The effect of glycosylation on the biological activity of eotaxin and related chemokines
A thesis submitted for the degree of Doctor of Philosophy University of London
by
Lee. G. Bodman B.Sc. (Hons)
Leukocyte Biology Section Biomedical Sciences Division Sir Alexander Fleming Building Imperial College South Kensington London SW7 2AZ
2004 For my parents
2 Acknowledgements
I would like to thank my co-supervisor Dr Dolores Conroy for her unfailing support and excellent teaching over the past years.
I would like to thank my co-supervisor Professor Timothy J Williams for enabling me to carry out my PhD in his department, and for his constant encouragement and ideas.
This work would not have been possible without the financial assistance of Pfizer (MA, USA) and I am indebted to them for their support.
I wish to acknowledge Dr Leonora Bishop for initially recognising my potential and recruiting me for this PhD project.
I thank Dr Mike Shelley at Velindre Hospital for allowing me to spend two months within his laboratory and stimulating my interest in medical research.
I would also like to thank all the other members of the Imperial College who have helped me along the way particularly:
Dr Pete Jose for his words of wisdom (especially regarding the state of Welsh rugby). Dr James Pease (Jimbo) for his encouragement (despite his incredibly poor jokes), Paul (Psuedo) Cameron and James (MQ) Hingston for making my time in London even more enjoyable particularly during the Clayponds years. Louise Jopling (now Dr Joppers aka Mrs Temple) for being such a great friend and supporting me all the way. Mrs Joy Dexter for all her help and assistance whilst I was writing up.
Last, but by no means least, I would like to thank Kathy and my family (Mum, Dad, Mark & Carrie-Ann) for pushing me on when times were difficult.
3 Abstract
Eotaxin, a C-C chemokine, has been identified as an important mediator of eosinophil trafficking. Glycosylation of guinea pig eotaxin, which occurs on the naturally produced protein, has been shown, using intravital microscopy, to be essential for the recruitment of eosinophils from the guinea pig mesenteric microvasculature. In contrast, recruitment of eosinophils in response to intradermally injected eotaxin was independent of glycosylation. Since eotaxin was applied topically to the surface of the mesentery these observations suggested that glycosylation was necessary for the movement of the chemokine through the surface mesothelial layer.
An in vitro chemotaxis assay was set up using freshly isolated guinea pig mesentery in a microBoyden chamber and chemotaxis of eosinophils across this tissue was compared to a polycarbonate filter. Both glycosylated guinea pig and human eotaxin induced a marked chemotaxis of guinea pig and human eosinophils, respectively, across the guinea pig mesentery. In contrast, the non-glycosylated variants showed no chemotactic activity. In contrast, both of the variants induced a similar degree of eosinophil migration across a polycarbonate filter.
To determine whether a chemoattractant gradient across the guinea pig mesentery could be established by both the glycosylated and non-glycosylated variants of eotaxin, the flux of the chemokine from the bottom wells to the upper wells of the chemotaxis chamber was measured by ELISA. The glycosylated, but not the non-glycosylated, eotaxin was transported rapidly through the mesentery reaching a near maximal concentration at 60 minutes. The flux of both variants across the polycarbonate filter was indistinguishable. Using confocal microscopy techniques, the presence of glycosylated eotaxin within the mesenteric tissue could be visualised. The non- glycosylated eotaxin could only be observed on the outside of the intact mesothelial cell layer.
The metabolic inhibitors 2-deoxyglucose (2-DOG) and sodium cyanide (NaCN) inhibited the flux of the glycosylated eotaxin across the guinea pig mesentery suggesting involvement of an active transport system. In addition transcytosis inhibitors
4 such as monensin and filipin inhibited the flux of the glycosylated eotaxin. Transport was not significantly inhibited by EGTA or by a guinea pig only CCR3 antibody. Gall31 _
3GalNAc was identified as the sugar present on human eotaxin using a plant lectin binding assay. GalNAc, but not GleNAc, inhibited the flux of the glycosylated eotaxin across the mesentery.
The flux of the two variants of eotaxin across the guinea pig mesothelium in vivo was investigated. Glycosylated eotaxin variant was detected in the blood 15 minutes following intraperitoneal injection, whereas the non-glycosylated variant was only detected after 120 minutes, possibly resulting from lymphatic clearance.
These data suggest that the eotaxin glycosylation is important for its biological activity on the guinea pig mesentery, and that this effect could be mediated by a specific receptor on the mesothelium recognising the carbohydrate, present on glycosylated eotaxin.
5 Content
1 GENERAL INTRODUCTION 19
1.1 THE ROLE OF LEUKOCYTES AND MAST CELLS IN INFLAMMATORY DISEASE 19 1.1.1 Eosinophils 19 1.1.2 T cells 20 1.1.3 Mast cells 21 1.1.4 Basophils 22 1.1.5 Monocytes and macrophages 22 1.1.6 Neutrophils 23 1.2 THE PROCESS OF LEUKOCYTE RECRUITMENT INTO TISSUE 24 1.2.1 Selectins 25 1.2.2 Integrins 25 1.2.3 I.mmun.oglobulin.s 26 1.3 MEDIATORS OF LEUKOCYTE RECRUITMENT 27 1.3.1 Chemokines 27 1.4 CHEMOKINE RECEPTORS 41 1.4.1 The eotaxin receptor: CCR3 42 1.5 FORMATION OF A CHEMOKINE GRADIENT 43 1.5.1 Glycosaminoglycans 43 1.5.2 The Duffy antigen/receptor for chemokines (DARC) 45 1.6 GLYCOSYLATION 46 1.6.1 Sequence motifs for glycosylation 46 1.6.2 Lepi.dopteron insect cell lines 47 1.6.3 Post-translational modification and cytokine biological activity 48 1.6.4 Eotaxin and glycosylation 49 1.7 PREVIOUS WORK THAT HAS LED UP TO THE PROJECT 50 1.8 THE MESENTERY AND MESOTHELIAL CELLS 51 1.8.1 Mediators released from mesothelial cells 51 1.8.2 Adhesion molecules 52 1.9 HYPOTHESIS OF THIS THESIS 53
2 MATERIALS AND METHODS 61
2.1 MATERIALS 61 2.1.1 Animals 61 2.1.2 General Reagents 61 2.1.3 Special Reagents 62 2.1.4 General. Buffers 63 2.1.5 ELBA Buffers 63 2.1.6 Antibodies 63 2.1.7 Chemokines 64 2.1.8 Other chemoattractants 65 2.2 METHODS 66
6
2.2.1 Gated Auto Fluoresce Scatter Assay (GAFS) 66 2.2.2 Chemotaxis 67 2.2.3 The Flux Assay 69 2.2.4 In vivo study 70 2.2.5 Enzyme Linked Immunosorbent Assay (ELTSA) 71 2.2.6 Protease Activity 74 2.2.7 Identification of the glycosylation present on chemokines 75 2.2.8 Glycosamin.oglycan competition assay 77 2.2.9 SDS-PAGE 77 2.2.10 Confocal microscopy 78 2.2.11 Preparation of mesentery for electron microscopy 79 2.3 STATISTICAL ANALYSIS OF DATA 79
3 COMPARISON OF THE EOSINOPHIL CHEMOATTRACTANT ACTIVITIES OF NATURALLY AND SYNTHETICALLY PRODUCED GUINEA PIG EOTAXIN 87
3.1 DETERMINATION OF THE .IN VIVO EOSINOPHIL RECRUITMENT ACTIVITY OF GLYCOSYLATED NATURAL AND NON-GLYCOSYLATED SYNTHETIC GUINEA PIG EOTAXIN USING INTRAVITAL MICROSCOPY 88 3.1.1 Cell adherence to the mesenteric venule wall 88 3.1.2 Cell migration from the mesenteric venule into the connective tissue 88 3.2 M IN LABELLED EOSINOPHIL MIGRATION INTO THE GUINEA PIG SKIN FOLLOWING INTRADERMAL INJECTION OF GUINEA PIG EOTAXIN 89 3.3 DETERMINATION OF THE MOLECULAR MASS DIFFERENCES BETWEEN THE GLYCOSYLATED NATURAL GUINEA PIG EOTAXIN VARIANTS AND NON-GLYCOSYLATED SYNTHETIC GUINEA PIG EOTAXIN 89 3.3.1 Time of flight mass spectroscopy 89 3.3.2 SDS-PAGE 89 3.4 DETERMINATION OF THE CHEMOTACTIC ACTIVITY OF GLYCOSYLATED AND NON- GLYCOSYLATED GUINEA PIG EOTAXIN ACROSS THE ISOLATED GUINEA PIG MESENTERY • 90 3.5 GUINEA PIG EOTAXIN FLUX ACROSS THE GUINEA PIG MESENTERY 91 3.6 SUMMARY OF RESULTS 93 3.7 DISCUSSION 106
4 DETERMINATION OF THE BIOLOGICAL ACTIVITY OF GLYCOSYLATED HUMAN EOTAXIN 112
4.1 HUMAN EOTAXIN DOES NOT INDUCE GUINEA PIG EOSINOPHILS TO MIGRATE ACROSS A POLYCARBONATE FILTER 113 4.2 A COMPARISON OF THE CHEMOTACTIC ACTIVITY OF GLYCOSYLATED AND NON- GLYCOSYLATED HUMAN EOTAXIN VARIANTS FOR HUMAN EOSINOPHILS ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 113 4.3 HUMAN EOSINOPHIL SHAPE CHANGE INDUCED BY GLYCOSYLATED AND NON- GLYCOSYLATED HUMAN EOTAXIN VARIANTS 114 4.4 THE TRANSPORT OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 114
7 4.4.1 Measurement of the flux of human eotaxin across an isolated guinea pig mesentery and a polycarbonate filter 114 4.4.2 Effect of glycosylated human eotaxin on the flux of glycosylated guinea pig eotaxin across the guinea pig mesentery 115 4.4.3 Effect of glycosylated guinea pig eotaxin on the flux of glycosylated human eotaxin across the guinea pig mesentery 115 4.5 THE BINDING AFFINITY OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN FORMS TO GLYCOSAMINOGLYCANS 116 4.6 ROLE OF MESOTI-IELIAL PROTEASES ON THE MOVEMENT OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 116 4.6.1 Effect of protease inhibitors on the flux of glycosylated and non- glycosylated human eotaxin 117 4.6.2 Determining the activity of mesentery endoproteases on glycosylated and non-glycosylated human eotaxin 117 4.7 MEASUREMENT OF DIPEPTIDYLPEPTIDASE IV (CD26) ACTIVITY IN THE GUINEA PIG MESENTERY 118 4.8 FLUX OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS. THE GUINEA PIG MESENTERY IN VIVO 118 4.9 SUMMARY OF RESULTS 119 4.10 DISCUSSION 136
5 IDENTIFICATION OF TIIE SA.CCHARIDE ON HUMAN EOTAXIN AND THE ROLE OF THE GLYCOSYLATION IN RELATION TO TRANSPORT ACROSS THE GUINEA PIG MESENTERY 147
5.1 IDENTIFICATION OF THE CARBOHYDRATE STRUCTURE ON GLYCOSYLATED HUMAN EOTAXIN 147 5.1.1 Detection of the carbohydrate structure present on glycosylated human eotaxin148 5.2 THE EFFECT OF THE PLANT LECTINS PNA AND WGA ON THE TRANSPORT OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 148 5.3 THE EFFECT OF SACCHARIDES ON THE FLUX OF HUMAN EOTAXIN ACROSS THE ISOLATED GUINEA PIG MESENTERY 149 5.3.1 The effect of the rnonoaccharides Gal.NAe and GleNAC on the flux of glycosylated human eotaxin across the guinea pig mesentery 149 - 5.3.2 The effect of the disaccharide Ga1131.3GalNAc, asialofetuin and fetuin on the flux of glycosylated human eotaxin. across the guinea pig mesentery 149 5.3.3 The effect of the disaccharide Ga1131.4G1cNAc and the monosaccharide GalNAc on the flux of human eotaxin across the guinea pig mesentery 150 5.4 THE EFFECT OF THE EGTA ON GLYCOSYLATED HUMAN EOTAXIN FLUX 150 5.5 THE EFFECT OF THE ANTI-GALECTIN 3 ANTIBODY ON THE FLUX OF HUMAN EOTAXIN 150 5.6 SUMMARY OF RESULTS 151 5.7 DISCUSSION 162
8 6 STUDIES ON THE MECHANISM OF TRANSPORT FOR THE GLYCOSYLATED EOTAXIN ACROSS THE GUINEA PIG MESENTERY —168
6.1 THE EFFECT OF COOLING THE GUINEA PIG MESENTERY TO 4°C ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 168 6.2 THE EFFECT OF THE METABOLIC INHIBITORS, TRANSCYTOSIS INHIBITORS AND A BLOCKING ANTIBODY TO CCR3 ON THE FLUX OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 168 6.2.1 Effect of the metabolic inhibitor 2-deoxyglucose (2-DOG) 168 6.2.2 Effect of the metabolic inhibitor Sodium Cyanide (Na.CN) 169 6.2.3 Effect of the transcytosis inhibitor monensin 169 6.2.4 Effect of the transcytosis inhibitor filipin 169 6.2.5 Effect of the blocking antibody to the eotax.in receptor CCR3 169 6.3 THE IMPORTANCE OF THE MESOTHELIAL CELL BARRIER 170 6.3.1 Physical disruption to the mesothelial cell layer 170 6.3.2 Localisation of glycosylated eotaxin within the mesentery tissue using confocal microscopy 170 6.3.3 Scanning electronmicrographs of an intact guinea pig mesentery 171 6.4 INVESTIGATING THE ABILITY OF OTHER CHEMOKINES TO MOVE ACROSS THE GUINEA PIG MESENTERY 171 6.4.1 Determining the molecular mass of the human eotaxi.n-2 forms 171 6.4.2 Identification of the carbohydrate moiety on human eotaxin-2 171 6.4.3 Comparison of the chemotactic activity of glycosylated and non- glycosylated human eotaxin-2 172 6.4.4 A comparison of the ability of glycosylated and non-glycosylated eotax in-2 to be transported across the isolated guinea pig mesentery 172 6.4.5 The ability of glycosylated and non-glycosylated MCP-1 to flux across the guinea pig mesentery 172 6.5 SUMMARY OF RESULTS 173 6.6 DISCUSSION 193
7 GENERAL DISCUSSION, FUTURE WORK AND CONCLUSIONS 202
7.1 GENERAL DISCUSSION 202 7.2 FUTURE WORK 203 7.3 CONCLUSIONS 205
8 BIBLIOGRAPHY 208
9 List of Figures
CHAPTER 1
FIGURE 1.1 - SCHEMATIC REPRESENTATION OF THE 73 AMINO ACID GUINEA PIG EOTAXIN 54 FIGURE 1.2 - PHOTO OF GUINEA PIG MESENTERY AND SMALL INTESTINE 55 FIGURE 1.3 - A DIAGRAMMATIC REPRESENTATION OF THE STRUCTURE OF THE GUINEA PIG MESENTERY (CROSS SECTION) 56
CHAPTER 2
FIGURE 2.1 DIAGRAMATIC REPRESENTATION OF THE MICROBOYDEN CHAMBER CHEMOTAXIS AND CHEMOKINE FLUX ASSAYS 80 FIGURE 2.2 DETECTION OF GUINEA PIG EOTAXIN VARIANTS BY ELISA 81 FIGURE 2.3 DETECTION OF GUINEA PIG VARIANTS USING BIOTINYLATED MATCHED ANTIBODY ELISA 82 FIGURE 2.4 DETECTION OF HUMAN EOTAXIN VARIANTS BY ELISA 83 FIGURE 2.5 DETECTION OF HUMAN EOTAXIN-2 VARIANTS BY ELISA 84
CHAPTER 3
FIGURE 3.1 REVERSE PHASE HPLC PURIFICATION OF EOTAXIN FROM BAL FLUID 94 FIGURE 3.2 EFFECT OF TOPICAL APPLICATION OF GLYCOSYLATED NATURAL AND NON- GLYCOSYLATED SYNTHETIC GUINEA PIG EOTAXIN ON CELL ADHERENCE TO A GUINEA PIG MESENTERIC VENULF 95 FIGURE 3.3 EFFECT OF TOPICAL APPLICATION OF GLYCOSYLATED NATURAL AND NON- GLYCOSYLATED SYNTHETIC GUINEA PIG EOTAXIN ON CELL EMIGRATION FROM A GUINEA PIG MESENTERIC VENULE 96 FIGURE 3.4 PHOTOMICROGRAPHS OF EOSINOPHIL RECRUITMENT IN GUINEA PIG MESENTERIC VENULE IN VIVO 97 FIGURE 3.5 M IN LABELLED EOSINOPHIL RECRUITMENT INTO GUINEA PIG SKIN INDUCED BY INTRADERMAL INJECTIONS OF GLYCOSYLATED NATURAL AND NON- GLYCOSYLATED SYNTHETIC GUINEA PIG EOTAXIN 98 FIGURE 3.6 SDS-PAGE OF REVERSE PHASE HPLC FRACTIONS OF BAL FLUID FROM SEPHADEX INJECTED GUINEA PIGS 100 FIGURE 3.7 CHLMOTACTIC RESPONSE OF' II INDIUM LABELLED GUINEA PIG EOSINOPHILS TO GLYCOSYLATED, NON-GLYCOSYLATED GUINEA PIG EOTAXIN AND LTB4 ACROSS A GUINEA PIG MESENTERY 101 FIGURE 3.8 CHEMOTAXIS OF GUINEA PIG EOSINOPFIILS INDUCED BY GLYCOSYLATED AND NON-GLYCOSYLATED GUINEA PIG EOTAXIN ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 102
10 FIGURE 3.9 CHEMOTAXIS OF GUINEA PIG EOSINOPHILS INDUCED BY GLYCOSYLATED GUINEA PIG EOTAXIN AND LTB4 ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 103 FIGURE 3.10 CHEMOTACTIC RESPONSE OF GUINEA PIG EOSINOPHILS TO NON- GLYCOSYLATED GUINEA PIG EOTAXIN ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 104 FIGURE 3.11 THE FLUX OF THE GUINEA PIG EOTAXIN VARIANTS ACROSS THE ISOLATED GUINEA PIG MESENTERY AND A POLYCARBONATE FILTER 105
CHAPTER 4
FIGURE 4.1 CHEMOTACTIC RESPONSE OF GUINEA PIG EOSINOPHILS TO GUINEA PIG EOTAXIN, HUMAN EOTAX1N AND LTB4 ACROSS A POLYCARBONATE FILTER 121 FIGURE 4.2 CHEMOTACTIC RESPONSE OF HUMAN EOSINOPHILS TO GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN AND LTB4 ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 122 FIGURE 4.3 SHAPE CHANGE OF HUMAN EOSINOPHILS IS INDUCED BY GLYCOSYLATED EOTAXIN AND NON-GLYCOSYLATED EOTAXIN 123 FIGURE 4.4 THE MOVEMENT OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 124 FIGURE 4.5 TIMECOURSE OF THE FLUX OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 125 FIGURE 4.6 EFFECT OF GLYCOSYLATED HUMAN EOTAXIN ON THE FLUX OF GLYCOSYLATED GUINEA PIG EOTAXIN ACROSS THE GUINEA PIG MESENTERY 126 FIGURE 4.7 EFFECT OF GLYCOSYLATED HUMAN GUINEA PIG EOTAXIN ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 127 FIGURE 4.8 BINDING OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN TO BIOTINYLATED HEPARIN 128 FIGURE 4.9 EFFECT OF CIIONDROITIN SULPHATE B AND HEPA.RAN SULPHATE ON THE BINDING OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN TO BIOTINYLATED HEPARIN 129 FIGURE 4.10 EFFECT OF PROTEASE INHIBITORS ON THE MOVEMENT OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY130 FIGURE 4.11 EFFECT OF PROTEASE INHIBITORS ON THE DEGRADATION OF EOTAXIN VARIANTS FOLLOWING EXPOSURE TO GUINEA PIG MESENTERY 131 FIGURE 4.12 SHAPE CHANGE OF HUMAN EOSINOPHILS IN RESPONSE TO GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN AFTER 1 HOUR EXPOSURE TO THE GUINEA PIG MESENTERY (GAFS ASSAY) 132 FIGURE 4.13 EFFECT OF PROTEASES FOUND IN GUINEA PIG MESENTERY LYSATES ON THE IMMUNOREACTIVITY OF HUMAN EOTAXIN 133 FIGURE 4.14 DETERMINATION OF THE PRESENCE OF THE PROTEASE DIPEPTIDYLPEPTIDASE IV IN THE GUINEA PIG MESENTERY AND KIDNEY LYSATES 134 FIGURE 4.15 TIME COURSE OF HUMAN EOTAXIN DETECTION IN GUINEA PIG BLOOD FOLLOWING INTRAPERITONEAL INJECTION 135
11 CHAPTER 5
FIGURE 5.1 BINDING OF PNA TO GLYCOSYLATED HUMAN EOTAXIN 152 FIGURE 5.2 LACK OF DSA LLCM BINDING TO GLYCOSYLATED HUMAN EOTAXIN 153 FIGURE 5.3 BINDING OF HUMAN EOTAXINS TO PNA AND WGA LECTINS 154 FIGURE 5.4 THE EFFECT OF .PNA ON THE FLUX OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 155 FIGURE 5.5 THE EFFECT OF WGA ON THE FLUX OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 156 FIGURE 5.6 THE EFFECT OF SACCHARIDES ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 157 FIGURE 5.7 THE EFFECT OF GALB1.3GALNAC , ASIALOFETUIN AND FETUIN ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE ISOLATED GUINEA PIG MESENTERY 158 FIGURE 5.8 THE EFFECT OF THE GALB14GLCNAC AND GALNAC ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 159 FIGURE 5.9 THE EFFECT OF THE EGTA ON THE FLUX OF HUMAN EOTAXIN ACROSS THE ISOLATED GUINEA PIG MESENTERY 160 FIGURE 5.10 THE EFFECT OF ANTI-GALECTIN 3 ANTIBODY ON THE FLUX OF HUMAN EOTAXIN ACROSS THE ISOLATED GUINEA PIG MESENTERY 161
CHAPTER 6
FIGURE 6.1 THE EFFECT OF COOLING THE TISSUE TO 4°C ON THE FLUX OF HUMAN GLYCOSYLATED EOTAXIN ACROSS THE GUINEA PIG MESENTERY 174 FIGURE 6.2 THE EFFECT OF 2-DEOXYGLUCOSE (2-DOG) ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 175 FIGURE 6.3 THE EFFECT OF SODIUM CYANIDE (NACN) ON THE FLUX OF HUMAN GLYCOSYLATED EOTAXIN ACROSS THE GUINEA PIG MESENTERY 176 FIGURE 6.4 THE EFFECT OF MONENSIN ON THE FLUX OF HUMAN EOTAXIN ACROSS THE GUINEA PIG MESENTERY 177 FIGURE 6.5 THE EFFECT OF FILIPIN ON THE FLUX OF HUMAN EOTAXIN ACROSS GUINEA PIG MESENTERY 178 FIGURE 6.6 THE EFFECT OF AN ANTI-GUINEA PIG CCR3 ANTIBODY ON THE FLUX OF GLYCOSYLATED HUMAN EOTAXIN ACROSS THE ISOLATED MESENTERY • 179 FIGURE 6.7 SCANNING CONFOCAL IMAGE OF INTACT AND DAMAGED MESOTHELIAL CELL LAYER ON A GUINEA PIG MESENTERY 180 FIGURE 6.8 THE EFFECT OF DISRUPTING THE GUINEA PIG MESOTHELIUM ON THE FLUX OF GLYCOSYLA.TED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS GUINEA PIG MESENTERY 181 FIGURE 6.9 THE FLUX OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN ACROSS GUINEA PIG MESENTERY AFTER OSMOTIC SHOCK TREATMENT 182 FIGURE 6.10 CONFOCAL MICROSCOPY SCANNING IMAGE OF A VENULE LOCATED WITHIN THE GUINEA PIG MESENTERY SHOWING GLYCOYLATED HUMAN EOTAXIN 183 FIGURE 6.11 CONFOCAL MICROSCOPY IMAGES OF THE GUINEA PIG MESENTERY AND MESOTHELIAL VENULE 184
12 FIGURE 6.12 CONFOCAL MICROSCOPY IMAGES OF THE GUINEA PIG MESENTERY AND MESOTHELIAL VENULE FOLLOWING OSMOTIC SHOCK 185 FIGURE 6.13 A SCANNING ELECTRON MICROGRAPH OF THE GUINEA PIG MESENTERY (X 5000) 186 FIGURE 6.14 TRANSMISSION ELECTRON MICROGRAPH ILLUSTRATING THE MESOTHELIAL VESICLES 187 FIGURE 6.15 SDS-PA GE OF THE HUMAN EOTAXIN-2 VARIANTS 188 FIGURE 6.16 DSA LECTTN BINDING TO HUMAN GLYCOSYLATED EOTAXIN-2 189 FIGURE 6.17 CHEMOTACTIC RESPONSE OF HUMAN EOSINOPHILS TO HUMAN EOTAXIN 2 ACROSS A GUINEA PIG MESENTERY AND POLYCARBONATE FILTER 190 FIGURE 6.18 THE FLUX OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN EOTAXIN-2 ACROSS THE GUINEA PIG MESENTERY 191 FIGURE 6.19 THE FLUX OF GLYCOSYLATED AND NON-GLYCOSYLATED HUMAN MCP-1 ACROSS THE GUINEA PIG MESENTERY AND POLYCARBONATE FILTER WITH TIME 192
CHAPTER 7
FIGURE 7.1 A DIAGRAM MONTAGE ILLUSTRATING THE PROPOSED STAGES OF GLYCOSYLATED HUMAN EOTAXIN TRANSPORT ACROSS THE GUINEA PIG MESOTHELIUM 206
13 List of Tables
CHAPTER 1
Table 1.1 Chemokine Nomenclature for the CC and CXC chemokine families
(Zlotnik and Yoshie, 2000) 57 Table 1.2 Chemokine receptor family and their agonists.... 58 Table 1.3 Amino acids and their relevant grouping .59
CHAPTER 2
Table 2.1 A list of the plant lectins used in this thesis to identify the saccharides present on glycosylated eotaxin (`The Lectins: Properties, Functions and Applications in Biology and Medicine') .85
CHAPTER 3
Table 3.1 Molecular weights of natural and synthetic guinea pig eotaxins ...... 99
CHAPTER 4
Table 4.1 Molecular mass of human eotaxin produced in Sf-9 insect cells using a baculovirus expression system and a E. coli expression system 120
CHAPTER 6
Table 6.1 Amino Acid sequence of chemokines for possible glycosylation sites as determined by O-glycbase 200
14 List of abbreviations
2-DOG 2-deoxyglucose A Ab Antibody AHR Airways hyperresponsiveness APMSF 4-(amidinophenyl)methanesulfonyl APC Antigen presenting cell B BAL Bronchoalveolar lavage BSA Bovine serum albumin C C5a Complement fragment 5a CCL CC-chemokine ligand CCR CC-chemokine receptor CD- Cluster of differentiation antigen designation CRD Carbohydrate Recognition Domain CSB Chondroitin sulphate B CXCR CXC-chemokine receptor D Da Dalton DARC Duffy antigen receptor for chemokines DIG digoxigenin DMEM Dulbeccos modified Eagle's medium DMSO Dimethyl sulphoxide E E.coli Escherichia. coli EAR Early asthmatic response ECM Extracellular matrix ECP Eosinophil cationic protein EDF Eosinophil differentiation factor EDN Eosinophil-derived neurotoxin EDTA Ethlenediaminetetraacetic acid EGFR Epidermal Growth Factor EGTA Ethyleneglycoltetraacetic acid ELISA Enzyme-linked immunosorbent assay EPO Eosinophil peroxidase ERK- Extracellular signal-regulated kinase F FACScan® Fluorescence-activated cell scanner FITC Fluorescein isothiocyanate FSC Forward scatter G GAFS Gated Auto Florescence/Forward Scatter GAGs Glycosaminoglycans Ga1NAc N-acetylgalactosamine Gal ii,_3GalNAc Galactose-P1.3Nacetylgalactosamine Gal131 _4GleNAc Galactose-f31.4Nacetylglucosamine GIcNAc N-acety]glucosamine G-CSF Granulocyte colony-stimulating factor GM-CSF Granulocyte-macrophage colony-stimulating factor H hr Hour HBSS Hank's balanced salt solution
15 HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HRP Horseradish peroxidase HUVEC Human umbilical vein endothelial cell HS Heparan sulphate i.d. Intradermal i.m. Intramuscular i.v. Intravenous ICAM Intracellular adhesion molecule IEC Ion-exchange chromatography IFN-y Interferon-y Ig Immunoglobulin IGF Insulin-like growth factor IGFBP Insulin-like growth factor binding protein IL- Interleukin- K kDa Kilodalton L 1 Litre LAR Late asthmatic response LEC Lymphatic endothelial cell LPS Lipopolysaccharide LT Leukotriene M p 1 Microlitre mAb Monoclonal antibody MBP Major basic protein MCP- Monocyte chemoattractant protein- M-CSF Macrophage colony-stimulating factor min Minute MIP-1 a Macrophage inflammatory protein-la ml Millilitre MPIF Myeloid progenitor inhibitory factor mRNA Messenger RNA mwt Molecular weight N NaCN Sodium Cyanide nM Nanomolar 0 O.D. Optical density OVA Ovalbumin P PAF Platelet activating factor PBS Phosphate buffered saline PDGF Platelet Derived Growth Factor PECAM-1 Platelet-endothelial cell adhesion molecule-1 PG Prostaglandins PI 3-KINASE Phosphatidylinositol 3-kinase PLC Phospholipase C PMN leukocyte Polymorphonuclear leukocyte PNA Peanut Agglutinin pM Picomolar R RANTES Regulated on activation, normal T-cell expressed and secreted S SCF Stem cell factor SCID Severe combined immunodeficiency SDF Stromal cell derived factor
16 SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM Standard error of the mean Sf-9 Spodopt era frugiperda sLex Sialyl Lewis antigen SSC Side scatter STAT- Signal transducers of activation T T.ni Trichoplusia.ni TCA Trichloroacetic acid TFA Trifluoroacetic acid TGF Tumour Growth Factor Thl Type 1 helper T- lymphocyte Th2 Type 2 helper T- lymphocyte TNF-ot Tumour necrosis factor a TRF T-lymphocyte replacing factor TRITC Tetramethylrhodamine isothiocyanate V VCAM Vascular cell adhesion molecule-1 VLA- Very late antigen W WGA Wheat Germ Agglutinin
17 Chapter 1
General Introduction
18 1 General Introduction
A vital component of local host defence reactions to infection or injury is the recruitment of leukocytes from the blood via particular regions of the microcirculation. Leukocytes are specialised for different functions and thus mechanisms have evolved to ensure that different types of leukocytes are recruited appropriately depending on the type of inflammatory stimulus and its location. These mechanisms involve a range of soluble chemical signals produced at the inflammatory site that act on receptors differentially expressed on various leukocyte types. Of most importance in this respect are the chemokines. There are now over 50 known chemokine ligands acting on approximately 20 closely related seven transmembrane G-protein coupled receptors. Chemokines, in addition to being chemotactic also regulate adhesion molecules that mediate attachment of leukocytes to the lumina' surface of the microvessel wall and stimulate other effector functions of leukocytes.
This thesis is concerned with one of these chemical signals, eotaxin, a CC chemokine that is potent in inducing the accumulation of eosinophils into tissues. Eosinophil accumulation is a characteristic feature of allergic reactions particularly allergic asthma.
1.1 The role of leukocytes and mast cells in inflammatory disease
1.1.1 Eosinophils
Infiltration of the lungs by the eosinophil leukocyte is an important pathogenic characteristic of allergic asthma. (Bentley et al, 1992; Wardlaw et al, 1988). The eosinophils that accumulate in tissue at sites of infection and inflammation are supplied by the bone marrow, the site of eosinophil haematopoiesis. Mature eosinophils are non- dividing, granule containing cells, approximately 81tm in diameter with a bilobed nucleus (Dvorak et al, 1991). Eosinophils are thought to contribute to airway hyperresponsiveness (AHR) through the activation, degranulation and release of proteins stored in their granules and the generation of oxidative products and lipid mediators. The granules are membrane bound with a crystalline core and consist mainly of major basic protein (MBP). Surrounding the core is eosinophil cationic protein
19 (ECP), eosinophil peroxidase (EPO) and eosinophil derived neurotoxin (EDN). It is believed that the evolutionary path of eosinophils began as a way for the host to eliminate helminthic infection. Indeed a highly efficient killing process exists where EDN paralyses the helminth, EPO and MBP damage the plasma membranes and ECP forms pores in the helminth resulting in death by osmotic lysis and ionic imbalance (Ackerman et al, 1990). Eosinophils can generate oxidative products and lipid mediators, including platelet activating factor (PAF) and leukotrienes (e.g. LTC4, LTD4) which can induce vasodilation and oedema formation and bronchoconstriction. The generation of all these products can cause microvascular leakage and may contribute to AHR as well as inducing extensive tissue damage and potentiating the accumulation of other inflammatory cells. In allergic asthma, the number of eosinophils in the airways, and the presence of extracellular cationic proteins resulting from eosinophil activation and degranulation, positively correlate with the severity of lung dysfunction (Kay et al, 1986; Bradley et al, 1991). It has been shown that eosinophils can act as antigen presenting cells (APCs), thus further integrating the eosinophil into the immune response (Shi et al, 1998; MacKenzie et al, 2001). It has been argued that these responses of eosinophils represent their role in host defence to helminth parasitic infection and thus, asthma and allergy may represent an aberration of such a defence reaction. Eosinophils have also been implicated in fibrosis and smooth muscle hyperplasia due to release of transforming growth factor-I3 (TGFI3), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) (Minshall et al, 1997; Tanaka et al, 2001). Activated eosinophils have been shown to induce mucin synthesis in human airway epithelial cells via EGF receptor activation and tumour growth factor-a (TGFa) (the ligand for the EGF receptor) produced by eosinophils and epithelial cells (Burgel et al, 2001).
1.1.2 T cells
T-cells constitute a large proportion of the inflammatory cells within the lungs of asthmatics and are believed to regulate the accumulation of eosinophils (Cohn et al, 1998). T-cells are small lymphocytes which originate in the thymus and are present in lymphoid tissues and blood. T-cells bear antigen specific receptors on their surface and only react to foreign antigens presented to them on the surface of another cell. The main types of T-cell are cytotoxic T-cells which recognise and kill cells of the body that have
20 become antigenically altered (e.g. infected by a virus), helper T-cells which are activated by foreign antigen presenting cells and which in turn activate appropriate B cells, and suppressor T-cells which are involved in suppressing the immune responses and the general regulation of the immune system. T-cell mediated immune responses are believed to be important contributors to AHR in asthmatic patients through the release of mediators that enhance lung inflammation by activating eosinophils and mast cells (Bradley et al, 1991; Shi et al, 1998). There are two T-cell subtypes involved in immune responses, T helper cell type 1 (Thl) and T helper cell type 2 (Th2), and the polarisation of the T-cells into the Thl or Th2 subtype involves cytokines that amplify one pathway whilst down regulating the other. Thl subtypes are associated with neutrophil recruitment and activation and produce interferon-y (IFN-y), interleukin-2 (IL-2) and tumour necrosis factor p (TNF- (3) and are required for effective eradication of intracellular pathogens. Th2 types are characterised by the profile of cytokines that they produce such as interleukin-4 (IL-4), IL-5, 1L-9 and IL-13 and are associated with helminth infections and allergic responses. Higher numbers of the Th2 type cells have been found in the blood of asthmatic patients (Robinson et al, 1992) and also in the lung (Del Prete et al, 1993).
1.1.3 Mast cells
Mast cells are derived form CD34+ haematopoietic progenitor cells and maturation which occurs in peripheral tissues is tightly regulated by various cytokines. Mast cells are regarded as key effector cells in IgE associated immediate hypersensitivity reactions and allergic disorders. Mast cells contain cytoplasmic granules within which are stored histamine and neutral proteases, mast cell tryptase, chymase and carboxypeptidase (Huang et al, 1998). Histamine is a primary amine and is stored within the granule via ionic association with the acidic residues of glycosaminoglycans (GAGs). A mast cell binds immunoglobulin E (IgE) which is secreted from activated B cells by its high affinity IgE receptor, FccRI. The IgE molecules bound to the mast cell serve as cell- surface receptors for antigen. When an antigen molecule binds to these membrane bound IgE antibodies so as to cross link them, this stimulates mast cell degranulation resulting in the release of preformed mediators such as histamine and proteases and de novo synthesis of inflammatory lipid mediators such as leukotrienes, cytokines and chemokines (Bradding et al, 1994; Galli & Nakae, 2003). Activation of the mast cell
21 results in acute bronchospasm and activation and attraction of other cells to the lung resulting in the early asthmatic response (EAR) associated with an allergic asthmatic attack (Schulman, 1986, Metcalfe et al, 1997; Wedemeyer et al, 2000, Williams & Galli, 2000; Shakoory et al, 2004). Mast cells upon activation produce IL-4, which in conjunction with IL-6 and TNF plays an important part in the inflammatory response (Bradding et al, 1994). Through the production of TNFa have been shown to contribute to the host defence against bacteria infection (McLachlan et al, 2003). The role of mast cells in the immune response has been reviewed by Gurish and Austen (2001).
