MOLECULAR PATHOGENESIS OF NON- EOSINOPHILIC ASTHMA Katherine Joanne Baines B.BiomedSci(Hons) A Thesis Submitted for the Degree of Doctor of Philosophy October 2007 Faculty of Health School of Biomedical Sciences University of Newcastle STATEMENT OF ORIGINALITY This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying subject to the provisions of the Copyright Act 1968. ACKNOWLEDGEMENT OF AUTHORSHIP/COLLABORATION I hereby certify that the work embodied in this Thesis is the result of original research, the greater part of which was completed subsequent to admission to candidature for the degree (except in cases where the Committee has granted approval for credit to be granted from previous candidature at another institution). Signature:……………………………….. Date:………………………………….. ii Acknowledgements When I first began thinking about writing this section of my thesis it dawned on me how lucky I am to have so many people I need to thank that have played an important role in both my PhD studies and my life. Doing a PhD has been a great challenge in which I have learnt so much and grown so much as a person and a researcher. First and foremost I would like to acknowledge the The Asthma Foundation of NSW and the Asthma CRC for providing funding for my PhD scholarship. I would like to thank my supervisors Prof Peter Gibson and Prof Rodney Scott for your ongoing support and encouragement throughout this process. I have a lot of respect for you both and thankyou for your sound advice and ideas, and for being patient with me, but also for pushing me when I needed it. Thanks Rodney for your guidance, confidence in me, and recommending me to the Respiratory group in the beginning, that change was the best thing I could have done. Peter, thankyou for all the opportunities you have offered and continue to offer me, I really admire your wealth of knowledge and ability to see ‘the big picture’. A big thankyou to all my colleagues and friends in the Respiratory Medicine and Childhood Cancer research groups at the Hunter Medical Research Institute. Thanks to Naomi Fibbens, Rebecca Oldham, Terry Grissell and Joanna Mimica for your lab work and skill, for teaching me methods, answering my many questions and also for a bit of a chat and laugh now and then. It’s loads of fun working with you guys, and you have become great friends. Thanks to Noreen Bell for your tireless work seeing the patients and collecting the samples for this study. Thanks to Deborah Hall for always getting me organised with everything I can think of, and for editing this thesis. Thanks to Glenda Walker for your help with the zymography. Thanks to Dr Nikola Bowden for your help with the microarrays. Thanks to Dr Vanessa Murphy for your help with formatting this thesis, and to Dr Lisa Wood for reviewing sections of this thesis. To Dr Jodie Simpson (Country Star), it’s great working with you, thanks for all your guidance and encouragement. Thanks for bringing me out of my ‘shell’ and for being such a great friend and workmate. To my office buddies Nicole Ryan and Heather Powell, thanks for always being ready for a coffee and for being so kind and supportive. iii To my dear friends Allison Thomas and Carolyn Brooks, thanks for always being there for me over the years; you are the best friends a girl could ask for. To the Uni girls (you know who you are), thanks for your friendship, for all the trips to Gloria’s, and for understanding the science talk and the PhD pressures. Thanks to my housemates Andrew and Bronwyn Rundle and Katie Brooker for putting up with me being stressed and having my papers everywhere, and for being so fun to live with. To Shane Nolland, thankyou for being my ‘rock’, and my best friend, I am very lucky to have you in my life. I don’t know if I would have got this far if it wasn’t for your love, patience and support. Thanks to my sister Penny Baines and to Ryan Chan, we really enjoy spending time with you guys. Pen, you are a cool little sister, thanks for never being afraid to have a go at anything, you always inspire me. To my Grandma, Audrey Johnson, thanks for always giving me perspective, you may not realise it, but you always remind me what is important in life. Finally I want to thank my parents, Lesley and Lee Baines, for your unconditional love and support in so many ways. Thankyou for always having faith in me and for encouraging me to go after whatever I want in life. Dad you can relax now, I have finally finished! iv Publications Simpson, J.L., Baines, K.J., Gibson, P.G. Chapter 17: The Biology of Neutrophils. Middleton’s Allergy 7th Edition. In Press August 2007. Abstracts Baines, K.J., Bell, N.V. Simpson, J.L., Scott, R.J., Boyle, M.J. Gibson, P.G. 2005. Enhanced IL-8 Release from Neutrophils in Non-Eosinophilic Asthma. Inflammation Research. 