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The role of SHIP in macrophage differentiation and function Rauh, Michael J. 2007

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THE ROLE OF SHIP IN MACROPHAGE DIFFERENTIATION AND FUNCTION by MICHAEL J. RAUH B.Sc. (Honours), Laurentian University, 1996 M.Sc, McMaster University, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF MEDICINE / DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA February 2007 © Michael J. Rauh, 2007 A B S T R A C T The SH2 containing inositol 5'-phosphatase (SHIP) is a hemopoietic-specific protein that catalyzes the hydrolysis of the phosphatidylinositol 3-kinase (PI3K)-generated second messenger PI-3,4,5-P3 (PIP3), to PI-3,4-P2 (PIP2) and thereby negatively regulates hemopoietic cell survival, proliferation, differentiation and activation. Herein, macrophage development and function were compared in SHIP+/+ and -/- mice. SHIP was found to restrain in vitro bone marrow-derived macrophage (BMMO) survival (or proliferation) and differentiation, consistent with the increased number of macrophages observed in SHIP-/- mice. We also compared the function of J2 virus-transformed SHIP+/+ and -/- BMMO cell lines and found that SHIP-/- J2M BMMO cell lines (-/-J2Ms) were functionally impaired in inducible nitric oxide (NO) synthase (iNOS) induction and high-output NO production, an important, classical (M1) macrophage activation strategy to combat the growth of tumours and microorganisms. This was ascribed to deficient nuclear localization of IRF1 and inhibition of iNOS transcription in these transformed macrophages. In contrast, primary SHIP-/- BMMOs routinely demonstrated enhanced LPS-stimulated iNOS/NO induction, likely as a result of PI3K-mediated enhancement of the p70S6K/IFNp/Stat1/iNOS pathway. Differential impacts upon this axis also provided an explanation for the opposite effects of the PI3K inhibitors, LY294002 and wortmannin, on iNOS/NO. We also found that SHIP-/- BMMOs failed to tolerize to a second dose of LPS, likely because SHIP protein levels were upregulated in wild-type BMMOs in an autocrine-acting, TGF(3-mediated tolerance loop. i i Analysis of in wVo-differentiated, resident peritoneal and alveolar macrophages (PM4>s, AM<t>s) from SHIP-/- mice revealed impaired NO generation, despite sufficient iNOS induction, due to constitutive arginase I-mediated L-arginine substrate competition, which redirected L-arginine metabolism away from cytotoxic NO and towards the production of healing/inflammation-resolving intermediates. These and other features were recognized as alternative (M2) macrophage activation. Consistent with pathological M2-skewing in SHIP-/- mice, their lungs were fibrotic and contained macrophage-associated Ym1 crystals. Moreover, implanted tumours grew more rapidly in the M2-skewed environment of SHIP-/- mice. Interestingly, BMMOs from SHIP-/- mice did not display this M2 phenotype unless exposed to TGF(3-containing mouse plasma early during in vitro differentiation, suggesting that an environment of elevated PIP3 and TGF|3 arising during in vivo macrophage differentiation may contribute to M2-skewing. i i i TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xv CO-AUTHORSHIP STATEMENT xxii ACKNOWLEDGEMENTS xxiii CHAPTER 1 - INTRODUCTION 1 1.1 HEMOPOIESIS 1 1.2 MYELOPOIESIS 2 1.3 THE MONONUCLEAR PHAGOCYTE SYSTEM (MPS) 2 1.4 CELL SURFACE CLASSIFICATION OF MONOCYTES, 4 MACROPHAGES AND THEIR PRECURSORS 1.5 MACROPHAGE ONTOGENY 7 1.6 REGULATION OF MONOCYTE/MACROPHAGE 8 DIFFERENTIATION: GROWTH FACTORS, SIGNAL TRANSDUCTION, AND TRANSCRIPTIONAL REGULATION 1.7 MATURE MACROPHAGE FUNCTION: DISTINCT 11 PROGRAMS REVOLVING AROUND L-ARGININE METABOLISM 1.8 THE SH2-CONTAINING INOSITOL 5'-PHOSPHATASE, SHIP .... 15 1.9 EXPERIMENTAL RATIONALE AND AIMS OF STUDY 16 iv CHAPTER 2 MATERIALS AND METHODS 18 2.1 LPS, CYTOKINES, REAGENTS AND ANTIBODIES 18 2.2 MICE 18 2.3 TISSUE CULTURE 19 2.3.1 PERITONEAL MACROPHAGES 19 2.3.2 ALVEOLAR MACROPHAGES 19 2.3.3 BONE MARROW PROGENITORS AND MACROPHAGES ....20 2.4 GENERATION OF MACROPHAGE CELL LINES 21 2.5 NITRIC OXIDE ASSAY 21 2.6 ARGINASE ASSAY 22 2.7 ELISAs 22 2.8 CYTOPLASMIC AND NUCLEAR EXTRACT PREPARATION 22 AND EMSA ANALYSIS 2.9 SDS-PAGE AND WESTERN BLOT ANALYSIS 24 2.10 FACS ANALYSIS 25 2.11 RNA PREPARATION, RNASE PROTECTION ANALYSIS, 25 NORTHERN BLOT ANALYSIS AND RT-PCR 2.12 TRANSIENT TRANSFECTIONS 27 2.13 MURINE TUMOUR MODEL 28 2.14 LPS SEPSIS MODEL 28 2.15 PLASMA AMINO ACID ANALYSIS 29 2.16 LUNG HISTOLOGY AND BAL ANALYSIS 29 2.17 LUNG CRYSTAL ISOLATION AND MASS 29 SPECTROSCOPY ANALYSIS 2.18 PERIPHERAL BLOOD PLASMA, LEUKOCYTE, 30 AND MONOCYTE ISOLATION CHAPTER 3 THE ROLE OF SHIP IN MACROPHAGE 31 DIFFERENTIATON AND FUNCTION IN VITRO 3.1 INTRODUCTION 31 3.2 RESULTS 32 3.2.1 THE ROLE OF SHIP DURING IN VITRO :....32 MACROPHAGE DIFFERENTIATION 3.2.2 DEVELOPMENT.VALIDATION AND CHARACTERIZATION 37 OF J2 RETROVIRUS-IMMORTALIZED SHIP+/+ AND -/-MACROPHAGE CELL LINES FOR IN VITRO FUNCTIONAL STUDIES 3.2.3 THE ROLE OF SHIP IN LPS-INDUCED RESPONSES OF....44 PRIMARY BONE MARROW-DERIVED MACROPHAGES 3.2.4 THE ROLE OF SHIP IN ENDOTOXIN TOLERANCE 60 OF PRIMARY BONE MARROW-DERIVED MACROPHAGES 3.3 DISCUSSION 69 CHAPTER 4 SHIP REPRESSES AN ALTERNATIVE PROGRAM 76 OF MACROPHAGE DIFFERENTIATION IN VIVO AND EX VIVO 4.1 INTRODUCTION 76 4.2 RESULTS 76 4.2.1 SHIP-/-MICE HAVE INCREASED NUMBERS OF 76 RESIDENT MACROPHAGES 4.2.2 LPS-STIMULATED SHIP-/- PMOS SECRETE LOW 78 LEVELS OF NO BUT CAN BE RESCUED BY EXOGENOUS L-ARGININE vi 4.2:3 LPS-STIMULATED SHIP-/-PMOS AND AMOS 79 SECRETE LOW LEVELS OF NO BECAUSE OF CONSTITUTIVELY HIGH ARGINASE ACTIVITY 4.2.4 ARGINASE I LEVELS ARE UPREGULATED BY 82 THE PI3K PATHWAY 4.2.5 THE M2 PHENOTYPE BECOMES MORE 85 PRONOUNCED AS SHIP-/- MICE AGE 4.2.6 M2 MO PROGRAMMING IN SHIP-/- MICE IS 88 ASSOCIATED WITH LUNG PATHOLOGY AND IMPLANTED TUMOUR SUSCEPTIBILITY 4.2.7 ROBUSTNESS OF SHIP-/- M2 MO PHENOTYPE 91 DURING PROLONGED IN VITRO CULTURE 4.2.8 IS HIGHLY INDUCIBLE EXPRESSION OF INOS 93 BY SHIP-/- PMOS INCOMPATIBLE WITH AN M2 PHENOTYPE? 4.2.9 A COMPARISON OF LPS SIGNALING, 97 ENDOTOXIN TOLERANCE AND PI3K-INHIBITORS IN SHIP+/+ AND -/- PMOS 4.2.10 ANALYSIS OF SHIP LEVELS IN PMOS 101 OF PROTOTYPICAL M1 AND M2 MOUSE STRAINS 4.3 DISCUSSION 104 CHAPTER 5 RECAPITULATION OF IN VIVO MACROPHAGE 108 DIFFERENTIATION IN VITRO 5.1 INTRODUCTION 108 5.2 RESULTS 109 5.2.1 MOUSE PLASMA SKEWS THE IN VITRO 109 DIFFERENTIATION OF SHIP-/- PROGENITORS TOWARDS M2 BMMOS 5.2.2 CHARACTERIZATION OF THE PLASMA 113 FACTOR(S) RESPONSIBLE FOR M2-SKEWING AND THE TIMING OF ITS ACTION vii 5.2.3 PI3K IS A UNIVERSAL REQUIREMENT FOR 118 M2 MO PROGRAMMING 5.2.4 FROM BENCH TO "CAGE-SIDE" AND BED-SIDE: 121 IN VIVO PREDICTIONS BASED ON IN VITRO ANALYSIS 5.3 DISCUSSION 126 CHAPTER 6 SUMMARY AND PERSPECTIVES 129 BIBLIOGRAPHY 138 APPENDIX I EPILOGUE: AUG 05-06 160 APPENDIX II THESIS-RELATED PUBLICATIONS 172 vm LIST OF TABLES CHAPTER 1 Table 1.1. Mononuclear phagocyte system (MPS) characterization according to cell surface characteristics. ix LIST OF FIGURES CHAPTER 1 Fig. 1.1. Schematic representation of MPS differentiation, 7 from bone marrow to blood and tissues. Fig. 1.2. LPS-induced MyD88-dependent and-independent 13 signal transduction in Ml macrophages leads to pro-inflammatory cytokine and NO synthesis. Fig. 1.3. Differential L-arginine metabolism as an axis for 14 M1 versus M2 macrophage phenotypic characterization. Fig. 1.4. Structure and function of SHIP 16 CHAPTER 3 Fig. 3.1. Accelerated and enhanced macrophage differentiation 33 of SHIP-/- bone marrow progenitors. Fig. 3.2. SHIP-/- macrophage differentiation is not appreciably 36 accelerated from more committed bone marrow progenitors, despite enhanced yield. Fig. 3.3. Enhanced FLT3-ligand-induced myeloid differentiation 37 in SHIP-/- mice. Fig. 3.4. Characterization of macrophage phenotypic features .38 in J2M+/+ and J2M-/- cell lines. Fig. 3.5. SHIP J2M-/- cells are impaired in NO production. 39 Fig. 3.6. Diminished LPS-induced NO production in 40 J2M-/- cells is associated with impaired iNOS induction. Fig. 3.7. Diminished LPS-induced iNOS transcription 40 and iNOS mRNA levels in J2M-/- cells. Fig. 3.8. IFN-y partially rescues the NO deficit of J2M-/- cells ..41 x Fig. 3.9. Deficient IRF-1 nuclear localization in J2M-/- cells 43 is associated with impaired iNOS induction. Fig. 3.10. The PI3K pathway is a positive regulator of iNOS/NO 45 in BMMOs and SHIP negatively regulates this process. Fig. 3.11. Wortmannin and LY294002 differentially affect 46 BMMO iNOS/NO induction in response to LPS. Fig. 3.12. The PI3K inhibition-inactive LY294002 analogue, 47 LY303511 has less negative impact upon LPS-induced iNOS and NO. Fig. 3.13. Differential effects on the p70S6K/IFNB/Stat1/iNOS 48 pathway may underlie differences in iNOS/NO induction between SHIP+/+ and -/- BMMOs and between wortmannin and LY294002. Fig. 3.14. A comparison of LPS-induced signal transduction 52 and pro-inflammatory mediator production in SHIP+/+ and -/- BMMOs in the presence of M-CSF. Fig. 3.15. A comparison of LPS-induced signal transduction 53 and pro-inflammatory mediator production in SHIP+/+ and -/- BMMOs in the absence of M-CSF. Fig. 3.16. Pooled analysis of LPS-induced TNFa, IL-6, and IL-10 54 production over time in SHIP+/+ and -/- BMMOs. Fig. 3.17. BMMO LPS-responsiveness is dependent upon cell 57 density and autocrine mediators. Fig. 3.18. PI3K inhibition inhibits LPS-induced Akt 59 phosphorylation and IL-10 production while it augments p65 NF-KB phosphorylation. Fig. 3.19. SHIP-/- BMMOs do not display endotoxin tolerance 61 Fig. 3.20. LPS dose-dependent tolerization in SHIP+/+ BMMOs 61 Fig. 3.21. SHIP-/- BMMOs that are LPS hypo-responsive 62 are also refractory to LPS-induced tolerance. Fig. 3.22. SHIP protein and mRNA levels are upregulated in .....64 SHIP+/+ BMMOs by LPS treatment. xi Fig. 3.23. SHIP upregulation is essential for endotoxin tolerance 65 and is mediated by LPS-stimulated TGFB. Fig. 3.24. Lack of endotoxin tolerance in SHIP-/- BMMOs is not 66 associated with perturbed TLR4/MD2 surface levels. Fig. 3.25. SHIP-/- BMMOs may be refractory to endotoxin 68 tolerance because of deficient control of Akt, Statl, and Erk1/2 activation. Fig. 3.26. SHIP-/-mice are hypersensitive to LPS-induced 68 mortality in vivo. CHAPTER 4 Fig. 4.1. SHIP-/- mice have increased numbers of mature 77 resident peritoneal macrophages. Fig. 4.2. LPS-stimulated SHIP-/- PMOs, unlike BMMOs 79 are deficient in NO production. Fig. 4.3. L-arginine supplementation rescues SHIP-/-PMO 80 NO production. Fig. 4.4. LPS-stimulated SHIP-/- PMOs and AMOs secrete 81 low levels of NO because of constitutively high arginase activity. Fig. 4.5. Constitutively elevated Argl expression is unique 82 to SHIP-/- resident MOs. Fig. 4.6. Arginase is upregulated by the PI3K pathway. 84 Fig. 4.7. The M2 MO phenotype becomes more pronounced 86 as SHIP-/- mice age. Fig. 4.8. Further M2 phenotypic characterization of 87 SHIP-/- PMOs. Fig. 4.9. Evidence for SHIP-/- M2 MO skewing in vivo 89 Fig. 4.10. Lewis lung carcinoma implants grow faster in 91 SHIP-/- mice. x i i Fig. 4.11. Assessment of the stability of the SHIP-/- M2 MO 92 phenotype and its responsiveness to TH1 and T H2 cytokines. Fig. 4.12. SHIP-/- PMOs demonstrate impaired LPS-induced 94 iNOS and are unable to sustain induced COX-2. Fig. 4.13. L-arginine supplementation or fresh media change 96 alleviate repression on LPS-induced SHIP-/- PMO iNOS and COX-2 levels. Fig, 4.14. Characterization of LPS-induced PMO signal transduction. ...98 Fig. 4.15. Wortmannin and LY294002 have opposite effects on 100 LPS-induced iNOS and NO synthesis in PMOs. Fig. 4.16. An analysis of endotoxin tolerance in SHIP+/+ 102 and -/- PMOs. Fig. 4.17. A reciprocal relationship exists between SHIP and Argl 103 protein levels in C57BL/6 (M1) and Balb/c (M2) PMOs. CHAPTER 5 Fig. 5.1. Mouse plasma skews the in vitro differentiation 110 of SHIP-/- myelomonocytic progenitors towards M2 BMMOs. Fig. 5.2. SHIP-/- mouse plasma has more arginase-inductive 110 capacity during SHIP-/- BMMO differentiation. Fig. 5.3. Differentiation of SHIP-/- BMMOs in mouse plasma 111 leads to increased Ym1 and Argl expression and decreased inducible NO synthesis. Fig 5.4. Mouse plasma-mediated M2-skewing of SHIP-/- 112 BMMOs occurs early during differentiation in a PI3K-dependent process. Fig. 5.5. An initial fractionation study of the M-2 skewing blood 113 factor suggests it is greater than 10 kDa and not positively charged at pH 7.2. x i i i Fig. 5.6. SHIP-/- bone marrow progenitors are hypersensitive 114 to PI3K-mediated arginase induction by TGFB and/or IFNy. Fig. 5.7. IL-10, but not IL-4, IL-13, or MSP, is another potential 115 candidate for the M2-skewing plasma factor. Fig. 5.8. TGFB in mouse plasma induces Argl and sustains 116 Ym1 in differentiating SHIP-/- BMMOs. Fig. 5.9. IL-4 can M2-skew both differentiating and mature 117 SHIP+/+ and -/- BMMOs while TGFB and MP can only affect differentiating SHIP-/- BMMOs. Fig. 5.10. Argl protein induction by mouse plasma appears 118 concomitant with c-fms induction during SHIP-/- BMMO differentiation. Fig. 5.11. PI3K is required for full arginase induction by IL-4 120 during differentiation and in mature BMMOs, independent of Stat6 phosphorylation. Fig. 5.12. PI3K is also required for full Ym1 induction by IL-4 121 or IL-13 in mature BMMOs from the prototypical M2 Balb/c mouse strain. Fig. 5.13. An analysis of cytokine levels in SHIP+/+and-/- 122 mouse plasma. Fig. 5.14. SHIP-/- peripheral blood contains elevated arginase 124 activity and expression in total leukocyte and monocyte fractions. Fig. 5.15. SHIP-/- peripheral blood leukocytes and monocytes 125 express elevated Argl but not Ym1 levels. Fig. 5.16. SHIP-/-peripheral blood contains reduced circulating 126 levels of L-arginine. CHAPTER 6 Fig. 6.1. The effects of SHIP on macrophage programming 133 and NO production are dependent upon conditions of differentiation and dynamic environmental influences. x i v LIST OF ABBREVIATIONS 4EBP1 = elongation initiation factor 4E-binding protein 1 Akt = protein kinase B = cellular homologue of v-Akt (AKT8 retrovirus prod AMO = alveolar macrophage AMCase = acidic mammalian chitinase AML = acute myelogenous leukemia Argl = arginase I ATRA = all-trans retinoic acid Bad = BCL-2 antagonist of cell death BAL = bronchoalveolar lavage BALF = BAL fluid BH 4 = tetrahydrobiopterin BM = bone marrow BME = 2-ME = P-ME = beta-mercaptoethanol BMMO = bone marrow-derived macrophage BSA = bovine serum albumin Btk = Bruton agammaglobulinemia tyrosine kinase CALT = coelome-associated lymphomyeloid tissue CaM = calmodulin CARS = compensatory anti-inflammatory response CCR = chemokine receptor CCL = chemokine ligand CD = cluster of differentiation C/EBP = CCAAT enhancer binding protein c-Fms = cellular Fms = macrophage colony stimulating factor receptor CFC = colony-forming cells CFSE = carboxyfluorescein succinimidyl ester CFL) = colony forming unit CFU-M = macrophage colony forming unit Chal = challenged Chitl = chitotriosidase 1 XV CHX = cyclohexamide c-Kit = cellular Kit = stem cell factor receptor tyrosine kinase CLP = common lymphoid progenitor CM = conditioned medium CML = chronic myelogenous leukemia CMP = common myeloid progenitor CMV = cytomegalovirus COX-2 = cyclo-oxygenase 2 CSF-1 = colony stimulating factor 1 = macrophage colony stimulating factor CX 3 CR = CX 3 chemokine receptor DC = dendritic cell Dev = developing dldC = deoxyinosine-deoxycytosine dpc = days post conception DMEM = Dulbecco's modified Eagle's medium DMSO = dimethyl sulfoxide DNA = deoxyribonucleic acid DTT = dithiothreitol ECL = Enhanced Chemiluminescence system E. coli = Escherichia coli EDTA = ethylenediaminetetraacetic acid ELISA = enzyme-linked immunosorbent assay EMSA = electrophoretic mobility shift assay eNOS = endothelial nitric oxide synthase Epo = erythropoietin ERK = extracellular signal-regulated kinase ESI = electrospray ionization F2 = second filial generation FACS = fluorescence-activated cell sorting FAD = flavin adenine dinucleotide FBS = fetal bovine serum = fetal calf serum xvi Fc = fragment crystallizable , of immunoglobulin FCS = fetal calf serum FITC = fluorescein isothiocyanate FIZZ1 = found in inflammatory zone 1 FKHR = Forkhead-related FL = FLT3L = Fms-like tyrosine kinase 3-ligand FMN = flavin mononucleotide GAPDH = glyceraldehyde-3-phosphate dehydrogenase GM = granulocyte/macrophage GMP = granulocyte/macrophage progenitor GPI = glycosylphosphatidylinositol Gr-1 = granulocyte antigen 1 = Ly6G Grb2 = growth factor receptor-bound 2 GSK3 = glycogen synthase kinase 3 HBSS = Hank's balanced salt solution H&E = hematoxylin and eosin HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HP = human plasma HPLC = high performance liquid chromatography HS = human serum HSC = hemopoietic stem cell hSHIP = human SH2 containing inositol 5-phosphatase I FN = interferon IgG = immunoglobulin G IKB = inhibitor protein kappa B IKK = IKB kinase IL = interleukin IL-1Ra = IL-1 receptor antagonist iMC = immature myeloid cell IMDM = Iscove's modified Dulbecco's medium iNOS = inducible nitric oxide synthase xv i i IP = intraperitonealy IRAK = interleukin 1 receptor associated kinase IRF = interferon regulatory factor J2M = J2 retrovirus-immortalized, bone marrow-derived macrophage JAK = Janus kinase L-arg = L-arginine Lin = lineage LLC = Lewis lung carcinoma L-NMMA = L-NG-monomethyl-arginine LPS = lipopolysaccharide LSK= LinSca-1 + c-Kit+ LT-HSC = long-term repopulating hemopoietic stem cell LY = LY294002 Lys = lysine M1 MO = classically activated macrophage M2 MO = alternatively activated macrophage Mac-1 = macrophage antigen 1 = CD11b/CD18 = a M S 2 integrin MALDl-TOF = matrix assisted laser desorption ionization - time of flight Mat = mature MCP-1 = monocyte chemoattractant protein 1 M-CSF = macrophage colony stimulating factor M-CSFR = macrophage colony stimulating factor receptor MD2 = myeloid differentiation protein 2 mDC = myeloid dendritic cell MEP = megakaryocyte/erythrocyte progenitor MeV = motheaten viable MMLV = Moloney murine leukemia virus M 0 = monocyte MOPS = morpholinepropanesulfonic acid MP = mouse plasma MPS = mononuclear phagocyte system xvi i i MR = mannose receptor MS = mass spectrometry MSB = Mason trichrome MSC = myeloid suppressor cell MSP = macrophage stimulating protein MTG = monothioglycerate mTOR= mammalian target of rapamycin MyD88 = myeloid differentiation factor 88 NADPH = nicotinamide adenine dinucleotide phosphate, reduced form N F - K B = nuclear factor kappa B NK = natural killer NLS = nuclear localization sequence nNOS = neuronal nitric oxide synthase NO = nitric oxide N0 2" = nitrite NOHA = Nw-hydroxy-L-arginine NPXY = amino acid sequence: asparagine, proline, any amino acid, tyrosine ODC = ornithine decarboxylase Op = osteopetrotic Orn = ornithine PB = peripheral blood PBL = peripheral blood leukocyte PBM 0 = peripheral blood monocyte PBS = phosphate buffered saline PCR = polymerase chain reaction pDC = plasmacytoid dendritic cell PDK = Pl-dependent protein kinase PE = phycoerythrin PH = pleckstrin homology PI = propidium iodide PI3K = phosphatidylinositol 3-kinase x i x PIP 2 = PI-3,4-P2= phosphatidylinositol-3,4-bisphosphate PIP 3 = PI-3,4,5-P3 = phosphatidylinositol-3,4,5-trisphosphate PKB = protein kinase B = Akt PLC = phospholipase C PMO = peritoneal macrophage PMSF = phenylmethylsulfonylfluoride PSB = phosphorylation solubilization buffer Ptase = phosphatase PTB = protein tyrosine binding PTEN = phosphatase and tensin homologue deleted on chromosome ten PVDF = polyvinylidene difluoride RES = reticuloendothelial system rh = recombinant human RLL) = relative light unit rm = recombinant murine RNA = ribonucleic acid RON = recepteur d'origine nantais = stem cell-derived tyrosine kinase RPA = RNase protection analysis RPM0 = resident peritoneal macrophage RT-PCR = reverse transcriptase polymerase chain reaction S6K = ribosomal S6 kinase SA = streptavidin SCF = stem cell factor = SF = Steel factor = Kit ligand SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM = standard error of the mean SH2 = Sre homology 2 domain SH3 = Sre homology 3 domain SHIP = SH2 containing inositol 5'-phosphatase SHIP2 = SH2 containing inositol 5'-phosphatase 2 SHP-1 = SH2 containing tyrosine phosphatase 1 SIRS = systemic inflammatory response syndrome X X S0CS1 = suppressor of cytokine signaling 1 SR-A = scavenger receptor A SSA = sulfosalicylic acid STAT = signal transducer and activator of transcription STK = stem cell-derived tyrosine kinase TAM = tumour-associated macrophage TCL = total cell lysate TGFB = transforming growth factor beta T/GPMO = thioglycollate-elicited peritoneal macrophage T H = Th = T cell helper TK = thymidylate kinase TLR = Toll-like receptor TNF = tumour necrosis factor Tol = tolerized Tris = Trizma = tris(hydromethyl)aminomethane hydrochloride v/v = volume/volume WM = wortmannin Y = tyrosine xxi CO-AUTHORSHIP STATEMENT In Chapter 3, the J2M macrophage cell line was produced by Dr. Jacqueline Damen (Terry Fox Laboratory, Vancouver) and the iNOS Northern blot was performed by Dr. Damen. The phenomenon of refractory endotoxin tolerance in SHIP-/- mice and bone marrow-derived macrophages was first described by Dr. Laura Sly (Terry Fox Laboratory) (Sly et al., 2004). Macrophages used in Figures 3.19. 3.20, 3.22, 3.23, 3.24, and 3.25 were obtained with my assistance, but experimentation was mostly conducted by Dr. Sly, and these were used with the permission of Dr. Sly. The SHIP-/- mice used in Chapter 4 were created previously (Helgason et al., 1998). Also in Chapter 4, the anti-Ym1 antibody was generated by Vivian Lam (Terry Fox Laboratory), and Vivian Lam also assisted in the CD204/CD206 FACS analysis. Histological lung sections were obtained with the technical assistance of Julie Chow (UBC Pathology, Vancouver). Lung crystal mass spectroscopy was conducted with the assistance of Dr. Suzanne Perry (UBC Laboratory of Molecular Biophysics Proteomics Core Facility, Vancouver). Lewis lung carcinoma studies were performed in the lab of Dr. Andrew I. Minchinton (BC Cancer Research Centre, Vancouver), with the assistance of Lynsey Huxham. C57BL/6 and Balb/c mice were provided by Brad Dykstra (lab of Dr. Connie Eaves, Terry Fox Laboratory). In Chapter 5, plasma amino acid analysis was conducted by HPLC with the assistance of Dr. Hilary Valance (BC Children's Hospital Biochemical Diseases Laboratory, Vancouver). Mice were housed in the Joint Animal Facility (JAF) with the assistance of the JAF staff. Finally, but importantly, many of the results presented in this thesis were obtained with the supervised technical assistance of UBC undergraduate co-operative students Michael Lane, Carla Pereira, Jessica Palmer, Anita Sham, and Victor Ho. For their dedication and assistance, I am deeply grateful. xxii ACKNOWLEDGEMENTS I sincerely thank Dr. Gerald Krystal for the opportunity to conduct these studies under his direction in his laboratory. More than that, I thank Dr. Krystal for his mentorship, guidance, patience, understanding, encouragement, support, humour, and friendship. Words cannot sufficiently express my gratitude. Thank you to members of my PhD Supervisory Committee, Dr. Alice Mui and Dr. Urs Steinbrecher, for their input, suggestions, guidance, and support throughout my PhD studies and during the process of presenting my findings in thesis format. I also wish to thank my MD/PhD directors, Dr. Anthony Chow and Dr. Lynn Raymond for their mentorship and guidance with respect to my clinical training and its integration with research. They have been inspirational to me. A sincere thank you is also in order to my lab mates, past and present, including Vivian Lam, Michael Hughes, Janet Kalesnikoff, Jackie Damen, Mark Ware, Michael Huber, Frann Antignano, Jens Ruschmann, Tom Buchse, Daisy Chow, Sandie Yew, and co-op students Victor Ho, Carla Pereira, Anita Sham, Michael Lane, and Jessica Palmer, (and the many others I did not directly supervise) for all their help and friendship in and out of the lab. I wish also to thank my fellow MD/PhD students, including Paul Yong, Claire Sheldon, Ryan Hung, Cheng-Han Lee, Jimmy Lee, and more current students for their support and advice in combining research and medical school. I next wish to thank Drs. Norman Wong, Vincent Duronio, Jackie Damen, Cheryl Helgason, Pamela Correll, Michael Huber, William J. Murphy, Manuel Modolell, and Zhou Zhu for helpful discussions. Lastly, I would like to thank my family: parents John and Jackie, sisters Mary Catherine and Stephanie, and extended family for their love, support and encouragement. Finally, thank you to Jennifer for being with me every step of the way. Together we've moved mountains. I dedicate this thesis to you. xxi i i CHAPTER 1 - INTRODUCTION 1.1 HEMOPOIESIS Blood contains an array of cell lineages with unique and important functions, but these cells generally have a short lifespan. Despite the loss of staggering numbers of cells each day, they are nonetheless replaced and maintained at a relatively constant level in a process known as hemopoiesis (Eaves, 2002). Seminal studies conducted in mice in the 1950's and 1960's, by Till, McCulloch and others, revealed that bone marrow (BM) contains the necessary cells capable of reconstituting the depleted hemopoietic system of an irradiated recipient (Main and Prehn, 1955; Ford et al., 1956; Till and McCulloch, 1961; Becker et al., 1963; Siminovitch et al., 1963). It is now widely accepted that hemopoiesis is ultimately maintained by a small and heterogeneous population of pluripotent (ie, able to reconstitute all hemopoietic lineages) long-lived, quiescent cells that are capable of self-renewal (Eaves, 2002; Huntley and Gilliland, 2005; Wang and Dick, 2005). These hemopoietic stem cells (HSCs) are succeeded by progressively more lineage-restricted and differentiated progenitors with less ability to self-renew, ultimately culminating in the amplification and specialization of mature blood cell types (Akashi et al., 2005). The rate of mature cell output is regulated by the complex coordination of cell survival, proliferation, and differentiation (Eaves, 2002) by intrinsic and extrinsic factors (Barreda et al., 2004; Akashi et al., 2005). In mouse bone marrow, HSCs have been identified within the population of lineage (Lin)", Sca-1+, c-Kit+ (LSK) cells (Akashi et al., 2005). Sca-1 (Ly6 A/E) is a cell surface GPI-linked adhesion protein necessary for normal HSC activity and for upregulation of c-Kit (stem cell factor receptor tyrosine kinase) expression in HSCs (Bradfute et al., 2005). HSC biology is an area of intense active investigation and heterogeneity of the LSK population has become increasingly apparent (Akashi et al., 2005; Wagers 2005). 1 1.2 MYELOPOIESIS The current model of hemopoiesis (Akashi et al., 2005) proposes that the first appreciable lineage-commitment step usually involves a bifurcation into the clonogenic common myeloid (GMP) and lymphoid progenitors (CLP) (Kondo et al., 1997; Akashi et al., 2000) with some notable exceptions (Takano et al., 2004; Adolfsson et al., 2005). In general terms, the myeloid pathway encompasses the production of mature erythrocytes, megakaryocytes/platelets, granulocytes, monocytes/macrophages, and myeloid dendritic cells (mDCs), while the lymphoid pathway leads to the production of B and T cell subsets, natural killer cells (NK cells), and plasmacytoid dendritic cells (pDCs) (Eaves, 2002; Akashi et al., 2005). Recently, the CMP (Akashi et al., 2000) has been divided into subpopulations that are enriched for committed granulocyte/macrophage progenitors (GMPs) or committed megakaryocyte/erythrocyte progenitors (MEPs) (Nutt et al., 2005). While eosinophils have been categorized as myeloid cells (Iwasaki et al., 2005), the origins of mast cells and basophils are less clear. Instead, they may actually arise directly from multipotential progenitors (Chen et al., 2005). As cells of the monocyte/macrophage lineage are the primary subjects of investigation in this thesis, the focus will now be shifted to this arm of the myeloid lineage. 1.3 THE MONONUCLEAR PHAGOCYTE SYSTEM (MPS) The Russian biologist llya Mechnikov (Elie Metchnikoff) is credited with the discovery of macrophages in 1882 (Bogdan, 2001b). Specifically, he recognized the presence of highly mobile mononuclear phagocytes in invertebrates that were able to ingest bacteria and organic manner, and coined the term "phagocytosis" to describe the process (in Greek phagon = eat, and kytos = cavity). He postulated that these cells were also present in higher vertebrates, were responsible for catching and destroying disease-producing 2 microbes that have entered the host, and for this he was awarded the 1908 Nobel Prize in Physiology or Medicine (Mechnikov, 1908; O'Neill 2004). In the early 1900's all phagocytic cells were classified together (mistakenly including non-phagocytic endothelial cells, due to their uptake of vital dyes) as the reticuloendothelial system (RES) (Aschoff, 1924). In the years that followed, mononuclear phagocytes were further characterized by their morphological features and expression of certain enzymes (like non-specific esterase) that could be easily detected using histochemical stains, and their ability to take up uncoated particles, or those coated by immunoglobulins or complement (Hume et al., 2002). These studies laid the foundation for subsequent seminal experimentation and classification by van Furth and Cohn (1968). The mononuclear phagocyte system (MPS) was proposed to be a specialized tissue distributed throughout the body and separated into two groups of cells: circulating mononuclear phagocytes (monocytes, M0s) and tissue macrophages (MOs) found in organs such as the spleen, lymph nodes, liver (Kuppfer cells), lung (alveolar MOs), peritoneal (serosal) cavity (peritoneal MOs), and the subcutaneous tissues (van Furth and Cohn, 1968). More recently, it has also become apparent that bone resorbing osteoclasts, brain microglia, MOs within reproductive organs, the gut lamina propria and those within the interstitium of the heart, pancreas and kidney, along with myeloid DCs and Langerhans cells are also components of the MPS (Taylor et al., 2005). These studies have confirmed the insightful proposal by Van Furth and Cohn that the MPS originated from progenitor cells in the bone marrow which differentiated to form blood M 0s that circulated in the blood, and entered tissues to become resident MOs (van Furth and Cohn, 1968; Hume et al., 2002). Moreover, it was also proposed that the majority of cellular proliferation of the MPS occurred in bone marrow monoblasts/promonocytes, and that the local proliferation of mature, tissue MOs was a very limited means of maintaining MO numbers (van Furth and Cohn, 1968). Thus, the following have become universally recognized as MPS characteristics (van Furth et al., 1972): 1) mononuclearity, as observed microscopically or by low orthogonal light scatter in flow cytometry, 2) myeloid 3 cell surface nature, as demonstrated by high expression of Mac-1 and other antigens (as discussed further below), 3) uniform phagocytic potential, and 4) if an immature MPS lineage cell, the capacity to differentiate into a mature MO upon exposure to MO-colony stimulating factor (M-CSF; discussed further below). The MPS classification has for the most part stood the test of time (Kennedy and Abkowitz, 1998) and this achievement has laid the foundation for more current studies. 1.4 CELL SURFACE CLASSIFICATION OF MONOCYTES, MACROPHAGES AND THEIR PRECURSORS Major advances in the understanding and further classification of the MPS arose with advances in antibody technology and flow cytometry (Hume et al., 2002). Specifically, it became possible to characterize particular populations according to their surface antigens (Taylor et al., 2005). Examples of such surface proteins used to identify cells of the mouse MPS have included: 1) Mac-1 (CD11b/CD18, aMB 2), an integrin important for interaction with extracellular matrices and other cells, whose engagement also transduces signals required for terminal MO differentiation (Shi et al., 2004); 2) F4/80 (EMR1), a protein of the epidermal growth factor-like seven-spanning membrane receptor (EGF-TM7) family of unknown function but recently identified as involved in the generation of peripheral tolerance (Lin et al., 2005); 3) CD14, a component of the LPS receptor complex (Wright et al., 1990); 4) CD68 (macrosialin), a member of the lamp/lgp family (Holness et al., 1993); 5) Sialoadhesin (SN, Siglec-1) (Taylor et al., 2005); 6) ER-MP12 (CD31, PECAM-1), a vascular endothelial adhesion molecule likely important for trans-endothelial migration (Ling et al., 1997); 7) ER-MP20 (Ly6C), one of two distinct antigens found within the Gr-1 antigen (the other being Ly6G, which is expressed almost exclusively on neutrophils) (Taylor et al., 2003); and antigens of undetermined proteins or proteins with as yet unknown functions, such as 8) ER-MP58 (Chan et al., 1998); 9) MUM-4 (Agger and Rhodes, 1994); and 10) 7/4 (Taylor et al., 2003). More recently, studies conducted in monocytes 4 have added two chemokine receptors, CX3CRI and CCR2, and one adhesion molecule, CD62L, to the MPS repertoire that have enabled the subdivision of M0s into "resident" and "inflammatory" sub-populations (Geissmann et al., 2003; Taylor and Gordon, 2003). Finally, despite the use of these markers in the characterization of the MPS, none of the above are completely specific to this lineage (Chan et al., 1998; Taylor et al., 2003) and it is important that they be used in combination. However, the most specific marker of cells of the MPS identified to date is the M-CSF receptor, c-fms (M-CSFR, CSF-1R, CD115) (Chan et al., 1998). Table 1.1 presents a summary of the current status of MPS classification from bone marrow precursors to M0s and finally MOs (including an analysis of some non-MPS cells to highlight shortcomings of individual markers), using information assimilated from the aforementioned studies and others (Lagasse and Weissman, 1996; Kennedy and Abkowitz, 1998; Tagoh et al., 2002; Cook et al., 2003; Leon et al., 2004; Sunderkotter et al., 2004). Table 1.1 expands upon M 0 classification, while Fig. 1.1 presents a summary of MPS differentiation in a simplified diagram. Of note, intraperitoneal injection of thioglycollate has long been used as a stimulatory agent for eliciting inflammatory MOs (T/G PMOs) (Gallily and Feldman, 1967; Cook et al., 2003), but more recent experimentation has revealed that this protocol induces the accumulation of a population of immature peritoneal MO-colony forming units (P-CFU-M) and mature T/G PMOs (however, T/G PMOs are less mature than resident, non-inflammatory RPMOs) (Chan et al., 1998; Cook et al., 2003). Moreover, T/G injection induces the out-migration of RPMOs in what has been called the MO disappearance reaction (Chan et al., 1998). Some of these features are also depicted in Fig. 1.1. Not depicted in Fig. 1.1 is the potential contribution made to peritoneal MO populations, both resident and inflammatory, by coelome-associated lymphomyeloid tissue (CALT), including the omentum, since this has received only limited recent attention (Broche and Felix, 2001; Pinho et al., 2002; Pinho et al., 2005). 