STUDIES ON THE INTRACELLULAR SIGNALING PATHWAYS TRIGGERED BY THE ANAPHYLATOXIN C5a IN HUMAN PHAGOCYTIC CELLS FARAZEELA BINTE MOHD IBRAHIM (B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I am extremely grateful and indebted to my supervisor, Dr Alirio J Melendez, for his advice and guidance throughout the course of my research in the lab. Regardless of his commitments and busy schedule, he always found time to supervise my work and was never short of comforting and motivating words for me, especially when the going was tough. Without his support and encouragement, this work would not have been accomplished. It was an eye-opening and wonderful experience to conduct research under his supervision. Thank you very much for introducing me to this fascinating world of research. Special thanks belong to my lab colleagues who have given me excellent cooperation and assistance throughout my stay in the Molecular and Cellular Immunology Lab in the Department of Physiology. I am honored to have had the opportunity to work with each and every one of them in different aspects of my research. In them, I have found firm friends and I truly cherish the friendship we share. I wish to acknowledge my deepest gratitude and appreciation to my husband, who has been my constant source of encouragement and moral support, my pillar of strength and my confidante, without whom this journey would have been that much harder. I am thankful to my parents, sisters and family members for their support and love throughout my life. The knowledge of them being there has been of great encouragement and importance to me. I would also like to thank Ms Anneke Melendez-Fraser for her helpful comments, invaluable advice and the time spent proofreading this thesis. i TABLE OF CONTENTS Acknowledgements i Table of Contents ii Summary vii List of Figures ix Abbreviations xii List of Publications xiv List of Posters and Abstracts presented xv CHAPTER I 1.1 1.2 INTRODUCTION The complement system 1.1.1 Complement proteins and nomenclature 1.1.2 Activation of the complement cascade 1.1.2.1 The classical pathway 1.1.2.2 The alternative pathway 1.1.2.3 The mannan-binding lectin (MBL) pathway 1.1.3 Functions of the complement system 1.1.4 Regulation of the complement system 1.1.5 Complement component C5a 10 1.1.5.1 Biological properties of C5a 10 1.1.5.2 Structure of ligand C5a 11 1.1.5.3 Complement 5a receptor (C5aR) 13 1.1.5.4 Role of C5a in diseases 16 Important downstream events triggered by C5a 17 1.2.1 NADPH oxidase 17 ii 1.3 1.4 1.5 1.2.2 Nuclear factor kappa B 18 1.2.3 Matrix metalloproteinases 20 1.2.4 Raf-Mitogen activated protein kinases 22 1.2.5 Cytokines 24 Sphingosine Kinase (SPHK) 26 1.3.1 Sphingolipid metabolism 26 1.3.2 Properties of SPHK 28 1.3.3 Activation and regulation of SPHK 29 1.3.4 Sphingosine-1-phosphate (S1P) 30 1.3.5 Role of SPHK and S1P in cellular processes 30 1.3.6 Role of SPHK and S1P in immune cells 32 Phospholipase D (PLD) 35 1.4.1 Transphosphatidylation reaction 35 1.4.2 Diversity and structure of PLD enzymes 37 1.4.3 Properties of mammalian PLD 38 1.4.4 Regulation of PLD enzymes 40 1.4.5 Downstream signaling of PLD products 41 1.4.6 43 PLD activity in immune cells Rationale of the project CHAPTER II 44 MATERIALS AND METHODS 2.1 Chemicals 45 2.2 Solutions and Buffers 47 2.3 Cell culture and differentiation of cells 50 iii 2.4 Transfection of cells with oligonucleotides 50 2.5 RNA extraction and reverse transcription-PCR 51 2.6 Isolation of primary human neutrophils 51 2.7 Isolation of primary human monocytes and differentiation to macrophages 52 2.8 C5a receptor stimulation 52 2.9 Measurement of sphingosine kinase activity in cell extracts 53 2.10 Measurement of sphingosine-1-phosphate generation in whole cells 53 2.11 Phospholipase D activity assay 54 2.12 Phospholipase C activity assay 55 2.13 Protein kinase C activity assay 55 2.14 Cytosolic calcium measurement 56 2.15 Chemotaxis assay 56 2.16 NADPH oxidative burst assay 57 2.17 Degranulation assay 57 2.18 Subcellular fractionation by differential centrifugation 58 2.19 Gel electrophoresis and western blotting analysis 58 2.20 Measurement of NF-κB activation 59 2.21 Measurement of cytokine release 61 2.22 Measurement of matrix metalloproteinase release 62 2.23 Fluorescence microscopy 63 2.24 Flow cytometry for cell viability 63 CHAPTER III 3.1 RESULTS Key role for sphingosine kinase in C5a signaling in human neutrophils 64 3.1.1 67 C5a stimulates SPHK activity in human neutrophils iv 3.1.2 Role of SPHK in C5a-triggered Ca2+ signals 70 3.1.3 Role of SPHK in C5a-triggered degranulation 73 3.1.4 Role of SPHK in C5a-triggered chemotaxis 73 3.1.5 Role of SPHK in C5a-triggered NADPH oxidative burst 74 3.1.6 SPHK is the enzyme activated by C5a. Antisense knockdown of SPHK1 78 3.1.7 SPHK1 mediates C5a-triggered Ca2+ release, degranulation, chemotaxis and NADPH oxidase activity 80 3.1.8 Effects of DMS and/or antisense oligonucleotides 83 3.1.9 Effects of sphingosine and sphingosine-1-phosphate on signaling 87 3.1.10 Discussion 3.2. 