The Role of Rasgrp1 and 4 in the Pathogenesis of Human Diseases

The Role of RasGRP1 and 4 in the Pathogenesis of Human Diseases

By Miao Qi B.Sc (Med.)

A thesis submitted in fulfillment

of the requirements for the degree of

Master of Science (Research)

Faculty of Medicine,

St George Clinical School,

University of New South Wales

Sydney, NSW, Australia, 2009 ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Miao Qi

15th January 2009 Abstract

Mast cells are known to play an important role in allergic events and in other inflammatory reactions through varied intracellular signaling transduction proteins.

RasGRP4 is a mast cell-restricted guanine nucleotide exchange factor (GEF) and diacylglycerol (DAG)/phorbol ester receptor. Interleukin (IL) -13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including

IL-4R, IL-13R1 and IL-13R2. IL-13R2 has been reported to be a decoy receptor for IL-13. My experiments indicate that the mast cell specific RasGRP4 protein regulates the level of IL-13R2 and controls IL-13/ IL-13R1-mediated intracellar signaling events in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate RasGRP4 could be used to modulate the IL-13-induced phosphorylation of

STAT-6 that may be used as therapy in patients with asthma.

SLE is a complex, heterogeneous systemic autoimmune disease characterized by the presence of high levels of autoantibodies. Dysregulation of RasGRP1, a Ras active gene, in mice resulted in a SLE-like disorder. Yasuda and coworkers demonstrated that a defective isoform of RasGRP1 (11) was present in a subset of patients with SLE. My experiments indicate that RasGRP1 upregulates the expression of IL2RG in T cells. In contrast the 11 RasGRP1 isoform expressed in a subset of SLE patients leads to defective expression of IL2RG. The IL2RG chain is a common chain which forms part of a number of different receptors eg. IL-2, 4, 7, 9, 15, 21. IL-2 as well as IL-21, which shares sequence homology with IL-2, has been reported to be involved in the generation of regulatory T cells (Tregs). In SLE patients, CD4 + CD25+ Tregs, which play an essential role in controlling immunologic tolerance to self-antigens and preventing autoimmunity, are significantly decreased when compared with healthy controls. The accumulative evidence suggests that the defective isoform of RasGRP1 (11) downregulates expression of IL2RG in SLE patients’ T cells and this could effect the generation of CD4 + CD25+ Tregs. This may be another immunological mechanism in loss of tolerance observed in patient with SLE.

Acknowledgments

During my term as a postgraduate student in The Immunology Laboratory at the St.

George Hospital Campus of the University of New South Wales over the two years, I would like to acknowledge the help and encouragement of my supervisor, Professor S

Krilis. He is very rigorous but very amiable to me. He has many good ideas that we have discussed which have helped me with my studies. He has helped me when I have encountered any difficulties during my experiment and he is always keen to help me to analyze my data to find out the best solution. I have found him an excellent supervisor.

At the same time, I also appreciate the support from my co-supervisor Dr. J.C. Qi. He supports me a lot not only in scientific research but also in living aspects. He has taught me many scientific techniques, which have been invaluable in my studies. He has also on a personal level encouraged me and helped me to adjust to a new and foreign environment.

I sincerely thank all of the individuals in this laboratory, who have helped me and supported me, including Dr Freda Passam, Dr Kumiko Tanaka, Dr John Ioannou, Dr

Jingyun Zhang, Dr Soheila Rahgozar, and Dr Bill Giannakopoulos. They provided me their friendship and expertise in the last two years. I have been very happy to study in this department and work here because of the harmonious working environment.

I would like to thank Dr Gareth Denyer who established the program for the microarray

database analysis system using Filemaker pro 9 which I have found invaluable in my studies. It is a very powerful and novel program that has allowed me to generate some novel and interesting data. Thanks also to Dr Gregory P Katsoulotos and Dr. J.C. Qi for establishing the hRasGRP4 trasnfectants which I also used in my experiments. I would like to acknowledge the help of Professor Richard L Stevens from Harvard Medical

School (Boston, MA, USA). Although I haven’t met him, I thank for him providing me with information on RasGRP which has been invaluable in my studies on this molecule.

Also I would like to thank Dr who kindly provides me with the DNA of RasGRP1 isoforms and information on his RasGRP1 studies.

Finally, I should say sincerely thank to my parents, my wife, and my whole family who have encouraged me throughout my postgraduate studies.

Table of Contents

Acknowledgments ...... 1

Table of Contents ...... 3

List of Figures and Tables ...... 6

List of Abbreviations ...... 8

List of Publications ...... 11

1. Chapter 1 Literature review ...... 12

1.1 Mast cells in asthma...... 12

1.1.1 Introduction ...... 12

1.1.2 Pathogenesis of asthma ...... 13

1.1.2.1 The immune response in airway inflammation ...... 13

1.1.2.2 Inflammatory cells in asthma ...... 14

1.1.2.3 Airway remodeling ...... 16

The epithelium in the asthmatic airway ...... 16

Airway remodeling ...... 18

1.1.3 Mast cell biology ...... 20

1.1.3.1 Features of mast cells ...... 20

1.1.3.2 Development of mast cells ...... 22

1.1.3.3 Migration of mast cells ...... 27

1.1.4 The role of mast cells in asthma ...... 28

1.1.4.1 Mast cell related mediator release ...... 29

1.1.4.2 The role of mast cells in the asthmatic airway ...... 30

1.1.5 RasGRP4: a novel mast cell-restricted gene ...... 33

1.1.5.1 Pathways of mast cell signal transduction ...... 33

1.1.5.2 RasGRP family ...... 37

1.1.5.3 RasGRP4 gene and protein ...... 38

1.2 The pathogenesis of systemic lupus erythematosus (SLE) ...... 42

1.2.1 Overview of SLE ...... 42

1.2.2 The role of T cells in SLE ...... 45

1.2.2.1 Evidence of involvement of T cells in SLE ...... 45

1.2.2.2 Dysfunction of T cell signaling transduction in SLE ...... 48

1.2.2.3 Genetic factors in SLE ...... 53

Autoimmunity genes ...... 53

Type I interferon (IFN) pathway ...... 54

1.2.3 Defective Expression of RasGRP1 in T cells associated with SLE... 55

2. Chapter 2. hRasGRP4 regulates the expression of

IL13R2 in a human mast cell line ...... 60

2.1 Introduction ...... 60

Aims: ...... 62

2.2 Materials and methods ...... 63

Equipment ...... 63

Reagents ...... 65

2.2.1 Cell culture ...... 68

2.2.2 PMA stimulation of RasGRP4 transfected HMC-1 cells ...... 69

2.2.3 PMA and IL-13 stimulates the RasGRP4 expressing HMC-1 cells . 70

Western blot ...... 71

2.3 Results ...... 75

2.4 Discussion ...... 78

3. Chapter 3. hRasGRP1 mediated changes of the expression of IL2R" in a human mast cell line ...... 89

3.1 Intruduction ...... 89

Aims: ...... 91

3.2 Materials and Methods ...... 91

Reagents ...... 91

3.2.1 Transfection of wild-type and exon 11 deletion (11) isoform of hRasGRP1 in HMC-1 cells ...... 93

Agarose gel electrophoresis: ...... 99

Transfection:...... 101

Limiting dilution: ...... 103

Clone characterization by PCR, SDS-PAGE Immunoblot and Immunohistochemistry

...... 103

3.2.2 Affymetrix GeneChip® Exon Arrays on non-PMA and PMA stimulated hRasGRP1 expressing HMC-1 cells ...... 109

3.2.3 Human RasGRP1 regulates expression of IL-2 Receptor Gamma 116

Quantitative Real-time PCR ...... 116

3.3 Results: ...... 117

3.4 Discussion ...... 124

Summary ...... 132

Future direction ...... 135

References ...... 136

List of Figures and Tables

Figures

Figure 1: Two models of mast cell-related hematopoiesis in mice ...... 24 Figure 2: The Ras Pathway ...... 35 Figure 3: RasGPP protein structure: a schematic domain structure map ...... 38 Figure 4: Novel defective RasGRP1 transcripts in the PBMCs of SLE patients 59 Figure 5: PMA-mediated translocation of hRasGRP4-GFP in HMC-1 cells...... 76 Figure 6: Evaluation of IL13R2 protein levels in HMC-1 cells, which differ in their hRasGRP4 expression before and after exposure of the cells to PMA, and evaluation of IL-13-dependent phosphorylation of STAT6 in these cells. . 77 Figure 7: IL-13 receptor systems and the regulation of IL-13 signaling pathway 85 Figure 8: RasGRP1 mRNA sequence Exon 1-18 & Exon 11 primers ...... 94 Figure 9: Map of pcDNA3.1/V5-His TOPO vector and the sequence of PCR product insertion point ...... 99 Figure 10: Digestion of two RasGRP1 transfectants...... 100 Figure 11: Clone characterization by PCR, SDS-PAGE Immunoblot ...... 108 Figure 12: Clone characterization by Immunohistochemistry ...... 108 Figure 13: Schematic for representation of Whole Transcript Sense Target Labeling Assay for 1 )g of total RNA ...... 111 Figure 14: Demonstration of filemaker pro 9 designed analytical program ...... 114 Figure 15: RasGRP1 microarray quality control data...... 115 Figure 16: The evaluation of RasGRP1 expression in RasGRP1 microarray data.119 Figure 17: Microarray data of expression of RasGRP1 in Full-length (F), 11 (D) and Mock (M) HMC-1 transfectants...... 120 Figure 18: RasGRP1 Microarray data analysis ...... 121 Figure 19: Microarray data of expression of IL2R" in Full-length (F), 11 (D) and Mock (M) HMC-1 transfectants...... 122 Figure 20: IL2R" expression in FL, 11 RasGRP1 and mock HMC-1 transfectants

measured by Quantitative Real-time PCR ...... 123 Figure 21: IL2R" expression in FL, 11 RasGRP1 and mock HMC-1 transfectants measured by RT- PCR ...... 123 Figure 22: The expression of IL-2R" in T cells of a subset of SLE patients and healthy individuals ...... 124 Figure 23: Protein structure of IL2 receptor systems and a schematic exon structure map of IL-2R" ...... 129