5 Bone Marrow (BM) Blood P Cavity Lung Other ! o ^~ CO <S B M - C F U - M B M r M b r i b b l a s t B M - P r o - M 0 BM -Stromal M<t> iriffam. P B - M 0 Resident P B - M 0 P - C F U - M T / G P M O R P M 0 AM<t> Neutrophil Eosinophil Q o o >> _j co s — Q si o o C; Q or or • — TO <!> 2 _ c 5>; to co s _ CO O O u. I CM O a. QL 00 to a. on UJ - hi + + +. - hi + Io + Io .+ hi + •+ hi hi Io + + - hi -• •+ •-+ -+ + + + - -• + -hi hi + - .+• '9: io + - + lb lo + -.+ + * - Io + hi .+ lo lo lo x o o o lo + -hi - . . . + Table 1.1. Mononuclear phagocyte system (MPS) characterization according to cell surface characteristics. P resented in tabular form is a s u m m a r y of the studies cited in C h a p t e r 1, Sec t ion 1.4. C o l u m n s depict unique cell sur face markers identified using ant ibodies, while rows are a r ranged accord ing to anatomical site a n d , where indicated by an arrow, cel ls are a r ranged from least to most mature. + (and hi or io) indicates that express ion of the marker in the particular cell type h a s been reported (and relative express ion levels are given). - indicates e x p r e s s i o n has specif ical ly not been detected, while blank s q u a r e s represent information that is lacking ( b a s e d upon the re ferences cited). Abbreviat ions u s e d : B M = b o n e marrow; C F U = co lony- forming unit; M 0 = monocyte ; M O = m a c r o p h a g e ; Inflam. = inflammatory; P B = peripheral b lood; P = peritoneal; T / G = thioglycollate-elicited; R = resident; A = alveolar. 6 Bone Marrow P r o - M , E R - M P 12 20 I / M a c - 1 , 0 / c - F m s l 0 Peripheral Blood Inflammatory M 0 4 G r - 1 + CX 3 CR1 ' ° C C R 2 + C D 6 2 L * Resident fvt ) G r - r C X 3 C R 1 h t C C R 2 " C D 6 2 L " M a c - 1 + / c - F m s 4 Peritoneal Cavity T / G P M O E R - M P 1 2 + c - F m s * M a c - 1 + F4/8G+ R P M 0 M a c - 1 h i / c - F m s ' hi Fig. 1.1. Schematic representation of MPS differentiation, from bone marrow to blood and t issues. Adapted from the references cited in Section 1.4, primarily Chan et al. (1998), Kennedy and Abkowitz (1998), and Taylor and Gordon (2003). 1.5 MACROPHAGE ONTOGENY The aforementioned studies on MPS classification and characterization were conducted in adult mice and humans, and pinpointed the bone marrow as the principal site of origin for cells of this lineage. However, the ontogeny of MOs in mouse and human embryos is quite different from that observed in adults (Lichanska and Hume, 2000; Shepard and Zon, 2000). Notably, during mouse embryogenesis MOs arise first in the yolk sac and then in the fetal liver. While these fetal MOs were identified as possessing phagocytic capability and sharing surface antigens with adult MOs, they were uniquely shown to possess 7 significant proliferative capacity and lacked some enzymatic activities found in adult MOs (Shepard and Zon, 2000). Moreover, it was suggested that their development was functionally distinct from those found in adults, possibly even bypassing the monocyte stage (Lichanska et al., 1999). Subsequent experimentation has clarified these issues and revealed three distinct waves of MOs in the mouse yolk sac (Bertrand et al., 2005). The first wave, beginning at 7.5-8 days post-conception (dpc), represent maternally-derived MOs, and perhaps account for the lack of an observable fetal M 0 stage (ie, these mature maternal MOs arrive at the yolk sac) (Bertrand et al., 2005). The second wave is of monopotent MO precursors (beginning at 8 dpc), while the third wave is of bipotent erythro-myeloid precursors (starting at 8.5 dpc), and both the second and third wave lead to the appearance of mature yolk sac MOs at 9-9.5 dpc (Bertrand et al., 2005). It is likely that the bipotent third wave precursors also colonize the fetal liver (Bertrand et al., 2005) and contribute to fetal hemopoiesis at this site (Lichanska and Hume, 2000; Shepard and Zon, 2000). Thus, in essence, yolk sac hemopoiesis follows a reverse course of events, compared to fetal liver and adult bone marrow: mature (albeit maternally-derived) MOs appear before immature precursors. 1.6 REGULATION OF MONOCYTE/MACROPHAGE DIFFERENTIATION: GROWTH FACTORS, SIGNAL TRANSDUCTION, AND TRANSCRIPTIONAL REGULATION As mentioned in Section 1.2, it is currently postulated that the HSC differentiates into a CMP, which in turn gives rise to more lineage-restricted progenitors: the GMP and MEP, which yield granulocyte/macrophage and megakaryocyte/erythrocyte lineages, respectively (Akashi et al., 2002; Nutt et al., 2005). Hemopoiesis is often conceptualized and presented as a static, linear hierarchy, but the process is actually plastic and dynamic (Quesenberry et al., 2002; Rosmarin et al., 2005). This is likely because fate determination in 8 hemopoiesis reflects the complex coordination by growth factors, the signals they generate, and the status of the cell upon which they act (Rosmarin et al., 2005). Myelopoiesis and monoctye/macrophage differentiation are known to be regulated by particular combinations of CSFs and growth factors. Specifically, early myeloid survival, proliferation, and differentiation are regulated in a synergistic fashion by stem cell factor (also called SCF, Steel factor, SF, or Kit-ligand), interleukin-3 (IL-3), Fms-like tyrosine kinase 3-ligand (FLT3L), and GM-CSF (Stirewalt and Radich, 2003; Barreda et al., 2004; Pixley and Stanley, 2004). M-CSF alone cannot support these aspects of early myelopoiesis, but does synergize with the aforementioned factors in this regard (Barreda et al., 2004; Pixley and Stanley, 2004). However, beyond this early stage, M-CSF is able to support the survival, proliferation, and differentiation of monocytic progenitors and the survival and proliferation of mature MOs (Pixley and Stanley, 2004). Myelopoeisis and M0/MO differentiation initiated by the aforementioned factors are thought to result from the coordinated actions of particular transcription factors that orchestrate the genetic program of differentiation (Valledor et al., 1998; Friedman, 2002). These transcription factors include PU.1, CCAAT enhancer binding proteins (C/EBPs), AML1, Maf-B, c-Jun, Egr-1 and Sp1 (Valledor et al., 1998; Friedman, 2002; Rosmarin et al., 2005), but it is important that their activation also be coordinated with chromatin remodeling and transcriptional coactivators and repressors to induce the MPS characteristics outlined in Sections 1.3 and 1.4 (Rosmarin et al., 2005). While no "master regulator" myeloid transcription factor has been identified, PU.1 and C/EBPa have emerged as two very important regulators of M0/MO differentiation (Friedman, 2002; Rosmarin et al., 2005). Specifically, PU.1 expression is essential for fetal hemopoiesis (Scott et al., 1994), while in the adult, graded PU.1 expression is the determinant for lineage commitment (DeKoter and Singh, 2000; Nutt et al., 2005). For instance, high concentrations of PU.1 promote MO differentiation while low concentrations are permissive for B cell differentiation (DeKoter and Singh, 2000). Moreover, while PU.1 and C/EBPa are required for 9 both MO and neutrophil differentiation, higher PU.1 expression relative to C/EBPa specifies MO fate (Dahl et al., 2003). This may be due, in part, to the observed repression of granulopoiesis relative to monopoiesis at the level of the GMP (Dakic et al., 2005). Interestingly, DCs have higher PU.1 expression relative to MOs, but this antagonizes MafB expression, which is required for MO differentiation (Bakri et al., 2005). Finally, consistent with the theme of antagonism, PU.1 and C/EBPa have also been demonstrated to favour MO differentiation by repressing transcription factors essential for alternate fates (ie, Gata-1 in the case of erythroid differentiation (Rhodes et al., 2005), and Pax5 in B cell determination (Xie et al., 2004)). Thus, PU.1 and C/EBPa have emerged as important transcription factors in myeloid and M0/MO differentiation. Much of what has been learned about M0/MO differentiation has emerged from the study of a natural M-CSF-deficient mouse strain (op/op), and engineered c-Fms-/- and c-Fms/GFP mice. Op/op mice have a striking decrease in tissue MOs and bone-resorbing osteoclasts, giving them a severe osteopetrotic (op) phenotype (Pixley and Stanley, 2004). Subsequent studies have revealed that M-CSF is synthesized by a variety of cell types, including stromal and endothelial cells (Barreda et al., 2004). Moreover, c-Fms is expressed primarily on cells of the M0/MO lineage (although it is also found in placental trophoblasts) and expression levels progressively increase from primitive precursors to mature cells (Barreda et al., 2004). Binding of M-CSF leads to the dimerization, tyrosine kinase domain activation, and autophosphorylation of c-Fms (Bourette and Rohrschneider, 2000; Pixley and Stanley, 2004). This provides docking sites for the recruitment and activation of proteins that initiate downstream signal transduction, culminating in cell survival, proliferation, differentiation, and motility (Bourette and Rohrshneider, 2000). However, in general, it is poorly understood how these pathways regulate these specific processes, particularly differentiation, and this is likely because most experiments have been conducted with mature MOs, or non-MO cells engineered to overexpress wild-type and mutant c-Fms protein (Pixley and Stanley, 2004). 10 1.7 MATURE MACROPHAGE FUNCTION: DISTINCT PROGRAMS REVOLVING AROUND L-ARGININE METABOLISM It is now appreciated that macrophages demonstrate remarkable heterogeneity and plasticity as exemplified by their ability to orchestrate both inflammation and its resolution (Mosser, 2001; Ma et al., 2003). While macrophages play important roles in host defense and homeostasis, overzealous macrophage responses contribute to host pathology in a wide array of human diseases (Gordon, 2003; Noel et al., 2004). Thus, it is crucial to understand and characterize the origins and control of macrophage heterogeneity in order to prevent disease and to harness macrophage plasticity for therapeutic applications. To this end, attempts have been made to characterize distinct macrophage subsets. This began with the description of 'classical' macrophage activation resulting from exposure to microbes or microbial-derived products, such as intracellular mycobacteria or lipopolysaccharide (LPS) of Gram-negative bacteria (Mackaness, 1964), and extended to exposure in the context of the T helper 1 ( T H 1 ) cell-derived cytokine, interferon-y (IFN-y) (Dalton et al., 1993). Classical MO activation is characterized by enhanced antigen presentation and antimicrobial activity, mediated by the production of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS)-generated nitric oxide (NO) (MacMicking et al., 1997; Bogdan, 2001; Hibbs et al., 2002). iNOS uses the amino acid, L-arginine, as a substrate and first oxidizes its guanidino nitrogen to produce Nw-hydroxy-L-arginine (NOHA) as an intermediate. Further oxidation of NOHA yields NO + L-citrulline (MacMicking et al., 1997). The reaction requires 2 additional co-substrates, molecular oxygen (0 2) and reducing equivalents in the form of NADPH, and five cofactors or prosthetic groups including FAD, FMN, calmodulin (CaM), tetrahydrobiopterin (BH4), and heme (Marietta, 1994; Nathan and Xie, 1994). As opposed to endothelial NOS (eNOS) and neuronal NOS (nNOS) which are constitutively expressed, low-output, elevated-Ca2+-sensitive 11 isoforms, iNOS is able to induce high-output, Ca -independent NO synthesis (likely due to the ability of iNOS to recruit CaM at resting Ca 2 + cellular concentrations) (MacMicking et al., 1997). Finally, iNOS is thought to impart classically activated MOs with cytostatic and cytotoxic properties as NO is a freely diffusible gas that reacts with a plethora of critical intracellular targets, leading to covalent modifications and oxidation events which halt cellular proliferation and metabolism, or even kill target cells (Stamler, 1994; Bogdan, 2001). The sequence of events leading to iNOS induction by LPS is presented in Fig. 1.2 and is discussed further in Chapter 3. In contrast to the aforementioned classical MO activation, MOs exposed to T H2 type cytokines, like IL-4 and IL-13, assume an 'alternative' activation phenotype (Stein et al., 1992) characterized by upregulation of broad-specificity pattern recognition and scavenger receptors, anti-inflammatory cytokines and chemokines, reduced NO synthesis due to L-arginine substrate competition with arginase, factors involved in tissue remodeling, wound healing, and angiogenesis, and novel secreted proteins Ym1/2 and FIZZ1 (Goerdt and Orfanos, 1999; Gordon, 2003) (Fig. 1.3). While Gordon has proposed limiting the definition of alternative macrophage activation to that induced by IL-4 and IL-13, others have broadened the definition to include those induced by IL-10, TGF - P , and glucocorticoids (Goerdt and Orfanos, 1999; Mantovani et al., 2004) rather than classify the latter macrophages as 'deactivated'. Adding complexity to the picture, engagement of receptor tyrosine kinases such as RON/STK and Mer, which recognize macrophage stimulating protein (MSP) and ligands associated with apoptotic cells, respectively, lead to phenotypes that overlap somewhat with alternative macrophage activation (Morrison et al., 2002; Correll et al., 2004; Freire-de-Lima et al., 2000; Scott et al., 2001; Tietzel and Mosser, 2001). Moreover, macrophage immune responses can also be altered by immune complex engagement of Fey receptors (Anderson and Mosser, 2002a; Anderson and Mosser 2002b) or by the intracellular thiol redox status (Murata et al., 2002). Finally, Mills and colleagues have taken a genetic approach and proposed that macrophages be classified as 'M-1' or 'M-2' 12 Fig. 1.2. LPS-induced MyD88-dependent and - independent signal transduction in M1 macrophages leads to pro-inflammatory cytokine and NO synthesis. depending on their propensity for arginine metabolism at the iNOS/arginase axis, to the killing/healing intermediates, NO/ornithine, as exemplified by macrophages from prototypical TH1 and T H2 strains of mice, respectively (Mills et al., 2000; Mills, 2001) (Fig. 1.3). However, as Mills and others concede (Gordon, 2003; Hume, 2000), such attempts at classification may result in oversimplification, and rather than succeed at identifying clonally separate macrophage populations they may likely point toward a developmental continuum of phenotypes between classical and alternative, M-1 and M-2. Nonetheless, recently the M1/M2 (dashes will now be excluded, ie, M1, not M-1) paradigm has gained acceptance as an operationally useful description of polar MO phenotypes (Mantovani et al, 2004). 13 t Collagen production Fig . 1.3. Differential L-arginine metabolism as an axis for M 1 versus M 2 macrophage phenotypic characterization. Macrophages are able to mount distinct M1 or M2 programs that utilize L-arginine for cytotoxic NO production, or healing intermediate production, respectively. Moreover, while M1 macrophages produce pro-inflammatory cytokines like IL-1, M2 counterparts produce anti-inflammatory mediators such as the IL-1 receptor antagonist (IL-1Ra). Please see Section 1.7 for a more detailed description. Despite this, others have suggested that determinist attempts to define macrophage subpopulations are futile since they propose macrophage heterogeneity results from gene-autonomous transcriptional probability of individual inducible genes (Hume, 2000; Ravasi et al., 2002). However, while it seems most likely that the actual mechanisms generating macrophage heterogeneity are complex (Rutherford et al., 1993; Hume et al., 2002; Stout and Suttles, 2004), definitive studies to resolve this controversy are lacking (Gordon, 2003; Stout and Suttles, 2004). Thus, there is an urgent requirement to unravel the mechanisms leading to macrophage heterogeneity in order to better understand and target macrophages in disease pathogenesis and therapeutics. 14 1.8 THE SH2-C0NTAINING INOSITOL 5'-PHOSPHATASE, SHIP The Src homology 2 domain (SH2)-containing inositol 5'-phosphatase (SHIP) is a hemopoietic cell-restricted, 145 kDa protein that is activated and tyrosine phosphorylated upon stimulation by multiple cytokines, and engagement of immunoglubulin Fc, B-cell or T-cell receptors (Damen et al., 1996; Lioubin et al., 1996; Ono et al., 1996). SHIP is recruited to tyrosine phosphorylated, plasma-membrane associated receptors and proteins where it negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway, at least in part by hydrolyzing the 5'-phosphate group of the critical PI3K-generated second messenger, PI-3,4,5-trisphosphate (PIP3) . In turn, this reduces the ability of pleckstrin homology (PH) domain-containing proteins such as protein kinase B (PKB)/Akt or Pl-dependent protein kinase 1 (PDK1), to be recruited to and activated at the plasma membrane (Krystal, 2000; Rohrschneider et al., 2000) (Fig. 1.4). SHIP has been demonstrated to be a master negative regulator of the immune system, controlling hemopoietic cell survival, proliferation, differentiation, and end cell activation (Krystal, 2000; Kalesnikoff et al., 2003). Targeted disruption of SHIP in mice results in multiple hemopoietic abnormalities, including chronic and infiltrative myeloid hyperplasia (Helgason et al., 1998; Liu et al., 1999), atopic, enhanced mast cell degranulation (Huber et al., 1998), B-cell hyperactivity associated with splenomegaly, lymphadenopathy, and elevated serum immunoglobulins (Brauweiler et al., 2000), perturbed natural killer (NK) cell development and deficient allograft rejection (Wang et al., 2002), and severe osteoporosis owing to the action of an increased number of Paget-like, hyperresorptive osteoclasts (Takeshita et al., 2002). The human form of SHIP has been cloned (Ware et al., 1996), and interestingly, perturbations in SHIP expression and activation have been implicated in human hemopoietic disorders which parallel the aforementioned SHIP-knockout phenotypes (Sattler et al., 1999). 15 degranuiation entry cell ceil into survival morphology S phase cell migration Fig. 1.4. Structure and function of SHIP. SHIP contains: 1) a C-terminal proline-rich region which facilitates interactions with SH3-containing proteins, 2) two NPXY motifs which become tyrosine-phosphorylated and recruit other proteins with PTB domains, 3) an N-terminal SH2 domain which recruits SHIP to tyrosine-phoshorylated sites of cytoplasmic receptor tails or intracellular proteins, and 4) a 5'-phosphatase (Ptase) domain which catalyzes the hydrolysis of PIP 3 to P IP 2 , and thereby dampens signaling pathways emanating from PIP 3 , including those which affect degranuiation (PLC-y), translation control and cell cycle entry (p70S6K), cell morphology and migration (Vav), and cell survival (GSK3, BAD, FKHR). 1.9 EXPERIMENTAL RATIONALE AND AIMS OF STUDY Prior to the commencement of this thesis, and despite much knowledge that had been gained on the role of SHIP in other hemopoietic cells, almost nothing was known about the role that SHIP played in macrophages. This was surprising given that the most obvious phenotype of the SHIP-/- mouse was a myeloproliferative disorder characterized by enhanced numbers of monocytes and MOs. Moreover, infiltration by these cells of vital organs, notably the lungs, was thought to contribute to the early mortality of SHIP-/- mice (Helgason et al., 16 1998). Thus, it appeared that there was much to gain by studying the role that SHIP plays in MOs. Specifically, I sought to determine: 1) why the absence of SHIP results in the expansion of this lineage and 2) what effect the absence of SHIP has on the phenotypic characteristics of MOs and 3) what effect the absence of SHIP in MOs has on the pathology observed in SHIP-/- mice. With respect to point 1), although it appeared that the absence of SHIP resulted in hypersensitivity of bone marrow progenitors to myeloid differentiation factors (Helgason et al., 1998), it had not been formally proven that cells in the resultant colonies were bona fide macrophages. Thus, our first hypothesis was that SHIP restrained macrophage differentiation. Just prior to the commencement of this thesis, it had been reported that the PI3K inhibitor, wortmannin, augmented LPS-induced NO secretion in peritoneal macrophages (Park et al., 1997). This piqued our interest, as it was unusual for the PI3K pathway to be implicated as a negative regulator (Krystal, 2000; Rohrschneider et al., 2000). Although it was appreciated PI3K was activated downstream of LPS in macrophages, the mechanisms involved in control of NO production remained to be fully elucidated. Since LPS-induced NO production was known to be a feature of classical macrophage activation, we thought that this would be an excellent starting point for comparing the phenotype of SHIP+/+ and SHIP-/- macrophages. Thus, our second hypothesis was that SHIP was a positive regulator of NO production in murine macrophages. Given the plastic and adaptable nature of MOs and their central roles in orchestrating inflammation and its resolution, our third hypothesis was that the phenotype of SHIP-/- MOs in vivo may be both a response to the environment of the SHIP-/- mouse and contribute to the pathologies observed therein. During the 6 years of this study, we had the good fortune to witness and participate in the growing body of knowledge surrounding macrophage innate immune responses and alternative activation states of these cells. As will become apparent, many of the findings that we initially classified as unexpected or paradoxical, later seemed more logical as an appreciation was gained for the influence of the environment on these plastic and adaptable cells. 17 CHAPTER 2 - MATERIAL AND METHODS 2.1 LPS, CYTOKINES, REAGENTS AND ANTIBODIES E. coli LPS from serotype 0127:B8, human serum and plasma, L-arginine, sulfonylamide, naphthylethylene diamine HCI, urea, isonitrosopropriophenone, MgCI2, and DMSO were purchased from the Sigma Chemical Company (St. Louis, MO). Unless otherwise indicated, all other reagents were also obtained from Sigma. The cytokines IFN-y, IL-4, IL-6, IL-10, IL-13, TGF-B1, GM-CSF, and M-CSF were purchased from StemCell Technologies (Vancouver, BC), while MSP was from Research Diagnostics Inc. The PI3K inhibitors LY294002 and wortmannin were from Calbiochem (La Jolla, CA) while the arginase inhibitors L-nor-NOHA and L-nor-valine were from Alexis Biochem (USA). Polyclonal rabbit antibodies raised against the following proteins were used: c-Fms, iNOS, kBa, C/EBPB, IRF-1, Grb2 (Santa Cruz), ERK1 (Stressgen), phospho-(p)Akt(S473), pAkt(T308), Akt, pStat1(S727), pERK1/2, pp38, pNF-KB p65(S536), pStat6, pp70S6K, p4EBP1 (Cell Signaling), pStat1(Y701) (Zymed Laboratories), COX-2 (Caymen Chemicals), SHIP (Damen et al., 1998). Monoclonal mouse antibodies were used to detect Arginase I (BD Transduction) and GAPDH (Research Diagnostics Inc.). Biotinylated neutralizing chicken anti-mouse TGFB1 and goat anti-mouse IL-10 were purchased from R&D Systems and 50 pg of each, reconstituted at 0.2 mg/ml in PBS, were conjugated to 300 pi streptavidin-agarose beads (Pierce) according to the manufacturer's instructions. Finally, anti-Ym1 antibody was generated in rabbits using the peptide sequence GYTGENSPLYK and affinity purified, as described previously for the SHIP antibody (Damen et al., 1998). 2.2 MICE Wild-type and SHIP-/- mice on a mixed C57BI6 x 129Sv background or wild-type mice on a pure C57BI6 or Balb/c background were housed in a 18 pathogen-free animal facility according to approved and ethical treatment standards of the University of British Columbia. 2.3 TISSUE CULTURE 2.3.1 PERITONEAL MACROPHAGES Primary macrophages were obtained from SHIP +/+ and SHIP -/- mice by peritoneal lavage using standard techniques. Briefly, 1.5 ml of Brewer's thioglycollate medium (Sigma) was injected into the peritoneal cavity four days prior to harvest (for T/GPMOs), or not injected (for RPMO), and peritoneal exudate cells collected by lavage with 2 x 5 ml IMDM (StemCell Technologies) containing 10% FCS (Hyclone), 0.00125% (v/v) monothioglycerate (MTG) (Sigma) and 100 U/ml penicillin/streptomycin (StemCell Technologies) (herein referred to as PMO medium). Macrophages were identified within the total lavage population based upon size and morphology, counted and centrifuged for 5 min at 200 * g. Cells were resuspended to give a final macrophage concentration of 0.5 to 1.0 x 106 cells/ml, and were plated at 105 cells/well in 96-well plates, 0.5 x 106 cells/well in 12-well plates, or 2.0 x 106/well in 6-well plates. Macrophages were allowed to adhere for a few h to overnight and non-adherent cells removed by repeated washings. 2.3.2 ALVEOLAR MACROPHAGES Alveolar macrophages (AMOs) were obtained as follows: 1 ml pre-warmed PBS was injected into the lungs via the trachea with a 26.5-gauge needle and a 1 ml syringe (Becton Dickson), and retrieved as bronchoalveolar lavage (BAL) fluid. The collected BAL was centrifuged at 200 * g and cells resuspended with 1-2 ml of PMO medium and plated on 12-well plates. AMOs were selected by adherence after repeated washings. 19 2.3.3 BONE MARROW PROGENITORS AND MACROPHAGES Bone marrow-derived progenitors and macrophages were obtained as follows: cells were flushed from the femurs and tibiae of each mouse using 40 ml PMO medium, plated in a 75 cm 2 Nunclon flask, and stromal cells were allowed to adhere for 3 h to overnight at 37°C. The non-adherent hemopoietic progenitor cells were then collected and resuspended in 60 ml PMO medium supplemented with 2% C127 cell-conditioned media (CM) (as a source of M-CSF) or 5 ng/ml recombinant mouse M-CSF (referred to as BMMO medium) in 175 cm 2 Nunclon flasks for 7-14 days at 37°C, with half medium changes at days 5 and 10. For experiments in which in vitro attempts were made to mimic the in vivo environment of macrophage differentiation, the aforementioned bone marrow progenitor cells were cultured for 7 days in BMMO medium without medium change at 0.5-2.0 x 106 cells/ml in 24-well plates, with or without the addition of cytokines, mouse or human serum or plasma at various stages of differentiation. Alternatively, for isolation of primitive bone marrow progenitors the aforementioned bone marrow cells from SHIP+/+ and -/- mice were treated with FITC-conjugated primary anti-lineage (Lin) marker (Mac-1, Gr-1, B220, Ter119, CD2) antibodies and PE-conjugated anti-Seal (as described in Section 2.10 below), and the Sca1+Lin- population was selected by FACS sort using a FACSort instrument (Becton-Dickson). Sca1+Lin- cells were then counted and added to Methocult GF M3434 methylcellulose medium (1% methylcellulose in IMDM, 15% fetal bovine serum, 1% bovine serum albumin (BSA), 10 mg/mL recombinant human (rh) insulin, 200 mg/mL human transferrin (iron - saturated), 10"4 M B-mercaptoethanol (B-ME), 2 mM L-glutamine, 50 ng/mL recombinant murine (rm) SF, 10 ng/mL rm IL-3, 10 ng/mL rh IL-6, 3 units/mL rh erythropoietin) (StemCell Technologies) in a 1:10 (v/v) ratio. Alternatively, cells were added to Methocult minus cyokines (M3234) but containing 10 ng/ml M-CSF. The mixture was then plated at 1.5 * 104 cells/well of 6 cm diameter plates using a 16-gauge blunt needle and a 10 ml syringe, and cultured at 37 °C for 0, 3, or 6 days prior to 20 counting visible colonies and harvesting for total cell counts and Mac-1 FACS (see below). 2.4 GENERATION OF MACROPHAGE CELL LINES Second filial generation (F2) C57BL/6J backcrosses of SHIP-/- mice and their wild-type littermates (Helgason et al., 1998) were used in the generation of macrophage cell lines and in the isolation of primary macrophages. Briefly, fresh bone marrow was isolated from the femurs and tibiae of 4- to 6-week-old mice and 1-5 x 107 cells were infected with the recombinant v-raf/v-myc retrovirus, J2, by resuspending in MJCREJ2 cell supernatant and 100 ng/ml of M-CSF (ONX packaging cells and ,+'CREJ2 cells had been maintained in DMEM + 10% FCS + 200 mM L-glutamate +100 U/ml penicillin streptomycin (Wessells et al., 2004)). After 24 h at 37°C, the supernatant was removed and the cells were maintained in PMO supplemented with 100 pg/ml of dextran-based cytodex 1 beads (CT beads) (Pharmacia) and M-CSF (2% C127 CM) for a further 5 d (Blasi et al., 1989). SHIP+/+ and SHIP-/- J2-macrophage (J2M) clones were subsequently isolated and maintained in PMQ> medium (with no M-CSF). 2.5 NITRIC OXIDE ASSAY NO production was determined indirectly by measuring the accumulation of the stable end product, NO2" in the tissue culture supernatant using a modification of the Griess reaction (Griess, 1879; Stuehr and Nathan, 1989). Briefly, 50 pi of supernatant was sequentially incubated with equal volumes of 1% sulfanilamide and 0.1% phenylnapthylethylenediamine dihydrochloride (both in 2.5% phosphoric acid) at 23°C. After 10 min, the absorbance of samples was determined at 570 nm and nitrite concentrations determined by comparison with a NaN02 standard curve. 21 2.6 ARGINASE ASSAY Arginase activity was assessed indirectly by measuring the concentration of urea generated by the arginase-dependent hydrolysis of L-arginine, as described by Corraliza et al. (1994). In brief, cells were lysed in a 1:1 mixture of 0.1% Triton X-100 and 25 mM Tris-HCl with 10 pg/ml aprotinin, 10 pg/ml leupeptin and 0.5 mM PMSF protease inhibitors. Protein concentration was determined using Bradford assay (BioRad) and lysates were subjected to SDS-PAGE or arginase assay. For arginase assay, the following reagents were sequentially added to 100 pi of the cell lysates: 10 pi of 10 mM MnCI2 at 55°C for 10 min to activate endogenous arginase, 100 pi of 0.5 M L-arginine (pH 9.5) at 37°C for 60 min to allow for hydrolysis of L-arginine, 800pl of H2SO4/H3PO4/H2O (1:3:7) to stop the reaction and 40 pi of 9% a-isonitrosopropiophenone at 100°C for 30 min to react with urea. The absorbance was read at 550 nm and the results compared to a urea standard curve. 2.7 ELISAs Mouse plasma or tissue culture supernatants of resting or stimulated macrophages were assessed for protein levels of TNFa, IL-6, IL-10, and IL-12 by ELISA (BD Biosciences, Mississauga, ON) according to the manufacturer's instructions. Where indicated, additional mouse plasma analysis was conducted using a FACS-based cytometric bead array IL-6, IL-10, IL-12, TNFa, IFNy and CCL2/MCP-1 Mouse Inflammation ELISA kit (BD Biosciences), according to the manufacturer's instructions. 2.8 CYTOPLASMIC AND NUCLEAR EXTRACT PREPARATION AND EMSA ANALYSIS Cells were treated, or left unstimulated, and following the indicated times, harvested from tissue culture plates. Briefly, cells were placed on ice, medium 22 was aspirated or collected, and cells were washed three times in ice-cold PBS. On the third wash, cells were gently scraped from the plate using a rubber policeman (Falcon), resuspended in the final wash volume with a pipette, and spun in a Heraeus #3325 microfuge at 500 * g and 4°C for 5 min. PBS wash was next aspirated and the cell pellet either frozen at - 70°C for future use, or placed directly in 0.5 ml/ 106 cells of hypotonic Buffer A [0.2% NP40, 10 mM MgCI2, 10 pg/ml aprotinin, 10 pg/ml leupeptin, and 0.5 mM PMSF in phosphorylation solubilization buffer (PSB) (Liu et al., 1994)(50 mM HEPES, 100 mM NaF, 10 mM NaPPj, 2 mM NaV0 4, and 4 mM EDTA)]. Cells were centrifuged 500 * g and 4°C for 3 min and the supernatant set aside as the cytoplasmic extract. The pellet was washed in an equal volume of Buffer B (0.05% NP40, 10 mM MgCI2, 0.25 M sucrose, 10 pg/ml aprotinin, 10 pg/ml leupeptin, and 0.5 mM PMSF in PSB) and centrifuged as with the Buffer A step. The supernatant was discarded, and the nuclear pellet resuspend in 50 pl/106 cells of high-salt Buffer C (0.1% NP40, 0.4 M NaCl, 10 pg/ml aprotinin, 10 pg/ml leupeptin, and 0.5 mM PMSF in PSB) followed by nutation on an Adams nutator (#1105) at 4 °C for 30 mins to 1.5 h. Nuclear extracts were then centrifuged for 15 min at 4°C and 16,000 * g. The supernatant was stored at - 70°C. For electrophoretic mobility shift analysis (EMSA), 2 pmol of oligonucleotide probe containing the N F - K B consensus binding sequence (KB element) (Santa Cruz) were end-labeled with 50 pCi [ a32]-P ATP (New England Nuclear) using T4 polynucleotide kinase (GibcoBRL) for 1 h at 37°C. Following phenol/chloroform extraction and ethanol precipitation, the probe was resuspended in 100 pi TE (10 mM Tris (pH 7.4), 1 mM Na 2EDTA (pH 8.0)). Nuclear extracts, 3 pl/reaction (or 6 x 105 cell-equivalents) were incubated at 23°C for 30 min with 7 pi of probe/dldC/binding buffer mix [per 7 pi: 1 pi 3 2 P-labelled probe, 1 pi poly-dldC (Pharmacia), and 5 pi 2* binding buffer ( 20 mM Tris, 200 mM KCI, 20 mM MgCI2, 0.1% NP40, 4 pM DTT, and 0.05% bovine serum albumin (BSA) (Sigma)) in a 1.5 ml Eppendorf tube. Samples were electrophoresed on acrylamide-TBE gels [3.75% acrylamide (37.5:1), 1* TBE (10.8 g/L Tris, 55 g/L boric acid, and 2 mM EDTA) at 150 V for 2.5 to 3 h, in 23 0.25* TBE using a Protean II gel tank (BioRad). Gels were then transfered to Whatman filter paper using a gel drying apparatus (BioRad), and either developed by autoradiography on Kodak X-OMAT film or with Phosphorlmager technology. In addition, cytoplasmic and nuclear extracts were analyzed for protein concentration by the Bradford assay and were subjected to SDS-PAGE and Western blot analysis as described below. 2.9 SDS-PAGE AND WESTERN BLOT ANALYSIS Where indicated, total cell lysates were prepared by boiling cell pellets for 5 min in 1 x SDS-PAGE sample buffer. Alternatively, for arginase assay lysates SDS-PAGE sample buffer was added to a final concentration of 1*, and samples prepared as above. For Western blot analysis, cell equivalents (total cell lysates) or equivalent Bradford quantitated protein amounts (arginase assay lysates) were loaded/well and separated by SDS-PAGE. Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon) using a BioRad wet transfer apparatus, and blocked for 1 h at 23°C or overnight at 4°C in 5% BSA in TBST [20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM KCI, and 0.01% Tween 20]. Western blots were performed as described previously (Damen et al., 1998). Briefly, blots were incubated in a 1:1000 dilution of primary antibody (unless otherwise indicated) in 2% BSA/0.1% azide in TBST, for 1 h at 23°C or overnight at 4°C. Blots were next washed for 4 * 5 min with TBST, and transfered to 1:10,000 dilution of the appropriate secondary antibody conjugated to HRP in TBST. Following another 4 * 5 min TBST wash, specific binding to target proteins was detected using the Enhanced Chemiluminescence system (ECL) and autoradiography on Kodak X-OMAT film. After exposure, blots were routinely washed in TBST, stripped for 30 min at 50°C in stripping buffer [62.5 mM Tris-HCl (pH 7.2), 2% SDS, and 100 mM B-mercaptoethanol, B-ME), blocked and reprobed as above. 