3.3 91 Key role for sphingosine kinase in C5a signaling in human macrophages 96 3.2.1 C5a stimulates SPHK activity in human macrophages 98 3.2.2 SPHK1 is the enzyme activated by C5a. Antisense knockdown of SPHK1 100 3.2.3 Role of SPHK1 in C5a-triggered Ca2+ signals 103 3.2.4 Role of SPHK1 in C5a-triggered PKC activity 105 3.2.5 Role of SPHK1 in C5a-triggered degranulation 105 3.2.6 Role of SPHK1 in C5a-triggered chemotaxis 108 3.2.7 Role of SPHK1 in C5a-triggered cytokine production 108 3.2.8 Discussion 112 Potential role for phospholipase D in C5a signaling in macrophages 116 3.3.1 C5a stimulates PLD activity in dbcAMP-differentiated U937 cells 119 3.3.2 C5a stimulates the translocation and redistribution of PLD1 isoform 119 3.3.3 Role of PLD in C5a-triggered Ca2+ signals 122 v 3.3.4 Role of PLD in C5a-triggered NADPH oxidase activity 122 3.3.5 Role of PLD in C5a-triggered degranulation 125 3.3.6 Role of PLD in C5a-triggered chemotaxis 125 3.3.7 Role of PLD in C5a-triggered NF-κB translocation and activation 128 3.3.8 Role of PLD in C5a-triggered cytokine production 131 3.3.9 Role of PLD in C5a-triggered matrix metalloproteinase (MMP) release 131 3.3.10 C5a induces Raf-1 translocation and phosphorylation of ERK1/2 and p38 135 3.3.11 C5a-triggered PLD activity is potentially upstream of SPHK, ERK1/2, p38 MAPK and PKC 138 3.3.12 Discussion 140 CHAPTER IV CONCLUSION 149 CHAPTER V REFERENCES 152 vi SUMMARY Anaphylatoxins play a key role in inflammatory responses, and in many diseases they contribute to the pathogenesis. Inflammation is the body’s natural response to tissue damage and injury, and is mediated by several interconnected enzymatic pathways. One such pathway is the complement cascade, through which the anaphylatoxin C5a, a potent stimulator of mediators of chronic and acute inflammation, is generated. Although the actions of C5a are well established, the mechanisms regulating C5atriggered intracellular signaling pathways are poorly understood. The phospholipidmodifying enzymes, sphingosine kinase (SPHK) and phospholipase D (PLD), are emerging as important signaling molecules, and have been suggested to function as crucial players in the physiological responses triggered by activated immune-effector cells. Hence, the objective of my study is to investigate the intracellular signaling pathways triggered by C5a, particularly the roles of SPHK and PLD, in mediating proinflammatory functions in human neutrophils and macrophages. The ultimate goal is to identify key molecules as candidates for novel therapeutic intervention. In this thesis, I provide evidence that demonstrate, for the first time, that the anaphylatoxin C5a activates the intracellular signaling molecule SPHK, and present data that support the role for SPHK in the proinflammatory responses triggered by C5a in human neutrophils and macrophages, showing that inhibition of this enzyme has potential anti-inflammatory properties. We demonstrate that C5a receptor activation stimulates SPHK activity in these cells. Moreover, the inhibition of SPHK by DMS inhibits C5a-stimulated Ca2+ mobilization, degranulation, chemotaxis, and NADPH activation in these cells. Furthermore, an antisense oligonucleotide specific for SPHK1 also inhibited the C5a-induced responses, suggesting that SPHK1 is the vii isoform triggered by C5a. We also show here that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate (S1P), suggesting a role for SPHK in removing a negative regulator (sphingosine), and generating a positive regulator (S1P). We also studied the effects of exogenously added sphingosine and S1P in the neutrophils. We found that sphingosine has no effect on C5a-triggered Ca2+ signals, chemotaxis and degranulation, but dual effect on C5a-stimulated NADPH oxidase activation and minimal effect on C5a-triggered PKC activity. S1P by itself did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and the oxidative burst. However, S1P showed a priming effect, enhancing all C5a-triggered responses. I also present data that suggest the potential role of PLD in C5a-induced proinflammatory responses in macrophage-differentiated U937 cells. In the presence of a primary alcohol (butan-1-ol), C5a-triggered Ca2+ signals, NADPH oxidative burst, chemotaxis, degranulation, NF-κB translocation and activation, cytokine release and MMP release are significantly inhibited, suggesting a role for PLD in triggering these responses. I also show that C5a induces Raf-1 translocation, which may activate MAPKs, and that PLD activity is potentially upstream of some signaling enzymes. Thus, our data contribute not only to the understanding of the intracellular molecular mechanisms utilized by C5a, suggesting that SPHK and PLD potentially play key roles in C5a-triggered proinflammatory functions, but also point out SPHK and PLD as novel candidates for potential therapeutic intervention to treat inflammatory and autoimmune diseases. viii LIST OF FIGURES Introduction: Figure A Complement activation pathways Figure B Ribbon diagram of the human C5a molecule 12 Figure C Model for the interaction of C5a with C5aR 15 Figure D Sphingolipid metabolic pathway 27 Figure E PLD-catalyzed hydrolysis and transphosphatidylation reactions 36 Figure F PLD metabolic pathway 42 Figure G C5a-triggered intracellular signaling in human phagocytic cells 148 Figure C5a triggers SPHK activity in primary human neutrophils and differentiated HL-60 cells (neutrophil model) 68 Figure C5a triggers S1P generation in primary human neutrophils and differentiated