CD137 ligand trigger in phospholipid signalling cascade in a monocyte cell line

CD137 LIGAND TRIGGER IN PHOSPHOLIPID SIGNALLING CASCADE IN A MONOCYTE CELL LINE H’NG SHIAU CHEN NATIONAL UNIVERSITY OF SINGAPORE 2013 CD137 LIGAND TRIGGER IN PHOSPHOLIPID SIGNALLING CASCADE IN A MONOCYTE CELL LINE H’NG SHIAU CHEN (B. Science with Merit, NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 i DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ______________________ H’NG SHIAU CHEN 1 APRIL 2013 ii ACKNOWLEDGEMENTS First and foremost, I would like to thank Department of Physiology, Yong Loo Lin School Of Medicine, National University of Singapore, especially to the Head of Department, A/P Soong Tuck Wah, for the opportunity to do my part time graduate studies in the department. Without the department’s support, I would not have realized my dream to pursue a higher degree in research. I would like to express my deepest gratitude to my supervisor, A/P Schwarz Herbert, for supporting me in completion of the project. I would like to thank him for his guidance, advice and patience throughout this project. I will not forget his words of encouragement and impromptu discussions whenever I needed one. I am also grateful to him for proof-reading my thesis. I also would like to thank all the past and present lab mates for all the help and assistance rendered to me throughout the studies especially Jane Pang, Dr. Shaqireen Kwajah, Dr. Shao Zhe, Dr. Angela Moh, Ho Weng Tong, Koh Liang Kai, Cheng Cheong Kin and Zulkarnain Harfuddin. I would like to thank A/P Lam Yulin and Dr. Wong Lingkai for providing compound 5c for my project. Special thanks go to Dr. Sheryl Tan and Dr. Wang Binbin for their friendships and supports throughout the years. I truly treasure the memories and wisdom shared in science and in life. I would like to thank Chong Oi Khuan, Teoh Chun Ming, Chin Chin Yein and Qiao Yong Kang for their encouragement and friendships. I would like to extend my appreciation to Dr Wong Boon Seng and Lim Mei Li for their kind understanding and support in the final year of my iii studies. I also wish to extend my warmest thanks to all colleagues in the Department Of Physiology for their understanding and assistance rendered to me throughout my studies. Last but not least, I would like to thank my parents, my siblings, my husband and my daughter for the love, patience and support. I could not have done this without them. This thesis is dedicated to my parents and my husband for their endless love and care. iv TABLE OF CONTENTS PAGE TITLE PAGE i DECLARATION ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii LIST OF ABBREVIATIONS xiii PRESENTATION xviii CHAPTER 1 INTRODUCTION 1 1.1 CD137 And CD137 Ligand Biology 1.1.1 CD137 Biology 1.1.2 CD137 Ligand Biology 1.1.3 Soluble CD137 And CD137 Ligand 1.1.4 CD137 Ligand Reverse Signalling 1.1.4.1 CD137 Ligand In Human Monocytes 1.1.4.2 CD137 Ligand In Human Monocyte-derived Dendritic Cell 1.1.4.3 CD137 Ligand In Murine Myeloid Macrophages 1.1.4.4 CD137 Ligand In Osteoclastogenesis v Cells And 1.1.5 1.2 CD137 And Its Ligand In Inflammation Sphingolipids 1.2.1 Sphingosine Metabolism Pathway 1.2.2 Sphingosine Kinase 1.2.3 Sphingosine Kinase Inhibitors 1.2.4 Natural Products As Sphingosine Kinase Inhibitors 1.2.5 Sphingosine Kinase In Inflammation 1.3 Phospholipase D, Protein Kinase C And Sphingosine Kinase 1.4 Monocytes/Macrophages And Chemokines 1.4.1 1.5 Chemokines In Diseases Rationale And Aims Of The Project CHAPTER 2 MATERIALS AND METHODS 2.1 Reagents And Chemicals 2.2 Solutions And Buffers 2.3 Cell Line 2.4 Stimulation Of Cells 2.5 2.4.1 Protein Immobilization 2.4.2 CD137 Ligand Stimulation Preparation And Treatment Of Inhibitors 2.5.1 Preparation Of DMS And Compound 5c 2.5.2 Preparation Of Bisindolylmaleimide I 2.5.3 Sphingosine Kinase Inhibition 2.5.4 Phospholipase D Inhibition 2.5.5 Protein Kinase C Inhibition vi 48 2.6 Morphological Changes 2.7 Preparation Of Cell Extracts 2.8 Western Blot 2.9 Measurement Of Sphingosine Kinase Activity Using Fluorometric Assay 2.10 Preparation Of Supernatants 2.11 Measurement Of Chemokines Using Enzyme-Linked Immunosorbent Assay (ELISA) 2.12 Statistics CHAPTER 3 RESULTS 3.1 57 CD137L Induced Adherence And Morphological Changes In THP-1 Cells 3.2 Involvement Of Sphingosine Kinases, Not Phospholipase D, In CD137L-Activated Cells 3.3 Expression Of Sphingosine Kinase 1 In Monocytic Cell Lines 3.4 CD137L Signalling Activates Sphingosine Kinase 1 3.5 CD137L Signalling Does Not Induce Sphingosine Kinase 1 Protein 3.6 Involvement Of Sphingosine Kinase In CD137L-Induced Inflammatory Chemokines Production 3.7 Involvement Of Protein Kinase C In CD137 Ligand Signalling CHAPTER 4 DISCUSSION 4.1 77 CD137 Ligand Induced Adherence And Morphological Changes In THP-1 Cells vii 4.2 Involvement Of Sphingosine Kinases, Not Phospholipase D, In CD137 Ligand-Activated Cells 4.3 CD137L Does Not Induce Expression Of Sphingosine Kinase 1 4.4 CD137 Ligand Signalling Activates Sphingosine Kinase 1 4.5 Involvement Of Sphingosine Kinases In CD137 Ligand-Induced Inflammatory Chemokines Production 4.6 Involvement Of Protein Kinase C In CD137 Ligand Signalling 4.7 Cross-linking Is Needed To Activate The CD137L Signalling Pathway 4.8 Controls In The Experiment CHAPTER 5 CONCLUSION 89 CHAPTER 6 FUTURE DIRECTION 92 REFERENCE 94 ABSTRACT FOR CONFERENCE 106 viii SUMMARY CD137 and its ligand (CD137L) are members of tumour necrosis factor (TNF) receptor and TNF superfamily, respectively, in which bidirectional signalling has been shown. CD137L is a transmembrane protein that is capable of transducing a downstream signalling pathway in the cells expressing it. CD137L is constitutively expressed by monocytes which are very pivotal in the host defence against infections. Monocytes circulating in the bloodstream migrate to inflammation sites under the guidance of chemokine gradients to support immune responses. A chemokine gradient is formed by the local release of chemokine at the inflamed tissues and its diffusion away from the site. Chemokine receptors on immune cells and chemokine gradients originating at the inflammation site are equally important in leukocyte homing. Sphingolipid metabolites have emerged as important second messengers, mediating signalling pathways triggered in immune cells. Sphingosine kinase (SphK) is implicated in inflammation, autoimmune diseases, allergies and cancer development. SphK phosphorylates sphingosine to yield sphingosine-1-phoshate (S1P) which acts as an intracellular second messenger in the cells, or as a ligand to S1P receptor (S1PR) after being transported out of the cells. In this study, we found that CD137L stimulation in the monocytic cell line THP-1 induced activation of SphK, which reached its peak activity at 10 minutes and remained active up to 30 minutes after stimulation when compared to the basal level of kinase activity. SphK is found to mediate the production of inflammatory chemokines, e.g. interleukin 8 (IL-8) by the CD137L activated cells. The most commonly used pharmacological SphK ix inhibitor, N, N-Dimethylsphingosine (DMS), and the SphK1 specific inhibitor, compound 5c, are found to inhibit the secretion of the inflammatory chemokine IL-8. CD137L activated THP-1 cells also produced other inflammatory chemokines such as monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein (MIP)-1α and MIP-1β, which are important in leukocyte homing. MCP-1 and its receptor on monocytes, CCR2, are proven to play an important role in recruiting monocytes to the inflamed tissues during bacterial infection. The secretions of MCP-1, MIP-1α and MIP1β were also inhibited by DMS and 5c. This study shows that SphK plays a role in the signal transduction induced by CD137L, and is pivotal in the production of inflammatory chemokines that mediate leukocyte homing. x LIST OF TABLES TABLE PAGE CHAPTER 1: INTRODUCTION 1 Inflammatory Chemokines 41 TABLE PAGE CHAPTER 2: MATERIALS AND METHODS 2 Volume of Protein added into tissue culture plates 50 3 Amount of THP-1 cells added into tissue culture plates 51 4 List of primary antibodies and corresponding secondary 54 antibodies 5 List of lower detection limit of all ELISAs xi 56 LIST OF FIGURES FIGURE PAGE CHAPTER 1: INTRODUCTION A Bidirectional signal transduction. 5 B Sphingosine metabolism pathway. 20 C Proposed CD137L signalling pathway. 91 FIGURE PAGE CHAPTER 3: RESULTS 1 CD137 ligand induced THP-1 cells activation. 59 2 Sphingosine kinase inhibitors decrease CD137L-induced 62 IL-8 secretion. 3 Phospholipase D inhibitor, Butan-1-ol, did not show any 63 inhibition in CD137L-induced THP-1 cell activation as shown by the release of IL-8. 4 Sphingosine kinase 1 was expressed in THP-1 cells and 65 U-937 cells. 5 Cross-linking of CD137 ligand induced sphingosine 67 kinase activity in THP-1 cells. 6 Sphingosine kinase 1 protein level remained unchanged 69 after 24 hours of cross-linking of CD137 ligand. 7 Cross-linking of CD137 ligand induced the production of 72 MCP-1. Sphingosine kinase inhibitors decrease the CD137L-induced MCP-1 secretion. 8 Cross-linking of CD137 ligand-induced the production of 73 MIP-1α. Sphingosine kinase inhibitors decrease the CD137L-induced MIP-1α secretion. 9 Cross-linking of CD137 ligand induced the production of 74 MIP-1β. Sphingosine kinase inhibitors decrease the CD137L-induced MIP-1β secretion. 10 Bisindolylmaleimide I decreases CD137-L induced IL-8 secretion. xii 76 LIST OF ABBREVIATIONS 5c Compound 5c 5-FU 5-fluorouracil A1AR A1 Adenosine Receptor ABC ATP Binding Cassette AIA Adjuvant-Induced Arthritis AICD Activation Induced Cell Death Akt Protein Kinase B (PKB) AML Acute Myeloid Leukaemia AP-1 Activator Protein-1 APC Antigen Presenting Cells APS Ammonium Persulfate ATCC American Type Culture Collection ATP Adenosine Triphosphate BAL Bronchoalveolar Lavage BD Behcet’s Disease Bis I Bisindolylmaleimide I BMM Bone Marrow-Derived Macrophages BSA Bovine Serum Albumin C/EBP CCAAT-Enhancer-Binding Proteins C5a Complement 5a Ca2+ Calcium Ions CCR CC-Chemokine Receptor CD137L CD137 Ligand CD137-Fc Recombinant CD137 Protein Fused To Fc Portion Of The Immunoglobulin CD95L CD95 Ligand CDK2 Cyclin-Dependent Kinase 2 CHF Chronic Heart Failure CIA Collagen-Induced Arthritis CK1 Casein Kinase 1 CNS Central Nervous System COX-2 Cyclooxygenase 2 xiii CREB Camp Response Element Binding CVB3 Coxsackievirus B3 CX3CR CX3C-Chemokine Receptor DC Dendritic Cells DC-SIGN DC-Specific Intercellular Adhesion Molecule-3-Grabbing Non-Integrin DHS Dihydrosphingosine DMF Dimethylformamide DMS N, N-Dimethylsphingosine DMSO Dimethyl Sulfoxide DSS Dextran Sulphate Sodium EAE Experimental Autoimmune Encephalomyelitis ECL Enhanced Chemiluminescence EDTA Ethylenediaminetetraacetic Acid EGF Epidermal Growth Factor ELISA Enzyme-Linked Immunosorbent Assay ER Endoplasmatic Reticulum ERK Extracellular Signal-Regulated Protein Kinases ESR Erythrosedimentation Rate FBS Fetal Bovine Serum Fc Fragment Crystallizable FcR Fc Receptor FCS Fetal Calf Serum FGF-2 Fibroblast Growth Factor-2 GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase GM-CSF Granulocyte Macrophage-Colony Stimulating Factor GVHD Graft-Versus-Host Disease HRP Horseradish Peroxidase HUVEC Human Umbilical Vein Endothelial Cells ICAM Intracellular Adhesion Molecule IFN Interferon IGF Insulin-Like Growth Factor IGFBP-3 IGF Binding Protein-3 xiv IL Interleukin IL-1RA IL-1 Receptor Antagonist ILA Induced By Lymphocyte Activation IP-10 IFN-γ-Inducible 10 kDa Protein IP3 Inositol 1, 4, 5-Triphosphate IRI Ischemia-Reperfusion Injury ITAC IFN-γ-Inducible T-Cell Chemoattractant I-κB NF-κB inhibitor JNK C-Jun N-Terminal Kinases KC Keratinocyte Chemoattractant KCl Potassium Chloride LPA Lysophosphatidic Acid LPS Lipopolysaccharide MAP Mitogen-Activated Protein Kinase MCP Monocyte Chemotactic Protein M-CSF Macrophage-Colony Stimulating Factor MEK MAPK/ERK Kinase MgCl2 Magnesium Chloride Mig Monokine Induced By Interferon-γ MIP Macrophage Inflammatory Protein MKP-1 Map Kinase Phosphatase-1 MMP Matrix Metalloproteinase MoDC Monocyte-Derived Dendritic Cell mRNA Messenger Ribonucleic Acid MS Multiple Sclerosis mTOR Mammalian Target Of Rapamycin MyD Myeloid Differentiation Primary Response Gene NADPH Nicotimamide Adenine Dinucleotide Phosphate (reduced Form) NFAT-2 Nuclear Factor Of Activated T Cells-2 NFAT-c1 Nuclear factor of activated T-cells, cytoplasmic 1 NF-κB Nuclear Factor Kappa-Light-Chain-Enhancer Of Activated B Cells xv NGF Nerve Growth Factor NHL Non-Hodgkin Lymphoma NK Natural Killer NPY Neuropeptide Y NSAIDs Non-Steroidal Anti-Inflammatory Drugs PA Phosphatidic Acid PAGE Polyacrylamide Gel Electrophoresis PB Peripheral Blood PBMC Peripheral Blood Mononuclear Cell PBS Phosphate-Buffered Saline PDGF Platelet-Derived Growth Factor PGE2 Prostaglandin E2 PI3-K Phosphoinositide 3-Kinase PKA Protein Kinase A PKC Protein Kinase C PLC Phospholipase C PLD Phospholipase D PMA Phorbol 12-Myristate 13-Acetate PVDF Polyvinylidene Fluoride RA Rheumatoid Arthritis RANKL Receptor Activator Of Nuclear Factor-Κb Ligand RANTES Regulated On Activation, Normal T Cell Expressed And Secreted RF Rheumatoid Factor ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute S1P Sphingosine-1-Phosphate S1PRs S1P Receptors sCD137 Soluble CD137 sCD137L Soluble CD137 Ligand SDS Sodium Dodecyl Sulphate siRNAs Small Interfering Ribonucleic Acid SLE Systemic Lupus Erythematosus xvi SMase Sphingomyelinases SphK Sphingosine Kinase spns2 Spinster 2 SPP S1P Phosphatase TBS Tris-Buffered Saline t-butanol Tertiary Butanol TEC Tubular Epithelial Cell TEMED Tetramethylethylenediamine TGF-β Transforming Growth Factor/Tumour Growth Factor TLR4 Toll-Like Receptor-4 TMS N, N, N-Trimethylsphingosine TN-C Tenascin C TNF Tumour Necrosis Factor TNFRSF TNF Receptor Superfamily TNFSF TNF Superfamily TRAF2 TNF Receptor-Associated Factor-2 TRIF TIR-Domain-Containing Adapter-Inducing Interferon-β VCAM Vascular Cell Adhesion Molecule VEGF Vascular Endothelial Growth Factor WT Wild Type xvii PRESENTATION 1. S.C. H’ng, H. Schwarz, A.J. Melendez. Role of Sphingosine Kinase in CD137L Signalling Pathway. Scandanavian Society of Immunology 2008, Sweden, 12th – 15th August 2008 xviii CHAPTER 1: INTRODUCTION CHAPTER 1 INTRODUCTION 1.1 CD137 And CD137 Ligand Biology 1.1.1 CD137 Biology CD137 (also known as “induced by lymphocyte activation” (ILA), 4- 1BB, TNFRSF9) is a member of tumour necrosis factor receptor superfamily (TNFRSF), and has been identified as T cell costimulatory molecule (Croft, 2003a, b). In 1996, at the 6th International Workshop on Human Leukocyte Differentiation Antigens held in Kobe, Japan, ILA or 4-1BB has received its nomenclature as CD137 (Kishimoto et al., 1997). CD137 was first identified as ILA in 1995 as a homologue to murine 4-1BB, found primarily in activated T and B lymphocytes, monocytes and non-lymphoid cell types such as epithelial and hepatoma cells (Schwarz et al., 1995). It was found to be the human counterpart of murine 4-1BB with 73.6% similarity and 59.6% identity to murine 4-1BB (Schwarz et al., 1995). CD137 is a type 1 transmembrane protein with three cysteine-rich repeats at the extracellular part, similar to other nerve growth factor/tumour necrosis factor (NGF/TNF) superfamily members (Schwarz et al., 1995). Human CD137 is found to be on chromosome 1p36, which also harbours genes of other TNFSF such as CD30 and OX40 and also a cluster of genes related to malignant diseases such as hematopoietic malignancies (Schwarz et al., 1997). It has been shown that this protein expression is inducible upon activation in T lymphocytes, B lymphocytes, monocytes (Schwarz et al., 1995), natural killer (NK) cells, NKT cells, mast cells, neutrophils, dendritic cells (DC), endothelial cells, eosinophils and osteoclast precursors (Croft, 2009; 1 CHAPTER 1: INTRODUCTION Croft et al., 2012). CD137 messenger ribonucleic acid (mRNA) and protein are also found present in astrocytes, neurons and microglia; CD137 expression is upregulated by fibroblast growth factor-2 (FGF-2) in neuron and astrocytes (Reali et al., 2003). CD137 is not expressed in the resting lymphocytes (Kienzle and von Kempis, 2000). In T lymphocytes, CD137 is said to be upregulated within 24 hours by stimulated T cells and to play a major role in costimulating T cells and sustaining the T cell response from a few hours to several days (Croft, 2003a, 2009). CD137 is constitutively expressed on monocytes and upon activation, it can induce monocyte activation. CD137 stimulated monocytes induce production of TNF-α, interleukin-8 (IL-8) but decrease production of IL-10 (Kienzle and von Kempis, 2000). 2 CHAPTER 1: INTRODUCTION 1.1.2 CD137 Ligand Biology CD137 ligand (CD137L) (also known as 4-1BBL, TNFSF9) is identified as a type II transmembrane protein since its carboxy-terminal domain is on the extracellular side (Alderson et al., 1994). Human CD137L is located on chromosome 19 in the region 19p13.3, and only shares about 36% amino acid sequence homology with murine CD137L, and even the cysteine residues are not conserved between these two species (Alderson et al., 1994). It has been speculated that there may be alternative ligands for CD137 (Alderson et al., 1994). CD137L is expressed by antigen presenting cells (APC) such as DCs, B cells, macrophages, and by T cells, NK cells, mast cells (Nishimoto et al., 2005), hematopoietic progenitors and osteoclast precursors (Croft, 2009; Croft et al., 2012) and during inflammation in non-immune cells such as endothelial cells and smooth muscle cells (Croft, 2009). CD137L is constitutively expressed by monocytes/macrophages but inducible in T lymphocytes (Alderson et al., 1994; Ju et al., 2003; Schwarz, 2005). Ectopically expressed CD137L induces the proliferation of T lymphocytes (Alderson et al., 1994). CD137L is also found in cells of the central nervous system (CNS) such as astrocytes and neurons, but not detected in microglia, and its expressions is not affected by FGF-2 treatment (Reali et al., 2003). However, recently, a report showed that CD137L is expressed in microglia cell lines and primary microglia (Yeo et al., 2012). As a transmembrane protein, CD137L can act as a co-receptor, and upon binding to CD137, CD137L too can trigger signalling cascade downstream in the cells expressing it (Figure A) (Shao and Schwarz, 2011). 3 CHAPTER 1: INTRODUCTION This is a unique feature of many receptor-ligand pairs in TNF superfamily that exhibits bidirectional signalling such as OX40L and CD40L (Lotz et al., 1996; Schwarz, 2005; Thum et al., 2009). CD137L is also found to be highly expressed in cancer cell lines and stimulation of CD137L on the tumour cell lines induces release of IL-8 but not IL-6, IL-10, IL-12, TNF-α or transforming growth factor β (TGF-β) (Salih et al., 2000). Activation of CD137 on T lymphocytes induces activation and proliferation of the lymphocytes; however, activation of its ligand by CD137 protein induces expression of CD95, inhibits proliferation and enhances apoptosis via activation induced cell death (AICD) in the lymphocytes (Lotz et al., 1996; Michel et al., 1999; Schwarz et al., 1996). CD137 stimulation on B lymphocytes induces apoptosis in the cells too (Kienzle and von Kempis, 2000). This is thought to be the regulation role displayed by CD137 and its ligand in the immune system. 