Tables Table 1: Equipment ...... 63 Table 2: Reagents used in Chapter 2 ...... 65 Table 3: PMA and IL-13 stimulation of transfected HMC-1 cells ...... 70 Table 4: Reagents used in Chapter 3 ...... 91 Table 5: RasGRP1 Microarray labeling ...... 113 Table 6: Signal intensity values of genes regulated by hRasGRP4 in non-PMA stimulated HMC-1 cells ...... 150

List of Abbreviations

AICD activation-induced cell death ANA antinuclear autoantibodies APCs antigen presenting cells BAL bronchoalveolar lavage BHR bronchial hyperreactivity BLyS B-lymphocyte stimulator bp base pairs BSA bovine serum albumin CCR,CXCR chemokine receptors CD Cluster Designation or Cluster of Differentiation cDNA complimentary DNA CAMREM cyclic adenine monophoshphate response element modulator CTLA-4 Cytotoxic T-lymphocyte antigen 4 DAG diacylglycerol DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxy nucleotide triphosphates dsDNA double-stranded deoxyribonucleic acid EBV Epstein-Barr virus EDTA ethylenediaminetetra-acetic acid EF calcium-binding elongation factor ERK Extracellular-signal-regulated kinase FBS fetal bovine serum FcRI high affinity receptor for the Fc fragment of IgE FL Full-length GEF guanine nucleotide exchange factors GFP green fluorescent protein GM-CSF Granulocyte-macrophage Colony Stimulating Factor HLA Human leukocyte antigen HMC human mast cells hnRNP heterogeneous ribonucleoprotein hRasGRP1 human Ras guanine releasing protein 1 hRasGRP4 human Ras guanine releasing protein 4 HRP horse radish peroxidase IFN Interferon IFNGR1,IFNGR2 IFN-" receptors 1,2 Ig Immunoglobulin IHC immunohistochemical IL Interleukin IL2R" Interleukin 2 receptor gamma

IMDM Iscove's Modified Dulbecco's Medium IRF5 Interferon regulatory factor 5 ISH in situ hybridization ITIM immunoreceptor tyrosine-based motif JAK Janus Kinase JNK c-Jun N-terminal kinase kd Dissociation constant kDa Kilodalton KL Kit ligand LYP lymphoid tyrosine phosphatase MAP Mitogen-activated protein MAPK Mitogen-activated protein kinase HMC-1 Human mast cell line -1 MITF Microphthalmia transcription factor Mock vector only transfectant mRasGRP1 mouse Ras guanine releasing protein 1 mRasGRP4 mouse Ras guanine releasing protein 4 mRNA messenger RNA PAGE Polyacrylamide gel electrophoresis PBMCs peripheral blood mononuclear cells PBS phosphate-buffered saline PCR Polymerase chain reaction PCD1 program cell death 1 PDGF Platelet-derived growth factor PFA-PBS paraformaldehyde-PBS PG Prostaglandin

PGDS PGD2Synthase PI Propidium Iodide

PI3 Phosphatidylinositol 3-kinase

PIP2 Phosphatidylinositol-4,5-bisphosphate PKA Protein Kinase A PKC Protein kinase C PLC Phospholipase C PMA Phorbol-12-myristate 13-acetate PTPN22 protein tyrosine phosphatase non-receptor 22 QC quality control RA rheumatoid arthritis REM Ras exchange motif RNA ribonucleic acid rpm revolutions per minute RT Reverse Transcriptase RT-PCR reverse transcriptase polymerase chain reaction RUNX1 runt-related transcription factor 1

SCF Stem cell factor SDS sodium dodecyl sulphate SH2 the Scr homology region 2 siRNA small interfering ribonucleic acid SLE Systemic lupus erythematosus SNP Single nucleotide polymorphism Sos Son-of-sevenless STAT Signal transducer and activator of transcription TAE tris-acetate-EDTA buffer Taq Thermus aquaticus TE tris-EDTA buffer TYK2 Tyrosine kinase 2 U units U.V ultra violet VEGF Vascular endothelial growth factor WSXWS tryptophan-serine-x(unconserved amino acid)-tryptophan-serine WT Whole Transcript

List of Publications

Journal article

[1] Qi, M., Katsoulotos, G.P., Qi, J.C., Tanaka, K., Hughes, W.C., Molloy, T.J., Adachi,

R., Stevens, R.L., Krilis, S.A.

The Diacylglycerol-dependent translocation of ras guanine nucleotide-releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor alpha2.

J Biol Chem, 2008. 283(3): p. 1610-21.

Poster presentations

1) Miao Qi, Jian Cheng Qi, Gregory P. Katsoulotos, Kumiko Tanaka, Richard L. Stevens and Steven A. Krilis

RasGRP4 a Mast Cell Specific Protein Regulates IL-13 Signaling and Function:

Implications for the Pathogenesis of Human Asthma

Persented at St George Hospital Medical Symposium 8 November, 2007

Winning Poster Award of 2007

2) Miao Qi, Jian Cheng Qi, Shinsuke Yasuda, Takao Koike, Richard L. Stevens, and Steven A. Krilis

A subset of patients with SLE has an abnormal Ras guanyl nucleotide-releasing protein 1 (RasGRP1) which Regulates the expression of IL-2 receptor gamma

(IL2RG)

Persented at St George Hospital Medical Symposium 13 November, 2008 11

1. Chapter 1 Literature review

1.1 Mast cells in asthma

1.1.1 Introduction

Asthma has been known as a chronic inflammatory disorder of airways which is accompanied with the clinical symptoms of wheezing, shortness of breath, chest tightness, cough, increased mucus production and dyspnoea associated with airflow obstruction or bronchial hyperreactivity (BHR). In the most severe cases, death from asphyxiation [2] can occur. Asthma is becoming a major public health issue in Australia.

Although there is no cure for asthma, there is an intense the therapy of research activity to understand the pathogenesis of asthma. A number of studies implied a variety of inflammatory cells such as macrophages, Eosinophils, T-lymphocytes and mast cells in the pathogenesis of asthma as well as many different mediators secreted by these cells such as chemokines and cytokines [3]. There is compelling evidence that mast cells contribute to the pathophysiology of asthma.

In this section, the role of mast cells in asthma will be primarily considered, mainly concentration on mast cell biology, its role in the pathogenesis of asthma. RasGRP4, a novel recently discovered guanine nucleotide-releasing protein which is restricted to mast cells and has been demonstrated to be an important factor in mast cell development and maturation will also be discussed.

1.1.2 Pathogenesis of asthma

Asthma is considered as a disorder of the conducting airway dominated by several different cells and mediators including Th2-type lymphocytes, IgE, mast cells, eosinophils, macrophages, cytokines. The airway epithelium vascular and neurological networks also play an important role. Although there are other heterogeneous respiratory disorders that may mimics asthma, the inflammatory disorder caused by exposure to a wide range of exogenous and endogenous stimuli such as allergens, microorganisms, pollutants is still considered as the primary pathogenesis of asthma [4].

Asthma is a heterogeneous disorder with clinical presentations leading to inflammatory airway smooth muscle contraction, mucus plugging and airway remodeling.

1.1.2.1 The immune response in airway inflammation

Airway inflammation in asthma is a multicellular process. Eosinophils, neutrophils,

CD4+ T-lymphocytes and mast cells participate in this process [5]. The fundamental mechanism of airway inflammation is associated with allergic sensitization and

IgE-mediator signaling transduction. Environmental allergens are recognized by airway epithelium and generate a Th2 cytokine response to them.

Dendritic cells located in the airway epithelium and submucosa and extend their processes to the airway surface can bind inhaled allergens [6, 7]. After being captured, 13

the allergen is bound by specific IgE on the dendritic cell surface which facilitates allergen internalization. Dendritic cell entrapping allergen which is processed by intracellular enzymes and the peptides are transported to the cell surface and presented to antigen-presenting cells in the context of MHC to T lymphocytes [8]. The dendritic cells receive signals to migrate to the local lymphoid tissue where antigen presentation takes place. In this migration processing, chemokine receptors, including CCR7 and its ligands CCL19 and CCL21 contribute to the antigen presentation to naive T cells.

Recent studies indicate that different immune response pathways may be involved in different clinical forms of asthma [9].

1.1.2.2 Inflammatory cells in asthma

In a certain extent, allergic inflammation ultimately depends on the presence of a terminally differentiated subset of T helper cells in lung, the T helper cell type 2 (TH2 cell) in mild to moderate asthma, but while asthma becomes more severe and chronic,

Th1 cells are recruited [10]. Once activated, T cell induces not only the chemokines like

CCL11, CCL24, CCL26, CCL7, CCL13, CCL17 and CCL22 (which interact with their reciprocal receptors including CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8) [11, 12], but also a range of cytokines, like IL-3, IL-4, IL-5,IL-6, IL-9, IL-13 and granulocyte–macrophage colony stimulating factor (GM-CSF) [13, 14]. However, the precise mechanism of T cell regulation in both mild to moderate and severe asthma is still unknown.

The significant reduction in sputum and tissue eosinophils after treatment of asthma with inhaled or oral corticosteroids indicates that the eosinophil is a very prominent cell in the inflammation of allergic asthma [15, 16]. It is present in the airway wall of uncontrolled asthma and is also found in large numbers in the sputum and bronchoalveolar lavage fluid [5, 17]. These cells are mostly recruited from the bone marrow as CD34+ precursors. The mediators IL-3, GM-CSF and eotaxins 1–3 contribute to the early derivation of eosinophils from CD34+ bone marrow precursor cells, and IL-5 induces their maturation and recruitment into the airways [18, 19].