HL-60 cells (neutrophil model) 69 Figure C5a-triggered cytosolic Ca2+ signals in neutrophils are inhibited by DMS: role for SPHK 71 Figure C5a-triggered phospholipase C activity in neutrophils is not inhibited by DMS 72 Figure Degranulation triggered by C5a in neutrophils is dependent on SPHK activity 75 Figure C5a-induced chemotaxis in neutrophils is inhibited by the SPHK inhibitor 76 Figure C5a-induced NADPH oxidase activity in neutrophils is inhibited by DMS 77 Results: ix • PLD running buffer: Ethyl acetate, iso-octane, acetic acid and distilled water in the ratio of 65:10:15:50 (v:v:v:v) • Lysis Buffer for nuclear extract preparation: 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, mM DTT and mM PMSF • Extraction Buffer for nuclear extract preparation: 20 mM HEPES (pH 7.9), 0.4 M NaCl, mM EDTA, mM EGTA, mM DTT and mM PMSF • Nuclear Preparation Buffer for subcellular fractionation: 10 mM Tris-HCl (pH 7.4), mM MgCl2, 0.14 M NaCl, 1% Triton X-100, mM EGTA, mM PMSF, 10 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin • Coating buffer for cytokine ELISA: NaHCO3 in 1X PBS • Assay buffer for cytokine ELISA: 1% FBS in 1X PBS • Washing buffer for cytokine ELISA: 0.05% Tween-20 in 1X PBS • Substrate solution for cytokine ELISA: tablet TMB in 10 ml 0.05 M citrate phosphate in 1x PBS and μl 30% H2O2 • Stop solution for cytokine ELISA: M H2SO4 • 12% Resolving gel for SDS-PAGE: distilled water, 30% bis-acrylamide, 1.5 M Tris (pH 8.8), 10% SDS, 10% APS, TEMED • 5% Stacking gel for SDS-PAGE: distilled water, 30% bis-acrylamide, 1.0 M Tris (pH 6.8), 10% SDS, 10% APS, TEMED • Running buffer for SDS-PAGE: 25 mM Tris base, 250 mM glycine (pH 8.3), 0.1% SDS • Transfer buffer for SDS-PAGE: 48 mM Tris base, 39 mM glycine, 0.037% SDS, 20% methanol • 1X SDS gel-loading buffer for SDS-PAGE: 50 mM Tris-HCl (pH 6.8), 100 mM βmercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol 48 • Washing buffer for SDS-PAGE: 1X TBS, 0.1% Tween-20 • Blocking buffer for SDS-PAGE: 5% non-fat milk in 1X TBST • 1X TBS for SDS-PAGE: 137 mM NaCl, 20 mM Tris-HCl (pH 7.6) • HEPES Ca2+-supplemented buffer for calcium assay: 145 mM NaCl, mM KCl, mM MgSO4, mM CaCl2, 10 mM HEPES, 0.18% Glucose, 0.2% BSA. • Substrate solution for macrophage degranulation assay: mM p-nitrophenyl-Nacetyl-D-glucosaminide in 0.1 M sodium citrate buffer (pH 4.5) • Substrate solution for neutrophil degranulation assay: mM 4-Nitrophenyl β-Dglucuronide in 0.1 M sodium citrate buffer (pH 4.5) 49 2.3 Cell culture and differentiation of cells The human promyelocytic leukemia (HL-60) and the human promonocytic lymphoma (U937) cell lines were used. All cells were maintained in RPMI 1640 (Sigma-Aldrich, Singapore) supplemented with 10% heat-inactivated FBS (Gibco, Invitrogen Singapore), 1% glutamine and 150 units/ml penicillin and 150μg/ml streptomycin. All cell lines were cultured in a 37°C incubator with 5% carbon dioxide in a watersaturated environment. The morphology of the cells was observed daily using a light microscope to ensure their health status before subsequently being used for experiments. Differentiation of cells into neutrophil-like cells for HL-60 cells, and into macrophage-like cells for U937 cells was induced by culturing the cells for 48 hrs in the presence of mM dbcAMP (Sigma-Aldrich, Singapore). 2.4 Transfection of cells with oligonucleotides Primary human macrophages and differentiated HL-60 cells were incubated for 48 hrs in the presence of antisense oligonucleotides for the transfection process. The antisense down-regulation was carried out as described (Melendez and Khaw, 2002). The antisense oligonucleotides were purchased from Oswell DNA Services (Southampton, U.K.). 20-mers were synthesized, capped at either end by the phosphorothioate linkages (the first two and last two linkages), and corresponded to the reverse complement of the first 20 coding nucleotides for SPHK1. A scrambled oligonucleotide was used as a control. The sequences of the oligonucleotides were as follows: 5’-CCCGCAGGATCCATAACCTC-3’ for antisense to SPHK1 5’CTGGTGGAAGAAGAGGACGT-3’ for the scrambled oligonucleotide control. The cells were transfected with μM of oligonucleotide mixed with FuGENE (Roche Molecular Biochemicals, Singapore) transfection reagent. 50 2.5 RNA extraction and reverse transcription – PCR mRNA from human-monocyte derived macrophages was isolated using the Qiagen midi kit (Qiagen, Valencia, CA) for mRNA extraction. Specific forward (5’TGAACCCGCGCGGCAAGGGC-3’) and reverse (5’- GGTCAGCCGGCGCCATCCACG-3’) primers were designed for the human SPHK1 to yield a 570-bp fragment, and specific forward (5’- CCATGGCGAGTTTGGCTCCT-3’) and reverse (5’-CATCCC CGGGCAGTGG AGTCCTCGG-3’) primers were designed for the human SPHK2 to yield a 356-bp fragment. 2.6 Isolation of primary human neutrophils Neutrophils were purified from healthy donors as described (Smith et al., 1991). Briefly, this was done using dextran sedimentation, followed by density gradient centrifugation with Lymphoprep (Nycomed, Norway) and hypotonic lysis of erythrocytes. Cells were resuspended in assay medium (RPMI 1640 medium with 10 mM HEPES and 2.5% FBS) before use. Cytological examination of stained centrifuged preparations showed 95% of the cells were neutrophils. Trypan Blue staining confirmed that >98% of these cells were viable. After purification, the cells (2 x 106/ml) were resuspended in RPMI 1640 medium supplemented with 2.5% FBS and allowed to recover for 30 mins at 37°C in 5% CO2 atmosphere. For experiments, the cells were left untreated or pretreated with DMS (10 μM) (Calbiochem, Merck Singapore) for 20 mins before stimulation. 