4 CHAPTER 1: INTRODUCTION CD137 Ligand CD137 Figure A. Bidirectional signal transduction. CD137 and its ligand are capable of inducing bidirectional signalling into the cells expressing them. 5 CHAPTER 1: INTRODUCTION 1.1.3 Soluble CD137 And CD137 Ligand Soluble CD137 (sCD137) and its ligand (sCD137L) have been reported in inflammatory diseases as well as cancers. sCD137 is released by activated lymphocytes through alternative splicing (Michel et al., 1998). The level of the protein has been reported to be low in the sera of healthy control donors as compared to rheumatoid arthritis (RA) patients (Jung et al., 2004; Michel et al., 1998), systemic lupus erythematosus (SLE) and Behcet’s disease (BD) (Jung et al., 2004), leukaemia and lymphoma patients (Furtner et al., 2005). The level of sCD137 is reported to correlate to the severity of the disease, and also to the degree of AICD induced in lymphocytes (Furtner et al., 2005; Jung et al., 2004; Michel and Schwarz, 2000). The soluble form of this molecule provides a negative feedback loop to the immune system to regulate the immune response as sCD137 is found to be expressed at later stage of the stimulation as compared to membrane bound CD137 (Michel and Schwarz, 2000). sCD137L too is elevated in SLE, BD (Jung et al., 2004), multiple sclerosis (MS) (Liu et al., 2006), Non-Hodgkin lymphoma (NHL), myelodysplastic syndrome and acute myeloid leukaemia (AML) (Salih et al., 2001). sCD137L is found to be cleaved from the membrane bound ligand, and remains functionally active in the system, as it would stimulate T lymphocytes to release IL-2 and interferon-γ (IFN-γ) (Salih et al., 2000). There is no inhibition of the T cells proliferation by the anti-CD137L antibody indicating that this may be just a regulatory role played by CD137L (Liu et al., 2006). 6 CHAPTER 1: INTRODUCTION 1.1.4 CD137 Ligand Reverse Signalling 1.1.4.1 CD137 Ligand In Human Monocytes CD137L is constitutively expressed on human monocytes/ macrophages (Alderson et al., 1994; Ju et al., 2003; Schwarz, 2005). Crosslinking of CD137L is required to trigger the signalling downstream in the cells because soluble antibody or recombinant CD137 protein do not activate the cells (Kang et al., 2007; Langstein et al., 1998; Langstein et al., 1999; Langstein and Schwarz, 1999; Yeo et al., 2012). CD137L has been identified as the novel stimulator that induces proliferation of monocytes (Ju et al., 2003; Langstein et al., 2000; Langstein et al., 1999). CD137L stimulation can also drive the cells into apoptosis (Langstein et al., 1999). However, a higher rate of proliferation that is simultaneously induced compensates the rate of apoptosis (Langstein et al., 1999). The lifespan of monocytes can be prolonged by the activation of CD137L via release of macrophage-colony stimulating factor (M-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-3 (Langstein et al., 2000; Langstein et al., 1999; Langstein and Schwarz, 1999). M-CSF also acts as the essential factor to support monocyte proliferation because neutralizing anti-M-CSF antibodies greatly reduced the viability of the cell (Langstein et al., 1999; Langstein and Schwarz, 1999). IL8 release, morphological changes and adhesiveness of the cells are indicators for the activation by CD137L (Langstein et al., 2000; Langstein et al., 1998; Langstein and Schwarz, 1999). The effects induced by CD137L in monocytes are comparable to that lipopolysaccharide (LPS), a potent monocyte activator, and CD137L can further augment the effects of LPS by inducing higher IL-8, M-CSF and myc expression (Langstein et al., 2000). CD137L stimulation has 7 CHAPTER 1: INTRODUCTION been found to increase the release of IL-6, IL-8, TNF-α and to inhibit IL-10 (Langstein et al., 1998). The expression of intracellular adhesion molecule (ICAM) is also induced, indicating the differentiation of monocyte to macrophages (Langstein et al., 1998). The CD137L activated signalling pathway induces the activation of tyrosine kinase, mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in monocytes (Söllner et al., 2007). The CD137L-activated IL-8 production in monocytes is mediated by Src kinase, MAPK p38, ERK1/2, phosphoinositide 3-kinase (PI3K), protein kinase A (PKA) but not protein kinase C (PKC) (Söllner et al., 2007). PI3-K is shown to be upstream of ERK1/2 as Wortmannin effectively inhibited the phosphorylation of ERK1/2 (Söllner et al., 2007). In the downstream signalling cascade, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is shown to be activated by CD137L in monocytes (Ju et al., 2009). CD137L engagement on monocytes induces monocytes to migrate to the site where CD137 is strongly expressed such as to the inflammatory tissues (Drenkard et al., 2007). CD137L is also shown to increase the number of monocytes rolling and adhering to ICAM-1 and E-selectin in a flow chamber simulating in vivo flow of blood in blood vessels (Quek et al., 2010). 8 CHAPTER 1: INTRODUCTION 1.1.4.2 CD137 Ligand In Human Monocyte-derived Dendritic Cell CD137L signalling also induces maturation of DC upon activation by the receptor (Ju et al., 2009; Lippert et al., 2008). CD137L activation upregulates the DC maturation marker CD83, the costimulatory molecules CD86 and HLA-DR on monocyte-derived dendritic cells (MoDC) which are also termed as classical DC (Ju et al., 2009; Lippert et al., 2008). The chemokine receptors, CXC-chemokine receptor 4 (CXCR4) and CCchemokine receptor 7 (CCR7), are also upregulated to mediate migration of DC (Lippert et al., 2008). TNF-α is shown to be important to drive the DC maturation as its neutralization suppressed the expression of CD83 (Lippert et al., 2008). Secretion of IL-12p70 and IFN-γ but not IL-10 upon tetanus toxoid and CD137L activation, suggests that the effects of CD137L signalling are driving Th cell differentiation to Th1 cells (Ju et al., 2009; Lippert et al., 2008). CD137L does not only drive the maturation of classical DC, it has also been shown to induce differentiation of monocytes into a different subset of DC (Kwajah M M and Schwarz, 2010). The CD137L-induced DCs are shown to be different from classical DC. CD137L-induced DCs upregulate CCR7 expression; however, the expression of CXCR4 is downregulated (Kwajah M M and Schwarz, 2010). The cells produce low level of IL-10 and high levels of IL-23 as compared to classic DC. CD137L-induced DCs do not secrete IL12, and do not express DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or CD1a (Kwajah M M and Schwarz, 2010). Differentiation of monocytes to DC by CD137L signalling inhibits NF-κB (Kwajah M M and Schwarz, 2010). However, another study demonstrated that 9 CHAPTER 1: INTRODUCTION CD137L-matured DCs induce the translocation of the NF-κB from cytosol to nucleus (Ju et al., 2009). However, there is a species difference reported recently according to which activation of CD137L signalling in murine immature classical DC did not induce maturation as compared to human immature classical DC (Tang et al., 2011). CD137L stimulated murine monocytes were shown to induce activation of the cells through the morphological changes and increased adherence of the cells onto the plate, but it did not induce expression of CD80 and CD86 (Tang et al., 2011) as reported by others for human immature DC (Ju et al., 2009; Lippert et al., 2008). The activation of CD137L in murine classical DC has the opposite effect compared to human classical DC, because IL-10 is produced instead of IL-12, and because addition of LPS further increases the release of IL-10 (Tang et al., 2011). 10 CHAPTER 1: INTRODUCTION 1.1.4.3 CD137 Ligand In Murine Myeloid Cells And Macrophages Another interesting finding is that CD137L is required to sustain the release of TNF-α, and that this activity of CD137L is independent of its receptor, CD137, but dependent on Toll-like receptor-4 (TLR4) (Kang et al., 2007). The cytoplasmic domain of CD137L and the TLR4 intracellular domain are found to associate. Surprisingly, myeloid differentiation primary response gene (MyD) 88 and TIR-domain-containing adapter-inducing interferon-β (TRIF) are not needed in the CD137L-TLR4 mediated TNF-α induction (Kang et al., 2007). LPS stimulates the expression of CD137L in macrophages, and this induction is dependent on TLR4, MyD88 and TRIF and is mediated by MAPK p38, c-Jun N-terminal kinases (JNK), MEK (Kang et al., 2007). CD137L activates the cAMP response element binding (CREB) and CCAAT-enhancer-binding proteins (C/EBP), which are also mediating the release of TNF-α upon LPS stimulation (Kang et al., 2007). MAPK p38, ERK1/2 and JNK are phosphorylated but not NF-κB upon CD137L stimulation, which then induce the production of TNFα in macrophages (Kang et al., 2007). Deletion of CD137L does not affect the activation of NF-κB, activator protein-1 (AP-1), JNK, ERK, and p38 in LPS stimulation (Kang et al., 2007). Another group has shown that CD137L stimulation in RAW264.7 cells is reported to be independent of MAPK p38, ERK1/2, JNK, NF-κB and CREB (Kim et al., 2009) which contradicts work done by Söllner et al. (2008) and Kang et al. (2007) (Kang et al., 2007; Söllner et al., 2007). Kim et al. (2009) showed that CD137L signalling induces the phosphorylation of tyrosine kinase, Akt (also known as protein kinase B) and p70s6K, indicating the involvement of PI3-K and mammalian target of rapamycin (mTOR) in the 11 CHAPTER 1: INTRODUCTION signalling cascade (Kim et al., 2009). However, bone marrow-derived macrophages (BMM) show responses similar to those observed in monocytes i.e. enhanced adherence (Kim et al., 2009; Langstein et al., 2000; Söllner et al., 2007) and increased levels of ICAM-1 (Kim et al., 2009; Langstein et al., 1998). Activation of CD137L in BMM increases the levels of IL-1β, IL-6 and M-CSF production (Kim et al., 2009). RAW264.7 cells also has increased adherence, proliferation, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-6, cyclooxygenase 2 (COX-2), tenascin C (TN-C), neuropeptide Y (NPY) and M-CSF mRNA in response to CD137L signalling (Kim et al., 2009). CD137L enhances the effect of LPS as reported in monocytes (Langstein et al., 2000) and of M-CSF in macrophages (Kim et al., 2009). Two independent signalling pathways were proposed based on the different effects implicated by Wortmannin and LY294002 (Kim et al., 2009). Akt is proposed to be the mediator in the release of IL-1β, which may involve a tyrosine kinase while the mTOR pathway is suggested to play a role in the survival and proliferation of macrophages through the release of M-CSF (Kim et al., 2009). 12 CHAPTER 1: INTRODUCTION 1.1.4.4 CD137 Ligand In Osteoclastogenesis In studies on bone formation, CD137L reverse signalling is shown to play a role in osteoclastogenesis in vitro and in vivo, through the use of CD137-/- mice, and cell lines (Saito et al., 2004; Shin et al., 2006a; Shin et al., 2006b; Yang et al., 2008). CD137L stimulation inhibits osteoclastogenesis induced by MCSF/receptor activator of nuclear factor-κB ligand (RANKL) (Saito et al., 2004; Shin et al., 2006a; Shin et al., 2006b). Wild type (WT) mice have high release of IFN-β, which enhances the release of IL-10 that leads to slower osteoclast formation as compared to CD137-/- mice (Shin et al., 2006a; Shin et al., 2006b). CD137, but not CD137L, protein expression was shown to increase in osteoblasts that had been infected by bacteria (Saito et al., 2004). CD137L was detected on the macrophage like cell line (RAW264.7) and on bone marrow cells (Saito et al., 2004). CD137L too induced proliferation and the release of M-CSF in primary bone marrow cells and BMM (Saito et al., 2004). CD137L activation inhibited the formation of osteoclasts in bone marrow (Saito et al., 2004). Casein kinase 1 (CK1) may be involved in this inhibition because the casein kinase 1 inhibitor (CKI7) suppressed the osteoclastogenesis inhibition by CD137L, and also the proliferation of the cells (Saito et al., 2004). The RANKL signalling pathway induces the phosphorylation of MAPK p44/42, p38, JNK, NF-κB inhibitor (I-κB) but CD137L activation did not affect the phosphorylation of these molecules (Saito et al., 2004). CD137L only suppresses the activation of Akt and nuclear factor of activated T cells-2 (NFAT-2) by RANKL (Saito et al., 2004). 13 CHAPTER 1: INTRODUCTION Despite all three reports describing an inhibitory role of CD137L in osteoclastogenesis, recently another group showed that CD137-/- mice have increased bone mass and a decreased formation of osteoclasts from BMM (Yang et al., 2008). Phosphorylation of JNK and p38 was lower and induction of c-Fos and NFAT-cytoplasmic 1 (NFAT-c1) was reduced in CD137-/- as compared to WT BMM. Again, in CD137-/- mice the phosphorylation state of MAPK p44/42 and I-κB were not affected (Yang et al., 2008). The difference in these reports could be due to the differences in the stimulation of the primary cells in the culture. 14 CHAPTER 1: INTRODUCTION 1.1.5 CD137 And Its Ligand In Inflammation More and more evidences have emerged showing that TNF and TNFR superfamily members are involved in inflammatory and autoimmune diseases such as sepsis, RA, atherosclerosis, myocarditis, graft-versus-host disease (GVHD), colitis (Croft et al., 2012) and MS (Liu et al., 2006). CD137 is strongly expressed on the wall of blood vessels in inflamed tissues such as the skin in vasculitis, the nasal septum in rhinitis, the colon of Crohn’s disease patients and the thyroid gland of Grave’s disease patients (Broll et al., 2001; Drenkard et al., 2007). CD137 expression on the endothelial cells is induced by TNF and IL-1 (Drenkard et al., 2007; Quek et al., 2010). Monocytes migrate to sites where CD137 is expressed as is shown by the infiltration of monocytes into the matrigel containing CD137-Fc protein in mice (Drenkard et al., 2007). Monocytes could be activated by the CD137 on the endothelial cells, causing extravasation of the monocytes into tissues (Broll et al., 2001). Monocytes roll and adhere to a flow chamber coated with CD137-Fc protein, a process which is mediated by ICAM-1 and E-selectin (Quek et al., 2010). CD137 protein expression increases during experimental autoimmune encephalomyelitis (EAE) in the CNS (Yeo et al., 2012). EAE, a murine model of human MS, is an autoimmune disease that is characterized by the destruction of the myelin sheats caused by inflammation, and subsequently the death of neurons. Activation of microglia through CD137L leads to the production of reactive oxygen species (ROS) which in turn induces apoptosis of the oligodendrocytes that contributes to the disease (Yeo et al., 2012). CD137L activation induces pro-inflammatory cytokines such as TNF, IL-1, 15 CHAPTER 1: INTRODUCTION IL-6, IL-12, and monocyte chemotactic protein-1 (MCP-1), and it also induces matrix metalloproteinase (MMP)-9 and soluble ICAM release from microglia (Yeo et al., 2012). In MS patients, membrane bound CD137L on CD14+ monocytes and sCD137L were found higher in the plasma (Liu et al., 2006). CD137L did not inhibit the proliferation of myelin basic protein-reactive T cells. The release of soluble forms of CD137L could be a way of regulation of the immune response in the patients (Liu et al., 2006). In Crohn’s disease, CD137 is expressed on the lamina propria in the inflamed tissue, and high levels of CDL137L are present in the mesenterial lymph nodes. Agonistic antibody against CD137 causes an elevated level of IFN-γ production. This study is suggesting that interaction of CD137 with its ligand may contribute to the chronic inflammation in patients (Maerten et al., 2004). In human atherosclerosis, CD137 is also highly expressed on T cells, endothelial cells, and smooth muscle cells, while CD137L is found on CD68+ macrophages found in the atherosclerotic lesions. Expression of CD137 on endothelial cells is enhanced by inflammatory cytokines (TNF-α, IL-1β and IFN-γ) (Olofsson et al., 2008). CD137 activation also induces vascular cell adhesion molecule (VCAM)-1 and ICAM-1 expression on the endothelial cells and decreases the proliferation of smooth muscle cells (Olofsson et al., 2008). In RA patients, sCD137 and sCD137L are released into the sera of the patients, and the levels in the sera correlate closely with the rheumatoid factor (RF) and Erythro Sedimentation Rate (ESR), a non-specific biomarker of inflammation, values (Jung et al., 2004; Michel et al., 1998). RA is a type of 16 CHAPTER 1: INTRODUCTION autoimmune disease that is characterized by a chronic inflammation at the synovial tissues of the joint that eventually leads to the destruction of the joint, including cartilages and bones (Lai et al., 2012). CD137L activation inhibits osteoclastogenesis and it was speculated that CD137L may play a role in the bone and cartilages destruction in final stage of RA (Saito et al., 2004; Shin et al., 2006a; Shin et al., 2006b; Yang et al., 2008). In a murine model of acute kidney ischemia-reperfusion injury (IRI), CD137 is expressed on NK cells and CD137L on the tubular epithelial cells (TECs) (Kim et al., 2012). NK cells activated CD137L reverse signalling in TECs, leading to the production of chemokine ligands, CXCL-1 and CXCL-2, in WT mice. Both the chemokine ligands are responsible for the recruitment of neutrophils into the inflamed kidney tissues during IRI (Kim et al., 2012). NFκB is activated by CD137 ligand activation in the TEC. Upregulation of CXCL-1 and CXCL-2 is mediated by p38 and JNK. Hence, CD137 and its ligand are involved in mediating the infiltration of the neutrophils and contribute to the inflammation of IRI model (Kim et al., 2012). CD137L, alongside with secretion of macrophage inflammatory protein (MIP)-1α and MIP-1β, is enhanced in peripheral blood mononuclear cell (PBMC) during chronic heart failure (CHF) (Yndestad et al., 2002). 17 CHAPTER 1: INTRODUCTION 1.2 Sphingolipids Sphingolipid metabolites have emerged as important second messengers, mediating signalling pathways triggered in immune cells including monocytes, macrophages, neutrophils and mast cells (Olivera and Spiegel, 2001b). Sphingosine kinase (SphK) is implicated in inflammation, autoimmune diseases, allergies and cancer development (Kee et al., 2005). SphK, in different types of cells, by different kinds of agonists, can initiate different signalling pathways through activation of several important second messengers including sphingosine-1-phosphate (S1P), PKC, ERK1/2, calcium ions (Ca2+), and phospholipase D (PLD) (Snider et al., 2010). SphK is activated by a number of stimuli including platelet-derived growth factor (PDGF), NGF, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), IGF binding protein-3 (IGFBP-3), lysophosphatidic acid (LPA), LPS, complement 5a (C5a), TNF-α, epidermal growth factor (EGF), activated immunoglobulin receptors (Fragment crystallizable (Fc)-γ receptor 1 (FcγR1) and FcεR1), acethylcholine (muscarinic agonists) and many more (Olivera and Spiegel, 2001b; Snider et al., 2010) 18 CHAPTER 1: INTRODUCTION 1.2.1 Sphingosine Metabolism Pathway Sphingomyelin, other than being a major sphingolipid in the plasma membrane, is also the main precursor of lipid signalling. In the metabolism pathway, sphingomyelin is hydrolysed by sphingomyelinases (SMase) to yield ceramide. Ceramide is then degraded by ceramidase to produce sphingosine, which is phosphorylated by SphK to yield S1P. In the salvage pathway, S1P phosphatase (SPP) can dephosphorylate S1P into sphingosine, and sphingosine is converted back to ceramide by ceramide synthase. S1P may also be converted into ethanolamine phosphate and hexadecenal irreversibly by S1P lyase (Figure B) (Olivera and Spiegel, 2001a, b; Snider et al., 2010; Spiegel, 1999). Ceramide and sphingosine have been linked to cell death, while their downstream molecule, S1P, is important for cell survival. Ceramide and sphingosine are generally produced under stress conditions and during apoptosis; S1P production is driven by growth factors, leading to proliferation and survival. The dynamic balance of ceramide - S1P determines the fate of the cell, and is often dubbed as the “sphingolipid rheostat” (Olivera and Spiegel, 2001b; Spiegel, 1999). The sphingolipid rheostat has been implicated in activating signalling cascades such as activation of calcium release from the internal store, activation of MAPK ERK pathway, and activation of AP-1mediated transcription factor pathway (Olivera and Spiegel, 2001b). 19 CHAPTER 1: INTRODUCTION Figure B. Sphingosine metabolism pathway. Sphingomyelin is hydrolysed by SMase to yield ceramide. Ceramide is then degraded to sphingosine by ceramidase. Sphingosine is phosphorylated by SphK to generate S1P. S1P can be either degraded into hexadecanal and phosphoethanolamine irreversibly by S1P lyase or dephosphorylated back to sphingosine by S1P phosphatase. Sphingosine then is converted back to ceramide by ceramide synthase. 