The Role of HLA Class I Antibody in Endothelial Cell Activation and Allograft Rejection Fatmah MA

309 Pages·2013·7.35 MB·English

The Role of HLA Class I Antibody in Endothelial Cell
Activation and Allograft Rejection

Fatmah M A Naemi

Thesis submitted in partial fulfilment of the requirements of the regulations
for the degree of Doctor of Philosophy

Applied Immunobiology and Transplantation Group
Institute of Cellular Medicine
Newcastle University, UK

September 2013
Abstract
Background: Antibody-mediated rejection is one of the major causes of acute and
chronic rejection. This is mediated by endothelium microvascular inflammation and
leukocyte migration to the graft. However, the role of donor specific HLA class I
antibodies in inducing allograft rejection in the absence of complement is not fully
understood. In this project, the mechanisms by which HLA class I antibodies induce
endothelial cell-leukocyte interactions were examined.

Methods: Human microvascular endothelial cells (HMEC-1) were stimulated with
HLA class I antibody either mouse monoclonal (W6/32) or from sensitized kidney
patients. The activation of cell signaling pathways was examined using Western
blotting. The expression of adhesion molecules and chemokines were determined
using flow cytometry and q-PCR, respectively. Monocyte adhesion and migration was
examined using chemotaxis and flow based adhesion assays.

Results: HMEC-1 cells stimulated with W6/32 antibody showed phosphorylation of a
transcription factor CREB by a mechanism dependent on the protein kinase A
pathway. W6/32 also induced significant expression of the adhesion molecules
VCAM-1 and ICAM-1 (P<0.001) in a mechanism dependent on the PI3K/Akt
pathway. Additionally, stimulated cells showed significant up-regulation in IL-6,
CXCL8, CXCL1, CCL5 and CXCL10. The expression of CXCL8 was significantly
reduced by knocking down CREB (p<0.001). Conditioned media from W6/32-treated
cells was able to induce significant THP-1 monocyte migration compared to control
(p<0.001). Furthermore, monocytes flowing at 0.5 dyne/cm2 significantly adhered to
HMEC-1 cells-treated with F(ab) -fragments of W6/32 (p<0.001). HLA class I
2
alloantibodies from patients induced phosphorylation of CREB and a significant
upregulation of VCAM-1, ICAM-1 and CXCL8. Monoclonal human HLA-B58
antibody was also able to induce CREB phosphorylation.

Conclusion: Exposure of microvascular endothelial cells to HLA class I antibodies
induce an activation of endothelial cell signaling that are responsible for upregulation
of adhesion molecules and chemokines. These mediators enhance the interaction
between donor endothelium and recipient leukocytes during cellular rejection.
Strategies that block endothelium-leukocyte interaction might reduce the incidence of
allograft rejection and improve allograft survival.

I
Acknowledgements
I would like to thank the funding body for this project, Ministry of Health in Kingdom
of Saudi Arabia. I am really grateful for their continuous help and support through my
study in the UK.
I would like to express my gratitude to my supervisors at Newcastle University
Professor John A. Kirby and Professor Simi Ali for their help, ideas, support and
useful comments through the project.
I would like to express my deepest appreciation to my supervisor Dr. Vaughan Carter,
NHS Blood and Transplant, Newcastle, for his collaboration in the project, his useful
advices, continuous support and time.
I would like also to thank all members who helped me during the project. From
Newcastle University, many thanks to Maureen Kirkley, JK laboratory previous
technician, Dr. Jeremy Palmer for his help and advice in antibody purification, Liz
Shiells for her help and all staff in flow cytometry facility.
Many thanks go to all staff in NHS Blood and Transplant at Newcastle for their
assistance during the project.
I would like to thank Dr. A. A. Alhasan and Dr. Eva-Maria for their help and support.
I would like to express a special gratitude to all my family members for their help,
support and understanding.