Eosinophils are the producer of granule basic proteins, such as eosinophil peroxidase, eosinophil cationic protein, and major basic protein. They also can generate eicosanoids such as prostacyclin (PGI2), cysteinyl leukotrienes and release potentially tissue-damaging superoxide as well as a range of cytokines and chemokines [20].

However, recent studies challenge the primary status of the eosinophil in the inflammatory milieux of asthma. Much evidence suggested that the eosinophil may play a more important role in airway remodeling than has hitherto been recognized [21].

Despite a plethora of experiments that have focused on eosinophil maturation and clearance as well as function in asthma, there still remains many unanswered questions.

Monocytes and macrophages play an important role in pathogenesis of chronic asthma.

They are the source of reactive oxygen, cysteinyl leukotrienes and a variety of lysosomal enzymes. Dendritic cells are induced from Monocytes with the presence of

GM-CSF [22] and IL-4 [23]. Nevertheless, the exact role of these cells in mediating tissue damage and airway pathology of asthma is still unclear.

The mast cell has long been considered to play an important role in asthma. Many granule-associated mediators, proinflammatory and profibrogenic cytokines and chemokines are released from mast cells in allergic asthma. In addition, mast cells contribute to airway remodeling and reactivation with Airway Smooth Muscle (ASM) in asthma. The relationship between asthma and mast cells will be discussed below.

1.1.2.3 Airway remodeling

The epithelium in the asthmatic airway

The epithelium in the airway plays an important role in protecting lupus from exogenous stimuli such as allergens. The function of the epithelium seems to be as a physical barrier between the exterior and internal environment. However, in the pathogenesis of asthma, much evidence demonstrates that metaplasia and damage of epithelium, thickening of the subepithelial basal lamina, and increased number of myofibroblasts as well as the airway remodeling are the cardinal issues, even in the mildest forms of asthma[24]. The downregulation of epithelium proliferative markers such as Ki67 suggests that the epithelium is easily damaged and cannot easily regenerate itself [25]. Another feature of damaged epithelium is that it loses the columnar cells due to disruption of both tight junctions and desmosomal attachments.

The permeability of asthmatic epithelial cells are remarkable increased compared to normals, based on in vitro culture experiments with epithelial cells from normal and asthmatic airways [26]. All of the experiment evidence indicates that the epithelium in asthmatic airways has lost its protective role and thus cannot act as a barrier to exogenous stimuli such as antigens, etc.

The reasons why the airway epithelium loses its ability of function as a barrier is still not fully understood. Recent developments have indicated that a number of genes contribute to this abnormality. For exanple, filaggrin and the S100 proteins which modulate epithelial integrity are found associated with the abnormal airways seen in asthma [27]. Furthermore, external noxious stimuli such as viral infections and air pollution might also contribute to the airway epithelial damage. The common cold virus such as rhinovirus have been reported to be associated with the development of asthma in both children and adults. Virally infected asthmatic epithelial cells may lose their ability to clear the virus due to the reduction of their ability to produce IFN-b and IFN-l

[28, 29].

Under normal physiological circumstance, after the epithelium is damaged, it needs to be repaired. However, as indicated above, there is a defect in the ability of the airway to repair. In this situation, the damaged airway stimulates the release of a number of cytokines and growth factors which attempt to repair this wound. One of these cytokines, epidermal growth factor (EGF) interacts with its specific receptor to activate

the epithelium and finally provide a mucus-secretory phenotype as well as alter the inflammatory response. All these features can be found in both chronic and severe forms of asthma [30]. Following the damage of the epithelium, many factors also induce the growth of fibroblasts and smooth muscle in the inflamed airway. For example,

TGF-b derived from the damaged epithelium not only can impair epithelial repair responses but also can promote the differentiation of fibroblasts into myofibroblasts

[31]. These changes are associated with the airway remodeling.

Airway remodeling

Airway remodeling in asthma is a complex multicellular process. The airway wall, vasculature, and neural networks are all involved in this process.

One of most obvious changes in the airway wall in asthma is airway smooth muscle hypertrophy and hyperplasia. This change in airway smooth muscle plays a major role in airway hyperresponsiveness. In chronic asthma, thickened airways are thought to be due to hypertrophy and hyperplasia of smooth muscle. However there is a contribution of fluid which is located between the smooth muscle cells. This is due to increased proliferation of microvessels and deposition of water-sequestrated proteoglycans. All together, this leads to increase in airway wall thickness. However, it is thought the physiological function of these components is to dampen the smooth muscle contraction in order to counteract the airway hyperactivity of smooth muscle seen in asthma [32].

Although how the abnormalities in the airway smooth muscle regulates asthma is not clear [33], there is strong experimental evidence for the important role of mast cells in the process of airway smooth muscle hypertrophy and its role in asthma pathogenesis.

The function of mast cells in airway remodeling will be discussed below.

Airway mesenchymal cells also promote the airway remodeling of asthma. One molecule of prime importance in this process ADAM33 is expressed by mesenchymal cells. It has been implicated in many studies examining the pathogenesis of airway hyperresponsiveness [34, 35]. Markers of airway wall remodeling such as increase in tenascin, procollagens 1 and 3, HSP-47 and a-smooth muscle actin can be detected before any obvious airway inflammation [36].

Vascular remodeling is one component contributing to the airway remodeling in patients with chronic asthma. As mentioned above, microvessels in the airway may function by downregulation the smooth muscle contraction in order to decrease the airway smooth muscle hyperactivity. A fundamental aspect of the pathogenesis of asthma is microvascular leakage. A wide range of different mediators have been implicated in the microvascular proliferation and reconfiguration of the airways. Major mediators in this process are vascular endothelial growth factor (VEGF) [37-39] and calcitonin gene-related peptide which is a potent vasodilator [40].

Another key to the pathogenesis of the asthmatic airway is the role of the neural

network. Varying factors derived from neural networks promote the asthmatic airway inflammation. Neurotrophins, for example, normally induce neural growth and maturation. On the other hand, they also contribute to the growth of mast cells and upregulate airway smooth muscle responsiveness. However, the exact mechanism of how the neural network induces an asthmatic airway is still poorly understood and requires further evaluation.

1.1.3 Mast cell biology

Mast cells play a pivotal role in immediate hypersensitivity and chronic allergic reactions and contribute not only to asthma but also other allergic diseases. Before I embark on the role of mast cells in asthma, it is important to examine the biology of mast cells, including development, migration and survival.

1.1.3.1 Features of mast cells

Human mast cells vary in shape, appearing in tissues as round, oval, or elongated spindle-shaped cells. The membrane has multiple folds and projections. The cytoplasm contains numerous cytoplasmic granules, mitochondria and rough endoplasmic reticulum. The nucleus is generally round, and the nuclear chromatin is often aggregated beneath the nuclear membrane but rarely appears as densely clumped as in basophils and other granulocytes.

Mast cells contain many granules containing substantial amounts of histamine, heparin, and the neutral proteases tryptase and chymase. Through the high-affinity IgE receptor

(FcRI)-mediated responses, mast cells activate multiple signaling pathways that lead to degranulation which can release their contents into the microenvironment and induce allergic reactions [41]. Based on the differences granule neutral protease composition, mast cells are generally divided into three subtypes in the human. The first two are designed MCT and MCTC. MCT which is the mucosal mast cell expresses tryptase and is T cell-dependent. MCTC which is the connective tissue mast cell expresses tryptase, chymase, cathepsin G, and CPA [42, 43] and is T cell-independent. The third type of mast cell which has recently been identified in-vivo, the MCC, lacks tryptase but expresses chymase [44]. At present, it is not clear what the relationship between these three mast-cell subtypes is. In addition, recent studies have attempted to answer the question of whether each subtype can reversibly alter its expression of neutral proteases.

However it has been demonstrated in-vivo in rodent systems that mast-cell subtypes can reversibly alter expression of their serine proteases [45, 46].

Mast cells express a wide range of surface antigens and receptors. As one of the most important receptors associated with mast cell development, mast cells express the high-affinity receptor IgE (FcRI) which can be activated by IgE specific antibodies and mediate the immediate hypersensitivity reactions that underlie allergic response. This reaction is considered as a major aspect of mast cell activation and the first step of a complex interaction of the allergic response [47]. FcRI activation induces multiple

signaling pathways which control diverse effector responses including allergic mediator release, relative gene transcription and secretion of a variety of molecules such IL-4,

IL-6, tumor necrosis factor (TNF-) and GM-CSF [48]. CD analyses can be used to identify different cell types in lymphocytes. Similar with basophils, mast cells express

CD43, CD55, CD59, CD63, CD40, histamine H2 and prostaglandin D2 receptors.

Furthermore, the c-kit receptor (CD117) is a critical receptor involved in mast cell growth and differentiation. Chemokine receptors which bind specific chemokines are also expressed on the surface of mast cells and play a crucial role in mast cell mediated immune and inflammatory reactions [49]. For example, mast cells express four chemokine receptors CXCR2, CCR3, CXCR4 and CCR5. In allergic disease, the transit of mast cells from the circulation to various sites within tissues is regulated by these receptors and their ligands.

1.1.3.2 Development of mast cells

Although mature mast cells are normally found in tissues, mast cells also exist as hematopoietic cells originating from CD34+ progenitors in peripheral blood and bone marrow. They normally do not mature before leaving the bone marrow. They circulate through the vascular system as immature progenitors and then complete their development peripherally within connective or mucosal tissues. Mast cell progenitors may either differentiate into mature mast cells or remain as progenitors depending on the microenvironmental milieu of cytokines and growth factors [50].