1.1.4 Basophils
Basophils are the smallest circulating granulocytes and comprise of <1% of the total circulating leukocytes in humans. The function of basophils is relatively the least known of all the leukocytes although it is believed that they are involved in combating parasitic infection. Similar to mast cells, basophils express the high-affinity receptor for IgE (FceRI). Basophils contain cytoplasmic granules which store mediators of inflammation e.g. histamine, PAF etc, however the range of basophil derived cytokines is limited compared to mast cells (Brunner et al, 1993). The contents of these granules are released by the binding to and cross linking of surface FccRI receptors. The cascade of basophil activation and granule release is similar to that described for mast cells (Wedemeyer et al, 2000).
1.1.5 Monocytes and macrophages
In 1924, Aschoff classified monocytes and macrophages as cells of the reticulo- endothelial system (RES). Van Furth et al, (1972) proposed the mononuclear phagocyte system (MPS), and monocytes and macrophages became basic cell types of this system. Monocytes are cells that possess migratory and phagocytotic functionality as well as receptors for IgG Fc domains. Upon migration into tissues, monocytes differentiate into macrophages and thus monocytes are considered to be immature macrophages. Macrophages can be divided into normal and inflammatory macrophages. Normal macrophages includes macrophages in connective tissue (histiocytes), liver (Kupffer's cells), lung (alveolar macrophages), lymph nodes (free and fixed macrophages), spleen (free and fixed macrophages), bone marrow (fixed macrophages), serous fluids (pleural
22 and peritoneal macrophages), skin (histiocytes, Langerhans' cell) and in other tissues. Inflammatory macrophages are present in various exudates. They may be characterized by various specific markers, e.g. peroxidase activity, and since they are derived exclusively from monocytes they share similar properties. Mononuclear phagocytes (monocytes and macrophages) are central to inflammation, as they produce many components which participate in, or regulate, the different plasma enzyme systems, and hence the mediators of the inflammatory response. They are also actively phagocytic and are involved in microbial killing, as are neutrophils. Macrophages can remove invading organisms by engulfment, however should the macrophage be unable to kill the organism it is capable of presenting antigenic peptides (small protein fragments) to T-cells. Macrophages are also capable of producing IL-1, IL-12 and TNF-a following engulfment of a pathogen thus altering the immune response (Ulich et al, 1991; Trinchieri and Gerosa, 1996).
1.1.6 Neutrophils
Neutrophils, which are sometimes referred to as polymorphonuclear leukocytes (PMN), represent 50 to 60% of the total circulating leukocytes and constitute the "first line of defence" against infectious agents or "nonself' substances that penetrate the body's physical barriers. Their targets include. bacteria, fungi, protozoa, viruses, virally infected cells and tumour cells. Their development occurs in the bone marrow where they undergo proliferation and differentiation. The neutrophil granules are of major importance for neutrophil- function. Neutrophils can be recruited by mediators such as PAF and C5a or neutrophil active chemokines such as IL-8. It is believed that neutrophils have the ability to amplify the inflammatory response and this is achieved by the production of cytokines such as IL-8 by the neutrophil itself (Bazzoni et al, 1991; Yamashiro et al, 2001). The ability of neutrophils to recognise invading pathogens is achieved via the complement receptor 3 (CR3) (also known as the CD11b/CD18 adhesion molecule) and pathogen recognition receptors (Toll-like receptors). The CR3 receptor is able to recognise components of the pathogen cell wall (Ross et al, 1985). Toll-like receptors are a type 1 transmembrane receptor that recognise a conserved molecular pattern on invading pathogens resulting in cytokine release and cellular activation. To date, there have been 10 Toll Like receptors (TLR1 _10) identified (Underhill and Ozinsky, 2002).
23 Following recognition of the pathogen via a CD1 lb/CD18-dependent or a Toll-like receptor IL-8 is generated (Bazzoni et al, 1991; Au et al, 1994) resulting in further neutrophil recruitment into the area and eventually resulting with phagocytosis of pathogen. Phagocytosis is a complex process composed of several biochemical steps. After recognition and particle binding to the phagocyte surface, ingestion (engulfment), phagosome origination, phagolysosome formation (fusion of phagosome with lysosomes), killing and degradation of ingested cells or other material proceed. Simultaneously with the recognition and particle binding a dramatic increase in oxygen consumption (the respiratory burst) is observed (Baldridge & Gerard, 1933; Sbarra & Karnovsky, 1959). It is responsible for the production of superoxide and other oxygen radicals, and also for the secretion of a variety of enzymes and biologically active substances controlling inflammatory and cytotoxic reactions (review by Faurschou & Borregaard, 2003). The respiratory burst in human neutrophils is reviewed by Dahlgren & Karlsson (1999).
1.2 The process of leukocyte recruitment into tissue
Leukocyte migration, regulated by specific molecular mechanisms, is a complex procedure involving multiple adhesive interactions between leukocytes and components of the blood vessel wall. Leukocyte recruitment into inflammatory sites requires the leukocyte to leave the blood vessel and pass through the endothelial cell layer and the basement membrane of the vessel wall. In order for leukocytes to migrate from the blood they must attach themselves to the vessel wall and resist the force of the blood flow. In response to an inflammatory stimulus, leukocytes form weak, reversible bonds with the endothelial cells which results in the leukocyte rolling along the vessel wall. The leukocytes then adhere firmly to the vessel wall and become stationary where they migrate through the cell-cell junction to the abluminal side of the endothelium. Although intercellular migration between the endothelial cell junctions is the generally accepted view of leukocyte extravasation (Johnson-Leger et al, 2000) it has been suggested that leukocytes can pass through the body of the endothelial cell rather than through the cell-cell junction (Feng et al, 1998). Finally the leukocyte moves through the basement membrane and into the extravascular tissue (Springer, 1994; Knol and Roos, 1996).
24 1.2.1 Selectins
Selectins are glycosylated membrane bound proteins that are associated with the initial leukocyte binding to the endothelial cell surface and subsequent rolling (Shimizu et al, 1999). Three selectin molecules have been characterised to date L-selectin (CD62L), P- selectin (CD62P) and E-selectin (CD62E). L-selectin is expressed exclusively on leukocytes (Gallatin et al, 1983; Lewinsohn et al, 1987) and it has been shown that it is found on the microvilli of the leukocyte where it is first likely to come into contact with the endothelium (Picker et al, 1991). E-selectin (Wellicome et al, 1990) is expressed on endothelial cells following activation by cytokines such as TNF-a (Bevilacqua et al, 1989). P-selectin is expressed on activated platelets and activated endothelial cells and is stored in Weibel-Palade bodies in endothelial cells (McEver et al, 1989). Upon activation by mediators such as histamine and thrombin. P-selectin is rapidly transported to the endothelial cell surface where the P-selectin containing granules fuse with the endothelial cell membrane. By observing eosinophils under flow conditions in vitro, Kitayama et al, (1997) have demonstrated that eosinophils use P-selectin but not E-selectin for initial tethering to activated endothelium. In vivo, allergic models in the mouse pleural cavity and skin have shown that eosinophil accumulation is largely dependent upon P-selectin and L-selectin (Henriques et al, 1996; Teixeira and Hellewell, 1998).
1.2.2 Integrins
Integrins expressed on the leukocyte surface have been associated with their firm adhesion to the microvascular endothelium. The stimulation of a leukocyte through its CC chemokine receptors results in an increase in integrin affinity and thus increased adhesion of the leukocyte to the endothelial cell surface (Seminaro and Bochner, 1997). Integrins are heterodimeric molecules consisting of a a and 13 polypeptide chain. The integrins are classified by differences in the [3 chains e.g. p, and (32, however more than one type of a chain can be associated with the same 13 chain. The most important integrins regarding leukocyte firm adhesion to the endothelium are the [31 (CD29) and 132 (CD18) integrins. (31 integrins expressed by leukocytes have been shown to act as receptors for endothelial cell matrix proteins such as fibronectin and vitronecin.
25 However, the a4131 integrin or very late antigen -4 (VLA-4) which is expressed by lymphocytes, monocytes and eosinophils also binds to vascular cell adhesion molecule- 1 (VCAM-1) expressed by endothelial cells. VLA-4 has been shown to be involved in lymphocyte, monocytes and eosinophil adhesion to cytokine-activated endothelial cells (Carlos et al, 1991; Dobrina et al, 1991; Vennegoor et al, 1992). The P2 integrins consist of four members CD11a/CD18, CD11b/CD18, CD11 c/CD18 and CD11 d/CD18. Lymphocytes have been shown to express CD11a, whereas monocytes and neutrophils express all four types. CD1 1 a and CD11 b are constitutively expressed on the surface of leukocytes, but the expression and binding affinity can be increased by various inflammatory mediators, such as fMLP, C5a, PAF, LTB4 and chemokines. Ligands for 132 integrins include adhesion molecules expressed by endothelial cells such as intracellular adhesion molecule-1 (ICAM-1), and also soluble proteins such as fibrinogen (Springer 1994).
1.2.3 Immunoglobulins
The immunoglobulin family encompass a number of cell-surface proteins, composed of one or more external Ig-like domains. Members of this family include ICAM-1, ICAM- 2, VCAM-1 and platelet endothelial cell adhesion molecule (PECAM-1). The adhesion molecule PECAM-1(CD31) is unique in that it the only adhesion molecule that does not have a role in the capture or arrest of leukocytes to the endothelial cell surface. PECAM-1 is expressed on leukocytes and endothelial cells. PECAM-1 expression appears to be concentrated at intercellular junctions and in vitro studies have shown that PECAM-1 is important for leukocyte migration across endothelial monolayers (Muller et al, 1993). PECAM-1 may be important in regulating both leukocyte migration across the endothelium and through the basement membrane (Wakelin et al, 1996). The four molecules are expressed constitutively; ICAM-1 and VCAM-1 expression can be transcriptionally upregulated by stimuli such as IL-113, TNF-a, LPS and hypoxia. Cytokine activation of the cell adhesion molecules can upregulate both ICAM and VCAM levels. Eosinophils and lymphocytes can bind to endothelial VCAM-1 via the VLA-4 integrin (Elices et al, 1990).
26 1.3 Mediators of leukocyte recruitment
Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators. Bacterial products and toxins can act as exogenous mediators of inflammation. Notable among these is endotoxin, or LPS of Gram-negative bacteria. Endotoxin can trigger complement activation, resulting in the formation of anaphylatoxins C3a and C5a which cause increased vascular permeability. In addition, endotoxin can elicit T cell proliferation (Tulic et al, 2002) and has been described as superantigen for T cells. Endogenous mediators of inflammation are produced from within the cells of the immune system itself e.g. histamine release from mast cells, lipid mediator production such as prostaglandins and cytokine/chemokine production e.g. IL-1 and eotaxin.
Leukocytes are recruited to sites of inflammation by chemoattractants, the most important of which are a family of low molecular weight proteins known as chemokines (chemotactic cytokines). Upon release by an inflammatory stimulus, chemokines induce leukocyte migration from the blood into the tissue. It is believed that chemokines bind to negatively charged portions of cell surface proteoglycans on the luminal surface of the endothelium creating a immobilised or haptotactic gradient (Rot, 1993; Tanaka et al, 1993; Hoogewerf et al, 1997; Middleton et al, 1997). Stimulation of chemokine receptors increases the affinity of integrin molecules for their complementary ligands that results in firm adhesion of rolling leukocytes to the microvascular endothelium (Tenscher et al, 1996). A chemoattractant gradient across the endothelium is thought to be required for migration across the endothelium (DeLisser and Albelda, 1998). The classical lipid chemoattractants LTB4 and PAF, and the complement fragment C5a stimulate directed migration of eosinophils. However, they also stimulate migration of other leukocyte types and thus are not selective for eosinophils.
1.3.1 Chemokines
The primary function of chemokines is the attraction of different types of leukocytes to specific sites, either under basal conditions or at sites of inflammation. Chemokines are a large family of low molecular mass proteins (8-10kDa) generally with four conserved cysteines forming two essential disulphide bonds (Cysl -Cys3 and Cys2-Cys4). The
27 chemokines are grouped according to the position of the NH2 terminal cysteine residues (Mackay, 1997; Baggiolini, 1998). Within the CXC (a) chemokines the first two cysteine residues are separated by one amino acid, whilst the first two cysteines are adjacent in the CC ((3) chemokines.
There are two other groups of chemokines that have been recently discovered. Fractalkine/neurotactin is a membrane bound chemokine and contains the sequence CX3C (8) where the first two cysteines are separated by three amino acids and is the only known member of this class. The chemokine lymphotactin has only a single cysteine near the N-terminus linking to another cysteine downstream via a single disulphide bond. Chemokines consist of a short amino terminal domain preceding the first cysteine, a backbone made of (3 strands and the connecting loops found between the second and fourth cysteines (first and second in the case of lymphotactin), and a carboxyl-terminal a-helix of 20-30 amino acids. The backbone has a well ordered structure whereas the N and C terminal domains are disordered (Rajarathnam et al, 1995).
A chemokine can often act on several different receptors and a particular cell type usually has more than one receptor type (Mantovani; 1999). Table 1.1 shows a list of chemokines, the receptor that they act on and the cell type that they attract. A standard chemokine nomenclature was introduced in 2000. Table 1.1 also summarises the new and old chemokine nomenclature as defined by Zlotnik and Yoshie, 2000. Chemokines play an important role in inflammatory reactions by recruiting leukocytes to the sites of inflammation (Baggiolini, 1998). It has been demonstrated that chemokines bind via a heparin-binding domain to proteoglycans on the surface of endothelial cells, which then presents the chemokine on the luminal surface of the endothelium to the intravascular leukocyte (Tanaka et al, 1993).
The CXC chemokines contain two subclasses, those with the glutamic acid-leucine- arginine (ELR) sequence within their N-terminal region and those without (non-ELR). CXC (ELR) chemokines are chemoattractant for neutrophils and examples are IL-8, GROa and NAP-2 (Clark-Lewis, et al, 1991; Taub et al, 1993). Non-ELR CXC chemokines are chemoattractant for lymphocytes and examples are SDF-1, MIG and IP- 10. The CXC chemokine genes are clustered on chromosome 4q12-q21.
28 CC chemokines differ from CXC chemokines in that they act on eosinophils, monocytes, basophils and mast cells and in general do not act on neutrophils. With respect to recruitment of eosinophils, the key CC chemokines are the monocyte chemoattractant proteins 3, 4 and 5 (MCP-3, MCP-4, MCP-5 - mouse only), macrophage inhibitory protein-la (MIP-la), regulated upon activation normal T cell expressed and secreted (RANTES), eotaxin, eotaxin-2 and eotaxin-3.
Lymphotactin, a C chemokine containing two cysteine residues, has been shown to be a potent chemoattractant for CD8+ T-lymphocytes (Kelner et al, 1994).
The CX3C chemokine, fractalkine, which has an extended mucin-like stalk has been shown to have chemoattractant activity for T-lymphocytes and monocytes (Bazan et al, 1997).
1.3.1.1 Eotaxin
Eotaxin is a member of the CC chemokine family and was originally purified from the bronchoalveolar lavage fluid (BAL) of sensitised guinea pigs following aerosolised allergen challenge (Griffiths-Johnson et al, 1993; Jose et al, 1994a). The BAL fluid was collected at different time points and injected into the skin of naïve guinea pigs previously injected intravenously with I 1 lIndium labelled eosinophils (Faccioli et al, 1991). Eosinophil migration was measured by counting the radioactivity in the relevant skin sites following intradermal injection of purified BAL fluid samples. Eosinophil migration was at its greatest in the BAL fractions collected 3-6 hours post challenge. Following purification, an active protein was sequenced and found to be a CC chemokine; this was termed eotaxin (Jose et al, 1994a). Subsequently, guinea pig (Jose et al, 1994b; Rothenberg et al, 1995a), murine (Rothenberg et al, 1995b; Gonzalo et al, 1996), human (Ponath et al, 1996; Garcia-Zepeda et al, 1996a; Kitaura et al, 1996) rat (Williams et al, 1998) and horse (Benarafa et al, 2000) eotaxins have all been cloned and all had up to 60% protein sequence similarity. Eotaxin has the highest homology to MCP-3 (Jose et al, 1994b) which although chemotactic for monocytes is also a potent eosinophil activator (Dahinden et al, 1994). The eotaxin gene is located on 17q11.2 in the CC chemokine cluster (Maho et al, 1999). Guinea pig eotaxin is an 8.3kDa protein
29 made up of 73 amino acids whereas the human eotaxin molecule contains 74 amino acids with 62% homology to the guinea pig protein (Kitaura et al, 1996; Ponath et al, 1996).
Through its action via CCR3, the eotaxin receptor, eotaxin exhibits chemotactic properties for peripheral blood eosinophils (Ponath et al, 1996; Garcia-Zepeda et al, 1996a), basophils (Yamada et al, 1997; Uguccioni et al, 1997), a subset of Th2 lymphocytes (Gerber et al, 1997; Sallusto et al, 1997) and endothelial cells (Salcedo et al, 2001). In vitro, eotaxin has been shown to stimulate eosinophil respiratory burst by releasing high amounts of reactive oxygen species comparable to the known eosinophil activator, C5a (Elsner et al, 1996). Furthermore, eotaxin induces transient increases in intracellular calcium concentration ([Ca2+]i) in human eosinophils (Elsner et al, 1996). Flow cytometric studies showed rapid and transient actin polymerisation on stimulation of eosinophils with eotaxin. At optimal concentrations, the changes induced by eotaxin were comparable with those induced with C5a, PAF, and RANTES. Eotaxin increases the expression of the 132 integrin CD11b, (Tenscher et al, 1996) and has also been demonstrated to enhance eosinophil adhesion to TNF-a stimulated lung microvascular endothelial cells through CD18 and VLA-4 (Burke-Gaffney et al, 1996). In addition, eotaxin has been shown to release preformed IL-4 from human eosinophils (Bandeira- Melo et al, 2001).
Aerosol exposure of naive guinea-pigs to eotaxin caused a selective increase in eosinophils in BAL fluid (Griffiths-Johnson et al, 1993). Eotaxin has been shown to stimulate local eosinophil accumulation following intradermal injection in guinea pigs (Griffiths-Johnson et al, 1993; Jose et al, 1994a; Collins et al, 1995; Humbles et al, 1997), mice (Teixeira, 1998), rats (Sanz et al, 1998) and rhesus monkeys (Ponath et al, 1996). The kinetics of eosinophil accumulation in the lungs of OVA challenged mice correlated with the expression of eotaxin. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA (Gonzalo et al, 1996). Intratracheal injection of eotaxin to IL-5 transgenic mice, but not the wild type, induced bronchial hyperresponsiveness (BHR) and increased the number of eosinophils detected in the BAL fluid (Hisada et al, 1999).
30 Human eotaxin has been found to be inactive on guinea pig CCR3 transfected cells when tested in a chemotaxis assay (Sabroe et al, 1998), although in contrast Fukuyama et al, (2000) have reported that human eotaxin was able to induce a Ca++ flux and chemotaxis of guinea pig eosinophils. Guinea pig eotaxin is able to induce chemotaxis of human eosinophils (Jose et al, 1994a).
1.3.1.2 Eotaxin-2
Eotaxin-2 (or myeloid progenitor inhibitory factor-2, MPIF-2) was discovered in an activated monocyte library as part of a large scale eDNA sequencing. Eotaxin-2 is only distantly related to eotaxin by its low amino acid homology (39%) and is located in different chromosomal position to eotaxin 7q11.23 (Nomiyama et al, 1998) whereas eotaxin is located on 17q11.2. Eotaxin-2 shares a high level of selectivity to activate eosinophils by the eotaxin receptor, CCR3, stimulating eosinophil respiratory burst, inducing a transient rise of intracellular calcium, cytoskeletal rearrangements and promote chemotaxis with similar potency as eotaxin (Patel et al, 1997; Forssmann et al, 1997; White et al, 1997; Dulkys et al, 2001). In addition, eotaxin-2 stimulates the chemotaxis of resting T-lymphocytes (Patel et al, 1997). Ying et al, (1999a) have shown that epithelial cells, CD31+ endothelial cells and CD68+ macrophages are the major sources of eotaxin-2 in bronchial biopsies of atopic and non-atopic asthmatics. Jahnsen et al (1999) found that mRNA expression for eotaxin-2 was significantly upregulated in nasal polyps. Peripheral blood monocytes have been shown to constitutively produce eotaxin-2 which can be upregulated by LPS, zymosan and IL-1I3, but not IL-4, IL-13 or TNF-a. Differentiation of monocytes into macrophages suppressed their constitutive generation of eotaxin-2. Moreover, IL-4, but not LPS, up- regulated the production of eotaxin-2 by macrophages (Watanabe et al, 2002).
Intradermal injection of eotaxin-2 in a rhesus monkey induced a marked local infiltration of eosinophils, which was most pronounced in the vicinity of postcapillary venules and was comparable to the effect of eotaxin (Forssmann et al, 1997). Eotaxin-2 mRNA expression correlated with eosinophil numbers detected at 24 hrs post allergen challenge in the skin of human atopic subjects, suggesting that eotaxin-2 could be involved in the later 24 hr infiltration of eosinophils (Ying et al, 1999b). Allergen challenge of mice with Aspergillus fumigatus or OVA revealed marked induction of eotaxin-2 mRNA (Zimmermann et al, 2000). Furthermore, eotaxin-2 mRNA was
31 strongly induced by both transgenic over-expression of IL-4 in the lung and administration of intranasal IL-4. Murine eotaxin-2 induced dose-dependent chemotactic responses in murine eosinophils (Zimmermann et al, 2000).
1.3.1.3 Eotaxin-3
Eotaxin-3 was cloned from IL-4 stimulated human vein endothelial cells (Shinkai et al, 1999; Kitaura et al, 1999). The predicted mature protein of 71 amino acids showed a 27-42% homology to other CC chemokines. Eotaxin-3 has been shown to be a functional ligand for CCR3 and is able to activate eosinophils to induce a transient rise of intracellular calcium, induce cytoskeletal rearrangements and promote chemotaxis with similar potency to eotaxin (Kitaura et al, 1999; Dulkys et al, 2001). Endothelial cell surface associated eotaxin-3 has been shown to be important for the CCR3 component of the sheer-dependent eosinophil migration across IL-4 stimulated HUVECs (Cuvelier and Patel, 2001). Increased eotaxin-3 mRNA expression in bronchial biopsies has been demonstrated at 24 hrs following allergen challenge of asthmatics (Berkman et al, 2001). It was shown that human dermal fibroblasts constitutively express mRNA for eotaxin-2 and that the expression levels after stimulation of fibroblasts with IL-4 and IL-4 and TNF-a the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. (Dulkys et al, 2001). The Th2 cytokines, IL-4 and IL-13 induced eotaxin-3 mRNA expression in human lung epithelial cells and dermal fibroblasts (Banwell et al, 2002). Furthermore, it has been shown that eotaxin-3 transcription levels and protein expression are increased following stimulation of human dermal fibroblasts with IL-13 and IL-4 (Hoeck and Woisetschlager, 2001).
1.3.1.4 Eotaxin in animal models of allergy
Eotaxin has been shown to be an important regulator of eosinophil trafficking into tissues during both allergic and non-allergic inflammatory responses. A detailed kinetic study has shown that allergen challenge of sensitised guinea pigs stimulates increased eotaxin generation in the lung tissue and BAL eotaxin peaked at 6 hrs. All of the eosinophil chemoattractant activity in the BAL fluid could be blocked by an antibody to eotaxin (Humbles et al, 1997). Fukuyama et al, (2000) have shown that eotaxin alone causes eosinophil accumulation in the airways but not BHR, and that additional factors
32 such as PAF are needed to activate eosinophils for the development of airway hyperresponsiveness.
In a murine model of allergic airway inflammation, blocking the action of eotaxin using anti-eotaxin antibodies significantly reduced the eosinophil recruitment into the lung by 50% (Gonzalo et al, 1996). Studies using eotaxin deficient mice have shown a reduced eosinophilia at 18hrs but not at 48hrs when compared to wild type mice (Rothenberg et al, 1997). However, another study demonstrated no difference in lung eosinophilia between wild type mice and eotaxin knockout mice following allergen challenge (Yang et al, 1998). Campbell et al, (1998) using a murine model of cockroach allergen induced airway hyperreactivity have also shown that eotaxin is a key mediator in the secondary rechallenge airway physiology. This was shown by the pre-treatment of mice with an anti-eotaxin antibody which abolished the airway hyperreactivity. Allergen induced eosinophil infiltration in the gastrointestinal tract was shown to be markedly impaired in eotaxin deficient mice (Matthews et al, 1998). Using allergen sensitised, eotaxin knockout and wild type mice, challenged with oral antigen it has been demonstrated that eotaxin is a critical regulator of antigen induced eosinophil inflammation in the gastrointestinal tract (Hogan et al, 2000). In allergic asthma, it is currently accepted that the driving force for airway eotaxin expression and eosinophilia is an allergen-specific Th2 immune response. In a mouse model of allergic asthma, inhaled allergens are processed by mucosal dendritic cells, which both initiate and maintain the activation of Th2 cells (Lambrecht et al, 2000).
Injection of eotaxin into the peritoneal cavity of mice has been shown to elicit an eosinophil response into the cavity and this response can be increased if the mice are pre-sensitised to ovalbumin (Harris et al, 1997; Das et al, 1997). Depletion of peritoneal mast cells or use of mast cell deficient mice has shown a reduction in eosinophil recruitment following intraperitoneal injection of eotaxin, which demonstrates that the mast cells have an important role in mediating the eosinophil recruitment actions of eotaxin (Harris et al, 1997; Das et al, 1998). a lipoxygenase inhibitor reduced the eosinophil accumulation into the peritoneal cavity suggesting the eotaxin may either be activating eosinophils to release leukotrienes or enhancing eosinophil responsiveness to leukotrienes (Harris et al, 1997). Using a murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens it
33 has been demonstrated that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils (Kim et al, 2001). Eotaxin is not only generated in allergic inflammation but can also be upregulated in non-allergic inflammation such as that induced by ozone inhalation (Ishii et al, 1998) or foreign body granulomatous reactions (Conroy et al, 1997; Ruth et al, 1998; Haddad et al, 2002).
1.3.1.5 Eotaxin in human disease
1.3.1.5.1 Asthma The number of cells that expressed eotaxin mRNA in the bronchial mucosa of asthmatic patients showed significant correlation with airway eosinophilia and bronchial hyperreactivity (Mattioli et al, 1997). In atopic asthmatic individuals the expression of eotaxin protein was upregulated in bronchial epithelial and endothelial cells (Ying et al, 1997). Atopic asthmatics are reported to have eotaxin mRNA expression and high eotaxin protein concentration in the epithelium and submucosa of their airways when compared to normal controls (Lamkhioued et al, 1997, Ying et al, 1997). Plasma eotaxin levels were significantly higher in patients with acute asthma symptoms and airflow obstruction than in subjects with stable asthma. Among the patients with emergency asthma flares, those who responded to asthma treatment with an increase in peak expiratory flow rate by an amount equal to at least 20% of their predicted normal value had lower eotaxin levels than those who did not (Lilly et al, 1999). Plasma eotaxin levels were associated with asthma and inversely related to lung function independent of age, race, sex, or smoking status (Nakamura et al, 1999). The concentration of eotaxin and eotaxin mRNA in the sputum of atopic patients has been demonstrated to be higher than that from the sputum of non-atopic patients (Zeibecoglou et al, 2000). Increased levels of eotaxin were found in the BAL fluid of patients with allergic asthma after allergen challenge which correlated with the number of eosinophils (Lilly et al, 2001). However, Berkman et al, (2001) have shown that eotaxin-3 mRNA and not eotaxin or eotaxin-2 mRNA expression was elevated in the later stages following allergen challenge, suggesting eotaxin is involved in the early time stages of airway inflammation.
34 1.3.1.5.2 Chronic sinusitis and allergic rhinitis and other inflammatory diseases
Eotaxin mRNA expression is increased in patients with chronic sinusitis when compared to normal controls (Minshall et al, 1997). In allergic rhinitis, eotaxin mRNA was upregulated in the epithelial and sub epithelial layers of the nasal mucosa following intra nasal allergen challenge (Minshall et al, 1997). This was direct evidence linking the increase in eotaxin mRNA expression with the number of infiltrating eosinophils into the nasal mucosa after an allergic response in atopic humans. Expression of eotaxin in nasal mucosa of grass-pollen allergic rhinitis patients was upregulated during pollen season (Pullerits et al, 2000).
Eotaxin and RANTES but not MCP-3 mRNA expression were elevated in non-atopic and atopic nasal polyps when compared to normal nasal mucosa (Bartels et al, 1997). Shin et al, 2000 showed no difference in mRNA expression for RANTES in nasal polyps, but eotaxin mRNA expression, which correlated with tissue eosinophilia and ECP levels, was markedly upregulated. Significantly higher levels of eotaxin and eotaxin-2 protein have been detected in the nasal polyp tissue compared to control middle turbinates (Caversaccio et al, 2000) while no difference in RANTES production was detected (Conroy, unpublished observations). In another study, mRNA expression for eotaxin, eotaxin-2 or MCP-4 was significantly increased in nasal polyps (Jahnsen et al, 1999). Eotaxin mRNA expression and protein concentration correlated with early (6 hrs) eosinophil infiltration when allergen was applied to the skin of atopic subjects (Ying et al, 1999a). Taha et al, (2000) demonstrate an increased expression of eotaxin and MCP-4 in acute and chronic lesions, suggesting that these chemotactic factors play a major role in the pathophysiologic mechanisms of atopic dermatitis. Spontaneous production of RANTES, MCP-1, MIP-1 a and MIP-10 by PBMC was augmented in atopic dermatitis patients compared with normal controls. It was suggested that augmented production of CC-chemokines correlates with inflammation associated with atopic dermatitis (Kaburagi et al, 2001).
The serum of patients of Crohn's disease demonstrates increased eotaxin levels and negatively correlates with eosinophil number in peripheral blood (Chen et al, 2001; Mir et al, 2002). Using a murine model of oral antigen-induced eosinophilic gastrointestinal allergy, eosinophil infiltration in the gastrointestinal tract was shown to be greatly reduced in eotaxin knockout mice (Hogan et al, 2000). Exposure of mice to enteric-
35 coated antigen promotes an extensive Th-2 associated eosinophilic inflammatory response involving the oesophagus, stomach, small intestine and Peyer's patches as well as the development of gastric dysmotility. Using electron microscopy eosinophils were _located in proximity to damaged axons which indicated that eosinophils were mediating a pathologic response. In addition, mice deficient in eotaxin have impaired eosinophil recruitment and are protected from gastric dysmotility (Hogan et al, 2001).
1.3.1.6 Cellular source of eotaxin
1.3.1.6.1 Animal Studies
Northern blot analysis showed eotaxin mRNA in the lungs of naïve and sensitised guinea pigs which was considerably increased post allergen challenge (Jose et al, 1994b, Rothenberg et al, 1995a). Immunohistochemistry of lung sections taken following aerosol allergen challenge of sensitised guinea pigs showed that airway epithelial and alveolar macrophages were the predominant cell sources of eotaxin (Humbles et al, 1997; Li et al, 1999). In a non-allergic model of lung inflammation characterised by a marked eosinophilia, alveolar macrophages appear to be the major source of eotaxin with no upregulation of eotaxin observed in epithelial cells (Conroy et al, 1997). Airway epithelial cells were identified as a major cellular source of eotaxin in the guinea pig pulmonary system (Cook et al, 1998). In addition, to constitutive eotaxin expression in the guinea pig lung (Jose et al, 1994b), RANTES has also been shown to be constitutively expressed (Asano et al, 2001). Allergen exposure in sensitized guinea pigs caused an increase in eotaxin mRNA, which correlated with pulmonary EPO activity, but not RANTES mRNA expression (Asano et al, 2001).
Using a mouse model of OVA induced pulmonary eosinophilia Gonzalo et al, (1996) demonstrated that there was an accumulation of eosinophils in the murine lung that correlated with increasing levels of eotaxin. It has been demonstrated that IL-13 is a potent inducer of eotaxin when administered intranasally into mice (Li et al, 1999). In addition overexpression of IL-13 in the lungs of mice induced an increase in eotaxin protein and mRNA (Zhu et al, 1999). Intranasal administration of IL-13 into the lungs of IL-5 knockout, IL-5/eotaxin knockout, wildtype and IL-5 transgenic mice resulted in significant lung eosinophilia (Pope et al, 2001). In a murine model of allergic asthma
36 the number of intraepithelial eosinophils was significantly increased 3 hours post exposure and had declined by 24 hours. In parallel, the amount of eotaxin detected in the airway epithelial cells was upregulated (Kumar et al, 2002).
1.3.1.6.2 Human Studies Eosinophils have been demonstrated to contain intracellular granule associated eotaxin, which could be released upon stimulation by C5a/Ionomycin, suggesting that eosinophil-derived eotaxin could contribute to the local accumulation of eosinophils at the site of inflammation (Nakajima et al, 1998). Eotaxin mRNA expression in human dermal fibroblasts was upregulated by IL-la and TNF-a within 6 hrs. IL-1 a induced eotaxin mRNA accumulation was transient; long-term stimulation with TNF-a resulted in a further increase of eotaxin mRNA expression (Bartels et al, 1996). IL-4 has been demonstrated to release three 0-glycosylated variants of human eotaxin from dermal fibroblasts (Mochizuki et al, 1998). Noso et al, (1998) have demonstrated that stimulation of dermal fibroblasts with TNF-a can result in human eotaxin generation. Miyamasu et al, (1999) could not detect generation of human eotaxin from peripheral leukocytes and vein endothelial cells or bronchial epithelial cells. However, similar to the observations of Mochizuki et al, (1998) high amounts of eotaxin were detected from dermal fibroblasts and this production was stimulated by IL-4. IL-4 and IL-13 induced eotaxin production in human nasal mucosal fibroblasts (Terada et al, 2000). Many cell types including airway epithelial cells (Stellato et al, 1995; Stellato et al, 1999), fibroblasts (Teran et al, 1999) and smooth muscle cells (Hirst et al, 2002; Moore et al, 2002) have defined distinct patterns of chemokine production in response to Thl and Th2 cytokines. IL-4 and IL-13 are potent inducers of eotaxin production whereas IFNI, is a stronger inducer of RANTES production. The effects of Thl and Th2 cytokines on RANTES and eotaxin secretion in these cells appears to be antagonistic. Human peritoneal mesothelial cells activated with IL-4 and TNF-a produced eotaxin whilst IFN-y inhibited eotaxin production (Katayama et al, 2002). Investigations on human peritoneal mesothelial cells (PMC) agree with these observations and these studies demonstrate that multiple cell lineages of distinct embryologic origin secrete eotaxin in a Th2 dependent manner (Georas et al, 2002).
37 1.3.1.7 Effects of eotaxin other than eosinophil recruitment
The constitutive presence of eotaxin in the many tissues such as the gut, lung, heart and placenta suggest that this chemokine may play a role in maintaining homeostasis (Rothenberg et al, 1995; Cheng et al, 2002b). In addition, a regulatory role for eotaxin in modulating the inflammatory response is now being recognised. Neutrophil dependent acute lung injury in rats was associated with an upregulation of eotaxin protein. Inhalation of eotaxin induced an increase in lung M1P-2 and cytokine induced neutrophil chemoattractant (CINC) which was associated with increased neutrophil accumulation and vascular injury (Guo et al, 2001). Eotaxin has been shown to suppress the production of IL-8, but not other mononuclear cell specific chemokines, from human endothelial cells (Cheng et al, 2002a). In addition, neutralisation of eotaxin in a murine model of endotoxemia has been shown to induce a significant increase in lung neutrophils, suggesting that eotaxin may play an inhibitory role (Cheng et al, 2002b).
1.3.1.8 Other eosinophil active chemokines
1.3.1.8.1 RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted)
RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) was initially described as a monocyte and CD4+ memory T-lymphocyte attractant (Schall et al, 1990) and was subsequently shown to be chemotactic for human peripheral blood eosinophils (Kameyoshi et al, 1992; Rot et al, 1992). RANTES has been shown to be expressed in various cell types including platelets (Kameyoshi et al, 1992), fibroblasts (Rathanaswami et al, 1993), macrophages and endothelial cells (Devergne et al, 1994) eosinophils (Ying et al, 1996), bronchial epithelial cells (Wang et al, 1996) and smooth muscle cells (Braciak et al, 1996). In vitro, in addition to stimulating chemotaxis, RANTES stimulates eosinophil respiratory burst and ECP release (Rot et al, 1992), and upregulates CD11 b expression (Alam et al, 1993). Priming of eosinophils by preincubation with IL-5 enhances the migratory response to RANTES (Schweizer et al, 1994; Ebisawa et al, 1994). It has been demonstrated that following intradermal injection of human RANTES into Rhesus monkeys eosinophil and macrophage recruitment is induced (Ponath et al, 1996). RANTES also induced eosinophil recruitment into the skin following intradermal
38 injection in human subjects (Beck et al, 1997). The guinea pig and human forms of RANTES induce human eosinophil migration but not that of guinea pig eosinophils (Campbell et al, 1997).