II
List of Abbreviations
ADCC Antibody-Dependent Cell Mediated Cytotoxicity
BCA BiCinchoninic Acid
BSA Bovine Serum Albumin
CaMK calcium-moduline kinases
cDNA complementary DNA
CNX Calnexin
CREB cAMP Responsive Element Binding protein
CREM cAMP-Responsive Element Modulators
DAPI 4, 6-Diamidino-2-Phenylindole
dcell donor cells
DMSO DiMethylSulphOxide
DNA Deoxyribose Nucleic Acid
EDTA EthyleneDiamineTetraAcetic acid
EGF Epidermal Growth Factor
ER Endoplasmic Reticulum
ERK Extracellular Regulatory Kinase
ELISA Enzyme Linked ImmunoSorbent Assay
FAK Focal Adhesion Kinase
FBS Foetal Bovine Serum
FGF Fibroblast Growth Factor
FITC Fluorescein IsoThioCyanate
FSC Forward side scatter
HLA Human Leukocyte Antigen
HMEC-1 Human Microvascular Endothelial Cells
HRP Horse Radish Peroxidase
HUVEC Human Umbilical Vein Endothelial Cells
ICAM-1 IntraCellular Adhesion Molecule-1
IFN-γ Interferon-γ
JAM-A Junctional Adhesion Molecule-A
LFA-1 Lymphocyte Function Associated Antigen-1
MAPK Mitogen Activated Protein Kinase
MFI Median Fluorescence Intensity
MHC Major Hisocompatibility Complex
MIC Major Histocompatibility Complex class I related chain A or B
MSK Mitogen/Stress Kinase
III
mTOR Mammalian Target of Rapamycin
NF-B Nuclear Factor- B
NK cells Natural Killer cells
Nrf2 Nf-E2-related factor 2
OPD O-phenylenediamine
PBMC Peripheral blood mononuclear cells
PBS phosphate buffered saline
PCR-SSP Polymerase Chain Reaction-Sequence Specific Primer
PCR-SSOP Polymerase Chain Reaction-Sequence Specific Oligonucleotide
Probe
PE PhycoErythrin
PECAM-1 Platelet Endothelial Cell Adhesion Molecules
PI Propidium Iodide
PI3K phosphatidylinositol-3-kinase
PKA protein kinase A
PSGL1 P-selectin Glycoprotein Ligand-1
PVDF Polyvinylidene fluoride
q-PCR Semi-quantitative Polymerase Chain Reaction
rAPC recipient Antigen Presenting Cells
RNA Ribose Nucleic Acid
SDS Sodium Dodecyl Sulphate
SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
siRNA Small Interfering RNA
SSC Side scatter
STS Staurosporine
TAE Tris-acetate EDTA electrophoresis buffer
TAP Transporter Associated with antigen Processing
TNF-α Tumor Necrosis Factor-α
TPBS 0.1 % tween 20 in PBS
VCAM-1 Vascular Cell Adhesion Molecule-1
VLA-4 Very Late Antigen-4

