavage of C5 by rat alveolar macrophages (AM) was found [58]. In the present study, the ability of OB and OLC to cleave C3 and C5 was investigated. It was shown that both OB and OLC were able to cleave C5 to C5a and C5b with OLC being much more efficient. A cell migration assay using the cell culture supernatants from these experiments revealed a strong chemotactic effect on PBMNC only for the supernatants of OLC cultures demonstrating that OLC were able to generate functionally active C5a. PMA a phorbol ester, which is often used as an activator for neutrophils and macrophages, was required to induce C5 cleavage activity of rat AM and human blood neutrophils [58]. As OB and OLC derive from different lineages, the present study compares the C5a generation in presence and absence of PMA. As expected, the cleavage activity of OB, which originate from the mesenchymal lineage was not influenced by the presence of PMA. In contrast PMA potently induced C5 cleavage by OLC, which derive from the monocytic lineage. By using inhibitors specific for different groups of proteases, Huber-Lang et al. demonstrated that C5 cleavage by AM depends of a serine protease as it was completely inhibited by two different specific serine protease inhibitors, SBTI and SLPI but not by other protease inhibitors. Neither SBTI nor SLTI interfered with CH50 activity of rat or human serum, indicating that the protease responsible for AM-dependent C5 cleavage is not a complement-derived C5 convertase [58]. C5a generation by OLC probably depends on a similar mechanism, as macrophages and OLC both derive from monocyte origin, and cell dependent cleavage was not yet described for other cell types. However, experiments to investigate the exact mechanism or the responsible protease in osteoblasts and osteoclasts were not yet performed.

68 4. Discussion

4.3. Functional Studies on Anaphylatoxin Receptors

Interactions of bone cells and the complement system are most likely mediated by the anaphylatoxins via their respective receptors that were found on MSC, OB and OLC in this study. Especially the significant up-regulation of C5aR and the strong increase in trend of the C3aR expression indicate an important role of anaphylatoxin signalling via these receptors (3.1.3). C3a and C5a were shown to modulate apoptosis, cell migration and cytokine release in several cell types [37, 51, 70, 125]. Currently, little is known about the functions of anaphylatoxin signalling in bone cells. Therefore, anaphylatoxin-triggered receptor internalisation, cell migration and pro-inflammatory response of bone cells were investigated in this study.

Internalisation of anaphylatoxin receptors Double immunostaining experiments were performed to elucidate the location of anaphylatoxin receptors on the cell surface or in the cytoplasm. These experiments showed that C3aR and C5aR were located mainly on the cell surface and only in small amounts inside the cells. Stimulation with the respective ligands led to complete internalisation of both receptors. Ligand-induced internalisation of the anaphylatoxin receptors was previously described for different cell types, including granulocytes [128, 142] and MSC [126]. There seem to be different effects of anaphylatoxin receptor internalisation. Internalisation of GPCR in general and anaphylatoxin receptors in particular was previously described as a regulatory mechanism involved in homologous desensitisation [16, 88, 91]. This regulation mechanism might be of high importance in excessive or systemic inflammation. Especially the externalisation of C5L2 following C5a stimulation is interesting, as C5L2 is guessed to be a regulatory scavenger receptor [63, 98]. Providing more C5L2 on the cell surface might enhance a possible regulatory effect of the receptor internalisation. However, a mechanism regulating externalisation of C5L2 in response to C5a is not yet known. In contrast, location in or translocation to the nucleus was also shown for different GPCR, leading to long-term effects including prolonged nuclear ERK1/2 activation [48, 49, 80, 151]. Internalisation accompanied by prolonged ERK1/2 activation was also demonstrated for C3aR and C5aR on MSC [126].

69 4. Discussion

Chemotaxis One of the roles of C5a is that of a powerful chemoattractant for many cell types including neutrophils, eosinophils, monocytes and mast cells [126]. This study revealed a strong dose-dependent cell migration of OB in response to C5a whereas only the highest dose of C5a induced migration of MSC. A checkerboard analysis performed with osteoblasts, confirmed that this cell migration was positive chemotaxis, which means directed cell migration towards a concentration gradient. Additionally, the chemotactic response was completely inhibited by pre-incubation with a C5aR antagonist, demonstrating that the observed effect was mediated directly by C5a binding to its receptor. In contrast to migration of OB, MSC migration was significantly lower at all tested C5a concentrations. MSC migration was only at the highest concentration used significantly increased in comparison to unstimulated controls. The lower sensitivity of MSC for the chemotactic effect of C5a is probably due to the lower receptor expression in MSC. Osteogenic differentiation of MSC increased both the expression of C5aR and the chemotactic response to C5a, suggesting a higher anaphylatoxin sensitivity of more mature cells within the osteogenic lineage. A previous study showed C5a induced migration in PBMNC but not in MSC [124], which is in line with the present results. In contrast, a recent study reported that human MSC strongly expressed C5aR and showed cell migration in response to C5a [126]. The contradiction between these findings may be explained by a divergent differentiation status of the cells since the MSC used in the work of Schraufstatter et al. were used in passages up to 7, whereas the MSC used in the present study were used only in passages 2 - 4. This might be of relevance as MSC of higher passages tend to spontaneous osteogenic differentiation [9].

