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Molecular and Cellular Mediators in Radiation-Induced Lung

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Molecular and Cellular Mediators in Radiation-Induced Lung MOLECULAR AND CELLULAR MEDIATORS IN RADIATION-INDUCED LUNG INJURY Xuebin Yang 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 Department of Pathology and Laboratory Medicine. Chapel Hill 2009 Approved by: Suzanne Kirby, M.D. Ph.D. Donald N. Cook, Ph.D. Virginia L. Godfrey, DVM. Ph.D. Nobuyo N. Maeda, Ph.D. Stephen Tilley, M.D. Abstract Xuebin Yang: Molecular and Cellular Mediators In Radiation-Induced Lung Injury (Under the direction of Suzanne Kirby, M.D. Ph.D) Radiation-induced lung injury is a common adverse effect in patients receiving thoracic irradiation and for which, there is currently no effective therapy. Using a murine model of thoracic irradiation, we found that mice deficient in either the chemokine CCL3 or one of its receptors, CCR1, are significantly protected from radiation lung injury. This protected phenotype includes improved survival, virtually no pneumonitis or fibrosis, and preserved lung function when compared to wild-type mice. We further showed that a specific CCR1 inhibitor, BX471 provided similar protection. Therefore, CCR1 is a promising target for reducing radiation lung injury. To investigate the mechanisms by which CCL3/CCR1 signalling mediates radiation lung injury, we evaluated their influence on lung inflammation after irradiation. When compared with irradiated WT mice, irradiated CCL3- and CCR1-deficient mice had less lung + + infiltration of CD4 and CD8 T cells; however, CD4-deficient mice showed only partial protection, while CD8-deficient mice had slightly worse fibrosis. We further analyzed + inflammatory cytokines and different subsets of CD4 lymphocytes, T 1, T 2, T 17 and H H H + Treg cells, in our model. We found no differences in lung Foxp3 Treg cells between WT and CCR1-deficient mice. Notably however, irradiated CCR1-deficient mice had less mRNA ii expression of the T 2 cytokines, IL-4 and IL-13, suggesting that T 2 cells may mediate H H radiation lung injury. In addition, the T 2-enhanced IL-10/12 double knockout mice (IL- H -/- 10/12 ) have increased expression of IL-13 and IL-4 with an associated earlier onset and enhanced degree of lung fibrosis. Even more striking was our finding that irradiated IL- -/- 10/12 mice had an earlier onset of increased IL-17 mRNA expression, as well as increased lung infiltration of T 17 cells, suggesting that IL-17 cells may also be important mediators in H radiation lung injury. In summary, our studies show that CCL3 and its receptor, CCR1, are key mediators of radiation lung injury that act at least in part by recruiting T 17 and T 2 cells into the H H irradiated lungs. Furthermore, our findings suggest that the specific inhibitor of CCR1, BX471, looks promising as a potential therapeutic agent for ameliorating radiation lung injury. iii ACKOWLEGEMENTS First, I feel grateful beyond measure to my advisor, Dr. Suzanne Kirby. Dr. Kirby has served a mentor to me since 2004. I am deeply indebted to her for believing in me and fostering my scientific career. I will never forget her tireless efforts to motivate and encourage me. I am always grateful to her for patience and understanding to foster me as a doctor of philosophy in molecular and cellular pathology. It has been a privilege and a pleasure to work with an advisor who has also been a good friend. My sincere gratitude also goes out to Dr. J. Charles Jennette, chair of the Pathology Department and Dr. William Coleman, director of Graduate Students, who have both been so supportive for the Pathology graduate students. I thank them for their guidance and support. I also want to specifically thank William Walton who has been always here to help and support me along the way. He provided me with most of my technical scientific training and enormous help in experiments. He also assisted me on a daily basis with everything from mouse room chores to tedious benchwork all throughout my doctoral training. I would like to thank the other members of my committee too! Thanks to Dr. Donald N. Cook, Dr. Virginia L. Godfrey, Dr. Nobuyo N. Maeda, Dr. James M. Samet, and Dr. Stephen Tilley for all of their great ideas, encouragment, and for supporting me through my doctoral training. I also thank past and present members of the Kirby/Serody Laboratoies, including but not limited to Christin Buehler, Joe Burgents, Dr. Meredith Burgents, Dr. Michael Carlson, Dr. Jay Coghill, Dr. Jennifer Gilner, Laura Greene, Karen Hogan, Robert Mango, Dr. Karen iv McKinnon, Dr. Timothy P. Moran, Dr. Jonathon Serody, Jenelle Vargas, Shelly West, Cole Wilson, and Dr. Chris Wysocki, who have taught me many new techniques and offered lots of advice and discussion. I am also