The development of mast cells has been an area of intense investigation. Although a number of investigator have used murine models of mast cell differentiation, it is clear that there are distinct differences between rodents and humans. In murine species, mast cells undoubtedly originate from hematopoietic stem cells. Recently, Chen et al. has challenged the current hypothesis paradigms that mast cells arise from a common myeloid progenitors (CMPs) and suggests that they are derived from multipotential progenitors (MPPs) [51] (Figue 1. A). In addition, these investigators postulate that a cell population identified as Lin− Kit+Sca-1−Ly6c−FcRI−CD27−!7+T1/ST2+ had been identified as mast cell progenitor (MCPs) in adult mouse bone marrow because these cells gave rise to mast cells in culture. However, other models of mast cell differentiation in mice have been described. Arinobu et al. suggested that mast cells originated mainly from GMPs in the bone marrow and this was a distinguishing feature between basophils and mast cells [52] (Figure 1. B). Unfortunately, the situation in humans with respect to identifying the mast cell committed progenitor which arises in the marrow compartment from pluripotent hematopoietic progenitors is still not clearly identified [53] and significant more research is required in this area.

Pro-T T cell A CLP NK cell

LT-HSC ST-HSC MPP Pro-B B cell

GMP Macrophage

CMP Granulocyte

MEP ? Eosinophil MK MCP Platelets

Mast cell

B Bone Marrow MEP Basophil BaP hi C/EBP>>> CMP

GMP C/EBP>>>

Mast cell MCP BMCP

C/EBP>>>

Intestine Spleen

Figure 1: Two models of mast cell-related hematopoiesis in mice

Stem cell factor (SCF, also known as Kit ligand) binds its receptor, Kit, and plays a

pivotal role in promoting the development of both murine and human mast cells [54-56].

SCF-mediated mast cell growth and differentiation also requires other growth factors for mast cell maturation and cytokines are involved. IL-3, which is a major mediator, which contributes to the differentiation of mast cells in mice but basophils in humans. In mice, mast cells can be developed at high efficiency by culturing mouse bone marrow cells in IL-3-containing medium. As a result, bone marrow-derived mast cells (BMMCs) are induced. Although BMMCs can be derived in vitro in relatively pure population, they are immature. But they have been widely used defining mouse mast cell biology.

IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) share the common receptor !-chain (CD131). However, they have the opposite effect on mast cell development: promoting and inhibiting mast cell development in mice and humans respectively [57, 58]. IL-3 mediator reveals controversial effect in human mast cell. The effect of IL-3 on progenitor cells is dose dependent. IL-3 at 1 ng/ml can enhance the growth of mast cell colonies without inducing granulocyte or macrophage (GM) colonies. At concentrations above 5 ng/ml, however, IL-3 induces a substantial number of GM colonies [59]. The Th2 T cell-derived cytokine IL-4 possesses mast cell growth factor activity in mice, whereas, in humans, it has been reported that IL-4 with SCF may have unknown effects on various mast cell subtypes. This suggests that the function of

IL-4 can be modulated depending on the microenvironment or the subtype of mast cells.

Because the pathology of mast cells in allergy is mainly controlled by Th2 T cells and the Th2 cytokines, not only includes IL-4, but also IL-5 [60, 61], IL-9 [61, 62],and

IL-16 [63] which have been shown to stimulate SCF-mediated proliferation of mast

cells from human cord-blood, bone marrow, and peripheral blood cells. Th1 cytokines have been reported to have a more complex effect on mast cell differentiation. IFN-" inhibits SCF-mediated differentiation of mast cell progenitors from both murine and human sources [64, 65], It induces apoptosis in mouse mast cells in a Stat1-dependent manner [66]. TNF- and IL-6 although are not mast cell growth factors, they can induce mast cell development. Nerve growth factor (NGF) can also induce the differentiation and proliferation of mouse BMMC in the presence of IL-3 [67], but do not effect human mast cell growth [68, 69].

Signal transduction through SCF-Kit plays a central role in mast cell development.

There are several pathways including PI3K, phospholipase C, protein kinase C,

Ras-MAPK cascade and the Jak-Stat pathway which participate. Furthermore, a number of signaling molecules and transcription factors have been identified that are important for mast cell development. The zinc-finger transcription factor GATA-1 which is expressed in mast cells as well as erythroid cells, eosinophils, and megakaryocytes is considered to be essential in development in these cell types [70]. Mast cells and

BMMCs can express GATA-1 protein after exposure to IL-3 and SCF. Experiments reveal that low GATA- expression in mast cells influences mast cell development [71].

Microphthalmia transcription factor (MITF) which has been recognized as basic-helix-loop-helix leucine zipper transcription factor is a necessary molecule in the mast cell development signaling pathway. In mice, MITF regulates expression of Kit, granzyme B and several mast cell-specific proteases. RasGRP4, a mast cell restricted

protein, has recently been reported to be important in mast cell development. RasGRP4 functions downstream of c-Kit and upstream of Ras. It is a guanine nucleotide exchange factor for the Ras family with a diacylglycerol-binding C1 domain and Ca2+-binding EF hand motif. This novel signal conductive protein plays a pivotal role in the regulation of many mast cell genes’ transcription and more detail will be given in the following chapter.

1.1.3.3 Migration of mast cells

The homing and recruitment of mast cells to various tissues are regulated by a variety of factors, such as integrins, chemokines, chemokine receptors and antigens. Mast cell progenitors and mature tissue mast cells express many chemokine receptors which contribute to the homing of mast cell progenitors from the circulation into the tissues. In humans, mast cell progenitors derived in vitro from cord blood express several chemokine receptors, including CXCR2, CCR3, CXCR4, and CCR5, and respond to the corresponding ligands in vitro [61, 72]. For example, CXCR3 is a chemokine receptor expressed on human lung mast cells and in the airway smooth muscle in patients with asthma and can activate airway smooth muscle [73]. Although antigen is generally concerned as a stimulant for the release of allergic mediators from mast cells, another function of it is a chemotactic factor for mast cells. Downstream of FcRI-IgE signaling pathway, sphingosine 1-phosphate (S1P) activates its receptors S1P1 and S1P2, which are expressed on mast cells, and this process engages different functions in migration of

mast cells toward antigen and mediating mast cell degranulation by S1P1 and S1P2 respectively [74].

1.1.4 The role of mast cells in asthma

Many studies provide support that human mast cells contribute to the pathophysiology of asthma. Mast-cell dependent allergen provocation leads to the early asthmatic reaction. Mast cells localize within the bronchial smooth muscle in patients with asthma but not in normal subjects or those with eosinophilic bronchitis. Activated mast cells contribute to the pathogenesis of asthma through the synthesis and release of numerous proinflammatory and profibrogenic mediators and cytokines depending on the initiation stimulus. Mast cells can secrete autacoid mediators: histamine, prostaglandin (PG) D2, and leukotriene (LT) C4, which can induce many of the features of asthma such as bronchoconstriction, mucus secretion and mucosal edema. Mast cells also can synthesize and secrete a remarkable array of proinflammatory cytokines (including IL-4,

IL-5, and IL-13) and profibrogenic cytokines (including TGF-b and basic fibroblast growth factor FGF-2). Drugs such as Sodium Cromoglycate and Nedocromil Sodium are believed to mediate their effects by inhibiting mast cell mediator secretion [75].

Mast cells present different functions in different phases of the asthmatic response.

Inhalation challenge with specific antigen induces lung mast cell activation, causing rapid development of airflow obstruction due to bronchial smooth muscle constriction

and bronchial wall edema that is called the early phase asthmatic response [76]. In this process, many mediators such as histamine, cys-LTs, PGD2, and tryptase, which are produced by mast cells, are detected in bronchial lavage fluids from asthmatic patients.

Moreover, studies indicate that mast cell-derived mediators also contribute to the late phase response of the allergic pulmonary response which follows the early phase response and is characterized as a more sustained period of airflow obstruction, accompanied by inflammatory cellular infiltration of the bronchial mucosa, persistent swelling of the bronchial wall, and potentiation of nonspecific AHR. Mast cell activation and mediator release as a result induces substantial alterations in airway function, as well as asthmatic bronchial inflammation [77].

1.1.4.1 Mast cell related mediator release

Through the IgE and FceRI interaction, several signal pathways are activated, as a result, the transcription of many cytokines and chemokines are induced and secreted after translation. Mast cells provide granule-associated mediators such as histamine, tryptase and other proteases, heparin, pre-formed cytokines, and proteoglycans which are released through exocytosis as well as newly formed eicosanoids that include PGD2, thromboxane (TX) A2, the cysteinyl leukotreines (LTC4 and LTD4) [78] and the parent molecule of the cysteinyl leukotrienes (cys-LTs) [79]. These mediators are potent smooth muscle contractile agents and also increase microvascular permeability. Mast cell activation through the high affinity IgE receptor (FceRI) contributes to the release

of above mediators and the cytokines which are located within mast cell granules, including TNF-a, IL-4, IL13, and IL-5. TNF- is a proinflammatory cytokine strongly implicated in the pathogenesis of asthma [80]. A number of studies have shown that

TNF- expression is markedly increased in severe asthma. Furthermore, TNF- can also induce mast cell mediator release. The evidence supports mast cell–derived TNF-a playing a major role in asthma pathophysiology, especially in patients with the severe form of the disease. In addition, tryptase, a neutral protease, which is exclusively found in mast cells, has a preferential action on G protein-coupled protease-activated receptor type 2 (PAR2). The activation of the PAR2 receptor in patients with asthma is supposed to involve in mesenchymal cell proliferation and airway wall remodeling [81].

