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Mast cells : homeostatic regulation, activation, gene expression, surface antigens, and role in allergic.. Haddon, David James 2009

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MAST CELLS: HOMEOSTATIC REGULATION, ACTIVATION, GENE EXPRESSION, SURFACE ANTIGENS, AND ROLE IN ALLERGIC DISEASE by DAVID JAMES HADDON A.A., Camosun College, 1997 B.Sc., The University of Victoria, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2009 © David James Haddon, 2009  ABSTRACT Overall, we aimed to discover more about mast cell physiology, focusing on their homeostatic regulation in vivo, their activation in vitro and in allergic disease, their gene expression patterns, and their surface antigens. In our first study, our objective was to establish the function of Src homology 2-containing inositol 5’-phosphatase (SHIPI), in mast cells in vivo. SHIP1 inhibits immune receptor signaling through hydrolysis of the phosphatidylinositol-3 kinase (P13K) product Pl-3,4,5-P , forming 3 . In mast cells, SHIPI represses FcERI- and cytokine-mediated activation in 2 P1-3,4-P vitro, but little is known regarding the function of SHIPI in mast cells in vivo, or the susceptibility of Shipl’ mice to mast cell-associated diseases. We found that Shipl “  mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF,  and IL-5, and a heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1’ or Shipl’ mast cells, we found that the above defects were due to loss of SHIPI in mast cells. Additionally, we found that mice reconstituted with Shipl’ mast cells suffered worse allergic asthma pathology than those reconstituted with Ship 1’ mast cells. In summary, our data show that SHIPI represses allergic inflammation and mast cell hyperplasia in vivo, and that SHIPI exerts these effects specifically in mast cells. In our second study we compared LinSca-1c-kit (LSK) cells, which are highly enriched for hematopoietic stem cells (HSC), and mast cells, using microarray expression analysis, and identified prion protein (PrPC) as a potentially novel marker of mast cells. Upon further investigation, we found that PrPC (1) is expressed on the surface of human and mouse mast cells, both in vitro and in vivo; (2) is not required  for mast cell differentiation or tissue homeostasis; (3) is released by mast cells at steady state and rapidly upon activation; and (4) is released in response to mast cell dependent allergic inflammation in vivo. Since mast cells are long-lived and known to traffic to the brain and central nervous system (CNS), our observations could have important implications for the transmission and pathology of prion diseases. Further, mast cells could be a unique system to investigate PrP°’s normal function.  III  TABLE OF CONTENTS ABSTRACT  .  TABLE OF CONTENTS  ii  iv  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  xii  DEDICATION  xiii  CO-AUTHORSHIP STATEMENT  xiv  CHAPTER 1. INTRODUCTION  I  1.1 MAST CELLS  I  1.1.1 Development  1  1.1.2 Activation and degranulation  4  1.1.3 In vivo models of mast cell function  7  1.1.4 Mast cells and disease  10  1.2 P13K AND SHIPI  13  1.2.1 Pl3Ksignalling  13  1.2.2SHIP  17  1.3 PRION PROTEIN  20  1.3.1 Distribution of prion protein  20  1.3.2 Prion diseases  22  1.4 AlMS OF STUDY  25  1.5 REFERENCES  26  CHAPTER 2. SHIPI IS A REPRESSOR OF MAST CELL HYPERPLASIA, CYTOKINE PRODUCTION, AND ALLERGIC INFLAMMATION IN WVO  34  2.1 INTRODUCTION  34  2.2 RESULTS  37  2.2.1 Shipr’ mice have mast cell hyperplasia in multiple tissues  37  2.2.2 Mast cell hyperplasia in Ship1 mice is mast cell autonomous .39 2.2.3 Ship’ mice have mast cell-dependent systemic inflammation .42 ..  2.2.4 Ship’ mice are hyperresponsive to passive systemic anaphylaxis 44 iv  2.2.5 Allergic airway inflammation is greater in Ship1-BMMC mice than Ship1’-BMMC controls  47  2.3 DISCUSSION  50  2.4 EXPERIMENTAL PROCEDURES  56  2.4.1 Mice  56  2.4.2 Cell culture  56  2.4.3 Reconstitution of mast cell-deficient KitW  Sh  mice  57  2.4.4 Cytokine assay  57  2.4.5 Passive systemic anaphylaxis  57  2.4.6 Asthma disease course  58  2.4.7 Flow cytometry  59  2.4.8 Statistics  59  2.5 REFERENCES  60  CHAPTER 3. PRION PROTEIN IS EXPRESSED ON MAST CELLS AND IS RELEASED UPON THEIR ACTIVATION  64  3.1 INTRODUCTION  64  3.2 MATERIALS AND METHODS  66  3.3 RESULTS AND DISCUSSION  68  3.4 REFERENCES  76  CHAPTER 4. CONCLUSION 4.1 REFERENCES  78 88  APPENDICES  90  APPENDIX A. SUPPLEMENTAL DATA ON SHIP IN MAST CELLS  90  A.1 RHEUMATOID ARTHRITIS  90  A.1 .1 Development of rheumatoid arthritis does not appear to be altered in Ship1-BMMC reconstituted mice  91  A.2 MIGRATION AND SURVIVAL OF SHIP1 MAST CELLS IN VITRO  93  A.3 HISTOLOGICAL ANALYSIS OF SHIPI’ MAST CELLS  95  A.4 REFERENCES APPENDIX B. SUPPLEMENTAL DATA ON PRPC IN MAST CELLS  101 102  V  APPENDIX C. UBC RESEARCH ETHICS BOARD CERTIFICATES OF APPROVAL 104  vi  LIST OF FIGURES Figure 1.1 Proposed model of hematopoiesis  .1  Figure 1.2 Schematic of FceRI signalling Figure 1.3 Reconstitution of  6 mice to study gene function in mast  cells specifically  9  Figure 1.4 P13K Signalling schematic and the domain structure of SHIPI  15  Figure 1.5 Structure of prion protein and the misfolding cascade  21  Figure 2.1 Mast cell hyperplasia in multiple tissues of Ship1 mice  38  Figure 2.2 Mast cell hyperplasia in Shipl’ mice is mast cell autonomous  40  Figure 2.3 Increased inflammatory cytokine levels in the serum of Shipl” mice, and mice reconstituted with Shipi’ mast cells Figure 2.4 Hyperresponsive anaphylaxis in Shipi”  42 mice, and mice  reconstituted with Ship 1’ mast cells  45  Figure 2.5 Increased allergic asthma pathology in mice reconstituted with Shipi’ mast cells  48  Figure 3.1 Gene expression analysis of mast cells and HSC identifies PrPC as a marker of human and mouse mast cells, in vitro and in vivo 69 Figure 3.2 Mast cells release PrPC at steady state and upon stimulation in vitro and invivo  72  Figure 4.1 Proposed mechanism of increased inflammatory cytokines, allergic asthma pathology, and anaphylaxis response in Ship 1 mice and mice reconstituted with Shipi’ mast cells 80 Figure 4.2 A model of PrPC expression and PrPSC conversion during IgE mediated mast cell activation and relation to laminin binding  84  Figure Al Similar rheumatoid arthritis pathology in mice reconstituted with wild-type and Shipi” mast cells  92  Figure A.2 Defective migration and similar survival of Shipi’ mast cells in  vitro Figure A.3 Histological analysis of tissue mast cells in Shipi’ mice  94 95  Figure A.4 Histological analysis of tissue mast cells in mice reconstituted with  Shipi” mast cells  97  VII  Figure A.5 Cell autonomous mucosal mast cell hyperplasia in the intestines of Shipi’ mice, and mice reconstituted with Shipi’ mast cells 99 Figure B.1 Mast cells express high levels of PrPC and downregulate Prnp transcription upon FcERI stimulation  102  VIII  LIST OF ABBREVIATIONS AA  amino acid  AHR  airway hyperresponsiveness  B6  C57B1/6  BAL  bronchoalveolar lavage  BMMC  bone marrow derived mast cells  CCL2  chemokine (C-C motif) ligand 2  CLP  common lymphoid progenitor  CMP  common myeloid progenitor  CNS  central nervous system  DAG  diacyiglycerol  DMEM  Dulbecco’s modified Eagle’s medium  DNP-HSA  dinitrophenol-human serum albumin  EIA  enzyme immunoassay  FACS  fluorescence activated cell sorting/scanning  FBS  fetal bovine serum  FCS  fetal calf serum  FcRI  Fc epsilon receptor I  FcyRIIb  Fc gamma receptor lIb  FDC  follicular dendritic cells  FYN  Fyn proto-oncogene  G6PI  glucose 6-phosphate isomerase  GAB2  growth-factor-receptor-bound protein 2-associated binding protein 2  GALT  gut-associated lymphoid tissue  GMP  granu locyte-macrophage progenitor  GPCR  G protein-coupled receptor  GPI  glycosylphosphatidylinositol  GPI-PLC  glycosylphosphatidylinositol-specific phospholipase C  GPI-PLD  glycosylphosphatidylinositol-specific phospholipase D  H&E  hematoxylin and eosin  HBSS  Hanks’ balanced salt solution  HSC  hematopoietic stem cells  HTRAI  HtrA serine peptidase I ix  i.n.  intranasal  i.p.  intraperitoneal  i.v.  intravenous  lg  immunoglobulin  IL  interleukin  IMDM  Iscove’s modified Dulbecco’s medium  3 1P  inositol triphosphate  ITIM  immu noreceptor tyrosi ne-based inhibitor motif  LAT  linker for activation of T cells  Lin  lineage markers  1pm  liters per minute  LPS  lipopolysaccharide  LSK  LinSca-1 c-kit  LT-HSC  long-term repopulating hematopoietic stem cells  LYN  Yamaguchi sarcoma viral oncogene homolog  M cells  microfold cells  MCP  mast cell progenitor  MCPTI  mast cell protease I  MEP  megakaryocyte-erythroid progenitor  MPP  multipotential progenitors  MTG  monothioglycerol  NBF  neutral buffered formalin  NK  natural killer  obj  objective  OVA  ovalbumin  PAF  platelet activating factor  PBS  phosphate buffered saline  2 PGD  prostaglandin D 2  2 PGE  prostaglandin E 2  PH  pleckstrin homology  P1  phosphatidylinositol  P13K  phosphatidylinositol-3 kinase  PKC  protein kinase C  x  PLCy  phospholipase Cy  PP  Peyer’s patches  Prnp PrPC  prion protein (gene) cellular prion protein  PrPSC  scrapie prion protein  PSA  passive systemic anaphylaxis  PTB  phosphotyrosine-binding  PTEN  phosphatase and tensin homolog  PWMC  peritoneal wash mast cells  SCF  stem cell factor  SH2  Src-homology 2 domain  SHIP  Src homology 2-containing inositol 5’-phosphatase  SSC  side scatter  ST-HSC  short-term repopulating hematopoletic stem cells  SYK  spleen tyrosine kinase  THI  Thelperl  TH2  T helper 2  TNF  tumour necrosis factor  TSE  transmissible spongiform encephalopathies  xi  ACKNOWLEDGEMENTS I would like to thank Dr Kelly McNagny, Dr Micheal Hughes, Frann Antignano, Dr Marie-Renée Blanchet, Lori Zbytnuik, Dr Julie Nielsen, Steven Maitby, Helen Merkens, Dr Erin Drew, Dr Jami Bennett, Mike Long, Andy Johnson, Jeff Duenas, Geoff Falk, George Gill, Nicole Voglmaier, Les Rollins, Taka Murakami, Jason Rogaiski, Krista Ranta, Dr Wilf Jefferies, Dr Fabio Rossi, Dr Jurgen Kast, Dr Hermann Ziltener Dr Brock Grill, Dr Doug Carlow, Suresh Chand, Dr Stephane Corbel, Bernhard Lehnertz, Aija White, Kurtis Wall, Mike Williams, Kenny and Wax-it histology, Dr Michael Rudnicki, Pearl Campbell, Dwayne Ashman, Alison Hirukawa, Jing Yang, Dr David Westaway, Dr Neil Cashman, and Dr Colby Zaph. Further, I would like to thank my committee, Dr Pamela Hoodless, Dr John Schrader, and Dr Gerald Krystal, for their supervision and advice. Finally, I would like to thank the Natural Sciences and Engineering Research Council of Canada, the University of British Columbia Faculty of Medicine, and Roman M. Babicki for financial support.  xii  DEDICATION This work is dedicated to my parents.  XIII  CO-AUTHORSHIP STATEMENT Chapter 2. D. James Haddon designed and performed research, analyzed and interpreted data, and wrote the manuscript; Frann Antignano and Michael R. Hughes designed and performed research, analyzed and interpreted data, and edited the manuscript; Marie-Renée Blanchet and Lori Zbytnuik performed research; Gerald Krystal and Kelly M. McNagny analyzed and interpreted data, and edited the manuscript. Chapter 3. D. James Haddon designed and performed research, analyzed and interpreted data, and wrote the manuscript; Michael R. Hughes designed and performed research, analyzed and interpreted data, and edited the manuscript; Frann Antignano performed research; David Westaway and Neil R. Cashman contributed vital new reagents; and Kelly M. McNagny analyzed and interpreted data, and wrote the manuscript.  xiv  CHAPTER 1. INTRODUCTION 1.1 MAST CELLS 1.1.1 Development Mast cells arise from hematopoietic stem cells (HSC) in the bone marrow, circulate in the blood as committed precursors, and infiltrate connective and mucosal tissues, where they complete differentiation into mature mast cells. Committed mast cell progenitors were recently identified in adult bone marrow and are thought to be derived directly from multipotential progenitors (MPP) (Chen, Grimbaldeston, et al, 2005) (Figure 1.1). In mice, mast cells are generally grouped into two subsets, connective tissue (CTMC) and mucosal type mast cells (MMC), based on their tissue of origin, morphology, staining characteristics, and biochemistry (Metcalfe, Baram, and Mekori, 1997; WeIle, 1997). CTMC can be found in the skin, peritoneal cavity, and central nervous system (CNS), while MMC mainly reside in the intestinal and urogenital tracts. Both mast cell subsets tend to be located in close proximity to nerves and blood vessels. Figure 1.1 Proposed model of hematopoiesis Multipotent, self-renewing long-term repopulating HSC (LT-HSC) can give rise to all of the differentiated blood cell lineages. They first form short-term repopulating HSC (ST-HSC), followed by multipotential progenitors (MPP), with limited and virtually absent self-renewal capacity, respectively. MPP in turn form common lymphoid and myeloid progenitors (CLP and CMP) or committed mast cell progenitors (MCP). It is unclear whether CMP can also form MCP. Adapted from (Chen, Grimbaldeston, et al, 2005).  I  NK cell  TceH  B cell  I LT-HSC  MEP Erythroid  2  Several cytokines promote or enhance the formation of mast cells in vitro and in vivo, including stem cell factor (SCF), IL-3, tumour necrosis factor (TNF), and lL-6. The requirement for c-kit/SCF signalling in mast cell development in vivo is evidenced by the fact that KitW and KitI&’ mice, with mutations in c-kit and SCF, respectively, are mast cell-deficient (Kitamura, and Go, 1979). Conversely, administration of recombinant SCF induces mast cell proliferation in vitro and in vivo (Maurer, Echtenacher, et al, 1998; Tsai, Shih, et al, 1991; Tsai, Takeishi, et al, 1991). SCF is a cytokine that is expressed in two forms, membrane bound and soluble, which both induce mast cell proliferation and act as growth factors for hematopoietic stem cells and progenitors (Roskoski, 2005). Macrophages and fibroblasts are the best known sources of SCF (Roskoski, 2005). IL-3 supports the growth of bone marrow derived mast cells (BMMC) in vitro, resulting in a homogenous population (>90%) of mast cells after four weeks in culture (IhIe, Keller, et al, 1983; Chiu, and Burrall, 1990). Further, administration (i.p.) of recombinant IL-3 induces an increase in mast cell numbers in the spleen, lymph nodes, and intestine (Abe, Sugaya, et al, 1993). The importance of TNF in mast cell development is illustrated by the fact that TNF-deficient mice have a 50% reduction in peritoneal mast cells and TNF-deficient bone marrow produces drastically reduced numbers of mast cells in vitro (Wright, Bailey, et al, 2006). Recombinant TNF also stimulates mast cell colony formation in vitro (Gounaris, Erdman, et al, 2007; Hu, Zhao, and Shimamura, 2007). IL-6 promotes mast cell development from mixed cultures, likely via a secondary mediator such as prostaglandin E (Hu, Zhao, and Shimamura, 2007). Since stimulated mast cells release both IL-6 and TNF, this suggests a positive feedback  3  system inducing mast cell expansion (Gordon, and GaIli, 1990; Plaut, Pierce, et al, 1989). 1.1.2 Activation and degranulation Mast cells are immune effector cells known for releasing their cytoplasmic granules when IgE antibodies, bound to the mast cell’s FcsRI receptors, are crosslinked by multivalent, exogenous antigen. Mast cells can also be induced to degranulate, independently of FcERI crosslinking, by inducing calcium mobilization with calcium ionophore (A23187) (Huber, Helgason, et al, 1998a). During degranulation, mast cells release stored inflammatory and immunomodulatory molecules including cytokines, chemokines, histamine, proteoglycans, and neutral proteases (reviewed in Galli, Grimbaldeston, and Tsai, 2008). In addition to the release of preformed mediators, stimulated mast cells synthesize and release a number of cytokines and lipid mediators, including IL-4, IL-5, prostaglandins, and arachidonic acid (Metcalfe, Baram, and Mekori, 1997). More recently, monomeric IgE, in the absence of crosslinking antigen, was found to induce cytokine secretion and survival in cultured mast cells, suggesting that binding IgE is more than passive presensitization (Kalesnikoff, Huber, et al, 2001). In addition to activation-dependent mediator secretion, mast cells constitutively secrete mast cell protease I (MCPTI), CCL2, and HtrA serine peptidase 1 (HTRAI), and when stimulated, they shed surface proteins including c-kit (Brown, Knight, et al, 2003; Cruz, Frank, et al, 2004; Gilicze, Kohalmi, et al, 2007). This could represent a mechanism to “soak up” nearby SCF, in turn reducing mast cell proliferation.  