The Role of TIMP3 in Models of Inflammation and Immunity By David Smookler March 2010 A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate department of Laboratory Medicine and Pathobiology University of Toronto © copyright by David Smookler 2010 ABSTRACT The Role of TIMP3 in Models of Inflammation and Immunity by David Smookler a PhD thesis for the Department of Laboratory Medicine and Pathobiology University of Toronto, 2010 The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells. ii ACKNOWLEDGMENTS I would like to thank Dr. Rama Khokha for her unwavering support over the years. Actually there has been a bit of wavering, but overall she has been a lighthouse to me and my thanks are deep and sincere. I would also like to thank Marco DiGrappa who by virtue of his intelligence and unstinting helpfulness coupled with a humorous state of pessimism and self-deprecation has taught me never to take myself too seriously, and to feel I will always have a sympathetic ear when something goes wrong. I would like to thank Hart Jackson, Alison Aiken and Sam Molyneux for the respect they pay me, probably as an older statesman of the lab but I hope respect earned by what I share with them. I’d like to thank Alex Beristain for reminding me that “older statesman” essentially means “old man”. I’d like to thank Adi Murthy for reminding me of the scientific enthusiasm that can be brought to my field, and I’d like to thank Virginie Defamie for the unconditional warmth with which I am greeted whenever I appear at the lab. I’d like to thank Purna Joshi for being an example of dogged focus and perseverance, not afraid to take on Herculean projects while other demands are made upon her. I would like to thank Iris Fang for her patience and generous helpfulness. I’d like to thank Trevor McKee for watching out for the post-docs’ welfare as I soon hope to join their ranks. I thank Gordon Duncan, whose practical advice was instrumental to my work, and Bill Sukloff who’s instrument, this laptop, made writing up my work practical. I would also like to thank former members of the Khokha lab Zam Kassiri and Geoff Wood for their guidance and support. I thank Swami Narala for reminding me that gratitude is payment in a currency that costs little but is worth much. And Linda, who put up with it all, I thank you. iii TABLE OF CONTENTS Chapter 1: INTRODUCTION The Extracellular Matrix--------------------------------------------------------------------------1 Collagens -------------------------------------------------------------------------------------2 Glycosaminoglycans and Proteoglycans -------------------------------------------------3 ECM glycoproteins -------------------------------------------------------------------------4 Proteases in the Mammalian Genome ----------------------------------------------------------6 Matrix Metalloproteinases -----------------------------------------------------------------6 MMP structure-------------------------------------------------------------6 Types of MMPs------------------------------------------------------------7 Roles of MMPs in normal physiology --------------------------------10 MMPs target growth factor signalling in several ways -------------12 Regulation of MMPs ----------------------------------------------------13 ADAMTSs----------------------------------------------------------------------------------14 ADAMs -------------------------------------------------------------------------------------14 TACE----------------------------------------------------------------------16 Metalloproteinases in inflammation-----------------------------------------------------19 Tissue Inhibitors of Metalloproteinases ------------------------------------------------------20 TIMP Structure-----------------------------------------------------------------------------21 TIMP3 ---------------------------------------------------------------------------------------21 Structural interaction of TIMP3 with TACE -------------------------22 iv Inflammation---------------------------------------------------------------------------------------26 Systemic Inflammatory Response Syndrome (SIRS) --------------------------------29 Lipopolysaccharide (LPS)----------------------------------------------------------------30 Tumour Necrosis Factor (TNF)----------------------------------------------------------31 Expression of TNF and TNF Receptors ------------------------------34 TNF Signal Transduction -----------------------------------------------34 TNF and IL-6-------------------------------------------------------------39 Nitric Oxide---------------------------------------------------------------------------------42 TIMP3 and Hematopoiesis ----------------------------------------------------------------------42 MPs targeted by TIMP3 affect hematopoietic growth factor signalling------------43 MPs are involved in the trafficking of hematopoietic cells --------------------------46 TIMPs influence hematopoiesis via MPi and non-MPi mechanisms---------------46 Rationale for the Thesis--------------------------------------------------------------------------47 Chapter 2: MATERIALS AND METHODS Mice --------------------------------------------------------------------------------------------------49 Reagents---------------------------------------------------------------------------------------------50 ELISAs ----------------------------------------------------------------------------------------------50 Statistical Analysis --------------------------------------------------------------------------------51 Tissue preparation for Western blots and TACE assay -----------------------------------51 Protein Quantification----------------------------------------------------------------------------52 TACE assay-----------------------------------------------------------------------------------------52 v Western blot antibodies --------------------------------------------------------------------------53 Quantitative PCR ---------------------------------------------------------------------------------54 Alanine aminotransferase (ALT) analysis----------------------------------------------------55 Synthetic metalloproteinase inhibitors--------------------------------------------------------56 Heart effluent---------------------------------------------------------------------------------------56 Nitric oxide analysis-------------------------------------------------------------------------------56 Peripheral blood analysis ------------------------------------------------------------------------57 Skin challenge --------------------------------------------------------------------------------------57 Memory cytotoxic lymphocyte analysis ------------------------------------------------------57 Hematopoiesis --------------------------------------------------------------------------------------58 Reproducibility and variation of results ------------------------------------------------------61 Chapter 3: TIMP3 Regulates TNF-Dependent Systemic Inflammation ABSTRACT ----------------------------------------------------------------------------------------63 INTRODUCTION---------------------------------------------------------------------------------64 RESULTS -------------------------------------------------------------------------------------------66 -/- Inactivated bacteria induces greater TNF release in Timp3 mice ----------------------66 -/- Timp3 mice are highly sensitive to LPS-induced septic shock ------------------------71 -/- p55 pathway is required for heightened inflammation in Timp3 mice ----------------73 -/- -/- Timp4 mice do not share the sensitivity to LPS found in Timp3 mice --------------75 DISCUSSION --------------------------------------------------------------------------------------77 vi Chapter 4: Investigation of the Organ Response to Inflammatory Challenge in Timp3-/- Mice ABSTRACT ----------------------------------------------------------------------------------------81 INTRODUCTION --------------------------------------------------------------------------------82 RESULTS -------------------------------------------------------------------------------------------84 -/- Timp3 livers show normal production of and sensitivity to IL-6 ----------------------84 -/- In response to LPS, JNK activation is greater in Timp3 livers -------------------------86 Comparison of TNF levels in Timp3-/- versus wild-type