Inflammation Triggering inflammation in response to a wide range of pathogens and other threats is another very important function of complement anaphylatoxins. Besides recruiting various immune cells, anaphylatoxin signalling also induces histamine release from mast cells, triggers the oxidative burst in macrophages and neutrophils and induces the release of pro-inflammatory cytokines by phagocytes [35, 36, 89]. Especially in chronic or excessive inflammation with high anaphylatoxin levels, bone cells might modulate the inflammatory response by

70 4. Discussion releasing pro-inflammatory cytokines. The present study demonstrates that OB treated with IL-1β and C5a or C3a respectively, in pathophysiological concentrations as they are found in major trauma, significantly increased the expression and release of the pro-inflammatory interleukins IL-6 and IL-8. In contrast, neither IL-1β nor the anaphylatoxins alone could induce a distinct increase of the interleukin expression and release. The interleukin release was as high as that induced by the positive control with LPS. Bacterial endotoxins such as LPS belong to the most important triggers of interleukin release in many cell types. Therefore, these results indicate that the interleukin concentrations released by MSC may be physiologically relevant. These findings were confirmed by the results of qRT-PCR demonstrating an increase of IL-6 mRNA following treatment with IL1β and C3a or C5a respectively. Similar effects were reported by Pobanz et al. who investigated IL-6 release by human osteosarcoma cell line MG-63 treated with C5a and IL1-β and found a synergistic induction [104]. However, Pobanz et al. also detected a significantly increased IL-6 release in MG-63 cells treated only with C5a that was, however, strongly increased by pre-incubation with IL-1β indicating a synergistic effect but not a complete dependency on co-stimulation as found in the present study. Complex interrelations of pro-inflammatory stimuli with anaphylatoxin effects have been previously reported for other cell types. IL-1β increased the expression of the C5aR on mononuclear cells [133]), whereas C5a stimulated IL-1β-release from PBMNC [99]. C5a and LPS synergistically induced IL-6 release from Kupffer cells [81]. The synergism of IL-1β and anaphylatoxins observed by Pobanz et al. and in the present study might be due to cross talks in the respective down-stream signalling pathways. IL-1β effects are mediated by activation of MEKK1 and proteins of the interleukin receptor associated kinase (IRAK) family, leading to activation of C-JUN or NF-κB [39]. C5aR signalling involves activation of ERK1/2, AKT and MAPK p38 [110]. C3aR activation was shown to induce ERK1/2 and NF- κB [77]. However, the exact mechanisms of the interactions of interleukins and anaphylatoxins remain unclear and should be subject to further investigations. The present data and the previous findings suggest that C3a and C5a can enhance the inflammatory response of osteoblasts in a pro-inflammatory environment as in inflammatory bone diseases or during bone healing.

71 4. Discussion

4.4. Influence of Anaphylatoxins on Maturation and Function of Bone Cells

A possible mechanism for impaired fracture healing caused by inappropriate complement activation is an impaired maturation or function of osteoblasts and/or an increased number or resorption activity of osteoclasts. Osteogenic differentiation of mesenchymal stem cells is essential for providing functional osteoblasts for bone repair or remodelling. The present study showed that alkaline phosphatase activity and matrix mineralisation of MSC after 21 days of osteogenic differentiation were not affected by the presence of 1 µg/ml C3a or 0.1 µg/ml C5a. Additionally the expression of osteogenic marker genes in presence of C3a or C5a was not altered compared to osteogenic cultures without C3a or C5a. Furthermore, in a single experiment there was no osteogenic effect of complement anaphylatoxins on MSC cultured without osteogenic supplements. These results demonstrate that complement anaphylatoxins neither enhance nor impair the osteogenic differentiation of osteoblasts. The effect of C3a and C5a on OLC formation from human PBMNC and the resorption activity of these cells were also investigated. As expected 10 µg/ml RANKL and 20 µg/ml M-CSF significantly increased the number of OLC formed after 28 days compared to control medium without RANKL and M-CSF. This is accordant with the finding that RANKL and M-CSF are essential for osteoclast formation [20, 72]. Intriguingly both C3a and C5a were found to significantly increase the number of OLC formed in absence of RANKL and M-CSF to a level comparable to the RANKL/M-CSF control. OLC generated from PBMNC by C3a or C5a in absence of RANKL and M-CSF did not show resorption of calcium phosphate coatings, indicating an essential function of RANKL and M-CSF for full functionality of OLC. Complement anaphylatoxins primarily seem to enhance fusion of osteoclast precursor cells. Together with the expression of complement zymogens by MSC, OB and OLC and the cell-dependent activation of C5 by OLC, these results suggest, that bone cells may locally produce and activate complement anaphylatoxins and that these anaphylatoxins may directly enhance osteoclast formation. Additionally, anaphylatoxins are known to have strong chemotactic effects on different cell types including osteoclast precursors [40]. In vivo, this may contribute to the recruitment of osteoclast precursor cells. Together these effects may locally contribute to osteoclast formation.

72 4. Discussion

The present study also showed an increase of RANKL and OPG expression under pro-inflammatory conditions in vitro, indicating additional indirect effects of IL- 1β/C3a and IL-1β/C5a co-stimulation on osteoclast formation. The increased RANKL expression may be due to direct effects or mediated by the increased expression of IL-6, which is known to enhance osteoclast formation and bone resorption via induction of RANKL [102].