51 2.7 Isolation of macrophages primary human monocytes and differentiation to Mononuclear cells were isolated from heparinized fasting venous blood by FicollPaqueTM (GE Healthcare Bio-Sciences, Singapore) centrifugation as described (Devaraj et al., 2001). 20 ml of blood (anticoagulated with 10 unit/ml heparin) was layered carefully on 15 ml of Ficoll-PaqueTM gradient and centrifuged at 500 x g, without brakes at room temperature for 30 mins. The mixed mononuclear band was aspirated and the cells were washed three times in phenol red RPMI 1640 medium containing 100 units/ml penicillin, 100 µg/ml streptomycin, and mM glutamine and suspended in a known volume. Leukocyte count was performed on a Coulter counter and then cells were plated (5-7 × 106 cells) in six-well culture plates in RPMI 1640 medium. Incubation was conducted at 37°C for h in 5% CO2/95% air, after which non-adherent cells were removed by washing the wells twice with RPMI 1640, and the remaining adherent cells were grown in the culture medium supplemented with 10% FBS and mM glutamine. The medium was replaced every 2–3 days. The cells were used after days of culture. Cell viability, determined by Trypan Blue exclusion, was >94% in all experiments. 2.8 C5a receptor stimulation The cells were resuspended in RPMI 1640 medium supplemented with 2.5% FBS. Depending on the particular experiments, the cells were left untreated or pretreated with DMS (10 μM), sphingosine (at the concentrations indicated), S1P (10 μM), butan-1-ol (0.5%), butan-2-ol (0.5%), PD98059 (50 μM), SB203580 (10 μM) or BisI (50 nM) for 20 mins, or with antisense oligonucleotides for 48 hrs. They were then stimulated by the addition of μg/ml of C5a (Sigma-Aldrich, Singapore) and incubated at 37°C for the times indicated. 52 2.9 Measurement of sphingosine kinase activity in cell extracts Sphingosine kinase was assayed as described (Melendez et al., 1998b). This assay is based on the SPHK-catalyzed transfer of the γ-phosphate group of ATP (using a mixture of cold ATP and [γ32P]ATP to a specific substrate. Briefly, after C5a stimulation, reactions were terminated at the times specified in the figures by the addition of ice-cold PBS. After centrifugation, primary human macrophages and neutrophils, and differentiated HL-60 cells (2 x 106/ml) were resuspended in ice-cold sphingosine kinase assay buffer. Cells were disrupted by freeze thawing and centrifuged at high speed. Supernatants were assayed for sphingosine kinase activity using sphingosine (Sigma-Aldrich, Singapore) and [γ32P]ATP (1 μCi/sample)(GE Healthcare Bio-Sciences, Singapore) as specified (Olivera et al., 1994). After incubation, the products were separated by TLC on silica Gel G60 (Whatman, Maidstone, U.K.) using the sphingosine kinase running buffer, and visualized by autoradiography. The radioactive spots corresponding to sphingosine-1-phosphate were scraped and counted in a scintillation counter. 2.10 Measurement of sphingosine-1-phosphate generation in whole cells Sphingosine kinase activity in intact cells was measured by assaying the amount of intracellular sphingosine-1-phosphate generated following receptor activation as described (Melendez et al., 1998a). Briefly, primary human macrophages and neutrophils, and differentiated HL-60 cells were preincubated overnight in media containing [3H]Serine (2 μCi/ml) (GE Healthcare Bio-Sciences, Singapore) to label cellular sphingolipids and free sphingosine pools. Following labeling, the cells (2 x 106/sample) were washed in ice-cold medium and resuspended in media containing 4deoxypyridoxine to inhibit pyridoxal-dependent sphingosine-1-phosphate lyase. Cells 53 were then stimulated by the addition of C5a and warming to 37°C, and the reactions were terminated at specified times. Cells were harvested by centrifugation and lipids were extracted and analyzed by TLC on silica Gel G60 (Whatman, Maidstone, U.K.) using the sphingosine kinase running buffer. Standard sphingosine-1-phosphate Avanti Polar Lipids Inc., Alabaster, AL) was applied with the samples, and the lipids were visualized using iodine vapors. Bands corresponding to S1P were excised from the plate and counted by liquid scintillation spectrometry. Results were calculated as a percentage of the total radioactivity incorporated in the lipids. 