IV
Table of Contents
Table of Contents ......................................................................................................... V
1. Chapter One-General Introduction .....................................................................1
1.1 Historical background .......................................................................................1
1.2 Forms of antibody-mediated rejection ..............................................................2
1.2.1 Hyperacute antibody-mediated rejection .............................................................. 2
1.2.2 Acute/accelerated antibody-mediated rejection .................................................... 3
1.2.3 Chronic antibody-mediated rejection .................................................................... 3
1.2.4 Subclinical antibody-mediated rejection ............................................................... 4
1.3 Clinical relevant antibody in solid organ transplantation .................................5
1.3.1 Blood group antibodies ......................................................................................... 5
1.3.2 HLA antibodies ..................................................................................................... 5
1.3.3 Polymorphic non-HLA antibodies ........................................................................ 6
1.3.4 Autoantibodies ...................................................................................................... 6
1.4 Mechanism of HLA class I antibody formation ...............................................7
1.5 Mechanisms of antibody action ......................................................................11
1.5.1 Complement-dependent mechanism ................................................................... 11
1.5.2 Complement-independent mechanism ................................................................ 13
1.6 Antibody-mediated graft accommodation ......................................................14
1.7 Human leukocyte antigen system ...................................................................16
1.7.1 Human leukocyte antigen classes ........................................................................ 16
1.7.2 Human leukocyte antigen structures ................................................................... 16
1.7.3 HLA class I assembly and antigen precursors..................................................... 18
1.8 HLA class I antibody and endothelial cell signaling ......................................22
1.8.1 Mechanism of HLA class I antibody signaling ................................................... 22
1.8.2 HLA class I antibody and Akt pathway .............................................................. 22
1.8.3 HLA class I antibody and mammalian target of rapamycin (mTOR) pathway.. 24
1.8.4 HLA class I antibody and cytoskeleton proteins ................................................. 24
1.8.5 HLA class I antibody and Growth factors ........................................................... 26
1.9 HLA class I antibody induce activation of transcription factors ....................26
1.9.1 HLA class I antibody and NF-B ....................................................................... 27
1.9.2 HLA class I antibody and E2F transcription factor ............................................. 28
1.9.3 HLA class I antibody and c-Jun transcription factor ........................................... 28
1.9.4 HLA class I antibody and Nf-E2-related factor 2 ............................................... 29
1.10 cAMP Responsive Element Binding protein (CREB) ....................................29
1.10.1 CREB structure ................................................................................................... 31
1.10.2 Activation of CREB ............................................................................................ 32
1.10.3 CREB function and disease ................................................................................. 33
1.11 Leukocyte migration in transplantation. .........................................................34
1.12 Mechanism of Leukocyte extravasation .........................................................35
1.12.1 Selectin-mediated rolling .................................................................................... 35
1.12.2 Integrin-mediated adhesion ................................................................................. 36
1.12.3 Leukocyte crawling and extravasation ................................................................ 37
1.13 Role of endothelial cells in rejection ..............................................................40
1.13.1 Endothelial cells as an initiator of rejection ........................................................ 40
1.13.2 Endothelial cells as a mediator of rejection......................................................... 41
1.13.3 Endothelial cells as a target during rejection....................................................... 42
V
1.14 Aim of the project .............................................................................................44
2. Chapter Two- General Material and Methods .................................................45
2.1 General practice ..............................................................................................45
2.2 Culture media ..................................................................................................45
2.2.1 MCDB-131 media ............................................................................................... 45
2.2.2 RPMI 1640 media ............................................................................................... 45
2.2.3 DMEM media ...................................................................................................... 46
2.2.4 DMEM F12 media .............................................................................................. 46
2.3 Cell lines and primary cells .............................................................................46
2.3.1 Human microvascular endothelial cell line (HMEC-1) ...................................... 46
2.3.2 Human large vessels endothelial cell line (EA.hy926) ....................................... 47
2.3.3 Human kidney epithelial cell lines (HK-2 & HKC-8) ........................................ 47
2.3.4 Human embryonic kidney 293 cell line (HEK 293 cells) ................................... 47