73 5. Summary

5. Summary

Fracture healing is impaired in patients with major trauma, accompanied by robust inflammatory response including systemic activation of the complement system. The reasons of this delay in healing remain incompletely understood and are presently subject to further studies. The tight interactions between bone and the immune system are the subject to osteoimmunology since several years. However the relevance of the complement system for these interactions is not yet fully elucidated. The aim of the present study was to investigate the mutual influences of the complement system and bone cells, including in this term mesenchymal stem cells (MSC), osteoblasts (OB) and osteoclasts (OC). Quantitative assessment of mRNA expression and immune fluorescence staining revealed the expression of several important components, regulators and receptors of the complement system in MSC, OB and OC. The membrane bound complement regulatory proteins, membrane cofactor of proteolysis (CD46, MCP), decay accelerating factor (CD55, DAF) and MAC inhibitory factor (CD59, MACIF), the complement components C3 and C5 and the anaphylatoxin receptors C3a receptor (C3aR), C5aR and C5a receptor like 2 (C5L2) where expressed in undifferentiated and differentiated MSC, OB and OC with a significant increase in the expression of CD46, CD59 and C5aR during osteogenic differentiation of MSC. Osteoclasts and to a certain degree osteoblasts where able to activate complement C5 as assessed by a C5a specific ELISA. The generated C5a was chemotatically active as shown by a migration assay with peripheral blood mononuclear cells (PBMNC). Analysis of mRNA expression and release of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 in OB showed no response to C3a or C5a alone. However in co-stimulation with IL-1ß both anaphylatoxins significantly induced the expression and release of IL-6 and IL-8 indicating a modulatory role of osteoblasts on the inflammatory response. Additionally the expression of the OC regulatory proteins receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) where significantly increased following co-stimulation with IL-1ß and C3a or C5a indicating an influence on the interactions of osteoblasts and osteoclasts. Both C3a and C5a had no influence on the osteogenic differentiation. However, C3a or C5a significantly enhanced osteoclast formation in absence of RANKL and macrophage-colony stimulating

74 5. Summary factor (M-CSF) suggesting a direct regulatory influence of complement anaphylatoxins on osteoclast formation. It can therefore be proposed, that complement contributes to the induction of an inflammatory response in osteoblasts and enhances osteoclast formation under pro-inflammatory conditions as they are present in inflammatory bone disorders or during fracture healing in particular after multiple trauma.

75 6. References

6. References

1. Akisaka T, Yoshida H, Suzuki R: The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane. Journal of electron microscopy 55: 53-61 (2006) 2. Aksamit RR, Falk W, Leonard EJ: Chemotaxis by mouse macrophage cell lines. J Immunol 126: 2194-2199 (1981) 3. Allen TD, Dexter TM: The essential cells of the hemopoietic microenvironment. Experimental hematology 12: 517-521 (1984) 4. Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Acker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber-Lang M: Molecular intercommunication between the complement and coagulation systems. J Immunol 185: 5628-5636 (2010) 5. Amaral JD, Xavier JM, Steer CJ, Rodrigues CM: The role of p53 in apoptosis. Discovery medicine 9: 145-152 (2010) 6. Andersson J, Ekdahl KN, Larsson R, Nilsson UR, Nilsson B: C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase. Journal of immunology 168: 5786-5791 (2002) 7. Andersson J, Bexborn F, Klinth J, Nilsson B, Ekdahl KN: Surface-attached PEO in the form of activated Pluronic with immobilized factor H reduces both coagulation and complement activation in a whole-blood model. Journal of biomedical materials research. Part A 76: 25-34 (2006) 8. Andrades JA, Nimni ME, Becerra J, Eisenstein R, Davis M, Sorgente N: Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization. Exp Cell Res 227: 208-213 (1996) 9. Banfi A, Bianchi G, Notaro R, Luzzatto L, Cancedda R, Quarto R: Replicative aging and gene expression in long-term cultures of human bone marrow stromal cells. Tissue engineering 8: 901-910 (2002) 10. Barry F, Boynton R, Murphy M, Haynesworth S, Zaia J: The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochemical and biophysical research communications 289: 519-524 (2001) 11. Barry FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J: The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochemical and biophysical research communications 265: 134-139 (1999) 12. Bellows CG, Heersche JN, Aubin JE: Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Developmental biology 140: 132-138 (1990) 13. Bellows CG, Aubin JE, Heersche JN: Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone and mineral 14: 27-40 (1991) 14. Bhandari M, Tornetta P, 3rd, Sprague S, Najibi S, Petrisor B, Griffith L, Guyatt GH: Predictors of reoperation following operative management of fractures of the tibial shaft. J Orthop Trauma 17: 353-361 (2003) 15. Bills CE, Eisenberg H, Pallante SL: Complexes of organic acids with calcium phosphate: the Von Kossa stain as a clue to the composition of bone mineral. The Johns Hopkins medical journal 128: 194-207 (1974) 16. Bock D, Martin U, Gartner S, Rheinheimer C, Raffetseder U, Arseniev L, Barker MD, Monk PN, Bautsch W, Kohl J, Klos A: The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization. European journal of immunology 27: 1522-1529 (1997) 17. Bokisch VA, Muller-Eberhard HJ: Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase. J Clin Invest 49: 2427-2436 (1970)

