Hapten-mediated contact allergy: a proteomic and immunological approach

210 Pages·2014·7.81 MB·English

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Hapten-mediated contact allergy: a
proteomic and immunological approach

By
Pete Boyd BSc. (hons)
Thesis for the degree of Doctor of Philosophy
January 2014

Supervisors – Dr Neil Smyth, Dr Paul Skipp & Prof. Eugene Healy

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UNIVERSITY OF SOUTHAMPTON
ABSTRACT
FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES
Biological science
Doctor of Philosophy
HAPTEN-MEDIATED CONTACT ALLERGY: A PROTEOMIC AND
IMMUNOLOGICAL APPROACH
By Peter Stewart Boyd
Allergic contact dermatitis (ACD) is a prevalent skin condition caused by chemical
haptens, which enter the epidermis, and by modifying self-proteins, render them
immunogenic via the activation of hapten-specific T-cells. It is currently not known
which specific protein modifications are responsible for sensitization.
This work investigates the extreme sensitizer DNCB, which confers a dinitrophenyl
(DNP) protein modification. Immortalised keratinocytes (HaCaT), incubated with
DNCB were compared to ex vivo human skin using immunofluorescence and western
blot detection of DNP-proteins, showing widespread protein modification and
similarities between tissue and cells when using a clinical dose. Proliferation assays
using lymphocytes from DNCB-sensitive donors showed responses to DNP proteins
isolated from DNCB-treated HaCaT cells and primary keratinocytes. While western blot
analysis of pH gradient separated fractions identified a number of DNP-proteins in the
DNP-HaCaT cell lysates, these were not immunogenic in lymphocyte assays. The
model protein human serum albumin (HSA) was used to investigate the modification
kinetics of DNCB by identifying which amino acid residues were changed more readily
using a range of DNCB concentrations and incubation times. Cysteine residues,
including those in disulphide bonds and particularly cysteine 34 are more readily
modified than lysine in HSA, suggesting that DNCB is able to alter the structure of
proteins. A novel process of hapten-reversal by a process termed ‘thiolysis’ was found
to remove DNP groups from the cysteine residues of synthetic peptides derived from the
sequence of HSA containing cysteine 34 using the reducing agent dithiothreitol.
Identical peptides with C34K34 showed no such hapten-reversal. This corresponds to
the unexpected immunogenicity of the cysteinyl peptide and also the DNP modified
tripeptide glutathione. Anti-DNP western blots show that the DNP group is transferred
to other proteins during incubation with human monocytes in culture. This suggests a
cellular process of removing DNP groups and GILT, a thiol reductase present in the
endosomes is presented as a candidate for this process. This work demonstrates that
DNCB can generate a wide variety of DNP protein adducts and that cysteinyl moieties
are able to stimulate lymphocyte proliferation by way of hapten transfer. This highlights
a potentially novel process involved in the mechanism of contact allergy.
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Contents

