REGULATION AND FUNCTION OF THE LEUKOCYTE
IMMUNOGLOBULIN-LIKE RECEPTORS (LILRS) IN
RHEUMATOID ARTHRITIS.
A thesis submitted to the University of New South Wales
by
OWEN ANTHONY HUYNH
For the degree of Doctor of Philosophy (Ph.D)
Inflammatory Diseases Research Unit, School of Medical Sciences
University of New South Wales
2008 ORIGINALITY / COPYRIGHT / AUTHENTICITY STATEMENTS
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th Owen Anthony HUYNH 24 November, 2008 B.MedSci (Hons) Date TABLE OF CONTENTS
ORIGINALITY / COPYRIGHT / AUTHENTICITY STATEMENTS ...... i
TABLE OF CONTENTS ...... ii
ABSTRACT ...... vi
ACKNOWLEDGEMENTS ...... viii
PERSONAL THANKS ...... ix
PUBLICATIONS ARISING FROM CANDIDATURE...... xi
CONFERENCES ATTENDED DURING CANDIDATURE...... xi
LIST OF IMPORTANT ABBREVIATIONS ...... xii
LIST OF TABLES...... xvi
LIST OF FIGURES...... xvii
CHAPTER 1. LITERATURE REVIEW...... 1
1.1 THE IMMUNE SYSTEM AND ITS REGULATION...... 1 1.2 PATHWAYS FOR NEGATIVE AND POSITIVE SIGNALLING BY IMMUNE CELLS...... 3 1.2.1 Immunoreceptor Tyrosine-based Activation Motifs (ITAMs)...... 3 1.2.2 Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs) ...... 4 1.3 THE LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTORS (LILRS)...... 8 1.3.1 Nomenclature of LILRs ...... 11 1.3.2 Structure of LILRs ...... 13 1.3.3 Expression of LILRs ...... 16 1.3.4 Ligands of LILRs...... 20 1.3.5 Function of LILRs...... 25 1.3.6 Pathological Role of LILRs ...... 34 1.4 LILR-RELATED RECEPTORS ...... 41 1.4.1 Killer Cell Immunoglobulin-like Receptors (KIRs)...... 41 1.4.2 LAIR-1 (CD305)...... 44 1.4.3 Murine Paired Immunoglobulin-like Receptor-B (PIR-B)...... 45 1.4.4 Murine LILRB4 (mLILRB4 or gp49B) ...... 45 1.5 TOLL-LIKE RECEPTORS (TLRS) ...... 48 1.6 RHEUMATOID ARTHRITIS ...... 52 1.6.1 Background...... 52
ii 1.6.2 Etiology...... 52 1.6.3 Pathology ...... 54 1.6.4 Treatments...... 61 1.6.5 LILRs in Rheumatoid Arthritis ...... 70 1.7 HYPOTHESIS AND AIMS ...... 71
CHAPTER 2. REGULATION OF LILRA2 AND LILRB4 BY CYTOKINES AND LPS ...... 72
2.1 INTRODUCTION ...... 72 2.2 MATERIALS AND METHODS...... 75 2.2.1 Antibodies and Reagents...... 75 2.2.2 Cell Culture...... 76 2.2.3 Peripheral Blood Mononuclear Cells (PBMCs) Extraction ...... 76
2.2.4 Differentiation of THP-1 Cells using Vitamin D3...... 77 2.2.5 Cell Stimulation ...... 77 2.2.6 Flow Cytometry ...... 77 2.2.7 RNA Extraction ...... 78 2.2.8 DNase-Treatment and Reverse Trascription (RT) ...... 78 2.2.9 Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) ...... 79 2.2.10 Statistical Analyses ...... 81 2.3 RESULTS ...... 82 2.3.1 Regulation of LILRA2 and LILRB4 Expression upon Differentiation of Monocytes to Macrophages...... 82 2.3.2 Regulation of LILRA2 and LILRB4 Expression by Cytokines and LPS on Monocytes and Macrophages...... 84 2.3.3 Regulation of LILRA2 and LILRB4 Expression by Cytokines and LPS on Primary Monocytes ...... 87 2.3.4 Regulation of LILRA2 mRNA by LPS on Primary Monocytes ...... 90 2.4 DISCUSSION ...... 92
CHAPTER 3. EFFECT OF LILRA2 AND LILRB4 ON CYTOKINE PRODUCTION IN MONOCYTES AND MACROPHAGES ...... 96
3.1 INTRODUCTION ...... 96 3.2 MATERIALS AND METHODS...... 99 3.2.1 Antibodies and Reagents...... 99 3.2.2 Cell Culture and Differentiation of THP-1 Cells ...... 99
iii 3.2.3 Expression of LILRA2 and LILRB4 on Monocytes and Lymphocytes...... 99 3.2.4 Cross-linking of Cell Surface LILRA2 ...... 100 3.2.5 Quantification of Cytokine Production ...... 100 3.2.6 Co-ligation of Cell Surface LILRA2 and LILRB4 ...... 101 3.2.7 Effect of Anti-Rheumatoid Drugs on Cell Viability ...... 102 3.2.8 Effect of DMARDs on LILRA2 Activation in Macrophages ...... 102 3.2.9 Statistical Analyses ...... 103 3.3 RESULTS ...... 104 3.3.1 Expression of LILRA2 and LILRB4 on Peripheral Blood Mononuclear Cells (PBMCs) ...... 104 3.3.2 Activation via LILRA2 Induces the Production of TNF-α in In vitro-derived Macrophages and Primary Monocytes...... 106 3.3.3 Inhibition of LILRA2-mediated Cytokine Production upon Co-ligation with LILRB4 ...... 108 3.3.4 Effects of Anti-rheumatoid Drugs on LILRA2-mediated Activation ...... 110 3.3.5 Activation of LILRA2 on PBMCs from Patients with RA ...... 113 3.4 DISCUSSION ...... 115
CHAPTER 4. EFFECTS OF LPS-STIMULATION ON THE FUNCTION OF LILRA2 AND LILRB4 ...... 120
4.1 INTRODUCTION ...... 120 4.2 MATERIALS AND METHODS...... 122 4.2.1 Antibodies and Reagents...... 122 4.2.2 Cross-linking Cell Surface LILRs...... 122 4.2.3 Cross-linking LILRA2 with LPS-stimulation...... 122 4.2.4 Sequential Activation with LILRA2 and LPS...... 123 4.2.5 Neutralisation of IL-10 in LILRA2 Cross-linking with LPS Co-stimulation...... 123 4.2.6 Measurement of TLR4 Expression by Flow Cytometry ...... 123 4.2.7 Cross-linking LILRB4 with LPS-stimulation ...... 124 4.2.8 Statistical Analyses ...... 124 4.3 RESULTS ...... 125 4.3.1 Effect of LPS on LILRA2-mediated Activation ...... 125 4.3.2 Effect of Sequential Activation with LILRA2 and LPS...... 130 4.3.3 Effect of LILRA2 Cross-linking on TLR4 Expression...... 131 4.3.4 Neutralisation of IL-10 during LILRA2/LPS Co-stimulation...... 134 4.3.5 Effect of LILRB4 on LPS-mediated Activation...... 134 4.4 DISCUSSION ...... 137
iv CHAPTER 5. EXPRESSION OF LILRS ON PERIPHERAL BLOOD LEUKOCYTES OF PATIENTS WITH RHEUMATOID ARTHRITIS ...... 141
5.1 INTRODUCTION ...... 141 5.2 MATERIALS AND METHODS...... 144 5.2.1 Patients...... 144 5.2.2 Antibodies and Reagents...... 144 5.2.3 Quantification of Leukocytes...... 145 5.2.4 Peripheral Blood Mononuclear Cells (PBMC) Extraction...... 145 5.2.5 Flow Cytometry ...... 145 5.2.6 Statistical Analyses ...... 146 5.3 RESULTS ...... 147 5.3.1 Patient Statistics...... 147 5.3.2 Quantitation of Circulating Leukocytes in Patients with RA vs. Control Subjects...... 150 5.3.3 Differential Expression of LILRs on Monocytes and Lymphocytes between Patients with RA and Control Subjects...... 154 5.3.4 Characterisation of LILR-positive Lymphocytes...... 162 5.4 DISCUSSION ...... 168
CHAPTER 6. DEVELOPING A MODEL TO INVESTIGATE THE SIGNALLING MECHANISM OF LILRB4...... 172
6.1 INTRODUCTION ...... 172 6.2 MATERIALS AND METHODS...... 174 6.2.1 Antibodies and Reagents...... 174 6.2.2 Cell Culture and Cross-linking FcεRI on RBL-2H3 Cells...... 174 6.2.3 Chimeric DNA Constructs and Transfection in RBL-2H3 Cells ...... 176 6.2.4 Statistical Analyses ...... 178 6.3 RESULTS ...... 179 6.3.1 Degranulation of RBL-2H3 Cells by Cross-linking of FcεRI...... 179 6.3.2 Stable Transfection of CD25/LILRB4 Chimera into RBL-2H3 Cells ...... 179 6.4 DISCUSSION ...... 183
CHAPTER 7. FINAL DISCUSSION...... 185
APPENDIX I. INHIBITION OF FCγRI BY LILRB4...... 191
CHAPTER 8. REFERENCES LIST...... 193
v ABSTRACT
The Leukocyte Immunoglobulin-like Receptors (LILRs) are a family of receptors that is broadly expressed on all leukocytes and have the ability to regulate their function. A substantial amount of evidence suggests that LILRs may be involved in immune homeostasis but also immune dysregulation. We therefore studied the role of LILRs in relation to the autoimmune disease, rheumatoid arthritis (RA). RA is a chronic and systemic inflammatory disease involving inflammation of the joints affecting the synovial membrane, cartilage and bone. Much of the tissue damage is a result of an inappropriate immune response within the joint space caused by the unwarranted activation of leukocytes. Here were report that LILRA2 (an activating receptor) that has been previously shown to be highly expressed in the rheumatoid synovium, induces the production of pro- inflammatory cytokines TNF-α, IL-1, IL-6, IFN-γ and IL-10 in primary monocytes. These cytokines are known to have an important role in the pathogenesis of RA indicating a pathway by which LILRA2 exacerbates RA. Co-ligation of LILRB4 (an inhibitory receptor) with LILRA2 abolishes cytokine production suggesting that LILRB4 is able to suppress the function of LILRA2. Expression of both LILRA2 and LILRB4 are regulated by inflammatory cytokines and LPS, indicative of a feedback mechanism. There is also cross-talk between LILRs and TLR4 as co-stimulation with LPS and either LILRA2 or
LILRB4 inhibits cytokine production. A differential expression of LILRs has also been identified on lymphocytes of patients with RA whereby an increase of LILRA1
(activating) and LILRB1 (inhibitory) expressing circulating lymphocytes is present in RA patients when compared to healthy control subjects. From these studies, we propose that
LILRs have a functional role in RA by regulating local and systemic inflammation. The
vi presence of LILRA2 in the RA joint is detrimental since its potent ability to induce inflammatory cytokines, particularly TNF-α, can initiate leukocyte recruitment and activation of proteases. Along with TLR4, LILRA2 and LILRB4 have the potential to moderate the innate immune system via regulation of cytokine production. Furthermore, suppression of LILRA2 function may serve as a therapeutic tool in many inflammatory diseases.
vii “It’s no wonder that truth is stranger than fiction. Fiction has to make sense.”
– Mark Twain
ACKNOWLEDGEMENTS
The patient study was done in collaboration with Professor H. Patrick McNeil, from the
Rheumatology Departments of Liverpool & Prince of Wales Hospitals. Many thanks to
Professor McNeil, Dr. Sean O’Neill and Dr. Jason Balgi, for the recruitment of patients as well as the collection of blood samples and clinical data. Also, thank you to the patients involved in this study (RA and control), for their kind participation.
LILRB4/CD25 mutant constructs used in this study were generated by Dr. Robert Liu and generously donated by Professor Jonathan P. Arm.
The results presented in Appendix I were taken from an honours thesis by Hao Kim Lu, titled; Leukocyte Immunoglobulin-Like Receptor B4 (LILRB4) Abrogates FcγRI (CD64)-
Mediated TNF-α Production by THP-1 cells. Graphs and figures were modified in line with the formatting of this thesis, though the raw data remains unchanged.
I would also like to acknowledge the following people; Dr. Kenneth Hsu, Dr. Yasumi
Endoh and Ms. Ainslie Mitchell for technical assistance in optimisation, validation of the real-time PCR protocol and DNA sequencing, Professor Andrew Lloyd for use of the differential cell counter, Dr. Ikuko Endoh for assistance on RBL-2H3 culture and stimulation, Dr. Taline Hampartzoumian for assistance on flow cytometry and multiplex
viii cytokine analysis, Dr. John E. Hunt for financial support and all IDRU members over the years for technical advice, moral support and friendships.
Many thanks to Arthritis Australia (board members and staff) for generously providing a scholarship during my candidature. I hope that my work will one day have some impact on achieving the goals of the foundation and that is, to make life better for those suffering from arthritis.
A very special acknowledgement to Pascal Carrive for without his hard work and support, this thesis would never have been completed. Pascal went beyond his call of duty to help me through all the disasters and so I would like to sincerely say, merci beaucoup.
PERSONAL THANKS
To all members of the IDRU over the years, I thank you all for making my post-graduate life so memorable. Being apart of this great research group has allowed me to understand how interactive research should be. The diverse amount of knowledge among the researchers is phenomenal and the critical reviews from the senior academics and fellow students have been greatly appreciated.
To my fellow students, you have all inspired me to go forward during the toughest times.
Together, we endured the hard hours, the challenging work and the minimal appreciation and thus, life as a PhD student. Thanks for all the technical advice over the years and the unconditional help. More importantly, thank you all for the personal advice too. Special
ix thanks to Anusha Hettiaratchi, Jessica Siegle, Natalia Cuan, Sharron Chow, Nicole
Jackson, Barbara Piraino, Beth Everett, Cristan Herbert and James Lazenby. Without you all, this journey would not have been as enjoyable and I probably would not have seen the end. You have all helped during the toughest time of my life and I am so proud to call you all friends.
Special thanks to Taline Hampartzoumian for being a surrogate supervisor by providing me with the most support and guidance, for which I will be forever grateful.
To my friends, past and present, I thank you all for helping me become the person I am today. I believe that we are defined by the friendships and bonds that we make during our lifetime so with that respect, I feel absolutely blessed with the friends that I have.
A mon cher partenaire, Ludovic Bernaudat. Tu as été mon inspiration durant cette thèse et je n’aurais jamais pu la réaliser sans toi. Tu m’as ouvert les yeux sur le monde et m’as changé en une personne meilleure. Merci du fond du cœur pour ton aide et ton support.
Je t’aime à jamais.
x PUBLICATIONS ARISING FROM CANDIDATURE
Huynh, O.A., Hampartzoumian, T., Arm, J.P., Hunt, J.E., Borges, L., Ahern, M., Smith,
M., Geczy, C.L., McNeil, H.P., Tedla, N. Down-regulation of Leucocyte Immunoglobulin- like Receptor Expression in the Synovium of Rheumatoid Arthritis Patients after
Treatment with Disease-Modifying Antirheumatic Drugs. Rheumatology. 2007 (May);
46(5):742-51.
CONFERENCES ATTENDED DURING CANDIDATURE
Huynh, O.A., Hampartzoumian, T., Arm, J.P., Borges, L., Geczy, C.L., McNeil, H.P.,
Tedla, N. Expression and Function of LILRA2 – Implications for Rheumatoid Arthritis
Pathogenesis. 8th World Congress on Inflammation (Oral Presentation), Copenhagen,
Denmark 2007.
Huynh, O.A., Hampartzoumian, T., Arm, J.P., Borges, L., Geczy, C.L., McNeil, H.P.,
Tedla, N. Expression and Function of LILRA2 – Implications for Rheumatoid Arthritis
Pathogenesis. Merck, Sharp and Dohme Poster Award (Poster Presentation), Research
Day, UNSW 2006.
Huynh, O.A. & Tedla, N. Regulation of the Leukocyte Immunoglobulin-like Receptors.
Australian Health and Medical Research Congress (Poster Presentation), Sydney
Exhibition Centre, Sydney, Australia 2004.
xi LIST OF IMPORTANT ABBREVIATIONS
μg Microgram
μL Microlitre
ANOVA Analysis of Variance
APC Allophycocyanin
APC Antigen-Presenting Cell
BSA Bovine Serum Albumin
CD Cluster of Differentiation
cDNA Complimentary Deoxyribonucleic Acid
CRP C-Reactive Protein
CsA Cyclosporin A
cysLT Cysteinyl Leukotriene
DAS Disease Activity Score 28
DC Dendritic Cell
Dex Dexamethasone
DMARD Disease Modifying Anti-Rheumatic Drug
D-PBS Dulbecco’s Modified Phosphate Buffered Saline
DNA Deoxyribonucleic Acid
EC Endothelial Cells
EDN Eosinophil-derived Neurotoxin
EDTA Ethylenediamine-tetraacetic Acid
ELISA Enzyme-linked Immunosorbant Assay
xii Etan Etanercept
FITC Fluorscein Isothiocynate
FcR Fc Receptor
HCMV Human cytomegalovirus
Hcq Hydroxychloroquine
HLA Human leukocyte antigen
IFN Interferon
IL Interleukin
ILT Immunoglobulin-like transcript
ITAM Immunoreceptor Tyrosine-based Activation Motif
ITIM Immunoreceptor Tyrosine-based Inhibitory Motif
KIR Killer Cell Immunoglobulin-like Receptor
LAIR Leukocyte-associated Immunoglobulin-like Receptor
Lef Leflunomide
LILR Leukocyte Immunoglobulin-like Receptor
LIR Leukocyte Immunoglobulin-like Receptor
LPS Lipopolysaccharide
LRC Leukocyte Receptor Complex
MFI Mean Fluorescence Intensity
MHC Major Histocompatability Complex
MIR Monocyte/Macrophage Immunoglobulin-like Receptors
mL Millilitre
xiii MMP Matrix Metalloproteinase
Mtx Methotrexate
NF-κB Nuclear Transcription Factor-kappa B
ng Nanogram
NK Natural Killer Cell
NSAID Non-Steroidal Anti-Inflammatory Drug
PBMC Peripheral Blood Mononuclear Cells
PE Phycoerythrin
PerCP Peridinin Chlorophyll Protein
pg Picogram
PMN Polymorphonuclear Cells
Pred Prednisone
PTK Protein Tyrosine Kinase
RA Rheumatoid Arthritis
RNA Ribonuclease
SEM Standard Error Mean
SD Standard Deviation
SHP SH2 domain-containing Protein Tyrosine Phosphatase
SHIP SH2 domain-containing Inositol Phosphate 5-phosphatase
SNP Single Nucleotide Polymorphism
Ssz Sulfasalazine
TLR Toll-like Receptor
xiv TNF Tumour Necrosis Factor
WBC White Blood Cell
xv LIST OF TABLES
Table 1.1 » Alternative names for LILRs...... 12
Table 1.2 » Structure of LILRs...... 14
Table 1.3A » Expression of LILRAs...... 17
Table 1.3B » Expression of LILRBs ...... 18
Table 1.4 » Ligands for LILRs ...... 21
Table 1.5 » LILRs grouped by MHC class I-binding potential...... 23
Table 1.6 » Ligands for each TLR and the signalling proteins that are utilised...... 49
Table 1.7 » Important effects of glucocorticoids on primary and secondary immune cells
...... 67
Table 2.1 » Base pair sequences of primer sets...... 80
Table 5.1 » Summary of age and sex for control subjects and patients with RA...... 148
Table 5.2 » Data for each individual RA patient...... 149
xvi LIST OF FIGURES
Figure 1.1 » ITAM-mediated signalling...... 6
Figure 1.2 » ITIM-inhibition of ITAMs upon co-ligation...... 7
Figure 1.3 » LRC on human chromosome 19q13.4 ...... 9
Figure 1.4 » Signalling pathways of KIRs ...... 43
Figure 1.5 » Intracellular signalling pathways of TLR4 ...... 51
Figure 1.6 » Pathogenesis of RA schematically represented in knee joint ...... 56
Figure 1.7 » Genomic actions of glucocorticoids...... 69
Figure 2.1 » Regulation of LILRA2 and LILRB4 expression by vitamin D3 ...... 83
Figure 2.2 » Expression of LILRA2 and LILRB4 on THP-1 monocytes after stimulation with cytokines and LPS ...... 85
Figure 2.3 » Expression of LILRA2 and LILRB4 on vitamin D3-differentiated- macrophages after stimulation with cytokines and LPS...... 86
Figure 2.4 » Expression of LILRA2 and LILRB4 on primary macrophages after stimulation with cytokines and LPS for 24 hours ...... 88
Figure 2.5 » Expression of LILRA2 and LILRB4 on primary macrophages after stimulation with cytokines and LPS for 48 hours ...... 89
Figure 2.6 » Expression of LILRA2 mRNA in primary monocytes after stimulation with
LPS ...... 91
Figure 3.1 » Expression of LILRA2 and LILRB4 on circulating lymphocytes and monocytes...... 105
Figure 3.2 » Antibody concentration-response and time-course for activation of cells via
LILRA2 ...... 107
xvii Figure 3.3 » Cytokine production induced by LILRA2 cross-linking and LILRA2-LILRB4 co-ligation...... 109
Figure 3.4 » Effects of anti-rheumatoid drugs on LILRA2-mediated activation in in vitro- derived macrophages ...... 111
Figure 3.5 » Analysis of apoptosis induced by anti-rheumatoid drugs ...... 112
Figure 3.6 » Cross-linking of LILRA2 on PBMCs from RA patients treated with or without glucocorticoids and in vitro dexamethasone stimulation ...... 114
Figure 4.1 » Cross-linking LILRA2 with LPS-stimulation in THP-1 cells ...... 126
Figure 4.2 » Cross-linking LILRA2 with LPS-stimulation in PBMCs...... 127
Figure 4.3 » Cytokine production induced by LILRA2 cross-linking and co-stimulation with LPS ...... 129
Figure 4.4 » Sequential effects of LILRA2 and LPS co-stimulation ...... 132
Figure 4.5 » Expression of TLR4 following LILRA2 cross-linking in primary monocytes
...... 133
Figure 4.6 » Neutralisation of IL-10 with LILRA2 and LPS co-stimulation...... 135
Figure 4.7 » Effect of LILRB4 on LPS-activation...... 136
Figure 5.1 » Analysis of white blood cell (WBC) counts and leukocyte subpopulations in patients with RA compared to healthy controls subjects...... 151
Figure 5.2 » Analysis of numbers of lymphocytes subpopulations in patients with RA .. 153
Figure 5.3 » Expression of LILRs on monocytes of patient with RA and healthy control subjects ...... 155
Figure 5.4 » Analysis of LILR expression on monocytes of patients with RA compared to healthy control subjects ...... 156
xviii Figure 5.5 » Expression of LILRs on lymphocytes of patient with RA and healthy control subjects ...... 158
Figure 5.6 » Analysis of LILR expression on lymphocytes of patients with RA compared to healthy control subjects...... 159
Figure 5.7 » Expression of LILRA1 and LILRB1 on lymphocytes of patients with RA compared to controls ...... 160
Figure 5.8 » Comparison of the percentage of LILRA1- and LILRB1-positive lymphocytes in patients with RA...... 161
Figure 5.9 » Correlation between LILR lymphocyte expression and disease activity...... 163
Figure 5.10 » Expression of LILRA1 and LILRB1 on lymphocyte populations of patients with RA ...... 166
Figure 5.11 » Expression of LILRA1 and LILRB1 on monocytes of patients with RA compared to controls ...... 167
Figure 6.1 » Schematic representation of LILRB4/CD25 chimeric protein and mutational changes of signalling tyrosines...... 177
Figure 6.2 » Degranulation of RBL-2H3 cells upon FcεR cross-linking...... 180
Figure 6.3 » Analysis of CD25/LILRB4 chimera expression on transfected RBL-2H3 cells
...... 182
xix Chapter 1. Literature Review
1.1 The Immune System and its Regulation
When the immune system is challenged by infectious microbes, it responds in an appropriate manner as to prevent the host from being overcome by the infection
(reactivity) (Ravetch et al., 2000). Under normal circumstances, this system is dormant
(quiescent) but at the same time remains alert for the unfortunate event in which microbes invade. Regulation of the immune system is a critical aspect of immune homeostasis, maintaining an equilibrious state between reactivity and quiescence (Ravetch et al., 2000).
Positive and negative signalling processes have an important role in the development of the immune system as well as immune responses to pathogens and these are attributed to activation and inhibitory receptors, respectively (reviewed in Healy et al., 1998; Bolland et al., 1999; Long, 1999; Ravetch et al., 2000). It is generally believed that the unchallenged immune system maintains a natural balance between the two opposing effects and as stated by Ravetch, pairing of activation and inhibitory receptors is necessary to modulate immune responses (Ravetch et al., 2000). However, there is insufficient understanding of how this system is regulated.
To overcome infection, there must be a rapid and specific response to eliminate the offending organism while protecting the host and once eliminated, responses must be counteracted to avoid unnecessary exhaustion (Long, 1999). Failure to maintain such tight regulation, thus resulting in excess reactivity, is proposed as one of the possible mechanisms which might contribute to the development of autoimmunity (Goodnow,
1 1997). In contrast, failure to mount an effective immune response may result in severe infection. Notably, there is no single event identified as being the cause of autoimmunity, so it is fair to assume that multiple factors would need to falter for the onset of an autoimmune disease to occur.
Commonly defined as an immune response directed towards “self antigens”, how and why autoimmunity is initiated is incompletely understood. It is generally accepted that autoimmunity occurs when there is a breakdown in tolerance involving certain major histocompatability complex (MHC)/human leukocyte antigen (HLA) haplotypes, co- stimulatory molecules, self-antigens as well as auto-reactive lymphocytes (reviewed in von
Bubnoff et al., 2002). Despite the strong correlation between MHC class II and autoimmune diseases, the role of MHC molecules on the onset of autoimmunity has not been well described. Certain HLA haplotypes are linked to autoimmune diseases, potentially altering the capacity of the receptor to present antigens and possibly favouring the maturation of self-reactive T cells whereas under normal circumstances, the deletion or negative selection of these populations of cells would lead to central tolerance (Ohashi et al., 1997; Healy et al., 1998). This all remains speculation as the neither the theory of a
‘self antigen’ nor the process of negative selection has been sufficiently proven.
This thesis focuses on a relatively new family of receptors named the leukocyte immunoglobulin-like receptors (LILRs) and their role in regulating immune responses.
There is a great deal of evidence to suggest that this family of receptors regulate leukocyte function in homeostasis and in diseases.
2 1.2 Pathways for Negative and Positive Signalling by Immune Cells
Intracellular signalling by immune receptors is a complex process involving the interaction of receptors with intracellular proteins and thus leading to either cellular activation or its inhibition (reviewed in Long, 1999). The motifs on the intracellular domain determine the function of the receptor upon ligation with its ligand. Immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are one class of immune-regulatory receptors that co-operatively control cellular activity and function.
1.2.1 Immunoreceptor Tyrosine-based Activation Motifs (ITAMs)
Upon cross-linking, ITAM-containing receptors such as the T cell receptor, B cell receptor and various Fc receptors, are able to induce positive signals which are crucial to immune responses. They have a dual role in that they activate leukocytes but at the same time are able to negatively select self-reactive lymphocyte clones (Healy et al., 1998). By comparing different types of activating receptors, a consensus sequence was identified for the ITAM: YxxL (Y-Tyrosine, L-leucine, x-any amino acid) (Reth, 1989).
ITAMs propagate cell signalling through tyrosine phosphorylation of the Src family kinases (Daeron et al., 1995). Initiation of activation by ligand interaction begins with poly-phosphorylation of ITAMs by src family of protein tyrosine kinases (PTKs), including fyn, blk and/or lyn. Consequently, phosphorylation opens the binding sites on
3 ITAMs for Src homology 2 (SH2)-domain containing kinases such as Syk family tyrosine kinase (Syk) in monocytes, or zeta-associated protein-70 (ZAP-70) in T cells. In the case of Syk-signalling in macrophages, Rho/Rac family of GTPases lead to reorganisation of the actin cytoskeleton while initiation of phopholipase C (PLC)-γ and phosphoinositide 3- kinase (PI3K) results in signalling via the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and calcium-dependent transcription factors pathways (Figure 1.1).
1.2.2 Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs)
Inhibitory receptors are crucial in terminating responses when they are no longer relevant or required. Potentially, a failure of inhibitory function could allow inflammatory cells to remain in an active state leading to unwanted tissue inflammation (Ravetch et al., 2000). It has been well established that the ability of ITIM-bearing inhibitory receptors to function is dependent on direct coupling with ITAM-bearing activation receptors to modulate immune responses (Daeron et al., 1995; Blery et al., 1999). It is no longer believed that termination of activating responses is due to down-regulation of the activating receptors but a dominant affect of inhibitory receptors to suppress ITAM-signalling (Bolland et al.,
1999; Long, 1999; Ravetch et al., 2000). Depicted in Figure 1.2 is the classical interaction of ITIM-mediated suppression of an ITAM-bearing receptor, whereby ITIMs inhibit
ITAM-dependent phosphorylation events via the recruitment of either SH2 domain- containing protein tyrosine phosphatase (SHP)-1, SHP-2 or SH2 domain-containing inositol phosphate 5-phosphatase (SHIP) (reviewed in Binstadt et al., 1996; Burshtyn et al., 1996; Malbec et al., 1998). SHP-1 binds ITIMs and functions by removing phosphate
4 groups added to ITAMs and other signalling intermediates by PTKs, thus reversing ITAM signalling (reviewed in Blery et al., 1998; Maeda et al., 1998; Maeda et al., 1999).
SHIP has multiple modes of inhibition, the first characterised mechanism being the removal of 5’ phosphate from phosphatidylinositol triphosphate (Wisniewski et al., 1999) and thus inhibition of downstream signalling the ITAM cascade via blocking of phopholipase C (PLC)-γ and Bruton's tyrosine kinase (Btk) (Figure 1.2). The net effect of
ITIM-mediated inhibition is an arrest of ITAM-triggered calcium mobilisation and cellular proliferation (Daeron et al., 1995; Ono et al., 1996; Malbec et al., 1998). Alternatively,
SHIP can inhibit signalling of the anti-apoptotic AKT and RAS-MAPK pathways though less is known about these mechanisms (Takai, 2002).
5
Figure 1.1 » ITAM-mediated signalling. Cross-linking of ITAMs induce phosphorylation of motifs and subsequent activation of Syk, Rho/Rac and SRC-family kinases leading to Ca2+-dependent transcriptional events and signalling via the MAPK pathway. DG = diacylglcerol, ADCC = Antibody- dependent cell-mediated cytotoxicity. Modified from (Takai, 2002).
6
Figure 1.2 » ITIM-inhibition of ITAMs upon co-ligation. ITIMs can recruit SHIP phosphatases that inhibit ITAM-associated kinases by dephosphorylation of adaptor proteins such as Btk and PLCγ.
BLNK = B cell linker protein, BCAP = B-cell PI3-K adaptor. Modified from (Takai, 2002).
7 1.3 The Leukocyte Immunoglobulin-like Receptors (LILRs)
The leukocyte immunoglobulin-like receptors (LILRs) are a family of immunoregulatory receptors that have emerged within the past decade. They belong to the immunoglobulin superfamily class of receptors, characterised by multiple highly homologous extracellular immunoglobulin-like domains. The current literature suggests that activating and inhibitory LILRs may have a role in maintaining control of immune responses. LILRs are broadly expressed on leukocytes and can regulate various effector cells functions during immune responses (reviewed in Long, 1999; Brown et al., 2004). For many of the LILRs, their ligand/s remains unidentified.
