REGULATION OF AIRWAY REACTIVITY: INTERACTIONS BETWEEN NEURONAL AND IMMUNE PATHWAYS IN THE LUNG Jaime Marie Cyphert A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology. Chapel Hill 2009 Approved by: Beverly H. Koller Stephen L. Tilley Martina Kovarova Ellen R. Weiss Mark W. Majesky Eva S. Anton ABSTRACT JAIME M. CYPHERT: Regulation of airway reactivity: Interactions between neuronal and immune pathways in the lung (under the direction of Beverly H. Koller) The prevalence of asthma has been on the rise since the 1970s, affecting more than 300 million people worldwide. In recent years, approximately 250,000 deaths per year have been attributed to the condition. The majority of these deaths are likely a direct result of airway obstruction, of which constriction of airway smooth muscle plays a significant role. Although several bronchodilators are currently on the market for alleviation of airway constriction, not all patients are responsive to these treatments. This underlies the importance of understanding the mechanisms triggering smooth muscle constriction so that additional drug targets can be discovered to help prevent or reverse airway constriction during asthmatic exacerbations. In this dissertation I utilize two approaches to analyze immune-related airway constriction in the mouse in an attempt to further understand the mechanism behind the human condition. The majority of asthmatics also have allergies, which can trigger both enhanced inflammation of the airways and airway constriction. Therefore, the first approach involved modeling allergic airway constriction in both the naïve and inflamed mouse lung to examine the mechanisms of IgE-mediated bronchoconstriction. Genetic, pharmacological, and surgical methods were then used to explore the cell types, mediators, and receptors involved in this response. ii The second approach involved the triggering of airway constriction through a non-allergic mechanism. In these studies, the thromboxane analog U46619 was used to elicit dose-dependent airway constriction. As the thromboxane receptor, Tp, is expressed on multiple cell types, mice carrying a tissue-specific deletion of this receptor were examined to define the cell types involved in U46619-mediated bronchoconstriction in both the naïve and inflamed lung. Asthma is a highly complex disease involving the combined effects of both genetic and environmental effects and therefore it is unlikely to be able to model the disease itself accurately in a laboratory animal that does not spontaneously develop the condition. However, it is possible to closely approximate several pathophysiological symptoms of asthma in the mouse, and using these models, the many mechanisms underlying the conditions of asthma can be teased away from the complexity of the condition as a whole. iii ACKNOWLEGEMENTS I would like to offer my heartfelt thanks to several individuals which have made the work described in this dissertation possible. First and foremost, I would like to thank my advisor, Bev Koller, not only for her training and advice but also for her willingness to believe in my abilities and my work. Under her guidance I have grown as a scientist, and have learned to think critically about my experiments and to be able to evaluate the results not only as to how they fit into the scope of my project, but to how they fit into a broader context. Her belief and support has helped me to develop skills that I would previously have thought impossible, and has enabled me to challenge myself both technically and mentally. Bev, I would like to express my sincere gratitude for all of the time, patience, and advice that you have given me over the years. I can only hope that my accomplishments in the future will make you proud. I would also like to thank the many members of the Koller Lab, both past and present, whose advice, encouragement, and friendship has made this work both possible and enjoyable. In particular, I would like to thank Coy Allen who has assisted greatly in my training as a graduate student, and whom has always been available for helpful discussion, comic relief, and a contagiously happy attitude; Leigh Jania and MyTrang Nguyen for vast quantities of technical advice and assistance; Anne Latour for genomic instruction and good conversation; Jay Snouwaert for all his patient assistance with cloning and construct design; Martina Kovarova for serving on my committee, sharing iv her knowledge of mast cells and for assisting me in several of my projects; and Rachel Coté for her willingness to assist me with tedious tissue collections and for a very valuable friendship and sympathetic ear. I also wish to thank the rest of my committee members, Steve Tilley, Mark Majesky, Ellen Weiss, and Eva Anton for their guidance and helpful discussions. Specifically, I would like to express my deepest thanks to Steve Tilley, who has been like a second mentor to me. Your discussion and assistance with projects and temperamental equipment has been a stabilizing factor in times of stress and exasperation. My love and gratitude go out to my family; my mother, father, and big sister for their love, enthusiasm, and unconditional support. Special thanks to my mother for being my best friend, my biggest fan, and for helping me to become the driven, independent person that I am today. You have given me the strength and confidence to succeed in any endeavor that I put my mind to. I am also very grateful for all of the good friends that I have made over the past 5 years, all of whom I think of as an extension of my family. Sabrina, Harmony, Rachel, Demetra, Kerry, and Colin; without you all the days would be longer. Thank you for keeping me sane and for lending a hand or an ear whenever needed. And finally, I cannot forget to thank all of the four-legged companions that have brought joy to me over the years. My special thanks goes out to Tigger who was with me since the third grade, and will be in my heart forever; Bentley for taking up where Tigger left off; and to Johnny, Indy, and Tucker for being sturdy partners and trusting friends in a sport that keeps me grounded, challenged, and happy despite any negative forces in my life. Again, my deepest thanks to everyone, without all of you none of this would have been possible. v TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. ix LIST OF FIGURES .................................................................................................................x LIST OF ABBREVIATIONS ............................................................................................. xiii CHAPTER 1: Introduction.....................................................................................................1 Structure, function, and regulation of the healthy lung .....................................................2 Epithelium.............................................................................................................4 Airway smooth muscle .........................................................................................6 Resident immune cells..........................................................................................6 Neural control.......................................................................................................8 Lung function measurements..............................................................................19 Regulation of the diseased lung .......................................................................................22 Epithelium...........................................................................................................24 Mucus production and edema .............................................................................25 Airway smooth muscle .......................................................................................26 Allergic inflammation.........................................................................................27 Lipid mediators ...................................................................................................41 Neural regulation................................................................................................44 Summary..........................................................................................................................55 Organization of dissertation.............................................................................................56 References........................................................................................................................57 vi CHAPTER 2: Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction .................................................82 Abstract............................................................................................................................83 Introduction......................................................................................................................84 Methods............................................................................................................................86 Results ............................................................................................................................92 Discussion......................................................................................................................101