2.11 Phospholipase D activity assay PLD activity was measured by the transphosphatidylation assay (Melendez et al., 2001a). Briefly, U937 cells (5 x 106/sample) were first differentiated with dbcAMP to macrophage-like cells. After 30 hrs of treatment, the cells were labeled with [3H]Palmitic acid (1 μCi/ml) (GE Healthcare Bio-Sciences, Singapore) in cell culture medium for 18 hrs. Following labeling, the cells were washed in ice-cold medium and resuspended in cell culture medium containing 1% ethanol. The cells were left on ice for 20 mins in the presence or absence of inhibitors before receptor stimulation for 30 mins at 37oC. Lipids were then extracted by Bligh–Dyer phase separation (Bligh and Dyer, 1959). An aliquot of the lower organic phase was removed to determine the content of radioactivity in the total lipid fraction. Another aliquot was also removed to quantitate the amount of product generated. Phosphatidylethanol (PtdEtOH) standard (MP Biomedicals Inc., Singapore) was added to these samples before being dried down under vacuum (Savant ISS110), then redissolved in 50 μl of 19:1 (v:v) chloroform/methanol and applied to pre-run, heat activated TLC silica Gel G60 plates (Whatman, Maidstone, U.K). The plate was developed in the organic phase of PLD 54 running buffer for approximately 90 mins. [3H]PtdEtOH was identified by the mobility of authentic standard visualized with iodine vapor. Lipids containing [3H]PtdEtOH were scraped and radioactivity was quantitated by liquid scintillation spectrometry (Wallac 1414 WinSpectral Liquid Scintillation Counter, PerkinElmer). Data were measured as the ratio of [3H]PtdEtOH to [3H]total lipids. 2.12 Phospholipase C activity assay Primary human macrophages and neutrophils, and differentiated HL-60 cells (2 x 106 cells/sample) were stimulated by the addition of C5a. Following stimulation, inositol1,4,5-trisphosphate (IP3) was measured using the BiotrakTM TRK 1000 kit (GE Healthcare Bio-Sciences, Singapore) as described (Melendez and Khaw, 2002). Briefly, this is a competition binding assay in which cellular generated (unlabeled) IP3 competes with a fixed, known amount of [3H]IP3 for binding to the IP3 receptor present in homogenates from bovine adrenal glands, which has a high affinity and specificity for IP3. 2.13 Protein kinase C activity assay PKC enzyme activity was measured using the BiotrakTM Protein Kinase C enzyme assay system (GE Healthcare Bio-Sciences, Singapore) as described (Melendez et al., 1999). Briefly, the system is based upon the PKC-catalyzed transfer of the γphosphate group of ATP, using a mixture of cold ATP and [γ32P]ATP (1 μCi/sample), to a peptide substrate specific for PKC. Primary human macrophages and neutrophils, and differentiated HL-60 cells (2 x 106 cells/sample) were stimulated by the addition of C5a for 15 mins. Following stimulation, PKC assays were conducted. Results are expressed as phosphorylation rate per picomolar of protein per minute. 55 2.14 Cytosolic calcium measurement The calcium assay was carried out as described (Melendez and Khaw, 2002). Cells (2 x 106/ml) were loaded with μg/ml Fura2-AM (Molecular Probes, Invitrogen Singapore) in PBS, 1% BSA and 1.5 mM Ca2+ to prevent depletion of calcium stores. After removal of excess reagents by dilution and centrifugation, the cells were resuspended in calcium assay buffer and warmed to 37°C in the cuvette, in the spectrofluorophotometer (Shimadzu RF-5301 PC). After the basal line was obtained, the cells (2 x 105/sample) were stimulated by the addition of C5a. Fluorescence was measured at 340 and 380 nm, and the background-corrected 340:380 ratio was calibrated using the method of Grynkiewicz (Grynkiewicz et al., 1985). 2.15 Chemotaxis assay Chemotaxis was assayed using the Chemicon QCMTM Chemotaxis 3μm 96-well Cell Migration Assay kit (catalog no. ECM 515, Temecula, CA) following the manufacturer’s instructions. Briefly, the assay is based on the 3-μm pore size of Boyden chambers. Cells (2 x 105/sample) are placed in the upper chamber, and C5a is placed in the lower chamber. The cells are then incubated at 37°C for hrs (neutrophils) or 24 hrs (macrophages). Migratory cells found in the lower chamber are collected, and migratory cells attached to the bottom of the insert membrane are dissociated from the membranes with the Cell Detachment Buffer provided. These cells are subsequently lysed and detected using the CyQuant GR dye (Molecular Probes) provided in the kit. This green fluorescence dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The number of migratory cells is determined by running a fluorescent cell dose curve, in which a known number of cells are lysed and detected using the CyQuant GR dye to generate a standard curve. 56 2.16 NADPH oxidative burst assay Primary human neutrophils, differentiated HL-60 cells and differentiated U937 cells (2 x 105/sample) were assayed for whole cell superoxide production using DiogenesTM (National Diagnostics, Atlanta, GA), a superoxide chemiluminescent enhancer. C5a was added to the cells at the same time as DiogenesTM, and luminescence was measured using a luminometer to quantify the light output (Wallac 1420 Multilabel counter). The intensity of light produced by DiogenesTM in the presence of superoxide is directly proportional to the superoxide O2- concentration. 