1.1.4.2 The role of mast cells in the asthmatic airway

Recent studies have indicated that mast cells present not only in the airway epithelium and submucosa but also in the deeper layers of airway wall. Mucosal-type mast cells that are tryptase positive and chymase negative have been found in the deeper airway wall and in the peripheral airways. These cells are under the control of mediators secreted by T lymphocytes specifically IL-3, IL-4 and IL-9. In this circumstance, mast cells are highly responsive to inhaled allergens and causing bronchoconstriction which is a major fundamental feature of chronic inflammatory responses in asthma [78]. Mast cell number are increased in airway smooth muscle and thus can interact directly with airway smooth muscle through the function of autacoid mediators (including

leukotriene (LT) D4, prostaglandin (PG) D2 and histamine) contributing to fibrogenesis and airway remodeling response [82, 83]. On the other hand, the connective tissue-type mast cells that are tryptase, chymase, and carboxypeptidase positive have also been found in the airway smooth muscle.

There is confusion between the clinical entities asthma and eosinophilic bronchitis (EB) because the immunopathology of asthma and EB is virtually identical [84, 85]. The clinical examinations including bronchoalveolar lavage, induced sputum, and airway biopsies reveal nearly the same results in the extent of T-cell’s and eosinophil’s infiltration and activation, mucosal mast cell numbers, IL-4 and IL-5 cytokine expression, epithelial integrity, subbasement membrane collagen deposition as well as mediator concentrations including histamine and PGD2 in both asthma and EB.

Fortunately, there are 2 key differences between the pathology of asthma and eosinophilic bronchitis. One is that the concentration of IL-13. Compared with EB⊛the number of IL-13+ cells increased in the airway mucosa in asthma. IL-13 is elevated in the induced sputum of subjects with asthma but not subjects with EB [86]. However, the essential difference between asthma and EB is the cellular component in ASM. In subjects with asthma, there are many mast cells between the ASM bundles, but none in the ASM from patients with EB or normal subjects [87]. As mentioned above, MCTC phenotypes (both tryptase and chymase positive) are the majority of mast cell phenotypes found in the ASM. They express IL-4 and IL-13, but not IL-5 [88]. Because of the lack of T cells or eosinophils in the ASM of asthmatic patients, it indicates that

mast cells infiltration of ASM is one of the critical determinants of the asthmatic phenotype.

As discussed above, mast cells in ASM in the asthmatic airway results in the development of ASM hypertrophy and hyperplasia, smooth muscle dysfunction which lead to BHR, and variable airflow obstruction. Mast cells localized in ASM can adhere to the ASM cells and this process is important for cell, cell interaction and retention of mast cells at this site [89]. Airway smooth muscle also secretes many chemokines and growth factors which have mast cell chemotactic activity, and include CCL11, CXCL8, and CXCL12. The interaction between CXCR3 which is expressed in human lung mast cells (HLMC) and its ligands CXCL9, CXCL10, and CXCL11 secreted by ASM is considered an important function in asthma because the chemotaxis of HLMC activated by these ligands is greatly enhanced in asthma compared with normal ASM [73]. SCF is critical in mast cell development and survival is produced by both ASM and mast cells.

In addition, after exposure to tryptase, ASM releases another mast cell chemoattractant

TGF-b. This mediator is involved with mast cell recruitment through an autocrine pathway [90].

The classic mast cell autacoid mediators: tryptase, histamine, PGD2, and LTC4 are all potent agonists for ASM contraction. In vitro and in vivo experiments indicate that tryptase can not only induce bronchoconstriction and BHR by itself, but also can enhance contractile responses induced by histamine [91, 92]. In humans, tryptase can

also potentiate stimuli for not only histamine but other cytokines released from mast cells. IL-4 and IL-13 are also important cytokines in the development of BHR [93]

Mast cells have been to also infiltrate into bronchial epithelium and submucosal glands in asthmatic airway. Mast cells are thus situated in the interface between the host and environment stimuli such as aeroallergens and are involved in the ongoing immunologic response. In addition, degranulation of mast cell has significant effects on epithelial function. Histopathological features such as mucus plugging, mast cell infiltration within airway submucosa glands are features of severe fatal as well as mild asthma. The remarkable increase in the number of mast cells within the mucosal gland stroma in nonfatal asthma and the increase in number of degranulated mast cells in both fatal and nonfatal asthma supports an important role for mast cells in the development of mucous gland hyperplasia and the mucus gland secretion characteristic of asthma.

1.1.5 RasGRP4: a novel mast cell-restricted gene

1.1.5.1 Pathways of mast cell signal transduction

Mast cell signaling pathways are considered following antigen induced aggregation of

IgE-bound high-affinity receptors for IgE (FcRIs) expressed at the mast-cell surface

[94]. Although FcRI is expressed on mast cells, other activation receptors for mast cells exist. KIT is of prime important in the regulation of mast cell signal transduction.

Both of these receptors initiate their signaling cascades by different mechanisms. The

final result for both these receptors is to stimulate the release of mast cell mediators, which presents as, ultimately, converging to the necessary downstream signaling events including degranulation, release of eicosanoids, and induction of expression of chemokines and cytokines. These receptors via their ligands can activate mast cells and contribute to the development, migration, survival and function of mast cells. For example, activation of T cell-dependent mucosal mast cell is mediated by IgE cross-linking the FcRI. It is the central mechanism of immediate allergic reaction such as allergen-induced bronchoconstriction [95]. SCF-dependent activation of KIT, which has been explained above, is crucial for the growth, differentiation, survival and homing of mast cell [96].

Figure 2: The Ras Pathway

A significant number of studies have implicated the Ras/Erk pathway as very important in cells division and survival [97]. Ras is a small G-protein. This group of GTP-binding 35

protein is composed of Ras (K-RasA, K-RasB, N-Ras and K-Ras), R-Ras (R-Ras,

TC21/R-Ras2, and M-Ras/R-Ras3), Rap (Rap1A, Rap1B, Rap2A and Rap2B) and Ra1

(Ra1A, Ra1B) proteins [98] (Figure 2). Ras proteins can oscillate between an activation state when bound to guanine nucleotide triphosphate (GTP), and an inactivation state when bound to guanine nucleotide drphosphate (GDP). Ras proteins are normally anchored to the cytoplasmic side of the plasma membrane. The existence of guanine nucleotide exchange factors (GEFs), which can replace GDP with GTP, Ras proteins regulate the inactivation state to activation state and in turn activate several signaling pathway, the end function of which is normally the regulation of genes expression. Sos is a common GEF associated with Ras activation. Sos exists in the cell in a preformed complex with adapter protein Grb2, which in turn associates via its SH2 domain to phosphorylated tyrosine residues within the consensus sequence p-YXN. The Grb2-Sos complex can act on Ras in the vicinity of the plasma membrane [99]. After activation,

GTP-bound Ras can bind downsream effectors.

The GTP-bound form of Ras binds to Raf, such as Raf-1, B-Raf and A-Raf [100], and transfers to the plasma membrane where its protein kinase activity is increased and the kinase cascade is activated [101-105]. Once activated, Raf phosphorylates and activates the dual specificity protein kinases MEK1 and MEK2. MEKs 1 and 2 are the activators of the MAP kinases ERK1 and ERK2. The mitogen activated protein (MAP) kinase cascade, leading to extracellular-regulated kinase (ERK) activation, is a key regulator of cell growth and proliferation. Recently discoveries imply that RasGRP proteins, which

are mediated by the PLC"-dependent generation of DAG binding with C1 domain, also act as guanine nucleotide exchange factors (GEFs) and regulate Ras/Erk pathway.

Phospholipase C (PLC) is also close related to Ras activation. PLC hydrolyses the phosphoinositide PIP2 to the second messengers DAG and inositol-1, 4, 5-trisphosphate

(IP3). DAG is an activator of the classical and novel forms of PKC with multiple downstream effects, including RasGRP activation.

1.1.5.2 RasGRP family

The Ras guanine nucleotide-releasing protein (RasGRP) family of signaling proteins is preferentially expressed in hematopoietic cells where these intracellular proteins play pivotal roles in the final stages of development of numerous immune cells. Four different members of the family have been identified in mice, rats, and humans:

RasGRP1 (known as CalDAG-GEF II, resides on human chromosome 15q15),

RasGRP2 (known as CalDAGGEF I, resides on human chromosome 11q13), RasGRP3

(known as KIAA0846 and CalDAGGEF III, resides on human chromosome 2p25.1) and RasGRP4 (resides on human chromosome 19q13.1). RasGRP1 is required for the final stages of T-cell development. It is abundantly expressed in T cells where it facilitates T-cell receptor- Ras signaling [106-108]. In addition, the defective expression of RasGRP1 gene and protein has been discovered in patients with systemic lupus erythematosus (SLE) [109]. The details of RasGRP1 in T cell regulating SLE will be

discussed in the following section. RasGRP2 [110-112] is expressed in megakaryocytes and platelets, and the disruption of its gene results in defective integrin-dependent signaling in these cells [113]. RasGRP3 [114, 115] is more abundant in B cells where it facilitates B-cell receptor-Ras signaling, and RasGRP3- null mice have low levels of

IgG2a [116, 117]. These homologous GEFs have additional Ca2+ and phorbol ester/diacylglycerol (DAG)-binding motifs in their C-terminal domains that implicate their involvement in multiple signaling pathways (Figure 3).

Figure 3: RasGPP protein structure: a schematic domain structure map

1.1.5.3 RasGRP4 gene and protein

In 2002, Yang and coworkers [118] discovered a new GEF member in mouse bone marrow-derived mast cells (mBMMCs). Culture of mouse bone marrow cells for 3 weeks in the presence of IL- 3 results in a population of immature cells [119]

(designated as mBMMCs) that can give rise to all known populations of tissue mast cells when adoptively transferred into MC-deficient WBB6F1-W/Wv mice [120-122].