The LILR genes are located on the leukocyte receptor complex (LRC) (Wagtmann et al.,
1997; Wende et al., 1999) at the chromosomal location of 19q13.4. LILRs are encoded in two inverted clusters containing sequences for 11 receptors and 2 pseudogenes (Figure 1.3) and the clusters are translated in opposite directions (Torkar et al., 1998; Volz et al., 2001).
Having some similarities to the MHC complex on chromosome 6 (Martin et al., 2002), the
LRC also encodes other cell surface receptors such as the killer cell immunoglobulin-like receptors (KIRs), Fc receptor for IgA (FcαR), platelet collagen receptor glycoprotein
(gpVI) and the leukocyte-associated immunoglobulin-like receptors (LAIRs) (Volz et al.,
2001), some of which are also involved in regulating cellular immune responses (reviewed in Martin et al., 2002; Bashirova et al., 2006). It is suggested that the formation of the two
LILR loci is a result of inverse duplication of a single locus; hence the high homology observed between receptors (Norman et al., 2003). It is likely that some LILRs may be redundant in function.
8
Figure 1.3 » LRC on human chromosome 19q13.4. Configuration of genes on LRC and the arrows indicate the direction of sequence, either centromeric (cen) or telomeric (tel). Modified from
(Bashirova et al., 2006).
9 Extensive variability in the LRC, particularly in the LILR segments, has prompted many studies to investigate functional polymorphisms and genetic variants (Wilson et al., 2000).
Polymorphisms in LILRB1, LILRB2, LILRB3, LILRB4, LILRA2 and LILRA3 have been identified (Colonna et al., 1997; Heinzmann et al., 2000; Norman et al., 2003;
Papanikolaou et al., 2004; Mamegano et al., 2008; Chang et al., 2008) as well as splice variants of LILRA1 (Borges et al., 1997), LILRA5 (Borges et al., 2003) and LILRB2
(Beinhauer et al., 2004). The significance of polymorphisms and variants have not been extensively studied, however for LILRB1 and LILRB2, changes in amino acid sequence are predicted to affect ligand binding but are yet to be shown (Colonna et al., 1997;
Norman et al., 2003). In the LILRB4 gene, at least 15 SNPs have been identified in a 3.6kb region, 12 of which result in an amino acid change and further linkage studies shows that many mutations are in linkage disequilibrium (Chang et al., 2008). Thus, it was hypothesised that certain polymorphisms/variants of LILRs could lead to the onset of autoimmune diseases such as psoriasis, coeliac disease, multiple sclerosis and systemic lupus erythematosus (SLE) (Moodie et al., 2002; Wisniewski et al., 2003; Koch et al.,
2005; Mamegano et al., 2008). One highly characterised mutation is a 6.7-kbp deletion identified in the exclusively soluble receptor, LILRA3 (Torkar et al., 2000; Wilson et al.,
2000). Population studies of genotype frequencies have found approximately 36% of
Caucasian people (Torkar et al., 2000) and 80% of Japanese people (Hirayasu et al., 2006) lack the complete LILRA3 DNA sequence. It is tempting to speculate that this deletion may alter their susceptibility to diseases, whether this may be beneficial or not.
10 A single nucleotide polymorphism (SNP) identified in the LILRA2 gene (GA) at the exon 6-intron 7 junction is thought to alter its translation and function of the receptor
(Mamegano et al., 2008). In a Japanese population, heterozygotes for the SNP (A/A) constituted less than 5% of the group studied and when screened in patients with either rheumatoid arthritis (RA), SLE or microscopic polyangitis (MPA), a significant association between the A/A genotype was made towards susceptibility of SLE and MPA
(Mamegano et al., 2008).
There are no distinct rodent homologues to many of the LILRs, though based on structure and function of the receptors, the murine paired immunoglobulin-like receptor (PIR)-B is classed as an orthologue to LILRB1 and LILRB3 (Kubagawa et al., 1997; Martin et al.,
2002) and the inhibitory gp49B1 receptor is an orthologue of LILRB4 (Arm et al., 1997) and is often referred to as murine LILRB4 (mLILRB4) (Katz, 2006).
1.3.1 Nomenclature of LILRs
A common obstacle encountered when studying the LILRs relates to the lack of use of the common nomenclature. Each receptor has multiple names and this is outlined in Table 1.1.
The alternate names for each receptor arose from independent studies and discoveries of the LILR family (Arm et al., 1997; Cosman et al., 1997; Samaridis et al., 1997; Wagtmann et al., 1997). In 2001, the various names were abandoned and officially renamed to LILRs and classified into 3 groups; LILRA (activating), LILRB (inhibitory) and LILRP
(pseudogenes) (Long et al., 2001). In this classification, the soluble LILRA3 receptor was grouped with the activating receptors.
11 Table 1.1 » Alternative names for LILRs.
LILR name Alternate names
LILRA1 LIR-6, CD85i
LILRA2 LIR-7, ILT-1, CD85h
LILRA3 LIR-4, ILT-6, CD85e, HM31/43
LILRA4 ILT-7, CD85g
LILRA5 LIR-9, ILT-11, CD85f
LILRA6 ILT-8, CD85b
LILRB1 LIR-1, ILT-2, CD85j, MIR-7
LILRB2 LIR-2, ILT-4, CD85d, MIR-10
LILRB3 LIR-3, ILT-5, CD85a, HL9
LILRB4 LIR-5, ILT-3, CD85k, HM18
LILRB5 LIR-8, CD85c
LILRP1 ILT-9, CD85l
LILRP2 ILT-10, CD85m
Each row represents the multiple names for the same receptor.
12 1.3.2 Structure of LILRs
The extracellular region of all LILRs is comprised of 2 or 4 highly homologous C-2 type immunoglobulin-like domains (Table 1.2). Each receptor is defined by their intracellular region, or lack thereof, and is described as being activation, inhibitory or soluble (Colonna et al., 1999). The activating group of LILRAs possess a positively-charged amino acid residue (arginine) in the transmembrane region and this allows ionic association with the
ITAM-bearing common γ-chain of Fc receptors (FcRγ) through which they have been shown to function (Nakajima et al., 1999). The inhibitory LILRBs encode 2-4 ITIMs on their intracellular domain (Table 1.2).
LILRA3 is an exclusively soluble receptor with the highest homology to the extracellular domain of LILRB1 and it is postulated to share ligands with some LILRs (Borges et al.,
2000). Other soluble forms have also been identified, either due to multiple gene variants
(LILRA5) (Borges et al., 2003) or post-translational cleavage of the membrane-bound protein (LILRB2) (Beinhauer et al., 2004) and these receptors have the potential to act as an antagonist to their cell-associated forms.
13 Table 1.2 » Structure of LILRs.
LILR Extracellular TM Intracellular Homology to LILRB1*
81% +
LILRA1
+
LILRA2 + 79%
LILRA3 84%
+ LILRA5 59%
LILRB1 100%
LILRB2 82%
LILRB3 70%
LILRB4 63%
LILRB5 -
14 Schematic representation of intracellular/extracellular domains of LILRs. LILRAs (activating) receptors have a positively charged arginine in the transmembrane region (TM) and LILRBs
(inhibitory) have multiple ITIMs on their intracellular domain. *Homology of each receptor compared to LILRB1 is based on the whole amino acid sequence of their respective extracellular domains
(Borges et al., 2000).
15 1.3.3 Expression of LILRs
LILRs are differentially expressed on various types of leukocytes (Table 1.3A and B) and the significance of this expression pattern is yet to be explored. An early study into LILR expression was conducted using cell lines and peripheral blood leukocytes where mRNA was isolated from cells of pure culture and analysed for expression (Borges et al., 1997). In this study, LILRs were identified on monocytes, B cells, NK cells and dendritic cells.
Following the production of antibodies, protein studies confirmed cell surface expression of LILRs on all primary leukocytes and this was performed primarily by flow cytometry
(Fanger et al., 1998; Nakajima et al., 1999; Tedla et al., 2003). It is likely that the pattern of LILR expression alters during the developmental stages and maturation of leukocytes which may be essential in balancing the threshold of activation of cells. Further characterisation and functional studies are required to determine this.
More recently, LILRs have been identified on endothelial cells (ECs) in transplantation and joint inflammation. Endomyocardial biopsies taken from heart transplant patients revealed a greater intensity of staining for LILRB4 in rejection-free patients compared to patients who exhibited signs of rejection (Manavalan et al., 2004). Synovial biopsies from
RA patients exhibited intense staining for LILRA2 (Tedla et al., 2002). This further supports their role in immune-regulation since ECs have key roles in inflammation via interaction with the leukocytes for activation signals, adhesion and migration into tissues
(reviewed in Cook-Mills et al., 2005). Potentially, LILRs can alter the expression of co- stimulatory or adhesion molecules and influence the leukocyte-EC interface.
16 Table 1.3A » Expression of LILRAs.
LILRA1 LILRA2 LILRA3 LILRA4 LILRA5
(Nakajima et al., (Tedla et al., 2003; monocytes 1999; Tedla et al., - - (Borges et al., 2003) Tedla et al., 2007) 2007)
(Nakajima et al., (Nakajima et al., macrophages 1999), RS (Tedla et 1999), RS (Tedla et - - - al., 2002) al., 2002)
T cells - - - - -
m (Borges et al., m (Borges et al., B cells (Fanger et al., 1998) - - 1997) 1997)
(Nakajima et al., m (Borges et al., NK cells - - - 1999) 1997)
eosinophils - (Tedla et al., 2003) - - -
m (Borges et al., neutrophils - (Tedla et al., 2003) - - 2003)
(Ju et al., 2004; Cao DCs - - - - et al., 2006)
ECs - (Tedla et al., 2002) - - -
Expression of LILR on different leukocytes and endothelial cells with corresponding references. Protein or mRNA expression of LILRs has been identified in following cell types. m = mRNA identified, i = protein expression is inducible, dash (-) = negative for LILR expression. 17 Table 1.3B » Expression of LILRBs.
LILRB1 LILRB2 LILRB3 LILRB4 LILRB5 monocytes (Fanger et al., 1998; (Fanger et al., 1998; m (Borges et al., m (Borges et al., (Tedla et al., 2003; Tedla et al., 2003; Tedla et al., 2007) 1997), (Tedla et al., 1997), (Tedla et al., Tedla et al., 2007) Tenca et al., 2005; 2003; Tedla et al., 2003; Tedla et al., Tedla et al., 2007) 2007) 2007) macrophages ND RS (Tedla et al., RS (Tedla et al., - - 2002) 2002) T cells (Fanger et al., 1998; - - - - Saverino et al., 2000) B cells (Fanger et al., 1998) - m (Borges et al., m (Borges et al., - 1997) 1997) NK cells (Fanger et al., 1998) - - m (Borges et al., m (Borges et al., 1997) 1997) eosinophils (Tedla et al., 2003) - (Tedla et al., 2003) - - neutrophils (Tedla et al., 2003) - (Tedla et al., 2003) - - DCs (Fanger et al., 1998; (Fanger et al., 1998) - m (Borges et al., - Tenca et al., 2005) 1997), (Cella et al., 1997; Cella et al., 1999) ECs - i (Manavalan et al., - i (Manavalan et al., - 2004) 2004)
Expression of LILR on different leukocytes and endothelial cells with corresponding references. Protein or mRNA expression of LILRs has been identified in following cell types. m = mRNA identified, i = protein expression is inducible, dash (-) = negative for LILR expression, ND = not done. 18 Little is known about the regulatory mechanisms of LILR expression. Promoter region analysis could reveal the transcription factors involved in inducing expression and give some insight to the biomolecular pathways that could regulate protein expression.
Moreover, investigations into the regulation of LILR expression may also help define some of the more specific function/s of LILRs. Active vitamin D3 (1α, 25-dihydroxyvitamin D3) is a major calcium mobilising hormone (Deluca et al., 2001), commonly used in in vitro cultures to differentiate cells into activated effector cells, e.g. monocytes to macrophages
(Kreutz et al., 1993). Vitamin D3 positively regulates cell surface expression of LILRB1 and LILRB4 on immature dendritic cells (DCs) but on DCs primed with lipopolysaccharide (LPS) or endotoxin, vitamin D3 increases only LILRB4 expression
(Manavalan et al., 2003; Penna et al., 2005). Immunoregulatory cytokines such as interleukin (IL)-10 and interferon (IFN)-α induce mRNA expression of LILRB2 and
LILRB4 on stimulated immature DCs (Kim-Schulze et al., 2006). Another study found both in vivo stimulation of psoriasis patients and in vitro stimulation of purified monocytes with IL-10 increased mRNA levels of LILRA1, LILRA2, LILRA3, LILRB2, LILRB3 and
LILRB4 as measured by an Affymetrix DNA chip (Jung et al., 2004). Stimulation of DCs with IL-10 and vitamin D3 induces tolerance suggesting that inhibitory LILRs may have a role in regulating T helper cell function (Steinbrink et al., 1997; Penna et al., 2000;
Levings et al., 2001; Jung et al., 2004). Similarly, IL-10 induces LILRB4 expression on
ECs and so LILRB4 may be potentially involved in inflammatory responses relating to leukocyte migration through the endothelium (Gleissner et al., 2007). Cytokine-regulation of LILR expression has been primarily investigated on DCs and ECs and so further studies on other cell types and with other cytokines is required.
19 1.3.4 Ligands of LILRs
There have been extensive investigations into the binding partners of LILRs and only
LILRB1, LILRB2 and LILRA1 have identified human ligands which are HLA class I molecules (Table 1.4). However for all other LILRs, their ligands still remain to be identified. By studying the binding site of LILRB1 to HLA-A and aligning the binding site sequence to other LILRs, certain LILRs are predicted to also bind HLA (Willcox et al.,
2003). Thus, LILRs are divided into 2 groups based on their binding potential to HLA class I (Table 1.5). It should be emphasised that this grouping is derived from amino acid sequence analysis and therefore, protein binding is required to support these hypotheses.
Upon scrutinising of these ligand-binding studies, there are some concerns regarding the specificity of LILR interactions with and HLA molecules. The recombinant LILRs used are often produced in non-mammalian systems, most often in bacteria and in particular,
Escherichia coli. Mis-folding of proteins is a primary concern and particularly in the case of LILRA2 and LILRB4, remains a great obstacle for determination of their ligand/s
(Garner et al., 2005; Chen et al., 2007). Some studies have also concentrated on the predicted binding domains of LILRs to produce a truncated protein in E.coli which consists of only the relevant immunoglobulins, mostly domains D1 and D2 (Willcox et al.,
2002; Shiroishi et al., 2003). It is difficult to determine whether exclusion of half the extracellular protein affects the receptor-ligand binding stability or the conformational structure of the receptor itself.
20 Table 1.4 » Ligands for LILRs.
LILRB1 LILRB2 LILRB3 LILRA1
(Colonna et al., 1997; Cosman et al., 1997; HLA-A Colonna et al., 1998; (Fanger et al., 1998) Fanger et al., 1998; Willcox et al., 2003)
(Colonna et al., 1997; Cosman et al., 1997; (Colonna et al., 1998; HLA-B (Allen et al., 2001) Fanger et al., 1998; Fanger et al., 1998) Chapman et al., 1999)
(Colonna et al., 1998; Fanger et al., 1998; HLA-C (Fanger et al., 1998) Chapman et al., 1999; Lepin et al., 2000)
(Chapman et al., 1999; HLA-E Navarro et al., 1999)
21 HLA-F (Lepin et al., 2000) (Lepin et al., 2000)
(Colonna et al., 1997; Chapman et al., 1999; (Allan et al., 1999; HLA-G Navarro et al., 1999; Shiroishi et al., 2006; Shiroishi et al., 2006; Shiroishi et al., 2006) Shiroishi et al., 2006) murine (Liang et al., 2006) MHC (Borges et al., 1997; UL18 Cosman et al., 1997; (Willcox et al., 2002) Chapman et al., 1999) E.coli (Nakayama et al., 2007) S.aureus (Nakayama et al., 2007) (Nakayama et al., 2007)
Known ligands for LILRs and corresponding references.
22 Table 1.5 » LILRs grouped by MHC class I-binding potential.
Group 1 Group 2
A1* A4
A2 A5
A3 A6
B1* B3
B2* B4
B5
LILRs are theoretically divided into 2 groups based on potential binding to MHC Class I. Group 1 receptors are likely to bind MHC class I while group 2 receptors are believed to have non-MHC ligands, as predicted by (Willcox et al., 2003). * confirmed binding. Adapted from (Willcox et al., 2003).
23 MHC class I receptors are responsible for the antigenic-presentation of endogenously processed peptides and self recognition by the hosts’ immune system (reviewed in Kumar et al., 2005; Parham, 2005; Aptsiauri et al., 2007). Cell death induced by HLA class I signalling is mediated via T lymphocytes and is dependent on the presence of co- stimulatory receptors (Diefenbach et al., 2002). This process is attributed to the function of natural killer (NK) cells and their recognition of HLA class I is via the KIR family of receptors (Section 1.4.1). A down-regulation of HLA class I expression on the surface of cells and can lead to specific killing by the NK cells and this is explained by the ‘missing self’ hypothesis which is a protective mechanism to prevent tumour growth and viral replication whereby HLA class I is down-regulated under both circumstances (Ljunggren et al., 1990). However, there are other mechanisms by which tumours and viruses evade the immune system, particularly by utilising various inhibitory receptors.
Both classes of MHC are inheritably polymorphic and the combination of particular polymorphisms in the MHC and KIR loci have been associated with various disease processes (reviewed in Parham, 2005). Variability in receptor-receptor binding due to polymorphisms could alter the threshold of antigen-recognition and activation of lymphocytes. HLA-B27 is a class I MHC that is linked to various inflammatory diseases and has the strongest correlation to the development of spondyloarthritis (reviewed in
Bowness et al., 1999). A UK study reported the presence of the HLA-B27 allele in 94% of patients with ankylosing spondylitis, compared to 9.5% of controls subjects, demonstrating a strong association (Brown et al., 1996). Although its role is not completely understood, a common theory is that HLA-B27 via its antigen-presenting function causes the expansion
24 of self-reactive T cell clones which allows progression of inflammation (Pazmany et al.,
1992; Hermann et al., 1993). Common questions raised, are: what is the athritogenic peptide and why HLA-B27 (Ivanyi, 1993; Bowness et al., 1999; de Castro, 2007)? HLA-
B27 signalling induces inhibition of IFN-γ production in target cells and is dependent on the peptide bound between the receptors (Kollnberger et al., 2007). HLA-B27 is able to bind LILRA1, LILRB1, LILRB2 (Colonna et al., 1997; Colonna et al., 1998; Allen et al.,
2001) and KIRs (Gumperz et al., 1995; Kollnberger et al., 2002), and their interaction could potentially induce autoreactivity in expressing T cells. Binding studies in which leukocyte subsets isolated from patients with spondyloarthritis were incubated with
+ recombinant dimeric HLA-B27 revealed an interaction with CD14 monocytes, subsets of
+ + + CD4 and CD8 T cells and CD19 B cells (Kollnberger et al., 2002). By using neutralising antibodies on T cells and macrophages, it was found that HLA-B27 binding was attributed to LILRB2 instead of LILRB1 (Kollnberger et al., 2002). However in B cells, the ligand could not be identified in this study but presumably, this binding is with
LILRA1 given that recombinant HLA-B27 complexes bind to LILRA1-transfected cells
(Allen et al., 2001).
1.3.5 Function of LILRs
The LILR family of receptors is believed to regulate immune responses by signalling through their respective intracellular motifs to modulate leukocyte function. However, their functions are not fully elucidated. Although extensive in vitro studies provides understanding of their regulatory potential, without complete knowledge of their ligands and signalling pathways, their pathological and molecular role in vivo remains to be
25 determined. Comparatively, less is known about the activating LILRs than their inhibitory counterparts. The broad expression of LILRs on both myeloid and lymphoid cells (Table
1.3) implies that they may function in both adaptive and innate immune responses.
1.3.5.1 Activating LILRs (LILRA)
There are limited studies into the expression and function of LILRA1 and therefore, little is known about its role in homeostasis or disease. LILRA1 is believed to have a role in HLA-
B27-associated diseases (Allen et al., 2001), but the extent of its expression and function requires further investigation.
LILRA2 is able to form a receptor-receptor complex with the FcεRI γ-chain in order to transduce ITAM-mediated signals (Nakajima et al., 1999). In transfected basophils,
LILRA2 is able to cause degranulation upon antibody ligation (Nakajima et al., 1999; Cao et al., 2006). A similar effect has been observed on peripheral blood-purified basophils
(Sloane et al., 2004) and eosinophils (Tedla et al., 2003) to initiate degranulation and the de novo production of cytokines. On monocytes and macrophages, LILRA2 cross-linking using monoclonal antibodies induces the production of acute phase proteins such as tumour necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 (Borges et al., 2003; Huynh et al., 2007) and can therefore function in acute immune responses.
The only exclusively soluble receptor within the family is LILRA3 which lacks the sequence of a transmembrane region and is therefore assumed to be putatively secreted by cells (Arm et al., 1997; Borges et al., 1997; Colonna et al., 1997). Protein has not been
26 identified and so its existence remains purely at the genetic level. The isolation of mRNA from B and NK cells suggests that protein exists but the extent of expression is yet to be determined (Borges et al., 1997). With 84% homology in amino acid sequence to the extracellular domain of LILRB1, it is hypothesised that LILRA3 may act as a receptor antagonist to LILRB1 by competitively binding HLA (Borges et al., 1997). The potential for LILRA3 to block LILRB1-mediated inhibition suggests that the absence of functional
LILRA3 could produce an excess of inhibitory signals and thus, LILRA3 mutations were investigated in coeliac disease (Moodie et al., 2002) and psoriasis (Wisniewski et al.,
2003), but no association was discovered. A weak association was however found in a
German cohort of patients with multiple sclerosis (MS) (Koch et al., 2005). It is suggested that homozygous deletion of LILRA3 could be one of many inherited predisposing genes which contributes to MS susceptibility.
LILRA4 is exclusively expressed on plasmacytoid DCs (pDCs) but not on monocyte- derived DCs (Rissoan et al., 2002; Ju et al., 2004). It also signals via its association with
FcεRI γ-chain (Cao et al., 2006) and cross-linking of cell surface LILRA4 on pDCs activates ITAM-mediated signalling by phosphorylation of Syk and Src family kinases to trigger calcium mobilisation (Cao et al., 2006). LILRA4 can counter responses induced by
Toll-like receptors (TLRs) in DCs by inhibiting IFN and TNF production after stimulation of TLR7 and TLR9 (Cao et al., 2006; Cho et al., 2008).
27 Assumed to also signal through the FcR-γ chain, cross-linking of LILRA5 induces TNF-α,
IL-1β and IL-6 production in monocytes (Borges et al., 2003). Further studies are required to investigate the function of this receptor.
1.3.5.2 Inhibitory LILRs (LILRB)
The role of inhibitory LILRs has been well described (reviewed in Katz, 2006) and most of the knowledge is based on extensive in vitro research on LILRB1. By binding to MHC class I, LILRB1 inhibits cellular function via recruitment of SHP-1 phosphatases to intracellular ITIMs (Colonna et al., 1997; Cosman et al., 1997; Fanger et al., 1998;
Dietrich et al., 2001; Sayos et al., 2004). Mutational analysis identified the most distal of the 4 ITIMs (i.e., the ITIM farthest from the membrane) is the most important motif for mediating the inhibitory function of LILRB1 (Bellon et al., 2002).
Inhibition via LILRB1 occurs on B cells and T cells, suggesting a role in adaptive immune responses. In these studies, ligation was done using monoclonal antibodies (mAbs). Co- ligation of LILRB1 to the B cell receptor (BCR) inhibits BCR-mediated activation
(Colonna et al., 1997) but also immunoglobulin class-switching which is essential for humoral immune development (Merlo et al., 2005). LILRB1 ligation directly blocks the clonal expansion of antigen-specific T cells, hindering adaptive immune responses and inducing tolerance (Saverino et al., 2000; Nikolova et al., 2002). This is interesting as T cell surface expression of LILRB1 never exceeds 4% of all CD3-positive lymphocytes extracted from whole blood (Fanger et al., 1998; Saverino et al., 2000) even though
LILRB1 is present intracelullarly in all T cells (Saverino et al., 2000). Surface expression
28 may potentially correlate with disease development. LILRB1 on antigen-presenting cells
(APCs) also has the potential to inhibit T cell receptor activation by co-localising to the immunological synapse and preventing both phosphorylation of associated kinases and actin cytoskeleton reorganisation (Dietrich et al., 2001). These are important events for cytokine production and subsequent T cell function (Valitutti et al., 1995; Holsinger et al.,
1998). On natural killer (NK) cells, LILRB1 is able to inhibit targeted cell lysis (Cerboni et al., 2006). In DC, LILRB1 can block the novel immune receptor, human osteoclast- associated receptor (hOSCAR) and indirectly inhibit T cell proliferation and T cell- mediated responses (Tenca et al., 2005). Like some LILRAs, hOSCAR associates with the
γ-chain for FcRs to stimulate T cell-dependent cytokines such as IL-8 and IL-12 to which
LILRB1 is able to inhibit (Merck et al., 2004; Tenca et al., 2005). The generation of
LILRB1-transgenic mice also provides insight to function. Cellular expression of LILRB1 on lymphocytes, NK cells and thymocytes of transgenic mice severely impairs T cell development and function (Liang et al., 2006). Interestingly, this has pathological relevance to transplantation whereby allogeneic-skin graft survival was greatly increased by the presence of LILRB1, not only delaying but preventing graft-rejection altogether
(Liang et al., 2006).
LILRB2 is 82% identical to LILRB1 in amino acid sequence (Borges et al., 2000) and also binds many MHC class I molecules (indicated in Table 1.4) but binds with much less affinity (Borges et al., 1997; Willcox et al., 2002; Shiroishi et al., 2003). Similarly,
LILRB2 is able to recruit SHP-1 to mediate inhibitory function (Fanger et al., 1998).
Given that LILRB1 and LILRB2 are co-expressed on monocytes, these receptors may
29 compete for ligands and may co-operatively or competitively influence cellular function.
2+ LILRB2 inhibits FcR-mediated signalling, Ca mobilisation (Fanger et al., 1998) and the release of serotonin in transfected RBL cells (Colonna et al., 1998). Like LILRB4,
LILRB2 is often studied in the context of transplant immunology. Soluble LILRB2 is produced by DCs following IL-10 treatment, rendering the cells tolerogenic by potentially blocking signalling via the membrane bound receptor (Beinhauer et al., 2004).
LILRB3 has 70% homology to LILRB1 (Borges et al., 2000) and fails to recognise any
HLA forms since differences are primarily in the HLA-binding site of domain D1 (Willcox et al., 2003). However, LILRB3 does inhibit the in vitro stimulation of LILRA2- and
FcεRI-mediated activation in primary basophils to produce and release allergic-mediators such as histamine, cysteinyl leukotrienes (cysLT) and IL-4 (Sloane et al., 2004). Like
LILRB2, polymorphisms in LILRB3 can potentially affect the threshold of its function
(Papanikolaou et al., 2004).
LILRB4 is an inhibitory receptor which has become increasingly studied, particularly in the context of transplant immunology. LILRB4 is a relatively small protein with only two immunoglobulin-like domains on its extracellular region (Table 1.2) and is primarily expressed on monocytes, macrophages and dendritic cells (Table 1.3B). Its inhibitory function was first characterised in monocytes and macrophages, inhibiting calcium mobilisation due to CD11b, MHC class II and CD16 in vitro stimulation (Cella et al.,
1997) and subsequently, inhibition of downstream NFκB signalling (Chang et al., 2002).
30 LILRB4 exerts inhibitory function via recruitment of SHP-1 and is thus likely to suppress associated-ITAMs via de-phosphorylation (Cella et al., 1997).
LILRB4 is a characteristic marker for DCs (Cella et al., 1997; Cella et al., 1999) and is believed to have an important role in the tolerisation of DCs (reviewed in Suciu-Foca et al., 2005; Vlad et al., 2008; Vlad et al., 2009). It is up-regulated on functional or
+ - + tolerogenic DCs following stimulation by CD8 CD28 FOXP3 T suppressor cells (TS)
(Chang et al., 2002; Manavalan et al., 2003; Suciu-Foca et al., 2003). LILTB4 is potentially involved in the uptake of antigens as it becomes internalised after cross-linking
(Cella et al., 1997). Downstream signalling of LILRB4 as well as LILRB2 on tolerogenic
+ + + DCs induces anergy in CD4 CD45RO CD25 T regulatory cells (TR) (Chang et al., 2002;
Manavalan et al., 2003). The functional capacity of LILRB2 and LILRB4 is extended to
+ + ECs, where similar interactions between LILRB2 LILRB4 ECs with TR and TS tolerises the ECs (Cortesini et al., 2004) and down-regulates expression of co-stimulatory and adhesion molecules on ECs, such as CD40, CD54, CD58, CD62E, CD83 and CD106
(Manavalan et al., 2004). Expression of LILRB2 and LILRB4 on DCs is tightly regulated by inflammatory stimuli, cytokines and growth factors (Manavalan et al., 2003; Ju et al.,
2004; Penna et al., 2005). It has been consistently shown that active vitamin D3 increases expression of LILRB4 on DCs, while IL-10 and IFN-α increase both LILRB4 and LILRB2 expression both on DCs and ECs (Manavalan et al., 2003; Manavalan et al., 2004; Penna et al., 2005; Gleissner et al., 2007). Interestingly, there is an inverse pattern of expression between inhibitory LILRs and the co-stimulatory molecules, CD86 (Chang et al., 2002),
CD40 and CD58 (Manavalan et al., 2004). Co-stimulatory molecules function by
31 providing necessary signals to antigen-specific lymphocytes and antigen-presenting cells
(APCs) to promote growth, differentiation and cytokine production, and these events are critical to immune responses (reviewed in Kroczek et al., 2004).
Extensive research implicates a role for LILRB4 and LILRB2 in tissue graft acceptance during transplantation, specifically host-versus-graft disease. In general, transplant rejection occurs as T cells and NK cells from the host recognise foreign MHC molecules expressed on the graft tissue (Shlomchik, 2007). The ability for MHC to bind and signal to allo-reactive cells is believed to be largely caused by inhibitory receptors inducing anergy in effector cells thus leading to unresponsiveness when ‘self’ MHC is encountered again, as seen with KIRs (Section 1.4.1). There is a potential for LILRB2 to directly function in this process since it is known that LILRB2 binds MHC (Colonna et al., 1998; Fanger et al.,
1998) and its signalling may be MHC-dependent. But for LILRB4, it either functions indirectly or independently of MHC as they do not bind. In studies of organ transplant recipients, the ability for the host to avoid graft-rejection is dependent on the ability for
+ - host Ts cells, namely CD8 CD28 T cells, to induce LILRB2 and LILRB4 expression in donor APCs (Chang et al., 2002; Suciu-Foca et al., 2003) and ECs (Manavalan et al.,
2004). In these studies, Ts cells were isolated from transplant recipients and the majority of rejection-free patients had Ts cells capable of inducing LILRB2 and LILRB4 mRNA and protein expression in APCs when co-cultured. Conversely, Ts cells from patients with symptoms of acute rejection failed to induce either LILRB2 or LILRB4 expression. These
LILRB2 and LILRB4-expressing DCs became tolerised and were able to induce the
+ + function of TR cells to suppress CD4 and CD8 T cells (Manavalan et al., 2003), thus
32 eliminating a host-versus-graft response. Preliminary studies show that administration of soluble LILRB4 to transplant recipients decreases graft rejection and may be useful for transplant therapy (Suciu-Foca et al., 2007). More recently, in a mouse model where allogeneic pancreatic islets were transplanted into non-obese diabetic/severe immunodeficiency (NOD/SCID) mice, soluble LILRB4-Fc was able to inhibit rejection of the pancreatic islets in 100% of mice and moreover, inhibits CD40-CD40L interaction
(Vlad et al., 2008). At the same time, LILRB4 may promote cancer progression by
+ inhibiting effector cells such as cytotoxic CD8 T cells and NK cells from eliminating malignant cells (Suciu-Foca et al., 2007). LILRB4 is not normally expressed on B cells but its expression can be induced on chronic lymphocytic leukaemia (CLL) B cells and expression of LILRB4 positively correlates with metastatic spread measured by lymphoid tissue involvement, suggesting that LILRB4 may have a prognostic role in CLL (Colovai
+ et al., 2007). LILRB4 expression was also been identified on CD64 macrophages surrounding metastatic melanoma, pancreatic carcinoma and colon carcinoma (Suciu-Foca et al., 2007). It is likely that LILRB4 expressed by malignant cells and inflammatory cells inhibit T cell responses towards cancer cells.
