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Regulation of mast cell activation Kalesnikoff, Janet 2003

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REGULATION OF MAST CELL ACTIVATION  By JANET KALESNIKOFF B.Sc, The University of British Columbia, 1997  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In THE FACULTY OF GRADUATE STUDIES Department of Medicine; Experimental Medicine Programme  We accept this thesjs-arr^forming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 2003 © Janet Kalesnikoff, 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or  by his  or  her representatives. It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of JExpoirirwQrAaA ^ oA\e>if\lLs The University of British Columbia Vancouver, Canada Date ^ C ^ . i ^ n ,  DE-6 (2/88)  3o/o3>  ABSTRACT  SH2-containing inositol 5'-phosphatase (SHIP) is a 145kDa protein that becomes both tyrosine phosphorylated and associated with the adapter protein She following the stimulation of hemopoietic cells with a variety of extracellular stimuli. SHIP typically acts as a negative regulator of hemopoietic cell activation, at least in part, by hydrolyzing the phosphatidylinositol 3'-kinase (PI3K) generated second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). To gain further insight into SHIP'S role in mast cell development and activation, we generated bone marrow-derived mast cells (BMMCs) from SHIP+/+ and -/- mice. We found that mature B M M C s from these mice exhibit comparable receptor expression profiles, total granularity and granular content, despite the faster rate of development observed in the absence of SHIP. Following stimulation of these cells, we found that IgE (Immunglobulin E) + antigen (Ag)induced degranulation, arachidonic acid (AA) metabolism, and proinflammatory cytokine production are substantially higher in SHIP-/- than +/+ B M M C s . Focusing on cytokine production, we demonstrate herein that SHIP negatively regulates interleukin (IL)-6 production by inhibiting nuclear factor K B ( N F K B ) activity. Using various pathway inhibitors we determined that the PI-3K/Protein Kinase (PK)B and P K C pathways, which are elevated in IgE+Ag-induced SHIP-/- cells, elevate IL-6 m R N A synthesis, at least in part, by enhancing the phosphorylation of k B and N F K B DNA binding. Conversely, the Erk and p38 pathways, which are also elevated in SHIP-/- cells, enhance IL-6 m R N A synthesis by increasing the transactivation potential of N F K B . Taken together, our results are consistent with a model in which SHIP negatively regulates N F K B activity and IL-6 synthesis by reducing IgE+Ag-induced P I P 3 levels and thus P K B , P K C , Erk and p38 activation.  Although IgE binding to mast cells is thought to be a passive pre-sensitization step in the current mast cell paradigm, we observed that SHIP-/- B M M C s degranulate in response to IgE alone, unlike their wildtype counterparts. We explored this phenomenon further and found that monomeric IgE (mlgE), in the absence of A g , stimulates multiple phosphorylation events in normal B M M C s . While mlgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE+Ag. Moreover, mlgE prevents the apoptosis of cytokine-deprived B M M C s , likely by maintaining.BCI-XL levels and inducing the production of autocrine-acting cytokines. Since IgE concentrations as low as 0.1 pg/ml enhance B M M C survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals. We also found that IgE alone triggers the adhesion of mast cells to the connective tissue component fibronectin (FN). This adhesion occurs to the same extent as  ii  that triggered by optimal levels of stem cell factor (SCF) or IgE+Ag and is mediated by an increased avidity of integrin aspY Moreover, IgE-induced adhesion requires PI3K, phospholipase C (PLC)y and extracellular calcium (Ca ) but not Erk or p38. We demonstrated, using a Ca channel blocker and Lyn-/- BMMCs, that 2+  2+  both IgE- and IgE+Ag-induced adhesion to FN require extracellular C a  2+  entry whereas SCF-induced  adhesion does not. Furthermore, our data suggest that FN acts synergistically with IgE to prolong intracellular phosphorylation events and enhance IgE-induced cytokine production and BMMC survival.  iii  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF FIGURES  viii  LIST OF TABLES  x  LIST OF ABBREVIATIONS  xi  CONTRIBUTIONS OF OTHERS  xvii  ACKNOWLEDGEMENTS  xviii  CHAPTER 1  1  INTRODUCTION  1.1  HEMOPOIESIS  1  1.2  MAST CELL DEVELOPMENT  3  1.2.1  Mucosal (MMCs) versus connective tissue mast cells (CTMCs)  1.3  5  MAST CELL FUNCTION  6  1.3.1  Antibodies (Abs)  7  1.3.2  IgE production  8  1.3.3  Type I hypersensitivity reactions  9  1.3.4  IgE-dependent mast cell activation  11  1.3.4.1 Degranulation 1.3.4.2 Arachidonic acid (AA) metabolism 1.3.4.3 Cytokine and chemokine production 1.3.5  IgE-independent mast cell activation  11 12 13 14  1.4  MAST CELLS AND DISEASE  1.5  SIGNAL TRANSDUCTION  17  1.6  RECEPTORS  17  1.6.1  c-kit  19  1.6.2  IL-3 receptor (IL-3R)  19  1.6.3  FcsRI  20  1.6.4  Integrins  21  1.7  KINASES 1.7.1  22 Protein tyrosine kinases (PTKs)  17.11 1.7.2 1.8  16  22  Src family kinases  23  Phosphatidylinositol (PI) kinases  24  1.7.2.1 Phosphatidylinositol  24  3'-kinases (PI3Ks)  PHOSPHATASES  26  1.8.1  PTEN  27  1.8.2  Inositol polyphosphate 5'-phosphatases (5-ptases)  28  Group I inositol polyphosphate 5-ptases  28  18.2.7  iv  1.8.2.2  1.8.3  Group II inositol polyphosphate 5-ptases  18.2.3 Group III inositol polyphosphate 5-ptases SH2-containing inositol 5'-phosphatase (SHIP) 1.8.3.1 SHIP knockout (-/-) mice  1.9  1.10 1.11  1.12 CHAPTER 2 2.1  PROTEIN-PROTEIN INTERACTIONS 1.9.1 Phosphorylation-dependent protein interaction modules 1.9.2 Phosphorylation-independent protein interactions modules PROTEIN-LIPID INTERACTIONS IgE RECEPTOR MEDIATED SIGNALING EVENTS 1.11.1 Immunoreceptor tyrosine based activation motifs (ITAMs) 1.11.2 Early FcsRI phosphorylation events 1.11.3 Negative regulation of FcsRI signaling AIMS OF STUDY MATERIALS AND METHODS  2.3  TISSUE CULTURE 2.1.1 Bone marrow-derived mast cells (BMMCs) 2.1.2 Swiss 3T3 fibroblasts 2.1.3 Connective tissue mast cells (CTMCs) 2.1.4 Sca-1 Lin-bone marrow isolation PROTEIN ANALYSIS 2.2.1 Cell stimulations, total cell lysates (TCLs), immunoprecipitations (IPs), and Western blot analysis 2.2.2 Inhibitors 2.2.3 Antibodies (Abs) 2.2.4 Plasma membrane preparation 2.2.5 Flow cytometry 2.2.6 Alcian blue/safranin staining BIOLOGICAL ANALYSIS OF BMMCs 2.3.1 Degranulation assays 2.3.2 LTC4/D4/E4 enzymeimmunoassays 2.3.3 RNase protection assays (RPAs) 2.3.4 ELISAs 2.3.5 Cell transfections and luciferase assays 2.3.6 Nuclear extract preparation 2.3.7 Electrophoretic mobility shift assays (EMSAs) 2.3.8 Preparation of monomeric IgE (mlgE) 2.3.9 Survival studies 2.3.10 CFSE labelling 2.3.11 Tritiated thymidine assays 2.3.12 Apoptosis/DNA fragmentation assays 2.3.13 Removal of IgE from BMMC conditioned medium 2.3.14 Adhesion assays 2.3.15 Intracellular Ca measurements SHIP MUTAGENESIS/ADDBACK BMMCs 2.4.1 Viral infection of bone marrow cells 2.4.2 SHIP point mutations 2+  2.4  34  35 36 37 38 39 39 40 44 46 47  +  2.2  29  31 32  \  V  47 47 47 47 48 48 48 49 50 50 51 51 51 51 52 52 53 53 54 54 54 54 55 55 55 55 56 56 57 57 57  CHAPTER 3 3.1 3.2  INTRODUCTION RESULTS 3.2.1 BMMCs differentiate faster in the absence of SHIP 3.2.2 Sca-1-Lin- cells differentiate faster in the absence of SHIP 3.2.3 FcsRI and c-kit expression levels are comparable in mature BMMCs derived from SHIP+/+and-/-mice 3.2.4 The granule content of mature BMMCs from SHIP+/+ and -/- mice is comparable DISCUSSION  3.3 CHAPTER 4 4.1 4.2  4.3 CHAPTER 5 5.1 5.2  5.3  A COMPARISON OF MAST CELLS DERIVED FROM SHIP+/+ AND -/- MICE  SHIP NEGATIVELY REGULATES IgE+Ag-INDUCED IL-6 PRODUCTION BY INHIBITING N F K B ACTIVITY  INTRODUCTION RESULTS 4.2.1 SHIP negatively regulates cytokine production in activated BMMCs 4.2.2 Addition of WT but not phosphatase deficient SHIP to SHIP-/- BMMCs reduces IL-6 production to that seen in SHIP+/+ BMMCs 4.2.3 IgE+Ag activates multiple pathways to a greater extent in SHIP-/-than+/+BMMCs 4.2.4 IgE+Ag-induced IL-6 production in BMMCs is dependent on the activation of the PI3K, PKC, Erk and p38 pathways 4.2.5 IKB phosphorylation/degradation and N F K B DNA binding and transactivation are higher in IgE+Ag-induced SHIP-/- BMMCs 4.2.6 PI3K/PKB and PKC enhance IKB phosphorylation/degradation and N F K B binding to DNA while Erk and p38 stimulate N F K B transactivation DISCUSSION  59 59 59  61 62 62 65 65 66 66 67 69 71 71 76 78  MONOMERIC IgE STIMULATES SIGNALING PATHWAYS IN MAST CELLS THAT LEAD TO CYTOKINE PRODUCTION AND CELL SURVIVAL INTRODUCTION RESULTS 5.2.1 IgE alone stimulates the phosphorylation of the Erks, p38, JNK, and PKB in normal murine BMMCs 5.2.2 IgE alone likely acts through the FcsRI and lipid rafts 5.2.3 IgE alone is more effective than IgE+Ag at increasing the levels of multiple cytokines 5.2.4 IgE enhances mast cell survival and does so by preventing apoptosis 5.2.5 The autocrine production of cytokines contributes to IgE-induced BMMC survival DISCUSSION VI  59 60  83 83 84 84 87 89 92 96 98  CHAPTER 6 6.1 6.2  IgE TRIGGERS THE ADHESION OF MAST CELLS TO FIBRONECTIN AND THIS ENHANCES CYTOKINE PRODUCTION AND MAST CELL SURVIVAL  INTRODUCTION RESULTS 6.2.1 IgE alone stimulates the adhesion of BMMCs and CTMCs to FN 6.2.2 IgE alone stimulates the adhesion of BMMCs to FN via an increase in the avidity of VLA-5 6.2.3 IgE-induced adhesion of BMMCs to FN requires PI3K but not Erk or p38 6.2.4 IgE, but not SCF, requires entry of extracellular C a to mediate BMMC adhesion to FN 6.2.5 VLA-5 activation acts together with IgE to prolong intracellular signaling and enhance cytokine production and BMMC survival DISCUSSION  103 103 104 104 105 107  2+  6.3  CHAPTER 7  SUMMARY AND PERSPECTIVES  107  112 113  118  REFERENCES  125  APPENDIX I  A  vii  LIST OF FIGURES  CHAPTER 1  Figure Figure Figure Figure Figure Figure  1.1 1.2 1.3 1.4 1.5 1.6  Figure 1.7 Figure 1.8  A tentative scheme of hemopoiesis and its growth factors A schematic drawing of an Ab IgE-dependent mast cell activation The structures of c-kit, IL-3R, FcsRI and asPi Phosphoinositide metabolism The structures of group I, II, and III inositol polyphosphate 5-ptases Inositol polyphosphate 5-ptase substrate specificity Early FcsRI phosphorylation events  2 8 10 20 25 29 31 42  CHAPTER 2  No Figures CHAPTER 3  Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4  Cultured bone marrow cells from SHIP-/- mice express FcsRI faster than their wildtype counterparts Cultured Sca-1i_in- cells purified from SHIP-/- bone marrow express FcsRI faster than their wildtype counterparts FcsRI and c-kit expression levels are comparable in mature BMMCs derived from SHIP+/+and-/-mice The granule content of mature BMMCs from SHIP+/+ and -/mice is comparable  60 61 61 63  CHAPTER 4  Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10  SHIP negatively regulates IgE+Ag-induced mast cell degranulation and LTCt production SHIP negatively regulates cytokine production in BMMCs Addition of wildtype, but not phosphatase deficient, SHIP reverts IL-6 production in SHIP-/- to SHIP+/+ BMMC levels SHIP represses multiple IgE+Ag-induced signaling pathways in BMMCs •. IgE+Ag-induced IL-6 production in BMMCs is dependent on the activation of the PI3K, PKC, Erk and p38 pathways N F K B regulates IL-6 production in BMMCs IKB phosphorylation and degradation are higher in SHIP-/- BMMCs SHIP negatively regulates IgE+Ag-induced N F K B DNA binding SHIP negatively regulates IgE+Ag-induced N F K B transactivation The PI3K/PKB, PKC, Erk and p38 pathways cross-talk in IgE+Agstimulated BMMCs viii  66 67 68 70 72 73 74 75 76 77  Figure 4.11  PI3K/PKB and PKC enhance IKB phosphorylation/degradation, N F K B DNA binding and transactivation while Erk and p38 only stimulate N F K B transactivation  79  A model of IgE+Ag-induced IL-6 mRNA synthesis  82  Figure 5.1  IgE alone causes mast cell degranulation in the absence of SHIP  83  Figure 5.2  IgE, in the absence of Ag, stimulates multiple phosphorylation events Phosphorylation events are delayed but far more prolonged in response to IgE alone versus IgE+Ag IgE dose response studies reveal an EC50 of 1 pg/ml Monomeric IgE stimulates Erk phosphorylation IgE likely acts through the FcsRI and lipid rafts IgE increases multiple cytokine mRNA levels IgE increases multiple cyotkine mRNA and protein levels more than IgE+Ag IgE alone enhances BMMC survival IgE alone maintains BCI-XL levels and prevents apoptosis of BMMCs Monomeric IgE enhances mast cell survival IgE-induced cytokines act in an autocrine manner to enhance BMMC survival A combination of cytokines (IL-2, -3, -4, -6, -13, & TNFa) can enhance BMMC survival, although to a lesser degree than that achieved with 10ug/ml IgE  Figure 4.12 CHAPTER 5  Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure Figure Figure Figure  5.9 5.10 5.11 5.12  Figure 5.13  85 86 87 88 90 91 93 94 95 96 97 98  CHAPTER 6  Figure 6.1 Figure 6.2 Figure 6.3 Figure 6.4 Figure 6.5  Figure 6.6 Figure 6.7 Figure 6.8 CHAPTER 7  No Figures  IgE alone stimulates the adhesion of both BMMCs andCTMCstoFN IgE alone stimulates the adhesion of BMMCs to FN via an increase in the avidity of VLA-5 IgE-induced adhesion of BMMCs to FN requires PI3K but not Erk or p38 IgE, but not SCF, requires the entry of extracellular C a to trigger BMMC adhesion to FN Lyn-/- BMMCs, which do not show increased intracellular C a with IgE, SCF, or IgE+DNP-HSA, display impaired adhesion to FN in response to IgE or IgE+DNP-HSA but not to SCF IgE, but not SCF, may utilize a Ca -dependent PKC to trigger BMMC adhesion to FN FN binding acts to enhance IgE-induced intracellular signaling events, cytokine production, and survival A model of IgE-stimulated adhesion to FN  105 106 108  2 +  109  2 +  110  2+  111 113 115  LIST OF TABLES  CHAPTER 1  Table 1.1  Granule content of mouse and human mast cells  5  Abs used in this thesis  50  CHAPTER 2  Table 2.1 CHAPTER 3  No Tables CHAPTER 4  No Tables CHAPTER 5  No Tables CHAPTER 6  No Tables CHAPTER 7  No Tables  X  LIST OF ABBREVIATIONS  •/•  knockout  +/+  wildtype  2-APB  2-amino ethoxydiphenyl borate  A  alanine  AA  arachidonic acid  Ab  antibody  aka  also known as  Ag APC  antigen  ATP  adenosine triphosphate  BCR  B cell receptor  BFU  burst forming unit  BMMC  bone marrow derived mast cell  BSA  bovine serum albumin  Btk  Bruton's tyrosine kinase  C  cysteine  C-  carboxy-  antigen presenting cell  Ca  calcium  cDNA  complementary deoxyribonucleic acid  CFSE  carboxyfluorescein diacetate succinimidyl ester  CFU  colony forming unit  CML  chronic myelogenous leukemia  COX  cyclooxygenase  cPKC  classical protein kinase C  CSF  colony stimulating factor  Csk  carboxy-terminal Src kinase  CTMC  connective tissue mast cell  D  aspartic acid  Dab1  Disabled 1  DAG  diacylglycerol  2+  xi  DNA  deoxyribonucleic acid  DNP-HSA  dinitrophenyl-human serum albumin  ECM  extracellular matrix  E. coli  Eschericia coli  EDTA  ethylenediaminetetraacetic acid  EGF  epidermal growth factor  EGTA  ethyleneglycol-bis ((3-animoethyl ether) N,N,N,N'-tetraacetic acid  EH  Eps15 homology  ELISA  enzyme-linked immunosorbent assay  Epo  erythropoietin  EpoR  erythropoietin receptor  ER  endoplasmic reticulum  Erk  extracellular regulated kinase  ES cell  embryonic stem cell  F  phenylalanine  Fab  fragment antigen binding  FACS  fluorescence activated cell sorting  FAK  focal adhesion kinase  Fc  fragment crystalline  FceRI  Fc epsilon receptor I  FcyR  Fc gamma receptor  FCS  fetal calf serum  FHA  forkhead association  FITC  fluorescein isothiocyanate  FN  fibronectin  FYVE  Fab-1, YOTB, Vad, and EEA1  G  glycine  Gab2  Grb2 associated binder 2  GAP  GTPase activating protein  G-CSF  granulocyte colony stimulating factor  GFP  green fluorescence protein  GM-CSF  granulocyte monocyte colony stimulating factor  gp  glycoprotein xii  GPCR  G protein-coupled receptor  Grb2  Growth factor receptor binding protein 2  GTP  guanosine triphosphate  HA  hemagglutinin  H chain  heavy chain  HIV  human immunodeficieny virus  HPLC  high performance liquid chromatography  hr  hour  I  isoleucine  IAP  integrin associated protein  IKB  inhibitor KB  IKK  IKB kinase  ICAM  intercellular adhesion molecule  IBD  inflammatory bowel disease  IFN  interferon  Ig  Immunoglobulin  IgE  Immunoglobulin E  IL  interleukin  IL-1(-2, -3,or-6)R  IL-1(-2, -3, or-6) receptor  IMDM  Iscove's modified Dulbecco's medium  IP  immunoprecipitation  IP3  inositol-1,4,5-trisphosphate  IP4  inositol-1,3,4,5-tetrakisphosphate  IRS  inhibitory receptor superfamily  ITAM  immunoreceptor tyrosine based activation motif  ITIM  immunoreceptor tyrosine based inhibition motif  Jak  Janus kinase  JNK  c-Jun N-terminal kinase  kb  kilobase  kDa  kilodalton  KIR  killer inhibitory receptor  L  leucine  LAT  linker for activation of T cells xiii  L chain  light chain  LO  lipooxygenase  LPS  lipopolysaccharide  LT  leukotriene  MAFA  mast cell function-associated antigen  MAPK  mitogen activated protein kinase  MC-CPA  mast cell carboxypeptidase  MCP  . monocyte chemotactic protein  M-CSF  monocyte colony stimulating factor  Mg  magnesium  MHC  major histocompatibility  mlgE  monomeric IgE  min  minute  MIP  macrophage inflammatory protein  MMAC  mutated in multiple advanced cancers  MMC  mucosal mast cell  MMCP  murine mast cell protease  mRNA  messenger ribonucleic acid  MS  multiple sclerosis  MTG  monothioglycerol  N  asparagine  N-  amino-  NFAT  nuclear factor of activated T cells  NFKB  nuclear factor K B  2+  NK cell  natural killer cell  NP-40  nonident P-40  NSAID  nonsteroidal anti-inflammatory drug  P  proline  PBS  phosphate buffered saline  PDGF  platelet derived growth factor  PDK1  phosphoinositide-dependent protein kinase 1  PE  phycoerythrin  PG  prostaglandin xiv  PH  pleckstrin homology  PI  phosphatidylinositol  PI3K  phosphatidylinositol 3'-kinase  PI-4,5-P  phosphatidylinositol 4,5-bisphosphate  PIAS1  protein inhibitor of activated STAT1  PIP  phosphatidylinositol 3,4,5-trisphosphate  PIR  paired Ig-like receptor  pITAM  tyrosine phosphorylated ITAM  pITIM  tyrosine phosphorylated ITIM  PKB  protein kinase B  PKC  protein kinase C  PLA  phospholipase A  PLC  phospholipase C  pS  phosphoserine  PSB  phosphorylation solubilization buffer  PT  phosphothreonine  PTB  phosphotyrosine binding  ptase  phosphatase  PTEN  phosphatase and tensin homologue deleted on chromosome ten  PTK  protein tyrosine kinase  PTP  protein tyrosine phosphatase  PX  phox homology  pY  phosphotyrosine  R  arginine  RPA  RNase protection assay  RTK  receptor tyrosine kinase  s  seconds  S  serine  SAM  sterile alpha motif  SCF  stem cell factor  SDS-PAGE  sodium dodecyl sulfate polyacrylamide gel electrophoresis  SH  Src homology  She  Src homology and collagen  2  3  XV  SHIP  SH2-containing inositol 5'-phosphatase  SHP  SH2-containing protein tyrosine phosphatase  SIRP  signal regulatory protein  SOCC  store operated calcium channel  Sos  Son of sevenless  s-SHIP  stem cell SHIP  S/T  serine/threonine  STAT  signal transducers and activators of transcription  Syk  spleen tyrosine kinase  T  threonine  Tc cell  cytotoxic T cell  TCL  total cell lysate  TCR  T cell receptor  TF  transcription factor  TGF  transforming growth factor  TH cell  helper T cell  T 1 cell  type 1 helper T cell  T 2 cell  type 2 helper T cell  TLR  toll like receptor  TNF  tumor necrosis factor  TPO  thrombopoietin  TX-100  Triton X-100  V  valine  VEGF  vascular endothelial growth factor  VLA  very late antigen  W  tryptophan  X  any amino acid  Y  tyrosine  H  H  xvi  CONTRIBUTIONS OF OTHERS  I would like to acknowledge those that contributed reagents and/or data vital to the completion of this thesis. I would like to acknowledge Dr. Jacqueline E. Damen and Dr. Mark D. Ware for the construction of the various SHIP mutants, the retroviral infection of SHIP-/- bone marrow cells, and the generation of the wildtype and D675G SHIP expressing BMMCs I used in chapter 4 (Figure 4.3). I would also like to recognize Dr. Jacqueline E. Damen's work in measuring the phospholipid levels in the wildtype and D675G addback mast cells (not a pretty procedure!). Thanks to Michael R. Hughes for the generation of data presented in Figure 4.4B and Dr. Michael Leitges (Max-Planck-lnstitute for Experimental Endocrinology, Hannover, Germany) for sending us supernatants from IgE+Ag-stimulated PKC5+/+ and -/- BMMCs (Figure 4.5B, right panel). I would like to acknowledge Dr. Michael Huber for his role in the initiation of the studies presented in chapter 5 and, more specifically, for the generation of Figure 5.2B. I would like to acknowledge Dr. Juan Zhang for the data presented in figure 5.4B and Dr. Reuben P. Siraganian for sending us the IgE producing hybridoma supernatants used in chapter 5 (both from the National Institutes of Health, Bethesda, MD). I would like to acknowledge Vivian Lam with whom I divided the work presented chapter 6. Vivian Lam played a vital role in the generation of the initial IgE-mediated adhesion data and the subsequent inhibitor studies. I would also like to thank Vivian for the generation of monomeric IgE via HPLC (Figure 5.5A), the tritiated thymidine assay shown in Figure 5.10C, the data presented Figure 5.9A, and the adhesion assays shown in Appendix I. I would like to recognize Nicole Baur and Corinna W.K. Lee, two extremely talented co-op students, for their help in the generation of data presented in chapter 4 and chapter 6, respectively. I would like to thank another excellent co-op student, Karen Chan, for her help in the generation of data presented in Appendix I. Finally, I would like to thank Dr. Janet M. Oliver for sending us the Lyn+/+ and -/- bone marrow cells that we used to generated BMMCs for the studies in chapter 6 (Figure 6.5), and our collaborator, Dr. Bridget S. Wilson, for sending us the Liu (Liu et al., 1980) IgE discussed in chapter 7 (both from the University of New Mexico, Albuquerque, NM).  xvii  ACKNOWLEDGEMENTS  Where to begin... First and foremost, I would like to acknowledge Graeme McLean for his love, support and understanding during the pursuit of this Ph.D. thesis. Graeme, you continuously helped me put my stresses and worries into perspective and encouraged me when I needed it most. Thank you for helping me enjoy life and for making me laugh. I would like to acknowledge my entire family for their support over the years. To my parents, John and Vi, thank you for always believing in me and for your unconditional love. You are truly amazing parents, role models, and friends. I hope I can make you proud in all my future endeavors. To my brother Mike and my wonderful new sister-in-law Denise, thank you for your continued encouragement and friendship. I would also like to thank Graeme's family, Sandy, Jean, Drew and Zoe, for their support and love over the years. Special thanks to my friends, both old and new (and to Jonny, who is probably my only friend that will actually read this entire thesis). You helped me get where I am today and gave me sanity along the way. Although our lives are quickly changing and some of you are now far away, I know you will always be an important part of my life and your continued friendship is invaluable. I would like to acknowledge those of you that I had the pleasure of working with over the years. In particular, thanks to the Krystal lab members (both past and present) for creating a fun and productive lab atmosphere. Special thanks to Jackie, Mark, the Mike's (1, 2 and 3), Vivian, and Laura, for helpful discussions, great lunches, candy and coffee runs, and valuable friendships. To Christine, thanks for all your help over the years, your sense of humor, and your friendship. I would like to thank the members of my supervisory committee for many helpful discussions. And last, but certainly not least, I would like to thank Gerry for his supervision and guidance over the past five years. Gerry, you provided me with the opportunity to begin my scientific career in an excellent research environment. I have learned so much from you over the years and I am excited about a future in science. And for the record, we are winning.  xviii  Chapter 1  INTRODUCTION 1.1  HEMOPOIESIS Hemopoiesis is the formation of blood cells. The hemopoietic stem cell, which exists in the bone  marrow, is capable of both self-renewal and differentiation into all the mature blood cells types and some other cell types as well, such as bone osteoclasts and dendritic cells (Alberts, 1994; Janeway, 2001, Zhu and Emerson, 2002). There are two major classes of blood cells: red and white. Red blood cells or erythrocytes, which function to transport oxygen and carbon dioxide, remain within the blood vessels. Conversely, white blood cells or leukocytes, which fight infection, must cross the walls of blood vessels and migrate into tissues to perform their functions (Alberts, 1994; Janeway, 2001). Additionally, platelets, which are small cell fragments derived from megakaryocytes, circulate in the blood and help repair damaged blood vessels and aid in blood clotting (Alberts, 1994). White blood cells are further divided into three groups: granulocytes, monocytes and lymphocytes. Granuloctyes and monocytes are myeloid cells that share a common multipotent precursor cell with erythrocytes and platelets (Figure 1.1) (Janeway, 2001). Granulocytes contain numerous granules or lysosomes and secretory vesicles, and are classified based on granule content. The most abundant type of granulocyte is the neutrophil or polymorphonuclear leukocyte, which has a multilobed nucleus and primarily functions to phagocytose and destroy invading bacteria. Other granulocytes include basophils and mast cells, which secrete histamine and serotonin from their granules to regulate inflammatory reactions, and eosinophils, which help destroy parasites and modulate allergic inflammatory responses (Alberts, 1994; Janeway, 2001). Monocytes, the next group of white blood cells, leave the blood vessels and mature into macrophages, which function as phagocytes along with neutrophils. In addition to phagocytosis of invading pathogens, macrophages remove dead or damaged cells in many tissues, and these cells are much larger and longer lived than neutrophils. Finally, there are two types of lymphocytes, B cells and T cells (Figure 1.1). B cells are responsible for the production of antibodies (Abs). T cells can be further divided into helper T cells (TH) or cytotoxic T cells (Tc), which regulate the activities of other leukocytes or kill virus infected cells, respectively (Alberts, 1994; Janeway, 2001). Additionally, natural killer (NK) cells are lymphocyte-like cells that can kill some types of tumor cells and virus infected cells.  l  BFU-E  CFU-E  EKLF F O G  Erythrocyte  Figure 1.1: A tentative scheme of hemopoiesis and its growth factors. The pluripotent stem cell divides infrequently to generate more stem cells (self-renewal; as indicated by^ ) or committed progenitor cells that can produce only one or a few types of blood cells. Progenitor cells are stimulated to proliferate and differentiate by specific growth factors (indicated on the arrows) but progressively lose their capacity for division and develop into terminally dfferentiated Wood cells, which live for only a few days or weeks. Only mature T cells, B cells, mast cells and macrophages are known to carry proliferative potential (as indicated by C ). Dotted arrows represent uncertain pathways. TFs involved in the dfferentiation of specfic pathways are indicated in italics under the arrows. GM-CSF = granulocyte monocyte colony stimulating factor; G-CSF = granulocyte colony stimulating factor; M-CSF = monocyte colony stimulating factor; TPO = thrombopoietin; EPO = erythropoietin. CFU = colony forming unit. BFU = burst forming unit Adapted from (Alberts, 1994) and (Zhu and Emerson, 2002).  2  The pluripotent stem cell gives rise to committed progenitor cells that, under the influence of various colony stimulating factors (CSFs) or growth factors (Figure 1.1), divide and differentiate into mature blood cells (Rane and Reddy, 2002). These mature cells usually die after several days or weeks, creating a hierarchy of hemopoietic cells. Both interleukin (IL)-3 and stem cell factor (SCF) play an important role in the proliferation and survival of numerous committed progenitor cells, whereas other factors specifically enhance the production and survival of a specific lineage. Erythropoietin (Epo), for example, selectively enhances erythroid cell proliferation and survival (Figure 1.1) (Alberts, 1994; Janeway, 2001; Rane and Reddy, 2002). Furthermore, we are beginning to understand the selective gene expression programs that are activated by transcription factors (TFs) and play a critical role in the development of different hemopoietic lineages (in italics in Figure 1.1) (Cantor and Orkin, 2001; Orkin, 1995; Zhu and Emerson, 2002). Evidence indicates that the development of a specific hemopoietic lineage depends on the crossantagonism of lineage-specific TFs (i.e. certain TFs actively repress alternate lineage gene programs) and the concentration at which a given TF is expressed plays a central role in this process (Figure 1.1) (Orkin, 1995). Overall, tight regulation of hemopoiesis is essential to prevent harmful proliferative disorders, such as leukemias, and to ensure the proper and timely expansion of lineages that carry out important effector functions, such as the initiation of an immune response against an invading pathogen.  1.2  MAST CELL DEVELOPMENT Mast cells were first identified in 1879 by Paul Ehrlich who called them "mastzellen" or "well fed  cells" because their cytoplasms were stuffed with prominent granules (Galli, 2000). As mentioned above, mast cells are hemopoietic cells of the myeloid lineage. Despite our growing understanding of hemopoiesis and its regulation by various growth factors (Figure 1.1), surprisingly little is known about mast cell development. For example, it is still debated whether the mast cell has its own lineage committed precursor or shares one with basophils (Figure 1.1) (Agis et al., 1993; Lunderius et al., 2000; Wedemeyer and Galli, 2000). However, it is widely accepted that the undifferentiated mast cell progenitor cell leaves the bone marrow and circulates in the blood and lymphatics. This allows mast cell progenitors to migrate to virtually all vascularized tissues, where they complete their maturation  under the control of the local  microenvironment and, under some circumstances, migrate into certain epithelia (Kawakami & Galli 2002; Galli, 2000; Gurish and Austen, 2001; Metcalfe et al., 1997; Welle, 1997).  During an inflammatory  response, existing mast cells are recruited to the site of inflammation where they can exert their effector functions. Mature mast cells also have proliferative potential, allowing for the rapid expansion of the local mast cell population during an inflammatory response (Galli, 2000). By contrast, basophils typically mature in the bone marrow then circulate in the blood, and mature basophils are recruited to tissues at sites of  3  immunological or inflammatory responses, but they cannot proliferate (Kawakami & Galli 2002; Galli, 2000). Of the mature hemopoietic cell types, only mature macrophages, T cells, B cells and mast cells are known to have proliferative potential (Figure 1.1). SCF (aka steel factor, kit ligand, or mast cell growth factor) has been identified as a critical growth factor for the survival, proliferation, differentiation, and enhanced functional activation of mast cells in both rodents and primates (Figure 1.1) (Ashman, 1999; Broudy, 1997; Galli, 2000). SCF, which exists in both soluble and membrane bound forms (Lev et al., 1992), binds to its receptor, c-kit, to enhance mast cell development. Dysregulation or mutations of either SCF or c-kit lead to mast cell disorders (Galli, 2000; Broudy, 1997). For example, gain-of-function mutations in c-kit occur in the vast majority of adult mastocytosis patients (Galli, 2000), and severe mast cell deficiencies (i.e. less than 1% of normal levels) are observed in W/W (mutations in both c-kit genes; (Kitamura et al., 1978)) and Sl/Sl (mutations in both v  d  SCF genes; (Kitamura and Go, 1979)) mice. Furthermore, the treatment of Sl/Sl  d  mice with SCF  dramatically increases mast cell numbers in the skin (Broudy, 1997). Studies with these mice also indicate that SCF is not required for basophil development nor does it significantly enhance basophil activation (Galli, 2000). By contrast, IL-3 appears to augment basophil production in mice and humans (Galli, 2000). Although IL-3 is a poor stimulator of mast cell development and proliferation under physiological conditions (Galli, 2000; Welle, 1997), studies in IL-3-/- mice show that IL-3 contributes to increased numbers of mast cells, in addition to basophils, following parasitic infection (Lantz et al., 1998). Furthermore, murine mast cell progenitors cultured in SCF plus IL-3 undergo optimal proliferation and maturation in vitro, and it is believed that cofactors, such as IL-3, IL-4 or IL-10 may be required for optimal SCF induced proliferation and differentiation in vivo (Broudy, 1997; Galli, 2000; Mekori and Metcalfe, 2000).  Mature mast cells can be identified by their receptor expression profile. As indicated above, the SCF receptor, c-kit, is expressed on these cells, since SCF is important for mast cell development and functional activation (section 1.6.1). Conversely, basophils do not express the SCF receptor (Lantz and Huff, 1995). The IL-3 receptor (IL-3R) is also expressed on mature mast cells, and IL-3 is the cytokine commonly used to culture mast cells from bone marrow in vitro (section 1.6.2). Finally, expression of the high affinity IgE receptor, FcsRI, is a useful marker of mast cell maturation (section 1.6.3). FcsRI is expressed exclusively on mast cells and basophils in rodents (Kinet, 1999; Lantz and Huff, 1995); however, this receptor is also found on monocytes, activated eosinophils, platelets, Langerhans cells and dendritic cells in humans, albeit at considerably lower levels than mast cells and basophils (Corry and Kheradmand, 1999; Kinet, 1999).  4  1.2.1  Mucosal (MMCs) versus connective tissue mast cells (CTMCs) Although mast cells can be identified by their receptor expression profile, mature cells exhibit  histochemical, biochemical and functional heterogeneity (Wedemeyer et al., 2000). For example, mast cells in different tissues vary markedly in granule content, which affects phenotype and function. Based on histochemical characteristics, two types of mast cells, mucosal (MMCs) and connective tissue mast cells (CTMCs), were originally reported in rodents (Gurish and Austen, 2001; Schwartz, 1994). It is now known that the histochemical difference between these mast cells reflects the presence or absence of the proteoglycan heparin in the mast cell granules; heparin is only found in CTMCs and can be visualized by staining with safranin (Table 1.1). However, it is unlikely that CTMCs express heparin during development and the neutral protease content of mast cells is now thought to be a more reliable way to distinguish these two mast cell types (Schwartz, 1994; Welle, 1997). This is especially true for human mast cells, which all express heparin, thus cannot be distinguished by safranin staining (Table 1.1) (Schwartz, 1994; Welle, 1997). Table 1.1: Granule content of mouse and human mast cells  Mouse  MMC  CTMCs  Biogenic amines  (low) histamine  (high) histamine & serotonin  Proteoglycans  chondroitin sulfate E  heparin  Proteases  MMCP-1,-2  MMCP-3,-4,-5,-6 & -7, MC-CPA  Human  MC  MCTC  T  Biogenic amines  histamine  histamine  Proteoglycans  heparin chondroitin sulfates A/E  heparin chondroitin sulfates A/E  Proteases  tryptase  tryptase, chymase, cathepsin G protease & C P A  Adapted from (Stevens etal., 1987), (Schwartz, 1994) and (Welle, 1997).  The majority of proteins found within rodent and human mast cell granules are neutral proteases (Schwartz, 1994; Welle, 1997). These proteases serve as the basis for mast cell classification in humans; human mast cells are classified as MCT (which contain the protease tryptase) or MCTC (which contain both tryptase and chymase) (Holgate, 1999; Schwartz, 1994). MCTC mast cells also contain carboxypeptidase (CPA) and cathepsin G protease (Table 1.1). The expression of murine mast cell proteases (MMCPs) and mast cell (MC)-CPA is indicated in Table 1.1; MMCP-1,-2, -3, -4 and -5 are chymases and MMCP-6 and -7 are tryptases (Schwartz, 1994; Welle, 1997). These MMCPs are stored in an active form; however, they cannot function in the acid pH of the granule. Other preformed mediators stored in mast cell granules include biogenic amines (histamine and serotonin) and proteoglycans (heparin and chondroitin sulfates); the distribution of these mediators is outlined in Table 1.1. In addition to imparting histochemical properties to rodent mast cells, heparin is required for the packaging of certain neutral proteases in murine 5  cytoplasmic granules (Schwartz, 1994; Wedemeyer et al., 2000). The distinct granular content of the different classes of mast cells explains, at least in part, the functional heterogeneity observed amongst mast cell populations. The two main types of mast cells are found at distinct sites in the body. MMCs are found in the mucosa of the gastrointestinal tract and the lamina propria of the respiratory tract, whereas CTMCs are located in the skin, the submucosa of the gastrointestinal tract and the peritoneum (Schwartz, 1994; Welle, 1997; Stevens et al., 1987). MCTS are thought to be the human equivalent of MMCs since they are also found in the intestinal mucosa and lung alveolar wall, and MCTC distribution mirrors that of CTMCs (Schwartz, 1994). Interestingly, nearly equivalent numbers of MCT and MCTC are found in the nasal mucosa (Schwartz, 1994). Both mast cell types are believed to arise from a common mast cell progenitor (Kobayashi et al., 1986). Furthermore, it has been shown that MMCs can differentiate into CTMCs and vice versa, suggesting that the local microenvironment plays a major role in determining mast cell phenotype (Welle, 1997).  1.3  MAST CELL FUNCTION As indicated above, mast cells are found in large numbers at sites where the body comes in  contact with the external environment (Abraham and Malaviya, 1997; Welle, 1997). Because these sites serve as the "portals of infection," where foreign material attempts to invade the host, mast cells are likely one of the first inflammatory cells encountered by invading pathogens (Abraham and Malaviya, 1997). Additionally, these sites are often proximal to blood vessels, lymphatic vessels and nerves, creating an ideal environment for mast cells to recognize invading pathogens or allergens and initiate first line defense mechanisms (Abraham and Malaviya, 1997; Welle, 1997). Mast cells are widely regarded as critical effector cells in IgE-dependent immune responses (Galli, 2000; Galli et al., 1999). On the one hand, this offers the body protective immunity in response to parasites. On the other hand, this same activation is responsible for the development of allergic or atopic disorders (Galli, 2000; Galli et al., 1999). "Allergic diseases, such as asthma, rhinitis, eczema and food allergies, are reaching epidemic proportions in both the developed and developing world" (Holgate, 1999); it is estimated that over half the population of the western world is atopic for one or more environmental allergen (Holgate, 1999; Janeway, 2001). The earliest known report describing an allergic reponse was written in 925 AD by Razi (Bungy et al., 1996). Since the 19 century, the fundamentals of allergy have gradually been th  elucidated, beginning with a description of the pathology of hay fever (1819), the discovery of a causative link with pollen grains (1873) and a transferable tissue sensitizing factor in serum (1921), later identified as 6  IgE (1967) (Holgate, 1999). Ironically, "the past 30 years have witnessed a spectacular increase in our knowledge of the cellular and molecular mechanisms of allergic disease, which has been paralleled by the rising trends in the incidence and health impacts of these diseases worldwide" (Holgate, 1999). Increased exposure to allergens and increased hygiene in western society are among the factors thought to be contributing to these rising trends (Holgate, 1999; Hopkin, 2002). The "hygiene hypothesis" currently finds most favor; it states that our "clean" societies deprive the immune system of important signals during critical periods of development (Holgate, 1999; Hopkin, 2002; Kinet, 1999). Due to the low rate of parasitic infection and the high incidence of allergic diseases, the mast cell is often referred to as the "appendix of the human immune system" in the industrialized world (Galli and Wershil, 1996). However, recent work shows that mast cells also contribute to the IgE-independent regulation of innate immunity (Galli et al.,  1999). To further explore and understand mast cell function, this section includes a brief description of Abs (section 1.3.1), including IgE, then focuses on the T cell-mediated immune response that regulates the production of IgE (section 1.3.2). This is followed by a detailed look at the IgE-mediated type I hypersensivity reaction (section 1.3.3) and the resulting proinflammatory mast cell mediators and their functions (section 1.3.4). This section ends with a look at the IgE-independent role of mast cells in innate and adaptive immune responses (section 1.3.5).  1.3.1  Antibodies (Abs)  Abs or immunoglobulins (Igs) are synthesized exclusively by B cell derived plasma cells and are one of the most abundant blood proteins, making up 20% of total plasma protein by weight (Alberts, 1994). Abs protect our bodies from infection by inactivating viruses and bacterial toxins and by recruiting white blood cells and the complement system to kill extracellular microorganisms and larger parasites (Alberts, 1994). Each Ab molecule consists of two identical light (L) chains and two identical heavy (H) chains held together by a combination of covalent and noncovalent interactions, creating a Y-shaped structure (Figure 1.2). Abs are bivalent; they have two identical antigen (Ag)-binding sites, or Fab (fragment antigen binding) portions. What happens to the Ab/Ag complex after binding is determined by the H chain, which makes up the Fc (crystalline) portion of the Ab molecule (Figure 1.2); this portion binds to specific Fc receptors that are differentially expressed in a cell type-specific manner (Alberts, 1994). The five classes of Abs found in higher vertebrates, IgA, IgD, IgE, IgG, and IgM, are distinguished by their heavy chains, a, 8, s, y, and \x, respectively. Additionally, IgG and IgA Abs can be further divided into subclasses (e.g. IgGl, which has a y1 chain) (Alberts, 1994). IgM is the first class of Ab produced by B 7  cells; pre-B cells synthesize u. H chains, which combine with L chains and insert into the membrane to serve as the B cell receptor (BCR). After IgM production, IgD can also be expressed on the B cell surface. Pentameric IgM is the major Ab class secreted during a primary Ab response and binding to Ag is followed by the Fc portion binding to and activating the complement system (Alberts, 1994). IgG, the most abundant Ab in the blood, is produced during secondary immune responses and can activate phagocytes in addition to the complement system following Ag binding (Alberts, 1994; Nielsen and Leslie, 2002). IgA is primarily found in secretions (milk, saliva, etc.) and IgE is found in extremely low levels in the blood due to an unusually high affinity for its receptor, FcsRI (section 1.6.3), expressed on mast cells and basophils. In addition to H chains, each Ab molecule consists of two identical L chains, which can be K or X chains, however, no functional differences between these two L chains have been defined to date (Pilstrom, 2002).  