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The Effect of Porphyromonas Levii on Macrophage Function and Pro-Inflammatory Cytokine Production

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The Effect of Porphyromonas Levii on Macrophage Function and Pro-Inflammatory Cytokine Production University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 2001 Macrophages in bovine footrot: the effect of porphyromonas levii on macrophage function and pro-inflammatory cytokine production Walter, Michaela Roylene Valerie Walter, M. R. (2001). Macrophages in bovine footrot: the effect of porphyromonas levii on macrophage function and pro-inflammatory cytokine production (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/19168 http://hdl.handle.net/1880/41129 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Macrophages in Bovine Footrot: The Effect of Purphyrontonas fevii on Macrophage Function and Pro-Inilammatory Cytokine Production. Michaela Roylene Valerie Walter A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA MAY, 200 1 O Michaela Roylene Valerie Walter 200 1 National Library Biiliothbque nationale du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive pennettant a la National Libmy of Canada to Bibliotheque nationale du Canada de reproduce, loan, distn'bute or sell reproduke, preter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d7auteurqui protege cette these. thesis nor substantial extracts fiom it Ni la these ni des edtssubstantiels may be printed or otherwise de celle-ci ne doivent iie imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Bovine footrot is an acute lameness disease that is characterized by inflammation of the interdigital tissues in the hoof. Porphyromonas levii an obligate anaerobic bacteria, has recently been isolated from interdigital biopsies of affected lesions. The objectives of this study were to evaluate the early immune responses of the host, specifically macrophages, in response to P. levii in vitro. Macrophages showed decreased chemotaxis, phagocytosis, and oxidative metabolism when exposed to P. levii in low concentrations. In high concentrations of P. levii, macrophages were more phagocytic and displayed significant oxidative metabolic responses. Phagocytosis by macrop hages was greatly enhanced when P. levii was opsonized with P. levii specific serum or affinity purified IgG. P. levii exposure to macrophages in wino, induced the mRNA expression of IL1 P, TNFa,and L8. Reduced clearance by macrophages, and the production of pro- inflammatory mediators, could contribute to the pathogenesis and the inflammation seen in bovine footrot. Acknowledgements Fiat of all I would like to thank my supervisor Dr. Doug Morck. During my time in his laboratory, I have had the independence and flexibility that has enabled me to truly appreciate and enjoy research. My ability to grow as a scientist has been insurmountable under his direction and I will always be gratefir1 for the opportunities he has given me. I would also like to thank my committee members Dr. R.L. Walker, Dr. H. Ceri. and Dr. J. Wallace, for accepting to serve on my thesis advisory committee. There are a number of individuals who have also contributed time out of their busy schedules to help me with some of the technical aspects of the project. Thanks to Kevin Garossino for looking after my calves and making my numerous trips out to the fann memorable and time-efficient. Thanks to Liz Middlerniss for her assistance with the TEM work that was done. I would also like to thank the Habibi lab for allowing me to use some of their equipment. I would also like to extend a huge thanks to the fellow students and researchers in my lab as well as the members of the Biofilm Research Group who have made the time in the lab enjoyable. I will always treasure the friendships and the moments that we have shared together. Finally I would like to acknowledge the financial support From the Natural Sciences and Engineering Research Council of Canada (NSERC). iv. Dedication To My Family: Mom, Dad, Cheryl and family, Tara, Sarah, and Matt For all the Family Dinners and especially for being supportive. Table of Contents .. Approval Page ......................................................... ...11 Ab~tfaCt............~~~~~~....~.~~~~..............~................. ..11i Acknowledgements .....................................................iv Dedication .............................................................v Table of Contents .*....................... ..............................vi List of Tables ..........................................................ix ListofFigum .........................................................x List of Abbreviations .. ..................... ............................ xii Chapter One: Literature Review .......................................... 1 A . Bovine Footrot .................................................1 Introduction ................................................ I 1. Clinical Signs and Diagnosis ............................... 2 2. Epidemiology and Predisposing Factors .......................6 3. Treatment ...............................................8 4 . Immunopathology ....................................... 10 5 . Role of Bacteria ........................................ .I I B . Anaerobic Bacterial Infections ................................... 17 C . Virulence Factors of Porplyromonas Species ....................... 18 D . inflammation .................................................24 E . Macrophages in Mammation ................................... 25 1. Development of Macrophages ............................. 27 2 . Chexmtaxis of Macrophages .............................. 28 3 . Phagocytosis by Mmphages ............................-30 4. Bactericidal Activity .....................................35 5 . Cellular Recruitment .....................................38 F . Cytokines ................................................... 41 1. Interleukin 1p .......................................... 41 2 . Turnour Necrosis Factor a ................................ 43 3. Interleukin 8 ...........................................45 4. Cytokine Induction by Porphyromonus Species ............... 47 G . Summary ...................................................-47 H . Objectives ...................................................48 I. Hypothesis ..................................................49 Chapter Two: Methods and Materials ....................................51 A . Monocyte Isolation & Macrophage Differentiation ...................51 1. Blood Collection & Mononuclear Cell Purification ............ 51 2. Mononuclear Cell Quantification & Viability ................. 52 3. Macrophage Culture & Differentiation ......................52 B . Macrophage Characterization .................................... 53 1. Monocyte to Macrophage: Change in Cell Size ............... 53 2. Non-Specific Esterase Staining of Mamphages .............. 54 vi . Table of Contents Continued 3 . CD 68 Antigen Staining .................................. 55 C .Bacterial Preparation ...........................................57 1. Growth .................. ...... .......................57 2 . Preparation ............................................58 3 . CultunMty ..........................................58 D. Macrophage Cell Movement .....................................58 1 . Macrophage Isolation ....................................58 2. T~tSolutiom......................................... .59 3 . Boyden Chambers ......................................60 4. Chemotaxis Assay ......................................60 5 . Chemokinesis Assay .................................... 63 6. Staining ..............................................63 7. Leading Front Method ...................................68 E . Phagocytosis ................................................ 69 1. Macrophages & Bacterial Preparation .......................69 2. Reparation of Control ...................................69 3 . Opsonizatioa of P. levii ..................................70 i . Immunization and Senun Collection ................... 70 ii .Titre Determination ... .. ...........................71 iii . Western Blot of Re=Immme.Low & High Titre Sem. -72 iv .Immunoglobulin G (IgG) Purification & Concentration ...73 v . IgG Quantification ................................74 vi .Opsonization of P . levii with Senun and IgG Samples ....77 4. Phagocytosis Assay ....................................77 i . Concentration and Time Studies ..................... -77 ii . Role of Opsonins in Phagocytosis of P. levii ........... 78 5 . Complement Analysis ..................................79 6 . Transmission Electron Microscopy ....................... 81 F . Oxidative Metabolism ....... ............... .................
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