2.3.5 Hybridoma cell line (W6/32) .............................................................................. 48
2.3.6 RAJI B cell line ................................................................................................... 48
2.3.7 Human monocytic cell line (THP-1) ................................................................... 48
2.3.8 MOLT-16 T-cell line ........................................................................................... 49
2.3.9 Peripheral blood mononuclear cells (PBMC) ..................................................... 49
2.4 Cell counting ...................................................................................................49
2.5 Cell cryopreservation ......................................................................................49
2.6 Mycolpasma screening....................................................................................50
2.7 Flow cytometry ...............................................................................................50
2.7.1 General principle ................................................................................................. 50
2.7.2 Indirect immunofluorescence .............................................................................. 51
2.7.3 Direct immunofluorescence ................................................................................ 52
2.8 Western blotting ..............................................................................................53
2.8.1 General principle ................................................................................................. 53
2.8.2 Preparation of cell lysate ..................................................................................... 53
2.8.3 Determination of protein concentration .............................................................. 54
2.8.4 SDS-PAGE electrophoresis................................................................................. 54
2.8.5 Gel staining ......................................................................................................... 56
2.8.6 Wet Protein transferring ...................................................................................... 57
2.8.7 Immunoblotting ................................................................................................... 57
2.9 Molecular biology ...........................................................................................58
2.9.1 General principle of RNA isolation .................................................................... 58
2.9.2 Procedure of RNA Isolation ................................................................................ 59
2.9.3 Analysis of RNA concentration and purity ......................................................... 59
2.9.4 Analysis of RNA integrity................................................................................... 60
2.9.5 cDNA synthesis. .................................................................................................. 60
2.10 Real time-polymerase chain reaction ..............................................................62
2.10.1 General principle ................................................................................................. 62
2.10.2 Taqman assay ...................................................................................................... 62
2.10.3 Primer efficiency ................................................................................................. 63
2.10.4 Semi quantitative real time-PCR ......................................................................... 65
2.10.5 Data analysis-comparative ∆∆CT value .............................................................. 65
2.11 Statistical analysis ...........................................................................................65
3. Chapter Three-Characterization of Cell Lines and Antibody Purification ...67
3.1 Introduction .....................................................................................................67
VI
3.2 Specific Aims ..................................................................................................69
3.3 Specific materials and methods ......................................................................70
3.3.1 Immunofluorescence staining on chamber slides ................................................ 70
3.3.2 Treatment of endothelial cell lines with IFN-γ and TNF-α ................................. 70
3.3.3 Direct immunofluorescence staining for HLA class II expression ..................... 70
3.3.4 Acid treatment assay ........................................................................................... 71
3.3.5 Effect of trypsin treatment on cell surface HLA class I expression .................... 71
3.3.6 Genotyping of cell lines for the HLA class I....................................................... 72
3.3.6.1 DNA Extraction ..................................................................................................... 72
3.3.6.2 HLA typing using PCR-SSP .................................................................................. 72
3.3.6.3 Agarose electropheresis ......................................................................................... 73
3.3.6.4 ABO genotyping of HMEC-1 cell line by PCR-SSP ............................................. 73
3.3.7 Purification of W6/32 antibody ........................................................................... 73
3.3.7.1 Media collection and processing ............................................................................ 73
3.3.7.2 Affinity chromatography ....................................................................................... 74
3.3.7.2.1 General principle ............................................................................................... 74
3.3.7.2.2 Antibody purification using Protein A column ................................................. 74
3.3.7.3 ELISA assay .......................................................................................................... 75
3.3.7.4 Reducing SDS-PAGE ............................................................................................ 75
3.3.7.5 Indirect immuno-fluorescence staining for class I expression ............................... 77
3.4 Results .............................................................................................................78
3.4.1 Expression of CD31 and CD34 on endothelial cells ........................................... 78
