UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE, HEALTH AND LIFE SCIENCE School of Medicine The Roles of Beta and Alpha Tryptases in Asthma: Genetic and Immunopharmacological Studies by Ahmed Asem Mahrous Abdelmotelb A thesis submitted for partial fulfilment for the degree of doctor of philosophy April, 2010 I DECLARATION OF AUTHORSHIP I, Ahmed Asem Mahrous Abdelmotelb I declare that the thesis entitled: The Roles of Beta and Alpha Tryptases in Asthma: Genetic and Immunopharmacological Studies, and the work presented in the thesis are both my own, and have been generated by me as the result of my own original research. I confirm that: this work was done wholly or mainly while in candidature for a research degree at this University; where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; where I have consulted the published work of others, this is always clearly attributed; where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; I have acknowledged all main sources of help; where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; none of this work has been published before submission, or [delete as appropriate] parts of this work have been published as: [please list references] Signed: ……………………………………………………………………….. Date:……………………………………………………………………………. II University of Southampton Abstract Faculty of Medicine Division of Infection, Inflammation and Immunity Doctor of Philosophy The Roles of Beta and Alpha Tryptases in Asthma: Genetic and Immunopharmacological Studies. By Ahmed Asem Mahrous Abdelmotelb Tryptases, the dominant secretory granular proteins from human mast cells, are emerging as important mediators in asthma and allergy. The β- and α- tryptases have highly similar nucleotide sequences and located on the same locus. While the entire population expresses β-tryptase, the α-tryptase gene exhibits copy number variation (CNV). We have studied the association of expression of these allelic variants with asthma or allergic diseases. We have investigated also the potential actions of β- and α-tryptases in vitro and in vivo. We have found that the one alpha tryptase copy allele was significantly associated with lower total serum IgE levels (Z= -2.39, p=0.01) and a tri-allelic architecture with alleles carrying no, one or two copies of the α-tryptase gene was postulated. The addition of β- tryptase to epithelial cells induced upregulation of mRNA for IL-8, IL-6 and TNF-α, while α-tryptase on the other hand was without effect in this model. Injection of β-tryptase into the mouse peritoneum induced great accumulation of neutrophils but accumulation of other cell types was less marked. Under the same conditions, injection of α- tryptase induced less neutrophilia but eosinophils, macrophages and mast cells numbers were significantly increased. The actions of β-tryptase seemed be independent of PAR-2 receptors but not the case for α-tryptase, where PAR-2 pathway might take the leads. In conclusion, recombinant α-tryptase may be a stimulus for the recruitment of inflammatory cells and altered cytokine gene expression with effects distinct from those of β-tryptase. III Table of Contents Title…………………………………………………………………………………………………I Abstract……………………………………………………………………………………………II Declaration……………………………………………………………………………………….III Table of contents……………………………………………………………………………….IV List of figures………………………………………………………………………………....IIIV List of tables……………………………………………………………………………………..X Abbreviations……………………………………………………………………………………XI Acknowledgement…………………………………………………………………………….XII List of publications……………………………………………………………………………XIII 1. General Introduction .............................................................................. - 2 - 1.1 The mast cell in asthma ......................................................................... - 2 - 1.2 Mast Cells .............................................................................................. - 4 - 1.3 Mast cell mediators ................................................................................ - 6 - 1.3.1 Histamine ........................................................................................... - 6 - 1.3.2 Proteoglycans ..................................................................................... - 6 - 1.3.3 Neutral proteases ............................................................................... - 6 - 1.3.4 Lipid mediators .................................................................................. - 9 - 1.3.5 Cytokines ......................................................................................... - 11 - 1.4 Mast cell tryptase ................................................................................. - 12 - 1.4.1 Alpha and β-tryptases ....................................................................... - 12 - 1.4.2 Biochemical characteristics ............................................................... - 14 - 1.4.3 Tryptase substrates and inhibitors .................................................... - 15 - 1.4.4 Role of tryptase in asthmatic disease ................................................ - 17 - 1.5 Protease activated receptor-2 (PAR-2) ................................................... - 18 - 1.5.1 Characteristics of PAR-2 ................................................................... - 18 - 1.5.2 Mechanisms of PAR-2 activation ....................................................... - 18 - 1.5.3 PAR-2 in the airways ......................................................................... - 19 - 1.6 Asthma genetics .................................................................................. - 22 - 1.6.1 Defining asthma in genetic studies ................................................... - 22 - 1.6.2 Gene copy variation and disease susceptibility.................................. - 22 - 1.6.3 Tryptase loci..................................................................................... - 23 - 1.6.4 Tryptase heterogeneity ..................................................................... - 25 - IV 1.7 Aim of the work ................................................................................... - 26 - 2. Materials and General Methods ............................................................ - 28 - 2.1 Materials .............................................................................................. - 28 - 2.2 Cell Culture .......................................................................................... - 29 - 2.3 RNA extraction..................................................................................... - 31 - 2.3.1 Tri RNA extraction ............................................................................ - 31 - 2.3.2 Qiagen RNeasy ................................................................................. - 32 - 2.3.3 RNA Quantitation ............................................................................. - 32 - 2.4 DNA extraction .................................................................................... - 32 - 2.4.1 Trizol extraction ............................................................................... - 32 - 2.4.2 DNA quantitation .............................................................................. - 33 - 2.5 Reverse-transcribed polymerase chain reaction (RT-PCR) ...................... - 33 - 2.6 Primer design ...................................................................................... - 33 - 2.7 Polymerase chain reaction (PCR) ........................................................... - 36 - 2.8 Electrophoresis .................................................................................... - 37 - 2.8.1 Polyacrylamide gel electrophoresis (Microplate Array Diagonal Gel Electrophoresis (MADGE) ................................................................................ - 38 - 2.8.2 Agarose gel electrophoresis ............................................................. - 38 - 2.9 Restriction digest ................................................................................. - 38 - 2.10 Fragment sizing ............................................................................... - 38 - 2.11 House keeping genes (HKGs) ............................................................ - 39 - 2.12 Real time PCR ................................................................................... - 39 - 2.13 DNA templates ................................................................................. - 41 - 2.14 The Southampton Phenotype characteristics ..................................... - 42 - 2.15 Purification recombinant mast cell β- tryptase .................................. - 44 - 2.15.2 Characterization of the purified protein ............................................ - 45 - 2.16 Mouse model .................................................................................... - 48 - 2.16.1 Animals ............................................................................................ - 48 - 2.16.2 Mouse tail biopsy for genotyping ...................................................... - 48 - 2.16.3 Study design .................................................................................... - 49 - 2.16.4 Peritoneal lavage .............................................................................