20 CHAPTER 1: INTRODUCTION 1.2.2 Sphingosine Kinase The first mammalian SphK, murine SphK1a and SphK1b, was cloned and characterized in 1998 (Kohama et al., 1998). Subsequently, in 2000, the human SphK1 was cloned and characterized (Melendez et al., 2000; Nava et al., 2000). In the same year, 2000, the second isoform of SphK, SphK2, was cloned, both in man and mouse (Liu et al., 2000). SphK1 and SphK2 share 80% of similarity and 45% identity which includes the putative catalytic domain and residues for adenosine triphosphate (ATP) and sphingosine binding (Pitman and Pitson, 2010). It is now being accepted that the two isoforms of SphK respond differently; almost opposite of each other, that activation of SphK1 leads to the synthesis of S1P, promoting survival while SphK2 promotes apoptosis through production of ceramide (Gangoiti et al., 2010). The opposite actions of the two isoforms are speculated to be caused by the different pathways that they may use: SphK1 is speculated to phosphorylate sphingosine produced by de novo synthesis, and SphK2 phosphorylates the salvaged sphingosine (Maceyka et al., 2005). SphK1 reduces ceramide level through phosphorylation of sphingosine into S1P, and S1P is degraded irreversibly by S1P lyase (Maceyka et al., 2005). Generated S1P may also negatively regulate ceramide synthesis (Maceyka et al., 2005). SphK2, on the other hand, may utilize the salvaged sphingosine as part of the effort of the cells to conserve resources, thus, promoting dephosphorylation of S1P by SPP, yielding sphingosine and ceramide (Maceyka et al., 2005). SphK1/SphK2 double knockout mice are embryonically lethal from severe defects in angiogenesis and neurogenesis but single knockout mice can 21 CHAPTER 1: INTRODUCTION develop normally, indicating that these two isoforms may play a compensatory role in the mice when the other isoform is absent (Pitman and Pitson, 2010). SphK1 is ubiquitously expressed in adult tissues mainly in the liver, lung, kidney, spleen, skeletal muscles, peripheral blood leukocytes and thymus (Melendez et al., 2000; Nava et al., 2000). Through alternative splicing, there are three forms of SphK1 namely SphK1a, SphK1b and SphK1c (Orr Gandy and Obeid, 2013). SphK1 specifically phosphorylates D-erythro-sphingosine and D-erythro-dihydrosphingosine in mammals (Kohama et al., 1998; Olivera and Spiegel, 2001b; Pitman and Pitson, 2010). Unlike SphK2, SphK1 does not phosphorylate phytosphingosine (Olivera and Spiegel, 2001b). SphK1 is generally located in the cytosol of the cells and translocate to the plasma membrane upon activation; translocation would bring the enzyme closer to its substrate, producing S1P (Ibrahim et al., 2004; Orr Gandy and Obeid, 2013; Pitson et al., 2003). Commercial inhibitors of ERK1/2 block the translocation and phosphorylation of SphK1 implying the upstream role of ERK1/2 in activating of SphK1 (Pitson et al., 2003). ERK1/2 and cyclin-dependent kinase 2 (CDK2) are shown to phosphorylate SphK1 specifically at Ser225 and induce the SphK activity in the cells (Pitson et al., 2003). However, ERK1/2 can be an upstream or downstream molecule of SphK in the TNF-α signalling cascade because phosphorylation of ERK1/2 is also inhibited upon activation of TNF-α in dominant-negative SphK cells (Pitson et al., 2003; Pitson et al., 2000). A site-directed mutagenesis of Gly82 to Asp was introduced in the catalytic domain of the human SphK to generate the dominant-negative SphK mutant, which blocks SphK activation in the cells (Pitson et al., 2000). 22 CHAPTER 1: INTRODUCTION The second isoform of SphK, SphK2, is expressed mainly in kidney, liver and brain (Liu et al., 2000). SphK2 localizes in endoplasmic reticulum (ER), nucleus and mitochondria, which is different from the SphK1 localization; and it is phosphorylated by ERK2 (Orr Gandy and Obeid, 2013). SphK2 has a broader range of substrate because it does not only phosphorylate D-erythro-sphingosine and D-erythro-dihydrosphingosine, but it also catalyses phosphorylation of phytosphingosine, ω-biotinyl the SphK inhibitors, FTY720 and D-erythro-sphingosine D, L-threo-dihydrosphingosine and (DHS) (Pitman and Pitson, 2010). SphK2 induces apoptosis through its catalytic domain, and this is shown to be a calcium dependent process (Maceyka et al., 2005). The sub-cellular localization and partly the generation of S1P are speculated to play a part in the apoptosis induction and the increase of the ceramide concentration in the cell (Maceyka et al., 2005). Serum starvation affects SphK2 localization in the cells so that it is reduced in the cytosol and plasma membrane but relatively increased in the internal membrane (Maceyka et al., 2005). SphK induces expression of the adhesion molecules VCAM-1 and Eselectin through activation of NF-κB upon TNF-α activation in human umbilical vein endothelial cells (HUVEC) (Xia et al., 1998). Activation of SphK increases the level of S1P in the cells, which in turn activates ERK1/2 phosphorylation but not JNK, and prevents apoptosis (Xia et al., 1998; Xia et al., 1999). Activation of SphK and production of S1P are shown to activate NF-κB and stimulate the expression of adhesion molecules through S1P (Xia et al., 1998). N, N-dimethylsphingosine (DMS) sensitizes HUVEC to apoptosis but this is rescued by exogenous S1P (Xia et al., 1999). Physical 23 CHAPTER 1: INTRODUCTION interaction of TNF receptor-associated factor-2 (TRAF2) with SphK is shown to be critical for SphK activation, the anti-apoptotic properties of TRAF-2, and also for the activation of the transcription factor NF-κB, but not JNK, in TNFα activated HEK293T cells though JNK is often activated in TRAF2-mediated signalling pathway (Xia et al., 2002). SphK is also shown to be activated in neutrophils and macrophages, stimulated by C5a (Ibrahim et al., 2004; Melendez and Ibrahim, 2004). An antisense oligonucleotide against SphK1 abolished SphK activity and the generation of S1P in human monocyte-derived macrophages (Melendez and Ibrahim, 2004). DMS and oligonucleotide against SphK1 also show inhibition of the release of Ca2+ from internal stores, degranulation, activation of nicotimamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase, chemotaxis and cytokines production (TNF-α, IL-6 and IL-8) induced by C5a. Increased production of inositol 1, 4, 5-triphosphate (IP3), a sign of activation of phospholipase C (PLC), and activation of PKC were observed in C5a-stimulated neutrophils but their activities are not regulated by SphK (Ibrahim et al., 2004). S1P is generated through phosphorylation of sphingosine by SphK in the cells. S1P is readily available in platelets at high concentration (Gangoiti et al., 2010). S1P, acting as a second messenger, induces cell proliferation and promotes cell survival, at the same time it inhibits apoptosis in the cells (Spiegel, 1999). S1P production is induced by many stimuli (PDGF, NGF or fetal calf serum (FCS) or cross-linking of Fc receptors) (Spiegel, 1999). It can also be exported out from the cells through the ATP Binding Cassette (ABC) family transporters and spinster 2 (spns2) transporters whereby it will bind to 24 CHAPTER 1: INTRODUCTION S1P receptors (S1PRs), S1P1R – S1P5R (Orr Gandy and Obeid, 2013). S1P is known to act in a paracrine or autocrine manner (Orr Gandy and Obeid, 2013). Microinjection of S1P into cells induces a rapid increase of Ca2+ which is independent of Ca2+ influx (Olivera and Spiegel, 2001b). S1P can be dephosphorylated to yield sphingosine or degraded by sphingosine phosphate lyase (Olivera and Spiegel, 2001b). 25 CHAPTER 1: INTRODUCTION 1.2.3 Sphingosine Kinase Inhibitors N, N-dimethylsphingosine (DMS), a competitive inhibitor to SphK, is demonstrated to be a more effective SphK inhibitor as compared to dihydrosphingosine (DHS) (Edsall et al., 1998) and N, DL-threo- N, N- trimethylsphingosine (TMS) (Igarashi, 1997). SphK activity is halved by low concentration of DMS (5µM) and completely abolished at 20µM to 25µM of DMS (Edsall et al., 1998). DMS decreases the formation of S1P, and induces accumulation of ceramide. In summary, DMS drives the cells to apoptosis (Edsall et al., 1998). DMS does not inhibit PKC activity at low concentration (10µM) nor does it inhibit the translocation of the PKCα and PKCδ at high concentration (50µM) (Edsall et al., 1998). D, L-threo-dihydrosphingosine (D, L-threo-DHS or safingol) is capable of inhibiting the kinase activity as effectively as the DMS at 5µM. However, at higher concentrations, L-threo-DHS may be phosphorylated by SphK and generate dihydrosphingosine 1-phosphate because it is a substrate for SphK2 (Edsall et al., 1998; Pitman and Pitson, 2010). Some effects of the L-threoDHS drug enantiomer is reported as an effect from PKC inhibition and in a prostate adenocarcinoma study, mice showed symptoms of hepatic toxicity after administration of the drug but which was not observed in human patients (Pitman and Pitson, 2010). N, N, N-trimethylsphingosine (TMS) is generated to replace DMS with enhanced water solubility and reduced cytotoxicity. However, this replacement is shown to be a less potent SphK inhibitor and its role as antitumour drug was shown through the inhibited PKC activity and not SphK (Pitman and Pitson, 2010). 26 CHAPTER 1: INTRODUCTION Compound 5c (5c) is synthesized as a sphingosine analogue, competing for SphK1 with a lower IC50 (3.3µM) relative to DMS (IC50=5.7µM) (Wong et al., 2009). 5c is less toxic to U-937 and HL-60 as compared to DMS. 5c only starts to show PKC activity inhibition at 100µM (Wong et al., 2009). In cancer research, 10µM 5C inhibits SphK activity in colorectal cell lines (HCT116, RKO, SW480, SW620) upon cell activation by 10% fetal bovine serum (FBS) (Tan S, 2011). In HCT-116 cells, 5c inhibits translocation of SphK1 from cytosol to plasma membrane and induces apoptosis in HCT-116 cells. The combination of 5c and 5-Fluorouracil (5-FU) increases the percentage of colorectal cancer cell death, thus, suggesting that 5c increases the sensitivity of cancer cell to 5-FU treatment (Tan S, 2011). 5FU is a chemotherapy agent that has been used to treat colorectal cancer. SK1-I (BML-258) is a sphingosine analogue generated by Spiegel and co-worker (2008), targeting specifically SphK1. SK1-I is a water soluble compound that does not inhibit other kinases such as PKC, SphK2, PKA, ERK2 or ceramide kinase (Paugh et al., 2008). It potently inhibits SphK1, reduces S1P production, and increases ceramide levels in the cells, hence directing the cells towards apoptosis (Paugh et al., 2008). This compound inhibits cell proliferation in leukaemia cell lines and is capable of reducing the AML xenograft tumour growth (Paugh et al., 2008). SKIs (I-IV) are non-lipid SphK inhibitors, identified through screening of synthetic compounds using purified human SphK (French et al., 2003). SKIs are shown to induce apoptosis in the tumour cells overexpressing the drug transport proteins P-glycoprotein or MRP1 (French et al., 2003) . SKI-II turns out to be the most selective SphK inhibitor and is an attractive 27 CHAPTER 1: INTRODUCTION compound because SKI-II is also orally bioavailable (French et al., 2006). SKI-I, -II and -V showed inhibition of tumour growth higher than 50% in the syngeneic BALB/C mouse solid tumour model that uses JC mammary adenocarcinoma (French et al., 2006). SKI shows potential in reducing airway inflammation in a murine asthmatic model and acute lung injury after trauma and haemorrhagic shock (Gangoiti et al., 2010). FTY720 is currently in a clinical trial under its trade name GilenyaTM to treat MS (Orr Gandy and Obeid, 2013). Gilenya (also known as Fingolimod) is now approved for the treatment of MS as it reduces relapse rates in the patients more efficiently than another drug, interferon (Loma and Heyman, 2011). FTY720 is a substrate of SphK2 that upon phosphorylation is exported out of the cells and binds to the S1PR. FTY720 is also a competitive inhibitor of SphK1 that leads to the proteosomal degradation of SphK1a (Orr Gandy and Obeid, 2013). ABC294640 is a water soluble SphK2 specific enzyme inhibitor, possesses a good oral bioavailability with moderate toxicity (Gangoiti et al., 2010). Its half-time plasma clearance is about 4.5 hours in mice (French et al., 2010). ABC294640 is a competitive inhibitor that acts as an antitumor agent since it induces cell death through autophagy in cancer cells, and inhibits metastasis of tumour cells (French et al., 2010; Orr Gandy and Obeid, 2013). It also reduces the severity of inflammation in colitis-driven colon cancer and in arthritis (Fitzpatrick et al., 2011; Maines et al., 2008). ABC294640 is also shown to be gentle to the stomach of the rat model of arthritis as compared to non-selective non-steroidal anti-inflammatory drugs (NSAIDs) (Fitzpatrick et al., 2011). 28 CHAPTER 1: INTRODUCTION (S)-FTY720-vinylphosphonate ((S)-vinyl-Pn) inhibits SphK1 in a noncompetitive manner. It leads to the degradation of SphK1, and also acts as antagonists to all the S1PRs (Orr Gandy and Obeid, 2013). SG14 or [N-((2S, 3R)-3-hydroxy-4-phenyl-1-(pyrrolidin-1-yl) butan-2-yl)stearamide)] inhibits SphK2 without affecting SphK1 or PKC at a high dose (50µM) while (R)FTY720 methyl ether ((R)-FTY720-Ome) induces degradation of SphK2 through ubiquitin-proteosomal degradation (Orr Gandy and Obeid, 2013; Pitman and Pitson, 2010). 29 CHAPTER 1: INTRODUCTION 1.2.4 Natural Products As Sphingosine Kinase Inhibitors F-12509a is a fungal metabolite that competes for SphK without discriminating any isoforms. However, methylation of this hydrophobic compound takes away its capability as SphK inhibitor (Pitman and Pitson, 2010). It blocks production of S1P and causes accumulation of ceramide that leads to apoptosis in HL-60 cells (Gangoiti et al., 2010). B-5354a, b, c, is isolated from novel marine bacterium SANK71896. B-5354c is a non-specific non-competitive inhibitor of SphK in human platelets; it inhibits SphK1 activity in a prostate cancer cell line when used singly or combined with camptothecin, a potent antitumor agent that targets topoisomerase I (Gangoiti et al., 2010). B-5354c does not inhibit other kinases such as PKC, PI3-K and ceramide kinase (Pitman and Pitson, 2010). It also sensitizes prostate cancer cells to be more responsive to chemotherapy drugs such as doxetaxel and camptothecin (Pitman and Pitson, 2010). 30 CHAPTER 1: INTRODUCTION 1.2.5 Sphingosine Kinase In Inflammation Sphingosine kinase has been implicated in numerous inflammatory diseases such as RA, kidney IRI, airway inflammation, and sepsis (Pitman and Pitson, 2010). COX-2 is an inflammation mediator that is only expressed upon stimulation by inflammatory stimuli such as TNF-α (Pettus et al., 2003). Sphingolipid metabolites such as ceramide, sphingosine and S1P increased protein levels of COX-2 and production of PGE2 when these metabolites were added separately into the fibroblast cells (L929 cell line) (Pettus et al., 2003). Inhibition of SphK1, not SphK2, by small interfering ribonucleic acids (siRNAs) abolished S1P production, COX-2 activation and prostaglandin E2 (PGE2) generation in the fibroblast cells (Pettus et al., 2003). In the lung injury inflammation model induced by LPS injection, SphK1 is shown to play an anti-inflammatory role to dampen the inflammation caused by the injury by inhibiting the activation of JNK and NADPH oxidase activation (Di et al., 2010). In SphK-/- mice, it was shown that there were high levels of cytokines and chemokines in the lungs, namely IL-6, MIP-1α, MIP-2 and TNF-α. Neutrophils isolated from SphK-/- mice had increased activation of NF-κB by TNF-α stimulation. Increased levels of ICAM-1 in endothelial cells induced higher neutrophil infiltration into the lungs and this induced higher levels of ROS in the SphK-/- mice as compared to the WT mice. Mice with SphK1 knocked out have a lower survival rate and a high lung vascular permeability (Di et al., 2010). In a murine model of allergic asthma, SphK1 is involved in the development of the disease, and inhibiting SphK signalling suppresses the 31 CHAPTER 1: INTRODUCTION development and severity of the disease (Lai et al., 2008a). DMS and siRNA against SphK1 greatly reduce infiltration of inflammatory cells into the lung and production of Th2 cytokines (IL-4, IL-5, eotaxin) in the bronchoalveolar lavage (BAL) (Lai et al., 2008a). Mucus production and goblet hyperplasia were also reduced in the mice treated with higher concentration of DMS in the study (Lai et al., 2008a). In RA patients, S1P levels are also shown to be higher in the synovial fluid as compared to synovial fluid of osteoarthritis patients (Lai et al., 2008b). In the murine collagen-induced arthritis (CIA) model, inhibiting SphK1 by DMS or siRNA decreases the severity of the disease (Lai et al., 2008b; Lai et al., 2009). However, inhibiting the Sphk2 isoform showed a totally opposite trend since the inhibition induced a more severe disease development (Lai et al., 2009). Treatment with SphK1 siRNA has markedly reduced the level of S1P released, decreased the amount of inflammatory cytokines such as TNF-α, IL-6 and IFN-γ, which in turn reduced the disease development and severity (Lai et al., 2008b; Lai et al., 2009). In the human TNF-α-induced chronic inflammatory arthritis mice model, the SphK1 isoform is shown to play a role in the inflammatory arthritis too (Baker et al., 2010). SphK-/- mice showed significantly lower synovial and periarticular inflammation, less paw swelling and deformity and reduced bone erosions; these mice had less articular COX-2 protein and synovial Th17 cells as compared to SphK+/+ mice (Baker et al., 2010). However, in SphK1-/- mice, CIA and peritonitis are not impaired, and no compensatory role is reported for SphK2. This leads to the conclusion that the role of SphK1 in inflammation is dispensable (Michaud et al., 2006). Another group reported that the SphK2 specific inhibitor, ABC294640, also 32 CHAPTER 1: INTRODUCTION slowed down the disease progression in the same murine CIA model (Fitzpatrick et al., 2011). In the rat adjuvant-induced arthritis (AIA) model, ABC294640 significantly reduced the severity of the disease as seen in the reduction of joint inflammation, synovial hyperplasia, pannus formation, cartilage destruction and bone deformation in the tibiotarsal joints of the rats (Fitzpatrick et al., 2011). Baker et al. (2012) reported otherwise in the TNF-αinduced arthritis using the same SphK2 inhibitor, ABC294640 (Baker et al., 2012). Treating the arthritic mice with ABC294640 had worsen the arthritis condition in the mice while the SphK2-/- mice did not show any difference in the disease progression (Baker et al., 2012). The group suggested that the difference between acute inhibition by ABC294640 and lifelong SphK2 deficiency may contribute to the different outcome of the disease progression in arthritis, apart from the drug dosage used in the experiment (Baker et al., 2012). In the murine renal IRI model, A1 adenosine receptor (A1AR) activation induces SphK activity in the kidney which leads to the production of S1P (Park et al., 2012). The activation of A1AR upregulates SphK1 protein, but not SphK2 protein. SphK1-/- mice have increased renal injury through necrosis and inflammation. Plasma creatinine levels are also higher in SphK1-/mice (Park et al., 2012). SphK1 seems to play a role in protecting the mice from severe renal injury as neutrophil infiltration in the kidneys, ICAM-1 and TNF-α mRNA expression are lower in WT mice as compared to SphK1-/- mice (Park et al., 2012). The high production of S1P and the exporting of S1P from the cells, leading to the binding to the S1P1R protect the cells from inflammation and necrosis (Park et al., 2012). 