2.17 Degranulation assay Degranulation was assessed by measuring the release of β-hexosaminidase from macrophages and β-glucuronidase from neutrophils in medium and cell lysates by a colorimetric assay as described (Melendez and Khaw, 2002). The difference between the two assays is the substrate used for the specific enzymes. In the β-hexosaminidase assay for macrophages, cells (2 x 105/sample) were stimulated by the addition of C5a for 30 mins. Following stimulation, 50 μl of the sample supernatant was incubated with 200 μl of the enzyme substrate, mM p-nitrophenyl-N-acetyl-D-glucosaminide (Sigma-Aldrich, Singapore) in 0.1 M sodium citrate buffer (pH 4.5), for hr at 37°C. The remaining buffer and cells were incubated with 1% Triton X-100 for 30 mins. This was done to lyse the cells so as to get the total β-hexosaminidase concentration. A 50 μl aliquot of the lysed cells was removed and analyzed as described above. The product p-nitrophenol, was converted to the chromophore, p-nitrophenate, by the addition of 0.1 M sodium carbonate buffer. Absorbance was read at 400 nm in a plate reader (Tecan SpectraFluor-Plus). Results are reported as the percentage of 57 intracellular β-hexosaminidase that was released into the medium after correction for spontaneous release. In the β-glucuronidase assay, the substrate used is 4-Nitrophenyl β-D-glucuronide (Sigma-Aldrich, Singapore) instead. 2.18 Subcellular fractionation by differential centrifugation Cells (2 x 106/sample) that have been stimulated with C5a were resuspended in nuclear preparation buffer (NPB). After lysis by freeze-thawing in liquid nitrogen, the cells were centrifuged at 13 000g for 15 mins at 40C, resulting in a pellet, which is the nuclear fraction. The supernatant obtained, which contains the plasma membrane and cytosol, was subjected to ultracentrifugation at 100 000g for 30 mins at 40C. The resultant supernatant is the cytosolic fraction while the pellet is the nuclear-free membrane fraction. The cytosolic and nuclear fractions were subsequently analyzed by western blotting techniques. 2.19 Gel electrophoresis and western blotting analysis Unless stated other ways, 40 μg of total cell lysate for each sample, prepared in RIPA buffer, was resolved on 12% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 μm nitrocellulose membranes. As for the expression of NF-κB analysis, 40 μg of each cytosolic and nuclear fraction, at each time point, was resolved on 10% SDS-PAGE. After blocking overnight at 4°C with SDS-PAGE blocking buffer, and washing, the membranes were incubated with the relevant antibodies for or hrs at room temperature. The membranes were washed extensively in SDS-PAGE washing buffer. For detection of SPHK protein expression levels, the blots were probed with specific, anti-SPHK1 polyclonal primary antibody, made in house (Olivera and Spiegel, 1993), and anti-rabbit HRP-conjugated 58 secondary antibody (Santa Cruz Biotech, Santa Cruz, CA). For equal loading of samples, the blots were probed with anti-Arf1 monoclonal primary antibody (Santa Cruz Biotech, Santa Cruz, CA) and anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotech, Santa Cruz, CA). For detection of phosphorylated MAPK protein expression levels, the blots were probed with specific, anti-phospho-ERK1/2 polyclonal or anti-phospho-p38 polyclonal primary antibodies (Cell Signaling Technology, Danvers, MA), and then anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA). For equal loading of protein samples, the blots were probed with anti-ERK1/2 polyclonal or anti-p38 polyclonal primary antibodies (Santa Cruz Biotech, Santa Cruz, CA), and then anti-rabbit HRPconjugated secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA). For detection of p50-NF-κB protein expression levels, the blots were probed with specific, antip50-NF-κB polyclonal primary antibody (Serotec, U.K.), and then anti-rabbit HRPconjugated secondary antibody (Santa Cruz Biotech, Santa Cruz, CA). For equal loading of protein samples, the blots were probed with anti-GAPDH monoclonal primary antibody (Santa Cruz Biotech, Santa Cruz, CA), and then anti-mouse HRPconjugated secondary antibody (Santa Cruz Biotech, Santa Cruz, CA). ECL Western Blotting Detection System (GE Healthcare Bio-Sciences, Singapore) was used for visualization of the bands. 2.20 Measurement of NF-κB activation Nuclear extract preparation: Differentiated U937 cells (10 x 106/sample) were activated with C5a, in the absence or presence of inhibitors, for 30 mins at 370C. The cells were washed twice with 1X PBS and later incubated with 200 μl of Cell Lysis Buffer for 15 mins on ice. The cells 59 were centrifuged and the cell pellet was resuspended once again in lysis buffer. After disrupting the cells by syringing, the cells were centrifuged at 10 500 rpm for 20 mins at 40C. The supernatant, which is the cytosolic extract, is transferred to a fresh tube, leaving behind the crude nuclear cell pellet, which is then resuspended in Extraction Buffer and incubated for 30 mins on ice, vortexed every mins. The cells are centrifuged at 13 000 rpm for mins at 40C and the supernatant obtained, which is the nuclear extract, is then transferred to a fresh tube and stored at -200C until use. The protein concentration of the nuclear extract is measured using the Bradford assay before doing the NF-κB activation assay. NF-κB Activation Assay: EZ-DetectTM NF-κB p65 Transcription Factor Kit (Pierce Biotech, Rockford, IL) was used to measure the activation of NF-κB. The 96-well plates are coated with streptavidin and bound to biotinylated-consensus sequence for NF-κB p65. 