Table of figures ............................................................................................................... 11
Acknowledgements ......................................................................................................... 15
Publications ..................................................................................................................... 17
Abbreviations .................................................................................................................. 19
1. GENERAL INTRODUCTION ............................................................................... 21
1.1 The public health and economic impact of ACD ............................................. 21
1.2 Skin biology ..................................................................................................... 22
1.2.1 Anatomy .................................................................................................... 22
1.2.2 Barrier function and basic skin immunology ............................................ 24
1.3 Pathologies of the skin ..................................................................................... 27
1.3.1 Innate responses to chemicals and pathogens ........................................... 27
1.3.2 Allergic contact dermatitis ........................................................................ 28
1.4 The mechanisms of ACD ................................................................................. 29
1.4.1 Penetration of a xenobiotic into the epidermis .......................................... 29
1.4.2 Haptenation of proteins ............................................................................. 29
1.4.3 Bioactivation: pro and pre-haptens ........................................................... 31
1.4.4 Antigen processing and MHC loading ...................................................... 32
1.4.5 Irritancy, innate immunity and DC migration ........................................... 32
1.4.6 Antigen presentation and T-cell activation ............................................... 33
1.4.7 The elicitation of ACD .............................................................................. 35
1.5 2,4-dinitro-1-chlorobenzene (DNCB) as a model sensitizer ............................ 37
1.6 The proteomics approach ................................................................................. 40
1.6.1 Sample fractionation ................................................................................. 42
1.6.2 Mass spectrometry ion sources ................................................................. 46
1.6.3 Quadrupoles .............................................................................................. 48
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1.6.4 Mass analysers .......................................................................................... 49
1.6.5 Tandem mass spectrometry ....................................................................... 52
1.6.6 Analysis of MS data .................................................................................. 54
1.7 Aims and objectives ......................................................................................... 57
2 MATERIALS AND METHODS ............................................................................ 59
2.1 Cell culture ....................................................................................................... 59
2.2 Treatment of HaCaT and primary keratinocyte cells with DNCB & [14C]-
DNCB.......................................................................................................................... 59
2.3 Culture of primary keratinocytes ...................................................................... 59
2.4 Preparation and treatment of human skin ......................................................... 60
2.5 Sectioning and fixation of ex vivo human skin................................................. 60
2.6 Separation of epidermis from ex vivo human skin and lysis of epidermal cells
60
2.7 Culture of HaCaTs on microscope slides and fixation of adherent cells ......... 61
2.8 Immunofluorescence detection of dinitrophenyl epitopes, dendritic cells and
nuclei in sections of human skin and HaCaT cells ..................................................... 61
2.9 Fluorescent and laser confocal microscope analysis of immune-stained human
skin and HaCaT cells .................................................................................................. 62
2.10 Cell lysis and protein extraction for SDS-PAGE ......................................... 62
2.11 Determination of protein concentration by bicinchoninic acid (BCA) assay
63
2.12 SDS-PAGE separation of proteins from lysates of cell lines and epidermis
from ex vivo skin ......................................................................................................... 63
2.13 Detection of dinitrophenyl adducts using western blot ................................ 63
2.14 Densitometry analysis of anti-dinitrophenyl western blots: qualitative
comparison of human skin with HaCaTs .................................................................... 64
2.15 Detection of dinitrophenyl adducts using autoradiography .......................... 64
2.16 Preparation of cell lysates for addition to lymphocyte assays ...................... 65
2.17 Isoelectric focussing fractionation of HaCaT proteins ................................. 65
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2.18 Isolation of PBMCs from human blood ........................................................ 66
2.19 Lymphocyte proliferation assays (LPA) ....................................................... 66
2.20 Precipitation, non-reducing enzymatic digestion and peptide de-salting of
protein lysates and fractions ........................................................................................ 67
2.21 De-salting of peptides ................................................................................... 67
2.22 MSE analysis of peptides from HaCaT lysates and fractions ....................... 68
2.23 Incubation of human serum albumin with DNCB ........................................ 68
2.24 In-gel protein digestion ................................................................................. 68
2.25 Nano-LC-MSE data analysis of DNP-HSA .................................................. 69
2.26 Isolation and culture of human monocytes from peripheral blood
mononuclear cells........................................................................................................ 70
2.27 Incubation of human monocytes with DNP-modified synthetic peptides in
culture 70
2.28 Thiolysis of DNP groups from synthetic peptides using dithiothreitol ........ 70
2.29 Mass spectrometric analysis of synthetic peptides ....................................... 71
3 DETECTION OF COVALENT PROTEIN MODIFICATION IN HUMAN SKIN
AND HACAT CELLS USING IMMUNOFLUORESCENCE AND
IMMUNOBLOTTING .................................................................................................... 73
3.1 Introduction ...................................................................................................... 73
3.2 Experimental design ......................................................................................... 75
3.3 Results .............................................................................................................. 77
3.3.1 High dose DNCB effects in ex vivo skin ................................................... 77
3.3.2 Penetration of DNCB into ex vivo skin during a 16 hour incubation........ 78
3.3.3 Clinical dose analysis of dinitrophenyl proteins in skin ........................... 79
3.3.4 Immunofluorescence of dinitrophenylated proteins in HaCaT cells......... 82
3.3.5 Comparison of modification dose-response in HaCaT cells using western
blot 84
3.3.6 Validation of western blots using [14C]-DNCB radiolabelling ................. 87
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3.4 Discussion ........................................................................................................ 89
3.4.1 Summary ................................................................................................... 92
4 ASSAYING THE IMMUNOGENICITY OF DNCB MODIFIED PROTEINS
EXTRACTED FROM HACAT CELLS......................................................................... 95
4.1 Introduction ...................................................................................................... 95
4.2 Experimental design ......................................................................................... 97
4.3 Results .............................................................................................................. 99
4.3.1 Lymphocyte proliferation in response to DNCB ...................................... 99
4.3.2 Lymphocyte proliferation in response to DNCB modified cell proteins 101
4.3.3 Lymphocyte proliferation responses to primary keratinocytes ............... 106
4.4 Discussion ...................................................................................................... 108
4.5 Summary ........................................................................................................ 110
5 THE EFFECTS OF DNCB CONCENTRATION AND INCUBATION TIME ON
THE MODIFICATION OF HUMAN SERUM ALBUMIN ........................................ 113
5.1 Introduction .................................................................................................... 113
5.2 Experimental design ....................................................................................... 117
5.3 Results ............................................................................................................ 119
5.4 Discussion ...................................................................................................... 133
5.4.1 Summary ................................................................................................. 138
6 A NOVEL PROCESS OF HAPTEN TRANSFER IN MEDIATING
IMMUNOGECITY TO DNCB .................................................................................... 141
6.1 Introduction .................................................................................................... 141
6.2 Experimental design ....................................................................................... 147
6.3 Results ............................................................................................................ 151
6.3.1 Differences in immunogenicity and antigen processing between DNP-Cys
and DNP-Lys......................................................................................................... 151
6.3.2 The buffer pH has a pronounced effect on the reduction properties of
dithiothreitol (DTT) .............................................................................................. 154
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6.3.3 Exploring the removal of DNP groups from DNP-Cys using DTT and
GILT 154
6.4 Discussion ...................................................................................................... 162
6.4.1 Summary ................................................................................................. 168
7 GENERAL DISCUSSION AND FUTURE WORK ............................................ 171
7.1 Summary of experimental work and results ................................................... 171
7.2 Can models be used to determine sensitizers? ............................................... 173
7.3 Is DNCB immunogenicity driven by specific or generic protein modifications?
174
7.4 Summary ........................................................................................................ 177
7.5 Further work ................................................................................................... 178
8 Appendix ............................................................................................................... 181
9 REFERENCES ...................................................................................................... 183

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