76 6. References

18. Boland GM, Perkins G, Hall DJ, Tuan RS: Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. Journal of cellular biochemistry 93: 1210-1230 (2004) 19. Boyce BF, Yoneda T, Lowe C, Soriano P, Mundy GR: Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. The Journal of clinical investigation 90: 1622-1627 (1992) 20. Boyle WJ, Simonet WS, Lacey DL: Osteoclast differentiation and activation. Nature 423: 337-342 (2003) 21. Burr DB: Targeted and nontargeted remodeling. Bone 30: 2-4 (2002) 22. Chakravarti A, Raquil MA, Tessier P, Poubelle PE: Surface RANKL of Toll-like receptor 4-stimulated human neutrophils activates osteoclastic bone resorption. Blood 114: 1633-1644 (2009) 23. Chellaiah MA: Regulation of actin ring formation by rho GTPases in osteoclasts. The Journal of biological chemistry 280: 32930-32943 (2005) 24. Chen D, Zhao M, Mundy GR: Bone morphogenetic proteins. Growth factors 22: 233- 241 (2004) 25. Chenoweth DE, Henderson LW: Complement activation during hemodialysis: laboratory evaluation of hemodialyzers. Artificial organs 11: 155-162 (1987) 26. Claes L, Ignatius A, Lechner R, Gebhard F, Kraus M, Baumgartel S, Recknagel S, Krischak GD: The effect of both a thoracic trauma and a soft-tissue trauma on fracture healing in a rat model. Acta orthopaedica 82: 223-227 (2011) 27. Clarke D, Frisen J: Differentiation potential of adult stem cells. Current opinion in genetics & development 11: 575-580 (2001) 28. Delaisse JM, Eeckhout Y, Neff L, Francois-Gillet C, Henriet P, Su Y, Vaes G, Baron R: (Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment. Journal of cell science 106 ( Pt 4): 1071-1082 (1993) 29. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8: 315-317 (2006) 30. Drake FH, Dodds RA, James IE, Connor JR, Debouck C, Richardson S, Lee- Rykaczewski E, Coleman L, Rieman D, Barthlow R, Hastings G, Gowen M: Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. The Journal of biological chemistry 271: 12511-12516 (1996) 31. Ehrengruber MU, Geiser T, Deranleau DA: Activation of human neutrophils by C3a and C5A. Comparison of the effects on shape changes, chemotaxis, secretion, and respiratory burst. FEBS letters 346: 181-184 (1994) 32. Ehrnthaller C, Ignatius A, Gebhard F, Huber-Lang M: New insights of an old defense system: structure, function, and clinical relevance of the complement system. Mol Med 17: 317-329 (2011) 33. Ek-Rylander B, Flores M, Wendel M, Heinegard D, Andersson G: Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate- resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. The Journal of biological chemistry 269: 14853-14856 (1994) 34. Ekdahl KN, Nilsson B, Golander CG, Elwing H, Lassen B, Nilsson UR: Complement activation on radio frequency plasma modified polystyrene surfaces Journal of Colloid and Interface Science 158: 121-128 (1993) 35. el-Lati SG, Dahinden CA, Church MK: Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells. J Invest Dermatol 102: 803-806 (1994) 36. Elsner J, Oppermann M, Czech W, Kapp A: C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G- proteins. Blood 83: 3324-3331 (1994)

77 6. References

37. Fernandez HN, Henson PM, Otani A, Hugli TE: Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions. J Immunol 120: 109-115 (1978) 38. Fiedler J, Brill C, Blum WF, Brenner RE: IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells. Biochem Biophys Res Commun 345: 1177-1183 (2006) 39. Flannery S, Bowie AG: The interleukin-1 receptor-associated kinases: critical regulators of innate immune signalling. Biochem Pharmacol 80: 1981-1991 (2010) 40. Flierl MA, Schreiber H, Huber-Lang MS: The role of complement, C5a and its receptors in sepsis and multiorgan dysfunction syndrome. J Invest Surg 19: 255-265 (2006) 41. Flierl MA, Perl M, Rittirsch D, Bartl C, Schreiber H, Fleig V, Schlaf G, Liener U, Brueckner UB, Gebhard F, Huber-Lang MS: The role of C5a in the innate immune response after experimental blunt chest trauma. Shock 29: 25-31 (2008) 42. Frank MM, Fries LF: The role of complement in inflammation and phagocytosis. Immunology today 12: 322-326 (1991) 43. Friedman MS, Long MW, Hankenson KD: Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. Journal of cellular biochemistry 98: 538-554 (2006) 44. Ganss B, Kim RH, Sodek J: Bone sialoprotein. Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists 10: 79-98 (1999) 45. Gao H, Neff TA, Guo RF, Speyer CL, Sarma JV, Tomlins S, Man Y, Riedemann NC, Hoesel LM, Younkin E, Zetoune FS, Ward PA: Evidence for a functional role of the second C5a receptor C5L2. FASEB J 19: 1003-1005 (2005) 46. Gebhard F, Strecker W, Brückner U, Kinzl L: Untersuchungen zur systemischen posttraumatischen Inflammation in der Frühphase nach Trauma. . Der Unfallchirurg 1-96 (2000) 47. Gerard NP, Lu B, Liu P, Craig S, Fujiwara Y, Okinaga S, Gerard C: An anti- inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2. J Biol Chem 280: 39677-39680 (2005) 48. Gobeil F, Fortier A, Zhu T, Bossolasco M, Leduc M, Grandbois M, Heveker N, Bkaily G, Chemtob S, Barbaz D: G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigm. Can J Physiol Pharmacol 84: 287-297 (2006) 49. Goetzl EJ: Diverse pathways for nuclear signaling by G protein-coupled receptors and their ligands. FASEB J 21: 638-642 (2007) 50. Gough AK, Lilley J, Eyre S, Holder RL, Emery P: Generalised bone loss in patients with early rheumatoid arthritis. Lancet 344: 23-27 (1994) 51. Haas PJ, van Strijp J: Anaphylatoxins: their role in bacterial infection and inflammation. Immunol Res 37: 161-175 (2007) 52. Harada S, Rodan GA: Control of osteoblast function and regulation of bone mass. Nature 423: 349-355 (2003) 53. Hartmann K, Henz BM, Kruger-Krasagakes S, Kohl J, Burger R, Guhl S, Haase I, Lippert U, Zuberbier T: C3a and C5a stimulate chemotaxis of human mast cells. Blood 89: 2863-2870 (1997) 54. Hed J, Johansson M, Lindroth M: Complement activation according to the alternate pathway by glass and plastic surfaces and its role in neutrophil adhesion. Immunology letters 8: 295-299 (1984) 55. Heng BC, Cao T, Stanton LW, Robson P, Olsen B: Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 19: 1379-1394 (2004) 56. Hong MH, Jin CH, Sato T, Ishimi Y, Abe E, Suda T: Transcriptional regulation of the production of the third component of complement (C3) by 1 alpha,25- dihydroxyvitamin D3 in mouse marrow-derived stromal cells (ST2) and primary osteoblastic cells. Endocrinology 129: 2774-2779 (1991)