3.4.2 Expression of HLA class II antigens on endothelial cells stimulated with IFN-
γ………………………………………………………………………………………….81
3.4.3 Expression of HLA class II antigens on endothelial cells stimulated with both
IFN-γ and TNF-α ............................................................................................................. 83
3.4.4 Effect of acid treatment on HLA class I expression on endothelial cells ............ 88
3.4.5 Effect of trypsin treatment on the expression of HLA class I antigen on
endothelial cells ................................................................................................................ 91
3.4.6 HLA class I and ABO genotyping on HMEC-1 cells ......................................... 93
3.4.7 Purification of W6/32 antibody ........................................................................... 95
3.5 Discussion .......................................................................................................97
4. Chapter Four- HLA Class I Antibodies and Endothelial Cell Signalling .....102
4.1 Introduction ...................................................................................................102
4.2 Specific Aims ................................................................................................104
4.3 Specific materials and methods ....................................................................105
4.3.1 Human phosphokinase array on endothelial cells ............................................. 105
4.3.2 Stimulation of endothelial cells for measurement of phosphorylated proteins
(Akt, ERK and CREB) ................................................................................................... 105
4.3.3 Stimulation of endothelial cells with forskolin ................................................. 106
4.3.4 Using specific inhibitors.................................................................................... 106
4.3.4.1 PI3K/Akt inhibitor ............................................................................................... 107
4.3.4.2 Protein kinase A inhibitor .................................................................................... 107
4.3.5 Trypan blue staining .......................................................................................... 107
4.3.6 Examination of biological functions ................................................................. 108
4.3.6.1 Cell proliferation assay ........................................................................................ 108
4.3.6.2 Cell apoptosis assay ............................................................................................. 108
4.3.6.2.1 General principle ............................................................................................. 108
4.3.6.2.2 Staurosporine treatment .................................................................................. 110
4.3.6.2.3 Sub-diploid peak assay .................................................................................... 111
4.4 Results ...........................................................................................................112
VII
4.4.1 Human phosphokinase array on endothelial cells following stimulation with
W6/32 antibody .............................................................................................................. 112
4.4.2 Endothelial ERK, Akt and CREB phosphorylation following stimulation with
W6/32 antibody .............................................................................................................. 114
4.4.3 Dose response of endothelial Akt and CREB phosphorylation following
stimulation with W6/32 antibody ................................................................................... 118
4.4.4 Effect of the PI3K/Akt pathway on CREB phosphorylation induced by W6/32
antibody .......................................................................................................................... 120
4.4.5 Effect of PKA pathway on endothelial CREB phosphorylation induced by
W6/32 antibody .............................................................................................................. 122
4.4.6 Effect of W6/32 antibody on cell proliferation ................................................. 125
4.4.7 Optimization of apoptosis of the HMEC-1 cells using staurosporine ............... 126
4.4.8 Optimization of apoptosis on EA.hy926 cells using staurosporine ................... 128
4.4.9 Effect of HLA class I antibody on cell cycle distribution of HMEC-1 cell line
130
4.5 Discussion .....................................................................................................131
5. Chapter Five-HLA class I Antibodies and Endothelial-Leukocyte Interaction
135
5.1 Introduction ...................................................................................................135
5.2 Specific Aims ................................................................................................137
5.3 Specific materials and methods ....................................................................138
5.3.1 Stimulation of endothelial cells for adhesion molecules expression ................. 138
5.3.2 Stimulation of endothelial cells for measurement of cytokine and chemokine
expression ....................................................................................................................... 138
5.3.3 Stimulation of endothelial cells for expression of CXCL8 ............................... 139
5.3.4 Transient transfection (siRNA technology) ...................................................... 139
5.3.4.1 General principle.................................................................................................. 139
5.3.4.2 Transfection efficiency using cy3 labelled GAPDH siRNA and lipid based
reagents . 141
5.3.4.3 Transfection efficiency using cy3 labelled GAPDH siRNA and electroporation
technique… .. …………………………………………………………………………………142
5.3.4.4 Evaluation of knockdown efficiency using different lipid based reagents and non-
fluorescently labelled GAPDH siRNA ................................................................................... 142
5.3.4.5 Knockdown efficiency at mRNA level ................................................................ 146
5.3.4.6 Knockdown efficiency at protein level ................................................................ 148
