The expression of potential molecular candidates for chloride ion channels in primary human granulocytes and granulocytic cell lines By Kirsty-Anne Kirk, RN (Dip) HE, BA (Hons), MSc, PGCME, FHEA A thesis submitted in complete fulfilment of the requirements of the University of East Anglia for the degree of Doctor of Philosophy Norwich Medical School Faculty of Medicine and Health Sciences University of East Anglia Submitted July 2014 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. DECLARATION I declare that the work submitted in this thesis was undertaken and completed by myself unless otherwise acknowledged, and has not been accepted in any previous application for a degree with any other institution. Kirsty Kirk July 2014 1 ABSTRACT INTRODUCTION AND HYPOTHESIS: The project aims to identify potential candidates for chloride (Cl-) ion channels in granulocytes, and granulocytic cell lines. It is hypothesised that Cl- ion channels, in particular hBest1, are implicated in the role of granulocytes in response to inflammation. METHODOLOGY: Two main methodologies were used; laboratory techniques and systematic review. Laboratory techniques included RT-PCR, flow cytometry and western blot analysis to characterise the expression of hANO1, hBest1 and hCLCA1 as potential chloride ion channels in granulocytes and granulocytic cell lines. Systematic review was performed to identify whether chloride ion channels are up-regulated in COPD and asthma. RESULTS: RT-PCR demonstrated hCLCA1 expression in granulocytes and eosinophils but not HL60. hBest1 and hBest3 was expressed in all 3 cell types. In granulocytes, flow cytometry demonstrated greater hCLCA1 protein expression intracellularly, compared to hBest1 protein, and greater hBest1 plasma membrane expression compared to hCLCA1 (P<0.05). There was a negative correlation between hBest1, and hCLCA1 but also a weak negative correlation between hBest1 and hANO1 (P<0.05). Granulocytes stimulated with IL-13 over 24 hours, had a greater protein expression both intracellularly and at the plasma membrane. There was increased migration of HL60s when transfected with hBest1, in response to fMLP (P<0.05). Systematic review did not support the project due to limitations. CONCLUSIONS: There is a complex relationship between hBest1, hCLCA1 and hANO1 which may contribute to the function of granulocytes. HBest1 protein expression peaked 24 hours after continuous stimulation with IL-13. This correlates with peak symptom expression in diseases such as COPD and asthma. It is suggested that hBest1 has a role in migration and activation of granulocytes, through regulation of cell shape and volume. It is concluded that hBest1 is a novel therapeutic target in the control of symptoms in chronic inflammatory lung diseases. 2 CONTENTS DECLARATION 1 ABSTRACT 2 CONTENTS 3 LIST OF FIGURES 10 LIST OF TABLES 15 ABBREVIATIONS 18 ACKNOWLEDGEMENTS 21 CHAPTER 1: INTRODUCTION 22 1.1 Inflammatory airway diseases and the role of 23 potential chloride ion channel candidates in granulocyte function within inflammatory airways 1.1.1 The role of granulocytes in inflammatory 32 airway diseases 1.1.1.1 Neutrophils 32 1.1.1.2 Eosinophils 36 1.1.1.3 Basophils 47 1.1.2 Chloride ion channels 54 1.1.3 Potential molecular candidates for chloride 73 ion channels in granulocytes 1.1.3.1 ClC 73 1.1.3.2 CFTR 74 1.1.3.3 CLCA 76 1.1.3.4 Bestrophin 82 3 1.1.3.5 TMEM16A and tweety (hTTYH3) as 84 potential candidates 1.1.3.6 CLIC 86 1.1.4 Evidence for Cl- channel candidates in 90 granulocytes 1.1.4.1 Volume gated Cl- channels 90 1.1.4.2 Voltage gated Cl- channels 93 1.1.4.3 Calcium-activated Cl- channels 96 1.1.4.4 cAMP-activated channels 97 1.1.5 Summarising remarks; Cl- channels and 99 granulocyte function 1.2 Hypothesis and rationale 100 CHAPTER 2: MATERIALS AND METHODS 103 2.1 Cell and tissue culture 104 2.1.1 Commercial cell lines 104 2.1.1.1 EoL-1 cell line 104 2.1.1.2 dEoL-1 104 2.1.1.3 HL60 cells 105 2.1.1.4 Epithelial cells 105 2.1.1.5 Trypan and kimura staining 107 2.1.2 Primary cells 108 2.1.2.1 Peripheral blood and PMN purification 108 2.1.3 Microscopy for cellular morphology 112 2.1.4 Cytokine stimulation 114 2.2 RNA expression and DNA sub-cloning 115 2.2.1 Reverse transcriptase polymerase chain 115 reactions 2.2.1.1 Preparation of mRNA 115 4 2.2.1.1.1 TRIzol® preparation of mRNA 115 2.2.1.1.2 Cell lysate preparation 116 2.2.1.1.3 Analysis of concentration and 116 purity of RNA 2.2.1.2 RT-PCR reaction 117 2.2.1.3 Analysis 124 2.2.1.3.1 Electrophoresis 124 2.2.1.3.2 Sequence confirmation 125 2.2.1.3.3 Semi-quantification of results 126 2.2.2 Sub-cloning 128 2.2.2.1 Transformations of