78 6. References

57. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A: Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7: 393- 395 (2005) 58. Huber-Lang M, Younkin EM, Sarma JV, Riedemann N, McGuire SR, Lu KT, Kunkel R, Younger JG, Zetoune FS, Ward PA: Generation of C5a by phagocytic cells. Am J Pathol 161: 1849-1859 (2002) 59. Huber-Lang M, Sarma JV, Zetoune FS, Rittirsch D, Neff TA, McGuire SR, Lambris JD, Warner RL, Flierl MA, Hoesel LM, Gebhard F, Younger JG, Drouin SM, Wetsel RA, Ward PA: Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med 12: 682-687 (2006) 60. Ignatius A, Ehrnthaller C, Brenner RE, Kreja L, Schoengraf P, Lisson P, Blakytny R, Recknagel S, Claes L, Gebhard F, Lambris JD, Huber-Lang M: The Anaphylatoxin Receptor C5aR Is Present During Fracture Healing in Rats and Mediates Osteoblast Migration In Vitro. The Journal of trauma (2011) 61. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP: Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. Journal of cellular biochemistry 64: 295-312 (1997) 62. Jin CH, Shinki T, Hong MH, Sato T, Yamaguchi A, Ikeda T, Yoshiki S, Abe E, Suda T: 1 alpha,25-dihydroxyvitamin D3 regulates in vivo production of the third component of complement (C3) in bone. Endocrinology 131: 2468-2475 (1992) 63. Johswich K, Martin M, Thalmann J, Rheinheimer C, Monk PN, Klos A: Ligand specificity of the anaphylatoxin C5L2 receptor and its regulation on myeloid and epithelial cell lines. J Biol Chem 281: 39088-39095 (2006) 64. Karladani AH, Granhed H, Karrholm J, Styf J: The influence of fracture etiology and type on fracture healing: a review of 104 consecutive tibial shaft fractures. Arch Orthop Trauma Surg 121: 325-328 (2001) 65. Karsdal MA, Neutzsky-Wulff AV, Dziegiel MH, Christiansen C, Henriksen K: Osteoclasts secrete non-bone derived signals that induce bone formation. Biochem Biophys Res Commun 366: 483-488 (2008) 66. Kassem M, Abdallah BM, Saeed H: Osteoblastic cells: differentiation and trans- differentiation. Archives of biochemistry and biophysics 473: 183-187 (2008) 67. Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA, 2nd, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA, Karsenty G, Chan L: Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. The Journal of cell biology 157: 303-314 (2002) 68. Kazanecki CC, Uzwiak DJ, Denhardt DT: Control of osteopontin signaling and function by post-translational phosphorylation and protein folding. Journal of cellular biochemistry 102: 912-924 (2007) 69. Kim DD, Song WC: Membrane complement regulatory proteins. Clin Immunol 118: 127-136 (2006) 70. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Kohl J: The role of the anaphylatoxins in health and disease. Mol Immunol 46: 2753-2766 (2009) 71. Konttinen YT, Ceponis A, Meri S, Vuorikoski A, Kortekangas P, Sorsa T, Sukura A, Santavirta S: Complement in acute and chronic arthritides: assessment of C3c, C9, and protectin (CD59) in synovial membrane. Ann Rheum Dis 55: 888-894 (1996) 72. Kreja L, Liedert A, Schmidt C, Claes L, Ignatius A: Influence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fetal calf serum on human osteoclast formation and activity. J Mol Histol 38: 341-345 (2007) 73. Kreja L, Brenner RE, Tautzenberger A, Liedert A, Friemert B, Ehrnthaller C, Huber- Lang M, Ignatius A: Non-resorbing osteoclasts induce migration and osteogenic differentiation of mesenchymal stem cells. J Cell Biochem 109: 347-355 (2010) 74. Kretzschmar T, Jeromin A, Gietz C, Bautsch W, Klos A, Kohl J, Rechkemmer G, Bitter-Suermann D: Chronic myelogenous leukemia-derived basophilic granulocytes