20 μg of the nuclear extract from the cells are added to the wells in triplicates and incubated for hr at room temperature in a plate shaker. The plate is washed three times with the wash buffer. The plate is then incubated with the anti-p65 primary antibody for hr at room temperature in the shaker. After three washes, the plate is incubated with the HRP-conjugated secondary antibody for hr at room temperature. For detection, a luminol-based chemiluminescent substrate is added to the wells. The signal is then detected using a luminometer. Since the biotinylated-consensus duplexes bind only the active forms of NF-κB p65, this chemiluminescent ELISA-based assay provides unsurpassed sensitivity, thus diminishing non-specific binding. Each run is also validated using a positive control lysate, as well as both wild type and mutant competitor duplexes, which ensures reliable results with a gauge for specificity. 60 2.21 Measurement of cytokine release Supernatant preparation: Primary human macrophages and differentiated U937 cells (2 x 106 cells/sample), pretreated or not with antisense oligonucleotides or inhibitors, were stimulated with C5a for 24 hrs. After stimulation, the supernatants were collected and stored at -200C until use. TNF-α, IL-6, and IL-8 levels in the supernatants were evaluated using the OptEIATM Kit (BD Biosciences, San Jose, CA) following the manufacturer’s instructions. Cytokine ELISA kit: The cytokine assay was carried out as described (Zhi et al., 2006). Briefly, a 96-well plate is first coated with capture antibody and incubated overnight at 40C. The next day, the plate is washed three times with ELISA washing buffer, after which it is blocked with ELISA blocking buffer for hrs at room temperature. After washing the plate three times, 50 μl of the prepared supernatants are added in triplicates and incubated for hrs at room temperature. The specific cytokine standards are also included in every run. The plate is washed three times and then incubated with detection antibody for hr. After another three washes, diluted enzyme concentrate (avidin-HRP) is added into all wells and the plate is incubated for 30 mins in the dark. The plate is washed three times once again and incubated with TMB substrate solution in the dark for approximately 30 mins or until the blue color intensifies. The reaction is quenched by adding M H2SO4. The OD of the samples is taken at 450 nm with reference range at 570 nm. From the standard curve generated, the concentration of cytokine in the samples are then determined. The specificity of these ELISA kits comes from the antibodies provided, which have been specifically fragmented to reduce the background and non-specific binding to other autoantibodies and proteins. 61 2.22 Measurement of matrix metalloproteinase release Supernatant preparation: Differentiated U937 cells (2 x 106/sample), pretreated or not with inhibitors, were stimulated with C5a for 24 hrs. Following stimulation, the supernatants were collected and stored at -200C until use. MMP release assay kit: MMP-9 -8, and -3 levels in the supernatants were evaluated using respective BiotrakTM MMP Activity Assay System (GE Healthcare Bio-Sciences, Singapore) following the manufacturer’s instructions. The MMP-9 assay plate is pre-coated with the anti-MMP-9 antibody while the MMP8 and MMP-3 assay plates are pre-coated with the (Fab’)2 goat anti-mouse antibody. For the latter two plates, the appropriate mouse anti-MMP antibody is first immobilized onto the plates for or hrs, respectively, at 370C, following which they are washed four times. The plates are now ready for sample analysis. The unknown samples are added into the appropriate wells, and the plates are incubated overnight at 40C. The next day, the plates are washed four times and paminophenylmercuric acetate (APMA) is added into all wells. Any bound MMPs in its pro form are activated using APMA, which has an artificial activation sequence recognized by MMPs. The plates are then incubated for 1.5 hrs, 1hr or 30 mins respectively, at 370C, after which, the detection reagent, consisting of detection enzyme concentrate and substrate, is added into all wells, and the plates are incubated once again for about hrs at 370C. Active MMP is detected through the activation of the modified pro detection enzyme and the subsequent cleavage of its chromogenic peptide. The resultant colour is read at 405 nm in a microtiter plate spectrophotometer. 62 The specificity of each assay kit is achieved through the use of the respective specific capture antibody, and by replacing the natural activation sequence in the detection enzyme with an artificial sequence recognized by specific MMPs. The range and sensitivity of each kit is equivalent to traditional MMP zymograms and ELISA. 2.23 Fluorescence microscopy After stimulation of cells (1 x 106/sample) with C5a, suspended cells were fixed in 4% paraformaldehyde and deposited on microscope slides in a cytospin centrifuge, then permeabilized for mins in 0.1% Triton X-100 in PBS. Fluorescent labeling was performed using the anti-SPHK1 polyclonal antibody made in-house as previously described (Olivera and Spiegel, 1993) as primary, and an anti-rabbit, FITC or TRITCconjugated as secondary antibody (Sigma-Aldrich, Singapore). For Raf-1 translocation experiments, anti-Raf-1 polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA) was used as primary antibody, and for PLD translocation experiments, anti-PLD1 polyclonal antibody and anti-PLD2 polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA) were used as primary antibodies. FITC- and TRITCconjugated antibodies were used as secondary antibodies (Sigma-Aldrich, Singapore). Staining was analyzed by fluorescence microscopy using a Leica DM IRB microscope, and images captured using a Leica DC 300F camera. 