1.3.1.8.2 Monocyte Chemoattractant Protein -2, -3 & -4 (MCP)
MCP-2 stimulates migration of human peripheral blood eosinophils, basophils, monocytes, T-lymphocytes and NK cells (Noso et al, 1994; Weber et al, 1995; Uguccioni et al, 1995). In terms of potency as chemoattractants for human peripheral blood eosinophils, MCP-2 is less effective at stimulating eosinophil chemotaxis than MCP-3 or MCP-4 (Weber et al, 1995).
MCP-3 was originally identified as a monocyte chemoattractant that was produced as a secretory product from cytokine stimulated osteosarcoma cells (Van Damme et al, 1992). MCP-3 is an eosinophil chemoattractant (Opdenakker et al, 1993; Dahinden et al, 1994) which also stimulates migration of monocytes, T-lymphocytes (Taub et al, 1995), basophils (Dahinden et al, 1994) and dendritic cells (Sozzani et al, 1995). Increased MCP-3 expression is observed in atopic skin 6 h after local allergen challenge (Ying et al, 1995), and in bronchial biopsies from asthmatics (Humbert et al, 1997) suggesting a role for MCP-3 in allergic disease. All aspects of MCP-3 have been reviewed by Menten et al, 2001.
The biological activities of MCP-4 were later characterised and it was demonstrated to be chemotactic for eosinophils, monocytes and lymphocytes (Uguccioni et al, 1996).The activity on monocytes was comparable to MCP-3 and considerably less than MCP-1 MCP-4 activity on eosinophils was comparable to eotaxin and thus more potent than MCP-3. It was also demonstrated that receptor desensitisation occurs when eosinophils are exposed to MCP-4 followed by eotaxin, MCP-3 or RANTES suggesting MCP-4 shares the same receptors as these CC chemokines (Uguccioni et al, 1996). MCP-4 induced calcium flux in HEK-293 cells transfected with CCR2B or CCR3 but not those transfected with CCR1 or CCRS suggesting that MCP-4 signals via CCR2 and CCR3 (Garcia-Zepeda et al, 1996b). MCP-4 was expressed in TNF-a and IL-1 stimulated epithelial and endothelial cells and also in the epithelial mucosa of patients with Th2 type allergic and Thl type non-allergic sinusitis (Garcia-Zepeda et al, 1996b).
39 In human eosinophils, MCP-4 induced the production of oxygen species and actin polymerisation leading to respiratory burst. In addition, stimulated human dermal fibroblasts expressed high levels of MCP-4 suggesting that fibroblasts are a source of MCP-4 (Petering et al, 1998). Murine MCP-5 (which is a structural and functional homologue of human MCP-1) is weakly chemoattractant for murine eosinophils (Sarafi et al, 1997), but may be important for regulating eosinophil migration through lung tissue during the allergic airways response (Gonzalo et al, 1998).
1.3.1.8.3 MIP1-a (Macrophage Inhibitory Protein 1a)
MIP-la is a chemoattractant for eosinophils (Rot et al, 1992), monocytes (Wang et al, 1993), CD8+T-lymphocytes and B-lymphocytes (Schall et al, 1993), NK cells and dendritic cells (Xu et al, 1996). Although it is chemotactic for human peripheral blood eosinophils, MIP-la is less potent than eotaxin, RANTES or MCP-4 (Rot et al, 1992). This may be related to the fact that human MIP-la signals through CCR1, which is expressed in low numbers on human eosinophils. It has been reported that murine MIP- 1 a is a potent chemoattractant for murine eosinophils (Post et al, 1995). The role of
MIP-la in regulating eosinophil accumulation in humans is not clear, but elevated levels of MIP-la protein are found in BAL fluid of asthmatics following allergen challenge (Cruickshank et al, 1995). Sabroe et al, (1999) have described a 'sub-group' of human blood donors that produce a potent eosinophil shape change response to MIP- I a via a non CCR3 pathway. This suggests that non-CCR3 signalling pathway may play a role in eosinophil recruitment in inflammatory disease.
1.3.1.9 Non eosinophil active chemokine
1.3.1.9.1 Monocyte Chemoattractant Protein-1 (MCP-1)
MCP-1 was purified from the culture fluid of a glioma cell line by rpHPLC (Yoshimura et al, 1989). MCP-1 lacks chemoattractant activity on eosinophils but was highly potent at induced monocytes chemotaxis, T-cells and basophils (Bischoff et al, 1992; Uguccioni et al, 1995; Taub et al, 1995). MCP-1 signals via the receptor CCR2 (Charo et al, 1994). Addition of an amino acid before MCP-1's NH2-terminal glutamine or deletion of this glutamine residue reduces the biological activity of MCP-1 on
40 monocytes approximately 100 fold (Gong and Clark-Lewis, 1995). Deletion of the NH2- terminal glutamine converts MCP-1 from an activator of basophils into an eosinophil chemoattractant (Weber et al, 1996). Murine MCP-1 was able to attract human monocytes in vitro with similar activity to human MCP-1 (Ernst et al, 1994). MCP-1 has been implicated in a number of allergic and chronic inflammatory diseases (Strieter et al, 1994), such as arthritis (Koch et al, 1992), artheriosclerosis (Nelken et al, 1991), and various lung diseases (Jones & Warren, 1992; Strieter et al, 1994).
1.4 Chemokine receptors
The effect of chemokines on leukocytes is mediated by specific receptors expressed on the leukocyte cell surface. All chemokine receptors are members of the G-protein coupled receptors family. Chemokine receptors contain seven a-helical structures that transverse the cell membrane. Three extracellular loops and the amino terminus are involved in ligand binding whereas three intracellular loops and the carboxyl terminus are involved in coupling to the G-proteins and instigating the signalling cascade. All chemokine receptors (except CCR8) contain a conserved amino acid sequence of aspartic acid, arginine and tyrosine (Asp-Arg-Tyr) more commonly known as the D-R- Y motif within the second intracellular loop (Ballesteros et al, 2001) and it is believed that this motif is important in G-protein signalling. It has been proposed that, following ligand binding to the chemokine receptor, phosphorylation of the carboxyl terminus occurs resulting in uncoupling to the G-protein cascade. The chemokine receptors are then internalised within the cell by an endocytotic pathway by clatharin-dependent pathway or vesicular mechanisms (Yang et al, 1999; Gilbert et al, 2001; Tsao et al, 2001; Meuller et al, 2002; Zimmerman & Rothenberg 2003). Following chemokine degradation and subsequent receptor dephosphorylation within the endosomes, the chemokine receptors are recycled and returned to the cell surface (Yang et al, 1999). Chemokine receptor nomenclature is defined with respect to the subfamily of chemokines that they bind (Sallusto et al, 2000) however chemokine receptors are `promiscuous' and can bind several chemokines within that class. Chemokine receptor classes of relevance to this thesis are the CXC receptors (CXCR), which can bind CXC chemokines, and the CC receptors (CCR), which can only bind CC chemokines (Powers and Wells, 1996). There has been six CXC receptors and eleven CC receptors identified to date. Eosinophils express only CCRI and CCR3 (Kitaura et al, 1996). CCR3 is
41 highly expressed on eosinophils, with approximately 40, 000 receptors per cell (Daugherty et al, 1996). It has been proposed that high numbers of cell surface chemokine receptors (>10,000 receptors per cell) are required to trigger the rapid increase in leukocyte adhesion that permits the arrest of rolling leukocytes under conditions of high blood flow, whereas lower levels of expression are required for a chemotactic response (Campbell et al, 1996).
1.4.1 The eotaxin receptor: CCR3
The CC chemokines eotaxin, eotaxin-2, eotaxin-3, RANTES, MCP-2, MCP-3 and MCP-4 are ligands for the CC chemokine receptor, CCR3. Eotaxin, eotaxin-2 and eotaxin-3 signal via CCR3 whilst RANTES, MCP-2, MCP-3 and MCP-4 can signal via other CC receptors (Heath et al, 1997). The human CCR3 receptor was cloned (Kitaura et al, 1996: Ponath et al, 1996) and subsequently mouse CCR3 (Gao et al, 1996), guinea pig CCR3 (Sabroe et al, 1998) and monkey CCR3 (Iino et al, 2002) have been cloned.
In addition to its expression on eosinophils CCR3 is also expressed on a subset of Th2 lymphocytes (Sallusto et al, 1997; Gerber et al, 1997), basophils (Uguccioni et al, 1997), microglial cells (He et al, 1997), endothelial cells (Hohki et al, 1997), dendritic cells (Rubbert et al, 1998), mast cells (Ochi et al, 1999; de Paulis et al, 2001) and epithelial cells (Stellato et al, 2001). Pertussis toxin can block the CCR3 signalling pathway demonstrating that this receptor couples to G,. CCR3 mRNA has also been identified in eosinophils, peripheral mononuclear cells, bronchial epithelial cells, human endothelial cells and in cells following nasal washing from patients with allergic rhinitis (Oyamada et al, 1999). Recently it has been shown that eotaxin was a partial agonist for CCR2b on THP-1 cells and thus does not exclusively signal via CCR3 as previously believed (Martinelli et al, 2001). In contrast, in human monocytes, eotaxin has been shown to be a natural antagonist for CCR2 and an agonist for CCR5 (Oglivie et al, 2001). In addition, eotaxin has been shown to act as an antagonist of the CXCR3 receptor (Weng et al, 1998), whereas CXCR3 ligands such as IP-10, Mig and ITAC are potent antagonists of CCR3 (Loetscher et al, 2000).
Blockade of murine CCR3 in ovalbumin sensitised mice by the use of a monoclonal antibody resulted in a significant reduction in eosinophil numbers in the airway lumen
42 post challenge (Justice et al, 2002). In a separate study, large numbers of 'trapped' eosinophils were located in the blood vessels of the airways of CCR3 knockout mice following ovalbumin challenge Compared to wildtype controls. This suggests that there are other mediators involved in the migration of eosinophils from the bone marrow into the blood. These trapped eosinophils in CCR3 knockout mice had surrounded the blood vessels and migrated through the endothelium but were unable to pass the lamina elastica of the endothelium and therefore failed to migrate into the inflamed lung following ovalbumin challenge (Humbles et al, 2002). However, the CCR3 deficient mice produced enhanced AHR results relative to wildtype mice controls. The numbers of mast cells detected in the airways of CCR3 knockout mice following ovalbumin challenge was significantly higher than the wildtype mice. It was believed that these increased numbers of mast cells release their complement of allergic mediators and thus counteract any potential benefit of blockading CCR3 (Humbles et al, 2002).
Another study underlined the importance of CCR3 and eosinophils in the effector phase of allergic asthma. Injection of ovalbumin epicutaneously into mice resulted in eosinophil infiltration into the skin. When CCR3 knockout mice were used in this model, it resulted in no eosinophil recruitment into the skin. However, mast cell numbers detected in the skin were comparable to wildtype controls. This suggests that the recruitment of eosinophils but not mast cells into the skin is CCR3 dependent. Eosinophil recruitment to the lung as detected in the BAL fluid was severely impaired in CCR3 knockout mice suggesting that CCR3 plays an important role in the development of AHR (Ma et al, 2002).
1.5 Formation of a chemokine gradient
1.5.1 Glycosaminoglycans
The precise role of chemoattractant gradients in vivo is contentious (Rot, 1993; Tanaka et al, 1993). The immobilisation of chemokines by glycosaminoglycans (GAGs) are believed to play a key role in the formation of a chemokine gradient across the endothelial cell and facilitating leukocyte migration into the tissue. The endothelial cell non-specific binding sites for IL-8, RANTES and MCP-1 were deduced to be GAGs by the use of competitive binding studies (Hoogewerf et al, 1997). Further experimental
43 evidence for this theory was produced by the observation that immobilised chemokines have been demonstrated to attract leukocytes in vitro (Hub and Rot, 1998). These chemokine binding sites are not specific to an individual class of chemokine as a CXC chemokine can be displaced by a CC chemokine. This suggests that the binding to the endothelium was not due to the relevant chemokine receptor but rather by a non-specific binding protein such as GAGs. The GAG binding-motifs of proteins has been identified as the following sequences BBXB and BBBXXB where B is a basic residue and X is any other amino acid (Cardin and Weintraub, 1989; Hileman et al, 1998). See Table 1.3 for a list defining basic and non basic amino acids.
GAGs are widely diverse disaccharide repeating units (polysaccharides) covalently linked to core proteins and contain sulphate and carboxyl groups producing an overall negative charge. GAGs are located throughout endothelial cell ECM and on the cell surface. The more commonly known GAGs include heparin, heparan sulphate and chondriotin sulphate A and B. The majority of the surface proteoglycans present on vascular endothelial cells have been identified as heparan sulphate (Mbemba et al, 2001). Different chemokines will bind with differing affinities to the GAGs present on vascular endothelial cells (Kuschert et al, 1999). It has been shown that interaction with GAGs is not required for a chemokine to maintain receptor functionality on a cell as chemokines retain their functionality on cells treated with glycanases (Ali et al, 2001; Laurence et al, 2001). GAGs however, can alter the biological function of chemokines and have been shown by Webb et al (1993) that heparin sulphate can enhance neutrophil response to--IL-8, whilst Kuschert et al, (1999) have shown that soluble GAGs inhibit the calcium flux of IL-8 in neutrophils.
The surface endothelial surface binding of IL-8 to GAGs has been shown to be vital for the recruitment of neutrophils to an inflammatory site (Hoogewerf et al, 1997). MIP-1 a has been shown to be able to stimulate Chinese Hamster Ovary cells (CHO) via the human receptors CCR1 and CCR5 and this process is independent of GAGs at high chemokine concentration. However, at low MIP-1 oc concentration the presence of cell surface GAGs increased the effectiveness of the chemokine to stimulate the cells (Ali et al, 2000). Proudfoot et al, 2003 have shown that following mutation of chemokines so that they are unable to bind to GAGs are unable to recruit cell in an in vivo assay, yet
44 are still biologically active in an in vitro assay. These results further suggest that GAG binding is a prerequisite for chemokine activity in vivo.
1.5.2 The Duffy antigen/receptor for chemokines (DARC)
Originally identified as an erythrocyte receptor, DARC was subsequently shown to be identical to the Duffy blood antigen (Horuk et al, 1993; Neote et al, 1993) which is a binding protein for the malarial parasite Plasmodium vivax (Miller et al, 1975). DARC contains seven transmembrane domains similar to the classical chemokine receptors, however DARC lacks a motif in the second intracellular loop that is associated with G- protein coupling (Neote et al, 1994) and thus ligand binding to DARC does not induce signal transduction. DARC is able to bind both CXC and CC chemokines except MIP- la and MIP-113 (Szabo et al, 1995). Binding studies have shown that DARC expressed by the venular endothelium binds chemokines in situ (Hub and Rot, 1998; Patterson et al, 2002, Rot, 2003). DARC has been identified at the endothelial cell surface (Chaudhuri et al, 1997) and both the CC and CXC chemokines RANTES and IL-8 cross compete for endothelial cell binding (in situ). The DARC-binding region on the chemokine MGSA is not the same region involved in binding of the chemokine to its receptor (Hesselgesser et al, 1995). These observations suggest that DARC could act as a chemokine presenting molecule and facilitate presentation to the leukocyte.
MIP-la did not bind to the endothelial cell and did not compete for the binding of RANTES suggesting that MIP-1 a does not bind DARC (Hub and Rot, 1998). MIP-1a which does not bind to DARC does induce leukocyte migration (Lee et al, 2000) suggesting that MIP-1a might bind via another molecule or that the immobilisation of a chemokine is not vital for biological activity. Using a model of LPS induced endotoxemia DARC knockout mice have an enhanced inflammatory response (Dawson et al, 2000) suggesting that DARC might act as a regulator of a chemokine mediated response. It has been proposed by Mantovani et al, 2001 that endothelial cell DARC could possess a chemokine neutralising role and the experimental data from Dawson would support this hypothesis. The potential chemokine presentation mechanism is reviewed by Middleton et al, 2002.
45 1.6 Glycosylation
The majority of extracellular proteins of higher animals are glycoproteins. The diversity of glycoproteins is matched by the breadth of functions that they perform in a wide range of important structural and biological processes-(Varki, 1993). Glycosylation of a protein is a post-translational modification. The two main types of glycosidic linkage in glycoproteins are an N-glycosidic link via the amide nitrogen of an asparagine residue (N-glycosylation) and 0-glycosidic link via the hydroxyl group serine, threonine, tyrosine, hydroxylyosine or hydroxyproline (0-glycosylation). Significant interest is currently focused on the function of carbohydrates as recognition determinants in a variety of physiological and pathological processes and the influence of carbohydrates on a proteins antigenicity, structural folding, solubility and stability.
Newly synthesised proteins enter the endoplasmic reticulum (ER) where the N-linked oligiosaccharides are attached en bloc to an asparagine residue with the sequence N-X- S/T. The glycoproteins are exported from the ER and enter the cis-Golgi processing compartment. They then move along to the medial compartment containing the central cisternae of the stack and finally they enter the trans compartment where N- glycosylation is completed (Yamashita et al, 1999).
0-linked glycosylation (0-glycosylation) is a post-translational modification whereby a carbohydrate is covalently linked to the hydroxyl group of serine or threonine. 0- glycosylation serves a variety of putative biological functions in biology. Perhaps the most studied is the role of 0-glycans as ligands for selectins thereby mediating cell adhesion and leukocyte migration across endothelial cells (Hansen et al, 1997).
1.6.1 Sequence motifs for glycosylation
A very low percentage (less than 5-10%) of serine and threonine residues present on peptides are modified by glycosylation, which suggests that a sequence context or conformational rules must exist which determine whether a given serine or threonine is 0-glycosylated. A rule has been deduced for the N-linked glycosylation of asparagine as the consensus tripeptide Asn-X-Ser/Thr, where X can be any residue except proline (Bause, 1983). Not every sequence triplet found within a protein is N-glycosylated but those that are contain this sequence (Hunt and Dayhoff, 1970; Opdenakker et al, 1993).
46 This makes prediction of N-glycosylation relatively simple compared to 0- glycosylation where there are at least seven types : mucin, intracellular, proteoglycan, collagen, clotting factor, fungal and plant.
The most common type of 0-glycosylation is the mucin type sub-class. No clear consensus motif exists for the mucin-type of 0-glycosylation although P-X1-X2-X3 where X1-X3 contains at least a hydroxyl amino acid (e.g. Thr) and either a small aliphatic residue or another proline has been suggested (Ku and Omary, 1995). If 0- glycosylation is a post translational process (Carraway and Hull, 1991) taking place after N-glycosylation, folding and oligomerisation, then 0-glycosylation is limited to residues present at the surface of the protein. Heterogeneity in 0-glycosylation is profound, since at some sites only a fraction of the glycoprotein molecules are glycosylated even if they are expressed in the same cell type. 0-glycosylation is initiated by the transfer of N-acetylgalactosamine to the hydroxyl amino acids serine and threonine by a family of differentially expressed UDP-GalNAc polypeptide N- acetylgalactosaminyl transferase in the Golgi department (Hansen et al, 1997). Elongation of the 0-linked oligiosaccharide chain occurs in the subsequent Golgi compartments in a stepwise process.
1.6.2 Lepidopteron insect cell lines
Glycosylated human eotaxin used in experiments described in this thesis was produced using a lepidopteron insect cell line. The lepidopteron insect cell lines, Spodoptera frugiperda (Sf-9) and Mamestra brassicae (Mb) are widely used as hosts for the expression of therapeutic glycoproteins in the baculovirus/insect cell system (Lucklow and Summers, 1988; Miller, 1988; O'Reilly et al, 1994; Kang, 1998). 0-glycans produced by these systems are non complex, short chained mannose glycans with terminal N-acetylglucosamine (G1cNAc) or N-acetylgalactosamine (Ga1NAc) residues (Noteborn et al, 1992; Grabenhorst et al, 1993; Veit et al, 1993; Lopez et al, 1997). The lepidopteron insect cell line lacks sialytransferases and therefore does not produce glycoproteins containing terminal sialyic acid residues (Altman et al, 1993; Velardo et al, 1993). Relatively little is known about 0-linked glycosylation in expression cell lines. When the virus envelope protein gp50 was expressed in Sf-9 or Chinese Hamster Ovary Cells (CHO) two different glycosylated variants were produced. In the Sf-9 cell
47 line the monosaccharide 0-GalNAc was expressed with no terminal sialyic acid residue while in the CHO cell line the disaccharide 0-Gal (31-3GalNac was expressed with some of the glycans containing a terminal sialyic acid residue (Thomsen et al, 1990). When human IL-2 was expressed in St-9 cells the glycan was GalNAc (Wathen et al, 1991). Human monocyte chemoattractant protein-1 (hMCP-1) produced using a Sf-9 baculovirus expression system contained three distinct bands of varying molecular mass and this heterogeneity was assigned to differences in carbohydrate processing (Jiang et al, 1991; Ueda et al, 1994; Ishii et al, 1995).
1.6.3 Post-translational modification and cytokine biological activity
Post-translational modification, such as glycosylation, of cytokines/chemokines has been shown to affect their biological activity. The lymphocyte proliferative activity of glycosylated IL-1(3 was shown to be reduced six fold compared to non-glycosylated IL- 1(3 et al, 1991). In contrast, the proliferation of NK-1 cells induced by glycosylated variants of IL-4 was significantly more active than the lower molecular weight non-glycosylated species (Thor and Brian, 1992). hMCP-1 produced using a St-9 baculovirus expression system had a higher molecular mass than hMCP-1 produced from E.coli due to the presence of a saccharide containing Ga1131-3GalNac (Ishii et al, 1995). Each glycosylated MCP-1 variant showed equipotent monocyte chemotactic activity, which was higher than that of the E coli derived one (Ueda et al, 1994; Ishii et al, 1995). In contrast, a higher molecular mass variant of hMCP-1 isolated from tumour cells containing Gal{31_3GalNac was shown to be as equipotent as the lower molecular mass non-glycosylated MCP-1 (Jiang et al, 1991). However, non-glycosylated MCP-1 has been shown to be three times more potent as a chemotactic agent for monocytes and THP-1 cells than the glycosylated form (Proost et al, 1998). A 'hyper' glycosylated variant of MCP-1 expressed in P.pastoris has been shown to lose all chemotactic activity for myelomonocytic cells (Ruggiero et al, 2003).
The 0-linked carbohydrate in G-CSF has been shown to contribute to the stability of the molecule and increase its activity by three fold when examined in a colony forming assay. Similarly, glycosylated IFN-y showed a difference in specific activity when compared to the non-glycosylated form (Riske et al, 1991). 0-glycosylated forms of human eotaxin have been shown to be released from dermal fibroblasts and have varying potencies on human eosinophil chemotaxis. Eotaxin which was 0-glycosylated
48 at threonine 71 was shown to have a similar potency to non-glycosylated eotaxin (Mochizuki et al, 1998; Noso el al, 1998).
Post-translational modification of serine and threonine hydroxyl groups by glycosylation has stimulated interest, not only because the 0-glycans are involved in many specific cell adhesion and recognition processes, but also because they are involved in protein folding (Vliegenthart and Casset, 1998). The contributions of glycosylation that have been identified include recognition of self/non-self, alteration of biological activity, affects on solubility, alteration of half life in vivo, and induction of resistance to protease attack (Elbein, 1991; Parekh, 1991; James et al, 1995; Hounsell et al, 1996; Shiyan & Bovin, 1997; Mackiewicz, 1997). Each of these functions however, applies to a few glycoproteins or a class of glycoproteins but none represents a universal role for glycosylation (Sasai, 1998).
1.6.4 Eotaxin and glycosylation
Purification by high performance liquid chromatography (HPLC) of BAL fluid from Sephadex injected guinea pigs (non-allergic inflammation) or ovalbumin sensitised and challenged guinea pigs (allergic inflammation) resulted in three distinct peaks of eotaxin, each of which was highly potent at recruiting eosinophils following intradermal injection (Jose et al, 1994b). It is believed that the differences in retention times for these peaks reflect different glycosylation patterns. These three peaks have different molecular weights and can be differentiated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). During protein sequencing of guinea pig eotaxin the amino acid at position 70 could not be identified. When eotaxin was cloned and the protein sequence deduced from the nucleotide sequence it became apparent that the unknown amino acid was a threonine (Figure 1.1). Although the exact amino acid sequence associated with 0-linked glycosylation is unknown a threonine or a serine residue are known to be required (Van de Steen et al, 1998). The amino acid sequence of guinea pig and human eotaxin was entered into the 0-GLYCBASE website (Gupta et al, 1999). The model predicted that the threonine at position 70 on guinea pig eotaxin or the corresponding threonine at position 71 on human eotaxin were the only possible 0- glycosylation sites. Indeed, glycosylated human eotaxin forms released from Th2
49 cytokine stimulated dermal fibroblasts have a saccharide moeity present on the amino acid threonine at position 71 (Noso et al, 1998; Mochizucki et al, 1998)
1.7 Previous work that has led up to the project
Two sources of guinea pig eotaxin were tested for their ability to induce eosinophil recruitment; natural (glycosylated) eotaxin, purified from guinea pig BAL fluid obtained six hours after intravenous injection of Sephadex beads, and chemically synthesised (non-glycosylated) eotaxin provided by Dr H. Showell (Pfizer, USA). The eosinophil recruitment activity of these two variants was examined using two different systems in vivo; eosinophil recruitment from the mesentery microvasculature following topical application of eotaxin viewed via intravital microscopy and the skin bioassay measuring the accumulation of labelled intravenously-injected 1"In eosinophils. Intravital microscopy describes the experimental approach in which the microvasculature of a transparent organ such as mesentery is viewed directly by light microscopy. This technique allows the rolling, firm adhesion and emigration of leukocytes from the microvasculature to be visualised as well as changes in rolling velocity. Muscle and skin microvessels have been observed by intravital microscopy in conscious animals, eliminating any possible changes in leukocyte behaviour induced by anaesthesia or surgery (Menger et al, 1993).
Following the intravenous injection of 1111n labelled eosinophils, glycosylated and the chemically synthesised non-glycosylated guinea pig eotaxin were injected intradermally and eosinophil accumulation in response to eotaxin quantified by counting "In in the skin sites. Both forms of eotaxin were potent at recruiting eosinophils from the skin microvasculature into the skin (Collins et al, 1995). When both the glycosylated and non-glycosylated forms of eotaxin were applied topically to the guinea pig mesentery and the eosinophil recruitment visualised by intravital microscopy, only the glycosylated form induced eosinophil recruitment (Walsh et al, unpublished observations). Thus, in the skin, both the glycosylated and non-glycosylated forms of eotaxin were able to induce eosinophil recruitment, whereas when applied topically to the mesentery only the glycosylated form of the guinea pig eotaxin was able to induce eosinophil recruitment. This difference may be related to the properties of the mesothelium, a monolayer of cells that covers the mesentery on both sides.
50 1.8 The mesentery and mesothelial cells
The mesentery is a simple squamous epithelium of mesodermic origin lining the peritoneal, pleural and pericardial cavities that functions as a slippery non-adhesive surface for internal organs. A vascular capillary network lies in close proximity to the mesothelium in the underlying connective tissue, which is primarily composed of collagen and elastin. Due to its anatomical location, mesothelial lining provides a selective barrier for molecules and cells trafficking between the blood and serous cavities. The guinea pig mesentery is a uniform, continuous, thin (18ium) sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces. Tight and gap junctions allow for cell to cell adhesion among mesothelial cells (See Figure 1.2 for a diagrammatic overview, and Figure 1.3 for an anatomical picture). These cells possess numerous micropinocytotic vesicles; a conspicuous basal lamina separates the mesothelium from the underlying connective tissue (Junqueirea et al, 1987). Mesothelial cells can actively participate in different physiopathological processes such as inflammation, and leukocyte traffic to and from the serous cavities. The high levels of cytokines, chemokines, growth factors and other soluble mediators that are found in the pleural or peritoneal cavity effusions of inflammatory, infectious or neoplastic origin are likely to be produced by resident or recruited leukocytes but also by the mesothelial cell itself (Topley et al, 1996).
1.8.1 Mediators released from mesothelial cells
Mesothelial cells are an important source of colony stimulating factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony- stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) (Lanfrancone et al, 1992; Demetri et al, 1989). In addition, IL-la and IL-1f3 have been found to be constitutively produced by mesothelial cells (Lanfrancone et al, 1992). Stimulation of mesothelial cells with IL-1(3 or TNF-a results in a time-dependent and dose-dependent increase in IL-6 generation and prostaglandin synthesis (Topley et al, 1993a; Topley et al, 1994; Offner et al, 1995). 1L-6, IL-1 and chemokine levels are increased in the peritoneal fluids of inflammatory conditions e.g. peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) (Hurst et al, 2001).
51 Mesothelial cells from pleural or peritoneal cavities express specific mRNA and synthesise the chemoattractants, IL-8 (Goodman et al, 1992; Topley et al, 1993b; Antony et al, 1993; Antony et al, 1995; Nasreen et al, 1998); MCP-1 (Mohammed et al, 1998a, Nasreen et al, 1998) and RANTES (Visser et al, 1998). IL-8 was one of the first chemokines identified in supernatants of mesothelial cell cultures (Boylan et al, 1992) and its generation can be stimulatedwith IL-1 or TNF-a (Robson et al, 2001). Interestingly, when mesothelial cells were stimulated with Staphylococcus aureus or IL- 1 at the basal or apical surface, significantly higher levels of IL-8 were released toward the apical surface (Nasreen et al, 2001). This suggests that mesothelial cells can regulate the direction of migration of neutrophils by polarised secretion of IL-8. Eotaxin and RANTES mRNA and protein were also shown to be induced following cytokine stimulation of mesothelial cells. Eotaxin production was induced by TNF-a and IL-4 and could be markedly upregulated by their combination. In contrast, eotaxin generation was inhibited by IFN-1 (Katayama et al, 2002).
1.8.2 Adhesion molecules
In a non-disease state the human mesothelial cell expresses CD44 and integrins (Ross et al, 1998; Witz et al, 2000). In recent years it has become clear that the mesothelium plays a prominent homeostatic role in the peritoneum, and can be profoundly altered in disease states. Mesothelial cells initiate pleural inflammation in response to various cytokines by the production and release of CXC and/or CC chemokines and the expression of adhesion molecules (Jonjic et al, 1992; Cannistra et al, 1994; Liberek et al, 1996; Nasreen et al, 1998). Adhesion molecules play a key role in cellular traffic through the mesothelium, particularly during the inflammatory response, when leukocytes from the peripheral circulation enter the peritoneal space (Holm et al, 1995). Nasreen et al (2001) demonstrated that the migration of neutrophils across the mesothelial cell monolayer was dependent upon ICAM-1 expression. Surface expression of ICAM-1 but not VCAM-1 was detected on freshly isolated mesothelial cells (Cannistra et al, 1994; Liang and Sasaki, 2000). LPS and cytokines such as IL-1 and TNF-a have been shown to induce the upregulation of the adhesion molecules ICAM-1 and VCAM-1 (Cannistra et al, 1994; Liberek et al, 1996; Liang and Sasaki, 2000). This increase in ICAM-1 and VCAM-1 expression could further facilitate leukocyte migration.
52 1.9 Hypothesis of this thesis
The work described in this thesis is based on the observation that natural, glycosylated eotaxin when topically applied to guinea pig mesentery induces eosinophil recruitment from the mesenteric microvasculature, whereas non-glycosylated eotaxin does not. The hypothesis is that glycosylation is necessary for the eotaxin to cross the mesothelial monolayer covering the mesentery. Possible mechanisms are addressed.
The main aims were: -
• to develop an in vitro technique that would reproduce the observations found in vivo following topical application of eotaxin to the guinea pig mesentery.
• to identify the saccharides found on naturally produced guinea pig eotaxin and St-9 expressed human eotaxin.
• to determine whether glycosylated and non-glycosylated human eotaxin produce similar responses to their guinea pig counterparts when applied to the guinea pig mesentery in vitro.
• to determine whether the glycosylation of eotaxin confers protection from mesentery ecto and endoproteases.
• to investigate if the glycosylation present on eotaxin affected the ability of the chemokine to bind to glycosaminoglycans
• to investigate the effects of glycosylation on the chemotactic activity of other CC and CXC chemokines.
53 disulphide bonds
0-glycosylation
73 Carboxyl terminus
Figure 1.1 - Schematic representation of the 73 amino acid guinea pig eotaxin Schematic representation of the 73 amino acid sequence of guinea pig eotaxin showing disulphide bonds at position 8-33 and 9-49 and the potential 0-glycosylation site at position 70 are shown.
54 mesentery blood vessel small intestine
Figure 1.2 - Photo of guinea pig mesentery and small intestine
55 ?'• 11%, •:
•••;'' r: .4" ?•1.7%.:4: .• N. :" •"p • ?' • ..11.
blood vessel connective tissue
Vascular endothelium mesothelial cell
Figure 1.3 - A diagrammatic representation of the structure of the guinea pig mesentery (cross section)
56 Systematic Systematic Name Human Ligand Human Ligand Name CC Family CXC Family CCL1 1-309 CXCL1 GROa CCL2 MCP-1 CXCL2 GROI3 CCL3 MIP-la CXCL3 GROy CCL4 MIP-113 CXCL4 PF4 CCL5 RANTES CXCL5 ENA-78 CCL7 MCP-3 CXCL6 GCP-2 CCL8 MCP-2 CXCL7 NAP-2 CCL11 Eotaxin CXCL8 IL-8 CCL13 MCP-4 CXCL9 MIG CCL14 HCC-1 CXCL10 IP-10 CCL15 MIP-16, Lkn-1 CXCL11 I-TAC CCL16 HCC-4 CXCL12 SDF- 1 a/I3 CCL17 TARC CXCL13 BLC/BCA-1 CCL18 PARC CXCL14 BRAK CCL19 MIP-3 [3 C Family CCL20 MIP-3a XCL1 Lptn CCL21 6Ckine XCL2 SCM-1[3 CCL22 MDC CX3C Family CCL23 MPIF-1, Ck138-1 CX3CL1 Fractalkine CCL24 Eotaxin-2 CCL25 TECK CCL26 Eotaxin-3 CCL2? CTACK
IL-8 (interleukin-8), GCP-2 (granulocyte chemotactic protein-2), GROa I3,y (growth related oncogene) ENA-78 (epithelial cell derived neutrophil activating factor), IP-10 (y-interferon inducible protein), MIG (monokine induced by y-interferon), SDF (stromal cell derived factor), BCA (B cell chemoattractant), MIP-X (monocyte inhibitory protein —X), MCP-X (monocyte chemoattractant protein —X), TARC (T cell and activation related chemokine), SLC (secondary lymphoid tissue chemokine) Table 1.1 Chemokine Nomenclature for the CC and CXC chemokine families (Zlotnik and Yoshie, 2000)
57 Name Main Agonists CXC subgroup CXCR1 CXCL8 CXCR2 CXCL1-3, CXCL5-8 CXCR3 CXCL9-11 CXCR4 CXCL12 CXCR5 CXCL13 CXCR6 CXCL16 CC subgroup CCR1 CCL3, CCL5, CCL7, CCL8, CCL13-16, CCL23 CCR2 CCL2, CCL7, CCL8, CCL13 CCR3 CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL24, CCL26 CCR4 CCL17, CCL22 CCR5 CCL3, CCL4, CCL5, CCL8, CCL14 CCR6 CCL20 CCR7 CCL19, CCL21 CCR8 CCL1, CCL4, CCL17 CCR9 CCL25 CCR10 CCL26-28 CX3C and C subgroups CX3CR1 CX3CL1 XCR1 XCL1-2
Table 1.2 Chemokine receptor family and their agonists (Murphy, 2002)
58 Amino Acid Symbol 1 Symbol 2 Group Arginine Arg R Basic Lysine Lys K Basic Histidine His H Basic Aspartic Acid Asp D Acidic Asparagine Asn N Acidic Glutamic Acid Glu E Acidic Glutamine Gln Q Acdic Glycine Gly G Aliphatic R group Alanine Ala A Aliphatic R group Valine Val V Aliphatic R group Leucine Leu L Aliphatic R group Isoleucine Ile I Aliphatic .R group Serine Ser S Non-aromtic with hydroxyl R groups Threonine Thr T Non-aromtic with hydroxyl R groups Cysteine Cys C Sulphur containing R groups Methionine Met M Sulphur containing R groups Phenylalanine Phe F Aromatic Ring Tyrosine Tyr Y Aromatic Ring Tryptophan Trp W Aromatic Proline Pro P Imino Acid
Table 1.3 Amino acids and their relevant grouping
59 Chapter 2
Materials and Methods
60 2 Materials and Methods
2.1 Materials
2.1.1 Animals
Male and female Dunkin-Hartley guinea pigs (300-350g), female Dunkin-Hartley ex- breeder guinea pigs (600g+) were purchased from Charles River (Margate, Kent,UK).
