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The Role of Kallikrein 5 and 7 and Protease Activated Receptor 2

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The Role of Kallikrein 5 and 7 and Protease Activated Receptor 2 UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Clinical and experimental sciences Effect of irritants in the epidermis: The role of kallikrein 5 and 7 and protease activated receptor 2 by Jo Underwood Thesis for the degree of Doctor of Philosophy September 2013 UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Dermatopharmacology Thesis for the degree of Doctor of Philosophy Effect of irritants in the epidermis: The role of kallikrein 5 and 7 and protease activated receptor 2 Joanne Marie Underwood Contact dermatitis affects 0.5-1.9 cases per 1,000 full-time workers annually, with 80% of cases accounted for by irritant contact dermatitis. The mechanisms underlying irritant contact dermatitis are incompletely understood and there is also a large need in industry for screening tools to identify the irritant potential of novel chemical compounds. The aim of this thesis was to investigate whether disruption of the stratum corneum barrier by irritants induces epidermal serine proteases such as KLKs which act through the PAR2 receptor to cause a pro-inflammatory response. Croton oil (3%) and SDS (5%) were applied to ex vivo human skin and expression of KLK5, KLK7 and PAR2 analysed. Both croton oil and SDS increased KLK5 (p=0.0001 and 0.0002 respectively), KLK7 (p=0.0027 and 0.0009 respectively), and PAR2 (p=0.0057 and p=0.0215 respectively). In addition, protease activity within the epidermis was concomitantly increased (p=0.0094 for croton oil and p=0.0185 for SDS). Although these findings help to provide mechanistic insight, and could have some use as a screening tool, the availability of human skin is a limiting factor in an industrial setting. Using Rho kinase-inhibition to increase the proliferative capacity of adult human keratinocytes to generate 3D epidermal equivalents, it was noted that the resulting stratum corneum was insufficiently resilient to withstand application of SDS, even at reduced concentrations. However, in monolayer cultures it was noted that, following initial screening using qPCR arrays, croton oil and dithranol caused upregulation of IL6, IL36A, CCL5 and CSF2 in Rho kinase-inhibited keratinocytes, and that CCL5 and CSF2 were similarly upregulated in normal human keratinocytes exposed to these compounds. Thus, in Rho kinase-inhibited keratinocytes, CCL5 and CSF2 (and possibly IL6 and IL36A) may form a useful screening tool for potential irritants generated in industry, although further work will be required to test sensitivity and specificity of this approach. iii Table of contents ABSTRACT ............................................................................................................................... iii Contents ..................................................................................................................................... v List of tables ............................................................................................................................ ix DECLARATION OF AUTHORSHIP ..................................................................................... xv Acknowledgements .......................................................................................................... xvii List of abbreviations .......................................................................................................... xix 1. Introduction .................................................................................................................. 1 1.1 Skin structure and function ................................................................................................... 1 1.1.1 Programming of the adaptive immune response ..................................................... 3 1.1.2 Dermis........................................................................................................................................ 3 1.1.3 Epidermis ................................................................................................................................. 4 1.1.4 Cellular components ............................................................................................................ 7 1.1.5 Melanocytes, Langerhans cells and Merkel cells ...................................................... 7 1.1.6 Keratinocytes .......................................................................................................................... 8 1.1.7 Desquamation ......................................................................................................................... 9 1.2 Contact Dermatitis.................................................................................................................. 11 1.2.1 Irritant Contact Dermatitis ............................................................................................. 11 1.2.2 Mechanism of irritant contact dermatitis ................................................................. 12 1.2.3 Allergic contact dermatitis ............................................................................................. 13 1.2.4 Mechanism of allergic contact dermatitis ................................................................ 14 1.2.5 Susceptibility to contact dermatitis ............................................................................ 16 1.3 The role of the skin barrier in irritant responses ...................................................... 17 1.4 Serine Proteases ...................................................................................................................... 18 1.4.1 Matriptase ............................................................................................................................. 19 1.4.2 Prostasin ................................................................................................................................ 20 1.5 Protease activated receptors.............................................................................................. 25 1.5.1 PAR2 ........................................................................................................................................ 26 1.6 PAR2 activation and its role in barrier homeostasis and skin immunity ......... 31 1.7 Ichthyosis ................................................................................................................................... 34 1.7.1 Atopic dermatitis ................................................................................................................ 35 1.7.2 Netherton’s syndrome...................................................................................................... 36 1.8 Current models of measuring sensitizing and irritant potential ......................... 37 1.8.1 In Silico, in chemico and in vitro methods ................................................................. 37 v Table of contents 1.9 3D skin modelling .................................................................................................................... 39 1.9.1 Immortalised keratinocytes ........................................................................................... 39 1.9.2 Rho Kinase inhibitors ........................................................................................................ 40 1.10 Summary ..................................................................................................................................... 41 1.11 Hypothesis .................................................................................................................................. 42 1.12 Aims .............................................................................................................................................. 42 2. Materials and Methods ........................................................................................... 43 2.1 Materials ..................................................................................................................................... 43 2.1.1 Buffers and gels ................................................................................................................... 43 2.1.2 Plasticware ............................................................................................................................ 44 2.2 Cell culture ................................................................................................................................. 44 2.2.1 Primary keratinocyte extraction .................................................................................. 44 2.2.2 Maintenance and subculture of primary keratinocytes ...................................... 45 2.2.3 Cell line culture and storage ........................................................................................... 46 2.3 Neutrophil isolation and shape change assay .............................................................. 46 2.4 Human skin explant model .................................................................................................. 47 2.5 Construction of 3D skin models ......................................................................................... 48 2.5.1 Creating 3D skin models using a polycarbonate scaffold ................................... 48 2.5.2 Creating 3D skin models using a collagen scaffold ................................................ 49 2.6 Immunofluorescent staining ............................................................................................... 51 2.6.1 Immunofluorescent staining of frozen sections ....................................................
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