Figure 1.2: A schematic drawing of an Ab. Each Ab consists of two identical heavy chains (•) and two identical light chains (•). Ag-binding sites are formed by the amino (N)-terminal regions of both the light and heavy chains. The carboxy (C)-terminal of the heavy chain comprises the Fc portion of the Ab, which binds to specific Ig receptors. Disulfide bonds are shown in pink.  c c  1.32  IgE production The production of IgE is regulated by the TH2 cell-mediated immune response. As indicated earlier,  T cells are classified as Tc cells, which kill cells harboring harmful microbes (e.g. viruses or intracellular bacteria), or TH cells, which activate other white blood cells by secreting specific cytokines (Janeway, 2001). TH cells are further classified into two functionally distinct effector subtypes: TH1 (type 1 helper) and TH2 (type 2 helper) cells. TH1 cells secrete IL-2, interferon (IFN)y, tumor necrosis factor (TNF)a, lymphotoxin and other cytokines that activate macrophages to destroy microorganisms that they have ingested. TH1 cytokines also antagonize the allergic response by suppressing IgE synthesis (Corry and Kheradmand, 1999; Janeway, 2001). TH2 cells, on the other hand, secrete IL-4, -5, -6, -9 and -13, which stimulate B cells to proliferate and secrete Abs in response to allergens or large, extracellular pathogens, such as parasites (e.g. helminthes) (Corry and Kheradmand, 1999; Janeway, 2001). When the body is first exposed to a foreign Ag or innocuous allergen, the allergen is ingested by Ag-presentjng cells (APCs), such as dendritic cells (Figure 1.3) (Holgate, 1999; Janeway, 2001). T 2 cells H  are selectively activated or "armed" when their T cell receptors (TCRs) encounter specific peptide 8  fragments  of the allergen  c o m p l e x e s on the A P C  in the form of p e p t i d e : M H C  (major  histocompatibility)  s u r f a c e (Corry a n d K h e r a d m a n d , 1 9 9 9 ; J a n e w a y ,  class  II  molecule  2 0 0 1 ) . Additionally,  APCs  e x p r e s s a n u m b e r of costimulatory m o l e c u l e s that s y n e r g i z e with allergen to activate T H 2 c e l l s (Holgate, 1 9 9 9 ; J a n e w a y , 2 0 0 1 ) . L i k e the A g rec e p tor s o n T c e l l s , B c e l l s e x p r e s s r e c e p tor s that r e c o g n i z e a n d bind to the a l l e r g e n ; the allergen is internalized a n d d e g r a d e d by the B cell, then returned to the B cell s u r f a c e a s p e p t i d e s b o u n d to M H C c l a s s II m o l e c u l e s ( J a n e w a y , 2 0 0 1 ) .  T h i s p e p t i d e : M H C c l a s s II c o m p l e x o n B  cells is r e c o g n i z e d by allergen s p e c i f i c " a r m e d " T H 2 c e l l s , c a u s i n g the T H 2 c e l l to p r o d u c e a restricted s u b s e t of c y t o k i n e s e n c o d e d by the IL-4 g e n e cluster on the long a r m of c h r o m o s o m e 5 ( b a n d s 3 1 - 3 3 ) (Holgate, 1 9 9 9 ; J a n e w a y , 2 0 0 1 ) . C o s t i m u l a t i o n of the B cell, through p h y s i c a l interaction with the activated TH2 cell a n d the resulting T H 2 c y t o k i n e s , is required for maturation of the B cell into a p l a s m a cell that s y n t h e s i z e s a n d r e l e a s e s a l l e r g e n - s p e c i f i c IgE A b s . IL-4 is often referred to a s the " p a r a d i g m a t i c type 2 c y t o k i n e " s i n c e it is the m o s t important c y t o k i n e that m e d i a t e s IgE s y n t h e s i s ; h o w e v e r , the c l o s e l y related I L - 1 3 (which utilizes the IL-4 receptor a chain) c a n a l s o stimulate IgE s y n t h e s i s (Figure 1.3) (Corry a n d K h e r a d m a n d , 1999). IL-4 receptor e n g a g e m e n t on B c e l l s a c t i v a t e s S T A T 6 , a m e m b e r of the S T A T (signal t r a n s d u c e r s a n d activators of transcription) family of T F s , w h i c h initiates transcription of s m R N A a n d induces s c l a s s switching  (Corry a n d K h e r a d m a n d ,  1999; Janeway,  2001). Additional  costimulatory  m o l e c u l e s p r e s e n t o n the B cell s u r f a c e include C D 4 0 a n d C D 8 0 / C D 8 6 , w h i c h bind to C D 1 5 4 a n d C D 2 8 , respectively, o n T H 2 c e l l s to e n h a n c e IgE production (Corry a n d K h e r a d m a n d , 1999).  T h e s y n t h e s i s a n d r e l e a s e of a l l e r g e n - s p e c i f i c IgE A b s is the key e v e n t in the d e v e l o p m e n t of allergy (Holgate, 1999). IgE binds to its receptor, F c s R I , present o n the s u r f a c e of m a s t c e l l s a n d b a s o p h i l s (Figure 1.3) with v e r y high affinity (Corry a n d K h e r a d m a n d , 1999). IgE binding p r i m e s m a s t c e l l s for s u b s e q u e n t e n c o u n t e r with the allergen a n d is often referred to a s a " p a s s i v e p r e s e n s t i z a t i o n " s t e p (Turner a n d Kinet, 1999). Interestingly, a positive correlation h a s b e e n reported b e t w e e n total s e r u m IgE levels a n d allergic d i s e a s e severity or protective immunity to p a r a s i t e s (Corry a n d K h e r a d m a n d , 1999). H o w e v e r ,  IgE  levels n e v e r r e a c h the titres reported for other A b c l a s s e s , e v e n in s e v e r e l y allergic patients w h e r e  IgE  levels o n e h u n d r e d t i m e s greater than n o r m a l h a v e b e e n reported. T h i s s u g g e s t s that tight regulation of IgE levels is e s s e n t i a l to avoid the potentially lethal c o n s e q u e n c e s a s s o c i a t e d with I g E - d e p e n d e n t inflammation (Corry a n d K h e r a d m a n d , 1999).  1.3.3  Type I hypersensitivity reactions T h e s y m p t o m s that w e a s s o c i a t e with atopic or allergic d i s e a s e s (such a s hay fever and- f o o d  allergies), uticaria (hives), a n d the m o r e s e r i o u s c o n d i t i o n s a s t h m a a n d a n a p h y l a x i s , are the result of type I  9  hypersensitivity reactions mediated by IgE (Corry and Kheradmand, 1999; Turner and Kinet, 1999). Hypersensitivity reactions are immune responses to innocuous Ags that result in tissue injury and often cause serious disease (Janeway, 2001). These reactions are classified into 4 groups based on mechanism of action. Type I hypersensitivity reactions involve IgE-dependent mast cell activation; type II hypersensitivity reactions are initiated by IgG Abs raised against cell surface or matrix Ags and are responsible for certain drug allergies (e.g. penicillin); type III hypersensitivity reactions involve immune Ag:Ab (IgG) complexes as reported in serum sickness and the Arthus reaction; and type IV hypersensitivity reactions are T cell-mediated responses that contribute to chronic dermatitis, chronic asthma, etc. (Janeway, 2001).  t  Y  Ag  (1 exposure)  Y  Y  -  A  9  (2" exposure)  s  IL-4 IL-13  —• Bcell  Mast cell  Histamine Leukotrienes Prostaglandins Cytokines Chemokines  Th2 cell signs and symptoms of atopic or allergic diseases  APC  Figure 1.3: IgE-dependent mast cell activation. When a foreign allergen or Ag enters the body, it is ingested and processed by various APCs. The processed Ag is then expressed in the form of a peptide:MHC class II complex on the surface of the APC and recognized by a Tcell of the TH2 subclass. Activated TH2 cells synthesize and release TH2 cytokines, such as IL-4 and IL-13, which stimulate B cells to synthesize and release Ag-specific IgE molecules. IgE circulates throughout the body and binds to its high affinity receptor, FcsRI, expressed on the surface of mast cells. Subsequent exposure to Ag, which is typically multivalent, results in the crosslinking of receptor-bound IgE molecules and activates the mast cell to release a wide array of inflammatory products that mediate the allergic response. Adapted from (Cony and Kheradmand, 1999).  An allergic reaction occurs when an individual, who previously produced IgE Abs to a specific allergen (as described in section 1.3.2), subsequently encounters the same allergen. This second exposure triggers the crosslinking of FcsRI-bound IgE molecules present on the surface of mast cells in the exposed tissue (Figure 1.3) (Janeway, 2001; Mekori and Metcalfe, 2000; Turner and Kinet, 1999). IgE plus allergeninduced activation of mast cells stimulates the release of a wide variety of inflammatory mast cell products that act together to elicit the familiar signs and symptoms of allergic disease (Figure 1.3) (Corry and Kheradmand, 1999; Janeway, 2001; Kinet 1999). Fortunately, the majority of these reactions, collectively referred to as type I hypersensitivity reactions, are not life threatening (Corry and Kheradmand, 1999; to  Holgate, 1999). For example, millions of allergy sufferers tolerate their symptoms with relatively little disturbance of daily activities; however, interference with sleep, intellectual functioning and recreational activities are common complaints of people with allergies (Galli and Wershil, 1996; Holgate, 1999). Conversely, asthmatic and anaphylactic reactions cause a number of deaths, often in young people, every year (Holgate, 1999). Due, in part, to the longevity of mast cells, which can survive for months or even years in the body, mast cells often participate in multiple type I hypersensitivity reactions (Abraham and Malaviya, 1997). Furthermore, mast cells are capable of synthesizing new mediators following release in response to a specific allergen (Abraham and Malaviya, 1997). The next section will take a closer look at some of the important mediators released by mast cells during type I hypersensitivity reactions.  1.3.4  IgE-dependent mast cell activation Crosslinking of FcsRI-bound IgE with multivalent Ag activates the mast cell to release 3 major  classes of proinflammatory mediators. Firstly, preformed granule associated bioactive amines, proteases and other mediators are released in a process referred to as degranulation (section 1.3.4.1). Secondly, activated mast cells synthesize and release arachidonic acid (AA) metabolites (section 1.3.4.2). Thirdly, these cells secrete a wide array of cytokines and chemokines (section 1.3.4.3) (Galli, 2000; Galli et al., 1999; Gurish and Austen, 2001). These mediators act on the vasculature, smooth muscle, connective tissue, mucous glands, and inflammatory cells to cause increased vascular permeability, changes in blood vessel tone, increased gastrointestinal motility, broncho-constriction, mucus production by goblet cells, leukocyte recruitment and activation, and other proinflammatory and immunoregulatory effects (Galli, 2000; Galli et al., 1999; Mekori and Metcalfe, 2000; Schwartz, 1994). Thus, mast cells contribute to both the early initiation phase of hypersensitivity reactions, which involves changes in vascular permeability, and the late phase, which involves leukocyte recruitment and wound healing. They also contribute to the pathogenesis of chronic inflammation and chronic tissue changes (Galli, 2000; Mekori and Metcalfe, 2000; Welle, 1997), as described below.  1.3.4.1 Degranulation Degranulation is the extraordinarily rapid process by which preformed mediators, stored in the cytoplasmic granules of mast cells, are released via exocytosis (Galli, 2000; Mekori and Metcalfe, 2000). The granule contents of the different types of human and mouse mast cells are listed in table 1.1 (section 1.2.1). The biogenic amine, histamine, is without a doubt the best known component of mast cell granules. Histamine is a short-lived vasoactive amine that causes an immediate increase in both vascular permeability and local blood flow, leading to the accumulation of fluid and blood proteins in the surrounding  n  tissue (Bachert, 2002; Janeway, 2001). These events set the stage for the recruitment of leukocytes; thus, histamine plays a key role in the initiation of allergic inflammation (Janeway, 2001). Interestingly, histamine is also reported to directly enhance the activity of numerous cells involved in the allergic inflammatory response, including TH2 cells, eosinophils, macrophages, epithelial and endothelial cells (Bachert, 2002). Histamine, which was discovered by Dale and Laidlaw in 1911, quickly became an attractive target for the management of type I hypersensitivity reactions and histamine antagonists (anti-histamines) are currently the first-line drugs used for the treatment of hay fever, uticaria, and anaphylaxis (Assanasen and Naclerio, 2002; Holgate, 1999). Unfortunately, anti-histamines are not effective in the management of asthma or eczema (Holgate, 1999). The large quantities of neutral proteases in mast cell granules suggest that these enzymes play an important role in type I hypersensitivity reactions (Holgate, 1999; Welle, 1997). For example, tryptase acts on endothelial and epithelial cells to stimulate cytokine production, such as IL-8, and the increased expression of adhesion molecules, such as ICAM (intercellular adhesion molecule)-1, that selectively recruit eosinophils and basophils to the site of inflammation (Holgate, 1999; Welle, 1997). The recruitment of these cells is important since eosinophils are the key mediators of chronic airway inflammation (Holgate, 1999). Tryptase also induces the cleavage of fibronectin and collagenase type IV in the epidermis, implicating this mast cell protease in tissue remodeling and matrix degradation (Welle, 1997). In fact, most matrix metalloproteinases (which cause tissue destruction by breaking down tissue matrix proteins) are secreted as inactive precursors that must be cleaved by mast cell proteases to be activated (Janeway, 2001; Welle, 1997). In addition to vasoactive amines and neutral proteases, mast cell granules contain some preformed cytokines, such as TNFa (Galli, 2000; Schwartz, 1994); the role of preformed and newly synthesized mast cell cytokines will be addressed in section 1.3.4.3.  1.3.4.2 Arachidonic acid (AA) metabolism AA is the 20 carbon fatty acid typically found at the second (sn-2) position of membrane glycerophospholipids. The aggregation of FcsRI by contact of cell-bound IgE with multivalent Ag stimulates the activation of phospholipase A2 (PLA2), which selectively hydrolyzes the sn-2 fatty acyl group of phospholipids to generate lysophopholipids" and AA (Alberts, 1994). PLA2 activity is regulated at both the transcriptional level (by inducible TFs such as  NFKB  (nuclear factor KB)) and post-translationally (by  phosphorylation events, calcium (Ca ) binding, and phospholipid binding) (Murakami and Kudo, 2002). AA 2+  Platelet activating factor, which induces platelet aggregation, vasodilation, bronchoconstriction, and increased vascular permeability, is one of the lipid mediators derived from lysophospholipids (Murakami and Kudo, 2002). 12  is a precursor of the four major classes of eicosanoids: prostaglandins (PGs); prostacyclins; thromboxanes; and leukotrienes (LTs). The synthesis of these lipid mediators is dependent on the enzymatic activity of cyclooxygenase (COX), which generates the first three products, and lipooxygenase (LO), which regulates the synthesis of LTs (Murakami and Kudo, 2002). Interestingly, different mast cell populations appear to favor the production of certain eicosanoids over others; for example, MMCs primarily generate LTs, whereas CTMCs are major sources of PGs (Schwartz, 1994; Welle, 1997). These lipid mediators are potent inducers of early phase responses, including smooth muscle contraction, increased vascular permeability and mucus secretion (Holgate, 1999; Schwartz, 1994; Welle, 1997). In fact,  LTC4  and  LTD4  are reported to be the most potent inducers of bronchoconstriction in humans (O'Byrne, 1997). LTs and PGs also regulate the migration and activation of leukocytes, which contribute to the late phase of hypersensitivity reactions (Holgate, 1999; Janeway, 2001; O'Byrne, 1997). Furthermore, certain PGs are important stimulators of blood clotting and wound healing through their induction of platelet aggregation and adhesion to blood vessel walls (Janeway, 2001). Since the eicosanoids are major inducers of pain, fever and inflammation, their synthetic pathways are common targets for therapeutic drugs. For example, NSAIDs (nonsteroidal anti-inflammatory drugs), such as aspirin and ibuprofin, block the oxidation step catalyzed by COX and thus inhibit PG synthesis (Alberts, 1994; Janeway, 2001). Interestingly, certain asthmatics are insensensitive to treatment with NSAIDs; these asthmatics demonstrate increased production of LTs via enhanced  LTC4  synthase activity  (Holgate, 1999; O'Byrne, 1997). Fortunately, LT modulating agents, such LO inhibitors and LT receptor antagonists, provide effective treatment for this group of asthmatics, and the potential use of these agents in other allergic inflammatory diseases, such as atopic dermatitis, is currently under investigation (Chari et al., 2001; Holgate, 1999). Finally, corticosteroid hormones (e.g. cortisone) target the first step of the eicosanoid synthesis pathway (i.e. PLA2 activity) and are successfully used in the treatment of various inflammatory disorders, including certain types of asthma and arthritis (Alberts, 1994; Ott and Cambier, 2000).  13.4.3 Cytokine and chemokine production As indicated above, activated mast cells synthesize and release a wide array of both pre-existing and newly generated cytokines and chemokines. These include the cytokines IL-1, -3, -4, -5, -6, -8, -10, 13, -16, SCF, TNFct, VEGF (vascular endothelial growth factor), TGF (transforming growth factor) p, and several chemokines, such as MIP (macrophage inflammatory nrotein)-1a and p, and MCP (monocyte chemotactic protein)-1 (Galli, 2000; Mekori and Metcalfe, 2000; Ott and Cambier, 2000; Wedemeyer and 13  Galli, 2000). By contrast, basophil cytokine production is quite limited and favors the production of IL-4 and IL-13 (MacGlashan et al„ 2002; Wedemeyer and Galli, 2000). Mast cell released cytokines and chemokines are key regulators of late phase events, such as the recruitment of leukocytes (e.g. eosinophils and basophils) to the site of inflammation. For example, TNFa modulates endothelial cell function by decreasing adhesion to each other and increasing the expression of adhesion molecules (e.g. E-selectin) that adhere to passing leukocytes and cause them to leave the blood vessel and enter the affected tissue (Galli, 2000; Mekori and Metcalfe, 2000). Additionally, mast cell chemokines and certain cytokines act as chemoattractants that attract specific leukocytes to the affected tissue; for example, IL-8 recruits neutrophils  (Alberts,  1994; Janeway, 2001).  Some mast  cell cytokines  also  play  important  immunoregulatory roles: IL-4 and -13 perpetuate the TH2 response by augmenting IgE synthesis; IL-5 promotes the growth, differentiation, survival and priming of eosinophils; and IL-6, which has a number of pro- and anti-inflammatory roles, stimulates T cells and augments Ab production (Galli, 2000; Janeway, 2001). Thus, mast cell cytokines and chemokines regulate diverse biological processes, including inflammation, tissue remodeling, pathologic fibrosis, blood clotting and wound repair, Ab production, hemopoiesis, and host responses to bacteria (section 1.3.5) (Galli, 2000; Janeway, 2001; Wedemeyer et al„ 2000).  1.3.5  IgE-independent mast cell activation In addition to IgE plus Ag (lgE+Ag)-induced activation of mast cells, various bacterial products and  non-immunogenic stimuli can activate mast cells (Abraham and Malaviya, 1997; Galli, 2000; Galli et al., 1999), allowing mast cells to contribute to several aspects of host defense during an innate immune response. As described above, mast cells are often referred to as the "appendix of the human immune system" in the western world, due to low rates of parasitic infection, high incidences of asthma and allergic diseases, and their catastrophic role in anaphylaxis; however, their newly discovered role in the innate immune response is beginning to cast a more favorable light on these cells (Galli and Wershil, 1996). Another clue that mast cells are valuable and beneficial to the host lies in the phylogenic preservation of mast cells through evolution (Abraham and Malaviya, 1997); mast cells or mast cell-like cells have been identified even among the lowest order of animals (Hakanson et al., 1986). Like other effector cells of the innate immune system, mast cells can recognize and bind to invading bacteria (Abraham and Malaviya, 1997). This binding can be direct, involving receptors on mast cells and the corresponding ligands on bacteria; for example, the fimbral adhesin, FimH, expressed on Eschericia coli (E. coli) and other enterobacteria binds to a mannose-containing receptor molecule on the 14  mast cell surface (Malaviya et al., 1994b; Norn et al., 1984). Alternatively, pathogens can bind to mast cells indirectly by first initiating a humoral immune response, which produces IgE Abs that facilitiate mast cell binding to the pathogen (e.g. parasitic helminths, Helicobacter pylori, and Staphylococcus aureus) (Abraham and Malaviya, 1997; Aceti et al., 1991; Leung et al., 1993), or by activating the complement system, since mast cells express the receptor for the iC3b fragment of complement which facilitates binding to Salmonella and Schistosoma mansoni (Abraham and Malaviya, 1997; Sher et al., 1979). Additionally, mast cells can simply be activated by the products of other complement activated cells (Galli, 2000). Finally, a physical interaction between the mast cell and the pathogenic bacteria is not necessary for mast cell activation. For example, bacterial toxins and cell wall components, such as lipopolysaccharide (LPS), can induce mast cell activation over a distance (Abraham and Malaviya, 1997; Galli, 2000). IgE-independent activation of mast cells results in the synthesis and release of the same mediators stimulated by IgE+Ag. However, some of these stimuli induce mast cells to release a restricted subset of mediators; for example, LPS, which binds to toll like receptors (TLRs), induces IL-6 production in the absence of mast cell degranulation (Galli, 2000; Leal-Berumen et al., 1994). Alternatively, some of these stimuli induce mast cells to release smaller amounts of mediators or stimulate slower release of mediators compared to IgE+Ag; for example, degranulation requires more than 1 hr to reach completion in response to type I fimbriated E. coli (Malaviya et al., 1994a). Studies in genetically mast cell-deficient W/W mice, and v  W/W knock-in mice selectively reconstituted with cultured mast cells, verify that mast cells are key v  regulators of host defense against bacterial infection (Abraham and Malaviya, 1997; Galli, 2000; Galli and Wershil, 1996). Studies using intraperitoneal challenge by enterobacteria or surgically induced bacterial peritonitis indicate that mast cells reduce the mortality in these mice, and that the recruitment of circulating leukocytes with bactericidal properties (e.g. neutrophils), is dependent on TNFoc production by mast cells (Echtenacher et al., 1996; Galli and Wershil, 1996; Malaviya et al., 1996). Furthermore, repetitive administration of SCF, which regulates mast cell development and function, to wildtype or knock-in mice can increase mast cell numbers, enhance mast cell dependent host responses and increase survival of mice subjected to cecal ligation and pucture, a model of acute bacterial peritonitis (Galli, 2000; Galli et al., 1999). In addition to their beneficial role in the innate immune response, mast cells are believed to be important for the initiation of acquired immune responses (Henz et al., 2001). The phagocytic potential of mast cells was recognized by Elie Metchnikoff as early as 1892 (Galli, 2000). Although this response is weaker than that observed with traditional phagocyes (i.e. neutrophils and macrophages), mast cells can phagocytose and eliminate bacteria, such as Salmonella and E. coli (Malaviya et al., 1994a; Sher et al.,  15  1979), via both nonoxidative and oxidative killing, the latter involving the production of superoxide anions (Abraham and Malaviya, 1997; Galli, 2000; Henz et al., 2001). Finally, bacterial activation of mast cells also contributes to the acquired immune response of the host via the secretion of immunoregulatory cytokines, such as IL-4, or via the processing and presentation of bacterial Ags to host immune cells on MHC class I or II molecules, i.e. mast cells can act as APCs (Abraham and Malaviya, 1997; Henz et al., 2001).  1.4  MAST CELLS AND DISEASE Mast cells are currently in the limelight in inflammatory research because of their ability to rapidly  initiate and regulate various inflammatory responses. This likely reflects the prime locations of these cells, at the portals between self and non-self, where they serve as a critical first line of defense against viral and bacterial infection (Abraham and Malaviya, 1997; Galli, 2000). In addition to their well characterized role in allergic inflammatory diseases, such as asthma and anaphylaxis, mast cells have been implicated in the development of multiple sclerosis (MS), cardiac hypertrophy, Alzheimer's disease, rheumatoid arthritis, inflammatory arthritis, inflammatory bowel disease (IBD), cancer, angiogenesis, sudden infant death syndrome, infection with human immunodeficiency virus (HIV), and numerous other disease states (Abraham and Malaviya, 1997; Galli, 2000; Panizo et al., 1995; Schwartz, 2001; Wedemeyer et al., 2000). More specifically, mast cells are reported to accumulate around the borders of MS plaques in the brain, along venules and capillaries, and contribute to the edema formation and myelin destruction characteristic of MS (Kruger, 2001; Secor et al., 2000). Studies conducted in mice and rats indicate that mast cell numbers in the heart increase, especially in the left ventricle, and contribute to the development of myocardial fibrosis with hypertensive cardiac hypertrophy (Hara et al., 2002; Panizo et al., 1995). The HIV glycoprotein (gp) 120 stimulates histamine, IL-4 and IL-13 release from human basophils and mast cells by interacting directly with IgE. This interaction may contribute to the TH2 pattern of cytokine production reported during the early stages of HIV infection (Marone et al., 2001; Wedemeyer et al., 2000). Mast cell proinflammatory mediators, such as tryptase and TNFa, are reported to contribute to a number of chronic inflammatory conditions, such as IBD and Crohn's disease, respectively (Lilja et al., 2000; Raithel et al., 2001). Finally, a myriad of products secreted by activated mast cells, including VEGF and histamine, are reported to enhance blood vessel permeability and promote angiogenesis (Galli, 2000; Norrby, 2002). In keeping with these findings, patients with increased numbers of mast cells at various tumor sites and those with systemic mastocytosis are reported to have a poorer prognosis due to increased rates of tumor vascularization, growth and metastasis (Tomita et al., 2001; Wimazal et al., 2002; Yano et al., 1999).  16  Despite the growing interest and recent advances in mast cell research, the signaling pathways that regulate the release of mast cell mediators in response to IgE+Ag or other stimuli are not fully defined. Because mast cells are involved in so many disease states, it is important to understand what activates and deactivates these cells and this can only be done by studying the signaling pathways involved.  1.5  SIGNAL TRANSDUCTION "The study of how cells communicate impinges on all aspects of biology, from development to  disease. At first glance it's a horrendously complicated business, but some simple themes are emerging" (Downward, 2001). Signal transduction is simply the mechanism by which a cell senses its external environment then interprets and reacts to that information (Downward, 2001). Multicellular organisms have developed elaborate signal transduction pathways essential for communication between both neighboring cells and cells separated by vast distances. Signal transduction begins when a receptor detects a change in the environment outside the cell by binding to its ligand. Information is then transferred to intracellular mediators that, through a series of protein-protein or protein-lipid interactions and post-translational modifications, change the activity of enzymes and regulate nuclear events, such as gene expression (Downward, 2001; Pawson, 1995; Pawson and Nash, 2000). At the individual cell level, signal transduction pathways determine whether a cell will become activated or inactivated, live or die, proliferate or differentiate, etc. At the level of the organism, signal transduction pathways regulate development, growth, etc. Due to the important roles of signaling pathways in individual cells and the organism as a whole, it is easy to see how alterations in one or more signaling pathway can lead to the development of disease states (Downward, 2001; Pawson, 1995; Pawson and Nash, 2000).  1.6  RECEPTORS Signal transduction pathways are initiated when a receptor, typically expressed on the cell surface,  binds its ligand and, as a result, undergoes some degree of dynamic change to transfer the signal into the cell (Downward, 2001; Pawson and Nash, 2000). Although all receptors function to transfer information from the outside to the inside of a cell, both the structures of different receptors and the mechanisms by which they transmit signals vary dramatically among the different receptor families. For example, steroid and ion-gated channel receptors exert their intracellular effects directly. Steroid or nuclear receptors, such as the estrogen receptor, are intracellular receptors that function as ligand activated TFs, allowing them to directly regulate the expression of target genes (Beato and Klug, 2000; Pawson and Nash, 2000). On the other hand, ligand binding to ion-gated channel receptors, such as the acetylcholine receptor, causes  17  receptor subunits (each with several transmembrane domains) to cluster, forming a gated channel that rapidly permits the influx or efflux of cations or anions (Reeves and Lummis, 2002). In contrast to the receptor families mentioned above, G protein-coupled receptors (GPCRs), serine/threonine (S/T) kinase receptors, and protein tyrosine kinase (PTK) receptors exert their intracellular effects indirectly by activating various intracellular signaling pathways. GPCRs or serpentine receptors, such as the p-adrenergic receptor, traverse the plasma membrane seven times and initiate signaling events via apy heterotrimeric G proteins that associate with their cytoplasmic tails (Ji et al., 1998). Ligand binding induces a conformational change in the receptor, which permits the a subunit to bind to guanosine triphosphate (GTP) and dissociate from the Py subunit, allowing both the a and Py subunits to activate target proteins (Ji et al., 1998). Conversely, the type I receptor subunits of S/T kinase receptor complexes, e.g. the TGF-p receptor, have intrinsic S/T kinase activity, which is activated by the type II receptor subunits following homodimeric ligand binding, and directly phosphorylates downstream targets, such as the Smads (Attisano and Wrana, 2002). PTK regulated receptors are further classified as growth factor receptors, cytokine receptors, or immunoreceptors. Only growth factor receptors or receptor tyrosine kinases (RTKs) possess intrinsic tyrosine kinase activity; ligand binding induces the dimerization of receptor subunits, allowing the tyrosine kinase domains to activate each other by cross phosphorylation. RTKs are subdivided into 3 subclasses. Subclass I RTKs, e.g. the epidermal growth factor (EGF) receptor, are activated by binding to monomeric ligand, which alters the extracellular conformation of the receptor to permit dimerization (Ullrich and Schlessinger, 1990). Subclass II RTKs, e.g. the insulin receptor, exist as disulfide linked dimers and ligand binding induces a conformational change required for tyrosine kinase activity. Subclass III RTKs, e.g. c-kit (section 1.6.1), utilize dimeric ligands to induce receptor dimerization (Ullrich and Schlessinger, 1990). Unlike RTKs, both cytokine receptors and immunoreceptors lack intrinsic catalytic activity and depend on associated PTKs to initiate signal transduction events. The cytokine receptor family can be further subdivided into four types. Type I cytokine receptors or hemopoietic receptors are activated by ligand induced dimerization, which permits the cross phosphorylation and activation of receptor associated Jak family tyrosine kinases. These type I receptors are further subclassified based on receptor subunit usage; they can homodimerize (e.g. EpoR), heterodimerize (pc and gp130 utilizing receptors; e.g. IL-3R (section 1.6.2) and IL-6R, respectively), or heterotrimerize (e.g. IL-2R) upon ligand binding (Bagley et al., 1997). Type II cytokine receptors are the IFN receptors and type III receptors trimerize upon binding to TNF family members to mediate apoptosis through various death domain conatining proteins (Moutoussamy et 18  al., or  1 9 9 8 ) . T y p e IV c y t o k i n e r e c e p t o r s , e . g . t h e I L - 1 R a n d t h e T L R s , d o n o t r e g u l a t e h e m o p o i e t i c c e l l g r o w t h differentiation,  but play  a  role  in i n f l a m m a t i o n  by  modulating  c e l l u l a r activity  i m m u n o r e c e p t o r s , s u c h a s t h e h i g h affinity I g E r e c e p t o r ( F c s R I ; s e c t i o n and  rely  tyrosine  on  associated  based  Src  activation  family  motifs  (ITAMs;  d o w n s t r e a m signaling e v e n t s (Isakov,  1.6.1  tyrosine  kinases  section  (section  1.11.1)  or  to  phosphorylate  more  receptor  Finally, subunits  immunoreceptor  subunits  and  initiate  1998).  c-kit The  r e c e p t o r f o r S C F , c - k i t , is w i d e l y e x p r e s s e d  b e l o n g s to t h e t y p e  c-kit c o n t a i n s  5  o n cell of h e m o p o i e t i c origin (Broudy, 1997).  ( M ) - C S F (Broudy, 1997;  Ig-like  motifs  ( F i g u r e 1.4),  Ullrich a n d S c h l e s s i n g e r ,  which  regulate  binding pocket a n d a phosphotransferase Heldin, 1996). S C F c a n be e x p r e s s e d S C F binding  induces  1990). T h e extracellular d o m a i n of  ligand binding and  t y r o s i n e k i n a s e d o m a i n in t h e c y t o p l a s m i c t a i l o f c - k i t is s e p a r a t e d  1992).  into a n  receptor dimerization. T h e  adenosine  region by a kinase insert s e q u e n c e  event  is  followed  triphosphate ( A T P )  ( F i g u r e 1.4)  (Broudy,  1997;  a s a s o l u b l e o r m e m b r a n e b o u n d n o n - c o v a l e n t l y l i n k e d d i m e r ( L e v et  c-kit  homodimerization, which  allows  the  cytoplasmic  d o m a i n s to i n t e r m o l e c u l a r l y " a u t o p h o s p h o r y l a t e " t h e r e c e p t o r c h a i n s ( B r o u d y , 1 9 9 7 ; phosphorylation  c-kit  III R T K f a m i l y , a l o n g w i t h t h e r e c e p t o r s f o r p l a t e l e t d e r i v e d g r o w t h f a c t o r ( P D G F ) , F l k -  2/Flt3, a n d m o n o c y t e  al.,  2000).  1.6.3), c o n s i s t of multiple  1.7.1)  in o n e  (O'Neill,  by  the  recruitment of  phosphotyrosine  tyrosine  kinase  L e v et al., 1992).  (pY) binding proteins  This  and  the  i n i t i a t i o n o f c - k i t s i g n a l i n g e v e n t s . In a d d i t i o n t o its i n t r i n s i c t y r o s i n e k i n a s e a c t i v i t y , c - k i t a s s o c i a t e s w i t h a n d activates  members  of  the  Src  kinase  family,  p h o s p h o r y l a t e s a n u m b e r of receptor a s s o c i a t e d  1.6.2  ( S c h r a d e r et receptor  is c o m m o n l y u s e d t o e n h a n c e al., 1981;  superfamily,  residues and W S X W S  1997).  as  Lyn,  and  this  receptor, The  Suda,  C  a  1985;  characterized  family  subsequently  p r o t e i n s ( T i m o k h i n a et al., 1 9 9 8 ) .  Lantz by  the proliferation a n d differentiation of m u r i n e m a s t cells et  cell  al., 1998). surface  The  IL-3R  glycoprotein  with the  subunit  receptors  (Figure  1.4)  for IL-5 a n d g r a n u l o c y t e is  unique  s u b u n i t is s h a r e d b e t w e e n t h e a  to  this  is  a  chains  m o t i f s in t h e i r e x t r a c e l l u l a r d o m a i n s ( F i g u r e 1.4)  along  IL-3R  C o n v e r s e l y , the B  member with  of the  four  type  conserved  monocyte and  (GM)-CSF confers  I cytokine cysteine  (Bagley  ligand  c h a i n s of the IL-3, IL-5, a n d G M - C S F  et  pc al.,  specificity.  r e c e p t o r s , b u t it  G u t h r i d g e et a l , 1 9 9 8 ) .  a d i s t i n c t B s u b u n i t (BIL-3) t h a t is u s e d e x c l u s i v e l y b y t h e I L - 3 R h a s b e e n  Interestingly,  r e p o r t e d in m i c e ( M o u t o u s s a m y  1 9 9 8 ) . T h e c y t o p l a s m i c tails o f t h e s e p s u b u n i t s c o n t a i n b o x 1 a n d b o x 2 d o m a i n s ( F i g u r e 1.4),  19  in vitro  ( I h l e e t a l . , 1 9 9 4 ) . T h e I L - 3 R is a  receptor  is a n i m p o r t a n t r e g u l a t o r o f l i g a n d b i n d i n g affinity ( B a g l e y et a l „ 1 9 9 7 ;  al,  kinase  IL-3 receptor (IL-3R) IL-3  utilizing  such  et  which  associate with Jak family PTKs. Following IL-3 induced dimerization, the associated Jak (Jak2) phosphorylates the p chain, resulting in the recruitment and subsequent phosphorylation of various signaling intermediates (Bagley et al., 1997; Ihle et al., 1994). Interestingly, the short intracellular tail of the IL-3R a subunit has been reported to be critical for IL-3 induced signaling events despite the lack of recognizable signaling motifs (Orban et al., 1999).  IL-3R  c-Kit  FcsRI  c^p,  I! fnnn 1  | ^ Kinase Domain  a  ! U|||  s  ,|  Hbox2  a  p  1  III  1 a  P  ITAM  7 T  p  Figure 1.4: The structures of c4tit IL-3R, FcsRI, and asBi. The extracellular portion of c-kit a type III RTK, consists of 5 Iglike motifs (•) that regulate binding to SCF and receptor homodimerization. A kinase insert sequence divides the c-kit intracellular kinase domain ( ) into an ATP binding region (membrane proximal) and a phosphotransferase region (membrane distal). The IL-3R is a f3c utilizing type I cytokine receptor; the IL-3R a subunit confers IL-3 binding specificity. Conserved WSXWS motifs are indicated (-). The box 1 and 2 domains (•) in the intracellular portion of the pc chain are involved in recruiting Jak family PTKs. FcsRI, the high affinity receptor for IgE, is a heterotetrameric immunoreceptor. The extracellular portion of the a chain consists of 2 Ig-like motifs (D1 and D2) that regulate binding to the Fc portion of IgE. The (3 and y chains contain ITAM motifs (•) that become phosphorylated by Lyn after receptor aggregation to initiate signaling events; the p chain also functions to amplify FcsRI signaling events. Integrin asPi binds to the ECM component, fibronectin (FN). Extracellular divalent cations, such as Ca *, are required for high affinity ligand binding; divalent cation binding domains are indicated (-). 2  1.6.3  FcsRI The high affinity receptor for IgE, FceRI, belongs to the immunoreceptor family, along with the  BCR, TCR, and the receptors for the Fc portions of other Abs (e.g. IgGs bind to FcyRs). Rodent FcsRI has an obligatory heterotetrameric structure composed of an a subunit, a four transmembrane-spanning p subunit and two identical disulphide linked y subunits (Figure 1.4) (Kinet 1999; Metzger, 1992; Rivera et al., 2002; Turner and Kinet, 1999). The p and y subunits are important for initiating signal transduction events downstream of this receptor since they each contain one ITAM motif (section 1.11.2) that is phosphorylated by Lyn following receptor aggregation; the p subunit also serves as an amplifier of IgE+Ag20  induced signaling events (Kinet, 1999; Saini et al, 2001; Turner and Kinet, 1999). The a subunit has a short intracellular tail that does not contribute to signal transduction events; however, the extracellular portion of this subunit is essential for binding to the Fc portion of the IgE molecule (Kinet, 1999; Turner and Kinet, 1999). Each a chain contains two extracellular Ig-related domains (Di and D2) (Figure 1.4); studies indicate that D2 binds to IgE and Di is required for high affinity binding (Kinet, 1999). Although most Fc receptors only demonstrate high affinity for the Fc portion of Abs that are bound to Ag, the FceRI binds to monomeric IgE at a very high affinity (Kd=10--10- ) (Turner and Kinet, 1999). Interestingly, while the Fc portion of each IgE is homodimeric, i.e. has the potential to bind to two separate a chains, the interaction stoichiometry between IgE and its receptor is 1:1 (Turner and Kinet, 1999). It is believed that binding of IgE to one FcsRI may result in a conformational change in the Fc portion of the Ab that prevents binding to a second receptor. Alternatively, the second FcsRI binding site on the IgE molecule may be masked upon binding to the first receptor (Kinet, 1999; Turner and Kinet, 1999). 9  10M  In addition to their role in signal transduction, the p and y subunits play an important role in cell surface expression of rodent FcsRI on mast cells and basophils, since the a chain contains an endoplasmic reticulum (ER) retention signal in its cytoplasmic domain (Kinet, 1999; Ott and Cambier, 2000). In humans, the p chain is not required for cell surface expression of the FcsRI, thus, both trimeric ay2 and tetrameric aPy2 forms exist (Kinet, 1999). Interestingly, human mast cells and basophils express only the tetrameric form of the IgE receptor, as observed in rodents, while only ay2 is expressed on human monocytes, Langerhans cells and dendritic cells (Kinet, 1999). 1.6.4  Integrins  The integrin family of cell surface receptors regulate cell-cell and cell-extracellular matrix (ECM) interactions (Houtman et al, 2001a; Schwartz, 2001). Adhesion to ECM proteins (such as fibronectin (FN), collagen, and laminin) and the subsequent signaling events initiated by integrins regulate numerous biological processes, including cell survival, growth, differentiation and motility (Alberts, 1994; Houtman et al, 2001a; Schwartz, 2001). These receptors consist of two noncovalently associated transmembrane glycoprotein subunits, called a and p, which both contribute to ligand binding (Figure 1.4) (Alberts, 1994; Schwartz, 2001). Eight a and fourteen p chain isotypes have been reported to date (Houtman et al, 2001a). The heterodimerization of particular isotypes regulates ligand specificity; for example, asPi binds preferentially to FN (Figure 1.4). On resting hemopoietic cells, these a and p subunits are associated, but exist in a low-affinity conformation (Houtman et al, 2001a; Schwartz, 2001); this may allow cells to explore 21  their environment by binding weakly to matrix molecules (Alberts, 1994). The stimulation of hemopoietic cells by various mediators (e.g. cytokines, chemokines, and growth factors) is required to activate integrin receptors and increase ligand binding affinity through a series of "inside-out" signaling events; these events alter the conformation or clustering of integrins to increase avidity (Alberts, 1994; Houtman et a l , 2001a). High affinity ligand binding is also dependent on extracellular divalent cations, such as C a  2 +  (Figure 1.4)  (Alberts, 1994; Houtman et a l , 2001a). The activation of integrins at the site of contact with the matrix or another cell leads to the activation of several intracellular pathways, referred to as "outside-in" signaling events (Alberts, 1994; Houtman et a l , 2001a). Because integrins lack intrinsic kinase activity, their carboxy (C)-terminal cytoplasmic tails associate with a number of adaptor proteins and kinases to trigger signaling events that reorganize the actin cytoskeleton and initiate the formation of complex protein structures, i.e. focal adhesion complexes, at the site of adhesion (Houtman et a l , 2001a; Schwartz, 2001). For example, many B chains bind to integrin linked kinase, focal adhesion kinase (FAK), actinin and filamin, while many a chains bind to paxillin to coordinate these events (Schwartz, 2001).  