Thousands of clones were arbitrarily selected from a BALB/c mBMMC cDNA library

in order to search the genes that contribute to final stage of mast cell development. As result, a novel and unique cDNA was identified, which was a new members of the

RasGRP family of GEFs, known as RasGRP4. Mouse RasGRP4 (mRasGRP4) consists of a 2.3-kb cDNA and 19-kb/18-exon gene. Applying homology-base screening approaches, rat and human RasGRP4 cDNAs and genes were isolated [118, 123].

RasGRP4 mRNA expression has been detected in some studies. All mouse, rat and human mast cells express RasGRP4 mRNA. In addition, mast cell-committed progenitors also express RasGRP4. However, even if macrophages and mast cells share a common hematopoietic progenitor, there is no evidence that macrophages express detectable amount of RasGRP4 [118, 124]. Not only macrophages but also other mature cells like lymphocytes, neutrophils, eosinophils, fibroblasts and epithelial cell lines have no appreciable amount of RasGRP4 protein. As a consequence, RasGRP4 is considered a mast cells restricted Ras guanine nucleotide-releasing protein [118].

The human RasGRP4 gene resids on chromosome 19q13.1 and > 15 kb in size consist of 18 exons. Immunohistochemical approaches showed that every tryptase+ mast cell in the interlobular connective tissue of human breast and the mucosa layer of human stomach contained hRasGRP4 protein and the similar results were obtained in the mouse. This indicates that RasGRP4 is selectively expressed in mature mast cells and their progenitors in humans and mice. The major RasGRP4 isoform expressed in normal human and mouse mast cells is 75kDa and consists of 637 amino acids. Like other

cytosolic proteins, RasGRP4 protein lacks a hydrophobic signal peptide at its N terminus. Thus RasGRP4 is an intracellular protein [118].

All RasGRPs have a N terminal domain that functions as a GEF and a C terminal domain that functions as a DAG/phorbol ester receptor [118]. Each RasGRP mainly contains four domains which engage in multiple signaling pathways: A Ras exchange motif (REM); a CDC25-like catalytic domain; at least one calcium-binding EF-hand motif and a DAG/ phorbol ester-binding domain (C1 domain).

The Saccharomyces cerevisiae cell division cycle 25 (CDC25) protein is required for the function of adenylate cyclase in yeast, and CDC25 promotes the exchange of guanine nucleotides bound to Ras [125, 126]. The most conserved region within

RasGRP1 and RasGRP4 corresponds to the catalytic domain of CDC25.

DAG/ phorbol ester-binding domain and calcium-binding EF-hand are the regulatory domains in RasGRP4. Ca2+ binding with calcium-binding EF-hand I RasGRP4 can inhibit the ability of recombinant RasGRP4 to activate H-Ras even in the present of a

15-fold molar excess of Mg2+ [118]. Also the data indicates that RasGRP4 signaling events are reduced when Ca2+ is increased during FcRI-mediated degranulation responses.

C1 domain serves as a membrane recruitment module and binds to DAG, which is the

hydrolysate of PIP2, with nanomolar affinity. Furthermore, the C1 domain plays a critical role in initiation of RasGRP4 signaling pathway. Kit ligand stimulation of mast cell via c-kit induces Phospholipase C enzymes to hydrolyse phosphatidylylinositol-4,

5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1, 4, 5-trisphosphate

(InsP3). The binding of DAG to the C1 domain in downstream signaling proteins enables the resulting hydrophobic complexes to bind to membranes. DAG has been linked to the pronounced morphological changes that occur in the cytokine-activated cells. RasGRP4 proteins provide C1 domain appears to have a similar structure as protein kinase C. The PMA-treated, RasGRP4-expressing fibroblasts lose their contact inhibition and increase their rate of proliferation. This suggests that C1 domain in its C terminus is used to translocate this signaling protein to undefined compartments inside mast cells where its targets small GTP-binding proteins reside. The characteristic that the DAG/ phorbol ester-binding C1 domain can receive the phorbol-12-myristate

13-acetatc (PMA) stimulation signal has been wildly engaged in in vitro RasGRP signal transduction experiments [118].

All previous studies indicate that RasGRP4 is the primary PMA/DAG-dependent signaling protein that regulates the development and morphological changes that occur when mast cells are activated through c-Kit pathway. Furthermore, RasGRP4 is of critical importance in mast cell development by regulating the transcription of many genes related to mast cell maturation.

1.2 The pathogenesis of systemic lupus erythematosus (SLE)

1.2.1 Overview of SLE

Systemic lupus erythematosus (SLE) is a complex, heterogeneous systemic autoimmune disease characterized clinically by protean manifestations, most commonly including arthralgia, arthritis, rash, alopecia, oral ulcers, serositis, leukopenia, central nervous system, and renal involvement [127]. The precise pathogenesis of SLE remains something of a mystery. Recently studies indicate that the etiology have been focused on a variety of abnormalities which cause disease susceptibility including abnormal genetic components and multiple putative genes, a number of environmental factors and immune dysfunction (hyperactivity of B cells, T cells and dysfunction of signaling transduction in both cell types).

The presence of antinuclear autoantibodies (ANA) is the immunological hallmark of

SLE. It is a result of immune dysfunction. B lymphocytes from SLE patients display a lack of self-tolerance, and an inappropriate overproduction of ANA. Nearly 98% of SLE patients have ANA in their blood [128]. Nowadays, the precise roles of antibodies in

SLE patients, such as anti-dsDNA antibodies, have been intensively investigated by a number of investigators. Anti-dsDNA antibodies are more specific than ANA and are detected in fewer (60%) patients with SLE. Anti-dsDNA antibodies may reflect disease activity in some cases [129], some of these antibodies are thought to be directly pathogenic. A higher percentage of SLE patients will have antibodies against

single-stranded DNA and histones, although the presence of these antibodies is not specific for SLE. In addition, a variety of other autoantibodies are often detected, for example antiphospholipid antibodies. The development of some autoantibodies (such as anti- ribonucleoprotein, Sm, and dsDNA) is temporally closely related to disease onset, so it may be contribute to the pathogenesis of the disease [130]. However, SLE still has lots of question marks around it, such as in anti-dsDNA antibodies, many patients can be found with high anti-dsDNA antibody levels without overt disease or with severe disease.

As mentioned above, there are genetic components related to disease susceptibility in

SLE. Using current technologies, many lupus susceptibility genes have been identified although there are contradiction findings in the literature. The genes encoding HLA antigens seem to be important for susceptibility. Interestingly, it seems different ethnicities present different HLA antigens. Moreover, chromosome 1q region also has been linked SLE susceptibility [131]. These targeted genes may have immune functions and may regulate and encode proteins, which are involved in antigen presentation, apoptosis, B and T cell function and also production of cytokines and complement, which induce abnormalities via varieties of mechanisms.

The combination of genetic susceptibility in SLE has been considered partly caused by environmental factors and viral infection[132]. Many viral pathogens are referred to associate with SLE pathogenesis, including Epstein-Barr virus (EBV), cytomegalovirus,

parvovirus B19 and the retroviruses. Nevertheless, there is no hard evidence to reveal one or any particular pathogenic virus inducing SLE. On the other hand, photosensitivity is a common presenting symptom of SLE. One hypothesis indicates that UV light exposure leads to the release of proinflammatory cytokines and increases the rate of keratinocyte apoptosis. As a result, the body is exposed to autoantigens including keratinocyte cytotoxicity [133].

The initial dysfunction of the immune response in SLE is B cell hyperactivity. An abundance of autoantibodies is generated by these B cells which are activated. A number of factors activate B cells, such as B-lymphocyte stimulator (BLyS), which are upregulated in SLE, further providing B-cell survival signal [134]. This may be related to the abnormal T cell signaling transduction that exists in patients with SLE. The high free intracellular calcium concentrations and the enhancement of tyrosine phosphorylated proteins in these cells may account in part for the contribution of these cells to SLE pathogenesis. Some studies support that genetic deficiency of complement has been found in SLE cases [135]. In addition, other modulators of the immune system, such as cytokines, also contribute to the autoimmune response seen in SLE. For IL-10

which is produced by TH cells may play a central role in the overproduction of autoantibodies via IL-10 induced B cell proliferation and autoantibody production.

Moreover, Tumour necrosis factor (TNF) also has been identified as a key factor in lupus nephritis. It seems that low concentration of TNF associates with an increase incidence of lupus nephritis, through the DR2 genotype [136]. Attentively,

DR3-positive patients have relatively high TNF production, and this may protect them from nephritis.

The overwhelming evidence is that female sex hormones are strongly associated with the development of autoimmune disease. SLE predominantly affects females ten times more than males. Interestingly, in mouse models, oestrogens and androgens have opposite effects on SLE.

1.2.2 The role of T cells in SLE

As stated above, SLE is associated with a dysfunction of the immune response. Many cells are involved in this process, including B cells, dendritic cells, nonlymphoid cells and T cells. B cells are important in SLE as they produce antibodies against nuclear and cell surface antigens. Antigen-presenting cells, including B cells and dendritic cells can present autoantigen to T cells. T cells participate in inflammation and tissue injury either directly or through recruitment of other cells. Studies, which have documented infiltration of affected organs from SLE patients, such as the salivary glands and kidneys with T cells, reveal that T cells appear to have a central role in the immune pathogenesis of SLE.

1.2.2.1 Evidence of involvement of T cells in SLE

There are many hypotheses on why there is immune hyperactivity in SLE. Exposure of 45

cryptic or neoepitopes, abnormal antigen processing, abnormal activation threshold for cell signaling, insufficient or inappropriate T regulatory cells are a few. Recently the concept of immunological self-tolerance is due to abnormal apoptosis.