33 CHAPTER 1: INTRODUCTION In the murine chronic and acute dextran sulphate sodium (DSS)induced ulcerative colitis model, SphK inhibition by ABC294640 and ABC747080 reduce the infiltration of neutrophils to the site of inflammation, and the production of inflammatory cytokines such as TNFα, IL-1β, IL-6, IFN-γ and S1P (Maines et al., 2008). PGE2 production and also the expression of adhesion molecules, ICAM-1 and VCAM-1, are markedly decreased (Maines et al., 2008). The effectiveness of ABC294640 and ABC747080 is comparable to that of Dipentum (olsolazine), a FDA-approved anti-colitis drug (Maines et al., 2008). ABC294640 and ABC747080 reduce disease progression and colon shortening (Maines et al., 2008). 34 CHAPTER 1: INTRODUCTION 1.3 Phospholipase D, Protein Kinase C And Sphingosine Kinase Phospholipase D (PLD) is an important phospholipid enzyme that catalyses phosphotidylcholine to produce phosphatidic acid (PA) and choline. TNF-α activates PLD as early as 2 minutes in monocytes and mobilizes PLD1 from the cytosol to the membrane (Sethu et al., 2008). PLD1 is also shown to induce activation of SphK and the release of cytosolic Ca2+ upon stimulation of monocytes with TNF-α. An antisense oligonucleotide against PLD1 inhibits activation of ERK1/2 and NF-κB and the secretion of cytokines such as IL-1β, IL-5, IL-6 and IL-13 (Sethu et al., 2008). In FcγR1-stimulated monocytes, PLD is activated and induces activation of SphK and calcium transients (Melendez et al., 2001). The signalling pathway downstream of FcγR1 in monocytes, through PLD leads to the activation of NADPH oxidase burst (Melendez et al., 2001). PKC is shown to mediate the translocation of SphK1 to the plasma membrane because treatment with the PKC inhibitors, Bisindolylmaleimide and Calphostin-c, abolishes the translocation of the enzyme after stimulation by phorbol 12-myristate 13-acetate (PMA) (Johnson et al., 2002). PMA, a PKC activator, also induces the phosphorylation and activity of SphK1, followed by S1P production (Johnson et al., 2002). A dominant negative G82D mutation also translocates to the membrane upon stimulation with PMA, indicating that activity of the kinase is not required for the translocation in HEK293 cells (Johnson et al., 2002). 35 CHAPTER 1: INTRODUCTION 1.4 Monocytes/Macrophages And Chemokines There are about 5-10% of mature monocytes among the circulating peripheral blood leukocytes. Monocytes, originating from the bone marrow, give rise to specialized cells such as DC or macrophages or osteoclasts under the different microenvironments they migrated to (Gordon and Taylor, 2005). Heterogeneity is common in the monocyte pool due to the different expression of surface markers on the monocytes such as CD14, CD16, CD32, CD64 and many more (Gordon and Taylor, 2005). Monocytes, may differentiate into macrophages or DC, are part of the first line defence of the immune system, and they are critical in eradicating pathogens from the body (Gordon and Taylor, 2005). Human monocytes differentiate into DC in the presence of GM-CSF and IL-4, and DC differentiation is said to favour monocytes with high expression of CD14 and CD16 (Gordon and Taylor, 2005). Differential expression of antigenic markers generates a heterogeneous pool of monocytes (Gordon and Taylor, 2005). There are two main subsets of monocytes mainly through their CD14 and CD16 expression, i.e. CD14HICD16- or CD14+CD16+. CD14HICD16- cells are generally called the classical monocytes, mainly expressing CCR2 (Gordon and Taylor, 2005). CD14+CD16+ monocytes are non-classical monocytes, expressing CCR5 (Gordon and Taylor, 2005). Classical monocytes are said to be the first to be recruited to the inflammation sites after neutrophils, and this extravasation is highly dependent on chemokine receptors, e.g. CCR2 (Ingersoll et al., 2011). Monocyte mobilization from bone marrow to the inflamed tissues during bacterial infection is shown to rely on the expression of the CCR2 on monocytes. Lacking of CCR2 expression on monocytes traps the cells in the bone marrow, 36 CHAPTER 1: INTRODUCTION unable to leave for the infection sites (Serbina and Pamer, 2006). Nonclassical monocytes are recruited to the inflammation site for tissue healing, and the CX3CR1 receptor is found to be the one responsible for such migration (Ingersoll et al., 2011). Macrophages are important for maintaining homeostasis, clearance of dead cells, remodelling and repair of tissues (Gordon and Taylor, 2005). They specialize in tissues they are residing in, such as the osteoclasts for bone remodelling, alveolar macrophages in lungs to clear the microorganisms and contaminants from environment, microglia in the central nervous system or Kupffer cells in the liver (Gordon and Taylor, 2005). During inflammation or infection, leukocytes are recruited to the inflamed tissues, guided by the chemokine gradients. Leukocytes move towards high chemokine concentrations (Deshmane et al., 2009). Certain chemokines are normally elevated at the site of inflammation to selectively attract the appropriate types of cells which express the matching chemokine receptors. This is viewed as a way to recruit the immune cells to the inflammation site as a host defence mechanism against infection (Deshmane et al., 2009). Initial migration of monocytes to the inflamed peritoneum is found to be highly dependent on the expression of CCR2 on the monocytes (Tsou et al., 2007). Progression of an inflammation or infection depends on the extent of the recruitment of immune cells to the infected sites, and how the inflammation is regulated (Power and Proudfoot, 2001). Leukocyte homing relies on the signals generated by the inflamed cells, through generation of chemokines and adhesion molecules (Power and Proudfoot, 2001). Lowering 37 CHAPTER 1: INTRODUCTION or inhibiting the level of chemokines is proven to decrease the level of inflammation because this prevents excessive numbers of leukocyte infiltrating into the inflamed tissues (Power and Proudfoot, 2001). Chemokines are divided into four subfamilies: (X) C, CC, CXC and CX3C and are subdivided into two main functions i.e. inflammatory and homeostatic chemokines (Deshmane et al., 2009; Zlotnik and Yoshie, 2012). In many cases, multiple chemokines are secreted within the inflammation site for recruitment of leukocytes, making the process of leukocyte mobilization seem complicated and complex (Table 1) (Rottman, 1999; Zlotnik and Yoshie, 2012). Another possible hypothesis is that only one particular chemokine is responsible for leukocyte homing through its chemokine gradient, after which leukocytes become desensitized and move on to another chemokine gradient until they reached the destination (Rottman, 1999). Chemokines are effective in leukocyte homing, and it has been established that lymphocyte maturation too may involve chemokine expression at different lymphoid tissues (Rottman, 1999). Th1 and Th2 are two different lymphocyte subsets which are characterised by expressing different sets of chemokine receptors which are important for their homing to different inflamed tissues (Rottman, 1999). Th1 cells mainly express CCR5 and CXCR3, Th2 cells express CCR3 while both subsets express CCR2 and CXCR4 (Rottman, 1999). CC chemokines such as MCP-1, regulated on activation, normal T cell expressed and secreted (RANTES), and MIP-1β are chemotactic for cells of the monocyte lineage and lymphocytes, with the majority encoded on chromosome 17 in humans (Cook, 1996; Deshmane et al., 2009). CXC 38 CHAPTER 1: INTRODUCTION chemokines such as IL-8 are chemo-attractants for neutrophils, and are located on chromosome 4 in man (Cook, 1996; Deshmane et al., 2009). MCP-1 (also known as CCL2), which binds to CCR2, was first identified as a chemotactic factor of monocytes. It is also found to mobilize memory T lymphocytes and NK cells. MCP-1 is found in MS, RA, psoriasis, atherosclerosis and diabetes (Deshmane et al., 2009). CCR2 is shared by a few ligands (e.g. CCL2, CCL7, CCL8, CCL16) and its expression is restricted to specific types of cells such as mononuclear cells, vascular smooth muscle cells, monocytes and NK cells (Deshmane et al., 2009; Zlotnik and Yoshie, 2012). CCR2 is postulated to be inflammatory or anti-inflammatory in its effects, depending on the type of cells it is expressed on. For instance, CCR2 on APCs and T cells acts inflammatory, and CCR2 on regulatory T cells acts antiinflammatory (Deshmane et al., 2009). MIP-1α and MIP-1β induce mobilization of monocytes and T cells with different resulting activities (Cook, 1996). MIP-1α mainly induces the chemotaxis of B lymphocytes, activated CD8+ T lymphocytes, NK cells and eosinophils. Further, MIP-1α stimulates ICAM-1 expression, mast cell degranulation, histamine release by basophils and the production of other inflammatory cytokines (Cook, 1996). Coxsackievirus B3 (CVB3)-induced myocarditis does not developed in MIP-1α-/- mice, proving that MIP-1α is an essential chemokine in developing myocarditis in mice (Cook, 1996). MIP-1α is also shown to play a role in the influenza virus-induced pneumonitis (Cook, 1996). In CCR2-/- mice, monocytes are retained in the bone marrow despite monocytosis which is induced by hypercholesterolemia. The population of 39 CHAPTER 1: INTRODUCTION monocytes residing in the bone marrow increases with the decrease in number of monocytes in the circulation (Tsou et al., 2007). MCP-1, MCP-3 and their receptor, CCR2 seem to be crucial in the monocyte exit from the bone marrow. MCP-1-/- and MCP-3-/- mice show fewer monocytes circulating in the peripheral blood whereas MCP-2-/- and MCP-2-/- MCP-5-/- mice have normal monocyte counts in the blood (Tsou et al., 2007). However, CCR2 expression on monocytes is found to be the most critical factor in the migration of monocytes out of the bone marrow (Tsou et al., 2007). 40 CHAPTER 1: INTRODUCTION Chemokine XC Subfamily XCL1 XCL2 CC Subfamily CCL1 CCL2 CCL3 CCL3L1 Other names Receptor Agonistic Lymphotactin, ATAC, SCM-1α SCM-1β XCR1 I-309 MCP-1 MIP-1α, LD78α LD78β CCR8 CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCL3L3 CCL4 CCL4L1 CCL4L2 CCL5 LD78β MIP-1β AT744.2 RANTES CCL7 MCP-3 CCL8 MCP-2 CCL11 CCL13 CCL17 CCL20 CCL22 CCL26 Eotaxin MCP-4 TARC MIP-3α, LARC MDC Eotaxin-3 Antagonistic XCR1 CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3, CCR5 CCR1, CCR2, CCR5 CCR3, CCR5 CCR2, CCR3 CCR4 CCR6 CCR4 CCR3, CXCR1 CCR5 CXCR3, CCR2 CCR1, CCR2, CCR5 CXC Chemokine CXCL1 GROα, MGSA CXCR2 CXCL2 GROβ CXCR2 CXCL3 GROγ CXCR2 CXCL5 ENA78 CXCR2 CXCL6 GCP2 CXCR1, CXCR2 CXCL7 NAP-2 CXCR1, CXCR2 CXCL8 IL-8 CXCR1, CXCR2 CXCL9 MIG CXCR3 CCR3 CXCL10 IP-10 CXCR3 CCR3 CXCL11 I-TAC CXCR3, CXCR7 CCR3, CCR5 CXCL16 SR-PSOX CXCR6 CX3C chemokine CX3CL1 Fractalkine CX3CR1 Table 1 Inflammatory Chemokines. Modified from (Zlotnik and Yoshie, 2012). 41 CHAPTER 1: INTRODUCTION 1.4.1 Chemokines In Diseases In many inflammatory diseases, such as MS, asthma or psoriasis, chemokines and/or their receptors are present at the inflammatory sites (Rottman, 1999). In RA patients, synovial tissues form the patients contain chemokines MCP-1, MIP-1α, IL-8 and RANTES, and the chemokine receptors CXCR3 and CCR5 are expressed on the lymphocytes that migrated to these tissues (Rottman, 1999). Chemokines binding to CCR1 may be the first of few that are secreted to initiate inflammatory responses because CCR1/- mice were spared from pulmonary granuloma formation and were protected from pulmonary inflammation when Schistosoma mansoni eggs were injected intravenously (Rottman, 1999). In kidney IRI model, monocytes infiltrate into the kidney through the binding of chemokine receptors on the monocytes to the chemokines generated at the injured kidney (Li et al., 2008). CCR2 and CXCR1 are responsible for the monocyte recruitment into the IRI kidney as CCR2-/- or CX3CR1-/- mice have lower numbers of macrophages compared to control mice (Li et al., 2008). Tubule necrosis is also reduced in CCR2-/- and CX3CR1 deficient mice (Li et al., 2008). In a murine model of endotoxemia or in acute lung injury induced by LPS, 20 inflammatory mediators were analysed closely using multiplex beadbased assay including the chemokines (Bosmann et al., 2012). In endotoxemia, endotoxic shock, which is done by administrating LPS into the peritoneum, most of the mediators were upregulated substantially by the third hour after administration of LPS including chemokines such as eotaxin, keratinocyte chemoattractant (KC), MCP-1, MIP-1α, MIP-1β and RANTES (Bosmann et 42 CHAPTER 1: INTRODUCTION al., 2012). Some of the mediators were sustained at high levels for up to 12 hours after induction (Bosmann et al., 2012). These six chemokines (eotaxin, KC, MCP-1, MIP-1α, MIP-1β and RANTES) were also found to be upregulated in LPS-induced lung injury from 4th hour to 18th hour after induction (Bosmann et al., 2012). MCP-1, but not MCP-2, MCP-3 or MCP-5, is proven to play a crucial role in the development of EAE. MCP-1-/- mice are resistant to the disease, and have substantially reduced numbers of macrophages infiltrating into the CNS (Huang et al., 2001). Other inflammatory cytokines such as MIP-1α, IFN-γ-inducible 10 kDa Protein (IP-10), RANTES and IFN-γ are significantly reduced in the MCP-1-/- mice upon EAE onset in the spinal cord (Huang et al., 2001). There were no compensatory roles played by MCP-2, MCP-3 or MCP5 in the MCP-1-/- mice (Huang et al., 2001). In another report, there were increased chemokine levels such as IP-10, monokine induced by IFN-γ (Mig) and RANTES in the cerebrospinal fluid in MS patients (Sørensen et al., 1999). Not only the chemokine levels were elevated, the corresponding receptors of the chemokine expressions were also enhanced such as CXCR3 (IP-10/Mig Receptor) on the lymphocytic cells, CCR5 (RANTES receptor) on lymphocytic cells, macrophages, and microglia in the actively demyelinating MS brain lesions (Sørensen et al., 1999). In Zymosan-induced inflammation in the peritoneal cavity, a large number of leukocytes and monocytes infiltrate into the cavity as the level of MCP-1, TNF-α, KC and MIP-1α increased (Ajuebor et al., 1998). The number of leukocytes infiltrating the cavity is markedly reduced when anti-mouse MCP-1 antibody is injected after the Zymosan administration (Ajuebor et al., 43 CHAPTER 1: INTRODUCTION 1998). MCP-1, when artificially administrated into the cavity, attracts leukocytes and monocytes as part of the host defence system (Ajuebor et al., 1998). However, MIP-1α secretion during the inflammation is affected by MCP-1 neutralization (Ajuebor et al., 1998). MCP-1 and MCP-3 are crucial in recruiting inflammatory monocytes to the infection sites during Listeria monocytogenes infection in mice (Jia et al., 2008). Manipulating knockout mice of MCP-1 and MCP-3, CCR2mediated recruitment of inflammatory monocytes to the infection site was much reduced. The inflammatory monocytes accumulated in the bone marrow of the mice during infection (Jia et al., 2008). In the absence of MCP-1 or MCP-2, mice are more susceptible to infection and take longer time to clear the bacteria from the system (Jia et al., 2008). Transplant rejection is characterized by production of multiple chemokines, and leukocyte infiltration at different stages of the rejection. MIP-2, KC, and MCP-1 are involved in the early stage followed by IP-10, MIG, IFN-γ-inducible T-cell chemoattractant (ITAC), MIP-1β and RANTES at the later stage. Production of these chemokines is responsible for the potential rejection of a transplant from the body (Power and Proudfoot, 2001). Power and Proudfoot (2001) proposed to treat acute and chronic inflammatory diseases by blocking the interaction of the chemokines with their receptors (Power and Proudfoot, 2001). Met-RANTES, an antagonist of CCR1 and CCR5, and anti-RANTES neutralizing antibodies are shown to be effective in reducing leukocyte infiltration and recruitment in the renal transplantation and lung allograft rejection respectively (Power and Proudfoot, 2001). 44 CHAPTER 1: INTRODUCTION A lot of research involving neutralizing or knocking out chemokine receptors was done to investigate the effectiveness of the approach on the model of cardiac allograft rejection (Power and Proudfoot, 2001). CCR-/- mice showed no signs of rejection of cardiac allografts. The same was achieved with treatment by a monoclonal antibody against the MHC class II co-receptor CD4 or by deletion of the CCR2 and CCR5 genes (Power and Proudfoot, 2001). CXCR3 deletion or anti-CXCR3 monoclonal antibodies postponed or completely abolish the cardiac allograft rejection (Power and Proudfoot, 2001). These studies demonstrate the critical and essential role that chemokines are playing in the inflammatory diseases and transplant rejection. 45 CHAPTER 1: INTRODUCTION 1.5 Rationale And Aims Of The Project CD137 and CD137L are found to play a role in inflammatory diseases such as RA, MS, EAE and kidney IRI (Croft et al., 2012). CD137 expression is elevated at the site of inflammation, and activation of CD137 on endothelial cells increases the expression of adhesion molecules such as ICAM-1, VCAM1 which are important for extravasation of monocytes into the inflamed tissues. Phospholipid modifying enzymes, like SphK and PLD, also regulate immune responses in inflammation, including RA, MS, EAE and kidney IRI. Inhibiting SphK enzymes suppresses the development of the inflammatory disease and dampens the degree of inflammation occurring in the tissues. At present, TNF-α has been shown to activate the PLD and/or SphK enzymes in monocytes and HUVEC (Sethu et al., 2008; Xia et al., 1998; Xia et al., 1999). In TNF-α activated monocytes, PLD1 is shown to induce secretion of inflammatory cytokines, phosphorylation of ERK1/2, and also activation of NF-κB, and these activities are mediated by SphK1 (Sethu et al., 2008). In monocytes, CD137L reverse signalling also activate ERK1/2, and NF-κB (Söllner et al., 2007). Would SphK and PLD then be involved in the CD137L reverse signalling in monocytes given that CD137 and CD137L belong to the TNF receptor and TNF superfamilies, respectively, in the manner as reported by Sethu et al. (2008) (Sethu et al., 2008)? Does CD137L reverse signalling enhance SphK and PLD activities as the downstream signalling cascade steps, or do they work in parallel to elicit robust inflammatory reactions in the tissues? 46 CHAPTER 1: INTRODUCTION In this research project, we are interested to investigate the potential involvement of phospholipid signalling in the CD137L reverse signalling pathway in leukocytes, particularly in monocytes. We would like to identify the phospholipids mediating the signalling pathway activated by CD137L in monocytes. In many signalling pathways, PKC is always being discussed for it is a classic crucial second messenger. Söllner et al. (2007) had reported that PKC is not involved in the CD137L reverse signalling pathway in monocytes as the inhibitor used, Chelerythrine chloride (up to 3µM), did not affect the production of IL-8 (Söllner et al., 2007). We would like to reinvestigate to confirm that this important classical second messenger, PKC, is not involved in the downstream signalling by CD137L as reported by Söllner et al. (2007) (Söllner et al., 2007). The importance of chemokines and their receptors in monocyte migration to the inflammation site is undeniable as demonstrated by chemokine or chemokine receptor knockout mice. Chemokine production at the inflammation site is crucial to generate chemokine gradients for recruitment of immune cells to the inflamed tissues. Different groups of chemokines are responsible for the recruitment of different types of immune cells to the site. Hence, we are also interested to understand the type of chemokines generated by monocytes in response to CD137L reverse signalling. 