2.1.2 General Reagents
BSA (RIA grade), phalloidin-TRITC, protease inhibitors AEBSF, E64, bestatin, leupeptin, aprotinin, pepstatin A, Plant lectins: Arachis hypogaea, Arachis hypogaea (biotinylated), Glycine max lectin, Glycine max (biotinylated) lectin, Conavalia A (biotinylated), Triticum vulgaris agglutinin, Triticum vulgaris agglutinin (biotinylated); sodium cyanide, monensin, nocodazole, filipin, 2-deoxyglucose (2-DOG), N-acetyl- galactosamMe (Ga1NAc), N-acetylglucosamine (G1cNAc), galactose, glucose, Tween 20 (polyoxyethlene sorbitan monaurate), thiomerosal, Cellfix buffered formaldehyde solution, tartrazine, carbonate buffer capsules, phosphate buffered saline tablets, phenol red, sodium azide, piperidine iodide, goat serum, Triton X-100, sodium azide, sodium phosphate (monobasic), heparin, chondroitin sulphate B, heparin, granular paraformaldehyde and phalloidin-labelled TRITC were purchased from Sigma Chemical Company, Poole, Dorset, UK. EDTA (made up to a 250mM stock and pH adjusted with NaOH), EGTA (made up to a 100mM stock and pH adjusted with NaOH), NaC1, trifluroacetic acid (TFA), AnalaR distilled water (HPLC grade), KH2PO4, K2PO4, H2SO4, were purchased from BDH, Poole, UK. Glycan differentiation kit was purchased from Boerhinger Mannheim (Germany). Horseradish peroxidase linked neutravidin was purchased from Pierce Chemical Company (Rockford, Illinois, USA). ELISA substrate tetramethylbenzidine/hydrogen peroxide (TMB H202) was purchased from Neogen Corp, (Kentucky, USA). Percoll and the PhastGel high density SDS-PAGE gels were purchased from Pharmacia Ltd (Upsalla, Sweden).
61 Hypnorm (fentanyl citrate 0.315mg/ml, fluanisone 10ing/m1) was purchased from Janssen Pharmaceuticals Ltd (Oxford, UK). Expiral (sodium pentobarbitone 200mg/m1) was purchased from May and Baker (Dagenham, UK). Heparin (1000units/m1) was purchased from Leo Laboratories, Bucks, UK. Hanks balanced salt solution without calcium and magnesium, Hanks balanced salt solution with calcium and magnesium, HEPES buffer solution, Dulbecco's sterile PBS solution were purchased from Life Technologies (Paisley, Scotland). The 48 wells (60uL volume) microBoyden chambers used in the chemotaxis assays were purchased from Neuro Probe Inc (Gaithersburg, MD, USA). The polycarbonate filters (51..tm) were purchased from Receptor Technologies, Oxon, UK. Immunofluore was purchased from ICN biomedicals Ltd (Aura, Ohio, USA). Mount Gene Frame gaskets were purchased from Advanced Biotechnologies (Leatherhead, UK).
2.1.3 Special Reagents
Hanks Balanced Salt Solution; D-Glucose 5mM, CaC12 3.33mM, MgC12.6H20 0.49mM, KC1 4.56mM, NaC1 120mM, Na2HPO4 0.7mM, NaH2PO4 1.5mM, NaHCO3 24mM,. Supplemented with BSA 0.25% and HEPES 30mM, pH adjusted to pH 7.4 with 1M NaOH (all reagents from Life Technologies, Paisley, Scotland, UK.). a) Kimura's stain Kimura's stain for positive identification of eosinophils (Kimura et al, 1973); 11 ml 0.05% Toluidine blue (Merck Ltd.), 5m1 0.0067M phosphate buffer, 0.03%`Light green (Merck Ltd.), 0.5m1 saturated solution of saponin in 50% ethyl alcohol (Sigma Chemical Co). Filtered before use. b) PhastGel High Density Buffer
SDS-PAGE of low molecular weight proteins using PhastGel High Density PhastGel high density is a homogeneous polyacrylamide gel designed for the separation of peptides and low molecular weight proteins.
62 The buffer system in the gel consists of :- 112mM acetate, 112mM Tris, pH 6.5. The SDS buffer strips contain 200mM tricine, 200mM Tris, 0.55%SDS and pH 8.1. Bands were visualised by staining with a Phast GelTM stain kit according to manufacturer instructions.
2.1.4 General Buffers
PBS (phosphate buffered saline): 8g/1 NaC1, 0.2g/1 KC1, 1.44g/1 Na2PO4, 0.24g/1 KH2PO4 and HC1 adjusted to pH 7.4. Filter before use.
2.1.5 ELISA Buffers
Coating Buffer :- Carbonate/bicarbonate buffer (pH 9.6) Coat/Block wash buffer(CB wash):- 1mM KPO4, 0.005% Tween20 and 0.02% thiomerosal Assay wash buffer (AB wash):- 0.15M NaC1, 1mM NaPO4, 2.7mM KC1, 0.02% tween20, 0.02% thiomerosal. Assay Buffer:- PBS, 0.1% BSA and 0.05% thiomerosal
2.1.6 Antibodies
10C11 - mouse anti-human eotaxin, monoclonal IgG was a gift from Leukosite Inc (Cambridge,MA, USA) #N3 - Polyclonal rabbit anti-human eotaxin IgG was produced in house by Dr P Jose. Mouse anti-human RANTES monoclonal IgG and biotinylated polyclonal IgG were purchased from R&D Systems (Oxon, UK). 72D - monoclonal mouse anti-guinea pig eotaxin purified IgG2b was a gift from Dr T Wells, Glaxo-Wellcome (Geneva, Switzerland). 3 #B - polyclonal rabbit anti-human eotaxin IgG was produced in house by Dr P Jose from rabbit serum 2A8 and 2A8 F(ab')2 fragments- IgG2a, mouse anti-guinea pig CCR3 from Dr P Ponath, Leukosite Inc. (Cambridge, MA, USA). #P3 - polyclonal rabbit anti-human IgG was donated from was produced in house by Dr P Jose 2G6 - mouse anti-human eotaxin monoclonal antibody was purchased from R&D Systems (Oxon, UK).
63 343 — non-biotinylated and biotinylated anti-human eotaxin-2 IgG was purchased as a matched pair from R&D Systems (Oxon, UK).. 679 — monoclonal mouse anti-human MCP-1 was purchased as matched pair from R&D Systems (Oxon, UK).. 279 — biotinylated goat IgG anti-human MCP-1 was purchased as a matched pair from R&D Systems (Oxon, UK). 208 — non-biotinylated and biotinylated anti-human IL-8 IgG was purchased as a matched pair from R&D Systems (Oxon, UK). 678 — mouse monoclonal anti-human RANTES was purchased as a matched pair from 278 — biotinylated anti-human RANTES goat IgG was purchased from R&D Systems (Oxon, UK). 17H4 (biotinylated) - monoclonal rabbit anti-human eotaxin-2 IgG was a gift from SmithKline Beecham. Goat-anti rabbit F'ab Alexa Fluor 488 was purchased from Molecular Probes, UK. NCL-GAL3 mouse monoclonal anti-human galectin-3 antibody was a gift from Novocastra Laboratories, Newcastle, UK. HRP-anti-rabbit IgG & HRP-anti-mouse IgG were purchased from Amersham International Plc (Buckinghamshire, UK). HRP-anti-goat and HRP-anti-guinea pig IgG were purchased from Euro-Path Ltd (Luton, UK).
2.1.7 Chemokines
Recombinant human eotaxin expressed in E.coli (non glycosylated) was purchased from Peprotech (London, UK). Synthetic human eotaxin was kindly provided by the late Dr I Clark-Lewis , Vancouver, Canada Guinea pig eotaxin (amide and carboxyl terminus) was kindly provided by Dr H Showell, Pfizer Central Research (Groton, USA). The synthetic guinea pig eotaxin with an Ala-Ala C terminal extension and the recombinant glycosylated human eotaxin produced in Sf-9 cells were gifts from Dr D Soler, Leukosite, MA (now Millennium Pharmaceuticals).
64 The recombinant glycosylated human eotaxin produced in Sf-9 cells using a baculovirus expression system was a gift from Dr D Soler, Leukosite, MA (now Millennium Pharmaceuticals).
The second source of glycosylated human eotaxin produced in St-9 cells using a baculovirus expression system was a gift from Fournier Laboratories, France. The natural glycosylated forms of guinea pig eotaxin were purified from the lungs of Sephadex injected guinea pigs as previously described by Jose et al, 1994a. Three peaks of activity were purified using reverse phase HPLC. Peak III was used in most of the studies described. Peaks I and II were compared for biological activity in the in vitro Gated Auto Florescence/Forward Scatter (GAFS) assay.
Human eotaxin-2 (C1((3-6) was a gift from Dr J.White, SmithKline Beecham, Philidelphia, USA
Human eotaxin-2 (non-glycosylated) was purchased from Peprotech (London, UK) Glycosylated MCP-1 was expressed in Sf-21 cells and was purchased from Pharminogen (CA, USA).
Non-glycosylated MCP-1 was purchased form Peprotech (London, UK).
2.1.8 Other chemoattractants
Human Complement fragment 5a (C5a) was produced in house by Dr P Jose. LTB4 was a gift from Ann Arbor, Cayman Chemicals, Michigan, USA.
65 2.2 Methods
2.2.1 Gated Auto Fluoresce Scatter Assay (GAFS)
2.2.1.1 Preparation of human eosinophils from peripheral blood
Volunteer blood donors were healthy normal subjects, or atopics. Platelet rich plasma was removed by centrifugation of citrated whole blood, after which the erythrocytes were removed by dextran sedimentation. The cell suspension was centrifuged for 20 min at 400g the mononuclear cells and platelets containing buffer coats were removed and the polymorphonuclear leukocyte (PMNs) was subsequently mixed with 1 vol gelatine solution (2.5% mass/vol in NaC1). After sedimaentation for 30 mins at 37°C the PMN rich supernatant was collected and the cells were re-suspended in 3m1 HBSS buffer containing 30mM HEPES, 0.25% BSA, pH 7.4. This was layered over discontinuous Percoll gradients of 1.095, 1.092, 1.090, 1.088, 1.085, 1.080g/m1 and subsequently centrifuged at 400g for 20min at 4°C. Cells were collected from each interface and contaminating erythrocytes were lysed by hypotonic shock. Fractions containing more than 90% eosinophils were used as eosinophil preparations.
2.2.1.2 Gated Auto Florescence/Forward Scatter Assay (GAFS assay) for
eosinophil activation
Mixed granulocytes were washed in buffer (PBS containing 10mM HEPES and 10mM glucose plus 0.1% BSA, pH 7.2-7.4) and pre-incubated for 30 mins at 37°C. The cells were washed in buffer and held for 5 mins at room temperature. Aliquots of cells (5x105 mixed granulocytes) were mixed with agonists or buffer in 1.2m1 polypropylene cluster tubes (Costar, Cambridge, MA) in a final volume of 100µ1. The tubes were placed in a 37°C shaking water-bath for 4 mins, after which they were transferred to an ice-water bath and 250111 of ice cold optimised fixative (a 1:4 dilution of lxlfix' buffered formaldehyde solution in PBS) added to terminate the reaction and maintain cell shape change until analysis. The samples were then analysed immediately on FACScan flow cytometer (Becton Dickinson, Mountainview, CA). Data was acquired using the FL-2 fluorescence channel on a sensitive setting allowing eosinophils to be distinguished
66 from neutrophils by the higher auto-fluorescence of the former. Forward light scatter (FSC), side scatter (SSC), and FL-2 data were acquired and acquisition terminated after 500 high fluorescence (eosinophil) events (Sabroe et al, 1999).
2.2.2 Chemotaxis
2.2.2.1 "11n-labelling of guinea pig eosinophils
10-20u1 of 111Indium chloride (approximately 4MBq) in 0.04M hydrochloric acid was chelated to 2-mercaptopyridine-N-oxide (4ug in 100u1) of 50mM PBSD, pH 7.5) and incubated with 2.0 — 5.0 x 107 eosinophils for 15 min at room temperature. The cells were then washed in 10m1 HBSS/PPP and centrifuged (380g, 7min 20°C). A 101a1 sample was taken from the supernatant and diluted in lml saline, this sample was labelled S1. Ther cells were then washed twice more in 10m1 HBSS/PPP removing 10µ1 aliquot eaxch time. These aliquots were counted and the radioactivity in counts per 111 minute (CPM) determined for calculation of the percentage binding 1n to the eosinophils in vitro. After the final wash, the cells were resuspended in lml HBSS/PPP and a 10 jul aliquot taken from suspension. This aliquot was diluted 1000-fold in saline and the radioactivity in lml determined. From this, the total number of eosinophil- bound 111In counts and the total CPM used in the labelling procedure was calculated. The eosinophils were then diluted to a final diluted concentration of lx107 111In- eosinophils/m1 for intravenous (i.v.) injection into an ear vein of recipient animals. Each 111 animal received 5x106 -eosinophils.
The number of eosinophil-bound 111In-counts was calculated as follows :
Total eosinophil-bound CPM = CPM in 10u1 x 1000
The CPM per eosinophil was then calculated as :
CPM per eosinophil = Total eosinophil-bound CPM / No of eosinophils labelled
The percentage binding of 1111n to the eosinophils in vitro was then calculated as :
% binding = (Total eosinophil-bound CPM x 100) / (Total CPM)
67 Where total CPM is calculated as :
Total CPM = (Total eosinophil-bound CPM) + CPM in washes 1+2+3
2.2.2.2 Preparation of guinea pig peritoneal eosinophils.
Peritoneal eosinophils were purified as previously described (Faccioli et al, 1991). Guinea pigs (ex breeder (600g+) were injected with lml horse serum into the peritoneal cavity every two days for a minimum of five injections. 18 hrs after the final injection the guinea pig was killed by asphyxiation with CO2. The peritoneal cavity was washed out with 35m1 of heparinised (100units/m1) saline. The cell suspension was centrifuged for 7 min at 300g. The cells were re-suspended in 3m1 buffer (HBSS buffer containing 30mM HEPES, 0.25% BSA, pH 7.4) and were layered over discontinuous Percoll gradients of 1.100, 1.095, 1.090, 1.085, 1.080g/ml that were then centrifuged at 1500g for 25min at 20°C. The purified eosinophils (found in the bands between 1.090/1.095g/ml and 1.095/1.100g/m1) were then removed and washed in 10m1 buffer (7min, 300g , 20°C). The eosinophils were re-suspended in lml buffer and an aliquot stained with Kimura counted in an improved Neubauer haemocytometer.
2.2.2.3 Chemotaxis of guinea pig eosinophils using guinea pig mesentery or polycarbonate filter (5itm pore) in a microBoyden chamber
Guinea pig peritoneal eosinophils were purified as described in section 2.2.2.2 (eosinophils were only used if the purity was greater than 95%). 281,t1 aliquots of guinea pig eotaxin (3-100nM) in HBSS containing 30mM HEPES, 0.25% BSA pH7.4 were placed into the bottom wells of a microBoyden chamber in duplicate and covered with a 51.tm polycarbonate filter or freshly isolated guinea pig mesentery. Any well that contained a visible blood vessel was excluded from the experiment. On average approximately 15 wells out of a possible 40 wells in the microBoyden chamber could be used. In each experiment, guinea pig eosinophils (1x105 cells in 28µ1 of assay buffer) were placed into the top wells. The microBoyden chamber was then incubated for 1 hr at 37°C in a humidified 5% CO2 95% 02 atmosphere. Cells migrating to the bottom chamber were counted using a flow cytometer (Beckton Dickinson FACScan®). Figure 2.1 shows a diagrammatic representation. The results are expressed as chemotactic index. For each experiment a mesentery from a freshly killed guinea pig was used.
68 The chemotactie index = stimulated migration random migration
2.2.2.4 Migration of human eosinophils measured in vitro using the MicroBoyden chamber.
Human blood eosinophils were purified as described in Section 2.2.1.1. The purity was greater than 99%. Assay buffer (24.1.1 aliquot) containing human eotaxin (3-1 00nM) was placed into the lower wells, and either a freshly isolated guinea pig mesentery or polycarbonate filter was placed over the lower chamber. Purified human eosinophils (1x105 eosinophils in 28111 of assay buffer) were placed in the top wells. The chamber was incubated for 1 hr at 37°C in a humidified 5% CO2 95% 02 atmosphere. Cells migrating through into the bottom chamber were counted using a flow cytometer (Beckton Dickinson FACScan®) and in some experiments for comparison the aliquots were stained with Kimura stain and counted using light microscopy. For each experiment a mesentery from a freshly killed guinea pig was used.
2.2.3 The Flux Assay
2.2.3.1 Guinea pig eotaxin movement across a polycarbonate filter or freshly isolated guinea pig mesentery
28µ1 aliquots of guinea pig eotaxin (3-100nM) in HBSS containing 30mM HEPES, 0.25% BSA pH7.4 were placed into the bottom wells of a microBoyden chamber, and either a freshly isolated guinea pig mesentery or a 51.tm pore polycarbonate filter was placed over the lower chamber. Assay buffer was placed in the top well and the chamber was incubated for 1 hr at 37°C in a humidified 5%CO2, 95%02 atmosphere. Eotaxin flux across the tissue into the top well was determined using a guinea pig eotaxin ELISA that recognised the glycosylated and non-glycosylated eotaxins with equal affinity.
69 2.2.3.2 Human cotaxin flux across the guinea pig mesentery or a
polycarbonate filter
The procedure was similar to the guinea pig eotaxin flux described in 2.2.3.1, with glycosylated and non-glycosylated human eotaxin (10-100nM) being used in place of guinea pig eotaxin. Human eotaxin was measuered using a human eotaxin ELISA that recognised the glycosylated and non-glycosylated variants with equal affinity.
2.2.3.3 Addition of inhibitors
The metabolic inhibitors sodium cyanide (100mM) and 2-deoxyglucose (10-1004m1), the transcytosis inhibitor monensin (1-30µM), the cholesterol binding agent filipin (1- 30µM), the saccharides N-acetylgalactosamine (10-100mM), N-acetylglucosamine (10- 100mM), galactose (10-100mM), glucose (10-100mM), the calcium ion chelator EGTA (10mM), the serine protease inhibitor 4-(amidinophenyl)methanesulfonyl (APMSF 30µM), the metallo protease inhibitor bestatin (50µM), the cysteine protease inhibitor leupeptin (30µM), the general protease cocktail inhibitor mix (containing metallo-, serine — and cysteine protease inhibitors) (100uM) and the anti-CCR3 antibody 2A8 (10µg/m1) were added to the top and bottom wells in the microBoyden chamber assays.
2.2.3.4 Cooling of the mesentery tissue
The procedure was similar to the human eotaxin flux described in 2.2.3.2, but, the freshly isolated guinea pig mesentery was washed for 5 mins with ice cold HBSS buffer containing 30mM HEPES, 0.25% BSA pH7.4. Glycosylated and non-glycosylated human eotaxin (10-100nM) was diluted using ice cold buffer and the experiment was performed at 4°C.
2.2.4 In vivo study
2.2.4.1 Intraperitoneal injections of glycosylated and non-glycosylated human eotaxin in the guinea pig.
Guinea pigs (250-300g) were sedated with Hypnorm and injected intraperitoneally with either 1 ml of 30nM glycosylated or non-glycosylated human eotaxin. At, 0, 15, 30, 60
70 and 120 mins following injection of human eotaxin, 400u1 of blood was drawn from the ear vein into heparin (100 units/m1). Following centrifugation (300g for 5 mins) the plasma was removed and stored at -20°C.
2.2.4.2 Preparation of plasma samples for human eotaxin ELISA
The plasma samples (see 2.2.4.1) were thawed at room temperature. An equal volume of 1.2% TFA/1.35M NaC1 was added to the plasma and left at room temperature for 5 mins. The samples were then centrifuged at 5000g for 5 mins. The supernatants were removed and 0.4 ml of neutralising solution (0.5m1 1M NaPO4 (pH 7.4) 0.45m1 1M NaOH, 0.05m1 Phenol Red made upto 50m1 with H20) was added to every 0.1m1 of supernatant ensuring that the final pH was 7.4. The samples were assayed for human eotaxin by ELISA.
2.2.5 Enzyme Linked Immunosorbent Assay (ELISA)
2.2.5.1 Guinea-pig eotaxin ELISA (monoclonal Ab coat)
96 well plates were coated with 100µ1/well of a monoclonal anti-guinea pig eotaxin antibody 72D+ (2µg/m1) in a carbonate/bicarbonate buffer (pH 9.6). After 12-15 hrs, the plates were washed four times with 250µ1 of coat/block (C/B) wash. The plates were blocked with 250111 of 1% milk protein in PBS for 1 hr, and washed as before with C/B wash buffer. 100[1 each of guinea pig eotaxin standard (10-320pM) or sample were added to the plate and incubated for 1 hr. The plate was then washed with assay wash buffer and incubated with rabbit anti-guinea pig eotaxin IgG B3 (1/boo dilution) for 1 hr. The plate was again washed with assay wash buffer and incubated with 100µ1 of goat anti-rabbit IgG linked HRP ('/,000 dilution). The plate was washed with assay wash buffer and l00µ1 of tetramethylbenzidine/H202 substrate added, after 30 mins the reaction was stopped by addition of 1001.t1 of 0.18M H2SO4. The absorbance was read within 30 mins at 450nm (A450). Standards, samples and antisera were diluted in assay buffer. The assay was performed at room temperature. The matched antibodies used in the guinea pig eotaxin ELISA had equal affinity for the glycosylated and non- glycosylated guinea pig eotaxin forms (Figure 2.2) and did not cross-react with any
71 other guinea pig or human chemokine. The lower and upper limit of detection for the guinea pig eotaxin was 32pM to 640pM respectively.
2.2.5.2 Guinea pig eotaxin ELISA (polyclonal Ab coat)
The ELISA was similar to that described above except the plates were coated with a polyclonal rabbit anti-guinea pig eotaxin IgG B3' ('/400 dilution), and left overnight at room temperature. After blocking the plates with 3% BSA in PBS for 2 hrs the samples and eotaxin standards (2-128pM) were added for 1 hr. The detection antibody was a biotinylated polyclonal rabbit anti-guinea pig eotaxin IgG B3' (1/1000 dilution). Neutravidin linked HRP was used at a 1/2000 dilution. This assay using the same polyclonal antibody for capture and detection was 3 times more sensitive than with the mAb capture (Figure 2.3)
2.2.5.3 Direct guinea pig eotaxin ELISA
96 well plates were coated guinea pig eotaxin variant (0.3-30nM, 100W/well) in a carbonate/bicarbonate buffer (pH 9.6). After 12-15 hrs, the plates were washed four times with 250R1 of coat/block (C/B) wash. The plates were blocked with 250111 of 5% milk protein in PBS for 2 hrs, and washed as before with C/B wash. 100111 of biotinylated rabbit polyclonal anti-guinea pig eotaxin B3 (1/300 dilution) were added to the plate and incubated for 2 hrs. The plate was then washed with assay wash with 100R1 of neutravidin linked HRP ('/i000 dilution) added for 30 mins. The plate was washed with assay wash and 1000 of tetramethylbenzidine/H202 substrate added, after 30 mins the reaction was stopped by addition of 100u1 of 0.18M H2SO4. The absorbance was measured within 30 mins at 450nm (A450). Samples were diluted in assay buffer (PBS, 0.1% BSA and 0.05% thiomerosal). The assay was performed at room temperature.
2.2.5.4 Human eotaxin ELISA
A similar ELISA protocol to that described in 2.2.5.1 was used to assay human eotaxin. The plates were coated with a monoclonal anti human eotaxin antibody 10C11 (2µg/ml) at room temperature overnight. The plates were blocked with 5% milk protein for a
72 minimum of 1 hr. The samples and standards (1.25-80pM) were left at 4°C overnight. The detector antibody rabbit anti-human eotaxin IgG (#N3 1/400 dilution) was added to the plates and left for 4 hrs at room temperature. The matched antibodies used in the human eotaxin ELISA recognised both the glycosylated and non-glycosylated forms with equal affinity (Figure 2.4).
2.2.5.5 Direct human Eotaxin ELISA
96 well plates were coated human eotaxin variant (0.3-30nM, 1O0µ1/well) in a carbonate/bicarbonate buffer (pH 9.6). After 12-15 hrs, the plates were washed four times with 250p.1 of coat/block (C/B) wash. The plates were blocked with 250µ1 of 5% milk protein in PBS for 2 hrs, and washed as before with CB wash. 100µ1 of biotinylated 10C11 (mouse mAb oc-human eotaxin) (2n/m1) were added to the plate and incubated for 2 hrs. The plate was then washed with assay wash with 100111 of neutravidin linked HRP (1 /1000 dilution) added for 30 mins. The plate was washed with assay wash and 100µ1 of tetramethylbenzidine/H202 substrate added, after 30 mins the reaction was stopped by addition of 100u1 of 0.18M H2SO4. The absorbance was measured within 30 mins at 450nm (A450). Samples were diluted in assay buffer (PBS, 0.1% BSA and 0.05% thiomerosal). The assay was performed at room temperature. The ELISA sensitivity for human eotaxin was 1000pM to 8000pM. The lower and upper limit of detection was 1.25pM to 80pM , respectively.
2.2.5.6 Human Eotaxin-2 ELISA.
As human eotaxin ELISA except the plates were coated using MAB343 (1/500 dilution) biotinylated anti-human eotaxin-2 IgG and human eotaxin-2 was detected using BAF343 biotinylated anti-human eotaxin-2 IgG (0.05n/m1). The matched antibodies recognised the glycosylated form of human eotaxin-2 approximately two fold less than the non-glycosylated form,
73 2.2.5.7 Human MCP-1 ELISA
Plated were coated with a monoclonal antibody to MCP-1, MAB679 (1/250 dilution and human MCP-1 was detected using a biotinylated anti-human MCP-1 IgG, BAF279 (0.054ml).
2.2.5.8 Human IL-8 ELISA
Plates were coated with a monoclonal antibody to 1L-8, MAB208 (1/250 dilution) and human IL-8 was detected with a biotinylated anti-human IL-8 IgG, BAF208 (0.054m1).
2.2.6 Protease Activity
2.2.6.1 Human eotaxin susceptibly to mesothelial ectoproteases
HBSS containing, 30mM HEPES, 0.25% BSA pH7.4 with either the protease inhibitors bestatin (50[tM), APMSF (30}1M) a protease inhibitor cocktail mixture (1001.1M) was placed into the bottom wells of a microBoyden chamber, and a freshly isolated guinea pig mesentery was placed over the lower chamber. 28µ1 aliquots of human eotaxin (30nM) in assay buffer containing either the protease inhibitors bestatin or APMSF was placed in the top well and the chamber was incubated for 1 hr at 37°C in a humidified 5%CO2, 95%02 atmosphere. The concentration of the recovered eotaxin forms was determined using the human eotaxin ELISA.
2.2.6.2 Preparation of tissue samples for protease activity
The kidney and mesentery were removed from the huinea pigs and washed with HBSS buffer (30mM HEPES, 0.25% BSA pH7.4). The kidney was placed into 5m1 HBSS/0.5%Triton X-100 buffer and the mesentery into lml of the same buffer and were cut into small sections using a surgical blade. The samples were gently shaken for 2 hrs and following centrifguation at 300g for 7 mins the supernatant were removed.
2.2.6.3 Assay for endomesothelial cell protease activity
Glycosylated and non-glycosylated human eotaxin (2004, 20nM) in HBSS buffer (30mM HEPES, 0.25% BSA pH7.4) was added to the mesentery lysate (200111) (Section
74 2.2.7.2) and incubated at 37°C for l hr in the presence of protease inhibitors APMSF (30µM), bestatin (50µM), leupeptin (30uM) or a protease inhibitor cocktail mix (100uM). After this time the samples were stored on ice and the concentration of immunoreactive eotaxin was determined by a human eotaxin ELISA.
2.2.6.4 Identification of CD26 activity within tissue samples
The tissue supernatants (51,t1) were prepared as described in Section 2.2.6.2 above were added to a 96 well plate and a dipeptidyl-peptidase photometric substrate glycyl-prolyl- p-nitroanilide (51.11/well, 3mM) in a glycine buffer (20111/well 6mM of glycine pH 8.7) with or without protease cocktail inhibitor (50µg/ml) and left at 37°C for 30 mins. The reaction was stopped with 270111 of 1M of Acetate buffer (pH 4.2) (1M Acetic Acid pH2 adjust with Na Acetate). The absorbance was measured within 30 mins at 420nm (A420).
2.2.7 Identification of the glycosylation present on chemokines
2.2.7.1 DIG glycan identification kit
For elucidation of the carbohydrate structures linked to eotaxin a glycan differentiation kit with digoxigenin labelled lectins were used according to the manufacturer's instructions (Boerhinger Mannheim).
2.2.7.2 Arachis hypogaea (peanut agglutinin, PNA) for the detection of Ga1p1_3Ga1Nac
96 well plates were coated with glycosylated and non-glycosylated eotaxin variants (3- 300nM, 100W/well) or asialofetuin (1[tg/m1) in a carbonate/bicarbonate buffer (pH 9.6). After 12-15 hrs, the plates were washed four times with 2501.11 of coat/block (C/B) wash. The plates were blocked with 250u1 of 5% milk protein in PBS for 2 hrs, and washed as before with C/B wash. 100µ1 of biotinylated PNA (21.1g/m1) were added to the plate and incubated for 2 hrs. The plate was then washed with assay wash with 100111 of neutravidin linked FIRP ('/i000 dilution) added for 30 mins. The plate was washed with assay wash and 100W of tetramethylbenzidine/H202 substrate added, after
75 30 mins the reaction was stopped by addition of 100u1 of 0.18M H2SO4. The absorbance was read within 30 mins at 450nm (A450). Samples were diluted in assay buffer (PBS, 0.1% BSA and 0.05% thiomerosal). The assay was performed at room temperature.
2.2.7.3 Glycine Max (GM) for the detection of terminal GalNac
The method was similar to the PNA solid phase binding assay described in section 2.2.7.2. 96 well plates were coated with glycosylated and non-glycosylated eotaxin variants (3- 300nM, 100µ1/well). Asialofetuin was used at a concentration of 3µg/ml. The plates were blocked with 3% chicken albumin for 2 hrs. 100[d of biotinylated Glycine max (2µg/ml) was added to the wells for 2 hrs.
2.2.7.4 Trilculum vulgaris (wheat germ agglutinin, \VGA) for the detection of Gal-al..4G1cNAc
The method was similar to the PNA solid phase binding assay described in section 2.2.7.2 :- 96 well plates were coated with glycosylated and non-glycosylated eotaxin variants (3- 300nM, 1000/well). The plates were blocked with 3% BSA for 2 hrs. 1001.t1 of biotinylated Tritculum vulgaris (2µg/ml) was added to the wells for 2 hrs.
2.2.8.5 Lens cullinaris (lentil lectin) for the detection of a-mannose The method was similar to the PNA solid phase binding assay described in 2.2.7.2:- 96 well plates were coated with glycosylated and non-glycosylated eotaxin variants (3- 300nM, 1001d/well) Carboxypeptidase Y was used at a concentration of 3µg/ml. 100µ1 of biotinylated Lens cullinaris (211g/m1) was added to the wells for 2 hrs.
2.2.7.5 Conavalia ensiformis for the detection of a-mannose
The method was similar to the PNA solid phase binding assay with following differences:- The plate was coated with glycosylated and non-glycosylated eotaxin variant (3- 300nM) or 3µg/ml of carboxypeptidase Y for 12-15 hrs. 100µ1 of biotinylated concavalin A (0.5µg/m1) was added to the wells for 2 hrs.
76 2.2.8 Glycosaminoglycan competition assay
96 well plates were coated with 100µ1/well with human eotaxin variant (50nM, 100111/well) in a carbonate/bicarbonate buffer (pH 9.6). After 12-15 hrs, the plates were washed four times with 250µ1 of coat/block (C/B) wash. The plates were blocked with 250111 of 3% BSA in PBS for 2 hrs, and washed as before with C/B wash. Biotinylated heparin (211g/m1) and non-biotinylated chondroitin sulphate B (0.1-100µg/m1) were added to the plate (final volume of 100µ.1 per well) and incubated for 2 hrs. The plate was then washed with assay wash with 100µ1 of neutravidin linked HRP (1/5000 dilution) added for 30 mins. The plate was washed with assay wash and 10411 of tetramethylbenzidine/H202 substrate added, after 30 mins the reaction was stopped by addition of 100u1 of 0.18M H2SO4. The absorbance was measured within 30 mins at 450nm (A450). Samples were diluted in assay buffer (PBS, 0.1% BSA and 0.05% thiomerosal). The assay was performed at room temperature.
2.2.9 SDS-PAGE
The separation of guinea pig and human eotaxins was achieved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using a Phast Gel system
2.2.9.1 Electrophoresis
Glass plates were assembled and placed into a casting tray according to the manufacturer's instructions. A 12% resolving gel solution was poured between the plates leaving sufficient space for the stacking gel and overlaid with dH2O saturated butanol to allow the production of a clean interface between the resolving and stacking gels. Once the resolving gel had polymerised the overlay was poured off and the top of the gel was washed with dH2O. A 5% stacking gel was then prepared and poured onto the surface of the resolving gel. A Teflon comb was inserted and the gel allowed to polymerise. After polymerisation was complete the comb was removed and the wells washed with dH2O. The gel was transferred from the casting tray to the electrophoresis apparatus. 1X Tris Glycine running buffer was added to the top and bottom reservoirs. The samples were prepared by denaturing at 95°C for 3 mins in SDS gel loading buffer
77 diluted to lX in the sample and then loaded into the wells of the gel. The system was run at 35mA/gel for approximately 45 mins until the bromophenol blue had almost reached the bottom of the gel. The gel was removed from the plates and stained with coomassie brilliant blue.
2.2.9.2 Visualisation of the protein
Proteins separated by SDS PAGE can be simultaneously fixed with methanol:acetic acid and stained with Coomassie brilliant blue. The gel is immersed in a least 5 volumes of staining buffer and incubated on a rotating platform at room temperature for 1 hr. The gel was then destained by soaking in destaining buffer on a rotating platform until the background had lost all colour but the protein bands remained stained. Stained gels were then washed in water several times and incubated in 5% glycerol for at least 1 hr and dried at room temperature between cellophane sheets.
2.2.10 Confocal microscopy
Guinea pigs were killed by CO2 asphyxiation and cervical dislocation and the mesentery removed. The tissue was washed with HBSS buffer containing 30mM HEPES, 0.25% BSA pH7.4 and spread open over a surface covered with parafilm. Biologically inert (13mm diameter, 0.22pm pore size) filters were placed underneath the tissue and those areas isolated. Human eotaxin variants (100nM) diluted in HBSS buffer containing 30mM HEPES, 0.25% BSA pH7.4 was added topically to the isolated mesentery area and incubated for 3 or 30 mins at 37°C in a humidified 5% CO2 95% 02 atmosphere. The tissue was then washed with PBS, containing 0.1% BSA and 0.02% sodium azide and fixed for 25 mins in 4% paraformaldehyde. The tissues were then washed in lml of PBS, containing 0.1% BSA and 0.02% sodium, four times at 15 min intervals. Permeabilisation of the samples was then achieved by the addition of TritonX-100 (0.5%) for 20mins, after which the samples were washed. The samples were then blocked overnight at 4°C with 10% goat serum and then washed, before 500µ1 of the mouse a-human eotaxin mAb 2G6 (51.1g/m1) or isotype matched control mouse IgGi (Sigma) was added for 4 hrs. The tissues were then washed again and incubated at 4°C 488TM overnight with 500111 alexafluor labelled of goat a-mouse F(ab')2 (101.tg/m1). To
78 localise actin and nuclei within the tissue we incubated with TR1TC labelled phalloidin (1µg/»1) and propidium iodide (0.8µg/m1) respectively. The samples were then washed eight times at 15 min intervals and mounted onto slides using mounting gaskets and Immunofluore. The slides were then kept in the dark at 4°C. Confocal images were collected on a Leica TCS NT system.
2.2.11 Preparation of mesentery for electron microscopy
A guinea pig was killed by CO2 asphyxiation. The peritoneal cavity was opened and the mesentery exposed. The tissue was washed with HBSS containing 30mM HEPES, 0.25% BSA and pH adjusted to 7.4 and spread open within the peritoneal cavity. The tissue was then fixed with 3% glutaraldehyde in cacodylate buffer (pH 7.4) for 10 mins, post fixed in osmium tetroxide, block stained in uranyl acetate dehydrated in ethanol and embedded in Spurr's resin before sectioning.
2.3 Statistical analysis of data
Data are presented as mean ± standard error of the mean (SEM) for n experiments, except where stated. For comparison of two groups, the student's t-test was used. For comparison of more than two groups, one-way analysis of variance (ANOVA) was performed, followed by the appropriate post-test. Dunnett's post-test was used for comparison of test groups with one control group. Bonferroni's modified t-test was used for multiple comparisons of up to five groups. All graphical and statistical analysis was performed using GraphPad Prism and Instat software.