very appreciative of the friends I have made within the Pathology Department, who provided me with support and inspiration: Hind Mullaen, Diane Bender, Jeremiah Hinson, Mehmet Karaca, Matt Medlin and Elizabeth Merricks. I would also like to recognize the supporting staff Dorothy Poteat who has provided me with encouragement, dependable administrative and technical support over the years. I also owe a great deal of thanks to my parents and sisters for their unwaiving support of me. I am proud to make them proud. I also thank my parents-in law who have provided me with a much needed helping hand in caring for my daughter, Lydia. I could not have completed my dissertation without their daily help. Lastly, I will forever be grateful to my wife Jane (Huaizhi) who has supported me and kept me motivated all along the way. Without her love, friendship and encouragement, none of this would have been possible. Finally, There are many others listed below who have helped, supported and taught me along the way. I never could have done this without all of you! Thanks! C. Robert Bagnell, Jr. PhD William Blackstock, MD W. June Brickey, PhD Yoshizumi Deguchi, MD. Hendrik Deventer, MD Christopher Haskell, PhD (Bayer) Xiaoyang Hua, MD Qingping Hu, MD Jenny Ting, PhD Tong Zhou, MD PhD v TABLE OF CONTENTS Page LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xvi Chapter I. Introduction………………………………………………………………………..1 I.A. General Principles of Radiation Injury………………………………………1 I.B. Radiation-induced Lung Injury………………………………………………2 1. Pathophysiology……………………………………………………………2 2. Chronic Inflammation and Fibrosis…………………………………….....5 3. Effects on Lung Function…………………………………………………..6 4. Management of Radiation Lung Injury…………………………………….7 5. Molecular Mediators of Radiation Pneumopathy………………………….8 6. Cellular Mediators of Radiation Pneumopathy…………………………..17 I.C. Chemokines and Chemokine Receptors…………………………………….21 1. Chemokine Structure………….……………………………………………21 2. Nomenclature………………………………………………………………21 3. Chemokine Receptors………………………………………………………21 4. Chemokine Sources and Regulation………..…………………………..….23 vi 5. Chemokine Functions……………………………………………………...26 6. Chemokines and Chemokine Receptors in Airway Diseases………………31 7. Chemokines Receptor Antagonists-New Perspective for Therapy…….…..33 I.D. Chemokines in Radiation-induced Lung Injury……………………….…….35 I.E. The chemokine receptor CCR1 in Radiation-induced Lung Injury……….…36 II. Murine Models of Radiation Lung Injury……………………………………….39 II.A. Introduction…………………………………………………………….…..39 II.B. Materials and Methods……………………………………………………..40 1. Mice………………………….…………………………………………….40 2. Thoracic Irradiation……………………………………………………....40 3. Hydroxyproline (HYP) and Pyridinoline (PYR) asaays…………………..40 4. Elastin Assay………………………………………….……………….…..41 5. Gene Expression Analyses…………………………………………….…..41 6. Histologic Analysis………………………………………………………..42 7. Measurements of Lung Mechanics….……………………………………..42 8. ELISA Analyses of TGF-β1…….……………………………………..…..42 9. Flow Cytometry……….……………………….…………………………..42 II.C. Results………………………………………….…………………………..43 1. Survival Analysis of C57BL/6J Mice after Thoracic Irradiation………..43 2. Increased Lung Content of Hydroxyproline and Cross-linked Collagen..43 3. Increased Gene Expression of Lung Collagen 1α…………………….....45 4. No Increase in Lung Elastin……………………………………………..45 5. Histologic Analyses……………………………..………………………..45 vii 6. Diminished Lung Function after Thoracic Irradiation…………………..46 7. Increased TGF-beta1 in lung after Thoracic Irradiation…...………..….51 8. Flow cytometric analyses of lung inflammatory cell infiltration…...…....51 III. Macrophage Inflammatory Protein-1 Alpha (MIP-1α) in Radiation-induced Lung njury………………………………………….………..56 III.A. Abstract……………………………………………………………………56 III.B. Introduction………………………………………………..………………57 III.C. Materials and Methods…………………………………..………..…….…57 1. Cell Cultures……………………………………………………………..57 2. Animals…………………………………………………………………..57 3. Irradiation………………………………………………….…………….58 4. ELISA and gene expression analyses of TGF-β1……...………..……….58 5. Quantification of Lung Hydroxyproline……….………..………..……....58 6. Flow Cytometry…………………….……………………………..……...58 7. Histologic Analyses…………….…………………………………..….…59 8. Antinuclear Antibody Assay……….…………………………………..…59 9. Statistical Analyses………………………………...………………….…59 III.D. Results…………………………….…………………………………….…59 1. Irradiation Induces MIP-1α Expression in lung-derived Cells in vitro...59 2. MIP-1α was Induced in the Lungs of Irradiated mice………………….60 3. MIP-1α -/- Mice have Improved Survival after Receiving Thoracic Radiation……………………………..……….…………..…..60 4. MIP-1α-deficient Mice have Decreased Fibrosis after Lung Irradiation………………………………………………………....60 viii 5. MIP-1α -/- Mice have Decreased TGFβ1 Production After Thoracic irradiation…………………….………………………...63
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