Of all the LILRs, the least is known about LILRB5. Interestingly, LILRB5 co-stains on the intracellular granules of mast cells but mobilises to the surface after activation via FcεR and is subsequently released from granules (Tedla et al., 2007). The functional reason for this event remains to be investigated though it is suggested that the release of LILRB5 during IgE-mediated responses may act as a decoy to down-regulate its inhibitory function
(Tedla et al., 2007).
33 1.3.6 Pathological Role of LILRs
1.3.6.1 Allergies and Autoimmunity
Basophils, eosinophils and mast cells are key inflammatory cells producing a spectrum of cytokines and mediators to initiate allergic inflammation (reviewed in Bochner et al.,
2001; Bochner et al., 2003; Prussin et al., 2006). One important pathway in activation of these inflammatory cells is via the FcεRI which is stimulated by IgE (reviewed in Kinet,
1999; Gould et al., 2003). Upon stimulation, cells are able to synthesise mediators or release mediators directly from preformed stores and these include histamine, leukotrienes, prostaglandins and cytokines such as IL-4, IL-5 and IL-13 (reviewed in Kinet, 1999;
Schroeder et al., 2001; Gould et al., 2003; Shi, 2004).
Basophils constitutively express LILRB1, LILRB2 and LILRA2 and eosinophils express
LILRB2, LILRB3 and LILRA2 (Table 1.3). Cross-linking of LILRA2 in eosinophils using mAb elicits the production and release of eosinophil-derived neurotoxin (EDN), leukotrienes (LTC4) and IL-12, but not IL-4 (Tedla et al., 2003) as well as release of histamine, LTC4 and IL-4 in basophils (Sloane et al., 2004). Thus LILRA2 can induce differential responses, mostly favouring Th2 responses as IL-12 and IL-4 have opposing
Th1 and Th2 skewing respectively (Romagnani, 2000). LILRB3 is able to diminish
LILRA2-mediated and FcεRI-mediated activation of basophils to produce such mediators and this is the only study to demonstrate the function of this receptor (Sloane et al., 2004).
It would be ideal to observe the natural interaction between LILRB3 and LILRA2 but this is difficult without identification of their respective ligands.
34 Studies of novel LILRB1 polymorphisms and susceptibility to rheumatic diseases revealed an association with a distinct polymorphism in patients with RA negative for the HLA-
DRB1 shared epitope but not with SLE (Kuroki et al., 2005). Furthermore, polymorphisms in the LILRB4 gene are not associated with atopic markers such as skin-prick response, antigen-specific IgE and total serum IgE levels (Heinzmann et al., 2000).
1.3.6.2 Mycobacterium Infection
The genus Mycobacterium encompasses many species of infectious bacteria that can cause severe and debilitating diseases. M. leprae and M. tuberculosis are responsible for the diseases leprosy and tuberculosis, respectively. In both diseases, there is a large spectrum of clinical and immunological manifestations (reviewed in Cosma et al., 2003).
In leprosy, the two extreme forms of the disease are tuberculoid leprosy (T-lep) and lepromatous leprosy (L-lep). One determining factor of the disease outcome is the immune response mounted by the host in relation to the Th1 and Th2 pathways. A Th1 response induces T-lep while a skew towards Th2 results in L-lep, the latter being associated with high bacterial dissemination, systemic disease and an increase in morbidity. The events that determine the response mounted by the host are not completely understood but a combination of factors are likely to be involved. These include class I and/or class II MHC antigen-presentation, T cell subset involvement and the types of cytokines produced
(reviewed in Meyer et al., 1998; Abulafia et al., 2001; Britton et al., 2004). TLRs are important receptors in antigen-presentation of mycobacterial lipoproteins to stimulate an immune response (Stenger et al., 2002). LILRA2 mRNA was found to be highly up-
35 regulated in skin biopsies from patients with L-lep compared to T-lep (Bleharski et al.,
2003). Activation via TLR2/1 and TLR4 down-regulates LILRA2 expression on monocytes and LILRA2 reduces IL-12 production via TLR2/1 and TLR4 stimulation but enhances IL-10 production via TLR4. This suggests LILRA2 expression skews towards a
Th2 profile and thus leads to the L-lep disease phenotype.
M. tuberculosis infects the lung and evades the immune system by residing intracellularly in macrophages. If the infection is not cleared, the host immune system develops a granuloma, consisting of leukocytes and connective tissue which isolates the bacteria
(reviewed in Ulrichs et al., 2006). Like leprosy, there is a complex network of cellular activity that is highly influenced by the cytokine profile which determines the progression of the disease (reviewed in Flynn, 2004; Raja, 2004). LILRB1 is expressed on T cell clones specific to M. tuberculosis and upon stimulation, inhibits antigen-specific lysis of target cells and blocks the proliferation of expressing T cell clones (Merlo et al., 2001).
Consequently, IL-2 and IFN-γ production is greatly suppressed, skewing towards a Th2 phenotype.
1.3.6.3 Human Immunodeficiency Virus (HIV) Infection
The human immunodeficiency virus (HIV) is a virus that constantly evades the hosts’ immune system and its ability to do so lies in the proteins encoded in the HIV genome and their interaction with inflammatory cells (reviewed in Stevenson, 2003). The virus gains entry into T cells, macrophages and DCs via the receptors; CD4, CCR5 and CXCR4, initiating an immunological battle against the virus which eventually results in morbidity
36 of the host (reviewed in Letvin et al., 2003; Alfano et al., 2005). The complexity of HIV pathogenesis will not be discussed in great detail here but it is important to note that HIV survival is dependent on the immune response of the host.
+ + + A higher percentage of LILRB1 CD3 CD8 T cells can be detected in HIV-infected and
HIV/HCV co-infected individuals compared to uninfected controls (Costa et al., 2001).
LILRB1 expression by these cytotoxic cells is able to inhibit cytolysis as blocking with mAb results in a substantial increase in HIV-specific lysis. Expression of LILRB1 decreased with anti-retroviral treatment though still remained higher than uninfected controls throughout the course of treatment up to 2 years after. Persistent expression of
+ LILRB1 may contribute to the inefficient killing of infected cells by CD8 T cells. Similar increases in LILRB2 expression were observed on monocytes of HIV patients and was up- regulated by ex vivo stimulation with recombinant and serum IL-10 (Vlad et al., 2003).
1.3.6.4 Human Cytomegalovirus (HCMV) Infection
While initial studies on the human cytomegalovirus (HCMV) surface protein UL18 led to the discovery of LILRs, these studies also provide much insight into their function. HCMV is a ubiquitous human β-herpesvirus which commonly manifests itself in immuno- compromised individuals. Lying latent in immuno-competent hosts, the virus readily evades the hosts’ immune system by means of molecular mimicry, such as the expression of UL18 (reviewed in Mocarski, 2004). UL18 is a structural homologue to human MHC class I and is expressed on infected cells shortly after infection. The human ligand to UL18 was identified as LILRB1 (Cosman et al., 1997) and limited expression of LILRB1 on T
37 cells (Fanger et al., 1998) and NK cells (Cosman et al., 1997; Fanger et al., 1998) indicates a potential role in HCMV pathogenesis since these are the key effector cells in controlling virus replication and clearance of infection (reviewed in Gandhi et al., 2004). During lung transplantation, opportunistic infection of the induced-immunosuppressed host allows uncontrolled replication of the virus.
+ There is an increase in LILRB1 lymphocytes during early HCMV infection after lung transplantation indicating that viral replication may induce the expression and/or cause
+ clonal expansion of LILRB1 cells (Berg et al., 2003). This is thought to be due to stimulation of transcription factors for the promoter region of the LILR gene cluster
+ + (Nakajima et al., 2003). There is also an expansion of HCMV-specific LILRB1 CD8 memory T cells during latent HCMV infection (Northfield et al., 2005) and viral reactivation (Antrobus et al., 2005) suggesting a long term role for LILRB1 in HCMV pathogenesis. It was predicted that the UL18 glycoprotein functions as a decoy to host
MHC which is down-regulated during infection, altering NK cell recognition of MHC on infected cells and consequently protecting cells from NK cell lysis (Reyburn et al., 1997).
However, an increase in LILRB1 expression correlates with an increase in NK lysis of target cells (Leong et al., 1998; Saverino et al., 2004) thus challenging this theory. Instead, interactions with UL18 and LILRB1 could be a host defence mechanism, leading to the lysis of HCMV-infected cells and in this case, the inhibitory receptor would be inducing
NK cell activity (Saverino et al., 2004). Conversely, LILRB1-inhibition of NK cell lysis can be abrogated by pre-treatment with soluble UL18 (Cerboni et al., 2006). IFN-γ is
+ produced by LILRB1 T cells and NK cells and this can be inhibited by the presence of
38 UL18 (Wagner et al., 2007). It must be acknowledged that LILRB1 is the only identified ligand for UL18 and binds with much greater affinity compared to its interaction with
MHC class 1 molecules (Chapman et al., 1999). Genetic variants and strains of HCMV, along with spontaneous genetic mutations of the HCMV genome have given rise to viral clones with differing capacity to bind LILRB1 but no conclusion could be drawn between affinity binding and its affect on NK-mediated killing (Vales-Gomez et al., 2005; Cerboni et al., 2006).
1.3.6.5 HLA-G-mediated Immunity
HLA-G is a non-classical HLA class I molecule with highly restricted tissue distribution and believed to have a specific function in immunity. To date, the understanding of HLA-
G function is that peptide binding and antigen-presentation results in negative selection of auto-reactive T cells during foetal immune development (reviewed in Rouas-Freiss et al.,
1997; Le Bouteiller et al., 1999; Carosella et al., 2003; McIntire et al., 2005). However, there is only a limited repertoire of peptides which HLA-G recognises (Diehl et al., 1996;
Munz et al., 1997; Munz et al., 1999). Functional expression of HLA-G has been identified on the extravillious cytotrophoblast during pregnancy (Jurisicova et al., 1996; Rouas-
Freiss et al., 1997) and some forms of cancer (Paul et al., 1998; Ibrahim et al., 2001;
Wiendl et al., 2002). In both immunological scenarios, the blocking of HLA-G results in an increase in direct NK-mediated lysis of cells, implying HLA-G expression is protective against cytotoxic activity. Thus in cancer, HLA-G expression is detrimental and possibly contributes to metastatic spread (Paul et al., 1999). But in pregnancy, HLA-G protects the cytotrophoblast from the maternal immune system (Rouas-Freiss et al., 1997). It is
39 therefore important to understand the molecular signalling aspect of HLA-G-immunity as a potential therapeutic target.
HLA-G serves as a ligand for inhibitory LILRB1 and LILRB2 (Table 1.4) but also the KIR receptor KIR2DL4 (Rajagopalan et al., 1999). Multimeric HLA-G binds LILRs and the association is dependent on stabilisation by β2-microglobulin (Gonen-Gross et al., 2005;
Shiroishi et al., 2006). Compared to classical HLA class I, LILRB1 and LILRB2 bind
HLA-G with a 3- to 4-fold greater affinity and it appears that both LILRs produce necessary inhibitory signals for HLA-G-mediated survival (Shiroishi et al., 2003).
Blocking of LILRB1 with blocking mAb in a model of NK cell-directed lysis restores cytolysis of targeted cells (Gonen-Gross et al., 2003). Mutation of HLA-G prevented
LILRB1 binding which also restores targeted lysis. HLA-G/LILRB2 interaction alters the phenotype of DCs by suppressing their maturation and leading to an increase in tolerogenic
DCs with down-regulated MHC class II expression to prevent antigen-presentation and antigen-specific immune responses (Ristich et al., 2005). In transgenic mice, in vivo co- stimulation of HLA-G and LILRB2 blocks T cell effector function and impairs IL-2
+ + production in both CD4 and CD8 T cells, leading to increased allograft survival (Ristich et al., 2005). Furthermore, there is a direct suppression of allo-reactive T cell proliferation during transplantation which is associated with a greater potential for graft acceptance (Le
Rond et al., 2006). In skin lesions of psoriasis, there is a co-expression of HLA-G and
+ LILRB2 and the receptors are expressed on macrophages and CD4 T cells, respectively
(Aractingi et al., 2001). It is hypothesised that binding and signalling of these receptors
40 + prevent CD8 T cells from becoming activated thus reducing cytotoxic T cell function.
Furthermore, HLA-G is able to stimulate expression of its ligands LILRB1, LILRB2 and
KIR2DL4 but also LILRB3 (LeMaoult et al., 2005) which HLA-G dose not bind. In response to HLA-G, LILRB2 and LILRB3 are up-regulated in APC lines while LILRB1 and LILRB2 increase in expression on monocytes. LILB1 and KIR2DL4 both increase on
+ + NK cells and CD4 cells but not CD8 cells. This suggests that HLA-G is able to regulate its own function by altering the availability of ligands.
1.4 LILR-related Receptors
1.4.1 Killer Cell Immunoglobulin-like Receptors (KIRs)
LILRs cannot be studied without first considering the function of other related immunoglobulin superfamily members and their involvement in immune responses. KIRs are structurally and functionally related to LILRs and modulate leukocyte responses, specifically in NK cells. Like LILRs, different KIRs have activating or inhibitory potential and also signal via ITAMs or ITIMs, depending on the receptor type. Inhibitory KIRs bind a broad range of HLA class I molecules in which they are able to induce specific responses
(reviewed in Lanier, 1998; Lanier, 2005).
The precise functions of KIRs are not completely understood but they are believed to influence the sentry function of NK cells along with many other regulatory receptors
(CD49, NKG2, NKR-P1, CD16 and CD28) via their association with MHC class I
(reviewed in Lanier, 1998; Lanier, 2005). NK cells are able to detect other cells with
41 abnormal expression of either MHC or other novel activating receptors and the proposed scenarios are illustrated in Figure 1.4 (from Lanier, 2005). In the absence of MHC class I and stimulatory receptor signalling, the leukocyte remains unresponsive (Figure 1.4A).
Similarly, if MHC class I receptor provides the sole signal, there is no response in the leukocyte (Figure 1.4B). Targeted lysis is induced if there is a down-regulation of MHC class I expression (Figure 1.4C). When signals are provided via both activating and inhibitory receptors, the outcome is determined by the strength of either signal (Figure
1.4D). Whether the strength of signals is quantitative or qualitative is yet to be determined.
In relation to diseases, KIRs have been associated to the development of RA, psoriasis, pre-eclampsia, some cancers and chronic infections (reviewed in Khakoo et al., 2006).
These diseases are not linked directly to KIR function but to the inheritable haplotypic and allelic diversity of both the KIR gene family and MHC class I (Middleton et al., 2005;
Khakoo et al., 2006). It is strongly believed that differences in affinity of binding between different KIR alleles with HLA-A, HLA-B or HLA-C variants can determine the responses of effector cells.
There is much more known about KIR signalling and function in comparison to LILRs and this can be attributed to knowledge of their ligands. It is tempting to speculate that LILRs function in a similar way to KIRs and that they differ in the cell types that they are expressed on.
42
Figure 1.4 » Signalling pathways of KIRs. Stimulation of NK cells (on the left) via KIRs can result in different outcomes depending the type of KIR that is engaged (activating or inhibitory) to its ligand.
From (Lanier, 2005).
43 1.4.2 LAIR-1 (CD305)
Another gene found on the LRC is the collagen receptor inhibitor called leukocyte- associated immunoglobulin-like receptor (LAIR)-1. Also found in mice and rats, human
LAIR-1 was identified and characterised as an inhibitory receptor for NK cell-mediated cytotoxicity (Meyaard et al., 1997). Its expression is not restricted like other inhibitory receptors and has been identified on virtually all cell types (reviewed in Meyaard, 2007).
Similar to the related LILRs, their inhibitory potential is demonstrated by in vitro cross- linking using monoclonal antibodies. Other than inhibiting NK cell function, LAIR-1 regulates the function of T cells, B cells, monocytes and granulocytes (van der Vuurst de
Vries et al., 1999; Fournier et al., 2000; Saverino et al., 2002; Verbrugge et al., 2003;
Merlo et al., 2005; Jansen et al., 2007). The intracellular ITIMs of LAIR-1 recruit SHP-1 and SHP-2 (Meyaard et al., 1997; Fournier et al., 2000) but not SHIP (Verbrugge et al.,
2006), providing insight into their downstream signalling mechanisms. LAIR-1 binds collagen by recognising specific amino acid motifs and its inhibitory potential is somewhat dependent on the presence of collagen (Lebbink et al., 2006; Jansen et al., 2007; Lebbink et al., 2007). Given the structural and functional similarities between LAIR-1 and inhibitory LILRs, there is great potential for LILRs to bind non-MHC ligands. With the continuing emergence of other immune regulatory receptors and pathways, we find that
LILRs can potentially function via alternative pathways but also contributing to the intricate balance of immune homeostasis.
44 1.4.3 Murine Paired Immunoglobulin-like Receptor-B (PIR-B)
Paired immunoglobulin-like receptor-B (PIR-B) is a murine encoded inhibitory receptor which binds MHC in a similar fashion to LILRB1 and LILRB2, and also signals against leukocyte activation (Nakamura et al., 2004). Broadly expressed on leukocytes, PIR-B functions via ITIMs to inhibit ITAM-mediated signalling, particularly those of the BCR
(reviewed in Katz, 2006). Expression of PIR-B (and PIR-A) is dependent on the maturity of cells, indicating it functions in the development of leukocytes (Kubagawa et al., 1999).
PIR-B knock-out mice have uncontrolled immune responses and this is linked to B cell
-/- function where PIR-B mice have a skewing towards Th2 responses and thus PIR-B
-/- appears to be critical for B cell suppression (Ujike et al., 2002). At the same time, PIR-B mice exhibit exaggerated graft-versus-host disease (Nakamura et al., 2004) and this further suggest that this inhibitory receptor is essential in specific immune responses and by extension in tissue graft acceptance during transplantation.
1.4.4 Murine LILRB4 (mLILRB4 or gp49B)
Recently, the inhibitory mouse receptor gp49B was renamed to mLILRB4 since it is highly homologous to human LILRB4 and it is primarily expressed on mast cells in which it exerts its inhibitory function (reviewed in Katz, 2007). Co-ligation of mLILRB4 to the Fc receptor for IgE (FcεR) inhibits the degranulation of mast cell associated mediators such as
β-hexosaminidase, histamine and cysteinyl leukotrienes (Katz et al., 1996). This inhibition is mediated by the recruitment of SHP-1 phosphatase (Lu-Kuo et al., 1999). From in vivo binding studies, mLILRB4 can associate with integrin αvβ3 as neutralising mAb prevent
45 the binding of recombinant mLILRB4 to cells but also prevents the membrane-bound form to confer its inhibitory function (Castells et al., 2001). Whether there are other receptors involved at the mLILRB4-αvβ3 synapse requires further investigation.
Significant knowledge of mLILRB4 function has been discerned from gene knock-out
-/- +/+ mice (Lilrb4 ) and comparing their inflammatory responses to wild-type mice (Lilrb4 ).
-/- Lilrb4 mice show an increase in sensitivity to IgE-mediated activation as their passive cutaneous responses to intradermal IgE injections are greatly exaggerated compared to
+/+ Lilrb4 mice (Daheshia et al., 2001). Similar observations were made with stem cell factor (SCF)-induced tissue responses following intradermal injections, with knock-out mice having greater swelling and edema (Feldweg et al., 2003). The same research group also investigated active cutaneous responses by sensitising mice with intraperitoneal ovalbumin (OVA). Following administration of intradermal OVA injections, similar responses were observed whereby mLILRB4-deficient mice had less controlled responses
(Daheshia et al., 2001). Moreover, mLILRB4 protected mice from anaphylactic shock
-/- since OVA-sensitised Lilrb4 mice were susceptible to morbidity upon intravenous OVA challenge (Daheshia et al., 2001).
-/- In a mouse model of inflammatory arthritis, Lilrb4 mice develop exaggerated symptoms when induced with the disease, compared to normal mice (Zhou et al., 2005). This is attributed to the inability of mLILRB4-deficient mice to down-regulate inflammatory responses by suppression of key pathological cytokines and chemokines but also the inability to prevent neutrophils from infiltrating the synovium and joint space. More
46 interestingly, LILRB4 influences innate responses to LPS and this is the first report of an
ITIM-bearing receptor inhibiting a non ITAM-signalling pathway. Differences were observed between wild-type and knock-out mice in their responses to local LPS challenge
-/- as Lilrb4 mice had increased neutrophilia, macroscopic hemorrhaging and thrombi
+/+ compared to Lilrb4 mice (Zhou et al., 2003). In this case, there was no correlation with these uncontrolled responses to cytokine and chemokine induction but instead the function of adhesion molecules (Zhou et al., 2003).
47 1.5 Toll-like Receptors (TLRs)
One of the most significant contributions to our understanding of innate immune responses is the discovery of the TLRs. This family of receptors (TLR1 – TLR10) serves as the primary pattern-recognition receptors (PPRs) for pathogen-associated molecular patterns
(PAMPs) by binding to a broad range of microbial and viral constituents as well as some synthetic compounds, as shown in Table 1.6 (reviewed in Akira et al., 2001; Takeda et al.,
2003; Broad et al., 2006; Bowie, 2007).
Interestingly, the signalling cascades for all TLRs converge at the activation of NF-κB and furthermore, the induction of AP-1- and NF-κB-dependent genes. However, intracellular signalling is more complicated than described here and determined by whether MyD88 is involved or not. TLRs are differentially expressed on leukocyte populations/subsets and upon binding with their ligands, initiate innate effector responses as well as inducing adaptive immune responses (reviewed in Iwasaki et al., 2004).
48 Table 1.6 » Ligands for each TLR and the signalling proteins that are utilised.
TLR Ligand(s) Signalling proteins
TLR2 Lipoproteins/lipopeptides (various pathogens) Peptidoglycan and lipoteichoic acid (Gram- (with either Mal and MyD88 TLR1 and positive bacteria) TLR6) Zymosan (fungi)
TLR3 Double-stranded RNA (viruses) TRIF
LPS (Gram-negative bacteria) Mal and MyD88 TLR4 Bacterial HSP60 TRAM and TRIF Respiratory syncytial virus coat protein
TLR5 Flagellin (bacteria) MyD88
TLR7 Imidazoquinolone antiviral drug MyD88
Single-stranded RNA (viruses) TLR8 Imidazoquinolone antiviral drug
TLR9 CpG DNA (bacteria) MyD88
TLR10 Ligand unknown
Known exogenous ligands to TLRs and respective signalling proteins recruited to mediate intracellular signalling. LPS = lipopolysaccharide, HSP = heat shock protein, TRAM = TRIF-related adapter molecule, TRIF = TIR domain-containing adapter inducing IFN-β. Modified from (Takeda et al., 2003;
Bowie, 2007).
49 The focus in this study is on the LPS-binding TLR4 which remains the most extensively studied TLR. TLR4 associates with CD14, MD-2 and the serum protein LPS-binding protein (LBP) to form a complex that is essential for the recognition of LPS and prevention of bacterial infections (Fitzgerald et al., 2004). TLR4 is unique among other TLRs in that it is able to signal via MyD88-dependent and -independent pathways (Figure 1.5). The important outcome of TLR4 signalling is the production of pro-inflammatory cytokines of the TNF and IFN families (reviewed in Albiger et al., 2007; Thompson et al., 2007).
There is increasing evidence to suggest that TLRs are able to cross-talk with Ig-receptors such as LILRs, where co-stimulation of both receptors can alter or divert immune responses (Daheshia et al., 2001; Bleharski et al., 2003; Cao et al., 2006; Hamerman et al.,
2006; Nakayama et al., 2007). This is described as a fine tuning mechanism of the immune system, to tailor responses for specific immunological challenge and conditions (Underhill et al., 2004; Taylor et al., 2005). Often, immune regulatory receptors are studied singularly and thus critical interactions between other receptors may be overlooked.
50
Figure 1.5 » Intracellular signalling pathways of TLR4. Ligand-stimulation of TLR4 can lead to signalling via 2 different pathways; TRAM (left) or MyD88 (right). Both pathways converge on the ubiquitination of TRAF-6 and lead to AP-1/NF-κB transcriptional activation. LRR = leucine rich repeat, TIR = Toll/IL-1R, TRAF = TNFR-associated factor, IRAK = interleukin-1 receptor associated kinase, IRF = IFN regulatory factor, AP = activator protein, JNK = Jun N-terminal kinase, Ub = ubiquitin, MKK = MAP kinase kinase, RIP = receptor-interacting protein. Modified from (Watters et al., 2007).
51 1.6 Rheumatoid Arthritis
1.6.1 Background
Rheumatoid arthritis (RA) is a debilitating disease with significant impact on those affected, their carers and the national economy. The disease affects approximately half a million Australians (Australian Bureau of Statistics, 2002) and approximately 1% of the population worldwide (Sangha, 2000). Notably, the percentage of Australians affected by
RA is higher than the world average and the reason behind this is unclear. In 2004, the disease accounted for over $340m of the total national health expenditure in Australia
(Australian Institute of Health and Welfare, 2004). Of the total health expenditure allocated to all arthritis and arthropathies, RA accounts for 10%, while osteoarthritis consumes almost half the total value (Australian Institute of Health and Welfare, 2004). On average, the total health costs for each patient is over $700 per year and on top of the immediate expenses, income loss and the constant need by patients for surgical intervention means
RA places a great burden on the public health system.
1.6.2 Etiology
RA is an autoimmune disease leading to chronic inflammation of the joints affecting the synovial membrane, cartilage and bone. Although extensive studies have revealed a great deal of knowledge regarding the etiology and pathogenesis of the disease, the direct pathways leading to clinical and pathologic manifestations are not completely understood.
As suggested recently, views concerning the mechanisms and pathways of the pathogenesis of RA have evolved over time and this is due largely to the multi-factorial
52 nature of the disease whereby different cell types interact via different pathways but each contributing to the overall disease development (Firestein, 2005). Extreme heterogeneity of the disease exists not only in its pathogenesis but also in the treatments of patients, and such factors must be considered when studying RA. Given that different patients are often on different treatments and usually multiple drugs, this often affects the outcome of patient studies. While different researchers have their own view of which pathway/s bear greater significance in the disease process, a universal definition for the pathogenesis of RA remains to be determined.
Studies involving genetic screening of patients with RA and their families have revealed a large quantity of data (often conflicting) regarding the genetic inheritability of RA. There is a strong genetic component to the development of the disease as it has been consistently shown that the MHC encoding region of the human genome is likely to be associated.
HLA-DR4 was one of the early genes found to have a strong correlation in patients with
RA (reviewed in Reveille, 1998) but currently, the HLA-DRB1 has been defined a more likely genetic culprit (Jawaheer et al., 2003). It is predicted that variability in amino acid sequences occurs in the antigen binding site and is likely to affect its ability to present antigens.
The involvement of microbial infection remains a topic of discussion in regards to its role in RA. Although no specific infectious agent has been identified to directly cause the onset of RA, there is evidence that the interplay between infection and genetic predisposition may be critical. Over time, a large number of bacteria and viruses have been suggested as a
53 causative agent, including EBV, E. coli, Parvovirus, Mycobacteria, Mycoplasma and
Proteus mirabilis (reviewed in Carty et al., 2004; Rashid et al., 2007). P. mirabilis is a bacterium of growing interest as increase in reactive antibodies is found to be elevated in patients with RA (reviewed in Rashid et al., 2007). The premise for implicating microbes is that highly conserved proteins such as glucose-6-phosphate isomerase, p205, and heat shock proteins (HSPs) produced by the microbes, induce antigen-specific T cells and antibodies that detrimentally cross-react with hosts’ athritogenic peptides (reviewed in
Corrigall et al., 2002).
Activated leukocytes and their mediators induce inflammation and tissue destruction in RA but the mechanisms and regulation of their activation are not fully understood. Because of their ability to activate or inhibit cellular function, LILRs may play a key role in the regulation of inflammatory cell function in RA. Dynamic changes in the balance between activating and inhibitory LILRs and/or their ligands might explain the recurrent nature of
RA involving episodes of remission and relapse.
1.6.3 Pathology
RA is a debilitating chronic inflammatory disease of joints largely due to an inappropriate immune response within the joint space (Brahee et al., 2003). Initially, inflammation develops within the joint capsule or synovium and this condition is called synovitis. The disease is progressive and there are distinct pathological hallmarks outlining each stage
(Figure 1.6) which also reflect clinically. Early stages are usually characterised by pain and swelling as they become inflamed. This is proceeded by an influx of inflammatory cells
54 and unless stopped, progresses to the advanced stages where extensive tissue inflammation occurs. Clinical presentation of advanced RA includes gross deformity and is partially due to formation of rheumatoid nodules that result in severe impairment of the affected joints
(Sayah et al., 2005). Generally, there is bilateral involvement of both the hands and wrists, and progression of the disease is relatively symmetric over time (Brahee et al., 2003). The combination of deformity and pain leads to severe disability in patients.
Although there is a great deal of knowledge about the general pathogenesis of RA, there is still much to be understood about the disease. The pathogenesis is multi-factorial in nature meaning there are many factors influencing disease onset, progression and severity, as well as the response of the patients to treatment. RA invokes both cellular and humoral immune responses, creating a complex interaction of inflammatory cells, cytokines, proteolytic enzymes, growth factors and cell surface receptors; all of which have been identified to be involved in the pathogenesis of RA. Substantial evidence illustrates that the multi-variant pathways are co-dependent and it is difficult to suggest that one particular component is more responsible than another. This also impedes the development of an effective treatment due to the presence of pleiotropic and redundant pathways.
55
Figure 1.6 » Pathogenesis of RA schematically represented in knee joint. RA has distinct developmental stages of disease beginning with leukocyte recruitment to the joint capsule mediating inflammation. In established disease, there is extensive tissue damage and the formation of a pannus.
From (Choy et al., 2001).
56 In the established stages of disease, much of the joint damage is due to the formation of a pannus on the synovial membrane which continually proliferates and destroys the underlying cartilage and bone (Tak et al., 2000). A pannus is a fibrocellular mass consisting of inflammatory cells; macrophages, lymphocytes, neutrophils and plasma cells but also granulation tissue and fibroblasts (Kobayashi et al., 1975). The interaction of all cell types is crucial in the pathogenesis of RA (reviewed in Kinne et al., 2000; Brennan et al., 2006).
Synovial macrophages originate from circulating monocytes and during infiltration of the synovial sub-lining and lining layers, differentiate to macrophages (Klareskog et al., 1982).
The infiltration and activity of macrophages positively correlate with the degree of joint inflammation (Tak et al., 1997) and articular destruction analysed by radiography
(Mulherin et al., 1996). Macrophages are responsible for the production of acute pro- inflammatory cytokines which undoubtedly have a crucial role in inflammatory responses and in the pathogenesis of RA (reviewed in Feldmann et al., 1996; Choy et al., 2001).