24 2.10 FACS ANALYSIS Macrophages or differentiating Sca+Lin" cells, at a density of 5-10 x 106 cells/ml were incubated on ice for 30 min with 3 pg/ml 2.4G2 (murine anti-IgG Fc receptor antibody) followed by incubation on ice for 40 min with the various FITC-labeled or phycoerythrine-conjugated antibodies or controls. Cells were washed twice in Hank's balanced salt solution (HBSS) (StemCell) containing 2% FBS at 4°C, and propidium iodide (PI) (Sigma) was included at a concentration of 1 pg/ml in the final wash. Cells were analyzed on a FACStar+ or FACSort (Becton-Dickson). Monoclonal antibodies used for FACS analysis were obtained from commercial sources and used according to the manufacturer's instructions: Mad , Mac2, Gr-1, Ter119, CD2, B220 (BD Transduction), TLR4, F4/80, CD204, CD206 (Serotec). Alternatively, for intracellular Ym1 FACS, SHIP+/+ and -/- total peritoneal lavage cells were intracellulary stained with isotype control or 1/25 anti-Ym1 using Fix and PermTM kit (Caltag Laboratories, Burlingame, CA), according to the manufacturer's instructions, and PMOs were analyzed using the side versus forward scatter gates established by Cook et al. (2003). Finally, for flow cytometric analysis of apoptosis (DNA fragmentation), cells were resuspended at 106 cells/ml in lysis buffer (0.1 % sodium citrate, 0.1 % Triton X-100, pH = 8) containing 20 pg/ml PI (Maguer-Satta et al., 1998) and analyzed as described previously (Nicoletti et al., 1991). 2.11 RNA PREPARATION, RNASE PROTECTION ANALYSIS, NORTHERN BLOT ANALYSIS AND RT-PCR RNA was isolated from fresh or frozen cell pellets using 1 ml TRIzol/107 cells as per the manufacturer's protocol (GibcoBRL). RNA was resuspended in DEPC-treated water and quantified by absorbance at 280/260 nm prior to use. Ribonuclease (RNase) protection analysis (RPA) of cytokine mRNA levels was performed using the RiboquantTM Multi-Probe RPA (BD Pharmingen) according 25 to the manufacturer's instructions. Briefly, 2-10 ug of total RNA was hybridized overnight to [a33P]-dUTP (NEN)-labelled anti-sense RNA probes generated from DNA template sets mCK2 or mCK3 (BD Pharmingen) by in vitro transcription. Excess probe and single-stranded RNA were then degraded by RNase treatment, while protected species were phenol-chloroform extracted and ethanol washed, resolved on denaturing polyacrylamide gels, and detected by autoradiography on large Kodak X-OMAT film. Northern blot analysis of RNA was performed as follows: 10 pg of total RNA in a volume of 5 pi was incubated with 0.6 pi of 10x MOPS (morpholinepropanesulfonic acid) buffer (200 mM MOPS, 50 mM sodium acetate, and 10 mM EDTA), 2 pi deionized formaldehyde, and 6 pi deionized formamide for 10 min at 65 C. Samples were chilled on ice, and 1.5 pi of loading dye (50% glycerol, 1 mM EDTA, 1 mg/ml xylene cyanol, and 1 mg/ml bromophenol blue) added. Samples were electrophoresed on formaldehyde gels (1% agarose, 1* MOPS, 5% deionized formaldehyde, and 0.5 pg/ml ethidium bromide) at 75 V in 1* MOPS for 2 h. Following electrophoresis, gels were washed briefly in distilled water, followed by one gentle, 20 min incubation in 10* SSC (1.5 M NaCl, 0.17 M sodium citrate). Gels were transferred overnight to Hybond-N membranes (Dupont) according to the method of Southern (1975) and UV cross-linked using a Stratalinker. Random-primed DNA probes were prepared according to the method of Feinberg and Vogelstein (1983), using the iNOS EcoRI cDNA fragment from pCLBS (provided by Dr. W. J. Murphy, University of Kansas) or a GAPDH cDNA fragment (provided by Dr. R. K. Humphries, Terry Fox Laboratory). [a-32P]CTP-labelled (NEN) probes were added to 8 ml hybridization buffer (50% deionized formamide, 5% SDS, 0.5 M NaH 2P0 4 , 1 mM EDTA, 1 mg/ml BSA, and 0.5 mg/ml ssDNA) and incubated with the Hybond-N membranes overnight. Membranes were washed 2 * 20 min each with 2xSSPE/0.3% SDS, 1xSSPE/0.5% SDS, and 0.3x SSPE/1.0% SDS at 55 C, then blotted dry, and DNA-RNA hybrids detected by autoradiography on Kodak X-OMAT film. 2 6 Reverse transcription reactions were performed using oligo (dT)-is and MMLV reverse transcriptase (Stratagene). PCR reactions were carried out using previously described primers and conditions specific for arginase I, B-actin (Morrison et al. 2002) and Ym1 (Nair et al., 2003), but cycle numbers were optimized for our own laboratory. Negative controls were minus reverse transcriptase and minus template. Black-white inverted images of PCR bands were obtained using a Kodak Digital Science DC40 Camera mounted to a Fisher Biotechnology Transilluminator. 2.12 T R A N S I E N T T R A N S F E C T I O N S SHIP +/+ and -/- J2M macrophages were seeded at a density of 5 x 106 cells/10 cm plate and transiently transfected using a modification of the DEAE-Dextran technique (McCutchan and Pagano, 1968). Briefly, 5 pg of the vector pGLH/H2 (Lowenstein et al., 1993), containing the luciferase reporter gene (Promega) driven by the wild-type mouse iNOS promoter (provided by Dr. W. J. Murphy, University of Kansas), and 1 pg of the lacZ reporter vector (from Dr. A. Mui, Jack Bell Vancouver Coastal Health Research lnstitute)/10 cm plate, were incubated with DEAE-dextran, at a final concentration of 0.5 mg/ml, in 3 ml IMDM-0.5* PBS for 15 min at 23°C. Following two washes of the cells with IMDM, the DNA/DEAE-dextran mixture was added and allowed to incubate for 15 min at 23°C. The mixture was then aspirated, and 3 ml of cold 20% glycerol was added for 2 min. Cells were washed once with IMDM, and placed in PMQ> medium overnight. The following day, cells were either left untreated or stimulated with 100 ng/ml LPS for 18 h. Lysates were prepared as per the manufacturer's protocol using a luciferase assay kit or dual-reporter luciferase kit (Promega), and firefly luciferase activity measured in 96-well assay plates using a luminometer (Turner Designs). Results were normalized for transfection efficiency using CMV- or TK-driven Renilla luciferase activity from the dual-luciferase kit. 27 SHIP +/+ peritoneal macrophages were seeded at a density of 5 * 105 cells/well in a 12-well plate and transiently transfected using a modification of the DEAE-Dextran technique described above. Briefly, 0, 2.5, or 5 pg of a vector containing dominant active PI3K (pSG5-myc-p110aCAAX) (provided by Dr. J. Downward) was incubated with DEAE-dextran, at a final concentration of 0.5 mg/ml, in 2 ml IMDM-0.5x PBS for 15 min at 23°C. Following two washes of the cells in IMDM alone, the DNA/DEAE-dextran mixture was added and allowed to incubate at 15 min at 23°C. The mixture was then aspirated, but the 2 min cold-shock step (i.e. 3 ml of cold 20% glycerol) was omitted. Cells were then washed once with IMDM and placed in PMO medium overnight. The following day, cells were lysed for arginase assays and Western analyses as described elsewhere in section 2.9. 2.13 MURINE TUMOR MODEL Six- to ten-week old wild type and SHIP-/- mice were subcutaneously injected with 2 x 105 M27 Lewis lung carcinoma cells. At appropriate intervals, tumor volume was determined using calipers and the formula for volume of an ellipsoid. Tumors were also harvested from several mice at day 17, minced with scissors and protein lysates prepared by homogenizing the tissue in 0.5 % NP-40 detergent in phosphorylation solublization buffer (PSB) plus protease inhibitors (Damen et al. 1998), using a syringe and progressively higher gauge needles (finest gauge was 26). Equivalent protein amounts were then prepared by boiling in 1 x final SDS-PAGE sample buffer, and subjected to Western blot analysis. 2.14 LPS SEPSIS MODEL Five week-old wild-type and SHIP-/- mice were injected intraperitonealy (IP) with 40 mg/kg of LPS or PBS vehicle control and survival followed over the course of 60 h. 28 2.15 PLASMA AMINO ACID ANALYSIS Pooled blood was obtained by cardiac puncture from overnight-fasted 5-6-week old wild-type and SHIP-/- mice, collected in Li-heparinized tubes and centrifuged at 1500 x g for 10 min at 4°C to obtain plasma. Sulfosalicylic acid (SSA) was added at 25 mg/500 ul of plasma to precipitate proteins, and the mixture was centrifuged at 1500 x g for 10 min at 4°C. Filtrates were collected and stored at -20°C for subsequent analysis by HPLC at the BC Children's Hospital Biochemical Diseases Laboratory. 2.16 LUNG HISTOLOGY AND BAL ANALYSIS Lungs were collected from wild-type and SHIP-/- mice, formalin fixed, ethanol washed, and paraffin embedded. Lung sections were obtained and stained with Masson's trichrome (MSB) or hematoxylin and eosin (H&E), using standard manual techniques (UBC Pathology). Photomicrographs of stained histological sections or BALF and cells were obtained using a Qlmaging QICAM Fast 1394 Cooled Colour 12-bit digit camera mounted on a Leica DMIL microscope, at 200 or 400 x original magnification. 2.17 LUNG CRYSTAL ISOLATION AND MASS SPECTROSCOPY ANALYSIS Crystals were purified from the lungs of SHIP-/- mice as previously described for MeV mice (Guo et al. 2000). 3 ml of BAL fluid (BALF) from SHIP-/-mice were mixed with 6 ml of Ficoll-Paque Plus (5.7 % w/v Ficoll 400 and 9.0 % diatrizoate sodium at density of 1.077 g/ml) (StemCell) and centrifuged at 250 x G for 5 min in a Beckman tabletop centrifuge. The Ficoll-Paque layer and first 2.8 ml of PBS were aspirated, the crystals resuspended in 2.8 ml fresh PBS followed by 6 ml of Ficoll-Paque, and the process was repeated 3 to 4 more times. Crystals were then boiled for 5 min in 1 x SDS sample buffer and subjected to SDS-PAGE, along with cell-free BALF from SHIP+/+ and -/- mice. 29 The purified 45 kDa band was excised and subjected to in-gel trypsin digestion and subsequent MALDI-TOF MS and ESI MS/MS at the UBC Laboratory of Molecular Biophysics Proteomics Core Facility (Vancouver, BC). 2.18 PERIPHERAL BLOOD PLASMA, LEUKOCYTE, AND MONOCYTE ISOLATION Peripheral blood was obtained via cardiac puncture into lithium-heparinized tubes immediately following carbon dioxide culling of adult SHIP+/+ and SHIP-/- mice. Following centrifugation for 10 min at 23°C in a Beckman micro-centrifuge at 2000 rpm, plasma was removed by pipette and placed at -70°C in Eppendorf tubes. The cell pellet was resuspended in PBS + 2mM EDTA + 2% FCS and red cells lysed by adding ammonium chloride (StemCell Technologies) at a 9:1 ratio (ammonium chloride:cells) for 10 min on ice. The lysis was quenched with PBS/EDTA/FCS and cells were washed once to remove residual red cell debris. This resulted in the isolation of peripheral blood leukocytes (PBLs). When monocytes were desired, the PBL fraction was subjected to negative selction for PB monocytes using the StemCell Technologies SpinSep system, according to the manufacturer's directions. Briefly, anti-CD2, -CD5, -CD19, and -F4/80 antibodies conjugated to dense beads were incubated with the PBLs, followed by washing, and layering over a density medium, which effectively depleted the PBLs of lymphocytes, eosinophils and granulocytes, leaving monocytes to be extracted from the interface, and washed. 30 CHAPTER 3 - THE ROLE OF SHIP IN MACROPHAGE DIFFERENTIATON AND FUNCTION IN VITRO 3.1 INTRODUCTION Prior to the commencement of this thesis, homozygous targeted disruption of the SHIP gene was achieved in a mouse model (Helgason et al., 1998). While SHIP-/- mice were viable and fertile, they demonstrated reduced survival associated with a myeloproliferative disorder, and in particular with the infiltration of vital organs by cells of the monocyte-macrophage lineage (Helgason et al., 1998; Liu et al., 1999). It was a goal of this chapter to gain an understanding of the basis for this myeloproliferative disorder and to test the hypothesis that SHIP restrains macrophage differentiation. Thus, hemopoietic progenitors were isolated from the bone marrow of SHIP+/+ and -/- mice, and in vitro attempts were made to recapitulate in vivo macrophage differentiation and to study the mechanisms involved in aberrant SHIP-/- myelopoiesis. Macrophages have the following key functions: 1) a) recognition and b) phagocytosis of 'infectious non-self and 'damaged self; 2) innate, direct mediation of microbicidal and tumoricidal effector responses; 3) direct and indirect orchestration of inflammation and its resolution; and 4) antigen presentation and bridging to the adaptive immune response (i.e. T cell stimulation) (Bogdan, 2001b). The choice as to which area to focus on was influenced by an earlier publication that implicated PI3K in Gram-negative bacterial lipopolysaccharide (LPS)-induced macrophage nitric oxide (NO) production (a macrophage effector response) (Park et al., 1997). Thus, emphasis was initially cast on functions 1) a) and 2) above. Specifically, we sought to test the hypothesis that SHIP is a positive regulator of murine macrophage NO synthesis. In this Chapter, retrovirally-immortalized SHIP+/+ and -/- macrophage cell lines are initially used to test this hypothesis. Analysis is extended to primary bone marrow-derived macrophages. Moreover, a summary of the insights gained into the role of SHIP during in vitro macrophage differentiation and function will be presented. 31 3.2 RESULTS 3.2.1 THE ROLE OF SHIP DURING IN VITRO MACROPHAGE DIFFERENTIATION The initial characterization of the SHIP-/- mouse phenotype revealed that the bone marrow and spleen of these mice contained elevated levels of granulocyte-macrophage colony forming cells (CFC) (Helgason et al., 1998), perhaps as a result of their hyper-responsiveness to the myeloid growth factors IL-3, IL-6, SF, GM-CSF and M-CSF, as demonstrated in vitro (Helgason et al., 1998; Liu et al., 1999). This was correlated in vivo with progressive, age-dependent increases in the number of splenic and circulating Mac-1 +Gr-1 + granulocyte-macrophage progenitors, increased proportion and number of peripheral blood monocytes and neutrophils, and consolidation of the lungs by massive infiltration of myeloid cells, particularly macrophages. Of note, these CFC studies were performed using total, heterogenous bone marrow cell aspirates. In order to gain further insight into the basis and nature of this myeloproliferative disorder, a more primitive and homogeneous population of bone marrow cells was chosen as a starting point. Thus, lineage marker (including Mac1)-depleted, Sca-1+ cells (Sca-1+Lin~) were FACS-sorted from total SHIP+/+ and -/- mice, as these have been demonstrated to contain pluripotent, long-term repopulating hemopoietic stem cells (LT-HSC) (Eaves, 2002). Sca-1+Lin" cells from both genotypes were then cultured in methylcellulose containing the myeloid growth factors Epo, IL-3, IL-6 and SF, and macrophage differentiation was assessed over time by FACS, using the Mac-1 surface marker. As can be seen in Fig. 3.1a (left panel), SHIP-/- macrophage differentiation was accelerated, compared to wild-type, when Sca-1+Lin" progenitors were exposed to a cocktail of myeloid differentiation factors. Moreover, as evident in Fig. 3.1b (left panel), this accelerated SHIP-/-differentiation was accompanied by a profound enhancement of cellular 32 Epo/IL-3/IL-67SF Epo/IL-3/IL-6/SF/TGF(J M-CSF 100 I T 80 a> aj 60 O +. 40 o (0 S 20 0 80 60 « 40 O otal 20 0 n/d n I 0 3 6 Epo/IL-3/IL-6/SF 0 3 6 Epo/IL-3/IL-6/SF/TGFp 3 M-GSF 6(d) I 0 6 0 6 0 6 (d) Fig. 3.1. Accelerated and enhanced macrophage differentiation of SHIP-/- bone marrow progenitors. Sca-1 + Lin ' BM progenitors were obtained from SHIP+/+ (black bars) and -/- (grey bars) mice and cultured for 0, 3, or 6 days in MethoCult G F M3434 (StemCell Technologies) containing Epo, IL-3, IL-6, and S F ± TGFB, or base MethoCult lacking the aforementioned factors but including M-CSF alone. Cells were subsequently harvested, counted (b), and subjected to Mac-1 F A C S as a marker of macrophage differentiation (a). Results shown are respresentative of at least 2 experiments (n/d = not determined). expansion. As methylcellulose colony sizes were not appreciably different between genotypes (as opposed to colony counts, which were increased in SHIP-/-), and as FACS-based carboxyfluorescein succinimidyl ester (CFSE) vital dye tracking revealed no difference in proliferation (data not shown), this suggested that enhanced SHIP-/- cell counts resulted from enhanced survival of SHIP-/- Sca-1+Lin" progenitors exposed to myeloid growth factors. Alternatively, or in addition, it may be possible that Sca-1+Lin" SHIP-/- progenitors display a skewed propensity for myeloid commitment. As previous studies conducted in this laboratory suggested that TGFB accelerated erythroid differentiation (Krystal et al., 1994), TGFB was included in the differentiation assay to determine if a similar phenomenon occurred with the 33 macrophage lineage. In contrast to the erythroid system, and as can be seen in Fig. 3.1a (middle panel), TGFB suppressed macrophage differentiation as assessed by Mac-1 surface staining. Moreover, TGFB suppressed cell numbers (Fig. 3.1b, middle panel), which abrogated the ability to measure Mac-1 surface staining on day 3. Thus, TGFB represses myeloid differentiation of Sca-1+Lin" progenitors in vitro, although in this setting the absence of SHIP still allows for augmented myeloproliferation relative to wild-type. Although exposure of Sca-1+Lin" progenitors to the myeloid cytokine cocktail, Epo/IL-3/IL-6/SF, does result in macrophage differentiation, it does so at least in part through the autocrine secretion and synergistic action of macrophage-colony stimulating factor (M-CSF) (Pixley and Stanley, 2004; Barreda et al., 2004). M-CSF alone cannot normally support the proliferation and differentiation of primitive progenitors. However, as revealed in studies performed using M-CSF-deficient mice (op/op mice), M-CSF has emerged as the critical factor for monocyte-macrophage lineage differentiation (Pixley and Stanley, 2004). Moreover, previous studies conducted in this laboratory have revealed an enhanced sensitivity of SHIP-/- BM aspirates to M-CSF-induced methylcellulose colony formation (Helgason et al., 1998). Thus, Sca-1+Lin" progenitors from wild-type and SHIP-/- mice were exposed solely to M-CSF. Although a similar pattern of enhanced cell-surface Mac-1 expression emerged in SHIP-/- cells at days 3 and 6 (compare Fig. 3.1a, right panel, to Epo/IL-3/IL-6/SF left panel), resultant cell numbers were significantly lower in M-CSF (Figure 3.1b, left) as opposed to the myeloid cocktail (Fig. 3.1b, right). Taken together, these results suggest that SHIP-/- Sca-1+Lin" bone marrow progenitors demonstrate accelerated and enhanced macrophage differentiation in response to typical, myeloid/macrophage cytokines, and that TGFB represses macrophage differentiation. While Mac-1 is expressed at highest levels on cells of the monocyte-macrophage lineage, it is by no means a specific marker since neutrophils and B lymphocytes are known to express Mac-1 (albeit at lower levels on B cells) (Cook et al., 2003). Thus, additional and more specific evidence of skewing to 34 macrophage differentiation was sought in SHIP-/- mice. Since primitive Sca+Lin" progenitors represent only a small fraction of total bone marrow cells (Eaves, 2002), and low starting numbers limit in vitro analysis, a more practical starting population was sought. As well, because M-CSF is a more commonly used macrophage differentiation-inducing factor (Cook et al., 2003), it was chosen for further analysis. Thus, wild-type and SHIP-/- Lin" bone marrow aspirate cells, (a more heterogeneous set of cells that still contain Sca-1+Lin" cells) were exposed to M-CSF in liquid tissue culture, and differentiation was assessed by SDS-PAGE and Western blot analysis of equal cell numbers harvested on successive days. As opposed to Sca-1+Lin" progenitors,'the utilization of more committed Lin" BM cells (or total BM) as a starting point did not result in a detectable acceleration of SHIP-/- macrophage differentiation (as assessed by c-fms, Gab2, and PU.1 protein expression) (data not shown). However, as was the case for more primitive Sca-1+Lin" cells, SHIP-/- Lin" BM yielded greater numbers of mature macrophages when cultured in M-CSF (Fig. 3.2a). Similarly, when total BM was used as a starting point, SHIP-/- macrophage yield was increased without the accelerated appearance or proportion of cells expressing macrophage differentiation markers (Fig. 3.2b and data not shown). Thus, SHIP-/-macrophage differentiation is not accelerated when more committed BM cells (Lin" BM, or total BM) are used as a starting point, despite enhanced macrophage yield. An alternative in vitro means of achieving macrophage differentiation is to first culture bone marrow in Flt3-ligand (FL) for 6 days before transferring the cells to M-CSF for a further 3 days (Bourgin et al., 2002). FL is reported to achieve myeloid progenitor expansion without terminal macrophage differentiation (Dannaeus et al., 1998; Nichols et al., 1999), although if cultured for prolonged periods in FL, cells do differentiate into dendritic cells (DCs) (Brasel et al., 2000). The combination of FL followed by M-CSF is reported to allow a greater yield of mature macrophages than with M-CSF alone. Thus, in order to expand upon the aforementioned results with M-CSF alone, SHIP+/+ and -/- BM cells were cultured in 5 ng/ml FL for 6 days and transferred to M-CSF-containing 35 a b Lin- BM 4 L i n * BM 5 o o 1 Time (d) in M - C S F 2 3 4 5 6 ° +/+ -/-7-day M - C S F culture Fig. 3.2. SHIP-/- macrophage differentiation is not appreciably accelerated from more committed bone marrow progenitors, despite enhanced yield. Equal starting numbers of Lin-B M progenitors from SHIP+/+ and -/- mice were differentiated in the presence of M-CSF, cells were harvested and counted on days 0, 2, 3, 4, and 5 (a)(closed squares, SHIP+/+; open triangles, SHIP-/-) . Shown is a representative experiment of two. (b) Equal starting numbers of total non-adherent BM cells were differentiated for 7 days in the presence of M-CSF , at which time adherent macrophages were collected and counted. This experiment is representative of greater than 20 such experiments (as yield of BMMcps was routinely determined). medium. As in the original study (Bourgin et al., 2002), day 6 FL cells from wild-type mice were approximately 50% Mac-1 positive and the same result was obtained for SHIP-/- cells (data not shown). Upon transfer to M-CSF for 5 days, both wild-type and SHIP-/- mice yielded 100% Mac-1 positive morphologically mature, adherent macrophages (data not shown). Moreover, c-Fms expression increased in both genotypes similar to the pattern seen with Mac-1 (data not shown) and consistent with the induction of terminal differentiation. However, as can be seen in Fig. 3.3b, day 6 SHIP-/- FL cells demonstrated enhanced survival and/or proliferation when transferred to M-CSF-containing medium, since their cell counts were significantly higher than wild-type. Thus, SHIP-/- BM cells also demonstrate enhanced sensitivity to the myeloid-promoting effects of FL/M-CSF. 36 12.5 04 , , . J. 0 1 2 3 4 f fme (cf) in M-CSF after 6d in F L Fig. 3.3. Enhanced FLT3-ligand-induced myeloid differentiation in SHIP-/- mice, (b) Equal starting numbers of total non-adherent BM cells (+/+ squares, -/- triangles) were differentiated for 6 days in the presence of 5 ng//ml FLT3-ligand (FL), and cell numbers determined after transfer to M - C S F for 0, 1, 2, or 3 days. This experiment is representative of 3 such experiments. 3.2.2 DEVELOPMENT, VALIDATION AND CHARACTERIZATION OF J2 RETROVIRUS-IMMORTALIZED SHIP+/+ AND -/- MACROPHAGE CELL LINES FOR IN VITRO FUNCTIONAL STUDIES In our initial studies to investigate the role of SHIP in mature macrophage functions, we generated J2 retrovirus (v-raf/v-myc)-immortalized cell lines from bone marrow-derived macrophages of SHIP+/+ and -/- mice (J2M+/+ and J2M-/-cells, respectively). This system has been successfully used by others to produce competent macrophage cell lines (Blasi et al., 1985; Blasi et al., 1989). However, in order to ascertain that the resulting cells phenotypically resembled macrophages, FACS surface staining was conducted on J2M+/+ and J2M-/- cells and compared to the established J774.1 murine macrophage cell line (Snyderman et al., 1977). As evident in Fig. 3.4a (and data not shown), J2M+/+ and J2M-/- cells were Mac-1 +, Mac-2+, CD14+, F4/80+ and B220-, and expressed similar surface levels as established J774.1 cells. Moreover, as can 37 be seen in the Western analysis conducted in Fig. 3.4b, J2M cells derived from SHIP-/- mice failed to express detectable protein levels of SHIP while they did express the M-CSF receptor (c-Fms) (albeit at lower levels) and the transcription factor necessary for its induction (PU.1). Since c-Fms levels are often downregulated in active MOs (Rovida et al., 2001) this did not detract from the MO character of J2M-/- cells. Furthermore, both J2M+/+ and -/- cells were competent at phagocytosis (J. Rey-Ladino and F. Takei, unpublished observations). Taken together, these results suggested that J2M+/+ and J2M-/-were bona fide macrophage cell lines. a b Mac-1 Mac-2 J2M BMMO Fig. 3.4. Characterization of macrophage phenotypic features in J2M+/+ and J2M-/- cell l ines, (a) The established J774.1 murine macrophage cell line and individual clones of the SHIP+/+ and -/- J2M cell lines (J2M+/+ and J2M-/-) were analyzed for surface expression of Mac-1 and Mac-2 macrophage phenotypic markers by F A C S (outline) or isotype control (shaded). Results are presented in histogram form, where the y-axis indicates the number of events and the x-axis indicates fluorescent intensity in log scale. Similar results were obtained for independent J2M+/+ and J2M- / - clones, (b) Total cell lysates were prepared from equal numbers of J2M+/+ and -/- cells cultured overnight in IMDM/10% F C S and subjected to Western analysis for c-Fms, SHIP, PU.1 , and G A P D H . n/s = non-specific band. In order to gain insight into the functional competency of J2M+/+ and -/-cell lines, the innate immune responsiveness to Gram-negative bacterial LPS was compared. Macrophages sense LPS via CD14/TLR4/MD2 and respond by secreting a host of cytokines, chemokines and reactive intermediates, including NO, that act to counter Gram-negative bacteria and tailor the adaptive arm of the immune response (Gordon, 2003). In macrophages, such LPS-generated, high output NO synthesis is mediated by iNOS (MacMicking et al., 1997). As can be seen in Fig. 3.5, J774.1, J2M+/+ and J2M-/- macrophage cell lines produced increasing amounts of the stable end-product of NO, N0 2" (Bogdan, 2001), in the tissue culture medium as the dose of LPS was escalated. Moreover, inclusion of the NOS inhibitor, L-NMMA, reduced NO output, suggesting that the LPS-induced NO observed was generated via iNOS. J774.1 and J2M+/+ macrophages produced similar amounts of NO in response to LPS, and this was greater than that in J2M-/- cells (Fig. 3.5). 80 Fig . 3.5. SHIP J 2 M - / - ce l ls are impaired in NO product ion. 10 5 J774.1, J2M+/+ or J2M- / - cells were treated with escalating concentrations of L P S for 20 h ± two concentrations of the N O S inhibitor, L-NMMA, and NO production was assessed by measuring the accumulation of N 0 2 " in tissue culture supernatants. Results are presented as the mean + S E M . Similar data were obtained in two replicate experiments. L P S (ug/ml; 20 h) L - N M M A (uM) 10 10 10 0 50 250 The production of NO by iNOS can be regulated at transcriptional, translation, and post-translational levels (Bogdan, 2001). In order to assess where the J2M-/- defect in NO production originated, iNOS Western blots were performed on LPS-stimulated J2M+/+ and -/- cell lines at various time points. As can be seen in Fig. 3.6 (right panels), J2M-/- cells had severely diminished LPS-stimulated iNOS protein levels, while they expressed similar levels of the LPS receptor complex component, CD14. In keeping with this, and consistent with previous analyses, J2M-/- cells demonstrated severely impaired NO production 3 9 J 2 M +/+ J 2 M -/-0 2 4 6 12 24 0 2 4 6 12 24(h) B l o t : INOS 2 4 6 12 ( L P S , 100 ng/ml) 24(h) B l o t : CD14 Fig. 3.6. Diminished LPS- induced NO production in J2M-/ - cells is associated with impaired iNOS induction. Equal numbers of J2M+/+ (black bars) and -/- cells (grey bars) were treated for the indicated times with 100 ng/ml LPS , supernatants collected and assessed for NO production (left) and total cell lysates prepared and subjected to Western analysis for iNOS or CD14 (right). Similar results were obtained in two independent experiments. a J 2 M +/+ J 2 M - / -0 12 24 0 12 24 (h) i N O S G A P D H C o n t r o l L P S Fig 3.7. Diminished LPS- induced iNOS transcription and iNOS m R N A levels in J2M- / - cells. (a) Equal amounts (10 ug) of total RNA were isolated from J2M+/+ and -/- cells and subjected to Northern blot analysis with radiolabeled iNOS or G A P D H cDNA probes, (b) J2M+/+ and -/- cells were transfected with CMV-Reni l la luciferase control vector ± an iNOS promoter-driven firefly luciferase vector (pGLH/H2), allowed to recover overnight, followed by stimulation with 100 ng/ml L P S for 18 h the next day. Lysates were prepared and relative luciferase activity of the iNOS promoter (RLU) was normalized to Renilla control. Data are presented as the mean ± S E M of triplicate determinations. Similar results were obtained in three independent experiments. 40 at the same time points (Fig. 3.6, left panel). Diminished J2M-/- iNOS mRNA induction by LPS, as assessed by Northern blot (Fig. 3.7a) suggested that the defect may lie at the level of transcription and this was confirmed using normalized dual-luciferase reporter assays with the murine iNOS promoter, which revealed diminished LPS-induced iNOS promoter activation in J2M-/- cells (Fig. 3.7b). While the J2M cell lines were able to produce NO (albeit at lower levels in J2M-/- cells) in response to LPS alone, maximal NO output was generated by prior or concurrent exposure of cells to the classical macrophage activating cytokine, IFN-y. In fact, IFN-y exposure can lead to modest NO production in its own right (Gordon 2003). As can be seen in Fig. 3.8, both J2M+/+ and J2M-/-cells were able to produce NO in response to IFN-y alone, and although J2M-/-cells still produced less NO than wild-type, the magnitude of the deficiency was less than that with LPS alone. Moreover, and importantly, co-treatment with LPS and IFN-y resulted in a dramatic improvement of J2M-/- NO production relative to LPS alone (Fig. 3.8). This suggested that the basis for the iNOS transcriptional defect by LPS alone in J2M-/- cells may be related to deficient IFN-B signaling. mm J2M+/+ J 2 M - / -r-Ii Fig. 3.8. IFN-y partially rescues the NO deficit of J 2 M - / - cel ls. J2M +/+ and -/- cells were treated for 20 h with the indicated concentrations of L P S (ng/ml), IFN-y (U/ml), or L P S + IFN-y and NO production was assessed. Representative results of duplicate determinations ± S E M are shown. L P S (|jg/ml; 20 h) 0 0 10 10 100 100 I F N - y (U/ml) 0 20 0 20 0 20 4 1 LPS does stimulate the production of autocrine-acting IFN-a/B, but it takes a few h for this to reach maximal levels in the tissue culture supernatant. The basis for enhanced LPS-induced NO production by IFN-y co-treatment is thought to result from earlier and more robust activation of Statl, a transcription factor activated by both IFN-a/B and IFN-y (Gao et al., 1997). Moreover, activation and binding of both Statl and IRF-1 (another transcription factor that is induced by Statl) to the iNOS promoter are crucial events for iNOS transcription (Kamijo et al., 1994; Martin et al., 1994; Ohmori and Hamilton, 2001) (Fig. 1.2). Thus, in Fig. 3.9, Western blots were conducted on cytoplasmic and nuclear extracts of LPS- or IFN-y-stimulated J2M cell lines to assess activation of Statl and IRF-1. The Statl target, IRF-1, was induced by LPS in both J2M+/+ and -/- (Fig. 3.9a). However, J2M-/- demonstrated a specific defect in IRF-1 nuclear localization, which correlated with deficient iNOS protein induction (compare cytosolic and nuclear extracts in Fig. 3.9a). Since COX-2 induction by LPS proceeded normally (actually enhanced in J2M-/- and addressed further in Chapter 3.3) under the same conditions, this highlighted the specificity of the IRF-1/iNOS defect. Since IFN-y was able to rescue J2M-/- NO production somewhat (Fig. 3.8), Statl and IRF-1 activation by this cytokine were also assessed. As can be seen in Fig. 3.9b, Statl was tyrosine phosphorylated, translocated to the nucleus, and mediated IRF-1 protein induction in both J2M+/+ and -/- cells. As with LPS, J2M-/- cells demonstrated an impaired ability to localize IRF-1 to the nucleus. However, some IRF-1 (albeit less) was able to localize to the nucleus in J2M-/-, perhaps explaining the ability of IFN-y to improve NO production by LPS (Fig. 3.8). Taken together, these results suggested that J2M-/- cells were impaired in LPS-stimulated NO production due to deficient iNOS transcription, associated with impaired IRF-1 nuclear localization. Thus, the J2M macrophage cell line model system supported the hypothesis that SHIP is a positive regulator of NO synthesis in murine macrophages. 4 2 J2M+/+ J 2 M - / -0 1 2 4 6 12 24 0 1 2 4 6 12 24(h) LPS (100 ng/ml) iNOS COX-2 IRF-1 IRF-1 * 1 Cytoplasm Nucleus J2M+/+ J 2 M - / -p-Stat1(Y701) IRF-1 0 10 2030 60120 240 0 10 2030 60120 240 min (IFN-y, 100 U/ml) Cytoplasm p-Stat1(Y701) IRF-1 Nucleus F ig . 3.9. Deficient IRF-1 nuclear local izat ion in J2M- / - ce l ls is assoc ia ted with impaired iNOS induct ion. J2M+/+ and -/- cells were stimulated with 100 ng/ml L P S (a) or 100 U/ml IFN-y (b) for the indicated times, cytoplasmic and nuclear extracts prepared, and equal protein amounts (100 ug cytoplamic extract or 25 ug nuclear extract) subjected to Western blot analysis for iNOS, C O X - 2 , and IRF-1 (a), or pStat1(Y701) and IRF-1 (b). Similar results were obtained in a second independent experiment. 43 3.2.3 THE ROLE OF SHIP IN LPS-INDUCED RESPONSES OF PRIMARY BONE MARROW-DERIVED MACROPHAGES In Chapter 3.2.2, the retrovirally-immortalized J2M cell line model revealed that SHIP is a positive regulator of LPS-induced NO synthesis. In order to determine if J2M results applied to primary cells, the role that SHIP plays in the LPS-induced innate immune response of primary cells was assessed using BMMOs derived from wild-type and SHIP-/- mice (as in Fig. 3.2b). As was the case with J2M cells (data not shown), LPS or LPS+IFNy-induced NO production was dose-dependently inhibited by the PI3K inhibitor, LY294002 (Fig. 3.10a, c), a phenomenon that was associated with reduced iNOS protein induction (Fig. 3.10b). However, unlike SHIP-/- J2M cells, SHIP-/- BMMOs demonstrated significantly enhanced LPS-induced NO production compared to wild-type BMMOs (Fig. 3.10c). Of note, co-stimulation with LPS + IFNy restored wild-type BMMO NO production to that of SHIP-/- BMMOs, suggesting that autocrine IFN production may be involved in PI3K-mediated enhancement of NO production by LPS (Fig. 3.10c, d). This hypothesis will be discussed further below. Taken together, these results suggest that SHIP is a negative regulator of NO induction by LPS in primary BMMOs, and PI3K is a positive regulator of this process. In order to determine if the enhanced NO production observed in LPS-treated SHIP-/- BMMOs was due to elevated levels of iNOS protein (associated with enhanced activation of events downstream of PI3K) Western blots were performed on these cells. As can be seen in Fig. 3.11a and b, respectively, 24 h LPS-treated (including DMSO vehicle) -/- BMMOs demonstrate elevated levels of phospho-Akt, iNOS, and NO compared to +/+ counterparts, with Statl serving as a loading control. Moreover, LY294002 dose-dependently reduced iNOS protein levels and NO production in both genotypes, while another PI3K inhibitor, wortmannin, resulted in a completely opposite outcome (i.e. enhancement of iNOS and NO levels; Fig. 3.11a, b). This result, although puzzling, was reminiscent of the result obtained in J2M cells (data not shown). While both LY294002 and wortmannin were able to reduce LPS-induced phosphorylation of 44 p70S6k and Akt, proteins downstream of PI3K, they displayed unique inhibitory profiles (Fig. 3.11a). For instance, wortmannin appeared less efficient at inhibiting p70S6k phosphorylation than LY294002. Taken together, given that LPS (100 ng/ml) I F N - Y (100 U/ml) DMSO (0.1 %) LY294002 (pM) BMMOs 100 2 3 75-| o" z 1 c 50 25 mm SHIP+/+ em SHIP-/-LPS (ng/ml) IFNy (100 U/ml) DMSO (0.1 %) LY294002 (pM) 10 s 10 3 1 0 4 1 0 4 1 0" 10 2 1 0 2 1 0 2 10 10 LPS/IFN-y INOS GAPDH Q 1 % LY294002. uM DMSO 5 10 20 50 40 30 20 10 0 BMMOs • SHIP+/+ A SHIP-/- J 2/ o r 0 10 20 30 40 50 Duration (h) of LPS + IFNy Fig . 