47 CHAPTER 2: MATERIALS AND METHODS CHAPTER 2 MATERIALS AND METHODS 2.1 Reagents And Chemicals - All reagents and chemicals unless stated otherwise, were purchased from Sigma-Aldrich. - Recombinant human CD137-Fc protein, which is a fusion protein consisting of the extracellular domain of human CD137, fused to the constant domain (Fc) of human IgG1 was purchased from ALEXIS Biochemicals (USA). - Human IgG1-Fc protein was purchased from Chemicon International (USA). - N,N-Dimethylsphingosine (DMS) was purchased from Calbiochem (USA) - Compound 5c (5c) was generated by Wong et al. (2009) (Wong et al., 2009) - Butan-1-ol and tertiary butanol (t-butanol) were purchased from MERCK (USA) - Bisindolylmaleimide I (Bis I) was purchased from Cell Signaling Technology, Inc., USA - 2.2 Dimethylformamide (DMF) was purchased from Merck, USA. Solutions And Buffers - RIPA buffer for total cell lysate preparation: 50mM Tris-HCl (pH 7.4), 1.0% NP-40, 0.25% sodium deoxycholate, 150mM sodium chloride, 1.0mM Ethylenediaminetetraacetic acid (EDTA). 48 CHAPTER 2: MATERIALS AND METHODS - Protease inhibitors cocktail for RIPA buffer: 1X complete, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, USA). - Phosphatase inhibitors cocktail for RIPA buffer: 1X phosSTOP (Roche, USA) - Resolving gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): Distilled water, 30% Bis-acrylamide (Bio-Rad Laboratories, USA), 1.5M Tris (pH 8.8), 10% SDS, 10% ammonium persulfate (APS), Tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, USA). - Stacking gel for SDS-PAGE: Distilled water, 30% Bis-acrylamide (Bio-Rad Laboratories, USA), 1.0M Tris (pH 6.8), 10% SDS, 10% APS, TEMED (Bio-Rad Laboratories, USA) - Running buffer for SDS-PAGE: 25mM Tris base, 250mM glycine, 0.1% SDS, pH 8.3. - Transfer buffer for SDS-PAGE: 25mM Tris base, 250mM glycine, 0.025% SDS, pH 8.3. - 1X SDS gel-loading buffer for SDS-PAGE: 50mM Tris HCl (pH 6.8), 100mM β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol. - Washing buffer for SDS-PAGE: 1X Tris-buffered saline (TBS), 0.1% Tween 20 (Bio-Rad Laboratories, USA). - Diluent buffer for ELISA: 10% bovine serum (Gibco, New Zealand). - Washing buffer for ELISA: 1X Phosphate-buffered saline (PBS), 0.05% Tween 20 (Bio-Rad Laboratories, USA). 49 CHAPTER 2: MATERIALS AND METHODS - SphK buffer for measurement of SphK activity: 50mM 4-(2hydroxyethyl) piperazine-1-ethanesulfonic acid (pH 7.4), 15mM magnesium chloride (MgCl2), 0.005% Triton X-100, 10mM potassium chloride (KCl). 2.3 Cell Line The THP-1 and U-937 cell lines were obtained from American Type Culture Collection (ATCC). Cells were cultured in polystyrene flasks (NUNC, USA) in Roswell Park Memorial Institute (RPMI) 1640 (Sigma Aldrich, USA) supplemented with 10% heat deactivated FBS (Gibco, USA) at 37°C and 5% carbon dioxide in water saturated atmosphere. 2.4 Stimulation Of Cells 2.4.1 Protein Immobilization Plates were coated overnight at 4°C with either 5µg/ml of human CD137-Fc or human IgG1-Fc (negative control) for stimulation of cells. Table 2 shows the volume of CD137-Fc used to coat the different sizes of plates. Plates were washed once with sterile 1X PBS before seeding cells onto the plate for stimulation. Plates Volume used per well (µl) 96-well plate 50 48-well plate 130 24-well plate 250 12-well plate 500 6-well plate 750 Table 2. Volume of protein added into tissue culture plates. 50 CHAPTER 2: MATERIALS AND METHODS 2.4.2 CD137 Ligand Stimulation Prior to experiments, cells (1.0 million/ml) were cultured in RPMI 1640 supplemented with 1% FBS for 18 hours. The cells were incubated with the Fc receptor blocking antibody (Miltenyi, Germany) according to the manufacturer’s recommendations, at 4°C for 45 minutes. Cells were then washed twice with ice cold 1X PBS, and resuspended in RPMI 1640 with 10% FBS at a density of 0.5million/ml for stimulation at 37°C. Table 3 shows the amount of cells used in different size of plates. CD137-Fc coated and human IgG-Fc coated plates were washed once with 1X PBS before cells were added. Upon the addition of cells into respective wells, the plates were kept in the 37°C incubator to start the reaction. Reactions were stopped with 1.0ml of ice cold 1X PBS at various time points and cell pellets were collected for subsequent experiments. Plates Amount of cells used per well 96-well plate 5x104 48-well plate 15x104 24-well plate 30x104 12-well plate 0.5x106 6-well plate 1.5x106 Table 3. Amount of THP-1 cells added into tissue culture plates. 2.5 Preparation And Treatment Of Inhibitors 2.5.1 Preparation Of DMS And Compound 5c DMS was prepared as a 10mM stock in 100% dimethyl sulfoxide (DMSO). A final concentration of 10µM DMS was used in all experimental set-ups. DMSO was kept at 0.1% (v/v) concentration in all experimental set-ups. 51 CHAPTER 2: MATERIALS AND METHODS Compound 5c was prepared as 50mM and 25mM stock respectively in 100% DMSO. A final concentration of 50µM and 25µM 5c were used respectively in all experimental set-ups. DMSO was kept at 0.1% (v/v) concentration in all experimental set-ups. 2.5.2 Preparation Of Bisindolylmaleimide I Bis I was prepared as a 500µM stock in 100% DMSO. A final concentration of 500nM Bis I was used in all experimental set-ups. DMSO was kept at 0.1% (v/v) concentration in all experimental set-ups. 2.5.3 Sphingosine Kinase Inhibition A final concentration of 10µM of DMS, 50µM and 25µM of 5c were used in all experimental set-ups. Inhibitors were added into the cell suspension and incubated on ice for 15 minutes. Cells were then transferred into the coated plates for stimulation; cells without any addition of inhibitors served as controls. 2.5.4 Phospholipase D Inhibition A final concentration of 0.3% (v/v) of primary alcohol, butan-1-ol, was used in all experimental set-ups. 0.3% (v/v) of t-butanol was used as control of the primary alcohol for this inhibition experiment. 0.3% of butan-1-ol or 0.3% of t-butanol were added into the cell suspension and incubated on ice for 15 minutes. Cells were then transferred into the coated plates for stimulation; cells without any addition of alcohols served as controls. 52 CHAPTER 2: MATERIALS AND METHODS 2.5.5 Protein Kinase C Inhibition A final concentration of 500nM of Bis I was used in all experimental set-ups. Inhibitor was added into the cell suspension and incubated on ice for 30 minutes. Cells were then transferred into the coated plates for stimulation; cells without any addition of Bis I served as controls. 2.6 Morphological Changes THP-1 cells were cultured in the CD137-Fc coated or human IgG-Fc coated plates. Uncoated and Fc coated plates served as control in this experimental set-up. Morphological changes were captured at 24 hours and 48 hours using the Olympus IX81 motorized inverted research microscope (Olympus) and DP71 microscope digital camera (Olympus). 2.7 Preparation Of Cell Extracts Cell pellets were lysed with RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors (Roche, USA). 70µL of RIPA buffer was used for every 1x106 THP-1 cells. Cells resuspended in RIPA buffer were subjected to vigorous vortexing followed by incubation at 4°C for 45 minutes. Cell nuclei and debris were removed by centrifugation at 13000 RPM, 4°C for 5 minutes. Protein concentrations were determined by the Bradford assay using Bio-Rad Protein Assay (Biorad, USA). 2.8 Western Blot Equal amount of protein lysates (15µg to 40µg) were prepared in 1X SDS Gel-loading buffer; samples were heated at 95°C for 5 minutes, separated on a 53 CHAPTER 2: MATERIALS AND METHODS 12% SDS PAGE gel under reducing conditions and blotted onto Polyvinylidene fluoride (PVDF) (Millipore, USA). Blots were probed with primary antibodies at 4°C overnight followed by appropriate secondary antibody conjugated to horseradish peroxidase (HRP). Immunocomplexes were visualized by enhanced chemiluminescence (ECL) (Thermo Scientific, USA) on films. Table 4 shows the list of primary antibodies and their corresponding secondary antibodies used. Primary Antibody Secondary antibody Mouse anti-α -tubulin Anti-mouse IgG HRP (Santa Cruz, Biotechnology Inc., USA) (Sigma Aldrich, USA) Mouse anti-GAPDH Anti-mouse IgG HRP (Santa Cruz Biotechnology Inc., USA) (Sigma Aldrich, USA) Rabbit anti-Sphingosine Kinase 1 Anti-rabbit IgG HRP (Cell Signaling Technology Inc., USA) (Thermo Scientific, USA) Table 4. List of primary antibodies and corresponding secondary antibodies. 2.9 Measurement Of Sphingosine Kinase Activity Using Fluorometric Assay This protocol was adapted from Billich and Ettmayer (2004) (Billich and Ettmayer, 2004). A total protein of 40μg from cell lysate was used for the reaction. The lysate was incubated with 20μM of 15-NBD-Sph (prepared as a complex with bovine serum albumin (BSA)) and ATP (1mM) in SphK buffer in the final volume of 100μL. After incubation at 37°C in water bath for 30 minutes, 100μl 1M potassium phosphate buffer, pH 8.5, was added to stop the reaction. 500μl chloroform/methanol (2:1) (Merck, USA) were added. The mixture was vortexed and the phases were separated by centrifugation at 5000 RPM for 5 minutes at room temperature. An aliquot of 75μl was removed 54 CHAPTER 2: MATERIALS AND METHODS from the upper aqueous layer and transferred into the wells of black 96-well polystyrene microplates (Greiner Bio-One, USA), followed by 75μl of dimethylformamide (DMF) (Merck, USA). Fluorescence intensity was measured with excitation wavelength at 485nm and emission wavelength at 535 nm. A reaction mixture containing no protein lysate served as blank. 2.10 Preparation Of Supernatants Cells were cultured in CD137-Fc-coated 48-well plate at 37°C for 24 hours. For sphingosine kinase inhibition, cells were cultured with and without the presence of DMS, compound 5c and DMSO; for PLD inhibition studies, cells were cultured with and without 0.3% of butan-1-ol and 0.3% of t-butanol; and for PKC inhibition studies, cells were cultured with and without Bis I and DMSO. Both uncoated wells and wells coated with human IgG1-Fc served as controls. Cells were spun down at 1000 RPM for 5 minutes and supernatants were collected for cytokine analysis. Each sample supernatant collected was prepared in triplicates within each experiment. 2.11 Measurement Of Chemokines Using Enzyme-Linked Immunosorbent Assay (ELISA) The concentration of IL-8 and MCP-1 in cell supernatants was determined by the BD OptEIATM human IL-8 enzyme-linked immunosorbent assay (ELISA) Set and BD OptEIA TM MCP-1 ELISA Set. The concentrations of MIP-1α and MIP-1β in cell supernatants were determined by the human MIP-1α and MIP-1β DuoSet ELISA kits (R&D Systems), respectively. ELISA 55 CHAPTER 2: MATERIALS AND METHODS was performed on Maxisorb plates (NUNC, USA) according to the manufacturers’ instructions. Chemokine Lower Detection Limit IL-8 (BD OptEIA ) 7.8pg/ml TM MCP-1 (BD OptEIA ) 7.8pg/ml MIP-1 α (R&D Systems DuoSet ELISA kits) 15.6pg/ml MIP-1 β (R&D Systems DuoSet ELISA kits) 7.8pg/ml Table 5. List of the lower detection limit of all ELISAs. TM 2.12 Statistics Statistical significance of the differences between the means of two groups was determined by the two sided unpaired Students’ t-test. Levels of significance were set at p < 0.05 and p < 0.01. 56 CHAPTER 3: RESULTS CHAPTER 3 RESULTS It is important to highlight here that the results presented are due to the effect of cross-linking of CD137L on THP-1 cells. Immobilized CD137-Fc protein was used to induce the CD137L signalling in THP-1 cells in all experimental set-ups. CD137-Fc is a recombinant fusion protein consisting of the extracellular domain of human CD137 linked to the constant domain of human IgG1 (Fc) (Langstein et al., 1998). Recombinant Fc protein and uncoated plates were used as negative controls. Plates were coated overnight at 4°C with either 5µg/ml of human CD137-Fc or human IgG1-Fc. CD137-Fc needed to be immobilized onto plates because it has been shown that soluble CD137-Fc is not able to induce CD137L signalling and monocyte activation (Langstein et al., 1998; Langstein et al., 1999). 57 CHAPTER 3: RESULTS 3.1 CD137L Induced Adherence And Morphological Changes In THP- 1 Cells Morphological changes induced by cross-linking of CD137L are a characteristic feature of activated monocytic cells as reported by Langstein et al. (1998, 1999) and Söllner et al. (2007) (Langstein et al., 1998; Langstein and Schwarz, 1999; Söllner et al., 2007). THP-1 cells were cultured in CD137Fc coated or Fc coated plates for 24 hours and 48 hours to observe the morphological changes. Uncoated plate and Fc coated plates were used as controls in this experimental set up. In CD137-Fc coated wells, THP-1 cells adhered strongly to the plate by 24 hours and some had changed their morphology from round and spheric to elongated and spread-out. Such features were not found in the two control wells (Figure 1). By 48 hours, these morphological changes in the CD137-Fc coated wells were even more profound and distinct. In a separate experiment, THP-1 cells were observed to attach to CD137-Fc coated plates as early as 30 minutes. A strong adherence was observed after 60 minutes of incubation of THP-1 cells in CD137-Fc coated plates. These observations were used in subsequent experiments as evidence that cells were indeed activated by CD137L signalling. 58 Fc CD137-Fc Uncoated CHAPTER 3: RESULTS 24 hours 48 hours Figure 1. CD137 ligand signalling induced THP-1 cells activation. 5x104 THP-1 cells were incubated in uncoated or CD137-Fc coated or Fc coated plates. Cells were viewed under the Olympus IX81 microscope and photographed after 24 hours and 48 hours. 59 CHAPTER 3: RESULTS 3.2 Involvement Of Sphingosine Kinases, Not Phospholipase D, In CD137L-Activated Cells Another key feature of CD137L activation in monocytic cells is the production of the pro-inflammatory chemokine, IL-8. High levels of IL-8 production were reported by Söllner et al. when cells were activated by CD137-Fc protein (Söllner et al., 2007). This piece of information has been used as a determinant of activation of the monocytic cells by the cross-linking of CD137L. THP-1 cells were cultured on uncoated or CD137-Fc protein coated or Fc coated plates. Uncoated and Fc protein coated wells served as negative control in the experiment. Figure 2 shows that cells in CD137-Fc coated wells had a high release of IL-8, measuring almost up to 11ng/ml, whereas there was only about 2ng/ml of IL-8 detected in uncoated or Fc coated wells. To study the involvement of phospholipids in CD137L signalling in THP-1 cells, general inhibitors against SphK and PLD were used. DMS was used to inhibit SphK, and butan-1-ol was used to inhibit PLD. THP-1 cells were incubated with inhibitors for 15 minutes on ice. Subsequently, THP-1 cells with and without pre-treatment of inhibitors were transferred into plates with immobilized CD137-Fc protein, or immobilized Fc protein or uncoated wells. Upon the addition of cells into the respective wells, the plates were incubated in the 37°C incubator for 24 hours. Supernatants were collected after 24 hours of incubation and subjected to IL-8 analysis using ELISA. For SphK inhibition studies, cells were pre-treated with 10µM of DMS. 0.1% of DMSO was included as vehicle control because DMS is dissolved in 60 CHAPTER 3: RESULTS DMSO at 0.1% of total volume. This is to ensure that the effect seen is a true inhibition by inhibitors and not a toxic effect by the solvent DMSO. t-butanol was used as a negative control for non-specific effect caused by alcohol, namely butan-1-ol, which was used to inhibit PLD in cells. The amount of IL-8 production in THP-1 cells was significantly reduced by 10µM of DMS from 11ng/ml to about 3ng/ml as compared to vehicle control (Figure 2). However, 0.3% of butan-1-ol did not inhibit the production of IL-8 in the CD137L-activated cells (Figure 3). Next, we were interested to investigate if the SphK1 isoform plays a role in the activation of the cells upon cross-linking of the CD137L. Compound 5c, was shown to inhibit specifically SphK1 but not SphK2 (Wong et al., 2009). Two concentration of 5c were chosen for the subsequent assays; 25µM and 50µM of 5c were selected to be used for the inhibition studies. There was no inhibition of IL-8 secretion in 25µM of 5c treated cells as compared to vehicle control (Figure 2). However, there was a substantial inhibition in the IL-8 secretion when cells were treated with 50µM of 5c. IL-8 release was inhibited from 11ng/ml to about 5ng/ml when cells were treated with 50µM of 5c (Figure 2). The amount of IL-8 inhibited by 50µM of 5c is less than what was inhibited by 10µM of DMS; this may lead to the hypothesis that SphK2 may be responsible for part of the IL-8 production induced by the CD137L in THP-1 cells. 61 CHAPTER 3: RESULTS 12 ** ** 10 IL-8 (ng/ml) 8 6 4 2 0 Untreated 10µM DMS 25µM 5c Uncoated CD137-Fc 50µM 5c 0.1% DMSO Fc Figure 2. Sphingosine kinase inhibitors decrease CD137L-induced IL-8 secretion. 1.5x104 THP-1 cells were pre-incubated for 15 minutes with a final concentration of 10µM of DMS, 50µM or 25µM of compound 5c or the vehicle control (0.1% DMSO). Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. IL-8 release was then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. ** p value < 0.01. 62 CHAPTER 3: RESULTS 12 10 IL-8 (ng/ml) 8 6 4 2 0 Untreated Uncoated 0.3% butanol CD137-Fc 0.3% t-butanol Fc Figure 3. Phopholipase D inhibitor, Butan-1-ol, did not show any inhibition in CD137L-induced THP-1 cell activation as shown by the release of IL-8. 1.5x104 THP-1 cells were pre-incubated for 15 minutes with a final concentration of 0.3% of butan-1-ol or control, 0.3% t-butanol. Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. IL-8 releases were then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 2 independent experiments with comparable results. 63 CHAPTER 3: RESULTS 3.3 Expression Of Sphingosine Kinase 1 In Monocytic Cell Lines Before proceeding to further investigations on the roles of SphK1 in CD137L-induced cells, we took a step back to look at the expression levels of SphK1 protein in the resting state of cells. The expression levels of SphK1 protein in THP-1 cells and U-937 cells were analysed by Western Blot analysis. Equal amounts of total protein from the cell extracts were used from each cell line and were probed for SphK1. Figure 4 shows that SphK1 protein was present in both cell lines at comparable amounts. A housekeeping gene protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as loading control in the Western Blot. 64 CHAPTER 3: RESULTS kDa 50 37 Sphingosine Kinase 1 37 GAPDH THP-1 U-937 Figure 4. Sphingosine kinase 1 was expressed in THP-1 cells and U-937 cells. Equal amounts of protein lysates were separated by SDS PAGE and blotted onto PVDF membrane. Blots were probed with rabbit anti-sphingosine kinase 1 antibody (Cell Signaling Technology Inc., USA) overnight followed by antirabbit IgG-HRP (Thermo Scientific, USA). GAPDH was used as loading control. Results are representative of 3 independent experiments with comparable results. 65 CHAPTER 3: RESULTS 3.4 CD137L Signalling Activates Sphingosine Kinase 1 THP-1 was shown to express SphK1 protein in amounts detectable by Western Blot analysis (Figure 4). But the presence of SphK1 protein does not imply that the enzyme is active in the cells. The measurement of enzyme activity is a more accurate way of investigating whether a protein plays a role in the signalling cascade upon cell activation. The sphingosine kinase activity was measured to determine if the enzyme activity was induced in THP-1 when CD137L was activated by the immobilized CD137-Fc protein. THP-1 cells were stimulated with immobilized CD137-Fc or Fc protein at time intervals from 2 to 30 minutes. Equal amounts of total cell lysates were used to investigate the sphingosine kinase activity. Figure 5 shows that cross-linking of CD137L in THP-1 cells increased the sphingosine kinase activity as early as 5 minutes. The activity of the enzyme remained high until 30 minutes after stimulation. Sphingosine kinase activity reached its peak at 10 minutes with a 40% higher activity as compared to basal level. In this case, basal was defined as unstimulated or resting cells and was expressed as 100% activity. The kinase activity was significantly higher at 10 minutes and 30 minutes as compared to Fc control too. The enzyme activity remained around 20% above the basal level at 15 minutes and 30 minutes (Figure 5). 