2.24 Flow cytometry for cell viability Cell viability was detected by propidium iodide (PI) staining, which stains dead or dying cells. 100 μl of PI (Sigma-Aldrich, Singapore) was added to a 1-ml cell suspension containing x 106 cells (PI final concentration 50 μg/ml), just before analysis using a COULTER EPICS-XL Flow Cytometer. 63 [...]... critical amino acid residues within the C-terminal, involved in the formation of the primary effector-binding site of C5aR (DeMartino et al., 19 94; Mery and Boulay, 19 94; Monk et al., 19 95) The N-terminal region of the receptor defines a secondary non-effector binding site (Morgan et al., 19 93) Features of the C5a molecule are consistent with the two-site model proposed for the C5aR, where the non-effector... by DMS, showing a role for SPHK Figure 16 C5a -triggered PKC activity in the monocyte-derived macrophages 10 6 is not inhibited by the SPHK1 antisense Figure 17 Degranulation triggered by C5a in macrophages is dependent on SPHK activity 10 7 Figure 18 C5a- induced chemotaxis is inhibited in macrophages pretreated with the SPHK1 antisense 10 9 Figure 19 TNF-α, IL-6, and IL-8 release triggered by C5a is inhibited... N-terminal portion is not required for the biologic activity of C5a, but it participates directly in receptor binding or in stabilizing binding sites elsewhere in the native C5a conformation (Gerard et al., 19 85) Furthermore, site-directed mutagenesis studies have confirmed and extended early protein chemical studies on the model of the C5a molecule, emphasizing the modulating role of the amino terminal... functions 88 Figure 12 C5a triggers S1P generation and SPHK activity in human macrophages 99 Figure 13 SPHK1 expression, subcellular localization and antisense knockdown in the human monocyte-derived macrophages 10 1 Figure 14 S1P generation and SPHK activity in SPHK1 antisense knockdown in human monocyte-derived macrophages 10 2 Figure 15 C5a -triggered cytosolic Ca2+ signals in macrophages are inhibited 10 4... dependent on PLD activity 12 6 Figure 25 PLD potentially mediates C5a- induced chemotaxis 12 7 Figure 26 C5a- induced NF-κB translocation and activation is inhibited by butan -1- ol 12 9 Figure 27 IL-6 and IL-8 release triggered by C5a in differentiated U937 cells is mediated by PLD 13 2 Figure 28 PLD plays a potential role in C5a- induced MMP release 13 3 Figure 29 C5a induces the translocation of Raf -1 in the macrophage-like... al., 19 91; Gerard and Gerard, 19 91) Essentially, C5aR is a typical 45-kDa G-protein coupled rhodopsin-type receptor, consisting of seven transmembrane-spanning regions and three extracellular loop regions (Figure C) The effector binding site of C5a has been located on the Cterminal half of C5aR, within the region containing the second and third extracellular loops (Pease et al., 19 94) Point mutation... intracellular signaling pathways triggered by C5a in macrophages Abstract presented at the 16 th Science Research Congress held in NUS, Singapore in Mar 2004 Ibrahim FB, Melendez AJ Study on the intracellular signaling pathways triggered by C5a in macrophages Abstract presented at the Postgraduate conference on Immunology and Cancer Biology held at the City University of Hong Kong, Hong Kong in February 2003... AJ Studies on the intracellular signaling pathways triggered by anaphylatoxin C5a on phagocytic cells: new targets for inflammatory and autoimmune diseases Abstract accepted for poster presentation in the 6th NUH-NUS Annual Scientific Meeting held in NUS, Singapore in August 2002 SCHOLARSHIP National University of Singapore Research Scholarship Jul 2002 – Jul 2006 xv CHAPTER I INTRODUCTION 1. 1 The. .. region and the importance of the carboxyl terminal pentapeptide (Mollison et al., 19 89) 11 Huber-Lang et al (2003) J Immunol 17 0, 611 5- 612 4 Copyright 2003 The American Association of Immunologists, Inc Figure B Ribbon diagram of the human C5a molecule This diagram shows the helices 1- 4 as thick gray bands and the less well-defined interconnecting interhelical loop regions as colored narrow regions The. .. importance in bridging the innate and adaptive immunity (Barrington et al., 20 01; Carroll, 2004; Dempsey et al., 19 96; Fearon and Carter, 19 95) 1. 1.4 Regulation of the complement system Just like any other biological system in the body, there exist control mechanisms to prevent the ongoing activation of the complement system Regulation is exceptionally critical here because of the amplifying capacity of the . of C5a 10 1. 1.5.2 Structure of ligand C5a 11 1. 1.5.3 Complement 5a receptor (C5aR) 13 1. 1.5.4 Role of C5a in diseases 16 1. 2 Important downstream events triggered by C5a 17 1. 2 .1. 1. 1.2.3 The mannan-binding lectin (MBL) pathway 5 1. 1.3 Functions of the complement system 7 1. 1.4 Regulation of the complement system 9 1. 1.5 Complement component C5a 10 1. 1.5 .1. I INTRODUCTION 1. 1 The complement system 1 1. 1 .1 Complement proteins and nomenclature 2 1. 1.2 Activation of the complement cascade 3 1. 1.2 .1 The classical pathway 3 1. 1.2.2 The