4  FcERI stimulation initiates a number of downstream signalling events, including phosphatidylinositol-3 kinase (P13K) activation and calcium flux (Gilfillan, and Tkaczyk, 2006) (Figure 1.2). Briefly, upon FcRl aggregation Yamaguchi sarcoma viral oncogene homolog (LYN), Fyn proto-oncogene (FYN), and spleen tyrosine kinase (SYK) kinases are activated. LYN and SYK phosphorylate linker for activation of T cells (LAT), which is bound by the Src-homology 2 (SH2) domain of phospholipase Cy (PLCy) (Gilfillan, and Tkaczyk, 2006). Active PLCy cleaves P1-4,52 forming 1P P , which binds to the lP3 receptor on the endoplamic reticulum inducing 3 release of calcium stores, and diacylglycerol (DAG), which activates the protein kinase C (PKC) pathway. Increased celluar calcium and PKC activity induce restructuring of microtubules and microfilaments, which in turn induce granule fusion to the plasma membrane (Gilfillan, and Tkaczyk, 2006). An alternative mechanism for PLC’ activation is that FYN phosphorylates growth-factor-receptor-bound protein 2-associated binding protein 2 (GAB2), which interacts with and activates P13K.  5  a  FcERI  LAT  Degranulation  Figure 1.2 Schematic of FceRl signalling Upon FcsRl aggregation (only a single FcERI receptor is shown) LYN, FYN, and SYK kinases are activated. LYN and SYK phosphorylate LAT, which activates PLCy. Alternatively, FYN can phosphorylate GAB2, activating P13K, which in turn activates PLC’. Active PLC’ cleaves P1-4,5-P 2 forming lP , which induces release of calcium 3 stores, and diacylglycerol (DAG), which activates PKC. Increased calcium levels and PKC activity induces granule release.  6  11.3 In vivo models of mast cell function Investigations of mast cell physiology in vivo have been facilitated through use of mast cell deficient mice, including KitW and  KitW  mice. Kitsh mice have  a chromosomal inversion, disrupting the 5’ regulatory sequences of Kit (Nagle, Kozak, et al, 1995), rendering adult mice mast cell-deficient, while other hematopoietic lineages (including basophils) are present at similar levels to Kit’ liffermates (Grimbaldeston, Chen, et al, 2005). Reconstituting Kitv( mice with Kit’ BMMC selectively repairs the mast cell deficiency of both CTMC and MMC,  while all other hematopoietic lineages are host-derived (Grimbaldeston, Chen, et al, 2005; Nakano, Sonoda, et al, 1985) (Figure 1.3). Further, if KitV/ mice are reconstituted with BMMC from knockout mice, they can be used to study the role of the gene of interest in mast cells in vivo (Hua, Kovarova, et al, 2007; Mallen-St Clair, Pham, et al, 2004; Nakae, Ho, et al, 2007). This approach is especially attractive when applied to knockout mice with phenotypes that affect many cell types, in that it eliminates bystander effects and allows investigation of the role of the gene of interest specifically in mast cell function (i.e. mast cell autonomous effects). Additionally, it is important to study mast cells in vivo, as mast cell physiology is especially dependent upon their microenvironment. Kit WJWV mice have a number of defects that suggest Kit¼ mice are superior  for in vivo investigations of mast cells. They include that KitwM mice suffer from mast cell-independent defects such as macrocytic anemia, sterility, spontaneous dermatitis, and intestinal abnormalities that are not observed in Kit mice (Grimbaldeston, Chen, et al, 2005). Recently a number of defects, namely  7  splenomegaly, neutrophilia, thrombocytosis, and cardiomegaly, were reported in KitI18h  mice (Nigrovic, Gray, et al, 2008). Our laboratory has preliminary data  suggesting the neutrophilia is reversed by reconstitution of Kit mice with mast cells. Further research will be required to determine whether the other Kit’ defects are also secondary to mast cell deficiency, and ultimately which of the Kit’ sh/W-sh  and K1tWMIV strains is most appropriate for investigating mast cells in vivo. Over  20 known genes are reversed by the W-sh inversion, including platelet-derived growth factor receptor (Pdgfra) (Nigrovic, Gray, et al, 2008). Further, one of the inversion’s breakpoints disrupts Corin, a cardiac transmembrane serine protease (Nigrovic, Gray, et al, 2008). The effects of this disruption will need to be investigated further, although expression of Pdgfra is at normal levels (Nigrovic, Gray, et al, 2008). Interestingly, Corin knockout mice suffer from cardiomegaly, which is also observed in Kit’ 6 mice and may be caused by disruption of Corin (Nigrovic, Gray, et al, 2008).  8  Knockout  Knockout BMMCKjtWS’t  Wild-type  lxi 0 BMMC/mouse  >  4 weeks  i.v. inject  Kitt1msh  Wild-type BMMCKitWsh1Ish  12 wes  Figure 1.3 Reconstitution of KItMsI7 mice to study gene function in mast cells specifically KIt81  mice are injected with I x i0 7 BMMC derived from wild-type or knockout  mice. After 12 weeks of engraftment, most mast cell compartments are reconstituted to wild-type levels and the mice can be subjected to mast cell-dependent immune reactions or disease models.  9  1.1.4 Mast cells and disease The physiological role of mast cells is unclear, with suggested roles including host defense and tissue repair (Echtenacher, Mannel, and Hultner, 1996; lerna, Scales, et al, 2008; Maurer, Theoharides, et al, 2003). Inappropriate or excessive activation of mast cells can result in pathological conditions, including allergic asthma and anaphylaxis. Many diseases disrupt mast cell homeostasis, resulting in mast cell hyperplasia. 1.1.4.1 Allergic asthma Allergic asthma is a complex, chronic inflammatory disease of the airways and lungs. Symptoms of allergic asthma include eosinophil recruitment, airway hyperresponsiveness (AHR), airway inflammation, mucus hyperplasia, and airway remodelling (Oh, Zheng, et al, 2007). Mast cell hyperplasia of the airway epithelia and smooth muscle is a common feature of asthma (Boyce, 2003). Investigations with Kit” ”’ and Kit’ mast cell-deficient mice revealed that mast cells 1 contribute to  multiple  features  of allergic asthma,  including  airway  hyperresponsiveness (AHR), chronic lung inflammation, airway remodelling, and mucus hyperplasia (Nakae, Ho, et al, 2007; Yu, Tsai, et al, 2006). Histamine, a biogenic amine that is preformed and stored in mast cell’s granules, and ), a lipid compound released by mast cells upon activation, 2 prostaglandin D 2 (PGD are both potent inducers of bronchoconstriction and likely mediate mast cell dependent AHR (Metcalfe, Baram, and Mekori, 1997). In addition to histamine and , mast cells produce cytokines, including IL-5, IL-6, and TNF, which can 2 PGD increase asthma pathology. Mast cells produce lL-5, which activates and increases  10  production of eosinophils, well-known effector cells in asthma (Takatsu, Kouro, and Nagai, 2009). Mast cells also release high levels of pre-formed IL-6 during degranulation, and IL-6 has been shown to increase asthma pathology through stimulation of effector-type T cells in asthma (Doganci, Eigenbrod, et al, 2005). Finally, mast cell-derived TNF was definitively shown to enhance lymphocyte recruitment, AHR, and TH2 cytokine production in allergic asthma through reconstitution experiments of Kit’ mice with Tnf’ BMMC (Nakae, Ho, et al, 2007). These studies illustrate the importance of mast cells and their secreted mediators in asthma pathogenesis. 1.1.4.2 Anaphylaxis Anaphylaxis is a type I hypersensitivity reaction mediated by IgE, FceRl, mast cells, histamine, and platelet activating factor (PAF) (Finkelman, 2007). It is primarily provoked when IgE antibodies, bound to FcERI receptors, are crosslinked by multivalent exogenous allergen, inducing degranulation. Symptoms of anaphylaxis include vascular permeability, hypotension, tachycardia, hypothermia, and mortality. The requirement for mast cells is illustrated by the fact that K1tWMFV and Kit’’ mast cell-deficient mice are resistant to FcERI-mediated anaphylaxis (Martin, Ando, et al, 1993; Zhou, Xing, et al, 2007). While mast cells are the primary effector cells in FcERI-mediated anaphylaxis, other cell types play a role in its pathology. For example, IL-4/IL-1 3 treatment increases the severity of anaphylaxis by enhancing the responsiveness of target cells, likely vascular endothelial cells, to the vasoactive mediators released during anaphylaxis (reviewed in Finkelman, Rothenberg, et al, 2005; Finkelman, 2007).  11  1.1.4.3 Mast cell homeostasis and hyperplasia Little is known about normal mast cell homeostasis, including what factors contribute to mast cell progenitor production in the bone marrow (Shelburne, and Ryan, 2001). Homeostasis of tissue mast cells must incorporate both positive signals, recruiting or expanding new mast cells, and negative signals, preventing over-accumulation. Positive signals may include mast cell growth factors, such as SCF or IL-3, as administration of either one induces an elevation in mast cell numbers in vivo (Abe, Sugaya, et al, 1993; Maurer Echtenacher, et al, 1998; Tsai, Shih, et al, 1991). Interestingly, injection of donor mast cells suppresses the recruitment of host mast cell progenitors to the peritoneum that is typically observed following local mast cell ablation (Kanakura, Kuriu, et al, 1988). This could be due to limiting amounts of growth factors, such as SCF, or negative signals arising from resident mast cells. Mast cell hyperplasia is observed in a wide variety of conditions, including chronic inflammation, fibrotic disorders, wound healing, neoplastic tissue transformation, and nematode (Trichinella spiralis) infection (reviewed in Bischoff, and SeIlge, 2002). lL-3 is involved in mast cell hyperplasia, as either anti-IL-3 antibodies or deletion of the gene encoding IL-3 suppress helminth-induced intestinal mastocytosis (Lantz, Boesiger, et al, 1998; Madden, Urban, et al, 1991). IL 3’s effects on mast cells appear to be specific to inflammatory hyperplasia, since JJ3 mice have normal numbers of tissue mast cells at steady state (Lantz, Boesiger, et al, 1998).  12  1.2 P13K AND SHIPI 1.2.1 P13K signalling A variety of immune cell receptors, including growth factor, cytokine, antibody, and inflammatory mediator receptors, activate phosphatidylinositol-3 kinase (P13K) signalling and, in turn, induce cell proliferation, survival, migration and differentiation (reviewed in Hawkins, Anderson, et al, 2006) (Figure 1 .4A). In mast cells, FcERI and c-kit receptors activate heterodimeric class IA PI3Ks, while G protein-coupled receptors (GPCR) activate class lB PI3Ks. Class IA P13K are tissue-specific regulators of mast cell homeostasis, as loss of the p85a regulatory subunit caused selective loss of gastrointestinal mast cells, while loss of the pllOô catalytic subunit resulted in reduced numbers of some dermal mast cells (Ali, Bilanclo, et al, 2004; Fukao, Yamada, et al, 2002). A role in mast cell homeostasis appears to be limited to class IA Pl3Ks, as PI3K’{’ (class IB) mice have normal tissue mast cell numbers (Laffargue, Calvez, et al, 2002). The P13K pathway also amplifies FcERI-mediated degranulation, since loss of either pllOô or PI3Ky reduced mast cell degranulation in vitro, however degranulation of p85a’ mast cells was unaffected. Accordingly, mice lacking pllOô or PI3Ky suffered less severe anaphylaxis reactions, while anaphylaxis was not affected in p85a” mice (Fukao, Yamada, et al, 2002). The P13K pathway also enhances mast cell cytokine production, as mast cells lacking pllOô exhibited decreased TNF and IL-6 secretion in vitro (Ali, Bilancio, et al, 2004). These studies highlight the functional specificity of members of the P13K family in mast cell homeostasis and activation. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that acts 13  through hydrolysis of the P13K product PI-3,4,5-P , forming Pl-4,5-P 3 , inhibiting 2 downstream activation of the P13K pathway (Figure 1 .4A). It is unique from the Src homology 2-containing inositol 5’-phosphatases SHIPI and SHIP2 in that it is widely expressed throughout the body, whereas SHIPI and SHIP2 expression is limited to specific tissues (Corns, Horan, et al, 2009). Further, PTEN activity is primarily regulated through inactivation via extracellular stimuli and in contrast, SHIPI and SHIP2 are relatively inactive until recruited to the inner leaflet of the membrane (Corns, Horan, et al, 2009). PTEN, SHIPI, and SHIP2 are expressed by mast cells and each participates in negatively regulating P13K-mediated activation and cytokine secretion in this cell type. Interestingly, while SHIPI and SHIP2 knockout mast cells show increased degranulation in vitro, PTEN knockdown mast cells do not (Furumoto, Brooks, et al, 2006). Whether this is related to functional specificities of these phosphatases, or differences in experimental systems (PTEN knockdown was perforrned in human cord blood-derived mast cells) remains to be determined.  14  Figure 1.4 P13K Signalling schematic and the domain structure of SHIPI (A) A simplified schematic of P13K signalling. Class IA and Class lB P13K are heterodimeric enzymes consisting of catalytic (blue) and regulatory subunits (pink). The regulatory subunit of Class IA P13K binds to tyrosine-phosphorylated motifs (YX)(M) of receptors, including the receptor tyrosine kinase c-kit, and adaptor proteins thus recruiting the catalytic subunit to the inner leaflet of the plasma membrane. Similarly, Class lB P13K bind f y G-protein subunits (purple), which are 3 activated by G protein-coupled receptors (GPCR) (orange). At the inner leaflet, P13K catalyze the formation of the second messenger P1-3,4,5-P 3 from Pl-4,5-P , which in 2 turn recruits pleckstrin-homology (PH) domain-containing P13K effectors (green), including AKT/PKB, BTK and ITK. Phosphatase and tensin homolog (PTEN) and SHIPI remove phosphate groups from PI-3,4,5-P , reducing downstream activation. 3 (B) The structure of SHIP1 consists of an N-terminal Src-homology 2 domain (SH2), phosphatase domain (phosphatase), tyrosine phosphorylation sites (NPXY) that interact with SH2 or PTB domains, and C-terminal proline-rich region that interact with SH3 domains.  