TNF-α and IL-1β are prominent cytokines in rheumatoid synovium perpetuating destruction of joints in RA through activation and regulation of other leukocytes
(macrophages and T cells) and inflammatory mediators (Arend et al., 1995). TNF-α induces the production of IL-1 as well as other pro-inflammatory cytokines such as IL-6,
IL-8 (Feldmann et al., 1996) and GM-CSF (Haworth et al., 1991). It is also likely that activated macrophages function in antigen-presentation to lymphocytes although the exact athritogenic antigens are yet to be identified (Pettit et al., 1999). Along with the production
57 of cytokines, synovial macrophages also regulate the activity of destructive proteinases
(Close, 2001; Szekanecz et al., 2007).
Matrix metalloproteinases (MMPs) are a family of matrix-degrading enzymes which contribute to tissue destruction in many pathological processes, as well as being involved in normal development. Associated with the extracellular matrix (ECM), MMPs are responsible for maintaining its composition and integrity in a tightly controlled and regulated system (reviewed in Chakraborti et al., 2003). One major method of controlling
MMP activity is via the interaction with tissue inhibitors of MMPs (TIMPs) and since both
MMPs and TIMPs are constitutively expressed, a common theory is that the unwanted activity of MMPs observed in pathological processes is due to a disruption in the balance between the two classes of enzymes (reviewed in Johnson et al., 1998; Bode et al., 1999).
The role of MMPs in RA is evident in the tissue destruction observed in affected joints.
Collagenase (MMP-1) was initially identified in abundance at the site of cartilage erosion
(Woolley et al., 1977) and subsequently, extensive proceeding studies have further established the expression of other MMPs and their inhibitors by most cell types within the rheumatoid synovium (reviewed in Tak et al., 2000). Not only defined in tissue, MMPs have been identified in rheumatoid synovial fluid (Walakovits et al., 1992; Sasaki et al.,
1994; Beekman et al., 1997) and serum (Sasaki et al., 1994; Yoshihara et al., 1995) of patients with RA at much higher levels compared to healthy and non-inflammatory arthritic controls. The MMPs present are also mostly in their activated state indicating an active role in ECM degradation (Beekman et al., 1997). There is a positive correlation between levels of the inflammatory cytokines, TNF-α and IL-1, and MMP expression
58 (MacNaul et al., 1990; Mauviel, 1993) but also TNF-α directly up-regulates the activity and production of tissue degrading MMP-9 in cells that are abundant in RA and facilitate migration of inflammatory cells through the extracellular matrix (Di Girolamo et al.,
2006).
Mast cells have been identified at the site of cartilage erosion (Bromley et al., 1984) and are able to produce a combination of proteases such as tryptase and chymase (Tetlow et al.,
1995). These proteases exacerbate tissue destruction by direct tissue degradation or by the activation of MMPs. Upon activation, mast cells release preformed granules or synthesise and release a range of mediators including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines (reviewed in Nigrovic et al., 2005). The downstream effects of these mediators include increasing the expression of adhesion molecules and recruitment of inflammatory cells to the joint (Gaboury et al., 1995; Gotis-Graham et al.,
1997; Gotis-Graham et al., 1998). Convincing data implicating the role of mast cells in the pathogenesis of RA comes from a study reporting that mast cell-deficient mice lack the ability to develop erosive inflammatory arthritis (Lee et al., 2002).
A major component of the inflamed rheumatoid synovium is B cells and plasma cells which have an active role in exacerbating RA. Commonly, B cells form follicle lymphoid- like structures within the inflamed synovium by recruiting follicular dendritic cells and organising aggregates with T cells (reviewed in Kim et al., 2000; Goronzy et al., 2005).
These B cells have the propensity to differentiate into immunoglobulin-producing plasma cells and this process is antigen-specific (reviewed in Kim et al., 2000). Antigen-
59 presentation via HLA-DR to B cells encourages cytokine production, proliferation and further recruitment in T cells (Takemura et al., 2001). In these aspects, T cell function has been shown to be highly dependent on B cell interaction suggesting that their presence maintains chronic inflammation within the synovial membrane. The lack of specific effector functions allows T cells to stimulate other leukocytes to perpetuate inflammation and tissue destruction.
Rheumatoid factors are auto-antibodies specific for the Fc region of self IgG and often used as a partial diagnostic marker of RA. Evidence suggests that auto-antibodies may be a prognostic marker for the development of RA based on a long-term study in which patients were screened for the presence of auto-antibodies before the onset of disease symptoms
(Nielen et al., 2004). In this study, approximately half the patients tested positive for IgM- rheumatoid factor many years before the onset of symptoms, the median time being 4.8 years. The presence of rheumatoid factor also correlates with a more severe and aggressive disease development with increased morbidity and mortality (van Zeben et al., 1992).
Although there is a high prevalence of rheumatoid factor in patients with RA, it does not occur in all patients and also occurs in other autoimmune diseases (Edwards et al., 1999;
Zhang et al., 2001). This supports the idea that RA is multifactorial in nature and the co- relationship between B cells and T cells mediate much of the disease pathogenesis. Other autoantibodies have been identified in RA including those specific for glucose-6-phosphate isomerase (Matsumoto et al., 1999), heat shock proteins, collagen, heavy chain binding protein and cyclic citrullinated peptides (van Boekel et al., 2002). It is suggested that these
60 antibodies have a significant role in the pathogenesis of RA due to their specificity against athritogenic peptides (Firestein, 2005).
1.6.4 Treatments
RA is an incurable disease and treatments are administrated with the objective to induce complete remission within the patient. However, most treatments are directed towards alleviating the immediate symptoms of disease to maximise the quality of life (American
College of Rheumatology Subcommittee on Rheumatoid Arthritis Guidelines, 2002). With the low percentage of patients achieving remission, current treatments for RA aim to reduce or inhibit the inflammatory process to prevent further tissue degradation (Cannella et al., 2003). The complexity of the pathogenesis of RA described earlier is reflected in the symptoms of patients. Constant waxing and waning in symptoms, and variation in disease progression place a burden on physicians to initiate the most effective treatment and this is observed in detail in the Guidelines for Management and Treatment of Rheumatoid
Arthritis (American College of Rheumatology Subcommittee on Rheumatoid Arthritis
Guidelines, 2002).
The drugs used in treating RA are broadly characterised into 4 groups: disease modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and biological therapies.
61 1.6.4.1 Diseases Modifying Anti-Rheumatoid Drugs (DMARDs)
DMARDs are a novel class of drugs used in slowing the progression of RA. There is an abundance of knowledge in regards to the efficacy of DMARDs and general mechanisms of action but in actual fact, many of the second-line DMARDs currently used have been discovered serendipitously with little knowledge of their specific mechanism of action in the pathogenesis of RA (Case, 2001). Many of these drugs were originally used as a therapeutic intervention for various other diseases but were incidentally found to retard joint destruction in active RA and to prevent further progression of the disease. There are numerous extensive studies investigating the biomolecular role of DMARDs in treating
RA however these studies do not question the efficacy of the drugs. The need for such studies is to assess long-term toxicity and/or complications which are often associated with long-term administration. It has been established that patients on various DMARDs over long periods of time exhibit at least one adverse effect.
Methotrexate is a commonly used DMARD in the treatment of RA. Despite its extensive use as an anti-inflammatory drug, the precise mechanism or mechanisms by which it inhibits an inflammatory response in RA is unclear (Whittle et al., 2004). Being a folate antagonist, methotrexate is able to block the synthesis of DNA and RNA by interfering in the de novo synthesis pathway of purine and pyrimidine bases (Jackson et al., 1973).
Methotrexate is effective in suppressing the proliferation of malignant cells but in the case of RA, it may inhibit the proliferation of inflammatory cells. A possible mechanism of action for methotrexate is the inhibition of an enzyme called 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR) transformylase, resulting in an accumulation of
62 AICAR which enhances release of adenosine into the extracellular space (Genestier et al.,
2000). Adenosine is believed to have an anti-inflammatory effect on neutrophils and mononuclear cells via interaction with adhesion molecules that are usually expressed during inflammation (Cronstein et al., 1991). There is also some evidence to suggest that methotrexate may inhibit the production of IL-1β (Genestier et al., 2000) or block IL-1β from binding to its receptor on target cells (Brody et al., 1993). Hepatocytotoxicity has been observed in patients with RA and psoriatic arthritis on a constant dose of methotrexate over a period of time (Weinblatt et al., 1999; Kremer et al., 2002; Helliwell et al., 2008).
Cyclosporin A is a fungal metabolite commonly used in preventing the rejection of organ transplants but also has a positive effect in treating RA. It is a potent immunosuppressive drug which acts predominantly on T lymphocytes, inhibiting the production of many cytokines including the T cell growth factor IL-2 (Jain et al., 1995) by blocking the binding of nuclear factors (Schreiber et al., 1992). In RA, cyclosporin A increases the number and function of regulatory T cells and in some patients, decreases the production of rheumatoid factor (reviewed in Ferraccioli et al., 2005).
To maximise the success of DMARD-treatment, they are administered in conjunction with either other DMARDs or other classes of drugs such as non-steroidal anti-inflammatory drugs (NSAIDs). Extensive studies have shown combination therapy to be highly effective as opposed to single-drug therapy (Mottonen et al., 1999; Korpela et al., 2004). Long-term blinded studies reveal over 80% of patients on a combination of drugs such as
63 methotrexate, leflunomide, sulfasalazine and/or hydroxychloroquine had minimal joint erosion over the 2 year observation with a significant proportion of patients achieving clinical remission (Cohen et al., 2001; Korpela et al., 2004 and reviewed in Doan et al.,
2005). Like other inflammatory diseases, early diagnosis and intervention is essential to achieving clinical remission. It is during the first 2-3 years of RA when the most rapid development of tissue destruction/erosion occurs and during this period, DMARD- treatment appears to be most effective. This small window of opportunity is a limiting factor which can impede effective intervention (Larsen et al., 1987; Fuchs et al., 1989;
Paimela, 1992; van der Heijde et al., 1992; Kaarela et al., 1997; Mottonen et al., 1998;
Finckh et al., 2006). It is also suggested that the longer patients are on DMARD-treatment, the less likely they are to attain remission since it more likely occurs within the earlier years of treatment (Sokka et al., 1999).
1.6.4.2 Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)
NSAIDs have limited efficacy in treating RA. The primary concern is the long-term use and the subsequent adverse effects that are associated. These drugs appear to be most effective during the early stages of disease, before extensive joint damage occurs. They do not slow the progression of disease but provide partial relief of pain and joint stiffness. The major adverse event following long-term use involves gastrointestinal problems (reviewed in Singh, 1998) and cardiovascular events (reviewed in Doan et al., 2005). Combination therapy of NSAIDs and DMARDs is considered a more aggressive approach to treatment and usually administered in the later stages of disease (American College of Rheumatology
Subcommittee on Rheumatoid Arthritis Guidelines, 2002).
64 1.6.4.3 Glucocorticoids
Glucocorticoids are a class of drugs with potent anti-inflammatory and immuno- suppressive function, broadly used for the treatment of inflammatory disorders.
Glucocorticoids were first introduced as an effective treatment for RA and drugs such as dexamethasone, prednisolone and prednisone have been extensively studied. These drugs have been shown to be effective in reducing joint destruction and disease progression
(Kirwan, 1995; Hickling et al., 1998; van Everdingen et al., 2002). A recent critical review validating the most significant clinical studies between 1957 and 2005 has determined that low-dose glucocorticoid treatment reduces the progression of RA over 1-2 years, as determined by radiography (Kirwan et al., 2007). Most studies outline that patients exhibit reduced bone/cartilage erosion and joint space narrowing within the first 6 months of treatment (Svensson et al., 2005; Wassenberg et al., 2005). It is most common for rheumatologists to administer glucocorticoid treatment during the initiation of therapy due to complications associated with long-term use (Townsend et al., 2004; Morand, 2007).
More crucial is the observation that glucocorticoid treatment appears most effective in patients already on DMARD-therapy (Kirwan et al., 2007).
The broad cellular effects of glucocorticoids involve mainly the inhibition of leukocyte trafficking and activation but also regulation of fibroblasts and endothelial cell function during inflammation (Table 1.7). It is because of these broad immuno-suppressive functions that glucocorticoids are used to treat a variety of inflammatory diseases.
65 Following diffusion through the plasma membrane, the central molecular role of glucocorticoids is attributed to binding with the cytoplasmic glucocorticoid receptor
(cGCR) and upon ligation, is able to influence transcriptional events (reviewed in Zhou et al., 2005; Stahn et al., 2007). Collectively, these events are known as the ‘genomic actions’ of glucocorticoids. It is proposed that by binding to either the promoter region up- stream to the transcription start site or binding to transcription factors (Figure 1.7), glucocorticoids can inhibit the transcription of inflammatory genes, such as those mentioned in Table 1.7. It should be highlighted that these events remain mostly hypothetical and ongoing research is still defining the mechanism/s of action of glucocorticoids.
The less defined ‘non-genomic actions’ of glucocorticoids can be dependent on either cGCR or membrane-bound glucocorticoid receptor (mGCR) though further investigations are required to determine their exact mechanism/s. Un-ligated cGCR are able to form complexes with heat-shock proteins (HSPs) and kinases such as MAPKs which can subsequently regulate the function of signalling molecules such as Src (reviewed in
Buttgereit et al., 2005; Stahn et al., 2007). It has been independently shown in T cells that the Src kinases, Lck and Fyn have been identified as targets for non-genomic glucocorticoid function (Lowenberg et al., 2005).
66 Table 1.7 » Important effects of glucocorticoids on primary and secondary immune cells.
Cell type Influence by glucocorticoid
↓ number of circulating cells (↓ myelopoiesis, ↓ release)
Monocyte/ ↓ expression of MHC class II molecules and Fc receptors macrophages ↓ synthesis of pro-inflammatory cytokines (e.g. IL-2, IL-6 and TNF-α) and prostaglandins
↓ number of circulating cells (redistribution effect) T cells ↓ production and action of IL-2 (most important)
↓ number of eosinophils and basophil granulocytes Granulocytes ↑ number of circulating neutrophils
↓ vessel permeability Endothelial ↓ expression of adhesion molecules cells ↓ production of IL-1 and prostaglandins
↓ proliferation Fibroblasts ↓ production of fibronectin and prostaglandins
Modified from (Buttgereit et al., 2005).
67 The adverse effects associated with glucocorticoid use still remain the largest obstacle and this has led to the development of preventative strategies (reviewed in Townsend et al.,
2004). The most common complication is glucocorticoid-induced osteoporosis (GIOP), caused by interference of glucocorticoid with the synthesis of type I collagen and subsequent bone formation (Canalis et al., 2002). Other adverse conditions include peptic ulcer disease, diverticulitis, hypertension, atherosclerosis, diabetes, cataract, glaucoma and susceptibility to infections (reviewed in Townsend et al., 2004).
1.6.4.4 Biological Therapies
Biological therapies have also been emerging in the past decade which includes the use of monoclonal antibodies and soluble receptors targeted towards key inflammatory factors in
RA. As the role of TNF-α in the pathogenesis of RA became well defined, the natural development was to inhibit its function to prevent ongoing disease. Anti-TNF-α mAb such as infliximab and etanercept are used in the treatment of not only RA but other inflammatory disorders also (reviewed in Case, 2001; Doan et al., 2005). Inhibition of
TNF-α is effective in retarding joint damage and degradation (Moreland et al., 1999;
Bathon et al., 2000). Known adverse effects of using TNF-α antagonists and inhibitors include susceptibility to infections and malignancies but also the production of antibodies against anti-TNF-α mAb themselves and thus decreasing efficacy.
68
Figure 1.7 » Genomic actions of glucocorticoids. Glucocorticoid-cGCR complexes can bind directly to
DNA at either; (A) the glucocorticoid response element (GRE) to induce transcription of immune- suppressive genes or (B) the negative glucocorticoid response element (nGRE) to inhibit transcription of pro-inflammatory genes. Alternatively, glucocorticoids may inhibit the function of transcription factors such as (C) the p65-p50 complex or (D) the AP-1-associated co-activator. Modified from
(Buttgereit et al., 2005). cGCR = cytosolic glucocorticoid receptor, AP-1 = activator protein-1.
69 1.6.5 LILRs in Rheumatoid Arthritis
The ability of LILRs to regulate immune responses supports the theory that they may contribute to inflammation whether it be in response to pathogens or in spontaneous auto- inflammation. LILRs can potentially determine or at the least, contribute to the threshold of immune equilibria that prevents autoimmunity and abnormal regulation of their expression and/or function could potentially initiate the onset of disease. In RA, LILRA2 is highly expressed within the inflamed synovium and primarily by macrophages and neutrophils (Tedla et al., 2002). The role of macrophages in RA has been greatly studied and their excessive activation mediates a great proportion of tissue degradation (reviewed in Kinne et al., 2000). Moreover, high LILRA2 expression is associated with active disease as patients who responded to treatment had decreased LILRA2 expression within the synovium (Huynh et al., 2007). Leukocyte activation by LILRA2 in vitro elicits an acute inflammatory response, inducing the production of cytokines that have significant roles in the pathogenesis of RA (Section 1.3.5.1). Therefore, determining the full extent of its function could provide new therapeutic strategies.
70 1.7 Hypothesis and Aims
Given the current evidence suggesting that LILRs function in immune homeostasis as well as inflammation via regulation of leukocyte function, we hypothesise that LILR expression is tightly regulated by inflammatory mediators and in turn, functions by producing cytokines to control immune responses. Furthermore, there may be a differential expression of LILRs (both activating and inhibitory) on circulating leukocytes of patients with RA and healthy control subjects that affects the development of the disease. This differential pattern of expression may be regulated by inflammatory mediators and may impact on cellular function. Therefore, the aims of this project are:
1. Determine whether LILRA2 and LILRB4 are regulated by inflammatory mediators
such as vitamin D3, LPS and the cytokines; TNF-α, IL-1β, IL-10 and IFN-γ.
2. Determine the function of LILRA2 and LILRB4 on monocyte/macrophages
activation and inhibition, and whether anti-rheumatoid drugs can influence
LILRA2-mediated activation.
3. Investigate the influence of LILRA2 and LILRB4 on LPS-mediated monocyte
responses.
4. Recruit a cohort of patients with active RA (and relevant control subjects) and
determine the expression pattern of LILRs on circulating leukocytes.
5. Elucidate the downstream signalling mechanisms of the inhibitory LILRB4.
71 Chapter 2. Regulation of LILRA2 and LILRB4 by Cytokines and LPS
2.1 Introduction
Activating and inhibitory LILRs are co-constitutively expressed on leukocytes (Table 1.4) and this supports the suggestion that they function in immune homeostasis. Activating signals produced by ITAMs lead to leukocyte activation and this is in part due to the lack of inhibitory signals. A dominance of ITIM-mediated signalling may result in a blockade, preventing ITAM-phosphorylation and subsequent initiation of calcium-dependent transcription factors (Section 1.4.7). The threshold which determines whether a cell becomes activated or inhibited remains unclear. A combination of quantitative and qualitative signals is likely to affect the cellular function and this is presumably ligand- dependent. As the function of a receptor often relates to level of its expression, understanding the regulation of LILR-expression could provide insight into their pathological role during inflammation.
In vivo regulation of LILRs has been observed in diseases such RA, psoriasis, infections
(tuberculosis, leprosy, HCMV and HIV) and cancer where variable expression of either activating or inhibitory LILR is associated with the outcome (Section 1.4.7). Similarly, expression of inhibitory LILRs during transplantation promotes graft acceptance (Section
1.4.8). In these diseases, an abundance in cells expressing inhibitory receptors is associated with immune evasion while a greater number of cells expressing activating receptors result
72 in a more exaggerated inflammatory response. In this regard, the underlying regulatory mechanisms of LILR-expression require further elucidation.
The regulation of LILR expression has not been extensively investigated (Section 1.3.3) and this limits the understanding of LILR function. LILRB4 is only known to be up- regulated on DCs after treatment with vitamin D3 (Manavalan et al., 2003; Penna et al.,
2005), IL-10 and IFN-α (Kim-Schulze et al., 2006). The functional relevance of this finding is that these mediators can tolerise DCs and consequently, induce T cell anergy.
For other LILRs, it is not known how their expression is induced.
The high expression of LILRA2 seen in the rheumatoid synovium is associated with the response of patients to treatment with anti-rheumatoid drugs but a direct regulation of
LILRA2 by DMARDs has been ruled out (Huynh et al., 2007). This leads us to believe that an indirect regulation of expression, possibly via cytokine signalling, could alter
LILRA2 expression. The pro-inflammatory cytokines, TNF-α and IL-1β have long been associated with RA and known to exacerbate joint inflammation by various means (Section
1.5.2). With the multitude of proteins regulated by TNF-α and IL-1 β signalling, it is fair to assume that LILRs could also potentially be regulated by similar means. IL-10 is also found within the synovium, being produced by mononuclear cells and having immunosuppressive activity (Katsikis et al., 1994). Understanding the mediators or factors that can alter LILR expression allows for further investigation into potential therapeutic targets.
73 The active form of vitamin D3 (1α,25-dihydroxyvitamin D3) is essential for calcium and bone metabolism but also has pronounced immunoregulatory functions particularly in abating autoimmunity (reviewed in Deluca et al., 2001; Mathieu et al., 2004). Affecting a multitude of cell types in many different ways (Dusso et al., 2005), one immunological importance of vitamin D3 is the skewing towards a Th2-response in T cells (reviewed in
Cantorna et al., 2004; Arnson et al., 2007). There are numerous studies which have investigated the potential benefits of vitamin D3 treatment.
Given the inflammatory role of LILRA2 and LILRB4 and their in vivo regulation of expression, we investigated the potential regulation of these receptors by inflammatory and immune regulatory mediators, such as active vitamin D3, LPS and the cytokines; TNF-α,
IL-1β, IL-10 and IFN-γ.
74 2.2 Materials and Methods
2.2.1 Antibodies and Reagents
Antibodies against LILRA2 and LILRB4 were generously donated by Amgen Inc. (Seattle,
WA) and irrelevant mouse IgG1 was purchased from Sigma-Aldrich (St Louis, MO).
FITC-conjugated anti-CD14, IgG2b-FITC and FITC-conjugated F(ab')2 goat anti-mouse
IgG [F(ab')2-specific] with minimum cross-reactivity to human, rat and bovine serum was from BD Biosciences (Mountain View, CA).
Recombinant human cytokines; TNF-α, IL-1β, IL-10 and IFN-γ were purchased from
R&D Systems (Minneapolis, MN), and vitamin D3, lipopolysaccharide (LPS), bovine serum albumin (BSA) and sodium azide (NaN3) from Sigma-Aldrich. Cell culture reagents; RPMI1640, Dulbecco’s phosphate-buffered saline (D-PBS), L-glutamine, penicillin/streptomycin, sodium pyruvate, N-2-Hydroxyethylpiperazine-N´-2-Ethane
Sulfonic Acid (HEPES) and 2-mercaptoethanol were from GIBCO-Invitrogen (Carlsbad,
CA), foetal bovine serum (FBS) purchased from JRH Biosciences (Lenexa, Kansas USA), and Ficoll-Paque™ PLUS was from GE Healthcare (Uppsala, Sweden).
® ® Tri Reagent and TURBO DNase were purchased from Sigma-Aldrich and Ambion
(Austin, TX), respectively while Polymerase Chain Reaction (PCR) reagents; PCR
Optimizer Kit™ SuperScript™ III Reverse Transcriptase and SYBRGreen™ were from
Invitrogen.
75 2.2.2 Cell Culture
The THP-1 pro-monocytic cell line from the American Cell Culture Collection (ATCC,
Manassas, VA) was used as in this model for regulation of LILR expression and cultured as suggested by ATCC. Cells were cultured in RPMI1640 medium supplemented with complement-inactivated FBS (10%), L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), sodium pyruvate (1 mM), HEPES (10 mM) and 2- mercaptoethanol (50 μM). FBS was complement-inactivated by heating to 56°C for 30 minutes. Further supplementation with L-glutamine was done as necessary. Cultures were
5 5 maintained between densities of 0.8 × 10 - 5 × 10 cells/mL and used experimentally at early passages (less than 8).
A chromogenic assay (Spectrozyme LAL assay) was periodically used to determine the level of LPS contamination in media and was obtained from American Diagnostica
(Stamford, CT).
2.2.3 Peripheral Blood Mononuclear Cells (PBMCs) Extraction
PBMCs were prepared from 40 mL of anti-coagulated whole blood obtained from healthy volunteers using standard Ficoll-Paque™ gradient. In brief, anti-coagulated blood was added to an equal volume of D-PBS and split into 30 mL lots. Ficoll-Paque™ PLUS (15 mL) was underlayed and then centrifuged at 420×g for 25 minutes. Buffy coats (PBMC) were carefully extracted and cells were washed twice with D-PBS. Cells were enumerated
® using a Coulter Ac·T Diff™ differential cell counter (Beckman Coulter, Fullerton, CA).
76 2.2.4 Differentiation of THP-1 Cells using Vitamin D3
THP-1 cells were differentiated to macrophages using active vitamin D3 by a standard
6 method as described elsewhere (Schwende et al., 1996). Cells (3 × 10 cells) were re- suspended in 15 mL media in a 100 mm diameter tissue culture dish (BD Falcon) containing 10 nM vitamin D3 in complete media for 48 hours at 37° with 5% CO2 in air.
Cells were washed with D-PBS and cells incubated for a further 24 hours in complete media containing vitamin D3. Cells were lifted by washing dishes with cold D-PBS then neutralised with warm D-PBS and counted before being used in further experiments.
2.2.5 Cell Stimulation
6 THP-1 cells (2 × 10 cells) were cultured in 3 mL media in 12 well tissue culture plates
(Greiner Bio-one, Monroe, NC). Cells were stimulated with LPS (100 ng/mL), TNF-α (25 ng/mL), IL-1β (25 ng/mL), IL-10 (10 ng/mL) or IFN-γ (25 ng/mL) and incubated at 37°C with 5% CO2 in air. After the optimised period of time, cells were either homogenised in
® TRI Reagent for RNA quantification or labelled for flow cytometry to assess protein expression.
2.2.6 Flow Cytometry
After stimulation, cells were washed with D-PBS and stained for LILR expression. Cells (1
5 × 10 ) were incubated with 0.5μg anti-LILR antibody for 30 minutes. Excess antibodies were removed by washing with 1.5 mL PAB buffer containing BSA (1%) and NaN3
(0.05%) in D-PBS. FITC-conjugated goat-anti-mouse IgG1 (Jackson) was pre-absorbed
77 with equal volume of goat serum for 30 minutes at room temperature and then added to cells (0.3 μg/μL) and incubated for 40 minutes on ice. Cells were fixed with 2%
paraformaldehyde in D-PBS, and analysed by using a FACScan™ flow cytometer and the
BD CellQuest™ software (BD Biosciences).
2.2.7 RNA Extraction
® RNA was extracted using TRI Reagent , as per manufacturer’s instruction. Cells were homogenised in 1000 µL Tri Reagent then one-fifth volume chloroform added and the mixture vortexed and spun at 14,000 rpm for 15 minutes (4°C). Equal volume of isopropanol was added to the aqueous layer and centrifuged for 8 minutes at room temperature. The RNA pellet was washed in 1000 µL 70% ethanol, air dried and re- suspended in 40 µL diethyl pyrocarbonate (DEPC)-treated H2O. RNA was quantitated
® using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE) and quality was assessed by separation of ribosomal subunits on a 1.5% agarose in Tris-
Acetate-EDTA (TAE) gel.
2.2.8 DNase-Treatment and Reverse Trascription (RT)
® Prior to reverse transcription, 1 μg RNA was subjected to DNase treatment using TURBO
DNase (Ambion) as suggested by the manufacturer. RNA was incubated with 1U of
DNase in the buffer provided and incubated at 37°C for 15 minutes. The reaction was stopped by the addition of 5 mM EDTA and heating samples at 75°C for a further 10 minutes. Complimentary DNA (cDNA) was synthesised using SuperScript™ III First-
78 Strand Synthesis SuperMix for qRT-PCR. RNA (1 µg) was reverse transcribed as per manufacturer’s instructions. The cDNA was diluted to a total volume of 50 µL in DEPC- treated H2O for use in downstream applications.
2.2.9 Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)
® PCR reactions contained the following: 5 µL of diluted cDNA, 12.5 µL SYBR
GreenER™ qPCR SuperMix Universal, 160 nM forward and reverse primers (Table 2.1) and remaining volume made up 25 µL with DEPC-treated H2O. Thermal cycling was carried out in an ABI PRISM™ 7700 Sequence Detector (Perkin Elmer, Waltham, MA) using the following conditions: 50°C for 2 minutes, 95°C for 10 minutes followed by 35 cycles of amplification (95°C denaturation for 15 seconds, 60°C annealing for 1 minute) and a final extension at 25°C for 1 minute. Data was analysed using the ABI PRISM™
Sequence Detection Systems: Sequence Detector (Version 1.7) program (Perkin Elmer).
Primer specificity was analysed after thermal cycling by using the following conditions:
95°C for 15 seconds, 60°C for 20 seconds and 95°C for 15 seconds with a ramping time of
20 minutes between the last 2 parameters. Melt curves were analysed for single products using the ABI PRISM™ Sequence Detection Systems: Dissociation Curves (Version 1.0) program (Perkin Elmer).
79 Table 2.1 » Base pair sequences of primer sets.
Transcript Sequence
(FP) 5’-GTT CGA GTC ATA AGC ATA GCA CC-3’ LILRA2 (RP) 5’-CAC CCA CAA CGC CTG AAC T-3’
(FP) 5’-CAT GTA CGT TGC TAT CCA GGC-3’ β-actin (RP) 5’-CTC CTT AAT GTC ACG CAC GAT-3’
(FP) = forward primer, (RP) = reverse primer
80 2.2.10 Statistical Analyses
Data for RNA and protein expression represents mean ± SEM. Changes in relative LILR expression were analysed by either a Student’s t-test or a one-way analysis of variance
(ANOVA) test with a Dunnett’s multiple comparison post test which compares treatment groups to an untreated control group. Statistical analyses were performed using GraphPad
® Prism 5 (San Diego, CA). A p value of <0.05 was considered significant.
81 2.3 Results
2.3.1 Regulation of LILRA2 and LILRB4 Expression upon Differentiation of Monocytes to Macrophages
After differentiation of THP-1 cells to macrophages, cells exhibited characteristic phenotypic changes having increased in size, developing dendritic processes and increasing in adherence (data not shown). THP-1 cells and differentiated-macrophages (or in vitro-derived macrophages) were stained for LILRA2 and LILRB4 expression as well as for CD14 as a positive control. Differentiated-macrophages were compared to a mock- treated control that was cultured under the same conditions without vitamin D3 and cell surface expression of LILRA2, LILRB4 and CD14 was measured by flow cytometry and the mean fluorescence intensity (MFI) was determined (Figure 2.1). Figure 2.1A illustrates how MFI shows changes in receptor expression between stimulated and un-stimulated cells. The histogram for LILRA2 expression on macrophages (red line) lies to the right of
THP-1 cells (blue line) and this reflects a greater fluorescence in macrophages as measured along the horizontal axis. Increase fluorescence correlates with a greater quantity of antibody bound to cells and therefore a greater expression of the receptor. The MFI is the mean value of the histogram curve. The average MFI for LILRA2 on un-stimulated cells is
12.80 ± 0.62 and after differentiation, increased to 23.33 ± 2.41 (Figure 2.1B, p=0.01).
Expression of LILRB4 also increased upon differentiation, from 78.20 ± 6.62 to 155.55 ±
12.11 (Figure 2.1C, p=0.005). Cell surface CD14 was used as a positive control as it has been already shown to be up-regulated upon differentiation of THP-1 cells and here, increased from 13.67 ± 5.28 to 50.60 ± 14.59 after differentiation (Figure 2.1D, p=0.001).