3.10. The PI3K pathway is a positive regulator of iNOS/NO in B M M O s and SHIP negatively regulates this process. SHIP+/+ BMMOs were treated for 24 h (black bars) or 48 h (grey bars) with L P S (100 ng/nl) + IFNy (100 U/ml) ± DMSO vehicle control (0.1%) or LY294002 (5, 10, or 20 uM) and (a) levels of NO were determined in duplicate (b) and total cell lysates were prepared at 48 h and subjected to Western analysis for iNOS and G A P D H . (c) SHIP+/+ and -/-B M M O s (10 6 cells/ml) were treated with the indicated concentration of L P S or L P S + IFNy for 24 h ± prior (30 min) addition of D M S O vehicle or LY294002, or (d) for the indicated times with 100 ng/ml L P S + 20 U/ml IFNy and the conditioned media analyzed for N0 2 " . Data points in all panels are the means ± S E M of at least triplicate determinations, except for duplicate determinations in (a). Significant difference between SHIP+/+ and SHIP-/- means: *p< 0.05, **p<0.01, using unpaired two-tailed student's f test. Similar results were obtained in at least 3 separate experiments. LY294002 doses used in these experiments were non-toxic (as assessed by trypan blue exclusion and data not shown). 4 5 genetic (SHIP-/-) and pharmacologic (LY294002) results are consistent (i.e. a positive role played by PI3K in iNOS/NO induction), it was tentatively assumed that the iNOS/NO-enhancing effects of wortmannin were due to a lack of efficient inhibition of a PI3K downstream target other than Akt and/or an inhibition of a non-PI3K-dependent iNOS/NO repressive pathway. SHIP+/+ BMMO WM LY29 D 200500 5 20 SHIP-/- BMMO WM LY29 D 200 500 5 20 SHIP INOS INOS (dk) Statl pp85S6K pp70S6K pAktT308 ... „ • • H i , • 60 1 40 20 In In 200 500 + LPS (100 ng/ml; 24 h) - DMSO (0.1 %) - Wortmannin (nM) 20 LY294002 (uM» F ig . 3.11. Wortmannin and LY294002 differentially affect B M M O iNOS/NO induct ion in response to L P S . SHIP+/+ and -/- BMMOs were treated for 24 h with 100 ng/ml L P S ± D M S O (0.1 %), wortmannin (200 or 500 nM), or LY294002 (5 or 20 pM) and (a) total cell lysates subjected to Western analysis for SHIP, iNOS (longer exposure indicated as dark (dk)), pp85S6K, pp70S6K, pAkt, and Statl loading control (b) 24 h supernatants from SHIP+/+ (black bars) or -/-(grey bars) B M M O s in (a) were assessed for NO production. Wortmannin is a natural compound whereas LY294002 is synthetic. Since the PI3K inhibition-inactive analogue of LY294002, called LY303511, was available, wild-type BMMOs were treated with LPS ± pre-treatment with DMSO vehicle, wortmannin, LY294002 or LY303511 and the effects on subsequent iNOS induction and NO synthesis examined (Fig. 3.12). It should be noted that a variable degree of iNOS/NO enhancement by wortmannin was observed in subsequent experiments on LPS-treated BMMOs. In Fig. 3.12, wortmannin led to a modest enhancement of LPS-induced iNOS protein levels and NO synthesis when LPS was used at a higher concentration (1 pg/ml), while LY294002 led to a dramatic decline in iNOS/NO. Inclusion of the PI3K inhibition-inactive LY analogue, LY303511, also diminished iNOS/NO, but not to the extent of LY294002, suggesting that the result may be partly due to PI3K non-specific effects of LY294002 (Fig. 3.12). Repeating this experiment with a lower concentration of the LY compounds (10 pM) resulted in a less obvious impact of LY303511 on iNOS protein levels (Fig. 3.13a and b). Of note, the degree of negative impact of the particular compound (wortmannin, LY294002, LY303511) on iNOS/NO appeared to be directly related to its ability to reduce levels of tyrosine-phosphorylated Statl (Y701) (Fig. 3.12, 3.13). Thus, the differential effects of wortmannin and LY294002 on LPS-induced iNOS/NO production in BMMOs may be related to their differential ability to inhibit Statl activation. iNOS P-Stat1(Y701) GAPDH T 30 LPS (1 ug/ml; 24 h) DMSO (0.1 % Wortmannin (nM) LY294002(25 uM) LY303511 (25 uM) Fig . 3.12. The PI3K inhibition-inactive LY294002 analogue, LY303511 has less negative impact upon LPS- induced iNOS and NO. SHIP+/+ B M M O s were treated with 1 ug/ml L P S for 24 h ± 30 min pre-treatment with DMSO, wortmannin, LY294002, or LY303511 at the indicated concentrations and total cell lysates were prepared and subjected to iNOS, pStat1(Y701) and G A P D H Western analysis or NO production was assessed in duplicate by Griess assay. 47 a LPS (200 ng/ml) 0 h + ^ 1 h +/+ -/-3 h 24 h +/+ +/+ -/-0 0 D W 29 D W 29 D W 29 30 D W 29 30 D W 29 30 D W 29 30 SHIP II I mm-iNOS — P-STAT1 (Y701) I I «*»•" P-STAT1 (S727) r : ' : " - " • l i r " T " J I M I " '~' L U - """"rr" p-p70S6K •:v:.:v .-• .. ^ GAPDH L P S (200 ng/ml; 24 h) DMSO (0.1 %) Wortmannin (100 nM) LY294002 (10 uM) LY303511 (10 uM) + + Fig. 3.13. Differential effects on the p70S6K/IFN6/Stat1/iNOS pathway may underlie differences in iNOS/NO induction between SHIP+/+ and -/- B M M O s and between wortmannin and LY294002. (a) 5 x 10 s +/+ or -/- BMMOs, at 1 * 10 6 cells/ml in 12-well plates, were pre-treated for 20 min with either D M S O (0.1%), wortmannin (100 nM), LY294002 (10 uM), or LY303511 (10 pM) prior to stimulation with L P S (200 ng/ml) for the indicated times, after which total cell lysates were subjected to Western analysis for iNOS, pStat1(Y701), pStatl (S727), pp70S6K, p4EBP1, k B a , and G A P D H . (b) Cells were treated as in (a) and supernatants collected after 24 h (SHIP+/+ (black bars) or -/- (grey bars)) and analyzed in duplicate for NO production by Griess assay. 48 LPS signals via the CD14/MD2/TLR4 complex in two major directions: the MyD88-dependent and MyD88-independent pathways (Dunne and O'Neill, 2005). The MyD88-dependent pathway leads to activation of the N F - K B family of transcription factors, which has been implicated in iNOS transcription (Hayden and Ghosh, 2004). The MyD88-independent pathway leads to activation of the transcription factor, IRF-3, which activates IFNB transcription (Moynaugh, 2005) (Fig. 1.2). While the PI3K pathway has not been implicated in IFNB transcription, it has been shown to control its translation and/or secretion in a positive manner (Weinstein et al., 2000; Rhee et al., 2003). Moreover, the IFNB protein thus produced is secreted from the cell, acts in an autocrine fashion via its receptor on the cell surface, and leads to the activation of two more transcription factors critical to iNOS induction: Statl and IRF-1 (Gao et al., 1997). Statl is serine phosphorylated in a PI3K-insensitive pathway downstream of TLR4, and this precedes tyrosine phosphorylation by type I IFN-receptor-associated Janus kinase (JAK) (Rhee et al., 2003). Although the identity of the PI3K effector(s) that controls the translation of IFNB have yet to be identified,the 70 kDa ribosomal S6 kinase (p70S6K) is a good candidate (Weinstein et al., 2000). p70S6K is activated downstream of both PI3K and mTOR (which is itself indirectly activated by PI3K), and mTOR also phosphorylates and relieves transcriptional repression mediated by the elongation initiation factor binding protein, 4EBP1 (Gingras et al., 2001). Of note, LY294002 has been demonstrated to have inhibitory effects on mTOR in some systems (Fingar and Blenis, 2004). With this in mind, a striking correlation was observed in Fig. 3.13 between phosphorylation of p70S6K (and less so, 4EBP1) and 24 h iNOS protein levels. LY294002 potently repressed phosphorylation of both p70S6K and 4EBP1 at all timepoints, while wortmannin was ineffective and LY303511 had only modest inhibitory effects on p-p70S6K at 24 h (Fig. 3.13a). Moreover, early (1 h), LPS-induced serine phosphorylation occured equally well in both genotypes and appeared independent of PI3K, while SHIP-/- BMMOs displayed enhanced tyrosine and serine phosphorylation of Statl at 3 h, at a time when these 49 processes are mediated by the autocrine action of IFNB (Gao et al. 1997; Rhee et al., 2003). These enhanced IFNB-dependent Statl outcomes were preceded in SHIP-/- BMMOs by a marked elevation of phospho-p70S6K (Fig. 3.13a, 1 h panel). Finally, LPS-induced degradation and re-synthesis of kBa proceeded normally in the presence of PI3K inhibitors. Taken together, these results suggested that SHIP-/- BMMOs had elevated activation (phosphorylation) of p70S6K in response to LPS. This might have led to enhanced translation and secretion of autocrine-acting IFNB, and subsequent enhanced Statl activation (phosphorylation) (simply due to more IFNB being produced, or possibly also due to enhanced IFNB-R signaling events in the absence of SHIP), culminating in enhanced iNOS transcription. While both wortmannin and LY294002 inhibited Akt phosphorylation, only the latter affected the phosphorylation of p70S6K. However, this might not have been a purely PI3K inhibition-specific effect of LY294002, since it also reduced phosphorylation of 4EBP1 (whereas, p4EBP1 levels were not elevated in SHIP-/- BMMOs). Thus, the potent effects of LY294002 on pp70S6K and p4EBP1 could have been due (in whole, or in part) to inhibition of mTOR. Finally, iNOS translation might also have been controlled at later time points (i.e. see 24 h panel in Fig. 3.13a) directly by p70S6K, since there was an identical pattern of pp70S6K and iNOS levels. Ultimately, while these results demonstrated that SHIP negatively regulates iNOS/NO induction by LPS, and suggested that it does so via a p70S6K/IFNB/Stat1 pathway, it was also apparent that identical modulation of the PI3K pathway was not achieved by two different pharmacological inhibitors and a mouse genetic model. The aforementioned studies on LPS-induced events in wild-type and SHIP-/- BMMOs focused primarily on the MyD88-independent pathway, leading to iNOS induction and NO synthesis. However, the CD14/MD2/TLR4 complex also leads to activation of MyD88-dependent signaling and induction of a host of other LPS target genes, including cytokines (Hayden and Ghosh, 2004). Thus, the focus was shifted in this direction to further elucidate the role that SHIP plays in BMMO innate immune responses to LPS. One conundrum in macrophage LPS signaling experiments is whether or not to include M-CSF in the tissue 50 culture medium. BMMOs are differentiated in M-CSF and, once mature, maintain a dependence on M-CSF for maximal survival. However, the presence of M-CSF, even at low levels, leads to basal macrophage signaling and may confound the interpretation of LPS-induced events (Sweet et al., 2002). Both approaches (M-CSF present, or not) were thus undertaken to compare and elucidate the role played by SHIP in BMMO LPS responses. LPS time course studies revealed that SHIP-/- BMMO TNFa and IL-6 production changed from relatively less or approximately equivalent to +/+ early on, while -/-BMMO production of these cytokines surpassed that of +/+ at later points (Fig. 3.14a). The pro-inflammatory LPS hypo-responsiveness of SHIP-/- BMMOs was extended to even later time points in the absence of M-CSF, as typified by the TNFa and IL-6 cytokine profiles observed in Fig. 3.15c. Coincident with this early pro-inflammatory hypo-responsiveness was the observation of enhanced SHIP-/-BMMO secretion of anti-inflammatory IL-10 (Fig. 3.15c). Pooled analysis of several such experiments, in which SHIP-/- LPS-induced cytokine production was recorded as a percentage of wild-type production, revealed that an early peak of enhanced -/- IL-10 production was associated with diminished pro-inflammatory cytokine production (Fig. 3.16). However, LPS-induced SHIP-/-cytokine production surpassed wild-type production beyond 12 h of stimulation (Fig. 3.16). This implied that the influence of SHIP on LPS-induced signaling and target induction, i.e. hypo-responsiveness versus hyper-responsiveness, was opposite during immediate-early versus indirect and/or later periods, respectively. Moreover, the presence of M-CSF favoured SHIP-/- BMMO hyper-responsiveness, while IL-10 production coincided with early hypo-responsiveness. Taken together, these results suggested a complex regulation of LPS responsiveness that is prone to modulation and feedback. 51 a 40 20 1 10 £ 7.5 0 5.0 Li. Z 2.5 0 125 75 I 25 0.50 °f Jj 0.25 0 50 40 2 n . 30 o 20 z 10 0 SHIP+/+ BMMO SHIP-/- BMMO 0 1 3 6 12 24 hLPS pErk1/2 I KBa GAPDH 0 0.25 0.5 1 3 6 12 24 0 0.25 0.5 1 3 6 12 24 h 24 h LPS F ig . 3.14. A compar ison of LPS- induced signal transduction and pro-inflammatory mediator production in SHIP+/+ and -/- B M M O s in the presence of M - C S F . (a) SHIP+/+ (black bars) and -/- (grey bars) B M M O s were treated for the indicated times with 100 ng/ml L P S and supernatants assessed for the production of TNFa , IL-6, and NO in the presence of 5 ng/ml M-CSF. (b) Cells were treated as in (a) and total cell lysates from equivalent cell numbers prepared at the indicated times and subjected to Western analysis for pSHIP(NPXY), SHIP, iNOS, pStat1(Y701), COX-2 , pAkt(T308), Akt, pErk1/2, k B a , and G A P D H . a SHIP+/+ BMM0 SHIP-/- B M M O 0 0.5 1 3 6 9 12 16 24 0 0.5 1 3 6 9 12 16 24 h pSHIP (NPXY) pSHIP (Btk) SHIP COX-2 pp65NFKB pAkt(T308) pErk1/2 I K B Q f mm %w • • » m-m mm«•> <** ^ mm mm mmwe-Wi Wm I H M mm nm mm m P?** «*, !t0m.,.mm. «||K **> <**' 1 «*. «fe «*. ' SHIP+/+ B M M O SHIP-/-BMM0 0 1 5 3 0 60 0 15 30 60 min iKBa (CHX pre-tx) 12 h LPS Fig . 3.15. A compar ison of LPS- induced signal transduction and pro-inflammatory mediator production in SHIP+/+ and -/- B M M O s in the absence of M-CSF. (a) SHIP+/+ and -/- B M M O s were treated for the indicated times with 100 ng/ml L P S and total cell lysates from equivalent cell numbers were prepared at the indicated times and subjected to Western analysis for pSHIP(NPXY), pSHIP(Btk homology site), SHIP, COX-2 , pp65 N F - K B , pAkt(T308), pErk1/2, and I K B O . Non-specific bands in the k B a panel were used as loading controls, (b) Cells were treated as in (a) except that L P S stimulated was preceded by a 20 min pre-treatment with 50 pg/ml cycloheximide (CHX), and Western analysis was only conducted for k B a . (c) Cells were treated as in (a) and SHIP+/+ (black bars) and -/- (grey bars) supernatants were assessed for the production of TNFa , IL-6, and IL-10. 53 250 Fig. 3.16. Pooled analysis of L P S -induced T N F a , IL-6, and IL-10 production over time in SHIP+/+ and -/- B M M O s . SHIP+/+ and -/- B M M O s were treated with 100 ng/ml L P S and the production of TNFa , IL-6, and IL-10 assessed over time by ELISA. SHIP-/-B M M O cytokine production is presented as the percent of +/+ B M M O production. The data set comprised 7 to 9 independent experiments, depending on the particular cytokine, and 2 to 8 independent, average experimental values were analyzed per time point. Results are presented as the mean of this analysis ± S E M . Undetectable +/+ B M M O IL-10 at later time points prevented a % comparison with detectable -/- values. However, in two experiments where sufficient +/+ IL-10 was produced, SHIP-/- IL-10 production was 230% (12 h) and 213 % (24 h) of wild-type production (not graphed, as n = 1). 0 5 10 15 20 25 30 Duration (h) of L P S (100 ng/ml) Having established the effects of SHIP on LPS-induced cytokine production, it was hypothesized that SHIP may affect intracellular signaling events in BMMOs, particularly those that were PI3K- and MyD88-dependent. First, suggesting an involvement of SHIP in LPS-induced signaling, it can be seen in Fig. 3.15a that SHIP became phosphorylated at two independent sites, with similar kinetics (peaking at 3-9 h), while at later times SHIP protein levels were induced but hypo-phosphorylated. The presence of M-CSF led to a basal level of SHIP phosphorylation which masked the inducibility seen in the absence of M-CSF (Fig. 3.14b). However, the hypo-phosphorylation of SHIP at later times was observed regardless of whether M-CSF was present, or not (Fig. 3.14b, 3.15a). Apart from differences in SHIP phosphorylation, the inclusion of M-CSF 54 did not result in any other apparent LPS signaling differences. For this reason all further signaling experiments were conducted in its absence. Not unexpectedly, basal and LPS-induced phosphorylation of Akt were enhanced in SHIP-/- BMMOs (Fig. 3.15a). This is in keeping with elevated levels of pp70S6K, as described earlier in Fig. 3.13a, and the fact that both Akt and p70S6K phosphorylation are triggered by PI3K-generated PIP 3 levels. Moreover, Akt phosphorylation levels fluctuated over time, possibly due to immediate-early LPS signaling and delayed indirect waves of signaling by the action of autocrine-acting, LPS-induced cytokines. Interestingly, Erk1/2 phosphorylation peaked early in -/- BMMOs, whereas +/+ counterparts were more able to sustain Erk1/2 phosphorylation after the early peak. Moreover, in SHIP-/- BMMOs Erk1/2 seemed to be equally phosphorylated, while in wild-type BMMOs Erk2 was more phosphorylated than Erkl (Fig. 3.15a). A hallmark of LPS-activated innate immune cells is the activation of the N F - K B family of transcription factors. In general the N F - K B family comprises a set of homo- and hetero-dimeric factors (most commonly p50NF-KB/RelA) which are sequestered in the cytoplasm by an inhibitory family of proteins, typified by kBa (Hayden and Ghosh, 2004). Upon LPS stimulation, the inhibitory kBa is phosphorylated and degraded in a MyD88-dependent fashion by the kBa kinase (IKK) complex, unmasking the nuclear localization sequence of N F - K B . This allows it to translocate to the nucleus, bind its consensus sequence in target promoters, and mediate transcription of a host of genes involved in inflammation and survival (Viatour et al., 2005). Most striking in Fig. 3.15a was the decreased p65 N F - K B phosphorylation on serine residue 536 in SHIP-/- BMMOs, particularly at early times (i.e. 0 to 9 h post-LPS). Phosphorylation of this residue has been demonstrated to be important for transcriptional activation of p65 N F - K B (Viatour et al., 2004). Thus, in the immediate-early period there appeared to be a reciprocal relationship between pAkt, pERK1 and pp65 N F - K B . Arguing against a more widespread inhibition of N F - K B in SHIP-/- BMMOs, the LPS-induced degradation and resynthesis of the inhibitory protein kBa, which functions to retain N F - K B in the cytosol, proceeded in a similar fashion in both genotypes. 55 Finally, as resynthesis of kBa can obscure differences in the kinetics of degradation, its LPS-induced degradation was compared in BMMOs following cycloheximide pre-treatment, and again revealed a similar pattern (Fig. 3.15b). Taken together, these results suggested that the hypo-responsiveness of SHIP-/-BMMOs to LPS at early points (with concomitant enhanced IL-10 secretion) might be related to enhanced Akt activation, perturbed Erk1:Erk2 phosphorylation, and a diminished level of phospho-p65 N F - K B . Given these results, it was hypothesized that stimulation of SHIP-/-BMMOs at a lower cell density, while not necessarily correcting direct, LPS-mediated signaling defects of SHIP-/- BMMOs, would alleviate some of the indirect negative feedback imposed upon pro-inflammatory cytokine production by autocrine-acting IL-10. Thus, in Fig. 3.17 SHIP+/+ and -/- BMMOs were treated with 2 different commercial preparations of LPS (to ensure that signaling results were not confounded by potential contaminants) at 2 different cell densities, and both signaling and cytokine responses compared. Consistent with the hypothesis and the results presented in Fig. 3.15a, stimulating SHIP-/-BMMOs at different cell densities did not affect their perturbed LPS signaling pattern: higher pAkt, higher pErk1:pErk2 ratio, and lower p-p65 N F - K B (Fig. 3.17a, left and right panels), suggesting that these were cell-autonomous, direct results of LPS recognition. Conversely, tyrosine phosphorylation of Statl, which is dependent upon the indirect autocrine action of LPS-induced IFNB, was diminished in both genotypes at lower cell density, likely as a result of a lowered extracellular concentration of IFNB (Fig. 3.17a, left and right panels). Consistent with previous observations, this perturbed SHIP-/- BMMO LPS signaling was associated with diminished and enhanced secretion of IL-6 and IL-10, respectively, compared to wild-type counterparts (Fig. 3.17b, left). Moreover, LPS-treating both genotypes at one quarter density led to diminished detectable tissue culture medium cytokine concentrations (Fig. 3.17b, right). Comparing IL-6 production on a per cell basis revealed, as expected, that SHIP-/- BMMO IL-6 production improved at a lower cell density, likely because autocrine IL-10 production was less likely to have as great a negative impact (Fig. 3.17c). 56 High Density ( 1 . 0 x 1 0 6 cells/ml) + / + s - '-s + / + c -'"c 1 3 1 3 1 3 1 3 L o w Density (0.25 x 10 s cells/ml) +/+s +8 + / + c -'-c 1 3 1 3 1 3 1 3h L P S pSHIP pStat1Y701 pp65NFKB pErk1/2 pAktT308 pp38 1 1 1 '"1 I"" - 1 1 1 I 1 i 1 1 1 1 1 1 1 i *" " i : i •mm | ~» |»» .. t_ 1 i t +/+s -/- s +/+c +/+s -l-s +/+c -/-c 1 3 h L P S High Densi ty L o w Densi ty L P S (100 ng /ml ; 3 h) F i g . 3 .17. B M M O L P S - r e s p o n s i v e n e s s is d e p e n d e n t u p o n ce l l d e n s i t y a n d a u t o c r i n e m e d i a t o r s . SHIP+/+ and -/- BMIvKPs were seeded in 12-well tissue culture plates at the usual density described for experiments thus far (i.e. 1 * 10 6 cell/ml) or at a reduced density (0.25 * 10 6 cell/ml), stimulated with 100 ng/ml L P S from Sigma (S) or Calbiochem (C) for the indicated times, and (a) subjected to Western analysis for pSHIP, pStat(Y701), pp65 N F - K B , pErk1/2, pAkt(T308), and pp38, or (b) SHIP+/+ (black bars) and -/- (grey bars) supernatants were collected and analyzed for IL-6 and IL-10 production by ELISA. (c) 3 h IL-6 production from (b) is presented on a per cell basis (fg/ml/cell). Interestingly, LPS-induced IL-6 production per cell in SHIP +/+ BMMOs was diminished at the lower cell density (Fig. 3.17c). It is known that the autocrine action of immediate-early TNFa and IL-1 (3 can positively influence the production of IL-6, and it is possible that this phenomenon was diminished at the lower SHIP+/+ cell density (Xaus et al., 2000; Shi et al., 2003). Thus, in the case of SHIP-/- BMMOs, the negative impact of autocrine IL-10 might have diminished the influence of the aforementioned positive autocrine mediators, and led to their immediate-early pro-inflammatory LPS hypo-responsiveness. Based upon these observations, it was hypothesized that inhibition of PI3K should augment LPS-induced phosphorylation of p65 N F - K B and also diminish the secretion of IL-10, thereby improving pro-inflammatory cytokine secretion. As can be seen in Fig. 3.18a, SHIP-/- BMMOs demonstrated elevated pAkt levels at 1 h post-LPS, compared to wild-type, and that PI3K inhibition with either wortmannin or LY294002 dose-dependently inhibited pAkt and reciprocally enhanced pp65 N F - K B (although more selectively in -/- BMMOs). Analysis of the supernatant from 1 h LPS-stimulated BMMOs reaffirmed the pattern of diminished -/- TNFa, associated with elevated IL-10 (Fig. 3.18b). As expected, both PI3K inhibitors dose-dependently decreased subsequent LPS-induced IL-10 secretion, suggesting that IL-10 was a bona fide target of PI3K in these cells. Consistent with this, wortmannin (particularly at the highest dose tested) enhanced TNFa secretion (especially in -/- BMMOs, where IL-10 production was diminished) (Fig. 3.18b). However, as was the case for LPS-induced iNOS/NO production, wortmannin and LY294002 displayed differential effects on TNFa secretion (enhancement versus inhibition, respectively). Although this has not been explored further, it could possibly reflect inhibition of TNFa translation by the dual PI3K/mTOR inhibitor, LY294002. Taken together, however, these results strengthened the hypothesis that diminished p65 N F - K B activation and enhanced IL-10 production in SHIP-/- BMMOs contributed to diminished immediate-early LPS pro-inflammatory responses. 58 a pRelA(S536) pAkt(T308) G A P D H SHIP-/- B M M O SHIP+/+ B M M O WM (nM) L Y (uM) WM (nM) LY(MM) D 5 50 500 10 100 D 5 50 500 10 100 LPS (100 ng/ml; 1 h) DMSO (0.1 %) Wortmannin (nM) LY294002 (uM)) F ig . 3.18. PI3K inhibition inhibits LPS- induced Akt phosphorylation and IL-10 production while it augments p65 N F - K B phosphorylation, (a) SHIP-/- (left) and +/+ B M M O s were pretreated for 30 min with 0.1 % D M S O (D), wortmannin (W) (5, 50, or 500 nM), or LY294002 (LY) (10 or 100 uM) prior to stimulation with 100 ng/ml L P S for 1 h. Total cell lysates were prepared and subjected to pp65 (RelA) NF -KB ( S 536 ) , pAkt(T308), or G A P D H Western analysis, (b) Supernatants from SHIP+/+ (black bars) and -/- (grey bars) B M M O s treated in (a) were collected and subjected to T N F a or IL-10 ELISA. 5 9 3.2.4 THE ROLE OF SHIP IN ENDOTOXIN TOLERANCE OF PRIMARY BONE MARROW-DERIVED MACROPHAGES The pattern of LPS responsiveness in SHIP-/- BMMOs commonly evolved from hypo- to hyper-pro-inflammatory, whereas +/+ BMMOs appeared to be more efficient at restraining a late, runaway LPS response. The +/+ pattern was reminiscent of what has been observed in a phenomenon known as endotoxin (LPS) tolerance. Specifically, after an initial exposure and pro-inflammatory response to LPS, wild-type BMMOs have been demonstrated to be refractory to a second LPS stimulus (Fan and Cook, 2004). This display of LPS tolerance is thought to have evolved to protect the host from overzealous responses to LPS, which may lead to host damage and septic shock (Beutler and Rietschel, 2003). Given that LPS responses in SHIP-/- BMMOs increased relative to wild-type at later stages (rather than being appropriately restrained), it was of great interest to determine how SHIP-/- BMMOs would respond to a second LPS challenge (i.e. would they demonstrate LPS tolerance?). As can be seen in Fig. 3.19, a 24 h pre-treatment of wild-type BMMOs with LPS severely attenuated the 3 h production of TNFa, IL-6, and IL-1B in wild-type BMMOs in response to a second LPS challenge. In contrast, SHIP-/-BMMOs failed to demonstrate LPS tolerance, since their 3 h LPS response was indistinguishable between those pre-exposed to a so-called 24 h tolerizing LPS dose, or not (Fig. 3.19). Moreover, SHIP-/- BMMOs failed to demonstrate LPS tolerance over a broader range of tolerizing and challenge LPS concentrations (Fig. 3.20). It should be mentioned that the aforementioned tolerance results were achieved in SHIP-/- BMMOs that displayed enhanced 3 h LPS-induced TNFa, IL-6, and IL-1B production compared to wild-type (as in Fig. 3.19). It was suggested in Section 3.2.3 that this may reflect a more rapid recovery from the repressed LPS pro-inflammatory responses usually observed, but incompletely understood, in SHIP-/- BMMOs. However, even in Fig. 3.21, where 60 SHIP+/+ SHIP-/-240 160 i f 80' n L P S - L P S L P S - LPS 1500 3600-1 L P S - L P S L P S - L P S 240' * 800' 160> J L =|- 600' S 400' 80' -7 200' o. n 0' Fig. 3.19. SHIP-/- B M M O s do not display endotoxin tolerance. SHIP+/+ (left) or - / -(right) B M M O s (as in F i g . 3.21) were untreated (clear bars) or tolerized for 24 h with 100 ng/ml L P S (black bars), w a s h e d , and cha l lenged for 3 h ± 100 ng/ml L P S . T h e levels of T N F a , IL-6, and IL-1B were determined by E L I S A . Resu l ts are p resented a s m e a n s ± S E M a n d *p<0.05, L P S - L P S LPS - L P S e SHIP+/+ ... i r J L _nfl.*i S H I P J -toteftzed 0 10 100 — . — . - — „ _ cha l lenged : o 4* \*c>«$> $ ^ c h a l l e n g e d : o *S> «Fo <SV*# *r 3O0Ch I I 600- 1 300- fl 1 0- .11 L a i to ienzed 0 ^ 10 ' 100 _ to ler ized: 0 10 100 cha l lenged o N * N * O - P ^ O s ^ c h a S e n g e d : ^ Fig. 3.20. L P S dose-dependent tolerization in SHIP+/+ BMMOs . SHIP+/+ (left) or - / - (right) B M M O were treated for 24 h with a tolerizing L P S concentrat ion of 0, 10, or 100 ng/ml (clear, grey, or black bars, respectively), w a s h e d , a n d cha l lenged for 3 h with the s a m e L P S concentrat ions u s e d in the tolerization step. Superna tants were a n a l y z e d for T N F a a n d IL-6 production. 61 SHIP-/- BMMOs displayed a diminished LPS-induced pro-inflammatory response associated with enhanced IL-10 production, it can be seen that LPS tolerance was not achieved. Moreover, as opposed to the indistinguishable SHIP-/-cytokine production with and without a first dose of LPS in Fig. 3.19, SHIP-/-BMMO pro-inflammatory cytokine production was in fact amplified by a second LPS dose in Fig. 3.21. Interestingly, IL-10 was an exception, in that it was tolerized somewhat in BMMOs of both genotypes. Taken together, these results suggested that SHIP-/- BMMOs failed to display endotoxin tolerance. This might have been related to the previous observation of an amplified late -/- LPS-response, relative to wild-type, and implied that the presence of SHIP was crucial to restraining late LPS-induced events, including responses to subsequent LPS challenge. T N F a IL-6 CD c T o l . (6h L P S , 100 ng/ml) Cha l . (12h L P S , 100 ng/ml) IL-12 c IL-10 Fig. 3.21. SHIP-/- BMMOs that are LPS hypo-responsive are also refractory to LPS-induced tolerance. SHIP+/+ (black bars) and -/- (grey bars) BMMOs (that fit the LPS hypo-responsive pattern observed in Figs. 3.15 - 3.17) were treated for 6 h ± 100 ng/ml LPS, washed, and challenged ± the same concentration of LPS for 12 h. Levels of TNFa, IL-12, IL-6, and IL-10 were determined in duplicate by ELISA. 62 The requirements for SHIP in restraining late LPS-induced inflammation and in mediating LPS tolerance while permitting immediate-early LPS inflammation, suggested that SHIP may be differentially regulated and/or interact with distinct partners during the time course of LPS stimulation. Thus, it was decided to further characterize and explore the mechanisms involved in LPS-induced SHIP protein induction. As shown in Fig. 3.22a, the induction of SHIP by LPS was maximal at 24 h, and induction was specific to SHIP, since levels of SHIP2 and PTEN were unaffected. Moreover, SHIP protein induction could be achieved with as little as 1 ng/ml LPS (Fig. 3.22b). Interestingly, while the SHIP protein induction was paralleled with a similar LPS induction of SHIP mRNA, this was uniquely preceded by a reduction of SHIP mRNA between 3 to 6 h post-LPS that was not reflected in SHIP protein levels (Fig. 3.22c and Fig. 3.15a). Finally, the elevation in SHIP mRNA levels by LPS was preceded by the induction of two other targets (SOCS1, peaking at 3 h, and IRAK-M, with induction beginning at 6 h) previously demonstrated to be critical for mediating LPS tolerance (Fig. 3.22c). Nonetheless, as tolerance could not readily be achieved in SHIP-/- BMMOs (Fig. 3.19), despite the fact that SOCS1 and IRAK-M were induced in the absence of SHIP (Sly and Krystal, unpublished observations), this suggested that the threshold to achieve tolerance was elevated in SHIP-/- BMMOs. Taken together these results suggested that the presence of SHIP, and particularly the late LPS-induced rise in its protein levels (and perhaps its hypo-phosphorylation), were essential for LPS tolerance. In other hemopoietic cell types, SHIP levels have been demonstrated to be increased by TGFB1 and other members of the TGFB superfamily (Valderrama-Carvajal et al., 2002). The late induction of SHIP by LPS suggested that this may not be achieved directly by LPS, but rather via the autocrine action of an LPS-induced factor. Thus, experiments were conducted to elucidate if TGFB was involved in SHIP upregulation. As can be seen in Fig. 3.23a, treatment of wild-type BMMOs with TGFB1 led to detectable SHIP induction after 4 h, and peak induction at 8 h. As BMMOs of both genotypes secreted detectable amounts of TGFB1 upon LPS stimulation (Fig. 3.23b), this suggested 63 BMmos time C 8 C 24 C 48 <h) k s H I P « S H I P 2 H 1 0 H 48 0.7 TT 1.0 1 t 1.0 08 O 0! 10 Q 7 0 ^ Of 0.$ 0ifS N G A P D H ' <*°se C 0 1 C 1 C 1 0 C 1 0 0 (ng/ml) ~~" '"" TST j-«SHlP . 1 Q ^ 2 0 12 5 3_ 1 0 _ 3 C r— • N G A P D H BMmo)s time 0 3 6 12 24 (h) JT~— | « S H I P "Hsocsi j - * IRAK-M <l f>actin F ig . 3.22. SHIP protein and m R N A levels are upregulated in SHIP+/+ B M M O s by L P S treatment, (a) SHIP+/+ B M M O s were treated for the indicated times ± 100 ng/ml L P S , total cell lysates prepared and subjected to Western analysis for SHIP, SHIP2, P T E N and G A P D H . Numbers below respective bands indicate relative levels compared to unstimulated control, assessed by densitometry, (b) +/+ B M M O s were treated for 24 h ± the indicated concentration of L P S and SHIP levels were determined as in (a), (c) total mRNA was isolated from SHIP+/+ B M M O s treated for the indicated times with 100 ng/ml L P S and the levels of SHIP, S O C S 1 , IRAK-M and p-actin mRNA were determined by semi-quantitative R T - P C R . Cycle numbers were optimized in prior experiments. that TGFB1 may be involved in an autocrine loop of SHIP upregulation, important for mediating LPS tolerance and absent in SHIP-/- BMMOs. This was further supported by the observation that co-treatment of LPS-stimulated SHIP+/+ BMMOs with a TGFB-neutralizing antibody, but not isotype control, led to the prevention of SHIP protein induction (Fig. 3.23c) and LPS tolerance, as assessed by TNFa and nitrite production (Fig. 3.23d). Finally, the lack of LPS tolerance in SHIP-/- BMMOs was not confounded by differences in cell surface expression of the LPS receptor complex TLR4/MD2, since similar FACS profiles were observed the two genotypes over the course of LPS stimulation (Fig. 3.24). a BMrrufrs rJ BMrrnfrs t ime C 4 C 8 C 24 «? 80T 1 b c C C atTGFp irrel F ig . 3.23. SHIP upregulat ion is essent ia l for endotoxin tolerance and is mediated by L P S -st imulated T G F p . (a) SHIP+/+ B M M O s were treated ± TGFB for the indicated times, and total cell lysates subjected to Western analysis for SHIP (upper panel) or She loading control (lower panel), (b) SHIP+/+ (clear bars) or -/- BMMOs (black bars) were treated ± 100 ng/ml L P S for 24 h after which time supernatants were assessed for TGFB. (c) SHIP +/+ B M M O s were treated as in (b) ± T G F B blocking antibody or irrelevant control antibody and levels of SHIP (upper panel) and She (lower panel) assessed as in (a), (d) SHIP+/+ B M M O s were not tolerized (clear bars) or tolerized (black bars) for 24 h with 100 ng/ml L P S ± TGFB-blocking or irrelevant control antibodies, washed and challenged for 3 h with a second 100 ng/ml dose of L P S . Supernatants were analyzed for the production of nitrite (upper panel) or T N F a (lower panel). 