1.7 KINASES Kinases catalyze the transfer of the y-phosphate from a nucleotide triphosphate, typically ATP, to their targets in a magnesium (Mg )-dependent manner (Cohen, 2002). Kinases play a pivotal role in the 2+  initiation, propagation and termination of all intracellular signaling events, thus, regulate every aspect of cellular life. The targets of kinases contain hydroxyl groups, which serve as the phosphate acceptor sites (Cohen, 2002). Common targets for phosphorylation include the amino acids serine (S), threonine (T) and tyrosine (Y), and certain membrane lipids, such as phosphatidylinositol (PI). Because kinase activity impinges on all aspects of cellular functioning, the activity of. kinases is tightly controlled. Common regulatory mechanisms include the association of second messengers or regulatory subunits, changes in subcellular location, and regulatory phosphorylation or dephosphorylation events (Johnson et a l , 1996). For example, most kinases autophosphorylate residues in the activation loop of their kinase domains to increase their own catalytic activity (Johnson et a l , 1996).  1.7.1  Protein tyrosine kinases (PTKs) v-Src (the protein product of the transforming gene of Rous sarcoma virus) was identified as a  kinase in the late 1970's, and by 1978 it had become the first identified PTK (Cohen, 2002; Collett and Erikson, 1978). Although greater than 99% of protein phosphorylation events in normal eukaryotic cells occur on S or T residues, the reversible phosphorylation of Y residues has proven to be essential for the activation of numerous signaling pathways (Alberts, 1994; Cohen, 2002). PTKs modulate transient protein22  protein interactions by creating docking sites for pY binding motifs (section 1.9.1) in signaling intermediates. The non-receptor PTKs commonly utilized by hemopoietic cells include the Src, Jak, Tec, Fes, and Abl kinase families (Bolen and Brugge, 1997). For example, Jak family kinases are activated following ligand induced dimerization of cytokine receptors and phosphorylate these receptors on Y residues. This is followed by the recruitment of Src homology (SH) 2 domain containing proteins, e.g. members of the STAT family of TFs, and their subsequent phosphorylation by Jak kinases (Liu et al., 1998a). Phosphorylated STAT molecules homo- or hetero-dimerize then translocate to the nucleus to inititate transcription of target genes (Ihleetal., 1994).  1.7.1.1 Src family kinases Members of the Src family of non-receptor PTKs are widely expressed in hemopoietic cells, and can be activated by immuno-, growth factor, and cytokine receptors, GPCRs, and ECM components (Bolen and Brugge, 1997; Thomas and Brugge, 1997). In mammals, the Src family consists of eight members that exhibit similar structural features and overlapping functions (Thomas and Brugge, 1997). Src, Fyn, and Yes are widely expressed, whereas Lyn, Blk, Hck, Fgr and Lck are expressed primarily in cells of hemopoietic origin (Bolen and Brugge, 1997). Src kinase family members contain six distinct functional regions (listed from amino (N)- to Cterminal end): the SH4 domain; the unique region; the SH3 domain; the SH2 domain; the catalytic domain (aka the SH1 domain); and the negative regulatory tail (Thomas and Brugge, 1997). The SH4 domain is myristylated; this modification, which occurs during protein translation, is essential for the association of Src kinases with the plasma membrane (Thomas and Brugge, 1997). The phosphorylation status of regulatory Y residues within the C-terminal tail and the kinase domain of Src family members regulates enzyme activity. In the unstimulated cell, Src kinases are found in a "closed" or inactive confomation; Csk (carboxyterminal Src kinase) phosphorylates the inhibitory Y residue in the C-terminal tail to create an intramolecular binding site for the SH2 domain and the SH3 domain interacts with a linker sequence between the SH2 and kinase domains (Pawson and Scott, 1997; Thomas and Brugge, 1997). Following stimulation of the cell, a tyrosine phosphatase, such as CD45, is activated and subsequently dephosphorylates the inhibitory C-terminal pY residue in Src family kinases. In addition, a proline rich sequence displaces the intramolecular SH3 interaction (Thomas and Brugge, 1997). These two steps permit the Src family kinase to adapt an "open" or active conformation and autophosphorylate the Y residue within the kinase domain (Thomas and Brugge, 1997). Activated Src family kinases can then phosphorylate associated receptors and downstream signaling intermediates. Src family kinases have been implicated in 23  signal transduction events downstream of every major class of cell surface receptor and it is believed that their unique domains confer some degree of specificity in terms of their interaction with receptors or other proteins (Thomas and Brugge, 1997). For example, both Lyn and Fyn associate with the FcsRI B chain (Parravicini et a l , 2002; Turner and Kinet, 1999). However, Lyn also binds to and regulates signaling processes downstream of c-kit (Ueda et a l , 2002) and the IL-3R (Adachi et a l , 1999).  1.7.2  Phosphatidylinositol (PI) kinases Phosphoinositides are minor constituents of the eukaryotic cell membrane inner leaflet  (approximately 10% of total membrane lipid), which suggests that they do not play a structural role. Instead, membrane phosphoinositides play an integral role in the regulation of signal transduction events by specifically interacting with a large number of proteins to regulate protein localization, conformation and activity (section 1.10) (Rameh and Cantley, 1999; Toker, 2002). The precursor of all phosphoinositides is PI (Figure 1.5). Unlike the headgroups of other phospholipids, the inositol headgroup is quite versatile because of its ability to be reversibly phosphorylated at three distinct positions (Sato et a l , 2001; Toker, 2002). PI kinases can phosphorylate PI or subsequent PI products on hydroxyl groups at the D-3, D-4, or D-5 position to generate seven additional lipids, as depicted in Figure 1.5 (Rameh and Cantley, 1999; Toker, 2002; Tolias and Cantley, 1999). Although the inositol head group contains five free hydroxyls, no phosphorylation has been reported at the D-2 or D-6 position to date. The hydroxyl group at the D-1 position forms a phosphodiester bond with the glycerol portion of DAG (diacylglycerol), which is required for the attachment of phosphoinositides to the membrane (Rameh and Cantley, 1999). In addition to PI kinases, various PI phosphatases can regulate the number and position of phosphates on the inositol head group to alter cell signaling events (Figure 1.5) (Toker, 2002), as described in section 1.8. Finally, phosphoinositides can be metabolized by phospholipases; for example, phospholipase C (PLC) hydrolyzes PI-4,5-P to generate inositol-1,4,5-trisphosphate (IP ) and DAG (Toker, 2002). 2  3  1.7.2.1 Phosphatidylinositol 3'-kinases (PI3Ks) Although less than 0.25% of the total inositol-containing lipids are phosphorylated at the D-3 position (Rameh and Cantley, 1999), great emphasis has been placed on the study of phosphatidylinositol 3'-kinases (PI3Ks) since they are activated following the stimulation of the vast majority of cell surface receptors. Furthermore, phosphorylation of Pis at the D-3 position has been shown to be essential for the initiation of events leading to cell survival, cell growth, cell cycle entry, and cell migration (Cantley, 2002). PI3K family members have been grouped into three classes based on substrate specificity. Class I PI3Ks are capable of phosphorylating multiple Pis (PI, PI-4-P, and PI-4.5-P2), however, PI-4.5-P2 appears to be 24  Figure 1.5: Phosphoinositide metabolism. PI is the precursor of all phosphoinositides. Arrows pointing to the right of the page represent reactions regulated by kinases and arrows pointing to the left represent reactions regulated by phosphatases. The enzymes involved in the synthesis of each phosphoinositide are designated on the arrows. The dotted arrows represent possible reactions that have yet to be confirmed in vivo. The positions of the lipid side chains are noted (Ri and R2). K = kinase, P = phosphate, ptase = phosphatase. Adapted from (Rameh and Cantiey, 1999).  their preferred substrate in vivo (Figure 1.5) (Tolias and Cantley, 1999). The primary class I PI3K product PI-3,4,5-P3 (PIP3), is required for the activation of PKB/Akt (protein kinase B), a S/T kinase that enhances cell survival and induces cell cycle entry, Btk (Bruton's tyrosine kinase), which plays a key role in regulating Ca flux and cell proliferation, Vav, a guanine nucleotide exchange factor that regulates cell migration, a variety of protein kinase C (PKC) family members, etc. (Rameh and Cantley, 1999). All class I PI3Ks exist as heterodimers containing a 110 to 120kDa catalytic subunit and a 50 to 100kDa regulatory subunit, the latter of which interacts with.various proteins following cell stimulation to regulate PI3K localization and activity (Cantley, 2002). Class I PI3Ks are further classified as la or lb based on regulatory subunit association. In mammals, class la PI3K catalytic subunits interact with one of seven regulatory subunits (p85a, p85cti, p55a, p55ai, p50a, p85(5, and p55y) capable of binding to pY residues (Vanhaesebroeck et al., 1997; Wymann and Pirola, 1998). Thus, class la PI3Ks are key mediators involved in PTK signaling pathways, such as those initiated by immuno-, growth factor, and cytokine receptors. Conversely, class lb PI3Ks utilize the p101 regulatory subunit and are activated by the free Py subunit of heterotrimeric G 2+  25  proteins, which interacts with both the regulatory and catalytic PI3K lb subunits, following GPCR engagement (Vanhaesebroeck et a l , 1997; Wymann and Pirola, 1998). The catalytic subunits of class la (p110ct, p110B, and p1105) and lb (p110y) share a similar structure, including a regulatory subunit binding domain, a Ras binding domain that interacts selectively with GTP-bound Ras and a C-terminal kinase domain (Vanhaesebroeck et a l , 1997). Class II PI3Ks are capable of phosphorylating PI and PI-4-P, but not PI-4.5-P2, and are believed to be a primary source for the generation of PI-3.4-P2 in vivo (Figure 1.5) (Toker, 2002; Vanhaesebroeck et a l , 1997). These enzymes contain a C2 domain (section 1.10) that binds weakly to phospholipids in a Ca -independent manner and has proven essential for catalytic function (Toker, 2002). Class II PI3K 2+  isoforms (a, p and y) do not associate with a regulatory subunit; the regulation of these enzymes, i.e. whether their activity is regulated by extracellular stimulation, remains to be determined, although the C2 domain is one candidate regulatory domain (Toker, 2002; Vanhaesebroeck et a l , 1997). Finally, the substrate specificity of class III PI3Ks is restricted to PI (Figure 1.5) (Toker, 2002; Wymann and Pirola, 1998). Class III PI3Ks are homologous to the yeast PI3K, Vps34p. These enzymes associate with an adaptor molecule that contains S/T kinase activity (Vps15p in yeast or the mammalian homologue p150); however, their regulation is hypothesized to be constitutive (Vanhaesebroeck et a l , 1997). Class III PI3Ks play an integral role in vacuole sorting, since PI-3-P serves as a docking site for various FYVE domain containing proteins (section 1.10) that are involved in the regulation of vesicle trafficking events (Rameh and Cantley, 1999).  1.8  PHOSPHATASES Phosphatases are the antithesis of kinases; they catalyze the removal of phosphate groups from their  targets via hydrolysis. Dephosphorylation of both proteins and lipids is of equal importance to protein and lipid phosphorylation for the regulation of cellular activities (Pawson and Scott, 1997). The majority of phosphatases identified to date function as negative regulators of cellular events by turning off the signaling pathways initiated by kinases. However, a number of phosphatases, such as CD45 (section 1.7.1.1), play positive roles in the initiation of signaling events, thus it is important to examine the function of each phosphatase individually. Like their kinase counterparts, protein phosphatases are grouped according to substrate specificity; S/T phosphatases, such as calcineurin (Crabtree and Olson, 2002), recognize pS and/or pT as substrates, whereas protein tyrosine phosphatases (PTPs), such as SHP-1 and SHP-2, target pY residues. SHP-1 and SHP-2 are unique amongst PTPs since they both contain two tandem SH2 domains (section 1.9) that allow these enzymes to interact with PTK signaling pathways. Intriguingly, these 26  closely related PTPs appear to play very different roles in the regulation of cell signaling events (Pawson and Scott, 1997; Tonks and Neel, 1996). SHP-1, which is expressed at highest levels in hemopoietic cells, negatively regulates signaling pathways downstream of immuno-, growth factor and cytokine receptors, whereas SHP-2, which is expressed ubiquitously, appears to act as both an adaptor/enhancer molecule and a phosphatase to positively regulate signaling pathways downstream of a wide variety of receptors (Feng, 1999; Tonks and Neel, 1996). The inositol polyphosphate phosphatases selectively remove phosphate groups from water soluble inositol phosphates and/or membrane integrated Pis (Figure 1.5) in a Mg -dependent manner. These 2+  enzymes are further subclassified as inositol polyphosphate 3'-, 4'-, or 5'-phosphatases (ptases), based on their abilities to remove the phosphate group from the D-3, D-4, or D-5 position of the inositol headgroup, respectively (Rameh and Cantley, 1999; Toker, 2002). The following sections will take a closer look at one recently characterized 3-ptase and the whole family of 5-ptases characterized to date.  1.8.1  PTEN The tumor suppressor PTEN (ghosphatase and tensin homologue deleted on chromosome ten) or  MMAC (mutated in multiple advanced cancers) is deleted or mutated in a large number of human cancers (Cantley and Neel, 1999; Krystal, 2000; Maehama and Dixon, 1999). Although PTEN can act as both a protein Y/S/T phosphatase and an inositol polyphosphate 3-ptase, evidence suggests that PTEN's tumor suppressor effects stem primarily from its ability to dephosphorylate PIP3 and downregulate the PI3K pathway (Cantley and Neel, 1999; Leslie and Downes, 2002). Homozygous disruption of PTEN is embryonic lethal. However, embryonic fibroblasts can be obtained from PTEN-/- mice and these cells display elevated PIP3 levels and constitutive activation of the protooncogene PKB (Krystal, 2000; Maehama and Dixon, 1999). In addition to early studies that showed PTEN promoted cell cycle arrest, induced apoptosis, and inhibited cell motility by downregulating PKB activity, more recent reports have identified PTEN as a negative regulator of angiogenesis since PKB positively regulates VEGF production (Leslie and Downes, 2002). Although PTEN displays the cysteine- and arginine-based signature motif found in all PTPs and dual specificity phosphatases, it is hypothesized that PTEN's preference for lipid substrates results from its' broader and deeper phosphatase domain that is tightly associated with a C2 domain (section 1.10) (Leslie and Downes, 2002). Although surprisingly little is known about the regulation of the cellular activity of this tumor suppressor, recent studies suggest that the C2 domain may regulate phospholipid binding in a C a 2+  independent manner (Leslie and Downes, 2002). Furthermore, phosphorylation on specific Y or S residues 27  can positively or negatively regulate the enzymatic activity of PTEN, respectively (Birle et a l , 2002; Koul et a l , 2002).  1.8.2  Inositol polyphosphate 5'-phosphatases (5-ptases) To date, "the most information concerning PI phosphatase function and regulation has come from  the study of inositol polyphosphate 5-ptases, which specifically dephosphorylate Pis containing a 5' phosphate, such as PI-5-P, PI-3,5-P , PI-4,5-P , and/or PIP " (Figure 1.5) (Toker, 2002). In addition to the 2  2  3  aforementioned Pis, 5-ptases can hydrolyze phosphate groups from the water soluble inositol phosphates, IP3 and inositol-1,3,4,5-tetrakisphosphate (IP4) (Majerus et a l , 1999). The catalytic domains of 5-ptases are characterized by two highly  conserved motifs  with the core sequences (F/I)WXGDXN(Y/F)R  (F=phenylalanine; l=isoleucine; W=tryptophan; G=glycine; D=aspartic acid; N=asparagine; R=arginine; X=any amino acid) and (R/N)XP(S/A)W(C/T)DR(I/V)(I/L) (P=proline; A=alanine; Ocysteine; V=valine; L=leucine), separated by 60-100 amino acids (Majerus et a l , 1999). Based on substrate specificity, these enzymes are subclassified into four groups. Groups I through III will be expanded on below (Figures 1.6 & 1.7). Group IV enzymes, which associate with PI3K and can only utilize PIP3 as a substrate (Figure 1.7), will not be discussed since this group remains poorly characterized (Jackson et a l , 1995; Majerus et a l , 1999). Worthy of note, the cDNA encoding a novel group IV 5-ptase was recently cloned and characterized; type IV 5-ptase mRNA is expressed primarily in brain and encodes a 70kDa protein that displays the highest affinity toward PIP3 of the known 5-ptases (Kisseleva et a l , 2000).  1.8.2.1 Group I inositol polyphosphate 5-ptases Group I 5-ptases selectively dephosphorylate the soluble inositol phosphates, IP3 and IP4, but not the corresponding phospholipids (Figure 1.7) (Majerus et a l , 1999; Toker, 2002). Since this group displays the highest activity of any 5-ptases towards these substrates (which are important regulators of cellular Ca  2 +  responses), it is hypothesized that Group I 5-ptases function primarily to downregulate C a 2+  dependent signaling events (Majerus et a l , 1999). These enzymes are anchored to the membrane through isoprenylation (De Smedt et a l , 1996). Relatively little is known about the regulation of this family of 5ptases, with the exception of the 43kDa type I 5-ptase (5-ptase I) originally identified in platelets (Figure 1.6) (Erneux et a l , 1998). 5-ptase I is found in a complex with pleckstrin and 14-3-3e, both of which contribute to 5-ptase I activation following cell stimulation (Majerus et a l , 1999). The activation of 5-ptase I by pleckstrin represents a negative feedback loop for PKC activity; activated PKCs phosphorylate pleckstrin on S/T residues and this leads to 5-ptase I activation, which subsequently reduces cellular IP3 levels that bind to IP3 receptor channels to facilitate the release, of intracellular C a  28  2 +  stores necessary for PKC  activation (Majerus et al., 1999). Finally, transfection of cells with antisense cDNA for 5-ptase I results in the elevation of IP3 and intracellular C a  2 +  levels, providing further support for the hypothesized negative  regulatory role of Group 15-ptases in Ca -dependent signaling events (Majerus et al., 1999). 2+  Group I  (43kDa)  5-ptase I Group II  5-ptase II  (115&104kDa)  OCRL-1  (105kDa)  Synaptojanin 1 |_  (145&175kDa)  Stop  (140kDa)  Synaptojanin 2 [J Group III  SHIP  (145kDa)  SHIP2  ] (150kDa)  Figure 1.6: The structures of group I, II and III inositol polyphosphate 5-ptases. Sptases are classified into groups (I to IV) based on substrate specificity. The major form of each protein is illustrated here; size is indicated in brackets. Several important regions are highlighted: 5'-ptase catalytic domains (•); SH2 domains (•); and proline rich regions (•). In synaptojanin 1, the position of the second stop site required for the formation of the shorter 145kDa form occurs within the prolinerichregion, as indicated. The thick black bars in SHIP and SHIP2 represent PTB consensus sequences (NPXY motifs).  1.8.2.2 Group II inositol polyphosphate 5-ptases Group II 5-ptases demonstrate broad substrate specificity; they catalyze the hydrolysis of IP3, IP4, PI-4.5-P2 and PIP3 to varying degrees in vitro (Figure 1.7) (Erneux et al., 1998; Toker, 2002). Additionally, members of this family often exist in several isoforms due to differential splicing of their mRNAs (Majerus et al., 1999). The first Group II 5-ptase cloned, type II 5-ptase (5-ptase II) or INPP5P, was originally isolated from platelets (Figure 1.6) (Erneux et al., 1998). In mice, two 5-ptase II isoforms exist a 104kDa isoform that is cytosolic and a larger 115kDa isoform that localizes to mitochondria and plasma membranes by means of C-terminal isoprenylation and N-terminal residues (Erneux et al., 1998; Matzaris et al., 1998). Based on the localization of these isoforms, it is predicted that the 104kDa isoform prefers the soluble inositol phosphates as substrates, whereas the 115kDa utilizes the membrane bound phospholipids. Furthermore, differential expression of these isoforms suggests that they play different roles in specific tissues; the major isoform in lung and testis is the 104kDa form, while the 115kDa form predominates in  29  brain and skeletal muscle, and comparable levels of both are found in the liver and kidney (Matzahs et a l , 1998). Interestingly, it is unknown whether these splicing events occur in humans (Erneux et a l , 1998). A second Group II family member, which exhibits significant homology to 5-ptase II, is OCRL-1, the 105kDa product of the X-chromosome gene mutated in Lowe syndrome (aka oculocerebrorenal dystrophy) (Figure 1.6) (Erneux et a l , 1998; Majerus et a l , 1999). OCRL-1 typically associates with lysosomes, although it was reported to associate with the Golgi apparatus in lymphocytes and fibroblasts; these associations occur independent of isoprenylation (Erneux et a l , 1998; Majerus et a l , 1999). OCRL-1 can dephosphorylate all potential inositol polyphosphate 5-ptase substrates (Figure 1.7), but displays a marked preference for PI-4.5-P2 (Zhang et a l , 1998). For example, renal proximal tubule cells from human patients with Lowe syndrome accumulate PI-4.5-P2 (Zhang et a l , 1998). PI-4.5-P2 is essential for the budding of membrane vesicles from lysosomes; thus, the accumulation of PI-4.5-P2 in Lowe syndrome may increase enzyme trafficking from lysosomes to the extracellular space, where these released enzymes are believed to cause tissue damage resulting in renal failure and blindness (Majerus et a l , 1999). Surprisingly, no abnormal phenotype was reported in OCRL-1 knockout (-/-) or 5-ptase II-/- mice (Janne et a l , 1998). However, crossing these mouse lines to create a double -/- mouse resulted in an embryonic lethal phenotype, suggesting that, unlike their human counterparts, these enzymes have overlapping functions in mice (Janneetal, 1998). Finally, synaptojanin 1 and 2 are closely related Group II 5-ptases that both contain an N-terminal region homologous to the yeast protein Sac I and a C-terminal proline rich region, in addition to a central 5ptase catalytic domain (Figure 1.6) (Erneux et a l , 1998; Majerus et a l , 1999). Although these proteins display high identity (over 50%) in the Sac I and 5-ptase domains, their proline rich regions show very little identity (Erneux et a l , 1998; Majerus et a l , 1999). It is predicted that the differences in their proline rich tails allow these proteins to interact with unique subsets of SH3 domain (section 1.9) containing proteins and are responsible for the different subcellular localization of these proteins (Erneux et a l , 1998; Majerus et a l , 1999). Interestingly, several alternatively spliced forms of the synaptojanins exist, and these alterations typically occur at the C-terminal end of these proteins (Erneux et a l , 1998); however, the cellular role of many of these isoforms remains to be determined. The synaptojanins are reported to prefer PI-4,5P2 as a substrate and primarily participate in synaptic vesicle trafficking (Erneux et a l , 1998; Majerus et a l , 1999; Toker, 2002). For example, the C-terminal end of synaptojanin 1 can form complexes with members of the endocytic machinery, such as dynamin and amphiphysin (Erneux et a l , 1998; Majerus et a l , 1999).  30  1-1,4,5-P,  Group II  Group IV  5-ptase II OCRL-1 Synaptojanin 1 Synaptojanin  5-ptase IV  l-1,3,4,5-P  PI-4.5-P,  4  PI-3A5-P,  Figure 1.7: Inositol polyphosphate 5-ptase substrate specificity. The four known substrates of the 5-ptases (groups I to IV) are illustrated; arrows indicate substrate specificity. Group I 5-ptases only remove the D-5 phosphate from the soluble inositol phosphates. Group II 5-ptases catalyze this hydrolysis reaction on all four substrates with varying efficiencies. Group III 5-ptases only recognize substrates phosphorylated at both the D-3 and D-5 positions of the inositol ring. Group IV 5-ptases only utilize PIP3 as a substrate. Ri and R2 represent the lipid side chains.  1.8.2.3 Group III inositol polyphosphate 5-ptases Group III 5-ptases only hydrolyze substrates that have a phosphate group at the D-3 position of the inositol ring, i.e. IP4 and PIP3 (Figure 1.7) (Majerus et a l , 1999)*. There are two group III enzymes: SHIP (SH2-containing inositol 5-p_tase) and SHIP2 (Figure 1.6). Both enzymes contain an N-terminal SH2 domain (that interacts with pY residues; section 1.9.1), a central 5-ptase domain, a C-terminal proline rich region (that interacts with SH3 domain containing proteins; section 1.9.2), and one (SHIP2) or two (SHIP) NPXY sequences (that, when phosphorylated, interact with PTB (phosphotyrosine binding) domain containing proteins; section 1.9.1) (Figure 1.6) (Erneux et a l , 1998; Krystal, 2000). These enzymes are over 38% identical at the amino acid level, with the lowest identity in their proline rich tails (Majerus et a l , 1999; Rohrschneider et a l , 2000). Despite the high sequence and structural similarity observed between these two proteins, they have distinct tissue distribution patterns. SHIP expression is primarily restricted to cells of hemopoietic origin (SHIP will be described in more detail in section 1.8.3). Conversely, SHIP2, the related but distinct gene product, has a more ubiquitous distribution; as such, it is found co-expressed with SHIP in many hemopoietic cells (Erneux et a l , 1998; Pesesse et a l , 1997). SHIP and SHIP2 function primarily as  * Worthy of note, it was recently reported that under certain conditions (i.e. in the presence of n-octyl (3-glucopyranoside but not cetyltriethylammonium bromide) SHIP can display activity towards PI-4.5-P2 in in vitro phosphatase assays (Kisselev et al, 2000); however, whether this reaction occurs in vivo or is physiologically relevant remains to be determined.  31  negative regulators of PI3K activity, since they both utilize PIP3 as a substrate in vivo (Damen et al., 1996; Pesesse et a l , 1998). Although they utilize the same substrate, their unique distribution patterns and different proline rich C-termini, which bind to Src and Grb2 (SHIP) or Abl (SHIP2) preferentially, suggest that SHIP and SHIP2 are not totally redundant in their functions (Krystal, 2000). SHIP2 was cloned in 1997 using degenerate primers coding for highly conserved amino acid regions shared between 5-ptase catalytic domains (Pesesse et a l , 1997). It has since been shown to become tyrosine phosphorylated in response to growth factors, such as insulin, EGF, and PDGF, cytokines, such as IL-3 and GM-CSF, and the co-ligation of BCR/FcyRIIB (Krystal, 2000; Majerus et a l , 1999; Muraille et a l , 2000). In addition to its role in the down regulation of PI3K activity, SHIP2 couples to the adaptor molecules She and Grb2 after stimulation to negatively regulate mitogen activated protein kinase (MAPK) pathways (Clement et a l , 2001; Toker, 2002). Generation of mice lacking the SHIP2 gene revealed that the "loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death" (Clement et a l , 2001). Because insulin is the primary regulator of glucose homeostasis and the impairment of insulin action and/or secretion plays a critical role in the development of diabetes, SHIP2 has become one potential target for the treatment of type II diabetes (Clement et a l , 2001; Marion et a l , 2002). A deletion in the 3' untranslated region of SHIP2 that encompasses a motif implicated in the control of protein synthesis was recently reported in type II diabetic subjects (Marion et a l , 2002). Furthermore, this deletion, which causes SHIP2 mRNA and protein overexpression in vitro, positively correlated with type II diabetes in a cohort of subjects (Marion et a l , 2002).  1.8.3  SH2-containing inositol 5'-phosphatase (SHIP) In the early 1990s, SHIP was identified as a 145kDa protein that became both tyrosine  phosphorylated and associated with the adaptor protein She after the stimulation of blood cells by a variety of cytokines and growth factors (Damen et a l , 1996; Krystal, 2000; Rohrschneider et a l , 2000). In 1996, Damen et al. (1996), Kavanaugh et al. (1996), and Lioubin et al. (1996) independently cloned the cDNA of SHIP. Their cloning strategies were based on: 1) the purification of SHIP from cytokine-stimulated hemopoietic cells using the C-terminal SH3 domain of Grb2 (Damen et a l , 1996); 2) expression cloning using Grb2 as a probe (Kavanaugh et a l , 1996); and 3) the interaction of tyrosine phosphorylated SHIP with the PTB domain of She (Lioubin et a l , 1996), respectively. When analyzed by SDS-PAGE, SHIP is detectable as multiple protein bands of approximately 145, 135, 125 and 110kDa in size. Some of the possible mechanisms responsible for the formation of these 32  different isoforms.include post-translational modification (such as phosphorylation), protein degradation, mRNA splicing, alternative transcriptional initiation and alternative translational initiation (Rohrschneider et al., 2000). The results of one study indicate that these different forms can be generated by proteolytic cleavage of the 145kDa SHIP, possibly by a member of the calpain protease family (Damen et al., 1998b). Other studies suggest that these isoforms are the result of specific mRNA splicing events that occur within various regions encoding the proline rich tail of SHIP (Lucas and Rohrschneider, 1999; Rohrschneider et al., 2000) or within the N-terminal coding region (resulting in the loss of the SH2 domain (Kavanaugh et al., 1996)). Intruigingly, a novel 104kDa SHIP isoform, s-SHIP (stem cell SHIP), that utilizes a transcriptional start site unique from that used by 145kDa SHIP was recently reported; it is expressed in embryonic and hemopoietic stem cells, but not lineage-committed hemopoietic cells (Tu et al., 2001). Overall, it remains controversial whether these mechanisms act alone or in concert to generate the various SHIP isoforms. Alternatively, some groups believe the smaller isoforms are simply the result of in vitro lysis conditions (Horn et al., 2001). Further characterization of the generation and roles of these different SHIP isoforms is essential since their expression may be cell type specific and they may play a key role in regulating the localization and activity of this enzyme, as described below.  SHIP becomes tyrosine phosphorylated following the activation of every major class of transmembrane receptor expressed by hemopoietic cells; however, it is still unclear whether phosphorylation positively or negatively regulates SHIP'S catalytic activity or cellular function (Krystal, 2000). In 1996, Damen et al. (1996) reported that SHIP'S 5-ptase activity did not change with stimulation in vitro. Based on this finding, it was suggested that SHIP'S 5-ptase activity is regulated primarily by translocation to its substrates after stimulation, and this appears to be the case (Krystal, 2000; Rohrschneider et al., 2000). Through its SH2 domain, SHIP is recruited to pY residues in plasma membrane bound proteins, such as the pITIM (tyrosine phosphorylated immunoreceptor tyrosine based inhibition motif) in the low-affinity IgG receptor, FcyRIIB (section 1.11.3); coaggregation of FcyRHB with the BCR, TCR or activating Fc receptors (e.g. FcsRI) leads to the inhibition of the PI3K pathway and thus cellular activation through SHIP'S hydrolysis of membrane bound PIP3 (Rauh and Krystal, 2002; Rohrschneider et al., 2000). In addition to SH2 domain mediated interactions, two groups recently reported that the C-terminal proline rich region of SHIP is essential for its catalytic function; this region may play a role in stabilizing the interaction of SHIP at the membrane by interacting with other proteins or it may be required for the efficient localization of SHIP to specific membrane compartments (Aman and Ravichandran, 2000; Damen et al., 2001).  33  In addition to pITIM sequences, the SH2 domain of SHIP can bind, at least in vitro, to tyrosine phosphorylated (p)ITAMs (section 1.10.1) in the cytoplasmic tails of various immunoreceptors (such as the FcsRI B chain (Gergely et al., 1999; Kimura et al., 1997) and y chains (Osborne et a l , 1996)) that typically lead to positive biological responses. This interaction, if it occurs in vivo, may be important for limiting immunoreceptor initiated responses, such as FcsRI-induced C a  2 +  flux and degranulation in mast cells, both  of which are dependent on the production of PIP3 (Huber et a l , 1998). Additionally, ligand engagement of various immunoreceptors (e.g. FcsRI (Huber et a l , 1998; Kimura et a l , 1997)), BCR (Harmer and DeFranco, 1999), TCR (Lamkin et a l , 1997), and FcyRI (Maresco et a l , 1999)) results in the tyrosine phosphorylation of SHIP, suggesting that pITAMs recruit SHIP to the membrane where it can be phosphorylated. This in turn could allow SHIP to function as an adaptor protein by interacting with and regulating the localization and activity of pY-binding proteins (Krystal, 2000; Rohrschneider et a l , 2000). For example, tyrosine phosphorylated SHIP can interact with the PTB domain of She (a ubiquitously expressed adaptor protein that regulates the MAPK pathway) (Lioubin et a l , 1996), the SH2 domain of p85 PI3K (the kinase responsible for the generation of SHIP'S primary target, PIP3; section 1.7.2.1) (Lucas and Rohrschneider, 1999), and the PTB domain of the adaptor proteins Dok-1 or -3 (Lemay et a l , 2000; Tamir et a l , 2000). In addition to its role in regulating PIP3 levels, SHIP is thought to play a key role in down regulating the MAPK pathway by competing with Grb2/Sos for She binding (Krystal, 2000; Rohrschneider et a l , 2000) or by binding to Dok-1 which recruits Ras-GAP (GTPase activating protein) to the membrane (Ott et a l , 2002; Tamir et a l , 2000). Other pY proteins that can interact with the SH2 domain of SHIP include members of the Dok family (Lemay et a l , 2000), the Gab (Grb2 associated binder) scaffolding proteins (Rohrschneider et a l , 2000), SHP-2 (Liu et a l , 1997), and Syk (Crowley et a l , 1996). Phosphorylation independent binding partners of SHIP include Dab1 and PIAS1 (protein inhibitor of activated STAT1) (Rohrschneider et a l , 2000). Additional studies will be required to determine the significance of most of these interactions with SHIP.  1.8.3.1 SHIP knockout (•/-) mice In collaboration with Dr. R. Keith Humphries' laboratory, our laboratory generated a SHIP-/- mouse to study the role of this protein in vivo. Targeted disruption was accomplished by replacing the first exon (254-bp) of the SHIP gene with the neomycin resistance gene in the antisense orientation (Helgason et a l , 1998). Briefly, 129 embryonic stem (ES) cells were electroporated with the linearized targetting vector and neomycin resistant colonies were selected in G418. DNA was isolated from positive colonies and subjected to Southern blot analysis to visualize the positively targeted allele. Positive ES cells were injected into  34  C 5 7 B L / 6 J b l a s t o c y s t s a n d i m p l a n t e d into a p s e u d o p r e g n a n t C 5 7 B L / 6 J f o s t e r m o t h e r to g e n e r a t e  germ-line  t r a n s m i s s i o n c h i m e r a s , w h i c h w e r e then b r e d onto a C 5 7 B L / 6 J b a c k g r o u n d ( H e l g a s o n et a l . , 1 9 9 8 ) .  H o m o z y g o u s S H I P - / - m i c e a r e both v i a b l e a n d fertile, revealed  that  these  mice  exhibit  pronounced  b u t f a i l to t h r i v e . A n a t o m i c a l  splenomegaly;  spleen  weights  and  total  examination  cellularity  i n c r e a s e d f i v e - t o s e v e n - f o l d in k n o c k o u t m i c e ( H e l g a s o n e t a l . , 1 9 9 8 ) . F u r t h e r m o r e , t h e l u n g s o f mice  are  enlarged  and  display  m a c r o p h a g e s and neutrophils).  patchy  white  discoloration  due  S i n c e S H I P e x p r e s s i o n is primarily  H e l g a s o n et al. (1998) e x a m i n e d this c o m p a r t m e n t  to  infiltration  by  myeloid  are  SHIP-/-  cells  limited to c e l l s of h e m o p o i e t i c  (i.e.  origin,  in t h e s e - / - m i c e a n d r e p o r t e d i n c r e a s e d g r a n u l o c y t e -  m a c r o p h a g e p r o g e n i t o r n u m b e r s in t h e b o n e m a r r o w a n d s p l e e n . I n t e r e s t i n g l y ,  these cells also displayed  h y p e r - r e s p o n s i v e n e s s to s t i m u l a t i o n b y M - C S F , G M - C S F , IL-3 o r S C F ( H e l g a s o n et a l . , 1 9 9 8 ) . T h e s e m i c e have  played  differentiation,  a  vital  role  in  defining  the  role  of  SHIP  in  mediating  hemopoietic  cell  proliferation,  activation, inactivation, s u r v i v a l a n d d e a t h d e c i s i o n s ( B r a u w e i l e r et al., 2 0 0 0 ; H u b e r et a l . ,  1998).  Furthermore, a n u m b e r of interesting  p a r a l l e l s h a v e b e e n o b s e r v e d b e t w e e n a variety of  d i s e a s e s and S H I P - / - phenotypes. For e x a m p l e , myeloproliferation  i s o b s e r v e d in h u m a n s w i t h  m y e l o g e n o u s l e u k e m i a ( C M L ) , w h i c h is c a u s e d b y a B c r / A b l t r a n s f o r m a t i o n  human chronic  that r e d u c e s e x p r e s s i o n  of  S H I P p r o t e i n ( R a u h a n d K r y s t a l , 2 0 0 2 ; R o h r s c h n e i d e r et a l . , 2 0 0 0 ) . T h i s s u p p r e s s i o n o f S H I P is d e p e n d e n t on  the  k i n a s e activity  identified  of A b l a n d  a c h e m i c a l inhbitor  as a successful drug therapy  of A b l  (STI-571  or  Gleevec) has  recently  been  a g a i n s t C M L ( R o h r s c h n e i d e r et al., 2 0 0 0 ; Sattler et al.,  1999).  Additionally, S H I P - / - B cells display e n h a n c e d function a n d S H I P - / - mice h a v e elevated s e r u m IgG a n d levels  (Helgason  characterized (Rauh  and  by  et IgG  al.,  2000);  lack  anti-nuclear  Krystal, 2002).  Abs  Finally, the  of  SHIP  and  appears  immune  bones  of  to  predispose  complex-mediated  SHIP-/-  mice  are  to  a  lupus-like  proliferative  IgM  autoimmunity  glomerulonephritis  severely osteoporotic  (described  in  c h a p t e r 7 ( T a k e s h i t a e t a l . , 2 0 0 2 ) ) . T h u s , t h e s e m i c e a l l o w u s to f u r t h e r e l u c i d a t e t h e r o l e o f S H I P in v a r i o u s h u m a n d i s e a s e s t a t e s a n d t h e y s e r v e a s a n e x t r e m e l y u s e f u l t o o l to s t u d y S H I P ' S p o t e n t i a l a s a t h e r a p e u t i c target.  1.9  PROTEIN-PROTEIN INTERACTIONS A  n u m b e r of s p e c i a l i z e d protein-protein  i n t e r a c t i o n d o m a i n s h a v e b e e n i d e n t i f i e d to d a t e .  These  d o m a i n s h a v e b e e n s h o w n t o p l a y a c r i t i c a l r o l e in t h e p r o p a g a t i o n o f s i g n a l s f r o m a n a c t i v a t e d r e c e p t o r b y mediating numerous m e m b r a n e , cytoplasmic, and nuclear p r o c e s s e s ( P a w s o n , 1995; Sudol, 1998). delineation  and characterization  of protein  modules exemplified  35  by S H d o m a i n s  has revolutionized  "The our  understanding of the molecular events underlying signal transduction pathways" (Sudol, 1998). Proteinprotein interaction domains are modular units, typically 40-150 amino acids in length, that can often function independently of their resident proteins (Sudol, 1998). These modules can be subdivided into two major classes: phosphorylation-dependent and phosphorylation-independent protein binding domains.  1.9.1  Phosphorylation-dependent protein interaction modules Although the majority of protein interaction domains are phosphorylation-independent domains, the  phosphorylation-dependent protein binding domains regulate reversible protein-protein interactions both spatially and temporally, thus, play a critical role in regulating the specificity of receptor initiated signaling events (Sudol, 1998; Yaffe, 2002). The first modular phosphorylation-dependent signaling domain identified was the SH2 domain (Sadowski et al., 1986). SH2 domains are modules of -100 amino acids, which bind to pY residues. Each SH2 domain consists of two conserved pockets: the first pocket contains several basic residues, including an invariant arginine, and binds to the pY residue by forming hydrogen bonds with the phosphate group; the second pocket is more variable in sequence, but confers binding specificity by recognizing three to six residues located C-terminal to the pY site (Pawson and Scott, 1997; Yaffe, 2002). "It has been estimated that the human genome encodes for eighty-seven proteins that contain ninety-five SH2 domains, whereas just one such protein has been found in Saccharomyces cervisiae" (Yaffe, 2002). The cellular functions of these SH2 domains include the recruitment of signaling molecules, e.g. PI3K, and adaptor molecules, e.g. Grb2 and She, to tyrosine phosphorylated receptors or downstream pY containing proteins (Pawson and Nash, 2000; Yaffe, 2002). SH2 domains also maintain cytoplasmic tyrosine kinases, e.g. Lyn, in an inactive state in the resting cell, and are important for the recruitment of substrates following kinase activation (Yaffe, 2002).  The second pY binding module discovered was the PTB domain. This domain was originally identified in the adaptor protein She as the region distinct from the SH2 domain that was capable of binding to tyrosine phosphorylated growth factor receptors (Blaikie et al., 1994; Kavanaugh and Williams, 1994). PTB domains are modules of-100-150 amino acids that bind to ligands tyrosine phosphorylated on a core NPXY sequence, although a subset of PTB containing proteins can bind to this core sequence in the unphosphoryated state (Sudol, 1998; Yaffe, 2002). Unlike SH2 domains, it is residues located N-terminal to the pY site that confer PTB domain binding specificity (Pawson and Scott, 1997; Yaffe, 2002). Twentyseven PTB domains have been identified in the human genome, whereas none have been found in Saccharomyces cerevisiae or Arabadopsis thaliana (Yaffe, 2002). The majority of PTB domain containing  36  proteins act as adaptors, such as She, or docking proteins, such as the Doks, to regulate cell signaling events (Pawson and Scott, 1997; Yaffe, 2002). Analogous to pY binding motifs, a growing family of protein modules that can bind to pS or pT residues and regulate protein-protein complex assembly in a context specific manner have been identified (Pawson and Nash, 2000; Tzivion et al., 2001). 14-3-3 was the first pS binding protein discovered (Muslin et a l , 1996). This 30kDa protein, which exists in several isoforms, mediates the localization and activation state of its binding partners (Tzivion et a l , 2001). 14-3-3 binding partners identified to date include the S/T kinase Raf-1, cdc25, Bad, histone deacetylase, and the forkhead TFs, e.g. FKHRL1 (Muslin, et a l , 1996; Tzivion et a l , 2001). The second pS binding domain to be identified was the FHA (Forkhead-association) domain. FHA domains are 65-100 amino acids in length and mediate pS/T interactions in eukaryotic nuclear proteins, such as Rad53, which controls the DNA damage checkpoint pathway (Tzivion et a l , 2001). Two additional pS/T binding domains have been identified, WD40 and WW domains; however, the binding of these modules is not restricted to pS/T residues, as will be discussed in the next section. The binding of WD40 domains in F-box proteins to pS or pT residues in proteins brings the phosphorylated protein into the ubiquitin ligase enzyme complex where it is ubiquitinated and subsequently degraded (Barinaga, 1999). WW domains in Pin1, which regulates cell division, and Nedd4, which regulates protein degradation, can also bind to pS residues (Barinaga, 1999; Lu et a l , 1999).  