Autoantigen-reactive cells are not presented uniquely in autoimmune diseases. On the contrary, this process occurs in the periphery of healthy individuals [137-139].

Normally, autoreactive cells are anergic or undergo activation- induced cell death upon stimulation and are thereby functionally eliminated. However, during apoptosis, if autoreactive cells are exposed to cryptic epitopes following abnormal antigen processing and with additional inappropriate T cell help and/or excess costimulation, these cells will contribute to overcoming immunological tolerance. Furthermore, abnormal T cell signaling and abnormalities of activation-induced cell death could also lead to the above.

Many nuclear autoantigens interactive with T cells have been found in the peripheral blood of SLE patients, such as DNA-histones, the small nuclear ribonucleoprotein antigenic proteins Sm-B, Sm-D, U1-70kD, U1-A, and heterogeneous ribonucleoprotein

(hnRNP). The first demonstration that SLE patients have anti-dsDNA inducing T cells was by Rajaogopalan et al. [140]. Moreover, CD4+ T cells can help B cells produce anti-dsDNA and antihistone antibodies. The reason why T cells can support anti-dsDNA/histone autoantibody producing B cells in lupus is now considered attributing to prolonged costimulation of T and B cells via CD40–CD40L interaction and signaling because of abnormal expression of CD40 ligand (CD40L, also known as

CD154) on lupus B and T cells [141, 142].

Abnormalities autoantigen processing and presentation in SLE patients may be also important in breaking T cell tolerance [143-145]. During apoptosis, some cryptic epitopes or neoepitopes are presented to the immune system by modifications of autoantigens induced by protease cleavage (caspases or granzyme B) or by oxidative damage. Because there is detection of apoptotic clearance in SLE cases, autoantigens including cryptic or neoepitopes have a prolonged exposure to the immune system. This could be an important factor which contributes to breaking of T cell tolerance in a genetically susceptible individual [146, 147]..

The dysfunction of signaling transduction in T cells is a significant factor in SLE pathogenesis. Several signaling pathways which have been implicated in this disragulation associate with abnormal expression of susceptible genes. Such as defects in C1b signaling identified by Yi et al. [142] and the mutant of type I protein kinase A regulatory subunit alpha reported by Kammer et al. are all considered to contribute to the pathogenesis of SLE. Another example is the decreased TCR/CD3-# expression in

SLE T cells. Simultaneously, FcR" chain upregulation has the function of replacing the

TCR/CD3-# chain and forms signaling aggregates on the cell surface [148]. These may come from the decrease of production of the # chain promoter binding form of the transcription factor Elf-1. Also, Transcription of the IL-2 gene is decreased because of the presence of transcriptional repressor cyclic adenine monophosphate response

element modulator (CREM) [149].

There are numerous studies that have focused on changes of T regulatory cells in SLE.

Even if it is not clear that the function of T regulatory cells are important in SLE pathogenesis, these cells (CD4+ CD25+ T cells) are decreased in peripheral blood of

SLE patients during active disease, when compared to control subjects and patients with inactive disease [150].

1.2.2.2 Dysfunction of T cell signaling transduction in SLE

Compared with healthy individuals, the balance of T cell function involving CD4 and

CD8 T cells is lost in SLE patients. The dysfunction is characterized by an exaggerated

CD4 and diminished CD8 T cell activities [151]. The mechanism causing T cell dysfunction has not been elucidated but is likely related to several defects of signal transduction. These defects can be roughly grouped into 3 regions: proximal, middle, and distal of the T cell signaling pathway.

The proximal pathway defect in SLE is decreased T cell receptor (TCR) #-chain expression. In contrast to normal and rheumatic disease control of T cells, # protein is deficient in the T cells in the majority of SLE patients [152, 153]. This deficiency is likely due to mutations and silent polymorphisms in the # protein coding region [154,

155]. In addition, there are also many alternatively spliced forms in one or more of the #

proteins 8 exons. These changes are considered to contribute to critical functional abnormalities of this protein. Nevertheless, why and how these changes occur is still needs to be elucidated. As a result, the two events, which are dependent on #-chain expression, activation-induced cell death (AICD) and NK cell activity, are decreased.

Although the decrease of the TCR #-chain has been reported in other diseases such as human immunodeficiency virus–infected and tumor-infiltrating T cells [156], the TCR # transcript is still present in these T cells. On the other hand, there is a significantly reduced amount of IFN " production, which is normally produced by T cells following

TCR-initiated signal transduction in SLE [157, 158]. The expression of another TCR chain, the $ chain which is similar to the # chain, but exon 8 has been replaced with exon 9 being truncated.

2+ Another proximal pathway defect in SLE is increased and prolonged Ca i. The regulation of extracellular and intracellular Ca2+ concentration is initiated by a signal from the TCR–CD3 complex. The second messenger Ca2+ is going to engage in functional activation of Ca2+-dependent enzymes such as calcineurin phosphatase and calcium/calmodulin kinases. For example, Ca2+ mediates calcineurin-catalyzed dephosphorylation of transcription factor, nuclear factor of activated T cells (NF-AT), resulting in its nuclear translocation. In SLE, T cells have an enhanced and prolonged

2+ rise in Ca i. The genes whose transcription is regulated by NF-AT, such as CD154, Fas ligand, and c-myc, are overexpressed in SLE T cells [141, 159-161]. However, although there are some possibilities that the heightened and prolonged Ca2+ is concerned as a

result of deficient PKA-catalyzed phosphorylation of PLC-"1 and inability to inactivate

PLC-"1 enzymatic activity, the precise mechanisms of aberrant Ca2+ response remains unclear [162].

One of the middle pathway defects is the defective cAMP dependent protein phosphorylation. It is the first reported abnormal signal transduction pathway in SLE T cells [163, 164]. This abnormity leads to a profound deficiency of cAMP-activatable

PKA activity in the cells as well as the first identification of a deficient protein kinase activity in SLE [165]. As a result, this results the disorders emerged that a deficiency of the PKA-I and PKA-II isozymes and followed by reduction of total PKA activity [165,

166]. PKA-I is composed of two holoenzymes: RI2C2 (also written as PKA-I) and

RI!2C2 (PKA-I!). (RI and RI! are the and ! isoforms of the type I regulatory [RI] subunit.) PKA-II also comprises two holoenzymes: RII2C2 (also written as PKA-II) and RII!2C2 (PKA-II!). In SLE patients, both PKA-I and PKA-II isozymes and their holoenzymes activities are dramatically changed compared with normal subjects

[166-168]. Investigations have reported that defects of both transcription and translation exist in SLE T cells. Especially at the translation level, T cells fail to express RI! protein. This leads to the decrease in cAMP-activatable PKA activity and IL-2 production which activate via the CD3 and CD28 cell surface receptors [169]. The mechanism that contributes to deficient RI transcript is the occurrence of transcript

(mRNA) mutations. In SLE T cells, heterogeneous transcript mutations, including deletions, transitions, and transversions have been found adjacent to GAGAG motifs

and CT repeats. These are considered as a Jhot spots” for transcript editing and/or molecular misreading. The mutant RI transcripts which are caused by these mutations may be pathophysiologically encode diverse aberrant RI proteins, and result in deficient PKA-I activity. This is the first transcript mutation identified in an autoimmune disease [170]. Because not only RI mRNA but also other genes in SLE undergo these kinds of mutation, such transcript editing are recognized as an important mechanism resulting in mutated proteins in SLE cells. The gene modification effects in

SLE disease will be mentioned in the following section.

Deficient Ras/mitogen-activated protein kinase (MAPK) activity is the one of distal pathway defect. There are 3 principal pathways in MAPK signaling: extracellular signal– regulated kinases (ERK-1/ERK-2), c-Jun N-terminal kinase (JNK), and p38

MAPK pathway. Deficient ERK pathway signaling is now considered to contribute to the immunopathogenesis of SLE. This has been discovered from studies on the regulation of DNA methylation in SLE T cells. The result of studies imply that the signaling defects in the proximal and middle pathways might be associated with abnormal signaling in the distal pathways [171-173]. In addition, T lymphocytes obtained from patients with active and inactive SLE, RA, and healthy controls were stimulated with phorbol myristate acetate/ionomycin (PMA/IO), and phosphorylation of

ERK-1/ERK-2 and JNK was analyzed. Compared with control and RA cells, MAPK kinase (MEK)–catalyzed ERK-1/ERK-2 phosphorylation in SLE peripheral blood mononuclear cells (PBMCs) and SLE CD4 T cells was significantly less, despite normal

levels of the ERK proteins. This decrease in ERK phosphorylation was directly proportional to disease activity [172]. The decrease of signaling through the ERK pathway contributes to the DNA hypomethylation which is characteristic of SLE T cells and possibly to the immunopathogenesis of SLE. Furthermore, this phenomenon has been explained by the studies from Yasuda and coworkers. The studies indicated that the defective expression of a protein, RasGRP1, which plays a crucial role in ERK signaling pathway, contributes to the low response of SLE T cells following stimulation by PMA [109].

Anergy to recall antigens by skin testing is a characteristic of SLE disease activity. The response to antigens or mitogens stimulation in vitro in SLE T cells is significantly less than T cells from healthy controls. In addition, studies have also implied that mitogen activated human SLE T cells also secrete significantly less IL-2 in vitro compared with healthy control cells [174-176]. It is likely that the low response to antigens skin testing is the result of the decrease of IL-2 production. The reason why IL-2 production is reduced most likely is attributed to 3 mechanisms: the absence of the p65/p50 NF-'B heterodimer, reduced AP-1 activity [177, 178] and inhibition of IL2 enhancer/promoter transcriptional activation. It has recently been reported that p-CREM binds to –180 region of the IL2 enhancer/promoter [179]. CREM is a transcriptional repressor that binds to cAMP response elements and downregulates the expression of genes having this binding site [180]. So probably p-CREM binding to this response element could impede IL-2 production by stimulated SLE T cells, contributing to T cell anergy.