79 6. References

express a functional active receptor for the anaphylatoxin C3a. Eur J Immunol 23: 558-561 (1993) 75. Kubota K, Sakikawa C, Katsumata M, Nakamura T, Wakabayashi K: Platelet- derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 17: 257-265 (2002) 76. Lhotta K, Wurzner R, Kronenberg F, Oppermann M, Konig P: Rapid activation of the complement system by cuprophane depends on complement component C4. Kidney international 53: 1044-1051 (1998) 77. Li K, Anderson KJ, Peng Q, Noble A, Lu B, Kelly AP, Wang N, Sacks SH, Zhou W: Cyclic AMP plays a critical role in C3a-receptor-mediated regulation of dendritic cells in antigen uptake and T-cell stimulation. Blood 112: 5084-5094 (2008) 78. Ljusberg J, Wang Y, Lang P, Norgard M, Dodds R, Hultenby K, Ek-Rylander B, Andersson G: Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts. The Journal of biological chemistry 280: 28370-28381 (2005) 79. Lories RJ, Luyten FP: The bone-cartilage unit in osteoarthritis. Nat Rev Rheumatol 7: 43-49 (2011) 80. Lu D, Yang H, Shaw G, Raizada MK: Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons. Endocrinology 139: 365-375 (1998) 81. Mack C, Jungermann K, Gotze O, Schieferdecker HL: Anaphylatoxin C5a actions in rat liver: synergistic enhancement by C5a of lipopolysaccharide-dependent alpha(2)- macroglobulin gene expression in hepatocytes via IL-6 release from Kupffer cells. J Immunol 167: 3972-3979 (2001) 82. Macsai CE, Foster BK, Xian CJ: Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair. Journal of cellular physiology 215: 578-587 (2008) 83. Marie PJ: Transcription factors controlling osteoblastogenesis. Archives of biochemistry and biophysics 473: 98-105 (2008) 84. Martin TJ: Paracrine regulation of osteoclast formation and activity: milestones in discovery. Journal of musculoskeletal & neuronal interactions 4: 243-253 (2004) 85. Maruotti N, Grano M, Colucci S, d'Onofrio F, Cantatore FP: Osteoclastogenesis and arthritis. Clinical and experimental medicine (2010) 86. Matsuo K, Irie N: Osteoclast-osteoblast communication. Archives of biochemistry and biophysics 473: 201-209 (2008) 87. McKay R: Stem cells--hype and hope. Nature 406: 361-364 (2000) 88. Meuer S, Hugli TE, Andreatta RH, Hadding U, Bitter-Suermann D: Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a). Investigations on their ability to induce platelet secretion. Inflammation 5: 263-273 (1981) 89. Murakami Y, Imamichi T, Nagasawa S: Characterization of C3a anaphylatoxin receptor on guinea-pig macrophages. Immunology 79: 633-638 (1993) 90. Murphy KM, Travers P, Walport M: Janeway Immunologie. In: (Hrsg) 7. Aufl, Spektrum Akademischer Verlag, S. (2009) 91. Naik N, Giannini E, Brouchon L, Boulay F: Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain. Journal of cell science 110 ( Pt 19): 2381-2390 (1997) 92. Nakagawa K, Sakiyama H, Fukazawa T, Matsumoto M, Takigawa M, Toyoguchi T, Moriya H: Coordinated change between complement C1s production and chondrocyte differentiation in vitro. Cell Tissue Res 289: 299-305 (1997) 93. Nataf S, Davoust N, Ames RS, Barnum SR: Human T cells express the C5a receptor and are chemoattracted to C5a. J Immunol 162: 4018-4023 (1999) 94. Neff JA, Tresco PA, Caldwell KD: Surface modification for controlled studies of cell- ligand interactions. Biomaterials 20: 2377-2393 (1999)