82 A. B. isotype 30 ** THP-1 macrophages 20
Counts 10 LILRA2 MFI LILRA2
0
LILRA2-FITC C. 200 **
150
100
LILRB4 MFI LILRB4 50
0
D. 80 **
60
40 CD14 MFI 20
0
untreated vitamin D3
Figure 2.1 » Regulation of LILRA2 and LILRB4 expression by vitamin D3. THP-1 monocytes were differentiated to macrophages by culturing cells with vitamin D3 (10 nM) for 72 hours and expression of LILRA2, LILRB4 and CD14 measured by flow cytometry. (A) The mean fluorescence intensity (MFI) was measured and macrophages had a shift in expression compared to THP-1 cells. There is a significant increase of (B) LILRA2 and (C) LILRB4 expression on differentiated-macrophages (n=5 and n=3, respectively). (D) CD14 also significantly increased on macrophages and was used as a positive control (n=5). Data represent mean ± SEM and analysed by a Student’s t-test. **p<0.01
83 2.3.2 Regulation of LILRA2 and LILRB4 Expression by Cytokines and LPS on Monocytes and Macrophages
To assess the potential regulation of LILR expression by inflammatory mediators such as
LPS, TNF-α, IL-1β, IL-10 or IFN-γ, cells were incubated with optimal doses for 24 hours
(Hu et al., 1996; Xu et al., 2000). Prior to culturing of cells, reagents including media and
D-PBS were periodically tested for endotoxin contaminants by Spectrozyme LAL assay
(data not shown). Only reagents where levels were below the level of detection were used.
Cells were harvested and stained for LILRA2 and LILRB4 expression for flow cytometry.
As previously done, changes in LILR expression were measured by observing the MFI.
Following stimulation of THP-1 monocytes, cells exhibited no change in LILRA2 or
LILRB4 expression for any cytokine or LPS (Figure 2.2A-B). On the other hand, differentiated-macrophages exhibited significantly altered expression of LILRs (Figure
2.3). LPS, TNF-α, IL-1β and IFN-γ down-regulated cell surface expression of both
LILRA2 (Figure 2.3A) and LILRB (Figure 2.3B).
84 A. 50
40
30
20 LILRA2 MFI LILRA2 10
0
B. 100
80
60
40 LILRB4 MFI LILRB4 20
0
β γ α 10 - LPS IL-1 IL- IFN reated TNF- nt u
Figure 2.2 » Expression of LILRA2 and LILRB4 on THP-1 monocytes after stimulation with cytokines and LPS. THP-1 cells were treated with either LPS (100 ng/mL), TNF-α (25 ng/mL), IL-1β (25 ng/mL), IL-10 (10 ng/mL) and IFN-γ (25 ng/mL) for 24 hours then (A) LILRA2 and (B) LILRB4 expression was measured by flow cytometry. The effects of inflammatory mediators did not change expression levels of (A) LILRA2 and (B) LILRB4. Data represent mean ± SEM (n=3 for each group) of mean fluorescence intensity (MFI) and analysed by an ANOVA with a Dunnett’s post test.
85 A. 70 60 50 ** ** 40 ** ** 30
LILRA2 MFI LILRA2 20 10 0
B.
200
150 * * * 100
LILRB4 MFI LILRB4 50
0 γ -α β ted F -1 a LPS N IL IL-10 IFN- re T unt
Figure 2.3 » Expression of LILRA2 and LILRB4 on vitamin D3-differentiated-macrophages after stimulation with cytokines and LPS. Macrophages were treated with either LPS (100 ng/mL), TNF-α (25 ng/mL), IL-1β (25 ng/mL), IL-10 (10 ng/mL) and IFN-γ (25 ng/mL) for 24 hours then LILRA2 and LILRB4 expression was measured by flow cytometry. LPS, TNF-α, IL-1β and IFN-γ all down- regulated both (A) LILRA2 and (B) LILRB4 cell surface expression. Data represent mean ± SEM (n=3 for each group) of mean fluorescence intensity (MFI) and analysed by an ANOVA with a Dunnett’s post test. *p<0.05, **p<0.01
86 2.3.3 Regulation of LILRA2 and LILRB4 Expression by Cytokines and LPS on Primary Monocytes
Following the observations that LPS and cytokines modulate LILRA2 and LILRB4 expression on in vitro-derived macrophages, the study was replicated using primary cells.
PBMCs were incubated with LPS and cytokines, TNF-α, IL-1β, IL-10 or IFN-γ, and expression of LILRA2 and LILRB4 were measured at 24 and 48 hours. The monocyte population was selected by gate and confirmed by analysis of CD14 expression.
As previously done, changes in LILR expression were measured by determining the MFI after flow cytometric analysis. Following stimulation of PBMCs, cells within the monocyte gate treated with IFN-γ had significantly increased in both LILRA2 and LILRB4 expression after 24 hours (Figure 2.4A-B). However at 48 hours, this measured difference is lost as untreated cells also exhibited up-regulation of LILRA2 and LILRB4 expression, though there was a significant difference between expressions of LILRB4 on untreated and
LPS-treated cells at this time-point (Figure 2.5A-B).
87 A. 60
* 40
20 LILRA2 MFI LILRA2
0
B. 60 ***
40
20 LILRB4 MFI LILRB4
0
S α β γ -1 -10 LP NF- IL IL IFN- T untreated
Figure 2.4 » Expression of LILRA2 and LILRB4 on primary macrophages after stimulation with cytokines and LPS for 24 hours. PBMCs were treated with either LPS (100 ng/mL), TNF-α (25 ng/mL), IL-1β (25 ng/mL), IL-10 (10 ng/mL) and IFN-γ (25 ng/mL) and monocytes analysed for LILRA2 and LILRB4 expression by flow cytometry. IFN-γ up-regulated both (A) LILRA2 and (B) LILRB4 cell surface expression while other mediators did not alter LILR expression. Data represent mean ± SEM (n=3 for each group) of mean fluorescence intensity (MFI) and analysed by an ANOVA with a Dunnett’s post test. *p<0.05, ***p<0.001
88 A. 100
80
60
40 LILRA2 MFI LILRA2 20
0
B. 100
80
60 * 40 LILRB4 MFI LILRB4 20
0
d S α β 0 γ e -1 -1 N- at LP IL IL IF tre TNF- n u
Figure 2.5 » Expression of LILRA2 and LILRB4 on primary macrophages after stimulation with cytokines and LPS for 48 hours. PBMCs were treated with either LPS (100 ng/mL), TNF-α (25 ng/mL), IL-1β (25 ng/mL), IL-10 (10 ng/mL) and IFN-γ (25 ng/mL) and monocytes analysed for LILRA2 and LILRB4 expression by flow cytometry. LPS did not alter (A) LILRA2 expression but down-regulated (B) LILRB4 and the other mediators did not alter LILR expression. Data represent mean ± SEM (n=3 for each group) of mean fluorescence intensity (MFI) and analysed by an ANOVA with a Dunnett’s post test. *p<0.05
89 2.3.4 Regulation of LILRA2 mRNA by LPS on Primary Monocytes
To assess the potential regulation of LILRA2 mRNA expression by LPS in primary monocytes, PBMCs were treated with LPS (10 ng/mL) and incubated for 2, 6 and 12 hours. Wells were gently washed to remove non-adherent cells and analysed for LILRA2 mRNA by real-time PCR. At 2 hours, there was no significant difference between untreated cells compared to cells stimulated with LPS (Figure 2.6). At 6 and 12 hours, LPS significantly increased the levels of LILRA2 mRNA.
90 0.10 * untreated 0.08 * LPS -actin) β 0.06
0.04
0.02 LILRA2 Fluorescence LILRA2 (normalised to (normalised 0.00 2 6 12 Hours
Figure 2.6 » Expression of LILRA2 mRNA in primary monocytes after stimulation with LPS. Cells were stimulated for 2, 6 and 12 hours with LPS and LILRA2 mRNA measured by real-time PCR. Expression of LILRA2 was normalised against the house-keeping gene, β-actin. There was no significant change in LILRA2 mRNA at 2 hours. However, at 6 and 12 hours there was a significant increase in LILRA2 mRNA in LPS stimulated cells (n=3 for each group). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. *p<0.05
91 2.4 Discussion
For stimulation of cells with LPS, it was essential to minimise potential contamination from media, wash buffers and reagents. Therefore, stringent measures were taken to ensure that cells were not prematurely stimulated during preparation. This included the use of commercially-tested media and D-PBS with undetectable levels of endotoxin. Reagents that were manually prepared were periodically tested using the Spectrozyme LAL assay and only reagents with endotoxin levels below the level of detection were used.
LILRA2 and LILRB4 may have a greater role on macrophages compared to monocytes since during differentiation their expression is induced (Figure 2.1). It has been previously shown that LILRB4 is up-regulated by vitamin D3 on immature DCs purified from peripheral blood (Penna et al., 2005). Consistent with the known literature, CD14 expression also increased when differentiating THP-1 monocytes to macrophages using vitamin D3 (Martin et al., 1994) and thus validating our findings. This up-regulation of
CD14 also occurs on primary monocytes differentiated to macrophages (Clavreul et al.,
1998). Signalling via LILRs could potentially function in myeloid development including the maturation of circulating monocytes to develop into tissue macrophages. Primary monocytes express both LILRA2 and LILRB4 so it would be interesting to observe their expression pattern in an in vivo model of inflammation. Culturing primary monocytes alone also promotes maturation to macrophages and so a change in LILR expression can potentially be detected here. Since LILRB4 is associated with a positive outcome for
92 transplantation, vitamin D3 supplementation with standard immuno-suppressants may delay or ablate acute graft rejection.
The role of cytokines has been well defined in the pathogenesis of RA. Cytokines such as
TNF-α and IL-1 recruit and activate cells to the site of inflammation in the joints. They also regulate the release of matrix-degrading enzymes MMPs (Section 1.2.4). Readily detected in the rheumatoid synovium (Fontana et al., 1982; Di Giovine et al., 1988;
Hopkins et al., 1988; Hopkins et al., 1988; Saxne et al., 1988), TNF-α and IL-1 have been of great interest for therapeutic treatment, with anti-TNF-α having greater success than anti-IL-1. Cytokine signalling induces leukocyte function through transcriptional regulation of other genes, mostly via NF-κB-dependent pathways (reviewed in Natoli et al., 1998; Van Antwerp et al., 1998). Interestingly, LILRA2 was down-modulated by
TNF-α on in vitro-derived macrophages, and also by LPS, IL-1 and IFN-γ (Figure 2.3).
Given that TNF-α promotes macrophage activation, down-regulation of an activating receptor is a likely to be the result of a feedback mechanism, to eliminate further activating signals. Concomitantly, there is a decrease in LILRB4 expression caused by the same mediators. Being closely related receptors, activating and inhibitory LILR genes may be regulated by the same transcriptional factors.
LPS was included in this investigation as earlier studies into the induction of reactive arthropathies implicated LPS as a potential triggering factor. Though its focus has waned over time, LPS was shown to induce reactive arthritis in animal models that closely resemble human RA (Stimpson et al., 1987; Noyori et al., 1994; Yoshino et al., 2000).
93 LPS induces the production of rheumatoid factor (Dresser et al., 1976; Izui et al., 1979), an important marker for RA and other autoimmune diseases. Expression of LPS ligands such as LPS-binding protein (LBP) (Heumann et al., 1995), CD14 (Yu et al., 1998) and TLR4
(Radstake et al., 2004) within the rheumatoid synovium further implicate the role of LPS in RA. In primary cells, LPS induces LILRA2 mRNA (Figure 2.6) but does not alter cell surface expression (Figure 2.4). It may be that intracellular expression is increased but fails to mobilise to the surface. LILRA2 expression by cell lines is down-regulated by LPS and this may be secondary to LPS induced TNF-α production which inhibits LILRA2 expression by these cells. It would be interesting to observe the effect of LPS on LILRA2 signalling. Also, further experimental studies into regulation of LILRA2 and LILRB4 mRNA expression by real-time PCR will be applied to cytokine-treated cells. Notably, a decrease in LILRA2 mRNA has been observed in culturing PBMCs over time and in the absence of LPS (Figure 2.6).
The cells predominantly expressing LILRs in the rheumatoid synovium are macrophages, neutrophils, mast cells, synovial fibroblasts and vascular endothelial cells (Section 1.6.5).
Importantly, LILRs are not expressed in the normal synovium and have low expression in the joints of patients with osteoarthritis. Among the patients with RA, those with a high
DAS, i.e. highly active disease, have a greater expression of LILRs compared to those with mild disease, and this was not attributed to a direct regulation by anti-rheumatoid drugs
(Huynh et al., 2007). It is likely that the drugs are affecting cytokine expression and indirectly modulating LILR expression.
94 Although it has been consistently shown that activation and inhibitory receptors are co- regulated, it is difficult to explain how they impact on cellular function. Given that activated macrophages express LILRA2 and LILRB4 and in rheumatoid synovial tissue,
LILRA2 is co-expressed with LILRB2 and LILRB3, there must be a failure of inhibitory signalling to result in an inflammatory response. Also, whether these LILRs directly interact is unknown. High homology between LILRs suggests that activating and inhibitory receptors could share or compete for ligands, so we cannot discount the regulation of receptor ligands and their role in controlling immune responses. Once again, we observe the co-regulation of activating and inhibitory receptors, emphasising the importance of counter-balancing signals in immune responses.
95 Chapter 3. Effect of LILRA2 and LILRB4 on Cytokine Production in Monocytes and Macrophages
3.1 Introduction
Each of the activating LILRs bear great resemblance in structure and sequence, the majority having 4 extracellular C-2 domains, a positively charged arginine residue in their transmembrane region and short cytoplasmic domains (Table 1.2). It was discovered in
LILRA2 that the activating LILRs, via a transmembrane arginine residue, form an ionic bond to the FcR γ chain and employ this adaptor protein for intracellular signalling purposes (Nakajima et al., 1999). The γ-chain is the principal signalling mechanism for Fc receptors, having 2 ITAMs on the intracellular domain (Takai, 2005). As ligands to
LILRA2 have not been identified, functional studies are performed by cross-linking of the cell surface receptor using monoclonal antibodies and this interaction mimics receptor-
2+ ligand binding. Cross-linking LILRA2 leads to intracellular calcium (Ca ) mobilisation
(Nakajima et al., 1999), the production and/or release of IL-3, IL-4, cysLT and histamine in primary basophils (Sloane et al., 2004), the production and/or release of IL-4, IL-12,
EDN and cysLT from primary eosinophils (Tedla et al., 2003) and TNF-α, IL1β and IL-6 production in primary monocytes and macrophages (Borges et al., 2003; Huynh et al.,
2007). Since these are early inflammatory mediators, it can be speculated that LILRA2 may function in acute inflammatory responses.
Of the inhibitory LILRs, LILRB4 is structurally unique in having 2 extracellular Ig-like domains (Table 1.2). Compared to LILRB1, this truncated protein lacks the HLA-binding
96 site and is therefore speculated not to bind HLA (Willcox et al., 2003). Despite not knowing its ligand, extensive studies have determined that LILRB4 on DCs functions in lymphocyte tolerance during allogeneic-recognition and inhibition of antigen-specific immune responses (Section 1.3.5.2). Since its inhibitory function is inferred on target cells during HLA-mediated recognition, identifying its ligand/s is a crucial factor to help understand the complete function of LILRB4.
LILRB4 is differentially expressed on DCs and is likely to signal via the immunological synapse formed between MHC and the TCR, though confirmation by molecular studies is required. By rendering effector T cells anergic, LILRB4 signalling is essential to antigen- specific immune responses (Chang et al., 2002; Manavalan et al., 2003). This has led to the implication of LILRB4 in transplant immunology whereby LILRB4 induces anergy in auto-reactive effector cells and eliminates a graft-versus-host response (reviewed in Suciu-
Foca et al., 2005). More recently, findings of abnormal LILRB4 expression on cancer cells has linked its role to tumour immune-evasion (Colovai et al., 2007; Suciu-Foca et al.,
2007). This is likely to occur by tolerisation of effector cells, such as NK cells, which are normally partially responsible for the elimination of anaplastic cells.
Classical ITIM-inhibition of ITAMs has been well described and so in the case of LILRs, many inhibitory LILRs have the potential to inhibit their activating counterparts. LILRB1,
LILRB2 and LILRB3 actively block the function of ITAM-bearing receptors upon co- ligation (Section 1.3.5.2) and whether this inhibitory potential applies to human LILRB4 is
97 yet to be defined. In mice, mLILRB4 has been shown to inhibit ITAM-mediated responses
(Section 1.4.4).
Cytokines such as TNF-α, IL-1 and IL-6 can be induced specifically by LILRA2 (Borges et al., 2003; Huynh et al., 2007) and are key mediators in the pathogenesis of RA (Section
1.6.3). As LILRA2 is highly expressed within the rheumatoid synovium (Tedla et al.,
2002), it is plausible that LILRA2-positive macrophages (via interaction with its ligand) contribute to inflammation of the joint by inducing the production of pro-inflammatory cytokines.
With the evident patterned expression of both LILRA2 and LILRB4 in various inflammatory conditions, we hypothesise that these receptors are functional on myeloid cells in regulating their activation during immune responses. We investigated the influence of LILRA2 and LILRB4 on the production of inflammatory cytokines.
98 3.2 Materials and Methods
3.2.1 Antibodies and Reagents
Antibodies against LILRs and irrelevant mouse IgG1 was previously stated (Section 2.2.1).
Coating antibody, F(ab’)2 goat anti-mouse IgG, Fcγ fragment-specific with minimal cross- reactivity to human, bovine and horse serum proteins were purchased from Jackson
ImmunoResearch. BSA Fraction V, 1α,25-dihydroxyvitamin D3 (active vitamin D3), dexamethasone, methotrexate and cyclosporin A was purchased from Sigma-Aldrich.
3.2.2 Cell Culture and Differentiation of THP-1 Cells
THP-1 cells were cultured and differentiated to macrophages as previously mentioned
(Section 2.2.2 and 2.2.4). A total of two washes with D-PBS were done prior to cross- linking/co-ligation experiments.
3.2.3 Expression of LILRA2 and LILRB4 on Monocytes and Lymphocytes
As PBMCs were used in cross-linking and co-ligation studies, we had to clearly define the expression of LILRA2 and LILRB4 on the cells that constitute PBMCs, namely monocytes and lymphocytes. PBMCs were isolated and stained for expression of LILRA2 and
LILRB4, as previously mentioned (Sections 2.2.3 and 2.2.6).
99 3.2.4 Cross-linking of Cell Surface LILRA2
THP-1 cells, THP-1-differentiated-macrophages and whole PBMCs were activated by cross-linking LILRA2 using plate bound anti-LILRA2 antibody as described elsewhere
® (Kim et al., 1997; Tedla et al., 2003). In summary, wells of 96-well flat-bottom Costar
3596 tissue culture plates (Corning Incorporated, Corning, NY) were coated overnight at
4°C with 50 μL (2.5 μg) F(ab’)2 goat anti-mouse IgG, Fcγ fragment-specific (with minimal cross-reactivity to human, bovine and horse serum proteins), in D-PBS. After aspiration,
50 μL LILRA2-specific mAb which was diluted to the desired concentrations in D-PBS containing 2.5% BSA-fraction V was added to wells. Irrelevant mouse IgG1 mAb was used as negative control. After incubation for 2 hours at 37°C with 5% CO2 in air, wells were washed twice with 0.9% NaCl before use. In the meantime, cells were harvested, washed twice with D-PBS and re-suspended in RPMI1640 supplemented with 10 mM
5 HEPES and 0.1% BSA. For THP-1 cells and macrophages, 1 × 10 cells in 200 μL were
5 added to wells and for PBMCs, 4 × 10 cells were used. After incubation at 37°C and 5%
CO2 in air, cell-free supernatants were collected at various time points and stored at -80°C for quantification of cytokines.
3.2.5 Quantification of Cytokine Production
Two methods were employed for the measurement of cytokines. Initially, supernatants were analysed for TNF-α levels by enzyme-linked immunosorbent assay (ELISA). TNF-α
® ELISA was performed using DuoSet ELISA Development Kit (R&D Systems,
Minneapolis, MN) according to manufacturer’s instructions. Cell-free supernatants (50 μL)
100 were analysed for TNF-α levels and assays performed in duplicate. In cross-linking/co- ligation assays, samples obtained from THP-1 cells were diluted 1:1 with ELISA dilution buffer, while supernatant samples from PBMCs were diluted 1:9 to fit into the standard curve of the ELISA.
Following optimisation of conditions by TNF-α ELISA, samples were also analysed using the 8-Plex™ Bio-Plex™ Cytokine Assay (Bio-Rad Laboratories, Hercules, CA), measuring TNF-α, IL-1β, IFN-γ, IL-4, IL-6, IL-10, IL-12 and IL-13, and was performed as per manufacturer’s instructions. In summary, supernatants harvested from receptor cross- linking/co-ligation studies were quantitatively analysed for the above mentioned cytokines in a 96 well filter plate. Bead-conjugated anti-cytokine antibodies were added to the plate and solution removed by vacuum filtration. Undiluted samples and standards were added to wells and incubated with beads for 30 minutes while shaking. Wells containing bead- conjugated cytokines were washed 3 times using wash buffer provided then cytokine detection antibodies were added and incubated for a further 30 minutes while shaking.
Wells were washed 3 times and detected with strepavidin-PE. The assay plate was analysed using a Bio-Plex™ 200 Suspension Array System (Bio-Rad Laboratories).
3.2.6 Co-ligation of Cell Surface LILRA2 and LILRB4
To assess the potential inhibition of LILRA2-activation by LILRB4, receptors were co- ligated on the surface of monocytes in whole PBMCs using mAb and supernatants measured for cytokine production. In brief, plates were coated with secondary antibody as previously described and then coated with a combination of LILRA2 and LILRB4
101 5 monoclonal antibodies at 1 and 10 μg/mL, respectively. PBMCs (4 × 10 ) were added to wells and incubated at 37°C and 5% CO2 in air. Cell-free supernatants were harvested after
24 hours and cytokines measured by 8-Plex™ Bio-Plex™ Cytokine Assay.
3.2.7 Effect of Anti-Rheumatoid Drugs on Cell Viability
To assess the cytotoxicity of the anti-rheumatoid drugs used in this study, THP-1 cells were treated with optimal doses of cyclosporin A (100 ng/mL), methotrexate (50 ng/mL) or dexamethasone (10 μM) for 72 hours and viability of cells in suspension assessed by flow cytometry using Annexin V-FITC Apoptosis Detection Kit I (BD BioSciences). Cells
5 (1 × 10 ) were stimulated in 1 mL of complete media for 72 hours then washed twice with
2 mL D-PBS and re-suspended in 1× binding buffer (provided in kit) at a concentration of
5 1 × 10 cells/mL. Cells were incubated with 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) for 15 minutes and then analysed by a FACScan™ flow cytometer and BD CellQuest™ software (BD Biosciences).
3.2.8 Effect of DMARDs on LILRA2 Activation in Macrophages
LILRA2-mediated TNF-α production was assessed at the maximal time point by cross- linking of LILRA2 on the surface of in vitro-derived macrophages with an optimal dose of anti-LILRA2 mAb (0.1 μg/mL) in the presence or absence of three doses of dexamethasone (1, 10, 100 μM), methotrexate (500, 50, 5 ng/mL) or cyclosporin A (100,
100, 10 ng/mL). Supernatants were harvested after 24 hours and TNF-α was measured by
ELISA.
102 3.2.9 Statistical Analyses
Data for cell activation measured by cytokine production is represented as mean ± SEM.
The quantity of TNF-α was normalised to the percentage of monocytes within PBMCs for each patient after a differential cell count. Changes in cytokine production between cell types (THP-1 cells, vitamin D3-differentiated-macrophages and PBMCs) was analysed by a two-way analysis of variance (ANOVA) while data obtained from drug-treatments were analysed by a one-way ANOVA with a Dunnett’s post test comparing each treatment group to the untreated control group. The effect of LILRB4 on LILRA2-activation was analysed by an ANOVA and Bonferroni’s post test. Statistical analyses were performed
® using GraphPad Prism 5. A p value of <0.05 was considered significant.
103 3.3 Results
3.3.1 Expression of LILRA2 and LILRB4 on Peripheral Blood Mononuclear Cells (PBMCs)
In order to study the function of LILRA2 and LILRB4 on primary leukocytes, their expression was first defined. Cell surface expression of LILRA2 and LILRB4 was analysed on lymphocytes and monocytes from purified PBMCs. PBMCs were purified from healthy, control subjects and stained using monoclonal antibodies against LILRA2 and LILRB4 along with the relevant IgG1 isotype control. On the forward-side scatter profile of PBMCs, lymphocytes and monocytes are presented in distinct populations based on size and granularity. Selecting lymphocytes by gate (Figure 3.1A), being smaller in size and less granular than monocytes, expression is analysed and represented in the frequency histograms. LILRA2 and LILRB4 staining, represented by the bold line is similar to the isotype control, represented by the thin line (Figure 3.1B-C), indicating that lymphocytes do not express these LILRs. Upon selection of monocytes by gate (Figure 3.1D), there is a rightward shift in the histograms for LILRA2 and LILRB4 when compared to the isotype control (Figure 3.1E-F) and therefore these cells express both LILRs.
104 A. B. C. SSC Counts Counts
D. E. F. SSC Counts Counts
FSC LILRA2 LILRB4
Figure 3.1 » Expression of LILRA2 and LILRB4 on circulating lymphocytes and monocytes. Purified PBMCs were stained for LILR expression and analysed by flow cytometry. (A) Lymphocytes were selected by gate and negative for both (B) LILRA2 and (C) LILRB4. (D) Monocytes were gated and stained positive for (E) LILRA2 and (F) LILRB4. Dot plot show forward scatter (FSC) vs. side scatter (SSC) profiles. Histograms show expression of LILRA2 and LILRB4 (bold lines) and relevant isotype controls (thin line).
105 3.3.2 Activation via LILRA2 Induces the Production of TNF-α in In vitro- derived Macrophages and Primary Monocytes.
Cell surface LILRA2 was cross-linked in a 96 well plate-based assay by coating plates with a capture secondary antibody followed by anti-LILRA2. Increasing the concentration of LILRA2 antibody induced a dose-dependent response and the peak concentration of
TNF-α was detected using 1 μg/mL antibodies producing an average of 632.9 ± 157.5 pg/mL (Figure 3.2A). Consistently, primary cells produced greater amounts of TNF-α than differentiated-macrophages. Time course analysis of LILRA2-mediated activation revealed maximal production of TNF-α at approximately 18 hours (Figure 3.2B). In contrast, substitution of anti-LILRA2 antibody with IgG1 isotype control antibody elicited negligible TNF-α production by THP-1 cells, differentiated-macrophages and PBMCs, ruling out non-specific activation. Moreover, the capture secondary antibody alone did not induce TNF-α production.
Cross-linking of LILRA2 on primary monocytes using increasing concentrations of antibodies also activated cells to produce TNF-α in a dose-dependent manner (Figure
3.2C). At the highest concentration of cross-linking antibody (1 ng/μL), 8210.0 ± 558.1 pg
4 4 of TNF-α per 10 monocytes was produced, significantly higher than 625.0 ± 375.0 pg/10 monocytes of TNF-α produced in response to the IgG1 isotype control antibody.
106 A. 700 *** 600 *** 500 400
(pg/mL) 300 ** α 200 TNF- 100 0 0.001 0.01 0.1 1 Concentration of antibody (μg/mL) B. 1500
1000
500 100 (pg/mL) α
50 TNF-
0 0 10 20 30 40 50 Time (hours) C. 10000 * 8000
6000 monocytes) 4 4000
(pg/10 2000 α
TNF- 0 0.001 0.01 0.1 1 Concentration of antibody (μg/mL)
Figure 3.2 » Antibody concentration-response and time-course for activation of cells via LILRA2. (A) LILRA2 was cross-linked on in vitro derived-macrophages for 24 hours with increasing concentrations of anti-LILRA2 mAb. (B) Supernatants collected at various time-points after cross-linking with 0.1 μg/mL of anti-LILRA2 mAb. (C) LILRA2 was cross-linked on PBMCs with increasing concentration of LILRA2 mAb. Graphs show data for cross-linking with anti-LILRA2 (▲) and IgG1 isotype control (●) (n=3 for each group). Data represents mean ± SEM. Data from (A) analysed by a two-way ANOVA and (C) was analysed by a Student’s t-test. *p<0.05, **p<0.01, ***p<0.001
107 3.3.3 Inhibition of LILRA2-mediated Cytokine Production upon Co- ligation with LILRB4
To assess its function, LILRA2 was cross-linked on monocytes of freshly purified PBMCs.
After 24 hours, supernatants were harvested and the production of TNF-α, IL-1β, IFN-γ,
IL-4, IL-6, IL-10, IL-12 and IL-13 was measured. LILRA2 cross-linking induced significant production of the cytokines, TNF-α, IL-6, IL-1β, IFN-γ and IL-10 when compared to IgG1-treated cells which elicited no detectable cytokine (Figure 3.3A-E).
In the same PBMC donors, LILRB4 was co-ligated with LILRA2 to investigate its inhibitory potential, using a mixture of 10 μg/mL and 1 μg/mL of mAbs, respectively. Co- ligation of LILRB4 abolished the production of all cytokines (TNF-α, IL-6, IL-1β, IFN-γ and IL-10) to below the level of detection (Figure 3.3A-E). The appropriate control for this co-ligation experiment are cells treated with IgG1 and LILRA2 mAb and this resulted in cytokine production comparable to LILRA2 cross-linking alone. A significant difference was observed when compared to LILRB4/LILRA2 co-ligation (Figure 3.3A-E). IL-4, IL-
12 and IL-13 were not detected in any wells of the assay suggesting that they were not produced (data not shown).
108 A. B. C. TNF-α IL-6 IL-1β 10000 4000 500 ** ** ** *** *** *** *** *** *** 8000 400 3000
6000 300 2000 (pg/mL) (pg/mL) (pg/mL) (pg/mL) αα
4000 ββ 200 IL-6 (pg/mL) (pg/mL) IL-6 IL-6
1000 IL-1IL-1 TNF-TNF- 2000 100
0 0 0 IgG1 + - - + + - - + + - - + αLILRA2 - + + + - + + + - + + + αLILRB4 - - + - - - + - - - + -
D. IFN-γ E. IL-10 80 100 ** ** *** * * * 80 60 60 40 (pg/mL) (pg/mL)
γγ 40 IL-10 (pg/mL) (pg/mL) IL-10 IL-10 IFN-IFN- 20 20
0 0 IgG1 + - - + + - - + αLILRA2 - + + + - + + + αLILRB4 - - + - - - + -
Figure 3.3 » Cytokine production induced by LILRA2 cross-linking and LILRA2-LILRB4 co-ligation. (A-E) Cross-linking of LILRA2 induced the production of TNF-α, IL-6, IL-1β, IFN-γ and IL-10 while LILRB4 co-ligated with LILRA2 inhibits cytokine production. IgG1 isotype control antibody did not induce cytokine production (n=3 for each group). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. *p<0.05, **p<0.01, ***p<0.001 109 3.3.4 Effects of Anti-rheumatoid Drugs on LILRA2-mediated Activation
Anti-rheumatoid drugs were added to the media of cells activated by LILRA2 to investigate whether these drugs impact LILRA2 function (Figure 3.4). Two DMARDs; cyclosporin A and methotrexate, and the corticosteroid, dexamethasone, were included in this study. Drugs were added to vitamin D-differentiated THP-1 cells at increasing log doses, the mid-dose being selected empirically from the existing literature (Hirohata et al.,
1999; Tsiavou et al., 2002). Dexamethasone inhibited LILRA2-mediated TNF-α production in a dose-dependent manner. With 100 μM of dexamethasone, a 70% reduction in TNF-α production was observed, reducing from 5183.3 ± 259.4 pg/mL to 1504.3 ±
171.3 pg/mL, (Figure 3.4A). Methotrexate and cyclosporin A failed to significantly alter
LILRA2-mediated TNF-α production at any concentration (Figure 3.4B-C).