1.9.2  Phosphorylation-independent protein interaction modules In addition  to the  phosphorylation-dependent  modules outlined  above, phosphorylation-  independent protein interaction modules play a key role in the regulation of signal transduction events and these modules co-exist in a variety of signaling proteins (Pawson and Nash, 2000; Sudol, 1998). A number of phosphorylation-independent modules have been identified and characterized to date, including PDZ, EH, SAM and WD40 domains. PDZ domains recognize and bind to C-terminal hydrophobic residues, such as valine, and function to localize cytosolic proteins to regions of cell-cell contact (Pawson and Scott, 1997; Sudol, 1998). EH (Eps15 homology) domains bind to core NPF sequences and are important regulators of vesicle trafficking and endocytosis (Confalonieri and Di Fiore, 2002; Sudol, 1998). The specificities of SAM (sterile alpha motif) and WD40 domains remain undefined, although both are found in a wide variety of proteins. SAM domains, which can be found in SHIP2 (Pesesse et a l , 1997), are believed to regulate the formation of homo- or heterotypic oligomers (Chi et a l , 1999). WD40 domains mediate a number of proteinprotein interactions, in addition to their pS/T binding role discussed above, such as G protein (3 subunit binding to a and y subunits (Sudol, 1998). 37  A growing family of modules, including SH3, GYF, WW, and EVH1 domains, recognize and bind to proline rich sequences (Sudol, 1998). SH3 domains bind to the minimum consensus sequence PXXP and function to regulate protein localization and enzymatic activity in addition to the formation of large signaling complexes (Ren et al., 1993; Sudol, 1998). A number of signaling proteins contain SH3 domains, including Src family members and Grb2. WW domains, which contain two highly conserved tryptophan (W) residues, are 35-40 amino acids in length and bind to PPXY or PPLP core sequences surrounded by three additional prolines (Pawson and Scott, 1997; Sudol, 1998). Only two WW domains identified to date exhibit pS binding specificity, as described above (Sudol, 1998). Finally, EVH1 (Ena/Vasp homology) domains are -110 amino acids in length and can be found in scaffolding proteins, such as vinculin, that regulate the formation of large protein complexes to modulate the actin cytoskeleton (Ball et al., 2002).  1.10  PROTEIN-LIPID INTERACTIONS A number of protein domains facilitate protein-lipid interactions, which play an important role in  regulating the localization of signaling molecules and/or altering the catalytic activity of enzymes (Pawson and Scott, 1997). Protein-lipid interactions are mediated by C1, C2, FYVE, PX, FERM and PH domains. C1 domains are cysteine-rich modules of -50 amino acids in length that function to recruit proteins to the membrane by binding to the PLCy product DAG or phorbol esters. For example, both classical and novel PKC S/T kinases contain C1 domains, which play the dual role of recruiting these proteins to the membrane (Feng et al., 2000; Vallentin et al., 2000) and increasing their catalytic activity (Jaken, 1996). C2 domains are -130 amino acids in length and regulate acidic phospholipid binding in a Ca -dependent or 2+  independent manner (Pawson and Scott, 1997). The cellular functions of C2 domains range from signal transduction (i.e. the C2 domain in PKC(3 regulates Ca -dependent binding to phosphatidylserine (Feng et 2+  al., 2000)) to vesicular trafficking (i.e. the C2 domain in synaptotagmin, a membrane protein in synaptic vesicles, regulates Ca -dependent binding to PI-4,5-P and PIP (Schiavo et al., 1996)). FYVE (Fab-1, 2+  2  3  YOTB, V a d and EEA1) domains are -60 amino acids in length and are structurally similar to C1 domains. These domains bind to PI-3-P and function to regulate signaling downstream of the TGFp receptor as well as vacuolar/lysosomal membrane trafficking events (Itoh et al., 2002; Misra et al., 2001). Finally, PX (Phox homology) domains bind to phosphoinositides in the membrane and regulate the location of proteins, such as NADPH oxidase (Sato et al., 2001), and FERM (Band 4.1, Ezrin, Radixin, and Moesin) domains regulate the membrane binding of cytoskeletal-associated proteins, such as Radixin (Hamada et al., 2000).  38  PH (Pleckstrin homology) domains regulate the recruitment of various signaling proteins to the plasma membrane by binding to the charged headgroups of specific phosphoinositides (e.g. PI-4.5-P2, PIP3 or PI-3.4-P2) (Pawson and Scott, 1997; Toker, 2002). PH domains have been identified in over one hundred eukaryotic proteins, but not plant or bacterial proteins, and appear to be related more by structural similarity than sequence homology (Bottomley et a l , 1998). Once at the membrane, PH domain containing proteins interact with regulators or targets; thus, both the recruitment and activation of PH domain containing proteins is mediated in large part by the regulation of membrane phospholipid levels. For example, the PH domain of PLCy binds preferentially to its substrate PI-4.5-P2, whereas the PH domain of Btk must bind to PIP3 in order to regulate downstream events such as C a  2 +  entry into the cell (section  1.11.2) (Sato et a l , 2001; Turner and Kinet, 1999). Overall, "PH domains couple the actions of PI kinases, inositol phosphatases and phospholipases to the regulation of intracellular signaling events" (Pawson and Scott, 1997).  1.11  IgE RECEPTOR MEDIATED SIGNALING EVENTS When a foreign multivalent Ag or allergen binds to FceRI-bound IgE on the surface of mast cells,  receptor crosslinking initiates a complex series of phosphate transfer events (Kinet, 1999; Metcalfe et a l , 1997; Ott and Cambier, 2000; Turner and Kinet, 1999). These signaling events are responsible for the generation and release of proinflammatory mast cell mediators, thus, regulate the allergic response (Ott and Cambier, 2000; Metcalfe et a l , 1997). This section will examine the early signaling events that regulate mast cell activation following antigenic crosslinking of the FceRI.  1.11.1 Immunoreceptor tyrosine based activation motifs (ITAMs) Immunoreceptors transduce activating signals through ITAMs in their cytoplasmic tails (Beaven and Metzger, 1993; Kinet, 1999; Turner and Kinet, 1999). These di-tyrosine based motifs, which were first described by Michael Reth (Reth, 1989), exist in one or more copies in each immunoreceptor signal transducing molecule (Gergely et a l , 1999; Turner and Kinet, 1999). For example, three ITAMs exist in the TCR <; subunit, while one copy is found in the TCR y, 8, and s subunits, and the BCR a and B subunits (Kinet, 1999). Similarly, the B and y chains of the FcsRI complex each contain one ITAM (Kinet, 1999; Turner and Kinet, 1999). The ITAM consensus sequence is D/EXXYXXLX7-11YXXL/I (E=glutamic acid) and the two Y residues, which, become rapidly and transiently phosphorylated by receptor-associated PTKs following receptor aggregation, serve as docking sites for cytoplasmic SH2 domain containing proteins (Gergely et a l , 1999; Ott and Cambier, 2000; Siraganian et a l , 2002). Interestingly, different pITAMs bind to distinct cytosolic effector molecules, allowing them to activate specific pathways downstream of each 39  immunoreceptor. For example, the FcsRI p and y chain ITAMs are distinct from each other in structure and function. Structurally, the p chain ITAM contains a third Y residue between the two YXXL consensus sequences and has a shorter spacer region than the y chain ITAM (Turner and Kinet, 1999). Functionally, the p pITAM associates with Lyn, while the y pITAM associates preferentially with Syk (as described in the next section) (Gergely et al., 1999; Turner and Kinet, 1999).  1.11.2 Early FcsRI phosphorylation events  00  Like all immunoreceptor family members, FcsRI lacks intrinsic tyrosine kinase activity. The Src family kinase, Lyn, which is found constitutively associated with the FcsRI p subunit, is well documented to initiate IgE+Ag-induced phosphate transfer events (Nadler and Kinet, 2002; Ott and Cambier, 2000; Turner and Kinet, 1999). a chain aggregation activates Lyn to phosphorylate the FcsRI p and y ITAMs (Figure 1.8A). Phosphorylation of the p chain ITAM is followed by the recruitment and activation of additional Lyn molecules, leading to enhanced phosphorylation events and the transphosphorylation of other receptors (Ott and Cambier, 2000; Turner and Kinet, 1999). The exact mechanisms by which FcsRI aggregation activates CD45 (section 1.7.1.1) phosphatase activity and inhibits Csk (section 1.7.1.1) kinase activity remain elusive. However, it is known that CD45 is excluded from the aggregated receptor context following the dephosphorylation and activation of Lyn; this exclusion, which may be caused by the segregation of receptor aggregates into lipid microdomains, prevents the continual dephosphorylation of Lyn or other tyrosine phosphorylated signaling intermediates by CD45 (Turner and Kinet, 1999). Interestingly, it also appears that Lyn becomes excluded from the aggregated FcsRI complex shortly (~2 minutes) after crosslinking (Wilson et al., 2000). Phosphorylation of the FcsRI y chain ITAMs by Lyn precedes the recruitment of Syk kinase, which contains tandem SH2 domains. Once Syk is recruited to the receptor complex, it becomes phosphorylated and activated by Lyn (Figure 1,8A) (Nadler and Kinet, 2002; Turner and Kinet, 1999; Wilson et al., 2000). Both Lyn and Syk phosphorylate specific target proteins to regulate a number of cellular functions, as described below.  PLCyl hydrolyzes the membrane phospholipid, PI-4.5-P2, to generate the soluble inositol phosphate, IP3, and membrane bound DAG. Both these products act as important second messengers in the regulation of mast cell degranulation, gene expression and cytoskeletal rearrangements; IP3 regulates intracellular C a  2 +  release, while DAG regulates the activity of various PKC isoforms (Nadler and Kinet,  The information in this section was used to generate a model of early FceRI phosphorylation events (Figure 1.8) based on existing literature at the time this thesis was written. However, this section will have to be amended as more information is accrued. w  40  2002; Turner and Kinet, 1999). To facilitate PI-4,5-P hydrolysis, the SH2 domains of PLCyl bind to LAT 2  (linker for activation of T cells), a membrane anchored protein that is phosphorylated by Syk (Figure 1.8B) (Nadler and Kinet, 2002; Rivera et al., 2002; Turner and Kinet, 1999). Once at the membrane, PLCyl itself must be phosphorylated on two distinct sites to become activated. Syk is responsible for one of these phosphorylation events, while the second event is catalyzed by the Tec family member, Btk (Figure 1.8B) (Ott and Cambier, 2000; Siraganian et al„ 2002; Turner and Kinet, 1999). However, to activate Btk, its PH domain must first bind to the PI3K product, PIP3 (PI3K activation is outlined below); this membrane binding facilitates Lyn-dependent phosphorylation of Btk and the subsequent autophosphorylation event required for full activation of Btk (Figure 1.8B) (Kinet, 1999; Turner and Kinet, 1999). As mentioned above, IP3 triggers the release of C a binding to IP3 C a  2 +  2 +  from intracellular stores; this is achieved by  channel receptors present on the surface of intracellular C a  1.8B). However, a sustained increase in intracellular C a  2 +  2 +  stores, e.g. the ER (Figure  levels is required for most FcsRI effector  functions, including the production of AA metabolites (since PLA2 activity requires C a  2 +  (Murakami and  Kudo, 2002)), and the transcription of cytokine genes (e.g. the TF NFAT (nuclear factor of activated T cells), which regulates IL-4 production, must be dephosphorylated by the Ca -dependent phosphatase, 2+  calcineurin, to be activated (Crabtree and Olson, 2002)). This second phase of C a dependent on the depletion of C a  2 +  by activating the store-operated C a  signaling, which is  2 +  from intracellular stores, is mediated by the entry of extracellular C a 2 +  2 +  channels (SOCCs) present on the plasma membrane (Figure 1.8B)  (Nadler and Kinet, 2002; Turner and Kinet, 1999). The resulting C a generates the majority of intracellular C a  2 +  and Kinet, 1999). Sustained intracellular C a  2 +  release activated current (ICRAC)  and mediates intracellular store refilling (Figure 1.8B) (Turner 2 +  is also important for the activation of Ca -dependent PKCs, 2+  which are thought to play a role in the regulation of mast cell degranulation (Nechushtan et al., 2000; Turner and Kinet, 1999); however, a number of Ca -independent PKCs have recently been reported to 2+  function as regulators of mast cell degranulation as well (Leitges et al., 2002; Liu et al., 2001; Parravicini et al„ 2002).  Another primary target of FcsRI signaling is PI3K. As indicated previously, the p85 regulatory subunit of PI3K mediates the recruitment of this enzyme to the membrane though its SH2 domains; this recruitment allows the associated p110 catalytic subunit to phosphorylate PI-4.5-P2 and generate PIP3 (Cantley, 2002). Our understanding of FcsRI-induced activation of PI3K has increased dramatically over the past two years. For example, the adaptor protein Gab2, which quickly becomes tyrosine phosphorylated after FcsRI aggregation, was recently reported to link the IgE receptor with PI3K activation  41  A <-L> a  B  a  Cytoskeletal targets Mitogen ic effects  Figure 1.8: Early FceRI phosphorylation events. A) Activation of Lyn and Syk. a chain aggregation (1) activates Lyn, which  is associated with the p chain, to phosphorylate the p (2) and y (4) chain ITAMs (*=tyrosine phosphorylation). The p chain pITAM binds to the SH2 domain of additional Lyn molecules (3), while the y chain pITAM recruits Syk (5) to the receptor complex where it is phosphorylated and activated by Lyn (6). B) Regulation of Ca flux Syk phosphorylates LAT (1), which then recruits the SH2 domains of PLCyl (2). At the membrane, PLCyl is phosphorylated by both Syk and Btk (3). These phosphorylation events activate PLCyl to hydrolyze the membrane bound lipid, PI-4.5-P2, to IP3 and DAG (4), the latter of which positively regulates PKC activity (5). IP3 binds to receptors (IP3R) on the ER (5) to release intracellular Ca * (6). This triggers the entry of extracellular Ca by activating the SOCCs (7) and the resulting ICRAC provides the majority of intracellular Ca * and mediates store refilling. C) PI3K activation. Fyn, which is associated with the p chain, phosphorylates Gab2 (1), which then serves as a docking site for additional Fyn molecules (2&3). Phosphorylated Gab2 binds the SH2 domains of the p85 subunit of PI3K (4), recruiting the p110 catalytic subunit to the membrane where it phosphorylates PI-4.5-P2 to generate PIP3 (5). PIP3 recruits the PH domain of Btk (6), which is then phosphorylated by Lyn (7) and autophosphorylates (8) for full activation. D) Roles of adaptor proteins. Syk phosphorylates LAT (1), which then recruits the SH2 domain of Grb2 and its associated effector molecules, Sos and Slp-76 (2). At the membrane, the guanine nucleotide exchange factor, Sos, activates Ras (3). Slp-76 is phosphorylated by Syk (3), then recruits the SH2 domain of the guanine nucleotide exchange factor, Vav (4), which is also 2+  2  2+  2  phosphorylated by Syk (5) prior to its activation of Rac (6). Adapted from (Turner and Kinet, 1999) and (Nadler and Kinet, 2002).  42  by binding to the p85 regulatory subunit (Gu et al., 2001; Parravicini et al., 2002; Rivera et al„ 2002). Studies in mast cells derived from Gab2-/- mice show an 80% reduction in PIP3 levels, compared to wildtype mast cells, following FcsRI aggregation; this reduction is thought to be responsible for the reduced degranulation and cytokine production (e.g. IL-6) observed in Gab2-/- mast cells (Gu et al., 2001). In addition, the Src family kinase, Fyn, was recently identified as the principal kinase that positively regulates Gab2 phosphorylation (independent of Lyn, Syk or LAT) (Figure 1.8C) (Nadler and Kinet, 2002; Parravicini et al., 2002). Similar to Lyn, Fyn appears to associate with the FcsRI p subunit in mast cells and this interaction is enhanced by FcsRI aggregation (Parravicini et al., 2002). Furthermore, Fyn associates with Gab2 in activated mast cells, which may serve to enhance Gab2 phosphorylation after FcsRI aggregation. Studies in mast cells derived from Fyn-/- mice reveal impaired degranulation following FcsRI aggregation (Parravicini et al., 2002). Interestingly, Lyn appears to negatively regulate Gab2 phosphorylation events (Figure 1.8C), since studies in mast cells from Lyn-/- mice reveal enhanced Gab2 phosphorylation, PI3K activity, PIP3 production, PKB phosphorylation and degranulation (Parravicini et al., 2002). While Ca flux 2+  is inhibited in the absence of Lyn, Fyn-deficient mast cells display normal Ca flux (Parravicini et al., 2002); 2+  thus, "Fyn mediated signals may affect Lyn mediated signals at the point of Btk recruitment" (Nadler and Kinet, 2002). Future studies on the role of Fyn in FcsRI-induced signaling will help clarify the roles of Fynversus Lyn-mediated signaling events downstream of this receptor, including areas of cross-talk, and may identify additional targets of Fyn.  Finally, a number of adaptor proteins that contain protein-protein interactions domains, but lack intrinsic tyrosine kinase activity, play important roles in the initiation of early FcsRI signaling events. For example, Grb2, which contains one SH2 domain and two SH3 domains, interacts with the phosphorylated adaptor protein She or LAT (Turner and Kinet, 1999). In mast cells, both Sos and Slp-76, which interact with Grb2's SH3 domains, have been identified as Grb2 effector molecules (Figure 1.8D) (Ott and Cambier, 2000; Turner and Kinet, 1999). Sos is a guanine nucleotide exchange factor for Ras that promotes GTP loading and Ras activation. Since Ras is membrane bound, Grb2 functions to recruit Sos to its substrate (Turner and Kinet, 1999). Sos also contains a PH domain, which may mediate membrane targeting. However, it is unknown whether this domain "assists, precedes, or follows the Grb2 interaction" (Turner and Kinet, 1999). One of the effector pathways downstream of activated Ras is the classical Raf/Mek/Erk (extracellular regulated kinase) cascade. Activation of this MAPK pathway regulates the expression of immediate early genes by activating the TF, Elk-1, the expression of numerous cytokines and chemokines by activating TFs such as N F K B , and the generation of AA metabolites by activating PLA2 (Miura et al., 1999; Ott and Cambier, 2000; Turner and Kinet, 1999). Other targets of activated Ras include JNK (c-Jun 43  N-terminal kinase) and p38 MAPKs, PI3K, and other GTPases (e.g. Rac and Rho) (Ott and Cambier, 2000; Turner and Kinet, 1999). Grb2-mediated recruitment of Slp-76 to the membrane is essential for Syk to phosphorylate Slp-76. Tyrosine phosphorylated Slp-76 recruits Vav, the guanine nucleotide exchange factor for Rac, to the membrane where it becomes tyrosine phosphorylated by Syk and subsequently activates Rac (Figure 1.8) (Rivera et al., 2001; Turner and Kinet, 1999). Dowstream effects of activated Rac include cytoskeletal rearrangements, which are critical for mast cell degranulation, and the activation of Rho (Rivera et al., 2001; Turner and Kinet, 1999). Additionally, Rac may regulate cytokine expression by activating the AP-1 class of TFs (together with Ras) and by regulating the subcellular location of NFAT, through an unknown effector pathway (Turner and Kinet, 1999).  1.11.3 Negative regulation of FcsRI signaling Signaling events initiated by the FcsRI and other ITAM-containing immunoreceptors are negatively regulated by coaggregation with ITIM-containing receptors (Daeron, 1997; Gergely et al., 1999; Ott and Cambier, 2000; Scharenberg, 1999). This recently discovered and rapidly growing family of ITIM-containing receptors is collectively referred to as the IRS (inhibitory receptor superfamily). (Daeron, 1997; Ott and Cambier, 2000). IRS members traverse the plasma membrane once, and their N-terminal extracellular domains are either C-type lectin (e.g. Ly49 and MAFA (mast cell function-associated antigen)) or Ig-like (e.g. FcyRIIB, KIR (killer inhibitory receptor), gp49B1, PIR-B (paired Ig-like receptor), and SIRPa (signal regulatory protein)) domains (Ott and Cambier, 2000; Scharenberg, 1999). Each of these receptors contains at least one cytoplasmic ITIM consensus motif (I/VXYXXL) that becomes phosphorylated after coaggregation and attenuates immunoreceptor-induced signaling events through the recruitment of specific SH2 domain containing phosphatases (i.e. SHP-1, SHP-2, SHIP or SHIP2) (Ott and Cambier, 2000). Because mast cells are critical mediators of IgE+Ag-dependent allergic responses, a number of laboratories are studying the molecular mechanisms by which IRS members negatively regulate mast cell activation in attempts to define new therapeutic targets (Ott and Cambier, 2000). This section will take a closer look at the negative regulatory roles of IRS family members expressed on mast cells. The low affinity IgG receptor, FcyRIIB, was the first identified IRS member. Expression of this receptor is restricted to cells of hemopoietic origin where it has been shown to inhibit signaling events when coaggregated with the BCR, TCR and activating Fc receptors, such as the FcsRI, and RTKs, such as c-kit (Ott and Cambier, 2000; Scharenberg, 1999). This receptor contains one ITIM that gets phosphorylated by Lyn following coaggregation with the FcsRI. This leads to the subsequent recruitment of SHIP and the inhibition of IgE+Ag-induced mast cell degranulation, C a 44  2 +  flux and cytokine production (e.g. TNFa);  however, coaggregation with FcyRIIB does not inhibit IgE+Ag-induced phosphorylation of the FceRI B and y ITAMs by Lyn (Gergely et al., 1999; Ott and Cambier, 2000). FcyRIIB is the only inhibitory receptor shown to recruit SHIP and coaggregate with FcsRI in vivo, making it a desirable therapeutic target. In fact, in certain cases, allergen-IgG complexes have proven successful in the treatment of allergic asthma (Ott and Cambier, 2000). A second IRS member, gp49B1, is preferentially expressed on mast cells and NK cells. Ab-induced coaggregation of gp49B1 with FcsRI inhibits IgE+Ag-induced degranulation and LTC4 synthesis (Daheshia et al., 2001; Ott and Cambier, 2000). This inhibitory receptor contains two ITIMs, which can bind SHP-1 and -2 in vitro; however, only SHP-1 binding has been observed in mast cells in vivo (Gergely et al., 1999; Ott and Cambier, 2000). Integrin a B 3 was recently reported to be a ligand for mouse gp49B1; this v  interaction is hypothesized to represent an innate pathway for the down regulation of IgE+Ag-induced mast cell activation (Castells et al., 2001). Interestingly, mast cell development and maturation is normal in gp49B1 knockout, mice (Rojo et al., 2000), but these mice display increased sensitivity to IgE+Agdependent passive cutaneous anaphylaxis, as assessed by mast cell degranulation (Daheshia et al., 2001). The inhibitory receptors, PIR-B and SIRPa, both contain four ITIMs that interact with SHP-1 and SHP-2 to down regulate IgE+Ag-induced signaling events, including degranulation, when they are expressed as chimeric receptors containing the extracellular domain of FcyRIIB and coaggregated with FcsRI (Ott and Cambier, 2000; Uehara et al., 2001). Although the ligand for PIR-B remains elusive, integrin-associated protein (IAP/CD47), the ubiquitously expressed cell surface glycoprotein that normally binds to ECM molecules to regulate adhesion, was identified recently as the ligand for SIRPa (Ott and Cambier, 2000). Finally, MAFA is typically found directly associated with or in close proximity to the FcsRI on the surface of mast cells (Gergely et al., 1999; Ott and Cambier, 2000). MAFA contains one ITIM in reverse orientation to the ITIMs in other IRS members. The ligand that initiates its inhibitory role in FcsRI signaling events remains to be identified, although MAFA is capable of binding oligosaccharides in a Ca -dependent 2+  manner (Ott and Cambier, 2000). Ab-induced crosslinking of MAFA alone or coaggregation with FcsRI stimulates the tyrosine phosphorylation of MAFA's ITIM, and it was recently published that SHIP is the primary enzyme that mediates MAFA's inhibition of IgE+Ag-induced responses in mast cells (Ott and Cambier, 2000; Xu et al., 2001).  45  1.12  AIMS OF STUDY  The initial aim of this study was to characterize the role of SHIP in IgE+Ag-induced mast cell activation. As a critical first step, we generated and characterized bone marrow-derived mast cells (BMMCs) from SHIP+/+ and -/- mice (chapter 3). We then stimulated these BMMCs with IgE+Ag and compared mast cell degranulation, AA metabolism, and pro-inflammatory, vasoactive cytokine production in the presence and absence of SHIP. Interestingly, all three endpoints were elevated in the absence of SHIP. Dr. Michael Huber, a postdoctoral fellow in our laboratory, characterized the role that SHIP plays in IgE+Aginduced mast cell degranulation (Huber et al., 1998; Huber et al., 1999), and the focus of my project became the role of SHIP in IgE+Ag-induced cytokine production. Because all the cytokines we examined were elevated in the absence of SHIP, we limited our studies to the role of SHIP in IgE+Ag-induced IL-6 production. We found that both IL-6 mRNA and protein levels were elevated in the absence of SHIP, thus, we further limited these studies to examine the role of SHIP in the regulation of IL-6 mRNA levels (chapter 4; the results in this chapter have been published (Kalesnikoff et al., 2002a)). Additional studies with these BMMCs revealed that IgE alone, in the absence of Ag, was capable of inducing mast cell degranulation in SHIP-/-, but not +/+, BMMCs (Huber et al., 1998). At this point, we decided to explore this phenomenon further, since these results challenge the current paradigm that IgE binding to the FceRI on the surface of mast cells is a passive pre-sensitization step. As described in chapter 5, we found that IgE alone was capable of signaling in normal BMMCs. The goal for the remainder of this thesis, therefore, became the elucidation of the role of these IgE-induced signaling events in mast cell survival, priming and activation (chapters 5 & 6). In chapter 5, we looked at typical mast cell endpoints (i.e. degranulation, AA metabolism, cytokine production and survival) and found that IgE alone was capable of inducing mast cell cytokine production and enhancing mast cell survival (chapter 5; the results in this chapter have been published (Kalesnikoff et al., 2001)). To further explore the repertoire of biological effects elicited by IgE alone, we next studied the ability of IgE alone to induce BMMC and CTMC adhesion to the connective tissue component, FN, the underlying signaling mechanisms that mediate this adhesion, and the biological effects of this IgE-induced adhesion (chapter 6; the results in this chapter have been submitted to Blood (October 2002)).  46  Chapter 2  MATERIALS AND METHODS  2.1  TISSUE CULTURE  2.1.1  Bone marrow-derived mast cells (BMMCs) Bone marrow cells were aspirated from the femurs and tibias of 4-8 week old SHIP+/+ and -/-  C57B6, Lyn+/+ and -/- C57B6, or PKC5+/+ and -/- 129/SV littermate mice using Iscove's modified Dulbecco's medium (IMDM) (StemCell Technologies Inc., Vancouver, BC) and a 21-gauge needle with syringe (6cc) (Huber et al., 1998; Leitges et al., 2001). Bone marrow cells were plated at 1x10 cells/ml in 4  methylcellulose (Methocult M3434; StemCell Technologies Inc.) containing 10ng/ml murine IL-3, 10ng/ml human IL-6, 50ng/ml murine SCF, and 3units/ml human Epo for 7-10 days. Cells were then harvested and grown  in suspension in  IMDM containing  15% FCS (StemCell Technologies Inc.), penicillin  (100U/ml)/streptomycin (100pg/ml) (P/S; StemCell Technologies Inc.), 150pM monothioglycerol (MTG) (Sigma, St. Louis, MO) and 30ng/ml IL-3 (StemCell Technologies Inc.). Cell cultures were maintained between 2x10 and 8x10 cells/ml with complete media replacement every 3-4 weeks. By 6 weeks in 5  5  culture, greater that 98% of the cells were c-kit and FcsRI positive, as assessed by fluorescein isothiocyanate (FITC)-labeled anti-c-kit Abs (BD PharMingen, Mississauga, ON) and FITC-labeled IgE (anti-Epo 26; StemCell Technologies Inc.), respectively.  2.1.2  Swiss 3T3 fibroblasts Contact-inhibited Swiss albino mouse-skin-derived 3T3 fibroblasts were a generous gift from Dr.  John Schrader (UBC, Vancouver, BC). These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; StemCell Technologies Inc.) supplemented with 10% FCS and P/S, in 100 x 20mm tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ).  Cells were trypsinized (1ml trypsin/plate; StemCell  Technologies Inc.) and split 1/10 every 3 days.  2.1.3  Connective tissue mast cells (CTMCs) CTMCs were generated by coculture of BMMCs with fibroblasts (Levi-Schaffer et al., 1986).  Briefly, Swiss 3T3 fibroblasts were resuspended at 5x10 cells/ml in DMEM + 10%FCS + P/S and plated 3  2ml/well in 6 well tissue culture dishes (Becton Dickinson). When the 3T3 fibroblasts reached confluence (approximately 3 days), they were washed twice in IMDM + 15% FCS + P/S + MTG + IL-3, prior to the  47  addition of BMMCs. 6 week old mature BMMCs (generated as in 1.1.1) were washed once, resuspended in IMDM + 15% FCS + P/S + MTG + IL-3 at 2.5x10 cells/ml and 2ml were seeded into each well containing 5  the confluent monolayer of fibroblasts. Cocultures were maintained in a humidified atmosphere of 6% CO2. The culture medium was aspirated every 48hr, and the monolayers were washed once with 2ml of medium to remove nonadherent mast cells. To enrich for adherent or CTMCs after 2 weeks of coculture, adherent cells were washed three times with IMDM + P/S, then treated for 20min at 37°C with C a - and Mg -free 2+  2+  Hank's Balanced Salt Solution (HBSS; StemCell Technologies Inc.) supplemented with 10% FCS and 1 mg/ml Pronase (protease type XIV; Sigma). Cells were then centrifuged at 275/g for 15min through 13% metrizamide (Sigma) in HBSS. Adherent CTMCs from the coculture were recovered at the bottom of the gradient.  2.1.4  Sca-1 Lin- bone marrow isolation +  Sca-1i_in- bone marrow cells were generated by a two step purification procedure. Firstly, bone marrow cells aspirated from SHIP+/+ or -/- mice were lineage depleted by StemSep™ (StemCell Technologies Inc.) column purification as per manufacturer's instructions, using the StemSep™ murine hemopoietic progenitor enrichment cocktail (StemCell Technologies Inc.). Secondly, lineage depleted cells were stained with phycoerythrin (PE)-conjugated anti-Sca-1 and FITC-labelled anti-lineage specific Abs (anti-Mac-1, anti-Gr-1, and anti-B220) (Table 2.1), then sorted by FACSort™ (Becton Dickinson), and the Sca-1 positive (Sca-1 ), lineage negative (Lin-) cells were collected. These cells were then cultured as in +  section 2.1.1 to generate BMMCs.  2.2  PROTEIN ANALYSIS  2.2.1  Cell stimulations, total cell lysates (TCLs), immunoprecipitations (IPs), and Western blot analysis To treat BMMCs with IgE alone or SCF, the cells were starved (i.e. incubated without IL-3) for 4hr  to overnight at 37°C in IMDM + 10% FCS + 150pM MTG, washed 3 times with IMDM + 0.1% BSA, resuspended in IMDM + 0.1% BSA, then equilibrated to 37°C for 5min prior to the addition of 5-20pg/ml anti-DNP IgE (ie monoclonal anti-dinitrophenyl (DNP) clone SPE-7 IgE (Sigma)) or 100ng/ml SCF (StemCell Technolgies Inc.) for the indicated times. To stimulate with IgE+Ag, BMMCs were preloaded with 1-5pg/ml IgE for 4hr to overnight at 37°C in IMDM + 10% FCS + 150 pM MTG, washed 3 times to remove unbound IgE, resuspended in IMDM + 0.1% BSA, then equilibrated to 37°C for 5min prior to the addition of 20ng/ml DNP-human serum albumin (DNP-HSA; 30-40 moles DNP/mole HSA; Sigma) for the indicated times. The cells were then washed with 4°C HBSS (StemCell Technologies Inc.) and solubilized either by 48  boiling for 1min in a 100uJ volume of SDS-sample buffer (10% glycerol, 2.3% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, plus a few crystals bromphenol blue; all from Sigma) using 5x10 BMMCs 5  per sample for total cell lysates (TCLs) or with 1% TritonX-100 (TX-100; using 10 x 10 BMMCs per 6  sample) in 1ml of 4°C phosphorylation solubilization buffer (PSB; 50mM HEPES (pH 7.5), 100mM sodium fluoride, 10mM tetrasodium pyrophosphate, 2mM sodium orthovanadate, 2mM sodium molybdate, 2mM EDTA; all from Sigma) (Liu et al., 1994) supplemented with protease inhibitors (10pg/ml aprotinin, 2p.g/ml leupeptin and 2mM phenyl methyl sulfonyl fluride (PMSF); all from Sigma). TX-100 lysates were subjected to immunoprecipitation (IP) using 5(ag-10|j.g of Ab per 10x10 cells as indicated in section 2.2.3 and 15pJ of 6  Protein A beads (Pierce, Rockford, IL) (Liu et al., 1994). TCLs and IPs were separated by SDS-PAGE followed by Western blot analysis, using the Abs listed in section 2.2.3 at the indicated concentrations. To compare IgE- versus IgE+fibronectin (FN)-induced signaling events, BMMCs were starved (as indicated above), resuspended at 1x10 cells/ml in IMDM + 0.1%BSA, aliquoted (2ml/well) into either Falcon 6 well 6  tissue culture plates (Becton Dickinson) or BIOCOAT® human FN-coated 6 well tissue culture plates (Becton Dickinson) and stimulated with 5|ag/ml IgE for the indicated times. To stop the stimulations, the plates were placed on ice, 4°C HBSS added and, in the case of the standard tissue culture plates, in which the cells were in suspension, the cells pelleted and lysed in PSB containing 0.5% NP-40 (Calbiochem, La Jolla, CA). For the FN-coated plates, the non-adherent cells were washed away and the adherent cells lysed in PSB + 0.5% NP-40. 50pg of protein (as determined by BCA™ Protein Assay Kit (Pierce) according to manufacturer's instructions) was loaded per lane and separated by SDS-PAGE.  2.2.2  Inhibitors For inhibitor studies, inhibitors were added at the indicated concentrations 15-30min prior to the  addition of IgE, if stimulating with IgE alone, or DNP-HSA, if stimulating with IgE+Ag. The inhibitors used were the PI3K inhibitor LY294002, the Mek inhibitors PD98059 and U0126, the p38 inhibitor SB203580, the NFKB  inhibitor Bay11-7082, the PKC inhibitor Compound 3 (Bisindolymaleimide I, HCI), the classical PKC  inhibitor G06976, and the PLC inhibitor U73122 (all from Calbiochem), wortmannin and 2-amino ethoxydiphenyl borate (2-APB) (both from Sigma), and cycloheximide (Boehringer Mannheim, Mannheim, Germany). The cholesterol depleting agents, filipin III and methyl-p-cyclodextrin, were from Sigma.  49  2.2.3  Antibodies (Abs)  Table 2.1: Abs used in this thesis Antibody  Type  FITC-a-c-kit (2B8) FITC-lgE (aEp26)  mAb (lgG2b)  PE-a-Sca-1 FITC-a-B220 FITC-a-Mac-1 FITC-a-Gr-1 2.4G2  mAb (lgG2a)  a-SHIP(P1C1) a-P-Erk1/2(T»W°<) a-Erk-1-CT 4G10 4G10 beads a-P-PKB (Ser ) a-P-PKB (Thr»s) a-PKB 473  a-PKC (a, 3, Y, 5) a-P-kB (Ser ) a-kB a-P-p38 (Thr^/Tyr ) 32  182  a-p38 a-P-JNK (Thr^/Tyr'®) a-JNK a-FceRI (3 chain a-Shc a-Bcl-X a-p65 (Rel A), Rel B, c-Rel, p50, p52 a-c-kit  mAb (lgG2b)  5 ng/ml  mAb mAb mAb mAb pAb (Rb) mAb(lgd) pAb (Rb) pAb (Rb) mAb(lgGi) mAb(lgGi) pAb (Rb) pAb (Rb) pAb (Rb)  10ug/ml 30ug/ml 10ug/ml 30 ug/ml 1 200 (IP), 1:2000 (W) 1 500 (IP), 1:2000 (W) 1 1000 1 1000  mAb (lgG2a)  a-CD49e PE-a-rat-IgG a-SHIP  L  mAb (lgG2b)  PAb (IgE)  FITC-mouse-IgG  Concentration 2ug/ml 100ug/ml 2ug/ml 2ug/ml 2ug/ml  mAb (lgG2b)  pAb (Rb) pAb (Rb) pAb (Rb) pAb (Rb) pAb (Rb) pAb (Rb) mAb pAb pAb pAb pAb  Procedure  Source  Flow Cytometry Flow Cytometry Flow Cytometry  BD PharMingen (Mississauga, ON) StemCell Technologies Inc. (Vancouver, BC) BD PharMingen (Mississauga, ON)  Flow Cytometry Flow Cytometry Flow Cytometry Flow Cytometry Flow Cytometry Flow Cytometry Flow Cytometry IP,W IP,W W W W IP W W W  BD PharMingen (Mississauga, ON) BD PharMingen (Mississauga, ON) BD PharMingen (Mississauga, ON) Dr. S. Szilvassy (Vancouver, BC) Dr. Peter Lansdorp (UBC, Vancouver, BC) BD PharMingen (Mississauga, ON) Jackson Laboratories (West Grove, PA) StemCell Technologies Inc. (Vancouver, BC) Santa Cruz Biotechnology Inc. (Santa Cruz, CA) Cell Signaling Technologies (Beverly, MA) Dr. Steven Pelech (UBC, Vancouver, BC)  W W W W W  Upstate Biotechnology Inc. (Lake Palcid, NY) Upstate Biotechnology Inc. (Lake Palcid, NY) Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA) Transduction Laboratories (Lexington, KY) Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA)  1 10000 (W), 10ug  W W W W W W, EMSA  Cell Signaling Technologies (Beverly, MA) Cell Signaling Technologies (Beverly, MA) Dr. Reuban Siriganian (NIH, Bethesda, MD) Transduction Laboratories (Lexington, KY) Transduction Laboratories (Lexington, KY) Santa Cruz Biotechnology Inc. (Santa Cruz, CA)  1:1000  W  Santa Cruz Biotechnology Inc. (Santa Cruz, CA)  0.5ug/ml 1 mg/ml 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000 1 1000  a=anti; P=phospho; IP=immunoprecipitation; W=Western blot; EMSA=electrophoretic mobility shift assay  2.2.4  Plasma membrane preparation BMMCs were stimulated as indicated above. To prepare plasma membrane-enriched membrane  fractions, cells were pelleted, resuspended at 1.5x10 cells/ml in 4°C hypotonic lysis buffer (20mM Tris-HCl, 7  pH 7.4, 5mM EDTA, 5mM EGTA, 5mM dithiotriotol (DTT), 5mM sodium orthovanadate, 0.5mM PMSF, 2pg/ml leupeptin, and 10pg/ml aprotinin; all from Sigma), allowed to swell for 5min on ice, and sonicated for 15 x 1 second bursts on ice using an ultrasonic cell disruptor (Heat Systems Ultrasonics, Faimingdale, NY) (Huber et al., 2000). After centrifugation at 2000xg for 5min at 4°C, the supernatant was centrifuged at 100,000xg for 10min in an airfuge (Beckman Instruments, Fullerton, CA). The pellet was resuspended in 400pl of hypotonic lysis buffer containing 1% NP-40 by repeated vortex mixing. After a 60min incubation at 4°C, the suspension was centrifuged at 100,000xg for 10min, and the supernatant was collected as the  50  plasma membrane fraction. This solubilized membrane fraction was then subjected to SDS-PAGE and Western blot analysis as indicated.  2.2.5  Flow cytometry Cells were resuspended in HFN (HBSS + 2% (v/v) FCS + 0.2% (w/v) sodium azide) at  1x10 cells/ml in Falcon 2058 test tubes (Becton Dickinson). Directly conjugated FITC- or PE-labelled Abs 7  were added at the appropriate concentration (see above) to 100pl of cells and incubated for 30min on ice. In the case of anti-CD49e, the cells were subsequently washed and incubated with PE-conjugated anti-rat IgG for 30min on ice. Stained cells were then washed twice with 4ml of HFN and resuspended in 1ml HFN plus 1 pg/ml propidium iodide (PI). Cells were analyzed on a FACSort™ (Becton Dickinson). For analysis of GFP expressing cells and/or CFSE analysis, cells were washed twice with HFN and resuspended in 1ml HFN plus 1 pg/ml PI. For 2.4G2 blocking experiments, BMMCs were pretreated ± 10pg/ml of 2.4G2 or isotype control (rat lgG2b anti-Mad a) Ab for 30min at 4°C and then either left unstained or exposed to 30pg/ml heat-aggregated (63°C, 30min, pH 8 (Daeron et al., 1980)) FITC-mouse IgG for 30min at 4°C and subjected to FACS analysis as described above.  2.2.6  Alcian blue/safranin staining BMMCs or CTMCs were resuspended at 1x10 cells/100pl Dulbecco's phosphate buffered saline 5  (PBS; StemCell Technologies Inc.) and cytospins were prepared by spinning at 400rpm, 5min. Cytospins were air dried overnight, then stained using the Alcian blue/safranin staining technique (Levi-Schaffer et al., 1986). Cytospins were incubated for 15min with a solution of 0.5% alcian blue (w/v)/0.3% acetic acid (v/v), washed with distilled water, and sequentially incubated for 20min with a solution of 0.1%safranin (w/v)/0.1% acetic acid (v/v). The stained cytospins were examined microscopically and pictures were taken at 200X magnification using a Nikon Coolpix 990 digital camera (Nikon, Tokyo, Japan).  2.3  BIOLOGICAL ANALYSIS OF BMMCs  2.3.1  Degranulation assays If stimulating with IgE alone, BMMCs (5x10 ) were washed and resuspended in 410pl/sample of 5  Tyrode's buffer (10mM HEPES (Sigma), pH 7.4,130pM sodium chloride, 5mM potassium chloride, 1.4mM Ca chloride, 1mM Mg chloride, 5.6mM glucose (all from Fischer Scientific, Ottawa, ON), and 0.1% (w/v) 2+  2+  BSA (Boehringer Mannheim)), equilibrated to 37°C for 5min before adding 5pg/ml anti-DNP IgE (Sigma) for 15min at 37°C. If stimulating with IgE+Ag, cells were washed and incubated at 1x10 cell/ml at 4°C for 6  51  1h in Tyrode's buffer containing 5pg/ml anti-DNP IgE, washed twice with 23°C Tyrode's buffer, equilibrated in Tyrode's buffer to 37°C for 5min, then treated for 15min ± 20ng/ml DNP-HSA (Sigma). Two equal fractions were collected (190pl) as duplicate samples and centrifuged in a microfuge (2500x g, 4°C, 5min). The supernatants (S/Ns) containing (3-hexosiminidase released during stimulation were transferred to new tubes on ice and 300pl Tyrode's buffer + 0.5% (v/v) NP-40 (Calbiochem) was added to the pellets to lyse the cells for collection of the unreleased p-hexosiminidase (Huber et al., 1998). The pellets were nutated at 4°C for 1hr in this lysis buffer then the insoluble fraction, containing mostly unsolubilized nuclei and cytoskeleton, was pelleted by centrifugation (13000xg, 10min, 4°C). 10pl of each S/N and lysed pellet was placed into wells of a 96 well, flat bottom Falcon plate (Becton Dickinson) along with 50pl of the phexosiminidase substrate p-nitrophenyl N-acetyl-p-D-glucosaminidine (Sigma). The p-hemoximinidase was allowed to react with the substrate for 90min at 37°C, at which time the reaction was stopped by the addition of 150pl, 0.2M glycine, pH 10.7. OD of each sample was measured at 405nm and % degranulation was determined using the following equation: % degranulation = (OD S/N * 19)/[(OD S/N *19)+(OD pellet *30)].  2.3.2  LTC4/D4/E4  For  LTC4  enzymeimmunoassays  analysis, BMMCs at 1x10 cells/ml were starved or preloaded (with 10pg/ml IgE) for 4hr 4  at 37°C in IMDM + 10%FCS. BMMCs were then washed and resuspended at 1x10 cells/ml in Tyrode's 4  buffer (section 2.3.1), followed by a 20min stimulation at 37°C with 10pg/ml IgE or 20ng/ml DNP-HSA, respectively. LTC4 levels in the supernatants were analyzed using the reagents and methodology supplied with the  LTC4/D4/E4  enzymeimmunoassay kit (Amersham Pharmacia Biotech, Piscataway, NJ).  2.3.3  RNase protection assays (RPAS) For mRNA analysis, BMMCs at 1x10 cells/ml were starved or preloaded (in the presence of 5pg/ml 6  IgE) for 4hr at 37°C in IMDM + 10%FCS. Starved BMMCs were washed then resuspended at 1x10 cells/ml 7  in IMDM + 0.1%BSA, followed by stimulation at 37°C with IgE (Sigma), IL-3, or SCF (StemCell Technologies Inc.), at the indicated concentrations for the indicated times. Similarly, preloaded BMMCs were washed then stimulated with DNP-HSA (Sigma) as indicated. RNA was isolated using TRIzol Reagent (Gibco-BRL, Burlington, ON), as per manufacturer's instructions. Cytokine mRNA levels were quantitated using a Riboquant™ Multi-Probe RNase Protection Assay (mCK-1, mCK-2, mCK-3, and mCK-4 template sets; BD PharMingen) according to the manufacturer's instructions, using [ P]dUTP 9NEG-307H from NEN 33  Life Science Products Inc. (Boston, MA). Quantitation of cytokine mRNA levels, using a phosphorimager 52  (Storm 860, Molecular Dynamics, Sunnyvale, CA), was standardized based on levels of the housekeeping gene GAPDH.  2.3.4  ELISAs For ELISA analysis, BMMCs at 1x10 cells/ml were starved or preloaded (in the presence of 5u.g/ml 6  IgE) for 4hr to overnight in IMDM + 10% FCS. BMMCs were then washed and resuspended at 1x10 cells/ml in IMDM + 0.1% BSA, followed by stimulation at 37°C with IgE or DNP-HSA, respectively, at 6  the indicated concentrations for the indicated times. Following stimulation, the BMMCs were pelleted (2500rpm, 5min, 4°C) by centrifugation and the S/Ns were collected for analysis. Mouse IL-3, IL-6, TNFoc, and IgE ELISAs (BD PharMingen) and IL-13 and SCF ELISAs (R&D Systems, Minneapolis, MN) were performed according to the manufacturers instructions. To compare IgE- versus IgE+FN-induced cytokine production, BMMCs were starved (as indicated above) then added to FN- or BSA-coated polystyrene beads (bead.cell ratio 1:1) ± IgE and incubated at 37°C for the indicated times. To coat the beads, Polybead® Polystyrene Microsphere 15p beads (Polysciences, Warrington, PA) were incubated for 1hr at 23°C with 1 mg/ml human FN (Sigma), then blocked with 3% BSA in HBSS for 30min at 23°C or incubated with 3% BSA for 1.5hr at 23°C, respectively, as described by Houtman (2000).  