80 6. References

95. Nesbitt SA, Horton MA: Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276: 266-269 (1997) 96. Ng KW: Future developments in osteoporosis therapy. Endocrine, metabolic & immune disorders drug targets 9: 371-384 (2009) 97. O' Gradaigh D, Ireland D, Bord S, Compston JE: Joint erosion in rheumatoid arthritis: interactions between tumour necrosis factor alpha, interleukin 1, and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclasts. Annals of the rheumatic diseases 63: 354-359 (2004) 98. Okinaga S, Slattery D, Humbles A, Zsengeller Z, Morteau O, Kinrade MB, Brodbeck RM, Krause JE, Choe HR, Gerard NP, Gerard C: C5L2, a nonsignaling C5A binding protein. Biochemistry 42: 9406-9415 (2003) 99. Okusawa S, Dinarello CA, Yancey KB, Endres S, Lawley TJ, Frank MM, Burke JF, Gelfand JA: C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma. J Immunol 139: 2635-2640 (1987) 100. Ottonello L, Corcione A, Tortolina G, Airoldi I, Albesiano E, Favre A, D'Agostino R, Malavasi F, Pistoia V, Dallegri F: rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues. J Immunol 162: 6510-6517 (1999) 101. Page G, Miossec P: RANK and RANKL expression as markers of dendritic cell-T cell interactions in paired samples of rheumatoid synovium and lymph nodes. Arthritis and rheumatism 52: 2307-2312 (2005) 102. Palmqvist P, Persson E, Conaway HH, Lerner UH: IL-6, leukemia inhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand, osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae. Journal of immunology 169: 3353-3362 (2002) 103. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 284: 143-147 (1999) 104. Pobanz JM, Reinhardt RA, Koka S, Sanderson SD: C5a modulation of interleukin-1 beta-induced interleukin-6 production by human osteoblast-like cells. J Periodontal Res 35: 137-145 (2000) 105. Recknagel S, Bindl R, Kurz J, Wehner T, Ehrnthaller C, Knoferl MW, Gebhard F, Huber-Lang M, Claes L, Ignatius A: Experimental blunt chest trauma impairs fracture healing in rats. J Orthop Res 29: 734-739 (2011) 106. Recknagel S, Bindl R, Kurz J, Wehner T, Schoengraf P, Ehrnthaller C, Qu H, Gebhard F, Huber-Lang M, Lambris JD, Claes L, Ignatius A: C5aR-antagonist significantly reduces the deleterious effect of a blunt chest trauma on fracture healing. Journal of orthopaedic research : official publication of the Orthopaedic Research Society (2011) 107. Reinholt FP, Hultenby K, Oldberg A, Heinegard D: Osteopontin--a possible anchor of osteoclasts to bone. Proceedings of the National Academy of Sciences of the United States of America 87: 4473-4475 (1990) 108. Ricklin D, Hajishengallis G, Yang K, Lambris JD: Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11: 785-797 (2010) 109. Robey PG, Termine JD: Human bone cells in vitro. Calcif Tissue Int 37: 453-460 (1985) 110. Rousseau S, Dolado I, Beardmore V, Shpiro N, Marquez R, Nebreda AR, Arthur JS, Case LM, Tessier-Lavigne M, Gaestel M, Cuenda A, Cohen P: CXCL12 and C5a trigger cell migration via a PAK1/2-p38alpha MAPK-MAPKAP-K2-HSP27 pathway. Cell Signal 18: 1897-1905 (2006) 111. Rutkowski MJ, Sughrue ME, Kane AJ, Ahn BJ, Fang S, Parsa AT: The complement cascade as a mediator of tissue growth and regeneration. Inflamm Res 59: 897-905 (2010) 112. Ryu J, Kim HJ, Chang EJ, Huang H, Banno Y, Kim HH: Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling. The EMBO journal 25: 5840-5851 (2006)

81 6. References

113. Sachs L: The molecular control of blood cell development. Science 238: 1374-1379 (1987) 114. Sakiyama H, Inaba N, Toyoguchi T, Okada Y, Matsumoto M, Moriya H, Ohtsu H: Immunolocalization of complement C1s and matrix metalloproteinase 9 (92kDa gelatinase/type IV collagenase) in the primary ossification center of the human femur. Cell Tissue Res 277: 239-245 (1994) 115. Sakiyama H, Nakagawa K, Kuriiwa K, Imai K, Okada Y, Tsuchida T, Moriya H, Imajoh-Ohmi S: Complement Cls, a classical enzyme with novel functions at the endochondral ossification center: immunohistochemical staining of activated Cls with a neoantigen-specific antibody. Cell Tissue Res 288: 557-565 (1997) 116. Salo J, Lehenkari P, Mulari M, Metsikko K, Vaananen HK: Removal of osteoclast bone resorption products by transcytosis. Science 276: 270-273 (1997) 117. Sample SJ, Hao Z, Wilson AP, Muir P: Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading. PLoS One 6: e20386 (2011) 118. Sato T, Hong MH, Jin CH, Ishimi Y, Udagawa N, Shinki T, Abe E, Suda T: The specific production of the third component of complement by osteoblastic cells treated with 1 alpha,25-dihydroxyvitamin D3. FEBS Lett 285: 21-24 (1991) 119. Sato T, Abe E, Jin CH, Hong MH, Katagiri T, Kinoshita T, Amizuka N, Ozawa H, Suda T: The biological roles of the third component of complement in osteoclast formation. Endocrinology 133: 397-404 (1993) 120. Sato T, Foged NT, Delaisse JM: The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 13: 59-66 (1998) 121. Sayah S, Jauneau AC, Patte C, Tonon MC, Vaudry H, Fontaine M: Two different transduction pathways are activated by C3a and C5a anaphylatoxins on astrocytes. Brain Res Mol Brain Res 112: 53-60 (2003) 122. Scadden DT: The stem-cell niche as an entity of action. Nature 441: 1075-1079 (2006) 123. Schiano de Colella JM, Barbarat B, Sweet R, Gastaut JA, Olive D, Costello RT: Rank ligand stimulation induces a partial but functional maturation of human monocyte-derived dendritic cells. European cytokine network 19: 81-88 (2008) 124. Schmal H, Niemeyer P, Roesslein M, Hartl D, Loop T, Sudkamp NP, Stark GB, Mehlhorn AT: Comparison of cellular functionality of human mesenchymal stromal cells and PBMC. Cytotherapy 9: 69-79 (2007) 125. Scholz W, McClurg MR, Cardenas GJ, Smith M, Noonan DJ, Hugli TE, Morgan EL: C5a-mediated release of interleukin 6 by human monocytes. Clin Immunol Immunopathol 57: 297-307 (1990) 126. Schraufstatter IU, Discipio RG, Zhao M, Khaldoyanidi SK: C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol 182: 3827-3836 (2009) 127. Segal LG, Lane NE: Osteoporosis and systemic lupus erythematosus: etiology and treatment strategies. Ann Med Interne (Paris) 147: 281-289 (1996) 128. Settmacher B, Bock D, Saad H, Gartner S, Rheinheimer C, Kohl J, Bautsch W, Klos A: Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a. J Immunol 162: 7409-7416 (1999) 129. Silver IA, Murrills RJ, Etherington DJ: Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Experimental cell research 175: 266-276 (1988) 130. Simmons DL, Satterthwaite AB, Tenen DG, Seed B: Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells. Journal of immunology 148: 267-271 (1992) 131. Sontheimer RD, Racila E, Racila DM: C1q: its functions within the innate and adaptive immune responses and its role in lupus autoimmunity. The Journal of investigative dermatology 125: 14-23 (2005)