plasmids into 128 bacteria 2.2.2.2 Purification of cDNA 129 2.2.2.3 Restriction digestion 130 2.2.2.4 Transfection of cDNA 132 2.3 Protein expression 133 2.3.1 Flow cytometry 133 2.3.1.1 Method for immunolabelling with 133 primary and secondary antibodies 2.3.1.2 Apoptosis assay 139 2.3.1.3 Analysis using flow cytometry 140 2.3.1.3.1 Beckman Coulter Epics flow 140 cytometer 2.3.1.3.2 Accuri, C6 flow cytometer 141 2.3.2 Western blot analysis 143 2.3.2.1 Samples and sample preparation 143 2.3.2.1.1 Preparation of protein lysates 143 2.3.2.1.2 Analysis of protein 144 concentration 5 2.3.2.1.3 Sample preparation for loading 147 onto Western gel 2.3.2.1.4 Loading of samples and 148 electrophoresis 2.3.2.2 Process of western blotting 149 2.3.2.2.1 Transfer of proteins onto 149 membrane and antibody labelling 2.3.2.2.2 Western blot membrane re- 153 probe 2.3.2.3 Analysis of results 154 2.4 Transmigration assay 155 2.5 Chloride and calcium flux assay 157 2.6 Statistical analysis 158 2.7 Systematic review 160 2.8 Experimental summary 161 CHAPTER 3: CHARACTERISATION OF 162 ANTIBODIES, SPECIFICTY OF RT-PCR PRIMERS AND METHODOLOGY OPTIMIZATION 3.1 Flow cytometry equipment 163 3.2 Cell lines selected as an effective model of human 166 cells 3.3 Preparation of mRNA and proteins 179 3.4 Specificity of primers for RT-PCR 182 3.5 Characterization and specificity of antibodies 185 CHAPTER 4: EXPRESSION OF POTENTIAL 192 CHLORIDE ION CHANNELS 4.1 Gene expression determination by RT-PCR 193 6 4.1.1 Results 193 4.1.1.1 hCLCA expression 194 4.1.1.2 Bestrophin expression 200 4.1.1.3 hANO expression 202 4.1.1.4 RT-PCR summary and densitometry 205 4.1.2 Discussion 213 4.1.2.1 hCLCA expression 213 4.1.2.2 Bestrophin expression 214 4.1.2.3 hANO expression 216 4.2 Protein expression determination by flow 217 cytometry 4.2.1 Summary of flow cytometry results 224 4.2.2 Results in context 231 4.3 Western blot analysis 232 CHAPTER 5: FUNCTIONAL ANALYSIS OF hBEST1 237 5.1 Cytokine stimulation 238 5.1.1 Results 238 5.1.2 Discussion 249 5.2 Transmigration assay 252 5.2.1 Transmigration results 252 5.2.2 Implications and discussion 257 5.3 Cl- and Ca2+ flux assays 263 CHAPTER 6: SYSTEMATIC REVIEW: IS THE GENE 265 AND PROTEIN EXPRESSION OF CHLORIDE ION CHANNELS UPREGULATED IN THE RESPIRATORY EPITHELIA, OR GRANULOCYTES, OF SUBJECTS WITH CHRONIC INFLAMMATORY LUNG DISEASE? 7 6.1 Rationale for systematic review 266 6.2 Objectives of systematic review 268 6.3 Methodology 269 6.3.1 Formulation of systematic review question 269 6.3.2 Inclusion/exclusion criteria 271 6.3.3 Identification of studies and search protocol 273 6.3.4 Assessing the risk of bias 276 6.3.5 Data extraction and synthesis of results 278 6.4 Results 279 6.4.1 Literature search results 279 6.4.2 Assessing the risk of bias 281 6.4.3 Characteristics of included studies 283 6.4.4 Data extraction 286 6.4.5 Summary of results 287 6.5 Discussion 289 6.5.1 Limitations of systematic review 290 6.5.2 Expression of chloride ion channels in asthma 293 6.5.2.1 Equine studies 293 6.5.2.2 Human studies 298 6.5.3 Expression of chloride ion channels in chronic 302 obstructive pulmonary disease (COPD) 6.5.4 Systematic review concluding remarks 303 CHAPTER 7: CONCLUSIONS 305 7.1 Limitations 306 7.2 Addressing the hypothesis 308 7.3 Concluding remarks and recommendations 313 REFERENCES 315 8 APPENDICES 368 Appendix 1: Letter of ethics approval for blood 369 harvest Appendix 2: Letter to laboratories for systematic 370 review Appendix 3: Inclusion/exclusion form for systematic 372 review Appendix 4: Risk of bias tool for systematic review 374 Appendix 5: Completed PRISMA checklist for 380 systematic review 9 LIST OF FIGURES Figure 1.1 Different pathophysiological 27 abnormalities observed in COPD and asthma Figure 1.2 Nucleus of neutrophils and eosinophils 33 under microscopy Figure 1.3 Summary of cytokine and chemokine 42 reactions in eosinophils Figure 1.4 Potential chloride ion channel structure 56 Figure 1.5 Five proposed chloride ion channel 58 categories Figure 1.6 Diagrammatical summary of postulated 98 chloride channel functions Figure 2.1 Protocol for preparation of peripheral 109 blood Figure 2.2 Analysis of primer pair hCLCA1 KW (mid) 122 using Primer-BLAST Figure 2.3 Process for DNA extraction from agarose 126 gel slice Figure 2.4 Protein standards used to measure 146 absorbance for calculation of unknown concentration of protein samples Figure 2.5 Acrylamide gel for western blot analysis 148 Figure 2.6 Summary of process for western blot 150 analysis 10 Figure 2.7 Transmigration assay equipment set up 156 Figure 3.1 Forward scatter and side scatter for 164 flow