15  A Class IA P13K  Class lB P13K  Metabolism Growth Proliferation  Survival Differentiation Migration  B SH2  Phosphatase  NPXY  NPXY  NI Proline-rich  16  1.2.2 SHIP Src homology 2-containing inositol 5’-phosphatase I (SHIP) inhibits immune receptor signaling through hydrolysis of the P13K product Pl-3,4,5-P , forming P1-3,43 2 (Figure 1.4B). Evidence suggests that SHIP1 also acts as a signalling adaptor P between FcyRllb and  62 p dok  with its SH2 domains binding phosphotyrosine in the  immunoreceptor tyrosine-based inhibitor motif (ITIM) of FcyRllb and its phosphotyrosine residues interacting with the phosphotyrosine-binding (PTB) domain of  62 p dok  (Tamir, Stolpa, et al, 2000). SHIPI is a repressor of activation,  survival, and/or proliferation in T cells, B cells, macrophages, NK cells, neutrophils, and mast cells (Gardai, Whitlock, et al, 2002; Huber, Helgason, et al, 1998a; Liu, Oliveira-Dos-Santos, et al, 1998; Rauh, Ho, et al, 2005; Tarasenko, Kole, et al, 2007; Trotta, Parihar, et al, 2005). While SHIPI is not required for mast cell differentiation,  Ship1 mast cells exhibit increased FcERI-mediated degranulation and cytokine secretion compared to Ship1’ mast cells (Huber, Helgason, et al, 1998a; Kalesnikoff, Baur, et al, 2002). Further, stimulation with stem cell factor (SCF) or IgE without crosslinking antigen induces aberrant degranulation in Shipl’, but not  Ship1’, mast cells (Huber, Helgason, et al, 1998a; Huber, Helgason, et al, 1998b). SHIPI activity appears to be controlled through modulation of protein levels, subcellular localization, and post-translational modifications. Macrophages upregulate SHIPI protein levels ten-fold following LPS treatment (Sly, Rauh, et al, 2003). SHIP1 contains an SH2 domain that can bind phospho-tyrosine residues, motifs that can be tyrosine phosphorylated and bound by SH2 or PTB domains, and proline-rich regions that can be bound by SH3 domains (Figure 1.4B). These  17  modifications and domains allow SHIPI recruitment to the inner leaflet of the plasma membrane upon stimulation via interaction with transmembrane proteins and associated adaptors (Sly, Rauh, et al, 2003). Shipl’ mice suffer from excessive granulocyte and macrophage production, profound splenomegaly, extramedullary hematopoiesis, massive myeloid infiltration of the lungs, osteoporosis, wasting, and a shortened lifespan (Helgason, Damen, et al, 1998; Takeshita, Namba, et al, 2002). Further, Shipl’ mice have increased numbers of granulocyte-macrophage progenitors (GMP) in their bone marrow, which exhibit enhanced sensitivity to multiple cytokines (Helgason, Damen, et al, 1998). While SHIPI’s role in mast cell activation has been investigated in vitro, little is known regarding its function in mast cells in vivo, or the susceptibility of Shipl’ mice to mast cell-associated diseases. This was the focus of our first study. SHIPI’s modulatory role in many inflammatory cells, as well as its limited distribution, have made it an attractive target for potentially treating human disease. Recently pelorol was identified as a small molecule activator of SHIPI by screening chemical libraries purified from tropical marine sponges (Ong, Ming-Lum, et al, 2007). Structural analogs of pelorol were synthesized with increased SHIPI activating ability and have been tested in vivo (Ong, Ming-Lum, et al, 2007). Pelorol analogs were found to decrease the severity of models of endotoxemia and cutaneous anaphylaxis in mice, but it is unclear whether these effects are via SHIPI activation, since they were not investigated in Ship1 mice (Ong, Ming-Lum, et al, 2007). Future investigations of Pelorol analogs in treatment of human disease will be of great interest. 18  SHIP2 is an inositol 5’ phosphatase with 47% protein sequence identity with SHIPI in mouse. Similar to SHIPI, SHIP2 is expressed by mast cell and negatively regulates IgE-mediated mast cell degranulation and cytokine secretion (Leung, and Bolland, 2007). It is interesting to note that loss of either phosphatase results in hyper-activation of IgE/FccRl signalling and it will be interesting to create compound knockouts in the future to evaluate SHIPI and SHIP2 redundancy in mast cells.  19  1.3 PRION PROTEIN 1.3.1 Distribution of prion protein In our second study we evaluated mast cells, using microarray expression analysis, and identified prion protein (PrPC) as a potentially novel marker of mast cells. Prion protein (PrPC) is a 32kDa, N-glycosylated, glycosyl phosphatidyl inositol (GPI)-anchored sialoglycoprotein (Figure 1 .5A). In addition to neurons and glial cells, many hematopoietic cells express PrPC, such as long term repopulating HSC (LT HSC), thymocytes, dendritic cells, erythroid cells, and granulocyte precursors (Liu, Li, et al, 2001; Zhang, Steele, et al, 2006). It is currently accepted that PrPC is downregulated during granulocyte differentiation (Dodelet, and Cashman, 1998). Upon activation, PrPC is upregulated on T cells, monocytes, and dendritic cells (Dung, Giese, et al, 2000; Li, Liu, et al, 2001; Martinez del Hoyo, Lopez-Bravo, et al, 2006). Expression of PrPC by mast cells has yet to be investigated.  20  A  96 SP  OH  I  Octapeptide  6H4  I’’  N[[  B  111 GPI PP  I  —  IC  220-231 Endogenous PrPC  •••  Interaction between PrPC and PrPSC  .  -----  Conversion of PrPC  Accumulation of PrPSC  Spontaneously formed or exogenous PrPSC  > ) >  Figure 1.5 Structure of prion protein and the misfolding cascade (A) The structure of prion protein consists of an N-terminal Signal peptide (SP), hydroxyproline (OH) residues, five repeats of the octapeptide sequence P(H/Q)GGG(—/G)WGQ (octapeptide), proteolytic cleavage sites (arrows with amino acid positions above), binding site of anti- PrPc mAb clone 6H4 (6H4), disulfide bond (179-214; black lines), N-linked glycosylation (circles), putative metalloprotease cleavage site (220-231), GPI anchor (GPI), and C-terminal propeptide (PP). (B) The scrapie isoform of PrPc (PrP&; pink shapes) arises in the host through genetic mutation, or by poorly understood sporadic mechanisms. Alternatively, exogenous PrPSC can be introduced through peripheral or CNS inoculation. PrPSC interacts with PrPc (blue circles), converting it into compact, protease-resistant aggregates of PrPSC and initiating a cascade of misfolding.  21  Neuroglial (Mov), epithelial (Roy), and neuroblastoma (N2a) cell lines release PrPC at steady state and recently investigators found that activated platelets release PrPC on exosomes (Alais, Simoes, et al, 2008; Fevrier Vilette, et al, 2004; Robertson, Booth, et al, 2006). Exosomes are small membrane vesicles (—50 nm diameter), which are released from the lumen of endosomes upon fusion with the plasma membrane (Thery, Zitvogel, and Amigorena, 2002). ADAM1O and TACE, two members of the disintegrin and metalloprotease family, proteolytically cleave PrPC (at AA Ill), releasing a soluble fragment of PrPC, termed Ni (Vincent, Paitel, et al, 2001) (Figure 1.5). Radical oxygen species (ROS) also cleave PrPC (at AA 96) (McMahon, Mange, et al, 2001). A third proteolytic site has been mapped, using polyclonal antibodies recognizing synthetic peptides of PrPC, to amino acids 220231, and the cleavage is inhibited by metalloprotease inhibitors (Parkin, Watt, et al, 2004). An additional mechanism of generating soluble PrPC could be via cleavage of its GPI anchor by GPI-specific phospholipase D (GPI-PLD) or GPI-PLC (Naghibalhossaini, and Ebadi, 2006; Yoon, Park, et al, 2007). 1.3.2 Prion diseases The function of PrPC under normal circumstances is unclear, although it has been proposed to be involved in copper binding, laminin binding, and calcium flux (reviewed in Linden, Martins, et al, 2008). PrPCs role as the causative agent in the transmissible spongiform encephalopathies (TSE), including scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease is more established. TSE are incurable, fatal, neurodegenerative diseases, thought to be caused by accumulation and aggregation of the scrapie isoform of PrPC (PrP) in  22  the brain. The prevalent hypothesis is that PrPSC converts the cellular isoform (PrPC) into compact, protease-resistant aggregates of PrPD, which ultimately form plaques in the brain (reviewed in Linden, Martins, et al, 2008) (Figure 1 .5B). 1.3.2.1 Transmission of the scrapie isoform of prion protein The mechanism by which PrPSC reaches the brain after entering the body, including the cell types involved, is poorly understood. There is a long latency period between introduction of infectious material and clinical manifestation of prion disease, likely because PrPS must first reach the CNS after entering the body at a peripheral site, and then spread infectivity within the CNS. This latent period may offer a therapeutic window in which to interfere with pathogenesis. Contagion of the prion diseases occurs when PrPSC is ingested and absorbed in the gastrointestinal tract. In vitro experiments suggest that microfold (M) cells transcytose PrP&, providing entrance from the lumen of the intestine (Aguzzi, Heppner, et al, 2003). Shortly after ingestion, PrPSC accumulates in the Peyer’s patches (PP) of the distal ileum and in the spleen (reviewed in Aguzzi, Heppner, et al, 2003). Follicular dendritic cells (FDC), present in the PP, are crucial to pathogenesis, since mice lacking FDC have delayed PrPS0 transmission to the brain and reduced susceptibility to disease (Prinz, Montrasio, et al, 2002). FDC are non hematopoietic in origin, non-migratory, and function in presenting antigen bound in immune complexes (Kosco-Vilbois, 2003). Transmission of PrPSC from the gut associated lymphoid tissue (GALT) to the brain is thought to proceed along the nerves enervating the gut (McBride, Schulz-Schaeffer, et al, 2001). Tnf’ and Tnfr1 mice have no FDCs, but are still susceptible to disease, suggesting other cell types 23  are capable of propagating prion disease (Aguzzi, Heppner, et al, 2003). In fact, temporary depletion of hematopoietic dendritic cells delayed onset of prion disease (Cordier-Dirikoc, and Chabry, 2008). Zrch I Prnp°’° mice, named for being derived in Zurich, Switzerland, have a Neomycin resistance gene inserted in exon three of prion protein (Prnp) and are resistant to scrapie (Bueler, Fischer, et at, 1992). They are otherwise unremarkable, but have been instrumental in identifying which organs and cell types are required for scrapie pathogenesis through transplantation and adoptive transfer experiments (Bueler, Fischer, et al, 1992). A less investigated, but interesting aspect of prion disease is the role of inflammation in its pathology. Chronic inflammation (in the form of pancreatitis, hepatitis, systemic lupus erythematosus, and autoimmune diabetes) allows accumulation of PrPSC in non-lymphoid, otherwise prion-free organs (Heikenwalder, Zeller, et al, 2005). Further knowledge of the cell types involved in transmission of prion disease and the role inflammation plays in prion disease pathogenesis will be critical to developing therapeutics for the prevention and treatment of prion disease.  24  1.4 AIMS OF STUDY Overall, we aimed to discover more about the physiology of mast cells, focusing on their homeostatic regulation in vivo, their activation in vitro and in allergic disease, their gene expression patterns, and their surface antigens. In our first study, our objective was to establish the function of SHIP1, a repressor of mast cell activation, in mast cells in vivo. While SHIPI’s role in mast cell activation has been investigated  in vitro, little is known regarding its function in mast cells in vivo, or the susceptibility of Shipl’ mice to mast cell-associated diseases. Since SHIPI is expressed in all hematopoletic lineages, we utilized mast cell-deficient Kit’1M/8h mice reconstituted with Ship1” or Shipl’ bone marrow derived mast cells to eliminate bystander effects. Given that SHIP1’s repressor activity in mast cells is conserved in humans, and agonists and antagonists of SHIPI function have recently been identified and tested in vivo, our findings could have implications for treatment of mast cellassociated diseases in humans (Langdon, Schroeder, et al, 2008; Ong, Ming-Lum, et al, 2007). In our second study, our initial aim was to compare the gene expression patterns of HSC and mast cells, as our group had previously found that mast cells share a number of surface markers with HSC, including CD34, Sca-1, and c-kit. 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Dual mechanisms for shedding of the cellular prion protein. J. Biol. Chem. 12, 11170-11178.  31  Plaut, M., Pierce, J.H., Watson, C.J., Hanley-Hyde, J., Nordan, R.P., and Paul, W.E. (1989). Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature 6219, 64-67. Prinz, M., Montrasio, F., Klein, M.A., Schwarz, R, Priller, J., Odermatt, B., Pfeffer, K., and Aguzzi, A. (2002). Lymph nodal prion replication and neuroinvasion in mice devoid offolliculardendritic cells. Proc. NatI. Acad. Sci. U.S.A. 2, 919-924. Rauh, M.J., Ho, V., Pereira, C., Sham, A., Sly, L.M., Lam, V., Huxham, L., Minchinton, A.I., Mui, A., and Krystal, G. (2005). SHIP represses the generation of alternatively activated macrophages. Immunity 4, 361-374. Robertson, C., Booth, S.A., Beniac, D.R., Coulthart, M.B., Booth, T.F., and McNicol, A. (2006). Cellular prion protein is released on exosomes from activated platelets. Blood 10, 3907-3911. Roskoski, R.,Jr. (2005). Structure and regulation of Kit protein-tyrosine kinase--the stem cell factor receptor. Biochem. Biophys. Res. Commun. 3, 1307-1 31 5. Shelburne, C.P., and Ryan, J.J. (2001). The role of Th2 cytokines in mast cell homeostasis. I mmunol. Rev. 82-93. Sly, L.M., Rauh, M.J., Kalesnikoff, J., Buchse, T., and Krystal, G. (2003). SHIP, SHIP2, and PTEN activities are regulated in vivo by modulation of their protein levels: SHIP is up-regulated in macrophages and mast cells by Iipopolysaccharide. Exp. Hematol. 12, 1170-1181. Takatsu, K., Kouro, T., and Nagai, Y. (2009). Interleukin 5 in the link between the innate and acquired immune response. Adv. Immunol. 191-236. Takeshita, S., Namba, N., Zhao, J.J., Jiang, Y., Genant, H.K., Silva, M.J., Brodt, M.D., Helgason, C.D., Kalesnikoff, J., Rauh, M.J. et a!. (2002). SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts. Nat. Med. 9, 943-949. Tamir, I., Stolpa, J.C., Helgason, C.D., Nakamura, K., Bruhns, R, Daeron, M., and Cambier, J.C. (2000). The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRllB signals in B cells. Immunity 3, 347-358. Tarasenko, T., Kole, H.K., Chi, A.W., Mentink-Kane, M.M., Wynn, T.A., and Bolland, S. (2007). T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Thl/Th2 and cytotoxic responses. Proc. NatI. Acad. Sci. U. S. A. 27, 11382-11387. Thery, C., Zitvogel, L., and Amigorena, S. (2002). Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 8, 569-579.  32  Trotta, R., Parihar, R., Yu, J., Becknell, B., Allard, J.,2nd, Wen, J., Ding, W., Mao, H., Tridandapani, S., Carson, W.E., and Caligiuri, M.A. (2005). Differential expression of SHIPI in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation. Blood 8, 3011-301 8. Tsai, M., Shih, L.S., Newlands, G.F., Takeishi, T., Langley, K.E., Zsebo, K.M., Miller, H.R., Geissler, E.N., and Galli, S.J. (1991). The rat c-kit ligand, stem cell factor, induces the development of connective tissue-type and mucosal mast cells in vivo. Analysis by anatomical distribution, histochemistry, and protease phenotype. J. Exp. Med. 1, 125-1 31. Tsai, M., Takeishi, T., Thompson, H., Langley, K.E., Zsebo, K.M., Metcalfe, D.D., Geissler, E.N., and Galli, S.J. (1991). Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc. Nati. Acad. Sci. U. S. A. 14, 6382-6386. Vincent, B., Paitel, E., Saftig, P., Frobert, Y., Hartmann, D., De Strooper, B., Grassi, J., Lopez-Perez, E., and Checler, F. (2001). The disintegrins ADAMIO and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein. J. Biol. Chem. 41, 37743-37746. Welle, M. (1997). Development, significance, and heterogeneity of mast cells with particular regard to the mast cell-specific proteases chymase and tryptase. J. Leukoc. Biol. 3, 233-245. Wright, H.V., Bailey, D., Kashyap, M., Kepley, C.L., Drutskaya, M.S., Nedospasov, S.A., and Ryan, J.J. (2006). lL-3-mediated TNF production is necessary for mast cell development. J. Immunol. 4, 2114-2121. Yoon, H.J., Park, S.W., Lee, H.B., Pm, S.Y., Hooper, N.M., and Park, H.S. (2007). Release of renal dipeptidase from glycosylphosphatidylinositol anchor by insulintriggered phospholipase C/intracellular Ca2+. Arch. Pharm. Res. 5, 608-61 5. Yu, M., Tsai, M., Tam, S.Y., Jones, C., Zehnder, J., and Galli, S.J. (2006). Mast cells can promote the development of multiple features of chronic asthma in mice. J. Clin. Invest. 6, 1633-1641. Zhang, C.C., Steele, A.D., Lindquist, S., and Lodish, H.F. (2006). Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal. Proc. NatI. Acad. Sci. U.S.A. 7, 2184-2189. Zhou, J.S., Xing, W., Friend, D.S., Austen, K.F., and Katz, H.R. (2007). Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. Med. 12, 2797-2802.  33  CHAPTER 2. SHIPI IS A REPRESSOR OF MAST CELL HYPERPLASIA, CYTOKINE PRODUCTION, AND ALLERGIC INFLAMMATION IN WVO’ 2.1 INTRODUCTION The Src homology 2-containing inositol 5’-phosphatase, SHIPI, inhibits immune receptor signaling by hydrolysis of the phosphatidylinositol-3 kinase (P13K) product , forming P1-3,4-P 3 Pl-3,4,5-P . SHIPI represses the activation, survival, or 2 proliferation of T cells, B cells, macrophages, NK cells, neutrophils, and mast cells (Gardai, Whitlock, et al, 2002; Huber, Helgason, et al, 1998a; Liu, Oliveira-Dos Santos, et al, 1998; Rauh, Ho, et al, 2005; Tarasenko, Kole, et al, 2007; Trotta, Parihar, et al, 2005). Shipf’ mice are viable and fertile, but suffer from excessive granulocyte and macrophage numbers, profound splenomegaly, extramedullary hematopoiesis, massive myeloid infiltration of the lungs, osteoporosis, wasting, and a shortened lifespan (Helgason, Damen, et al, 1998; Takeshita, Namba, et al, 2002). Since SHIP1 is expressed in all hematopoietic cell lineages, it has been difficult to determine which cell types are responsible for given phenotypes. Mast cells are immune effector cells known for releasing their cytoplasmic granules when IgE antibodies, bound to FccRI receptors, are crosslinked by multivalent exogenous antigen. During degranulation, mast cells release inflammatory and immunomodulatory molecules, including cytokines, chemokines, lipid-derived mediators, histamine, proteoglycans, and neutral proteases (reviewed in Galli, Grimbaldeston, and Tsai, 2008). Although SHIPI is not required for mast cell  A version of this chapter has been accepted for publication. Haddon, D.J., Antignano, F., Hughes, M.R., Blanchet, M.R., Zbytnuik, L., Krystal, G. and McNagny, K.M. (2009) SHIP1 is a repressor of mast cell hyperplasia, cytokine production, and allergic inflammation in vivo. J Immunol.  