2.3.5  Cell transfections and luciferase assays BMMCs were incubated for 4hr in fresh growth medium, washed and resuspended in IMDM + 10%  FCS, then aliquoted (10 cells/250pl) into electroporation cuvettes (4mm gap; Bio-Rad Laboratories, CA). 7  Cells were incubated at 23°C for 10min, then the reporter gene construct (10jag/ml, pNFicB-LUC; Stratagene, LaJolla, CA) was added together with 2pg/ml of pRL-TK (thymidine kinase promoterdependent renilla luciferase construct; Promega, Madison, Wl) to assess transfection efficiency and the cells were electroporated (Bio-Rad Gene Pulser) at 280V and 960pF (O'Laughlin-Bunneret al., 2001). The electroporated cells were incubated in tissue culture flasks at 1x10 cells/ml in IMDM + 10% FCS + 1 pg/ml 6  IgE overnight. For IgE alone stimulations, starved cells were washed and resuspended at 1x10 cells/ml in 6  IMDM + 0.1%BSA then treated ± 5pg/ml IgE for 4hr. For IgE+Ag stimulations, preloaded cells were washed twice to remove unbound IgE, then treated ± 20ng/ml DNP-HSA for 4hr. Luciferase assays were performed according to the manufacturer's instructions (Dual Luciferase Reporter Assay; Promega).  53  2.3.6  Nuclear extract preparation BMMCs (5x10 cells/condition) were stimulated as indicated in section 2.2.1. After washing with 6  PBS, cells were resuspended in 250pl PSB (section 2.2.1) supplemented with 0.2% NP-40, 10mM M g  2+  chloride, 10pg/ml aprotinin, 2pg/ml leupeptin and 2mM PMSF. After 1min on ice, the nuclei were pelleted and washed in the same buffer, but with 0.05% NP-40 and 0.25M sucrose. The nuclei were again pelleted and then extracted with vigorous agitation at 4°C in PSB containing 0.1% NP-40, 0.4M sodium chloride, and protease inhibitors as above.  2.3.7  Electrophoretic mobility shift assays (EMSAs) 5pg of nuclear protein (as determined by BCA™ Protein Assay Kit (Pierce) according to  manufacturer's instructions) were incubated with 1pmol P-labeled N F K B consenus oligonucleotide probe 32  (Santa Cruz Biotechnology Inc.) in binding buffer (10mM Tris-HCl pH 8.0,100mM potassium chloride, 5mM M g - chloride, 1mM DTT, 0.5% NP-40) and 1pg of poly-dldC (BD PharMingen) for 15min at 23°C and then 2  electrophoresed on 5% polyacrylamide gels in 0.25X Tris-borate-EDTA (Mui et al., 1995). Ab supershifts were performed by preincubating with 10pg of anti-NFKB Abs (Rel A, Rel B, c-Rel, p50, p52; Santa Cruz Biotechnology Inc.) 15min before the addition of oligonucleotide probe. These Abs were also used for Western blot analysis with nuclear extracts (5pg/lane on SDS-PAGE; Table 2.1).  2.3.8  Preparation of monomeric IgE (mlgE) Commercial preparations of clone SPE-7 anti-DNP IgE (Sigma) were fractionated by size using a  Waters HPLC system with a BioSep SEC S3000 gel filtration column (300 x 7.8mm, Phenomenex, Torrance, CA) equilibrated and run with PBS. Fractions (0.5 ml) were collected and IgE monomer and aggregate peaks, based on optical density (OD) readings at 280nm, were pooled separately. Monomeric IgE (mlgE) was also prepared by the standard 60min airfuge method (100,000xg or 24psi; Beckman Instruments Air-Driven Ultramicrofuge), which removes larger aggregates, to verify the results obtained using HPLC separated IgE.  2.3.9  Survival studies BMMCs were washed with IMDM and incubated at 5x10 cells/ml in IMDM + 10% FCS or 0.1% 5  BSA, ± test substance, e.g. IgE (Sigma), as indicated, in Falcon 3047 96 well flat bottom plates (total volume 200pl/well). Viability was assessed by trypan blue exclusion. The murine cytokines, IL-2, IL-4 and IL-13 were from R&D Systems, IL-3, SCF and IL-6 were from StemCell Technologies Inc., IL-4 was a  54  generous gift from Dr. K. Humphries (UBC, Vancouver, BC) and TNFa was from Peprotech (Rocky Hill, NJ). For survival on FN, Falcon 1172 96 well flat bottom plates were first treated for 1 hr at 37°C ± 50pg/ml human FN (Sigma), then treated for 1hr with 3% BSA, prior to the addition of BMMCs.  2.3.10 CFSE labelling BMMCs (5x10 /ml PBS) were stained with 5pM carboxyfluorescein diacetate succinimidyl ester 6  (CFSE; Molecular Probes, Eugene, OR) for 10min at 37°C, washed with 4°C HBSS and resuspended at 5x10 cells/ml in IMDM + 10% FCS for 5hr at 37°C (Glimm and Eaves, 1999). A 20 channel gate from an 5  FL-1 histogram was collected on a FACStar Plus cell sorter (Becton Dickinson) and the collected cells set up in survival assays (section 2.3.9) and analyzed daily by Flow Cytometry (section 1.2.4).  2.3.11 Tritiated thymidine assays BMMCs were washed with IMDM and incubated at 5x10 cells/ml in IMDM + 10% FCS or 0.1% 5  BSA, ± IL-3 or IgE, as indicated, in Falcon 3047 96 well flat bottom plates (total volume 200pl/well). After a 3 day incubation, the BMMCs, at 2.5x10 cells/well, were then aliquoted into 96 well U-bottom microtitre 4  plates (ICN) to give a final volume of 100uL/well and the plates incubated for 22hr at37°C in a humidified atmosphere of 5% CO2, 95% air. A total of 20uL of a 50uCi/mL solution of tritiated thymidine ( H-Tdr) in 3  RPMI was then added to each well to give a final concentration of 1uCi/well (Damen et al., 1998a). After another 6hr at 37°C, the contents of each well were harvested onto filtermats and counted using an LKB Betaplate Harvester and Liquid Scintillation Counter (LKB Wallac, Turku, Finland).  2.3.12 Apoptosis/DNA fragmentation assays 5x10 BMMCs were washed with IMDM and cultured at 5x10 cells/ml for 48hr in IMDM + 10% FCS 6  5  ± IL-3 or IgE. Cells were lysed in 1% NP-40 in 20mM EDTA, 50mM Tris-CI (pH 7.4). Lysates were brought to 1% SDS and digested at 37°C for 3hr with RNase A (5pg/pl) followed by 12hr with Proteinase K (2.5pg/pl). 10M ammonium acetate precipitated DNA was separated by electrophoresis on a 1% agarose gel to visualize apoptotic DNA fragments (Herrmann et al., 1994).  2.3.13 Removal of IgE from BMMC conditioned medium DNP-beads were made by coupling 2,4-dinitrobenzenesulfonic acid (Aldrich, Oakville, ON) to hydrazide CarboLink™ Coupling Gel (Pierce). 1ml of gel was washed in saturated Na2B4O7-10H 0 (pK'9.3) 2  and incubated at 23°C for 1 hr with 2.5ml of 20mg/ml 2,4-dinitrobenzenesulfonic acid in Na2B4O7-10H20 55  (Inman and Dintzis, 1969). DNP-bead preparations were then washed consecutively with 0.1 N acetic acid, water, and 0.1 N NH4OH, followed by storage in PBS + 0.02% sodium azide. Conditioned medium was generated by incubating BMMCs at 5x10 cells/ml in IMDM + 10%FCS in the presence of 10pg/ml IgE for 3 5  days. After 3 days, the BMMCs were spun down (at 2500rpm, 5min) and IgE was depleted from the media by 2 consecutive 1 hr incubations at 23°C with 100uJ DNP-beads/ml conditioned media.  2.3.14 Adhesion assays 96 well Nunc Maxisorp plates were coated with 50pg/mL FN (Sigma) in PBS for 2hr at 37°C or 16hr at 4°C, washed, blocked with 3% BSA (Sigma) in HBSS for 1hr at 37°C, then washed 3X with HBSS + 0.03% BSA. For stimulation with IgE or SCF, BMMCs were washed, resuspended at 1x10 cells/mL in 6  RPMI (without phenol red; Gibco-BRL) + 0.1%BSA and labelled with 3jag/mL Calcein AM (Molecular Probes) for 20min at 37°C. For stimulation with IgE+Ag, BMMCs were preloaded with 1u,g/ml IgE overnight, washed twice, then resuspended and labelled as above. After labelling, the cells were washed once then resuspended at 1 X 10 cells/mL in HBSS + 0.03% BSA. 50pL of test substances, e.g. IgE, SCF 6  or DNP-HSA, in HBSS + 0.03% BSA were added to the wells followed by 50p± of labelled cells (50,000cells/well). Adhesion assays were carried out at the indicated concentration of test substance for the indicated times. The degree of adhesion was quantitated using a Cytofluor 2300 Microplate Reader (Millipore, Bedford, MA) and is expressed as the percentage fluorescence remaining in the wells after washing away unbound cells. Adhesion to 3% BSA coated wells was performed as a control. Inhibitors were added to Calcein AM labelled cells for 30min at 37°C prior to the addition of test substance. The peptides GRGDSP and GRGESP (Invitrogen Life Technologies, Carlsbad, CA) were used at a final concentration of 400pg/ml. For experiments without C a , Calcein AM loaded cells were washed three 2+  times with PBS (which does not contain Ca ) and the assays were carried out in this medium. 2+  2.3.15 Intracellular Ca measurements 2+  BMMCs were washed once in Tyrode's buffer (Section 2.3.1) then resuspended at 5x10 cell/ml in 5  Tyrode's buffer containing 2pM fura-2/AM (Molecular Probes). Cells were incubated with mixing at 23°C for 45min in the dark, allowing fura-2/AM to accumulate in the cytoplasm of the cell. For stimulation with IgE+Ag, 5pg/ml IgE was added during the fura-2 loading step. Cells were then washed twice to remove extracellular fura-2/AM and resuspended at 5x10 cells/ml in Tyrode's buffer. Next, 1ml of cells was placed 5  into a quartz cuvette and stimulated with IgE, SCF or DNP-HSA as indicated. For the 2-APB studies, cells were labelled with fura-2/AM at 37°C ± 50pM 2-APB, then stimulated as above. For EGTA studies, 5mM 56  E G T A was added immediately prior to the addition of the stimulus. Cytoplasmic C a  2 +  influx was measured  in real-time by monitoring the fluorescence intensity at 510nm (after excitation of the sample at 340nm and 380nm) using an MC200 spectrophotometer/monochromator (SLM-Aminco, Urbana, IL), controlled by the 8100 V3.0 software program.  2.4  SHIP MUTAGENESIS/ADDBACK BMMCs  2.4.1  Viral infection of bone marrow cells Bone marrow aspirated from three 4-8 week old SHIP-/- and +/+ littermates (section 2.1.1) was  resuspended in complete medium (IMDM + 15% F C S (v/v) + 3U/ml Epo + 10ng/ml IL-3 + 10ng/ml human IL-6 + 30ng/ml S C F + 150pM MTG) at 4x10 nucleated bone marrow cells/ml. Approximately 10x10 bone 6  6  marrow cells were then mixed, in a 10ml tissue culture flask, with 5ml of 0.45p filtered, 36hr, viral supernatants collected from B O S C 23 cell cultures transiently transfected with M S C V - P a c  retroviral  constructs (section 2.4.2) and 6pg/ml polybrene (or 5mg/ml protamine sulfate) for 4-5hr then resuspended in 5ml of complete medium and incubated overnight (Damen et al., 2001). The next day, cells were infected two more times with 36hr and 40hr viral supernatants, as indicated above. After infection, cells that remained in suspension were resuspended at 1x10 cells/ml in complete medium (2.5ml total). Cells were 6  then added to 25ml (10 volumes) of methylcellulose (Metholcult M3434; Stem Cell Technologies Inc.) containing 2pg/ml puromycin and the mixture was vortexed and plated in 1ml aliquots (~1x10 cells/ml) on 5  Grenier dishes (Stem Cell Technologies Inc.). After 10 days, cells were harvested and cultured for 6 weeks as described in section 1.1.1 and tested for maturity by flow cytometry (see sections 1.1.1 & 1.2.4). Mature B M M C cultures were also analyzed for expression of G F P by flow cytometry and those colonies found to have low expression were sorted by FACSort™ (Becton Dickinson), collecting only the top 30% of G F P positive cells.  2.4.2  SHIP point mutations SHIP point mutations were generated by Dr. Jacqueline E. Damen and Dr. Mark D. Ware (Damen  et al., 2001). Briefly, murine SHIP cDNA, with a 27-bp hemagglutinin (HA)-tag fused at the 5' end in frame with the translation start site, was subcloned into BSKS+ (Stratagene) using Xho\ (5') and E c o R l (3') and used for the production of the wild type (WT) SHIP construct and the D675G SHIP point mutant. D675G was  generated using the QuickChange™ site-directed mutagenesis kit (Stratagene), according to the  manufacturer's instructions. The primers used were 5 ' - G C C G T C C T G G T G C G ^ C C G A G T C C T C T G G A A G T - 3 ' A  5'-CTTCCAGAGGACTCGG  T_>C  G  and  C G C A C C A G G A C G G C - 3 ' (superscript indicates the nucleotide substitutions made).  57  The following primers were used to replace the C-termini of the SHIP mutants with a PCR product lacking the stop codon: 5'-ATGACTGGCCACTTCAGGGGAGAGATT-3' (Mscl restriction enzyme site is bolded) and 5 ' - G T C G M T T C ^ C T G C A T G G C A G T C C T - 3 ' (EcoRl site is bolded). ELONGase™ enzyme A  mix (Invitrogen Life Technologies) was used for all PCR reactions as per manufacturer's instructions. The PCR product was digested with Msc\ and EcoRl, gel purified and subcloned into the corresponding unique restriction sites within the pBSKS -SHIP vectors. The resultant cDNAs were digested with +  Xhol (5') and  EcoRl (3'), and subcloned into the corresponding unique sites in pEGFP-N1 (Clontech) to create HA-SHIP cDNAs in-frame with the enhanced green fluorescence protein (GFP). The HA- and GFP-tagged WT and D675G constructs were subsequently cloned into MSCV-Pac for retroviral transfection (section 2.4.1).  58  Chapter 3  A COMPARISON OF MAST CELLS DERIVED FROM SHIP+/+ AND -/- MICE  3.1  INTRODUCTION Murine mast cells are classified as MMCs or CTMCs based on histochemical differences (section  1.2.1). The two most common sources of mast cells from mice are the peritoneal cavity and bone marrow. The peritoneal cavity can be flushed to obtain mature CTMCs; however, resulting mast cell numbers are low on a per mouse basis when this method is used. Alternatively, a large, uniform population of in vitro differentiated bone marrow-derived mast cells (BMMCs) can be generated by culturing bone marrow cells in the presence of IL-3 (Levi-Schaffer et al., 1986). The generation of BMMCs is well documented in the literature (Razin et al., 1982; Razin et al., 1983), and BMMCs are the presumptive in vitro counterpart of MMCs, since they do not stain with safranin (section 1.2.1) (Levi-Schaffer et al., 1986; Stevens et al., 1987). Supporting the idea that the local environment plays a key role in determining mast cell phenotype (Welle, 1997), mature BMMCs can be further differentiated into CTMCs by coculturing with 3T3 fibroblasts for 2 weeks (Levi-Schaffer et al., 1986; Stevens et al., 1987). Mature BMMCs are also reported to undergo partial differentiation into CTMCs following 6 days of culture in the presence of SCF and IL-4 (Karimi et al., 2000). Because we were interested in studying the role of SHIP in the regulation of mast cell activation, we generated BMMCs from SHIP+/+ and -/- mice. Before comparing these cells, we wanted to ensure that the phenotypes of SHIP+/+ and -/- BMMCs were not so different that they would confound interpretation of our activation studies. Thus, we characterized the development, receptor expression and granule content of SHIP+/+ and -/- BMMCs. Fortunately, despite the observed difference in the rate of development between SHIP+/+ and -/- BMMCs, we found that the receptor expression profile, level of receptor expression, total granularity and the granular content of mature SHIP+/+ and -/- BMMCs was similar.  3.2  RESULTS  3.2.1  BMMCS differentiate faster in the absence of SHIP One of the hallmarks of mast cell development is expression of the high affinity IgE receptor, FceRI  (section 1.2). Using FITC-coupled IgE, we monitored the expression of functional FcsRI on SHIP+/+ and -/mast cells over time by flow cytometry. Bone marrow was aspirated from SHIP+/+ or -/- littermate controls  59  and cultured in IL-3, as described in Chapter 2. In accordance with existing literature, we consistently observed that bone marrow from wildtype mice differentiated into a uniform population of FcsRI positive BMMCs (-99% FcsRI positive) by 4-6 weeks in culture (Figure 3.1) (Razin et al., 1983). Interestingly, our studies indicated that FcsRI expression was detectable earlier in the absence of SHIP (Figure 3.1). FcsRI expression was detectable on a small percentage of +/+ cells as early as 1-2 weeks; however, expression was consistently higher at these earlier time points on cells derived from SHIP-/- mice (Figure 3.1). Furthermore, we were capable of generating a uniform population of SHIP-/- FcsRI positive BMMCs (-99% FcsRI positive) by 3-4 weeks in culture, in 5 separate experiments (Figure 3.1).  w  100-  0  Figure 3.1: Cultured bone marrow cells from SHIP-/mice express FceRI faster than their wildtype  u  counterparts. Bone marrow from SHIP+/+ (•) and -I(•) mice was aspirated and plated in complete media for one week, followed by culture in media containing IL-3 only. FceRI expression was monitored by FACs for 5 weeks. Results are representative of 5 separate experiments.  "55 o  >  75-  Q. 50-  5 w  u  25-  0  ill  1  2  3  4  5  Week  3.2.2  Sca-1 Lin- cells differentiate faster in the absence of SHIP +  Although we consistently observed faster BMMC development from total bone marrow in the absence of SHIP, we could not rule out that this result simply reflected differences in the cellular content of bone marrow from SHIP+/+ and -/- mice. In attempts to ensure our starting cell populations were similar, we purified lineage negative progenitor cells from SHIP+/+ and -/- marrow.  Bone marrow was lineage  depleted using the StemSep™ murine hemopoietic progenitor Enrichment Cocktail (StemCell Technologies Inc.) and StemSep™ negative selection column. The lineage depleted cells were then stained for Sca-1 (LY-6A/E), an 18kDa phosphatidylinositol-anchored protein expressed on multipotent hemopoietic stem cells in mice (Okada et al., 1992; van de Rijn et al., 1989). The same cells were also stained with FITClineage specific Abs (Mac-1, Gr-1, and B220) (see section 2.2.3), and sorted by FACSort™ (Becton Dickinson); only the Sca-1 positive (Sca-1 ), lineage negative (Lin-) cells were collected. The purified Sea+  l-tin-cells were cultured in complete medium for 1 week, then switched to medium containing IL-3 only for the remaining 5 weeks in culture (section 2.1). These cells were monitored for FcsRI expression two times per week by flow cytometry. Again, we found that IgE receptor expression occured faster in the absence of SHIP (Figure 3.2). 60  j/)  Figure 3.2: Cultured Sca-1 Lirr cells purified from SHIP-/- bone marrow express FceRI faster than their wildtype counterparts. Bone marrow from +  100-  8  a> 75H >  SHIP+/+ (•) and -/- (•) mice was aspirated, lineage depleted using StemSep™ purification, then sorted for Sca-1 Lin- cells using the FACSort™ (Becton Dickenson). Sca-1Un- cells were plated in complete media for one week, followed by culture in media containing IL-3 only. FcsRI expression was monitored by FACS 2 times per week. Results are representative of 3 separate experiments.  o  Q.  +  50-  E  ou  25H  0  _Q  1.5  1  2  2.5  3  3.5  4  5  Week  3.2.3  FceRI and c-kit expression levels are comparable in mature BMMCs derived from SHIP+/+ and -/- mice Since bone marrow and Sca-1 Lin- cells from SHIP-/- mice expressed IgE receptors faster than +  their wildtype counterparts, we wanted to ensure that the mature mast cell populations that developed following culture in IL-3 were comparable. To address this question, we compared the protein expression levels of two well documented mast cell markers, FcsRI and c-kit, on 6 week old populations of SHIP+/+ and -/- BMMCs. As indicated earlier, c-kit is the receptor for SCF, a critical mast cell development factor in  vivo (Austen and Boyce, 2001; Galli et al., 1993). As shown in Figure 3.3A, mature SHIP+/+ and -/- BMMCs  B  +/+  +/+ -/W: anti-c-kit io  u  io'  i<r  i<r  io  H  W: anti-SHIP  W: anti-Fc E R I p chain  icr  io  «^ io io J  FceRI  fl  •  10"  10'  10'  10  c-kit  Figure 3.3: FceRI and c-kit expression levels are comparable in mature BMMCs derived from SHIP+/+ and -/- mice. A) 6 week old mature SHIP+/+ and -/- BMMCs were analysed for FcsRI (left panels) and c-kit (right panels) expression by FACS (shaded histograms represent unstained BMMCs). B) Total cell lystates from 1x10 SHIP+/+ or -/- BMMCs were subjected to Western blot analysis using anti-c-kit (top panel), anti-SHIP (middle panel), and anti-FcsRI p chain (bottom panel) Abs. 6  61  expressed approximately equai FcsRI and c-kit receptor levels at the cell surface, as assessed by flow cytometry. Western blot analysis showed that total cell levels were also similar (Figure 3.3B).  3.2.4  The granule content of mature BMMCs from SHIP+/+ and -/- mice is comparable Once we had established that receptor levels were comparable on mature SHIP+/+ and -/-  BMMCs, we wanted to determine that our BMMC populations were developmentally comparable (i.e. since the SHIP-/- BMMCs developed faster, we wanted to ensure they did not develop differently or further (to a more differentiated state) than their wildtype counterparts). As indicated earlier, CTMCs and BMMCs (or MMCs) can be defined by their distinct immunohistochemical and biochemical properties (section 1.2.1) (Stevens et al., 1987). Furthermore, CTMCs are reported to have larger granules and increased histamine content than BMMCs (Stevens et al., 1987). To determine if the total granularity of the SHIP+/+ and -/BMMCs was similar, we lysed equal numbers of SHIP+/+ and -/- BMMCs and measured total B hexosiminidase activity (the enzyme activity that we measure in our degranulation assays). As can be seen in Figure 3.4A, three separate batches of SHIP+/+ and -/- BMMCs showed similar activity of this acid hydrolase. To further study the development of mast cells in the absence of SHIP, cytospins were performed on 6 week old SHIP+/+ and -/- BMMCs. These cytospins were then stained with alcian blue (which binds to chondroitin sulfate present in the granules of MMCs) and safranin (which binds to heparin present in the granules of CTMCs) (Levi-Schaffer et al., 1986; Stevens et al., 1987). As shown in Figure 3.4B, BMMCs from both SHIP+/+ and -/- littermate controls stained with alcian blue, but not safranin, and were comparable in size and granularity, indicating that SHIP-/- BMMCs do not develop further than their wildtype counterparts. As indicated above, BMMCs can be further differentiated into CTMCs by coculture with mouse skin-derived fibroblasts (Swiss 3T3s) (Levi-Schaffer et al., 1986; Stevens et al., 1987). BMMCs adhere to the fibroblast layer where they divide and differentiate into CTMCs, presumably under the influence of surface bound SCF present on the 3T3s. SHIP+/+ and -/- BMMCs were cocultured with Swiss 3T3s for 14 days, purified through a metrizamide gradient, then stained with alcian blue/safranin. As shown in Figure 3.4C, both SHIP+/+ and -/- BMMCs were capable of differentiating into safranin positive CTMCs.  3.3  DISCUSSION Although major disruptions have been observed in the hemopoietic systems of SHIP-/- mice  (section 1.8.2), relatively little is known about the role of SHIP in regulating hemopoietic cell differentiation. We consistently observed that, when cultured in IL-3, bone marrow cells from SHIP-/- mice 62  Figure 3.4: The granule content of mature BMMCs from SHIP+/+ and -/- mice is comparable. A) Total (3-hexosiminidase content from 5x10 SHIP+/+ P ) and -/- (•) BMMCs was analysed by lysing cells in Tyrode's buffer + 0.5% NP40. Lanes 1-3 represent the averages of triplicate determinations from 3 separate batches of BMMCs derived from littermates. B) BMMCs or C) CTMCs were resuspended at IxlO^ells/IOOul PBS and cytospins were prepared by spinning at 400rpm, 5min. Cytospins were incubated for 15min with a solution of 0.5% alcian blue/0.3% acetic acid, washed with distilled water, and sequentially incubated for 20min with a solution of 0.1%safranin/0.1% acetic acid. The stained cytospins were examined microscopically and pictures were taken at 200X magnification using a Nikon Coolpix 990 digital camera (Nikon, Tokyo, Japan). 5  20,  •in m .S  Si  8 2" at  i  tc-  Jg  5-5.  05|  00  2  J 3  B  -/-  +/+  (200X)  +/+  (200X)  express higher levels of the FcsRI at earlier time points than their +/+ counterparts, and that w e could generate a pure population of SHIP-/- FcsRI positive B M M C s faster than SHIP+/+ B M M C s . These results were also obtained when we started with purified Sca-1 Lin- bone marrow cells. In support of our findings, it +  was recently reported that B cells from SHIP-/- mice develop more rapidly than their +/+ counterparts (Brauweiler et al., 2000). Our findings suggest that SHIP, likely through its ability to dephosphorylate the PI3K product PIP3, negatively regulates mast cell differentiation. To determine if this effect is due to the catalytic activity of SHIP, we plan to infect SHIP-/- bone marrow cells with various SHIP mutants, including a phosphatase dead mutant and monitor their differentiation rate into FcsRI positive B M M C s . Furthermore,  63  SHIP mRNA and protein levels are reported to fluctuate during B cell (Kerr et al., 1996) and T cell (Liu et al., 1998b) development, respectively; whether similar changes occur in developing mast cells remains to be determined. Finally, as the specific TFs involved in the regulation of mast cell development are characterized (e.g. PU.1 or YY1 and GATA-1 positively regulate FceRI a chain mRNA transcription, whereas Elf-1 represses a chain expression (Nishiyama et al., 2002)), it would be interesting to examine the role of SHIP in the regulation of these TFs. Since one of the primary goals of this thesis was to study the role of SHIP in mast cell signaling events initiated through the IgE receptor, we characterized mature BMMCs derived from SHIP+/+ and -/littermate controls to ensure they were comparable. Despite the faster expression of the FceRI observed in the absence of SHIP, our results showed that mature SHIP+/+ and -/- BMMCs express comparable numbers of both IgE and SCF receptors. Our finding that FceRI expression levels were the same on mature SHIP+/+ and -/- BMMCs was critical to rule out the possibility that any differences we observe following stimulation simply reflect differences in receptor expression levels. We also showed that SHIP+/+ and -/- BMMCs are developmental^ comparable, i.e. they demonstrate similar granule content and reach a similar maturation state (as assessed by total p-hexosminidase activity and alcian blue/safranin staining). Furthermore, both SHIP+/+ and -/- BMMCs were capable of differentiating into heparin containing CTMCs when cocultured with 3T3 fibroblasts, providing further evidence that SHIP+/+ and -/- BMMCs are developmental^ similar. It would be interesting in the future to study the rate of CTMC differentiation in the presence and absence of SHIP, by monitoring heparin synthesis, to determine if SHIP negatively regulates this differentiation event as well. The next data chapter utilizes these characterized cells to study the role of SHIP in the regulation of IgE+Ag-induced mast cell degranulation, AA metabolism, and cytokine production.  64  Chapter 4  SHIP NEGATIVELY REGULATES IgE+Ag-INDUCED IL-6 PRODUCTION BY INHIBITING N F K B ACTIVITY 4.1  INTRODUCTION The three major classes of proinflammatory mediators released by IgE+Ag-activated mast cells are  preformed granule-associated chemical mediators, newly synthesized AA metabolites, and proinflammatory vasoactive cytokines (e.g. TNFa and IL-6) (section 1.3.4) (Galli, 2000; Mekori and Metcalfe, 2000; Schwartz, 1994). IgE initiates these processes by binding to mast cells via the high-affinity IgE receptor, FcsRI. Multivalent Ag-induced crosslinking of IgE bound FcsRIs leads to the activation of FcsRI-associated Src kinase family members, which phosphorylate the B and y chain ITAMs (section 1.11.2) and initiate a variety of intracellular signaling cascades that work together to activate the mast cell (Beaven and Metzger, 1993; Turner and Kinet, 1999). One of the proteins that becomes tyrosine phosphorylated in mast cells in response to IgE+Ag is SHIP (Damen et al., 1996; Huber et al., 1998; Rohrschneider et al., 2000). While this phosphorylation event does not appear to increase the enzymatic activity of SHIP (Damen et al., 1996), it may be involved in the localization of SHIP to the plasma membrane (Ono et al., 1997) where it cleaves the 5'-phosphate from the PI3K-generated product, PIP3, to yield PI-3,4-P2. This in turn reduces the ability of certain PH domain containing proteins (e.g. PKB/Akt, phosphoinositide-dependent protein kinase 1 (PDK1), and Btk) to target to the plasma membrane and be activated (Bolland et al., 1998; Liu et al., 1999; Marte and Downward, 1997).  Using SHIP+/+ and -/- BMMCs, we recently demonstrated that SHIP is a key negative regulator of IgE+Ag-induced mast cell degranulation (Huber et al., 1998) and AA metabolism (as assessed by measuring LTC4 production) (Figure 4.1). To further explore the role that SHIP plays in regulating IgE+Aginduced mast cell activation, we have now compared cytokine production in murine BMMCs from SHIP+/+ and -/- littermates. This chapter demonstrates that SHIP negatively regulates IgE+Ag-induced IL-6 mRNA and protein levels and requires its phosphatase activity to exert this negative effect. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP-/- BMMCs, we found the PI3K/PKB, Erk, p38, JNK and PKC pathways are all elevated in IgE+Ag-induced SHIP-/- cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE+Ag-induced IL-6 production is dependent on the activity of the  TF  NFKB  and that 65  IKB  phosphorylation/degradation and  NFKB  translocation, DNA binding and transactivation are much higher in SHIP-/- BMMCs. Interestingly, using various pathway inhibitors it appears that the PI3K/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of IKB and N F K B DNA binding, while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of N F K B . Taken together, our data are consistent with a model in which SHIP negatively regulates N F K B activity and IL-6 synthesis by reducing IgE+Ag-induced PIP3 levels and thus PKB, PKC, Erk and p38 activation.  Figure 4.1: SHIP negatively regulates lgE+Ag4nduced mast cell degranulation and LTC4 production. A) SHIP+/+ (•) and -/- (•) BMMCs were preloaded with 10ug/ml IgE for 1hr, then stimulated for 15min ± 20ng/ml DNP-HSA and percent degranulation determined by assaying supernatants and cell pellets for p-hexosaminidase activity. Each bar represents the mean ± SEM of duplicates after subtracting the percent degranulation obtained in the absence of DNP-HSA. B) SHIP+/+ (•) and -/- (•) BMMCs were preloaded with 5ug/ml IgE for 4hr, washed and resuspended at 1x10 cells/ml in Tyrode's buffer, followed by a 20min stimulation at 37°C with 20ng/ml DNP-HSA. L T & levels in the supernatants were analyzed using the reagents and methodology supplied with the LTC4/D4/E4 enzymeimmunoassay kit (Amersham Pharmacia Biotech). 4  4.2  RESULTS  4.2.1  SHIP negatively regulates cytokine production in activated BMMCs To explore the role that SHIP plays in regulating the IgE+Ag-induced synthesis and release of  proinflammatory cytokines from BMMCs, we first carried out RNase protection assays (RPAs) with IgE+Agstimulated SHIP+/+ and -/- BMMCs. As shown in Figure 4.2A, these studies revealed that the mRNA levels of IL-4, IL-5, IL-6, IL-13, and TNFa were substantially higher in SHIP-/- than in +/+ BMMCs (Figure 4.2A). This difference in cytokine mRNA levels was observed not only in response to IgE+Ag but in response to SCF and IL-3 as well (shown for IL-6 mRNA, Figure 4.2B). To investigate how SHIP negatively regulates cytokine expression at the signal transduction level, we focused our attention on IL-6 since a great deal was already known about the regulation of this cytokine in mast cells (Marquardt and Walker, 2000). As shown in Figure 4.2C, we found that IgE+Ag-induced IL-6 protein levels, as assessed by ELISA, were 66  significantly higher in the conditioned medium from SHIP-/- BMMCs, consistent with the elevated mRNA levels observed in Figure 4.2A.  IL-4  IL-5  IL-6  IL-13  TNFct  B IL-6  100-  4000-  $ 75-\  g  c  <  = !5  «. 25H  J6  0'  Q  150 IL-3  400  i  25  3000-  CD  o  O) 2000-  a. to  -j  1000-  100 0'  SCF  60'  3hr  24hr  48hr  Figure 4.2: SHIP negatively regulates cytokine production in BMMCs. A) SHIP+/+ (•) and -/- (•) BMMCs were preloaded with 5ug/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 30min and subjected to RNase protection assays (RPAs). The relative band intensities of IL-4, IL-5, IL-6, IL-13 and TNFct mRNAs were quantified using a phosphorimager and standardized using levels of the housekeeping gene, GAPDH. Results are representative of 3 separate experiments. B) SHIP+/+ (•) and -/(•) BMMCs were starved for 4hr in 10% FCS, then stimulated ± IL-3 or S C F at the indicated conentrations (ng/ml) for 30min and subjected to RPAs as in (A). IL-6 mRNA results are shown. C) SHIP+/+ (•) and -/- (•) BMMCs were preloaded with IgE, washed to remove IL-6 produced in response to IgE alone, and stimulated ± DNP-HSA as in (A) for the indicated times. IL-6 protein levels in the cell supernatants were assessed by ELISA. Each bar represents the mean ± SEM of 8 determinations.  4.2.2  Addition of WT but not phosphatase deficient SHIP to SHIP-/- BMMCs reduces IL-6 production to that seen in SHIP+/+ cells To determine if the observed difference in IL-6 production between SHIP+/+ and -/- BMMCs was a  result of SHIP'S enzymatic activity or its ability to serve as an adaptor protein (Rohrschneider et al., 2000; Tamir et al., 2000), bone marrow cells from SHIP-/- mice were retrovirally infected with N-terminal HA- and C-terminal GFP-tagged versions of wildtype (WT) or phosphatase deficient (D675G) SHIP (Figure 4.3A, top panel) (Damen et al., 2001). Following 10 days in methylcellulose containing puromycin, mast cell colonies were pooled and put in suspension culture for 8 weeks (Damen et al., 2001). At this time the two cultures were greater than 98% FcsRI and c-kit positive and Western blot analysis, using anti-SHIP Abs, revealed 67  that the WT and D675G SHIP constructs expressed approximately the same amount of SHIP protein and this level was about half that present in SHIP+/+ BMMCs (Figure 4.3A, bottom panel). Studies with these BMMCs revealed that the introduction of WT SHIP, which reduced IgE+Ag-induced PIP3 (Damen et al., 2001) and degranulation (Figure 4.3B) to levels approaching those observed in SHIP+/+ BMMCs, reverted  B  HA  5-Ptase  WT  ^ _ Proline-rich G F P (1-1190)  ZL"  D675G  (1-1190)  +/+ -/- W T D675G  +/+  -/-  WT D675G  W: anti-SHIP  +/+  -/-  WT  D675G  0' 30' 60' 0' 30'60' 0' 30'60' 0' 30' 60'  L32 GAPDH  WT  D675G  Figure 4.3: Addition of wildtype, but not phosphatase deficient, SHIP reverts IL-6 production in SHIP-/- to SHIP+/+ BMMC levels. A) Full length (1190 amino acid) wild type (WT) and D675G SHIP constructs HA-tagged at the N-terminus (I) and GFP-tagged at the C-terminus (•). Total cells lysates from 1x10 SHIP+/+, -/-, WT and D675G BMMCs were subjected to Western analysis using anti-SHIP Abs (bottom panel). B) SHIP+/+, -/-, WT and D675G BMMCs were preloaded with 10ug/ml IgE for 1 hr, then stimulated for 15min ± 20ng/ml DNP-HSA and percent degranulation determined by assaying supernatants and cell 6  pellets for p-hexosaminidase activity. Each bar represents the mean ± S E M of duplicates after subtracting the percent degranulation obtained in the absence of DNP-HSA. C) SHIP+/+, -/-, WT, and D675G BMMCs were preloaded with 5u.g/ml IgE for 4hr, treated ± 20ng/ml DNP-HSA for the indicated times and subjected to R P A (IL-6 mRNA levels are boxed). Results are representative of 2 separate experiments. D) SHIP+/+, -/-, WT, and D675G BMMCs were preloaded and stimulated as in (C) and IL-6 protein levels in the supernatants determined by ELISA. The bars represent the mean ± SEM of 4 separate experiments.  68  the IgE+Ag-induced increase in IL-6 mRNA (as assessed by RPA (Figure 4.3C)) and protein expression (as assessed by ELISA (Figure 4.3D)) to close to those observed in SHIP+/+BMMCs. The D675G SHIP, on the other hand, did not revert any of the responses examined (Damen et al., 2001) (Figures 4.3B, C & D). These results suggested that the difference in IL-6 production was dependent on the phosphatase activity of SHIP, and thus, likely, PIP levels. 3  4.2.3  IgE+Ag activates multiple pathways to a greater extent in SHIP-/- than +/+ BMMCs To explore which PIP3-regulated pathways might be responsible for the elevated IL-6 mRNA and  protein levels seen in IgE+Ag-induced SHIP-/- BMMCs, we first compared the activation states of various pathways known to be triggered by IgE+Ag in BMMCs. Specifically, we compared the phosphorylation state of PKB, a PH domain-containing S/T kinase that is recruited to the plasma membrane by the transient IgE+Ag-induced increase in PIP3 and activated via phosphorylation at Thr , by the PIP3 binding PH308  containing S/T kinase, PDK1, and at Ser  473  by either an as yet unidentified PDK2 or by autophosphorylation  in a manner similar to PKCs (Alessi et al., 1997; Downward, 1999; Stokoe et al., 1997; Toker and Newton, 2000). As expected, since SHIP down regulates PIP3 levels and thus the recruitment of PKB (and likely PDK1), we observed more intense and prolonged phosphorylation of PKB in IgE+Ag-induced SHIP-/BMMCs (Figure 4.4A, upper panel). Reprobing with anti-FcsRI p-subunit Abs demonstrated equal loading (lower panel).  We then compared members of the PKC family of S/T kinases since several isoforms of this multigene family may bind to PIP , in addition to DAG (Chou et al., 1998; Singh et al., 1993), and 3  subsequently become activated/phosphorylated by PDK1 (Balendran et al., 2000; Belham et al., 1999; Le Good et al., 1998). To assess the activation state of various PKC isoforms, we measured their levels in plasma membrane preparations since cell stimulation has been shown to trigger the translocation of PKCs from the cytosol to the plasma membrane where they become activated (Mochly-Rosen, 1995; Nishizuka, 1992) and gain access to their substrates (Feng et al., 2000). As can be seen in the left panel of Figure 4.4B, IgE+Ag treatment resulted in a substantially greater recruitment of PKC a , p, y, and 8 to the plasma membrane of SHIP-/- than +/+ BMMCs. Reprobing with anti-FcsRI p chain Abs demonstrated equal levels of membrane protein. Importantly, total cell lysates showed comparable levels of these PKC isoforms in SHIP+/+ and -/- BMMCs (Figure 4.4B, right panel).  Since PKC has been shown to phosphorylate/activate Raf-1 in hemopoietic cells (Carroll and May, 1994), we then compared the IgE+Ag-induced phosphorylation of Erk in SHIP+/+ and -/- BMMCs and found  69  B  +/+ 0' 2' 5' 10' 30'  +/+  0' 2' 5' 10' 30'  C  0' 3'  -/-_ 0'  TCL  3'  W: anti-P-PKB (Ser )  +/+./.  473  PKCa  PKCP  W: anti-FceRI p chain  PKCy C  +/+ 0' 2' 5' 10' 30'  -/PKC8  0' 2' 5' 10' 30'  FceRI P chain  W: anti-P-Erk1/2 W: anti-FceRI p chain  +/+ 0' 2' 5' 10' 30'  +/+  •/•  0' 2'  5' 10'  30'  -*•  •»  «  0'  5' 10'  •/•  30'  W: anti-P-p38  W: anti-P-JNK  W: anti- FceRI p chain  W: anti-JNK  0'  5'  10'  30'  Figure 4.4:  SHIP represses multiple IgE+Ag-induced signaling pathways in BMMCs. A) SHIP+/+ and -/- BMMCs were preloaded with 5u.g/ml IgE for 4hr and stimulated ± 20ng/ml DNP-HSA for the indicated times. Total cell lysates were subjected to Western analysis using anti-phospho-PKB (Ser ) Abs (top panel). The blot was reprobed with anti-FceRI (3 chain Abs to show equal loading. B) SHIP+/+ and -/- BMMCs were preloaded as in (A), then treated ± DNP-HSA for 3min and Western analyses carried out with plasma membrane preparations using anti-PKCcc, B, y and 5 Abs and the blot reprobed with anti-FceRI B chain Abs to show equal loading (left panel). Total cell lysates were subjected to Western analysis with the same Abs (right panel). In (C, D & E) SHIP+/+ and -/- BMMCs were preloaded and stimulated as in (A). Total cell lysates were subjected to Western analysis using C) anti-phospho-Erk1/2, D) anti-phospho-p38, and E) anti-phospho-JNK Abs (top panels). These blots were reprobed with anti- FcsRI p chain and anti-JNK Abs to show equal loading. These blots are representative of 3 separate experiments. 473  much more intense and prolonged phosphorylation in SHIP-/- BMMCs (top panel of Figure 4.4C). Reprobing with anti-FcsRI p-subunit Abs demonstrated equal loading (bottom panel, Figure 4.4C). We also looked at the effect of SHIP on IgE+Ag-induced activation of p38 MAPK, since, depending on the stimulus and cell type, this S/T kinase has been shown to be activated (Madrid et al., 2001) or inhibited (Gratton et al., 2001) by the PI3K/PKB pathway and also activated by members of the PKC family (Rahman et al., 2001). Interestingly, we found that p38 phosphorylation was both more intense and more prolonged in response to IgE+Ag in SHIP-/- BMMCs (top panel, Figure 4.4D). Reprobing with anti-FcsRI p-subunit Abs 70  demonstrated equal loading (bottom panel, Figure 4.4D). Lastly, we looked at the effect of SHIP on IgE+Ag-induced phosphorylation/activation of J N K since the activation of this stress-activated protein kinase, has been shown to enhance IL-6 production in mast cells (Song et al., 1999). As shown in the top panel of Figure 4.4E, JNK phosphorylation was both more intense and more prolonged in response to IgE+Ag in SHIP-/- BMMCs. Reprobing with anti-JNK Abs demonstrated equal loading (bottom panel, Figure 4.4E).  4.2.4  IgE+Ag-induced IL-6 production in BMMCs is dependent on the activation of the PI3K, PKC, Erk and p38 pathways To determine which of these pathways, if any, contributed to the elevated IL-6 production observed  in SHIP-/- BMMCs, we added pathway specific inhibitors to SHIP+/+ and -/- BMMCs and then stimulated the cells with IgE+Ag for 3hr and performed IL-6 ELISAs on the conditioned media. As can be seen in Figure 4.5A, addition of the PI3K inhibitor LY294002 (25pM) or wortmannin (25nM), the PKC inhibitor Compound 3 (bisindolylmaleimide) (10pM), the Mek inhibitor PD98059 (50pM), or the p38 inhibitor SB203580 (2pM) completely abrogated IgE+Ag-induced IL-6 production in both SHIP+/+ (left panel) and -/(right panel) BMMCs.  Thus it appeared that all of these signaling pathways were essential for IL-6  production and, given that they were all elevated in IgE+Ag-induced SHIP-/- BMMCs, could contribute to the elevated IL-6 production observed in these cells. To gain some insight into which PKC isoform(s) were involved, we also examined IL-6 production from IgE+Ag-induced SHIP+/+ and -/- BMMCs in the presence and absence of the classical PKC (cPKC) inhibitor, G06976. As can be seen in Figure 4.5B (left panel), this inhibitor blocked IL-6 production, indicating a role for a Ca -dependent PKC in IL-6 regulation. This is 2+  consistent with a recent finding showing decreased IL-6 production from PKCp-deficient BMMCs (Nechushtan and Razin, 2001). To determine if PKC-mediated regulation of IgE+Ag-induced IL-6 production was limited to cPKC isoforms, we compared IL-6 production from PKC5+/+ and -/- BMMCs. As shown in Figure 4.5B (right panel), IL-6 production from PKC5-/- BMMCs was significantly reduced compared to their +/+ counterparts, suggesting a role for a novel PKC isoform in the regulation of IL-6 production as well.  4.2.5  phosphorylation/degradation and IgE+Ag-induced SHIP-/- BMMCs  IKB  NFKB  Because it had been shown recently that the mRNA  in  BMMCs  by  binding  directly  DNA binding and transactivation are higher in  TF NFKB  is a key regulator of IgE+Ag-induced IL-6  to K B elements within the IL-6 promoter and activating  transcription (Marquardt and Walker, 2000), we next compared the activity of 71  •  NFKB  in IgE+Ag-induced  5000  750-  4000 500-  3000  a.  