82 6. References

132. Stein GS, Lian JB: Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocrine reviews 14: 424-442 (1993) 133. Takabayashi T, Shimizu S, Clark BD, Beinborn M, Burke JF, Gelfand JA: Interleukin-1 upregulates anaphylatoxin receptors on mononuclear cells. Surgery 135: 544-554 (2004) 134. Takayanagi H: Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 7: 292-304 (2007) 135. Teitelbaum SL: Bone resorption by osteoclasts. Science 289: 1504-1508 (2000) 136. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-1147 (1998) 137. Townsend CM, Beauchamp RC, Evers BM, Mattox KL: Sabiston Textbook of Surgery. Saunders Elsevier, S. (2008) 138. Toyoguchi T, Yamaguchi K, Nakagawa K, Fukusawa T, Moriya H, Sakiyama H: Change of complement C1s synthesis during development of hamster cartilage. Cell Tissue Res 285: 199-204 (1996) 139. Tu Z, Bu H, Dennis JE, Lin F: Efficient osteoclast differentiation requires local complement activation. Blood 116: 4456-4463 (2010) 140. Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T: Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proceedings of the National Academy of Sciences of the United States of America 87: 7260-7264 (1990) 141. Vaananen HK, Laitala-Leinonen T: Osteoclast lineage and function. Archives of biochemistry and biophysics 473: 132-138 (2008) 142. Van Epps DE, Simpson S, Bender JG, Chenoweth DE: Regulation of C5a and formyl peptide receptor expression on human polymorphonuclear leukocytes. J Immunol 144: 1062-1068 (1990) 143. Vincent F, de la Salle H, Bohbot A, Bergerat JP, Hauptmann G, Oberling F: Synthesis and regulation of complement components by human monocytes/macrophages and by acute monocytic leukemia. DNA and cell biology 12: 415-423 (1993) 144. Wang Q, Rozelle AL, Lepus CM, Scanzello CR, Song JJ, Larsen DM, Crish JF, Bebek G, Ritter SY, Lindstrom TM, Hwang I, Wong HH, Punzi L, Encarnacion A, Shamloo M, Goodman SB, Wyss-Coray T, Goldring SR, Banda NK, Thurman JM, Gobezie R, Crow MK, Holers VM, Lee DM, Robinson WH: Identification of a central role for complement in osteoarthritis. Nature medicine (2011) 145. Watt FM, Hogan BL: Out of Eden: stem cells and their niches. Science 287: 1427- 1430 (2000) 146. Weissman IL, Anderson DJ, Gage F: Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annual review of cell and developmental biology 17: 387-403 (2001) 147. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA: Novel regulators of bone formation: molecular clones and activities. Science 242: 1528-1534 (1988) 148. Yamaguchi K, Sakiyama H, Matsumoto M, Moriya H, Sakiyama S: Degradation of type I and II collagen by human activated C1-s. FEBS Lett 268: 206-208 (1990) 149. Yavropoulou MP, Yovos JG: Osteoclastogenesis--current knowledge and future perspectives. Journal of musculoskeletal & neuronal interactions 8: 204-216 (2008) 150. Zhao C, Irie N, Takada Y, Shimoda K, Miyamoto T, Nishiwaki T, Suda T, Matsuo K: Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell metabolism 4: 111-121 (2006) 151. Zhao M, Mueller BM, DiScipio RG, Schraufstatter IU: Akt plays an important role in breast cancer cell chemotaxis to CXCL12. Breast Cancer Res Treat 110: 211-222 (2008)

83 6. References

152. Zhou W: The new face of anaphylatoxins in immune regulation. Immunobiology (2011)

84

Acknowledgements

I would like to thank Prof. Dr. Anita Ignatius for the chance to write my dissertation in this fascinating field of research at the border between basic and clinical research and for all her support throughout my doctoral thesis.

I also would like to thank Prof. Dr. Huber-Lang for acting as 2nd reviewer and for the inspiring cooperation in the KFO200.

I am especially grateful to Dr. Ludwika Kreja for the support and patience and for the invaluable scientific advice.

I want to thank everyone in the institute for all the help I got with every single problem I encountered and for the convenient working atmosphere. I learned a lot for writing, scientific work and for life in general.

I am very grateful for all the friends I found in the institute. Especially Andrea Tautzenberger, Antje Mietsch, Anja Lubomierski, Katharina Gruchenberg, Stefan Recknagel, Ronny Bindl, Andi Seitz and Malte Steiner have to be mentioned for all the good times we had inside and outside the institute.

Finally, I am indebted to my family for always trusting in me and supporting me all the years. Furthermore, special thanks goes to Bettina Titz for all her help and for being a very important part of my life.

85

Curriculum vitae

For reasons of data protection the curriculum vitae is not included in the online version.

86