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A Study of the Molecular Pharmacological Properties of C5a and C5a Des Arg: Defining Their Biased Agonist Profile and Contrasting Biological Function”

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A Study of the Molecular Pharmacological Properties of C5a and C5a Des Arg: Defining Their Biased Agonist Profile and Contrasting Biological Function” Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool & Inflammation and Immunology Research Unit Pfizer Worldwide Research and Development “A study of the molecular pharmacological properties of C5a and C5a des Arg: defining their biased agonist profile and contrasting biological function”. Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by: Simeon Jon Ramsey October 2017 Declaration This thesis is the result of my own work. The material contained within this thesis has not been presented, nor is currently being presented, either wholly or in part for any degree or other qualification. All research was conducted in the laboratories at Pfizer Worldwide Research and Development, 610 Main Street, Cambridge, Massachusetts, USA. Simeon Ramsey Student number: 201001573 Table of contents Abstract ........................................................................................................................... i Acknowledgements ....................................................................................................... iii Abbreviations ................................................................................................................ iv Chapter 1 ..................................................................................................................... 1 1. Introduction ............................................................................................................ 2 1.1. Overview of the complement system ............................................................. 5 1.2. The pathways of complement activation ........................................................ 6 1.2.1. The Classical pathway .............................................................................. 6 1.2.2. The Lectin pathway .................................................................................. 8 1.2.3. The Alternative pathway .......................................................................... 9 1.2.4. The Extrinsic mechanism of complement activation ............................. 11 1.3. Regulatory proteins of the complement system .......................................... 13 1.4. Disorders associated with the complement system ..................................... 15 1.4.1. Complement and rheumatoid arthritis .................................................. 17 1.4.2. Complement and sepsis ......................................................................... 18 1.5. C5a and the cleaved isoform C5a des Arg ..................................................... 21 1.6. The two C5a receptors .................................................................................. 22 1.6.1. The multi domain interaction between C5a and its receptors .............. 23 1.6.2. Functional signalling of the C5a1 receptor ............................................. 25 1.6.3. The enigmatic role of the C5a2 receptor ................................................ 27 1.7. Therapeutically targeting the complement system ...................................... 32 1.7.1. Preventing C5 cleavage .......................................................................... 33 1.7.2. Neutralizing C5a ..................................................................................... 34 1.7.3. Antagonizing the C5a1 receptor ............................................................. 34 1.8. Rationale, aims, hypotheses and experimental strategy. ............................. 37 Chapter 2 ................................................................................................................... 39 Materials and methods ................................................................................................ 40 2.1. Reagents and assay kits ................................................................................. 40 2.2. Assay buffer ................................................................................................... 40 2.3. C5a1 and C5a2 receptor expressing cell lines ................................................ 40 2.3. Assessment of recombinant C5a1 and C5a2 receptor sequences ................. 41 2.4. Quantitation of receptor gene expression .................................................... 42 2.5. Detection of protein expression using gel electrophoresis and immunoblotting ............................................................................................. 43 2.6. Characterization of human serum derived C5a and C5a des Arg ................. 45 2.7. 125I-C5a receptor-ligand binding studies ....................................................... 46 2.8. Isolation of neutrophils from human whole blood ....................................... 47 2.9. Neutrophil purity and activation state .......................................................... 48 2.10. Detection of cell surface C5a receptor expression using flow cytometry . 49 2.11. The impact of inflammatory agents on neutrophil receptor expression .. 50 2.12. Agonist induced intracellular calcium mobilization .................................. 51 2.13. Agonist induced CD11b up regulation on human isolated neutrophils .... 51 2.14. Agonist induced human isolated neutrophil respiratory burst ................. 52 2.15. Detection of intracellular ROS generation in the human isolated neutrophil .................................................................................................. 53 2.16. Agonist induced human isolated neutrophil chemotaxis .......................... 54 2.17. Agonist induced cAMP accumulation in CHO cells expressing the C5a1 receptor ..................................................................................................... 55 2.18. Agonist induced ERK 1/2 phosphorylation in CHO cells expressing the C5a1 receptor ..................................................................................................... 55 2.19. Agonist induced C5a1 or C5a2 receptor internalization ............................. 56 2.20. Agonist induced C5a1 or C5a2 receptor -arrestin recruitment ................ 57 2.21. Expression of wild type and mutant C5a1 receptors in CHO cells ............. 58 Chapter 3 ................................................................................................................... 59 The characterization of reagents and pharmacological tools to enable accurate interrogation of C5a, C5a des Arg and their receptors, C5a1 and C5a2 ....................... 60 3.1. Abstract ......................................................................................................... 60 3.2. Introduction ................................................................................................... 61 3.3. Chapter specific methods .............................................................................. 63 3.3.1. Data analysis and model fitting ............................................................. 63 3.4. Results ........................................................................................................... 65 3.4.1. Sequence analysis of recombinant C5a1 receptor and C5a2 receptor overexpressed in CHO and U2OS cells ................................................... 65 3.4.2. Quantifying the expression of each C5a receptor in the CHO and U2OS cells......................................................................................................... 66 3.3.3. Characterisation of human purified C5a and C5a des Arg ..................... 70 3.3.4. Determining the specificity of ligands and pharmacological tools for the C5a1 and C5a2 receptors ........................................................................ 71 3.5. Discussion ...................................................................................................... 82 Chapter 4 ................................................................................................................... 87 Characterization of a C5a sensitive biological test system, the human isolated neutrophil..................................................................................................................... 88 4.1. Abstract ......................................................................................................... 88 4.2. Introduction ................................................................................................... 89 4.3. Chapter specific methods .............................................................................. 92 4.3.1. Isolation of neutrophils from human whole blood using the Miltenyi Biotec MACSexpress® kit ....................................................................... 92 4.3.2. Statistical analysis. ................................................................................. 92 4.4 Results. .......................................................................................................... 94 4.4.1. The MACSxpress® kit isolates a pure population of neutrophils from human whole blood with minimal erythrocyte contamination ............ 94 4.4.2. Gene expression of neutrophil C5a receptors and other proteins associated with the innate immune system .......................................... 95 4.4.3. Both C5a receptors are expressed on the surface of the human isolated neutrophil............................................................................................. 102 4.4.4. Regulation of C5a receptor expression on human isolated neutrophils ... .............................................................................................................
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