66 CHAPTER 3: RESULTS 160 * 140 SphK Activity (%) 120 100 # # 80 CD137-Fc Fc Control 60 40 20 0 0 5 10 15 20 Time (min) 25 30 35 Figure 5. Cross-linking of CD137 ligand induced sphingosine kinase activity in THP-1 cells. 0.5x106 THP-1 cells were seeded on CD137-Fc protein coated and Fc protein coated 12 well plates for stimulation for indicated times. Cells were lysed and equal amounts of protein lysates were used for each SphK activity measurement. The levels of sphingosine kinase activity at the indicated time points are expressed as percentage change over the basal level (0 minute). Basal level activity is expressed as 100%. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. * p value < 0.05 between CD137-Fc stimulation at the indicated time points and basal level (0 minute). # p value < 0.05 between CD137-Fc stimulation and Fc control at the indicated time point. 67 CHAPTER 3: RESULTS 3.5 CD137L Signalling Does Not Induce Sphingosine Kinase 1 Protein It has been shown that SphK1 protein expression is detectable by Western Blot analysis in THP-1 cell at the resting state (Figure 4). It has also been shown that SphK1 was involved in the production of the proinflammatory chemokine, IL-8, within 24 hours after the cells were activated by CD137L signalling (Figure 2). Following up on these two sets of data, it was interesting to find out if activation of the CD137L signalling cascade in THP-1 cells would increase the level of SphK1 protein over 24 hours. THP-1 cells were cultured on plates with immobilized CD137-Fc or Fc protein or on uncoated plates for 24 hours. Cells were collected and lysed for protein analysis by Western Blotting for SphK1. Cross-linking of CD137L on THP-1 cells did not induce an increase of SphK1 protein expression in the cells after 24 hours (Figure 6). SphK1 protein was shown to remain at the same level of expression, either in CD137-Fc stimulated or Fc control or unstimulated THP1 cells (Figure 6). GAPDH was used as loading control in this experimental set-up. 68 CHAPTER 3: RESULTS Figure 6. Sphingosine kinase 1 protein level remained unchanged after 24 hours of cross-linking of CD137 ligand. THP-1 cells were cultured on uncoated (Un.) plates or on plates which were coated with Fc (Fc) or CD137Fc protein for 24 hours. Equal amounts of protein lysates were separated by SDS PAGE and blotted onto Polyvinylidene fluoride membrane. Blots were probed with rabbit sphingosine kinase 1 antibody (Cell Signaling Technology Inc., USA) overnight, followed by anti-rabbit IgG-HRP (Thermo Scientific, USA). GAPDH was used as loading control. Results are representative of 3 independent experiments with comparable results. 69 CHAPTER 3: RESULTS 3.6 Involvement Of Sphingosine Kinase In CD137L-Induced Inflammatory Chemokines Production IL-8, MCP-1, MIP-1α and MIP-1β belong to the inflammatory chemokine family. IL-8 is classified under the chemokine subfamily CXC chemokine, whereas MCP-1, MIP-1α and MIP-1β are classified under the chemokine subfamily CC chemokine (Deshmane et al., 2009; Zlotnik and Yoshie, 2012). As demonstrated above, IL-8 was secreted by THP-1 cells upon activation of CD137L (Figure 1). Next we moved on to investigate if other chemokines, namely MCP-1, MIP-1α and MIP-1β are secreted by CD137L-activated THP-1 cells. And, if they are produced by the stimulated cells, are they also being regulated by SphKs? Cross-linking of CD137 ligand is shown to induce the production of MCP-1 (Figure 7). About 1.3ng/ml of MCP-1 had been detected in the supernatants of CD137L-activated cells. However, this amount of cytokine was greatly reduced from 1.3ng/ml to 0.2ng/ml, 0.4ng/ml and 0.1ng/ml of MCP-1 when cells were pre-treated with 10µM of DMS, 25µM of 5c or 50µM of 5c respectively. These reductions of the MCP-1 release were significant as compared to vehicle control, DMSO. Activation of CD137 ligand signalling downstream not only induced the production of IL-8 and MCP-1 in the cells, but CD137L-activated cells were also producing MIP-1α (Figure 8) and MIP-1β (Figure 9). The secretion of both chemokines was suppressed by the SphK inhibitors, DMS and 5c. Cross-linking of CD137L on THP-1 cells produced about 2ng/ml of MIP-1α and this amount was reduced by 10µM of DMS, 25µM of 5c and 50µM of 5c to about 1ng/ml, 1.8ng/ml and 1.3ng/ml respectively (Figure 8). 70 CHAPTER 3: RESULTS About 1.4ng/ml of MIP-1β were secreted in CD137L-induced THP-1 cells and this was also suppressed by the SphK inhibitors, DMS and 5c, to 0.4ng/ml (by 10µM of DMS), 1.2ng/ml (by 25µM of 5c) and 0.7ng/ml (by 50µM of 5c) (Figure 9). 71 CHAPTER 3: RESULTS ** ** ** 1.6 1.4 1.2 MCP-1 (ng/ml) 1.0 0.8 0.6 0.4 0.2 0.0 Untreated 10µM DMS 25µM 5c Uncoated CD137-Fc 50µM 5c Fc 0.1% DMSO Figure 7. Cross-linking of CD137 ligand induced the production of MCP-1. Sphingosine kinase inhibitors decrease the CD137L-induced MCP-1 secretion. 1.5x104 THP-1 cells were pre-incubated for 15 minutes with a final concentration of 10µM of DMS, 50µM or 25µM of compound 5c or the vehicle control (0.1% DMSO). Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. MCP-1 release was then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. ** p value < 0.01. 72 CHAPTER 3: RESULTS 2.5 ** ** MIP-1α (ng/ml) 2.0 1.5 1.0 0.5 0.0 Untreated 10µM DMS 25µM 5c Uncoated CD137-Fc 50µM 5c 0.1% DMSO Fc Figure 8. Cross-linking of CD137 ligand induced the production of MIP-1. Sphingosine kinase inhibitors decrease the CD137L-induced MIP-1 secretion. 1.5x104 THP-1 cells were pre-incubated for 15 minutes with a final concentration of 10µM of DMS, 50µM or 25µM of compound 5c or the vehicle control (0.1% DMSO). Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. MIP-1 release was then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. ** p value < 0.01. 73 CHAPTER 3: RESULTS 1.8 1.6 ** ** 1.4 MIP-1β (ng/ml) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Untreated 10µM DMS 25µM 5c Uncoated CD137-Fc 50µM 5c 0.1% DMSO Fc Figure 9. Cross-linking of CD137 ligand induced the production of MIP-1. Sphingosine kinase inhibitors decrease the CD137L-induced MIP-1 secretion. 1.5x104 THP-1 cells were pre-incubated for 15 minutes with a final concentration of 10µM of DMS, 50µM or 25µM of compound 5c or the vehicle control (0.1% DMSO). Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. MIP-1 release was then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. ** p value < 0.01. 74 CHAPTER 3: RESULTS 3.7 Involvement Of Protein Kinase C In CD137 Ligand Signalling PKC is a very important mediator in signalling cascade in immune cells (Tan and Parker, 2003). IL-8 production induced by the CD137L signalling cascade in THP1 cells was halved by 500nM of the general PKC inhibitor; a total amount of 4ng/ml of IL-8 was reduced to 2ng/ml of IL-8 by Bis I as shown in Figure 10. The vehicle control, 0.1% DMSO, had no effect on the secretion of the IL-8 by the cells, implying that this reduction in IL-8 is solely due to an inhibition by the Bis I, and not due to any toxicity of the solvent. 75 CHAPTER 3: RESULTS 5.0 4.5 ** 4.0 3.5 IL-8 (ng/ml) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Untreated Uncoated 500nm Bis I CD137-Fc 0.1% DMSO Fc Figure 10. Bisindolylmaleimide I decreases CD137L-induced IL-8 secretion. 1.5x104 THP-1 cells were pre-incubated for 30 minutes with a final concentration of 500nM of Bisindolylmaleimide I or the vehicle control (0.1% DMSO). Cells were then transferred into plates coated with Fc or CD137-Fc protein or uncoated plates for stimulation for 24 hours. Cells without any addition of inhibitors served as controls. IL-8 release was then determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. Results are representative of 3 independent experiments with comparable results. ** p value < 0.01. 76 CHAPTER 4: DISCUSSION CHAPTER 4 DISCUSSION 4.1 CD137 Ligand Induced Adherence And Morphological Changes In THP-1 Cells Here, we have shown that THP-1 cells cultured in CD137-Fc coated plates had a higher percentage of adherent cells. THP-1 cells had shown morphological changes from round and spheric to elongated and a spread-out shape by 24 hours and continued to adapt to the elongated and spread-out by 48 hours. Cells grown on Fc coated or uncoated plates did not have such changes. Both Langstein et al. (1999) and Söllner et al. (2007) showed that monocytes underwent morphological changes upon activation by CD137-Fc protein on the plates; morphological changes were found to be more pronounced in primary monocytes than in THP-1 cells (Langstein et al., 1998; Langstein and Schwarz, 1999; Söllner et al., 2007). In a separate experiment, cross-linking of CD137L by CD137-Fc protein induced adherence of THP-1 cells as early as 30 minutes and strong attachment was observed at 60 minutes. It was reassuring to observe cells to adhere to the CD137-Fc coated plates because this simply showed that CD137L on the THP-1 cell surface had bound to the CD137-Fc protein on the plate. This observation is in line with what was reported that CD137L activation causes increased cell adherence in monocytic cell line, THP-1, primary monocytes (Langstein et al., 2000; Langstein et al., 1998; Söllner et al., 2007), bone marrow-derived macrophages, murine myeloid cell line, RAW264.7 (Kim et al., 2009). 77 CHAPTER 4: DISCUSSION 4.2 Involvement Of Sphingosine Kinases, Not Phospholipase D, In CD137 Ligand-Activated Cells CD137L-induced THP-1 cells secreted large amounts of the proinflammatory chemokine, IL-8, at 24 hours and this is consistent with what was reported by literatures (Langstein et al., 2000; Langstein et al., 1998; Söllner et al., 2007). Around 11ng/ml of IL-8 were detected when cells were activated by CD137-Fc protein. IL-8 production was only detected around 2ng/ml in Fc coated plates or uncoated plates; the effect of high release of IL-8 is mainly due to the cross-linked CD137L on the monocytic cells. Our group has shown that CD137L reverse signalling in monocytes involves a few important classical kinases such as protein tyrosine kinase, MEK, MAPK p38, MAPK p44/42, PI3-K and PKA (Söllner et al., 2007). In this current study, the data that we have gotten so far suggest that SphK, particularly SphK1, is involved in the downstream signalling cascade in THP1 cells. DMS, a potent pharmacological inhibitor of SphK, inhibited around 70% of the total release of IL-8 induced by CD137L-activated THP-1 cells. The compound 5c was generated by Wong et al. (2009), and specifically targets the SphK1 isoform and not SphK2, also showed inhibition towards the secretion of IL-8 by CD137L-induced THP-1 cells (Wong et al., 2009). A lower dosage of 25µM of 5c did not affect the production of IL-8 by the cells; however, there is a significant drop in the IL-8 release when cells were treated with 50µM of 5c. Only 5ng/ml of IL-8 was detected in the supernatant of the cells pre-treated with 50µM of 5c. 78 CHAPTER 4: DISCUSSION DMS is a general inhibitor that is widely used to study the involvement of SphK. At 10µM, DMS is shown to be a competitive inhibitor to SphK only, and does not affect PKC activity (Edsall et al., 1998). In this study, the DMS concentration had been kept at 10µM in order not to affect other the activity of kinases, especially not PKC activity. However, perhaps a lower range of DMS concentration (2µM to 5µM) could be used in future experiments to minimise the side effects of the inhibitor; moreover, Edsall et al. (1998) had shown that SphK activity was reduced to 50% when DMS was used at 5µM (Edsall et al., 1998). 10µM of DMS inhibited a higher percentage (~70%) of IL-8 release (from 11ng/ml to 3ng/ml) as compared to only ~55% inhibition by 50µM of 5c (from 11ng/ml to 5ng/ml). The remaining 2ng/ml that was not inhibited by 50µM of 5c may be contributed by the SphK2 isoform. But as whether the SphK2 is playing a compensatory role to the inhibited SphK1 or SphK2 is actually contributing to the production of IL-8 in the normal physiological condition, further investigation is needed to answer this question. PLD has been an important regulatory enzyme that catalyses phosphatidylcholine to yield another important second messenger, phosphatidic acid (PA), and choline. Sethu et al. (2008) have shown that PLD1 plays a major role in TNF-α signalling in monocytic cells, specifically U-937 and primary monocytes. TNF-α stimulation on monocytes activates PLD1, which subsequently activates SphK and calcium release. PLD1 is also demonstrated to play a part in cytokine release such as IL-1β and IL-6 (Sethu et al., 2008). This study had encouraged us to look at the possibilities that PLD may play a role in the CD137L signalling pathway given that CD137L and TNF-α belong to the same superfamily of TNF. If PLD is shown to also 79 CHAPTER 4: DISCUSSION contribute to the production of IL-8 in the CD137L signalling pathway, we would be wondering if PLD is also upstream of SphK as reported by Sethu et al. (2008). However, we have shown that 0.3% of butan-1-ol did not affect the release of IL-8 upon cross-linking of CD137L. This may imply that PLD is not activated by the reverse signalling of CD137L in THP-1 cells. There has always been a concern that CD137-Fc recombinant protein may activate Fc receptor (FcR) through its Fc portion of the protein, hence effect seen by CD137-Fc activation may be caused by the FcR. Melendez et al. (2001) showed that PLD1 is coupled to the activation of FcγR1 and subsequently activates SphK and transient release of calcium in U-937 cells (Melendez et al., 2001). In our studies, we showed that PLD is not involved in the downstream signalling cascade for IL-8 production upon CD137L stimulation in THP-1 cells. This may lend another supportive evident that the results that we have seen so far are mainly due to the CD137L activation, and not the FcR activation. 80 CHAPTER 4: DISCUSSION 4.3 CD137L Does Not Induce Expression Of Sphingosine Kinase 1 Under normal physiological conditions where cells are at a resting state, we have shown that SphK1 protein was present in THP-1 and U-937 cells. The amounts of SphK1 in the unstimulated cells are abundant in cytosol of both cell lines as also seen in macrophages (Melendez and Ibrahim, 2004). As shown by ELISA, CD137L activation induced high production of IL-8 and this is largely contributed to the SphK enzyme, particularly SphK1. DMS and 5c have markedly reduced the amount of this chemokine released by THP-1 cells upon the cross-linking of CD137L. Based on this observation, we were interested to find out if the readily available SphK1 in the cytosol is responsible for the whole production of IL-8 in the signalling pathway or do the cells produce more SphK enzyme in response to the activation of the CD137L signalling pathway. In other words, do the cells have a positive feedback mechanism to increase the amount of the enzymes in the cells to aid the chemokine production upon the activation of CD137L? CD137L activation did not induce SphK1 expression in THP-1 cells in 24 hours culture. There is no increase of the amount of protein in the activated cells as compared to the untreated or Fc treated cells. Michel J. et al. (1999) have reported the same, that CD137L activation in lymphocytes did not induce higher expression of CD95 ligand (CD95L) in the Western Blot analysis in 24 and 48 hours stimulation (Michel et al., 1999). Together, this may just indicate that CD137L activated cells do not activate the transcriptional machinery to produce proteins, be it SphK1 enzyme or CD95L, to drive an even more robust response within 24 hours. 81 CHAPTER 4: DISCUSSION 4.4 CD137 Ligand Signalling Activates Sphingosine Kinase 1 Stimulation of CD137L on THP-1 cells was shown to induce SphK activity. The SphK activity started to increase from 5 minutes reached its highest point at 10 minutes and remained higher than basal level even at 30 minutes. Our results showed that SphK activity reached its peak at 10 minutes at 140% when compared to basal level which was expressed as 100%. The SphK enzyme remained active, its activity measured higher than 20% above basal level. This enzyme activity is found to be consistent with data from Xia et al. group (1999); the group was stimulating HUVEC with TNF-α for a longer time interval, measuring the activity up to 60 minutes (Xia et al., 1999). Xia et al. (1999) showed that SphK activity was induced by TNF-α in HUVEC. The SphK activity was also shown to reach its peak at 10 minutes, measuring higher than 150% and the enzyme remained active and higher than basal at 30 minutes. SphK activity was shown to only return to resting state activity at 60 minutes (Xia et al., 1999). In two other studies, SphK activities were shown to peak at earlier time point i.e. at 5 minutes instead. Xia et al. (1998) reported that TNF-α stimulated SphK activity in HUVEC, with its highest activity at 165% at 5 minutes interval. This observation was found to be accompanied by S1P production in the cells at the same time (Xia et al., 1998). Sethu et al. (2008) also showed that SphK activity peaked at 5 minutes, with a much higher activity after stimulation with TNF-α in U-937 and primary monocytes. SphK activity was higher than 200% and remained around that level for up to 30 minutes (Sethu et al., 2008). 82 CHAPTER 4: DISCUSSION 4.5 Involvement Of Sphingosine Kinases In CD137 Ligand-Induced Inflammatory Chemokines Production MCP-1 (CCL2), as by the name of it, is a monocyte chemoattractant that mediates the migration and infiltration of monocytes or macrophages as a response to inflammation (Deshmane et al., 2009). Here, we have shown that cross-linking of CD137L in monocytes increased the release of MCP-1 at a substantial amount. From a basal level of less than 0.1ng/ml, CD137L-induced cells produced more than 1.3ng/ml of MCP-1 over 24 hours. Such an increment in the MCP-1 level is hypothesized to mediate the recruitment of monocytes to migrate to the site with higher levels of MCP-1, following its chemokine gradient. Ajuebor and co-workers (1998) have shown that in a murine inflammation model, administration of Zymosan into the peritoneal cavity attracted a huge number of polymorphonuclear leukocytes and monocytes to the site, mainly caused by the increase of MCP-1 (Ajuebor et al., 1998). They further confirmed this data by injection of murine recombinant MCP-1 protein into the peritoneal cavity, mimicking an artificial high secretion of MCP-1; this had specifically attracted migration of monocytes into the cavity. The migration of monocytes was significantly reduced to 40% when anti-mouse MCP-1 antibody was injected into the peritoneal cavity after the administration of Zymosan (Ajuebor et al., 1998). The production of MCP-1 by CD137L activation (~1.3ng/ml) was clearly suppressed by DMS and 5c. MCP-1 production was very sensitive to 5c since at 25µM, MCP-1 secretion was inhibited to around 0.4ng/ml. For other chemokine profiles, namely IL-8, MIP-1α and MIP-1β, 25µM of 5c did 83 CHAPTER 4: DISCUSSION not reduce the releases significantly upon stimulation of CD137L. This may be an indication that especially MCP-1 production could be tightly regulated by SphK1 as shown in the data. In a cell-contact assay between fixed activated Jurkat T cells and U937 cells, Lai et al. (2008) showed that MCP-1 was secreted at a very high level (~9ng/ml) by the monocytic cell line (Lai et al., 2008). The same was observed when T lymphocytes derived from RA patients were cultured with autologous peripheral blood (PB) monocytes (MCP-1 secretion was about 4ng/ml). This chemokine was shown to be inhibited by DMS. There was no sign of apoptosis observed in the inhibitor conditions, confirming that SphK1 played a role in the MCP-1 production (Lai et al., 2008). High levels of MIP-1α and MIP-1β in terms of nanograms were detected in the supernatant of CD137L-activated cells. These chemokines were inhibited significantly by 10µM of DMS and 50µM of 5c. 25µM of 5c did not significantly reduce these chemokine levels. In the acute inflammatory peritoneal model by Ajuebor et al. (1998), MIP-1α was also reported to be elevated but the release was not affected by MCP-1 release. The chemokine was shown to only be secreted during inflammation (Ajuebor et al., 1998). 