34  differentiation, it functions as a repressor of mast cell degranulation and cytokine secretion in vitro. Specifically, Shipl’ mast cells exhibit increased degranulation and cytokine secretion compared to Ship  mast cells in response to lgE crosslinking  (Huber, Helgason, et al, 1998a; Kalesnikoff, Baur, et al, 2002). Further, stimulation with stem cell factor (SCF) or IgE without crosslinking antigen induces aberrant degranulation in Shipl’, but not Ship1’, mast cells (Huber, Helgason, et al, 1998a; Huber, Helgason, et al, 1998b). Mast cells contribute to a wide variety of inflammatory diseases, including anaphylaxis and allergic asthma (reviewed in Okayama, Ra, and Saito, 2007 and Grimbaldeston, Metz, et al, 2006). Anaphylaxis is a type I hypersensitivity reaction primarily provoked by allergen-induced mast cell degranulation. Symptoms of anaphylaxis include vascular permeability, tachycardia, hypothermia, and mortality. Allergic asthma is a complex, chronic inflammatory disease of the airways and lungs. Naïve Ship1 mice have symptoms of allergic asthma under steady state conditions, including severe airway inflammation, mucus hyperproduction, and symptoms of airway remodeling, but the contribution of Shipl’ mast cells to these symptoms is unknown (Oh, Zheng, et al, 2007). Mast cell physiology can be studied in vivo by using mast cell-deficient mice, which lack mast cells due to a chromosomal inversion that disrupts the 5’ regulatory sequences of Kit (Nagle, Kozak, et al, 1995; Nigrovic, Gray, et al, 2008). Reconstituting Kit mice with Kit’ bone marrow derived mast cells (BMMC) selectively repairs their mast cell deficiency (Grimbaldeston, Chen, et al, 2005). Further, if KitW/ mice are reconstituted with BMMC from knockout mice, they 35  can be used to study gene function in mast cells in vivo (Hua, Kovarova, et al, 2007; Mallen-St Clair, Pham, et al, 2004; Nakae, Ho, et al, 2007). While SHIPI’s role in immune receptor signaling has been established in mast cells in vitro, little is known about SHIPI’s function in mast cells in vivo, or the susceptibility of Shipl’ mice to mast cell-associated diseases. In this study, we found that Ship1 mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened susceptibility to anaphylaxis. Further, by reconstituting mast cell deficient Kit’’ mice with Ship1 or Shipf’ BMMC, we established that loss of SHIPI in the mast cell lineage was responsible for these defects (with the exception of increased IL-5). In addition, we found that mice reconstituted with Shipl’ mast cells suffered worse allergic asthma pathology than those reconstituted with Ship 1’ mast cells. In summary, our data show that SHIP1 is a repressor of allergic inflammation and mast cell hyperplasia in vivo, and that SHIPI exerts these effects specifically in mast cells.  36  2.2 RESULTS 2.2.1 Shipl 4 mice have mast cell hyperplasia in multiple tissues We suspected the chronic inflammatory phenotype of Ship1 mice could be due in part to increased tissue mast cell numbers. With this in mind, we enumerated the tissue mast cells of Ship 1’ and Ship1 mice and found significantly higher numbers in the spleen, lung, ileum, mesentery, and back skin of Shipl’ mice (Figure 2.IA). We also found Shipl’ mice had higher numbers of colon mast cells, although the counts were more variable than for other tissues assessed. Similarly, Shipl’ mice had over two times more peritoneal mast cells than wild type controls (Figure 2.IB). The frequency of peritoneal mast cells was similar between Shipl’ and wild type mice, reflecting a greater number of total peritoneal cells in Ship1 mice than Ship1 mice (6.8 SD 2.8 vs. 2.0 SD 0.13 cells (x 106), P< 0114). Thus, Shipl’ mice have mast cell hyperplasia in the spleen, lungs, colon, ileum, mesentery, peritoneal cavity, and back skin.  37  A  Spleen  Lung  100  Back skin  3 P=.0009  80  200 P= 0005  ..  60  100 40  •.•  1 .. .  20 E E  0  0  ...  .  P=.0909  •  1.0  U  Mesentery 8  P=.0003  80  6 4  40  0.0  2 .  .  F  Peritoneal wash  104:1  0  2.05% (SD 0.58)  U,1 1 (  6 P=.0037  . •  4  102 a)  •:•  2 •.  —  C a)  101  0  S..——  a)  0 I-  C.) C’) Co  .  .  I  F  B U, 0  .  ••.•  0  F  .. .  .  20  (I) a)  .  .ee  .  a) V  .  p<.0001  .  60  0.5  a)  .‘ •e. .  ••.••  100  1.5  •  . ••  Ileum  Colon  C) a) a)  .  P=.0622  150  2  101 C)  Shipi-!  Shipl+/+  .  0 i0  0  102  i0 100 C-kit  01  io2  io  i0  Figure 2.1 Mast cell hyperplasia in multiple tissues of Shipi’ mice (A) Toluidine blue histological staining revealed that Shipf’ mice (n=11) had significantly higher numbers of metachromatic tissue mast cells than Ship 1’ controls (n=1O). (B) Shipl’ mice (n=1O) had a higher total number (graph), but similar frequency (gate statistics) of SSChu/ckituh1 peritoneal wash mast cells to  Ship 1’ mice (n9), as determined by flow cytometry. Data represent pooled results from two independent experiments.  38  2.2.2 Mast cell hyperplasia in Shipi’ mice is mast cell autonomous Since SHIPI is expressed in many hematopoietic cell types, it was unclear whether the mast cell hyperplasia we observed in ubiquitous Ship1 mice was mast cell autonomous. To answer this question, we intravenously reconstituted Kit’ mast cell deficient mice with Ship1’ or Ship1 BMMC (designated Ship 1’-BMMC and Ship1-BMMC mice) and, following 12 weeks of engraftment, found mast cell hyperplasia in the spleen, lung, colon, ileum, mesentery, and back skin of Ship1BMMC mice (Figure 2.2A). Likewise, Ship1-BMMC mice also had a greater number and frequency (0.92% SD 0.52 vs. 0.10% SD 0.13, P<.0015) of peritoneal mast cells than Ship1-BMMC controls (Figure 2.2B). Kitv  JSh  mice were mast cell deficient  in all tissues assessed, and the mast cell numbers in the tissues of B6, and SI?  mice reconstituted with wild type mast cells were similar to previous reports  (Grimbaldeston, Chen, et al, 2005). Thus, the mast cell hyperplasia observed in Shipf’ mice is a mast cell autonomous defect.  39  Figure 2.2 Mast cell hyperplasia in Shipi’ mice is mast cell autonomous KitWs  mice were injected with 1 x iO Ship1’ or Shipl’ BMMC via the tail  vein and, after 12 weeks reconstitution, the mice were sacrificed and their tissue mast cells were enumerated. (A) The number of metachromatic tissue mast cells was significantly higher in Shipl’-BMMC mice (n7) than Ship1’-BMMC mice (n=7) and, in most cases, B6 mice (n=6), while 1 Kit” ” ’ mice (n=9) were mast cell deficient, as determined by toluidine blue histological staining. (B) Similar results were observed for the total number (graph) and frequency (gate statistics) of SSChu/ckith1 peritoneal mast cells from B6 (n=3), Ship 1-BMMC (n=3), Ship1’BMMC (n=2), and KIt’’ (n=3) mice by flow cytometry. Data represent pooled results from two independent experiments.  40  A  Spleen 1500  Lung 40  P=.0033  Back skin 50  P=.0009 ••  30 1000  30  . •• 500  U  20  •. 1  E E  .  10  —•1•—  o  • •  Ileum P=.0093  80 . P=.0214  60  40  40  20  20  10 —  Colon 80  20  •  U  60  P=.0113  40  •.  0  -  Mesentery P.0051  60 •  •  40  20  •  .a  • •  U  •:  6 x  \X  ——  -  ?  / /  —  x,  1—  1.24%(SDO.23)  Peritoneal wash P=.0204  If)  0  •  7 B  •  • r.  ue.r  0.01% (SD 0.01)  • 4  2  •  101  0  —?-—— 1’  •  %X  _  •  $%  •• \  U)  KitW  B6  ..n  ••  0.92% (SD 0.52) S  3’4r.  o.1o(sDo.i3)_______ .:  •j  101 Shipl-/- BMMC  100  hiPl÷/+ BMMC  ,,,,,..  i°  101  io2  io  ioio°  101  io2  io  C-kit  41  2.2.3 Ship’ mice have mast cell-dependent systemic inflammation Since Shipl’ mast cells exhibit enhanced degranulation and cytokine secretion in response to FcsRI stimulation and aberrantly to c-kit stimulation in vitro, we investigated the levels of inflammatory cytokines in the serum of adult Shipl’ mice (Huber, Helgason, et al, 1998a; Huber, Helgason, et al, 1998b; Kalesnikoff, Baur, et al, 2002). Shipl’ mouse serum contained significantly increased levels of IL-5, lL-6, and TNF (and a small increase in CCL2) compared to Ship 1’ controls (Figure 2.3A). To determine whether Shipl’ mast cells were responsible for the increased levels of inflammatory serum cytokines observed in Ship1 mice, we evaluated the serum of Ship f’-BMMC reconstituted mice. We detected significantly higher levels of IL-6 and TNF in the serum of Ship1-BMMC mice, while IL-5 and CCL2 were only marginally increased compared to controls (Figure 2.3B). We concluded that Shipl’ mast cells induce increased serum levels of the inflammatory cytokines lL-6 and  TNF in vivo. Figure 2.3 Increased inflammatory cytokine levels in the serum of Shipi’ mice, and mice reconstituted with Ship 11 mast cells (A) Cytometric bead arrays of mouse cytokines were employed to assay the serum of both F2-Shipl’ (n=7) and F2-Ship1” (n=7) mice. Data represent pooled results from two independent experiments. (B) Cytometric bead arrays were also used to assay the serum of B6 (n=4), Ship1-BMMC (n=8), Ship1’-BMMC (n=8), and Kit sh,Wsh  (n=6) mice. Data represent pooled results from two independent experiments.  42  A  IL-6  IL-5 50  50  P=.0094  40  P=.0280  40  30 20 Q  20  10  10  .  0  •._  —•.  0  •e.,i.•  CCL2  TNF-alpha  250  50 P.4643  200 150 •I  50  .  0  40 ‘.  30  ‘  20  •  10  20  IL-5 P=.2490  10 •. •  —••I-  .••—  e  •  •e  :  •  •  • —  •  •  •  ‘ •  —— •.  100  :  20 0  :  .  “  TNF-alpha P=.0477  80 60] •  -  -f 40  •  I  204  ••  0  40  •,.  :. -_;_  80 60  CCL2 P=.1322  500 — 400  •  P=.0435 •  0  •  ••  •.•  IL-6 15  5  •  0  •  B  D C-  P=.0131  •  •  J%X  —S  43  2.2.4 Ship’ mice are hyperresponsive to passive systemic anaphylaxis Since Shipl’ mice possess mast cell hyperplasia in multiple tissues, and Shipf’ mast cells exhibit increased degranulation in response to IgE crosslinking in vitro, we suspected that Shipl’ mice would be more susceptible to anaphylaxis (Huber, Helgason, et al, 1998a). To test this hypothesis, we subjected Shipl’ mice and littermate controls to passive systemic anaphylaxis (PSA) by sensitizing them with anti-DNP IgE, challenging them with DNP-HSA the next day, and monitoring their rectal temperature over 90 minutes (Figure 2.4A,B). Interestingly, the body temperature of Shipl’ mice (prior to challenge) was on average 1°C lower than wild type controls. This may reflect low-level degranulation induced by the IgE sensitization, as Shipl’ mast cells aberrantly degranulate in response to IgE without crosslinking antigen in vitro (Huber, Helgason, et al, 1998a). More importantly, we found that Ship1 mice suffered increased mortality, more severe hypothermia (at 120, 5°C SD 0.6 vs. 3°C SD 0.7, P<.0001), and no body temperature recovery, compared to wild type controls (Figure 2.4A,B).  44  Figure 2.4 Hyperresponsive anaphylaxis in Ship 1’ mice, and mice reconstituted with Shipf’ mast cells To induce passive systemic anaphylaxis, Shipl’ (n=1O, red line) and Ship1 (n=9, blue line) mice were sensitized with anti-DNP IgE and challenged the following day with DNP-HSA. Increased mortality (A) and hypothermia (B), measured rectally, were observed in Ship1 mice. Anaphylaxis was induced and monitored in Shipi’ BMMC (n=5, red line) and Ship1’-BMMC (n=6, blue line) mice (C), as well as B6 (n=5, black line) and Kit (n=5, gray line) controls (D) as described above. Data for (C) and (D) was measured concurrently, but separated for clarity. Data in A, B, C, and D each represent pooled results from two separate experiments. (*, P<.05; Pc 01;  Pc 001). Bars represent SE.  45  A 100• .  P=.0163  60 40. I.  2O 0  I  I  15  30  B  I  I  45 60 Time (minutes)  I  -  75  -t  90  39.  * **  31  ***  29.  ****  27.  C —‘  39 **  ,37  **  ;33 m  31  D 39  -r  -r  37  -5  15  35  55  75  95  Time (minutes)  46  It was unclear whether the increased severity of anaphylaxis suffered by Shipl’ mice was due to increased mast cell activation and mast cell numbers, or reactivity of other cell types to the mediators released during the PSA challenge (reviewed in Finkelman, 2007). Therefore we subjected reconstituted Ship1-BMMC mice to PSA and found that they also suffered a much larger temperature drop than Ship BMMC mice (at t35, 7°C SD 2.2 vs. 1°C SD 2.5, P=.0037) (Figure 2.4C). Notably, unlike ubiquitous Shipl’ mice, Ship 1-BMMC mice exhibited body temperature recovery 40 minutes post-challenge. While Kit mice were resistant to PSA, B6 mice suffered a similar temperature drop to Ship 1’-BMMC mice (Figure 2.4D). In summary, we find that the increased severity of anaphylaxis experienced by Shipl’ mice is largely mast cell autonomous. 2.2.5 Allergic airway inflammation is greater in Shipl’-BMMC mice than Ship1-BMMC controls Naïve Shipl’ mice have allergic asthma-like symptoms, including severe airway inflammation, mucus hyperproduction, and symptoms of airway remodeling (Oh, Zheng, et al, 2007). To determine the role of Shipl’ mast cells in allergic asthma pathology, we induced an acute form of the disease in Ship 1’-BMMC and Ship 1’BMMC reconstituted mice (Figure 2.5A). We found that asthmatic Ship1-BMMC mice had over two times more infiltrating cells in their airways than Ship1’-BMMC mice, including increased eosinophils, which are the primary effector cells in asthma (Figure 2.5B,C). Perivascular, peribronchial, and parenchymal infiltration, as well as epithelial damage were most prominent in the lungs of asthmatic Ship 1’-BMMC mice, and clinical scores of asthmatic Ship 1’-BMMC lung sections were significantly  47  higher than Ship 1’-BMMC mice (Figure 2.5D,E). Kit’ mice suffered comparable symptoms to B6 mice, similar to a study that also used an OVA-induced (with alum adjuvant) model of allergic asthma published after we completed our asthma course (see Nakae, Ho, et al, 2007 Figure El). Ship1’-BMMC mice had a slightly reduced number of infiltrating bronchoalveolar lavage (BAL) cells compared to KitWs  Sh  mice, highlighting the importance of wild type reconstituted controls.  We concluded that Shipl’-BMMC mice suffered worse allergic asthma pathology than Ship1’-BMMC mice. Figure 2.5 Increased allergic asthma pathology in mice reconstituted with Shipi’ mast cells (A) Asthma was induced in B6 (n=l0), Ship1-BMMC (n=13), Ship1’-BMMC (n=l0), and KIt’ (n=l1) mice via two i.p. sensitizations (with alum adjuvant; days I and 7) and five intranasal (i.n.) challenges (days 21, 22, 23, 26, and 27) of OVA prior to sacrificing (Sac.) the mice (day 28). (B) Inflammation was assessed by enumerating cellular infiltrate in the BAL using flow cytometry. (C) Differentials were performed with a modified Wright-Giemsa stain; B6 (n=7), Ship1-BMMC (n=9), Ship1’-BMMC (n=6), and KitW (n=7). BAL data represent pooled results from three independent experiments, and differentials from two. (D) Selected, representative micrographs of H&E stained lungs from PBS or OVA-treated Shipl’ BMMC reconstituted mice and controls (bars  =  100 Lm). (E) Clinical scores were  assigned to each lung based on the following parameters: perivascular infiltration, peribronchial infiltration, parenchymal infiltration, and epithelial damage (maximum score of 16). Data represent pooled results from three independent experiments.  48  CD  U,  •  Shipl-/- BMMC •  •.I  •.  •.  sits  •..I  •s ‘Isi  • 5  I  •  1• 4  Shipl+/+ BMMC  B6  Kit Wsh/Wsh  Shipl-/- BMMC  Shipl+/+ OMMC  06  0  •.  S.  Clinical score  •  0  C’)  0)  m KitW55h  •  .1I  ,sh  Kit  •.•. •l  Shipi-?- BMMC  Shipl+/÷ BMMC  06  KitW5u1,  Shipl-/- BMMC  Shipl+/÷ BMMC  B6  0  • •  0  m  0  a  0  Shipl-/- BMMC  Cells/mi (x10 ) 5 0  0  Shipl+/+ BMMC  If •.:•  e4..  *1  01  Cells/mi (x10 ) 5  •  r\)  UI  0  UI  -  4-  (..  0  C’,  4— Cl) 2) p  B6;  0)  4—  1—  4—  2.3 DISCUSSION Our results show that (1) Ship1 mice have systemic mast cell hyperplasia, increased serum levels of the inflammatory cytokines IL-6, TNF, and lL-5, and increased susceptibility to anaphylaxis; (2) the above phenotypes are mast cell autonomous (with the exception of increased serum lL-5); and (3) mice reconstituted with Ship1 mast cells suffered worse allergic asthma pathology than Ship 1’ controls. Previous studies have focused on SHIPI’s function in mast cells in vitro. Here we demonstrate that SHIPI is also a repressor of mast cells in vivo, and that loss of SHIPI increases the symptoms of two mast cell associated diseases. Further, we demonstrate a cell autonomous role for SHIPI in mast cell homeostasis. Mast cell hyperplasia is observed in chronic inflammatory processes, fibrotic disorders, wound healing and neoplastic tissue transformation, but little is known about normal mast cell homeostasis (reviewed in Bischoff, and SelIge, 2002). Our results demonstrate that SHIPI is a cell autonomous repressor of mast cell hyperplasia and key player in mast cell homeostasis. Ship1 mice have increased numbers of granulocyte-macrophage progenitors (GMP) in their bone marrow, which exhibit enhanced sensitivity to multiple cytokines (Helgason, Damen, et al, 1998). In our studies, since the reconstituted BMMC were composed exclusively of mast cells and their committed precursors, and the numbers of mast cells observed in the tissues of Ship 1-BMMC mice were comparable or greater than those in ubiquitous  Shipl’ mice, we concluded that the mast cell hyperplasia observed in Ship1 mice is not due to the increased number or sensitivity of GMP. Further we found that loss of SHIPI specifically in mast cells was not sufficient to induce the excessive  50  granulocyte and macrophage numbers previously observed in the blood, spleen and lungs of Ship1 mice (data not shown) (Helgason, Damen, et al, 1998). Interestingly, we found that Shipl’ mice have elevated serum levels of the inflammatory cytokines IL-5, IL-6, and TNF compared to Ship 1’ controls. This is in agreement with a previous report that Shipl’ mice have increased serum IL-6 (Takeshita, Namba, et al, 2002). We demonstrated that the increased levels of lL-6 and TNF were due to loss of SHIPI specifically in mast cells. Since Shipl’ mast cells produce more IL-6 and TNF than wild type mast cells upon FcRl-mediated stimulation in vitro, we suspect that the increased serum levels of these cytokines arose directly from Ship1 mast cells via stimulation with endogenous lgE (Kalesnikoff, Baur, et al, 2002). Although the high level of IL-6 in the serum of Shipl “  mice was previously attributed to macrophages, here we demonstrate that Shipl’  mast cells also contribute to the increased IL-6 levels in vivo (Takeshita, Namba, et al, 2002). Further, the increased IL-6 was previously proposed to inhibit B cell, and enhance myeloid cell development, but we found the frequency of B cells and myeloid cells in the bone marrow, peripheral blood, and spleen of Ship1-BMMC reconstituted mice were similar to controls (data not shown) (Nakamura, Kouro, et al, 2004). Thus, our data suggest that increased serum lL-6 is not sufficient to cause the hematopoietic lineage skewing observed in Ship1 mice and a local increase of IL-6 in the bone marrow may be required. Our studies also suggest that the systemic mast cell hyperplasia observed in Ship1 mice is due to the high levels of IL-6 and TNF produced by Shipl’ mast cells. TNF is required for mast cell development and stimulates mast cell colony 51  formation in vitro (Gounaris, Erdman, et at, 2007; Hu, Zhao, and Shimamura, 2007). In addition, IL-6 promotes mast cell development from mixed cultures, likely via a secondary mediator such as prostaglandin E (Hu, Zhao, and Shimamura, 2007). We suspect that the systemic mast cell hyperplasia observed in Shipl’ mice is also due to Ship1 mast cells’ hypersensitivity to stimulation with SCF or IgE (without crosslinking antigen) (Huber, Helgason, et al, 1998a; Huber, Helgason, et al, 1998b). SCF and IgE induce mast cell proliferation and survival, respectively, and endogenous levels of these factors may be capable of sustaining higher levels of hypersensitive Shipl’ mast cells in vivo (Kalesnikoff, Huber, et at, 2001; Tsai, Takeishi, et al, 1991). We also found that Shipl’ mice suffer considerably more severe anaphylaxis than controls. While mast cells are the primary effector cell in the PSA model of anaphylaxis, other cell types also play a rote in its pathology. For example, IL-4 or IL13 treatment increases the severity of anaphylaxis by enhancing the responsiveness of target cells to the vasoactive mediators released during anaphylaxis (reviewed in Finkelman, 2007). We found that Ship 1-BMMC mice also suffered more severe anaphylaxis than controls, suggesting that this effect is largely mast cell autonomous. We suspect that the increased severity of anaphylaxis observed in Ship1 (and Ship 1’-BMMC) mice is due to their systemic mast cell hyperplasia and the enhanced degranulation of the Shipl’ mast cells, but have not ruled out the possibility that the increased levels of IL-6 and TNF in their serum could have also contributed to this effect (Huber, Helgason, et at, 1998a). In our studies, we also found that Ship 1-BMMC mice suffered worse allergic 52  asthma pathology than Ship1”-BMMC mice, in an acute, OVA-induced (with alum adjuvant) model of allergic asthma (Blanchet, Maltby, et al, 2007). Since Kit’ mice also developed symptoms of asthma, it made it difficult to determine to what extent Shipf’ mast cells were pro-inflammatory or instead lacked anti-inflammatory activity present in Ship 1’ mast cells. Indeed, asthmatic Ship1’-BMMC mice had lower numbers of infiltrating cells and clinical scores than KitW controls, suggesting an anti-inflammatory role for mast cells in this model. With this in mind, we believe Shipl’ mast cells also contributed to inflammation for the following reasons. First, mast cells amplify allergic asthma symptoms in a similar model of asthma (without alum adjuvant) (Williams, and Galli, 2000). Second, mast cells are hyperplasic in the lungs of asthmatic mice, and the pre-existing mast cell hyperplasia of the lungs in Ship 1-BMMC mice would likely predispose them to heightened allergic asthma pathology (Yu, Tsai, et al, 2006). Third, lL-6 and TNF, inflammatory cytokines associated with increased asthma pathology, are elevated in the serum of Shipl’-BMMC mice (Doganci, Eigenbrod, et al, 2005; Nakae, Ho, et al, 2007). Finally, Shipl’ mast cells intensified the severity of anaphylaxis, another mast celldependent inflammatory disease model. The fact that Shipf’-BMMC mice exhibit greater allergic asthma pathology than Ship1’-BMMC mice strongly suggests a mast cell autonomous role for SHIPI in allergic asthma response. The P13K pathway is involved in cytokine production and amplifying FcERI mediated degranulation in mast cells in vitro, and enhances anaphylaxis in vivo (reviewed in Gilfillan, and Tkaczyk, 2006). In mast cells, FcERI and c-kit receptors activate heterodimeric class IA PI3Ks, while GPCR, including the adenosine A3  53  receptor, activate class lB Pl3Ks. SHIPI negatively regulates P13K activation via its phosphatase activity and can also act as a signaling adaptor (Tamir, Stolpa, et al, 2000). Other investigators have reported that the loss of the p85a subunit of class IA P13K causes selective loss of gastrointestinal mast cells, but does not affect mast cell degranulation in vitro or passive systemic anaphylaxis in vivo (Fukao, Yamada, et aI, 2002). Loss of the p11O subunit of P13K (class IA) results in reductions in numbers of some dermal mast cells, degranulation, cutaneous anaphylaxis, and TNF and IL-6 production (Au, Bilanclo, et al, 2004). PI3Ky’ (class IB) cultured mast cells, on the other hand, have reduced degranulation and Pl3Ky mice experience less severe passive systemic anaphylaxis (Laffargue, Calvez, et al, 2002). The P13K inhibitor LY294002 has also been reported to reduce asthma symptoms in vivo (Duan, Aguinaldo Datiles, et al, 2005). In combination with our results, the studies outlined above indicate that SHIPI may repress p85a subunit-dependent P13K activity (class IA) during the formation or maintenance of gastrointestinal mast cells, pllOô-dependent P13K activity (class IA) in the formation or maintenance of some dermal mast cells and TNF and IL-6 production, and both PI3K’{’ (class IB) and pllOô-dependent (class IA) P13K activity in degranulation and anaphylaxis. This suggests that SHIPI’s phosphatase activity (rather than adaptor function) is central to negative regulation of these processes. It is interesting to note the breadth of SHIPI’s activity compared to the functional specificity of the P13K family, potentially making SHIPI a more extensive target for therapy. We propose that Ship1-BMMC reconstituted mice can serve as an in vivo model of hyperactive and hyperplasic mast cells, for investigation of mast cell-associated 54  diseases and immune reactions. In this study we investigated harmful roles that Shipl’ mast cells play in inflammatory diseases. It would be interesting to test whether Ship1 mast cells also have enhanced function in beneficial roles of mast cells, such as clearing peritoneal and helminth infections (Echtenacher, Mannel, and Hultner, 1996; lerna, Scales, et al, 2008). SHIPI’s repressor activity in mast cells is conserved in humans, and agonists and antagonists of SHIPI function have recently been identified and tested in vivo (Langdon, Schroeder, et al, 2008; Ong, Ming-Lum, et al, 2007). Our study suggests that SHIPI agonists could reduce the severity of anaphylaxis, allergic asthma, and other mast cell-associated diseases in humans. Given that mast cells also have a role in such diverse diseases as rheumatoid arthritis, inflammatory bowel disease, and tumor progression, and conversely aid in clearing peritoneal and helminth infections, modulators of SHIPI function in mast cells may offer a variety of therapeutic strategies to key human diseases (Echtenacher, Mannel, and Hultner, 1996; Gounaris, Erdman, et al, 2007; lerna, Scales, et al, 2008; Lee, Friend, et al, 2002; Rijnierse, Koster, et al, 2006).  55  2.4 EXPERIMENTAL PROCEDURES 2.4.1 Mice All mice were housed in specific pathogen free mouse facilities at The Biomedical Research Centre or BC Cancer Research Centre, with approval from the University of British Columbia Animal Care Committee and according to the Canadian Council on Animal Care guidelines. C57BI/6 (B6) and B6.Cg-Kit’/HNihrJaeBsmJ (KIt1 SI?)  mouse strains were acquired from Jackson Laboratory (Bar Harbor, ME), and  each were maintained as homozygous colonies by sibling breeding. B6-congenic Ship1 mice were a kind gift from Dr F. R. Jirik (University of Calgary, Calgary, AB) and were backcrossed at least 12 times. F2 Ship1 mice (F2-Shiplj were maintained on a B6 x 129/Sv mixed background. All mice used were at least four weeks old and controls were littermates or age- and sex-matched from the same colony. 2.4.2 Cell culture Bone marrow cells were flushed from the femurs of six week old Ship  and  Shipl’ mice and placed in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% FCS (StemCell Technologies, Vancouver, BC), 150 tM monothioglycerol (MTG, Sigma), 100 U/mI penicillin, 100 tg/ml streptomycin (Sigma), and 30 ng/ml rmlL-3 (R&D Systems). Cells were maintained at 37°C in a 5% C02 humidified atmosphere, selected for non-adherence, and considered to be BMMC after six weeks in culture, as verified by Wright-Giemsa staining and flow cytometry (data not shown).  56  243 Reconstitution of mast cell-deficient Kit KitWss  M4sh  mice  7 Ship1’ or Ship1 BMMC in 200 III of mice were injected with 1 x i0  HBSS via the tail vein (designated Ship1’-BMMC and Ship1-BMMC mice). After 12 weeks, the mice were bled for serum collection, subjected to passive systemic anaphylaxis, or sensitized for allergic asthma, and then euthanized via CO 2 inhalation. After euthanizing the mice, peritoneal washes were taken for subsequent analysis by flow cytometry. Samples of back skin, spleen, lung, mesentery, ileum, and colon were fixed in 10% neutral buffered formalin (NBF). Fixed samples were paraffin-embedded, sectioned, and stained by Wax-it Histology Services Inc. (Vancouver, BC, Canada). All tissues were toluidine blue, stained. Mast cells were enumerated by counting the number of toluidine blue positive cells in each section at 40x or lOOx objective on a Zeiss Axioplan 2 microscope. The area of the section was then determined using the polygon area tool with OpenLab 4.0.4 software (Improvision, Boston, MA) at 5x objective. 2.4.4 Cytokine assay Serum (50 iI) was collected from the saphenous vein and assayed using BD Mouse Inflammation and Mouse Thl/Th2 Cytometric Bead Array kits, according to manufacturer’s instructions. 2.4.5 Passive systemic anaphylaxis Mice were sensitized by i.v. injection with 20 g of IgE SPE-7 (Sigma) in 100 li.I of HBSS  +  0.1% BSA. The following day, anaphylaxis was induced by i.v. injection with  I mg DNP-HSA in 100 !l PBS. Rectal temperature was taken five minutes prior to,  57  and immediately preceding injection, and then monitored every five minutes for 40 minutes, and again at 75 and 90 minutes, at which time all mice were sacrificed. Temperature measurements were performed with a traceable expanded range metric digital thermometer (VWR). 2.4.6 Asthma disease course Female mice were sensitized with 100 il of 2 mg/mI OVA in alum 3 (Al(OH) Sigma) ; via Lp. injection on days I and 8. Following sensitization, asthma was induced by introducing 50 tl of 20 mg/mI OVA in HBSS intranasally on days 22, 23, 24, 27, and 28. During intranasal sensitizations, mice were anesthetized via inhalation of 3% isoflurane in 0.8 1pm oxygen. Mice were sacrificed by CO 2 inhalation on day 29. Bronchoalveolar lavages (BAL) were performed by completing three separate additions and removals of 1 ml PBS. Total CD45.2 BAL cells were enumerated using flow cytometry, and differentials were performed on Wright-Giemsa stained cytospins. A portion of the right lower lobe of the lung was taken from each mouse for histopathological grading and mast cell enumeration. Lung portions were fixed in 10% NBF, paraffin-embedded, sectioned, and hematoxylin and eosin (H&E) stained by Wax-it Histology Services Inc. Samples were blinded and clinically scored from zero (no infiltration) to four (profound infiltration) on each of the following parameters: perivascular infiltration, peribronchial infiltration, parenchymal infiltration, and epithelial damage (maximum score of 16).  58  2.4.7 Flow cytometry Samples were blocked with 10% goat serum and anti-CDI6/32, followed by staining with anti-c-kit-PE (Pharmingen), or anti-FcERI-biotin (eBioscience), followed by SA-FITC (Pharmingen), anti-CD45.2-PE (eBioscience). 10 m latex beads (Invitrogen) were added to HBSS with 0.1% BSA prior to peritoneal washes, and to BAL to calculate total cell recovery via flow cytometry. All flow cytometry experiments were performed on a BD FACSCalibur with CeliQuest software and post-collection analyses were performed with FlowJo (TreeStar). 2.4.8 Statistics The unpaired t-test (two-tailed) was used for analysis of tissue mast cell numbers, asthma BAL cellular infiltrate, BAL differentials, lung clinical scores, serum cytokine levels, and PSA assays. A Kruskal-Wallis test was used for the mesentery of BMMC reconstituted mice (due to all zero values in the Ship 1’ condition). A log-rank (Mantel-Cox) test was used to compare the PSA survival rates. Separate allergic asthma experiments were normalized based on their OVA treated B6 levels prior to analysis. All of the above tests were performed using GraphPad Prism 5 or Microsoft Excel X software. 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Ong, C.J., Ming-Lum, A., Nodwell, M., Ghanipour, A., Yang, L., Williams, D.E., Kim, J., Demirjian, L., Qasimi, P., Ruschmann, J. et al. (2007). Small-molecule agonists of SHIPI inhibit the phosphoinositide 3-kinase pathway in hematopoietic cells. Blood 6, 1942-1 949. 