79 a) MicroBoyden chamber chemotaxis assay
Eosinophils from donor Guinea pig mesentery or polycarbonate filter
Migrated eosinophils Test chemokine
b) MicroBoyden chamber chemokine flux assay
Chemokine fluxed across the mesentery Guinea pig mesentery or U W polycarbonate filter P E P L E L R L W 0 E W L E L Test chemokine CHEMokINE
Figure 2.1 Diagramatic representation of the microBoyden chamber chemotaxis and chemokine flux assays
80 The monoclonalantiguineapigeotaxinantibody (72D)andthepolyclonalantiguinea glycosylated peak Figure 2.2Detectionofguineapigeotaxinvariants byELISA guinea pigeotaxintothesameasevidencedbysimilar absorptionprofiles. pig eotaxinantibody(B3)usedintheELISA for guineapigeotaxinrecognisedthe Absorbance (450nm) 10
I (■), peak II(A),peak Guinea pigeotaxin(pM) 100 81
III (•) and non-glycosylatedforms( 1000 ❑ ) of ELISA Figure 2.3Detectionofguineapigvariantsusing biotinylatedmatchedantibody eotaxin withthesensitivityapproximatelythree timesgreaterthancapturingnon- eotaxin recognisedtheglycosylated(.)andnon-glycosylated forms( anti guineapigeotaxinantibody(N The polyclonalantiguineapigeotaxinantibody (N glycosylated eotaxinusingthemousemonoclonal antibody(72D,♦) Absorbance (450nm) Guinea pigeotaxin(pM) 10
3- biotinylated) usedintheELISAforguineapig 82 100
3 ) andthebiotinylatedpolyclonal 1000 ❑ ) ofguineapig The monoclonalantihumaneotaxin(10C1 Figure 2.4Detectionofhumaneotaxinvariants byELISA (N glycosylated forms( absorption profiles. 3 ) usedinthehumaneotaxinELISArecognised theglycosylated(.)andnon- Absorbance (450nm) ❑ ) ofhumaneotaxintothesameasevidenced bysimilar Human eotaxin(pM) 10
83 1) and thepolyclonalantihumaneotaxin 100 4
0nm 3 45 (
nce 2 ba or 1 Abs
0 10 30 100 Human eotaxin-2 (pM)
Figure 2.5 Detection of human eotaxin-2 variants by ELISA
The polyclonal anti human eotaxin-2 IgG (343) and the biotinylated polyclonal human eotaxin-2 IgG used in the human eotaxin-2 ELISA recognised the glycosylated (.) form approximately 2 fold less than the non-glycosylated forms RnD (0) and Peprotech (X) of human eotaxin-2.
84 Taxonomic Name Common Name Sugar Specificity
Arachis hypogaea Peanut agglutinin (PNA) Gal {31_3GalNAc (Lotan et al, 1975)
Datura stramonium Jimson Weed (DSA) GlcNAc (Crowley et al, 1984)
Glycine Max Soybean (GM) Ga1NAc (Lis et al, 1970) (Lotan et al, 1974) Triticum vulgaris Wheat Germ Agglutinin GlcNac (WGA) (Nagata and Burger, 1974) Conavalia ensiformis Concanavalin A (Con A) a-mannose (Reeke et al, 1974)
Lens cullinaris Lentil a-mannose (Howard et al, 1971)
Table 2.1 A list of the plant lectins used in this thesis to identify the saccharides present on glycosylated eotaxin (`The Lectins: Properties, Functions and Applications in Biology and Medicine')
85 Chapter 3
Comparison of the eosinophil chemoattractant activities of naturally and synthetically produced guinea pig eotaxin
86 3 Comparison of the eosinophil chemoattractant activities of naturally and synthetically produced guinea pig eotaxin
In 1994, a novel chemokine, eotaxin, was isolated and purified from the bronchoalveolar lavage (BAL) fluid of allergen sensitised/challenged guinea pigs (Jose et al, 1994a). Following digestion of eotaxin with trypsin, the intact molecule and tryptic fragments were microsequenced and the amino acid sequence deduced. However, the amino acid at position 70 could not be identified at that time and it was only after the subsequent cloning of the protein that it was revealed to be a threonine (Jose et al, 1994b). Purification by reverse phase high performance liquid chromatography (rpHPLC) of BAL fluid from Sephadex injected guinea pigs (non- allergic inflammation) or ovalbumin sensitised and challenged guinea pigs (allergic inflammation) resulted in three peaks of eotaxin, each of which was highly potent in inducing eosinophil migration in an in vivo skin assay (Figure 3.1). The inability of the amino acid threonine to be determined from the microsequenced fragments of naturally produced eotaxin together with each purified fraction being of higher molecular weight relative to the synthetically produced eotaxin (Dr H Showell, Pfizer, Conneticut, USA) suggested that the naturally produced eotaxin was glycosylated. The amino acid threonine has been suggested as a likely amino acid to allow the attachment of an 0- linked saccharide moiety (Ku and Omary, 1995). This was confirmed when the amino acid sequence was analysed by means of a glycosylation computer simulation, which predicted that the amino acid threonine at position 70 on guinea pig eotaxin was the only amino acid with a high likelihood of 0-linked glycosylation occurring (Gupta et al, 1999).
In the present chapter, the biological activity of naturally produced guinea pig eotaxin (purified from the BAL fluid of Sephadex-injected guinea pigs) was compared to two chemically synthesised non-glycosylated guinea pig eotaxins. The chemically synthesised guinea pig eotaxin possessed either a CO-NH2 (amide) or a CO-OH (carboxyl) group at the C-terminus.
87 3.1 Determination of the in vivo eosinophil recruitment activity of glycosylated natural and non-glycosylated synthetic guinea pig eotaxin
using intravital microscopy
3.1.1 Cell adherence to the mesenteric venule wall
Glycosylated natural and non-glycosylated synthetic guinea pig eotaxin (10pmol) were topically applied to the guinea pig mesentery and the number of cells adhering along a 1001,tm length of mesenteric venule was determined using intravital microscopy. It can be seen in Figure 3.2 that the number of adherent cells (later determined to be eosinophils by haematoxylin and eosin staining) significantly increased with time when glycosylated natural guinea pig eotaxin was topically applied to a mesenteric venule. The non-glycosylated synthetic guinea pig eotaxin did not induce any significant eosinophil adherence to the mesenteric venule wall compared to the saline/BSA control. (These experiments were performed by Dr D Walsh).
3.1.2 Cell migration from the mesenteric venule into the connective tissue
Glycosylated natural guinea pig eotaxin, when topically applied to a guinea pig mesenteric venule, induced a significant recruitment of eosinophils into the surrounding connective tissue compared to the non-glycosylated synthetic guinea pig eotaxin and the saline control (Figure 3.3). The glycosylated and non-glycosylated guinea pig eotaxin did not induce a significant neutrophil recruitment. The photomicrographs in Figures 3.4a and 3.4b show cells adhering and migrating into the surrounding connective tissue following topical application of glycosylated eotaxin (10 pmol, 20 min). In contrast, topical application of non-glycosylated guinea pig eotaxin did not result in any adherence or migration of cells into the tissue (Figure 3.4c). (These experiments were performed by Dr D Walsh).
88 3.2 "'In labelled eosinophil migration into the guinea pig skin following intradermal injection of guinea pig eotaxin
Intradermal injection of both glycosylated natural and non-glycosylated synthetic guinea pig eotaxin (3pmol) induced a significant recruitment of 1 1 l in labelled eosinophils into the skin sites when compared to saline/BSA control (Figure 3.5). (These experiments were performed by Dr D Walsh).
3.3 Determination of the molecular mass differences between the glycosylated natural guinea pig eotaxin variants and non-glycosylated synthetic guinea pig eotaxin
3.3.1 Time of flight mass spectroscopy
The molecular weights of the three peaks of eotaxin purified from BAL fluid from sensitised/challenged guinea pigs by reverse phase HPLC was determined by time of flight mass spectroscopy as described by Jose et al, 1994b (Table 3.1). The molecular mass of peak I was calculated as 9.03kDa, peak II as 8.81kDa and peak III as 8.38kDa. The molecular mass of synthetic guinea pig eotaxin was calculated by time of flight mass spectroscopy as 8.15kDa (personal communication H.Showell, Pfizer, Connecticut, USA).
3.3.2 SDS-PAGE
The three glycosylated forms can clearly be seen to have different molecular weights as determined by their migration in SDS-PAGE (Figure 3.6). Each of the three forms has a higher molecular weight than the non-glycosylated synthetic guinea pig eotaxin (8.15kDa) which is consistent with the time of flight mass spectroscopy observations. The higher molecular weight protein observed for the synthetic eotaxin on the SDS- PAGE gel could be a dimer of eotaxin (Figure 3.6).
89 3.4 Determination of the chemotactic activity of glycosylated and non-
glycosylated guinea pig eotaxin across the isolated guinea pig
mesentery
The above experiments using intravital microscopy to visualise eosinophil recruitment from guinea pig mesenteric vasculature induced by eotaxin were carried out by Dr D Walsh (unpublished observations). To investigate further the difference in chemotactic activity between natural and synthetic eotaxin an in vitro chemotaxis assay using either an isolated guinea pig mesentery or a polycarbonate filter in a 48 well microBoyden chamber was set up. In a preliminary experiment, guinea pig mesentery was removed from the small and large intestines and placed between the upper and lower compartments of microBoyden a chemotaxis chamber. 111In labelled guinea pig peritoneal eosinophils are added to the top well and the two forms (glycosylated and non-glycosylated) of eotaxin were added in the bottom wells. Glycosylated guinea pig eotaxin (Peak III, 50nM) was able to induce migration of the labelled eosinophils across the isolated mesentery. Non-glycosylated eotaxin (25nM) did not induce a significant recruitment compared to the control (Figure 3.7). LTB4 was used as a positive control in these experiments. Although the concentration of non-glycosylated eotaxin was lower, this preliminary experiment demonstrated that eosinophils are able to migrate across the guinea pig mesentery in response to glycosylated guinea pig eotaxin and LTB4.
Having established that 1111n labelled eosinophils could migrate across the mesentery the next series of experiments were designed to investigate the chemotactic activity of glycosylated and non-glycosylated guinea pig eotaxin for unlabelled guinea pig peritoneal eosinophils across a guinea pig mesentery or a polycarbonate filter. The experiments were again performed in a 48 well microBoyden chemotaxis chamber with eotaxin added to the lower wells, and purified guinea pig peritoneal eosinophils added to the upper wells. After 60 mins, the migration of the eosinophils into the lower wells across a guinea pig mesentery or polycarbonate filter was determined by counting the migrated cells using a flow cytometer (and a haemocytometer).
It can be seen from Figure 3.8a that glycosylated guinea pig eotaxin (Peak III) was able to induce a significant guinea pig eosinophil migration across the guinea pig mesentery with 30nM glycosylated guinea pig eotaxin producing a chemotactic index of
90 approximately 7. Non-glycosylated guinea pig eotaxin failed to induce any significant eosinophil migration across the guinea pig mesentery. In contrast both the glycosylated and the non-glycosylated forms of guinea pig eotaxin induced a similar degree of eosinophil migration across a polycarbonate filter (Figure 3.8b).
The chemotactic activity of all three naturally produced glycosylated guinea pig eotaxin forms (peak LII and III) was compared using the in vitro mesentery chemotaxis assay system. Figure 3.9a demonstrates that all three forms of glycosylated guinea pig eotaxin were equipotent in inducing eosinophil migration across the guinea pig mesentery. The three glycosylated forms of guinea pig eotaxin also had similar eosinophil chemotactic potencies across a polycarbonate filter (Figure 3.9b). Leukotriene B4 (LTB4, 100nM) was used as a positive control and was able to induce a similar degree of eosinophil migration across both the isolated mesentery and polycarbonate filter (Figure 3.9a and Figure 3.9b).
The effect of amidation of the C-terminus of the non-glycosylated synthetic guinea pig eotaxin on its chemotactic activity was investigated using the guinea pig mesentery (Figure 3.10a) and polycarbonate filter (Figure 3.10b). Neither the amidated nor the non-amidated synthetic guinea pig eotaxins (3-100nM) induced eosinophil migration across the guinea pig mesentery. However, they both induced a similar eosinophil migration across a polycarbonate filter, producing a chemotactic index of approximately
6.
3.5 Guinea pig eotaxin flux across the guinea pig mesentery
The following experiments were carried out using a microBoyden chamber where guinea pig eotaxin was added to the lower wells which were separated from the upper wells by either guinea pig mesentery or a polycarbonate filter. The concentration of eotaxin in the upper well was measured after 1 hour by ELISA.
As shown in Figure 3.11a all three forms of naturally produced glycosylated guinea pig eotaxin were detected in the upper wells of the microBoyden chamber containing the mesentery. The concentration in the upper wells increased as the concentration of glycosylated guinea pig eotaxin in the lower chamber was increased. In contrast, non-
91 glycosylated cotaxin did not appear to move across the guinea pig mesentery since it could not be detected in the upper wells. Similar levels of glycosylated (Peak I, II and III) and non-glycosylated guinea pig eotaxin were measured in the upper wells of the microBoyden chamber in the experiments employing the polycarbonate filter (Figure 3.1 lb).
92 3.6 Summary of results
1. The glycosylated natural guinea pig eotaxin, but not the non-glycosylated synthetic guinea pig eotaxin, was able to induce adherence of guinea pig eosinophils to the microvascular endothelium of the guinea pig mesentery in vivo as viewed by intravital microscopy.
2. Both the glycosylated and non-glycosylated guinea pig eotaxin, when injected intradermally, induced 1111n labelled eosinophils to migrate into the guinea pig skin in vivo.
3. The natural guinea pig eotaxins (peaks I, II and III) purified from the BAL fluid of allergen sensitised/challenged guinea pigs or Sephadex-injected guinea pigs each had a molecular mass higher than that of the non-glycosylated synthetically produced guinea pig eotaxin as determined by mass spectroscopy and SDS- PAGE.
4. All three peaks of the glycosylated natural guinea pig eotaxin induced guinea pig eosinophil migration in an in vitro assay across an isolated guinea pig mesentery and there was no significant difference in chemotactic activity between these peaks.
5. Using an in vitro chemotaxis assay, non-glycosylated eotaxin did not induce eosinophil migration across the guinea pig mesentery, but both the glycosylated and non-glycosylated eotaxins had equal eosinophil chemotactic activity across the polycarbonate filter.
6. The glycosylated guinea pig eotaxin, but not the non-glycosylated guinea pig eotaxin, fluxed across the isolated guinea pig mesentery in an in vitro assay.
93
0.10
)
80 ] [-- 14nm 2 ile ( itr
0.05 - n nce -- 40 to ba r Ace % Abso —0
10 20 30 40 Retention Time (min)
Figure 3.1 Reverse phase HPLC purification of eotaxin from BAL fluid
Purification by reverse phase high performance liquid chromatography (rpHPLC) of BAL fluid from ovalbumin sensitised and challenged guinea pigs showing absorbance at 214nm and the acetonitrile gradient. I, II and III represent the three glycosylated natural forms of eotaxin
Taken from Jose et al, 1994 J Exp Med 179, 881-887
94 **
*
0 10 20 Time (minutes)
Figure 3.2 Effect of topical application of glycosylated natural and non- glycosylated synthetic guinea pig eotaxin on cell adherence to a guinea pig mesenteric venule
The number of cells adhering to 100µm length of guinea pig mesenteric venule, after topical application of saline/BSA (*), glycosylated natural (I) or non-glycosylated synthetic (0) guinea pig eotaxin (10pmol) was determined using intravital microscopy. Data shown as mean ± SEM of 5 separate experiments where 5 individual sections of the venule were counted and averaged. *P<0.05 and ** P<0.01 compared to BSA/saline control.
(These experiments were performed by Dr D Walsh).
95 80 - ** vi a) szu Ez_ 45 (3) .E E 60 - o co (0 715 40 - _ca
n C15 20 - Z
0 Eosinophils Neutrophils
Figure 3.3 Effect of topical application of glycosylated natural and non- glycosylated synthetic guinea pig eotaxin on cell emigration from a guinea pig mesenteric venule
The number of cells emigrating from the mesenteric venule into the connective tissue 60 minutes after topical application of glycosylated natural (black), non-glycosylated synthetic (white) guinea pig eotaxin (10pmol) or saline/BSA (hatched) was determined. Migrated cells were quantified in 5 high power fields around different venules stained with haemotoxylin and eosin. Data shown as mean ± SEM of 5 separate experiments. **P<0.01 compared to BSA/saline control.
(These experiments were performed by Dr D Walsh).
96 b)
c)
Figure 3.4 Photomicrographs of eosinophil recruitment in guinea pig mesenteric venule in vivo
Video photomicrographs of a guinea pig mesenteric venule showing a) and b) eosinophil margination and migration at 5 min post topical application of 10 pmol glycosylated guinea pig eotaxin, compared to c) application of 10 pmol non- glycosylated guinea pig eotaxin.
(These experiments were performed by Dr D Walsh).
97 15
•c x 10 •(7) o o so -co— o c aD E w 0- Z
0 0.3 1.0 3.0 pmol/site
Figure 3.5 inlin labelled eosinophil recruitment into guinea pig skin induced by intradermal injections of glycosylated natural and non-glycosylated synthetic guinea pig eotaxin
The number of eosinophils migrating into the guinea pig skin sites 120 minutes after intradermal injection of BSA/saline (.), glycosylated natural (M) or non-glycosylated synthetic (D) guinea pig eotaxin (0.3-3pmol) was determined. Data shown as mean ± SEM of 5 separate experiments. Data shown as mean ± SEM of 5 separate experiments. *P<0.01 compared to BSA/saline control.
(These experiments were performed by Dr D Walsh).
98 Mass (according to Mass Spectroscopy) kilo Daltons
Guinea pig eotaxin peak I 9.03 (glycosylated)
Guinea pig eotaxin peak II (glycosylated) 8.81
Guinea pig eotaxin peak III (glycosylated) 8.38
Guinea pig eotaxin Synthetically produced 8.15 (non-glycosylated)
Table 3.1 Molecular weights of natural and synthetic guinea pig eotaxins
The molecular weights of peaks I, II and III of naturally produced glycosylated guinea pig eotaxin purified from the BAL fluid of ovalbumin sensitised/challenged guinea pigs and synthetically produced non-glycosylated guinea pig eotaxin as determined by time of flight mass spectroscopy analysis.
Jose el al, 1994 J Exp Med 179, 881-887
99 21.0
Molecular GpE-NG GpE-G GpE-G GpE-G IL-8 Weight (synthetic) (peak I) (peak II) (peak III) Markers (kDa)
Figure 3.6 SDS-PAGE of reverse phase HPLC fractions of BAL fluid from Sephadex injected guinea pigs
A protein band was observed for each of the glycosylated natural guinea pig eotaxin (GpE-G) fractions labelled peak 1, II and III. And the non-glycosylated synthetic guinea pig eotaxin (GpE-NG). The fractions appear to have different molecular weights as evidenced by their migration in SDS gels.
100 100-
co
4i5 >, 75- 0 >
0 Eot-NG Eot-G LTB4 Control (25nM) (50nM) (100nM)
Figure 3.7 Chemotactic response of Indium labelled guinea pig eosinophils to glycosylated, non-glycosylated guinea pig eotaxin and LTB4 across a guinea pig mesentery
Migration of 1 il indium labelled guinea pig peritoneal eosinophils across an isolated guinea pig mesentery in a microBoyden chamber to non-glycosylated synthetic guinea pig eotaxin (Eot-NG, 25nM), glycosylated guinea pig eotaxin (Eot-G, peak 111, 50nM), and LTB4 (100nM). The control used in this experiment was HBSS buffer. Data shown as mean chemotactic index ± SEM for 1 experiment carried out in triplicate.
101
a) Mesentery
10 -
8 - dex In
ic 6 - t
tac 4 - mo 2 - Che
0 1 3 10 30
Filter 10 -
8 -
dex
In 6 -
ic t 4 - tac
mo 2 - Che 0 1 3 10 30 Guinea pig eotaxin (nM)
Figure 3.8 Chemotaxis of guinea pig eosinophils induced by glycosylated and non- glycosylated guinea pig eotaxin across a guinea pig mesentery and polycarbonate filter
Migration of guinea pig peritoneal eosinophils across a) isolated guinea pig mesentery and b) polycarbonate filter in a microBoyden chamber in response to glycosylated guinea pig eotaxin peak 111 (II, 3-30nM) non-glycosylated synthetic guinea pig eotaxin (❑, 3-30nM). The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index ± SEM for 5 separate experiments carried out in duplicate. *P<0.05 compared to non-glycosylated synthetic guinea pig eotaxin.
102 a) Mesentery
8 -
x
de 6 In ic t 4 - tac mo 2 - Che
0 1 3 10 30 100
Filter
6
sc - a) E 4 - U
0 2
0 1 3 10 30 100 Guinea pig eotaxin (nM)
Figure 3.9 Chemotaxis of guinea pig eosinophils induced by glycosylated guinea pig eotaxin and LTB4 across a guinea pig mesentery and polycarbonate filter
Migration of guinea pig peritoneal eosinophils across a) isolated guinea pig mesentery and b) polycarbonate filter in a microBoyden chamber in response to glycosylated guinea pig eotaxin peak I (N,), peak II (•,) and peak III (7,) (3-30nM) and LTB4 (X, 100nM). The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index + SEM for 5 separate experiments carried out in duplicate.
103
Mesentery
10 -
dex 8 - In
ic t c ta mo Che
10 30 100 b) Filter
10 -
x 8 - de In ic
t 6 tac 4 - mo
Che 2 -
0 1 3 10 30 100
Guinea pig eotaxin (nM)
Figure 3.10 Chemotactic response of guinea pig eosinophils to non-glycosylated guinea pig eotaxin across a guinea pig mesentery and polycarbonate filter
Migration of guinea pig eosinophils induced by non-glycosylated synthetic guinea pig eotaxin with a carboxyl terminus (❑, 3-100nM), and an amide terminus (L, 3-100nM) across a isolated guinea pig mesentery and b) across a polycarbonate filter in a microBoyden chamber. The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index mean + SEM for 5 separate experiments carried out in duplicate.
104
a) Mesentery
2500 -
2000-
1500-
1000-
500 - EL 0 10 30 100
Filter
2500 - in n M) io
t 2000 - (p tra ber 1500 -
ham 1000 - c concen r e in
x 500 ta upp
Eo 0 10 30 100
Guinea pig eotaxin (nM)
Figure 3.11 The flux of the guinea pig eotaxin variants across the isolated guinea pig mesentery and a polycarbonate filter
Glycosylated guinea pig eotaxin variants peak I (NO, peak II (A,), peak III (V) ,and non- glycosylated amidated eotaxin (0), all 30 and 100nM were placed into the lower wells of microBoyden chamber. Flux of eotaxin into the upper wells across (a) across an isolated guinea pig mesentery or (b) a polycarbonate filter was measured after 1 h by ELISA. Data shown as mean ± SEM for 5 separate experiments carried out in duplicate. *P<0.05 compared to non-glycosylated amidated eotaxin
105 3.7 Discussion
Glycosylated natural guinea pig eotaxin, but not non-glycosylated synthetic eotaxin, induced eosinophil migration from the guinea pig mesenteric microvasculature as observed by intravital microscopy. However, both the glycosylated and non- glycosylated forms of guinea pig eotaxin, when injected intradermally into naïve guinea pigs that had previously received "In labelled eosinophils, were equally potent at inducing eosinophil migration from the microvasculature into the skin sites. Intratracheal instillation of non-glycosylated Escherichia.coli (E.coli) expressed guinea pig eotaxin has been shown to induce eosinophil accumulation into naïve guinea pig lungs (Fukuyama et al, 2000). Following, intraperitoneal injection of ovalbumin- sensitised mice with E.coli expressed non-glycosylated eotaxin a significant eosinophil accumulation was also observed (Das et al, 1997; Harris et al, 1997). Similarly, intraperitoneal injection of guinea pigs with E.coli expressed non-glycosylated eotaxin resulted in significant increase in eosinophil numbers in the peritoneal cavity (internal observation Leukocyte Biology). Thus, non-glycosylated eotaxin was able to induce the migration and accumulation of eosinophils when injected into guinea pig skin, when instilled into the guinea pig lung and following injection into the peritoneal cavity. In the experiments described in this thesis non-glycosylated eotaxin was shown to be unable to induce eosinophil recruitment when topically applied to the guinea pig mesentery. In contrast, glycosylated guinea pig eotaxin induced eosinophil migration from the mesenteric venules into the surrounding connective tissue.
It has been suggested that the different molecular masses of naturally produced eotaxin purified from the BAL fluid of ovalbumin sensitised/challenged guinea pigs was due to glycosylation (Jose et al, 1994a). The mass signals obtained from the mass spectroscopy data of the glycosylated natural eotaxin was 8.38kDa, 8.81kDa, and 9.03kDa and each are different from each other by multiples of approximately 220Da (Jose et al, 1994a). Furthermore, this glycosylation was believed to be 0-linked because guinea pig eotaxin does not contain the consensus sequence for N-linked glycosylation (Kornfeld and Kornfe]d, 1985; Hirschberg and Snider, 1987). When the amino acid sequence of guinea pig eotaxin was analysed by 0-GLYCBASE (Gupta et al, 1999) a predicted 0- glycosylation site was deduced on the threonine at position 70, the same amino acid that
106 could not be sequenced following trypsin digestion of the naturally produced guinea pig eotaxin (Jose et al, 1994a).
In a preliminary experiment, 1111n labelled guinea pig eosinophils were shown to migrate across the guinea pig mesentery in response to glycosylated natural guinea pig eotaxin and LTB4. However, the non-glycosylated synthetic guinea pig eotaxin (25nM) was unable to induce a significant eosinophil migration across the guinea pig mesentery. Although a higher concentration of glycosylated natural guinea pig eotaxin than non- glycosylated synthetic eotaxin was used in this experiment, it successfully demonstrated that eosinophils could migrate across an isolated guinea pig mesentery in a microBoyden chamber. The experiment was repeated using unlabelled guinea pig eosinophils with the migrating cells counted by flow cytometry. Glycosylated natural guinea pig eotaxin (Peak III) was able to induce eosinophil chemotaxis across the isolated guinea pig mesentery, whereas identical concentrations of the non-glycosylated synthetic guinea pig eotaxin were inactive. In contrast, both the glycosylated and non- glycosylated variants of eotaxin had similar eosinophil chemotactic activity using a polycarbonate filter. All three rpHPLC peaks of guinea pig natural eotaxin were equiactive in their ability to induce eosinophil chemotaxis across both an isolated guinea pig mesentery and a polycarbonate filter. This would suggest that the different molecular mass of the three guinea pig eotaxin variants does not alter their chemotactic activity for eosinophils.
It is important to briefly describe the structure of the guinea pig mesentery and highlight the differences between the eosinophil chemotaxis assay using an isolated guinea pig mesentery and eosinophil recruitment from the mesenteric microvasculature as observed by intravital microscopy. The guinea pig mesentery is a uniform, thin (8-35um) continuous sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces, thus forming a continuous mesothelium. The cytoplasm can become very thin (60nm) at the mesothelial cell periphery. The free surface of mesothelial cells shows few, irregularly disposed microvilli of variable sizes. Pinocytic vesicles are frequently observed within the cell suggesting a low metabolite transferring activity and a highly active macromolecule transcellular transport mechanism. The existence of tight and gap junctions provides for both and a permeability barrier and intercellular communication among mesothelial cells (Junqueira et al, 1987). Coated
107 vesicles, pinocytotic vesicles and transcellular channels have all been observed in the rat mesentery (Gotloib et al, 1988), but the rat mesentery in contrast to the guinea pig mesentery, contains naturally occurring holes that have been shown to permit the passage of physiological India ink (Easty and Easty, 1974). Thus, the rat mesentery will allow the passive diffusion of molecules and will not present itself as a physiological tight barrier.
Eosinophil recruitment, following topical application of eotaxin to a mesenteric venule involves the migration of eosinophils from the guinea pig mesenteric vasculature as viewed by the intravital microscopy. The distance by which eotaxin must transverse the guinea pig mesentery in order to induce eosinophil migration differs between the in vivo eosinophil recruitment from the mesenteric vasculature and the in vitro eosinophil chemotaxis assay using the isolated guinea pig mesentery. In the in vivo assay the topically applied eotaxin must cross the mesothelial cell barrier, the connective tissue of the mesentery and the endothelial cells of the mesenteric blood vessel. The eotaxin must then bind to the endothelial cells in order to prevent the chemokine being washed away with the blood flow. The chemokine must also be bound in such a way that it does not interfere with its ability to recruit leukocytes from the blood into the connective tissue. The non-specific chemokine binding sites present on the endothelial cell surface were identified as glycosaminoglycans (GAGs) (Hoogewerf et al, 1997). It has subsequently been shown that chemokines bound to GAGs are able to induce leukocyte migration (Hoogewerf et al, 1997; Hub and Rot, 1998; Ali et al, 2000). Thus, in this experimental model eotaxin presentation on the endothelial cell surface is believed to be vital for the chemokine to induce eosinophil recruitment.
In the isolated guinea pig mesentery chemotaxis assay, the eotaxin must form a chemokine gradient across the whole width of the mesentery tissue e.g. the lower mesothelial cell barrier, the connective tissue and the upper mesothelial barrier before being presented to the guinea pig eosinophils in the upper microBoyden chamber. In this assay, the relative distance that the eotaxin and subsequently the eosinophils must travel is greater than the distances involved in the leukocyte recruitment as viewed by intravital microscopy. Presentation of eotaxin by GAGs within the mesenteric blood vessel does not play a significant role within this assay. It could be postulated that
108 binding of the eotaxin within the blood vessel would hinder the eosinophil migration process in this in vitro model.
Chemotaxis is defined as the 'reaction of motile cells to a chemical stimuli by movement towards the source of the chemical'. Upon encountering a chemotactic molecule, eosinophils begin to migrate directionally from regions of low chemoattractant concentration towards a higher concentration (the site of chemoattractant production being the area of highest concentration). Chemokines are able to bind to GAGs within the extracellular matrix (ECM) of the endothelial cell and this property can create a proposed solid phase gradient or haptotactic gradient. The binding affinity of various chemokines for these ECM GAGs correlated with the avidity of the chemokine to bind to heparin (Patel et al, 2001). In this thesis the inability of non-glycosylated eotaxin to induce eosinophil chemotaxis across the guinea pig mesentery appears to be due to the lack of a chemokine gradient being formed since in contrast to glycosylated guinea pig eotaxin, the non-glycosylated form could not be detected in the upper chamber in the flux assay. Results in this chapter clearly show that only the glycosylated natural guinea pig eotaxin was able to be detected in the upper microBoyden chamber across an isolated guinea pig mesentery. This suggests that the inability of the non-glycosylated synthetic guinea pig eotaxin to induce eosinophil chemotaxis across the guinea pig mesentery is because a concentration gradient was not established across the tissue. However, as non-glycosylated eotaxin induced eosinophil recruitment when injected intradermally and instilled intratracheally (Collins et al, 1995) this would suggest that a either a soluble or haptotactic gradient of non-glycosylated eotaxin can be formed across endothelial cells in the skin, and across epithelial and endothelial cells of the guinea pig lung.
To investigate further the observation that the non-glycosylated guinea pig eotaxin is unable to induce eosinophil migration from the guinea pig mesenteric blood vessels when applied topically, the two synthetic eotaxins (CO-NH2 and the CO-OH) were tested in the chemotaxis assay using an isolated guinea pig mesentery. Both forms of the non-glycosylated guinea pig eotaxin failed to induce eosinophil migration across the guinea pig mesentery, but induced a potent eosinophil chemotactic response across a polycarbonate filter. This result together with the observation that injection of natural or recombinant E.coli expressed eotaxin injected intradermally induces eosinophil
109 recruitment (Figure 3.5), strongly suggests that the lack of biological activity of the non- glycosylated synthetic guinea pig eotaxin when either applied topically to the mesentery or using the chemotaxis assay across an isolated guinea pig mesentery is not due to the difference in the C-terminus of the protein. Collectively these results suggest that a selective mechanism that can distinguish between glycosylated and non-glycosylated guinea pig eotaxin exists on the guinea pig mesentery.
In conclusion, the data presented in this chapter demonstrate that eosinophil adherence to, and migration from the mesenteric venule into the surrounding connective tissue occurs rapidly following topical application of glycosylated natural guinea pig eotaxin. This is in contrast to the non-glycosylated synthetic guinea pig eotaxin which failed to induce guinea pig eosinophil adherence or migration across the mesentery. When the glycosylated and non-glycosylated guinea pig eotaxins were injected intradermally, they exhibited similar eosinophil recruitment activity to the guinea pig skin. The three peaks of glycosylated guinea pig eotaxin, purified from BAL fluid of Sephadex injected guinea pigs identified by reverse phase HPLC, induced guinea pig eosinophil migration across the guinea pig mesentery with equal potency in the microBoyden chamber chemotaxis assay. In addition, only the glycosylated guinea pig eotaxins were detected in the upper chamber when a flux assay was performed using the guinea pig mesentery. The non-glycosylated guinea pig eotaxin was unable to induce eosinophil migration across the guinea pig mesentery and was not detected in the upper wells in the flux assay suggesting that it did not form a chemokine gradient across the mesentery.
110 Chapter 4
Determination of the biological activity of the glycosylated human eotaxin
111 4 Determination of the biological activity of glycosylated human eotaxin
The data presented in Chapter 3 shows that the glycosylation present on the naturally produced guinea pig eotaxin molecule is important for the migration of guinea pig eosinophils across the mesentery.
The glycosylated natural guinea pig eotaxin forms used in the experiments described in Chapter 3 were purified from the BAL fluid from Sephadex injected guinea pigs. Further studies using the natural forms of guinea pig eotaxin were not feasible since it was difficult to produce these proteins in sufficient quantities. The experiments described in this chapter were designed to investigate whether glycosylated human eotaxin could produce similar effects to the naturally produced glycosylated guinea pig eotaxin forms described in Chapter 3.
For the following experiments glycosylated human eotaxin (produced using Spodoptera fi-ugiperda (Sf-9) insect cell line hosting a baculovirus expression system) (Dr D Soler, Leukosite Inc, MA) and recombinant non-glycosylated human eotaxin (an E.coli expression system) were used. E.coli expressed human eotaxin has a molecular mass of 8.361kDa while the recombinant human eotaxin produced using the Sf-9 insect cell line/baculovirus expression system has a molecular mass of 8.726kDa and 9.091kDa (See Table 4.1). The difference in molecular mass between the E.coli expressed and Sf-9 insect cell/baculovirus expressed eotaxin was 365Da and 730Da. Human eotaxin does not contain the amino acid consensus sequence for N-glycosylation, but contains one potential 0-glycosylation site on the amino acid at position 71 (threonine) as calculated by 0-GLYCBASE (Gupta et al, 1999). The Sf-9 insect cell/baculovirus expression system has the capacity to N- or 0- glycosylate the expressed protein at a potential N- or 0- glycosylation site (Lopez et al, 1999; Wolff et al, 1999). The recombinant E.coli expression system does not contain the biochemical pathways required to N- or 0- glycosylate proteins. Consequently, the difference in molecular weight between the Sf-9 recombinant expressed human eotaxin and the E.coli expressed human eotaxin was likely to be due to the presence of an 0-linked saccharide moiety at position 71 on the Sf-9 expressed protein.
112 Unless otherwise stated the glycosylated human eotaxins used in this and future chapters is Sf-9 insect cell/baculovirus expressed as supplied by LeukoSite lnc,USA
The ability of glycosylated human eotaxin and non-glycosylated human eotaxin to induce human eosinophil migration across either an isolated guinea pig mesentery or a polycarbonate filter in a microBoyden chamber was investigated in the following experiments. Eotaxins were added to the lower wells of the microBoyden chamber and human eosinophils to the upper wells and after 1 hr, the number of eosinophils migrating across either a guinea pig mesentery or a polycarbonate filter into the lower wells were counted by flow cytometry.
4.1 Human eotaxin does not induce guinea pig eosinophils to migrate across a polycarbonate filter
The chemotactic activity of non-glycosylated human eotaxin (E.coli expressed), and non-glycosylated guinea pig eotaxin (synthetically produced) (10-100nM) for guinea pig peritoneal eosinophils was determined across a polycarbonate filter. Non- glycosylated guinea pig eotaxin produced a classical bell shaped dose response curve with a maximal chemotactic activity at 30nM. The non-glycosylated human eotaxin up to 100nM failed to induce chemotaxis of guinea pig eosinophils. LTB4 (100nM), used as a positive control, produced a chemotactic index of 6 which was comparable to that induced by 30nM guinea pig eotaxin (Figure 4.1).
4.2 A comparison of the chemotactic activity of glycosylated and non- glycosylated human eotaxin variants for human eosinophils across a guinea pig mesentery and polycarbonate filter
The chemotactic activity of glycosylated and non-glycosylated human eotaxin (10- 100nM) for human eosinophils (purified from peripheral blood) was determined across the guinea pig mesentery. Glycosylated human eotaxin induced a dose-dependent eosinophil migration across the guinea pig mesentery showing maximal chemotactic activity at 100nM (Figure 4.2a). These results contrast with the non-glycosylated form, which failed to induce eosinophil chemotaxis across the tissue at doses up to 100nM. When the chemotaxis assay was performed with a polycarbonate filter, both the
113 glycosylated and non-glycosylated eotaxin forms had equal chemotactic activity, which peaked at 30nM (Figure 4.2b). LTB4 (100nM) induced a similar degree of eosinophil chemotaxis (chemotactic index of 3) across both the guinea pig mesentery and the polycarbonate filter (Figures 4.2a and 4.2b).
4.3 Human eosinophil shape change induced by glycosylated and non- glycosylated human eotaxin variants
The ability of the glycosylated and non-glycosylated human eotaxin forms to induce shape change of human eosinophils was determined by flow cytometry. The glycosylated and non-glycosylated human eotaxin forms both produced similar responses with maximal activity at 1nM (Figure 4.3). A second source of glycosylated eotaxin (Sf-9 insect cells/baculovirus expressed produced by Fournier, France) also induced eosinophil shape change and produced a similar dose-response curve to the glycosylated human eotaxin provided by Leukosite, MA (Figure 4.3).