84 CHAPTER 4: DISCUSSION 4.6 Involvement Of Protein Kinase C In CD137 Ligand Signalling It has previously been shown that Chelerythrine, a PKC specific inhibitor, did not inhibit production of IL-8 in primary monocytes and THP-1 upon stimulation of CD137L (Söllner et al., 2007). The concentrations of Chelerythrine used were up to 3µM, but this inhibitor did not show any effect towards IL-8 secretion by the cells and it was also shown that at such high concentration, the inhibitor did not cause any reduction in cell viability (Söllner et al., 2007). However, in this present study, utilizing another commonly used PKC inhibitor, Bis 1, also known as GF109203X, we showed that IL-8 release initiated through the CD137L reverse signalling pathway is inhibited. Bis 1 inhibited the release of IL-8 by half. This is not surprising because a differential effect by different PKC inhibitors was also observed in IL-1α-induced chemokine (IL-8) release in synovial fibroblasts (Jordan et al., 1996). Jordan and co-worker (1996) reported that Chelerythrine chloride (concentration at 0.1 – 3µM) induced a slight increase in the mRNA and protein level of the chemokine. In the same study, this group has also shown that Bis 1 has a biphasic effects in IL-1αinduced IL-8 release, that at low concentration (less than 3µM), Bis 1 caused higher chemokine release but at higher concentration (10 – 30µM), Bis 1 effectively inhibited the chemokine production (Jordan et al., 1996). There are another two groups that reported lower concentration of Bis 1 is sufficient for PKC inhibition. 10µM of Bis 1 was sufficient to inhibit PMA-induced phosphorylation of Raf-1, a PKC target protein, substantially (Han et al., 2000) and at 2.5µM, Bis 1 could inhibit PMA-induced MAP Kinase Phosphatase-1 (MKP-1) protein (Beltman et al., 1996). MKP-1 is an immediate early gene 85 CHAPTER 4: DISCUSSION product induced by growth factor. The inhibitory effect shown was not due to cell death because it was demonstrated that Bis 1 did not induce apoptosis in the cells up to 4 days of incubation (Han et al., 2000). There are two reports showing Chelerythrine does not inhibit PKC (Davies et al., 2000; Lee et al., 1998). Lee et al. (1998) reported that in their experimental set-up, Chelerythrine did not inhibit the PKC activity of calf brain at IC50=60-80µg/ml, it was shown that this compound stimulated PKC activity in the cytosol instead (Lee et al., 1998). In the same experimental setup, another PKC inhibitor, staurosoprine was shown to inhibit PKC activity very significantly (Lee et al., 1998). PKC has two functional moieties, the regulatory domain and the catalytic domain. Apart from different domain inhibition, enzyme inhibitors can be divided into competitive inhibitors and non-competitive inhibitors (Pajak et al., 2008). Chelerythrine was reported as a potent and specific inhibitor of PKC at IC50=0.66µM, acting on the substrate site of the PKC and not on the regulatory site of the kinase. It has been shown to be a competitive inhibitor to phosphate acceptor and a non-competitive inhibitor towards the ATP binding site. It does not block the phorbol esther binding to the PKC (Herbert et al., 1990; Pajak et al., 2008). On the other hand, Bis 1 inhibits PKC activity specifically at the ATPbinding site as a competitive inhibitor against ATP. Bis 1 inhibits soluble and membrane forms of PKC and does not inhibit binding of [3H] phorbol dibutyrate to PKC (Pajak et al., 2008). 86 CHAPTER 4: DISCUSSION 4.7 Cross-linking Is Needed To Activate The CD137L Signalling Pathway CD137-Fc is a recombinant fusion protein consisting of the extracellular domain of human CD137 linked to the constant domain of human IgG1 (Fc). Immobilizing the CD137-Fc onto the plates for CD137L stimulation is critical and essential because Langstein et al. reported that soluble CD137-Fc was neither capable of inducing adherence of the cells on the plate (Langstein et al., 1998) nor the proliferation of the monocytes (Langstein et al., 1999). Soluble CD137-Fc protein is only capable of dimerization of the ligand on the cell surface through its Fc domain but not enough to achieve multimerization of the ligand (Langstein et al., 1999). Higher level of multimerization is needed to activate the ligand signalling cascade downstream. In order to achieve higher level of multimerization of the ligand, one can immobilize the protein onto the plate or cross-link the antibody with a secondary antibody. One may cross-link the CD137-Fc protein by the addition of anti-Fc antibody. An anti-Fc antibody is shown to be capable of cross-linking the ligand but to the lesser extent of the activation state by immobilizing the protein on the plate (Kang et al., 2007) Immobilizing the protein on the plate created a higher level of multimerization of the ligand, achieving the optimal activation of CD137L in the cells. 87 CHAPTER 4: DISCUSSION 4.8 Controls In The Experiment Recombinant CD137 protein that is available commercially is fused to Fc portion of the immunoglobulin (referred as CD137-Fc in this thesis) in order to facilitate protein purification. Fcγ receptors (FcγR) are present on immune cells including monocytes and engagement of FcγR on monocytes can trigger downstream signalling pathways including PLD, PKC, PI3-K and MAPK (Jovanovic et al., 2009). FcγR has been reported to activate PLD and SphK (Melendez et al., 1998). As an additional control to eliminate the possibilities that Fc receptors on THP-1 cells are activated by the CD137-Fc protein, cells were first incubated with Fc blockers reagents to ensure that Fc receptors are blocked before cells were stimulated with CD137-Fc. Excessive Fc blocker reagents were washed away before cells were seeded onto the immobilized CD137-Fc or Fc protein wells. It is not unreasonable to include a negative control consisting of human IgG-Fc to eliminate any possibilities that the results obtained and analysed were the effects of the Fc protein and not the CD137 domain of the CD137-Fc protein. Uncoated plates were also used as negative control to observe the activities of untreated cell without any addition of proteins. As monocytes are heterogeneous in the culture, THP-1 cells were grown in low level of FBS (controlled at 1% v/v) media for 18 hours to synchronize the cells to the same phase i.e. G0 to obtain a homogenous culture for the experiments. 88 CHAPTER 5: CONCLUSION CHAPTER 5 CONCLUSION We showed that stimulation of CD137L induces adherence and morphological changes in THP-1 cells, and release of IL-8 as reported in the literature by several groups. We observed that cells had started to attach to the plate as early as 30 minutes and strong adherence was observed within 1 hour. Of the two phospholipid enzymes investigated, only SphK1, but not PLD plays a part in the release of IL-8. As shown, the SphK inhibitor DMS reduced IL-8 secretion from 11ng/ml to 3ng/ml whereas the SphK inhibitor 5c reduced IL-8 secretion from 11ng/ml to 5ng/ml. This observation led us to speculate that SphK2 may be involved in IL-8 production. CD137L stimulation activates SphK activity. SphK activity reached its peak after 10 minutes and maintained higher than basal level for up to 30 minutes. However, CD137L stimulation does not increase the SphK1 protein level in the cells within 24 hours. CC chemokines favours the recruitment of monocytes (Cook, 1996; Deshmane et al., 2009). Stimulation of CD137L in THP-1 cells induces high production of CC chemokines, namely MCP-1, MIP-1α, MIP-1β. We suspected that the increase of these chemokines is mainly to attract monocytes into the inflamed tissues for a more robust inflammatory response. DMS and 5c substantially inhibit the secretion of MCP-1, MIP-1α and MIP-1β by THP-1 cells. Inhibition of the secretion of these chemokines indicates that SphK, particularly SphK1, is mediating the production of chemokines in the cells. MCP-1 secretion is significantly inhibited by a lower concentration of 5c, a hint that shows MCP-1 production may be tightly 89 CHAPTER 5: CONCLUSION regulated by SphK1. Targeting SphK in CD137L activated inflammation would be expected to reduce the release of inflammatory chemokines which in turn would reduce the influx of monocytes/macrophages. A reduction of leukocyte homing to the site of inflammation may decrease the inflammation severity especially in chronic inflammatory disease such as RA, atherosclerosis and etc. PKC is involved in production of IL-8 by CD137L stimulated THP-1 cells as showed by the inhibition of IL-8 production when the PKC inhibitor Bis I was used. The fact that the PKC inhibitor Chelerythrine was previously shown not to inhibit the release of IL-8 was perhaps caused the ineffectiveness of Chelerythrine as a PKC inhibitor as reported by others. Figure C shows the proposed CD137L signalling pathway based on the findings by Söllner et al. (2007) and current findings in this study (Söllner et al., 2007). 90 CHAPTER 5: CONCLUSION Figure C. Proposed CD137L signalling pathway. 91 CHAPTER 6: FUTURE DIRECTION CHAPTER 6 FUTURE DIRECTION Dissecting the signalling cascade stimulated by CD137L is crucial to manipulate the molecules involved for the generation of novel therapeutic drugs, particularly to dampen excessive inflammation that may lead to autoimmune diseases. Phospholipid modifying enzymes like SphK that regulate immune responses triggered by CD137L are of most interest. In this project, SphK1 is identified as one of the key players in the release of chemokines. The next stage that we could go into would be to investigate the involvement of SphK in the regulation of the MAPK pathway and PI3-K pathway, and how SphK can affect the downstream signalling molecules in eliciting immune responses. It would be interesting to understand the mechanisms that are being employed by SphK. Is SphK involved in the regulation of the MAPK signalling pathway or is the MAPK the one responsible for the activation of SphK? How is SphK related to PKC in the CD137L reverse signalling, if there is any connection? NF-κB is responsible for the production of many cytokines and chemokines. Is this transcription factor also involved in the CD137L reverse signalling pathway? Or are there other transcription factors responsible for the production of the chemokines and cytokines? There is a possibility that SphK2 may also contribute to the effect of the reverse signalling of CD137L, either by compensating for the reduction of SphK1 activity, or it may be a normal physiological process that SphK2 is also involved in the chemokine production. Specific inhibitors for SphK2 such as ABC294640 could be used as valuable tools to understand the role of SphK2 92 CHAPTER 6: FUTURE DIRECTION in the reverse CD137L signalling in THP-1 cells. The two variants’ roles can be further confirmed by knocking down the genes using siRNA or antisense oligonucleotides against SphK1 or SphK2. 93 REFERENCES REFERENCES Ajuebor, M.N., Flower, R.J., Hannon, R., Christie, M., Bowers, K., Verity, A., and Perretti, M. (1998). Endogenous monocyte chemoattractant protein-1 recruits monocytes in the zymosan peritonitis model. J Leukoc Biol 63, 108116. Alderson, M.R., Smith, C.A., Tough, T.W., Davis-Smith, T., Armitage, R.J., Falk, B., Roux, E., Baker, E., Sutherland, G.R., and Din, W.S. (1994). Molecular and biological characterization of human 4-1BB and its ligand. Eur J Immunol 24, 2219-2227. Baker, D.A., Barth, J., Chang, R., Obeid, L.M., and Gilkeson, G.S. (2010). Genetic sphingosine kinase 1 deficiency significantly decreases synovial inflammation and joint erosions in murine TNF-alpha-induced arthritis. J Immunol 185, 2570-2579. Baker, D.A., Eudaly, J., Smith, C.D., Obeid, L.M., and Gilkeson, G.S. (2012). Impact of sphingosine kinase 2 deficiency on the development of TNF-alphainduced inflammatory arthritis. Rheumatol Int. Beltman, J., McCormick, F., and Cook, S.J. (1996). The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem 271, 27018-27024. Billich, A., and Ettmayer, P. (2004). Fluorescence-based assay of sphingosine kinases. Anal Biochem 326, 114-119. Bosmann, M., Russkamp, N.F., and Ward, P.A. (2012). Fingerprinting of the TLR4-induced acute inflammatory response. Exp Mol Pathol 93, 319-323. Broll, K., Richter, G., Pauly, S., Hofstaedter, F., and Schwarz, H. (2001). CD137 expression in tumor vessel walls. High correlation with malignant tumors. Am J Clin Pathol 115, 543-549. Cook, D.N. (1996). The role of MIP-1 alpha in inflammation and hematopoiesis. J Leukoc Biol 59, 61-66. Croft, M. (2003a). Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat Rev Immunol 3, 609-620. 94 REFERENCES Croft, M. (2003b). Costimulation of T cells by OX40, 4-1BB, and CD27. Cytokine Growth Factor Rev 14, 265-273. Croft, M. (2009). The role of TNF superfamily members in T-cell function and diseases. Nat Rev Immunol 9, 271-285. Croft, M., Duan, W., Choi, H., Eun, S.Y., Madireddi, S., and Mehta, A. (2012). TNF superfamily in inflammatory disease: translating basic insights. Trends Immunol 33, 144-152. Davies, S.P., Reddy, H., Caivano, M., and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95-105. Deshmane, S.L., Kremlev, S., Amini, S., and Sawaya, B.E. (2009). Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 29, 313-326. Di, A., Kawamura, T., Gao, X.P., Tang, H., Berdyshev, E., Vogel, S.M., Zhao, Y.Y., Sharma, T., Bachmaier, K., Xu, J., et al. (2010). A novel function of sphingosine kinase 1 suppression of JNK activity in preventing inflammation and injury. J Biol Chem 285, 15848-15857. Drenkard, D., Becke, F.M., Langstein, J., Spruss, T., Kunz-Schughart, L.A., Tan, T.E., Lim, Y.C., and Schwarz, H. (2007). CD137 is expressed on blood vessel walls at sites of inflammation and enhances monocyte migratory activity. FASEB J 21, 456-463. Edsall, L.C., Van Brocklyn, J.R., Cuvillier, O., Kleuser, B., and Spiegel, S. (1998). N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide. Biochemistry 37, 12892-12898. Fitzpatrick, L.R., Green, C., Frauenhoffer, E.E., French, K.J., Zhuang, Y., Maines, L.W., Upson, J.J., Paul, E., Donahue, H., Mosher, T.J., et al. (2011). Attenuation of arthritis in rodents by a novel orally-available inhibitor of sphingosine kinase. Inflammopharmacology 19, 75-87. French, K.J., Schrecengost, R.S., Lee, B.D., Zhuang, Y., Smith, S.N., Eberly, J.L., Yun, J.K., and Smith, C.D. (2003). Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 63, 5962-5969. 95 REFERENCES French, K.J., Upson, J.J., Keller, S.N., Zhuang, Y., Yun, J.K., and Smith, C.D. (2006). Antitumor activity of sphingosine kinase inhibitors. J Pharmacol Exp Ther 318, 596-603. French, K.J., Zhuang, Y., Maines, L.W., Gao, P., Wang, W., Beljanski, V., Upson, J.J., Green, C.L., Keller, S.N., and Smith, C.D. (2010). Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2. J Pharmacol Exp Ther 333, 129-139. Furtner, M., Straub, R.H., Krüger, S., and Schwarz, H. (2005). Levels of soluble CD137 are enhanced in sera of leukemia and lymphoma patients and are strongly associated with chronic lymphocytic leukemia. Leukemia 19, 883-885. Gangoiti, P., Camacho, L., Arana, L., Ouro, A., Granado, M.H., Brizuela, L., Casas, J., Fabriás, G., Abad, J.L., Delgado, A., et al. (2010). Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease. Prog Lipid Res 49, 316-334. Gordon, S., and Taylor, P.R. (2005). Monocyte and macrophage heterogeneity. Nat Rev Immunol 5, 953-964. Han, Z., Pantazis, P., Lange, T.S., Wyche, J.H., and Hendrickson, E.A. (2000). The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C. Cell Death Differ 7, 521-530. Herbert, J.M., Augereau, J.M., Gleye, J., and Maffrand, J.P. (1990). Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172, 993-999. Huang, D.R., Wang, J., Kivisakk, P., Rollins, B.J., and Ransohoff, R.M. (2001). Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J Exp Med 193, 713-726. Ibrahim, F.B., Pang, S.J., and Melendez, A.J. (2004). Anaphylatoxin signaling in human neutrophils. A key role for sphingosine kinase. J Biol Chem 279, 44802-44811. Igarashi, Y. (1997). Functional roles of sphingosine, sphingosine 1-phosphate, and methylsphingosines: in regard to membrane sphingolipid signaling pathways. J Biochem 122, 1080-1087. 96 REFERENCES Ingersoll, M.A., Platt, A.M., Potteaux, S., and Randolph, G.J. (2011). Monocyte trafficking in acute and chronic inflammation. Trends Immunol 32, 470-477. Jia, T., Serbina, N.V., Brandl, K., Zhong, M.X., Leiner, I.M., Charo, I.F., and Pamer, E.G. (2008). Additive roles for MCP-1 and MCP-3 in CCR2-mediated recruitment of inflammatory monocytes during Listeria monocytogenes infection. J Immunol 180, 6846-6853. Johnson, K.R., Becker, K.P., Facchinetti, M.M., Hannun, Y.A., and Obeid, L.M. (2002). PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1phosphate induced by phorbol 12-myristate 13-acetate (PMA). J Biol Chem 277, 35257-35262. Jordan, N.J., Watson, M.L., Yoshimura, T., and Westwick, J. (1996). Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts. Br J Pharmacol 117, 1245-1253. Jovanovic, V., Dai, X., Lim, Y.T., Kemeny, D.M., and MacAry, P.A. (2009). Fc gamma receptor biology and systemic lupus erythematosus. Int J Rheum Dis 12, 293-298. Ju, S., Ge, Y., Qiu, H., Lu, B., Qiu, Y., Fu, J., Liu, G., Wang, Q., Hu, Y., Shu, Y., et al. (2009). A novel approach to induce human DCs from monocytes by triggering 4-1BBL reverse signaling. Int Immunol 21, 1135-1144. Ju, S.W., Ju, S.G., Wang, F.M., Gu, Z.J., Qiu, Y.H., Yu, G.H., Ma, H.B., and Zhang, X.G. (2003). A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 41BBL. Hybrid Hybridomics 22, 333-338. Jung, H.W., Choi, S.W., Choi, J.I., and Kwon, B.S. (2004). Serum concentrations of soluble 4-1BB and 4-1BB ligand correlated with the disease severity in rheumatoid arthritis. Exp Mol Med 36, 13-22. Kang, Y.J., Kim, S.O., Shimada, S., Otsuka, M., Seit-Nebi, A., Kwon, B.S., Watts, T.H., and Han, J. (2007). Cell surface 4-1BBL mediates sequential signaling pathways 'downstream' of TLR and is required for sustained TNF production in macrophages. Nat Immunol 8, 601-609. Kee, T.H., Vit, P., and Melendez, A.J. (2005). Sphingosine kinase signalling in immune cells. Clin Exp Pharmacol Physiol 32, 153-161. 97 REFERENCES Kienzle, G., and von Kempis, J. (2000). CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes. Int Immunol 12, 73-82. Kim, D.K., Lee, S.C., and Lee, H.W. (2009). CD137 ligand-mediated reverse signals increase cell viability and cytokine expression in murine myeloid cells: involvement of mTOR/p70S6 kinase and Akt. Eur J Immunol 39, 2617-2628. Kim, H.J., Lee, J.S., Kim, J.D., Cha, H.J., Kim, A., Lee, S.K., Lee, S.C., Kwon, B.S., Mittler, R.S., Cho, H.R., et al. (2012). Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation. Proc Natl Acad Sci U S A 109, E13-22. Kishimoto, T., Goyert, S., Kikutani, H., Mason, D., Miyasaka, M., Moretta, L., Ohno, T., Okumura, K., Shaw, S., Springer, T.A., et al. (1997). Update of CD antigens, 1996. J Immunol 158, 3035-3036. Kohama, T., Olivera, A., Edsall, L., Nagiec, M.M., Dickson, R., and Spiegel, S. (1998). Molecular cloning and functional characterization of murine sphingosine kinase. J Biol Chem 273, 23722-23728. Kwajah M M, S., and Schwarz, H. (2010). CD137 ligand signaling induces human monocyte to dendritic cell differentiation. Eur J Immunol 40, 19381949. Lai, W.Q., Chia, F.L., and Leung, B.P. (2012). Sphingosine kinase and sphingosine-1-phosphate receptors: novel therapeutic targets of rheumatoid arthritis? Future Med Chem 4, 727-733. Lai, W.Q., Goh, H.H., Bao, Z., Wong, W.S., Melendez, A.J., and Leung, B.P. (2008a). The role of sphingosine kinase in a murine model of allergic asthma. J Immunol 180, 4323-4329. Lai, W.Q., Irwan, A.W., Goh, H.H., Howe, H.S., Yu, D.T., Valle-Oñate, R., McInnes, I.B., Melendez, A.J., and Leung, B.P. (2008b). Anti-inflammatory effects of sphingosine kinase modulation in inflammatory arthritis. J Immunol 181, 8010-8017. Lai, W.Q., Irwan, A.W., Goh, H.H., Melendez, A.J., McInnes, I.B., and Leung, B.P. (2009). Distinct roles of sphingosine kinase 1 and 2 in murine collageninduced arthritis. J Immunol 183, 2097-2103. 