62  Rauh, M.J., Ho, V., Pereira, C., Sham, A., Sly, L.M., Lam, V., Huxham, L, Minchinton, A.l., Mui, A., and Krystal, C. (2005). SHIP represses the generation of alternatively activated macrophages. Immunity 4, 361-374. Rijnierse, A., Koster, A.S., Nijkamp, F.P., and Kraneveld, A.D. (2006). Critical role for mast cells in the pathogenesis of 2,4-dinitrobenzene-induced murine colonic hypersensitivity reaction. J. Immunol. 7, 4375-4384. Takeshita, S., Namba, N., Zhao, J.J., Jiang, Y., Genant, H.K., Silva, M.J., Brodt, M.D., Helgason, C.D., Kalesnikoff, J., Rauh, M.J. et al. (2002). SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts. Nat. Med. 9, 943-949. Tamir, I., Stolpa, J.C., Helgason, C.D., Nakamura, K., Bruhns, P., Daeron, M., and Cambier, J.C. (2000). The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRllB signals in B cells. Immunity 3, 347-358. Tarasenko, T., Kole, H.K., Chi, A.W., Mentink-Kane, M.M., Wynn, T.A., and Bolland, S. (2007). T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating ThlITh2 and cytotoxic responses. Proc. NatI. Acad. Sci. U. S. A. 27, 11382-11387. Trotta, R., Parihar, R., Yu, J., Becknell, B., Allard, J.,2nd, Wen, J., Ding, W., Mao, H., Tridandapani, S., Carson, W.E., and Caligiuri, M.A. (2005). Differential expression of SHIPI in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation. Blood 8, 3011-301 8. Tsai, M., Takeishi, T., Thompson, H., Langley, K.E., Zsebo, K.M., Metcalfe, D.D., Geissler, E.N., and Galli, S.J. (1991). Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc. NatI. Acad. Sci. U. S. A. 14, 6382-6386. Williams, C.M, and Galli, S.J. (2000). Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice. J. Exp. Med. 3, 455462. Yu, M., Tsai, M., Tam, S.Y., Jones, C., Zehnder, J., and Galli, S.J. (2006). Mast cells can promote the development of multiple features of chronic asthma in mice. J. Clin. Invest. 6, 1633-1 641.  63  CHAPTER 3. PRION PROTEIN IS EXPRESSED ON MAST CELLS AND IS RELEASED UPON THEIR ACTIVATION 1 3.1 INTRODUCTION Mast cells are highly granular immune effector cells that develop from hematopoietic stem cells (HSC), circulate as committed precursors, and mature in their target tissues. Mast cells are located in many tissues, including the skin, gut, brain, and peritoneal cavity, and tend to be in close proximity to nerves and blood vessels. When mast cells degranulate, they release an assortment inflammatory mediators that can trigger anaphylaxis. Our group previously reported that mast cells share a number of surface markers with HSC, including CD34, Sca-1 and c-kit (Drew, Merkens, et al, 2002). The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored sialoglycoprotein, and is the substrate for post-translational, template-directed misfolding process implicated in transmissible spongiform encephalopathies (TSE), including scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease. TSE are incurable, fatal, neurodegenerative diseases, associated with accumulation and aggregation of the scrapie isoform of PrPC (PrP) in the brain. The mechanism by which infectious prions reach the brain after peripheral inoculation, including the cell types involved, is incompletely understood. PrPD is expressed by many cell types, including hematopoietic cells, but expression of PrPC by mast cells has yet to be investigated (Cashman, Loertscher, et al, 1990; Dodelet,  1  A version of this chapter has been accepted for publication. Haddon, D.J., Hughes, M.R., Antignano, F., Westaway, D., Cashman, N.R., and McNagny, K.M. (2009) Prion protein is expressed on mast cells and is released upon their activation. J Infect Dis.  64  and Cashman, 1998). In this study we compared LinSca-1c-kit (LSK) cells, which are highly enriched for HSC, and mast cells, using microarray expression analysis, and identified PrPC as a potentially novel marker of mast cells. Upon further investigation, we found that PrPC (1) is expressed on the surface of human and mouse mast cells, both in vitro and in vivo; (2) is not required for mast cell differentiation or tissue homeostasis; (3) is released by mast cells at steady state and rapidly upon activation; and (4) is released in response to mast cell-dependent allergic inflammation in vivo. Since mast cells are long-lived and traffic to the CNS, our observations could have important implications for the transmission of PrPSC and the pathology of prion disease.  65  3.2 MATERIALS AND METHODS C57Bl/6 (B6) and B6.Cg-Kit’/HNihrJaeBsmJ (Kit”) mice were acquired from Jackson Laboratory (Bar Harbor, ME). Zrch I Prnp°’° mice were maintained on a B6 background (Bueler, Fischer, et al, 1992). Mice were at least four weeks old at experimentation, age- and sex-matched, and kept according to the Canadian Council on Animal Care guidelines. Bone marrow mast cells (BMMC) for microarray analysis were prepared as previously described (Drew, Merkens, et al, 2002). Total RNA was isolated from three independent BMMC cultures at 14-16 days (immature) or 8-15 weeks in culture (mature) using a Qiagen RNeasy Mini RNA isolation kit. Total RNA samples were amplified by Affymetrix GeneChip Eukaryotic Small Sample Target Labeling, and hybridized to Affymetrix GeneChips (MOE43OA and MOE43OB). The resulting gene expression data, as well as data retrieved for LinSca-1c-kit cells (LSK), are available from  StemBase  (http://www.stembase.ca)  and  NCBI’s  GEO  (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3776 and GSE3234) (Edgar, Domrachev, and Lash, 2002; Perez-lratxeta, Palidwor, et al, 2005). Differentially expressed transcripts were those that were called “present” (Affymetrix MAS5.0 algorithm) and had a greater than two-fold, statistically significant (P<.05) difference in expression between conditions (t-test with Benjamini and Hochberg False Discovery Rate correction performed with Genespring GX). For all other experiments, BMMC were prepared from bone marrow harvested from the femurs of B6 (Prnp’) or Pm p°’° mice and placed in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% FCS, 150 jtM monothioglycerol 66  (MTG), 100 U/mI penicillin, 100 gIml streptomycin, and 30 ng/ml rmlL-3. At six weeks BMMC were >90% pure, as determined by flow cytometry (FcERI and c-kit). HMC-1 cells, maintained as previously described, were a gift from Joseph H. Butterfield M.D. (Mayo Clinic, Rochester, MN) (Hartmann, Henz, et al, 1997). To induce degranulation, we incubated BMMC overnight in IMDM containing 0.1% BSA, and 150 tM MTG with or without I g/ml mouse anti-DNP (dinitrophenol) lgE SPE-7. We washed and resuspended the cells in Tyrode’s buffer containing 0.1% BSA, and stimulated with 10 ng/ml DNP-HSA or I 1 tM A23187 for the indicated times. I-hexosaminidase activity was determined as previously described (Nishizumi, and Yamamoto, 1997). PrPC enzyme immunoassay (EIA; SPI-bio, France) was used to determine the relative levels of PrPC release. Flow cytometry samples were blocked with goat serum and anti-CD16/32, labeled with anti-c-kit-PE (Pharmingen), antiPrPC 6H4 (Prionics), or anti-FcRl-biotin (eBioscience), followed by SA-FITC (Pharmingen). AntiPrPC was labeled with a 1 Labeling Kit (Molecular Probes) or goat anti Zenon AlexaFluor488 Mouse lgG mouse lgG-FITC (Serotec). Acquisition was performed on a BD FACSCalibur and analyzed with FlowJo (TreeStar).  67  3.3 RESULTS AND DISCUSSION To further compare mast cells and HSC, we evaluated the gene expression of Lin Sca-1c-kit cells (LSK), which are highly enriched for HSC, to immature and mature mast cells using microarray (Figure 3.IA). We identified 1228 differentially expressed transcripts between mature mast cells and LSK (Figure 3.IB). Mast cells expressed higher levels of mast cell-associated genes (including Fcorla, Mcpt5, and Mcpt6; Figure 3.IC). For the most part, LSK expressed higher levels of HSC associated genes (such as Cd34, EPCR, and F1t3), while mast cells expressed higher levels of Sca-1, c-kit, and, surprisingly, Prnp (Zhang, Steele, et al, 2006). We were intrigued to find that mast cells express high levels of Prnp transcript, as, to our knowledge, PrPC expression by mast cells has not been reported. We found that BMMC, PWMC, and the HMC-1 human mast cell line all expressed high levels of surface PrPC (Figure 3.1 D-F). To test whether PrPD is required for the formation of mast cells, we attempted to derive BMMC from Prnp’ and Pm p°’° bone marrow. After four weeks of culture, both had formed FcERlc-kit mast cells (Figure 3.IG,H). We also found that Prnp°’° PWMC were present at normal numbers, and had normal levels of c-kit expression (Figure 3.11).  68  Figure 3.1 Gene expression analysis of mast cells and HSC identifies PrPC as a marker of human and mouse mast cells, in vitro and in vivo (A) Representative flow cytometry histograms of immature (14-16 days in culture) and mature mast cells (8-15 weeks in culture) from which total RNAwas isolated for microarray analysis. C-kit expression is indicative of mast cell differentiation (blue, unstained/isotype; red, anti-c-kit-PE). (B) A scatter plot of 1228 differentially expressed transcripts between mature mast cells and LSK (each circle represents a transcript, diagonal lines represent one- and two-fold cutoffs). (C) Levels of selected HSC- and mast cell-associated transcripts in LSK, immature mast cells, and mature mast cells as determined by microarray. Flow cytometry analysis of BMMC (D), HMC-1 (E), and ckith1SSCh1 peritoneal wash mast cells (F) for surface expression of PrPC (blue, isotype; red, antiPrPC 6H4). (G) Prnp’ and Prnp°’° BMMC had similar morphology, as shown by Wright-Giemsa stained cytospins (bars  =  1Om). Images  were acquired on a Zeiss Axioplan 2 microscope (Zeiss, Toronto, ON) with a 40x10.75 dry objective and a Qimaging Retiga EX CCD camera (Minneapolis, MN)  using Openlab 4.0.4 software (Improvision, Boston, MA). (H) Flow cytometry analysis revealed that Prnp°’° BMMC (blue line) had similar expression of FcERI and c-kit to Prnp’ BMMC (red line), but lacked surface PrPC (dashed line, isotype). (I) The total number (graph) and frequency (gate statistics) of ckithh1SSCh1 peritoneal wash mast cells were comparable between Prnp’ (red line) and Prnp°’° (blue line) mice. Flow cytometry data are representative of three independent experiments.  69  C  x  ‘4’ F .  C) 0  Total mast cells (x10 ) 4  -U -U C)  Relative frequency  Granularity (SSC)  -U C)  -o  Relative frequency  C)  -c  -i:  0  0 0 0  -  P  ‘1  Relative frequency  -U C)  -ci  -  -  0 0  0 0 0  m  Relative frequency  0  -  Mast cells normalized intensity  Granularity (SSC)  Z  CD—  z  0) N  3  0—  p 0  a  0  w  Mcpt6  114 116  Adr  c-kit Sca-1 Cd34 F1t3 Mpl EPCR Notchi t-’rnp Abcg2 Tek CXCR4 HoxalO Hoxa9 Fcerla Fcerlg lL4ra Hrh4  —-  —,  —  C)  (DE  DI  Normalized intensity  Relative frequency  CD  C  0)  3 3  Some cell lines release PrPC into the culture supernatant at steady state (Alais, Simoes, et al, 2008; Fevrier, Vilette, et al, 2004). We found that Prnp BMMC also released high levels of PrPC into the culture supernatant at steady state (Figure 3.2A). Further, we found that upon activation, BMMC rapidly released 30% of their total cellular PrP’ (Figure 3.2B,C). Activation-dependent release of PrPC likely occurred through a previously reported proteolytic or lipolytic loss of its GPI-anchor, as the majority of PrPC in the releasate segregated to the aqueous phase when subjected to a Triton X-114 phase extraction, while PrPC in BMMC lysates segregated to the detergent phase (Figure 3.2D) (Parkin, Watt, et al, 2004). Additionally, since the EIA relies upon binding within PrPCs octapeptide repeats and to amino acids 144-153 (DYEDRYYREN), it rules out intervening cleavages at 96 and 111 (McMahon, Mange, et al, 2001; Vincent, Paitel, et al, 2001). To investigate whether mast cell-dependent allergic inflammation can provoke release of PrPC j vivo, we induced passive systemic anaphylaxis (PSA) in B6 and mast cell-deficient Kit”’  mice. While Kit’’ mice were resistant to PSA, B6 mice suffered  severe hypothermia (Figure 32E) (Zhou, Xing, et al, 2007). Strikingly, anaphylaxis induced a two-fold increase in serum PrPC (Figure 3.2F).  71  Figure 3.2 Mast cells release PrPC at steady state and upon stimulation in vitro and in vivo (A) Prnp°’° and Prnp’ BMMC were seeded at I x i0 5 cells/mi, cultured for one  week, and high levels of PrPC were found in the supernatants of Prnp’ BMMC by PrPD enzyme immunoassay (EIA). (B) BMMC were stimulated with A23187 (solid squares), and PrPC was detected in the supernatant within two minutes (PrPC EIA, red lines) and prior to maximal degranulation (13-hexosaminidase activity, blue lines) compared to vehicle controls (open circles). Data are representative of three experiments. (C) EIA of whole cell lysates (one volume of Tyrode’s with 0.1% BSA, 0.5% NP-40, and tissue culture protease inhibitor cocktail) from Prnp’ BMMC stimulated with A231 87 or IgE  +  DNP for 15 minutes revealed a loss of 30% of total  cellular PrPC, compared to vehicle and IgE-alone controls. Data represent pooled results from two (IgE  +  DNP) or three (A23187) independent experiments. (D)  BMMC were stimulated with A23187 for 2 minutes, and a phase extraction was performed on the pellets (106 cells) and releasates (from I 0 cells) in Tyrode’s with 0.1% BSA, 0.5% Triton X-114, and tissue culture protease inhibitor cocktail. PrPC EIA of the aqueous and detergent phases revealed that, in contrast to the lysates, the majority of PrPC in the releasate segregated to the aqueous phase. (E) To induce passive systemic anaphylaxis, B6 (n=3, blue line) and KItw (n=3, red line) mice were sensitized (at t=0) with anti-DNP IgE (20 tg, i.v.) and challenged the following day with DNP-HSA (1 mg i.v.). (F) PrPC EIA of pre-sensitization and post anaphylaxis serum revealed that anaphylaxis induced a two-fold increase in serum PrPC in B6, but not Kitw  l’Sh  mice. Bars represent SE. (*, P<.05;  Pc 01;  72  sesFeue Ie3!1S!1iS fl8 JOJ posn OJOM (peie-oivq) S1SO4- POJ!8dUfl (.oo>d  •-.1  CD CI  3  3  —I  C.,  0)  01  C  I—’  01  PJ  I-  * * *  0)  03 0)  0) .  0)  0) 01  0) 0)  0)  ‘1  0) CO  m  C  F  H  P3  COD  0) CD  >  -uc  C..)  PrPC concentration (A ) 405  0) C)  Rectal temperature( C)  Detergent  Aqueous  Detergent  Aqueous  lgE+DNP  IgE  lgE+DNP  IgE  P3 C> 4 C)  0) 0  ]  H  0  CO  I  0  (Fl  b  0  -  9  0 (11  ‘  0  U’  CD  C,)  CD  Cl)  CD CD  (Fl  9k) )  C, C 0  PrPC concentration (A ) 405  A23187 -1  Vehicle  A23187  Vehicle  C)  % of total cellular PrPC  C CD C,,  3  CD  3  01  01  -  -o,.  -I  -o  4  I_  0  p  o  I  -  P3  w  C)  (%)  0  -  I  01 I  p  o  ()  I  01  uotenuei6eQ  0  r%)  I  I  0  -•  .  o  C) I  PrPC concentration (A ) 405  0  PrPC concentration (A405)  In summary, we report that (1) PrPC is expressed on the surface of human mast cells and mouse mast cells, both in vitro and in vivo; (2) PrPC is not required for mast cell differentiation, or tissue homeostasis; (3) PrPC is released by mast cells at steady state and rapidly upon activation; and (4) PrPC is released in response to mast cell-dependent allergic inflammation in vivo. Via the oral route, prions are ingested, and initially infect the gut-associated lymphoid tissue before propagation to the CNS. Since mast cells are migratory, located in gut, brain, CNS, and in close proximity to nerves and blood vessels, and express high levels of PrPC, they are well suited to be infected with prions and may present a unique target to disrupt the propagation of infectivity to the CNS. Further, mast cells may be a unique system to investigate the normal function of PrPC.  75  3.4 REFERENCES Alais, S., Simoes, S., Baas, D., Lehmann, S., Raposo, G., Darlix, J.L., and Leblanc, P. (2008). Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol. Cell. 10, 603-61 5. Bueler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H.P., DeArmond, S.J., Prusiner S.B., Aguet, M., and Weissmann, C. (1992). Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 6370, 577582. Cashman, N.R., Loertscher, R., Nalbantoglu, J., Shaw, I., Kascsak, R.J., Bolton, D.C., and Bendheim, P.E. (1990). Cellular isoform of the scrapie agent protein participates in lymphocyte activation. Cell 1, 185-192. Dodelet, V.C., and Cashman, N.R. (1998). Prion protein expression in human leukocyte differentiation. Blood 5, 1556-1 561. Drew, E., Merkens, H., Chelliah, S., Doyonnas, R., and McNagny, K.M. (2002). CD34 is a specific marker of mature murine mast cells. Exp. Hematol. 