65 Thus, the elevation of SHIP levels achieved late in LPS signaling likely resulted from the action of TGFB, and this phenomenon was important for mediating LPS tolerance. BMm<t>s SHIP+/+ SHIP-/-Fig. 3.24. Lack of endotoxin tolerance in SHIP-/- BMMOs is not associated with perturbed TLR4/MD2 surface levels. SHIP+/+ and -/- B M M O s were treated with 100 ng/ml L P S for the indicated times and subjected to flow cytometric analysis of the TLR4/MD2 surface complex over time. Specifically, cells were stained with rabbit anti-TLR4/MD2 and FITC-labelled anti-rabbit secondary antibody (rightmost histogram peaks) or secondary antibody alone as control (leftmost histogram peaks). Results are representative of 3 independent experiments. TLR4 * TLR4 * 66 To elucidate the role of SHIP in secondary LPS exposure signaling, wild-type and -/- BMMOs were stimulated for 8 h with LPS, after which time the cells were repeatedly washed to remove residual LPS, followed by a second LPS stimulus at 24 h. This was not an artificial attempt to dampen 1 s t LPS dose signalling, as LPS tolerance has been demonstrated to last both in vitro and in vivo for a period of several days, after which time initial signaling has subsided (West and Heagy, 2002). Thus, using this procedure in Fig. 3.25, it could be seen that in wild-type BMMOs the secondary pAkt response was dampened, or in effect, tolerized. However, in SHIP-/- BMMOs this process did not occur, and pAkt became phosphorylated to an even greater extent by the second LPS dose (Fig. 3.25). Moreover, SHIP-/- BMMOs displayed similar inabilities to dampen Statl and Erk1/2 phosphorylation following the,second LPS dose (Fig. 3.25). Taken together, these results suggested that the upregulation of SHIP by LPS/TGFB was necessary to dampen Akt, Statl, and Erk1/2 activation, and that failure to do so in SHIP-/- BMMOs precipitated the failure of the cells to LPS tolerize. These results were consistent with the hypersensitivity of SHIP-/- mice to LPS-induced mortality, observed in a model of LPS-induced septic shock (Fig. 3.26). 67 • /+ loter ized BMm^s to le rued time C S 1 3 6 9 12 16 24 5 1 3 6 (h) tone C 5 1 .......... j^^t -±Li^jjs- jgttttrib iHMtk. Jtfli'nY ~ .im^ t HgHft ^ flg&fe zMfitth iVIiVlrn — • «SHtP 3 6 912 16 24 5 1 3 6_{hJ •4P.SHIP •«P-Stat1 « P - N F K B «P-Akt «P-Erk1/2 "•GAPDH «PAkt V exp <iP-Erk1/2 1* exp «P-SHIP «P-St3l1 « P - N F K B ««P~Akt «P-Erk1/2 ••GAPDH Fig. 3.25. SHIP-/- B M M O s may be refractory to endotoxin tolerance because of deficient control of Akt, Sta t l , and Erk1/2 activation. SHIP+/+ and -/- B M M O s were treated for the indicated times with 100 ng/ml L P S for up to 8 h, after which time L P S was washed from the medium. Cells were then re-stimulated with a second 100 ng/ml L P S dose where indicated (tolerized), total cell lysates prepared and subjected to Western analysis for SHIP, pSHIP(NPXY), pStat1(Y701), pAkt(S473), pErk1/2, and G A P D H . Longer exposures are shown for SHIP+/+ B M M O s (bottom two panels on left). LPS 40 mg/kg 100 t Time (h) F ig . 3.26. SHIP-/- mice are hypersensitive to LPS- induced mortality in vivo. 5 week-old SHIP+/+ (open circles) (n = 5) and SHIP-/- mouse littermates (closed squares) (n = 5) were injected i.p. with 40 mg/kg L P S and survival observed over 60 h. Similar results were obtained in an independent experiment. 68 3.3 DISCUSSION SHIP has been demonstrated to be a master negative regulator of hemopoietic cell survival, proliferation, differentiation and activation by virtue of its ability to hydrolyze the 5'-phosphate of the PI3K-generated product, PIP3 (Krystal, 2000). At the commencement of this thesis, despite the observation of a myeloproliferative disorder in SHIP-/- mice and the consolidation of their lungs by macrophages in a process deemed incompatible with survival (Helgason et al., 1998), not much focus had been paid to the role of SHIP in cells of this lineage. It was known that the bone marrow and spleen of SHIP-/- mice contained elevated numbers of CFC-GM and this was attributed to hyper-responsiveness to IL-3, SF, GM-CSF, and M-CSF observed in colony assays, in vitro (Helgason et al., 1998). While providing insight into the basis for the myeloproliferative disorder, these studies were conducted using heterogeneous, total bone marrow and did not characterize the cells formed in the colonies. Thus, it was decided to extend this analysis, using more primitive Sca-1+Lin" bone marrow progenitors (Eaves 2002), and to track the process of myeloid/macrophage differentiation using the Mac-1 surface marker (Springer et al., 1979; Beller et al., 1982; Gordon et al., 1992). Using this analysis, it was determined that SHIP-/- Sca-1+Lin" progenitors displayed accelerated and enhanced macrophage differentiation (Fig. 3.1) compared to wild-type in the presence of Epo/IL-3/IL-6/SF and M-CSF-alone, likely as a result of their enhanced survival. This was in keeping with a subsequent study which also concluded that SHIP enhanced myeloid cell survival, although this study was performed using mature cells (Liu et al., 1999). Alternatively, or in addition, it could not be ruled out that SHIP-/- progenitors demonstrated a cell-autonomous propensity to skew down this lineage, as has been described for SHP-1 mutant mice (Paling and Welham, 2005), which phenotypically resemble SHIP-/- mice (Ward, 1978; Tsui et al., 1993; Shultz et al., 1993). 69 Finally, since Mac-1 was by no means a specific marker of macrophage differentiation, this process was next assessed using a more specific marker: the M-CSF receptor, c-fms (Cook et al., 2003). To facilitate analysis, more study-amenable Lin" or total BM populations were chosen, and differentiation was assessed in the presence of M-CSF (Fig. 3.2). This led to the observation of enhanced resultant SHIP-/- BMMO numbers, but not accelerated differentiation. It was possible that any survival advantage gained in the absence of SHIP was masked using this more heterogeneous and committed starting population (as opposed to the accelerated differentiation observed using SHIP-/- Sca-1+Lin" progenitors). Alternatively or in addition, these differences might also have reflected different roles of SHIP in responses to Epo/IL-3/IL-6/SF versus M-CSF. Nonetheless, taken together, these studies demonstrated accelerated and enhanced SHIP-/- macrophage differentiation in vitro, recapitulating what was observed in vivo (Helgason et al., 1998). Given these findings, the phenotype of the resultant in vitro-expanded SHIP-/- MO pool was determined. In order to facilitate analysis, immortalized BMMO cell lines were prepared by J2 (v-raf/v-myc) retroviral-mediated transformation (Blasi et al., 1985; Blasi et al., 1989) (Chapter 3). This approach had successfully been taken by others to derive the GG2EE MO cell line from C3H/HeJ mice (Blasi et al., 1987), INF-3A and HeNC2 from C3H/HeN (Cox et al., 1989; Jin et al., 1997), and ANA-1 from C57BL/6 mice (Cox et al., 1989). While both SHIP J2M+/+ and J2M-/- cell lines were c-fms-positive and expressed similar surface Mac-1 and Mac-2 levels as the established v-abl-transformed J774.1 murine MO cell line (Fig. 3.4), only the J2M+/+ and J744.1 cells were able to significantly induce NO production in response to LPS (Fig. 3.5). However, rather than being completely unresponsive to LPS, it became clear that the J2M-/- cell line produced greater levels of IL-1, TNFa, IL-6, IL-12, and COX-2 than J2M+/+ cells (Fig. 3.9 and data not shown). Moreover, J2M-/- displayed enhanced basal activation of Akt, Erk1/2, p38, and N F - K B pathways and elevated basal levels of anti-inflammatory IL-1Ra and kBa (data not shown). Importantly, J2M-/- cells displayed deficient nuclear localization of IRF-1 in response to LPS 70 or IFNy (Fig. 3.9) (the former, mediated by autocrine IFNB (Gao et al., 1997)) and since binding of IRF-1 to the iNOS promoter has been shown to be essential for inducing iNOS (Kamijo et al., 1994; Martin et al., 1994), this was consistent with their inability to effectively transcribe iNOS mRNA and produce protein (Fig. 3.6, 3.7). Given this IRF-1 nuclear localization deficiency in J2M-/- cells, it was surprising that induction of COX-2 by LPS was not impaired, since it has also been identified as an IRF-1 target (Blanco et al., 2000). However, COX-2 induction by LPS is also regulated by other transcription factors, like the CCAAT-enhancer binding protein B (C/EBPB) and those downstream of mitogen-activated protein kinase (MAPK) cascades (Wadleigh et al., 2000). Moreover, transformation of MOs by the v-raf/v-myc J2 retrovirus has been demonstrated to occur through a constitutive C/EBPB-mediated autocrine loop (Wessells et al., 2004) . Taken together with the observed constitutive Akt, MAPK, and N F - K B activation observed in J2M-/- cells (data not shown), this may suggest that their perturbed constitutive and LPS-induced expression pattern, relative to J2M+/+, reflects the unique sensitivity of target promoters to the altered transcription factor milieu. In this regard, the iNOS promoter might have been particularly sensitive to the loss of nuclear IRF-1, as would be expected (Kamijo et al., 1994; Martin et al., 1994). Although the nuclear localization sequence (NLS) of IRF-1 has been identified (Schaper et al., 1998), not much is known about how its localization is regulated, apart from a very recent paper demonstrating enhancement of IFNy-induced IRF-1 nuclear localization by all-trans retinoic acid (ATRA) (Luo and Ross, 2005). It is conceivable that competition by aberrantly over-activated transcription factors in J2M-/- cells with IRF-1 for limiting nuclear co-activators may hasten the shuttling of IRF-1 out of the nucleus, as this phenomenon has been described for other transcription factors (Martin et al., 2005) . However, this awaits further study. Thus, although SHIP appeared to positively regulate iNOS/NO in J2M cell lines by permitting the timely appearance of IRF-1 in the nucleus, and negatively regulate other pro-inflammatory factors, it was decided that these could be 71 artifacts of transformed cell lines. However, similar results obtained with independent J2M-/- clones suggested it was not an isolated phenomenon (data not shown). Analysis was therefore shifted to primary SHIP+/+ and -/- BMMOs to determine the suitability of the J2M model system. In contrast to J2M-/- cells, primary SHIP-/- BMMOs were able to produce more NO in response to LPS, compared to wild-type (Fig. 3.10). This was attributed to enhanced activation in SHIP-/- BMMOs of two targets downstream of PI3K: Akt and p70S6K (Fig. 3.11 and 3.13). Specifically, LPS signaling is known to lead to the secretion of IFNB, which acts in an autocrine manner to activate the Statl (tyrosine phosphorylation) and, subsequently, IRF-1 transcription factors necessary for iNOS induction (Gao et al., 1997). PI3K has been implicated in the translation and/or secretion of IFNB by LPS (Weinstein et al., 2000; Rhee et al., 2003). Although not directly demonstrated, it was suggested that enhanced LPS-induced p70S6K activation in SHIP-/- BMMOs led to enhanced IFNB secretion. This would be consistent with the observed enhancement of Statl tyrosine phosphorylation observed in SHIP-/- BMMOs. Interestingly, the PI3K inhibitors wortmannin and LY294002 gave very different results when applied to LPS-treated BMMOs. While both inhibitors dampened down LPS-induced pAkt levels, only LY294002 potently inhibited pp70S6K. Although this initially suggested LY294002 was a better PI3K inhibitor, another protein implicated in translation control, 4EBP1, also had its LPS-induced phosphorylation significantly reduced by LY294002 (Gingras et al., 2001)). This raised some concern, as both p70S6K and 4EBP fall under the control of mTOR (while only p70S6k has been suggested to be regulated more directly by PI3K) (Fingar and Blenis, 2004). Moreover, LY294002 has been suggested to impact directly upon mTOR in some systems (Fingar and Blenis, 2004), and the fact that the PI3K inhibition-inactive analogue, LY303511, did not result in severe inhibition of pp70S6k or p4EBP (Fig. 3.13) lent support to this hypothesis. In summary, it is thus likely that the LPS/p70S6K/IFNB/Stat1/iNOS axis was enhanced in SHIP-/- BMMOs via bone fide enhanced activation downstream of PI3K, while LY294002 very potently affected this axis due to 72 inhibition of PI3K and mTOR. This is not to say that wortmannin was a perfect PI3K inhibitor (even though it more potently inhibited pAkt), as it has been shown to inhibit other non-PI3K targets and has been suggested to be unstable (Fingar and Blenis, 2004). Similar differential effects of wortmannin and LY294002 on LPS-induced NO production were observed by another group using v-Abl transformed RAW cell lines, although they postulated this was due to differential phosphorylation of iNOS (Salh et al., 1998). Analysis was next shifted to LPS-induced cytokine production that has been attributed to the MyD88-dependent pathway downstream of TLR4 (Dunne and O'Neill, 2005). Intriguingly, it initially appeared that SHIP-/- BMMOs were able to mount two very distinct early LPS response patterns: hyper-responsive pro-inflammatory cytokine induction (Fig. 3.19) versus hypo-responsive (Fig. 3.15). Pooled analysis of several independent experiments clarified this apparent conundrum by suggesting that the pattern of SHIP-/- BMMO LPS responsiveness changed from hypo-responsive at early times, to hyper-responsive at later times (Fig. 3.16). Moreover, it was suggested that this pattern was related to enhanced LPS-induced secretion of anti-inflammatory IL-10 in SHIP-/- BMMOs at early times, and a possible reciprocal relationship between early pAkt and pp65 N F - K B levels (Fig. 3.15, 3.17 and 3.18). Since both wortmannin and LY294002 pre-treatment reduced IL-10 secretion and improved pp65 N F - K B levels in SHIP-/- BMMOs, it was suggested that these were bona fide outcomes of enhanced PI3K activation in these cells (Fig. 3.18). In short, it appeared that PI3K-mediated cell-autonomous and autocrine effects were responsible for the early LPS (pro-inflammatory) hypo-responsiveness of SHIP-/-BMMOs. Whether PI3K is a positive or negative regulator of LPS-induced pro-inflammatory outcomes is a subject of much contention, with some claiming a positive role (Hattori et al., 2003; Hirsch et al., 2000; Li et al., 2003; Madrid et al., 2001; Weinstein et al, 2000), and others suggesting a negative role (Diaz-Guerra et al., 1999; Guha and Mackman, 2002; Pahan et al., 1999; Park et al., 1997). It is likely that much of this controversy has arisen from the use of different cell 73 types, different measurement times, and relying too heavily on data obtained with pharmacological inhibitors. The improvement of SHIP-/- BMMO pro-inflammatory cytokine production observed over time and, in fact, their transition to a hyper-responsive state relative to wild-type could reflect a differential impact of SHIP/PI3K on early versus late TLR4 signaling or on signaling initiated by autocrine-acting cytokines. For instance, a recent seminal publication has concluded that the relative impact of PI3K/Akt on N F - K B may be related to the ratio of the IKKa and IKKB isoforms in the IKK complex (Gustin et al., 2004). Specifically, it was determined that PI3K/Akt impacts in a positive manner upon IKKa and does not affect IKKB. This is particularly interesting in light of studies conducted in our laboratory demonstrating enhanced N F - K B activation in IgE + antigen-treated SHIP-/- mast cells (Kalesnikoff et al., 2002). A subsequent study has revealed that this process is mediated by IKKa (Peng et al., 2005). In contrast, and lending support to cell type-specific differences, IKKa has been demonstrated to limit IKKB and N F - K B activation in macrophages (Lawrence et al., 2005; Li et al., 2005). Thus, if IKKa activity is elevated in SHIP-/- BMMOs, then this might help explain their hypo-responsiveness. Moreover, Neil Reiner's group has demonstrated PI3K-mediated early degradation of IRAK, a positive regulator of MyD88-dependent N F - K B activation (Noubir et al., 2004) and another group has implicated PI3K in the recruitment of the negative TLR4 regulator, Tollip, to the plasma membrane via PIP3 (Li et al., 2004). Finally, consistent with what has been reported here, another recent paper has suggested that LPS-induced PI3K/Akt leads to enhanced IL-10 secretion and diminished pro-inflammatory cytokine secretion (Martin et al., 2005). Akt-mediated glycogen synthase kinase 3B (GSK3B) deactivation (phosphorylation) was suggested to cause this phenomenon (Martin et al., 2005). It remains to be seen whether this also explains the similar association in SHIP-/- BMMOs (i.e. enhanced IL-10 and diminished TNFa), but is likely worthy of future experimentation. Thus, the role of PI3K/Akt in LPS responses is very likely complex and dependent upon many parameters. PI3K is not unique in this regard, as two recent high-profile publications have proposed opposite roles for the signaling 74 adaptor, DAP12, in LPS-mediated responses (Hamerman et al., 2005; Turnbull et al., 2005). Thus, fully elucidating LPS/TLR4 signaling may prove to be a challenging task. Returning to the enhanced LPS responses observed in SHIP-/- BMMOs at later times, it appeared that these cells were less able to restrain a runaway LPS response, compared to wild-type. It was thus decided to determine if SHIP-/-BMMOs would respond in a phenomenon known as endotoxin (LPS) tolerance. Specifically, after an initial exposure and pro-inflammatory response to LPS, wild-type BMMOs have been demonstrated to be refractory to a second LPS stimulus (Fan and Cook, 2004). It was discovered that SHIP-/- BMMOs were refractory to endotoxin tolerance regardless of whether they demonstrated hyper- or hypo-responsive patterns to the initial LPS dose (Fig. 3.19, 3.20, and 3.21). This was likely because SHIP was normally upregulated in wild-type BMMOs by LPS in an autocrine TGFB-mediated fashion (Fig. 3.22 and 3.23). At the signaling level, SHIP-/- BMMOs appeared less able to restrain Akt, Erk1/2, and Statl activation upon exposure to a second LPS challenge (Fig. 3.25). Moreover, despite their ability to induce known mediators of endotoxin tolerance, IRAK-M and SOCS1 (Kinjyo et al., 2002; Kobayashi et al, 2002; Nakagawa et al., 2002), SHIP-/-BMMOs remained refractory to this phenomenon, suggesting that elevated P I P 3 levels raise the threshold required for hyporesponsiveness (Sly et al., 2004). Taken together, these properties were thought to contribute to the enhanced mortality observed in SHIP-/- mice upon LPS challenge in vivo (Fig. 3.26). Upon this discovery, it was decided to shift the focus to in vivo-differentiated macrophages from SHIP+/+ and -/- mice, such as peritoneal MOs (PMOs), and this will be described in the next chapter. 75 CHAPTER 4 - SHIP REPRESSES AN ALTERNATIVE PROGRAM OF MACROPHAGE DIFFERENTIATION IN VIVO AND EX VIVO 4.1 INTRODUCTION Previous studies conducted in this laboratory (Helgason et al., 1998) and studies conducted in Chapter 3 suggested that SHIP-/- mice have increased numbers of granulocyte/macrophage progenitors and that these progenitors demonstrate enhanced sensitivity to growth factors in vitro. Moreover, this was suggested to account for the observed elevated number of granulocytes and monocytes/macrophages in circulation and infiltrating the tissues of SHIP-/- mice. In Chapter 3, studies were conducted on macrophages differentiated in vitro from bone marrow precursors. In order to extend and determine the applicability of these in vitro macrophage findings to an in vivo counterpart, peritoneal (PMOs) and alveolar macrophages (AMOs) were obtained from the lavage fluid (peritoneal and broncho-alveolar, respectively) of wild-type and SHIP-/- mice, as these represent a previously validated, reliable and easily retrievable source of in vivo-differentiated macrophages (Bogdan, 2001b). As will become evident, the phenotype of these in vivo-differentiated SHIP-/- MOs was quite distinct from in vitro-differentiated J2M-/- cells and SHIP-/- BMMOs. In fact, it became clear that SHIP repressed an alternative program of macrophage differentiation and activation in vivo versus in vitro. 4.2 RESULTS 4.2.1 SHIP-/- MICE HAVE INCREASED NUMBERS OF RESIDENT MACROPHAGES Resident cells were obtained from the peritoneal cavity of wild-type and SHIP-/- mice by repeated lavage, and PMOs were selected from the heterogenous population based on their ability to adhere to the tissue culture plate after 3 to 18 h incubation at 37 °C and remain after repeated washings. As can be seen in Fig. 4.1a, on average, more PMOs were retrieved from SHIP-/-76 Fig. 4.1. SHIP-/- mice have increased numbers of mature resident peritoneal macrophages. (a) PMOs were obtained from 17 paired SHIP+/+ and -/- mice selected by adherence to tissue culture plates for 3 h to overnight and repeated washings to remove non-adherent cells, and counted. Equal numbers of SHIP+/+ and -/- PMOs were collected and subjected to Western analysis for (b) c-fms and SHIP or (c) SHIP and PU.1 or (d) were subjected to FACS histogram analysis for F4/80, M a d and Gr1 surface expression (blue) or isotype control (red). Similar results were obtained in at least one independent experiment. 77 mice compared to wild-type littermates. This suggested that the myeloproliferative disorder of SHIP-/- mice was also manifested in the peritoneal cavity. Although the cells of both genotypes morphologically resembled macrophages (data not shown), surface marker analysis was conducted to further determine the phenotype of the cells. FACS analysis revealed that adherent cells from both genotypes were Mac1+F4/80+Gr1" (Fig. 4.1d) and CD204/scavenger receptor-A (SR-A)+ (Fig. 4.8 c), while Western blot analysis revealed similar expression of the M-CSF receptor, c-Fms (Fig. 4.1b), consistent with the surface phenotype of PMO (Taylor et al., 2005) and the PU.1 transcription factor implicated in its induction (Fig. 4.1c). Analysis of broncho-alveolar lavage fluid also revealed increased numbers of adherent AMOs in SHIP-/- mice (data not shown), consistent with their observed elevated presence in SHIP-/- lung histological sections (Helgason et al., 1998; Liu et al., 1999). Thus, SHIP-/- mice have elevated numbers of resident PMOs and AMOs, as compared to wild-type. 4.2.2 LPS-STIMULATED SHIP-/- PMOS SECRETE LOW LEVELS OF NO BUT CAN BE RESCUED BY EXOGENOUS L-ARGININE In contrast to the results obtained in Chapter 3 with in vitro-differentiated BMMOs (where -/- cells were often able to produce more NO than +/+ cells), when the ability of in wVo-differentiated PMOs from SHIP+/+ and -/- mice to produce NO in response to LPS or LPS + IFNy was compared, it was found that the SHIP-/- PMOs produced far less NO (Fig. 4.2a and b). We sought to determine if this was because LPS was less efficient at upregulating inducible nitric oxide synthase (iNOS) in SHIP-/- PMOs. As can be seen in Fig. 4.2c this was not the case and, in fact, the increase in iNOS typically occurred faster and in some cases, reached higher levels in SHIP-/- PMOs. Next I tested if the iNOS substrate, L-arginine, was limiting in SHIP-/- PMOs by comparing NO production from LPS + IFNy-stimulated SHIP+/+ and -/- PMOs in the presence and absence of exogenously added L-arginine. Of note the level of L-arginine in the culture medium used, i.e., IMDM, is approximately 400 LIM. A S shown in Fig. 4.3a and b, 78 the addition of 2 mM L-arginine had a negligible effect on NO production from LPS + IFNy-stimulated SHIP+/+ PMOs but dramatically increased NO production from SHIP-/- PMOs. Interestingly, this effect was not observed with in vitro-derived, SHIP-/- BMMOs (Fig. 4.3b). IFN-y (U/ml) - - - 50 20 Duration (h) of stimulation LPS (100 ng/ml) + IFN-y (20 U/ml) Fig. 4.2. LPS-stimulated SHIP-/- PMOs, unlike BMMOs, are deficient in NO production. SHIP+/+ and -/- resting (a) or thioglycollate-elicited (b) P M O s were treated ± the indicated concentrations of L P S and IFNy for 24 h (a) or L P S (100 ng/ml) + IFNy (20 U/ml) (b) and NO production measured, (c) Total cell lysates (TCLs) were also prepared from equal numbers of the P M O s in (b) and subjected to Western analysis for iNOS (right panel). Data points in all panels are the means +/- S E M of at least triplicate determinations. Significant difference between SHIP+/+ and SHIP-/- : *p< 0.05, **p<0.01, ***p<0.001, using unpaired two-tailed student's Hest. Similar results were obtained in at least 3 separate experiments. 4.2.3 LPS-STIMULATED SHIP-/- PMOS AND AMOS SECRETE LOW LEVELS OF NO BECAUSE OF CONSTITUTIVELY HIGH ARGINASE ACTIVITY It has been reported that M2, healer MOs possess very high levels of arginase I (Argl) and this effectively competes with iNOS for L-arginine (Gordon 2003; Noel et al. 2004; Goerdt and Orfanos 1999 and Mills 2001). To confirm that high Argl levels might explain the high arginine requirement in SHIP-/-PMOs, Argl Western blots were performed. Protein levels of Argl increased slightly in wild-type PMOs with time following LPS + IFNy treatment, but they were markedly and constitutively elevated in SHIP-/- PMOs (Fig. 4.4a). To determine if the constitutively elevated Argl levels in SHIP-/- PMOs were responsible for the low LPS-induced NO production from these cells, the effects 79 Fig. 4.3. L-arginine supplementation rescues SHIP-/- PMO NO production, (a) SHIP+/+ (squares) and -/- (triangles) P M O s were treated with 100 ng/ml L P S + 100 U/ml IFNy for the indicated times and NO production measured with (open symbols) or without (closed symbols) 30 min prior addition of 2 mM L-arginine. (b) SHIP+/+ (black bars) and -/- (grey bars) P M O s (left) and B M M O s (right) were treated for 48 h with L P S + IFNy (as in C) ± 2 mM L-arginine and NO production measured and expressed as % increase in NO of L-arginine supplemented/non-supplemented controls. Data points in all panels are the means ± S E M of at least triplicate determinations, except for duplicate determinations in (b). Significant difference between SHIP+/+ and SHIP-/- : *p< 0.05, **p<0.01, ***p<0.001, using unpaired two-tailed student's f test. Similar results were obtained in at least 3 separate experiments. of the arginase inhibitors, L-nor-NOHA and L-nor-valine on LPS-induced NO production from SHIP+/+ and -/- PMOs were compared. As shown in Fig. 4 . 4 b , these inhibitors significantly increased NO production from SHIP-/- but not +/+ PMOs. To address whether these constitutively high Argl levels were restricted to PMOs, AMOs were isolated from SHIP+/+ and -/- bronchoalveolar lavage fluid. As shown in Fig. 4.5a, Argl levels (left panel) and activity (right panel) were also dramatically elevated in SHIP-/- AMOs. Consistent with this, it was found that SHIP-/- AMOs produced less NO than their wild type counterparts in response to LPS + IFNy .(data not shown). Moreover, within the hemopoietic compartment, SHIP+/+ P M O s 0 6 12 24 SHIP-/-P M O s 0 6 12 24 h I KBa *» mm mmm ^tmt ^MP^  ^AP^ M^to tm, 50 _ f — . 40 O > z z 30 | i S - i 20 S -«= C 00 - S , 10 P M O s mm SHIP+/+ C Z Z 3 SHIP-/-L-nor -NOHA L-nor -Valine Fig. 4.4. LPS-stimulated SHIP-/- P M O s and A M O s secrete low levels of NO because of constitutively high arginase activity, (a) SHIP+/+ and -/- P M O s (10 6 cells/ml) were stimulated for the indicated times with 100 ng/ml L P S + 20 U/ml IFNy and T C L s subjected to Western analysis for Argl and IKBO . (b) SHIP+/+ and -/- P M O s were stimulated for 48 h with 100 ng/ml L P S + 100 U/ml IFNy ± the arginase inhibitors L-nor-NOHA (100 uM) or L-nor-valine (20 mM) and NO production measured as a percent of non-inhibited controls. Results shown are the mean ± S E M of duplicate determinations and are representative of 3 separate experiments. Argl was not detected in any significant amount in SHIP+/+ or -/- bone marrow-derived mast cells (BMMCs), neutrophils, or dendritic cells (data not shown). Lastly, it was asked if this elevated Argl was restricted to MOs or was also present in SHIP-/- liver hepatocytes. Related to this, Argl expression is restricted primarily to hepatocytes (where it is expressed in a constitutive fashion to generate urea as part of the urea cycle) and to MOs (where it is expressed in an inducible fashion) (Wu and Morris 1998). As shown in Fig. 4.5b, there was no detectable difference in Argl levels (left panel) or activity (right panel) in hepatocyte-rich liver homogenates from SHIP+/+ and -/- mice, suggesting that the differences were MO-specific. 81 a Argl GAPDH Argl GAPDH A MOs +/+ -/-Liver +/+ -/-> i 0 > <D — 1 5" ro 13 I, £ **. SHIP+/+ SHIP-/-A M 0 S SHIP+/+ SHIP-/-Llver Fig. 4.5. Constitutively elevated Argl expression is unique to SHIP-/- resident M O s . (a) Unstimulated A M O s obtained from SHIP+/+ or -/- BAL fluid by adherence, were lysed after overnight incubation and subjected to Argl and G A P D H Western analysis (left panel) or arginase activity assays (right panel) in which the amount of urea produced from the hydrolysis of L-arginine in 1 h was normalized to the amount of protein in the lysate ( * * p < 0 . 0 1 ) . (b) Liver homogenates, prepared from SHIP+/+ and -/- mice in arginase assay lysis buffer, were subjected to Argl and G A P D H Western analysis (left panel) and arginase activity assays (right panel) as in (a). 4.2.4 ARGINASE I LEVELS ARE UPREGULATED BY THE PI3K PATHWAY To gain some insight into why Argl levels were elevated in SHIP-/- PMOs and AMOs, wild-type PMOs were stimulated with LPS in the presence and absence of LY294002 or wortmannin and it was found that these PI3K inhibitors effectively reduced LPS-induced Argl levels (Fig. 4.6a). Next, wild-type PMOs 82 were transiently transfected with a constitutively active PI3K (pHOCAAX) and this was found to cause a modest, dose-dependent increase in both arginase activity and Argl levels in the absence of LPS stimulation (Fig. 4.6b, left panels). Furthermore, SHIP-/- PMOs were exposed to low doses of LY294002 for 48 h in the absence of LPS and this resulted in a reduction of constitutive arginase activity and Argl levels (Fig. 4.6b, right panels) (similar results were obtained with SHIP+/+ PMOs, data not shown). These results were consistent with PI3K being a positive regulator of Argl and suggested that the increased PIP3 levels in SHIP-/- PMOs were responsible for the increased arginase activity. It was next asked if the increased Argl protein levels in SHIP-/- PMOs were due, at least in part, to increased transcription and/or stability of Argl mRNA by carrying out semi-quantitative RT-PCR analysis of unstimulated SHIP+/+ and -/- PMOs. As shown in Fig. 4.6c, higher Argl mRNA levels were present in SHIP-/- PMOs. Finally, since the transcription factors Stat6 and C/EBPB have been implicated in mediating macrophage Argl transcription (Gray et al. 2005), these factors were compared in PMOs from both genotypes. As can be seen in Fig. 4.6d, SHIP-/-PMOs have elevated levels of tyrosine-phosphorylated (active) Stat6 and increased C/EBPB isoforms at rest, suggesting that the basis for elevated Argl is at least partly due to effects on transcription. 83 SHIP+/+ P M O s A r g i n a s e I G A P D H L P S (200 ng/ml , 24h) D M S O (0.1 %) W o r t m a n n i n (nM) LY294002 (uM) ~ 1.00 > fS 0.75 <t> <^ <u ? 0.50 (0 re "2 c ra — 4> cn i -0.25 — 0.00 A r g i n a s e I Erkl SHIP+/+ P M O s m 0 2.5 5.0 p 1 1 0 C A A X ( u g ) 50 500 A r g i n a s e I Grb2 0 5 2 LY294002 (uM) P M O s +/+ -I-A r g i n a s e I p-actin P M O s +y* c-fms SHIP pStat6 A r g i n a s e I C / E B P p ^ La I 40 kDa r 33 kDa L 24 kDa Fig. 4.6. Arginase is upregulated by the PI3K pathway, (a) SHIP+/+ PMOs were pretreated for 30 min with wortmannin, LY294002 or DMSO and treated ± 200 ng/ml LPS for 24 h. Total cell lysates from equal numbers of cells were subjected to Argl and GAPDH Western analysis, (b) SHIP+/+ PMOs (2 x 106 cells/6 cm plate) were transiently transfected with the indicated amount of pHOCAAX using DEAE-dextran, allowed to recover overnight (left), or SHIP-/- PMOs were incubated for 48 h with the indicated concentration of LY294002 (right), and lysates prepared for arginase assays (upper panels) or Argl ,Erk1, and Grb2 Western analysis (lower panels), (c) Total RNA was obtained from SHIP+/+ and -/- PMOs using Trizol and mRNA levels of Argl and (3-actin determined by semi-quantitative RT-PCR, using specific primers and after titrating optimal cycle numbers, (d) Total cell lysates were prepared from resting SHIP+/+ and -/- RPMOs after only a few h in culture and subjected to Western analysis for SHIP, pStat6, C/EBP3, arginase I, or c-fms (as a loading control and confirmation of macrophage phenotype). Results are representative of 2 separate experiments. 84 4.2.5 THE M2 PHENOTYPE BECOMES MORE PRONOUNCED AS SHIP-/-MICE AGE Interestingly, while SHIP-/- PMOs consistently displayed higher arginase activity than +/+ PMOs from 6-10 week-old mice, considerable variability was found in SHIP-/- PMO Argl activity and levels. It appeared that PMOs from older SHIP-/- mice possessed higher Argl levels. To determine if this was indeed the case, the arginase activity in PMOs from SHIP+/+ and -/- mice sacrificed at different ages was examined. Intriguingly, it was found that PMOs from young SHIP+/+ and -/- mice (3-4 weeks of age) possessed similar, low arginase activity (Fig. 4.7a). The mean SHIP-/- PMO arginase activity in this age group was only 1.5-fold higher than +/+ PMOs. However, as the SHIP-/- mice aged, the arginase activity increased dramatically in their PMOs (Fig. 4.7a). This age-induced Argl increase in SHIP-/- mice correlated nicely with a reduced ability to produce NO in response to LPS + IFNy (Fig. 4.7b). Further features characteristic of M2 MOs were next examined, to see if these also became more pronounced as SHIP-/-mice aged. Specifically, LPS-stimulated levels of the pro-inflammatory cytokines, TNFa, IL-6 and IL-12 were compared from PMOs isolated from 4 week and 10 week-old SHIP+/+ and -/- mice. While there was little difference in the production of these cytokines from 4 week-old mice, there was a marked reduction in the ability of the SHIP-/- PMOs to secrete these same cytokines, compared to littermate controls, when they were taken from 10 week-old mice (Fig. 4.8a). As well, LPS-stimulated levels of IL-1 receptor antagonist (IL-1Ra) were elevated in PMOs from 10 week-old SHIP-/- mice (Fig. 4.8b). Also, unstimulated PMOs from 10 week-old SHIP-/- mice produced substantially more IL-6 and IL-10 than their littermate controls (Fig. 4.8b). Finally, PMOs from adult SHIP-/- mice demonstrated enhanced surface staining for an established M2 MO marker (Stein et al., 1992), the mannose receptor (MR/CD206) (Fig. 4.8c). Taken together, these findings were consistent with a skewing towards an M2 MO phenotype as SHIP-/- mice age (Gordon 2003 and Goerdt and Orfanos 1999). 8 5 PMOs 3-4 weeks 5 weeks 7-10 weeks PMOs 4 week-old mice 125 100 1 75 c 18 50 c \_ 4) a. 25 • • SHIP+/+ • SHIP-/-Lu i 24 48 P M O s 10 week-old mice Duration (hj of LPS + IFNy stimulation Fig. 4.7. The M2 M O phenotype becomes more pronounced as SHIP-/- mice age. (a) SHIP+/+ (solid squares ) and - / - (open squares ) P M O arg inase activity w a s recorded a s a function of m o u s e a g e (***p<0.001). (b) L P S (100 ng/ml) + IFNy (100 U /ml ) - induced N O product ion is c o m p a r e d between P M O s from 4 week (left panel) and 10 week-o ld (right panel) SHIP+/+ (black bars) and - / - (grey bars) mice. Data shown are the m e a n + S E M of at least triplicate determinat ions a n d similar results were obtained in at least 3 separa te exper iments . 