4.4 The transport of human eotaxin across the guinea pig mesentery
4.4.1 Measurement of the flux of human eotaxin across an isolated guinea pig mesentery and a polycarbonate filter
The following series of experiments were carried out using a microBoyden chamber using either a guinea pig mesentery or a polycarbonate filter as a barrier. Glycosylated and non-glycosylated forms of human eotaxin were added to the lower wells and the concentration in the upper wells was measured by ELISA after 60 minutes unless otherwise stated.
In the presence of the guinea pig mesentery the concentration of glycosylated eotaxin measured in the upper wells increased in a dose-dependent fashion to a maximum of 400pM, which was achieved on addition of 100nM to the lower well (Figure 4.4a). In contrast, the non-glycosylated human eotaxin could not be detected in the upper wells. When a polycarbonate filer was used in the assay both the glycosylated and non- glycosylated human eotaxin forms could be detected in the upper chamber in similar
114 concentrations (Figure 4.4b). Similar amounts of glycosylated eotaxin were transported across the mesentery and the filter.
To determine whether the non-glycosylated eotaxin could be detected in the upper chamber over an increased time period, human eotaxin (30nM) was added to the lower wells and the concentration in the upper wells was determined after 30, 60 and 120 mins. When the flux of human eotaxin across the guinea pig mesentery was measured, a significant increase in glycosylated human eotaxin could be detected in the upper wells at 60 and 120 mins. At all times points, the non-glycosylated human eotaxin remained below the accurate limit of detection of the ELISA (Figure 4.5).
4.4.2 Effect of glycosylated human eotaxin on the flux of glycosylated guinea pig eotaxin across the guinea pig mesentery
Experiments were carried out to investigate if human eotaxin and guinea pig eotaxin would compete to inhibit the flux across the mesentery when they were added to the lower chamber of the microBoyden chamber. The inclusion of glycosylated human eotaxin (30nM) in the lower chamber inhibited the flux of glycosylated guinea pig eotaxin (peak III, 30nM) across the mesentery (Figure 4.6). The concentration of glycosylated guinea pig eotaxin detected in the upper well was reduced from 420pM to 160pM in the presence of glycosylated human eotaxin. Non-glycosylated human eotaxin did not reduce the concentration of guinea pig eotaxin detected in the upper well.
4.4.3 Effect of glycosylated guinea pig eotaxin on the flux of glycosylated human eotaxin across the guinea pig mesentery
The addition of glycosylated guinea pig eotaxin (peak 111, 30nM) to the lower chamber inhibited the flux of glycosylated human eotaxin (30nM) across the guinea pig mesentery (Figure 4.7). The concentration of glycosylated human eotaxin detected in the upper well was reduced by approximately 50% when glycosylated guinea pig eotaxin was present in the lower wells. Non-glycosylated guinea pig eotaxin did not have this inhibitory affect.
115 4.5 The binding affinity of glycosylated and non-glycosylated human eotaxin forms to glycosaminoglycans
Cell surface glycosaminoglycans (GAGs) have been shown to sequester chemokines, increasing their local concentrations and facilitating binding to chemokine receptors expressed on leukocytes (Hoogewerf et al, 1997). The guinea pig mesentery has been shown by Junquirea et al, (1987) to contain sulphated GAGs. Therefore it was postulated that these GAGs could offer glycosylated eotaxin an advantage enabling it to have chemotactic activity across the mesentery.
Due to the design of the GAG binding assay it was important to determine whether the glycosylated and non-glycosylated human eotaxin forms bound biotinylated heparin with equal affinity. It can be seen from Figure 4.8 that both forms of eotaxin bound to biotinylated heparin with equal affinity. Using biotinylated heparin in a competition assay, the binding affinities of the two eotaxin forms for either chondroitin sulphate B (CSB) or heparan sulphate (HS) were examined. A ratio of 1000:1 CSB to biotinylated heparin was found not to displace the biotinylated heparin from binding to either the glycosylated or non-glycosylated human eotaxin forms (Figure 4.9a). In contrast, heparan sulphate was able to compete with the biotinylated heparin, and bound to the glycosylated and non-glycosylated human eotaxins in a dose dependent manner, which was significant from a ratio of 1000:1 HS to heparin. However, there was no significant difference in displacement between the glycosylated and non-glycosylated human eotaxin variants (Figure 4.9b).
4.6 Role of mesothelial proteases on the movement of glycosylated human eotaxin across the guinea pig mesentery
The next series of experiments were designed to investigate the effect of proteases found on and/or within the mesentery, on the flux and immunoreactivity of glycosylated and non-glycosylated human eotaxin.
116 4.6.1 Effect of protease inhibitors on the flux of glycosylated and non-
glycosylated human eotaxin
Figure 4.10a demonstrates the lack of effect of the protease inhibitors, bestatin, 4- (amidinophenyl)methanesulfonyl (APMSF) and a protease inhibitor cocktail mixture on the flux of glycosylated human eotaxin across the guinea pig mesentery. The non- glycosylated human eotaxin remained below the accurate level of detection in the presence of the protease inhibitors (Figure 4.10b).
This experiment was designed to determine whether proteases present on the mesothelial cell surface affected either the glycosylated or non-glycosylated forms of human eotaxin sufficiently to alter its ability to be detected by ELISA. This was achieved by topically applying the eotaxin forms to the guinea pig mesentery in the presence of protease inhibitors betstatin and APMSF. As is shown in Figure 4.11, the protease inhibitors did not affect the amount of immunoreactive glycosylated or non- glycosylated human eotaxin measured in the upper chamber.
Both the glycosylated and non-glycosylated eotaxin which had been exposed to the mesentery for 1 hr (recovered from the wells containing no protease inhibitor of the previous experiment described above) were biologically active. They both induced an eosinophil shape change comparable to eotaxin that had not been exposed to the mesentery (Figure 4.12)
4.6.2 Determining the activity of mesentery endoproteases on glycosylated and non-glycosylated human eotaxin
Guinea pig mesentery lysates were prepared as described in the methods section and were incubated with glycosylated and non-glycosylated human eotaxin (10nM) for lhr at 37°C. As shown in Figure 4.13 the amount of both glycosylated and non-glycosylated human eotaxin detected after incubation with the guinea pig mesentery lysate was greatly reduced from the initial concentration of 1 OnM to below 2nM. The protease inhibitor APMSF (30µM) increased the amount of immunoreactive eotaxin recovered from the lysates to approximately 7nM and 8nM in the case of glycosylated eotaxin and non-glycosylated eotaxin respectively.The inhibitors bestatin (50uM), leupeptin (30pM)
117 and a cocktail of protease inhibitors (100pM) did not prevent protease degradation of the eotaxin forms
4.7 Measurement of dipeptidylpeptidase 1V (CD26) activity in the guinea
pig mesentery
Dipeptidylpeptidase IV (CD26) has been shown to cleave two amino acids from the N- terminus region of human eotaxin and consequently reduce its chemotactic activity for human eosinophils (Struyf et al, 1999). Using glycyl-prolyl-p-nitroanilide which is a specific substrate for dipeptidylpeptidase IV (Nagatsu et al, 1976) the presence of this enzyme in the guinea pig mesentery and kidney was investigated. Dipeptidylpeptidase IV was detected in the guinea pig mesentery lysate and kidney lysate (Figure 4.14). The activity of dipeptidylpeptidase IV could be blocked by the use of a protease inhibitor cocktail (100pM).
4.8 Flux of glycosylated and non-glycosylated human eotaxin across the guinea pig mesentery in vivo
Following intraperitoneal injection of lml of 30nM (30pmoles) of glycosylated or non- glycosylated human eotaxin into guinea pigs, blood was taken from the marginal ear vein at various time intervals. When glycosylated human eotaxin was injected intraperitoneally, a significant increase in glycosylated human eotaxin could be detected in the blood as early as 30 mins. In contrast, following injection of non-glycosylated human eotaxin it was not detected in the blood until after 60 mins. However, at 120mins, similar concentrations of glycosylated and non-glycosylated human eotaxins could be detected in the guinea pig blood (Figure 4.15).
118 4.9 Summary of results
1. Glycosylated human eotaxin induced human eosinophil migration across an isolated guinea pig mesentery. Non-glycosylated human eotaxin was inactive in this assay. In contrast, both the glycosylated and non-glycosylated human eotaxin forms were equipotent at inducing eosinophil migration across a polycarbonate filter.
2. Glycosylated human eotaxin but not non-glycosylated human eotaxin could be transported across the guinea pig mesentery.
3. The transport of glycosylated human eotaxin across the mesentery was inhibited in the presence of glycosylated guinea pig eotaxin, while the transport of glycosylated guinea pig eotaxin was reduced in the presence of glycosylated human eotaxin.
4. Biotinylated heparin bound both forms of human eotaxin with equal affinity. Heparan sulphate but not chondroitin sulphate B could compete with the biotinylated heparin for the binding of both forms of eotaxin.
5. Protease inhibitors did not affect the flux of glycosylated or non-glycosylated human eotaxin across the guinea pig mesentery.
6. The enzyme dipeptidylpeptidase IV was found to be present in the guinea pig mesentery and kidney lysates.
7. Glycosylated human eotaxin could be rapidly detected in the guinea pig blood (30 mins) following intraperitoneal injection. In contrast, non-glycosylated human eotaxin could only be detected in significant concentrations at 120 mins, when it reached a similar concentration in the blood as the glycosylated eotaxin.
119 Human eotaxin' for)/ Molecular Mass (kilo Daltons)
St-9 insect cells/baculovirus expressed 8.361 human eotaxin (Leukosite, MA) 8.726 9.091
St-9 insect cells/baculovirus expressed 8.743 human eotaxin (Fournier, France)
E.coli expressed human eotaxin 8.360 (Peprotech, UK)
Table 4.1 Molecular mass of human eotaxin produced in St-9 insect cells using a baculovirus expression system and a E.coli expression system
The molecular mass of Sf-9 insect cell or E.coli expressed human eotaxin as determined by time of flight mass analysis. Data supplied as personal communication from Dr D.Soler, Leukosite, MA and Dr Paquet, Founier, France.
120 ** X
ic t 4 - tac
mo 2 Che
10 100 Eotaxin (nM)
Figure 4.1 Chemotactic response of guinea pig eosinophils to guinea pig eotaxin, human eotaxin and LTB4 across a polycarbonate filter
Migration of guinea pig eosinophils across a polycarbonate filter (51tm pore) in a microBoyden chamber in response to non-glycosylated guinea pig eotaxin (m, 10- 100nM), non-glycosylated human eotaxin (o 10-100nM) and LTB4 (X, 100nM). The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index ± SEM for 3-5 separate experiments carried out in duplicate **P<0.01 compared to human eotaxin control.
121
Mesentery 8 -
0 3 110 30 100 b) Filter
10 -
8 dex
In 6 ic t
tac 4
2 Chemo
0 3 10 30 100
Human eotaxin (nM)
Figure 4.2 Chemotactic response of human eosinophils to glycosylated and non- glycosylated human eotaxin and LTB4 across a guinea pig mesentery and polycarbonate filter
Migration of human eosinophils across a) isolated guinea pig mesentery b) polycarbonate filter in a microBoyden chamber in response to glycosylated human eotaxin (II, 10-100nM), non-glycosylated human eotaxin (E, 10-100nM) and LTB4 (X, 100nM). The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index ± SEM for 5 separate experiments carried out in duplicate. *P<0.05 compared to non-glycosylated human eotaxin.
122
350
325
300 (/) 275
250
225
200 0 0.1 1 10 100
Human eotaxin (nM)
Figure 4.3 Shape change of human eosinophils is induced by glycosylated eotaxin and non-glycosylated eotaxin
Human eosinophil shape change measured as change in forward scatter (FSC) in response to glycosylated human eotaxin (Leukosite, USA) Or 0.1 -30nM), glycosylated human eotaxin (Fournier, France) (A, 0.1 - 30nM) and non-glycosylated human eotaxin (Peprotech, UK) (El, 0.1 — 30nM). Data shown as mean FSC for 1 experiment carried out in triplicate
123 Mesentery
600 ** cl• ** 400 = 4-p a) a)
c a .>7 0- co 200 O
10 30 100
Filter
800 -
in d M)
te 600 - ll (p tec
we 400 - de r e in tax
upp 200 - Eo
10 30 100 Human eotaxin (nM)
Figure 4.4 The movement of glycosylated and non-glycosylated human eotaxin across a guinea pig mesentery and polycarbonate filter
The movement of glycosylated 10-100nM) and non-glycosylated (D, 10-100nM) human eotaxin across a) an isolated guinea pig mesentery and b) 51.tm pore polycarbonate filter. Immunoreactive eotaxin in the upper wells was measured after 60 mins by ELISA. Dotted line represents the lower limit of detection for the ELISA. Data shown as mean concentration ± SEM for 5 separate experiments performed in duplicate. **P<0.01 compared to the non-glycosylated human eotaxin.
124 800
** 0. 600 (.3 a)- ** 400 .a Q ct. o 200
20 40 60 80 100 120 140 Time (mins)
Figure 4.5 Timecourse of the flux of glycosylated and non-glycosylated human eotaxin across the guinea pig mesentery
The movement of the glycosylated (II, 30nM) and non-glycosylated (❑, 30nM) human eotaxin across the isolated guinea pig mesentery at 30, 60 and 120 mins. Dotted line represents lower limit of detection for the ELISA. Data shown as mean concentration ± SEM for 5 separate experiments carried out in duplicate. "P<0.01 compared to non- glycosylated human eotaxin.
125 C -a 600 E o_ mi 0 . 400 X E 2, CU 0 CD t) .C3) Q.). 200 a a. ca 0=- a) C CD 0 GpE—G GpE-G + GpE-G + alone HuE-nG HuE-G
Figure 4.6 Effect of glycosylated human eotaxin on the flux of glycosylated guinea pig eotaxin across the guinea pig mesentery
The flux of glycosylated guinea pig eotaxin alone (GpE-G) (M, peak III, 30nM) across an isolated guinea pig mesentery or in the presence of non-glycosylated human eotaxin (HuE-nG) (D, 30nM) or glycosylated human eotaxin (HuE-G) ( , 30nM). Dotted line represents the lower limit of detection of the ELISA. Data shown as mean concentration + SEM for 3 separate experiments performed in duplicate. *P<0.05 compared to glycosylated guinea pig eotaxin alone.
126 Figure 4.7Effectofglycosylatedhuman guineapigeotaxinonthefluxof glycosylated humaneotaxinacrosstheguinea pig mesentery (GpE-nG) ( The fluxofglycosylatedhumaneotaxinalone (HuE-Galone)(II,30nM)acrossan isolated guineapigmesenteryorinthepresence ofnon-glycosylatedguineapigeotaxin Dotted linerepresentsthelowerlimitofdetection oftheELISA.Datashownasmean compared toglycosylated humaneotaxinalone. concentration ±SEMfor3separateexperiments performedinduplicate.*P<0.05
Human eotaxin detected ❑
, 30nM)orglycosylatedguineapigeotaxin(GpE-G) ( in upper chamber (pM) 300 200 100 HuE-G + alone GpE-nGGpE-G 127
peak III,30nM).
0 inE
c 2 co 8 U) .C2
1.0 10.0 100.0 Human Eotaxin (nM)
Figure 4.8 Binding of glycosylated and non-glycosylated human eotaxin to biotinylated heparin
Biotinylated heparin binding to glycosylated (■, 1-100nM) and non-glycosylated (0, 1- 100nM) human eotaxin. Data shown as mean absorbance ± SEM for 2 separate experiments carried out in duplicate.
128 4
3 - 450nm) (
nce 2 - ba r Abso 0 0 1:1 10:1 100:1 1000:1 Chondroitin Sulphate B:Heparin
U co 2
O
0 ' 0 1:1 10:1 100:1 1000:1 Heparan Sulphate:Heparin
Figure 4.9 Effect of chondroitin sulphate B and heparan sulphate on the binding of glycosylated and non-glycosylated human eotaxin to biotinylated heparin
Competition with a) chondroitin sulphate B (CSB:heparin 1:1 to 1000:1) and b) heparan sulphate (HS:heparin 1:1 to 1000:1) for the binding of biotinylated heparin to glycosylated (N, 30nM) and non-glycosylated (D, 30nM) human eotaxin. Data shown as absorbance SEM for 3 separate of experiments carried out in duplicate. *P<0.05 compared to heparin alone
129 d --- 500-
te E
tec .-0 400 - de
in 300- =R tax
eo ir) 200- n a_
ma c 100- ._ Hu 0 Control Betstatin APMSF Cocktail b)
70 500 - Ect. 4- 03, 400- -a
- o 4. 3 200- C CU E 100
0 Control Bestatin APMSF Cocktail
Figure 4.10 Effect of protease inhibitors on the movement of glycosylated and non- glycosylated human eotaxin across the guinea pig mesentery
The flux of a) glycosylated (II, 30nM) and b) non-glycosylated (0, 30nM) human eotaxin (control) in the presence of bestatin (501AM), APMSF (30µM) or protease cocktail mixture (100uM) across an isolated guinea pig mesentery in a microBoyden chamber. Dotted line represents the ELISA lower limit of detection. Data shown as mean concentration + SEM for 3 separate experiments performed in duplicate.
130 •• •" • E 15 in d
te 0)
c .0
te E io co de in
x 0 0- 5 ta O. Eo
Control APMSF 1BESTATIN Protease Inhibitor
Figure 4.11 Effect of protease inhibitors on the degradation of eotaxin variants following exposure to guinea pig mesentery
Glycosylated (II, 30nM) and non-glycosylated (0, 30nM) human eotaxin variants alone or in the presence of APMSF (30µM) or bestatin (50µM) were applied to the upper wells of a microBoyden chamber containing guinea pig mesentery. The eotaxin concentration in the upper wells was measured after 1 hour by ELISA. Data shown as average for 2 separate experiments carried out in duplicate.
131 325
0 300
L- 275
250
225
200 I I ► 0 0.03 0.1 0.3 1.0
Human eotaxin (nM)
Figure 4.12 Shape change of human eosinophils in response to glycosylated and non-glycosylated human eotaxin after 1 hour exposure to the guinea pig mesentery (GAFS assay)
Human eosinophil shape change measured as change in forward scatter (FSC) in response to glycosylated 0.03-1nM), non-glycosylated human eotaxin (0, 0.03-1nM) after exposure to the guinea pig mesentery for 1 hour and non-glycosylated human eotaxin that had not been exposed to guinea pig mesentery (0.03 — 1nM) (-x—). Data shown as mean FSC ± SEM for 3 separate experiments carried out in duplicate.
132 2 10 c --, 8 -
2 o 6 - w -o L.co - 0 > o (..) 2 0 ce El 0 ii ri • •ri In Buffer APMSF Bestatin Leupeptin Cocktail
Figure 4.13 Effect of proteases found in guinea pig mesentery lysates on the immunoreactivity of human eotaxin
Guinea pig mesentery lysates were incubated with glycosylated (10nM, or non- glycosylated (10nM, 1=1) human eotaxin at 37°C for lhr in the presence of APMSF (30µM), bestatin (50µM), Leupeptin (301.1M) or a protease inhibitor cocktail mix (100uM). The concentration of eotaxin was then determined by ELISA. Data shown as average concentration for 2 separate experiments performed in duplicate.
133 0.5 m)
0n 0.4 42
( 0.3 nce 0.2 - ba or 0.1 - Abs 0.0 Control Mesentery Mesentery Kidney Kidney Lysate Lysate + Lysate Lysate + protease protease inhibitor inhibitor cocktail cocktail
Figure 4.14 Determination of the presence of the protease dipeptidylpeptidase IV in the guinea pig mesentery and kidney lysates
Dipeptidylpeptidase IV activity was determined using a photometric assay with the substrate glycyl-prolyl-p-nitroanilide incubated with either HBSS buffer (control), mesentery lysate or kidney lysate with and without protease inhibitor cocktail (100µM). Data shown as mean absorbance ± SEM for 3 separate experiments performed in duplicate. *P<0.05 compared to control
134 150 cu
$3 0 ' 0 0 100 C
0 a) coa) c c 50 co -5 E .c
20 40 60 80 100 120 140 Time (mins)
Figure 4.15 Time course of h uman eotaxin detection in guinea pig blood following intraperitoneal injection
Human eotaxin was measured by ELISA in the guinea pig plasma at different time points following the intraperitoneal injection of either glycosylated 30nM, 1m1), or non-glycosylated 30nM, 1m1) human eotaxin to guinea pigs. Dotted line represents the lower detection limit of the ELISA. Data shown as mean concentration of eotaxin SEM for 5 separate experiments. "P<0.01 compared to non-glycosylated human eotaxin.
135 4.10 Discussion
The results presented in this chapter have demonstrated that glycosylated human eotaxin was able to induce eosinophil migration across the guinea pig mesentery whereas the non-glycosylated form was inactive. However, both the glycosylated and non- glycosylated human eotaxin forms were equipotent at inducing eosinophil chemotaxis across a polycarbonate filter. In addition, only the glycosylated human eotaxin could be detected in the upper wells of the guinea pig mesentery flux assay, whilst both the glycosylated and non-glycosylated eotaxin could be detected using a polycarbonate filter.
There have been conflicting reports regarding the effect of glycosylation on a protein and its biological activity. Comparison of the E.coli expressed and the cell-derived human IL-2 revealed no difference in activity when compared in a proliferation assay (Roifman et al, 1985). The 0-linked carbohydrate on human granulocyte colony- stimulating factor (G-CSF) has been shown to contribute to the stability of the molecule and increase its activity three fold when examined in a colony-forming assay (Oh-eda et al, 1990). Human granulocyte macrophage colony stimulating factor (hGM-CSF) contains two potential N-glycosylation sites and several 0-glycosylation sites and when expressed in human lymphoblastoid Nambala cells various glycosylated forms were produced (Okamoto et al, 1991). The non-glycosylated (lower molecular weight) form of murine IL-4 produced from an activated D10 cell line was demonstrated to be significantly more active than the glycosylated (higher molecular weight) species when tested in a proliferation assay (Thor and Brian, 1992). Jiang et al, (1991) and Ueda et al, (1994) have both demonstrated that glycosylated MCP-1 of varying mass exhibit no difference in the chemotaxis of monocytes. This was in contrast to Proost et al, (1998a and 1998b) who demonstrated that 0-glycosylated MCP-1 (molecular weight 12 and 13.5kDa) was three times less potent at inducing chemotaxis of THP-1 cells when compared to the non-glycosylated MCP-1 (10kDa). In contrast to all of the above observations, Ishii et al, (1995) have shown that MCP-1 expressed in St-9 insect cells was 0-glycosylated, and that the 0-glycosylated form of MCP-1 had a higher monocyte chemotactic activity than E.coli derived MCP-1. It would appear that the effect of glycosylation on the monocyte chemotaxis activity of MCP-1 remains unclear.
136 0-glycosylation of insulin-like growth factor binding protein-6 (1GFBP-6) has been shown to inhibit its binding to GAGs, thereby maintaining it in a high affinity soluble form (Marinaro et al, 2000a). In addition, 0-glycosylation has been shown to delay its clearance from the circulation and may contribute to its role as a circulating inhibitor of insulin-like growth factor II (IGF-II) actions (Marinaro et al, 2000b). With relevance to human eotaxin, Noso et al, (1998) have shown that human dermal fibroblasts stimulated with TNF-a produced different glycosylated forms of human eotaxin which had identical chemotactic activity to a non-glycosylated (E.coli expressed) eotaxin. Mochizucki et al, (1998) have also demonstrated that stimulation of human dermal fibroblasts with IL-4 and/or TNF-ct resulted in the release of glycosylated eotaxin variants, with similar eosinophil chemotactic activity to the recombinant expressed eotaxin. However, one glycosylated variant of eotaxin demonstrated a slightly decreased eosinophil recruitment activity than the other glycosylation variants.
For chemoattraction of leukocytes from the blood into the tissue to occur a chemokine gradient must be formed, along which the leukocytes will migrate. The in vitro chemotaxis assay used in this thesis involves the establishment of an artificial chemokine gradient. Eosinophils are placed in the upper well in buffer whilst the lower well contains a high concentration of chemokine. The upper and lower wells are separated by a membrane with a pore size (5[tm) that prevents cells dropping through e.g. the cells need to migrate through the membrane. In this system chemokine mutants incapable of binding to glycosaminoglycans (GAGs) have been shown to induce chemotaxis (Proudfoot et al, 2003). When the polycarbonate filter was replaced with a freshly isolated mesentery only the glycosylated eotaxin was able to induce an eosinophil chemotaxis. It was hypothesised therefore that the glycosylation on the eotaxin was facilitating the chemokine binding to GAGs on the mesentery surface and thus recruiting eosinophils. It is interesting to note that increasing the glycosylated eotaxin concentration in the bottom wells of the microBoyden chamber from 10, 30 and 100nM results in a near linear increase in eosinophil chemotaxis (Figure 4.2a). Similarly, the concentration of glycosylated eotaxin detected in the upper wells of the microBoyden chamber increases linearly (Figure 4.4a). This could suggest that there is sufficient GAG present on the mesothelial cell surface to facilitate transport across the tissue.
137 It has been postulated that by the action of blood flow, a soluble chemokine gradient across the endothelium would be removed, and indeed the chemokine could indiscriminately activate cells within the blood. It is thought therefore, that chemokines are immobilised whilst allowing presentation of the chemokine to the leukocyte, thus maintaining a chemokine gradient across the endothelium (Rot et al, 1993). It has previously been reported that chemokines can bind to GAGs and form an insoluble solid phase gradient across a cell (Patel et al, 2001). GAGs are located both on the cell surface and within the ECM and consist of polysaccharides covalently linked to core proteins. These core proteins contain sulphate and carboxyl groups which yield a large negative charge. (The role of GAGs and leukocyte extravasation is reviewed by Middleton et al, 2002). Gilat et al, (1994) demonstrated that the chemokines RANTES and MIP-10 are able to stimulate leukocyte activation whilst bound to the subendothelial ECM by the GAG, heparan sulphate (HS). Indeed, binding of the chemokine IL-8 to immobilised HS has been shown to enhance neutrophil chemotaxis (Webb et al, 1993). The Ca2+ flux and chemotaxis of GAG depleted CHO cells is reduced compared to wild type cells in response to RANTES and MIP-1 PI, whilst the binding affinity to the receptors were similar (Ali et al, 2001). In contrast, MIP-lia had a reduced binding affinity for the receptor CCR1 on cells stripped of GAGS (Hoogewerf et al, 1997; Ali et al, 2001).
Soluble GAGs e.g. GAGs that are not immobilised are also able to bind to chemokines and have been shown to reduce/abolish biological activity (Gilat et al, 1994; Douglas et al, 1997; Kuschert et al, 1999; Ali et al, 2000; Hirose et al, 2002). It is suggested that a haptotactic gradient is formed across the endothelial cell with IL-8 immobilised by GAGs (Middleton et al, 2002). It has been shown by Hardy et al, (2004) that a chemokine gradient in vitro is not required in order for leukocytes to migrate across an endothelial cell layer. It was proposed that the chemokine MCP-1 will bind to the GAGs present on the apical surface of the vascular endothelium in sufficient concentration as to incite leukocyte migration. Whilst this is interesting phenomenon, in an in vivo inflammatory situation the chemokine is rarely produced at the apical surface of the endothelium, but further back within the tissue and thus a gradient must be formed across the endothelial cell in order for the leukocyte to migrate from the blood.
138 The GAG binding-motifs of proteins has been identified as the following sequences BBXB where B is a basic residue and X is any other amino acid (Cardin and Weintraub, 1989; Hileman et al, 1998). It has been shown that the N-terminal region of chemokines is involved in receptor binding (Hebert et al, 1991; Clark-Lewis et al, 1991; Proudfoot et al, 1996; Pakianthan et al, 1997),while the GAG binding region is typically toward the C-terminus (Mayo et al, 1995; Kuschert et al, 1996; Chakravarty et al, 1998; Hirose et al, 2002). However, the GAG binding site for IL-8, SDF-1 and RANTES has been shown to be towards the N-terminus, suggesting possible overlap of receptor binding and the GAG binding domains (Proudfoot et al, 2001). It has been suggested that chemokines whose GAG binding site overlaps with the receptor binding site may require oligomerisation to be active in vivo. Proudfoot et al (2003) postulated, that some subunits of the oligiomer would bind GAGs whilst the remaining units would bind the receptor. They have also demonstrated that multimerisation of RANTES, MCP-1 and MIP-113 was required for leukocyte recruitment activity in vivo. In contrast, eotaxin is a naturally occurring monomer (Crump et al, 1998) and when injected into the guinea pig skin (Jose et al, 1994a), the mouse peritoneal cavity (Harris et al, 1997; Das et al, 1997) or as data from this thesis shows, applied topically to the guinea pig mesentery is able to recruit leukocytes. Thus, oligomerisation of eotaxin does not appear to be required for its biological activity in vivo.
The binding of glycosylated and non-glycosylated human eotaxin to GAGs was an important question to address, since the guinea pig mesentery has been shown to contain CSB (78%) and HS (22%) (Junqueirea et al, 1987). The binding affinities of chemokines for GAGs can vary greatly. It is has been shown that the size of the GAGs, and the level of sulphation on the GAG are important in its ability to bind chemokines (Kuschert et al, 1999). For the CXC chemokine IL-8 the order of binding affinity has been shown to be HS>>CSB=CSA, for the CC chemokines, MIP-1 a, HS>CSA>CSB, for MCP-1, HS>CSB>CSA and for RANTES CSB=HS>>CSA (Kuschert et al, 1999). Experiments in this chapter have shown that the glycosylated and non-glycosylated forms of human eotaxin bind to the GAG heparin with equal affinity. The binding affinity of CSB and HS to the two forms of human eotaxin was compared in a competition assay using biotinylated heparin. CSB was unable to compete with the biotinylated heparin for the binding of either of the two forms of eotaxin, even at 1000
139 fold excess. This suggests that neither the glycosylated nor non-glycosylated form of human eotaxin bound to CSB with higher affinity relative to heparin. In contrast, HS were able to compete with the biotinylated heparin for the binding to eotaxin. Similar concentrations of I-1S was required to compete with heparin for the binding to both the glycosylated and non-glycosylated eotaxin forms. These results suggest that HS but not CSB was able to bind both forms of eotaxin to the same extent. This observation was in agreement with other groups who have investigated the binding of chemokines to GAGs (Middleton et al, 1997; Hoogewerf et al, 1997; Kuschert et al, 1998, Kuschert et al, 1999). The experiments presented in this thesis would suggest that the GAG binding order for eotaxin is HS>>CSB which is similar compared to the CC chemokines MIP- la and MCP-1 but differs from the order for RANTES a chemokine of similar size and charge to eotaxin.
Since the C terminal region of eotaxin contains a consensus site for 0-glycosylation (Patel et al, 1999), it could therefore be postulated that glycosylation could interfere with the binding affinity of human eotaxin to GAGs. The results presented in this thesis suggest that the glycosylation on the St-9 expressed human eotaxin does not interfere with the binding of eotaxin to the shorter GAG, heparin, and the longer chain GAG, HS. From these results it can be concluded that eotaxin binding to GAGs is not responsible for the difference in activity observed across the guinea pig mesentery between the glycosylated and non-glycosylated human eotaxin. However, the results show that both forms bind with equal affinity to HS which has been shown to be present in the guinea pig mesentery and thus both forms are potentially capable of forming a haptotactic gradient.
It is interesting to note that the concentration of both the glycosylated and non- glycosylated forms of eotaxins detected in the upper chamber is relatively low whether a guinea pig mesentery or polycarbonate filter flux assay was performed. This suggests that both forms of eotaxins are significantly binding to the plastic of the microBoyden chamber despite pre-blocking the wells with a BSA/PBS solution. The binding is most probably due to the ionic charge of the eotaxins and the concentration of the bound eotaxins could have been determined by washing the wells with a high concentration of salt solution and the washes assayed by an ELISA to determine eotaxin concentration.
140 Protein glycosylation has been shown to protect against protease activity and thus prevent loss of biological activity (Van Berkel et al, 1995; Carter et al, 2004; Van Veen et al, 2004). It was hypothesised that the glycosylation present on eotaxin was conferring resistance to protease degradation. The observations made using intravital microscopy involved applying eotaxin topically to the mesothelium and thus exposed to proteases present on the tissue surface. The method used to determine chemotactic activity using the mesentery microBoyden eosinophil chemotaxis assay also involved application of eotaxin to the mesentery surface. In this chapter the role that both proteases present on the mesothelium surface and those within the mesentery tissue have on this observation were investigated.
Results from this thesis demonstrate that the addition of protease inhibitors did not significantly increase the concentration of non-glycosylated human eotaxin detected in a guinea pig mesentery flux assay. This suggests that protease activity was not the reason why non-glycosylated eotaxin was unable to flux or direct eosinophil chemotaxis across the mesentery. In addition, when non-glycosylated and glycosylated eotaxin were exposed to the guinea pig mesentery for 60 mins, the concentrations of the two eotaxin forms detected by ELISA, were similar to each other but reduced. Following 60 mins incubation on the guinea pig mesentery, the eosinophil shape change activity of the recovered glycosylated and non-glycosylated human eotaxins, were also similar and correlated well with the measured ELISA concentration. This suggests that the glycosylation on eotaxin does not protect the chemokine from mesothelial surface proteases.
Sereneva et al, (1994) have demonstrated that the bioactivity of the non-glycosylated, but not the glycosylated, form of IFN-y was greatly reduced when exposed to a crude granulocyte protease, suggesting that glycosylation of IFN-y was critical for maintaining resistance against the protease. Glycosylated and non-glycosylated human eotaxin were incubated with guinea pig mesentery lysates the concentrations of both forms detected by ELISA were greatly reduced. This was shown to be due to protease degradation of eotaxin as it was prevented when eotaxin was coincubated with the serine protease inhibitor, 4-(amidinophenyl)methanesulfonyl (APMSF). This suggests that the proteases found within the mesentery are serine proteases and that the
141 glycosylation present on human eotaxin does not protect the molecule from serine protease degradation.
The protease dipeptidyl-peptidate IV (DPP IV also known as CD26) is an aminopeptidase that cleaves NH2-terminal dipeptides from proteins that contain the amino acid sequence proline, hydroxyproline or alanine at its penultimate position (Vanhoof et al, 1995). DPP IV has been shown to be present on endothelial cells, epithelial cells and fibroblasts (Reynaud et al, 1992, Darmoul et al, 1994). During the process of leukocyte recruitment, eotaxin and other chemokines are likely to be exposed to DPP IV present on these cell types. DPP IV has also been found in a soluble form in the plasma further increasing the likelihood of chemokines encountering this enzyme (Durinx et al, 2000) and has also been detected in BAL fluid (Van der Velden et al, 1998). The ranking (starting with the highest affinity) of chemokine substrates for cleavage by DPP IV has been shown to be the following : SDF-la, MDC, I-TAC, IP- 10, Mig, eotaxin, RANTES (Lambeir et al, 2001). Chemokine activity following exposure to DPP IV varies greatly. However, not all chemokines contain the correct penultimate amino acid motif to be substrates, whilst those which have the correct sequence might still be resistant to degradation by this protease. Granulocyte chemotactic protein-2 (GCP-2) following exposure to DPP IV is converted into the truncated chemokine GCP-2 (3_77).which maintains its neutrophil chemotactic activity (Proost et al, 1998a). This is in contrast to SDF-la which following exposure to DPP IV was processed to SDF-la (3_68) which lacked lymphocyte chemotactic activity (Proost et al, 1998b). Interestingly, the MCP chemokine subgroup contain the correct penultimate amino acid residue sequence to be processed by DPP IV however, cyclisation of the amide terminal glutamine residue into pyroglutamate occurs resulting in protection from DPP IV (Van Coillie et al, 1998).
Non-glycosylated human eotaxin has been shown to be a substrate for DPP IV cleaving the chemokine at the N-terminus resulting in a 30 fold reduction in eosinophil chemotactic activity but only a 6 fold reduction in binding to its receptor CCR3 (Struyf et al, 1999) suggesting that truncated eotaxin could act as a natural antagonist for CCR3. Glycosylation on human eotaxin is believed to occur at amino acid position 71 which is towards the C-terminus, away from the site of DPP IV cleavage. Stimulation of dermal fibroblasts with IL-4 or TNF-a has been demonstrated to produce eotaxin
142 variants, glycosylated at amino acid position 71. In addition, full length (1_74) and truncated (3_74) eotaxin forms were also produced suggesting eotaxin had undergone protease degradation by DPP IV (Mochizucki et al, 1998; Noso et al, 1998). The presence of DPP IV was detected in the guinea pig mesentery by use of p-nitroanilides which are substrates for this enzyme (Nagatsu et al, 1976).
