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Modulation of Neutrophil Degranulation by Hypoxia Modulation of neutrophil degranulation by hypoxia Kim Hoenderdos Submitted towards the degree of Doctor of Philosophy UNIVERSITY OF CAMBRIDGE Hughes Hall Department of Medicine 2014 Declaration This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. This thesis was composed on the basis of work carried out under the supervision of Professor Edwin Chilvers and Dr. Alison Condliffe in the Division of Respiratory Medicine, Department of Medicine, University of Cambridge. This thesis (excluding figures, tables, appendices and bibliography) does not exceed the word limit imposed by the Clinical Medicine and Clinical Veterinary Medicine degree committee. Kim Hoenderdos September 2014, Cambridge i | Acknowledgements First and foremost I would like to thank my supervisors Alison Condliffe and Edwin Chilvers for giving a crazy Dutch girl a chance to come and study in Cambridge and for their support and enthusiasm throughout the course of my Ph.D. studies. Their doors were always open if I needed advice and I could not have asked for better supervisors! Next I would like to thanks my colleagues from the Morrell and Chilvers group and especially my “office mates” for their help along the way! Your enthusiasm, scientific discussions and banter made the lab a joy to work in! A special thanks to Linsey, Ross and Jatinder for all their help and to Jo for all our fun coffee breaks and chats. During my Ph.D. I supervised 2 students; Charlotte and Cheng and I would like to thank both of them for all the hard work they put in. Even if it was a bit chaotic at times, I really enjoyed working with both of you and it sometime felt more like a very chatty “tea party” then work! Away from the lab I spend most of my time in a dance studio and I made some amazing friends there and in Cambridge in general. Fil, Tanya, Zara, Michael, Sylvie, Lukasz, Melanie, Sanna and of course my dance partner Chris, thank you so much for your support (in the form of a willing ear, cards/presents or alcohol), and providing me with a place where I could forget about everything but dance, during the inevitable ups and downs of doing research. I would like to express my gratitude to the British Lung Foundation for funding me and to all the blood donors that made this work possible. Finally a very special thanks goes out to my family for their continuous love and support. You were always there for me and provided me with a place where I could recharge and relax. I could not have done this without any of you! ii | Table of Contents Declaration....................................................................................................... i Acknowledgements ....................................................................................... ii Table of Contents ......................................................................................... iii List of Figures ............................................................................................. viii List of Tables .................................................................................................xi List of Abbreviations .................................................................................. xii Abstract ......................................................................................................... xv 1. Introduction .............................................................................................. 17 1.1 The immune system ................................................................................................... 17 1.2 Neutrophils ................................................................................................................. 17 1.2.1 Neutrophil recruitment ...................................................................................... 19 1.2.2 Neutrophil intracellular bacterial killing ........................................................... 23 1.2.3 Neutrophil extracellular traps ............................................................................ 25 1.2.4 Role of neutrophils in disease: a double-edged sword ...................................... 27 1.3 Neutrophil granules: development and function .................................................... 28 1.3.1 Secretory vesicles .............................................................................................. 32 1.3.2 Gelatinase granules ............................................................................................ 32 1.3.3 Specific granules................................................................................................ 33 1.3.4 Azurophilic granules.......................................................................................... 34 1.4 Lung epithelial dysfunction and damage ................................................................. 37 1.4.1 Structure of the respiratory tract ........................................................................ 37 1.5 The airway epithelium ............................................................................................... 38 1.5.1 Role of neutrophil proteases in airway epithelial injury and lung disease ........ 39 1.6 Mechanisms of neutrophil degranulation ................................................................ 44 1.6.1 Receptors regulating degranulation ................................................................... 46 1.6.2 Phospholipid and kinase signalling in degranulation ........................................ 47 1.6.3 Calcium signalling in degranulation .................................................................. 47 1.6.4 Src family kinases in degranulation .................................................................. 48 1.6.5 GTPase signalling in degranulation ................................................................... 50 iii | 1.6.6 SNAP/SNARE proteins in degranulation .......................................................... 52 1.7 Effects of cigarette smoke on neutrophils ................................................................ 53 1.8 Hypoxia ....................................................................................................................... 54 1.8.1 Physiological and pathological hypoxia ............................................................ 54 1.8.2 Cellular requirements for oxygen ...................................................................... 57 1.8.3 Mechanism of oxygen sensing .......................................................................... 58 1.8.4 Neutrophil function under hypoxic conditions .................................................. 63 1.8.5 Neutrophil degranulation under hypoxic conditions ......................................... 64 1.9 Summary ..................................................................................................................... 65 2. Hypothesis & Aims .................................................................................. 66 2.1 Hypothesis ................................................................................................................... 66 2.2 Specific aims ............................................................................................................... 66 3. Materials and Methods ............................................................................ 68 3.1 Materials ..................................................................................................................... 68 3.2 Neutrophil isolation ................................................................................................... 70 3.3 Working under hypoxic conditions .......................................................................... 72 3.3.1 The hypoxic incubator ....................................................................................... 72 3.3.2 Analysis of media pH, pO2 and pCO2 ............................................................... 74 3.4 Preparation of cigarette smoke medium .................................................................. 74 3.5 Neutrophil extracellular reactive oxygen species production ................................ 75 3.6 Measurement of intracellular reactive oxygen species (ROS) production ........... 76 3.7 Apoptosis assays ......................................................................................................... 76 3.8 Assessment of degranulation ..................................................................................... 78 3.8.1 Assessment of the effect of hypoxia on secreted NE activity ........................... 78 3.8.2 Quantification of NE ......................................................................................... 78 3.8.3 Optimisation of the NE activity assay ............................................................... 81 3.8.4 Investigation of the mechanism of hypoxia-induced augmented neutrophil degranulation .............................................................................................................. 83 3.8.5 Quantification of myeloperoxidase ................................................................... 83 3.8.6 Quantification of lactoferrin release by ELISA ................................................. 83 3.8.7 Quantification of MMP-9 (gelatinase) release by ELISA ................................. 85 3.8.8 Quantification of secreted MMP-9 activity by gelatin zymography ................. 86 3.9 Western blot analysis of neutrophil granule
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