X  250H  2000H 1000  LY  0'  W  C3  PD SB  LY  W  3hr  B  C3  PD SB  3hr PKC8 +/+  5000 -,  PKC8 -/-  5000 4000 -  4000  "35  o  CO  IL-6 (pgj  O  3000 -  i  2000 1000 0 -  0'  C3  3000 2000 1000 0  GO  3hr  0'  3hr  Figure 4.5: IgE+Ag-induced IL-6 production in BMMCs is dependent on the activation of the PI3K, PKC, Erk and p38 pathways. A) SHIP+/+ (•; left panel) and -/- (•; right panel) BMMCs were preloaded with 5ug/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 3hr in the absence (-) or presence of 25uM LY294002 (LY), 25nM wortmannin (W), 10u.M Compound 3 (C3), 50uM PD98059 (PD), and 2uM SB203580 (SB), added 15min before the DNP-HSA. IL-6 production was assessed by ELISA. Results shown are the mean ± SEM of 6 determinations. B) SHIP+/+ (•) and -/- (•) BMMCs were stimulated as in (A) in the absence (-) or presence of 10uM C3 or 1u.M G66976 (Go) and IL-6 levels measured by ELISA (left panel). PKC5 +/+ (•) and -/- (•) BMMCs were preloaded as above and then stimulated ± DNP-HSA for 3hr and IL-6 levels in the supernatants assessed by ELISA (right panel). Results shown are the mean ± SEM of 4 determinations.  SHIP+/+ and -/- BMMCs.  NFKB  which binds and masks the  activity is tightly regulated by  NFKB  Following IgE+Ag-stimulation,  IKB  IKBOC  (Gilmore, 1999; Zandi et al., 1997)  nuclear localization signal and thus sequesters  NFKB  in the cytoplasm.  kinase (IKK) is activated and phosphorylates  IKBOC,  which targets the  latter for ubiquitination and degradation by the proteasome, and frees  NFKB  to translocate to the nucleus to  activate target gene transcription (Koul et al., 2001; Zandi and Karin, 1999). As expected, we found that the NFKB  inhibitor Bay 11-7082 (Bay11), which irreversibly inhibits the phosphorylation of  IKB  (Pierce et al.,  1997), was a potent inhibitor of IL-6 mRNA and protein production in both SHIP+/+ and -/- BMMCs (Figure 4.6A). As a test of the specificity of this inhibitor we checked its effect on IgE+Ag-induced IL-4 synthesis, 72  IL-6  8000  c3 £ 50  <25  E „  n  0'  D CL  30'  + + + 5 10  B  + + + 5 10 20  20  Bay 11 (nM)  +  Bay11  20  (nM)  IL-4  ~ 50 tf) § 40 £• ra 30  XX  3.  <  z a E  EL 0'  30'  0'  + + + 5 10  20  IL-5  _ . 50  30' + + + 5 10 20  3hr Bay 11 (nM)  IL-13  40  +  +  10  20  Bay11 (nM)  TNFa  -g 20  3. < 10  z E  nnnn  00  ELQi 30'  0'  30' Bay 11  5 10 20  5 10 20  5 10 20  10  5 10 20  (MM)  20  Figure 4.6: N F K B regulates IL-6 production in BMMCs. A) SHIP+/+ (•) and -/- (•) BMMCs were preloaded with 5ng/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 30' (left panel) or 3hr (right panel) in the presence (+) or absence (-) of Bay 11 7082 (Bay 11) at 5, 10, or 20uM, added 15 min before the DNP-HSA. IL-6 mRNA levels were assesed by R P A (left panel) and corrected based on the mRNA levels of the housekeeping gene GAPDH. IL-6 levels in the supernatants were assessed by ELISA (right panel). Results shown are the mean ± S E M of 4 determinations. B) SHIP-/- (•) BMMCs were stimulated and analyzed as in (A) for IL-4 mRNA and protein levels. Only IL-4 mRNA levels were examined in SHIP+/+ (•) BMMCs since these cells do not produce IL-4 protein levels detectable by ELISA. C) SHIP-/- (•) BMMCs were stimulated and analyzed as in (A) for IL-5, IL-13 and T N F a mRNA levels (top panels). Protein levels of IL-13 and T N F a were measured by ELISA (bottom panels). Only mRNA levels were examined in SHIP+/+ (•) BMMCs. Results in (B & C) are representative of 2 separate experiments.  73  since the IL-4 promoter does not possess a KB element (Marquardt and Walker, 2000; Stassen et al., 2001), and found no inhibition (assessed by RPA and ELISA, Figure 4.6B). As a point of interest, we also found that Bay11 had no effect on IL-5 production (assessed by RPA, Figure 4.6C, left panel) as expected (Marquardt and Walker, 2000), but was capable of inhibiting IL-13 and TNFa production (assessed by RPA and ELISA, Figure 4.6C) (Kitaura et al., 2000; Stassen et al., 2001). Next, using phospho-specific IKBOC Abs, we examined IgE+Ag-induced IKB phosphorylation and found much higher phosphorylation in SHIP-/- than +/+ BMMCs (Figure 4.7A). Moreover, re-introduction of WT SHIP into SHIP-/- cells reduced this phosphorylation close to the levels seen in SHIP+/+ BMMCs (Figure 4.7A). In keeping with this increased IKB phosphorylation in SHIP-/- BMMCs, IKB degradation was significantly greater in these cells, but this was the case only when the protein synthesis inhibitor, cycloheximide was added (Figure 4.7B). In the absence of cycloheximide, IgE+Ag-induced degradation of IKB was similar in SHIP+/+ and -/- BMMCs (Figure 4.7C), most likely because of a compensatory increase in the transcription/translation  of the NFKB target, IKB, in the -/- cells that masks the increased IKB  degradation (Gilmore, 1999; Zandi and Karin, 1999; Zandi et al., 1997).  +/+  -/- WT  0' 5' 0' 5' 0' 5' •<w  < p j•  W: anti-P-kB (S!er )  +/+  •/•  0' 10' 30' 60'  0' 10'30'60'  W: anti-hcB  32  - - —Z W: anti-kB  W: anti-She Figure 4.7: I K B phosphorylation and degradation are higher in SHIP-/- BMMCs.  +/+ 0' 5' 10" 30'60'90'  1: HmZm —  A) SHIP+/+, -/-, and WT BMMCs were preloaded with 5ug/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 5min. Total cell lysates were subjected to Western analysis using anti-phosphoIKB (Ser ) Abs (top panel). The blot was reprobed with anti-kB Abs to show equal loading (bottom panel). B) SHIP+/+ and -/- BMMCs were preloaded as in (A) and stimulated with 50ug/ml cycloheximide for 15min prior to the addition of  0' 5' 10' 30' 60' 90'  W: anti-hcB  32  W: anti-FceRI B chain  DNP-HSA for the indicated times. Total cell lysates were subjected to Western analysis using anti-kB Abs (top panel). The blot was reprobed with anti-She Abs to show equal loading (bottom panel). C) SHIP+/+ and -/- BMMCs were preloaded as in (A) and stimulated ± DNP-HSA for the indicated times. Total cell lysates were subjected to Western analysis using anti-kB Abs (top panel). The blot was reprobed with anti-FcsRI p chain Abs to show equal loading (bottom panel). All blots are representative of 3 separate experiments. 74  We next looked at N F K B translocation to the nucleus by carrying out anti-NFKB p65 and p50 immunoblots with nuclear extracts and found, interestingly, that p50, but not p65 levels were substantially higher in IgE+Ag-induced SHIP-/- than +/+ BMMCs (Figure 4.8A, left panels). Total cell levels of NFKB (p50), on the other hand, were comparable (Figure 4.8A, right panel). A higher concentration of nuclear N F K B , however, is not necessarily synonymous with more IL-6 mRNA synthesis (Saccani et al., 2001). In order to initiate transcription of target genes, NFKB TFS must bind first as dimers to KB sites (Gilmore,  1999). Using EMSAs, we therefore investigated whether N F K B  DNA binding was higher in IgE+Ag-induced  SHIP-/- than +/+ BMMCs and found, as shown in Fig 8B, that this was indeed the case. We then carried out supershift studies using Abs to the members of the N F K B family (p50, p52, p65 (Rel A), RelB, and c-Rel (40)) and found that only anti-p50 and anti-p65 supershifted the NFKB/oligonucleotide complex (Figure 4.8C). Thus, a p50/p65 heterodimer was likely the predominant species that bound to KB sites in activated mast cells. As expected, the addition of Bay11 (20pM) to SHIP+/+ and -/- BMMCs completely abrogated the nuclear localization of N F K B , as assessed by EMSA on nuclear preparations from both cell types (Figure 4.8B).  B +/+  +/+  •/•  DNP Bay11  0' 30' 60' 120' 0' 30' 60' 120'  -/-  0' 30' 60' 120' 60' 0' 30' 60' 120' 60' . . . . + . . . . +  TCL  W: anti-NRcB p65  +/+ -/•  W: anti-NFKB p50  [  p65  B C p50 p52  p50  - p65  Figure 4.8: S H I P negatively regulates IgE+Ag-induced N F K B D N A binding. A ) Nuclear extracts (5ug protein/lane) from SHIP+/+ and -/- BMMCs, preloaded with 5ug/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for the indicated times were subjected to Western analysis using anti-p65 (top panel) and anti-p50 NFKB Abs (bottom left panel). Total cell lysates from SHIP+/+ and -/- BMMCs demonstrated equal levels of p50 (right panel). These blots are representative of 3 separate experiments. B ) Nuclear extracts from SHIP+/+ and -/- BMMCs preloaded as in (A) and stimulated ± DNP-HSA for the indicated times ± 20u.M Bay 11-7082 (Bay11) were incubated for 30min at 23°C with a radiolabeled oligonucleotide probe that contained an N F K B DNA binding site. Protein/DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography. This autoradiogram is representative of 3 separate experiments. C ) Nuclear extracts from SHIP-/- BMMCs preloaded as in (B) and stimulated with DNP-HSA for 2hr were split into 6 conditions (left panel) or 4 conditions (right panel) and incubated for 15min ± 10ug anti-p65/RelA (p65), anti-RelB (B), anti-c-Rel (C), anti-p50 (p50) or anti-p52 (p52) Abs (as indicated) then incubated for 30min at 23°C with a radiolabeled oligonucleotide probe that contained an NFKB DNA binding site. Protein/DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography. Supershifted bands are indicated by an arrow. 75  While phosphorylation-induced degradation of IKB and the subsequent nuclear translocation and DNA binding of released  NFKB  is regarded as the principle mechanism for activating  gene expression, some recent studies have suggested that the transcriptional activity of highly regulated (Madrid et al., 2001). We thus carried out for increased  NFKB  NFKB  dependent  NFKB  NFKB  is also  luciferase assays to obtain direct evidence  transactivation in IgE+Ag-induced SHIP-/- BMMCs. Specifically, SHIP+/+ and -/-  BMMCs were electroporated with an NFKB-firefly luciferase reporter gene construct, together with a thymidine kinase promoter-dependent renilla luciferase construct (pRL-TK) to assess transfection efficiency and the cells were then preloaded with 1 ug/ml IgE for 18hr and subjected to 20ng/ml DNP for 4hr. As shown in Figure 4.9A, the transactivation potential of Interestingly, the high  NFKB  NFKB  was significantly higher in SHIP-/- BMMCs.  transactivation observed in the SHIP-/- BMMCs in the absence of crosslinker  is consistent with our previous data showing IgE alone is capable of inducing  NFKB  transactivation (Figure  4.9B) (Kalesnikoff et al., 2002b) and IL-6 production (Kalesnikoff et al., 2001) (the IgE alone results will be expanded in chapter 5 of this thesis). Both of these processes are negatively regulated by SHIP.  A  0  IgE  IgE +  Figure 4.9: SHIP negatively regulates IgE+Ag-induced NFKB transactivation. A) SHIP+/+ (•) and -/- (•)  hr  4 DNP  BMMCs were  electroporated with PNFKB-LUC (firefly luciferase) and pRL-TK (ren/7/a luciferase) constructs, preloaded overnight at 1 ug/ml IgE and stimulated ± 20ng/ml DNP-HSA for 4hr. N F K B activity was determined using a luminometer and normalized to renilla luciferase activity. Results shown are the mean ± S E M of 6 determinations. B) SHIP+/+ BMMCs were electroporated as in (A), starved or preloaded overnight at 1u.g/ml IgE and stimulated ± 5ug/ml IgE (•) or 20ng/ml DNP-HSA (•), respectively, for 4hr. N F K B activity was determined as in (A).  4.2.6  PI3K/PKFJ and PKC enhance I K B phosphorylation/degradation and N F K B binding to while Erk and p38 stimulate N F K B transactivation  DNA  Since we found that the PI3K/PKB, PKC, Erk and p38 pathways were all elevated in SHIP-/BMMCs and they were all required for IL-6 production, we asked if and how these pathways elevated NFKB  to  activity. However, a complication in delineating the relative contributions of these upstream pathways  NFKB  activation is that they "cross-talk" with each other. To examine the contribution of "cross-talk" in 76  IgE+Ag-induced SHIP-/- BMMCs we first looked at the effect of LY294002 and wortmannin on the activation of PKB, Erk, and p38 and found that these PI3K inhibitors reduced all 3 phosphorylation events (Figure 4.10A). Thus PIP3 levels affect all 3 pathways in these cells. We next looked at the effect of Compound 3 on these 3 pathways and found that it not only inhibited Erk phosphorylation, as expected (Carroll and May, 1994), but PKB and, to a lesser extent p38, phosphorylation as well (Figure 4.10B & C).  B  5'  0'  LY W  B  C3  1.6 4.6 0.2 0.8  PD  0.1 17.9 12.6 20.0 0.3  W: anti-P-PKB (Ser ) 473  W: anti-P-Erk1/2  0.3 32.3 1.1 2.5  19.9 23.1 21.0 18.3 19.0  W: anti-P-Erk1/2  W: anti-Erk1  0.8 33.9 18.7 12.3  0.9 17.6  W: anti-P-p38  4.7  15.0 15.4  W: anti-P-PKB (Ser ) 473  20.9 17.1 14.4 17.5  9.5  W: anti-FceRI B chain  8.6  8.8  8.9 9.6  W: anti-FceRI p chain  D SB C3  B SB  0'  PD  5.7  18.9 4.2  W: anti-P-p38 8.4  23.6 18.3 22.9 4.5  0.4 9.7 9.4  W: anti-P-p38  0.2  2.8  2.2  W: anti-P-PKB (Ser ) 473  22.2 18.8 19.7 19.9 19.6  10.7 10.8 11.1  W: anti-FcsRI p chain  0.4  3.2  2.8  W: anti-P-Erk1/2 6.9  8.7  5.6  W: anti-FceRI p chain Figure 4.10: The PI3K/PKB, PKC, Erk and p38 pathways cross-talk in IgE+Ag-stimulated BMMCs. A) SHIP-/- BMMCs were preloaded with 5(ig/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 5min in the absence (-) or presence of 25uM LY294002 (LY) or 25nM wortmannin(W), added 15min prior to DNP-HSA. Total cell lysates were subjected to Western analysis using anti-phospho-PKB (Ser ) (top panel), anti-phospho-Erk1/2 (second panel), and anti-phospho-p38 (third panel) Abs. The blot was reprobed with anti-FcsRI p chain Abs to show equal loading. B, C & D) SHIP-/- BMMCs were preloaded and stimulated as in (A) in the absence (-) or presence of 10uM Compound 3 (C3), 20uM Bay11-7082 (B), 50uM PD98059 (PD) or 2uM SB203580 (SB). Total cell lysates were subjected to Western analysis using B) anti-phospho-Erk1/2 Abs and reprobed with antiErk1 Abs (top panels) or anti-phospho-PKB (Ser ) Abs and reprobed with anti-FceRI p chain Abs (bottom panels) or C) antiphospho-p38 Abs and reprobed with anti-FceRI p chain Abs or D) anti-phospho-p38, anti-phospho-PKB (Ser473), and antiphospho-Erk1/2 Abs then reprobed with anti-FcsRI p chain Abs to show equal loading. The blots shown in (A-D) are representative of at least 3 separate experiments. 473  473  77  This might be due to non-specificity of this PKC inhibitor at this concentration (10u,M) or, at least in the case of PKB, a PI3K-independent, PKC-mediated activation of PKB, as has been reported by Kroner et al. (2000). As expected, PD98059 completely inhibited Erk1/2 phosphorylation, but had no effect on PKB (Figure 4.10B) or p38 phosphorylation (Figure 4.10C). SB203580, at a concentration (2u.M) that completely blocked p38 phosphorylation, had no effect on Erk or PKB phosphorylation (Figure 4.10D). Of interest, Bay11 had no effect on Erk, PKB or p38 phosphorylation (Figures 4.10B & C). Similar results were obtained with these inhibitors using SHIP+/+ BMMCs (data not shown). With this "cross-talk" information in hand, we then asked if IgE+Ag-induced IKB phosphorylation or NFKB  DNA binding was affected by the inhibitors of these upstream pathways. Specifically, we examined  the effects of Compound 3, Bay 11, PD98059 and SB203580 on IKB phosphorylation. As can be seen in Figure 4.11 A (top panel), IKB phosphorylation was markedly inhibited by Bay 11, as expected, and by Compound 3 but only slightly inhibited by PD98059 and not at all by SB203580. Reprobing with anti-FcsRI B chain Abs demonstrated equal loading (Figure 4.11 A, bottom panel). Since the concentration of PD98059 used in this study totally abrogated Erk phosphorylation while that of Compound 3 only partially inhibited Erk phosphorylation (Figure 4.11B, top two panels), this indicated that the PKC-mediated phosphorylation of IKB likely occurred independent of the Erk pathway. EMSAs of nuclear extracts confirmed and extended these findings by showing that Compound 3, as well as LY294002, but neither PD98059 nor SB203580 inhibited  NFKB  DNA binding (Figure 4.11B). The numbers below each lane represent relative band  intensities, determined by densitometry. As mentioned earlier,  NFKB  activity can also be regulated at the transactivation step and one of  the players involved in this regulation is p38 (Madrid et al., 2001; Wesselborg et al., 1997). We therefore tested the effects of SB203580, as well as PD98059, Compound 3, LY294002 and Bay11 in  NFKB-  luciferase assays and found that they all totally abrogated the IgE+Ag-induced increase in luciferase activity (Figure 4.11C). These results suggested that the p38 and Erk pathways, unlike the PKC and PI3K pathways, enhance N F K B activity via increasing N F K B transactivation independent of NFKB  4.3  IKB  degradation and  DNA binding.  DISCUSSION  We demonstrate herein that SHIP negatively regulates IgE+Ag-induced production of proinflammatory cytokines in BMMCs. Focusing on IL-6 production, we show that this repression is  78  5' 0'  -  C3  B  5' B  PD  0'  SO-  - SB  % 4.6  16.5  6.3  1.6  13.3  1.2 6.7 6.0  18.5  15.1  CS  PD  LY  W: anti-P-kB (Ser ) 32  7.6 17.4  17.5  17.1  18.4  13.6  15.6  7.8  18.9  7.9  10.1  W: anti- FcsRI p chain Figure 4.11: PI3K/PKB and PKC enhance I K B phosphorylation/degradation, N F K B D N A binding and transactivation while Erk and p38 only stimulate NFKB transactivation. A ) SHIP-/- BMMCs were preloaded with 5ug/ml IgE for 4hr, then stimulated ± 20ng/ml DNP-HSA for 5min in the absence (-) or presence of 10uM Compound 3 (C3), 20u.M Bay11 (B), 50uM PD98059 (PD), or 2uM SB203580 (SB) added 15min prior to the addition of DNP-HSA. TCLs were analyzed using anti-phospho-kB (Ser ) Abs and the blot was reprobed with anti-FcsRI B chain Abs to show equal loading. B) Nuclear extracts from SHIP-/- BMMCs Dreloaded as in (A), then stimulated ± DNP-HSA for 32  4hr  60min in the absence (-) or presence of 10uM C3, 50uM PD, and 25u.M LY (left panel) or 2u.M SB (right panel), added 15min prior to DNP-HSA, were incubated for 30min at 23°C with a radiolabeled oligonucleotide probe that contained an N F K B DNA binding site. Protein/DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography. The numbers below each lane represent band intensities determined by densitometry. Blots shown in (A) and the autoradiogram shown in (B) are representative of at least 3 separate experiments. C) SHIP-/- BMMCs were electroporated with PNFKB-LUC (firefly luciferase) and pRL-TK (renilla luciferase) constructs, preloaded overnight at 1 ug/ml IgE and stimulated + DNP-HSA for 4hr in the absence (-) or presence of LY, C3, B, PD or B. N F K B activity was determined using a luminometer and normalized to renilla luciferase activity. Results shown in (C) are the mean ± SEM of 4 determinations.  dependent on the phosphatase activity of SHIP and thus, most likely, on restraining PI3K-induced PIP3 levels. This is consistent with our finding that the PI3K inhibitors, LY294002 and wortmannin, not only block IL-6 production but also inhibit the upstream pathways that we found were both elevated in SHIP-/- BMMCs and contributed to IL-6 production. These results contrast with a previous study in which wortmannin failed to inhibit IgE+Ag-induced IL-6 production in BMMCs (Marquardt et al., 1996) and also with a report that PI3K may even inhibit IL-1-induced IL-6 production in myeloid cells (Birkenkamp et al., 2000). To address how SHIP regulates IL-6 production, we honed in on the regulation of IL-6 mRNA synthesis since IL-6 mRNA levels correlated nicely with secreted IL-6 protein levels. Narrowing our window of investigation even further, we focused exclusively on  NFKB  activity since this TF has been shown to be a  major positive regulator of IgE+Ag-induced IL-6 mRNA synthesis in BMMCs (Marquardt and Walker, 2000). However, we fully appreciate that SHIP may also regulate the activity of other TFs involved in IL-6 mRNA 79  synthesis (Song et al., 1999) and/or the secretion of the IL-6 protein from these cells (Baumgartner et al., 1994;  Nechushtan and Razin, 2001). As predicted, the N F K B inhibitor, Bay11, blocked IL-6 production in  both SHIP+/+ and -/- BMMCs. To determine how SHIP negatively regulates N F K B activity, we examined four major levels of N F K B regulation: IKB phosphorylation/degradation, N F K B translocation to the nucleus, DNA binding and transactivation. Our finding that IKB is phosphorylated to a greater extent in SHIP-/- BMMCs is consistent with several recent reports showing that PKB acts as a postive regulator.of N F K B activity by transiently binding and phosphorylating/activating IKK (Bertolini et al., 2000; Kane et al., 1999; Kitaura et al., 2000; Madrid et al., 2001; Madrid et al., 2000; Ozes et al., 1999; Reddy et al., 1997; Romashkova and Makarov, 1999). Relevant to our finding that IKB degradation is increased in SHIP-/- BMMCs in the presence of cycloheximide, Koul et al. (2001) recently reported that the tumor suppressor PTEN, which also hydrolyses PIP3,  inhibits N F K B DNA binding without affecting IKB degradation. However, they did not examine IKB  degradation in the presence of cycloheximide. Gustin et al. (2001), on the other hand, found, in support of our findings, that PTEN inhibits the activation of IKK and phosphorylation of IKB in response to TNF, but they did not examine IKB degradation.  Interestingly, we observe an increased translocation of the p50 but not the p65 subunit of N F K B into the nucleus of IgE+Ag-stimulated SHIP-/- BMMCS, as assessed by Western analysis of nuclear extracts. Using EMSAs, we show that SHIP negatively regulates N F K B DNA binding though this may be explained entirely by the reduced nuclear translocation of p50 in the presence of SHIP. As well, our finding that N F K B DNA binding is inhibited by LY294002 (confirming the work of Gustin et al. (2001)) strengthens the case that SHIP represses IL-6 production via its hydrolysis of PIP3. Related to this, the 3'-phosphatase, PTEN, has also been shown to negatively regulate N F K B DNA binding (Gustin et al., 2001; Koul et al., 2001). To gain some insight into the SH IP-regulated upstream pathways that modulate IL-6 mRNA levels in BMMCs and to delineate the contribution of each of these pathways to N F K B activation, we used specific inhibitors to pathways that were elevated in the absence of SHIP. As mentioned earlier, the elevated PKB activity in SHIP-/- BMMCs likely contributes to the increased N F K B activity in these cells by directly phosphorylating/activating IKK. Complicating the picture, the PIP3-dependent PDK1, besides playing a critical role in activating PKB, has also been shown to phosphorylate/activate various PKC isoforms (Balendran et al., 2000; Belham et al., 1999; Le Good et al., 1998). Related to this, many PKC isoforms, 80  such as a , s, 0, and 4, have been shown to positively regulate  NFKB  activity in a variety of cell types  (Lallena et al., 1999; Lin et al„ 2000; Rahman et al., 2001) and the cPKC isoform, PKCp, is known to be a positive regulator of IgE+Ag-induced mast cell degranulation and IL-6 production (Nechushtan et al., 2000). Herein, we show that the novel PKC isoform, PKC8, also acts as a positive regulator of IL-6 production. As to  how PKC isoforms regulate  IL-6  production, we show that PKC isoforms  regulate  IKB «  phosphorylation/degradation, and thus subsequent N F K B DNA binding and transactivation, and this finding is supported by Lellena et al. (1999) who showed that P K C a and atypical PKC isoforms bind to IKKs in vitro and in vivo.  NFKB  activity can also be regulated at the transactivation step and several recent reports support  our finding that p38 is capable of upregulating NFKB  nuclear translocation or  NFKB  NFKB  transactivation without affecting  IKB  degradation,  DNA binding (Carter et al., 1999; Madrid et al., 2001; Rahman et al.,  2001; Wesselborg et al., 1997). Looking at p38 activation, Madrid et al. (2001) found that PKB mediates IL1-induced activation of  NFKB  by activating p38 in an IKK-dependent manner. However, we found that  Bay11, which irreversibly inhibits IKK and the subsequent phosphorylation of IKB, had no effect on p38 phosphorylation in response to IgE+Ag. We thus propose that IKK and p38 enhance  NFKB  activity by  separate pathways and this is supported by studies showing that PKC5 regulates parallel IKK and p38 pathways to enhance  NFKB  activity in response to thrombin (Rahman et al., 2001). Since we found that  PKC inhibitors partially block IgE+Ag-induced p38 phosphorylation in BMMCs it is conceivable that p38 activity is regulated in these cells via a PI3K/PDK1/PKC pathway in response to IgE+Ag. Related to this, the phosphorylation of p38 and Erk are significantly reduced in PKC8-/- BMMCs (Dr. Michael Leitges; personal communication) and this may contribute to the decreased IL-6 production observed in these cells.  While p38 is a well established regulator of  NFKB  transactivation (Madrid et al., 2001; Vanden  Berghe et al., 1998; Wesselborg et al., 1997), the role of Erk in  NFKB  regulation is more controversial. Our  inhibitor studies suggest that IgE+Ag-induced Erk activation is highly dependent on PI3K and PKC activation. As well, using the Mek inhibitor, PD98059, we show that Erk activity is required for transactivation independent of  IKB  phosphorylation and  NFKB  Vanden Berghe et al. (1998) found that PD98059 inhibited  NFKB  DNA binding. In support of our finding,  NFKB  transactivation in response to TNFa.  However, Madrid et al. (2001) did not find any significant decrease in  NFKB  luciferase in 3T3 cells in the  presence of PD98059, albeit using very low (2pM) inhibitor levels. As to how PD98059 inhibits the transactivation of  NFKB,  Chen et al. (2001) recently showed that 81  NFKB  can be acetylated and that  acetylated N F K B p65 interacts weakly, if at all, with k B a . Related to this, Vanden Berghe et al. (2000) found that both p38 and Erk promoted the acetylation capacity of the enhanceosome and thus stimulated TNF-induced, NFKB-mediated, IL-6 gene expression. While we also found that SHIP acts as a negative regulator of IgE+Ag-induced JNK phosphorylation, we were unable to study the role of this MAPK family member in the regulation of N F K B activity and IL-6 production due to the lack of JNK-specific inhibitors. However, it has been reported that the PIP3-dependent tyrosine phosphorylation of the PH-containing Rac guanine nucleotide exchange factor, Vav, stimulates IL-6 production in mast cells by a Rac/JNK-dependent pathway, and we observe increased membrane recruitment and phosphorylation of Vav in SHIP-/- BMMCs (unpublished, M. Ware & G. Krystal). Taken together our results suggest a model, shown in Figure 4.12, in which SHIP represses IL-6 production in BMMCs, at least in part, by reducing PI3K generated PIP3 levels induced by IgE+Ag. This in turn inhibits PKB- and PKC-mediated IKB phosphorylation/degradation and the nuclear translocation and DNA binding of released N F K B as well as p38- and Erk-mediated N F K B transactivation. Thus, these upstream pathways which synergize to stimulate IL-6 mRNA synthesis are all negatively regulated by SHIP.  Figure 4.12: A model of IgE+Ag-induced IL-6 mRNA synthesis. P K B and P K C enhance IKK-mediated phosphorylation/degradation of IKB and the nuclear translocation/DNA binding of N F K B while p38 and Erk, which are activated by P K B and P K C , enhance N F K B transactivation without increasing IKB phosphorylation or N F K B D N A binding.  82  Chapter 5  MONOMERIC IgE STIMULATES SIGNALING PATHWAYS IN MAST CELLS THAT LEAD TO CYTOKINE PRODUCTION AND CELL SURVIVAL  5.1  INTRODUCTION IgE+Ag-induced mast cell activation is responsible for both allergic disorders, such as hay fever  and asthma, and for host resistance to parasites via the secretion of proinflammatory mediators (Galli, 2000; Schwartz, 1994; Yamaguchi et al., 1999). IgE binding to the FcsRI is referred to as a "passive presensitization" step in the current mast cell signaling paradigm. FcsRI activation and signaling is thought to occur only when receptor-bound IgE is subsequently crosslinked via multivalent Ag (Beaven and Metzger, 1993; Kinet, 1999). Although IgE binding to mast cells is thought to be a passive pre-sensitization step, we have previously shown that SHIP-/- BMMCs degranulate in response to IgE alone, unlike their wildtype counterparts (Figure 5.1) (Huber et al., 1998). Furthermore, we found that SHIP prevents this signaling from progressing to degranulation by hydrolysing IgE-induced generation of PIP3 (Huber et al., 1998). The massive degranulation response we observed in SHIP-/- BMMCs in response to IgE alone was our first indication that IgE alone is capable of signaling in mast cells, thus we decided to explored this phenomenon further in normal BMMCs.  50-1  +/+  -/-  +/+  IgE + DNP  -/-  IgE  Figure 5.1: IgE alone causes mast cell degranulation in the absence of SHIP. SHIP+/+ and -/- BMMCs were preloaded with 10ug/ml IgE for 1 hr then stimulated for 15min ± 20ng/ml DNP-HSA (•) or stimulated ± 5u.g/ml IgE alone (•) and percent degranulation determined by assaying supernatants and cell pellets for p-hexosaminidase activity. Each bar represents the mean ± SEM of duplicates after subtracting the percent degranulation obtained in the absence of DNP-HSA or IgE, respectively.  83  In this chapter, we demonstrate that monomeric IgE (mlgE), in the absence of Ag, stimulates multiple phosphorylation events in normal murine BMMCs. Our results indicate that while mlgE does not induce degranulation or leukotriene synthesis in normal BMMCs, it leads to a more potent production of cytokines than IgE+Ag. Moreover, mlgE acts as a survival factor and prevents the apoptosis of cytokinedeprived BMMCs, likely by maintaining  BCI-XL  levels and producing autocrine-acting cytokines. Since IgE  concentrations as low as 0.1 pg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.  5.2  RESULTS  5.2.1  IgE alone stimulates the phosphorylation of the Erks, p38, JNK and PKB in normal murine BMMCs We recently reported that a 3min exposure to 5pg/ml IgE, in the absence of Ag, triggered the  phosphorylation of the FceRI B and y subunits, She and SHIP in normal murine BMMCs and that the presence of SHIP prevented IgE-induced signaling from progressing to degranulation (Huber et al., 1998). To further explore the signaling potential of IgE in normal BMMCs, we carried out time course studies with IgE alone (monoclonal anti-DNP clone SPE-7 IgE, Sigma) and examined the phosphorylation of the mitogen activated protein kinases, Erk1/2, p38 (aka HogK) and JNK (aka SAPK). As can be seen in Figure 5.2A, 5pg/ml of IgE alone stimulated near maximal phosphorylation of the Erks within 5min and this phosphorylation was still at maximal levels at 60min of stimulation. The stress-activated kinases, p38 and JNK, were also phosphorylated in response to IgE alone, with p38 again being maximally phosphorylated within 5min of stimulation and remaining at maximal levels following 60min of exposure. Interestingly, JNK phosphorylation was relatively delayed, not peaking until 15min post-stimulation. In addition, as shown in Figure 5.2B, IgE alone stimulated the phosphorylation of the survival-enhancing kinase, PKB, at both Ser  473  and Thr . This dual phosphorylation, which is required for full activation of PKB (Alessi et al., 1997), 308  indicated that IgE alone was also capable of recruiting PI3K to the plasma membrane since PI3K is upstream of PKB phosphorylation and generates PIP3 at the plasma membrane to attract the PH domain containing kinases, PDK1 and PKB (Alessi et al., 1997). A reprobing of the blots shown in Figures 5.2A & B with anti-Erk1, anti-p38, anti-JNK and anti-PKB Abs, respectively, established equal loading. Since it had been reported previously that IgE+Ag triggers the phosphorylation of these same signaling intermediates (Lu-Kuo et al., 2000), we compared the intensity and kinetics of phosphorylation of the Erks and PKB in response to IgE alone versus IgE+Ag (DNP-HSA). As shown with Erk1/2 in Figure 5.3A, we found that phosphorylation events were slightly delayed but far more prolonged in response to IgE  84  B igE 0'  5'  15'  0' 5'  60'  15'  igE 60'  0"  5'  15'  igE 60'  0'  5'  15'  60'  Erk 1 Erk 2  •  W:anti-P-Erk1/2  W:anti-P-p38  W: anti-P-JNK  W: anti-P-PKB (Ser )  W: anti-Erk1  W: anti-p38  W: anti-JNK  W: anti-P-PKB (Thr°8)  473  3  W: anti-PKB Figure 5.2: IgE, in the absence of Ag, stimulates multiple phosphorylation events. Normal BMMCs were treated with 5ug/ml IgE alone for the indicated times and the SDS-solubilized total cell lysates subjected to A) Western analysis with phospho-specific Abs to Erk1/2 (left panel), p38 (middle panel) or JNK (right panel). The blots were then reprobed with Abs to Erk1, p38 and JNK, respectively, to confirm equal loading. In B) Western analysis were performed with anti-phosphoSer specific PKB (top panel), and reprobed with anti-phosphoThr specific PKB (middle panel) and anti-PKB Abs (to confirm equal loading; bottom panel). These blots are representative of 5 separate experiments. 473  308  alone. Specifically, 5pg/ml of IgE for 4hr followed by an optimal concentration of Ag (20ng/ml) for the indicated times, triggered a maximal phosphorylation of the Erks by 2min but this phosphorylation was barely detectable at 15min and was back to baseline levels by 1 hr. With 5pg/ml IgE alone, on the other hand, Erk1/2 phosphorylation was barely detectable at 2min, peaked at 15min, remained clearly visible at 2hr and was back to baseline levels at 4hr (the zero time for IgE+Ag). A longer time course of signaling events with IgE alone is shown in Figure 5.3B, to show that these events have returned to baseline by 4hr after stimulation. Similar results were obtained with PKB (data not shown; experiments performed by Dr. Michael Huber). Worthy of note is that the level of Erk1/2 phosphorylation stimulated by 5pg/ml IgE alone was similar to that attained with 5pg/ml IgE+Ag (4hr preload). Similar results were obtained when the cells were preloaded overnight at 1 or 5pg/ml IgE, ruling out that the similar phosphorylation intensities were simply due to a transient desensitization by IgE during the preloading step (Figure 5.3C).  Having established that signaling events are initiated by 5pg/ml IgE alone and with a similar intensity to those elicited with IgE+Ag, we compared the tyrosine phosphorylation of the FcsRI itself. BMMCs were stimulated as above and the cell lysates subjected to immunoprecipitation with antiphosphotyrosine (4G10) beads and Western analysis with anti-FcsRI p chain Abs (Figure 5.3A, bottom  85  panel). Interestingly, phosphorylation of the FcsRI p chain by IgE alone was less intense than that observed in response to IgE+Ag, but phosphorylation levels remained clearly detectable at 4hr (i.e. the zero time for IgE+Ag). With IgE+Ag, on the other hand, phosphorylation of the FcsRI p chain was clearly faster and more intense, peaking at 2min, but returning to its baseline levels by 1 hr.  A  IgE + DNP  igE 0'  2'  5' 15' 60' 120' 0'  2'  5' 15' 60' 120' • Erkl •Erk 2  TCL  W: anti-P-Erkl/2 W: anti-Erk1  4G10IP |  - ~  W; anti-FcsRI p chain  B  IgE 0  1  2.25 2.5 2.75  3  3.25 3.5 3.75 4  hr 4-Erk1 4-Erk2  W: anti-P€rk1/2  «*»«-» W: anti-Erkl 5ug/ml IgE IgE  1u.g/mllgE  5jig/ml IgE  (4hr) + DNP (O/N) + DNP (O/N) + DNP  0' 2' 5' 15' 0' 2' 5'15' 0' 2' 5'15' 0' 2' 5'15'  W: anti-P£rk1/2 W: anti-Erkl Figure 5.3: Phosphorylation events are delayed but far more prolonged in response to IgE alone versus IgE+Ag.  A) BMMCs, treated ± 5ug/ml IgE alone for the indicated times or treated with 5u.g/ml IgE for 4hr then ± 20ng/ml DNP-HSA for the indicated times, were lysed with TX-100 and subjected to immunoprecipitation with anti-phosphotyrosine (4G10) beads and Western analysis with anti-FceRI p chain Abs. (lower panel). Total cell lysates from these stimulations were subjected to Western analysis with anti-phospho-specific Erk1/2 (top panel) and then anti-Erkl Abs (middle panel). Results are representative of 6 separate experiments. B) BMMCs were stimulated with 5ug/ml IgE for the indicated times and SDS-solubilized total cell lysates subjected to Western analysis with phospho-specific Abs to Erk1/2 and then reprobed with Abs to Erk1 to show equal loading. C) BMMCs were starved for 4hr then stimulated ± 5ug/ml IgE or preloaded with 5ug/ml IgE for 4hr, 1 ug/ml IgE overnight or 5u,g/ml IgE overnight then stimulated ± 20ng/ml DNP-HSA for the indicated times and analyzed as in (B). Blots in (B & C) are representative of 2 separate experiments. 86  5.2.2  IgE alone likely acts through the FcsRI and lipid rafts We then carried out IgE dose response studies in the presence or absence of Ag, using Erk  phosphorylation as an endpoint. As can be seen in Figure 5.4A, 2-5pg/ml of IgE (for 60min) was required for maximal Erk phosphorylation whereas only 0.01 pg/ml of IgE was required for maximal phosphorylation when a 4hr pretreatment with IgE was followed with 2min of 20ng/ml Ag. Densitometric analyses of these two blots suggested an EC50 of 1 pg/ml for IgE alone and 0.005pg/ml for IgE+Ag. Similar results were obtained with the rat basophilic cell line, RBL-2H3 (Figure 5.4B). This 200-fold difference in potency raised the possibility that IgE alone was stimulating these cells because of a small number of aggregates in the commercial IgE anti-DNP preparation. To test this, we fractionated this Sigma IgE via BioSep SEC S3000 gel filtration HPLC and found, depending on the lot number, that these anti-DNP IgE preparations contained from 5-20% aggregates (dimers plus higher molecular weight aggregates) (Figure 5.5A, left panel). The purified monomers were rechromatographed immediately following purification, or 7 days after incubation at 37°C in Tyrode's buffer and in all cases remained in the monomeric state (i.e. contained less than 0.5% aggregates, our lower limit of detection) (Figure 5.5A, middle and right panels).  IgE + DNP igE  0 0.1 0.5 1.0 2.0 5.0  (ng/ml) 0 <^ ^ ^  0.1 1.0  -Erk1-  «-Erk2-H  W: anti-P-Erk  W: anti-Erk1  B IgE 0  0.01 0.03  0.1  DNP 0.3 1.0  5.0  0.1  IgE (ng/ml) 0  0.01 0.03 0.1  DNP 0.3  1.0  5.0  0  0.1  W: anti-P-Erk  W: anti-Erk1 Figure 5.4: IgE dose response studies reveal an EC50 of 1u.g/ml. A) BMMCs, treated with the indicated concentrations of IgE alone (left panels) for 60min or the indicated concentrations of IgE for 4hr and then 20ng/ml Ag for 2min (right panels), were SDS-solubilized and total cell lysates subjected to Western analysis with anti-phospho-specific Erk1/2 (upper panels) and then anti-Erk1 Abs (lower panels) to confirm equal loading. Results are representative of 2 separate experiments. B) RBL-2H3 monolayers were cultured overnight ± 1 ug/ml of 60min airfuged IgE. Cells not exposed to IgE overnight were stimulated for 4min (left panels) or 30min (right panels) with the indicated concentrations of IgE. Cells sensitized with IgE were stimulated with 0.1 ng/ml DNP-HSA. The cells were then lysed and analysed as in (A). 87  To determine if the IgE-induced Erk phosphorylation was due to the presence of these aggregates, freshly prepared mlgE (at 5pg/ml, as assessed by IgE ELISA), aggregates alone (at the level present in Spg/ml of unfractionated IgE, ie, 0.5pg/ml) or aggregates + 5pg/ml mlgE were tested. As can be seen in Figure 5.5B, 5pg/ml mlgE could still robustly stimulate the phosphorylation of Erk1/2. While this stimulation was slightly reduced compared to unfractionated IgE, the aggregates were very poor stimulants and the addition of aggregates to mlgE did not increase the response over that seen with mlgE alone (Figure 5.5B). These results suggest that the aggregates in these commercial preparations of IgE are for the most part in inactive conformations. Of note, column eluate alone did not stimulate Erk phosphorylation (Figure  5.5B).  We also assessed the levels of endotoxin in both our unfractionated and HPLC purified mlgE preparations  UJ  LU O  ^MONOMER  o z <  z <  co  CQ  C£  a:  o  O CO  co <  DIMER LARGE \ AGGREGAT^  5  CO CQ  <  L. 10  20  0  TIME (min)  5  10  20  B IgE 0' 2' 5'  5' 2' 5' 2' 5' 2' 5'  10  TIME (min)  3  1  mlgE B mlgE + agg agg  5  TIME (min)  4  5  12.5 12.5 LPS LPS 0 +0.5 IgE Erk1  (pg/ml) 0.5 5 5 IgE IgE LPS  (pg/ml) • Erk1 • Erk2  Erk2  W: anti-P-Erkl/2  W: anti-P-Erk1/2  W: anti-Erkl  W: anti-Erkl  Figure 5.5: Monomeric IgE stimulates Erk phosphorylation. A) Clone SPE-7 anti-DNP IgE (Sigma) was fractionated using a BioSep SEC S3000 column (left panel) and the monomer peak either rechromatographed immediately (middle panel) or after incubation for 7 days at 37°C in Tyrode's buffer (right panel). Results are representative of 10 separate experiments. B) BMMCs were treated with 5u.g/ml unfractionated IgE, HPLC buffer eluate (B), 5(j.g/ml mlgE, 5ug/ml mlgE+0.5 ug/ml aggregates (agg) or 0.5ug/ml aggregates alone for the indicated times and SDS-solubilized and subjected to Western analysis with anti-phospho Erk1/2 Abs. The blot was reprobed with anti-Erkl Abs to demonstrate equal loading. C) BMMCs were stimulated for 5 min as indicated and the SDS-solubilized lysates subjected to Western analysis as in (B). Results in (B & C) are representative of 2 separate experiments. S8  since endotoxin has been shown to stimulate mast cells (section 1.3.5) (Leal-Berumen et al., 1994). Using the highly sensitive LAL reagent clotting assay (Sullivan et al., 1983), we found the level of endotoxin was below the limit of detection (1.25pg/ml). Concentrations 10-fold higher than this had no effect on Erk phosphorylation, either alone or together with a suboptimal IgE concentration (0.5pg/ml) (Figure 5.5C, lanes 2 & 3). Even 5pg/ml LPS gives a barely detectable phosphorylation of Erk (Figure 5.5C, lane 6). The 200-fold difference in potency between IgE alone and IgE+Ag also raised the possibility that IgE alone was acting through a low affinity IgE receptor. Of the known low affinity IgE receptors on BMMCs, the most likely candidate, given that IgE alone stimulates the phosphorylation of the p subunit of FcsRI, was FcyRIII. Although FcyRIII is expressed at relatively low levels at the surface of BMMCs (Katz and Lobell, 1995), it also utilizes the p subunit (Kurosaki et al., 1992) and has been shown to be a low affinity receptor for both IgG and IgE (Takizawa et al., 1992). To test if IgE was signaling through this receptor, we pre-incubated BMMCs with the FcyRIII/FcyRII blocking Ab, 2.4G2 (Unkeless, 1979), under conditions which completely blocked binding to these receptors (Figure 5.6A), and found that it had no effect on subsequent IgE-induced Erk phosphorylation (Figure 5.6B, left panel). Another low affinity IgE receptor that has been shown to be present RBL-2H3s (Frigeri and Liu, 1992), and thus may be present on BMMCs, is the lectin Mac-2 (aka sBP). This receptor binds to p-galactoside on the cell surface and thus can be efficiently eluted from cells with 25mM lactose (Frigeri and Liu, 1992). However, this treatment did not diminish IgE-induced Erk phosphorylation (Figure 5.6B, right panel). These results suggest that IgE alone triggers Erk phosphorylation through the FcsRI.  