10, 1211. Edgar, R., Domrachev, M., and Lash, A.E. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 1, 207-210. Fevrier, B., Vilette, D., Archer, F., Loew, D., Faigle, W., Vidal, M., Laude, H., and Raposo, G. (2004). Cells release prions in association with exosomes. Proc. NatI. Acad. Sci. U. S. A. 26, 9683-9688. Hartmann, K., Henz, B.M., Kruger-Krasagakes, S., Kohl, J., Burger, R., GuhI, S., Haase, I., Lippert, U., and Zuberbier, 1 (1997). C3a and C5a stimulate chemotaxis of human mast cells. Blood 8, 2863-2870. McMahon, H.E., Mange, A., Nishida, N., Creminon, C., Casanova, D., and Lehmann, S. (2001). Cleavage of the amino terminus of the prion protein by reactive oxygen species. J. Biol. Chem. 3, 2286-2291. Nishizumi, H., and Yamamoto, T. (1997). Impaired tyrosine phosphorylation and Ca2+ mobilization, but not degranulation, in lyn-deficient bone marrow-derived mast cells. J. Immunol. 5, 2350-2355. Parkin, E.T., Watt, N.T., Turner, A.J., and Hooper, N.M. (2004). Dual mechanisms for shedding of the cellular prion protein. J. Biol. Chem. 12, 11170-11178. Perez-lratxeta, C., Palidwor, G., Porter, C.J., Sanche, N.A., Huska, M.R., Suomela, B.P., Muro, E.M., Krzyzanowski, P.M., Hughes, E., Campbell, P.A., Rudnicki, M.A., and Andrade, M.A. (2005). Study of stem cell function using microarray experiments. FEBS Lett. 8, 1795-1 801. 76  Vincent, B., Paitel, E., Saftig, P., Frobert, Y., Hartmann, D., De Strooper B., Grassi, J., Lopez-Perez, E., and Checler, F. (2001). The disintegrins ADAMIO and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein. J. Biol. Chem. 41, 37743-37746. Zhang, C.C., Steele, A.D., Lindquist, S., and Lodish, H.F. (2006). Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal. Proc. Nati. Acad. Sci. U. S. A. 7, 2184-21 89. Zhou, J.S., Xing, W., Friend, D.S., Austen, K.F., and Katz, H.R. (2007). Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. Med. 12, 2797-2802.  77  CHAPTER 4. CONCLUSION Overall, we aimed to discover more about the physiology of mast cells, focusing on their homeostatic regulation in vivo, their activation in vitro and in allergic disease, their gene expression patterns, and their surface antigens. In our first study, we demonstrated that SHIPI represses allergic inflammation and mast cell hyperplasia  in vivo, and that SHIPI exerts these effects specifically in mast cells. Further, our results demonstrate that SHIPI is a cell autonomous repressor in mast cell homeostasis. Our findings could have implications for treatment of mast cellassociated diseases in humans, since SHIPI is a repressor of human mast cells and agonists and small molecule antagonists of SHIPI function have recently been identified and tested in vivo (Langdon, Schroeder, et al, 2008; Ong, Ming-Lum, et al, 2007). While we suspect that the systemic mast cell hyperplasia observed in Shipl’ mice is due to the high levels of IL-6 and TNF produced by Shipl’ mast cells, it could be mediated by other mechanisms. Two of our findings provide evidence against alternative mechanisms, namely enhanced migration or survival of Shipl’ mast cells. We found that Ship1 mast cells migrate less efficiently than wild-type controls  in vitro (Figure A.2A). To our knowledge, this is the first report of a migration defect in Shipl’ mast cells. Defective migration and homing defects have been described in Ship1 neutrophils, peritoneal macrophages, and BM progenitors, while enhanced migration has been observed in Ship1 bone marrow derived macrophages, BM progenitors, B cells, and T cells in vitro (Desponts, Hazen, et al, 2006; Kim, Hangoc, et al, 1999; Nishio, Watanabe, et al, 2007; Vedham, Phee, and Coggeshall, 2005).  78  The discrepancy likely reflects differences in experimental systems and in SHIPI’s activities within specific cell types. A likely mechanism for the defective migration of Shipl’ mast cells is that SHIPI is required to limit localization of PIP 3 to the leading edge of the migrating cell, translating the direction of the chemoattractant gradient to the inner leaflet of the plasma membrane, as reported in migrating neutrophils (Nishio, Watanabe, et al, 2007). Next we found that Shipl’ mast cells, when deprived of IL-3, have virtually identical survival kinetics to controls In vitro (Figure A.2B). Taken together, these results suggest that the mast cell hyperplasia is a result of increased expansion of Ship 1 mast cells in response to high levels of lL-6 and TNF (Figure 4.1). A potential experiment that could test this hypothesis would be to competitively transplant wild-type and Shipl’ BMMC into Kit’” mice, utilizing the Ly5.1/Ly5.2 hematopoietic transplantation marker system. The Ly5.1/Ly5.2 marker system utilizes alternative alleles of CD45 to allow tracking and identifcation of donor and host hematopoietic cells after transplant via FACS. Heterozygous mice, double positive for Ly5.1 and Ly5.2, can be used to identify a second competitive donor if necessary. If the mechanism of the hyperplasia is that IL-6 and TNF produced by Ship1 BMMC support increased mast cell numbers, one would predict that wild-type mast cells would also be hyperplastic in the competitive transplant.  79  Anaphylaxis  Asthma  1  SHIP TNF  Figure 4.1 Proposed mechanism of increased inflammatory cytokines, allergic asthma pathology, and anaphylaxis response in Ship1 mice and mice reconstituted with Shipi’ mast cells Ship1 mast cells produce higher levels of the inflammatory cytokines IL-6 and TNF, which in turn induce increased numbers of mast cells. lL-6 likely mediates mast cell hyperplasia via an intermediate, such as PGE , produced by macrophages or 2 fibroblasts. The increased mast cell numbers confer increased allergic response and the higher levels of lL-6 and TNF additionally worsen asthma pathology.  80  To evaluate whether the hypersensitivity to anaphylaxis we observed in mice reconstituted with Ship1 BMMC was due to hyperplasia or increased degranulation we considered transplanting higher numbers of wild-type mast cells to equilibrate mast cell numbers between genotypes. We ruled that plan out for the following technical reason  -  the hyperplasia observed in mice reconstituted with Shipl-/- mast  cells is unique to each tissue type. For instance, two-fold more mast cells are observed in the spleens of mice reconstituted with Shipl’ mast cells than Ship 1’ mast cells, while there are 20-fold more in the ileum, and 60-fold more in the peritoneal cavity. Thus, increasing the number of wild-type mast cells (or decreasing the number of Shipl’ mast cells) may match mast cell numbers for a single tissue, but would not faithfully recreate the unique tissue distribution observed. We reported that Ship 1’-BMMC mice exhibited increased allergic asthma pathology, but it was unclear whether Shipl’ mast cells caused increased inflammation, or failed to inhibit inflammation associated with the Kitw? host. This could be clarified by conducting the asthma induction without alum adjuvant, a model that is more mast cell-dependent (Kit’’’ mice suffer significantly reduced symptoms, which are returned to wild-type levels upon reconstitution with mast cells) (Nakae, Ho, et al, 2007). We used the alum adjuvant model of asthma because at the time of initiating our experiments it had yet to be investigated in KitW  lW-si,  mice.  A similar mast cell-independent trend had been observed in Kit’’ mice, but they are on a mixed genetic background and we assumed that since KitW/ mice are on a pure B6 background, they might respond differently. Proteins are thought to adsorb to alum, which is suspected to extend their release in vivo, and may reduce  81  the threshold for T cell activation. When alum is removed, T cells may rely more heavily on mast cells and their TH2 cytokines for activation and promotion of asthma (Nakae, Ho, et al, 2007). To determine whether loss of SHIP1 in mast cells would also confer increased pathology in other mast cell-associated inflammatory diseases, we induced an antibody-dependent model of rheumatoid arthritis (RA), and preliminary results show Shipl’-BMMC mice had near identical symptoms to controls (Figure A.1). Surprisingly, KitM/ mice not only developed arthritis, they suffered more severe arthritis than B6 controls. Similarly, Kit8l8h mice were susceptible to an anti-collagen antibody-mediated model of arthritis, but had nearly equivalent symptoms to wild-type controls (Zhou, Xing, et al, 2007). Kit” mice were largely protected from anti-collagen antibody-mediated arthritis, and therefore future investigations of Shipf’ mast cell’s role in arthritis would be more informative in KIt’v mice (Zhou, Xing, et al, 2007). A potential pattern to the combined findings that mice reconstituted with Shipl’ mast cells are hypersensitive to anaphylaxis, suffer worse allergic asthma pathology, but do not appear to confer increased susceptibility to arthritis is that perhaps Shipl’ mast cells increase susceptibility to TH2 type diseases (Asthma and anaphylaxis), but not THI type diseases (Arthritis). This interpretation is likely incomplete, since mice reconstituted with Shipl’ mast cells have high levels of TNF, a THI cytokine, and lL-6, typically a TH2 cytokine. Further investigation is necessary as the levels of TNF and IL-6 were measured at steady state and it is likely that the THI/TH2 cytokine levels would change during different disease courses, potentially giving a better explanation of the above results. A potential limitation of our findings using Kit1M’ mice reconstituted with  82  Ship1’ or Shipl’ BMMC is the artificial aspects of the model, including culture and transplantation steps. Indeed we suspect the anaphylaxis-induced hypothermia in mice reconstituted with Ship  BMMC was mild compared to B6 controls because,  as was observed by other groups, not all tissues are reconstituted to wild-type levels in this model (Grimbaldeston, Chen, et al, 2005). This is likely the explanation for why ubiquitous Shipl’ mice did not exhibit body temperature recovery during anaphylaxis, while mice that were reconstituted with Shipl’ BMMC did. Further, it is likely that defects in mast cell progenitors would not be identified. A method to circumvent some of these limitations would be to use conditional knockout mice. Two strains of mast cell-specific Cre-expressing mice and mice with a IoxP-flanked allele of Ship I recently became available (Karlsson, Guinamard, et al, 2003; Musch, Wege, et al, 2008; Scholten, Hartmann, et al, 2008). Crosses between the Greexpressing strains and the IoxP-Ship mice have not been reported in the literature to date  —  they will be of great interest.  In our second study, we identified prion protein (PrPC) as a novel marker of human and mouse mast cells, both in vItro and in vivo. Further, we found that PrPC is released by mast cells upon activation and in response to mast cell dependent allergic inflammation in vivo (Figure 4.2). Since mast cells are long-lived and present in the brain and CNS, these findings could have important implications for the transmission and pathology of prion diseases. Further, the function of PrPC under normal circumstances is not known and mast cells could present a unique system to investigate PrPCs normal function.  83  Figure 4.2 A model of PrPC expression and PrP conversion during IgE mediated mast cell activation and relation to laminin binding (A) Mast cells actively transcribe Prnp and PrPC is present on their surface. When lgE antibodies, bound to FcERI receptors, are crosslinked by allergen, soluble PrPC is rapidly released via loss of its GPI anchor and transcription of Prnp is vastly reduced. Future studies into whether PrPC on the surface of mast cells can be converted to PrP&, whether mast cells participate in PrPSC transmission to the CNS, and whether mast cell-dependent inflammation can accelerate TSE pathology will be of great interest. (B) PrPC on the surface of mast cells may function in binding to laminin present in basement membranes or ECM. The rapid transcriptional downregulation of Prnp and release of PrPC following IgE-mediated stimulation could release mast cells from the laminin, in concert with granule protease-mediated digestion of the ECM.  84  A  ••  •oo \ TSE transmission to CNS  PrPC (GPI-linked)  Allergen  PrPSC (GPI-linked)  IgE antibody  FceRl receptor —-‘----‘  Laminin  Laminin receptor  85  Our results suggest that upon activation BMMC release PrPC via proteolytic cleavage within amino acids 220-231, a cleavage that is inhibited by metalloprotease inhibitors, or lipolytic cleavage of its GPI anchor (Parkin, Watt, et al, 2004). Interestingly, while IgE crosslinking caused a decrease in cell-associated PrPC, we didn’t detect a corresponding increase in soluble PrPC. This suggests that IgE crosslinking induces destruction of PrPCs reactive epitope, possibly by a mast cell protease or creation of radical oxygen species (Figure B.1 D). 3-hexosaminidase was also degraded, as evidenced by reduced enzymatic activity, following IgE crosslinking, but not ionophore treatment (data not shown). This, in addition with the reduced levels of total cellular PrPC following IgE stimulation (Figure 3.2), provides circumstantial evidence for a proteolytic mechanism instead of internalization. It would be interesting to identify the protease responsible (especially since PrPS is unusually protease resistant). In line with the reduction in surface PrPC that we observed in activated mast cells, we found that Prnp transcript levels are also dramatically downregulated during mast cell activation (Figure B.1C). Further work will be required to determine the significance of PrPC release during degranulation, as Prnp°’° BMMC do not have any obvious defect in degranulation (Figure B.1 E). The mechanism and cell types involved in transmission of PrP& to the brain after ingestion are poorly understood. Further, little is known regarding the role of inflammation in PrPSC transmission and prion disease pathology. Experiments using mast cell-deficient mice (Kit”’ and Kith!1 strains) could be used to determine the role of mast cells and mast cell-dependent inflammation in prion disease. Additionally, it would be interesting to examine prion disease in mice with  86  hyperactive mast cells, such as Ship1-BMMC reconstituted mice. Mast cells share a number of surface markers with HSC, and we identified potential markers (including F1t3, MpI, and EPCR (Procr)) for distinguishing HSC and mast cells. Recent interest has focused on the committed mast cell precursors and their relation to HSC and myeloid progenitors, and the above markers may be useful in further defining and purifying these cell types (Arinobu, Iwasaki, et al, 2005; Chen, Grimbaldeston, et al, 2005). Further, we have provided a unique set of gene expression data that will be useful for future investigation of mast cell biology. Previous microarray expression analyses of mast cells have focused on other species, cell lines, less common culturing methods, or exclusively on stimulated mast cells (Gilicze, Kohalmi, et al, 2007; Jeffrey, Brummer, et al, 2006; Valadi, Ekstrom, et al, 2007; Wiener, Pocza, et al, 2008). Our analysis is unique in that it is on primary mast cells from two stages of development, in biological triplicate, and on a highly comparable, common microarray platform.  87  4.1 REFERENCES Arinobu, Y., Iwasaki, H., Gurish, M.F., Mizuno, S., Shigematsu, H., Ozawa, H., Tenen, D.C., Austen, K.F., and Akashi, K. (2005). Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis. Proc. Nati. Acad. Sd. U. S. A. 50, 181 05-1 8110. Chen, C.C., Grimbaldeston, M.A., Tsai, M., Weissman, l.L., and Galli, S.J. (2005). Identification of mast cell progenitors in adult mice. Proc. NatI. Acad. Sci. U. S. A. 32, 11408-11413. Desponts, C., Hazen, A.L., Paraiso, K.H., and Kerr, W.G. (2006). SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation. Blood 11, 4338-4345. Gilicze, A., Kohalmi, B., Pocza, P., Keszei, M., Jaeger, J., Gorbe, E., Papp, Z., Toth, S., Falus, A., and Wiener, Z. (2007). HtrAl is a novel mast cell serine protease of mice and men. Mol. Immunol. 