It has been shown that human eotaxin binds to the guinea pig eotaxin receptor, CCR3, (Sabroe et al, 1998) but was unable to cause a functional response in the guinea pig CCR3 stably transfected 4B4 cell line. Any cross functional activity of the guinea pig CCR3 by the human eotaxin would be prevented, thus the movement of eotaxin across the mesothelium could be studied without inducing an immunological response which could effect the flux of eotaxin across the tissue. Interestingly, Fukuyama et al (2000) have shown that human eotaxin can induce a Ca++ flux and chemotaxis of guinea pig eosinophils. However in this thesis human eotaxin did not induce guinea pig eosinophils to migrate across a polycarbonate filter. Since human eotaxin was inactive on guinea pig CCR3, it was not possible to study its effects on eosinophil recruitment from the guinea pig mesenteric microvasculature using intravital microscopy.
The kinetics of different glycosylation forms of human eotaxin crossing the guinea pig mesentery in vivo following i.p. injection and entering the blood were studied. It could clearly be observed that the glycosylated human eotaxin was detected in the blood at a significant concentration as early as 30 mins following i.p injection. Interestingly, the non-glycosylated human eotaxin was detected in the blood at a similar concentration to the glycosylated form after 60 mins. The peritoneum has been classically considered to be an inert passive membrane which allows the fluid in the peritoneal cavity to come into osmotic equilibrium with the extracellular fluid, following the kinetics of Fick's first law of diffusion (Schechter et al, 1933). However it has been demonstrated that the peritoneal mesothelium as well as the submesothelial interstitium and microvessels are negatively charged (Gotloib et al, 1988). Since the glycosylated and non-glycosylated human eotaxins have the same negative charge (-7), the possibility that the mesothelium differentiates between the two molecules can be eliminated. A possible explanation for the delay in non-glycosylated human eotaxin entering the circulation could be an inability to cross the guinea pig mesentery as has been shown by the mesentery flux assay, whilst entering the circulation via the lymphatic system. Lacunae are large
143 terminal lymphatics located beneath the mesothelium of the peritoneal surface which remove fluid, cells and particles from the peritoneal cavity and into the blood (Tsilibary and Wissig, 1983). Solute, to be removed from the peritoneal cavity enters the lacunae via openings known as stomata located between mesothelial cell borders (Yoffey and Courtice, 1970). Tsilibary and Wissig (1983) have shown that the clearance time of the peritoneal cavity can be altered by various factors. The role of the lymphatic system on the transport of glycosylated and non-glycosylated human eotaxin from the peritoneal cavity into the blood warrants further study.
Differences in systemic clearance of the glycosylated and non-glycosylated eotaxin could be another explanation for the detection of glycosylated and not the non- glycosylated human eotaxin in the guinea pig blood. The duffy antigen/receptor for chemokines (DARC) is present on red blood cells and venular endothelium (Hub and Rot, 1998; Patterson et al, 2002, Rot, 2003) and is able to bind chemokines. The difference in binding of glycosylated and non-glycosylated human eotaxin to GAGs has been addressed in this thesis; however any binding differences to DARC have not been investigated. A difference in binding affinity between the glycosylated and non- glycosylated human eotaxin could explain the difference in concentration profiles in the blood. It could also explain the low detectable concentration of eotaxin in the circulation (less than 0.5% of the i.p. administered protein was detected) as it is unknown if the methodology used would enable the ELISA to detect eotaxin if bound to DARC. Flesher et al, (1995) demonstrated that IgG containing higher degrees of glycosylation has a slower clearance profile from the blood than that of IgG with lower amounts of glycosylation when injected into the mouse. However, the two forms of IgG had similar binding characteristics in vitro. The difference in the clearance rates between the glycosylated and non-glycosylated forms of human eotaxin from guinea pig blood has not been addressed.
The results presented in this chapter suggest that glycosylation of the human eotaxin does not offer any significant protection against the proteases found on or within the guinea pig mesentery. They have also shown that glycosylation of eotaxin does not significantly alter the binding affinity of the eotaxin protein to GAGs. An in vivo model has also been produced that can mimic the results from the observations made using the in vitro mesentery microBoyden chamber flux assay. This chapter has also shown that
144 glycosylated human eotaxin appears to use the same or similar mechanism to move across the mesentery as glycosylated guinea pig eotaxin since each glycosylated variant can inhibit the flux of the other across the guinea pig mesentery.
145 Chapter 5
Identification of the saccharide on glycosylated human eotaxin and its role in transport across the guinea pig mesentery
146 5 Identification of the saccharide on human eotaxin and the role of the glycosylation in relation to transport across the guinea pig mesentery
Results presented in Chapter 4 demonstrate that the glycosylation on the human eotaxin protein was necessary for its eosinophil chemoattraction across the guinea pig mesentery. The glycosylated human eotaxin used in the experiments described in this thesis was produced in a St-9 insect cell expression system. This system contains the enzymes N-acetylgalactosaminyltransferase and UDP-Gal:core-1131,3- galactosyltransferase which can result in the addition of N-acetylgalactosamine containing sugars (Lopez et al, 1999).
The experiments described in this chapter were designed to identify the saccharide present on glycosylated human eotaxin and to study its role in the transport of eotaxin across the guinea pig mesentery. To determine the sugar present on the glycosylated human eotaxin, plant lectins which bind specific sugar residues were used. The plant lectins used in this thesis are identified shown in Table 2.1 (Page 83).
5.1 Identification of the carbohydrate structure on glycosylated human
eotaxin
In order to identify the carbohydrate present on the glycosylated human eotaxin a glycan differentiation kit with various digoxygenin-labelled lectins was used enabling immunological detection of the bound lectins. Lectins are structurally diverse and are known to vary in molecular size, amino acid composition, metal requirement and three dimensional structure. The PNA lectin (derived from Arachis hypogaea or peanut), recognises the terminal sequence of Galf31 _3GalNAc (Lotan et al, 1975). PNA cannot bind to the saccharide Galf31 _3GalNAc if it contains a terminal sialic acid residue (Lotan et al, 1975). As can be seen in Figure 5.1, PNA, which recognises the terminal disaccharide Gal(31 _3GalNAc, clearly bound to glycosylated human eotaxin (1 and 3µg) and asialofetuin (l µg) when the proteins were immobilised on a nitrocellulose filter. Asialofetuin is a glycoprotein which contains both 0-glycosylated Ga1131 _3Ga1NAc and 0-glycosylated Ga1131 _4GlcNAc linked chains. In contrast, PNA did not bind the non- glycosylated human eotaxin (31.1g) or the sialic acid containing protein, fetuin
147 DSA derived from Jimson Weed or Datura stramonium recognises terminal Galf31 _
4G1cNAc (Crowley et al, 1984). DSA did not bind either the glycosylated or non- glycosylated human eotaxin (Figure 5.2). The glycosylated form of eotaxin-2 (C1(136) gave a positive signal with DSA, suggesting the presence of Ga1131 _4G1cNAc. Asialofetuin, gave a clear positive signal with both PNA and DSA, whereas fetuin (the same protein containing a terminal sialic acid residue) did not give a positive signal with either PNA or DSA (Figure 5.1 and 5.2).
5.1.1 Detection of the carbohydrate structure present on glycosylated human eotaxin
The ability of PNA and WGA to bind to glycosylated and non-glycosylated human eotaxin was determined using a lectin solid phase binding assay as described in Methods. It can be seen that PNA bound to the glycosylated eotaxin (Figure 5.3a). Another St-9 insect cell/baculovirus expressed glycosylated human eotaxin (Fournier, France) was tested in this assay and, as can be seen in Figure 5.3a, also bound to PNA with approximately 3-fold greater affinity than the human glycosylated eotaxin from LeukoSite. The non-glycosylated human eotaxin did not bind to PNA. WGA did not bind the glycosylated or non-glycosylated forms of human eotaxin (Figure 5.3b). Asialofetuin, gave a positive signal with both PNA and WGA at In/mi.
5.2 The effect of the plant lectins PNA and WGA on the transport of human eotaxin across the guinea pig mesentery
In the following experiments glycosylated human eotaxin (30nM) was added to the lower well of a microBoyden chamber and the flux (i.e. the amount in the upper well) across the guinea pig mesentery was measured in the presence and absence of PNA or WGA (3-100nM) after 1 hour by ELISA.
The concentration of glycosylated human eotaxin in the upper well significantly decreased when PNA at 100nM was added to the lower well (Figure 5.4a). The presense of PNA did not facilitate the flux of non-glycosylated eotaxin (Figure 5.4b). It has previously been shown in this thesis that the non-glycosylated human eotaxin was not transported across the guinea pig mesentery (Figure 4.4a) and this was not affected by
148 the addition of PNA. In contrast to PNA, WGA at 3-100nM did not inhibit the flux of glycosylated human eotaxin across the guinea pig mesentery (Figure 5.5a) and did not induce flux of non-glycosylated human eotaxin across the guinea pig mesentery. The presense of PNA did not facilitate the flux of non-glycosylated eotaxin (Figure 5.5b).
5.3 The effect of saccharides on the flux of human eotaxin across the isolated guinea pig mesentery
5.3.1 The effect of the monoaccharides Ga1NAc and G1cNAC on the flux of glycosylated human eotaxin across the guinea pig mesentery
The monosaccharide N-acetylgalactosamine (Ga1NAc), at 30 and 100mM, significantly decreased the flux of human glycosylated eotaxin across the guinea pig mesentery, while its enantiomer N-acetylglucosamine (GleNAc) had no significant effect (Figure 5.6a). Ga1NAc at 100mM gave a 50% inhibition of the transport of glycosylated human eotaxin across the guinea pig mesentery. As can be seen in Figure 5.6b galactose also did not affect the transport of glycosylated human eotaxin across the guinea pig mesentery. In contrast, glucose at 100mM significantly increased the concentration of glycosylated human eotaxin detected in the upper chamber. Non-glycosylated human eotaxin was used as a negative control to ensure that the increased concentration of saccharides had not disrupted the cellular barrier of the guinea pig mesentery. The non- glycosylated human eotaxin did not flux across the guinea pig mesentery suggesting that the mesothelia] cell barrier had not been disrupted.
5.3.2 The effect of the disaccharide Galpi_3GalNAc, asialofetuin and fetuin on the flux of glycosylated human eotaxin across the guinea pig
mesentery
The disaccharide Galf31 _3Ga1NAc (10011M) and asialofetuin (1gg/nil) inhibited the flux of human glycosylated eotaxin across the guinea pig mesentery (Figure 5.7). The concentration of glycosylated human eotaxin detected in the upper chamber was decreased from 170pM to 70pM in the presence of Galf31 _3Ga1NAc (100µM). Asialofetuin (1iig/m1) also significantly inhibited the flux of glycosylated human
149 eotaxin. In contrast, fetuin (1µg/m1) had no effect on the flux of glycosylated human eotaxin.
5.3.3 The effect of the disaccharide Ga1f31_4G1cNAc and the monosaccharide Ga1NAc on the flux of human eotaxin across the guinea pig mesentery
As can be seen in Figure 5.8 the concentration of glycosylated human eotaxin detected in the upper chamber significantly decreased from 350pM to 200pM in the presence of Gall31 _4G1cNAc. In addition, the monosaccharide, GalNAc (100mM) significantly reduced the levels of glycosylated human eotaxin levels from 322pM to 180pM.
5.4 The effect of the EGTA on glycosylated human eotaxin flux
To determine if the transport of glycosylated eotaxin required calcium, EGTA (1- 10mM) was added to the mesentery flux assay. The transport of glycosylated human eotaxin across the guinea pig mesentery was not inhibited in the presence of EGTA, suggesting that the initial binding of the glycosylated human eotaxin to the 'putative' receptor was calcium independent (Fig 5.9).
5.5 The effect of the anti-galectin 3 antibody on the flux of human eotaxin
Galectins are calcium independent lectins and have been shown to be involved in several in vitro physiopathological processes requiring carbohydrate recognition, such inflammation (Yamaoka et al, 1995). Galectin-3 has been shown to be present on eosinophils (Hughes 1999) and therefore this experiment was designed to determine if Galectin-3 was involved in the flux of glycosylated eotaxin across the mesentery. The flux of glycosylated human eotaxin across the guinea pig mesentery was not significantly altered in the presence of a mouse monoclonal anti-human galectin-3 IgG antibody (1 µg/ml) or by the control mouse anti-human IgG (Figure 5.10). The concentration of non-glycosylated eotaxin in the upper chamber remained below the level of detection of the ELISA (data not shown).
150 5.6 Summary of results
1. The saccharide moiety present on St-9 expressed glycosylated human eotaxin was identified as Ga1131.3GalNAc.
2. The movement of the glycosylated human eotaxin across the guinea pig mesentery was significantly decreased by the lectin PNA, which recognises the sugar Galf31 _3 GalNAc.
3. Glycosylated human eotaxin was inhibited from fluxing across the guinea pig mesentery by the addition of the monosaccharide GalNAc, the disaccharides Ga1i31_3Ga1NAc and Gal[31 _4G1cNAc and the protein asialofetuin, which contains both Ga1131.3GalNAc and Ga1131 _4G1cNAc side chains.
4. EGTA and mouse monoclonal anti-human galectin-3 antibody did not affect the transport of glycosylated human eotaxin across the mesentery.
151 Asialofetuin
Human eotaxin[G] 3µg
Human eotaxin[G] 1
Human eotaxin[NG] 3 lig
41 Fetuin
Asialofetuin
Figure 5.1 Binding of PNA to giycosyiated human eotaxin
Slot blot showing positive reaction of glycosylated human eotaxin [G] (1 and 3i_tg) and asialofetuin (1p0 with digoxygenin labelled PNA, which recognises the saccharide Ga1131 _3GalNAc.
152 Asialofeutin 1 pg
Human eotaxin[NG] 3pg
Transferrin 1pg
Human eotaxin[G] 3pg - Fournier
Feutin 1 pg
Human eotaxin[G] 3pg - Leukosite
Human eotaxin2[G] (CkP6) 3pg
Human eotaxin2[NG] 3pg
Asialofeutin 1 pg
Figure 5.2 Lack of DSA lectin binding to glycosylated human eotaxin
Slot blot showing negative reaction with glycosylated and non-glycosylated human eotaxin (3[1g) and non-glycosylated human eotaxin-2 (3µg) together with a positive reaction of glycosylated human eotaxin-2 (314) and asialofetuin (11.tg) with digoxygenin labelled DSA that recognises Galf11 _4G1cNAc.
153
1 10 100 1000 b) WGA
0nm) 45 ( nce ba r o
Abs 10 100 1000 Human eotaxin (nM)
Figure 5.3 Binding of human eotaxins to PNA and WGA lectins
Binding of glycosylated (Sf-9 insect cell/baculovirus expressed (LeukoSite Inc,USA ■) and (Fournier, France ♦), non-glycosylated E.coli expressed, (❑) and synthetically produced (o) human eotaxin to a) PNA (211g/m1) and b) WGA (2µg/m1). A representative of two experiments performed in duplicate is shown.
154
a)
E-5 - a 400 - a
CO 1_300 _ O a Lijc 1200 ca E • 1:3culoo 0 Control 3 10 30 100
a 500 - b) -
a) (,) 400-
•-• — 300- CO
w 200 a 0- Al a oo T
0 Control 3 10 30 100
PNA (nM)
Figure 5.4 The effect of PNA on the flux of human eotaxin across the guinea pig mesentery
The flux of a) glycosylated human eotaxin 30nM) and b) non-glycosylated human eotaxin (0, 30nM) across the isolated guinea pig mesentery was determined in the presence of PNA (3 - 100nM). Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 4 separate experiments carried out in duplicate. *P<0.05 compared to control.
155 a) -a 500 a) Q. 400
300 1- 12. UJ 200 c c E •- 100
0 Control 3 10 30 100
._C 500 - ci) 400 a) 2 -a C-300 - )7 1) o 0 as 200 - c ca a. loo T T
0 Control 3 10 30 100 WGA (nM)
Figure 5.5 The effect of WGA on the flux of human eotaxin across the guinea pig mesentery
The flux of a) glycosylated human eotaxin (N, 3OnM) and b) non-glycosylated human eotaxin (❑, 30nM) across the isolated guinea pig eotaxin was determined in the presence of WGA (3 - 100nM). Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 4 separate experiments carried out in duplicate.
156
a) b) across theguineapigmesentery Figure 5.6Theeffectofsaccharidesontheflux ofglycosylatedhumaneotaxin The fluxofglycosylatedhumaneotaxin(30nM) mesentery wasdeterminedwiththesaccharides a)(• Galactose) and(0Glucose)at3-100mM.Datashown detected intheupperchamber +SEMof5separateexperiments "P<0.01 and*P<0.05compared tocontrol. -41 75 E C ca 0 o a) 6- co
Human eotaxin detected -0 300 • _c 200 — E E I O c Q. a3 0. 0. z . 100 400 300 400 200 100 0 A 0 Saccharide concentration(mM) - - - -
0 1 0 1 i ✓ A
10 10
157 100 100 * * as meanconcentrationofeotaxin across theisolatedguineapig Ga1NAc),
carried outinduplicate.
GlcNAc), andb)(f)
0. 250 - a_ a) a_ 200 -0 6. a)a) c.) 150 -
-a u ▪ax 100 ea
50 -
0
I 1
M E /m '72 /m 0, z ug
0 3ug 1 100u in 0. Ac tu in IN fe tu lo fe Ga 3 ia _ lo 1 3 ia As li As Ga
Figure 5.7 The effect of Ga1131 _3GalNAc , asialofetuin and fetuin on the flux of glycosylated human eotaxin across the isolated guinea pig mesentery
The flux of glycosylated human eotaxin (I, 30nM) across the isolated guinea pig mesentery was determined in the presence and of the disaccharide Galf31 _3GalNAc (100µM), asialofetuin (0.3µg/ml and 1pg/m1) and fetuin (0.31.1g/m1 and I tg/ml). Data shown as mean concentration of glycosylated eotaxin detected in the upper chamber of one experiment carried out in duplicate.
158 400 -
*
0 Control Ga1131.4GIcNAc GaINAc 100µM 100mM
Figure 5.8 The effect of the Galf31 _4G1cNAc and GalNAc on the flux of glycosylated human eotaxin across the guinea pig mesentery
The flux of glycosylated human eotaxin (I, 30nM) across the isolated guinea pig mesentery was determined in the presence of the disaccharide Galf31 _4G1cNAc (100µM) and the monosaccharide GaINAc (100mM). Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 3 separate experiments carried out in duplicate. *P<0.05 compared to control.
159 Figure 5.9TheeffectoftheEGTAonflux of humaneotaxinacrosstheisolated guinea pigmesentery The fluxofglycosylatedhumaneotaxin(M, 30nM) acrossanisolatedguineapig concentration ofeotaxindetectedintheupper chamber±SEMof3separate mesentery wasdeterminedinthepresenceofEGTA (1-10mM).Datashownasmean experiments carriedoutinduplicate.
Human eotaxin detected Q. .Q .E 50- ▪ E c2 I s_ t 250 c co Q = .) 300— 200 — 350 — 150 — 100- 0 - Control
EGTA [mM] 1
160 3
10 c
u. 400 — O ce.c1)
c E 300 E• m4_3a) as_- .— 200 cc 4-a _c 0 CD • 4-, 1 00 O CD • "C; >t (.7 0 Control anti-gal3 Control IgG mAb Ab
Figure 5.10 The effect of anti-galectin 3 antibody on the flux of human eotaxin across the isolated guinea pig mesentery
The flux of glycosylated human eotaxin 30nM) across the isolated guinea pig mesentery was determined with an mouse monoclonal anti human anti-galectin 3 IgG antibody (1 i_tg/m1) or control mouse anti human IgG. Data shown as mean concentration of eotaxin detected in the upper chamber + SEM of 3 separate experiments carried out in duplicate.
161 5.7 Discussion
The Sf-9 insect cell line has been shown to contain UDP-GalNAc:polypeptide N- acetylgalactosaminyltransferase and UDP-Gal:corel [31,3-galactosyltransferase enzyme activity which, allows the expression system to modify proteins by the addition of the monosaccharide N-acetylgalactosamine (Ga1NAc) and the disaccharide galactose(3i_3N- acetylgalacatosamine (Gal[31 _3GalNAc). The molecular mass difference between the Sf-9 insect cell/baculovirus expressed human eotaxin and E.coli expressed eotaxin was 365Da and 730Da as determined from the mass spectroscopy data, which corresponds to the molecular mass of Ga1f31_3Ga1NAc and Galr31.3Ga1NAc-Galf31_3Ga1NAc respectively. This suggests that the glycosylation present on the Sf-9 expressed human eotaxin was Galf31_3GalNAc or Galf31 _3GalNAc-Galf31.3GalNAc. However, this does not discount the possibility that an enantiomer of Ga1131_3GaINAc, namely Galactosef31_4N- acetylglucosamine (Galf3i4G1cNAc) also with the molecular mass of 365Da, could be the saccharide present on the Sf-9 insect cell/baculovirus expressed human eotaxin. The glycosylation present on the MCP-1 forms expressed in a Sf-9 expression system was identified as Galf31_3GalNAc using the enzyme 0-glycanase (Ishii et al, 1995). Similarly, human IL-2 and IFN-y expressed in Sf-21 insect cells using a baculovirus expression system has been shown to contain the disaccharide Galf31_3GalNAc (Grabenhorst et al, 1993; Sugyiama et al, 1993).
To identify the composition of the glycan on glycosylated human eotaxin, plant lectins which recognise and bind to specific saccharide residues were used. The PNA lectin which recognises the terminal sequence of Galf31_3GalNAc (Lotan et al, 1975) bound with high affinity to the nitrocellulose immobilised Sf-9 expressed glycosylated human eotaxin, but not to the non-glycosylated E.coli expressed or synthetically produced human eotaxins. PNA cannot bind to the saccharide Ga1131_3GalNAc if it contains a terminal sialic acid residue (Lotan et al, 1975). This was confirmed as PNA bound to asialofetuin (a glycosprotein containing both 0-glycosylated Galf31.3GalNAc and 0- glycosylated Ga1( 1 _4G1cNAc linked chains with no terminal sialic acid) but not fetuin (which contains terminal sialic acid). This suggests that the glycosylation present on Sf- 9 expressed human eotaxin is Gall31 _3GalNAc without sialic acid. Lectins for the saccharides Galf31_40cNAc, GlcNAc and a-mannose were used to confirm that the
162 glycosylated human eotaxin did not contain other glycosylation variants. Noso el al, (1998) had shown that the human eotaxin variants generated by TNF-a stimulated dermal fibroblasts expressed the 0-glycosylated saccharide Ga1131 _3GalNAc both with and without sialic acid residues. Thus, the glycosylation present on the Sf-9 expressed human eotaxin is similar to the eotaxin produced by stimulated human cells.
In addition to the glycosylated human eotaxin supplied by LeukoSite (MA) another Sf-9 insect cell baculovirus expressed eotaxin was obtained from Fournier, France. Mass analysis provided from Fournier demonstrated that the eotaxin protein contained one mass peak of 8.743 kDa. Both forms of human eotaxin were further tested in the solid phase binding assay which, unlike the slot blot assay, is able to quantify the percentage of protein that contains a saccharide relative to a known positive control. The eotaxin provided by Fournier induced a maximal absorbance with the biotinylated PNA using one third less chemokine compared to the eotaxin from Leukocyte. This suggests firstly that the glycosylated human eotaxin provided by Fournier contains three fold more glycosylated protein relative to the eotaxin from LeukoSite. This can be postulated because human eotaxin only contains one glycosylation site and a difference in mass between glycosylated and non-glycosylated eotaxin was approximately 365Da. Secondly, approximately 33% of the eotaxin protein provided by Leuoksite contains glycosylation i.e. it is a mixture of glycosylated and non-glycosylated protein. This can be postulated because the eotaxin provided by Fournier contains one mass peak demonstrating that 100% of the protein is glycosylated. Of the eotaxin variants (glycosylated and non-glycosylated) none bound to the WGA lectin suggesting that a terminal GleNAc residue was not present.
In order for glycosylated human eotaxin to move across the guinea pig mesentery, a mechanism must exist that is capable of differentiating between glycosylated and non- glycosylated eotaxin. It could be postulated that this might involve a 'receptor' that recognises the saccharide moiety present on the glycosylated eotaxin. This 'receptor' must be able to selectively bind the glycosylated human eotaxin and it could facilitate the uptake of glycosylated human eotaxin into the mesothelial cell. Experiments in this chapter have attempted to identify the characteristics of a 'putative' receptor on the mesentery. The lectin PNA, but not WGA, was shown to decrease the flux of glycosylated human eotaxin across the isolated mesentery. This suggests that the
163 presence of a specific lectin molecule which binds the saccharide Ga1131 _3GalNAc on the glycosylated eotaxin, could prevent its movement across the guinea pig mesentery. This further demonstrates that the saccharide present on the glycosylated human eotaxin was important for it to flux across the mesentery.
Galf31 _3Ga1NAc, the sugar shown to be present on glycosylated human eotaxin, significantly inhibited the flux of glycosylated human eotaxin across the guinea pig mesentery. Interestingly, its enantiomer Ga1131 _4G1cNAc (100µM) which is not present on human eotaxin also significantly inhibited the transport of glycosylated eotaxin. A concentration of 100mM of the monosaccharide Ga1NAc was required to inhibit the flux of glycosylated human eotaxin to the similar level as achieved by 100µM of the disaccharides Ga1131_3GalNAc or Ga101.4G1cNAc. Interestingly, the monosaccharide N- acetylglucosamine (G1cNAc, 100mM) did not reduce the concentration of glycosylated human eotaxin crossing the mesentery which was unexpected as the disaccharide Ga1131-
4G1cNAc was able to inhibit the glycosylated eotaxin flux across the guinea pig mesentery. As the concentrations for inhibition were much lower for the disaccharide molecule (collectively known as 13-galactoside) than the monosaccharide, it suggests that P-galactosides are important for the binding of glycosylated eotaxin to the mesothelial cell 'receptor'. The flux of glycosylated eotaxin was reduced by the addition of asialofetuin and not fetuin. This suggests that the mechanism by which eotaxin transverses the mesentery is not protein specific but recognises 13-galactosides without terminal sialic acid. Interestingly, the flux of glycosylated human eotaxin across the guinea pig mesentery significantly increased in the presence of the monosaccharide glucose (100mM) but not galactose. This strongly suggests that the concentration of glucose in the medium (5mM) could potentially be rate limiting for the transport of glycosylated human eotaxin across the mesentery, and that the passage of glycosylated eotaxin was an active process. Further study is required to confirm this observation. Glucose at 100mM despite increasing flux of glycosylated human eotaxin did not permit the passage of non-glycosylated eotaxin across the guinea pig mesentery showing that it was not affecting the integrity of the mesentery.
The results presented in this chapter suggests that a 'receptor' may exist on the guinea pig mesentery with a specificity for 13-galactosides of at least two saccharide molecules
164 in length. Lectins which are carbohydrate-binding proteins, recognising specific oligosaccharide structures or ligands were considered to be likely candidates. In vertebrates, two broad classes of lectins have been identified. The C-type lectins, which require calcium for carbohydrate binding (Cummings and Smith, 1992; Drickamer, 1993) and S-type lectins, which are calcium independent in their ligand binding (Drickamer and Taylor, 1993). Since EGTA had no effect on the movement of glycosylated human eotaxin across the guinea pig mesentery a role for C-type lectins was ruled out. The more potent calcium and magnesium chelator EDTA was not used as removal of magnesium ions would block the glycolysis pathway, therefore preventing any energy dependent pathways.
Calcium independent, S-type lectins, more commonly known as galectins ((3-01actoside binding lectins), are evolutionarily conserved proteins widely distributed from lower invertebrates to mammals (Barondes et al 1994a, Barondes et al 1994b, Kasai and Hirabayashi, 1996) and to date 10 subtypes have been identified (for an overview see Lefler H, 1997). Galectins are defined by their sequence and structural similarities in the carbohydrate recognition domain (CRD) and their specificity for polylactosamine- enriched glycoconjugates (the P-galactoside Ga1(31_4G1cNAc) (Kasai and Hirabyashi, 1996). Hirabayashi and Kasai classified galectins into proto-type, chimera-type and tandem repeat type depending on their molecular structure. Galectins have been involved in several in vitro physiopathological processes requiring carbohydrate recognition, such as cell adhesion (Cooper and Barondes, 1999; Sato et al, 2002), apoptosis (Perillo et al, 1995; Rabinovich et al, 1998) inflammation (Yamaoka et al, 1995) and tumour distribution (Raz and Lotan, 1987). Galectin-1 has been demonstrated to have an anti-inflammatory effect in an acute inflammation model in the rat (Rabinovich et al, 2000). The presence of galectin-3 on activated macrophages, eosinophils, neutrophils, mast cells and the epithelium of gastrointestinal and respiratory tracts (Hughes, 1999), suggests that it may play a role in inflammatory conditions. Galectin-3 participates in the allergic response by binding IgE receptors (Kimata, 2002) and promotes IL-1 production by human monocytes, activates human neutrophils and induces superoxide release (Perillo et al, 1998). Galectin-3 has been shown to down-regulate the expression of the inflammatory cytokine, IL-5, (Cortegano et al, 1998) and inhibits bronchial obstruction and inflammation in antigen-challenged rats by down-regulated 1L-5 production (del Pozo et al, 2002).
165 Using a blocking antibody to galectin-3, a role for this S-type lectin in the transport of glycosylated human eotaxin was ruled out as the flux of glycosylated human eotaxin across the guinea pig mesentery was not inhibited. However, a potential explanation for the inability of the antibody to reduce the transport of glycosylated human eotaxin was that the experiment was performed using guinea pig tissue and the antibody was raised against human galectin-3. Despite a high percentage of conservation of galectins between species (Rabinovich et a/, 1999) there is no experimental evidence available to determine whether this anti-human antibody binds to guinea pig galectin-3. All galectin sub types bind 13-galactoside sugars particularly N-acetyllactoasmine (Gal(31_4G1cNAc). There is however, an order of affinity of the galectins for the saccharides they bind. Galectin-1 binds shorter ligands such as disaccharides, whereas galectin-3 has an extended binding site that can accommodate longer oligiosaccharides such as GalNAcoci_3[Fucal_3]Galf31.4G1cNAc and thus has a higher affinity for longer saccharides (Henrick et al, 1998). This suggests that galectin-3 would have a low affinity for the disaccharide Ga1131 _3Ga1NAc present on the human eotaxin and would be unlikely to be involved in the transport of glycosylated eotaxin across the guinea pig mesentery. Further work to determine if the galectins play any role in the transport of glycosylated human eotaxin across the guinea pig mesentery is required.
The results presented in this chapter have identified that the saccharide moiety present on St-9 insect cell/baculovirus expressed glycosylated human eotaxin as Ga1131..
3GalNAc. This disaccharide was shown to inhibit the flux of glycosylated human eotaxin across the guinea pig mesentery.
166 Chapter 6
Studies on the mechanism of transport of glycosylated eotaxin and other chemokines across the guinea pig mesentery
167 6 Studies on the mechanism of transport of glycosylated eotaxin across the guinea pig mesentery
6.1 The effect of cooling the guinea pig mesentery to 4°C on the flux of glycosylated human eotaxin across the guinea pig mesentery
The tissue was equilibrated in ice-cold buffer for 10 minutes prior to performing the microBoyden chamber flux assay on ice (see methods 2.2.3.4). As can be seen in Figure 6.1, the concentration of glycosylated eotaxin detected in the upper wells at 1 hr was significantly reduced when the assay was carried out at 4°C. When the experiment was performed at 4°C approximately 80pM eotaxin was measured in the upper wells compared to approximately 350pM when the experiment was carried out at 37°C The concentration of non-glycosylated human eotaxin remained below the limit of detection for the ELISA in all cases.
6.2 The effect of the metabolic inhibitors, transcytosis inhibitors and a blocking antibody to CCR3 on the flux of human eotaxin across the
guinea pig mesentery
6.2.1 Effect of the metabolic inhibitor 2-deoxyglucose (2-DOG)
To determine if the flux of glycosylated human eotaxin across the mesentery was an active process, the effect of the metabolic inhibitor, 2-DOG, (30mM) was studied at 37°C. These experiments were carried out using a microBoyden chamber where the chemokines were added to the lower well and the concentration in the upper well was measured after 1 hour by ELISA. The HBSS buffer in both the upper and lower wells contained 2-DOG. The concentration of glycosylated human eotaxin in the upper well significantly decreased from 210pM to 50pM (an inhibition of 75%) in the presence of 2-DOG. Again, the concentration of non-glycosylated eotaxin in the upper wells remained below the accurate detection level of the ELISA and this was not affected by the addition of 2-DOG (Figure 6.2).
168 6.2.2 Effect of the metabolic inhibitor Sodium Cyanide (NaCN)
The addition of the metabolic poison NaCN (100mM) to the flux assay using the guinea pig mesentery significantly reduced the concentration of glycosylated human eotaxin detected in the upper wells from 236pM to 75pM after 1 hr, an inhibition of 68%. The concentration of non-glycosylated eotaxin in the upper wells remained below the detection limit of the ELISA and was not affected by NaCN (Figure 6.3).
6.2.3 Effect of the transcytosis inhibitor monensin
Figure 6.4a shows that the concentration of glycosylated human eotaxin detected in the upper chamber was significantly reduced in the presence of 10 and 30µM monensin a Na+/K4- ion pump inhibitor. The non-glycosylated human eotaxin remained below the accurate level of detection of the assay in the presence or absence of monensin (Figure 6.4b).
6.2.4 Effect of the transcytosis inhibitor filipin
The cholesterol binding agent, filipin, at 5 and lOpM significantly inhibited the flux of glycosylated human eotaxin across the guinea pig mesentery. The concentration detected in the upper chamber significantly decreased by 66% in the presence of lOpM of filipin (Figure 6.5).
6.2.5 Effect of the blocking antibody to the eotaxin receptor CCR3
To determine whether guinea pig CCR3, the eotaxin receptor, was involved in the transport of glycosylated human eotaxin across the guinea pig mesentery, a monoclonal anti guinea pig CCR3 antibody, 2A8, was used. This antibody had previously been shown to inhibit guinea pig eosinophil chemotaxis at a concentration of 10µg/m1
(Sabroe et al, 1998). 2A8 (10µ.g/ml) was unable to block the transport of glycosylated human eotaxin across the guinea pig mesentery (Figure 6.6). In the same experiment both filipin (10pM) and monensin (10µM) significantly inhibited the transport of
glycosylated human eotaxin.
169 6.3 The importance of the mesothelial cell barrier
6.3.1 Physical disruption to the mesothelial cell layer
To investigate the importance of the integrity of the mesothelial cell barrier, the guinea pig mesentery was rubbed gently with a moist cotton wool bud on both sides of the tissue to disrupt the cell monolayer. Using confocal scanning microscopy, the mesothelial cell layer was shown to be intact before undergoing disruption, with a continuous mesothelial cell layer (Figure 6.7a). Following gentle rubbing with a moistened cotton bud, the mesothelial cell layer can clearly be seen to be disrupted with large gaps in the mesothelial cell layer seen (Figure 6.7b). In the microBoyden chamber mesentery flux assay, the concentration of glycosylated human eotaxin detected in the upper chamber significantly increased from 350pM to 800pM when the mesothelial cell layer was disrupted (Figure 6.8). In addition, the concentration of non-glycosylated eotaxin detected in the upper chamber was significantly increased from below detectable levels to 750pM.
In other experiments, the tissue was exposed to ice cold water (osmotic shock), which resulted in the intact mesothelial cell layer becoming substantially damaged (Fig 6.12a). The concentration of glycosylated human eotaxin detected in the upper chamber increased from 250pM to 1600pM following treatment of the tissue with ice cold water (Figure 6.9). Similarly the concentration of non-glycosylated eotaxin had increased from below detectable levels to 1500pM.
6.3.2 Localisation of glycosylated eotaxin within the mesentery tissue using
confocal microscopy
Scanning confocal microscopy was used to visualise the location of glycosylated human eotaxin within the mesenteric tissue. The tissue was incubated with human eotaxin (30nM), followed by a mouse monoclonal anti-human eotaxin antibody (2G6) and subsequently by a goat anti-mouse IgG F'ab2 fragment labelled with a (green) fluorescent marker Alexafluor 488 (See methods 2.2.11). Propidium iodide and TRITC- labelled phalloidin were used to visualise cell nuclei and f-actin, respectively, (both of which appear as red staining). It can be seen in Figure 6.10 that the glycosylated human eotaxin (green fluorescent staining) was only visible in blood vessels located within the
170 guinea pig mesentery and not in the surrounding connective tissue. The non- glycosylated human eotaxin was visualised on the surface of the intact upper mesothelial cell layer (Figure 6.11a), but could not be detected in any mesenteric blood vessels within the guinea pig mesentery or in the surrounding connective tissue (Figure 6.11b). After exposing the mesentery to ice cold water, the mesothelial cell layer was clearly damaged (Figure 6.12a). The non-glycosylated eotaxin could now be clearly visualised within the mesenteric blood vessels (Figure 6.12b).
6.3.3 Scanning electronmicrographs of an intact guinea pig mesentery
Transmission electron microscopy of the guinea pig mesentery were taken at following exposure to glycosylated human eotaxin to visulaise the internalisation process. Small flask-shaped invaginations of the plasma memebrane can be observed (Figure 6.13 and 6.14). This work was carried out in collaboration with Dr Jill Moss, Dept of Histopathology, Imperial College, London.
