The role of the metalloproteases ADAM8, 9, 10 and 17 in leukocyte migration in vitro Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Humanbiologen Franz Martin Heß aus Darmstadt Berichter: Universitätsprofessor Dr. rer. nat. Andreas Ludwig Universitätsprofessor Dr. rer. nat. Martin Zenke Tag der mündlichen Prüfung: 26.06.2012 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Index 1. Introduction............................................................................................................1 1.1. Aim of this study............................................................................................18 2. Materials and Methods...........................................................................................20 2.1. Materials, Chemicals and Equipment................................................................20 2.2. Cell culture methods.......................................................................................33 2.3. Protein biochemistry methods .........................................................................35 2.4. Molecular biology methods..............................................................................37 2.5. Functional assays...........................................................................................40 2.6. Statistics .......................................................................................................42 3. Results.................................................................................................................43 3.1. Comparison of ADAM8, 9, 10 and 17 expression in different leukocytic, endothelial and epithelial cell culture lines....................................................................................43 3.2. The effect of ADAM-inhibitors on leukocyte migration........................................44 3.3. Lentiviral shRNA-based knock-down of ADAM 8, 9, 10 and 17............................48 3.4. Effect of ADAM10 and 17 silencing on shedding activity.....................................55 3.5. Role of ADAM 10 and ADAM 8, 9, 17 for transmigration and chemotaxis of THP1 cells 58 3.6. Role of ADAMs for the sensitivity of LV-THP1 cells towards different MCP-1 concentrations ..........................................................................................................70 3.7. The effect of ADAM8, 9, 10 and 17 knock-down on the Ca2+ response of THP1 cells to MCP-1 ..................................................................................................................72 3.8. Influence of ADAM8, 9, 10 and 17 knock-down on adhesion of THP1 cells to VCAM-1 and fibronectin under static conditions ...........................................................76 3.9. Role of ADAM8, 9, 10 and 17 gene silencing on epithelial cell migration..............79 3.10. Remarks........................................................................................................82 4. Discussion ............................................................................................................83 4.1. Conclusion.....................................................................................................97 I 5. Summary............................................................................................................ 104 6. Literature ........................................................................................................... 106 7. List of abbreviations ............................................................................................ 122 8. List of figures...................................................................................................... 128 9. List of tables....................................................................................................... 131 10. Appendix ........................................................................................................ 132 10.1. Plasmid maps .............................................................................................. 132 10.2. Complementary figure: Comparison of ADAM8, 9, 10 and 17 expression in different leukocytic, endothelial and epithelial cell culture lines................................................. 136 10.3. Complementary figures: Relative number of migrated ADAM8, ADAM9 or ADAM17- KD LV-THP1 in transmigration and chemotaxis assay. ................................................ 137 11. Publications..................................................................................................... 139 12. Curriculum Vitae.............................................................................................. 140 13. Declaration ..................................................................................................... 141 14. Acknowledgements.......................................................................................... 142 II 1. Introduction Inflammation is the organism’s defence response to invading pathogens such as bacteria, virus or parasites as well as to traumas caused by tissue damage, wound injury or tumours. Without inflammation the organism would be unable to fend off infections or induce tissue repair after a trauma. However, the coin has two faces: excessive inflammatory responses are responsible for a variety of disorders including septic shock, autoimmune diseases or chronic inflammatory diseases such as asthma, atherosclerosis, fibrosis, rheumatoid arthritis and even cancer. Given its importance and the consequences for the organism, inflammation is as tightly controlled as embryogenesis, metabolism, cell proliferation and other crucial complex biological processes. Metaphorically speaking, the vasculature constitutes the front line in the war of inflammation. It provides a barrier for the circulatory system, which transports the troops – the leukocytes – to the battle being waged by the inflammatory response. One of the first responses in the inflammation process is the dilatation of the vasculature near the site of inflammation. This results in a reduction of the blood flow rate. The permeability of the endothelium in the microvessels increases and leukocytes are recruited to the site of inflammation. Later stages of inflammation include neovascularisation and the repair of the affected tissue. Different cell vascular cells take part in these processes: endothelial cells, smooth muscle cells, resident macrophages, platelets and the leukocytes of the blood flow. The process of leukocyte recruitment itself is subdivided into several steps: the inflammatory activation of the tissue at the inflammation site, the rolling of the leukocytes on the activated endothelium, their tight adhesion and finally the transmigration or diapedesis of the leukocytes through the endothelium. Having overcome the endothelial barrier the leukocytes migrate, guided by the gradient of chemotactic stimuli, to the inflammation site, where they do their duty fighting in the cause of inflammation. Since leukocyte recruitment to the site of inflammation is one of the critical hallmarks in inflammation, it is tightly controlled and finely tuned. Proteins that are involved in the modulation of these events belong to the families of cytokines, chemokines, growth factors and adhesion molecules. To coordinate this critical process most of the mediators of inflammation are tightly regulated on the transcriptional level. Several of the mentioned molecules undergo posttranslational modifications as well. 1 Many of the effector proteins of inflammation are initially expressed as transmembrane proteins on cells of the vasculature or leukocytes. During the process of limited proteolysis or shedding, soluble variants of these molecules are released into the surrounding tissue. Several proteins of the A Disintegin And Metalloproteinase (ADAM) family are involved in the shedding of proteins that regulate inflammation responses and leukocyte recruitment1–4. The most prominent member of the ADAM family, ADAM17 actually received its common name TACE (tumour necrosis factor-α-converting enzyme) because it activates one of the most important mediators of inflammation – the cytokine TNF-α – by proteolytic cleavage5. Figure 1: The process of leukocyte recruitment. In the early inflammatory activation cytokines and chemokines are released by resident macrophages and smooth muscle cells of the affected tissue. Cytokines like TNFα and IL-1 lead to the activation of the adjacent endothelial cells which shed cell-cell adhesion molecules and express adhesion molecules on their surface. The endothelial permeability increases, chemokines diffuse easier into the vascular space and are presented on the activated endothelium. Leucocytes of the vasculture interact via their adhesion molecules with the adhesion molecules of the activated endothelial cells. This initiates the leukocytes’ process of slow rolling on the endothelium. The interaction of leukocytic integrins with endothelial adhesion molecules VCAM-1, ICAM-1 and membrane bound chemokines leads to the firm adhesion of the leukocytes. The increased permeability of the endothelium facilitates the subsequent transendothelial migration or diapedesis of the leukocytes. Cytokines The first and most crucial step in almost all inflammatory responses is the release of cytokines by cells in the affected tissue.