To gain some insight into how IgE alone triggers these signaling events, we examined the effects of the cholesterol depleting agents, filipin III (Aman and Ravichandran, 2000) and methyl-p-cyclodextrin (Vereb et al., 2000), on IgE-induced Erk phosphorylation. As can be seen in Figure 5.6C, both markedly inhibited IgE-induced Erk phosphorylation. This may suggest that signaling induced by IgE alone, like IgE+Ag (Holowka et al., 2000), involves a critical detergent resistant membrane (DRM) step.  5.2.3  IgE alone is more effective than IgE+Ag at increasing the levels of multiple cytokines Since stimulating normal BMMCs with IgE+Ag has been shown to trigger mast cell degranulation,  the generation of AA metabolites and the synthesis of various cytokines (Mekori and Metcalfe, 2000), we compared the ability of IgE alone versus IgE+Ag to elicit these three biological responses. Specifically, we compared p-hexosaminidase release to monitor degranulation, LTC4 production (using enzyme immunoassays) to monitor AA metabolite production, and cytokine mRNA levels via RNase protection  89  2.4G2 + Unstained  "io°  3'  io  io  2  3  IgG-FITC  IgG-FITC  io**  FL1-H  ~\o°ST  10  2  Isotype C + IgG-FITC  '10  3  io  4  2  |Jo  ,> 0  FL1-H  10  2  "i'o  3  '  10  FL1-H  B IgE  2.4G2 + IgE  0' 15' 0' 15'  lactose IgE + IgE  igE 0'  0' 15' 0' 15'  5'  IgE + DNP F  M  0'  5'  F  M - Erk 1 •Erk 2  • Erk 1 Erk 2  W: anti-P-Erkl/2  W: anti-P-Erkl/2  W: anti-Erkl  W: anti-Erkl  -  T ~  T~T I  Figure 5.6: IgE likely acts through the FceRI and lipid rafts. A) BMMCs were pretreated ± 10ug/ml of 2.4G2 or isotype control (rat lgG2b anti-Mad a) Ab for 30min at 4°C and then either left unstained or exposed to 30ug/ml heat-aggregated (63°C, 30min, pH 8) FITC-mouse IgG for 30min at 4°C and subjected to FACS analysis. The 1 panel shows the unstained control, the 2 panel, the non-pretreated FITC-IgG sample, the 3 panel, the 2.4G2 pre-treated sample and the 4 panel, the isotype control pre-treated sample. B) BMMCs were either pretreated ± 10ug/ml 2.4G2 for 30min at 4°C and then treated ± 5ug/ml IgE for 15min (left panel) or pretreated ± 25mM lactose for 10min at 37°C and then treated ± 5ug/ml IgE for 15min (right panel). C) BMMCs were pretreated for 15min ± 1.6u.g/ml filipin III (F) or 7mM methyl-B-cyclodextrin (M) following a 4hr starve or preload (with 5ug/ml IgE), then treated ± 5ug/ml IgE or 20ng/ml DNP-HSA for 5min, respectively. Total cell lysates in (B & C) were subjected to Western analysis with anti-phospho-specific Erk1/2 and then anti-Erkl Abs to show equal loading. Results are representative of 2 separate experiments. st  nd  rd  th  assays (RPAs). As expected, IgE+Ag stimulated all 3 responses (Figure 5.7). Surprisingly, however, while IgE alone was incapable of triggering degranulation or significant LTC4 release (Figure 5.7A), it dramatically increased the mRNA levels of multiple cytokines (e.g. IL-6, TNFa, IL-4 and IL-13) in a dose dependent manner (Figure 5.7B). In fact, further analysis revealed that IgE alone was more potent than IgE+Ag at increasing the mRNA levels of these cytokines. A typical result, shown with IL-6 mRNA, is depicted in Fig 8A. Reminiscent of our protein phosphorylation data (Figure 5.3A), the vast majority of cytokine mRNA levels remained elevated longer in response to IgE alone than IgE+Ag (Figure 5.8A). However, this was not true for all cytokines examined. IL-5 mRNA levels, for example, were substantially higher following stimulation with IgE+Ag than IgE alone (Figure 5.8A, insert).  90  20  75i  «  501  § 10  ,2H  Q  0'  0.5  15'  0'  0.5  0.5  20'  0.5  Figure 5.7: IgE increases multiple cytokine mRNA levels. BMMCs, treated ± 10ug/ml IgE (•) for the indicated times or preloaded with 10ug/ml IgE for 4hr and then treated ± 20ng/ml DNP-HSA (•) for the indicated times, were analyzed for A) degranulation (left panel) and LTCt production (right panel). The values shown in the left panel are the means ± SEM of duplicate determinations and both panels are representative of at least 3 separate experiments. B) BMMCs, treated with IgE for 15min at the indicated concentrations (in ug/ml) were subjected to RNase protein assay (RPAs; Riboquant) and relative band intensities of IL-6, TNFa, IL-4 and IL-13 mRNA (arbitrary units) were quantitated using a phosphoimager and standardized using levels of the housekeeping gene, GAPDH. Results are representative of 4 separate experiments.  To determine if cytokine levels secreted by BMMCs in response to IgE alone versus IgE+Ag paralleled their mRNA levels, we compared IL-6 protein levels, by ELISA, in medium conditioned by BMMCs. As shown in Figure 5.8B, this was indeed the case with secreted IL-6 levels being 5-10 fold higher following stimulation with IgE alone than with IgE+Ag. Similar results were obtained with IL-13 and TNFa (Figure 5.8C). Of note, identical results were obtained when a protease inhibitor cocktail was added to the medium (Sigma, P-8340), suggesting that this difference is not due to a differential secretion of proteases or protease inhibitors. Also of note, mlgE was only slightly less potent than unfractionated IgE while IgE aggregates were very poor stimulators of cytokine secretion (Figure 5.8B). We repeated our IL-6 secretion studies with monomeric-enriched preparations obtained via a 60min airfuge procedure, bearing in mind the caveat that this procedure only removes larger aggregates, and found, once again, that all the IgE-induced production of IL-6 could be attributed to mlgE (Figure 5.8B). 91  Interestingly, while IgE-induced IL-6 mRNA levels peaked at 60 min, IL-6 protein levels did not reach plateau levels until 3hr and this discrepancy could suggest, as has been shown with TNFa production from RBL-2H3 cells (Baumgartner et al., 1994), that secretion from BMMCs is actively regulated via a C a - and PKC-mediated pathway and that this pathway may be more active in response to IgE 2+  alone. An IgE dose response study was then carried out and revealed a similar EC50 for IL-6 and IL-13 secretion (Figure 5.8D) to that seen for Erk phosphorylation. To gain some insight into the pathways involved in eliciting this robust production of inflammatory cytokines, BMMCs were stimulated with IgE alone or IgE+Ag for 3hr ± the PI3K inhibitor, LY294002, the MEK inhibitor, PD98059, or the p38 inhibitor, SB203580. As can be seen in Figure 5.8E, all 3 inhibitors blocked IL-6 production, suggesting that the PI3K, Erk and p38 pathways all play a role in mediating IgEas well as IgE+Ag-induced IL-6 production/secretion (described in chapter 4). Of note, these inhibitors did not affect cell viability at the concentrations used, even after 24hr of incubation with IgE-stimulated BMMCs.  5.2.4  IgE enhances mast cell survival and does so by preventing apoptosis Since PKB, the Erks and the cytokines, IL-4, IL-6 and IL-13 have all been shown to enhance  survival in some cell systems (Deng et al., 2000; Downward, 1998; Lai and Mosmann, 1999; Yanagida et al., 1995), we asked if IgE alone might be a pro-survival factor for BMMCs. As can be seen in Figure 5.9A, a 48hr incubation of these cells with 5pg/ml IgE alone was as effective at preventing cell death as maximal levels of IL-3 or SCF, two well documented mast cell survival factors (Gommerman and Berger, 1998). Similar results were obtained with a hybridoma supernatant from a different IgE (anti-Epo lgE-26; StemCell Technologies Inc.), at a final concentration of 10pg/ml, but not from an equal volume of an IgGi-producing hybridoma supernatant produced in the same medium (anti-glycophorin A at 20pg/ml; StemCell Technologies Inc.) (Figure 5.9B), ruling out the possibility that the purification procedure used in producing the commercial anti-DNP IgE altered its properties. Moreover, we observed equally potent IgE-induced survival of BMMCs from both C57B6 and BalbC mice, indicating that our results are not mouse strain specific (Figure 5.9C).  To determine if IgE was enhancing survival by preventing apoptosis, DNA was isolated from BMMCs following 48hr of incubation ± 5pg/ml IgE or 30ng/ml IL-3. As can be seen in Figure 5.1 OA, IgE alone was as effective as IL-3 at preventing DNA fragmentation. To gain some insight into the mechanism(s) underlying IgE's ability to prevent apoptosis, we compared the levels of the anti-apoptotic protein,  BCI-XL,  in BMMCs following 24hr of incubation ± IgE or IL-3. As shown in Figure 5.1 OB, IgE, like 92  B ID-  0 15 60 120 240  Hi  in  0 15 60 120 240  IgE + DNP  igE  s-  S'  0) p O O-  O O)  4-  0' 15' 60' 120' 240' 15' 60' 120' 240'  0  in rl a 1 hr 3hr  24hr 48hr mlgE agg algE  IgE + DNP  igE  O 2000  cn o. 1000  0  n  3 24 48  0  EL  3 24 48 hr  0 3 24 48  0 3 24 48 hr  igE  IgE + DNP  IgE + DNP  igE  2H  $1 0 0.1 0.5 1 2 IgE (ug/ml)  5  100^  in 0 0.1 0.5 1 2 IgE (ug/ml)  5  0  If!  IH  in  in  3hr LY  PD SB  Figure 5.8: IgE increases multiple cytokine mRNA and protein levels more than IgE+Ag. A) BMMCs, treated with 5u.g/ml IgE (•) for the indicated times or preloaded with 5ug/ml IgE for 4hr, then washed 3 times and exposed to 20ng/ml DNP-HSA (•) for the indicated times were subjected to RPAs as in Figure 5.7B. Results are representative of 4 separate experiments. The insert shows the levels of IL-5 mRNA for the same samples. B) BMMCs were treated as in (A) and the levels of IL-6 protein in the conditioned medium were assessed by IL-6 ELISA. The last 3 lanes show the level of IL-6 secreted by 3hr in response to 5ug/ml mlgE (HPLC), 0.5ug/ml aggregates (HPLC), or 5ug/ml IgE following a 60min airfuge at 100,000xg (algE), respectively. Values are the mean ± SEM of 8 determinations (duplicates of 4 separate experiments). C) The levels of TNFct (left panel) and IL-13 (right panel) in the conditioned medium from (B) were determined by ELISA. D) BMMCs were treated with the indicated concentrations of IgE for 24hr and the levels of secreted IL-6 and IL-13 determined. E) 50uM LY294002 (LY), 50uM PD98059 (PD) and 10uM SB203580 (SB) were added to BMMCs 30min before the addition of 5ug/ml IgE (•) or 20ng/ml DNP-HSA (•) and the production of IL-6 assessed after 3hr of incubation. Values for (D & E) are the mean ± SEM of triplicate determinations and are representative of 2 separate experiments. 93  B  Day  Day Figure 5.9: IgE alone enhances BMMC survival. A) BMMCs derived from 4 separate mice were incubated separately for 72hr with 10% FCS alone (•) or + 5ug/ml IgE (A), 40ng/ml SCF (O) or 30ng/ml IL-3 (V) and viable cells counted. Values are the mean ± SEM of 8 determinations (duplicates for the 4 BMMC preparations). B) BMMCs were cultured with 5% FCS alone (•) or in anti-Epo lgE-26 hybridoma supernatant (A) at 10ug/ml IgE (as determined by IgE ELISA), or in an equal volume of an IgGi-producing supernatant (V) (final concentration of 20ug/ml Igd) produced in the same medium, or in 5% FCS + 10ug/ml purified anti-Epo lgE-26 (O). C) BMMCs generated from BalbC mice were cultured with 10% FCS alone (•) or + 10ug/ml IgE (A) and viable cells counted on the days indicated. Values in (B & C) are the mean ± SEM of 4 determinations.  Day  IL-3, prevented  BCI-XL  levels from falling to those seen in starved cells. For this study a 24hr time point  was chosen to ensure that the majority of cells in the starved culture were still viable (Figure 5.9). An antiShe reprobing of the blot confirmed equal loading of the gel (Figure 5.1 OB). The maintenance of this prosurvival Bcl-2 family member suggests at least one pathway by which IgE may be preventing BMMC death. To determine if IgE was acting primarily as a survival factor or a mitogen, we carried out both H3  thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) vital dye (Glimm and Eaves, 1999) studies. As can be seen in Figure 5.10C, a 6hr labeling of BMMCs following 3 days with IgE, IL-3 or control medium (10% FCS), revealed that IL-3-containing cultures incorporated substantial levels of thymidine while IgE-containing cultures did not. At this time there were 4.0 (IgE), 1.8 (control) and 6.25 (IL3) x 10 viable BMMCs/ml. BMMCs were also stained with 5pM CFSE, flow cytometry sorted to obtain a 5  homogeneous population of stained cells and incubated for 3 days ± IL-3 or IgE. Similar to our H 3  thymidine results, the CFSE results suggested that IgE acts primarily a survival factor (Figure 5.10D). 94  1kb C IL-3 IgE  B C IL-3 IgE 2000CH  111  15000T3  W: anti-Bcl-X  _  — - »  L  H  ' 100005000o-l  W: anti-She  FCS  IL-3  IgE  Figure 5.10: IgE alone maintains Bcl-Xj. levels and prevents apoptosis of BMMCs. A) BMMCs were incubated for 48hr with 10% FCS alone (C) or + 30ng/ml IL-3 or 5ug/ml IgE and the DNA was then extracted and subjected to agarose gel electrophoresis. 1 kb = 1 kb DNA ladder (Gibco-BRL). Results are representative of 2 separate experiments. B) BMMCs were cultured for 24hr with 10% FCS alone (C) or + 30ng/ml IL-3 or 10ug/ml IgE and total cell lysates subjected to Western analysis with anti-Bcl-Xi and reprobing with anti-She Abs to show equal loading. Results are representative of 3 separate experiments. C) H-fhymidine incorporation was measured after a 6hr labeling with 1uCi/50,000 cell sample (2Ci/mmole) following 3 days of culture in 10% FCS alone ± 30ng/ml IL-3 or 10u.g/ml IgE. Each point is the mean ± SEM of 3 determinations. D) Representative FACS profiles of CFSE-stained BMMCs after 3 days of culture in 10% FCS alone (left panel) or + 30ng/ml IL-3 (middle panel) or 10|ig/ml IgE (right panel). This experiment was repeated 3 times. 3  Freshly prepared mlgE was also tested for its effect on BMMC survival. Dose response studies revealed that concentrations as low as 0.1 ja.g/ml were capable of significantly enhancing survival (Figure 5.11A). IgE aggregates, on the other hand, at 0.5Lig/ml, were found to be relatively poor enhancers of survival (i.e. approximately equal to O.Vg/ml monomeric IgE, Figure 5.11 A). This strongly suggested that if there were low levels of contaminating IgE aggregates in our mlgE preparations they were not playing a significant role in enhancing BMMC survival. Also of interest was our finding that mlgE did not have to synergize with factors in FCS to enhance survival since similar survival enhancement was observed with medium containing 0.1% BSA instead of 10% FCS (Figure 5.11B). Interestingly, no enhancement in BMMC 95  survival was observed when Ag was added to 5pg/ml mlgE (Figure 5.11C). However, it was conceivable that we were already at the maximal levels of survival attainable and so carried out mlgE dose response studies ± Ag and again found no enhancement of survival (Figure 5.11C).  B  A 7-,  7-, O  6-  able eel Is/ml  5. 54 432-  > 1-i  0  1  1  1  2  1  3  1  4  1  5  1  6  1  7  8  0-  r  0  c  2  3  4  5  6  7  8  Day  5.2.5  3  4  5  6  7  Figure 5.11: Monomeric IgE enhances mast cell survival. A) BMMCs were cultured with 10% FCS alone (•) or + 0.1 (0), 1(A), 5 (•) or 10 (•) ng/ml mlgE or + 0. 5ug/ml IgE aggregates ( - • - ) and viable cells counted on the days indicated. B) BMMCs were cultured with 0.1% BSA alone (•) or + 10ug/ml mlgE (•). C) BMMCs were cultured with 10% FCS alone (•) or + 0.1, 1, 5ug/ml mlgE in the presence (0,A,O) and absence (•>,•) of 20ng/ml DNP-HSA. Viable cell counts were determined on the days indicated. For (A, B & C) the values shown are the mean ± SEM of duplicate determinations and similar results were obtained in 2 or more separate experiments.  7-|  1  2  Day  Day  0  1  The autocrine production of cytokines contributes to IgE-induced BMMC survival To determine if cells cultured in the presence of IgE were producing cytokines or other factors that  contributed to the enhancement of BMMC survival, medium conditioned by a 3 day incubation of BMMCs with IgE (10pg/ml) was treated with DNP-agarose beads to remove the IgE. This purged conditioned medium.-which retained approximately 0.5pg/ml of IgE (sample 3; Figure 5.12A, insert), was then added to naive BMMCs and survival monitored. As can be seen in Figure 5.12A, this conditioned medium enhanced survival to the same degree as medium containing 10pg/ml IgE. Thus, IgE-stimulated cells were producing autocrine-acting factors that were sufficient to mediate the IgE-induced cell survival. This was also suggested by our finding that IgE-induced survival was cell density dependent (Figure 5.12B). Interestingly, 96  if BMMCs were treated with lOpg/ml IgE for only 4hr and the cell medium was treated with DNP-agarose then added back to the cells, the resulting survival was similar to that obtained with 3 day conditioned media (Figure 5.12C). This suggests that exposure to mlgE for 4hr is sufficient to enhance survival.  Figure 5.12: IgE-induced cytokines act in an autocrine manner to enhance BMMC survival. A) BMMCs were cultured with 10% FCS alone (•) or + 0.5ug/ml mlgE (•), 10ug/ml mlgE (•), 3 day conditioned medium (A) or 3 day conditioned medium treated with DNP-agarose to reduce IgE levels (as assessed by IgE ELISA) to 0.5ug/ml (O) and viable cells counted on the days indicated. The insert in (A) shows IgE levels in fresh medium containing 10ug/ml IgE (1) and in this same medium following 3 days of incubation with BMMCs before (2) and after (3) treatment with DNP-agarose. B) BMMCs were cultured at 1x10cells/ml (left panel), 5x10cells/ml (middle panel), or 1x10cells/ml (right panel) with 10% FCS alone (•) or + 2ug/ml IgE (O) or 5ug/ml IgE (•). C) BMMCs were cultured with 10% FCs alone (•) or + 0.5ug/ml (•), 10ug/ml IgE (•) or 4hr conditioned medium treated with DNP-agarose to reduce IgE levels to 0.5ug/ml (O). The insert in (C) shows IgE levels in fresh medium containing 10ug/ml IgE (1) and in this same medium following 4hr of incubation with BMMCs before (2) and after (3) treatment with DNPagarose. Values in (A, B, & C) are the mean ± SEM of duplicate determinations and similar results were obtained in 2 separate experiments. 5  5  6  97  Lastly, to see if the autocrine-acting factors could be the cytokines produced in response to IgE, we added a combination of cytokines (i.e. IL-2, -3, -4, -6, -13, and TNFa) to BMMCs cultured in the absence (Figure 5.13A) or presence (Figure 5.13B) of 0.5u.g/ml IgE. The cytokine concentrations used were based either on levels in conditioned media from 3 day IgE-stimulated BMMCs cultures (for IL-3, -6 and TNFa, based on ELISAs) or from extrapolating RPA results (for IL-2, -4 and -13). As can be seen in Figure 5.13A, these cytokines significantly enhanced BMMC survival although to a lesser degree than that achieved with 10u,g/ml IgE (survival in the presence of 0.5pg/ml IgE alone is shown as a dashed line to compare to Figure 5.13B). However, as shown in Figure 5.13B, the combination of these cytokines with 0.5|ag/ml IgE yielded a survival equal to that obtained with 10u.g/ml IgE, at least for the first 3 days of culture. The subsequent drop in survival could be due to the omission of either one or more secreted cytokines or certain non-cytokine secreted factors.  A  B  Figure 5.13: A combination of cytokines (IL-2, -3, -4, -6, -13, & TNFa) can enhance BMMC survival, although to a lesser degree than that achieved with 10ug/ml IgE. A) BMMCs were cultured with 10% FCS alone (•) or + 0.5ug/ml mlgE ( - • - ) , 10ug/ml mlgE (A) or a combination of 500pg/ml IL-2, 10pg/ml IL-3, 500pg/ml IL-4, 5ng/ml IL-6, 5ng/ml IL-13 and 2.5ng/ml TNFa (A) and viable cells counted on the days indicated. B) BMMCs were cultured with 10% FCS alone (•) or + 0.5ug/ml mlgE (•), 10ug/ml mlgE (A) or a combination of the same cytokines as in (A) + 0.5ug/ml mlgE (A) and viable cells counted on the days indicated. Values are the mean ± SEM of duplicate determinations and similar results were obtained in 2 separate experiments.  5.3  DISCUSSION We demonstrate herein that IgE alone triggers multiple signaling pathways and a more robust  secretion of cytokines than IgE+Ag in primary murine BMMCs. Moreover, we show that IgE alone enhances the survival of these BMMCs and does so, at least in part, via the secretion of autocrine-acting cytokines. This challenges, to some extent, the current paradigm which states that binding of IgE to FcsRI is a passive pre-sensitization step that does not lead to intracellular signaling unless receptors are subsequently  98  crosslinked (Beaven and Metzger, 1993). Our findings are not totally unprecedented, however, since earlier studies have shown that IgE alone increases FcsRI expression both in vitro and in vivo in mouse and human mast cells (Hsu and MacGlashan, 1996; MacGlashan et al., 1997a; Yamaguchi et al., 1997; Yamaguchi  et  al.,  1999)  internalization/degradation  and  this  upregulation  consists  of  an  initial  protection  phase and a subsequent cyclohexamide-sensitive upregulation  from phase  (Yamaguchi et al., 1997). Importantly, our results are not totally in conflict with the current paradigm since they are still consistent with signaling being mediated via aggregation of FcsRI. Related to this, while our studies with 2.4G2 and lactose strongly suggest that mlgE is initiating signaling via FcsRI, future experiments with BMMCs from FcsRI a chain deficient mice will be required to confirm this.* To address the major question of how IgE alone is stimulating BMMCs, it is highly unlikely that the aggregates in the commercial IgE anti-DNP preparations are responsible since they are very poor stimulators of Erk phosphorylation, IL-6 production and survival. Even if trace amounts of active aggregates are present in our mlgE preparations, more should be present in our aggregate preparations and yet we always observe far less activity in them. Given the extensive evidence in the literature, we favor a model in which mlgE activates BMMCs by triggering FcsRI aggregation. The only question is whether mlgE does this by first aggregating in solution within the BMMC milieu, perhaps in response to cell secreted factors, and then binding to the FcsRI or by binding to the FcsRI first and then triggering aggregation. Against the former, we do not see any detectable aggregation upon storage of mlgE in PBS or in medium containing 0.1% BSA (the medium in which we stimulate BMMCs with mlgE) and we do not see any increased activity when we pre-incubate mlgE with BMMCs. We thus hypothesize that mlgE binds to the FcsRI and reduces, to a modest extent given its EC50 of Ipg/ml, an inherent repulsion between neighboring receptors, allowing them to slowly form small clusters in lipid rafts and trigger a low but prolonged signaling of various intracellular pathways. Relevant to the concept of IgE reducing an inherent repulsion between neighboring FcsRI, we found in our survival studies with various IgE producing hybridomas (data not shown) that the supernatants from 3 different IgE-anti-DNP hybridomas were more potent (based on IgE ELISAs) than those of 5 other IgE producing hybridomas. On the other hand, one IgE to an as yet unknown epitope was even more potent than the IgEs against DNP. This suggests perhaps that the charge/conformation within the Fab hypervariable region of IgE influences its ability to aggregate FcsRI. Related to this, anti-Epo IgE-  * The data presented in this chapter was published in the June 2001 issue of Immunity (volume 14) along with a second group (Asai et al., 2001) reporting that IgE alone was capable of enhancing mast cell survival. This group had access to an FceRI a chain -/- mouse and showed that IgE-mediated enhancement of mast cell survival is indeed dependent on functional FceRls. The results from this paper will be discussed in chapter 7.  99  26 was significantly less potent than anti-DNP IgE at inducing IL-6 secretion and this correlated with a reduced ability to enhance survival. Interestingly, we found that adding DNP-lysine to BMMCs 1 hr after the addition of anti-DNP mlgE abruptly halted IL-6 secretion (Kalesnikoff et al., 2002b). Addition of DNP-lysine to anti-Epo lgE-26, on the other hand, had no effect, ruling out non-specific inhibition. This is consistent with a model in which the binding of DNP-lysine to IgE abrogates the ability of IgE to reduce the inherent repulsion between neighboring IgE receptors and both prevents the formation of and disrupts IgE/FceRI cell surface aggregates. This is both compatible with and sheds new light on early studies in which DNPlysine was used to disrupt signaling initiated by DNP-albumin induced aggregates (Kawakami et al., 1992). We also found that if we washed mlgE-treated BMMCs free of mlgE after 1 hr and then added DNP-lysine to the cells, it still abruptly halted IL-6 secretion. This suggests that DNP-lysine is disrupting aggregates at the cell surface rather than in solution and this effect is consistent with IgE triggering signaling via receptor aggregation. As to why IgE alone stimulates cytokine secretion to a much greater extent than IgE+Ag but is incapable of triggering degranulation or LTC4, production, it is possible that intensity and kinetic differences in signaling are responsible. Thus, while we cannot rule out qualitative differences, it may not be necessary to invoke them. IgE+Ag triggers a much more rapid and robust tyrosine phosphorylation of the receptor B subunit and more rapid Erk phosphorylation and this higher signaling intensity early on may allow several signals to converge with sufficient intensity to surpass the threshold for degranulation and leukotriene production. The weaker, longer lasting signal triggered by IgE alone, due perhaps to the relatively unsynchronized, slow on rate of IgE (Kulczycki et al., 1974), the possible requirement for a sufficient saturation of receptors to facilitate IgE-lgE aggregation and/or the far slower internalization rate of uncrosslinked IgE/FceRI (Mao et al., 1993), may play a pivotal role in allowing cytokines to be actively secreted from BMMCs. Related to this, Kituara et al. (2000) recently proposed that the enhanced secretion of TNFa and IL-2 they observe in IgE+Ag-stimulated Lyn-deficient BMMCs might be due to a prolonged activation of the Erks and JNK.  As to how the prolonged signals we observed with IgE alone might lead to enhanced survival via the maintenance of  BCI-XL  and the induction of cytokine production, the Erks have been shown to play an-  anti-apoptotic role in some cell types (Deng et al., 2000) and PKB is a well established pro-survival kinase (Downward, 1998). PKB activation leads, amongst other things, to the activation of NFAT, which is a well known activator of cytokine gene expression in mast cells (Kitaura et al., 2000; Ozes et al., 1999), and NFKB,  which upregulates the synthesis of many cytokines, including IL-6 (Kitaura et al., 2000; Marquardt  100  and Walker, 2000), and directly upregulates the anti-apoptotic protein,  BCI-XL  (Downward, 1998; Khoshnan  et al., 2000). As well, both p38 and JNK have been shown to enhance inflammatory cytokine synthesis (Kozawa et al., 1999), the latter via upregulating c-Jun (Kitaura et al., 2000). Related to this, our finding that IgE-induced IL-6 secretion is inhibited by LY294002, PD98059 and SB203580, implicates the PI3K/PKB, Erk and p38 pathways in this process. Cytokine production has also been shown to be regulated in mast cells at the level of secretion and the prolonged signaling induced by IgE may preferentially enhance this step as well. Specifically, Baumgartner et al. (1994) found there is an Ag-induced C a - and PKC2+  dependent secretion step, independent of protein synthesis, that determines the release of TNFoc from RBL-2H3 cells. It is thus possible that IgE alone, perhaps because of its prolonged signaling, stimulates the release of IL-6 for a longer period of time than IgE+Ag. Related to this, these authors found that the addition of DNP-lysine 1 hr after the addition of DNP-BSA, to disaggregate the residual cell surface complexes, immediately halted secretion (and promoted intracellular degradation of TNFa), suggesting that a continuous Ag-induced signal is required to maintain secretion (Baumgartner et al., 1994).  In attempts to identify the autocrine-acting cytokine(s) that enhances IgE-induced BMMC survival, we assessed the level of SCF in medium conditioned by 3 days with IgE, since de Paulis et al. (1999) showed that SCF is released from human mast cells. However, it was below the limit of detection of the ELISA (less than 250 pg/ml) and levels of neutralizing anti-SCF Ab that could inhibit 30ng/ml of SCF from enhancing BMMC survival did not reduce the survival effects of IgE. Thus, SCF does not appear to be an important player in IgE-induced BMMC survival. Similarly, neutralizing anti-IL-6 Ab did not reduce the survival effects mediated by IgE. However, SCF and/or IL-6 may act in concert with a combination of other IgE-induced cytokines to enhance BMMC survival. Complicating matters further, the autocrine-acting factor(s) may not be a cytokine(s) or both a cytokine(s) and non-cytokine(s) may be required to enhance survival.  An important outstanding issue, of course, is whether IgE alone enhances mast cell survival in mice and humans. In this regard, it has been known for some time that serum IgE levels are elevated in patients with allergic disorders or parasitic infections (Bennich and Johansson, 1970) and correlate with both disease severity and protective immunity to parasites (Corry and Kheradmand, 1999). This can be explained, at least in part, by the finding that mast cells undergo IgE-dependent FcsRI upregulation, and this enables the cells to bind more IgE and thus be activated to release mediators with lower concentrations of a given Ag (Galli, 2000; Kinet, 1999; Yamaguchi et al., 1997; Yamaguchi et al., 1999). Our results suggest that elevated levels of IgE may also be stimulating the release of proinflammatory cytokines. While  101  this cytokine release may not lead, in the absence of degranulation and leukotriene production, to an inflammatory response by itself in vivo, it may contribute to a subsequent, Ag-initiated inflammatory response. As well, since the concentration of IgE that promotes survival in our BMMC assay (0.1-10 pg/ml) is present in mouse and human sera exposed to parasites and allergens (Bennich and Johansson, 1970), it may be responsible, at least in part, for the elevated numbers of mast cells observed under these circumstances.  102  Chapter 6  IgE TRIGGERS THE ADHESION OF MAST CELLS TO FIBRONECTIN AND THIS ENHANCES CYTOKINE PRODUCTION AND MAST CELL SURVIVAL  6.1  INTRODUCTION Mature mast cells originate from pluripotent hemopoietic stem cell derived progenitors in the bone  marrow that are recruited out of the circulation and into connective tissues (Galli, 2000; Gurish and Austen, 2001). This recruitment, via adhesion of integrins on the surface of mast cell progenitors to components of connective tissue, like fibronectin (FN), is thought to play a critical role in mast cell retention and the subsequent proliferation, differentiation, survival, priming and activation of these cells (Hamawy et al., 1994; Okayama, 2000). Various stimuli have been shown to stimulate the adhesion of BMMCs to FN, via the integrins  cuPi (very  late antigen (VLA)-4) and  aspi (VLA-5)  (Dastych et al., 1991). These include  physiologically relevant stimuli, such as SCF (Dastych and Metcalfe, 1994; Kinashi and Springer, 1994) and IgE+Ag-induced crosslinking of the high affinity IgE receptor, FcsRI (Ra et al., 1994). While the cell surface level of VLA-4 decreases with BMMC differentiation, that of VLA-5 remains high (Fehlner-Gardiner et al., 1996) and appears to be the predominant integrin involved in mature mast cell adhesion to FN (Houtman et al., 2001a). We recently demonstrated that IgE alone (i.e. in the absence of crosslinking agents) was capable of stimulating multiple signaling pathways in normal BMMCs grown in suspension cultures and that this led to an increased survival of these cells in the absence of exogenous cytokines (chapter 5) (Kalesnikoff et al., 2001). In this chapter we have explored the possibility that IgE alone might also affect biological responses other than survival. Specifically, we asked if IgE alone could enhance the adhesion of mast cells to the connective tissue component, FN. Our data suggest that IgE alone does indeed trigger adhesion to FN and does so to the same extent as the well documented inducer of adhesion, SCF. Like SCF, it does so via inside-out-signaling to increase the avidity of the FN-binding integrin, VLA-5. However, unlike SCF, IgEinduced adhesion appears to require the entry of extracellular C a  2 +  and may be positively regulated by a  Ca -dependent PKC. We also show that this IgE-induced increase in avidity is dependent upon activation 2+  of PI3K and PLCy, but not Erk or p38. Intriguingly, IgE-induced FN binding acts synergistically with IgE to prolong intracellular phosphorylation events in BMMCs and to enhance proinflammatory production and mast cell survival.  103  cytokine  6.2  RESULTS  6.2.1  IgE alone stimulates the adhesion of both BMMCs and CTMCs to FN We recently demonstrated that IgE, in the absence of crosslinking agents or exogenous growth  factors, was capable of enhancing the survival of BMMCs and did so, at least in part, by maintaining BCI-XL levels and by inducing the production of autocrine-acting cytokines (Kalesnikoff et al., 2001). To determine if IgE alone had any other biological effects on BMMCs, we tested its ability to stimulate the adhesion of these cells to the connective tissue component, FN. As can be seen in Figure 6.1A (left panel), IgE (SPE-7) alone stimulated the adhesion of Calcein-AM loaded normal C57B6 mouse derived BMMCs to FN in a dose dependent fashion. Utilizing a 60min adhesion assay, adhesion typically plateaued between 50-60% of input cells. The IgE concentration yielding half-maximal adhesion was found to be between 100 and 500ng/ml IgE. When BSA-coated wells were substituted for FN-coated wells, no adhesion was observed in response to IgE (Figure 6.1 A, left panel, black bars). Identical results were obtained with mlgE, derived by HPLC fractionation of purchased SPE-7 IgE (Kalesnikoff et al., 2001), indicating that this was not due to low levels of IgE aggregates in the commercial IgE preparation, and using an anti-Epo lgE-26, demonstrating that these results were not restricted to the anti-DNP IgE, SPE-7 (data not shown; experiments performed by Vivian Lam).  For comparison, we carried out dose response studies with SCF (Figure 6.1A, middle panel) and a similar plateau was observed with optimal levels of SCF, shown in previous studies to be highly effective at inducing adhesion of BMMCs to FN (Dastych and Metcalfe, 1994). We also found that IgE+Ag was capable of inducing BMMC adhesion to FN (Figure 6.1 A, right panel), in keeping with previous reports (Kinashi et al., 1999; Ra et al., 1994), and this adhesion reached a plateau similar to that obtained with IgE alone or with SCF. As predicted, based on previous reports, the concentrations of SCF and Ag that gave half maximal adhesion (i.e. approximately 0.5ng/ml and 1.5ng/ml, respectively) were far less (10-100 fold) than that required for half maximal stimulation of proliferation (Kinashi and Springer, 1994) or degranulation (Houtman et al., 2001b; Wyczolkowska et al., 1994), respectively. It is now well documented that SCF and other agents that enhance adhesion of mast cells to FN do so in a transient way (Kinashi and Springer, 1994; Wyczolkowska et al., 1994). We therefore carried out time course studies with IgE alone to determine if the kinetics of adhesion and/or release were similar to that obtained with SCF or IgE+Ag. As can be seen in Figure 6.1 B, IgE-induced adhesion of BMMCs to FN was slower (i.e. IgE-induced adhesion was undetectable at 5min) and plateaued later than that induced by SCF or IgE+Ag. However, IgE-induced adhesion remained elevated significantly longer (i.e. was still at plateau levels at 60min) than with SCF or IgE+Ag. 104  We next asked if IgE alone could stimulate the adhesion of CTMCs to FN. As shown in Figure 6.1C, although background adhesion was consistently higher with these cells, IgE alone, as well as SCF and IgE+Ag, triggered CTMC adhesion. The concentration of IgE alone that gave half maximal adhesion was approximately 500ng/ml. Thus, IgE-induced adhesion was not restricted to BMMCs.  A  Time (min)  IgE (ng/ml)  SCF DNP  Figure 6.1: IgE alone stimulates the adhesion of both BMMCs and CTMCs to FN. A) Adhesion of normal BMMCs to FN  following a 60min adhesion assay with increasing concentrations of IgE alone (left panel), SCF (middle panel), or IgE+DNP-HSA (right panel). The black bars in the left panel indicate the level of adhesion to wells coated with BSA instead of FN. B) A time course of BMMC adhesion to FN in the presence of 1 ng/ml IgE alone (•), 5ng/ml SCF (A), or lgE+2ng/ml DNP-HSA (•). C) Adhesion of normal CTMCs to FN following a 60min adhesion assay with IgE alone at indicated concentrations, 5ng/ml SCF, or lgE+5ng/ml DNP-HSA. Results shown are the mean + SEM of triplicate determinations. Similar results were obtained in 5 (A), 3 (B), and 2 (C) separate experiments.  6.2.2  IgE alone stimulates the adhesion of BMMCs to FN via an increase in the avidity of VLA-5 To gain some insight into the nature of the receptors on BMMCs that bind to FN in response to IgE  alone, we carried out adhesion assays in the presence and absence of the peptide GRGDSP (which contains the RGD consensus sequence within FN that binds to a subset of integrins (Houtman et al., 2001a)) or a control peptide, GRGESP. As expected from previous reports (Dastych and Metcalfe, 1994), the RGD-containing peptide markedly inhibited SCF-induced adhesion while the control RGE-containing peptide did not, and the same results were obtained with IgE (Figure 6.2A). To hone in on which integrin 105  was involved in IgE-induced adhesion to FN, we next examined the effect of blocking VLA-5 with the antia5 integrin Ab MFR-5 (anti-CD49e) since it had been shown previously that this integrin was involved in SCF- and IgE+Ag-induced adhesion of BMMCs to FN (Kinashi et al., 1999; Kinashi and Springer, 1994). As can be seen in Figure 6.2B, this Ab also inhibited IgE-induced adhesion to FN. To determine if IgE, in the absence of Ag, was enhancing BMMC adhesion to FN by upregulating the cell surface level of VLA-5, we carried out flow cytometry using anti-CD49e. As can be seen in Figure 6.2C (top panel) there was no increase in the cell surface level of a5 integrin following a 1 hr treatment with IgE (when binding to FN is maximal (Figure 6.1 B)). Thus IgE alone, similar to what has been reported previously with SCF and IgE+Ag (Kinashi et al., 1999), appeared to enhance adhesion not by upregulating integrin receptors but by increasing the avidity of VLA-5 via inside-out-signaling. Also worthy of note, there was no change in the cell surface level of oc5 integrin following a 4hr treatment with IgE in the presence of either BSA (Figure 6.2C, middle panel) or FN (Figure 6.2C, bottom panel). This demonstrates that the detachment of IgE-stimulated BMMCs from FN at this time was not due simply to a reduction in the level of cell surface VLA-5.  B 80 60  in  <u  40  < 20  IgE SCF  in*.  i  IgE SCF  c  Figure 6.2: IgE alone stimulates the adhesion of BMMCs to FN via an  increase in the avidity of VLA-5. A) Adhesion of normal BMMCs to FN in response to assay medium alone (C), 1 ug/ml IgE, or 5ng/ml SCF in the absence (•) or presence of 400u.g/ml RGD-containing peptide (•) or 400ug/ml control RGE-containing peptide ( 0 ) for 15min. B) Adhesion of BMMCs to FN in response to assay medium alone (C), 0.5ug/ml IgE, or 5ng/ml SCF for 15min in the absence (•) or presence (•) of 40ug/ml anti-CD49e added 30min prior to stimulation. Results shown in (A & B) are the mean + SEM of triplicate determinations and similar results were obtained in 3 separate experiments. C) BMMCs were incubated on BSA (top and middle panels) or FN (bottom panel) coated wells for 1 hr (top panel) or 4hr (middle and bottom panels) in the absence (—) or presence (---) of 2ug/ml IgE. The cells were then stained with 1 ug/ml anti-CD49e Ab for 30min at 4°C and analyzed by FACs. The blackened area profiles were obtained with isotype control Ab.  106  6.2.3  IgE-induced adhesion of BMMCs to FN requires PI3K but not Erk or p38 To gain some insight into the intracellular pathways through which IgE alone triggers adhesion to  FN, we first explored the role of the PI3K pathway since it had been shown previously to be involved in SCF- and IgE+Ag-induced BMMC adhesion to FN (Kinashi et al., 1999; Serve et al., 1995; Vosseller et al., 1997). To do this, we utilized the PI3K inhibitor, LY294002, and found that it inhibited both SCF- and IgEinduced adhesion of BMMCs to FN (Figure 6.3A). Similar results were obtained with wortmannin (data not shown). It is worthy of note that we carried out these and subsequent inhibitor studies at concentrations of IgE and SCF that were suboptimal for adhesion to FN in order to maximize the sensitivity of the assay to potential inhibitors. As well, we titrated the various inhibitors used via Western analyses of BMMCs stimulated for 5min with 5pg/ml IgE or 10ng/ml SCF, so that we employed the lowest concentration that completely inhibited the target pathway. To explore the role of the PI3K pathway further, we compared the IgE-induced adhesion of SHIP+/+ and -/- BMMCs to FN since SHIP deficient BMMCs have been shown to have elevated PIP3 levels (i.e. a more active PI3K pathway) (Damen et al., 2001). Specifically, we carried out dose response (Figure 6.3B) and time course (Figure 6.3C) studies using SHIP+/+ and -/- BMMCs and found that the SHIP-/BMMCs displayed both more rapid and increased IgE-mediated adhesion to FN than the SHIP+/+ cells. This was also seen when these two cell types were stimulated with SCF (Figures 6.3B & C). Since the cell surface expression of VLA-5 was comparable on SHIP+/+ and -/- BMMCs (data not shown), the increased adhesion of SHIP-/- BMMCs suggested that SHIP plays a role in restraining the increase in the avidity of VLA-5.  We then asked whether the Erk or p38 pathways, which we found previously to be more active in IgE+Ag-induced SHIP-/- BMMCs (Kalesnikoff et al., 2002a), were playing a role in mediating the IgEinduced adhesion to FN. Specifically, we tested the Mek inhibitors, U0126 and PD98059, and the p38 MAPK inhibitor, SB203580, using concentrations that totally blocked IgE- or SCF-induced Erk and p38 phosphorylation (Kalesnikoff et al., 2002a). As can be seen in Figure 6.3D, these inhibitors had no effect on either IgE- or SCF-induced adhesion of BMMCs to FN.  6.2.4  IgE, but not SCF, requires entry of extracellular Ca to mediate BMMC adhesion to FN 2+  Since the binding of C a  2 +  to the extracellular domain of the VLA-5 dimer has been shown to be  essential for integrin-mediated adhesion induced via inside-out signaling (Houtman et al., 2001b; Leitinger etal., 2000), we next asked if extracellular C a  2 +  was also required for IgE-triggered BMMC adhesion to  107  50-  40-|  c  •§ 30<u |  20" 10-  0  25  50  100 _0  25_ LY (nM)  SCF  igE  IgE (u,g/ml)  SCF (ng/ml)  100 80H  c  I III I I  § 60-  3 3 5  30  60  Time (min)  4020-  80  U0126  PD  SB  SCF  "igE  Figure 6.3: IgE-induced adhesion of BMMCs to FN requires PI3K but not Erk or p38. A) Adhesion of normal BMMCs to FN in response to 1 ng/ml IgE or 5ng/ml SCF in the presence of indicated concentrations of LY294002 (LY). All wells contained the same level of DMSO (vehicle for LY). B) Adhesion of SHIP+/+ (•) and -/- (•) BMMCs to FN following a 60min exposure to the indicated concentrations of IgE or SCF. C) A time course of SHIP+/+ (•) and -/- (•) BMMC adhesion to FN in response to 1 ng/ml IgE and, for comparison, a 15min exposure to 1ng/ml SCF. D) Adhesion of normal BMMCs to FN in the presence of 1 ng/ml IgE (•) or 2ng/ml SCF (•) in the absence (C) or presence of 1uM U0126, 20nM PD98059 (PD), or 10nM SB203580 (SB). All wells contained the same level of DMSO (vehicle for the inhibitors). Results shown are the mean ± SEM of triplicate determinations. Background adhesion was subtracted from the values graphed in (A, C & D). Similar results were obtained in 4 (A), 5 (B), 3 (C & D) separate experiments.  FN. Specifically, IgE alone or SCF was added to BMMCs and adhesion to FN monitored in the presence and absence of C a  2 +  in the medium. As shown in Figure 6.4A, both agonists required extracellular C a  2 +  to  trigger adhesion (as did IgE+Ag, data not shown). This confirms and extends previous reports showing that extracellular C a  2 +  is critical for the SCF- and IgE+Ag-induced increase in the avidity of VLA-5 (Dastych and  Metcalfe, 1994; Houtman et al., 2001b; Wyczolkowska et al., 1994). However, complicating the interpretation of these findings is the fact that extracellular C a  2 +  can also enter the cell to enhance cPKC  activity as well as other, Ca -dependent processes in the cell (Houtman et al., 2001b). To determine 2+  whether the entry of extracellular C a , which we have shown previously occurs in response to IgE alone 2+  (Huber et al., 1998), is required for IgE-mediated adhesion, we tested the C a  2 +  channel blocker, 2-amino  ethoxydiphenyl borate (2-APB). This blocker (Gregory et al., 2001) has been shown previously, at the levels we used, to reduce IgE+Ag-induced C a  2 +  entry into RBL-2H3 cells (Ching et al., 2001). As shown in  108  Figure 6.4B, 2-APB effectively reduced extracellular C a  2 +  entry into cells stimulated with IgE alone or SCF  (or IgE+Ag, data not shown). However, while IgE-induced adhesion to FN (as well as IgE+Ag induced adhesion, data not shown) was reduced in the presence of 2-APB, SCF-induced adhesion was not (Figure 6.4C). Thus IgE, but not SCF, appears to require an influx of extracellular C a  2 +  to increase the avidity of  VLA-5 on BMMCs.  