54: S137. (Poster) Baines, K.J., Simpson, J.L., Scott, R.J., Bell, N.V., Boyle, M.J. Gibson, P.G. 2006. Enhanced IL-8 Release from Neutrophils in Non-Eosinophilic Asthma. Respirology. 11(2): A29. (Poster) Baines, K.J., Bowden, N.A., Scott, R.J., Simpson, J.L., Gibson, P.G. 2007. Molecular Analysis of Neutrophils in Asthma Subtypes. Respirology. 12(1): A20. (Oral) Baines, K.J., Bowden, N.A., Scott, R.J., Simpson, J.L., Gibson, P.G. 2007. Molecular Analysis of Neutrophils in Asthma Subtypes. American Journal of Respiratory and Critical Care Medicine. 175: A683. (Poster) Gibson, P.G., Baines, K.J., Simpson, J.L., Scott R.J. 2007. Gene Expression Profiling Identifies Neutrophilic and Eosinophilic Asthma as Distinct Subtypes. European Respiratory Journal. 30(51): A3169. (Oral) v TABLE OF CONTENTS Table of Tables………………………………………………………….…………….xi Table of Figures……………………………………………………………………....xv Abbreviations……………………………………………………………………......xvii Abstract……...………………………………………………………………..………..1 CHAPTER 1: GENERAL INTRODUCTION .............................................................3 1.1 ASTHMA.............................................................................................................3 1.1.1 Allergy.......................................................................................................4 1.2 INFLAMMATORY SUBTYPES OF ASTHMA............................................................4 1.2.1 Eosinophilic Asthma .................................................................................5 1.2.1.1 Adaptive Immune Response .................................................................5 1.2.1.2 Eosinophils............................................................................................7 1.2.2 Non-Eosinophilic Asthma .........................................................................8 1.3 NEUTROPHILS IN ASTHMA................................................................................12 1.4 NEUTROPHILS IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)......13 1.5 BIOLOGY OF NEUTROPHILS ..............................................................................14 1.5.1 Neutrophil Migration ..............................................................................15 1.5.1.1 Myeloid Development.........................................................................16 1.5.1.2 Neutrophil Trafficking and Margination.............................................18 1.5.1.3 Cellular Adhesion Molecules..............................................................18 1.5.1.4 Integrins ..............................................................................................19 1.5.1.5 Inflammatory Stimulus .......................................................................20 1.5.1.6 Endothelial Cell Interactions...............................................................20 1.5.1.7 Epithelial Cell Interactions..................................................................20 1.5.1.8 Chemotactic Mediators.......................................................................22 1.5.1.8.1 Interleukin-8..................................................................................23 1.5.2 Neutrophil Phagocytosis .........................................................................24 1.5.3 Innate Immune Activation .......................................................................24 1.5.3.1 Toll Like Receptors (TLRs)................................................................25 1.5.3.2 Nucleotide-Binding Oligomerisation Domain (NOD) Molecules ......27 1.5.4 Neutrophil Granules ...............................................................................27 1.5.4.1 Mechanisms of Degranulation ............................................................29 1.5.4.2 Proteolytic Enzymes ...........................................................................29 1.5.5 Respiratory Burst ....................................................................................31 1.5.5.1 Reactive Oxygen Species....................................................................32 1.5.6 Neutrophil Clearance and Death............................................................33 1.5.7 Cytokine Synthesis...................................................................................34 1.5.8 Neutrophil Priming and Activation.........................................................36