To exclude the possibility that the drugs are inducing cell death, cells were analysed for apoptosis by standard Annexin V staining (Figure 3.5). Cells were incubated with drugs at optimal concentrations for 24 hours, including an untreated control. Cells were harvested and stained with Annexin V and propidium iodide (PI) to identify cells undergoing apoptosis and necrosis, respectively. In untreated cells, 7.5% of cells positively stained for both Annexin V and PI while 7.9% were positive for Annexin V alone, a combined total of
15.4% of dead cells (Figure 3.5). Comparable to the DMARD-treated cells, dexamethasone-treated exhibited 13.7%, methotrexate, 14.9% and cyclosporin A, 16.5% of apoptotic cells, respectively (Figure 3.5B-D). The dot plots shown are representative of 3 individual experiments and there were no significant differences between the treated groups compared to the untreated control.
110 A. 600 * 500
400 **
300 (pg/mL) α 200 ** TNF 100
0 0 1 10 100 Dexamethasone (μM) B. 600
500
400
300 (pg/mL) α 200 TNF 100
0 0 5 50 500 Methotrexate (ng/mL) C. 600
500
400
300 (pg/mL) α 200 TNF 100
0 0 10 100 1000 Cyclosporin A (ng/mL)
Figure 3.4 » Effects of anti-rheumatoid drugs on LILRA2-mediated activation in in vitro-derived macrophages. LILRA2 was cross-linked on macrophages with an optimised dose of 0.1μg/mL anti- LILRA2 in the presence of the anti-rheumatoid drugs, dexamethasone, methotrexate and cyclosporin A at varying doses. (A) Dexamethasone inhibited LILRA2-mediated TNF-α production in a dose- dependent manner while no difference was observed in cells treated with (B) methotrexate and (C) cyclosporin A (n=3 for each group). Data represents the mean ± SEM and analysed by an ANOVA with a Dunnett’s post test. *p<0.05, **p<0.01
111 A. B. untreated dex 7.5% 5.8%
7.9% 7.9%
C. mtx D. csA 5.6% 6.8%
9.3% 9.7%
E. F. negative positive 0.2% 95.3%
0.0% 4.4%
Figure 3.5 » Analysis of apoptosis induced by anti-rheumatoid drugs. Differentiated-macrophages were incubated with drugs at optimal concentrations, including an untreated control, for 24 hours. Cells were harvested and stained with Annexin V and propidium iodide (PI). (A) Untreated cells exhibited some cell death in culture. Cells treated with (B) dexamethasone (dex), (C) methotrexate (mtx) and (D) cyclosporin A (csA) did not demonstrate significantly increased cell death compared to the untreated control. (E) Negative (no antibodies) and (F) positive controls (treated with DMSO) verified true staining in treated groups. Dot plots shown are representative of 3 individual experiments.
112 3.3.5 Activation of LILRA2 on PBMCs from Patients with RA
Given that ex vivo steroid treatment of in vitro-derived macrophages inhibited LILRA2- mediated production of TNF-α, we tested whether leukocytes from patients treated with steroids had a differential response to LILRA2 stimulation compared to patients who were on treatments other than steroids. Cells from patients with RA produced greater amounts of
4 TNF-α after LILRA2 cross-linking (7876.2 ± 773.0 pg/10 monocytes) when compared to
4 healthy control subjects (5810.2 ± 1579.1 pg/10 monocytes), though this difference did not reach statistical significance (Figure 3.6A). Between the two RA groups (those treated with or without steroids), TNF-α production was similar upon LILRA2 cross-linking
4 (8154.3 ± 1064.6 and 7598.4 ± 1334.4 pg/10 monocytes, respectively). To investigate whether primary cells exhibit the same inhibitory response to in vitro dexamethasone treatment as seen with differentiated-macrophages, LILRA2 was cross-linked with various doses of dexamethasone added. Cells from healthy control subjects had diminished
LILRA2-mediated TNF-α production with 100 μM concentration of dexamethasone
(Figure 3.6B). Similarly in both groups of patients, there was a dose-dependent inhibition in response to ex vivo dexamethasone (Figure 3.6C-D).
113 A. B. Controls 10000 10000
7500 7500 monocytes) monocytes) 4 5000 4 5000 *
(pg/10 2500 (pg/10 2500 α α
TNF- 0
TNF- 0 normal RA (steroids) RA (no steroids)
C. RA - no steroids 10000
7500 ** monocytes)
4 5000 **
(pg/10 2500 α
TNF- 0
D. RA - steroids 10000
7500 * monocytes)
4 5000 **
(pg/10 2500 α
TNF- 0 0 1 10 100 Dexamethasone (μM)
Figure 3.6 » Cross-linking of LILRA2 on PBMCs from patients with RA treated with or without glucocorticoids and in vitro dexamethasone stimulation. (A) LILRA2-activation was compared between healthy control subjects, patients treated with glucocorticoids and patients without. RA patients had a slightly higher production of TNF-α upon LILRA2 cross-linking (n=3 for each group). PBMCs were also treated with in vitro dexamethasone along with LILRA2 cross-linking from patients (B) with steroid treatment and (C) patients without steroid treatment. In vitro dexamethasone continued to inhibit LILRA2-mediated TNF-α production (n=3 for both groups). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test.
114 3.4 Discussion
Acute inflammatory cytokines such as TNF-α, IL-1β and IL-6 are essential for immune responses towards microbial infections and a primary source of these cytokines are from cells of myeloid lineage, especially monocytes and macrophages. TNF-α is a multifunctional cytokine that induces the expression of other immune regulatory cytokines, chemokines and adhesion molecules, all of which are important for leukocyte activation and trafficking to the site of injury (reviewed in Sedgwick et al., 2000). TNF-α was first defined as a tumour killing agent and its highly pleiotropic nature provides many complications for targeted therapy. Anti-TNF-α therapy is often associated with increased risks of infection (both primary and secondary) and malignancies (reviewed in Bongartz et al., 2006; Strangfeld et al., 2006) not to mention, an immune response towards the anti-
TNF-α antibodies leading to autoimmunity (Doan et al., 2005). Paradoxically, TNF-α-
-/- deficient mice (TNF-α ) exhibit some protection from cancer progression (Suganuma et al., 1999), however, this artificial absence of TNF-α altogether has systemic implications.
Moreover, many inflammatory disorders are exacerbated by TNF-α in particular, autoimmune diseases such as RA and inflammatory bowel disease.
Cross-linking and co-ligation of cell surface receptors using antibodies is a useful tool, particularly when the ligand is unidentified, as it often mimics natural ligand-receptor interaction to stimulate the downstream signalling mechanisms of the receptor. This technique has been applied to studies on numerous immune-regulatory receptors including the T cell Receptor (Garcia et al., 1998), FcεRI (Yamasaki et al., 2005) and mLILRB (Lu-
Kuo et al., 1999), just to name a few. We applied the plate-based assay to our
115 monocyte/macrophage studies as activation of these cells induces adherence, allowing easy extraction of cell-free supernatants.
Considering LILRA2 is highly expressed within the inflamed synovium and cross-linking induces TNF-α release from monocytes and macrophages (Figure 3.2 and Figure 3.3), this suggests that LILRA2 may be involved in the pathogenesis of RA. It is well known that
TNF-α is abundant in the rheumatoid synovium (Fontana et al., 1982; Saxne et al., 1988) and potentially, LILRA2 is inducing its production in synovial macrophages and thus exacerbating the disease by further activation and recruitment of cells via TNF-α- dependent signalling. Understandably, there are many other regulators of TNF-α and
LILRA2-mediated production is not the sole source. However, its high expression in the synovium of patients with RA indicates that LILRA2 to some degree, may contribute to the abundant quantity of TNF-α found in the rheumatoid synovium. Also, it is unlikely that
LILRA2 exclusively functions in the pathogenesis of RA but may also have a role in other inflammatory disorders such as inflammatory bowel disease. Screening of other inflamed tissues can provide further insight into the inflammatory role of LILRA2.
Since LILRA2 and LILRB4 are expressed on monocytes and not on lymphocytes (Figure
3.1), stimulation of these receptors occurs on the monocytes in PBMCs. We cannot, however, rule out secondary effects of lymphocytes present in PBMCs. In the time-course of cytokine production upon LILRA2 cross-linking (Figure 3.2B), a single peak is observed and so we assume that the activity of lymphocytes is negligible to the activation monocytes. Typically, a second peak in cytokine production would reflect a secondary
116 immune response. This study attempts to explore the immunosuppressive function of
LILRB4 which has been observed in vivo. As previously reviewed, LILRB4 signalling is associated with induction of T cell tolerance and prevention of immune responses and its expression is associated with an increased chance of tissue graft acceptance after transplantation as well as tumour growth and progression (Section 1.3.8).
LILRB4 inhibits cytokine production (TNF-α, IL-6, IL-1β, IFN-γ and IL-10) via LILRA2 stimulation upon co-ligation of these receptors (Figure 3.3). Whether this occurs in vivo is yet to be explored however in vitro, LILRB4 has the potential to completely inhibit cytokine production by LILRA2. A direct interaction of these two receptors is difficult to determine without knowledge of their binding partners.
It is likely that LILRB4-mediated inhibition occurs locally and that TNF-α suppression occurs where LILRB4 is expressed. In the case of LILRB4 tumour-expression, this results in the lack of tumour killing by caspase-dependent and -independent programmed cell death which is attributed to TNF-α. Thus, the ability of LILRB4 to inhibit TNF-α production correlates with the observation that LILRB4-expression on tumours is associated with cancer progression. LILRB4 can also inhibit CD64 (FcγRI)-mediated
TNF-α production (Appendix I) suggesting a broader role for its inhibitory function.
Further molecular studies are required to determine the signalling molecules involved in
LILRB4-mediated inhibition.
117 LILRA2-mediated induction of TNF-α production was significantly abrogated by dexamethasone in a dose-dependent manner but not by the DMARDs tested (Figure 3.4 and Figure 3.6) and none of these drugs caused significant cell death by either apoptosis or necrosis of the THP-1-derived macrophages and PBMCs (Figure 3.5). Glucocorticoids are thought to exert anti-inflammatory effects via the GCR by blocking NFκB and AP-1 transcription factors and subsequent inhibition of genes which encode inflammatory mediators (Adcock et al., 1999; Newton, 2000) and their immunosuppressive function impacts on lymphocytes, monocytes/macrophages and endothelial cells (Boumpas et al.,
1993). Other mechanisms of action include; blocking LPS-mediated TNF-α production
(Han et al., 1990; Amano et al., 1993), inducing apoptosis of inflammatory cells (Fauci et al., 1976) and inhibiting the production of nitric oxide (Korhonen et al., 2002). Our results clearly indicate that inhibition of LILRA2-induced TNF-α production by macrophages and
PBMCs in response to dexamethasone is not due to increased cell death or down- regulation of LILRA2 expression on the cell surface. Whether dexamethasone inhibits
LILRA2-mediated cell activation by blocking NFκB and/or AP-1 remains to be explored but it is known that LILRA2 transduces stimulatory signals by recruiting protein tyrosine kinases through a cytoplasmic (ITAM) (Borges et al., 1997). It is therefore possible that dexamethasone inhibits LILRA2-mediated cell activation by interfering with the recruitment of protein tyrosine kinases. The cGR has shown to recruit Src in the presence of HSPs and MAPK (Section 1.6.4.3) and so a down-regulation of either may lead to reduced LILRA2 activity. Interestingly, patient treatment with steroids does not affect ex vivo stimulation of LILRA2, and cells are still responsive to further steroid treatment. It would be interesting to observe serum levels of TNF-α between patient groups to
118 determine the impact of glucocorticoids on systemic TNF-α production. The blocking of
LILRA2-mediated TNF-α production by dexamethasone is consistent with the known potent and pleiotropic effects of glucocorticoids in regulating inflammation. The inability for other DMARDs to significantly reduce TNF-α levels suggests that the efficacy of these drugs does not relate to a direct effect on LILR expression and/or function.
Although the studies presented here explore the function of LILRA2 and LILRB4, ex vivo, there would be more benefits in studying their functions using an in vivo experimental model of inflammation. The production of LILRA2-expressing transgenic mice may be useful to studying the systemic effects of LILRA2 activation and the potential therapeutic benefit of inhibiting the receptor by mAb or drugs.
Undoubtedly, the ability of ITIMs to inhibit cellular function is reliant to physical coupling of ITIM- and ITAM-bearing receptors (Bolland et al., 1999; Long, 1999). Without identification of the ligands, the exact function of LILRA2 and LILRB4 still remains elusive. The urgency for ligand identification of LILRA2 and LILRB4 is being recognised and the issue is currently being addressed however, with some difficulty (Garner et al.,
2005; Chen et al., 2007). Despite this, LILRA2 may be a therapeutic target for inflammatory disorders such as RA and help overcome the problems of anti-TNF-α therapy while LILRB4-induction could assist with the prevention of organ transplant rejection.
119 Chapter 4. Effects of LPS-stimulation on the Function of LILRA2 and LILRB4
4.1 Introduction
The innate immune system is essential for the elimination of infectious microbes by invoking the function of pattern recognition receptors (PRRs). TLRs are a family of PRRs and have been extensively studied in the context of innate immune responses. Collectively, they recognise a wide range of ubiquitous microbial antigens by their pathogen-associated molecular patterns (PAMPs). The focus in this study is on the function of TLR4 which binds LPS along with CD14.
CD14 and TLR4 (expressed on monocytes and macrophages) are the recognition receptors for LPS and bind with great affinity to induce immune responses (Section 1.5). Signalling of TLR4 and other TLRs occur via MyD88, TRAM and TRIF to induce NF-κB and interferon response factor (IRF)-3 and IRF-5 transcription factor-dependent pathways
(reviewed in Akira et al., 2006; Kawai et al., 2006; O'Neill, 2006; Uematsu et al., 2006;
Bowie, 2007). Downstream of TLR4-activation is the production of TNF-α, IL-6, IL-12,
IFN-β and RANTES (reviewed in Bowie, 2007). Extensive studies have indicated that
TLR-function on APC, DCs and macrophages are critical for transition from innate to adaptive immune responses (Schnare et al., 2001 and reviewed in Akira et al., 2001; Iwasaki et al., 2004). Moreover, some Ig receptors including activating LILRs can influence TLR- mediated responses. This cross-talk between receptor families may be the functional link between innate and adaptive immune responses.
120 LILRA2 is able to divert immune response in presence of TLR1/2 and TLR4 ligands as
LPS stimulation of primary monocytes induces both IL-12 and IL-10 production but in conjunction with LILRA2 stimulation, IL-12 is down-regulated and IL-10 production is enhanced (Bleharski et al., 2003). IL-10 is a product of both Th1 and Th2 cells but primarily functions in down-regulating the Th1 response (Del Prete et al., 1993) and thus,
LILRA2 cross-linking in the presence of LPS induces a Th2 immune response by altering
IL-10 to IL-12 ratios. LILRA2 has a broad role in inflammation by stimulating cytokine production that influences both acute and chronic inflammatory responses. Similarly,
LILRA4 affects TLR stimulation where LILRA4-cross-linking with CpG (TLR9 ligand) inhibits TNF-α and IFN-γ production in DCs, compared to individual stimulation (Cao et al., 2006). Again, there is a diversion of the immune response by skewing cytokine expression ratios. By altering cytokine patterns, LILRA2 and LILRA4 have an impact on
TLR function and influencing downstream responses. Whether interaction of LILRs and
TLRs is on a molecular level or regulated by ligand interaction, is currently unknown.
Potentially, activating receptors can act in synergism to enhance activation or have an antagonistic effect which may result in inhibition. Cytokine profiling of LILR-stimulation can provide insight into the function of LILRs.
The interaction of LILRA2 and TLR4 on cytokine production warrants further investigation. Although LILRA2 induces acute inflammatory cytokines, we hypothesise that it also influences adaptive and chronic responses through TLR4 by altering cytokine production.
121 4.2 Materials and Methods
4.2.1 Antibodies and Reagents
Antibodies against LILRs, irrelevant mouse IgG1, F(ab’)2 goat anti-mouse IgG, Fcγ fragment-specific, LPS, vitamin D3 and cell culture reagents used were previously stated
(Section 2.2.1). Anti-TLR4-PE and IgG2a-PE antibodies were from eBioscience (San
Diego, CA). Anti-IL-10 and IgG2b isotype control antibodies were purchased from R&D
Systems.
4.2.2 Cross-linking Cell Surface LILRs
THP-1 cells, THP-1-differentiated-macrophages and whole PBMCs were activated by cross-linking LILRA2, as described previously (Section 3.2.4). Supernatants were harvested after 24 hours and cytokine levels were measured by ELISA and Multi-Plex™ cytokine analysis, as described previously (Sections 3.2.4 and 3.2.5).
4.2.3 Cross-linking LILRA2 with LPS-stimulation
To assess the effects of LPS on LILRA2-mediated cytokine production, LILRA2 was cross-linked in the presence of LPS in the culture medium. In summary, cell surface
LILRA2 was cross-linked as previously mentioned with LPS added in the culture supernatant. LPS was added at varying concentrations (1, 10, 100, 1000 pg/mL) against a fixed concentration of anti-LILRA2 antibody. Supernatants were harvested after 24 hours and cytokine levels were measured by ELISA.
122 4.2.4 Sequential Activation with LILRA2 and LPS
Following co-stimulation of cross-linking LILRA2 in the presence of LPS, sequential activation was done to ascertain whether the order of events affects the outcome. Primary monocytes (in whole PBMCs) were either pre-treated with LPS (1 or 10 ng/mL) for 30 minutes prior to cross-linking of LILRA2, by standard method or LILRA2 was cross- linked for 30 minutes, prior to addition of LPS (1 or 10 ng/mL). Supernatants were harvested after 24 hours and cytokine levels were measured by ELISA. The method for this study was based on another published report observing the effects of CpG (TLR9 ligand) on LILRA7 function (Cao et al., 2006).
4.2.5 Neutralisation of IL-10 in LILRA2 Cross-linking with LPS Co- stimulation
To investigate the potential role of IL-10 in LILRA2 and TLR4 (via LPS) co-stimulation,
IL-10 was neutralised using mAb in cross-linking experiments. In brief, LILRA2 was cross-linked with LPS in the culture supernatant, as well as anti-IL-10 antibody or isotype control antibody at a final concentration of 1 μg/mL, based on suggestion by manufacturer.
Supernatants were harvested after 24 hours and cytokine levels were measured by ELISA.
4.2.6 Measurement of TLR4 Expression by Flow Cytometry
The potential for LILRA2 cross-linking to alter TLR4 expression was investigated by cross-linking LILRA2 on PBMCs in 96 well plates the measuring TLR4 expression. Cells were harvested by gently washing the wells 3 times with 300 μL D-PBS. Cells were
123 stained for flow cytometry, as previously done (Section 2.2.6). Monocytes were selected by gate and analysed for TLR4 expression by determining the mean fluorescence intensity.
4.2.7 Cross-linking LILRB4 with LPS-stimulation
To assess the potential inhibition of LPS-mediated TNF-α production by LILRB4, LILRB4 was cross-linked on PBMCs with 10μg/mL antibodies, with or without LPS added simultaneously in the culture medium.
4.2.8 Statistical Analyses
Data depicting cytokine production by LILRA2 cross-linking with or without LPS is represented as mean ± SEM and was analysed by an ANOVA and Bonferroni’s post test.
The effect of LILRB4 on LPS-mediated cytokine production was measured by a Student’s t-test. Graphs showing the effect of LILRA2-activation on TLR4 expression represents mean of MFI ± SEM and analysed by a Student’s t-test. Statistical analyses were
® performed using GraphPad Prism 5. A p value of <0.05 was considered significant.
124 4.3 Results
4.3.1 Effect of LPS on LILRA2-mediated Activation
To assess the effect of LPS of LILRA2-mediated activation, LILRA2 was cross-linked on
THP-1 cells and PBMCs with the addition of LPS in the culture medium. A synergistic effect was observed between LPS and LILRA2 in THP-1 cells. LILRA2 cross-linking alone induced 10.0 ± 4.3 pg/mL of TNF-α while LPS stimulation at the highest dose of 10 ng/mL induced 23.3 ± 7.4 pg/mL. A combination of both stimuli induced 110.1 ± 14.2 pg/mL of TNF-α, much greater than the addition of each individual stimulus (Figure 4.1).
However, the converse was seen in PBMCs as LPS had an inhibitory effect on LILRA2 activation. LILRA2 activation elicited 798.8 ± 166.6 pg/mL of TNF-α and LPS produced
4 123.0 ± 52.8 (Figure 4.2). In co-stimulated PBMCs, 364.4 ± 41.9 pg/10 monocytes of
TNF-α was measured.
125 *** 150 IgG1 αLILRA2 100 (pg/mL)
α * 50 TNF-
0 0 0.01 0.1 1 10 LPS (ng/mL)
Figure 4.1 » Cross-linking LILRA2 with LPS-stimulation in THP-1 cells. Cell surface LILRA2 is cross- linked in the presence of LPS within the culture media and TNF-α production measured. With increasing doses of LPS, a dose-dependent increase in TNF-α production was observed. At 10 ng/mL of LPS with LILRA2 cross-linking, there is a synergism in TNF-α production (n=3). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. *p<0.05, ***p<0.001
126 ** 10000 IgG1 8000 αLILRA2
6000 monocytes) 4 4000
(pg/10 2000 α
TNF- 0 0 1 10 100 1000 LPS (pg/mL)
Figure 4.2 » Cross-linking LILRA2 with LPS-stimulation in PBMCs. Cell surface LILRA2 is cross- linked in the presence of LPS within the culture media and TNF-α production measured. With increasing doses of LPS, a dose-dependent increase in TNF-α production was observed. At 1000 pg/mL of LPS with LILRA2 cross-linking, TNF-α production is significantly inhibited (n=3). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. **p<0.01
127 PBMCs co-stimulated by LILRA2 cross-linking with LPS (at 1 μg/mL) was further analysed for the production of TNF-α, IL-1β, IFN-γ, IL-4, IL-6, IL-10, IL-12 and IL-13.
As previously seen in Figure 3.3, LILRA2 cross-linking induces the production of TNF-α,
IL-6, IL-1β, IFN-γ and IL-10 but not IL-4, IL-12 or IL-13 (Figure 4.3A-E). However, co- stimulation with LPS suppressed TNF-α and IL-1β levels while significantly enhancing the production of IL-6 and IFN-γ. LPS-stimulated cells induced cytokine production comparable to LILRA2 cross-linked cells except for TNF-α which was much less in LPS- stimulation alone (Figure 4.3A).
128 A. B. C. TNF-α IL-6 IL-1β 10000 4000 300 *** * *** * * * * 8000 3000 200 6000 monocytes) monocytes) monocytes) monocytes) 44 monocytes) monocytes) 2000 44 44 4000 100
(pg/10 (pg/10 1000 2000 (pg/10 (pg/10 αα ββ IL-6 (pg/10 (pg/10 IL-6 IL-6 IL-1IL-1
TNF-TNF- 0 0 0 IgG1 + - - + + - - + + - - + αLILRA2 - + + - - + + - - + + - LPS - - + + - - + + - - + +
D. IFN-γ E. IL-10 200 400 * *** ** ** 150 300 monocytes) monocytes) monocytes) monocytes)
44 100 200 44
50 100 (pg/10 (pg/10 γγ IL-10IL-10 (pg/10 (pg/10 IFN-IFN- 0 0 IgG1 + - - + + - - + αLILRA2 - + + - - + + - LPS - - + + - - + +
Figure 4.3 » Cytokine production induced by LILRA2 cross-linking and co-stimulation with LPS. To determine effect of LPS on LILRA2-mediated cytokine production, LILRA2 was cross-linked on monocytes of PBMCs with LPS. (A-E) Cross-linking of LILRA2 induced the production of TNF- α, IL-6, IL-1β, IFN-γ and IL-10 while co-stimulation with LPS diminished TNF-α and IL-1β but enhanced IL-6 and IFN-γ (n=3 for all groups). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. *p<0.05, **p<0.01, ***p<0.001 129 4.3.2 Effect of Sequential Activation with LILRA2 and LPS
To assess whether the inhibitory effect of LPS on LILRA2-mediated TNF-α production is dependent on the sequence of stimulation, cells were either pre-treated with LPS for 30 minutes before LILRA2 cross-linking or alternatively, LILRA2 was cross-linked and LPS added 30 minutes later (Figure 4.4). PBMCs pre-treated with LPS (1 and 10 μg/mL) still inhibited LILRA2-mediated TNF-α production, from 2048.5 ± 255.8 to 869.3 ± 83.4 and
4 269.0 ± 177.4 to 1198.0 ± 159.7 pg/10 monocytes, respectively (Figure 4.4A). Similar observations was made when LILRA2 was first cross-linked before LPS stimulation (1 and
10 μg/mL) decreasing from 1974.2 ± 317.2 to 757.0 ± 164.9 and 3928.4 ± 952.4 to 178.1 ±
4 418.2 pg/10 monocytes, respectively (Figure 4.4B) though a significant difference was only observed in cells treated with 10 μg/mL LPS. This indicates that it is not only LPS which affects LILRA2-mediated activation but also the converse is true where LILRA2 impacts LPS-mediated activation and subsequent TNF-α production.
130 4.3.3 Effect of LILRA2 Cross-linking on TLR4 Expression
One possible mechanism that may cause LILRA2 to modulate LPS function is the potential regulation of TLR4 expression upon induction of LILRA2 signalling. Therefore, TLR4 expression was measured after cross-linking of LILRA2 for 24 hours by flow cytometric analysis and monocytes were selected by gate. In IgG1 cross-linked cells, the mean fluorescence intensity of TLR4 on monocytes was 11.9 ± 5.65 arbitrary fluorescence units and after LILRA2 cross-linking, reduced to 8.9 ± 1.6 (Figure 4.5). However, there was no significant difference in TLR4 MFI between the IgG1 or LILRA2 cross-linked groups
(p=0.694).
131 A. LPS (30 min) → LILRA2
5000 * LPS (1 ng/mL) 4000 * LPS (10 ng/mL) 3000 monocytes) 4 2000
(pg/10 1000 α
TNF- 0
1 2 G Ig IgG1 αLILRA2 αLILRA
B. LILRA2 (30 min) → LPS * 5000
4000
3000 monocytes) 4 2000
(pg/10 1000 α
TNF- 0
1 G Ig IgG1 ILRA2 αL αLILRA2
Figure 4.4 » Sequential effects of LILRA2 and LPS co-stimulation. (A) Pre-treatment with LPS (at 1 and 10 μg/mL) for 30 minutes prior to LILRA2 cross-linking inhibited TNF-α production. (B) Conversely, LILRA2 cross-linking 30 minutes prior to LPS stimulation also inhibited TNF-α production (n=3 for each group). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test. *p<0.05
132 20
15
10 TLR4 MFI TLR4 5
0 IgG1 LILRA2 Cross-linking
Figure 4.5 » Expression of TLR4 following LILRA2 cross-linking in primary monocytes. Cell surface LILRA2 was cross-linked for 24 hours and monocytes were analysed for TLR4 expression by flow cytometry. No significant change in TLR4 expression was measured between IgG1 and LILRA2 cross- linked cells (n=3). Data represents mean ± SEM and analysed by a Student’s t-test.
133 4.3.4 Neutralisation of IL-10 during LILRA2/LPS Co-stimulation
Following the observed inhibition in LILRA2 and LPS co-stimulated cells, the potential role of IL-10 was investigated. LILRA2 was cross-linked in the presence of LPS as previously done and neutralising anti-IL-10 antibodies or appropriate isotype control antibody (IgG2b) was added in the media. With LILRA2 cross-linking alone, anti-IL-10 antibodies did not affect the production of TNF-α (Figure 4.6). Also, neutralisation of IL-
10 did not alter cytokine production with LILRA2 and LPS co-stimulation indicating that suppression of LILRA2-mediated TNF-α production by LPS is independent of IL-10.
4.3.5 Effect of LILRB4 on LPS-mediated Activation
The ability of LILRB4 to inhibit LPS-mediated TNF-α production was assessed by cross- linking the receptor in the presence of LPS in the culture media. Treatment of PBMCs with
LILRB4 mAb and LPS caused a 25.3% reduction in TNF-α production, from 5752.0 ±
472.3 to 4299.8 ± 758.6 pg/mL, when compared to LILRB4-ligated cells alone (Figure 4.7, p=0.044).
134 15000 LILRA2 LILRA2 + LPS
10000 (pg/mL) α 5000 TNF-
0 IgG2b αIL-10
Figure 4.6 » Neutralisation of IL-10 with LILRA2 and LPS co-stimulation. Anti-IL-10 antibodies were added to LILRA2 cross-linked cells with or without LPS. As previously seen, LPS is able to inhibit LILRA2-mediated TNF-α production. Anti-IL-10 antibodies did not alter the quantity of TNF-α between LILRA2 cross-linked cells and LILRA2/LPS co-stimulated cells (n=2). Data represents mean ± SEM.
135 8000 *
6000
4000 (pg/mL) α
2000 TNF-
0 LPS LPS + LILRB4
Figure 4.7 » Effect of LILRB4 on LPS-activation. Ligation of LILRB4 using 10 μg/mL of antibodies in the presence of LPS (10 ng/mL), significantly reduced LPS-mediated TNF-α production in PBMCs (n=6). Data represents mean ± SEM and analysed by a Student’s t-test.
136 4.4 Discussion
There is growing interest in TLR function in relation to other immune regulatory receptors and evidence suggests that TLRs may interact with LILRs. Increasing concentrations of
LPS in the presence of LILRA2 cross-linking significantly reduces TNF-α production, indicating cross-talk between LILRA2 and TLR4/CD14. In another study, a similar decrease in IL-12 production has been reported while IL-10 production is augmented
(Bleharski et al., 2003). This observation was explained as LILRA2 diverting the balance of innate immunity-associated cytokines from a pro-inflammatory response (Bleharski et al., 2003), as the production of acute phase cytokines is bypassed. In any case, LILRA2 co-stimulation may increase the chance of survival to lethal doses of LPS.
LPS is a more potent inducer of the TLR signalling pathway via MyD88 compared to other
TLR ligands and TNF-α is a primary by-product of its stimulation (Bjorkbacka et al.,
2004). The function of TNF-α in response to bacterial constituents such as LPS has been well described and as LPS regulates the expression of LILRA2, endotoxin tolerance can be induced whereby repeated exposure to LPS dampens secondary responses upon re- exposure (Cavaillon et al., 2006). Given that in primary monocytes, LPS induces LILRA2 expression and furthermore, TLR4 and LILRA2 co-stimulation down-modulates the LPS response, LILRA2 could be functional in endotoxin tolerance. Co-stimulation enhances
IFN-γ production via which is influences chronic inflammatory responses. This suggests a possible skew towards a Th1 phenotype when LILRA2 is activated in the presence of LPS.
Down-regulation of TLR4 by LILRA2 signalling has been excluded as the cause for down- modulation of TNF-α production as well as the possible induction of IL-10. This suggests
137 that an alternative mechanism of interaction is involved, most likely via their respective intracellular signalling molecules.
