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Inflammatory Regulation of Cysteine Cathepsins Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2008 Inflammatory Regulation of Cysteine Cathepsins Blaine Creasy Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medicine and Health Sciences Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1586 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Blaine Madison Creasy, 2008 All Rights Reserved 2 A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by BLAINE MADISON CREASY B.S in Biology, James Madison University, 2002 Director: KATHLEEN L. MCCOY PROFESSOR, DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY Virginia Commonwealth University Richmond, Virginia May, 2008 3 - -ii Dedication This work is dedicated in loving memory of my mother Delores Martin Creasy. Her emphasis on the importance of education was a main influence on my decision to continue my education for so many years. She always encouraged me to thrive in school, in life, to climb mountains and achieve my goals no matter what. She is forever in my heart and constantly reminding me to be the person she always knew I could be. ii - -iii Acknowledgement Throughout the years that it required to complete this body of work I have gone through many thoughts, feelings and emotions. There have been times of extreme stress, excitement, disappointment, frustration and feelings of defeat. I want to thank my family and friends for all their support and encouragement, as I am sure without it I would have long ago given up. I would like to thank my parents for their support both emotional and financial, which allowed me to continue my education over the years. I would like to thank my undergraduate research mentor Dr. Chris Lantz. Not only did he give me my first exposure to scientific research but, without his encouragement to attend graduate school I would not be receiving this degree today. I would also like to extend my gratitude to the members of my graduate committee, Dr. Suzanne Barbour, Dr. Daniel Conrad, Dr. Phil Hylemon, and Dr. Kimber White. Their suggestions, instruction, and mentorship through the years have been invaluable to my training. Additionally, I would like to acknowledge the many faculty members here at VCU who always have an open door policy and have been a great resource for a student to have. I would also like to acknowledge Constance Hartmann, who I have worked beside for 4 years and has contributed to the work presented here. Last, and most importantly, I would like to thank my graduate advisor Dr. Kathleen McCoy. As a student the amount of work becomes overwhelming at times and iii - -iv we forget how much work we also create for our mentor. There are no words to describe how much I appreciate her mentorship, helpful insights, and countless efforts towards my training as a scientific researcher. I have been lucky to have such a talented and dedicated mentor. iv - -v Table of Contents Page List of Tables ................................................................................................................. ix List of Figures................................................................................................................. x Abbreviations...............................................................................................................xiii Abstract....................................................................................................................... xvii Chapter 1: Introduction…………………...........................................................................2 Antigen-Presenting Cells and Antigen Processing...................................... 3 Macrophages ............................................................................................. 6 Cathepsins ............................................................................................... 11 Cathepsin B ............................................................................................. 15 Cathepsin L ............................................................................................. 18 Cathepsin S ............................................................................................. 19 Toll Like Receptors ................................................................................. 22 Cannabinoids........................................................................................... 29 Rationale and Objectives ......................................................................... 36 2 Materials and Methods................................................................................. 39 Mice ........................................................................................................ 39 Cell Lines................................................................................................ 39 Monoclonal and Polyclonal Antibodies.................................................... 39 v - -vi Splenocyte Preparation ............................................................................ 40 Cathepsin Substrates and Inhibitors ........................................................ 41 Confocal Microscopy .............................................................................. 41 Cathepsin Enzymatic Activity in Live Cells Using Magic Red Substrates 42 Substrate in Stimulated Cells Not Limiting.............................................. 43 Protease Activity in Cell Lysates ............................................................. 43 MR Substrate Specificity......................................................................... 44 Cathepsin Activity in Live Cells Using AMC Substrates.......................... 44 Cathepsin Assays Combined with Immunofluorescence Staining............. 45 TLR Ligands ........................................................................................... 46 TLR Stimulation...................................................................................... 46 ImageStream® Cell Analysis................................................................... 46 Cathepsin Secretion ................................................................................. 47 Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction ...... 48 Co-culture Experiments ........................................................................... 49 Activation of Non-responsive Bystander Cells with Culture Supernatants 50 Cytokine Neutralization Studies............................................................... 50 Cannabinoid Receptor Expression ........................................................... 51 Cannabinoid Treatment of Cells .............................................................. 51 Characterization of CB2-/- Macrophages ................................................. 51 vi - -vii Apoptosis Studies .................................................................................... 52 TLR Cell Surface Expression................................................................... 52 Statistical Analysis .................................................................................. 53 3 New Assay Staining to Measure Cysteine Cathepsin Activity in Live Cells.. 54 Introduction............................................................................................. 54 Results..................................................................................................... 56 Discussion ............................................................................................... 68 4 Toll-Like Receptor Regulation of Cysteine Cathepsins ................................ 76 Introduction............................................................................................. 76 Results..................................................................................................... 79 Discussion ............................................................................................... 95 5 Cannabinoid Inhibition of Cysteine Cathepsin Upregulation During an Inflammatory Response ........................................................................... 99 Introduction............................................................................................. 99 Results................................................................................................... 101 Discussion ............................................................................................. 110 6 Discussion ................................................................................................. 117 TLR Regulation of Cysteine Cathepsins ................................................ 117 Cannabinoid Inhibition of Cysteine Cathepsin Upregulation.................. 125 References .................................................................................................................. 134 vii - -viii Appendix .................................................................................................................... 155 viii - -ix List of Tables Page Table 1: Tissue expression of cathepsins and phenotypes of cathepsin deficient mice.... 13 Table 2: TLR pathogen associated molecular patterns. .................................................
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