B  3.2 C O C O  2.8  s 2.4 n o  ro 2.0 1.6 0  100  200  300  400  0  100  Time (s)  200  300  400  Time (s)  80  .1 in  60  a>  % 40 <  3  cS 20 -|  0  IgE  Figure 6.4: IgE, but not SCF, requires the entry of extracellular C a to trigger BMMC adhesion to FN. A) Adhesion of normal BMMCs to FN in Ca -free media in response to medium alone (C), 1 ng/ml IgE, or 5ng/ml SCF for 30min with (•) or without (•) the addition of 1.8mM Ca . B) Intracellular Ca measurements in BMMCs stimulated with 5ug/ml IgE (—; left panel) or 50ng/ml SCF (—; right panel) alone, or in the presence of 50uM 2-APB (--) or 5mM EGTA (—). The 2-APB was pre-incubated with the cells for 30min while the EGTA was added immediately prior to the addition of IgE or SCF at 100s (I). C) Adhesion of BMMCs to FN in response to 1 ng/ml IgE or 5ng/ml SCF for 30min in the presence of vehicle control (•), 25nM (•), or 50nM (0) 2-APB added 30min prior to stimulation. Background adhesion was subtracted in (C). Results in (A & C) are the mean ± SEM of triplicate determinations. Similar results for (A, B & C) were obtained in at least 3 separate experiments.  II  2+  2+  2+  SCF  To confirm the role of extracellular C a  2 +  2+  entry in IgE-induced, but not SCF-induced, adhesion we  utilized BMMCs from Lyn+/+ and -/- mice. As shown in Figure 6.5A, we did not observe any C a  2 +  influx into  Lyn-/- BMMCs in response to IgE, SCF or IgE+Ag while responses were normal with BMMCs from wildtype littermate controls. This corroborates and expands on the very recent findings that IgE+Ag-induced C a  2 +  influx is dependent on the presence of Lyn in BMMCs (Parravicini et al., 2002) and that SCF-induced C a  2 +  entry is dependent on the recruitment of Lyn to tyrosine phosphorylated c-kit (Ueda et al., 2002). We then compared the ability of IgE, IgE+Ag and SCF to induce the adhesion of Lyn+/+ and -/- BMMCs to FN and found, as predicted from our 2-APB results, that IgE- and IgE+Ag-induced adhesion to FN was significantly lower with Lyn-/- than with Lyn+/+ BMMCs (Figure 6.5B). Also in support of our 2-APB findings, the impaired C a  2 +  mobilization observed in the absence of Lyn did not reduce SCF-induced adhesion of these  109  BMMCs to FN. In fact we reproducibly observed a slight increase in SCF-induced adhesion with the Lyn-/BMMCs (Figure 6.5B). It is worthy of note that the cell surface FcsRI and c-kit levels were similar on Lyn+/+ and -/- BMMCs (data not shown).  10  2  1 . 0  100  200  300  400  0  Time (s)  100  200  300  3  400  100  Time (s)  200  300  400  Time (s)  80 • c o  60-  -e  40-  <  _  20-  ft 0.5  i  i i  1 2 5 10 IgE (pg/ml)  20  0.5  1 2 5 10 S C F (ng/ml)  20  0.1  0.5 1 10 20 50 D N P - H S A (ng/ml)  Figure 6.5: Lyn-/- BMMCs, which do not show increased intracellular Ca with IgE, SCF, or IgE+DNP-HSA, display impaired adhesion to FN in response to IgE or IgE+DNP-HSA but not to SCF. A) Intracellular Ca measurements in Lyn+/+ (—) and -/- (-) BMMCs in response to 10ug/ml IgE (left panel), 100ng/ml SCF (middle panel), or lgE+20ng/ml DNP-HSA (right panel) injected at 100s (I). B) Adhesion of Lyn+/+ (•) and -/- (•) BMMCs to FN following a 60min exposure of the cells to the indicated concentrations of IgE, SCF, or IgE+DNP-HSA. Background adhesion was subtracted. Results shown are the mean ± SEM of triplicate determinations. Similar results were obtained in 2 (A) and 3 (B) separate experiments. 2+  2+  To further explore the signaling requirements for IgE-induced adhesion, we tested the phospholipase Cy (PLCy) inhibitor, U73122. PLCy cleaves PI-4.5-P2 to generate two second messengers; DAG, which binds and activates a subset of PKC family members (Toker et al., 1994), and IP3, which binds to IP3 receptors on ER and mitochondria to release intracellular stores of C a release in turn triggers the entry of extracellular C a  2 +  2 +  (Ching et al., 2001). This  and the subsequent activation of many C a 2+  dependent processes. As can be seen in Figure 6.6A, U73122 but not its inactive analog, U73343, markedly inhibited both IgE-induced and SCF-induced adhesion of BMMCs to FN. The 1u,M concentration used blocked both intracellular C a  2 +  release and subsequent extracellular C a  DAG and/or IP3 (i.e. extracellular C a  2 +  2 +  entry. This suggested that  entry) were required for IgE-induced adhesion and that DAG was  110  required for SCF-induced adhesion (since extracellular C a  2 +  entry had been ruled out as necessary in the  previous experiment). To probe the role of DAG and C a might be involved since both C a  2 +  2 +  further, we asked if one or more of the 12 isoforms of. PKC  and DAG are known activators of certain PKCs (Toker et al., 1994). To  test this we first added the pan-specific PKC inhibitor, Compound 3 (bisindolylmaleimide I) to our adhesion assay. Surprisingly, as shown in Figure 6.6B, no inhibition was observed with Compound 3 at a concentration which completely blocked IgE+Ag-induced phosphorylation of I K B at Ser (Kalesnikoff et al., 32  2002a). In fact a modest stimulation was consistently observed, both in the absence and presence of IgE alone or SCF. This is consistent with a previous report demonstrating that Compound 3 did not inhibit SCFinduced adhesion of BMMCs (Dastych et al., 1998). Thus, it is likely that one or more isoforms of PKC may actually play a negative role in regulating the avidity of VLA-5 on BMMCs for FN. We then tested the cPKC inhibitor, G66976, and found, consistent with the 2-APB results described above, that it potently inhibited IgE-induced (and IgE+Ag-induced) adhesion but had no effect on SCF-induced adhesion to FN (Figure 6.6C). This suggested that a Ca -dependent PKC may positively regulate IgE- but not SCF-induced 2+  adhesion. Alternatively, G66976 may be acting through a different kinase to inhibit IgE-induced adhesion.  A  B 80  80-r  c o  60-  in £  40-  C  IgE  SCF  Figure 6:6: IgE, but not SCF, may utilize a Ca dependent PKC to trigger BMMC adhesion to FN. A) Adhesion of normal BMMCs to FN in response to 1 ug/ml IgE or 5ng/ml SCF with vehicle control (•), 1uM U73122 (•), or 1uM U73343 (FJ2) added 30min prior to stimulation. B) Adhesion of BMMCs to FN in response to media alone (C), 1 ug/ml IgE or 5ng/ml SCF in the presence of vehicle control (•) or 25uM Compound 3 (•) added 30min prior to stimulation. C) Adhesion of BMMCs to FN in response to 1 ug/ml IgE or 5ng/ml SCF in the presence of vehicle control (•) or 1uM G66976 (•) added 30min prior to stimulation. All adhesion assays were 30min and the results are the mean ± SEM of triplicate determinations. Similar results were obtained in 4 (A & B), and 3 (C) separate experiments. Background was subtracted in (A &C). 2+  IgE  SCF  111  6.2.5  VLA-5 activation acts together with IgE to prolong intracellular signaling and enhance cytokine production and BMMC survival Engagement of integrins by their ligands is known to activate various signaling pathways and this is  referred to as "outside-in" signaling. These "outside-in" signals have been shown to modulate signals coming in from other receptors and alter biological responses triggered by these other receptors (Miranti and Brugge, 2002; Schwartz and Ginsberg, 2002; Yamada and Even-Ram, 2002). To investigate the downstream ramifications of IgE-induced adhesion of BMMCs to FN, we first asked if there were any apparent differences in intracellular signaling events when cells were stimulated with IgE alone versus IgE+FN (i.e. do IgE-induced adherent BMMCs display a different signaling pattern because of input from the activated VLA-5?). To test this we first compared the overall tyrosine phosphorylation pattern of BMMCs in suspension versus attached to FN, at different exposure times to IgE. As can be seen in Figure 6.7A (top panel), tyrosine phosphorylation was more intense and substantially prolonged with FN-adherent cells. We then carried out similar time course studies to specifically examine the effect of IgE alone versus IgE+FN on Erk1/2 phosphorylation and found that phosphorylation was both prolonged and more intense when the cells were attached to FN (Figure 6.7A, middle panel). A reprobe with anti-Erkl Abs demonstrated equal loading (Figure 6.7A, bottom panel).  To look at the biological ramifications of IgE-induced adhesion to FN we first asked if IgE alone, which does not trigger detectable degranulation in suspension cultures of BMMCs (Kalesnikoff et al., 2001), might now trigger degranulation of FN-adhered cells. However, no significant degranulation was observed (data not shown). We then compared the levels of proinflammatory cytokines secreted into the medium from non-adherent versus FN-adherent IgE-treated BMMCs. For these experiments, BMMCs were stimulated with 5|ag/ml IgE for 3hr in the presence of 15JJ, polystyrene beads previously coated with BSA or FN (Houtman 2000). IL-6 and TNFoc ELISAs revealed that IgE-induced activation of VLA-5 substantially increased the levels of these cytokines (Figure 6.7B). Since we had previously shown that the production of autocrine acting cytokines may contribute to IgE-mediated enhancement of BMMC survival (Kalesnikoff et al., 2001) and we observed that FN binding enhanced IgE-induced cytokine production, we next performed survival studies with IgE in the presence and absence of FN. Two concentrations of IgE were initially tested, 2Lig/ml and 5Lig/ml, and viable cells were counted after four days. We observed, as seen in Figure 6.7C (left panel), that adhesion to FN significantly enhanced this IgE-mediated survival, and this enhancement appeared to be greatest at  112  suboptimal concentrations of IgE. Even after one week in the presence of 2u,g/ml IgE alone, IgE-induced binding to FN consistently enhanced BMMC survival by about two-fold (Figure 6.7C, right panel).  IgE 0  IgE + FN  B  15 30 60 90 0 15 30 60 90  500  ^ 12 " 10  "5>  0  1  2.5  igE (ug/m I)  0  1 2.5 5 IgE (ug/ml)  W: 4G10 IgE + FN  !SJL  0 15 30 60 90 120240 0 15 30 60 90 120240 (min  W: anti-P-Erk1/2  0 1 2 3 4 5 6  IgE (ng/ml)  0  2  IgE (ng/ml)  W: anti-Erkl  Figure 6.7: FN binding acts to enhance IgE-induced intracellular signaling events, cytokine production, and survival. A ) Total cell lysates (50ug as assessed by BCA assays) from IgE-stimulated suspension and FN-adhered BMMCs were subjected to Western analysis using anti-phosphotyrosine (4G10) Abs (top panel) or anti-phospho-specific Erk1/2 Abs (middle panel) and reprobed with anti-Erkl Abs (bottom panel) to demonstrate equal loading. Blots are representative of 3 separate experiments. B) BMMCs were stimulated for 3hr at the indicated concentrations of IgE in the presence of 15u polystyrene beads coated with BSA (•) or FN (•). IL-6 (left panel) and TNF-a (right panel) levels in the supernatants were detected by ELISA. C ) BMMCs were plated at 5x10cells/ml in IMDM + 0.1% BSA ± IgE at 2 and 5u.g/ml in FN (•) or BSA (•) coated wells. On day 4, viable cells were counted by trypan blue exclusion (left panel). In the right panel, BMMCs were set up as above ± 2ug/ml IgE and viable cells counted on day 7. Data points are the mean ± SEM of 6 (B), 2 (C; left panel), and 3 (C; right panel) determinations. Similar results for (B & C) were obtained in at least 3 separate experiments. 5  6.3  DISCUSSION  Mast cells congregate in connective tissue and are especially numerous beneath the epithelial surfaces of the skin and in the respiratory, gastrointestinal and genito-urinary tracts. This tissue localization, which plays a critical role in enabling mast cells to respond rapidly and vigorously to invading parasites, bacteria and environmental antigens, is thought to involve the binding of mast cell progenitors via their integrins to FN and other components of connective tissue. One approach, therefore, to modify allergic and anti-microbial responses would be to modulate tissue localization and to do this it is first necessary to understand the various factors that regulate this process. We demonstrate herein that IgE alone (at  113  approximately 2^ig/ml), is capable of maximally triggering adhesion of both BMMCs and CTMCs to FN and does so to the same extent as optimal levels of SCF (approximately 10ng/ml) or IgE+Ag (5ng/ml of Ag). Interestingly, these levels of SCF and Ag are substantially lower than those required to induce BMMC proliferation and degranulation, respectively, confirming earlier reports (Dastych and Metcalfe, 1994; Kinashi and Springer, 1994; Wyczolkowska et al., 1994) and may suggest that, in vivo, low levels of these stimuli play an important role in mast cell recruitment. Relevant to this, the concentration of SCF in normal human serum is approximately 3ng/ml (Langley et al., 1993) while that of IgE ranges from less than 0.1u.g/ml in normal individuals to more than 30Lig/ml in highly atopic individuals (Bennich and Johansson, 1970). Thus it is possible that IgE alone plays a significant role in mediating recruitment, adhesion and subsequent cytokine production and survival of mast cells during infections or allergic reactions. We also show that IgE-induced binding to FN, like that induced by SCF and IgE+Ag, is mediated by inside-out signaling that increases the avidity of the integrin VLA-5. Interestingly, this adhesion is transient with all three stimuli, consistent with earlier studies with IgE+Ag (Wyczolkowska et al., 1994) and SCF (Kinashi and Springer, 1994), but is significantly slower and more prolonged with IgE than with SCF or IgE+Ag. This could be due in part to the slow on rate of IgE (Kulczycki et al., 1974) and the slow internalization rate of uncrosslinked IgE/FcsRI (Mao et al., 1993), respectively. Since cell surface levels of VLA-5 remained constant for 1 and 4hr following stimulation with IgE or SCF (in the presence or absence of FN), this suggests that the induced adhesion and subsequent release are a result of avidity modulation of VLA-5 via inside-out signaling. Taken together, our inside-out signaling studies suggest that the activation of both the PI3K pathway and the PLCy-generated IP3 pathway (and subsequent draining of intracellular C a extracellular C a  2 +  2 +  stores,  entry and possibly activation of one or more cPKCs) are critical to IgE-mediated (and  IgE+Ag-mediated) adhesion. SCF-mediated adhesion, on the other hand, appears to be independent of extracellular C a  2 +  entry but dependent on the activation of both the PI3K pathway and PLCy-generated  DAG (see model in Figure 6.8). These results suggest that SCF-induced adhesion may be positively regulated by one or more Ca -independent, DAG-dependent PKCs. Toker et al. (1994) have shown that 2+  PKCs is most activated by PIP3 in vitro, therefore, this Ca -independent, DAG-dependent isoform might be 2+  a good candidate for the PKC involved in SCF-induced adhesion. Alternatively, SCF-induced production of DAG may be required to activate other pathways, such as chimaerins, RasGRPs, and Munc13 isoforms (Kazanietz, 2002), to increase VLA-5 avidity. The role of PKCs in mediating IgE- and SCF-induced adhesion events remains controversial; for example, as reported by Dastych et al. (1998), we did not  114  observe any inhibition of adhesion in the presence of Compound 3, whereas Vosseller et al. (1997) reported that the PKC inhibitor, Calphostin C, inhibits SCF-induced adhesion of BMMCs. Complicating matters further, we observed that the cPKC inhibitor, G66976, inhibited IgE-, but not SCF-, induced adhesion. Based on our Compound 3 results, this finding suggests that one or more PKC isoform may play a negative role in regulating VLA-5 avidity, thus, masking the effects of Compound 3 inhibition on the postive regulatory PKC isoforms, or that G66976 is exerting its effects by acting though a different kinase. The use of BMMCs from various PKC knockout mice remains essential to define the roles of the different PKC isoforms on IgE- and SCF-induced adhesion events.  Figure 6.8: A model of IgE-stimulated adhesion to FN. IgE binding to the FceRI on BMMCs may reduce an inherent repulsion between neighboring FceRI molecules, allowing them to aggregate at a low frequency. This results in a relatively low (compared to IgE+Ag) but prolonged signal that activates, among other pathways, the PI3K and PLCy pathways. These two pathways play a critical role in IgE- and IgE+Ag-induced adhesion to FN by inducing the entry of extracellular Ca and the subsequent activation of a cPKC or other Ca -dependent pathway. SCF-induced adhesion of BMMCS to FN, on the other hand, appears to require the stimulation of the PI3K and PLCy pathways and the subsequent activation of a DAG-dependent, Ca -independent pathway. 2+  2+  2+  Our results are consistent with earlier studies showing that the PI3K pathway plays a critical role in SCF- and IgE+Ag-induced adhesion of BMMCs to FN (Dastych et al., 1998; Kinashi and Springer, 1994; Serve et al., 1995). Intriguingly, Kinashi et al. (1999) found that BMMC adhesion to FN could be triggered via a constitutively active PI3K but not a constitutively active Akt/PKB, suggesting that elevated PIP3 but not the downstream Akt pathway is critical to inside-out signaling. Although Vosseller et al. (1997) also concluded that a Ca -independent, PIP3-dependent PKC might be involved in SCF-mediated adhesion to 2+  115  FN, they did not, in contrast to our findings, observe an increase in C a  2 +  entry in response to SCF. In  keeping with our results, however, Ueda et al. (2002) recently reported that SCF does induce C a  2 +  entry  and that Lyn is a key mediator of this influx. One caveat with their studies, however, is that they were carried out with c-kit transfected Ba/F3 cells rather than primary BMMCs. Also relevant to our model, Lorentz et al. (2002) very recently reported that SCF-stimulated adhesion of human intestinal mucosa derived mast cells to FN is blocked with 100nM wortmannin or 20\M apigenin but not with 2|aM G66976. Apart from their results with apigenin, a putative Erk pathway inhibitor, this is in agreement with our SCF findings. Our results with the Mek inhibitors, PD98059 and U0126, suggest that IgE-, SCF- and IgE+Aginduced adhesion does not require the Erk pathway. Moreover, we found that apigenin was not a specific Erk pathway inhibitor, in fact, it inhibited many pathways at 20LJ.M, including the SCF-induced tyrosine phosphorylation of c-kit, while at 5^iM it inhibited neither Erk phosphorylation nor SCF-induced adhesion (data not shown). The role that extracellular C a  2 +  plays in mast cell adhesion is complicated because it not only binds  directly to the extracellular domains of the integrins to modulate heterodimer formation and affinity for FN and other ligands, but it can also enter the mast cell to activate various intracellular pathways (Ching et al., 2001; Houtman et al., 2001a; Houtman et al., 2001b; Leitinger et al., 2000). As far as the latter is concerned, it has been known for some time that intracellular C a  2 +  is involved in the inside-out signaling  that leads to integrin-mediated adhesion and studies with LFA-1 mediated adhesion to ICAM-1 suggest, perhaps, a general role for intracellular C a  2 +  in increasing the avidity of integrins by activating the C a 2+  dependent protease, calpain which then cleaves the attachment between the integrin and the cytoskeleton (Leitinger et al., 2000). This allows the integrin to move in the membrane and gather in clusters which may or may not become tethered to the cytoskeleton again (Leitinger et al., 2000). One major question that arises from our data is how SCF, which does not appear to require entry of extracellular C a  2 +  to increase  adhesion, mediates integrin clustering. As far as outside-in signaling is concerned, our observation that tyrosine phosphorylation events are prolonged and of greater intensity when VLA-5 activation acts in concert with IgE stimulation is reminiscent of earlier studies by Hamawy et al. (1993) who showed that FcsRI aggregation-induced adhesion of RBL-2H3 cells to FN increased the tyrosine phosphorylation of FAK. Furthermore, the enhanced proinflammatory cytokine production that we observed with IgE-induced adhesion is somewhat consistent with earlier studies showing that adhesion of mast cells to FN via VLA-5 enhances IgE+Aginduced degranulation (Ra et al., 1994; Wyczolkowska et al., 1994) and release of proinflammatory  116  cytokines (Ra et al., 1994). Interestingly, the enhanced IgE-induced survival we observe with FN-bound B M M C s may further explain, at least in part, the elevated mast cell numbers seen in atopic individuals with elevated plasma IgE levels. Related to this, R a et al. (1994) found that the IgE+Ag-induced adhesion of rat and mouse cultured mast cells to FN led to enhanced survival (perhaps via the autocrine action of IL-3) than that observed with IgE+Ag alone.  Our findings increase the repertoire of biological effects elicited by IgE alone and raise the possibility that IgE, in the absence of Ag, may help in vivo to concentrate mast cells at sites of inflammation and promote, in concert with F N , proinflammatory cytokine release and mast cell survival.  117  Chapter 7  SUMMARY AND PERSPECTIVES  In summary, we have shown in chapter 3 that both total bone marrow cells and Sca-1 -Lin- purified bone marrow cells from SHIP-/- mice become FcsRI positive faster than their wildtype counterparts when cultured in IL-3. Coincident with this enhanced rate of differentiation we find that it is much more difficult to obtain SHIP-/- than +/+ BMMCs, and this may be explained, at least in part, by the impaired proliferation we observe in SHIP-/- cells in the face of enhanced differentiation. For example, during the first 4 weeks in culture, the proliferation of SHIP-/- suspension cells (which contain mast cell progenitors and/or mast cells) in culture is quite low, and these cells have to be concentrated often. Furthermore, even when cultured in IL-3 alone, conditions that typically favor BMMC development, macrophages are the predominant cell type in SHIP-/- bone marrow cultures for the first 2-4 weeks in culture. Thus, this difficulty generating SHIP-/BMMCs may also reflect the preferential development of -/- bone marrow cells towards the macrophage lineage or perhaps the production of molecules by macrophages that inhibit mast cell progenitor proliferation. Related to this, mast cell numbers are relatively normal in SHIP-/- mice (with the exception of the foot pads where preliminary results indicate elevated mast cell numbers; Dr. Cheryl Helgason, personal communication), while macrophage numbers are significantly elevated. In fact, many of the characteristics of SHIP-/- mice, such as shortened life span, can be attributed, at least in part, to the myeloproliferative disorder we observe in these mice, and the resulting consolidation of the lungs caused by infiltration of large numbers of macrophages and neutrophils (Helgason et al., 1998). In future studies, it would be interesting to look at the levels/activity of certain TFs known to regulate myeloid cell differentiation; for example, PU.1, is a key regulator of macrophage differentiation (Anderson et al., 1999; Yamada et al., 2001). Preliminary data from our laboratory suggest that PU.1 levels and activity are elevated in SHIP-/bone marrow cells in culture (Michael J. Rauh, personal communication).  Elevated PU.1 levels/activity  favor macrophage development, whereas medium or low activity is found in developing mast cells and erythrocytes (Yamada et al., 2001; Cantor and Orkin, 2002), respectively, offering one potential mechanism for enhanced macrophage development in the absence of SHIP. Our laboratory is expanding these studies to examine the role of SHIP in regulating proliferation/differentiation decisions within all hemopoietic lineages. Similar to our results with mast cells, both cultured bone marrow cells and Sca-1 Lin- purified bone marrow cells express mature macrophage +  markers (i.e. Mac-1) faster than their wildtype counterparts following culture in complete medium or M-CSF  118  (Michael J. Rauh, personal communication). Interestingly, however, the presence or absence of SHIP does not appear to have any effect on erythropoiesis, perhaps because SHIP expression is turned off during normal erythropoiesis in mice (Michael R. Hughes, personal communication). Similar results have been obtained when differentiation  has been initiated with SHIP-/- ES cells (Dr. Laura Sly, personal  communication). Our preliminary findings suggest that SHIP, likely through its ability to dephosphorylate the PI3K product, PIP3, negatively regulates myeloid cell differentiation. As indicated in chapter 3, we intend to infect bone marrow and/or ES cells with various SHIP mutants (Damen et al., 2001) and monitor subsequent myeloid cell differentiation/proliferation decisions. To further verify the role of the PI3K pathway in myeloid cell development, we would like to compare the proliferation and differentiation of SHIP+/+ bone marrow cells and ES cells infected with a constitutively active PI3K construct (which consists of a p110 catalytic subunit fused to a CAAX sequence to keep this protein embedded in the membrane) versus a dominant-negative kinase dead p110 construct.  As described in chapter 4, mature SHIP+/+ and -/- BMMCs were utilized to study the role of SHIP in traditional IgE+Ag-induced mast cell activation. Studies conducted by Dr. Michael Huber, a postdoctoral fellow in our laboratory, revealed that SHIP acts as a gatekeeper of mast cell degranulation by hydrolyzing the PI3K generated second messenger PIP3 (Huber et al„ 1998; Huber et al., 1999). Although we also observed increased LTC4 production in response to IgE+Ag in SHIP-/- BMMCs (chapter 4), we have not yet characterized SHIP'S role in the regulation of AA metabolism. Based on current literature, we predict that this elevated L T C 4 production is due, at least in part, to the elevated N F K B activity (since PLA2 activity is regulated by N F K B at the level of transcription (Murakami and Kudo, 2002)), the elevated Erk and p38 activity (since both these proteins have been shown to phosphorylate and activate PLA2 in human eosinophils (Zhu et al., 2001)) and the elevated C a  2 +  flux (since PLA2 binds to Ca (Murakami and Kudo, 2+  2002)) we observe in SHIP-/- BMMCs (Huber et al., 1998; Kalesnikoff et al., 2002a). Furthermore, it was recently published that IgE+Ag-induced LTC4 production is positively regulated by PI3K activity in human basophils (Miura et al., 2001). The role of SHIP in the regulation of IgE+Ag-induced cytokine production was the focus of the results presented in chapter 4. More specifically, we investigated how SHIP negatively regulates IL-6 production, since a great deal is already known about the regulation of this cytokine in mast cells (Marquardt and Walker, 2000). IL-6, which was originally identified as a differentiation factor for B cells, is a multifunctional cytokine that regulates hemopoiesis, immune responses, acute phase responses, and inflammation (Ishihara and Hirano, 2002). We found that the repression of IL-6 mRNA and protein production in SHIP+/+ BMMCs requires the enzymatic activity of SHIP, since SHIP-/- BMMCs expressing 119  wildtype, but not phosphatase-deficient, SHIP revert the IgE+Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP+/+ BMMCs (Kalesnikoff et al., 2002a). Dysregulation of IL-6 production has been implicated in the pathology of several diseases, such as rheumatoid arthritis, osteoporosis, and psoriasis, as well as several experimentally induced autoimmune diseases, such as Ag-induced arthritis (Ishihara and Hirano, 2002). We assessed total serum IL-6 levels in SHIP-/- mice, and found they are significantly elevated compared to those in wildtype mice (Figure 4C in (Takeshita et al., 2002)). IL-6 is known to have important effects on the formation and function of osteoclasts (the bone resorbing cells of the myeloid lineage; Figure 1.1), and, in collaboration with Dr. Patrick Ross' laboratory (Washington University, St. Louis, MO), we have recently shown that SHIP-/- mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts (Takeshita et al., 2002). The two-fold increase in osteoclast numbers in these mice likely reflects the elevated serum IL-6 levels and the increased numbers of osteoclast precursors (i.e. macrophages) in these mice.  Since we found that both IL-6 mRNA and protein levels are elevated in IgE+Ag-activated SHIP-/BMMCs, we focused our studies on the role of SHIP in the regulation of IL-6 mRNA synthesis. We initiated our studies by looking at N F K B activity, since this TF is known to regulate IL-6 production in mast cells (Marquardt and Walker, 2000), and found that IKB phosphorylation/degradation, N F K B translocation to the nucleus, DNA binding, and transactivation are much higher in SHIP-/- cells (Kalesnikoff et al., 2002a). In addition, inhibitor studies revealed that a number of pathways that are elevated in the absence of SHIP regulate IgE+Ag-induced N F K B activity. The PI3K/PKB and PKC pathways positively regulate N F K B nuclear translocation and DNA binding, likely through the regulation of IKK activity, whereas Erk and p38 positively regulate N F K B transactivation (Kalesnikoff et al., 2002a). It would now be interesting to study the role of SHIP in the regulation of other TFs involved in IL-6 mRNA synthesis (e.g. the Ca -dependent 2+  nuclear factor IL-6 (NFIL-6)), its role in the regulation of IL-6 protein secretion, and its role in the production of other mast cell pro-inflammatory, vasoactivate cytokines. The important role of N F K B in the regulation of inflammation and the initiation and coordination of innate and adaptive immune responses is well documented in the literature (Caamano and Hunter, 2002; Ghosh and Karin, 2002). Recent evidence also indicates that N F K B and the pathways involved in its activation are important modulators of tumor development (Karin et al., 2002). Thus, our results expand the current state of knowledge on the regulation of this important TF and may have additional widespread implications in the future. PIP3 is the well documented lipid substrate of SHIP in vivo (Krystal, 2000; Majerus et al., 1999); however, SHIP is reported to display 5'-phosphatase activity towards the soluble IP4 in in vitro phosphatase 120  assays (Damen et al., 1996). Intriguingly, York et al. (1999) recently reported that the generation of inositol 1,2,3,4,5,6-hexakisphosphate (IPs) from PI-4.5-P2 is important for efficient mRNA export from the nucleus in yeast, and this mechanism is thought to be conserved across species, i.e. in mice and humans (Feng et al., 2001; Ives et al., 2000). Because IP4 is a precursor for IP6 generation, we hypothesize that, if SHIP can utilize  IP4  as a substrate  in vivo, \Pe  levels will be elevated in SHIP-/- compared to +/+ BMMCs following  stimulation. Thus, we have initiated a collaborative study with Dr. Christina Mitchell (Monash University, Clayton, Victoria, Australia) to examine the levels of IP6 in SHIP+/+ and -/- BMMCs, and preliminary results indicate that IP6 levels are higher in the absence of SHIP. These results suggest that mRNA export may be more efficient in SHIP-/- BMMCs and may also contribute to the elevated cytokine production we observe in the absence of SHIP.  In summary, all the endpoints examined in our laboratory (i.e. degranulation, AA metabolism and cytokine production) indicate that SHIP functions as a critical negative regulator of mast cell activation. In keeping with our findings, it was recently published that the hyper-releasability of basophils from a subpopulation of highly allergic donors is associated with lower than normal levels of SHIP protein (but not mRNA). Interestingly, SHIP protein levels were not decreased in these patients' lymphocytes or monocytes. These results implicate SHIP as an important negative regulator of secretion in human basophils (Vonakis et al., 2001; MacDonald and Vonakis, 2002; Rauh and Krystal, 2002;). We have initiated a collaborative study with Susan MacDonald (Johns Hopkins Asthma and Allergy Center, Baltimore, MD) to study this phenomenon further in murine and human cells and believe our findings may influence the design of future therapies for allergies.  Although it is generally accepted that mast cell activation requires crosslinking of IgE bound FceRls with multivalent Ag (Beaven and Metzger, 1993; Kinet, 1999), our finding that SHIP-/- BMMCs degranulate in response to IgE alone (Huber et al., 1998) set the stage to challenge this paradigm. In chapter 5, we demonstrated that mlgE, in the absence of Ag, stimulates multiple phosphorylation events in normal BMMCs. While mlgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE+Ag. Moreover, mlgE acts as a survival factor to prevent the apoptosis of cytokine-deprived BMMCs, likely by maintaining BCI-XL levels and inducing the production of autocrineacting cytokines. The data presented in chapter 5 was published back to back with a second group that also observed enhanced BMMC survival in the presence of mlgE (Asai et al., 2001). Our studies are complementary in many respects. For example, we both agree that mlgE acts as a survival factor, not a mitogen, to protect BMMCs from growth factor deprivation-induced apoptosis. Furthermore, we both agree that mlgE alone acts through the FcsRI to elicit these effects; as indicated earlier, Asai et al. (2001) had 121  access to an FcsRI a chain -/- mouse and did not observe an IgE-induced enhancement of survival in BMMCs derived from these mice. Along with these similarities, numerous differences exist between our papers. For example, Asai et al. (2001) did not observe any signaling in BMMCs stimulated with IgE alone. However, they only looked at very late time points, i.e. 3, 6, 9 and 24hr, and we have shown that IgE-induced signaling events return to baseline levels 3-4hr post-stimulation (Figure 5.3B, chapter 5). We have since obtained the Liu anti-DNP IgE (Liu et al., 1980) used by Asai et al. and find that this IgE is capable of inducing signaling events (assessed by Western blot analysis using anti-P-Erk1/2 and anti-P-PKB (Ser ) Abs and calcium flux; 473  Appendix I), albeit to lower levels than those observed following stimulation with the SPE-7 IgE used in our studies. Furthermore, Asai et al. (2001) did not observe any cytokine production in response to the Liu IgE alone and reported that the IgE-induced survival of BMMCs requires the continuous presence of the Liu IgE (Asai et al., 2001). Our preliminary studies indicate that cytokine production is quite low when BMMCs are stimulated with the Liu IgE. Intruigingly, in keeping with decreased signaling and cytokine production, we observe significantly less survival of growth factor-deprived BMMCs cultured with the Liu IgE compared to the SPE-7 IgE. Although we have not yet compared the levels of the anti-apoptotic proteins  BCI-XL  and Bcl-  2 in response to these two IgEs, studies with these (and additional IgE molecules) are currently underway in our laboratory. Preliminary results indicate a positive correlation between the ability of various IgEs to signal and their ability to enhance mast cell survival.  Mast cell numbers undergo dramatic increases during the course of Tri2-mediated immune responses and these numbers typically return to baseline levels at the end of the response (Galli et al., 1999; Metcalfe et al., 1997). Since the concentrations of IgE that promote survival in our BMMC assay have been reported in the serum of mice and humans exposed to parasites and allergens (Bennich and Johansson, 1970; Matsuda et al., 1997), IgE may be responsible, at least in part, for the elevated numbers of mast cells observed under these conditions. Furthermore, numerous studies indicate that IgE binding to FcsRI enhances FcsRI expression on the surface of mast cells and basophils (Borkowski et al., 2001; Kubo et al., 2001; MacGlashan et al., 1999; Yamaguchi et al., 1997). This IgE-mediated upregulation of FcsRI expression serves to enhance the effector and immunoregulatory functions of these cells, since elevated FcsRI levels enable mast cells to bind more IgEs and be activated at lower concentrations of a given Ag (Galli, 2000; Yamaguchi et al., 1997; Yamaguchi et al., 1999). We propose that the ability of IgE to enhance mast cell survival further contributes to the IgE-mediated amplification of the allergic response (Asai et al.,  122  2001; Kalesnikoff et al., 2001). Finally, the IgE-induced production of cytokines that we observe may contribute to, or prime the body for, a subsequent, Ag-induced allergic response. These studies represent the first look into the function and priming ability of IgE alone. However, besides the aforementioned dependence on the FcsRI, neither our group nor Asai et al. (2001) has elucidated the mechanism by which IgE alone initiates these effects. Based on microarray data, Asai et al. (2001) proposed that IgE alone activates signaling pathways distinct from those initiated by IgE+Ag. Conversely, we observed that many of the same pathways are activated in response to IgE alone and IgE+Ag, albeit with different kinetics (Figure 5.2 & 5.3, chapter 5). To further understand the signaling events initiated in response to IgE alone, we are currently studying IgE- versus IgE+Ag-induced signaling events in Lyn+/+ and -/- BMMCs (a kind gift from Dr. Janet M. Oliver, University of New Mexico, Albuquerque, NM), since Lyn is one of the key players in the initiation of signaling downstream of the FcsRI. Based on the results presented in chapter 5, we propose that mlgE binding to the FcsRI reduces, to a modest extent given its EC50 of 1 pg/ml, an inherent repulsion between neighboring receptors, allowing them to slowly form small clusters and trigger a low but prolonged signaling of various intracellular pathways. Alternatively, IgE binding may induce a conformational change in the FcsRI to initiate signaling events. To differentiate between these two models, we have initiated a collaborative study with Dr. Bridget S. Wilson (University of New Mexico, Albuquerque, NM). Her group has developed a technique, using transmission electron microscopy and gold particle-coupled Abs, to observe early FcsRI signaling events at the level of the plasma membrane (Wilson et al., 2002; Wilson et al., 2000). Thus, we are utilizing this technique to compare the aggregation of FcsRIs and the recruitment of various signaling molecules in response to IgE alone versus IgE+Ag.  Because the aforementioned studies were all conducted in vitro on cells derived from mice (Asai et al., 2001; Kalesnikoff et al., 2001), the most important outstanding issue at this time remains whether IgE alone can enhance mast cell survival in vivo in mice and humans. Interestingly, IgE-/- mice express comparable levels of mast cells to lgE+/+ mice, suggesting that baseline levels of IgE do not contribute significantly to mast cell numbers; however, IgE-mediated enhancement of mast cell survival may only contribute to increased mast cell numbers during times of elevated IgE levels, e.g. following exposure to parasites or allergens. Treatment of IgE-/- mice with IgE is reported to increase FcsRI expression levels on mast cells and basophils (Yamaguchi et al., 1997; Yano et al., 1997) and to cause a statistically significant, albeit modest, increase in circulating basophil numbers (Lantz etal., 1997). In human cells, there is one preliminary report indicating that human IgE can induce chemokine production from human umbilical cord 123 '  blood-derived mast cells in vitro (Gilchrest, 2002); however, the in vivo relevance in humans remains to be determined. One of the current therapies being used to treat moderate to severe cases of allergic asthma and rhinitis is omalizumab (Genentech Inc./Novartis AG/Tanox), a humanized monoclonal anti-lgE Ab. Treatment with this Ab causes a marked reduction in circulating free IgE levels (to 1% of pretreatment levels) and it has been shown to reduce symptoms and decrease the need for other medications, such as inhaled corticosteroids, especially in patients at high risk of serious asthma-related morbidity (Babu and Holgate, 2002; Chung, 2002; Johansson et al., 2002; MacGlashan et al., 1997b). These results can be explained, at least in part, by the significant down regulation of FcsRI observed on human basophils. However, it remains to be determined whether anti-lgE therapy is also effective because it contributes to decreased mast cell and basophil survival. In chapter 6, we looked at the ability of IgE alone to induce a biological effect distinct from survival; i.e. adhesion. We found that IgE alone induces the adhesion of BMMCs and CTMCs to the connective tissue component, FN. As reported for two documented inducers of mast cell adhesion, SCF and IgE+Ag (Kinashi and Springer, 1994; Ra et al., 1994), this IgE-induced adhesion is mediated via PI3K- and PLCydependent increases in VLA-5 avidity. Furthermore, we found that both IgE- and IgE+Ag-induced adhesion require the entry of extracelluar C a  2 +  and a Ca -dependent PKC, unlike SCF-induced adhesion. 2+  Intriguingly, IgE-induced FN binding acts synergistically with IgE to prolong intracellular phosphorylation events in BMMCs and to enhance inflammatory cytokine production and mast cell survival. Preliminary results indicate that the Liu IgE used by Asai et al. (2001) also induces BMMC adhesion to FN in normal BMMCs, but not Lyn-/- BMMCs, and this adhesion can be blocked using intracellular signaling pathway inhibitors (Appendix I). Adhesion is documented to regulate the proliferation, differentiation, survival, priming and activation of mast cells. Thus, in addition to enhancing mast cell survival and numbers, IgEmediated adhesion to FN may increase the subsequent responsiveness of these cells. In conclusion, these studies have revealed some new and important insights into the regulation of mast cell development, survival, priming and activation. Moreover, our results have increased our understanding of the role of SHIP in the regulation of hemopoietic cell proliferation, differentiation, and activation. Furthermore, they suggest that the continued exploration of this intriguing molecule will prove invaluable for a comprehensive understanding of the immune system as a whole. Our results with IgE alone provide a starting point for future studies to examine the ability of IgE to enhance mast cell survival and contribute to the amplification of allergic responses.  124  REFERENCES  Abraham, S.N. and Malaviya, R. 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J Immunol, 167,461-8.  150  APPENDIX I  A  SPE-7  Liu  aEp26  Liu  0 5 15 60 5 15 60 5 15 60 min  S mlgE  0 2 5 10 20 5 5  OT5SS ; MZ> W: anti-P-Erk  W: anti-P-Erk  - . - - * W: anti-FceRI B chain  B  W: anti-FceRI B chain  10 8  —  SPE-7  8  .o  <0  4  or  0  100  200 300 400  Time (s)  80-  40 L  .11.1111 ni 0  1 10 SPE-7  1  10 20 Liu  2 5 ug/ml aEpo26  i  10ug/ml Liu  1  1ug/ml SPE-7  1  2(ig/ml aEpo26  A) BMMCs were starved overnight in IMDM + 10%FCS, then stimulated with 5ng/ml anti-DNP IgE (SPE-7 or Liu) or anti-Epo26 IgE for the indicated times (left panels). Alternatively, starved BMMCs were stimulated for 10min with anti-DNP IgE (Liu, SPE-7 (S) or monomeric SPE-7 (mlgE)) at the indicated concentrations (right panels). Total cell lysates were separated by SDS-PAGE and subjected to Western blot analysis using anti-phospho-Erk1/2 antibodies and reprobed with anti-FcsRI p chain antibodies to show equal loading. B) Intracellular Ca measurements in normal BMMCs in response to 10ug/ml IgE (SPE-7 or Liu) injected at 100s (1). C) Adhesion of normal BMMCs to FN following a 60min adhesion assay with IgE alone (SPE-7, Liu, or anti-Epo26) at the indicated concentrations (left panel). Alternatively, BMMCs were incubated with DMSO (vehicle control; •) or 50mM LY294002 (•) for 30min at 37°C prior to the addition of the cells to FN coated wells ± IgE (Liu (L), SPE-7 (S) or anti-Epo26) at the indicated concentrations for 30min at 37°C (right panel). Results in (C) are the mean ± SEM of triplicate determinations. Similar results were obtained in 3 (A), 2 (B), and 2 (C) separate experiments. 2+  A  

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