86 PMOs 4 week-old mice PMOs 10 week-old mice 125 100 125 100 125 100 12 h LPS (1 ug/ml) 12 h LPS (1 |jg/ml) SHIP+/+ PMOs 0 12 24 h Ra SHIP-/-PMOs 0 12 24h 600 I £ 400 200 0 100 §1 o -a 'P • Ra SHIP+/+ PMOs SHIP-/-PMOs C *f+ Cdotrol J- Control %of I 4 Of 2 m a s t rl/ j-coaoe COJMJMR) Fig. 4.8. Further M2 phenotypic characterization of SHIP-/- P M O s . (a) PMOs from 4 and 10 week-old SHIP+/+ and -/- mice were treated with LPS (1 ug/ml) and the secreted levels of M1 factors TNFa, IL-6, and IL-12 determined by ELISA and recorded as percent of wild-type production, (b) Total RNA was obtained using Trizol from SHIP+/+ and -/- PMOs stimulated for the indicated times with 1 pg/ml LPS. RNA (5 ug/condition) was subjected to RNase protection analysis for IL-1 a, IL-1 (3 and IL-1Ra. Shown is a representative autoradiograph of 2 independent experiments, (c) Resting SHIP+/+ and -/- PMOs were stained with fluorochrome-conjugate anti-CD204/SR-A and anti-CD206/MR antibodies and fluorescent intensity compared by event histograms to control autofluoresence. 87 4.2.6 M2 MO PROGRAMMING IN SHIP-/- MICE IS ASSOCIATED WITH LUNG PATHOLOGY AND IMPLANTED TUMOUR SUSCEPTIBILITY As further evidence for M2 MO skewing in vivo it was observed that histological sections of the lungs from >8 week old SHIP-/- mice showed considerable consolidation and fibrosis (Fig. 4.9a, compare SHIP+/+ panel 1 with SHIP-/- panel 2), consistent with M2-MO-induced proliferation (Gordon 2003; Noel et al. 2004; Goerdt and Orfanos 1999 and Song et al. 2000) and reminiscent of human TH2-skewed asthmatic lungs (Zimmermann et al. 2003). As well, SHIP-/- lungs as well as BAL fluids (BALF) from these lungs revealed the presence of large, hexagonal, MO-associated crystals (Fig. 4.9a, panels 3 to 6). Similar crystals have been reported in the lungs of motheaten viable (MeV) mice (Guo et al. 2000), which lack functional tyrosine phosphatase 1 (SHP1), and have been shown in these mice to be composed of the chitinase-like protein, Ym1 (Nio et al. 2004). To determine if MOs from SHIP-/- mice also over-express Ym1, semi-quantitative RT-PCR was carried out with both PMOs and AMOs and it was found that mRNA levels of this protein were indeed elevated in SHIP-/-mouse MOs (Fig. 4.9b). To confirm this at the protein level, in the absence of commercially available Ym1 antibodies, SDS-PAGE was carried out with both cell-free BALF from SHIP+/+ and -/- mice and with purified crystals from the BALF of SHIP-/- mice and, as shown in Fig. 5C, a prominent 45 kDa Coomassie Blue band was found, corresponding to the molecular mass of Ym1 (Guo et al. 2000 and Hung et al. 2002), only in the SHIP-/- fluids and markedly enriched in the crystals from SHIP-/- mice. Mass spectral analysis of this purified band confirmed that it was Ym1 (Rauh et aL, 2005). Finally, given the elevated levels of Ym1 in cells and fluids from SHIP-/- mice, anti-Ym1 antibodies were generated by immunizing rabbits with a peptide corresponding to the sequence GYTGENSPLYK in murine Ym1. Western blot studies conducted with anti-Ym1 confirmed that Ym1 levels were indeed elevated in SHIP-/- AMOs, BAL fluid, and PMOs (Fig. 4.9d and e). Interestingly, this peptide is predicted to also recognize the acidic mammalian chitinase (AMCase, 52 kDa) and chitotriosidase 88 PM** A M O s Fig 4.9. Evidence for SHIP-/- M2 M O skewing in vivo, (a) Panels 1 and 2 are photomicrographs of Masson trichrome stained histological sections of SHIP+/+ (1) and -/- (2) lungs (200X). Panel 3 is a photomicrograph of an H&E-stained SHIP-/- lung histological section revealing a bronchiolar lumen occluded with MO-associated, extracellular, eosinophilic crystals. Panel 4 is an image of tissue culture plate-adherent SHIP-/- AMOs and MO-associated crystals. Panel 5 is a photomicrograph of H&E-stained SHIP-/- lung showing a hexagonal, eosinophilic crystal almost occluding a lumen, and panel 6 is a similar MO-associated crystal observed in BAL fluid (400X for panels 3-6). (b) Total RNA was obtained from SHIP+/+ and -/- PMOs or AMOs, and subjected to Ym1 RT-PCR, using specific primers and after titrating optimal cycle numbers. 3-actin was also optimally titrated as a control, (c) 40 pg of total BALF protein from SHIP+/+ and SHIP-/- mice, together with purified crystals obtained from 3 ml of pooled BALF from SHIP-/- mice by repeated centrifugations through Ficoll-diatrizoate, were boiled for 10 min in SDS-sample buffer and resolved by SDS-PAGE. A 45 kDa Coomassie stained protein band enriched in SHIP-/- cell-free BALF that co-migrated with purified crystals is indicated by an open arrow head, (d) Normalized protein equivalents from SHIP+/+ and -/- AMO lysates, BALF, or PMOs (e) were subjected to Western analysis using antibodies to Ym1, Argl, and Grb2. 8 9 (Chitl, 50 and 39 kDa) and bands of the appropriate size (and an additional band at approximately 30 kDa) are also present in greater proportions in SHIP-/- MOs and BALF (Fig. 4.9d and e). Further study will be necessary to confirm the identity of these bands. However, taken together these studies provide further evidence for M2 MO programming and associated lung pathology in SHIP-/-mice. Given the presence of M2, healing MOs in SHIP-/- mice it was asked if these mice were more amenable to tumour growth (Mantovani et al. 2002 and Rodriguez et al. 2004) by subcutaneously injecting M27 Lewis lung carcinoma cells into 6-10 week old SHIP-/- mice and their littermate controls. As shown in the Fig. 4.10a, the tumours grew substantially faster in the SHIP-/- mice. The tumours were also analyzed for their Argl levels and it was found that they were significantly higher in SHIP-/- mice (Fig. 4.10b). Since levels of the MO-specific marker, c-fms, were similar in tumours from both genotypes, this suggested that the elevated arginase levels were not due to pronounced differences in macrophage recruitment, but resulted from an enhanced M2 macrophage phenotype in SHIP-/- tumors (Fig. 4.10b). Consistent with these results, a very recent mouse study showed that within subcutaneously injected Lewis lung tumours, Argl was only expressed in the infiltrating mature macrophages (Rodriguez et al. 2004). Finally, in contrast to SHIP-/- PMOs and AMOs, tumour-associated MOs (TAMs) from SHIP-/- mice did not display elevated Ym1 levels, compared to wild-type (Fig. 4.10b). Thus, as has been suggested by other groups, TAMs may represent a unique population of M2 MOs. Nonetheless, these results suggest that enhanced M2 MO programming in SHIP-/- mice make these mice more susceptible to tumour growth in vivo. 90 Time (days) F ig . 4.10. Lewis lung carc inoma implants grow faster in SHIP-/- mice, (a) 6-10 week-old SHIP+/+ (filled squares) (n = 11) and -/- mice (open triangles) (n=10) were subcutaneously injected with 2x10 5 M27 Lewis lung carcinoma cells and tumour volume (mm 3) measured over time (upper panel) (**p<0.01). (b) Tumours were also harvested from several mice on day 17, and lysates with equivalent protein levels subjected to Western analysis for c-fms, Argl and G A P D H . 4.2.7 ROBUSTNESS OF SHIP-/- M2 MO PHENOTYPE DURING PROLONGED IN VITRO CULTURE Controversy exists in the literature as to whether macrophage phenotype results from the commitment of progenitors to specific subsets during differentiation, or whether phenotype results from the functional adaptivity of mature macrophages to an ever changing micro-environment. Thus, experiments were designed to address the stability of the SHIP-/- M2 MO phenotype and its influence on LPS-responsiveness in these cells. First, SHIP-/-PMOs were obtained by lavage and either lysed on the first day, or cultured for an additional 5 days in vitro in the presence or absence of additional factors, including the TH1 cytokines, IFNy, IL-12, or TNFa before lysing to compare Ym1 and Argl expression. As can be seen in Fig. 4.11a, SHIP-/- PMO Argl expression did not diminish over the course of 5 days, while intracellular Ym1 expression was lost during this time. Moreover, the addition of TH1 cytokines (which are thought to promote M1-programming and antagonize M2) did diminish Argl expression, but levels still remained detectably high (Fig. 4.11a). Since replacing the tissue culture media on day 2 had the most dramatic effect on reducing Argl levels in SHIP-/- PMOs, this suggested that they may produce an autocrine factor(s) that sustains Argl protein levels. Finally, NO production in SHIP-/- PMOs remained undetectable under any of these conditions, suggesting that the slight diminution of Argl levels was not sufficient to alleviate the block on NO production (data not shown). Taken together, these results suggest that the high M2 Argl phenotype of SHIP-/- PMOs is robust and stable under prolonged in vitro culture, while Ym1 expression may require certain, as yet, unidentified in vivo cues that are not present in vitro. D 0 Day 5 C C M A 12 y a IL-4 +/+ -/- +/+ -I-Arginase i mmm -mmm mmm mmm I f5-act i n Ym1 p-actin SHIP+/+ SHIP-/-RPMOs R P M * s 3 days 3 days C HS I M IL 13 C HS IM IL 13 Ym1 Arg l GAPDH Grb2 Fig. 4.11. A s s e s s m e n t of the stability of the SHIP-/- M2 M O phenotype and its responsiveness to T H1 and T H 2 cytokines, (a) P M O s from 5 w e e k old S H I P - / - m i c e were cultured in vitro for a few h or 5 d a y s with 1 0 % F C S in I M D M (C), + either 5 ng/ml M - C S F (M), a m e d i u m a c h a n g e on day 2 (A), 40 U/ml IL-12 (12), 100 U/ml IFNy (y) or 4 ng/ml T N F a (a) a n d W e s t e r n blot ana lys is c o n d u c t e d on T C L s for A r g l , Y m 1 or G r b 2 . (b) SHIP+/+ a n d - / - P M O s were cultured overnight in IL-4 (10 ng/ml) and Arg l , Y m 1 , and 3-actin m R N A levels determined by R T -P C R , or (c) in vitro for 3 d a y s ± 5% h u m a n s e r u m (HS) , IL-4 (10 ng/ml) or IL-13 (20 ng/ml) a n d T C L s a s s e s s e d for Y m 1 , Arg l , a n d G A P D H by W e s t e r n analys is . 92 In contrast to TH1 cytokines, T H 2 cytokines like IL-4 and IL-13 are known to promote the alternative (M2) activation of PMOs, specifically the induction of Argl and Ym1 (Welch et al., 2002; Pauleau et al., 2004). Thus, it was next asked if the constitutive M2 phenotype of -/- PMOs could be further induced.by IL-4 or IL-13. As can be seen in Fig. 4.11b, freshly isolated SHIP-/- PMOs, but not +/+ PMOs, demonstrate constitutively detectable mRNA levels of Argl and Ym1. IL-4 treatment was able to induce transcript levels further in both genotypes. A similar pattern was observed with both IL-4 and IL-13 at the Argl protein level (Fig. 4.11c). However, in this case the PMOs had been cultured for 3 days in the absence or presence of IL-4 or IL-13, and during this time constitutive -/- PMO Ym1 expression was likely lost (consistent with Fig. 4.11a). Nonetheless, IL-4 and IL-13 were able to induce Ym1 protein to a similar extent in PMOs of both genotypes (Fig. 4.11c). Of note, PMOs were also treated with human serum, and this led to a modest induction of Argl (this will be discussed further in Chapter 5). Taken together, these results suggest that although SHIP-/- PMOs demonstrate constitutive M2 programming, typified by Argl and Ym1 expression, they can be further induced to a plateau level, similar to wild-type, by the T H2 cytokines, IL-4 and IL-13. 4.2.8 IS HIGHLY INDUCIBLE EXPRESSION OF INOS BY SHIP-/- PMOS INCOMPATIBLE WITH AN M2 PHENOTYPE? While low LPS- or LPS+IFNy-induced NO production has been established as a consistent feature of M2 MOs, it is not clear if this is always associated with lower iNOS protein levels. As reported in this Chapter, despite their low NO production, SHIP-/- PMOs were not deficient in LPS+IFNy-induced iNOS and, in fact, were often able to induce iNOS more rapidly and to a greater extent than wild-type counterparts (Fig. 4.2c). While this was a consistent finding, results obtained with LPS alone as a stimulus were more variable. Specifically, SHIP-/- PMOs demonstrated variable induction of iNOS with LPS alone - in some short-term experiments they achieved equal to greater iNOS levels, compared to wild-type, while in others they demonstrated poor and/or 93 non-sustainable iNOS levels (Fig. 4.12a and data not shown). Another peculiar finding was that SHIP-/- PMOs were able to induce COX-2 with similar kinetics to levels seen in SHIP+/+ PMOs, but were often unable to sustain such induction with LPS alone or with IFNy co-stimulation (even when iNOS levels were sustainable) (Fig. 4.12b). SHIP+/+ P M O s S H I P - / -P M O s 0 24 48 84 0 24 48 84 h L P S i N O S A r g i n a s e I i N O S C O X - 2 SHIP+/+ P M O s 0 6 12 24 48 S H I P - / -P M O s 0 6 12 24 48 h F i g . 4 .12. S H I P - / - P M O s d e m o n s t r a t e i m p a i r e d L P S - i n d u c e d i N O S a n d a re u n a b l e t o s u s t a i n i n d u c e d C O X - 2 . (a) Protein lysates were prepared from L P S - t r e a t e d (100 ng/ml) SHIP+/+ a n d - / - P M O s and equa l protein a m o u n t s were subjected to W e s t e r n ana lys is for i N O S a n d Arg l following S D S - P A G E gel resolution, (b) A n a l y s i s w a s co n d u c t ed a s in (a), except L P S + IFNy (100 U/ml) co-st imulation w a s u s e d , and blots were probed for C O X - 2 . Possible insight into these observations was gained with the recent publication of three studies that suggested iNOS translation is specifically blocked by the actions of arginase (Lee et al., 2003; El-Gayar et al., 2003; Bussiere et al., 2005). Specifically, it was found that both the depletion of L-arginine by Argl, and the subsequent downstream generation of the polyamine spermine led to a block in the translation of iNOS mRNA to protein. Examination of LPS+IFNy-induced iNOS protein levels in SHIP +/+ and -/- PMOs revealed that they were not affected by the addition of exogenous L-arginine, while this treatment did appreciably rescue SHIP-/- NO production (Fig. 4.13a, top and bottom panels). In contrast, supplementation with L-arginine augmented iNOS protein levels in LPS alone-stimulated SHIP-/- PMOs, but not in wild-type counterparts (Fig. 4.13b, L-arg supplementation indicated by an asterisk). 94 Unexpectedly, a similar rescue of COX-2 protein levels was also observed under exogenous L-arginine supplementation (Fig. 4.13b), suggesting that this enzyme was somehow also sensitive to levels of this amino acid and/or its metabolites. As this was not the initial intention of the experiment in Fig. 4.13b, attempts were made to reproduce these results. As can be seen in Fig. 4.13c and 4.13d, supplementation of the tissue culture medium with exogenous L-arginine after overnight culture, or replacing this medium with fresh medium at the time of LPS stimulation led to a specific augmentation of SHIP-/- PMO iNOS and COX-2 induction. However, it could not be excluded that replacing the tissue culture medium might have also removed other non-arginine related intermediates (like IL-1Ra and IL-10) that were produced in greater concentration by SHIP-/- PMOs and could also have negatively impacted iNOS and COX-2 levels. Thus, taken together, these results suggested that iNOS and COX-2 levels in SHIP-/- PMOs were reduced due to their sensitivity to depleted arginine and/or arginine metabolites like spermine (in addition to possible other negative regulators), due to high constitutive arginase levels and activity in SHIP-/- PMOs. Thus, these results suggested that while it was possible to achieve high iNOS levels in LPS+IFNy-stimulated SHIP-/- PMOs, variable iNOS expression (particularly with LPS alone, under conditions of prolonged culture) was related to the action of the M2 enzyme arginase, and this was consistent with M2 programming. 9 5 a +/+ -i-RPM<D RPM<D INOS +/+ P M O s -/- PM<Ds SHIP+/+ PM<Ds SHIP-/- P M O s 0 6 12 24 48 72 96 48* 0 6 12 24 48 72 96 48* iNOS COX-2 Arg I mmmmmmm L P S F M +/+ P M O s + + - + - / -P M O s + + - + C O X - 2 Grb2 A r g l L P S - + + L-Arg - - + + + - + INOS C O X - 2 A r g l Grb2 Fig . 4.13. L-arginine supplementation or fresh media change alleviate repression on L P S -induced SHIP-/- P M O iNOS and COX-2 levels, (a) SHIP+/+ (black bars) a n d - / - (grey bars) P M O s were treated for 48 h with 100 ng/ml L P S + 100 U/ml IFNy ± supplementat ion with e x o g e n o u s L-arginine (2 m M ) before subjecting to i N O S and Arg l W e s t e r n ana lys is (top panels) or N 0 2 " a s s a y (bottom), (b) SHIP+/+ a n d - / - P M O s were treated over 96 h with 100 ng/ml L P S ± 2 m M L-arginine at the 48 h time point (* indicates L-arginine supp lemented) a n d subjected to i N O S , C O X - 2 , a n d Arg l W e s t e r n analys is , (c) P M O s were treated with L P S for 24 h ± L-arginine a n d a n a l y s e d by W e s t e r n blot for C O X - 2 , G r b 2 , and Arg l . (d) P M O s were a l lowed to condit ion their m e d i a for 48 h prior to an additional 18 h L P S stimulation period ± f resh m e d i a c h a n g e , a n d W e s t e r n ana lys is w a s c o n d u c t e d on i N O S , C O X - 2 , A r g l , and G r b 2 loading control. 96 4.2.9 A COMPARISON OF LPS-INDUCED SIGNALING IN SHIP+/+ AND -/-PMOS AND THE EFFECTS OF PI3K INHIBITORS ON THESE TWO CELL TYPES To complement earlier studies with SHIP+/+ and -/- BMMOs, LPS-induced signaling was compared in SHIP+/+ and -/- PMOs and the effects of PI3K inhibitors on SHIP+/+ and -/- PMOs were also determined. Specifically, LPS signal transduction of wild-type and SHIP-/- PMOs was first assessed by Western blots (Figs. 4.14a, b, c, and d) and it was observed that: 1) SHIP became tyrosine phosphorylated in response to LPS, as early as 1 h post-stimulation, but SHIP levels tended to rise and SHIP became hypo-phosphorylated at later time points, 2) Akt phosphorylation was stimulated to a greater degree in SHIP-/- PMOs, 3) induction of serine phosphorylation of Statl was similar and preceded its tyrosine phosphorylation, which was at least equivalent, but sometimes enhanced, in SHIP-/- PMOs, 4) c-fms levels decreased upon LPS stimulation but returned at later points (as previously reported (Rovida et al., 2001)), 5) in SHIP-/- PMOs an increased ratio of phospho- Erk1:Erk2 was observed, but not consistently and, 6) p38 phosphorylation levels and dynamics were equivalent. These findings were similar to those observed with BMMOs (Figs. 3.13a, 3.14b, 3.15a and data not shown). On the other hand, the following differences were noted between BMMOs and PMOs: 1) Argl levels were constitutively elevated and remained so in SHIP-/- PMOs, while wild-type counterparts displayed only slight induction at 24 h post-LPS, 2) resting kBa levels were slightly elevated in SHIP-/- PMOs, but its degradation and re-synthesis proceeded with similar dynamics, 3) in contrast to SHIP-/- BMMOs, SHIP-/- PMOs displayed similar induction of phosho-p65 N F -K B , and 4) while SHIP-/- PMOs showed slightly elevated resting levels of C/EBPB isoforms (arrows in C/EBPB panel of Fig. 4.14b), consistent with Fig. 4.6d, LPS induction was similar in both genotypes. Taken together, these results suggested that LPS-induced signaling proceeded in a relatively normal fashion, at least according to the parameters examined, in cells with a pronounced resting 9 7 SHIP+/+ PMOs SHIP-/- PMOs 0 0.25 0.5 1 3 8 24 0 0.25 0.5 1 3 S 24h " * » | | | pStat1(Y701) pErkl SHIP+/+ PMOs SHIP-/-PMOs 0 0.5 1 3 6 24 0 0.5 1 3 6 24 h LPS |pStat1(Y701) m <mm%,tm w mms • — - - -pErk1/2 pp38 Arginase I SHIP+/+ PMOs SHIP-J-PMOs 0 0.5 1 3 6 12 24 0 0.5 1 3 6 12 24 h LPS pSHIP(2NPXY) SHIP pStat1(Y701) pStat1(S727) pAkt(T308) C/EBPP Arginase I I K B a '•  SS5 *W 49 W p» '<m % mm* •*# wmm « - - - - S I * SHIP+/+ PMOs SHIP-/- PMOs c-fms SHIP pp65NFKB pAk1T308 Akt pp38 p38 Arginase I IKBQ 0 0.5 2 4 0 0.5 2 4 h LPS Fig. 4.14. Characterization of LPS- induced P M O signal transduction, (a, b, c, and d ) 5 " 105 SHIP+/+ and -/- PMOs were treated with 100 ng/ml LPS for the times indicated, resolved by S D S - P A G E and subjected to Western analysis as indicated. 98 M2 phenotype (particularly elevated Argl). It thus did not appear that these differences could account for the profound suppression of LPS-induced pro-inflammatory outcomes in SHIP-/- PMOs. Moreover, these results were consistent with the observations of the preceding section, ie that the primary negative impact on LPS-induced NO, COX-2, and cytokine production in SHIP-/-PMOs was due, at least in a major part, to the depletion of L-arg by profound expression of Argl and/or increased downstream production of polyamines. As was the case with BMMOs and J2MOs, the pharmacological inhibitors of PI3K, wortmannin and LY294002, had opposite effects on LPS-induced NO synthesis in PMOs. Specifically, as can be seen in Figs. 4.15a and b, wortmannin dose dependently augmented LPS-induced iNOS levels and NO production in PMOs of both genotypes, while LY294002 potently inhibited production. As was observed with BMMOs in Chapter 3 (Figs. 3.11a, 3.12, and 3.13a), reduced iNOS protein levels in LY294002-treated PMOs were associated with lower levels of tyrosine-phosphorylated Statl (Fig. 4.15a). The potential differential impact of wortmannin and LY294002 on translational control machinery (notably p70S6K and 4EBP1), as reported in Chapter 3, was not examined in these PMOs, but lower pStatl with LY294002 treatment was consistent with these conclusions. What was noted, however, was that treatment with both PI3K inhibitors reduced the 24-h levels of Argl in both genotypes (Fig. 4.15a - it is worthy of note that resting Argl activity was 10-fold higher in SHIP-/-PMOs, data not shown). Taken together with the results of Figs. 4.13a, b, c, and d, where it was suggested that pronounced Arg1-mediated reduction of L-arginine and/or increased production of Argl metabolites reduced iNOS levels in SHIP-/- PMOs, these results suggested that augmented iNOS levels and NO production by wortmannin might also have resulted from its ability to relieve Argl-mediated repression. Moreover, since wortmannin had no impact on Statl phosphorylation and less effect than LY294002 on p70S6K (which appeared in Figs. 3.11-3.13 to be critical to achieve maximal iNOS levels), while it also reduced LPS-induced synthesis of anti-inflammatory IL-10 (Fig. 3.18b), this suggested a rationale for the opposite effects of wortmannin and LY294002 99 SHIP INOS P-Stat1(Y701) P-Akt(T308) Arginase I GAPDH LPS (200 ng/ml) DMSO (0.1%) Wortmannin (nM) LY294002 (uM) SHIP+/+ PMOs Akt <*»«»•*• SHIP-/-PMOs mwkM mm m yfgf Hip w 50 500 -- - 10 - + 50 500 -- - 10 LPS (200 ng/ml) DMSO (0.1%) Wortmannin (nM) LY294002 (uM) PMOs F ig . 4.15. Wortmannin and LY294002 have opposi te effects on LPS- induced iNOS and NO syn thes is in P M O s . (a) SHIP+/+ and -/- P M O s were treated for 24 h with L P S (200 ng/ml) ± D M S O vehicle control, Wortmannin, or LY294002 at the indicated concentrations, and subjected to Western analysis for SHIP, iNOS, pStat1(Y701), pAkt(T308), Akt, Argl, and G A P D H . (b) SHIP+/+ and -/- P M O supernatants from (a) were collected and analyzed for NO production by Griess assay. Similar results were obtained in an independent experiment. on iNOS/NO. In short, wortmannin augmented LPS-induced iNOS/NO, at least in part, due to relief of inhibition imposed by Argl and IL-10, whose induction was PI3K-dependent, without significant impact upon Statl/p70S6K (it should be noted that additional PI3K-independent augmentation of iNOS could not be ruled out). Although LY294002 also prevented induction of Argl and IL-10 by LPS, it negatively impacted Statl and p70S6K activation and was thus incompatible with iNOS synthesis. Thus, studies in PMOs have shed more light on the possible reasons for the peculiar opposite effects of wortmannin and LY294002 on iNOS/NO. Studies in Chapter 3 revealed that SHIP-/- BMMOs did not display endotoxin tolerance, likely because LPS-induced upregulation of SHIP by the autocrine action of TGFB was necessary to suppress activation of PI3K, pro-inflammatory cytokine and NO synthesis. As SHIP-/- PMOs were M2 programmed (not necessarily synonymous with non-endotoxin tolerized), already displaying a propensity to reduced LPS-induced pro-inflammatory cytokine and NO production, it was next asked how these PMOs would respond to a second dose of LPS (i.e. would they LPS-tolerize?). To minimize the influence of M2 programming, PMOs were obtained from 4 week-old mice. As can be seen in Fig. 4.16, SHIP-/- PMOs, like their wild-type counterpars, did display endotoxin tolerance for TNFa production using a variety of tolerizing/challenging LPS doses. However, they were uniquely more refractory to tolerizing effects on IL-6 output. As can be seen in Fig. 4.16, while SHIP+/+ PMOs displayed LPS-tolerance for IL-6 production at a variety of LPS concentrations and at both 4 and 18 h post secondary challenge, SHIP-/- PMOs were refractory to tolerance at 4 h, but did display some evidence of tolerance for IL-6 production at 18 h (albeit less than +/+). Thus, although SHIP-/- PMOs did not display endotoxin tolerance in regard to IL-6, they did for TNFa production. This likely reflected different thresholds for tolerance of IL-6 versus TNFa. Moreover, as SHIP-/- BMMOs were refractory to tolerance, ie both IL-6 and TNFa, this suggested that M2 programming in SHIP-/- PMOs might have contributed to an intermediate endotoxin tolerance-refractory phenotype (between wt MOs and -/- BMMOs). 4.2.10 ANALYSIS OF SHIP LEVELS IN PMOS OF PROTOTYPICAL M1 AND M2 MOUSE STRAINS Our interpretation of the results to this point was that in vivo-differentiated SHIP-/- MOs display a constitutive M2 phenotype and that SHIP functions in vivo to repress this phenotype. Of note, these studies have been conducted with mice on a mixed genetic background (F2: 129Sv * C57BL/6). It has been proposed that genetic differences may be responsible for the tendency of C57BL/6 and Balb/c mice to be prototypical Th1/Th2 and M1/M2 strains, respectively. However, the genetic polymorphisms that may be involved in skewing MO programming to M1 versus M2 have yet to be fully elucidated. This 101 4 h E • f t LL z r- 1 H S H I P + / + r s S H I P -/- T _L •a 0 100 0 100 1,000 10 10 100 100 100 0 C h a l . 0 T o l . 0 0 100 0 100 1,000 10 10 100 100 100 Fig. 4.16. A n analysis of endotoxin tolerance in SHIP+/+ and -/- P M O s . P M O s from 4 week-old SHIP+/+ and -/- mice were challenged (Chal.), or not, for 24 h with an initial dose of L P S at the indicated concentrations (ng/ml), after which time media were washed and cells treated with a second L P S dose (Tol.) for 4 h (left) or 18 h (right) and the levels of T N F a and IL-6 were determined by ELISA. The results are representative of two similar experiments. seemed an ideal dichotomy in which to examine any relationship that may exist between M1/M2 programming and SHIP. Thus, the findings of others were first confirmed with respect to macrophage phenotype in these mouse strains. As can be seen in Fig. 4.17a, PMOs from Balb/c mice produced less NO when stimulated with LPS or LPS+IFNy, compared to PMOs from C57BL/6 mice, consistent with what has been reported (Mills et al., 2000). This has been attributed to increased arginase activity in Balb/c PMOs, and indeed it was observed that Balb/c PMOs expressed more Argl protein at rest than C57BL/6 PMOs (Fig. 4.17b). Intriguingly, Balb/c PMOs also expressed less SHIP protein than C57BL/6 counterparts (Fig. 4.17b). These results suggested that reduced 102 SHIP expression in Balb/c PMOs could at least contribute to their elevated Argl levels and their tendency to be M2-skewed. a PMOs LPS (200 ng/ml) - + + IFNy (100 U/ml) - - + Tlme(h) 24 24 24 48 + + 48 Resting PMOs C57BL/6 Balb/C SHIP A r g l Grb2 Resting PMOs C57BL/6 Balb/c 1 ' ••  — — SHIP Argl GAPDH Fig . 4.17. A reciprocal relationship exists between SHIP and Argl protein levels in C57BL/6 (M1) and Balb/c (M2) P M O s . (a) Resident P M O s were obtained from peritoneal lavage fluid of adult C57BL/6 and Balb/c mice, selected by adherence and repeated washing, plated at 10 5 cells/well (100 pi) of a 96-well plate, and duplicate determinations of NO production were recorded following L P S (100 ng/ml) or L P S + IFNy (100 U/ml) treatment for 24 and 48 h. (b) 5 * 10 s overnight, resting, 12-well cultured P M O s from C57BL/6 and Balb/c were washed once with P B S prior to in-well total cell lysis using 1* S D S - P A G E sample lysis buffer, were boiled, resolved by S D S - P A G E , and subjected to Western blot analysis for SHIP, Argl, and Grb2 or G A P D H loading control. Two representative experiments are shown. 103 4.3 DISCUSSION Macrophages play important roles both in host defense against microbial infections and in the subsequent healing that follows the eradication of the invading microorganism (reviewed in Gordon 2003 and Noel et al. 2004). These two functions appear to be carried out by distinct subsets of MOs. The killer, classically activated, M1 MOs are thought to develop in the presence of the T H1-derived cytokine, IFNy and are characterized by enhanced antigen presentation and the secretion of high levels of pro-inflammatory cytokines and iNOS-generated NO (reviewed in (MacMicking et al. 1997 and Bogdan 2001)). Macrophages exposed to T H2 type cytokines like IL-4 and IL-13, on the other hand, are thought to assume a healer, alternative, M2 activation phenotype (Stein et al. 1992 and Mantovani et al. 2004). These M2 MOs are characterized by upregulation of broad-specificity pattern recognition and scavenger receptors, anti-inflammatory cytokines and chemokines, novel secreted proteins Ym1/2 and FIZZ1 and a reduced NO production because of a marked elevation in arginase levels (reviewed in Gordon 2003 and Noel et al. 2004). It is likely that these M2 MOs are themselves composed of distinct subsets, with differing abilities to promote cell proliferation and immunosuppression (Mantovani et al. 2004). Currently, there is a great deal of controversy concerning the factors that regulate the development of these different MO subsets, the time they arise during the differentiation process and the degree to which these cells can switch phenotypes (Ravasi et al. 2002; Stout and Suttles 2004 and Gordon 2003). In order to harness these M1 and M2 MOs for future anti-cancer and anti-inflammatory therapies it is very important to resolve these issues. The studies presented herein with SHIP-/- mice provide some unexpected insights into the mechanisms that govern MO programming. Specifically, our finding that in wVo-derived PMOs and AMOs from SHIP-/- mice are M2-programmed while these same cells from SHIP+/+ mice have an M1 propensity suggests that SHIP plays a key role in preventing M2 skewing. As well, our findings that M2 phenotypic features become more prominent as SHIP-/- mice 104 age suggest that extracellular factors also play an important role in M1 versus M2 MO decisions. With regard to the shift to a more pronounced M2 phenotype with age, previous studies with SHIP-/- mice have shown that natural killer (NK) cells become more tolerant as these mice age and this prevents them from rejecting mismatched bone marrow grafts (Wang et al. 2002). Moreover, Coggeshall's group recently showed that IL-6 production by SHIP-/- myeloid cells leads to an age-dependent suppression of early B cell development in the bone marrow (Nakamura et al. 2004). Thus, the SHIP-/- mouse may become more tolerant with age and this may be an attempt to counter an environment of chronic inflammation in these mice (Nathan 2002). This may explain the variability in LPS-induced iNOS levels we observed in SHIP-/- PMOs since the lowest levels were observed with cells from the oldest SHIP-/- mice. This is consistent with reports suggesting that the translation of iNOS may be negatively regulated by Argl-mediated L-arginine depletion and/or polyamine production (Lee et al. 2003; El-Gayar et al. 2003; Bussier et al. 2005). Related to this, both the PI3K pathway and M2 MOs have been implicated in the compensatory anti-inflammatory response (CARS) (Bone 1996) observed in sepsis (Learn et al., 2001; Williams et al. 2004), in non-infectious systemic inflammatory response syndrome (SIRS) and in the susceptibility of hosts to nosocomial infections (Adib-Conquy et al., 2003; Takahashi et al. 2004 and Dal Pizzol 2004). Recently, the SHIP-/- mouse has been reported to contain elevated numbers of so called immature myeloid cells (iMCs)/myeloid suppressor cells (MSCs) in the spleen and secondary lymphoid tissue which contributes to diminished allogenic T cell responses (Ghansah et al., 2004). iMCs/MSCs are a heterogeneous and poorly defined population of Mac1/Gr1 double-positive cells found in lymphoid organs that impair lymphocyte responses to antigen during inflammation and tumour growth. Moreover, they do so via the iNOS or Argl-mediated metabolism of L-arginine, by simultaneously and robustly expressing both iNOS and Argl, and via the production of superoxide (Bronte and Zanovello, 2005; Kusmartsev and Gabrilovich, 2005). However, it seems unlikely that the 105 PMOs isolated herein from SHIP-/- mice have been mistaken for iMCs/MSCs, as they are c-fms+Mac-1+F4/80+, like bona fide +/+ PMOs (Fig. 4.1). The relationship between iMCs/MSCs and M2 MOs is an area of great interest (Bronte and Zanovello, 2005) and the SHIP-/- mouse may prove to be a valuable model for this field. One in vivo consequence of enhanced M2 MO programming is likely the presence of fibrosis and Ym1 crystals in the lungs of SHIP-/- mice. Ym1 is a chitinase-like secretory lectin that spontaneously crystallizes under high concentrations (eg, in chronic TH2-type inflammation like asthma or parasitic infections), particularly in the lung, and is associated with the presence of M2 MOs (Raes et al. 2002; Welch et al. 2002 and Nair et al. 2003). Ym1 also has been postulated to function in myelopoiesis (Hung et al. 2002) and allergic lung remodeling (Webb et al. 2001 and Hung et al. 2002). Interestingly, Ym1 lung crystals have also been isolated from MeV mice (Guo et al. 2000), which share many phenotypic characteristics with SHIP-/- mice (Tsui et al. 1993 and Shultz et al. 1993), including chronic lung inflammation (Ward 1978 and Helgason et al. 1998). These crystals may represent an exaggerated manifestation of immune tolerance and healing, and may, in fact, be contributing to the lung pathology in both mouse types. Taken together, our results suggest that MOs within SHIP-/-, and perhaps MeV, mice become constitutively M2 programmed and that this contributes to their lung pathology and shortened lifespan. Solid tumours are known to exploit the M1/M2 MO balance, tipping macrophage programming towards M2 to avoid cytotoxic NO and to take advantage of pro-proliferative polyamines and mediators of tissue remodeling and angiogenesis (Mantovani et al. 2002). It was therefore not surprising to find that subcutaneously injected Lewis lung carcinomas grow faster in SHIP-/- than +/+ mice and display elevated levels of Argl. Related to this, a recent elegant study demonstrated that host TAMs are the sole source of arginase in the Lewis lung carcinoma model (Rodriguez et al. 2004). Moreover, in this study it was shown that the high Argl expressing macrophages depleted the extracellular tumour milieu of arginine and this, in turn, reduced the expression of CD3£ on 106 infiltrating T cells, resulting in T cell anergy and tumor growth (Rodriguez et al. 2004). Consistent with this, they also found that the injection of an arginase inhibitor blocked the growth of the tumour. In summary, the results presented in this chapter and elsewhere (Rauh et al., 2005) suggest that while SHIP+/+ and -/- PMOs express similar cell surface levels of SR-A, Mad (CD11b), c-Fms, F4/80, and no Gr-1, the SHIP-/- PMOs constitutively express higher than normal levels of Argl, Ym1, IL-1Ra, IL-10, IL-6, TGFB and MR and, in response to LPS, higher than normal levels of IL-1Ra, IL-10 and TGFB but lower levels of TNFa, IL-6, IL-12, CCL3 and NO. These findings are consistent with SHIP-/- PMOs possessing an M2 MO phenotype (Gordon, 2003; Mantovani et al., 2004). Moreover, consistent with this, SHIP-/-mice display M2-mediated lung pathology and enhanced tumor implant growth. 