11, 2961-2968. Grimbaldeston, M.A., Chen, C.C., Piliponsky, A.M., Tsai, M., Tam, S.Y., and Galli, S.J. (2005). Mast cell-deficient W-sash c-kit mutant Kit W-shIW-sh mice as a model for investigating mast cell biology in vivo. Am. J. Pathol. 3, 835-848. Jeffrey, K.L., Brummer, T., Rolph, M.S., Liu, S.M., Callejas, N.A., Grumont, R.J., Gillieron, C., Mackay, F., Grey, S., Camps, M. et al. (2006). Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1. Nat. Immunol. 3, 274-283. Karisson, M.C., Guinamard, R., Bolland, S., Sankala, M., Steinman, R.M., and Ravetch, J.V. (2003). Macrophages control the retention and trafficking of B lymphocytes in the splenic marginal zone. J. Exp. Med. 2, 333-340. Kim, C.H., Hangoc, C., Cooper S., Helgason, C.D., Yew, S., Humphries, R.K., Krystal, G., and Broxmeyer, H.E. (1999). Altered responsiveness to chemokines due to targeted disruption of SHIP. J. CIin. Invest. 12, 1751-1759. Langdon, J.M., Schroeder, J.T., Vonakis, B.M., Bieneman, A.R, Chichester, K., and Macdonald, S.M. (2008). Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-i knockdown in cultured human mast cell and basophil models. J. Leukoc. Biol. 4, 1151-1158. Musch, W., Wege, A.K., Mannel, D.N., and Hehigans, T. (2008). Generation and characterization of aipha-chymase-Cre transgenic mice. Genesis 3, 163-166. Nakae, S., Ho, L.H., Yu, M., Monteforte, R., likura, M., Suto, H., and Galli, S.J. (2007). Mast cell-derived TNF contributes to airway hyperreactivity, inflammation,  88  and TH2 cytokine production in an asthma model in mice. J. Allergy Clin. Immunol. 1, 48-55. Nishio, M., Watanabe, K., Sasaki, J., Taya, C., Takasuga, S., lizuka, R., Balla, T., Yamazaki, M., Watanabe, H., Itoh, R. et al. (2007). Control of cell polarity and motility by the Ptdlns(3,4,5)P3 phosphatase SHIPI. Nat. Cell Biol. 1, 36-44. Ong, C.J., Ming-Lum, A., Nodwell, M., Ghanipour, A., Yang, L., Williams, D.E., Kim, J., Demirjian, L., Qasimi, P., Ruschmann, J. et a!. (2007). Small-molecule agonists of SHIP1 inhibit the phosphoinositide 3-kinase pathway in hematopoietic cells. Blood 6, 1942-1949. Parkin, E.T., Watt, N.T., Turner, A.J., and Hooper, N.M. (2004). Dual mechanisms for shedding of the cellular prion protein. J. Biol. Chem. 12, 11170-11178. Scholten, J., Hartmann, K., Gerbaulet, A., Krieg, T., Muller, W., Testa, G., and Roers, A. (2008). Mast cell-specific Cre/loxP-mediated recombination in vivo. Transgenic Res. 2, 307-31 5. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., and Lotvall, J.O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 6, 654-659. Vedham, V., Phee, H., and Coggeshall, K.M. (2005). Vav activation and function as a rac guanine nucleotide exchange factor in macrophage colony-stimulating factorinduced macrophage chemotaxis. Mol. Cell. Biol. 10, 4211-4220. Wiener, Z., Pocza, R, Racz, M., Nagy, G., Tolgyesi, G., Molnar, V., Jaeger, J., Buzas, E., Gorbe, E., Papp, Z., Rigo, J., and Falus, A. (2008). lL-18 induces a marked gene expression profile change and increased Ccli (1-309) production in mouse mucosal mast cell homologs. Int. Immunol. Zhou, J.S., Xing, W., Friend, D.S., Austen, K.F., and Katz, H.R. (2007). Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. Med. 12, 2797-2802.  89  APPENDICES APPENDIX A. SUPPLEMENTAL DATA ON SHIP IN MAST CELLS  A.1 RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that causes progressive erosion and destruction of the joint surface. It is associated with leukocyte invasion, synovial inflammation and thickening, pannus formation, and cartilage and bone destruction. The KIBxN transgenic mouse line is a model for RA and spontaneously develops symptoms at three to four weeks of age (Ditzel, 2004). KIBxN mice express a transgene that encodes a TCR that is specific for glucose 6phosphate isomerase (G6PI) self-peptide. With help from G6PI-reactive T cells, KIBxn B cells produce high titres of anti-G6PI Igs. (Maccioni, Zeder-Lutz, et al, 2002). Serum from KIBxN can be transferred to unaffected mice via i.p. injection to induce RA, with immune complex-mediated damage occuring within days. The role of mast cells in RA is contentious based on recent findings with mast cell deficient mice. The Kit’ mouse line is resistant to KIBxN RA, but upon reconstition of mast cells, becomes symptomatic (Lee, Friend, et al, 2002). A recent study using anti-type II collagen mAbs followed by LPS treatment to induce RA found that mast cell deficient Kit’” mice were fully susceptible, casting controversy on mast cells’ role in this painful disease (Zhou, Xing, et al, 2007).  90  A.1 .1 Development of rheumatoid arthritis does not appear to be altered in -BMMC reconstituted mice 4 Shipl To determine whether Shipl’-BMMC mice are more susceptible to RA than Ship1’-BMMC mice, we subjected them to the KIBxN serum-transfer model of arthritis (KIBxN serum was a kind gift from Dr David Lee, Harvard). Mice were injected with 80 tI filter-sterilized (0.2 !m) KIBxN serum i.p. and measurement of joints was performed on days 0, 2, 4, 6, 8, and 11 with a Mitutoyo Dial Thickness Gage (0O1 mm, Japan). Surprisingly, Kit’” mice were most susceptible to RA. Shipl’ and Ship 1’ reconstituted mice had virtually identical inflammation, followed by B6 controls (Figure A.1). Thus, Ship1 mast cells did not appreciably affect the pathology of RA In vivo. In the future, it would be interesting to determine whether ubiquitous Ship1 mice suffer increased RA symptoms in this model, potentially mediated by mast cell-independent mechanisms.  91  100 —f— B6 KIt’5tIWth  —--Ship÷/+ BMMC —.— Ship-/- BMMC  80  E  :  $20  ci) U  0  -20 0  2  4  6 Day  8  10  12  Figure A.1 Similar rheumatoid arthritis pathology in mice reconstituted with wild-type and Shipi’ mast cells Arthritis was induced in age- and sex-matched B6 (n=3), Ship1-BMMC (n=3), Ship1’-BMMC (n=3), and Kit” (n=3) mice via ip. injection of KIBxN serum. Thinkness of the hind limbs was measured on days 0, 2, 4, 6, 8, and 11. Bars represent SE.  92  A.2 MIGRATION AND SURVIVAL OF SHIPI’ MAST CELLS IN VITRO We suspected that the mast cell hyperplasia of Shipl’ mice was due to the expansion of mast cells in response to IL-6 and TNF each of which can act as mast cell growth factors (Gounaris, Erdman, et al, 2007; Hu, Zhao, and Shimamura, 2007). To determine whether enhanced migration or survival of ShIpl’ mast cells were also responsible for the mast cell hyperplasia, we first performed in vitro transwell migration assays with Shipl’ BMMC. We found that Shipl’ mast cells had impaired migration to SCF, with over three times more wild-type cells migrating than Ship1 mast cells (Figure A.2A). Briefly, Ship 1” and Shipl’ BMMC were starved for 18 h and then 5 x iü cells were placed in the upper well of an uncoated transwell migration chamber (8.0 im pore size; Corning Costar). A range of rmSCF concentrations were added to the lower chamber and the transwell plates were then incubated in a 37°C CO 2 incubator for 4 h. The cells that reached the lower well were enumerated using a flow cytometer (10 m latex bead standards were added to each well for quantification). The number of Ship1 BMMC that migrated to 10 ng/ml rmSCF was defined as maximum (100%), and all other conditions were expressed as a percent thereof. Next, to determine whether Shipl’ mast cells have enhanced survival, we subjected Ship1 BMMC to suboptimal growth conditions (removal of IL-3) and monitored their survival by manually counting viable cells. We found that Ship1 mast cells and wild-type mast cells had comparable in vitro survival kinetics in response to cytokine withdrawal (Figure A.2B).  93  A E 100 c  E  -  80-  0  o  40  -  0)  E 0.01  B  1 0.1 100 10 SCF concentration (ngfml)  14 12 10 06 0  E  z  2 0 0  2  4 Day  6  8  Figure A.2 Defective migration and similar survival of Ship1 mast cells in  vitro (A) Ship1 (blue line) and Shipl’ BMMC (red line) migration to rmSCF was assessed using in vitro transwell assays. Values are expressed as percentages of the number of Ship1’ BMMC that migrated to 10 nglml rmSCF. (B) Ship1’ (blue line) and Shipl’ BMMC (red line) were subjected to suboptimal growth conditions (culture in BMMC media lacking IL-3) and their survival was monitored via trypan blue counts using a hemacytometer. Data in A and B represent pooled results from  two independent experiments. Bars represent SE.  94  A.3 HISTOLOGICAL ANALYSIS OF SHIP1 MAST CELLS Figure A.3 Histological analysis of tissue mast cells in Shipl’ mice Highly metachromatic, toluidine blue stained mast cells are visible in the lung, mesentery, skin, and spleen of Shipl’ and Ship1 mice. CAE positive (red) mast cells are visible in the colon and ileum (bars  =  25 pm). Image insets show digitally  enlarged views of tissue mast cells.  95  c,)  CD  Colon Ileum  Mesentery •  -  ,  e.  ‘:  Lung ‘  ‘I  •5  -‘J  [  5-..  ,•  ..  1  .  .  •:  Skin  -s  Spleen  Figure A.4 Histological analysis of tissue mast cells in mice reconstituted with Ship f’ mast cells Highly metachromatic, toluidine blue mast cells are visible in the lung, mesentery, skin, and spleen of Ship1-BMMC and Ship1’-BMMC mice. CAE positive (red) mast cells are visible in the colon and ileum (bars  =  25 !tm). Image insets show  digitally enlarged views of tissue mast cells.  97  CD 0,  Colon  Ileum  “  ‘,  •  -  .  Mesentery  ,-  lb  S  ..:.‘.  ‘‘  I  r  ••••••  ‘•:,  •c._-*4_-_.  .,••1  -  Lung Skin Spleen  C)  C,)  C)  + +  Cd)  0)  Figure A.5 Cell autonomous mucosal mast cell hyperplasia in the intestines of Ship f’ mice, and mice reconstituted with Ship f’ mast cells Tissue mast cells of the muscularis and mucosal layer of the ileum (A) and colon (B) of age- and sex-matched Shipl’ (n6) and Ship 1’ (n=6) mice were enumerated using chioroacetate esterase (CAE) histological staining revealed that Shipf’ mice also have more mast cells in the mucosa of their ileum and colon. CAE stains were performed on the mucosal layer (C, D) and muscularis (E, F) of the colon and ileum of B6 (n=3), Shipl’-BMMC (n=3), Ship1’-BMMC (n=2), and Kit sh/Wsh  (n=3) mice mast cell hyperplasia in the spleen, lung, colon, ileum, mesentery,  and back skin of Shipl’-BMMC mice, with both connective and mucosal type mast cells contributing to this phenotype. Bars represent SE.  99  0 0  -  0  0)  C  U) C-)  C  U)  -I  0)  C  C Co C)  -  -  I  I  C)  C C) 0 Co  Ship-I- BMMC  KItW&/sh  Ship-/- BMMC  Ship+/+ BMMC  I  C)  KjtWsh/WSh  Ship-I- BMMC  Ship+/+ BMMC  I  C) C  (DC)  B6  I  CD  1’3 C)  I  I  B6  11 ° t Kit’’ ’  Ship-I- BMMC  CD  I  Ship+/+ BMMC  I  Ship+I+ BMMC  I  B6  I  B6  I  00000000C)  m  F  I  [iI  I  (31  I  -  I  0)  I  CD  .a  [I  J-H  } 1  C)  C  I  CO  Mast celts (/mm ) 2 —  I  C  I  (31  C  0  1  0)  C  Cl) C-)  C) 0 Cl) 0)  C  3  0)  8Co  C  C)  Ship-I-  Ship÷/+  Ship-I  Ship+/+  Ship-I  Ship+/+  Ship-I  Ship+/+  F  C  p  C)  -  CD  I  p  0 0 0  M C)  -  1  I  b  (.) C  0  0  1?0  C)  .  D  0 0  C-)  C  CD  A.4 REFERENCES Ditzel, H.J. (2004). The K/BxN mouse: a model of human inflammatory arthritis. Trends Mol. Med. 1, 40-45. Gounaris, E., Erdman, S.E., Restaino, C., Gurish, M.F., Friend, D.S., Gounari, F., Lee, D.M., Zhang, G., Glickman, J.N., Shin, K. et al. (2007). Mast cells are an essential hematopoietic component for polyp development. Proc. NatI. Acad. Sci. U. S. A. 50, 19977-1 9982. Hu, Z.Q., Zhao, W.H., and Shimamura, T. (2007). Regulation of mast cell development by inflammatory factors. Curr. Med. Chem. 28, 3044-3050. Lee, D.M., Friend, D.S., Gurish, M.F., Benoist, C., Mathis, D., and Brenner, M.B. (2002). Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science 5587, 1689-1692. Maccioni, M., Zeder-Lutz, G., Huang, H., Ebel, C., Gerber, P., Hergueux, J., Marchal, P., Duchatelle, V., Degott, C., van Regenmortel, M., Benoist, C., and Mathis, D. (2002). Arthritogenic monoclonal antibodies from K/BxN mice. J. Exp. Med. 8, 1071-1077. Zhou, J.S., Xing, W, Friend, D.S., Austen, K.F., and Katz, H.R. (2007). Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J. Exp. Med. 12, 2797-2802.  101  APPENDIX B. SUPPLEMENTAL DATA ON PRPC IN MAST CELLS Figure B.1 Mast cells express high levels of PrPC and downregulate Prnp transcription upon FcERI stimulation (A) Western blot of whole cell lysates of Prnp’ and Prnp°’° BMMC with anti- PrPC 6H4. (B) Flow cytometry analysis of peritoneal wash cells collected from Prnp’ and Prnp°’° mice indicates that PWMC express the highest levels of PrPC (cells in quadrant IV of both plots are false positive B lymphocytes). (C) Gene expression analysis revealed that mast cells rapidly downregulated levels of Prnp transcript following stimulation with IgE, or IgE  +  Ag. (D) BMMC were stimulated with anti-DNP  IgE, followed by DNP-HSA, or with A23187, for 15 minutes. While IgE crosslinking induced a reduction in PrPC levels in the pellets (Figure 2C), PrPC wasn’t detected in the supernatant, suggesting that the reactive epitope is destroyed. (E) Prnp°’° and Prnp’ BMMC were stimulated as above and their degranulation was measured by -hexosaminidase release. Data are representative of two separate experiments.  102  C  cx,  -  N) C,)  -U  z  c5  A23187  Vehicle  lgE+DNP  gE  IgE÷Ag 6hr  IgE+Ag lhr  01  .  -.  0  -‘  01  1’) 0  C)  o  I  01  C)  -  01  -  r1  I  N) C)  Degranulation  I  (71  N)  (%) (71 C  -  C)  C)  m  PrPC concentration (normalized) pp p p p : o r’ a a o I’.)  }  IgE]  Untreated  c,  Normalized intensity (x 103)  C)  -  --  01 01  c-kit  .  c  -4  APPENDIX C. UBC RESEARCH ETHICS BOARD CERTIFICATES OF APPROVAL  104  iJjj1  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A06-1483 Investigator or Course Director: Kelly McNagny Department: Biomedical Research Centre (BRC) Animals:  Mice WBB6F-1 J-Kit wI Kit w-v 120 Mice C57B116-CD34 120 Mice PHIL 120 Mice C57Bl/6 280  Start Date: July 21, 2003  Approval  August 11, 2008  Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title:  Michael Smith Foundation for Health Research CD34 in inflammatory cell migration and function Canadian Institutes of Health Research (CIHR) Role of mast cells and eosinophils in allergic inflammation and fibrosis of the lung Stem Cell Network (SCN) Networks of Centres of Excellence (NCE) -  Cell therapy for muscular disease Allergy, Genes and Environment Network (AllerGen) Networks of Centres of Excellence (NCE)  -  CanGoFar Programme B diagnostics and therapeutics -  105  Funding Agency: Funding Funding Agency: Funding Title Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title:  Unfunded title:  Canadian Institutes of Health Research (CIHR) CD34 in inflammatory cell migration and function Stem Cell Network (SCN) Networks of Centres of Excellence (NCE) -  Cell theraphy for muscular disease  Taplow Ventures Ltd. Cell Therapy for Muscular Disease Canadian Institutes of Health Research (CIHR) CD34 as a marker of murine mast cells: expression, regulation and function Canadian Allergy, Asthma and Immunology Foundation Role of CD34 and CD43 in mast cells migration in asthma, occupational asthma and hypersensitivity pneumonitis Taplow Ventures Ltd. Characterization of mouse models of hematopoietic stem cell disease  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project.  This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  106  1  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A07-0376 Investigator or Course Director: Kelly l4cNagny Department: Biomedical Research Centre (BRC) Animals:  Mice C57B116 wt 16 Mice C57B1/6-CD34 16 Mice WBB6F-l J-Kit wI Kit w-v 50 Mice C57B116 Ly5.1 60  Start Date: July 1, 2007  Approval  January 31, 2008  Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding  Michael Smith Foundation for Health Research A role for CD34 in the development of inflammatory and allergic diseases Canadian Institutes of Health Research (CIHR) Role of mast cells and eosinophils in allergic inflammation and fibrosis of the lung Canadian Institutes of Health Research (CIHR) .  .  .  CD34 in inflammatory cell migration and function  Taplow Ventures Ltd.  Characterization of mouse models of hematopoietic stem cell 107  Title:  disease  Unfunded title:  N/A  The Animal Care Committee has examined and approved th use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  108  

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