6.4 Investigating the ability of other chemokines to move across the guinea
pig mesentery
6.4.1 Determining the molecular mass of the human eotaxin-2 forms
The molecular mass of both glycosylated eotaxin-2 produced in a Drosophilia expression system, and non-glycosylated eotaxin-2 produced in an E.coli expression system was determined by SDS-PAGE. As can be seen in Figure 6.15 the glycosylated eotaxin-2 had a higher molecular mass than both non-glycosylated eotaxin-2 and glycosylated human eotaxin. The molecular mass of glycosylated eotaxin-2 and non- glycosylated eotaxin-2 was determined to be approximately 12kDa and 8.5kDa respectively.
6.4.2 Identification of the carbohydrate moiety on human eotaxin-2
In order to identify the carbohydrate present on the human eotaxin-2 a glycan differentiation kit with various digoxygenin-labelled lectins enabling immunological detection of the bound lectins was used. As can be seen in Figure 6.16, DSA, which
171 recognises the terminal disaccharide Galf31 _4G1cNAc, clearly bound to glycosylated human eotaxin-2 (311g) and asialofetuin (l jig) when the proteins were immobilised on a nitrocellulose filter.
6.4.3 Comparison of the chemotactic activity of glycosylated and non- glycosylated human eotaxin-2
The ability of glycosylated and non-glycosylated human eotaxin-2 to induce chemotaxis of human eosinophils across a guinea pig mesentery or a polycarbonate filter was investigated. Neither the glycosylated nor the non-glycosylated human eotaxin-2 (10- 100nM) were able to induce chemotaxis of human eosinophils across the guinea pig mesentery (Figure 6.17a). However, both the glycosylated and non-glycosylated eotaxin-2 had equal chemotactic activity across the polycarbonate filter with a classical bell shaped dose response curve and peak chemotactic activity at 30nM (Figure 6.17b).
6.4.4 A comparison of the ability of glycosylated and non-glycosylated eotaxin-2 to be transported across the isolated guinea pig mesentery
Using the mesentery and polycarbonate filter flux assay the glycosylated and non- glycosylated human eotaxin-2 forms were compared. Figure 6.18 shows that both the glycosylated and non-glycosylated forms of human eotaxin-2 were detected in the upper wells. There was no significant difference in the detectable concentration between the glycosylated and non-glycosylated eotaxin-2.
6.4.5 The ability of glycosylated and non-glycosylated MCP-1 to flux across
the guinea pig mesentery
Glycosylated MCP-1 was produced in Trichoplusia.ni insect cells hosting a baculovirus expression system. The molecular mass of glycosylated MCP-1 was 14kDa compared to 8.5kDa for non-glycosylated MCP-1 as determined by SDS-PAGE. Both the glycosylated and non-glycosylated human MCP-1 could be detected in equal concentrations in the upper chamber after 60 minutes and the concentration of both forms of MCP-1 (Figure 6.19).
172 6.5 Summary of results
1. Metabolic blockade of the guinea pig mesentery with NaCN or 2-DOG inhibited the transport of glycosylated human eotaxin across the guinea pig mesentery.
2. Monensin and filipin inhibited the transport of glycosylated human eotaxin across the guinea pig mesentery.
3. Blockade of CCR3 did not inhibit the transport of glycosylated human eotaxin across the guinea pig mesentery.
4. Disruption of the continuous guinea pig mesothelial cell layer increased the flux of glycosylated eotaxin across the mesentery. In addition, non-glycosylated human eotaxin could also be detected in similar amounts following mesothelial disruption.
5. Neither glycosylated or non-glycosylated human eotaxin-2 induced eosinophil migration across an isolated guinea pig mesentery despite both forms being capable of transport across the guinea pig mesentery.
173 Control (37°C) 4°C
Figure 6.1 The effect of cooling the tissue to 4°C on the flux of human eotaxin across the guinea pig mesentery
The flux of glycosylated human eotaxin (N, 30nM) and non-glycosylated human eotaxin (❑, 30nM) across the isolated guinea pig mesentery was determined at 4°C at lhr. The control values were taken from a separate set of experiments performed at 37°C at lhr. Dotted line represents the lower limit of detection of the ELISA. Data shown as concentration of eotaxin detected in the upper chamber ± SEM of 4 separate experiments carried out in duplicate. "P<0.01 compared to glycosylated human eotaxin at 37°C.
174 across theguineapigmesentery (30mM) atlhr.Dottedlinerepresentsthelower limitofdetectiontheELISA.Data ( Figure 6.2Theeffectof2-deoxyglucose(2-DOG)onthefluxhumaneotaxin The fluxofglycosylatedhumaneotaxin(I,30nM) andnon-glycosylatedhumaneotaxin experiments carriedoutinduplicate.*P<0.05compared tocontol shown asconcentrationofeotaxindetectedinthe upperchamber±SEMof4separate ❑ , 30nM)acrosstheisolatedguineapigmesentery withthemetabolicblocker2-DOG Human eotaxin detected in upper chamber (pM) Control 175 2-DOG
**
-1-
Control NaCN
Figure 6.3 The effect of sodium cyanide (NaCN) on the flux of human eotaxin across the guinea pig mesentery
The flux of glycosylated human eotaxin (II, 30nM) and non-glycosylated human eotaxin 30nM) across the isolated guinea pig mesentery was determined in the presence of the metabolic blocker NaCN (100mM). Dotted line represents the lower limit of detection of the ELISA. Data shown as concentration of eotaxin detected in the upper chamber ± SEM of 5 separate experiments carried out in duplicate. **P<0.01 compared control
176 a)
— 350 - E a 300 - 42) 'cr) "C3 250 - c r 200 - 6. c CL 150 - IS cp. E z c 100 - 50 - 0 Control 10 30
b) o., M 350 - -..- 300 -o .0 E E 250 - x n) o200 - 0 ct c o_ 150 - ca ct. E 100 - x 50 0 Control 3 10 30 Monensin (pM)
Figure 6.4 The effect of monensin on the flux of human eotaxin across the guinea pig mesentery
The flux of a) glycosylated human eotaxin 30nM) and b) non-glycosylated human eotaxin (0, 30nM) across the isolated guinea pig mesentery was determined in the presence of monensin (3-30µM) at lhr. Dotted line represents the lower limit of detection of the ELISA. Data shown as mean concentration of eotaxin detected in the upper chamber SEM of 5 separate experiments carried out in duplicate. The dashed line represents the lower limit of detection of the ELISA. *P<0.05 and **P<0.01 compared to control.
177 500 TC CU 0. +0 _ a) 400 R• E E m • -c 300 10 a) Q. si 200
O .— >,0 100 or) $3 w -0 Control 3 5 10
Filipin (pM)
Figure 6.5 The effect of filipin on the flux of human eotaxin across guinea pig mesentery
The flux of glycosylated human eotaxin (II, 30nM) across the isolated guinea pig mesentery was determined in the presence of filipin (3-10µM). Dotted line represents the lower limit of detection of the ELISA. Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 5 separate experiments carried out in duplicate. *P<0.05 and **P<0.01 compared to control
178 ,400
s- 22 300 — a) E c 2 I„ "0 200 — S 0 a.) a c '11 0 100
C 0 Monensin Control 2A8 Filipin 10µg/m1 10µM 10µM
Figure 6.6 The effect of an anti-guinea pig CCR3 antibody on the flux of glycosylated human eotaxin across the isolated mesentery
The flux of glycosylated human eotaxin (II, 30nM) across the isolated guinea pig mesentery was determined in the presence of an anti-guinea pig CCR3 antibody, 2A8, (10m/m1), filipin (10µM) and monensin (101.1,M) at lhr. Dotted line represents the lower limit of detection. Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 5 separate experiments carried out in duplicate. *13<0.05 compared to control.
179 b)
Figure 6.7 Scanning confocal image of intact and damaged mesothelial cell layer on a guinea pig mesentery
A confocal image of guinea pig mesentery taken at a magnification power of 63X. The cell actin is bound by TRITC labelled phalloidin. Figure 6.8a illustrates an intact mesothelial cell layer. Figure 6.8b demonstrates damage to the mesothelial cell layer casued by subjecting the tissue to gentle rubbing with a moist cotton bud.
180 150
CL a) 0 -c) • E 100 T< 0 .c ••F, O L. 0 • 0- CUE 500
Control Disrupted
Figure 6.8 The effect of disrupting the guinea pig mesothelium on the flux of glycosylated and non-glycosylated human eotaxin across guinea pig mesentery
The flux of glycosylated human eotaxin (III, 30nM) and non-glycosylated human eotaxin (D, 30nM) across intact guinea pig mesentery and a mesentery disrupted by gently rubbing with a moist cotton wool bud at lhr. Dotted line represents the lower limit of detection of the ELISA. Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 3 separate experiments carried out in duplicate. *P<0.05 compared to control.
181
c 2000 -02a) a. 06. a) 0 E c 1000 O 0 O 0- c Q- E• °'
Control After osmotic shock
Figure 6.9 The flux of glycosylated and non-glycosylated human eotaxin across guinea pig mesentery after osmotic shock treatment
The flux of glycosylated human eotaxin (N, 30nM) and non-glycosylated human eotaxin (0, 30nM) across intact guinea pig mesentery and after exposing the mesentery to ice cold water for 30 seconds. Dotted line represents the lower limit of detection of the ELISA. Data shown as mean concentration of eotaxin detected in the upper chamber ± SEM of 3 separate experiments carried out in duplicate. **P<0.01 compared to control.
182 Figure 6.10 Confocal microscopy scanning image of a venule located within the guinea pig mesentery stained for glycosylated human eotaxin
A confocal image of a blood vessel located deep within the guinea pig mesentery following topical application of glycosylated human eotaxin. The red staining is phalloidin-TRITC which binds to f actin located within blood vessels. The green staining is produced by Alexafluor 488 conjugated to anti-eotaxin antibody. The yellow colour represent co-localisation.
183 Figure 6.11 Confocal microscopy images of the guinea pig mesentery and mesothelial venule
Figure 6.11a shows a confocal image of the guinea pig mesothelial cell surface after topical exposure to non-glycosylated human eotaxin. Figure 6.12b is a confocal image of a blood vessel within the guinea pig mesentery. The red staining is phalloidin-TRITC which binds to f actin located within blood vessels. The green staining is Alexafluor 488 conjugated to an anti-eotaxin antibody. The yellow colour represent co-localisation.
184 a)
b)
Figure 6.12 Confocal microscopy images of the guinea pig mesentery and mesothelial venule following osmotic shock
Figure 6.12a show a confocal image of the guinea pig mesothelial cell surface following osmotic shock treatment. Images taken deep within the mesenteric tissue shows localisation of non-glycosylated human eotaxin within mesenteric blood vessels (Figure 6.12b).The red staining is phalloidin-TRITC which binds to f actin located within blood vessels. The green staining is produced by Alexafluor 488 conjugated to an anti-eotaxin antibody.
185 Figure 6.13 A scanning electron micrograph of the guinea pig mesentery (x 5000)
A scanning electron micrograph of guinea pig mesentery where vesicle like structures are clearly visible (A) with an approximate diameter of 111.tm. These vesicles can be seen fusing with the apical mesothelial cell membrane (B). Scale bar = 50t.tm.
This work was carried out in collaboration with Dr Jill Moss, Dept of Histopathology, Imperial College, London.
186 Mesothelial Vesicles
Figure 6.14 Transmission electron micrograph illustrating the mesothelial vesicles This work was carried out in collaboration with Dr Jill Moss, Dept of Histopathology, Imperial College, London.
1R7
t I I Molecular Human Human Human Weight Eotaxin2 Eotaxin Eotaxin2 Markers (NG) (G) (G) (kDa)
Figure 6.15 SDS-PAGE of the human eotaxin-2 variants
The glycosylated eotaxin-2 (Drosophila expressed) and non-glycosylated eotaxin-2 (E.coli expressed) have different molecular weights as evidenced by their migration in SDS gels. The molecular mass of glycosylated human eotaxin-2 and non-glycosylated human eotaxin-2 were approximately 121(Da and 8.5kDa respectively.
188 Asialofeutin 1 pg
Human eotaxin[NG] 3pg
Transferrin 1µg
Human eotaxin[G] 3pg - Fournier
Feutin 1µg
Human eotaxin[G] 3pg - Leukosite
Human eotaxin2[G] (Ck[36) 3pg
Human eotaxin2[NG] 3pg
Asialofeutin 1 pg
Figure 6.16 DSA lectin binding to human glycosylated eotaxin-2
Slot blot showing a positive reaction of glycosylated human eotaxin-2 (3pg) and asialofetuin (1µg) and a negative reaction with human non-glycosylated eotaxin-2 (31t0, glycosylated and non-glycosylated human eotaxin (31.1g) with digoxygenin labelled DSA that recognises Gal[31 _4G1cNAc.
189
a) Mesentery
0 a 6 0 4 - 6 E
(.) 2 -
0 3 100
Filter
8
x
de 6 In ic t 4 tac
mo 2 - Che 0 I 3 10 30 100
Eotaxin-2 (nM)
Figure 6.17 Chemotactic response of human eosinophils to human eotaxin 2 across a guinea pig mesentery and polycarbonate filter
Migration of human eosinophils across a) guinea pig mesentery and b) 51,1m polycarbonate filter in a microBoyden chamber induced by glycosylated human eotaxin- 2 (A, 10-100nM) and non-glycosylated human eotaxin-2 10-100nM). The dashed line represents the basal migration to buffer (chemotactic index of 1). Data shown as mean chemotactic index ± SEM of 4 separate experiments carried out in duplicate.
190 Figure 6.18Thefluxofglycosylatedandnon-glycosylated humaneotaxin-2across the guineapigmesentery The fluxofglycosylatedhumaneotaxin-2(R SKB)andnon-glycosylatedhuman eotaxin-2 (DR&D)and(Peprotech)(30nM)across theisolatedguineapigmesentery in theupperchamber±SEM of3separateexperimentscarriedoutinduplicate. limit ofdetectionthe EL1SA. Datashownasmeanconcentrationofeotaxin detected was determinedusingthemicroBoydenchamber assay.Dottedlinerepresentsthelower
Human eotaxin-2 detected I a_ 0. co • @ a. L. c 300
500 400 200 100 SKB Human eotaxin-2(nM)
R&D 191
Peprotech Mesentery
4000 c fa- 7:12 3000 An E -c 2000 -o L. o. 0 a_ 1000 E
20 40 60 80 100 120 140
Filter
4000
in Ea 3000 d te sa tec E 2000
1 de 0
CP- st; 1000
M a
20 40 60 80 100 120 140 Time (mins)
Figure 6.19 The flux of glycosylated and non-glycosylated human MCP-1 across the guinea pig mesentery and polycarbonate filter with time
The flux of glycosylated human MCP-1 (A, 30nM) and non-glycosylated human MCP-1 30nM) across a) the isolated guinea pig mesentery and b) a polycarbonate 51.tm filter was determined using the microBoyden chamber assay. Data shown as mean concentration of MCP-1 detected in the upper chamber ± SEM of 3 separate experiments carried out in duplicate.
192 6.6 Discussion
To determine whether an active or passive transport system was involved in the flux of glycosylated human eotaxin across the guinea pig mesentery, the effect of cooling was studied (Fig 6.1). Mammals require a constant temperature of 37°C for energy production to occur most efficiently and this energy would be required by an active system. The inhibition of the flux of glycosylated eotaxin across the guinea pig mesentery following tissue cooling indicated that the transport of glycosylated eotaxin was indeed an active process. It has been demonstrated that the transport of BSA which occurs in the apical to basolateral direction across an epithelial cell layer was temperature sensitive with the cessation of transport occurring at temperatures lower than 20°C (Daffenbach et al, 1996). However, passive diffusion is also temperature sensitive, as defined by Brownian motion where the random movement of particles or molecules is dependent on temperature.
To eliminate the possibility that passive diffusion was involved, experiments were carried out 37°C and energy production was blocked by the metabolic inhibitors 2-DOG and NaCN (Figs 6.2 and 6.3). 2-DOG acts by competing with glucose in the glycolysis pathway and thus preventing ATP production. NaCN binds and uncouples the electron transport chain and blocks electron flow. Shasby and Shasby, 1985 demonstrated that NaCN (1mM) was sufficient to abolish the active cellular transport of albumin across cultured porcine pulmonary artery endothelial cells. After treatment of the mesentery with NaCN and 2- DOG, the flux of glycosylated human eotaxin decreased by approximately 75% showing that the metabolic inhibitors successfully penetrated the tissue and terminated ATP production. Cascarno et al, (1964) have demonstrated that uptake of proteins by both the endothelium and mesothelium is controlled by oxidative metabolism and ATP formation is an important factor, which is in agreement with the data using metabolic inhibitors presented in this chapter. Thus, the flux of glycosylated human eotaxin across the guinea pig mesentery was not due to diffusion through the gaps in tight junctions on the mesothelial cell surface.
193 Disruption of the mesothelial cell barrier (Fig 6.7) of the guinea pig mesentery resulted in a higher concentration of glycosylated human eotaxin being detected in the upper wells in a mesentery flux assay (Figs 6.8 and 6.9). In addition, a similarly high amount of non-glycosylated human eotaxin was detected in the upper wells. These results suggest that an intact, continuous mesothelial cell layer was able to selectively allow the glycosylated human eotaxin to be transported across the guinea pig mesentery. This was visualised as glycosylated human eotaxin could be detected within a mesenteric venule of an intact guinea pig mesentery (Fig 6.10). These results were visulised via confocal microscopy whereby non-glycosylated eotaxin could only be detected on the mesothelial surface and not with the intact mesentery tissue (Fig 6.11). Following, disruption of the mesothelial cell layer non-glycosylated eotaxin could be detected within the tissue (Fig 6.12). Interestingly, once inside the guinea pig mesentery, eotaxin could only be observed within the guinea pig venules, presumably due to accumulation of eotaxin into these vessels and the sensitivity of the confocal microscopy assay.
The CXC chemokine IL-8 (which does not possess any potential glycosylation sites) has been shown to be transported across endothelial cells via a transcytotic route (Middleton et al, 1997). These authors demonstrated by immunoelectron microscopy that IL-8, following intradermal injection into rabbits bound to the abluminal endothelial cell surface, was internalised and incorporated into smooth membrane bound plasmalemmal vesicles containing the 21-24kDa protein caveolin (hence these vesicles were tenned caveolae) and the lipid cholesterol. Caveolae are plasmalemmal vesicles (60-80nm in diameter), formed from plasma membranes, which shuttle between cell luminal and basal surfaces discharging their contents to the plasma or interstitial fluid compartments (Anderson, 1998). Transmission electron microscopy of the guinea pig mesentery showed a layer of collagen bundles containing blood microvessel sandwiched between monolayers of mesothelial cells, interfaced by a basement membrane (Fig 6.13). Higher power views of the mesothelial cells showed small flask-shaped invaginations of the plasma membrane and numerous intracellular vesicles (Fig 6.14). Caveolae vesicles can shuttle molecules across cells without fusing with an intermediate compartment (Simionescu et al, 1973; Simionescu et al, 1975; Ghitescu and Bendayan, 1992). Transcytosis is defined as 'the passage of materials or solutes across a cell by endocytosis at one face, transport in vesicles, and release at the other face'. The shuttle theory of transcytosis is simply that vesicles fuse at one end of
194 the cell, take up the protein to be transported and shuttle it across to the other side. The protein is then released and the process can start again (Palade et al, 1979; Ghitescu et al, 1986; Parton et al, 1994; Schnitzer et al, 1996). The active transcytosis process is inhibited by N-ethylmaleimide (NEM) and filpin (Schitzer et al, 1994; Predescu et al, 1994; Schnitzer et al, 1995). Caveolae have also been shown to be involved in the entry of viral pathogens into the endoplasmic reticulum of cells (Kartenbeck et al, 1989; Anderson et al, 1996; Stang et al, 1997; Meuller et al, 2002). Caveolae are implicated in specific transport of a molecule initialised by the activation of a receptor. Receptors for endothelin (Chun et al, 1994), acetylcholine (Feron et al, 1997) and bradykinin (de Weerd & Leeb-Lundberg 1997) have all been identified within caveolae membranes. The internalisation of the chemokine receptor CCR5 in CHO cells was affected by the introduction of the sterol binding agents filipin and nystatin. These drugs are inhibitors of cholesterol metabolism and are essential for maintaining the shape of caveolae and their ability to form intracellular vesicle (Rothberg et al, 1992; Smart et al, 1994). This suggests that caveolae are involved in this internalisation process of this receptor (Meuller et al, 2002). The chemokine receptor CXCR1 has been shown to co-localise with caveolin-1 suggesting that the CXCR1 was expressed within caveolae on the surface of intestinal epithelial cells (Meuhlhoefer et al, 2000).
Another mechanism of vesicular transcytosis involves a clathrin-dependent process. Following receptor activation, arrestin-2 binds the phosphorylated receptor and initiates the internalisation process by binding to clathrin. The receptor-arrestin-2 complex is sequestered by clathrin coated pits which are then pinched off and become clathrin coated veesicles (Mosuavi et al, 2004). This clathrin dependent pathway can be prevented by the use of sucrose (Heuser & Anderson, 1989). The internalisation of the chemokine receptor CCR5 was affected by sucrose, which suggests a role of a clathrin- dependent pathway (Meuller et al, 2002). Zimmermann and Rothenberg (2003) have demonstrated that pre-treatment of peripheral blood-derived human eosinophils with sucrose can inhibit actin polymerisation of CCR3 suggesting that receptor internalisation occurs via the clathrin-dependent pit pathway.
It was postulated that the eotaxin receptor, CCR3, could be present either within caveolae or clathrin-coated pits on the guinea pig mesothelial cell surface. Thus, CCR3 was considered to be a likely candidate for the transport of glycosylated eotaxin across
195 the mesentery. It has been demonstrated that human eotaxin can bind to guinea pig CCR3 although it was unable to signal, so internalisation within the mesothelial cell could occur (Sabroe et al, 1999). The transport of glycosylated human eotaxin across the guinea pig tissue was unaffected by a blocking antibody to the guinea pig CCR3 receptor (Fig 6.6), suggesting that guinea pig CCR3 does not play a role in the transport of glycosylated human eotaxin.
There are currently two theories regarding the internalisation and transport of proteins by caveolae. Firstly, there is vesicular transcellular transport, where the vesicle containing caveolae are free to move through the cell. This process is inhibited by the cholesterol binding agent, filipin, (Schnitzer et al, 1994), monensin, an ionophore that interferes with Na+/H+ ion exchange, (Mollenhauer et al, 1990) and nocodazole, an antimitotic agent that disrupts microtubules by binding to P-tubulin (Luduena & Roach 1991; Vasquez et al, 1997). Nocodazole has also been shown to inhibit transcytosis and uptake of BSA in epithelial cells (Hunziker et al, 1990; Hastings et al, 1994). There is evidence that movement of the vesicle requires the hydrolysis of guanosine triphosphate and is therefore energy dependent (Schnitzer et al, 1996). The second mechanism suggests that the formation of a tube-like structure occurs by the fusion of numerous vesicles. This tube structure can span the length of the cell and connect luminal with abluminal surfaces. The transport molecule would then passively diffuse through the tube and across the cell (Dvorak et al, 1996; Feng et al, 1996). Endothelial cell caveolae have been demonstrated to fuse to form transcellular channels that allow the passage of small molecules across the cell (Simionescu et al, 1975). Electron micrographs of the guinea pig mesentery (Fig 6.13 and Fig 6.14) show numerous intracellular vesicular structures resembling caveolae suggesting that these vesicles might be involved in the transport of glycosylated human eotaxin across the mesentery. However, it was not possible to conclusively determine if these vesicles are involved, and whether the transport mechanisms (vesicle shuttle or tubular formation) was important for the transport of glycosylated human eotaxin.
Eotaxin-2 is another potent eosinophil chemotattractant, which also activates eosinophils via CCR3 (Forssman et al, 1997). Eotaxin-2 does not contain an 0-linked glycosylation site (Table 6.1; Gupta et al, 1999), but it has a potential N-glycosylation site at amino acid position 89 (White et al, 1997). Glycosylated human eotaxin-2 was
196 shown to have a higher molecular weight (approximately 12kDa) than non-glycosylated human eotaxin-2 (approximately 8.5kDa) as determined by SDS-PAGE (Fig 6.15) .The terminal sugar moiety present on glycosylated eotaxin-2 was identified as Ga1131..
4G1cNAc using the lectin, DSA, in the DIG glycan identification assay (Fig 6.16). The disaccharide Galf31.4G1cNAc has been shown in Chapter 5 to inhibit the transport of glycosylated human eotaxin across guinea pig mesentery. Thus, chemokines containing Ga1131 _4G1cNAc could possibly utilise the same transport mechanism as glycosylated human eotaxin.
Neither the N-linked glycosylated nor the non-glycosylated form of human eotaxin-2 were able to induce eosinophil migration across an isolated guinea pig mesentery (Fig 6.17a), whilst both forms demonstrated eosinophil chemoattractant activity when using the polycarbonate filter (Fig 6.17b). However, both glycosylated and non-glycosylated forms of eotaxin-2 were detected in the mesentery flux assay. A possible explanation for the lack of chemotactic activity across the mesentery could be that eotaxin-2 was susceptible to protease attack which removed its biological activity but not its immunoreactivity. The protease dipeptidyl-peptidase was found to be present within the guinea pig mesentery; however, eotaxin-2 is not a substrate for this protease as it does not contain the amino acid sequence Xaa-Pro or Xaa-Ala (White et al, 1997) at the N- or C-terminus which is known to be important for dipeptidyl-peptidase activity (Vanhoof et al, 1995). Experiments in this thesis have demonstrated the existence of serine proteases within the guinea pig mesentery (Fig 4.13). The functional domain of eotaxin-2 involved with interaction and binding with CCR3 is located towards the N- terminus of the peptide (Mayer and Stone, 2000). The N-linked glycosylation of eotaxin-2 occurs on asparagine at position 89 which is towards the C-terminus of the peptide. It could be postulated that the glycosylation present on the eotaxin-2 does not confer protection against the mesothelial serine proteases. It is possible that the peptide fragments of the degraded eotaxin-2 contain the intact epitopes required for recognition by the antibodies used within the ELISA.
Human MCP-1 contains two potential glycosylation sites, an N-linked consensus sequence at the amino acid at position 14 and an 0-linked glycosylation site at amino acid 73 (Beall et al, 1998; and as determined by 0-glycbase, Patel et al, 1999). Native MCP-1 produced from a human tumour cell line was 0-glycosylated with Galf31..
197 3GalNAc as determined using the lectin PNA, however, modification by N- glycosylation was not detected (Jiang et al, 1991). Recombinant MCP-1 produced in Sf- 21 and Sf-9 cells has been modified by the addition of the 0-linked glycan Gal[3]..
3GalNAc (Ueda et al, 1994; Ishii et al, 1995). In the mesentery flux assay non- glycosylated MCP-1 was detected in similar concentrations to glycosylated MCP-1 (Fig 6.19) suggesting use of the same mode of transportation. Interestingly, the amounts of glycosylated and non-glycosylated MCP-1 transversing the mesentery were much greater than the concentrations of glycosylated eotaxin. This would suggest that glycosylated and non-glycosylated MCP-1 have either a greater affinity for the same transport mechanism as utilised by glycosylated human eotaxin or that the MCP's are using a different mechanism. Damage to the mesothelium during the experiment could be excluded as non-glycosylated eotaxin did not flux across the mesentery in the same experiments.
Eotaxin-2 and MCP-1, which contain potential N-glycosylation sites, appear to be transported across the guinea pig mesentery in higher amounts than glycosylated eotaxin which does not have an N-glycosylation site. This could suggest that N- glycosylated chemokines and their non-glycosylated counterparts may utilise different transport mechanisms to those of 0-glycosylated chemokines. One such mechanism could be the recently described D6 decoy receptor, which has a high structural similarity with chemokine receptors and has been shown to bind only the inflammatory CC chemokines (Bonini et al, 1997; Nibbs et al, 1997; Bonecchi et al, 2004). D6 does not couple to the G-protein linked pathway associated with CC chemokine receptors (Nibbs et al, 1997). Recently, it has been reported that the chemokines that bind D6 are internalised and degraded by lysomes (Fra et al, 2003). An alternative destination for the chemokine is a possibility whereby it has been shown that the endocytosis of D6- chemokine complex is via a clathrin coated pit pathway and that D6 is then predominantly localised in recycling endosomes (Weber et al, 2004) It was also shown that the lysosome degradation can be prevented by altering the pH of the vesicle (Fra et al, 2003). D6 has not been detected on peripheral blood cells where it could potentially regulate chemokine activity, but instead has been detected on LECs lining a subset of lymphatic channels (Nibbs et al, 2001). Both the glycosylated and non-glycosylated eotaxin-2 and MCP-1 could be ligands for D6, degraded during transcytosis whilst the breakdown products still contain the epitope responsible for antibody binding which
198 would explain why immunoreactive MCP-1 and eotaxin-2 could be detected by ELISA. However this does not explain why eotaxin-2 failed to induce eosinophil chemotaxis across the mesentery. The endocytotoic D6 pathway shares similar features with the mechanism described within this thesis responsible for the flux of eotaxin across the mesentery.
The results presented in this chapter suggest that the mesothelial cells covering the guinea pig mesentery actively transport glycosylated human eotaxin by a transcytotic mechanism. Electron microscopy has produced images of vesicles within the guinea pig mesothelial cell and vesicular transport disruptors, when applied to the mesentery tissue, block glycosylated eotaxin transport. It has been established that a continuous intact mesothelial cell layer is essential in order for the tissue to differentiate between glycosylated and non-glycosylated human eotaxin.
199 0-linked glycosylation
Human Eotaxin
GPASVPTTCCFNLANRKI PLQRLE SYRRITSGKC PQKAVIFKTKLAKDICADPKKKWVQ DSMKYLDQKSPTPKP
0-Glycosylation site
N- and 0- linked glycosylation
Human MCP-1 N-Glycosylation site
Q PDAINAPVTCCYNFTNRK I SVQRLASYRR I TS SKC PKEAVI FKT IVAKE I CADPKQKW VQDSMDHLDKQTQTPKT
0-Glycosylation site
N-linked glycosylation
Eotaxin-2
SVVI PS PCCMFFVSKRI PENRVVSYQLS SRSTCLKGGVIFTTKKGQQFCGDPKQEWVQR YMKNLDAKQKKAS PRARAVAVKGPVQRYPGNQTTC
N-Glycosylation site
No potential glycosylation sites
Human IL-8
SAKELRCQC IKTYSKPFHPKFIKELRVIESGPHCANTE I IVKLSDGRELCLDPKENWVQ RVVEKFLKRAENS
Table 6.1 Amino Acid sequence of chemokines for possible glycosylation sites as determined by 0-glycbase
200 Chapter 7
General Discussion and Conclusions
201 7 General Discussion, Future Work and Conclusions
7.1 General Discussion and Future Work
Animal cells characteristically glycosylate many secreted proteins and this modification can have a significant impact on their properties in terms of biological activity (Berg et al, 1993; Ishii et al, 1995; Noso et al, 1998; Beall et al, 1998; Ruggiero et al, 2003;) protein folding (Helenins, 1995) and clearance rate in vivo (Flesher et al, 1995; Marinaro et al, 2000b).
The work presented in this thesis shows that glycosylation can also be important in facilitating the movement of a protein through a cellular monolayer that creates a barrier in vivo. Natural eotaxin has been shown to be secreted by cells in a glycosylated form or forms. Eotaxin with an 0-linked sugar was shown to cross the mesothelium via an active transport process, whereas non-glycosylated eotaxin was unable to cross the barrier. When added topically to guinea pig mesentery glycosylated, but not non- glycosylated, eotaxin could induce eosinophil recruitment from venules. In contrast, intradermal injection of the two forms produced identical effects on eosinophil accumulation. This would suggest that movement of the chemokine through the microvascular endothelium employs a different mechanism from that involved in movement through the mesothelium.
Other laboratories have explored the process of movement of a chemokine through the endothelium by studying the flux of IL-8, a chemokine that is non-glycosylated in its natural state (Middleton et al 1997). Similarly, the uptake of certain chemokines by lymphatic endothelial cells involving binding to the D6 receptor has been demonstrated (Webster et al 2004) where glycosylation does not appear to have a role.
The mechanisms that distinguish glycosylated from non-glycosylated eotaxin by mesothelial cells have not been determined in detail. One possibility relates to the studies of Ruggiero et al, (2003) who showed that glycosylation of MCP-1 increased its biological stability. However, when the two forms of guinea pig eotaxin were incubated with mesothelial cells and then tested for biological activity, there was no detectable difference. Therefore, the difference is likely to be related to differential binding of the
202 chemokine to the surface of the mesothelial cell, associated with a transport mechanism and all the results with inhibitors presented in this thesis support this hypothesis. It is suggested that glycosylated eotaxin binds to a receptor on the surface of the mesothelial cell that recognised the sugar, i.e. a receptor that is a lectin. From the results shown here, this process does not distinguish between human and guinea pig eotaxin. This is unlike responses of eosinophils where guinea pig, but not human, eotaxin stimulates response by acting via the CCR3 receptor. Indeed, a monoclonal antibody blocking guinea pig CCR3 was shown to be unable to block the transport process in mesothelial cells, although this antibody is able to block stimulation of eosinophils by eotaxin. Work is underway in an attempt to identify the unknown receptor and establish the signalling process involved.
7.2 Future Work
Attempts to identify a putitive receptor responsible for the transport of glycosylated eotaxin are currently underway in the Leukocyte Biology Section.at Imperial College. Preliminary data show that glycosylated eotaxin appears to be associated with an 150kDa protein present on the mesothelial cell surface. Whether this is the 'receptor' that binds the Ga1131 _3GalNAc present on the glycosylated human eotaxin remains to be determined. Studies of eotaxin transport across the mesentery of other species has not been examined in this thesis. Non-glycosylated human eotaxin has been shown to induce eosinophil recruitment in rat skin following intradermal injection (Sanz et al, 1998). In addition, it induced a rapid firm adhesion and extravasation of eosinophils from the rat mesenteric vasculature (Nagai et al, 1999) which is in contrast to the results presented in this thesis using the guinea pig mesentery. The reason for this could be due to the anatomical differences between the guinea pig and rat mesentery. The rat mesentery has been shown to contain naturally occurring holes that can permit the passage of physiological India ink (Easty and Easty, 1974). Thus, rat mesentery, in contrast to the guinea pig mesentery will therefore allow the passive diffusion of molecules and will not present itself as a physiological tight barrier.
Further work employing confluent layers of guinea pig mesothelial cells could be used to examine further the mechanisms of transport and the receptor involved. The in vitro system employed during this study is limited in that you are restricted to detecting
203 eotaxin movement across the entire tissue which emcompasses two mesothelial layers and a connective tissue section. Single cell, confluent layer of guinea pig mesothelial cells and could determine specific factors of the transport mechanism e.g. whether the uptake of eotaxin was polarised. Mesothelial cells have been shown to secrete IL-8 in a polarised fashion (from basal to apical) which would promote the transmesothelial migration of neutrophils into the pleural cavity (Nasreen et al, 2001).
Future work could utilise a single cell, confluent layer of guinea pig mesothelial cells and could determine specific factors of the transport mechanism e.g. whether the uptake of eotaxin was polarised. Mesothelial cells have been shown to secrete IL-8 in a polarised fashion (from basal to apical) which would promote the transmesothelial migration of neutrophils into the pleural cavity (Nasreen et al, 2001).
204 7.3 Conclusions
These results show that an active transport system is employed by mesothelial cells to enable eotaxin to cross mesothelial barriers and reach its target on the eosinophil eotaxin (CCR3) receptor. This process is critically dependent on eotaxin glycosylation and suggests that the initial stage is Galf31_3GalNAc binding to a lectin on the mesothelial cell surface, triggering calcium-independent, caveolae-mediated transcytosis. In the context of host defence, this mechanism would facilitate the accumulation of eosinophils, effector cells that target parasitic worms, around such parasites located in cavities lined with mesothelial cells. As illustrated in Figure 7.1 appropriately stimulated peritoneal macrophages could, for example, secrete eotaxin and recruit eosinophils, not only from the microvasculature, but also potentially from the resident eosinophil population in the gut wall (Gleich and Adolphson 1986; Mishra et al, 1999). The chemokine transport system is only necessary because of the diffusion barrier presented by the mesothelium, which raises broader questions. The mesothelium lines the peritoneal cavity and covers all organs contained within it. This effectively segregates the intestinal microcirculation, which drains via the portal vein to the liver, from the microcirculation of the other abdominal organs, which drain directly into the vena cava. Thus, the mesothelial barrier may ensure that certain macromolecules from the gut lumen (including bacterial proteins that may cross a compromised epithelial lining) are channelled to the liver for processing, rather than entering the general circulation.
The mechanism of glycosylation dependent transcytosis described in this thesis may apply to other cellular barriers to enable eotaxin to pass from its site of generation to its site of action. The glycosylation of many different cell-cell signalling molecules suggests the mechanism utilised by eotaxin may represent the first example of a more general phenomenon that assists an extracellular signalling molecule to reach its target.
205 Proposed mechanism of glycosylated eotaxin transcytosis through guinea pig mesothelial cell
Macrophage -
Peritoneal Cavity 1< Eotaxin Vesicle 4.z __,LectiniReceptor
Eotaxin: de-glycosylated within.the vesicle
Mesenteric venule
Eosinophil Chapter 8
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207 8 Bibliography
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