Although cross-talk between activating LILRs and TLRs is evident, the mechanism by which this occurs is less clear. Observing the respective intracellular signalling pathways, it appears that activating LILRs and TLRs share late signalling molecules via the MAPK transcriptional pathway, though differ in earlier molecules. It is possible that one signalling pathway could dominate over the other by sequestration of substrates. This effect could occur later, whereby signalling via TNF-R1 also overlaps with TLR4 and a loss of secondary TNF-α function is induced with TLR4 stimulation, given that LPS is a potent inducer of TNF-α. Recently, it has been shown that PIR-A1 and PIR-B (ortholog to
LILRB1 and LILRB3) binds to S. aureus (Nakayama et al., 2007), revealing another possible mechanism by which LILRs and TLRs could influence each other. Further investigations will help to define the critical transition from innate and adaptive immune responses.
Interestingly, LILRB4 was able to inhibit LPS-induced TNF-α production and thus documenting a non-conventional function for ITIMs. The underlying intracellular mechanisms driving this inhibition are unknown. It is possible that the response to LPS is merely delayed by LILRB4 but we are inclined to believe that this is complete inhibition as cytokine levels were measured relatively late in LPS response. Alternatively, LILRB4 may inhibit the cell surface expression (and function) of LPS binding receptors. In general,
ITIM-bearing receptors function by blocking ITAM-signalling. However, there was some
138 precedence for studying the effects of LILRB4 on LPS responses. The murine ortholog of
LILRB4, gp49B, is able to retard the development of LPS-associated synovitis (Zhou et al., 2005) and LPS-induced angiopathy (Zhou et al., 2003). Both studies were conducted using gp49B knock-out mice where knock-out mice had early exaggerated symptoms of joint swelling (Zhou et al., 2005) and thrombo-haemorrhagic responses (Zhou et al., 2003).
However, it could not be determined whether LILRB4 directly affects the local production of cytokines and chemokines. Although human and murine LILRB4 are expressed on different cell types, their role may be to maintain a low responsiveness to ubiquitous foreign substances such as LPS. By extension, LILRB4 may also prevent septic shock, the potentially fatal sequela induced by an acute overproduction of cytokines (Tracey et al.,
1990).
Again, we see a contradiction in results from studies on cell lines versus primary cells. The opposite effect was seen in THP-1 cells where LPS enhanced LILRA2 response, in a synergistic manner. It is crucial to understand that THP-1 cells are essentially pro- monocytic cells arising from malignant development of myeloid cells, taken from a child
(Tsuchiya et al., 1980). This difference in responses between cell types may correlate to either the developmental stage of the cells or the type of mutations that have been acquired during malignant transformation. In THP-1 cells, LPS consistently down-regulates
LILRA2 expression at the protein level (Figure 2.3) while in primary monocytes, mRNA is significantly up-regulated (Figure 2.6).
139 Although the link between LPS and the development of RA is weak, the presence of LPS ligands in the RA synovium cannot be ignored (Heumann et al., 1995; Yu et al., 1998)
(Radstake et al., 2004). It may be early speculate, but the changing expression of LILRA2 in the RA synovium in conjunction with TLR4 stimulation could be crucial to the pathogenesis of RA. Further investigations into the interaction of these receptors may be important to understanding the development of RA. It is evident that LILRA2 suppression of LPS responses is not IL-10-dependent as neutralising antibodies failed to significantly restore TNF-α production during co-stimulation of LILRA2 with LPS. IL-10 is known to negatively regulate immune responses via its intracellular interactions with NF-κB and inhibiting pro-inflammatory responses, including those induced by LPS (reviewed in
Grutz, 2005). We speculate that their involvement is at the intracellular level.
140 Chapter 5. Expression of LILRs on Peripheral Blood Leukocytes of Patients with Rheumatoid Arthritis
5.1 Introduction
In autoinflammatory disorders such as RA, tissue damage is mediated by the uncontrolled activation of leukocytes and in most diseases, the initiating factors of leukocyte activation are currently unknown. There are implications of auto-antigens and super-antigens in the primary response (reviewed in Blass et al., 1999; VanderBorght et al., 2001; van Boekel et al., 2002) though there is a lack of hard evidence outlining any single factor. Undoubtedly, chronic inflammation is sustained by the constant accumulation, activation and proliferation of leukocytes in the inflamed joint (Section 1.6.3).
Expression of LILRs in the rheumatoid synovium of patients with active RA (Tedla et al.,
2002) led us to speculate about their role in the pathogenesis. Co-expression of activating
(LILRA2) and inhibitory (LILRB2, LILRB3) receptors on a wide variety of cell types in the inflamed joint supports to the hypothesis that a balance between activating and inhibitory signals regulate immune responses. LILR-expression has also been shown in
+ other pathological settings including; increase of LILRB1 lymphocytes in HCMV and
HIV infections, increase in LILRA2 expression in lepromatous skin and an increase of
LILRB2 expression in psoriatic skin lesions (Section 1.3.5.1). However, their function in these diseases is not known. The in vitro function of both activating and inhibitory LILRs have been well studied (discussed in Section 1.3.6) and there is substantial evidence indicating that LILRs and other related molecules may determine the threshold and extent
141 of leukocyte activation (Tedla et al., 2002; Sloane et al., 2004). It is likely that the role
LILRs in these inflammatory diseases is to regulate leukocyte function.
Characterisation of LILR-expression on circulating leukocytes has also been well documented, however, modulation of their expression is poorly understood (Section 1.3.3).
Given their role in leukocyte function, expression is likely to be regulated by inflammatory mediators. Granulocytes and monocytes express a wide variety of LILRs (Tedla et al.,
2003) but less is known about their expression on lymphocytes. There is also a lack of knowledge about whether LILR-expression alters on circulating leukocytes in disease processes.
Since RA has systemic components to its pathogenesis, it is likely that there is an altered pattern of receptor expression on circulating cells. Chronic inflammation of the joints releases mediators both locally and systemically, potentially altering expression of LILRs and other immune regulatory receptors. Changes in the relative surface expression of activating to inhibitory receptors may be a triggering factor in leukocyte activation, transitioning cells across a threshold from homeostasis to an activated state. Relative expression ratios of each counterpart could affect severity and/or duration of disease.
Whether LILR-expression on circulating leukocytes alters in RA could provide insight into the pathogenesis of this disease. Profiling of LILR expression may uncover potential markers for various inflammatory diseases, including RA. Notably, ligands for most LILRs are unknown and their identification remains a crucial task.
142 By understanding the pattern expression of activating and inhibitory receptors on inflamed tissue and peripheral blood leukocytes, we can speculate on which receptors may be involved in the development of RA. Expression of LILRA2, LILRB2 and LILRB3 is already documented to be present in the rheumatoid synovium (Tedla et al., 2002) and high
LILRA2-expression was associated with active disease while patients in remission had lower expression (Huynh et al., 2007). Variation in expression could not be correlated with any other factors such as disease activity score (DAS) 28 or C-reactive protein (CRP) levels. We were interested to observe whether LILR-expression in inflamed tissue correlates with expression on circulating leukocytes.
We hypothesise that relative expression of LILRs on PBMCs can attribute to disease progression and/or severity by altering the relative expression of activating and inhibitory receptors to favour a pro-inflammatory environment. The aim of this study was to characterise the expression of LILRs on circulating leukocytes.
143 5.2 Materials and Methods
5.2.1 Patients
Patients were recruited during a follow-up consultation and all fulfilled the 1987 ACR
criteria for RA, (Arnett et al., 1988), had active RA [swollen and tender joint count >5, C-
reactive protein (CRP) >20 mg/L] with involvement of at least one knee joint and were willing to donate whole blood. Serum CRP concentration, measured by rate nephelometry
(normal <6 mg/L), was used as a laboratory measure of inflammation. Response to
treatment was assigned on the basis of the clinical and laboratory parameters, using the
ACR criteria for improvement (Felson et al., 1995) and remission (Pinals et al., 1982) in response to drug treatment. In addition, a disease activity score 28 (DAS) was calculated for each patient as previously described with low disease activity defined as a DAS<2.6 as defined by the European League Against Rheumatism (EULAR) criteria (van Gestel et al.,
1996). Blood was collected from patients into Acid Citrate Dextrose (ACD) for use in this
study. This project was approved by the Human Research Ethics Committee, University of
New South Wales (HREC 05020) and informed consent was obtained from each patient.
5.2.2 Antibodies and Reagents
Antibodies against LILRs, irrelevant mouse IgG1 and F(ab’)2 goat anti-mouse IgG, Fcγ fragment-specific were previously reported. Fluorochrome-conjugated antibodies used for flow cytometry including; CD4-Phycoerythrin (PE), CD8-Peridinin Chlorophyll Protein
(PerCP), CD56-PE, CD19-PerCP, CD3-Allophycocyanin (APC), IgG1-PE, IgG1-PerCP and IgG1-APC as well as FACS lysis buffer were from BD Biosciences.
144 5.2.3 Quantification of Leukocytes
® Leukocytes were enumerated using a Coulter Ac·T Diff™ differential cell counter
(Beckman Coulter). Data provided included absolute concentration of white blood cells
(WBC) as well as absolute concentration and percentage proportions of lymphocytes, monocytes and granulocytes.
5.2.4 Peripheral Blood Mononuclear Cells (PBMC) Extraction
PBMC were prepared from 40 mL of anti-coagulated whole blood obtained from a new group of RA patients (n=14) and healthy volunteers (n=10) using standard Ficoll-Paque™ gradient. In brief, anti-coagulated blood was added to an equal volume of D-PBS and split into 30mL lots. Ficoll-Paque™ PLUS (15 mL) was underlayed and then centrifuged at
420×g for 25 minutes. Buffy coats of PBMCs were carefully extracted and cells were washed twice with D-PBS.
5.2.5 Flow Cytometry
Initially, whole blood from patients was stained for expression of LILRs (B1, B2, B3, B4,
A1, and A2) in separate tubes. Analysis of cell types was done by staining cells for leukocyte markers; CD45 (pan-leukocyte marker), CD14 (monocytes), CD19 (B cells),
CD56 (NK cells), CD3 (T cells) and CD16 (neutrophils). On scatter profiles, cell populations were gated and analysed for LILR expression. Fifty-microlitres of whole blood
was incubated for 30 minutes at room temperature with mAb to all LILRs or control mouse
IgG1 (0.5 µg). Red blood cells were lysed with 1.5mL FACS lysis buffer, mixed
145 vigorously and incubated at room temperature for 5 minutes. After two washes with cold
PAB buffer, 3 µg of FITC-conjugated F(ab')2 goat anti-mouse IgG [F(ab')2-specific] was added to samples and then placed on ice for 45 minutes. Cells were washed twice with
PAB buffer, fixed with 2% paraformaldehyde in D-PBS and analysed by using a
FACSort™ flow cytometer and the BD CellQuest™ software (Becton Dickinson).
Multiple staining of cells for LILRs and CD markers was performed on purified PBMCs.
Cells were first incubated with mAb against LILRs for 1 hour at room temperature. Cells were washed once with PAB buffer and incubated with 3 µg FITC-conjugated F(ab')2 goat anti-mouse IgG [F(ab')2-specific] on ice for 1 hour. After 2 washes, cells were incubated with fluorochrome-conjugated antibodies; CD4-PE, CD8-PerCP and CD3-APC or CD56-
PE, CD19-PerCP and CD3-APC, on ice for 20 minutes. Cells were washed fixed in 2% paraformaldehyde in D-PBS. Samples were analysed on a FACSCalibur™ flow cytometer.
5.2.6 Statistical Analyses
Cell counts (determined as absolute numbers or percentages of whole white blood cell count) are presented as dot plots and the bar represents the mean value. Comparison of data from patients with RA to healthy control subjects were analysed by Student’s t-test.
Statistical analyses were performed using GraphPad Prism® 5. A p value of <0.05 was considered significant.
146 5.3 Results
5.3.1 Patient Statistics
Statistics for RA patients and control subjects are comparable; the mean age for each group being 51.7± 14.3 and 53.8 ± 8.2 respectively (Table 5.1). Although fewer control subjects were recruited, the ratio of females to males is similar: RA – 12/6 and controls – 7/3. Most patients were on combination therapy (Table 5.2), in particular the DMARDs: methotrexate (mtx), sulfasalazine (ssz), leflunomide (lef), hydroxychloroquine (hcq) and/or cyclosporin A (csA). The steroid, prednisone (pred), was supplemented in 4 patients and only one patient was on etanercept, a TNF-α inhibitor. Most patients had active disease indicated by a DAS greater than 2.6, except for patient RA11 with a DAS of 0.6.
147 Table 5.1 » Summary of age and sex for control subjects and patients with RA.
Patient numbers Mean age ± S.D. Sex ratio (M/F)
Control 10 53.8 ± 8.2 3/7
RA 18 51.7 ± 14.3 6/12
148 Table 5.2 » Data for each individual RA patient.
Patient Age/sex treatment DAS28
RA1 64/F mtx 4.4
RA2 56/F mtx, ssz 4.2
RA3 36/F mtx, ssz, hcq 4.2
RA4 37/F mtx, lef, pred 4.7
RA5 60/M mtx, pred 8.5
RA6 70/F mtx, lef, pred 3.2
RA7 22/F mtx, lef 4.3
RA8 65/M mtx 5.0
RA9 68/M mtx, lef, pred 6.0
RA10 56/F mtx, lef, csA 7.3
RA11 58/F ssz 0.6
RA12 58/M mtx 4.4
RA13 25/F lef, pred 4.3
RA14 38/F mtx, ssz 4.8
RA15 50/M mtx 3.7
RA16 58/F mtx 4.1
RA17 60/F pred, etan 4.7
RA18 50/M mtx, lef 2.7
Methotrexate (mtx), leflunomide (lef), sulfasalazine (ssz), prednisone (pred), etanercept (etan), cyclosporin A (csA), hydroxychloroquine (hcq).
149 5.3.2 Quantitation of Circulating Leukocytes in Patients with RA vs. Control Subjects
Differential leukocyte counts were obtained from whole blood of each patient using an automated differential cell counter to identifying lymphocytes, monocytes and
6 granulocytes. Leukocyte subsets were represented as whole counts (× 10 cells/mL) or as a percentage of the whole white blood cell (WBC) count. The overall WBC counts between patients with RA and control subjects were not significantly different (Figure 5.1A) however, a significant reduction in lymphocytes was observed in both cell counts
(p=0.0126) and as a percentage (p=0.0002) of WBC (Figure 5.1B-C). Monocyte numbers and percentages remain unchanged between groups (Figure 5.1D-E) but granulocytes increased in both absolute numbers (p=0.031) and percentage (p=0.0046) in patients with
RA (Figure 5.1F-G).
Since changes in lymphocyte numbers were observed, the lymphocyte population was further analysed and quantitative analysis of subsets was done by staining cells for the CD markers; CD3, CD56 and CD19 to identify T lymphocytes, NK cells and B lymphocytes, respectively. There was a significant decrease in the numbers of circulating T cells
(p=0.0063) and B cells (p=0.0025) in patients with RA when compared to control subjects
(Figure 5.2A and Figure 5.2C) though there was no significant change when expressed as a percentage of whole lymphocytes (Figure 5.2B and Figure 5.2D). The numbers and percentages of NK cells between RA patients and control subjects remained relatively similar (Figure 5.2E-F).
150 A. 20 ) 6 15 10 ×
10
5 WBC count ( WBC count
0
B.* C. *** 4 50 /mL)
6 40 3 10
× 30 2 20 1 10 Lymphocytes (%)Lymphocytes Lymphocytes ( 0 0 D. E.
2.0 30
/mL) 1.5 6 20 10 × 1.0
10 0.5 Monocytes (%)Monocytes Monocytes ( Monocytes 0.0 0 F. G. * ** 15 100 /mL)
6 80
10 10 × 60
40 5
20 Granulocytes (%) Granulocytes ( Granulocytes 0 0 control RA control RA
Adobe error. Adobe error. Adobe error. Adobe error. Adobe error. Adobe error. Adobe error.
151 Figure 5.1 » Analysis of white blood cell (WBC) counts and leukocyte subpopulations in patients with RA compared to healthy controls subjects. Absolute numbers of circulating WBC and subpopulations (lymphocytes, monocytes and granulocytes) were quantitated for each individual patient using an automated differential cell counter and percentages of lymphocytes, monocytes and granulocytes were also determined. (A) WBC counts between patients with RA and healthy subjects were similar. (B-C) Absolute numbers and percentage of lymphocytes were significantly reduced in patients with RA. (D- E) Monocyte numbers and percentage remained unchanged between the patient groups. (F-G) Absolute numbers and percentage of granulocytes were significantly increased in patients with RA compared to healthy controls. Data is represented as a scatter plot for each group and bar represents mean. Differences between groups were analysed by Student’s t-test. * p<0.05 ** p<0.01 *** p<0.001
152 A. B. ** 2.5 90
2.0 80 ) 6
10 1.5 70 ×
1.0 60 T cells (%)T cells T cells ( 0.5 50
0.0 40
C. D. ** 0.5 20
0.4 ) 15 6
10 0.3 × 10 0.2
B Cells (%) B Cells 5 B Cells ( B Cells 0.1
0.0 0
E. F.
0.5 25
0.4 20 cells/mL)
6 0.3 15 10
× 0.2 10 NK Cells (%) NK Cells 0.1 5
NK Cells ( NK Cells 0.0 0 control RA control RA
Figure 5.2 » Analysis of numbers of lymphocytes subpopulations in patients with RA. PBMCs were harvested and stained with fluorochrome-conjugated antibodies against CD3, CD19 and CD56 to identify T cells, B cells and NK cells, respectively. (A-B) There was a significant reduction in the absolute numbers of circulating T cells in patients with RA compared to controls but no significant change in the percentage of cells. (C-D) Absolute numbers of B cells were significantly reduced in RA patients but the percentage of B cells was similar between the two groups. (E-F) No difference was observed in numbers or percentages of NK cells. Data is represented as a scatter plot for each group and bar represents mean. Differences between groups were analysed by Student’s t-test. ** p<0.01
153 5.3.3 Differential Expression of LILRs on Monocytes and Lymphocytes between Patients with RA and Control Subjects
To investigate whether there is a differential pattern of LILR-expression on circulating leukocytes of patients with RA compared to healthy control subjects, whole blood was stained for LILR expression (A1, A2, A5, B1, B2, B3, B4, B5) by a standard two-step staining protocol and analysed by flow cytometry. The different cell types were analysed by gating on the corresponding population on the forward-scatted/side-scatter profiles.
Monocytes were first analysed by gating the population (Figure 5.3A) and purity was measured by positive staining for the marker CD14 at 97.6% (Figure 5.3B). Isotype staining of cells revealed minimal cross-reactivity with cells (Figure 5.3C) and monocytes from all patients exhibited positive staining for all LILRs studied (Figure 5.3D-J). Similar profiles were obtained for 4 patients with RA and 6 healthy control subjects and the proportions of LILR-positive monocytes were compared between the two groups. Analysis revealed no significant difference in the percentage of positive cells for any of the LILRs tested (Figure 5.4A-G).
154 A. B.
97.6%
C.D. CD14-FITC E. F.
0.8% 94.6% 92.9% 92.5%
IgG1-FITC LILRA1-FITC LILRA2-FITC LILRA5-FITC G. H. I. J.
96.7% 96.5% 95.4% 93.3%
LILRB1-FITC LILRB2-FITC LILRB3-FITC LILRB4-FITC
Figure 5.3 » Expression of LILRs on monocytes of patient with RA and healthy control subjects. Whole blood was stained for LILR expression and analysed by flow cytometry. (A) Monocytes were selected by gate and (B) stained for CD14 for confirmation. Cells were stained with anti-LILR and relevant IgG1 isotype control antibodies using a standard two-step protocol. (C) Isotype staining shows minimal non-specific binding of antibodies. (D-J) Almost all monocytes were positive for expression of the LILRs tested (activating and inhibitory) as percentage of positive cells are indicated in each dot plot. The dot plots shown in this figure is representative of a healthy control subject in the study.
155 A. B. C.
100 100 100
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0 LILRA1-positive monocytes (%) (%) monocytes monocytes LILRA1-positive LILRA1-positive LILRA2-positive monocytes (%) (%) monocytes monocytes LILRA2-positive LILRA2-positive (%) (%) monocytes monocytes LILRA5-positive LILRA5-positive D. E. F. G.
100 100 100 100
80 80 80 80
60 60 60 60
40 40 40 40
20 20 20 20
0 0 0 0 control RA control RA control RA control RA LILRB4-positive monocytes (%)(%) monocytes monocytes LILRB4-positive LILRB4-positive LILRB1-positive monocytes (%)(%) monocytes monocytes LILRB1-positive LILRB1-positive (%)(%) monocytes monocytes LILRB2-positive LILRB2-positive LILRB3-positive monocytes (%) (%) monocytes monocytes LILRB3-positive LILRB3-positive
Figure 5.4 » Analysis of LILR expression on monocytes of patients with RA compared to healthy control subjects. The percentage of LILR-positive cells was quantitated for each patient by flow cytometry. (A-G) There was no significant differences seen the percentage of monocytes positive for LILRA1, LILRA2, LILRA5, LILRB1, LILRB2, LILRB3 nor LILRB4. Bar represents mean and data was analysed by an ANOVA and Bonferroni’s post test.
156 Lymphocytes were analysed for LILR expression (Figure 5.5 and Figure 5.6), similar to monocytes as seen in Figure 5.3 and Figure 5.4. Lymphocytes were selected by gate, based on their profile on a forward-scatter/side-scatter plot (Figure 5.5A) and further analysed for
LILR expression. IgG1 isotype control staining showed minimal non-specific binding of antibodies (Figure 5.5B). Of the activating LILRs, only LILRA1 showed expression with
16.6% cells positive (Figure 5.5C-E) and 14.7% of cells were positive for LILRB1 of the inhibitory LILRs (Figure 5.5F-I) After screening of 7 patients with RA and 5 controls for
LILR expression, LILRA2, LILRA5, LILRB1, LILRB2, LILRB3 and LILRB5 remained negative and focus was directed towards LILRA1 and LILRB1. From the preliminary study was a significant difference in LILRA1 and LILRB1 between patients with RA and healthy control subjects (Figure 5.6A and Figure 5.6D) while no significant difference was observed in other LILRs.
A further 18 RA patients and 10 controls subjects were screened for expression of LILRA1 and LILRB1 on lymphocytes. A significant increase in the percentage of LILRA1
(p=0.0017) and LILRB1 (p=0.0446) lymphocytes was observed inpatients with RA compared to the control group (Figure 5.7A-B). Healthy control subjects had 15.2 ± 1.3 % and 14.5 ± 1.9 % of cells positive for LILRA1 and LILRB1, respectively while RA patients showed 33.7 ± 4.5 % and 23.4 ± 3.5 % in positive staining. Moreover, most patients with higher percentages of LILRA1-positive lymphocytes also had a higher percentage of LILRB1-positive lymphocytes (Figure 5.9).
157 A.
A.
B.C. D. E.
1.1% 16.6% 2.4% 1.9%
IgG1-FITC LILRA1-FITC LILRA2-FITC LILRA5-FITC F. G. H. I.
14.7% 3.7% 1.6% 0.7%
LILRB1-FITC LILRB2-FITC LILRB3-FITC LILRB4-FITC
Figure 5.5 » Expression of LILRs on lymphocytes of patient with RA and healthy control subjects. Whole blood was stained for LILR expression and analysed by flow cytometry. (A) Lymphocytes were selected by gating on the population. Cells were stained with anti-LILR and relevant IgG1 isotype control antibodies using a standard two-step protocol. (B) Isotype staining shows minimal non-specific binding of antibodies. (C) LILRA1 is positive on 16.6% of cells within lymphocytes gate. (D-E) Lymphocytes were negative for LILRA2 and LILRA5. (F) Only LILRB1 of the inhibitory LILRs stained positive on lymphocytes and 14.7% of the cells were in the positive quadrant. (G-I) Lymphocytes were negative for LILRB2, LILRB3 and LILRB4. Dot plots shown in this figure is representative of a healthy control subject in the study.
158 A. B. C. ** 100 100 100
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0 LILRA2-positive lymphocytes (%)(%) lymphocytes lymphocytes LILRA2-positive LILRA2-positive LILRA5-positiveLILRA5-positive(%)(%) lymphocytes lymphocytes LILRA1-positive lymphocytes (%)(%) lymphocytes lymphocytes LILRA1-positive LILRA1-positive D. E. F. G. ** 100 100 100 100
80 80 80 80
60 60 60 60
40 40 40 40
20 20 20 20
0 0 0 0 control RA control RA control RA control RA LILRB4-positiveLILRB4-positive lymphocytes (%) lymphocytes (%) LILRB1-positiveLILRB1-positive lymphocytes lymphocytes (%)(%) LILRB2-positiveLILRB2-positive (%) (%) lymphocytes lymphocytes LILRB3-positiveLILRB3-positive lymphocytes (%) lymphocytes (%)
Figure 5.6 » Analysis of LILR expression on lymphocytes of patients with RA compared to healthy control subjects. The percentage of LILR- positive cells was quantitated for each patient by flow cytometry. Of the LILRAs, there was a significant increase in the percentage of monocytes positive for (A) LILRA1 but not (B) LILRA2 nor (C) LILRA5. An increase was also observed in the expression of (D) LILRB1 but not (E) LILRB2, (F) LILRB3 nor (G) LILRB4. Bar represents mean and data was analysed by an ANOVA and Bonferroni’s post test. *p<0.05
159
A. ** 90 80 70 60 50 40 30 20 10 0 control RA LILRA1-positive lymphocytes (%) B.
70 * 60 50 40 30 20 10 0 control RA LILRB1-positive lymphocytes(%)
Figure 5.7 » Expression of LILRA1 and LILRB1 on lymphocytes of patients with RA compared to controls. In the same patients, whole lymphocytes were analysed for expression of LILRA1 and LILRB1by flow cytometry. The numbers of positive cells were and expressed as a percentage. Patients with active RA have a significantly higher percentage of (A) LILRA1- and (B) LILRB1-positive lymphocytes. Data is represented as a scatter plot for each group and analysed by a Student’s t-test. Bar represents the mean. * p<0.05, **p<0.01
160 100
80
60
40
20
0
LILR-positive (%) lymphocytes LILRA1 LILRB1
Figure 5.8 » Comparison of the percentages of LILRA1- and LILRB1-positive lymphocytes in patients with RA. The bar represents data from a single patient, connecting the percentage of LILRA1-positive lymphocytes to the percentage of LILRB1-positive detected for that patient.
161 To determine whether LILRA1 and LILRB1 expression of lymphocytes correlated with the level of disease activity of the patient, the percentage of LILR-positive lymphocytes was plotted against the DAS of each patient. There was no correlation observed between the percentage of circulating LILRA1-positive lymphocytes and DAS (Figure 5.9A, p=0.95), or LILRB1-positive cells and DAS (Figure 5.9B, p=0.72).
5.3.4 Characterisation of LILR-positive Lymphocytes
In depth analysis of cells expressing LILRs aimed to determine which subset of T cells were expressing LILRA1 and LILRB1. PBMCs from 6 patients with RA as well as age- and sex-matched controls were co-stained for LILRs and CD markers to identify helper T cells (CD4), cytotoxic T cells (CD8), NK cells (CD56) and B lymphocytes (CD19). Cells were quantitated and compared between RA patients and control subjects (Figure 5.10).
The percentage of LILR-positive cells was determined by enumerating LILR-positive cells against the total number of cells for each subset. The percentage of LILRA1- and LILRB1-
+ positive CD4 lymphocytes was significantly increased in the RA group (Figure 5.10A-B, p=0.0174 and 0.0096, respectively) and a similar increase in positive cells are observed for
+ CD8 lymphocytes (Figure 5.10C-D, p=0.0057 and 0.0001, respectively). The percentage of LILRA1- and LILRB1-positive NK cells remained relatively unchanged (Figure 5.10E-
F) and also for LILRA1-positive B cells (Figure 5.10H). However, the percentage of B cells expressing LILRA1 was significantly reduced in patients with RA (Figure 5.10G, p=0.0001).
162 A.
60 r2=0.0003
40
20
0 0 2 4 6 8 10 LILRA1-positive lymphocytes (%) lymphocytes LILRA1-positive
B.
60 r2=0.01
40
20
0 0 2 4 6 8 10 LILRB1-positive lymphocytes (%) lymphocytes LILRB1-positive DAS
Figure 5.9 » Correlation between LILR lymphocyte expression and disease activity. In patients with RA, the percentage of LILR-positive cells was plotted against DAS. No correlation was observed between the percentage of (A) LILRA1- or (B) LILRB1-positive lymphocytes and DAS. Dot plot represents percentage of LILR expression and DAS for each individual patient. Line graph represents linear regression of the data. Data was analysed by a Pearson’s correlation.
163 In the same patients, monocytes populations were also analysed by flow cytometry to investigate whether LILR-expression also alters on these cells. As previously done, monocytes were selected by gate and confirmed for CD14 expression while isotype staining exhibited minimal non-specific cross-reactivity (as seen in Figure 5.3). Almost all monocytes were positive for LILRA1 in control patients and similarly in RA (Figure
5.11A) and the same trend was observed for LILRB1 (Figure 5.11B). Upon analysis of expression for 6 RA and controls, no significant change in the percentage of LILRA1- and
LILRB1-positive monocytes was observed between patients with RA and healthy control subjects. Moreover, there was no significant difference in the MFI for each receptor (data not shown).
164 LILRA1 LILRB1 A. * B. **
100 100
75 75
50 50 T cells (%) cells T T cells (%) cells T + + 25 25 CD4 CD4
0 0 C. ** D. *** 100 100
75 75
50 50 T cells (%) cells T (%) cells T + + 25 25 CD8 CD8
0 0
E. F. 100 100
75 75
50 50
NK Cells (%)NK Cells 25 (%)NK Cells 25
0 0
G. *** H.
100 100
75 75
50 50 B Cells (%) B Cells 25 (%) B Cells 25
0 0
control RA control RA
165 Figure 5.10 » Expression of LILRA1 and LILRB1 on lymphocyte populations of patients with RA. Lymphocytes were analysed from PBMCs for LILR expression by double staining cells for LILRA1 and LILRB1 along with lymphocyte markers; CD4 (helper T cells), CD8 (cytotoxic T cells), CD56 (NK cells) or CD19 (B cells). (A-B) The percentage of both LILRA1- and LILRB1-positive CD4+ helper T cells are increased in patients with RA and a similar increase is seen on (C-D) CD8+ cytotoxic T cells as well. (E-F) The percentage of LILRA1- and LILRB1-positive NK cells remains unchanged between the two groups. (G-H) On B cells, only LILRA1 shows a significant change and decreasing in the RA group. Data represents the percentage of LILR-positive cells of the given lymphocyte population by staining for CD markers. Bar represents mean. Data was analysed by Student’s t-test. * p<0.05 ** p<0.01 *** p<0.001
166 A.
100
95
90
85
LILRA1-positive cells (%) 80
B. 100
95
90
85
LILRB1-positive cells (%) 80 control RA
Figure 5.11 » Expression of LILRA1 and LILRB1 on monocytes of patients with RA compared to controls. Monocytes were analysed for expression of LILRA1 and LILRB1by flow cytometry. The numbers of positive cells were and expressed as a percentage. No significant change was observed in (A) LILRA1- and (B) LILRB1-positive monocytes between patient groups. Data is represented as a scatter plot for each group and analysed by a Student’s t-test. Bar represents the mean.
167 5.4 Discussion
Autoinflammatory diseases such as RA are difficult to study as there is great variability between patients in terms of disease onset, progression, severity and treatments. Extreme heterogeneity within cohorts often reflects in experimental assays and this is difficult to avoid. The recruitment of proper control subjects is essential to minimising error in comparing groups.