98 REFERENCES Langstein, J., Becke, F.M., Söllner, L., Krause, G., Brockhoff, G., Kreutz, M., Andreesen, R., and Schwarz, H. (2000). Comparative analysis of CD137 and LPS effects on monocyte activation, survival, and proliferation. Biochem Biophys Res Commun 273, 117-122. Langstein, J., Michel, J., Fritsche, J., Kreutz, M., Andreesen, R., and Schwarz, H. (1998). CD137 (ILA/4-1BB), a member of the TNF receptor family, induces monocyte activation via bidirectional signaling. J Immunol 160, 24882494. Langstein, J., Michel, J., and Schwarz, H. (1999). CD137 induces proliferation and endomitosis in monocytes. Blood 94, 3161-3168. Langstein, J., and Schwarz, H. (1999). Identification of CD137 as a potent monocyte survival factor. J Leukoc Biol 65, 829-833. Lee, S.K., Qing, W.G., Mar, W., Luyengi, L., Mehta, R.G., Kawanishi, K., Fong, H.H., Beecher, C.W., Kinghorn, A.D., and Pezzuto, J.M. (1998). Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C. J Biol Chem 273, 19829-19833. Li, L., Huang, L., Sung, S.S., Vergis, A.L., Rosin, D.L., Rose, C.E., Lobo, P.I., and Okusa, M.D. (2008). The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia-reperfusion injury. Kidney Int 74, 1526-1537. Lippert, U., Zachmann, K., Ferrari, D.M., Schwarz, H., Brunner, E., MahbubUl Latif, A.H., Neumann, C., and Soruri, A. (2008). CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response. Eur J Immunol 38, 1024-1032. Liu, G.Z., Gomes, A.C., Putheti, P., Karrenbauer, V., Kostulas, K., Press, R., Hillert, J., Hjelmström, P., and Gao, X.G. (2006). Increased soluble 4-1BB ligand (4-1BBL) levels in peripheral blood of patients with multiple sclerosis. Scand J Immunol 64, 412-419. Liu, H., Sugiura, M., Nava, V.E., Edsall, L.C., Kono, K., Poulton, S., Milstien, S., Kohama, T., and Spiegel, S. (2000). Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J Biol Chem 275, 19513-19520. Loma, I., and Heyman, R. (2011). Multiple sclerosis: pathogenesis and treatment. Curr Neuropharmacol 9, 409-416. 99 REFERENCES Lotz, M., Setareh, M., von Kempis, J., and Schwarz, H. (1996). The nerve growth factor/tumor necrosis factor receptor family. J Leukoc Biol 60, 1-7. Maceyka, M., Sankala, H., Hait, N.C., Le Stunff, H., Liu, H., Toman, R., Collier, C., Zhang, M., Satin, L.S., Merrill, A.H., et al. (2005). SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem 280, 37118-37129. Maerten, P., Geboes, K., De Hertogh, G., Shen, C., Cadot, P., Bullens, D.M., Van Assche, G., Penninckx, F., Rutgeerts, P., and Ceuppens, J.L. (2004). Functional expression of 4-1BB (CD137) in the inflammatory tissue in Crohn's disease. Clin Immunol 112, 239-246. Maines, L.W., Fitzpatrick, L.R., French, K.J., Zhuang, Y., Xia, Z., Keller, S.N., Upson, J.J., and Smith, C.D. (2008). Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase. Dig Dis Sci 53, 997-1012. Melendez, A., Floto, R.A., Cameron, A.J., Gillooly, D.J., Harnett, M.M., and Allen, J.M. (1998). A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium. Curr Biol 8, 210221. Melendez, A.J., Bruetschy, L., Floto, R.A., Harnett, M.M., and Allen, J.M. (2001). Functional coupling of FcgammaRI to nicotinamide adenine dinucleotide phosphate (reduced form) oxidative burst and immune complex trafficking requires the activation of phospholipase D1. Blood 98, 3421-3428. Melendez, A.J., Carlos-Dias, E., Gosink, M., Allen, J.M., and Takacs, L. (2000). Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution. Gene 251, 19-26. Melendez, A.J., and Ibrahim, F.B. (2004). Antisense knockdown of sphingosine kinase 1 in human macrophages inhibits C5a receptor-dependent signal transduction, Ca2+ signals, enzyme release, cytokine production, and chemotaxis. J Immunol 173, 1596-1603. Michaud, J., Kohno, M., Proia, R.L., and Hla, T. (2006). Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice. FEBS Lett 580, 4607-4612. Michel, J., Langstein, J., Hofstädter, F., and Schwarz, H. (1998). A soluble form of CD137 (ILA/4-1BB), a member of the TNF receptor family, is 100 REFERENCES released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. Eur J Immunol 28, 290-295. Michel, J., Pauly, S., Langstein, J., Krammer, P.H., and Schwarz, H. (1999). CD137-induced apoptosis is independent of CD95. Immunology 98, 42-46. Michel, J., and Schwarz, H. (2000). Expression of soluble CD137 correlates with activation-induced cell death of lymphocytes. Cytokine 12, 742-746. Nava, V.E., Lacana, E., Poulton, S., Liu, H., Sugiura, M., Kono, K., Milstien, S., Kohama, T., and Spiegel, S. (2000). Functional characterization of human sphingosine kinase-1. FEBS Lett 473, 81-84. Nishimoto, H., Lee, S.W., Hong, H., Potter, K.G., Maeda-Yamamoto, M., Kinoshita, T., Kawakami, Y., Mittler, R.S., Kwon, B.S., Ware, C.F., et al. (2005). Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factor receptor superfamily, with the high-affinity IgE receptor. Blood 106, 4241-4248. Olivera, A., and Spiegel, S. (2001a). Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins 64, 123-134. Olivera, A., and Spiegel, S. (2001b). Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins Other Lipid Mediat 64, 123-134. Olofsson, P.S., Söderström, L.A., Wågsäter, D., Sheikine, Y., Ocaya, P., Lang, F., Rabu, C., Chen, L., Rudling, M., Aukrust, P., et al. (2008). CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice. Circulation 117, 1292-1301. Orr Gandy, K.A., and Obeid, L.M. (2013). Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: Review of sphingosine kinase inhibitors. Biochim Biophys Acta 1831, 157-166. Pajak, B., Orzechowska, S., Gajkowska, B., and Orzechowski, A. (2008). Bisindolylmaleimides in anti-cancer therapy - more than PKC inhibitors. Adv Med Sci 53, 21-31. Park, S.W., Kim, M., Kim, J.Y., Brown, K.M., Haase, V.H., D'Agati, V.D., and Lee, H.T. (2012). Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia. Kidney Int 82, 878-891. 101 REFERENCES Paugh, S.W., Paugh, B.S., Rahmani, M., Kapitonov, D., Almenara, J.A., Kordula, T., Milstien, S., Adams, J.K., Zipkin, R.E., Grant, S., et al. (2008). A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia. Blood 112, 1382-1391. Pettus, B.J., Bielawski, J., Porcelli, A.M., Reames, D.L., Johnson, K.R., Morrow, J., Chalfant, C.E., Obeid, L.M., and Hannun, Y.A. (2003). The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha. FASEB J 17, 14111421. Pitman, M.R., and Pitson, S.M. (2010). Inhibitors of the sphingosine kinase pathway as potential therapeutics. Curr Cancer Drug Targets 10, 354-367. Pitson, S.M., Moretti, P.A., Zebol, J.R., Lynn, H.E., Xia, P., Vadas, M.A., and Wattenberg, B.W. (2003). Activation of sphingosine kinase 1 by ERK1/2mediated phosphorylation. EMBO J 22, 5491-5500. Pitson, S.M., Moretti, P.A., Zebol, J.R., Xia, P., Gamble, J.R., Vadas, M.A., D'Andrea, R.J., and Wattenberg, B.W. (2000). Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase. J Biol Chem 275, 3394533950. Power, C.A., and Proudfoot, A.E. (2001). The chemokine system: novel broad-spectrum therapeutic targets. Curr Opin Pharmacol 1, 417-424. Quek, B.Z., Lim, Y.C., Lin, J.H., Tan, T.E., Chan, J., Biswas, A., and Schwarz, H. (2010). CD137 enhances monocyte-ICAM-1 interactions in an E-selectindependent manner under flow conditions. Mol Immunol 47, 1839-1847. Reali, C., Curto, M., Sogos, V., Scintu, F., Pauly, S., Schwarz, H., and Gremo, F. (2003). Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2. J Neurosci Res 74, 67-73. Rottman, J.B. (1999). Key role of chemokines and chemokine receptors in inflammation, immunity, neoplasia, and infectious disease. Vet Pathol 36, 357-367. Saito, K., Ohara, N., Hotokezaka, H., Fukumoto, S., Yuasa, K., Naito, M., Fujiwara, T., and Nakayama, K. (2004). Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on 102 REFERENCES M-CSF/RANKL-induced in vitro osteoclastogenesis. J Biol Chem 279, 13555-13563. Salih, H.R., Kosowski, S.G., Haluska, V.F., Starling, G.C., Loo, D.T., Lee, F., Aruffo, A.A., Trail, P.A., and Kiener, P.A. (2000). Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J Immunol 165, 29032910. Salih, H.R., Schmetzer, H.M., Burke, C., Starling, G.C., Dunn, R., PelkaFleischer, R., Nuessler, V., and Kiener, P.A. (2001). Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J Immunol 167, 4059-4066. Schwarz, H. (2005). Biological activities of reverse signal transduction through CD137 ligand. J Leukoc Biol 77, 281-286. Schwarz, H., Arden, K., and Lotz, M. (1997). CD137, a member of the tumor necrosis factor receptor family, is located on chromosome 1p36, in a cluster of related genes, and colocalizes with several malignancies. Biochem Biophys Res Commun 235, 699-703. Schwarz, H., Blanco, F.J., von Kempis, J., Valbracht, J., and Lotz, M. (1996). ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival. Blood 87, 2839-2845. Schwarz, H., Valbracht, J., Tuckwell, J., von Kempis, J., and Lotz, M. (1995). ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood 85, 1043-1052. Serbina, N.V., and Pamer, E.G. (2006). Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat Immunol 7, 311-317. Sethu, S., Mendez-Corao, G., and Melendez, A.J. (2008). Phospholipase D1 plays a key role in TNF-alpha signaling. J Immunol 180, 6027-6034. Shao, Z., and Schwarz, H. (2011). CD137 ligand, a member of the tumor necrosis factor family, regulates immune responses via reverse signal transduction. J Leukoc Biol 89, 21-29. Shin, H.H., Lee, E.A., Kim, S.J., Kwon, B.S., and Choi, H.S. (2006a). A signal through 4-1BB ligand inhibits receptor for activation of nuclear factor103 REFERENCES kappaB ligand (RANKL)-induced osteoclastogenesis by increasing interferon (IFN)-beta production. FEBS Lett 580, 1601-1606. Shin, H.H., Lee, J.E., Lee, E.A., Kwon, B.S., and Choi, H.S. (2006b). Enhanced osteoclastogenesis in 4-1BB-deficient mice caused by reduced interleukin-10. J Bone Miner Res 21, 1907-1912. Snider, A.J., Orr Gandy, K.A., and Obeid, L.M. (2010). Sphingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation. Biochimie 92, 707-715. Spiegel, S. (1999). Sphingosine 1-phosphate: a prototype of a new class of second messengers. J Leukoc Biol 65, 341-344. Söllner, L., Shaqireen D O Kwajah, M.M., Wu, J.T., and Schwarz, H. (2007). Signal transduction mechanisms of CD137 ligand in human monocytes. Cell Signal 19, 1899-1908. Sørensen, T.L., Tani, M., Jensen, J., Pierce, V., Lucchinetti, C., Folcik, V.A., Qin, S., Rottman, J., Sellebjerg, F., Strieter, R.M., et al. (1999). Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J Clin Invest 103, 807-815. Tan, S.L., and Parker, P.J. (2003). Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem J 376, 545-552. Tang, Q., Jiang, D., Shao, Z., Martínez Gómez, J.M., and Schwarz, H. (2011). Species difference of CD137 ligand signaling in human and murine monocytes. PLoS One 6, e16129. Thum, E., Shao, Z., and Schwarz, H. (2009). CD137, implications in immunity and potential for therapy. Front Biosci 14, 4173-4188. Tsou, C.L., Peters, W., Si, Y., Slaymaker, S., Aslanian, A.M., Weisberg, S.P., Mack, M., and Charo, I.F. (2007). Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J Clin Invest 117, 902-909. Wong, L., Tan, S.S., Lam, Y., and Melendez, A.J. (2009). Synthesis and evaluation of sphingosine analogues as inhibitors of sphingosine kinases. J Med Chem 52, 3618-3626. 104 REFERENCES Xia, P., Gamble, J.R., Rye, K.A., Wang, L., Hii, C.S., Cockerill, P., KhewGoodall, Y., Bert, A.G., Barter, P.J., and Vadas, M.A. (1998). Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc Natl Acad Sci U S A 95, 14196-14201. Xia, P., Wang, L., Gamble, J.R., and Vadas, M.A. (1999). Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells. J Biol Chem 274, 34499-34505. Xia, P., Wang, L., Moretti, P.A., Albanese, N., Chai, F., Pitson, S.M., D'Andrea, R.J., Gamble, J.R., and Vadas, M.A. (2002). Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-alpha signaling. J Biol Chem 277, 7996-8003. Yang, J., Park, O.J., Lee, Y.J., Jung, H.M., Woo, K.M., and Choi, Y. (2008). The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling. Eur J Immunol 38, 1598-1609. Yeo, Y.A., Martínez Gómez, J.M., Croxford, J.L., Gasser, S., Ling, E.A., and Schwarz, H. (2012). CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species. J Neuroinflammation 9, 173. Yndestad, A., Damås, J.K., Geir Eiken, H., Holm, T., Haug, T., Simonsen, S., Frøland, S.S., Gullestad, L., and Aukrust, P. (2002). Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure. Cardiovasc Res 54, 175-182. Zlotnik, A., and Yoshie, O. (2012). The chemokine superfamily revisited. Immunity 36, 705-716. Ph.D Thesis Reference Tan, S.L.S. (2011). Role of Sphingosine Kinase 1 in colorectal cancer. National University Of Singapore, Ph.D Thesis. 105 ABSTRACT FOR CONFERENCE   ABSTRACT FOR CONFERENCE Role of Sphingosine Kinase in CD137L Signaling Pathway Background: CD137 (ILA/4-1BB), a member of tumour necrosis factor (TNF) receptor family, is a potent regulator of T cells activities. Both CD137 receptor and CD137 ligand (CD137L) are expressed as cell membrane proteins which allow bidirectional signalling. The signalling displayed by the CD137L is not well established especially the signalling mechanism and the molecules mediating it. It has been shown that cross-linking the CD137L induces proliferations, expression of M-CSF and the expression of several proinflammatory cytokines such as IL6, IL8 and TNF-α. In this study, we investigated the signal transduction pathways triggered by CD137L on human monocytic cells, with particular emphasis on the role of sphingosine kinase (SphK). Our laboratory has shown that SphK is activated by several immunereceptors, on various immune-effector cells, and that SphK mediates various pro-inflammatory responses. SphK phosphorylates sphingosine to sphingosine-1-phosphate and can be inhibited by the sphingosine-analogue N,N-dimethylsphingosine (DMS). Methods: CD137-Fc protein and anti-human 4-1BB ligand clone 5F4 antibody were used to crosslink the CD137L on THP1 cells. 10µM of DMS were used to inhibit SphK activity. We measured the CD137L-mediated cytokine release (IL-8, IL-6) by ELISA, performed western blot for protein expression of IκB and phosphorylated p38 MAPK. We also analysed cell degranulation by measuring beta-hexosaminidase release. Results: Our findings showed that crosslinking CD137L triggers SphK activity and leads to the production of IL6 and IL8. Inhibition of SphK by 106 ABSTRACT FOR CONFERENCE   DMS inhibited the CD137L-triggered IL-6 production at 24 hours. Interestingly, the production of IL-8 was not inhibited by DMS. Western blot results showed that IκB is degraded following CD137L stimulation. Degradation of IκB is an indirect indicative of the activation of NF-κB. In cells pre-treated with DMS, there was a substantial inhibition on IκB degradation. Phosphorylated p38 was found to be inhibited by DMS. CD137L does not appear to trigger degranulation. Taken together the results presented here demonstrate that CD137L triggers intracellular signalling cascades in human monocytic cells, and suggest a key role for SphK in the signal transduction pathway leading to the activation of p38 MAPK, NF-κB and the subsequent production of IL-6 production. 107 [...]... 5-fluorouracil A1 AR A1 Adenosine Receptor ABC ATP Binding Cassette AIA Adjuvant-Induced Arthritis AICD Activation Induced Cell Death Akt Protein Kinase B (PKB) AML Acute Myeloid Leukaemia AP-1 Activator Protein-1 APC Antigen Presenting Cells APS Ammonium Persulfate ATCC American Type Culture Collection ATP Adenosine Triphosphate BAL Bronchoalveolar Lavage BD Behcet’s Disease Bis I Bisindolylmaleimide... system 4 CHAPTER 1: INTRODUCTION CD137 Ligand CD137 Figure A Bidirectional signal transduction CD137 and its ligand are capable of inducing bidirectional signalling into the cells expressing them 5 CHAPTER 1: INTRODUCTION 1.1.3 Soluble CD137 And CD137 Ligand Soluble CD137 (sCD137) and its ligand (sCD137L) have been reported in inflammatory diseases as well as cancers sCD137 is released by activated lymphocytes... and primary microglia (Yeo et al., 2012) As a transmembrane protein, CD137L can act as a co-receptor, and upon binding to CD137, CD137L too can trigger signalling cascade downstream in the cells expressing it (Figure A) (Shao and Schwarz, 2011) 3 CHAPTER 1: INTRODUCTION This is a unique feature of many receptor -ligand pairs in TNF superfamily that exhibits bidirectional signalling such as OX40L and CD40L... to increase the release of IL-6, IL-8, TNF-α and to inhibit IL-10 (Langstein et al., 1998) The expression of intracellular adhesion molecule (ICAM) is also induced, indicating the differentiation of monocyte to macrophages (Langstein et al., 1998) The CD137L activated signalling pathway induces the activation of tyrosine kinase, mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated... of IL-8 4 Sphingosine kinase 1 was expressed in THP-1 cells and 65 U-937 cells 5 Cross-linking of CD137 ligand induced sphingosine 67 kinase activity in THP-1 cells 6 Sphingosine kinase 1 protein level remained unchanged 69 after 24 hours of cross-linking of CD137 ligand 7 Cross-linking of CD137 ligand induced the production of 72 MCP-1 Sphingosine kinase inhibitors decrease the CD137L-induced MCP-1... osteoblasts that had been infected by bacteria (Saito et al., 2004) CD137L was detected on the macrophage like cell line (RAW264.7) and on bone marrow cells (Saito et al., 2004) CD137L too induced proliferation and the release of M-CSF in primary bone marrow cells and BMM (Saito et al., 2004) CD137L activation inhibited the formation of osteoclasts in bone marrow (Saito et al., 2004) Casein kinase 1... and it was speculated that CD137L may play a role in the bone and cartilages destruction in final stage of RA (Saito et al., 2004; Shin et al., 200 6a; Shin et al., 2006b; Yang et al., 2008) In a murine model of acute kidney ischemia-reperfusion injury (IRI), CD137 is expressed on NK cells and CD137L on the tubular epithelial cells (TECs) (Kim et al., 2012) NK cells activated CD137L reverse signalling. .. stimulating factor (GM-CSF) and IL-3 (Langstein et al., 2000; Langstein et al., 1999; Langstein and Schwarz, 1999) M-CSF also acts as the essential factor to support monocyte proliferation because neutralizing anti-M-CSF antibodies greatly reduced the viability of the cell (Langstein et al., 1999; Langstein and Schwarz, 1999) IL8 release, morphological changes and adhesiveness of the cells are indicators... Ischemia-Reperfusion Injury ITAC IFN-γ-Inducible T -Cell Chemoattractant I-κB NF-κB inhibitor JNK C-Jun N-Terminal Kinases KC Keratinocyte Chemoattractant KCl Potassium Chloride LPA Lysophosphatidic Acid LPS Lipopolysaccharide MAP Mitogen-Activated Protein Kinase MCP Monocyte Chemotactic Protein M-CSF Macrophage-Colony Stimulating Factor MEK MAPK/ERK Kinase MgCl2 Magnesium Chloride Mig Monokine Induced By Interferon-γ... Bidirectional signal transduction 5 B Sphingosine metabolism pathway 20 C Proposed CD137L signalling pathway 91 FIGURE PAGE CHAPTER 3: RESULTS 1 CD137 ligand induced THP-1 cells activation 59 2 Sphingosine kinase inhibitors decrease CD137L-induced 62 IL-8 secretion 3 Phospholipase D inhibitor, Butan-1-ol, did not show any 63 inhibition in CD137L-induced THP-1 cell activation as shown by the release of ... 5-fluorouracil A1 AR A1 Adenosine Receptor ABC ATP Binding Cassette AIA Adjuvant-Induced Arthritis AICD Activation Induced Cell Death Akt Protein Kinase B (PKB) AML Acute Myeloid Leukaemia AP-1 Activator... Expression Of Sphingosine Kinase 4.4 CD137 Ligand Signalling Activates Sphingosine Kinase 4.5 Involvement Of Sphingosine Kinases In CD137 Ligand- Induced Inflammatory Chemokines Production 4.6 Involvement... expressed in microglia cell lines and primary microglia (Yeo et al., 2012) As a transmembrane protein, CD137L can act as a co-receptor, and upon binding to CD137, CD137L too can trigger signalling cascade