107 CHAPTER 5 - RECAPITULATION OF IN VIVO MACROPHAGE DIFFERENTIATION IN VITRO 5.1 INTRODUCTION Macrophages are found in many parts of the body and demonstrate heterogeneous, tissue-specific functions (Hume et al., 2002). Although readily obtainable from areas like the peritoneal cavity, such in vivo-differentiated macrophages have limited ability to proliferate in vitro in the absence of stimulation (Martens et al., 1999), and their use in experiments can therefore be constrained by limited cell numbers (Chan et al., 1998). Culturing bone marrow progenitors in M-CSF for several days in vitro is a widely used technique to obtain an expanded population of bone marrow-derived macrophages (BMMOs), amenable to study (Hume et al., 2002). However, although such in vitro-generated MOs do express MHC class II, accessory and adhesion molecules, they have been suggested to be less efficient at antigen presentation than in vivo counterparts (Lee et al., 2005). Moreover, while murine PMOs express the STK receptor tyrosine kinase, its expression is notably absent from BMMOs (Correll et al., 2004). Despite the aforementioned deficiencies and the lack of exhaustive comparisons between in vitro- versus in vivo-generated MOs, BMMOs continue to be a mainstay of MO experimentation. Given the alternative activation (M2) phenotype described for SHIP-/- PMOs in Chapter 4, and the different propensities of SHIP-/- BMMO and SHIP-/- PMOs for NO production, the relevance of the SHIP-/- in vitro MO differentiation system was called into question. This chapter will describe efforts to study differences between in vitro and in vivo MO differentiation and attempts to recapitulate, in vitro, the in vivo SHIP-/- PMO phenotype. 108 5.2 RESULTS 5.2.1 MOUSE PLASMA SKEWS THE IN VITRO DIFFERENTIATION OF SHIP-/- PROGENITORS TOWARDS M2 BMMOS Having established that in wVo-derived, SHIP-/- PMOs and AMOs possessed an M2 phenotype, it was asked if MOs derived from the bone marrow of SHIP-/- mice under standard in vitro culture conditions (ie, M-CSF + 10% FCS) were also M2-skewed and found that this was not the case, ie., SHIP-/- BMMOs, like SHIP+/+ BMMOs, possessed undetectable Argl levels (Fig. 5.1a). This was consistent with the finding that LPS-induced NO production from SHIP-/- BMMOs was not impaired (Fig. 3.10, 3.11, 3.13, and 3.14) and that L-arginine supplementation did not enhance it (Fig. 4.3b). In an attempt to mimic the in vivo differentiation environment in SHIP-/-mice, 10% mouse plasma (MP) was added to SHIP-/- in vitro bone marrow cultures and it was found, after 7 days of culture, that this dramatically increased Argl expression (Fig. 5.1b). Supplementation with an additional 10% FCS, on the other hand, had no effect. Moreover, 10% MP did not induce Argl expression in SHIP+/+ BM cultures (Fig. 5.1b). Next, a MP dose response study was carried out and it was found that the inclusion of as little as 2% SHIP+/+MP (+MP) or SHIP-/-MP (-MP) dramatically increased Argl levels (Fig. 5.2a) and activity (Fig. 5.2b) in the fully developed SHIP-/- BMMOs but had no effect on SHIP+/+ BMMOs. This mimicked in vivo MO differentiation in SHIP+/+ and -/- mice and suggested that the in vivo skewing of SHIP-/- MOs required both intrinsically elevated PIP 3 levels and an extracellular factor(s) present in the plasma of both genotypes. The fact that -MP was about twice as potent as +MP may reflect an attempt to counter the inflammatory phenotype of the SHIP-/- mouse (Nathan 2002). We next sought to determine if high Argl expression induced by differentiating SHIP-/- BMMOs in the presence of MP led to diminished production of NO. A comparison of LPS + IFNy-induced NO production revealed that while control differentiated SHIP-/-BMMOs produced more NO than wild-109 a B M M O s P M O s +/+ -/- +/+ -/-SHIP-/- B M M O s +/+ F C S M P M P Argl Grb2 Argl G A P D H F ig . 5.1. Mouse plasma skews the in vitro differentiation of SHIP-/- myelomonocyt ic progenitors towards M2 B M M O s (a) T C L s (2.5 pg) from SHIP+/+ and - / - B M M O s a n d P M O s were subjected to W e s t e r n ana lys is using anti-Argl a n d ant i -Grb2. (b) S H I P - / - B M M O s , genera ted after 7 d a y s in I M D M + 1 0 % F C S + 5ng/ml M - C S F (-), + 1 0 % supp lementa l F C S ( F C S ) , or + 1 0 % + M P (MP) , or SHIP+/+ B M M O s generated in +MP, were subjected to Argl and G A P D H W e s t e r n analys is . SHIP-/ -B M M O s % % % F C S +MP - M P SHIP+/+ B M M O s % % % F C S +MP - M P 5 2 5 2 5 2 5 2 5 2 5 2 Arg l c- fms 10 B M M O s 7.5 • > 15 act! lys act! lys • ™ 1 -5" to 13 5 • c O 2.5 • < Ut mm SHIP+/+ i — i SHIP-/-J L I [ J % F C S % +MP % - M P Fig . 5 .2. SHIP-/- mouse plasma has more arginase-inductive capacity during SHIP-/- B M M O differentiation, (a) S H I P - / - and +/+ b o n e marrow progenitors were cultured for 7 d a y s in s tandard differentiation m e d i u m ± supplementa l F C S , +MP, - M P a d d e d 1 day after the initiation of the cultures (ie, day 1). After 7 d a y s , adherent cel ls with M O morphology were subjected to Arg l a n d c - f m s (loading control and M O differentiation marker) W e s t e r n analys is , (b) S H I P - / - (grey bars) a n d +/+ (black bars) B M M O lysates from (a) were subjected to an a rg inase a s s a y a n d results s h o w n are the m e a n + S E M of dupl icates. Resul ts are representat ive of 3 independent exper iments . 110 type counterparts (Fig. 5.3), as has been observed in Chapter 3, the so-called M2 SHIP-/- BMMOs differentiated in the presence of MP produced much less NO, consistent with higher Argl levels and activity (Fig. 5.3). In contrast, inclusion of MP during +/+ BMMO differentiation led to only a reproducibly modest drop in NO synthesis (Fig. 5.3 and data not shown). Thus, exposing SHIP-/- BM cultures to MP during differentiation led to a phenotype which mimicked the M2 programming observed in SHIP-/- PMOs (i.e. higher Argl and lower NO). SHIP+/+ SHIP-/-Fig . 5.3. Differentiation of SHIP-/- B M M O s in mouse plasma leads to decreased inducible N O synthesis . SHIP+/+ (black) and -/- (grey) B M M O s differentiated in the absence (sold bars) or presence of +MP (hatched bars) for 7 days were treated for 48 with L P S (100 ng/ml) and IFNy (100 U/ml) and NO production was assessed. Results shown are the mean ±SEM of duplicates and are representative of 3 independent experiments. To gain some insight into when during differentiation this plasma factor(s) acted, delayed addition studies were carried out with +MP and it was found that +MP was no longer capable of producing high arginase SHIP-/- BMMOs if addition was delayed until day 2 (Fig. 5.4a). This suggested that it was acting early during MO differentiation. Ym1 was also induced by +MP and maximal Ym1 levels were also achieved in SHIP-/- BMMOs when +MP was added to the differentiation culture prior to day 2 (Fig. 5.4b). Moreover, inclusion of the PI3K 111 inhibitors, wortmannin or LY294002, with +MP in the 7 day assay diminished arginase induction, suggesting that the plasma factor(s) was dependent upon PI3K for M2-skewing (Fig. 5.4c). Subsequent experimentation revealed that the factor(s) was also present in human plasma, and in the serum from humans, goats, or chicken but, peculiarly, not bovine FCS (Fig. 5.4a and data not shown). a SHIP-/- BMMOs 12.5 10.0 > 'S 7 <l> SHIP-/- BMMOs SHIP-/- BMMOs MP © d a y FCS © d a y C 0 1 2 3 0 1 2 3 mm mmm *m m«m Ym1 GAPDH -MP (5%) - + DMSO (0.02%) + WM(100nM) + LY(2uM) + Fig 5.4. Mouse plasma-mediated M2-skewing of SHIP-/- B M M O s occurs early during differentiation in a PI3K-dependent process, (a) SHIP-/- bone marrow progenitors were cultured for 7 days in standard differentiation medium ± 5% F C S , 5% +MP, or 5% human serum (HS) added on days 0, 1, 2, or 3 and lysates assayed for arginase activity or (b) lysates were subjected to Western blot analysis for Ym1, G A P D H and Grb2. (c) SHIP-/- bone marrow progenitors were treated as in (a) except that the day 1 addition of 5% +MP was preceded by a 30 min pre-incubation with one dose of DMSO, wortmannin, or LY294002, which remained in the medium for the 7 day culture period. 112 5.2.2 CHARACTERIZATION OF THE PLASMA FACTOR(S) RESPONSIBLE FOR M2-SKEWING AND THE TIMING OF ITS ACTION In order to gain some insight into the identity of the -/- M2-skewing blood factor, human serum was chosen for fractionation studies, given that it was readily available in large quantities. Thus, total human serum, dialyzed serum, or four different column fractions from a carboxymethylated-AffiGel Blue (CM-AffiGel Blue) column (BioRad) (flow-through fraction #1, and pooled fractions #2-4), were obtained and assessed for their ability to induce arginase activity in differentiating SHIP-/- BMMOs. As can be seen in Fig. 5.5, dialyzed human serum (using a 10 kDa pore size dialysis membrane) and the flow-through fraction retained, for the most part, the M2-skewing activity, suggesting that the factor(s) was greater than 10 kDa and not positively charged at pH 7.2. Fig. 5.5. An initial fractionation study of the M-2 skewing blood factor suggests it is greater than 10 kDa and not positively charged at pH 7.2. SHIP-/-bone marrow progenitors were cultured for 7 days in differentiation medium ± 5% HS, 10% dialyzed HS, or 25% final volume of CM-AffiGel Blue column fractions 1 through 4 and lysates were assayed for arginase activity. In addition, arginase activity was assessed in mature SHIP-/- BMM<t>s treated for 24 h (at day 7) with total, dialyzed, or fraction 1 HS as above. In order to identify the factor(s) in MP responsible for the selective upregulation of Argl in SHIP-/- MO progenitors, response studies were carried out with the prototypical type1/TH1 and type2/TH2 cytokines, IFNy and TGFB, respectively. It was not unexpected that SHIP-/- BMMOs preferentially increased Argl levels (Fig. 5.6a) and activity (Fig. 5.6b) in a PI3K-dependent manner in response to TGFB. However, the supposed negative control, IFNy, had a similar 6 2? « > 15 t o — in 3" t o 7a C 0) < cn SHIP-/- BMMOs 5 4 3 2 1 • I c o u fi # • 4 I S E JO uL W O c H > • -re o o o o o • - ° q re re to re ^ Q r e LL LL LL LL LL Human Serum @ day 1 H. Serum @ day 7 113 effect in SHIP-/- BMMOs, and actually appeared to synergize with TGFB (Fig. 5.6a and b). These results suggested that SHIP-/- BM progenitors were hypersensitive to Argl induction and promiscuous in their responsiveness, and/or that a previously unrecognized, specific, PI3K-dependent connecting pathway existed between IFNy and TGFB. +/+ -/-BMMOs BMMOs +/+ -/-BMMOs BMMOs c-Fms Arg I GAPDH M l i l l m 9 m * • w • M-CSF + + + TGF-p - + + IFN-y - - -LY294002 - - + + + + - + • + + + + + + + + - - + + - -+ + + + + + + + + + 15 1110 ^ o >» 1 ra — o °> at = L ra — e r a -£ 5 < ™ 0 M-CSF + TGF-p -IFN-y " LY294002 -lD •n I Fig . 5.6. SHIP-/- bone marrow progenitors are hypersensitive to PI3K-mediated arginase induction by T G F p and/or IFNy. (a) SHIP-/- and +/+ bone marrow progenitors were cultured for 7 days in M - C S F medium ± supplemental TGFB (5 ng/ml), IFNy (100 U/ml), or Ly294002 (2 pM) added 1 day after the initiation of the cultures (ie, day 1). After 7 days, adherent cells with M O morphology were subjected to c-fms, Argl, and G A P D H Western analysis, (b) SHIP+/+ (black bars) and -/- (grey bars) B M M O lysates from (a) were subjected to an arginase assay and results shown are the mean ± S E M of duplicates. Results are representative of 2 independent experiments. In order to distinguish between promiscuous versus specific Argl hyperinduction in SHIP-/- BMMOs, BM cultures from both genotypes were exposed to a further panel of TH1/TH2 cytokines and other factors during differentiation, and Argl activity compared in the resultant mature BMMOs. Interestingly, although IL-4, and to a lesser degree, IL-13, were potent inducers of Argl, they increased the levels of this enzyme to the same degree in the resulting SHIP+/+ and -/- BMMOs (Fig. 5.7a). In addition, MSP had an equal, albeit modest effect in the two genotypes (Fig. 5.7b and data not shown). IL-6 and GM-CSF, on the other hand, had no effect on either cell type, and GM-CSF was less effective at supporting MP-mediated arginase induction than M-CSF (data not shown). Interestingly, TGFB and IL-10 only induced Argl in the resulting SHIP-/- BMMOs, making them potential candidates for the MP factor (Fig. 5.7a). Therefore, +MP was pre-incubated with biotinylated anti-TGFB1 or biotinylated anti-IL-10, prebound to streptavidin beads, and the depleted plasma samples were added to the differentiating SHIP-/- progenitor cultures. As can be seen in Fig. 5.8a, while this depletion protocol effectively reduced the ability of both exogenously added TGFB1 and IL-10 to induce arginase activity in the resulting BMMOs, only TGFB1 depletion of MP resulted in a reduction (50%) of arginase activity. Similarly, depletion of exogenously added TGFB1, but not IL-10, led to a reduction of SHIP-/- BMMO Ym1 levels (Fig. 5.8b). However, pre-clearing TGFB1 from MP led to only a very modest reduction of Ym1 levels (Fig. 5.8b), suggesting that, while sufficient on its own to augment Ym1 levels, TGFB1 may synergize with another very important factor in MP in this regard. The identity of this other factor(s) may warrant future study. Thus, TGFB1 in plasma was at least partially responsible for Argl and Ym1 induction in SHIP-/- MOs. •* 6 >M 5 > to J B ? 3 2 BMMOs Jj £ P> 3 I + / + rill C 1 10 100 1 10 20 1 10 100 1 10 100 IL-4 TGF-(J1 IL-13 IL-10 MSP (ng/ml) TGFpl (ng/ml) BMMOs F ig . 5.7. IL-10 but not IL-4, IL-13, or M S P is another potential candidate for the M2-skewing p lasma factor, (a) Bone marrow progenitors from SHIP+/+ (black bars) and -/- (grey bars) mice were exposed to T H 2 cytokines at the indicated doses (ng/ml) or (b) the indicated dose (ng/ml) of macrophage-stimulating protein (MSP) + 5 ng/ml TGFB and arginase activity measured on day 7. 115 SHIP-/- BMMOs SA Beads a - T G F p i a-IL-10 SHIP-/- BMMOs MP T G F p IL-10 ® D a y 1 S A c p d O - SAnp - SAccIO (Ab pre-clear) Ym1 (SA - s t reptav ld in b e a d s only) +MP T G F p l IL-10 Fig. 5.8. T G F p in mouse plasma induces Argl and sustains Ym1 in differentiating SHIP-/-B M M O s . (a) 50ug of biotinylated neutralizing chicken anti-mouse TGFB1 or goat anti-mouse IL-10, at 0.2mg/ml in P B S , was conjugated to 300ul S A beads. After washing in P B S to remove unbound antibody, 300ul aliquots of 5% +MP, 2.5ng/ml TGF81 , or 20ng/ml IL-10 were incubated for 1h at 4°C with 100ul of S A beads alone, or antibody-conjugated beads. Suspensions were pulse-centrifuged and the supernatants added to SHIP-/- bone marrow progenitors on day 1. Arginase assays were performed on day 7. (b) Cells were treated as in (a) except lysates were subjected to Western analysis for Ym1, G A P D H and Grb2. Less of the TGFB-treated lysates were available for Westerns and therefore only 1/4 protein levels were analyzed relative to MP and IL-10 samples. Returning to IL-4 and IL-13, which induced arginase equally well in SHIP+/+ and -/- bone marrow cultures (Fig. 5.7a), it was found that IL-4 (and IL-13, data not shown) could induce Argl in fully mature macrophages (Fig. 5.9a), in keeping with previously published results (Munder et al. 1999 and Pauleau et al. 2004), while MP or TGFB could not. Also, it was found, by removing IL-4, IL-13 (data not shown) or MP at different times during the 7 day culture period that they were all capable of skewing progenitors towards high arginase-expressing mature BMMOs to some extent, even if they were only present during the first 2 days of culture (Fig. 5.9b). Taken together with the results of the delayed addition studies (Fig. 5.4a), these washout studies suggested that the factors were acting at an early stage of MO differentiation. 116 Dev. Mat. Dev. Mat. Dev. Mat. w a s n w a s n w a s n wash ^sr , w a s h +MP TGFpl IL-4 IL-4 +MP Fig. 5.9. IL-4 can M2-skew both differentiating and mature SHIP+/+ and -/- B M M O s while T G F p and MP can only affect differentiating SHIP-/- B M M O s . (a) Bone marrow progenitors (Dev.) or mature B M M O s (Mat.) from SHIP +/+ and SHIP-/- mice were treated ± 5% +MP or IL-4 (10ng/ml) for 7 days (Dev.) or 3 days (Mat.), and arginase activity determined, (b) Bone marrow progenitors from SHIP+/+ (left panel) and -/- (right panel) mice were treated with IL-4 (10ng/ml) and 5% +MP, respectively, on day 0, and sham washed on day 2, or washed twice and replaced with non-IL-4 or non+MP-containing medium on day 2 or day 4. Arginase assays were performed on day 7. Shown are the mean ± S E M of duplicate determinations and similar results were obtained in 2 independent experiments. Consistent with this, Argl induction (assessed by Western blot) on consecutive days of BM differentiation in M-CSF with or without the addition of MP or IL-4, revealed that Argl expression was first detected 24 h post-addition, and maximal at 48 h (Fig. 5.10a and b). Whereas IL-4 was able to induce Argl in both genotypes, MP displayed selectivity for SHIP-/- BM cells (Fig. 5.10a and data not shown). Moreover, Argl induction coincided with that of the MO differentiation marker, c-fms, suggesting that progenitor and not mature cells were responding to the factors (Fig. 5.10a and b). This was further supported by the observation that fully mature SHIP-/- BMMOs could not respond to MP or TGFB by inducing Argl (Fig. 5.9a). However, as the total non-adherent BM starting population consisted of a heterogeneous mix of cells at various stages of lineage commitment, it could not be ruled out that only more mature cells, and not the progenitors within the population, were the responsive cell type. 117 Interestingly, wild-type BM cells expressed more Ym1 than SHIP-/-, and Ym1 levels declined in both genotypes during M-CSF-induced BMMO differentiation (Fig. 5.10a and b). As opposed to Argl, which was induced by MP, Ym1 levels instead appeared to be sustained by MP, and further induced by IL-4. Thus, although MP acts at an early stage of differentiation to produce SHIP-/-BMMOs that have high Argl and Ym1 M2 phenotypic characteristics, the means by which this is achieved are unique. SHIP+/+ SHIP+/+ BMMOs BMMOs M-CSF M-CSF + IL-4 0 1 2 3 4 5 6 1 2 3 4 6 6 (d) mmm m m m m , m t c-Fms Ym1 (Y1 Ab) Argl GAPDH SHIP-/- SHIP-/- SHIP-A BMMOs BMMOs BMMOs M-CSF M-CSF + IL-4 M-CSF + MP 0 1 2 3 4 6 6 1 2 3 4 6 6 1 2 3 4 5 6 (d) c-Fms Ym1 Arg I GAPDH Fig. 5.10. Argl protein induction by mouse plasma appears concomitant with c-fms induction during SHIP-/- B M M O differentiation. SHIP+/+ (left) and -/- (right) bone marrow progenitors were cultured in M-CSF-containing differentiation medium for the indicated number of days ± 2% +MP or 10 ng/ml IL-4 added on day 0, arginase assay lysates were prepared and equivalent amounts of protein were used in subsequent Western analysis for c-fms, Ym1, Argl and G A P D H . 5.2.3 PI3K IS A UNIVERSAL REQUIREMENT FOR M2 MO PROGRAMMING Our interpretation of the results to this point was that the absence of SHIP reduced the threshold for M2 MO induction by MP, TGFB, and IL-10 during M-CSF-induced differentiation and revealed the existence of a previously unappreciated PI3K-dependent process of developmental macrophage programming. That fact that SHIP seemed dispensable for IL-4 and IL-13-mediated M2 programming suggested that SHIP or PI3K may not be involved in 118 this process, or that even in the presence of SHIP a threshold level of required PI3K signaling and PIP 3 levels were achieved. To distinguish between these possibilities and to determine if PI3K played a limited or more universal role in M2 MO programming, the PI3K inhibitors wortmannin or LY294002 were included during a 5 day IL-4 + M-CSF differentiation protocol. This reduced the arginase activity in mature BMMOs of both genotypes (Fig. 5.11). Similarly, wortmannin and LY294002 reduced Argl levels and activity in IL-4-treated mature SHIP+/+ and -/- BMMOs (Fig. 5.11b and data not shown for -/-). Moreover, Ym1 induction was also prevented with PI3K inhibitors, albeit most dramatically with LY294002. Importantly, PI3K inhibition did not appear to diminish activation of Stat6 by IL-4, as measured using phospho-tyrosine-specific Stat6 antibody (Fig. 5.11b bottom panel). This suggested that PI3K was not exerting its effects by preventing Stat6 activation. Since these results were obtained at 24 h post stimulation, earlier signaling events were examined and this also revealed unimpaired Stat6 tyrosine phosphorylation in the presence of PI3K inhibitors, while PIP3-dependent signaling was dampened (Fig. 5.11c). As these studies were conducted with BMMOs on a mixed 129Sv/C57BL/6 background, and in order to rule out mouse strain-specific effects, the experiments were repeated in Balb/c BMMOs. In BMMOs from this strain, Ym1 has been reported to be the most heavily induced target of IL-4 and IL-13 (Welch et al., 2002), and in Fig. 5.12 it can be seen that PI3K inhibition impaired Ym1 induction by these T H2 cytokines. Moreover, Argl and Ym1 induction by IL-4/-13 is prevented in the Stat6-/- mouse (Rutschman et al., 2001) (where it is presumed but not known that PI3K activation may still take place). Taken together with these findings, it would appear that PI3K activation by IL-4/-13 represents a hitherto unappreciated necessary, but not sufficient, step in M2 programming by these T H2 cytokines. Moreover, since TGFB and IL-10 are not known to activate Stat6, and since induction of Stat6 phosphorylation by TGFB, IL-10, or MP has not been detected (data not shown), this would suggest that PI3K is necessary for the novel Stat6-independent M2 MO differentiation induced by these factors. 119 devBMMQs SHIP+/+ BMMO SHIP-/-BMMO D W L IL-4 D W L + IL-4 (10 ng/ml, 5 days) DMSO (0.1%) WM (100 nM) + LY (10L IM) 5* * " W\ pStat6 pAkt pGSK3(3 Grb2 » 12.5 SHIP+/+ BMMOs IL-4 (10 ng/ml) - + + + + + DMSO (0.1 %) - + - - - -WM (nM) - - 50125 - -LY(pM) - - - - 2 5 Argl Ym1 pStat6 24 h Fig . 5.11. PI3K is required for full arginase induction by IL-4 during differentiation and in mature B M M O s , independent of Stat6 phosphorylation, (a) Non-adherent bone marrow cells from SHIP+/+ (black bars) and -/- (grey bars) mice were differentiated for 5 days in M - C S F + IL-4 + wortmannin (WM) or LY294002 (LY) and duplicate determinations were made of the arginase activity in the resultant mature BMMOs. (b) Mature, SHIP+/+ B M M O s were pre-incubated for 30 min with DMSO, W M or LY and then treated ± IL-4 for 24 h. Duplicate arginase assay lysates were then prepared and used for arginase activity determinations (top panel) or pooled for Argl and phospho-Stat6 Western blot analysis (bottom panels), (c) Mature SHIP+/+ and -/- B M M O were pre-incubated ± 0.1% D M S O (D), 100 nM wortmannin (W), or 10 pM LY294002 for 30 min prior to a further 30 min stimulation with 10 ng/ml IL-4, total cell lysates were prepared and subjected to pStat6, pAtk, pGSK3B, and Grb2 Western analysis. Balb/c BMMOs IL-13 WM LY* WM L Y ' C D 50 500 5 20 C D 50 500 5 20 pAkt Ym1 Grb2 Fig. 5.12. PI3K is also required for full Ym1 induction by IL-4 or IL-13 in mature B M M O s from the prototypical M2 Balb/c mouse strain. Mature Balb/c B M M O s were left untreated, or treated for 24 h with IL-4 (10 ng/ml) or IL-13 (20 ng/ml) ± 20 minutes of pre-incubation with D M S O (D), W M , or LY at the indicated concentrations, and Western blot analysis was conducted for pAkt, Ym1, and Akt. 5.2.4 FROM BENCH TO "CAGE-SIDE" AND BED-SIDE: IN VIVO PREDICTIONS BASED ON IN VITRO ANALYSIS The novel PI3K-dependent M2 BMMO in vitro differentiation program induced by TGFB, and perhaps other plasma factors, mimicked to some degree the M2 phenotype differences of in vivo-differentiated SHIP+/+ and -/- PMOs from Chapter 4. Although definitively proving the validity of the model was beyond the scope of this thesis, it was thought that returning to SHIP-/- mice and addressing questions raised from the in vitro differentiation model in this setting could at least yield insight into its robustness. The first area of translational interest pertained to the identity of the M2-skewing factor found in mouse plasma. The factor was attributed, at least in part, to TGFB, while IL-4 was ruled out. Coupled with the observation that SHIP-/- MP was on average about twice as potent as +/+ MP, this raised the question as to possible differences in the plasma of wild-type and SHIP-/-mice. Given that TGFB was implicated in vitro, it was decided to compare plasma levels of TGFB in the plasma of both genotypes. However, as can be seen in Fig. 5.13, levels of this cytokine were actually lower in SHIP-/- mice. Since platelets represent a 121 , m mm mm. ... 1 major source of plasma TGFB, and since SHIP-/- mice have been reported to be thrombocytopenic (Moody et al., 2004), then this was at least consistent with the observed result. SHIP-/- BM cells were more sensitive to the arginase-inductive effects of TGFB, but given its lower levels in SHIP-/- mice, it seemed unlikely that this accounted for the increased M2 potency of SHIP-/- MP. As had been reported for serum (Takeshita et al., 2002), IL-6 plasma levels were also significantly elevated in SHIP-/- mice, compared to wild-type. However, IL-6 did not have significant M2-inductive capacity in the differentiation assay on its own (data not shown), although it cannot be ruled out that it might synergize with another factor. Finally, consistent with the prediction that IL-4 was not the differential MP constituent, levels of IL-4 were very low in the plasma of SHIP+/+ and -/- mice (Fig. 5.13). However, the highest IL-4 readings, albeit still in the low range, were found in SHIP-/- mice, and it is possible that a higher average level of IL-4 in the plasma could account for the slightly increased basal level of pStat6 in the PMOs of SHIP-/- mice (Fig. 4.6d). Thus, the identity of the factor(s) that differentially conveyed more M2-skewing potency to SHIP-/- plasma remains elusive and warrants further investigation. ~ 6 E "a +/+ -/-Fig. 5.13. An analysis of cytokine levels in SHIP+/+ and -/- mouse plasma. Plasma samples from 6-10 week-old SHIP+/+ and -/- mice were analyzed for TGFB (latent + active), IL-6, and IL-4 levels by ELISA. Lower SHIP-/- plasma TGFB levels have been confirmed in an independent experiment while in the same experiment IL-4 levels in both genotypes were undetectable (Rauh etal . , 2005). 122 A second prediction that arose from the in vitro differentiation model was that Argl induction likely occurs at an early stage of MO differentiation in vivo, and not in fully mature MOs. Given that MO differentiation in vivo begins in the BM, proceeds at the monocyte (M0) stage to the peripheral blood where further maturation occurs, and is followed by terminal differentiation in different tissues (eg, in the lung and peritoneal cavity) where SHIP-/- MOs have an Argl-high phenotype, this prediction was explored by asking if SHIP-/- M 0s display an M2 phenotype. Thus, red blood cell-depleted peripheral blood leukocytes (PBLs) were obtained from adult wild-type and SHIP-/- mice and were assessed for arginase activity before and after isolation of PBM 0s using a commercial negative selection procedure (see Materials and Methods, Chapter 2.18). As can be seen in the left panels of Fig. 5.14 (a = scatter plot and b = bar chart), at least 6-fold more arginase activity was detected in SHIP-/- PBLs, compared to wild-type PBLs. Interpretation of the fold-difference was complicated by the knowledge that SHIP-/- mice have increased numbers of circulating M0s (Helgason et al., 1998). Thus, M0s might contribute a greater proportion of the SHIP-/- PBLs. However, SHIP-/- mice were reported by Helgason et al. (1998) to have an average 1.5 to 2.5-fold increase in circulating M0s, and this suggested that the aforementioned arginase activity might be indeed increased in SHIP-/- PBLs on a per-cell basis. This was further suggested by the observation that isolated SHIP-/- PBM 0s also displayed increased normalized arginase activity, compared to wild-type (Fig. 5.14, right panels). Next, in order to confirm that the increased arginase activity corresponded to an increased level of Argl in SHIP-/- PBLs and PBM 0s, these cells were isolated from both genotypes and subjected to Western blot analysis. As can be seen in Fig. 5.15a, SHIP-/- PBLs displayed enhanced levels of Argl protein but, surprisingly, not Ym1. Moreover, as was evident in Fig. 5.15b, the enhanced SHIP-/- Argl levels were due, at least in part, to the PBM 0 fraction. Interestingly, although PBM 0s have been reported to express low levels of c-fms, it appeared that expression was enhanced in the SHIP-/- M 0 fraction (Fig. 5.15b). The significance of this observation requires further analysis. 123 PBL +/+ -/-PBM„ Fig. 5.14. SHIP-/- peripheral blood contains elevated arginase activity and expression in total leukocyte and monocyte fractions. Red cell-depleted peripheral blood leukocytes (PBLs) were obtained in three separate experiments from a total of 7 adult SHIP +/+ and SHIP-/- mice, arginase assay lysates were prepared and arginase activity was assessed. Results are presented (left) as (a) scatter plots or (b) the mean ± S E M . Three independent monocyte fractions (PBM 0 s) were obtained from these peripheral blood samples using a commercial monocyte negative selection kit, and arginase assays were performed and presented (right) as was for the PBLs . As was observed with the total PBL fraction, PBM 0s from both genotypes expressed similar levels of Ym1 (Fig. 5.15b). Since wild-type mature PMOs did not express Western-detectable Ym1 levels (Chapter 4), this suggested that Ym1 expression is normally lost in the wild-type PBM 0 to mature MO transition, as 124 might have been predicted by the loss of Ym1 in the in vitro differentiation model (Figs. 5.10 a and b). Moreover, this also suggested that in SHIP-/- mice, expression of Ym1 is not lost in the M0-MO in vivo transition perhaps because factors found within the tissue microenvironments sustain or induce Ym1 further. The similar PBM 0 Ym1 levels in both genotypes, despite enhanced SHIP-/- PBM 0 Argl levels, were reminiscent of the pattern observed in the Lewis lung carcinoma protein extracts (Fig. 4.10b) and suggested that the tumour-infiltrating myeloid/M0/MO cells were similar, but not identical to mature tissue SHIP-/- M2 PMOs. PBL PBL +/+ -/-PBM 0 +/+ -/-Ym1 Argl Grb2 +/+ -c-Fms Ym1 GAPDH Fig . 5.15. SHIP-/- peripheral blood leukocytes and monocytes express elevated Argl but not Ym1 levels as compared to wild-type. Protein lysates were prepared from two sets of SHIP+/+ and -/- PBLs (a) and (b) or one set of P B M 0 s (b) and subjected to Western blot analysis for c-fms, Ym1, Argl, Grb2, or G A P D H , where indicated. Given the observation of elevated arginase activity and Argl levels in SHIP-/- PBM 0s, it was hypothesized that SHIP-/- mice might display reduced circulating L-arginine levels. As can be seen in Fig. 5.16a, SHIP-/- mouse plasma did contain less L-arginine, as assessed by HPLC, very near the threshold of statistical significance. Given that HPLC was performed on samples at several independent times, a more relative measure of L-arginine levels was sought, to control for experimental variation. Thus, as has been validated elsewhere (Wu and Morris, 1998; Morris et al., 2004), L-arginine levels were assessed relative to lysine and ornithine, two amino acids whose transport is regulated in a similar manner to L-arginine (Fig. 5.16b). Using this technique, a significantly lower relative plasma L-arginine concentration was found in SH IP-A-mice. a b F i g . 5.16. S H I P - / - p e r i p h e r a l b l o o d c o n t a i n s r e d u c e d c i r c u l a t i n g l e v e l s o f L - a r g i n i n e . Plasma was obtained from 5 to 6 adult overnight-fasted SHIP+/+ and -/- mice, deproteinated, filtered, and subjected to H P L C analysis for amino acids on three separate occasions, (a) The left panel presents L-arginine concentrations, while the right panel (b) presents relative L-arginine (L-arginine/lysine + ornithine, as these represent a sub-class of amino acids with similar transport), in part to control for variation of measurement on the three separate occasions. Results are presented as the mean ± S E M and statistical analysis was conducted using the unpaired, one-tailed, student's t test. 5.3 DISCUSSION The data presented in this chapter suggests a model in which elevated PIP3 levels predispose early MO progenitors towards an M2 phenotype and SHIP acts as a potent negative regulator of this skewing. In support of this, it was found that PI3K inhibitors prevented the induction of Argl in SHIP-/- MO progenitors by MP (Fig. 5.4c) or TGFB (Fig. 5.6). It was also found that IL-4 and IL-13 were capable of skewing both SHIP+/+ and -/- mature MOs as well as their progenitors towards an M2 phenotype (Fig. 5.7a, 5.9, and data not shown) and it is proposed that they can do so because they are not only robust activators of the PI3K pathway (Kelly-Welch et al. 2003; Montaner et al. 1999 and Ruetten 126 and Thiemermann 1997) but can activate Stat6 as well (Pauleau et al. 2004 and Kelly-Welch et al. 2003). Related to this, it was found that PI3K inhibitors blocked IL-4-induced Argl and Ym1 induction in mature SHIP+/+ BMMOs, without having any significant effect on Stat6 phosphorylation (Fig. 5.11). This suggests that PI3K activation may be a universal requirement for M2 programming and this may help to explain some of the controversy surrounding the role of PI3K in TLR4 signaling (see Chapter 3). Thus, although we (Sly et al., 2004) and others (Weinstein et al, 2000; Rhee et al., 2003) have shown that PI3K can stimulate LPS-induced TLR4 signaling under some circumstances, it also subsequently enhances an anti-inflammatory response. It is also proposed that TGFp and IL-10 can only skew SHIP-/- progenitors to an M2 phenotype because they are weak PI3K-inducers (Kim et al. 2004; Conery et al. 2004 and Bhattacharyya et al. 2004) and are not known to activate Stat6. In fact, there is growing evidence that, at least in some cell types, TGFp and IL-10 may actually antagonize the PI3K pathway (Kim et al. 2004; Valderrama-Carvajal et al. 2002; Sly et al. 2004; Conery et al. 2004; Remy et al. 2004; and Ghanipour and Mui, unpublished data). Given that MP was incapable of skewing SHIP+/+ progenitors towards an M2 phenotype in our in vitro differentiation assays it is also tentatively proposed that IL-4 and IL-13 do not play a major role in determining M1/M2 MO levels in the absence of microbial infections and that under normal homeostatic conditions intrinsic PIP 3 levels and TGFB levels play the dominant role. As far as the identity of the MP factor(s) is concerned, these studies have identified TGFP as one component. Since TGFp mimics in vivo differentiation, ie, it skews SHIP-/- but not SHIP+/+ progenitors towards an M2 phenotype (Fig. 5.6 and 5.9a), it is tentatively assumed TGFp is at least partially responsible for the skewing that occurs in vivo in SHIP-/- mice. Since -MP was about twice as potent as +MP in Argl induction, it was asked if TGFP levels were elevated in the plasma of SHIP-/- mice, but this was not the case. In fact, TFGP levels (both total (Fig 5.13) and active (data not shown)) were consistently lower in SHIP-/-plasma. The levels of IL-4, IL-10, IL-12, and IFNy, on the other hand, were 127 similar in SHIP+/+ and -/- mouse plasma and present at very low levels (Fig. 5.13 , Rauh et al., 2005, and data not shown). However, SHIP-/- plasma did contain higher IL-6 (Fig. 5.13) and TNFa levels (75 pg/ml vs 20 pg/ml in pooled SHIP+/+ plasma, assessed by cytometric bead array (Rauh et al., 2005)) and it is possible one or both are involved. Interestingly, the delayed addition and washing studies suggest that M2-skewing may be occurring during an early well defined period of MO differentiation (Fig. 5.4a and 5.9b). Consistent with this, MP-induced Arg I concomitant with c-fms during differentiation of SHIP-/- BM cells in vitro (Fig. 5.10), and elevated Argl levels and activity were detected in peripheral blood monocytes from SHIP-/- mice in vivo (Fig. 5.14). This suggested that at least some features of M2 programming are attained in SHIP-/- mice prior to terminal differentiation in tissues. In summary, while most studies on MO programming thus far have been conducted with naive, in wiVo-differentiated, mature BMMOs, these studies with SHIP-/- progenitors have revealed that the combination of intrinsic (ie, elevated PIP3 levels) and extrinsic factors (ie, cytokine environment) during a critical early period of MO differentiation can determine the MO phenotype. As 'naive', mature MOs can be M2-skewed by factors such as IL-4 and IL-13, this suggests that the MO phenotype can arise both during differentiation and once it is completed. Although these studies do not distinguish between distinct M1 and M2 progenitors or a plastic common progenitor, this in vitro system may more accurately reflect the conditions of in vivo MO differentiation and programming and be amenable to future studies. Importantly, these studies caution against extrapolating findings obtained with in wYra-derived BMMOs to more complex and dynamic in vivo systems. Finally, it is suggested that targeting the SHIP/PI3K axis in situ, or ex vivo, may represent a viable future means of harnessing and manipulating the macrophage phenotype for cancer, infection, and chronic inflammation therapies. This will be expanded upon in the next Chapter. 