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