5.3.4.7 Transfection of CREB siRNA ............................................................................. 148
5.3.5 Preparation of F(ab) fragment .......................................................................... 151
2
5.3.5.1 General principle.................................................................................................. 151
5.3.5.2 F(ab) fragment preparation ................................................................................. 151
2
5.3.5.3 Flow cytometry analysis of F(ab') fragment ....................................................... 153
2
5.3.6 Chemotaxis assay .............................................................................................. 155
5.3.6.1 General principle- the Boyden chamber .............................................................. 155
5.3.6.2 Method for chemotaxis assay ............................................................................... 155
5.3.7 In vitro flow based adhesion assays .................................................................. 157
5.3.7.1 General principle-Cellix platform ........................................................................ 157
5.3.7.2 Flow based adhesion assay using recombinant human adhesion molecule ......... 158
5.3.7.3 Flow based adhesion assays using a monolayer of endothelial cells .................. 160
5.4 Results ...........................................................................................................161
5.4.1 Effect of TNF-α stimulation on the expression of adhesion molecules on HMEC-
1 cells 161
5.4.2 Effect of W6/32 antibody on the expression of adhesion molecules on
endothelial cells .............................................................................................................. 167
VIII
5.4.3 Effect of the PI3K/Akt pathway on the expression of adhesion molecules
induced by W6/32 antibody ........................................................................................... 173
5.4.4 Effect of W6/32 antibody on the expression of endothelial cytokines and
chemokines ..................................................................................................................... 176
5.4.5 Dose and time response of W6/32 antibody on endothelial CXCL8 expression.
178
5.4.6 Effect of PI3K/Akt and PKA/CREB pathways on CXCL8 expression induced by
HLA class I antibody ..................................................................................................... 180
5.4.7 Effect of W6/32 conditioned media on monocyte migration ............................ 182
5.4.8 In vitro flow based adhesion assay using VCAM-1/Fc and THP-1 cells .......... 184
5.4.9 In-vitro flow based adhesion assay on TNF-α activated HMEC-1 using THP-1
cells and chemokines ...................................................................................................... 187
5.4.10 In vitro flow based adhesion assay using whole molecule of W6/32
antibody……………………………………………………………………………..….190
5.4.11 Expression of VCAM-1 and ICAM-1 by endothelial cells following stimulation
with F(ab) fragment of W6/32 antibody .................................................................. ….193
2
5.4.12 In vitro flow based adhesion assay using F(ab) fragment of W6/32
2
antibody……………………………………………………………………………..….195
5.5 Discussion .....................................................................................................197
6. Chapter Six-Allospecific Antibodies and Endothelial Cell Activation ..........203
6.1 Introduction ...................................................................................................203
6.2 Specific Aims ................................................................................................204
6.3 Specific materials and methods ....................................................................205
6.3.1 Sources of allospecific antibodies ..................................................................... 205
6.3.2 Antibody purification ........................................................................................ 205
6.3.3 Gel electrophoresis ............................................................................................ 205
6.3.4 Indirect immunofluorescence ............................................................................ 207
6.3.5 Antibody screening using Luminex cytometer ................................................. 207
6.3.5.1 General principle.................................................................................................. 207
6.3.5.1 Antibody screening using Luminex assay ........................................................... 209
6.3.6 Stimulation of endothelial cells using allospecific antibodies .......................... 209
6.3.7 Assessment of the specificity of isolated IgG antibodies to endothelial HLA class
I antigens. ....................................................................................................................... 212
6.3.7.1 Acid treatment of HMEC-1 cells ......................................................................... 212
6.3.7.2 Knockdown efficiency of HLA class I antigens (siRNA) using W6/32 antibody 213
6.3.7.3 Knockdown efficiency using human monoclonal HLA class I antibody ............. 213
6.3.8 p-CREB Cell based ELISA ............................................................................... 215
6.4 Results ...........................................................................................................217
6.4.1 Effect of allospecific antibodies on endothelial cell signalling ......................... 217
6.4.1.1 Effect of allospecific antibodies from multiparous females on endothelial cell
signalling .......... ……………………………………………………………………………217
6.4.1.2 Effect of allospecific antibodies from sensitized patients on endothelial cell
signalling .. …………………………………………………………………………………221
6.4.2 Effect of allospecific antibodies on adhesion molecules expression................. 225
6.4.2.1 Effect of antibodies from Multiparous females on adhesion molecules
expression..……………………………………………………………..………………….…225
6.4.2.2 Effect of allospecific antibodies from sensitized patients on the expression of
endothelial adhesion molecules .............................................................................................. 228
6.4.3 Effect of allospecific HLA class I antibodies on CXCL8 expression ............... 231
6.4.4 Examination of the specificity of the purified IgG antibodies by acid treatment
…………………………………………………………………………………..…234
IX