128 CHAPTER 6 - SUMMARY AND PERSPECTIVES SHIP is a hemopoietic-restricted protein that is recruited to sites of active signal transduction by growth factors, cytokines, and immune receptors, and catalyzes the hydrolysis of the PI3K-generated second messenger plasma membrane lipid PIP3, to PI-3,4-P2. By virtue of its ability to deplete PIP 3 levels at the membrane, and the subsequent recruitment of PH domain-containing signaling proteins, SHIP has been demonstrated to be a master negative regulator of hemopoietic cell differentiation, proliferation, survival, and activation (Krystal, 2000). Targeted disruption of SHIP in mice results in a chronic and infiltrative myeloproliferative disorder characterized by enhanced numbers of granulocyte-macrophage (GM) progenitors and increased numbers of mature GM cells in the periphery (Helgason et al., 1998; Liu et al., 1999). At the commencement of this thesis, the role of SHIP in macrophage development and function remained to be fully elucidated. The SHIP-/- mouse model provided a unique opportunity for study (Helgason et al., 1998). It was demonstrated in Chapter 3 that Sca-1+Lin" bone marrow progenitors differentiated into Mac1+ cells faster and in greater proportion when exposed to IL-3, IL-6, Epo, and SF in the absence of SHIP. Moreover, SHIP-/- total or Lin" bone marrow cells demonstrated enhanced proliferation in response to M-CSF, a growth factor important for myeloid/macrophage differentiation (Pixley and Stanley, 2004). These findings were consistent with the hypothesis that SHIP restrains macrophage differentiation. As macrophages are important components of the innate immune system (Mosser, 2003), we also sought to determine the role of SHIP in macrophage function. As a model system amenable to experimentation, an immortalized set of bone marrow-derived macrophages (J2M-BMMOs) were derived from SHIP+/+ and -/- mice (Blasi et al., 1985) (Chapter 3). Inducible nitric oxide (NO) synthase (iNOS)-mediated high-output production of NO by macrophages is an important strategy to combat the growth of tumours and microorganisms, and is a feature of classical, or M1, macrophage activation 129 (MacMicking et al., 1997), and this was chosen as the major point for comparison. At the commencement of this thesis, it had been reported that the PI3K inhibitor, wortmannin, augmented LPS-induced NO secretion in peritoneal macrophages (Park et al., 1997). Thus, it was hypothesized that SHIP was a positive regulator of NO production. Consistent with this hypothesis, SHIP-/-J2M-BMM0S failed to significantly produce NO due to inhibition at the level of iNOS transcription, resulting at least in part, from deficient nuclear localization of IRF1. In contrast, primary bone marrow-derived macrophages (BMMOs) from SHIP-knockout mice were not routinely impaired in iNOS induction as long as negative feedback mechanisms were minimized, and in fact demonstrated enhanced LPS-stimulated iNOS induction. This was likely because a primary effect of the PI3K pathway in LPS-induced BMMOs was to enhance the autocrine-acting p70S6K/IFNB/Stat1 pathway leading to iNOS. Differential impacts upon this axis also provided a likely explanation for the opposite effects of the PI3K pharmacological inhibitors LY294002 and wortmannin on iNOS/NO synthesis, being inhibition and augmentation, respectively. Moreover, these studies cautioned against extrapolation of results obtained with immortalized cell lines and pharmacological inhibitors. Consistent with a negative role for SHIP in LPS-induced signaling and pro-inflammatory outcomes in BMMOs, it was also discovered in Chapter 3 that SHIP-/- BMMOs were refractory to a phenomenon known as endotoxin tolerance (West and Heagy, 2002). Specifically, SHIP-/- BMMOs did not appropriately dampen down the production of pro-inflammatory cytokines when exposed to a second dose of LPS (ie, they did not tolerize), likely because SHIP protein levels were normally upregulated in wild-type BMMOs in an essential, autocrine TGFB-mediated tolerance loop (Sly et al., 1994). Extending the analysis to in wVo-differentiated primary resident peritoneal macrophages (PMOs) from SHIP-/- mice, it was discovered in Chapter 4 that they were also not normally impaired in iNOS induction, and instead showed accelerated and enhanced iNOS induction in response to LPS + IFN-gamma. 130 However, these macrophages were impaired in subsequent NO generation due to L-arginine substrate competition by constitutively high levels of the enzyme arginase I, which redirected L-arginine metabolism away from cytotoxic NO and likely towards the production of L-ornithine, proline and polyamines, which are important intermediates in healing and the resolution of inflammation (Goerdt and Orfanos, 1999). These were recognized as features of alternatively activated (M2) macrophages (Stein et al., 1992; Gordon et al., 2003; Mantovani et al., 2004) and subsequent experimentation expanded and confirmed this SHIP-/-PMO phenotype. Consistent with skewing to a M2 macrophage phenotype in SHIP-/- mice, studies in Chapter 4 also revealed that SHIP-/- lungs were fibrotic and contained macrophage-associated Ym1 crystals. Moreover, Lewis lung carcinoma cells grew more rapidly in the M2-skewed environment of SHIP-/- mice. Thus, it appeared that M2-skewing in SHIP-/- mice arose in part as a homeostatic attempt to counter an environment of chronic inflammation and that the exaggerated SHIP-/- M2 MO responses were also implicated in pathological processes in these mice. In keeping with this, Chapter 5 suggested that BMMOs from SHIP-/- mice did not display this M2 phenotype unless exposed to TGFB-containing mouse plasma early during in vitro differentiation. The data in Chapter 5 suggested a model in which elevated PIP 3 levels predisposed MO progenitors towards an M2 phenotype and that SHIP acted as a potent negative regulator of this skewing. On the other hand, since IL-4 and IL-13 could skew both SHIP+/+ and -/- mature MOs as well as their progenitors towards an M2 phenotype, it was proposed they could do so because they were not only robust activators of the PI3K pathway (Kelly-Welch et al. 2003; Montaner et al. 1999; Ruetten and Thiemermann, 1997) but could also activate Stat6 (Pauleau et al. 2004 and Kelly-Welch et al. 2003). Furthermore, it was also concluded in Chapter 5 that PI3K activation represented a previously unappreciated universal requirement for M2 programming. Moreover, this was put forth as at least one explanation for the controversy 131 surrounding the role of PI3K in TLR4 signaling (ie, PI3K participates in both pro-inflammatory and anti-inflammatory cascades in macrophages). These results highlighted a critical role for SHIP and the PI3K pathway in M1/M2 MO programming and suggested that polarization of macrophage phenotype could arise during differentiation and upon maturity, but was particularly sensitive to the microenvironment (ie. retroviral transformation, in vitro differentiation conditions, inhibitory profiles of pharmacological PI3K inhibitors, and the in vivo environment, including mouse genetics and age). Moreover, these studies underscored the pitfalls of extrapolating findings obtained with in vitro-derived MO cell lines and BMMOs to more complex and dynamic in vivo systems. These findings are summarized in a simplified form in Fig. 6.1. Many unanswered questions remain but the next section will highlight a few areas I feel are worthy of future investigation. Chapter 3 revealed that SHIP-/- J2MOs have a specific inability to maintain nuclear levels of IRF-1 and this might account for their inability to efficiently transcribe iNOS. These results were obtained using a retrovirally transformed cell line and raised a red flag regarding studies which rely too heavily on cell line data to extrapolate findings to normal in vivo processes. That said, these results may actually have some application in abnormal in vivo situations, particularly transformation. In this regard, it is appreciated that IRF-1 has tumour suppressive functions in myeloid and other cells (Passioura et al., 2005), and that activation of IRF-1 can revert the transformed phenotype (Kroger et al., 2003). It may be interesting to explore the mechanisms involved in the proposed SHIP-/- J2MO IRF-1 inactivation. Finally, highlighting another peculiar feature of these cells is their differential regulation of iNOS and COX-2 (ie, they are able to induce COX-2 in response to LPS, but are unable to induce iNOS). Should the mechanisms underlying this difference be further elucidated, then it may be possible to selectively and specifically target these important inflammatory mediators for future therapeutics. 132 MyD88 T R I F N F - K B I R F - 3 TNFa IFNB ± iNOS • GSK3B? | IL-10 Akt, p70S6K •specific iNOS deficit •low NO production TNFa IFNf3 ? iNOS •variable early repression •absent late LPS tolerance •usually higher NO levels I F N B IFN6-R r • MyD88 TRIF N F - K B IRF -3 | f A r g l Jak Statl/ IRF-1 TNFa IFNB * fA rg l iNOS • constitutive M2 phenotype • low NO production due to inhibition by Argl Fig. 6.1. The effects of SHIP on macrophage programming and NO production are dependent upon condit ions of differentiation and dynamic environmental influences. The phenotype of SHIP-/- MOs are presented relative to SHIP+/+ MOs, and processes which augment wild-type outcomes are indicated in green, while those that impair it are indicated in red. Please see the text of Chapter 6 for a more detailed description of these events. Chapter 3 also revealed a complex and dynamic regulation of primary macrophage responses to LPS. Specifically, SHIP appeared to exert regulatory control on both pro-inflammatory and anti-inflammatory pathways. Moreover, it was suggested that these differential effects were dependent upon cell type, cell density (both during differentiation and during stimulation), and the amount of time that had elapsed after LPS stimulation. This description is very reminiscent of the complex picture that is emerging of sepsis and septic shock in humans (Annane et al., 2005). Specifically, rather than this process simply reflecting an 133 overzealous pro-inflammatory response, it is now clear that sepsis also contains compensatory anti-inflammatory components, which can cause their own problems (ie, increased susceptibility of hosts to nosocomial infections) (Dal Pizzol, 2004). The time window for sepsis intervention in humans is thus very short and dependent upon elapsed time and host genetic factors (Annane et al., 2005). The key to effective intervention will be an increased understanding of the dynamics of molecular pathways and host responses. Thus, furthering the understanding of SHIP-/- BMMO responses to LPS in vitro and of SHIP-/- mice to LPS in vivo may lead to the identity of more specific targets and techniques for future therapeutics. For example, in the future it may be possible to identify host genetic factors associated with pro- and/or anti-inflammatory phenotypes and subsequent outcomes, novel diagnostic and prognostic biomarkers, including immune cell subsets, which together may allow for real-time tracking and tailoring of specific therapies. Chapter 4 uncovered a profound, constitutive M2 program of in vivo-differentiated SHIP-/- MOs. While M2 MOs are beneficial in normal inflammatory resolution and healing, they have been implicated in human pathology (Gordon et al., 2003). It may be of interest in the future to manipulate arginine metabolism in the SHIP-/- mouse and observe its consequences. For instance, it may be interesting to feed SHIP-/- mice L-arginine, arginase inhibitors, or chitinase inhibitors and compare outcomes such as lifespan, body weight, plasma L-arginine levels, lung histology, etc., with non-treated SHIP-/- control mice. Alternatively, or in addition, it may be interesting to cross SHIP-/- mice with Argl-/- mice, macrophage-specific and/or conditional Argl-/- mice, or Argl transgenic overexpressing mice and determine the consequences. The goal of these studies would be to further elucidate the role of M2 MOs and L-arginine metabolism in normal and disease processes. (Please also see Appendix I for an expansion on potential diagnostic, prognostic, and therapeutic implications for M2 MOs). Chapter 5 revealed that TGFB in mouse plasma is at least partially responsible for its M2-inductive effects in SHIP-/- MOs. These studies also 134 established an in vitro system for deriving M2 MOs. Perhaps with further validation and experimentation it may be possible to attempt adoptive transfer of such M2 cells into hosts, and determine how they may affect disease processes in vivo. However, studies conducted in Chapter 5 also suggested that as yet unidentified factor(s) in plasma may also contribute to M2-skewing. Thus, it would be worthwhile to pursue the identity of these factors, using at least two approaches. One could involve a general scheme to fractionate and separate plasma components and then test them in vitro. If crude fractions with enhanced activity are isolated, then attempts to further fractionate and purify components should be pursued. In conjunction with this approach, it may be possible to identify lead candidates based upon the literature. For instance, Rowley and Stach (1998) have identified a complex of IgG and TGFB (ie, IgG-TGFB) that is elevated in the plasma of chronically-inflamed or tumour-bearing hosts that has potent immunosuppressive activity. It acts upon myeloid progenitors and endows them with myeloid suppressor cell-like (MSC-like) activity against adaptive (ie, T cell) responses (Rowley and Stach, 1998). It is entirely speculative at present, but MSCs are known to suppress via arginase (Bronte and Zanovello, 2005), and it is tempting to speculate that this may be downstream of IgG-TGFB. Finally, unpublished observations in this laboratory have suggested an M2-inductive effect of IgG in mouse plasma. Thus, it may be fruitful to pursue the identity of the remaining M2-skewing plasma factors, to reveal potentially new factors or new roles for common factors. Ultimately, it is hoped that the insights gained from SHIP-/- mice may lead to translational findings that may aid in human disease (Rauh and Krystal, 2002). It is therefore of interest that reduced human SHIP (hSHIP) (Ware et al., 1996) levels or SHIP phosphatase activity have been discovered in patients with chronic myelogenous leukemia (CML) (Sattler et al., 1999; Jiang et al., 2003) and acute myelogenous leukemia (AML) (Luo et al., 2003; Luo et al., 2004) and have been implicated in disease pathogenesis. Since SHIP-/- mice have a myeloproliferative disorder characterized by expansion of immature MSCs (Ghansah et al., 2004) and mature M2 MOs (Rauh et al., 2005), it may be 135 interesting to ask if M2 features are evident in cells from human CML and AML patients. This may be more than speculative since it has been reported that in human CML and/or AML: 1) peripheral blood monocytes express elevated levels of ornithine decarboxylase (ODC), an enzyme downstream of arginase in the polyamine biosynthetic pathway (Tripathi et al., 2002); 2) monocytes spontaneously produce IL-6 leading to elevated plasma levels and have impaired pro-inflammatory cytokine production in response to LPS stimulation (Anand et al., 1998); 3) monocytes express elevated levels of the Ym1/FIZZ family member, FIZZ3/resistin (Yang et al., 2003); 4) T cells have diminished T cell receptor zeta chain surface expression (Chen et al., 2000), which has been associated with decreased L-arginine concentrations (Baniyash, 2004); and 5) macrophages in the bone marrow, including so-called pseudo-Gaucher cells, have been observed to contain intracellular crystals and associated extracellular crystals (Kuto et al., 1984; Busche et al., 1997; Rubin et al., 1997). These features are very reminiscent of SHIP-/- M2 MOs. Perhaps features of the M2 phenotype, if adopted by human AML and/or CML cells, could aid in proliferation and immunosuppression and thus represent targets for future therapeutics. (Please also see Appendix I for an expansion on the role of M2 MOs in malignancy and the potential for therapeutic interventions that target these cells). Of note, while elevated Argl expression has been detected in some human disease states (Bruch-Gerharz et al., 2003; Zimmermann et al., 2003; Morris et al., 2005; Zea et al., 2005), it has been reported that humans do not express Ym1 (Raes et al., 2005), but instead have two highly homologous proteins called acidic mammalian chitinase (AMCase) and chitotriosidase 1 (Chitl) (Boot et al., 2001). Preliminary studies (data not shown) using an anti-mYm1/hAMCase/hChit1 antibody and anti-hArg1, have demonstrated that differential expression of proteins corresponding to the appropriate size of AMCase, Chitl, and Argl are detected in human AML peripheral blood cell protein lysates. These preliminary results await confirmation using appropriate healthy subject controls. 136 It has been a fascinating experience to gain some insight into the regulation of macrophages during the course of this thesis. As conductors of an immune orchestra with a broad performance repertoire that extends from inflammation to its resolution, macrophages are truly remarkable and adaptable cells. 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J.Clin.Invest. 777, 1863-74. 159 APPENDIX I - EPILOGUE: AUG 05-06 This thesis was drafted in August, 2005, but its formal submission did not occur until approximately one year later due to my return to full-time medical school studies and clinical clerkship. Herein, I will present studies published during this interval that I feel contribute further insight into this thesis and its potential ramifications. Hemopoiesis Hume (2006) has recently reviewed the concept of a unified mononuclear phagocyte system (MPS) and has called for studies to expand and revise its scope. Fogg et al. (2006) have taken a great step towards our understanding of the MPS by identifying a CX3CR1+CD117+(c-kit+)Lin" common macrophage and dendritic cell progenitor (MDP), devoid of granulocytic, erythroid, megakaryocytic and lymphoid potential. DCs from SHIP-/- mice are currently being characterized in the lab of Dr. Krystal (Antignano and Krystal, unpublished observations). The identification of this MDP may aid in attempts to modulate MO and DC differentiation toward particular subsets with the goal of utilizing these cells for therapeutic purposes. SHIP The Kerr group has found that SHIP-/- NK cells over-express the 2B4 MHC-independent inhibitory receptor and this compromises key NK cytolytic functions mediated by NKG2D and Ly49H activating receptors, including killing of tumour targets (Wahle et al., 2006). This is reminiscent of the Argl/iNOS balance in SHIP-/- MOs. Moreover, Rothman's group have reported that SHIP-/- mice have an increased proportion of regulatory CD4+CD25+ T cells (Tregs) that produce TGFB and lead to anergy to T cell receptor (TCR) stimulation (Kashiwada et al., 2006). It will be interesting to determine how pervasive the 160 expansion of immunosuppressive populations is in the SHIP-/- hemopoietic compartment and the degree of interrelatedness of these cells. TLR and PI3K Signal Transduction Recent studies have begun to elucidate the roles played by phosphatidylinositol metabolism in TLR signal transduction. Medzhitov's group have found that the TIRAP/Mal adaptor localizes to the plasma membrane by binding to phosphatidylinositol-4,5-bisphosphate. In turn, MyD88 is recruited to TLR4 to initiate signal transduction (Kagan and Medzhitov, 2006). Similarly, myristoylation of the TRAM adaptor targets it to the plasma membrane where it can recruit TICAM-1/TRIF and initiate MyD88-independent signaling (Rowe et al., 2006). Thus, plasma membrane engagement of "sorting adaptors" (TIRAP/Mal and TRAM) facilitates recruitment of "signaling adaptors" (MyD88 and TICAM-1/TRIF) (Fitzgerald and Chen, 2006). Moreover, recruitment and function of Tollip, a negative regulator of TLR signaling that controls the magnitude of the pro-inflammatory response (Didierlaurent et al., 2006), is dependent upon its ability to bind to phosphatidlyinositol-3-phosphate and PIP3 (Li et al., 2004). Finally, Bruton's tyrosine kinase (Btk), recruited to sites of active signal transduction by PH domain-mediated binding to PIP 3 in a process regulated by SHIP (Boland et al., 1998), has recently been demonstrated to tyrosine phosphorylate TIRAP/Mal downstream of TLR2 and 4 (Gray et al., 2006). Initially, this leads to transcriptional activation of N F - K B (Gray et al., 2006) but at later timepoints tyrosine-phosphorylated TIRAP/Mal is specifically targeted for SOCS-1-mediated degradation (Mansell et al., 2006). Therefore, it may be of interest to determine how SHIP affects plasma membrane recruitment of TLR adaptors, Btk, and Tollip, and to potentially correlate this pattern with downstream observations. Two groups have recently identified TNF receptor-associated factor 3 (TRAF3) as a key component of the MyD88-independent pathway leading to the induction of type I interferons and anti-inflammatory IL-10 (Hacker et al., 2006; 161 Oganesyan et al., 2006). Furthermore, using systems biology approaches, two other groups have identified activating transcription factor 3 (ATF3) as an LPS-induced component of a negative feedback loop that regulates inflammation (Gilchrist et al., 2006; Nilsson et al., 2006). Given that SHIP has been implicated in the regulation of LPS-induced type I interferon, IL-10 production, and tolerance, it will be interesting to investigate if this may be related to the impact of SHIP on TRAF3 or ATF3. It is apparent that the roles played by PI3K/SHIP in TLR signaling are complex. Kuo et al. (2006) have also reported that the effects of PI3K are dependent upon the particular isoform: class III PI3K impacts upon TLR9 signaling upstream of MyD88, while class I PI3K regulation is downstream of MyD88 for both TLR4 and 9. Moreover, as has been the case with TLR4, contradictory roles have been proposed for PI3K in TLR5 signal transduction (Rhee et al., 2006; Yu et al., 2006). A result that does not appear to be in dispute is the role of SHIP/PI3K in TLR-mediated IL-10 production. Two other groups have confirmed that LPS-induced IL-10 production is positively regulated by PI3K and negatively regulated by SHIP in murine macrophages (Pengal et al., 2006; Ghahipour and Mui, manuscript in preparation). Moreover, PI3K positively regulates IL-10 production stimulated by TLR9 in murine macrophages, and autocrine-acting IL-10 subsequently inhibits pro-inflammatory cytokine production (Saegusa et al., 2006). Interestingly, while it has been appreciated for some time that IFNy synergizes with TLR ligands during inflammation, it does so at least in part by suppressing IL-10 production downstream of PI3K (Hu et al., 2006). M2 MO Classification and Biology Groups led by Raes and Mosser have utilized comparative gene expression profiling to identify signatures for M1, M2, and type-ll (LPS + immune complex-stimulated) murine macrophages (Ghassabeh et al., 2006; Edwards et al., 2006). Type-ll murine MOs specifically upregulate sphingosine kinase 1 and 162 the TNF superfamily member, LIGHT (Edwards et al., 2006). Moreover, a common signature for M2 MOs, independent of disease model, mouse strain, or source of in vivo MOs, was identified by Ghassabeh et al. (2006), which included some genes that could not be induced by M2 cytokines under in vitro conditions. It may be of interest to expand the analysis of SHIP-/- M2 MOs to include members of this signature. It is also tempting to speculate that the M2-inducing plasma factor identified by our group may account, in part, for the discrepancies between in vitro and in vivo induction observed by Ghassabeh et al. (2006). Heterogeneity of the macrophage lineage is conserved in mice and humans (Gordon and Taylor, 2006). While it has been suggested that human M2 MOs cannot induce Argl in response to IL-4 and IL-13 (Raes et al., 2005), these studies were performed on cells differentiated in vitro. Erdely et al. (2006) have subsequently demonstrated that human macrophages can induce Argl by combining IL-4 with agents that induce cyclic adenosine monophosphate (cAMP) levels. Moreover, Argl is also expressed in human granulocytes and has T cell suppressive functions, as it does in murine macrophages (Munder et al., 2006). Furthermore, tumour-associated myeloid cells in renal carcinoma patients express Argl, leading to tumour immune evasion (Zea et al., 2005). Thus, while important differences may exist between murine and human M2 MOs, I feel it is premature to conclude that Argl is not a marker of human M2 MOs. Advancements in the characterization of human MO populations have included the identification of CCL1 as a specific marker of type-ll activated MOs (Sironi et al., 2006), and stabilin-1 interacting chitinase-like protein (S1-CLP) as a marker of human M2 MOs (Kzhyshkowska et al., 2006a). It has been proposed that S1-CLP and stabilin-1 facilitate M2 MO-mediated extracellular matrix remodeling, angiogenesis and tumour progression (Kzhyshkowska et al., 2006b). The repertoire of cytokines that control M2 MO activation has recently been expanded to include IL-21 (Pesce et al., 2006) and IL-27 (Wirtz et al., 2006). While not able to induce M2 MOs individually, IL-21 and IL-27 can synergize with IL-4, IL-10, and IL-13 in this capacity. Consistent with this, 163 expression of the IL-27 receptor, WSX-1, is upregulated on alternatively activated MOs (Ruckerl et al., 2006). Several groups have recently shed light on the relationship between M2 MOs and T cells. Anthony et al. (2006) and Reece et al., (2006) have found that T H 2 cells induce M2 MOs during helminthic parasite infections. Importantly, it has been demonstrated for the first time that M2 MO Argl mediates host protective responses against these parasites (Anthony et al., 2006). However, as parasitic infections evolve over time, M2 MOs induce CD4 + T cell hyporesponsiveness (Taylor et al., 2006). While this phenomenon was partly due to M2 MO production of TGFB (Taylor et al., 2006), it has also recently been discovered that macrophages can drive the differentiation of regulatory T cells (Tregs) (Hoves et al., 2006). Thus T cell-induced alternative macrophage activation can mediate protective responses against parasites and likely reduce host pathology by controlling the magnitude of adaptive immunity (Herbert et al., 2004). Since SHIP-/- mice have elevated numbers of M2 MOs and Tregs (Kashiwada et al., 2006), it may be informative to determine how these mice respond to parasitic infections, and to further elucidate relationships between M2 MOs and T cell anergy/Tregs in these mice. MSCs and M2 MOs in Disease and Therapeutics Evidence is mounting for the ability of tumours to perturb and skew myeloid and macrophage lineage differentiation, both locally at the tumour site (M2 MOs/tumour-associated MOs (TAMs)) and in peripheral blood and lymphoid organs (immature myeloid cells (iMCs)/myeloid suppressor cells (MSCs), in order to facilitate tumour growth, progression and immune evasion (Lewis and Pollard, 2006; Serafini et al., 2006; Van Ginderachter et al., 2006b). Moreover, links have been established between chronic "smoldering" inflammation, the induction of MSCs, M2 MOs and the promotion of malignant disease (Bunt et al., 2006; Balkwill et al., 2005). It has also been proposed that some tumour cells are even 164 able to fuse with TAMs, leading to aneuploidy, metastatic potential and immune evasion (Pawelek, 2005). Studies performed within the last year on human patients with ovarian cancer and mouse models of the disease have yielded insights into the roles of MSCs and M2 MOs in ovarian cancer and potential therapeutic interventions. Gordon and Freedman (2006) have reported defective anti-tumour functions of ovarian cancer TAMs, consistent with TAM M2-skewing by tumour-derived factors reported by Hagemann et al. (2006). Ovarian cancers are also able to induce MSCs (Yang et al., 2006). However, targeting CD80 expression on MSCs reverses Treg CD152-mediated immunosuppression (Yang et al., 2006) and blocking the negative T cell co-stimulatory B7-H4 molecule expressed on ovarian cancer TAMs may ameliorate T cell anergy (Kryczek et al., 2006). De Baetselier's group has demonstrated that peroxisome proliferator-activated receptor gamma (PPARy) ligands (such as thiazolidinediones currently used for diabetes treatment) reverse cytotoxic CD8 + T lymphocyte (CTL) suppression by M2 TAMs (Van Ginderachter et al., 2006a). Moreover, Luo et al. (2006) have identified legumain as a novel TAM protein and have successfully targeted TAMs using a legumain-based DNA vaccine. This led to suppression of tumour growth, angiogenesis and metastasis in mouse models of breast, colon, and non-small cell lung cancers (Luo et al., 2006). Thus, targeting MSCs and M2 MOs may represent feasible strategies in human cancer therapeutics. In addition to utilizing them as diagnostic and prognostic tools, potential points of intervention include preventing their differentiation and recruitment, blocking their effector functions, and targeting them in situ (for re-polarization or elimination). Results presented with SHIP-/- mice have suggested a role for M2 MOs in pulmonary fibrosis. Both in a mouse model of idiopathic pulmonary fibrosis (IPF) and in human patients with the disease, M2-skewed alveolar macrophages have been implicated in the perpetuation of pulmonary fibrosis (Mora et al., 2006; Prasse et al., 2006). Thus, it will be of interest to determine if perturbations in PI3K/SHIP are found in human IPF alveolar MOs, and if this may offer any promise towards treatment. 165 Finally, in this thesis it was suggested that M2 MO features may be shared by myeloid leukemia cells and that this may be of clinical relevance. It has subsequently been reported by Bergmann et al. 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TLR5-mediated phosphoinositide 3-kinase activation negatively regulates flagellin-induced proinflammatory gene expression. J Immunol. 176:6194-201. 171 APPENDIX II - THESIS-RELATED PUBLICATIONS Rauh, MJ, Ho, V, Pereira, C, Sham, A, Sly, LM, Damen, JE, Huxham, L, Minchinton, Al, Mui, A L-F, and Krystal G. SHIP represses the generation of alternatively activated macrophages. Immunity. 2005 Oct;23(4): 361-74. PMID: 16226502 Rauh MJ, Sly LM, Kalesnikoff J, Hughes MR, Cao L-P, and Krystal G. The role of SHIP in macrophage programming and activation. Biochem. Soc. Trans. 2004 Oct;32(Pt 5):785-8. PMID: 15494015 Sly LM, Rauh MJ, Kalesnikoff J, Song CH, Krystal G. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity. 2004 Aug;21(2):227-39. PMID: 15308103 Sly LM, Rauh MJ, Kalesnikoff J, Buchse T, Krystal G. SHIP, SHIP2, and PTEN activities are regulated in vivo by modulation of their protein levels: SHIP is up-regulated in macrophages and mast cells by lipopolysaccharide. Exp Hematol. 2003 Dec;31 (12): 1170-81. PMID: 14662322 Rauh, MJ. Canadian bacon: straight goods on the MD/PhD program experience from a student's perspective. Science (Next Wave) online October 24, 2003. http://sciencecareers.sciencemag.org/career development/previous issues /articles/2660/canadian bacon straight goods on the md phd program experience from a student s perspective/(parent)/12095 Kalesnikoff J, Sly LM, Hughes MR, Buchse T, Rauh MJ, Cao LP, Lam V, Mui A, Huber M, Krystal G. The role of SHIP in cytokine-induced signaling. Rev Physiol Biochem Pharmacol. 2003 Apr 12 [Epub ahead of print] PMID: 12692707 172 Rauh MJ, Kalesnikoff J, Hughes M, Sly L, Lam V, Krystal G. Role of Sre homology 2-containing-inositol 5'-phosphatase (SHIP) in mast cells and macrophages. Biochem Soc Trans. 2003 Feb;31(Pt 1):286-91. PMID: 12546703 Takeshita S, Namba N, Zhao JJ, Jiang Y, Genant HK, Silva MJ, Brodt MD, Helgason CD, Kalesnikoff J, Rauh MJ, Humphries RK, Krystal G, Teitelbaum SL, Ross FP. SH IP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts. Nat Med. 2002 Sep;8(9):943-9. PMID: 12161749 Rauh MJ, Krystal G. Of mice and men: elucidating the role of SH2-containing inositol 5-phosphatase (SHIP) in human disease. Clin Invest Med. 2002 Jun;25(3):68-70. PMID: 12137253 Rauh MJ. The forecast is bright for budding clinician-scientists on Canada's west coast. Clin Invest Med. 2002 Jun;25(3):66-7. PMID: 12137252 Rauh MJ. An inspiring model for translational research: nuclear factor-kappaB (NF-kappaB), bench to bedside. Clin Invest Med. 2002 Jun;25(3):65-6. PMID: 12137251 173 

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