Lymphopenia, the reduction in circulating lymphocytes, is commonly seen in patients with
RA and is attributed to treatment with cytotoxic drugs (Weinblatt et al., 1985; Weinblatt et al., 1988; Symmons et al., 1989; Wachi et al., 2005). This is confirmed in this study as reduction in the whole numbers and percentage of lymphocytes was observed (Figure
5.1B-C). Upon further analysis of lymphocyte subset numbers, it was specifically T and B lymphocytes that were diminished (Figure 5.2A-D). The absolute numbers and percentage of granulocytes increased correspondingly (Figure 5.1F-G) to compensate for the loss of lymphocytes and hence, no change is seen in total WBC counts (Figure 5.1A). This suggests that treatments for RA may selectively target lymphocytes, hence the reduction in their numbers in the circulation. Cytotoxic functions have already been defined in a wide range of anti-rheumatoid drugs including methotrexate and cyclosporin A.
Although in patients with RA there is a marked decrease in T lymphocytes, LILRA1- and
LILRB-positive cells were significantly higher than those observed in normal subjects
(Figure 5.7). One possible mechanism is that cytotoxic anti-rheumatoid drugs selectively kill LILRA1- and LILRB1-negative T cells. In B cells, there is a reduction in LILRB1-
168 positive cells suggesting selection against these cells is occurring during RA treatment.
The type of treatment among patient within the group could determine the level of expression although in vitro regulation has not been observed (Huynh et al., 2007).
Alternatively, the drugs may regulate the expression of both LILRs to induce their expression on T cells but at the same time, down-regulating expression on B cells.
Regulation of LILR-expression on T cells must be tightly regulated since similar alterations in LILRA1 and LILRB1 expression is not observed on monocytes (Figure
5.10). But whether there is a direct regulation of these LILRs by the drugs is yet to be determined. An earlier study has reported an increase in the percentage of peripheral blood
+ + CD4 CD25 T lymphocytes in patients with RA and this was explained as a consequent of an enhanced immunoregulatory mechanism due to chronic inflammation (van Amelsfort et al., 2004). It is possible that these are the same population of cells expressing LILRA1 and
LILRB1 observed in this study though further investigation is required to determine this.
Another study has reported a slight, yet insignificant increase in LILRB1-positive cells in patients with RA and SLE (Monsivais-Urenda et al., 2007), comparable to the results seen in this study. Interestingly, patients with RA had corresponding numbers in the percentage of LILRA1- and LILRB1-positive lymphocytes (Figure 5.9). The genes for LILRA1 and
LILRB1 lie next to each other in the LRC (Figure 1.3) and it is likely that they are co- regulated by the same transcription factors. This could explain why we consistently see a co-up-regulation of both cell surface proteins on lymphocytes and regulation of neither receptor not on monocytes.
169 Interestingly, the only identified ligand for LILRA1 is HLA-B27 (Allen et al., 2001) and this HLA class I allele has a strong association to spondyloarthropathies (Brewerton et al.,
1973). HLA-B27 is able to present athritogenic peptides to cytotoxic lymphocytes
(Scofield et al., 1993; Schumacher et al., 1998) and may be initiating factor of joint inflammation and other systemic complications. Classical activation would occur with peptide bound-HLA-B27 presented to the T cell receptor but the disease may also be attributed to LILRA1-HLA-B27 interaction. The role of LILRA1 remains unknown but may serve as a co-receptor or docking protein for HLA-B27-mediated antigen presentation.
Being an ITAM-bearing receptor, activation via LILRA1 is possible in specific T cell clones and blocking of LILRA1 may prevent activation of autoreactive T cells. Screening of patients with ankylosing spondylitis for LILRA1 expression on leukocytes could prove useful in explaining the role of HLA-B27 in the pathogenesis of this disease.
LILRA1 function has not been extensively studied but it is assumed via association with
ITAM-bearing adaptor proteins to activate leukocytes, although this is yet to be shown.
+ Whether LILRA1 is able to induce either cytokine production in CD4 T cells, production
+ of cytotoxic granules in CD8 T cells or cellular proliferation, needs to be evaluated. On the other hand, LILRB1 has pronounced functions in inhibiting leukocyte activation. Given that both receptors are co-expressed, LILRB1 could potentially inhibit LILRA1-mediated activation. A direct interaction of these receptors may help to explain the co-regulation of their expression which has been observed.
170 The mechanisms of action of many drugs currently used in the treatment of RA, including
DMARDS, are not completely understood. Studies have examined the effects of treatment on synovial membrane pathology and demonstrated changes in numbers of T lymphocytes, macrophages, microvessels and/or synovial fibroblasts (Walters et al., 1987; Rooney et al.,
1989; Soden et al., 1991; Yanni et al., 1994; Tak et al., 1996; Dolhain et al., 1998; Smith et al., 2001). There are limited studies on the effect of these drugs on circulating leukocytes with variable conclusions. In any case, the mechanisms responsible for these changes are poorly defined. Selective killing of lymphocytes appears to be one of the many functions of anti-rheumatoid drugs.
LILRA1 and LILRB1 are potential markers for the development of RA, given the consistent increase in positive-staining T lymphocytes. Since there is preferential expansion of these cells and not of other subsets, this suggests that these receptors serve a particular function in the disease. LILRB1 and LILRA1 are known to bind HLA class I molecules (Section 1.4.6) and it is likely that their signalling in RA may be MHC- dependent. Functional studies are essential to determine the exact role of LILRA1 and
LILRB1 in the pathogenesis of RA. Co-regulation of expression of an activating and inhibitory receptor emphasises the hypothesis that the immune system relies on expression of counter-signalling receptors. By studying other auto-inflammatory diseases, we could determine whether these receptors are disease-specific or a general marker for inflammation.
171 Chapter 6. Developing a Model to Investigate the Signalling Mechanism of LILRB4
6.1 Introduction
LILRB4 is an inhibitory receptor of increasing interest, as discussed in Section 1.3.5.2. It is structurally unique among other LILRBs in that it only has 2 extracellular immunoglobulin-like domains (Table 1.2). Although its exact functions are not entirely understood, extensive research indicates that LILRB4 plays a role in DC function, immune regulation and immune suppression. The suggestion that LILRB4 is an important transducer of tolerising signals to dendritic cells implicates this receptor in the process of antigen-specific immune responses (reviewed in Suciu-Foca et al., 2005).
The intracellular ITIMs of LILRB4 is known to recruit SHP-1 (Cella et al., 1997) but other adaptor proteins recruited for downstream signalling has not been identified. Stimulation of LILRB4 inhibits calcium mobilisation (Cella et al., 1997) and downstream signalling via the NFκB pathway (Chang et al., 2002).
Studies presented in previous chapters indicate that LILRB4 may have similar function/s to that of mLILRB4. Not only does LILRB4 suppress the function of ITAM-bearing receptors such as LILRA2 (Section 1.3.5.2) and CD64 (Figure A.2), its inhibitory potential extends to TLR signalling (Figure 4.7). Ligands for LILRB4 are yet to be identified and this prevents full understanding of its function/s. The main difference between human and murine LILRB4 is that mLILRB4 is primarily expressed on mast cells and neutrophils, and
172 is a potent regulatory of mast cell function by inhibiting activation via the FcεR (Section
1.4.4). Co-ligation of mLILRB4 and FcεR inhibits many processes including degranulation, de novo production of leukotrienes and secretion of cytokines. This is indicative that mLILRB4 is essential to countering immune responses, particularly those mediated by IgE (Katz et al., 1996).
In a well established model for the study of inhibitory receptors, the immunoregulatory function of LILRB1, LILRB2, LILRB5 and mLILRB4 have been investigated. Receptors were transfected into the rat basophilic leukaemia (RBL-2H3) cell line which expresses the high affinity IgE receptor (FcεRI), at higher levels than on other cell types (Conrad et al.,
1975). Cross-linking of the IgE receptor activates cells and induces the degranulation of inflammatory mediators and the most potent being histamine (DeLisi et al., 1979). Co- ligation of transfected cells containing LILRB1 (Colonna et al., 1997; Bellon et al., 2002),
LILRB2, LILRB5 (Colonna et al., 1998) and mLILRB4 (Lu-Kuo et al., 1999) to FcεRI prevents degranulation and thus directly inhibiting the IgE response. In the signalling of
LILRB1 and mLILRB4, the ITIM farthest from the membrane is the most significant residue to inhibit ITAM-activation as determined by mutation of tyrosine residues on the
ITIMs (Lu-Kuo et al., 1999; Bellon et al., 2002).
We speculate that LILRB4 signals via ITIMs and recruitment of relevant phosphatases, similar to those observed in studies on other inhibitors LILRs including mLILRB4. This study aims to establish a model for elucidating the downstream signalling mechanisms of
LILRB4 by replicating similar studies that have been previously conducted.
173 6.2 Materials and Methods
6.2.1 Antibodies and Reagents
Unconjugated anti-human CD25 and FITC (fluorescein isothiocyanate) conjugated anti- human CD25 were from Affinity Bioreagents (location), IgG1 control and anti- dinitrophenyl (DNP) IgE were from Sigma-Aldrich and AffiniPure F(ab')2 fragment goat- anti-mouse IgG (H+L) was from Jackson Immunoresearch. Cell Culture reagents including minimum essential medium (MEM) containing Earle’s salts, L-glutamine and D-PBS were purchased from GIBCO-Invitrogen, and non-essential amino acids, sodium pyruvate,
EDTA and Hank’s Balanced Salt Solutions (HBSS) were from Sigma-Aldrich.
® Transfection reagents Lipofectamine™ 2000 and Opti-MEM were purchased from
® Invitrogen, and G-418 (Geneticin ) from GIBCO-Invitrogen. Restriction enzymes BamHI and XbaI with appropriate reaction buffers were purchased from Promega (Madison, WI).
® Triton X-100 and 4-Nitrophenyl N-acetyl-β-D-glucosaminide were from Sigma-Aldrich.
6.2.2 Cell Culture and Cross-linking FcεRI on RBL-2H3 Cells
RBL-2H3 were cultured as suggested by ATCC, in minimum essential medium (MEM) containing Earle’s salts, L-glutamine (1 mM), sodium pyruvate (1 mM) and non-essential amino acids (1 mM). Cells were washed twice with D-PBS and non-enzymatically dissociated from flasks using 20 mM EDTA in HBSS (pH 7.2) and incubating at 37°C/5%
CO2 for 5 minutes. Flasks were gently tapped to dislodge loosely adherent cells and EDTA
2+ was neutralised by adding 4 times volume of culture medium (containing Ca to neutralise
EDTA).
174 RBL-2H3 cells were activated by cross-linking FcεRI, as reported elsewhere (Lu-Kuo et al., 1999). Cells were activated in 96 well, flat bottom tissue culture plates (Greiner Bio-
5 one) by stimulating 1 × 10 cells with 20 ng anti-DNP IgE overnight. The following day, cells were washed once with 200 μL PGE buffer containing 25 mM PIPES, 120 mM NaCl,
5 mM KCl, 40 mM NaOH, 5.6 mM glucose, 1 mM CaCl2 and 0.1% BSA. IgE-bound FcεR was cross-linked by adding 2.5 μg of cross-reactive F(ab')2 fragment goat-anti-mouse IgG
(H+L) in 100 μL PGE and incubated at 37°C with 5%CO2 in air, for 40 minutes.
Degranulation is measured by analysis of β-hexasominidase (β-hex) released in supernatants and this was performed in 96 well plates. Supernatants were harvested and
® cells washed once with PGE then lysed with 100μL 0.5% Triton X-100 in PGE buffer.
Cell lysis and supernatant samples were incubated with equal volume of 5μM 4-
Nitrophenyl N-acetyl-β-D-glucosaminide dissolved in 50mM sodium citrate solution (pH
4.5), in a total volume of 50 μL. Samples were incubated at 37°C for 2 hours and the reaction was stopped by the addition of 250μL of 0.2M glycine solution (pH 10.6).
® 384 Absorbance was read at 405nm using a SpectraMax Plus Spectrophotometer.
Degranulation was calculated using the absorbance values for cell lysis (P) and supernatant
(S) samples and the following formula:
% of degranulation = P/(P+S) × 100.
175 6.2.3 Chimeric DNA Constructs and Transfection in RBL-2H3 Cells
DNA constructs encoding a human CD25 extracellular domain fused to the transmembrane and intracellular regions of LILRB4 was sub-cloned into an expression vector (pMH-Neo) using BamHI and XbaI restriction enzymes. Single base pair mutations were introduced into the sequence of each of 3 tyrosine-codons to create amino acid substitution to phenylalanine (Y360F, Y412F and Y442F). Combinations of either a tyrosine or phenylalanine for each residue produce 8 different combinations; YYY (wild-type), YYF,
YFY, FYY (single mutants), YFF, FYF, FFY (double mutants) and FFF (triple mutant).
This is shown schematically below in Figure 6.1.
Large scale plasmid DNA was produced using a Plasmid Maxi Kit (QIAGEN) as per manufacturer’s instructions. Plasmid DNA was re-suspended in Tris-EDTA (TE) buffer
® and quantitated using the NanoDrop ND-1000 Spectrophotometer (NanoDrop
Technologies). DNA was digested using BamHI and XbaI, and products separated on a
0.8% agarose gel to confirm sizes of insert and vector. Bands corresponding to the size of the insert were cut and sequenced for further quality control.
Plasmid constructs were chemically transfected into the RBL-2H3 cell line using
Lipofectamine™ 2000 (Invitrogen) and optimised according to manufacturer’s suggestions. Cells were seeded the day before in 6 well tissue culture plates (Greiner Bio-
5 one) in complete media with 2.5 × 10 cells added to each well. Four hours prior to transfection, media was removed and replaced with serum reduced media containing 7.5%
FBS.
176 Y - tyrosine F - phenylalanine CD25 extracellular domain
TM
LILRB4 transmembrane 360 Y Y Y F Y F F F region and intracellular mutation combinations domain 412 Y Y F Y F Y F F 442 Y F Y Y F F Y F
Figure 6.1 » Schematic representation of LILRB4/CD25 chimeric protein and mutational changes of signalling tyrosines. A chimeric protein was constructed to have the extracellular domain of CD25 and the transmembrane (TM) and intracellular regions of LILRB4. Combinations of tyrosine motifs (Y) were mutated to phenylalanine (F) to produce 8 different mutant constructs.
177 For each of the 8 construct (YYY, YYF, YFY, FYY, YFF, FYF, FFY, FFF), purified
® plasmid DNA (13.2 μg) was incubated with 660μL Opti-MEM (Invitrogen) and at the
® same time, 39.6 μL Lipofectamine™ 2000 was diluted up to 660μL with Opti-MEM and both incubated at room temperature for 7 minutes. A mock control containing no DNA was included. Respective tubes were pooled and incubated for a further 20 minutes at room temperature. Plasmid/Lipofectamine™ 2000 mixtures were added to cells in the 96 well
2 plates and grown at 37°C/5% CO2 in air, overnight. Cells were seeded into 75cm flasks
® the following day. Selection antibiotic G-418 or Geneticin (GIBCO) was added at 800
μg/mL 70 hours post-transfection. Fresh media with new G-418 was added every 4 days and once cells grew in log-phase, concentration of G-418 was reduced to 400 μg/mL.
Stably-transfected cells were screened for CD25 cell surface expression by staining with anti-CD25-FITC (Affinity BioReagents, Golden, CO) for 30 minutes. Cells were washed twice with PAB buffer and fixed with 2% paraformaldehyde in D-PBS. Cells were analysed by a FACScan™ flow cytometer and the BD CellQuest™ software.
6.2.4 Statistical Analyses
Degranulation of RBL-2H3 is represented as mean ± SEM. Differences in percentage of degranulation was analysed by a one-way ANOVA with a Bonferroni’s post test.
® Statistical analyses were performed using GraphPad Prism 5. A p value of <0.05 was considered significant.
178 6.3 Results
6.3.1 Degranulation of RBL-2H3 Cells by Cross-linking of FcεRI
To ensure that RBL-2H3 cells were a suitable cell line to test the inhibitory potential of
LILRB4, their ability to degranulate upon FcεR cross-linking was firstly assessed. Cells were pre-treated with IgE to bind FcεR and cross-linked using a secondary goat-anti- mouse mAb. β-hex was measured following stimulation of cells to determine relative levels of activation (Figure 6.2). Resting RBL-2H3 cells exhibited minimal degranulation of β-hex without stimulation (1.83 ± 0.50 %) while cells treated with IgE and cross-linking mAb had significantly degranulated at 29.33 ± 1.97 %. Relative controls of cells treated with IgE or cross-linking mAb alone did not induce significant degranulation (2.03 ± 0.15
% and 1.93 ± 0.30 %, respectively).
6.3.2 Stable Transfection of CD25/LILRB4 Chimera into RBL-2H3 Cells
To assess the signalling mechanisms of LILRB4, a chimeric mutant construct was created by conjugating the intracellular signalling domain of LILRB4 to the extracellular domain of human CD25, the IL-2Rα chain, as shown in Figure 6.1. DNA constructs were created by site-directed mutagenesis and to ensure correct sequences, DNA was verified by sequencing.
179 40 *** *** *** 30
20
10 -hex dengranulation (%) dengranulation -hex
β 0 IgE - + + -
cross-linking mAb - - + +
Figure 6.2 » Degranulation of RBL-2H3 cells upon FcεR cross-linking. Cells were incubated with IgE overnight and then FcεR-bound IgE were cross-linked using a secondary mAb. Cross-linking of FcεR induced significant amounts β-hex compared to controls containing IgE alone, cross-linking mAb alone or neither antibodies (n=3). Data represents mean ± SEM and analysed by an ANOVA and Bonferroni’s post test.
180 A total of 8 constructs were created by mutating each possible signalling tyrosine (Y) residue to phenylalanine (F). These are; YYY (wild-type), YFF, YFY, FYY (single mutants), YFF, FYF, FFY (double mutants) and FFF (triple mutant). Transfected cells were maintained under high concentration of selection, G-418, to produce stable tranfectants and expression was analysed by staining for CD25 for flow cytometry.
Expression of CD25 for untransfected RBL-2H3 cells and each mutant is represented by the bold lines in the frequency histograms (Figure 6.3) while the IgG1 isotype-stained cells are depicted by the thin line. RBL-2H3 cells do not normally do not express human CD25
(Figure 6.3A). Each of the wild-type and mutant-transfected RBL-2H3 cells express similar levels of cell surface CD25 (Figure 6.3B-I).
181 A. B. C. RBL YYY FFF
D. E. F. YYF YFY FYY
G. H. I. YFF FYF FFY
Figure 6.3 » Analysis of CD25/LILRB4 chimera expression on transfected RBL-2H3 cells. Each of the wt and mutant constructs for the CD25/LILRB4 chimeric protein were transfected into RBL-2H3 and maintained under selection to produce stable transfectants. Expression was measured by flow cytometry using anti-CD25-PE (bold line) and compared to the isotype control (thin line). (A) Untransfected RBL-2H3 cells do not express human CD25. Transfection of cells for (B) wt (YYY) and (C) triple mutant (FFF) highly express CD25. (D-F) Single mutants (YYF, YFY and FYY) as well as (G-I) double mutants (YFF, FYF and FFY) also express CD25 at a similar intensity.
182 6.4 Discussion
From this study, we have developed the tools for studying LILRB4 function by utilising a well established model used for the investigating the function of inhibitory receptors
(Colonna et al., 1997; Colonna et al., 1998; Lu-Kuo et al., 1999; Bellon et al., 2002). The extracellular domain of human CD25 was used in this study as specific antibodies are commercially available and do not cross-react with rat CD25. Although there is much work still to be done for this project, we now have the resources for determining the most functionally relevant ITIM residue and the intracellular signalling molecules recruited by
LILRB4 to confer inhibition.
LILRB4 has broad functions in modulating immune responses in transplantation, autoimmunity and cancer. Therefore, identification and understanding of the signalling pathways of LILRB4 may be useful in developing therapeutic interventions in relevant diseases. It would also be interesting to compare the signalling mechanisms of human and murine LILRB4, given that mLILRB4 has a significant role in controlling innate and allergic immune responses in mice. Studies on relevant receptors reveal that more often, the most distal motif is usually the most functional and similar findings are expected for
LILRB4.
It would be curious to observe any potential relationship between LILRB4 and ZAP-70 as independent studies have shown that LILRB4-expression CLL B cells have increased survival (Colovai et al., 2007) yet CCL B cells preferentially signal via ZAP-70-dependent pathways (Cutrona et al., 2006). ZAP-70 is an SH2-containing PTK that is essential in
183 TCR signalling as it is recruited by the phosphorylated ITAMs of the γ-chain of CD3
(Bene, 2006). In the case of CLL B cells, the presence of ZAP-70 enhances proliferation as they typically signal with Syk. We can almost rule out an inhibitory effect of LILRB4 on
ZAP-70 function but studies are required to confirm this. It is likely that LILRB4 inhibits anti-tumour responses via tolerisation of T helper cells and subsequent suppression of cytotoxic T cell function (Colovai et al., 2007) as up-regulation of inhibitory receptors such as LILRB4 is an important event for development of tolerogenic DCs (Manavalan et al., 2003). However, the resident function of LILRB4 on expressing CLL B cells still remains to be determined.
As previously determine, LILRB4 is able to inhibit LPS-responses (Figure 4.7), marking a function of ITIMs on non-ITAM activating pathways. Results from this experiment may answer the question of how might the ITIMs become phosphorylated if the receptor is not brought close to a source of src family kinase.
It is strongly suggested that LILRB4 functions in antigen-specific responses (Chang et al.,
2002) and predicted to have a non-MHC ligand. Although its ligand remains unidentified, neutralisation by mAb confirms the role of LILRB4 in controlling immune responses
(Manavalan et al., 2003). Once its ligand is discovered, we will have a better understanding of LILRB4 and its role in the immune system.
184 Chapter 7. Final Discussion
“Balance” is a word often used when discussing immune-regulatory receptors and the studies presented here reinforces the hypothesis that a balance between reactivity and quiescence remains the key between auto-reactivity and immune-suppression. Though many attempts have been made to define this balance, there is still much to be understood.
From this thesis, we find a significant contribution of LILRs to regulation of immune responses and their role in autoimmune diseases such as RA. There are very few studies exploring the function of LILRs in autoimmune diseases.
Activating and inhibitory receptors are almost always co-expressed on leukocytes and
LILRs are no exception (Table 1.3A and Table 1.3B). Activating and inhibitory LILRs are often co-regulated as seen in this study (Chapter 3) and another (Jung et al., 2004). It may be that counter-acting receptors are co-regulated in tandem to maintain the essential balance between activation and inhibition. It is likely that the regulation of LILRA2 and
LILRB4 by inflammatory mediators is required to modulate their function during immune responses. This study is the first to investigate the regulation of LILRA2 by various inflammatory mediators.
Although the exact functions of LILRA2 are unknown, there is substantial evidence suggesting that it functions in inflammation via the induction of cytokine production
(Section 1.3.5.1) and the studies presented here support this (Figure 3.3). LILRA2 induces the production of acute inflammatory cytokines; TNF-α, IL-6 and IL-1β but also immune-
185 regulatory cytokines such as IFN-γ and IL-10 (Figure 3.3). The ability for LILRA2 to potently induce pro-inflammatory cytokines (Figure 4.3), particularly TNF-α, is comparable to LPS-stimulation and supports the suggestion that LILRA2 functions in inflammation. The pathophysiological role of TNF-α has been well described since its purification and cloning in the mid 1980s and one of its major roles is in the elimination of infectious microbes via its ability to initiate the cytokine network (Feldmann et al., 2001).
The quantity of cytokines produced upon LILRA2 cross-linking most probably has pathological significance and identification of its ligand/s would help to determine its exact function. Furthermore, our findings that LILRA2 expression is modulated by such cytokines, is indicative of a feedback mechanism of regulation. As LILRA2 induces the production of TNF-α and IL-1 and is then down-regulated by these cytokines, it is likely that this autocrine regulation helps to prevent excessive activation of cells.
At the present, LILRA2 is believe to have a functional role in the pathogenesis of leprosy
(Section 1.3.6.2) and RA (Section 1.6.5) and given the potent ability of LILRA2 to induce the production of inflammatory cytokines, it may also have a role in many other inflammatory disease. Screening for the presence of LILRA2 in inflamed tissue from other diseases such as inflammatory bowel disease, SLE or other localised infections could determine whether LILRA2 is disease-specific or its presence is merely a consequence of leukocyte infiltration/activation, on which it is constitutively expressed. Within the rheumatoid synovium, the high expression of LILRA2 on macrophages most probably induces the local production of inflammatory cytokines which perpetuates inflammation of the joint by recruiting and activating other cells. It is tempting to speculate that LILRA2
186 may also regulate protease activity and possibly the expression of co-stimulatory receptors and adhesion molecules.
It is no surprise that glucocorticoids block LILRA2-mediated TNF-α production (Figure
3.4) as glucocorticoids are well known for suppressing cytokine production (Section
1.6.4.3) and this is one main reason why they are used in treating many inflammatory disorders. This inhibition is most likely attributed to the genomic function of glucocorticoids and blocking the promoter region for the genes encoding inflammatory cytokines (Section 1.6.4.3). Although glucocorticoids block the function of LILRA2, the development of a drug to decrease LILRA2 expression may also be beneficial in treating associated diseases.
Current in vivo studies indicate that LILRB4 function by blocking the activation of inflammatory cells and these events can influence tissue graft acceptance but also cancer survival (Section 1.3.5.2). It was therefore necessary to investigate the regulation of its expression and determine its function upon signalling. To date, the ability for LILRB4 to influence monocyte activation and subsequent cytokine production has not been elucidated. From these studies, we understand that LILRB4 expression can be altered by inflammatory mediators such as endotoxin, TNF-α and IL-1β but also immune-regulatory mediators; IL-10, IFN-γ and vitamin D3 (Figure 2.3).
There have been extensive studies into the function of murine LILRB4 but there has since been little correlation between the murine and human forms. We have now demonstrated
187 inhibitory function of human LILRB4 to block ITAM-bearing receptors but also
LPS/TLR4-signalling, as seen with the murine receptor mLILRB4. This broad mechanism of inhibition suggests that LILRB4 may be able to block general inflammatory responses to prevent excessive tissue destruction that is associated with ongoing inflammation.
The study shown in Chapter 6 is evidently unfinished and due to time constraints could not be completed prior to the finalisation of this report. As experiments continue with respect to this study, there are hopes that details of the signalling mechanisms of LILRB4 will be ascertained in regards to the role of each individual ITIM, the down-stream signalling molecules recruited and other suppressive functions of the receptor.
The relationship between LILRs and TLRs has been increasingly studied and it is evident that LILRA2 and LILRB4 are able to influence the LPS-mediated immune responses and vice versa. In PBMCs, the inhibitory effect of LILRA2 monocyte activation on LPS function was not caused by changes in cell surface of either receptor and so their interaction is most likely occurring at the intracellular level. Competition for signalling substrates may block effective transcription of genes and hence, the suppression of TNF-α upon co-stimulation. We have ruled out immune suppression via the production of IL-10 as neutralising antibodies against IL-10 have failed to restore responses (Figure 4.6).
Interestingly, in the THP-1 monocyte cell line, synergism is observed rather than inhibition whereby co-stimulation with LPS and LILRA2 cross-linking induced a response that was substantially greater than each individual stimulus. In these cells, LPS did not alter
LILRA2 expression suggesting that this enhancement in LILRA2 function is independent
188 of cell surface expression. The conflicting data observed in PBMCs compared to THP-1 cells may be explained by the presence of lymphocytes in PBMC. Lymphocytes do not express LILRA2 (Figure 3.1) but can potentially regulate the function of LILRA2 by indirect means. Repeat studies on negatively selected populations of pure monocytes could determine whether lymphocytes influence this inhibition.
Like most autoimmune diseases, the pathological processes of RA remain elusive. By teasing out possible mechanisms involved in the disease process, there is a potential to improve disease maintenance through new therapeutic targets. The increase LILRA1- and
LILRB1-positive circulating T lymphocytes found in patients with RA is the first step to exploring the role of these LILRs in the pathogenesis of this disease. Further studies are required which would involve the purification of these cells, further phenotypic characterisation of lymphocyte sub-population and then downstream functional studies upon receptor cross-linking/co-ligation. Potentially, these cells could be antigen-specific immunoregulatory T cells mediating disease via persistent activation of other inflammatory cells, though this remains to be investigated. Should these cells be functionally significant in RA, development of drugs against these receptors may provide therapeutic benefit.
Regulation of LILRA2 in the RA joint is supportive of a functional role. Although this alteration in expression may be incidental rather than integral, the results here suggest that
LILRA2 at the least exacerbates local inflammation via its ability to induce the production
189 of pro-inflammatory cytokines, such as TNF-α. Therapeutic targeting of ITAM-bearing receptors may alleviate inflammatory responses.
To reiterate one crucial point, unidentified ligands still remains an obstacle to understanding the full potential of LILRs, including their role in maintaining health and initiating diseases. It is tempting to speculate that LILRs to share ligands and subsequent responses that are induced are dependent on the type of LILR which is bound. Thus differential responses may be controlled by type of LILR expressed. Whether differences in affinity/avidity of LILR-ligand interaction are influencing factors, remain to be determined.
Expression of LILRs may be critical in immune homeostasis, immune responses and disease development. From our understanding of their expression and function, it is likely that LILRs have a functional role in the pathogenesis of RA. Nevertheless LILRs may be potential novel therapeutic targets in pathological inflammatory processes such as RA.
Definitive demonstration of a role for LILRs in regulating inflammation in vivo requires identification and/or development of specific agonists and antagonists.
190 Appendix I. Inhibition of FcγRI by LILRB4
The high affinity IgG receptor, CD64 or FcγRI, is an ITAM-bearing receptor which is primarily expressed on cells of myeloid lineage and essential during chronic immune responses via its recognition of IgG antibodies (reviewed in Takai, 2005). Upon ligation with antigen-bound IgG antibodies, CD64 activation and signalling induces cellular immune responses such as phagocytosis, granule secretion and the generation of reactive oxygen species (reviewed in Cohen-Solal et al., 2004). CD64-signalling prevents re- infection of pathogenic organisms by stimulating a quick antigen-specific response.
In this study, the THP-1 monocyte cell line was used and expression of both CD64 and
LILRB4 was confirmed by standard 2-step staining for flow cytometry. THP-1 cells exhibited relatively high expression of both receptors (Figure A.1A-B) and this expression remained unaltered during culture over time (data not shown).
It has been established that TNF-α is a released upon CD64 cross-linking (Loegering et al.,
2004) and also confirmed in this study. Co-ligation of CD64 with LILRB4 using monoclonal antibodies (10 μg/mL each) inhibited TNF-α production by 64.7% from 58.87
± 9.29 to 20.78 ± 3.63 pg/mL (Figure A.2).
This is the first observation of LILRB4 directly affecting a well established inflammatory process. It is speculated that LILRB4 inhibits CD64 by classical ITIM-ITAM interactions.
191 A. B.
CD64-FITC LILRB4-FITC
Figure A.1 » Expression of CD64 and LILRB4 on THP-1 cells. Cells were cultured and stained for - receptors using mAb and analysed by flow cytometry. Positive expression of both (A) CD64 and (B)
LILRB4 is shown (bold lines) in comparison to isotype controls (thin lines). Histograms are representative of 3 individual experiments.
** 100
80 ** *
60 (pg/mL)
α 40
TNF- 20
0
IgG1 + - - + αCD64 - + + + αLILRB4 - - + -
Figure A.2 » Cross-linking of CD64 and co-ligation with LILRB4 on THP-1 cells using mAbs. CD64 cross-linking significantly induced TNF-α production in cells and co-ligation of CD64 with LILRB4 significantly inhibited cytokine production (n=3). Data represents mean ± SEM and analysed by an
ANOVA and Bonferroni’s post test.
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