1.2.2.3 Genetic factors in SLE

It is well known that the SLE predominantly affects women at a ratio of nine women per one man. It is also more prevalent among families of affected patients in a non-Mendelian inheritance pattern [181]. This phenomenon implies that ethnic and genetic heterogeneity contributes to the complexity in this disease. Eight chromosomal regions with significant linkage to SLE have been identified through genome scan studies. They are divided into specific groups which engage in different functions, such as autoimmunity genes, type I interferon (IFN) pathway, and so on.

Autoimmunity genes

Cytotoxic T-lymphocyte antigen 4 (CTLA-4 in Chromosome 2q33) encoded protein

CTLA-4 is a structural homologue of CD28. It downregulates T-cell function and prevents autoimmune diseases through its function that competes with the binding of

CD28 on antigen presenting cells (APCs), and transduces inhibitory signals by activation of serine/threonine phosphatases. Some SLE cases have increased levels of soluble CTLA-4, whereas other studies are contradiction. In addition, different mutations were found within the CTLA4 promoter (−1722T/C, −1661A/G, −319C/T) and exon 1 (+49G/A) in various ethnic groups [182].

Program cell death 1 (PDCD1 in Chromosome 2q37) encoded protein PDCD1 is an 53

immunoreceptor of the CD28/CTLA4/ICOS co-stimulatory receptor family that bears an inhibitory immunoreceptor tyrosine-based motif (ITIM). An intronic PDCD-1 SNP

(PD1.3A, the minor A allele of 7146 G/A) was found to be related with SLE susceptibility in Europeans and Mexicans [183]. This leads to a loss of the binding affinity to runt-related transcription factor 1 (RUNX1) an intron enhancer, which induces lymphocytic hyperactivity in SLE. The loss of a RUNX1 binding site may result in susceptibility to autoimmune disease.

Rotein tyrosine phosphatase non-receptor 22 (PTPN22 in Chromosome 1p13) encoded protein PTPN22 is a negative regulator for T-cell signal transduction in cellular immunity. PTPN22 gene encodes lymphoid tyrosine phosphatase (LYP) whereas a missense polymorphism of PTPN22 in the proximal protein-rich SH3-binding domain

(C1858T) has been found and results in limiting the function of LYP which can suppress T cell activation [184]. Interestingly, this is easily found in Caucasians rather than in East Asian populations.

Type I interferon (IFN) pathway

High levels of type I IFN (IFN-) and type II IFN (IFN-") are reported in patients with

SLE. So it seems play a significant role in the pathogenesis of SLE. Expression of genes which induce IFN- and IFN-" are also implicated in the pathogenesis of SLE [185]. In particular, in human lupus nephritis, pathogenesis-related genetic polymorphisms within

IFN-" receptors 1 and 2 (IFNGR1 and IFNGR2) have been identified.

Tyrosine kinase 2 (TYK2) in chromosome 19p13.2 displays a strong association with

SLE susceptibility and is linked to the formation of anti-dsDNA antibodies in

Scandinavian and Caucasian populations. Missense variants in TYK2 have been found in SLE patients. This may reduce the biological function of TYK2 and may lower the susceptibility to SLE [182].

Interferon regulatory factor 5 (IRF5 in Chromosome 7q32) encoded protein IRF5 is an innate immune response transcription factor during viral infections [186]. Studies demonstrate that polymorphism of IRF5 interferes with IFN- signaling, leading to changes in cellular functions related to the development of SLE, including apoptosis, cell signaling, and cell-cycle regulation [186]. In addition, it also leads to an increase expression of IL-10.

1.2.3 Defective Expression of RasGRP1 in T cells associated with SLE

Recently, Yasuda and coworkers reported that several defective isoforms of human

RasGRP1, which plays a crucial role in ERK signaling pathway, have been found in a cohort of SLE patients but not in other rheumatic disease patients and normals.

RasGRP1 is a member of the RasGRP family. As described above, the four RasGRP

family members have a similar domain structure and function. RasGRP1, which is predominately expressed in T cells, is a guanine nucleotide exchange factor [187].

RasGRP1 as well as SOS can activate Ras by exchanging Ras-GDP to Ras-GTP which binds to different effector molecules, such as phosphoinositide 3’ kinase (PI3 kinase),

Ral guanine nucleotide dissociation stimulator, Ras interference gene 1, mitogen-activated protein (MAP) kinase kinase kinase 1, and Raf, leading to activation of divergent signaling pathways [188, 189]. One of the Ras downstream signaling pathways, MAP kinases pathway, is activated by active-Ras in T cells, however, this pathway has reported to be blocked in SLE patients. The human RasGRP1 gene

Genebank number NC_000015.8 and GeneID 10125 is 76.7 kb in size, contains 17 exons and encodes a ~95 kDa protein of 797 amino acids. Like other RasGRP members,

RasGRP1 contains a DAG-binding domain, a pair of calcium-binding elongation factor

(EF) hands, a catalytic domain consisting of a Ras exchange motif and CDC25 box.

This is the reason why synthetic DAG analogues, like phorbol myristate acetate (PMA) can activate Ras in lymphocytes, without Grb2-SOS involvement [190, 191].

The activation of Ras seems to occur at several different sites in the endoplasmic reticulum and Golgi membranes, in addition to the plasma membrane [192]. Meanwhile, studies reveal that RasGRP1 is unique in its ability to localize to the Golgi apparatus in the existence of calcium signals [193, 194]. So the localization of Ras activation could be very important and can explain why different GEFs are involved in Ras activation. In animal models, RasGRP1 null mice have inhibition in thymocyte development as well

as low CD4+/CD8- and CD4-/CD8+ T cells. On the contrary, the overexpression of

RasGRP1 in mice leads to the increase of CD4+/CD8- T cells. Therefore, it is concluded that RasGRP1 plays a crucial role in T cell maturation [106].

In order to study SLE, an animal model was established. The lag mouse, which was engineered to be deficient in GDID4, a GDP-dissociation inhibitor of the RhoGTPase family of small G proteins, has reduced numbers of mature T cells. Old lag mice display high titre autoantibodies and develop SLE-like disorder. Non-functional RasGRP1 proteins have been identified in these mice. An Exon 4 deletion mutant of RasGRP1 has been identified. The present of these RasGRP1 mutants in mice with SLE promoted studies in humans with SLE to determine if RasGRP1 mutant could be detected

In order to study the different isoforms of RasGRP1 transcripts in SLE patients, Yasuda and coworkers chose twenty apparently healthy and 32 unrelated Japanese patients with

SLE. In addition, twelve patients with other autoimmune diseases rheumatoid arthritis

(n=5), polymyositis/ dermatomyositis (n= 2), systemic sclerosis (n=3), and Sjogren’s syndrome (n=2) were selected as autoimmune control patients. RT-PCR was performed to amplify RasGRP1 cDNA from each patient’s PBMCs, and primers were designed from the beginning to the end of the coding domain in the normal RasGRP1 transcript present in human T cells. As a result, the normal isoform of RasGRP1 which is 2.4-kb cDNA was found in all healthy individuals as well as in those patients with non-SLE autoimmune disorders. However, in addition to the normal isoform of RasGRP1 found

in the PBMCs of SLE patients, abnormal RasGRP1 transcripts were present in nearly all

SLE patients. Moreover, in some SLE patients there was no detectable normal

RasGRP1 transcript. A number of different sized PCR products were detected in this cohort of SLE patients. Based on the accumulated data, 13 new isoforms of human

RasGRP1 named splice variant A – M were identified due to alternate splicing of exons

5–17 in its precursor transcript (Figure 4). Nevertheless, splice variant A which is due to a exon 11 deletion of RasGRP1 transcripts was frequently observed. Furthermore, other splice variants induced deletion of exon 11. Splice variant A does not cause a frame-shift abnormality or a premature translation termination codon in the processed transcript. Nevertheless, the abnormal translated RasGRP1 protein loses the 35-mer sequence of PLTPSKPPVVVDWASGVSPKPDPKTISKHVQRMVD that links the functional GEF domain to the regulatory DAG/phorbol ester- and calcium-binding domains. It is considered that the loss of this 35-mer domain results in a defective signaling protein [109].

Figure 4: Novel defective RasGRP1 transcripts in the PBMCs of SLE patients [109]

2. Chapter 2. hRasGRP4 regulates the expression of

IL13R2 in a human mast cell line

2.1 Introduction

In previous RasGRP4 functional studies, RasGRP4+ HMC-1 cells contained > 100-fold more PGD2 synthase mRNA than did the starting population of HMC-1 cells that express nonfunctional forms of RasGRP4. Even if HMC-1 cells express the transcripts that encode the surface receptors for IL-4, IL-10, IL-13, and KL they also express three distinct receptors that recognize TNF- and its family members. None of these cytokines were able to induce PGD2 synthase expression in RasGRP4- HMC-1 cells.

This indicated that RasGRP4 is the dominant intracellular signaling protein that controls

PGD2 expression in mast cells downstream of CD117/c-kit [195]. So RasGRP4 was considered as a novel signaling transduction protein which may regulate expression of many downstream signaling proteins which involved in the development of mast cells.

Two kinds of transfected HMC-1 cell including Mock cells which are RasGRP4- vector-GFP only HMC-1 cells and C7 cells which are RasGRP4+-GFP HMC-1 cells have been previously established in order to investigate the function of RasGRP4 in human mast cells. Many studies have reported that RasGRP1, RasGRP2, and RasGRP3 are able to translocate from the cytosol to the plasma membrane in the presence of phorbol ester, but only RasGRP1 and RasGRP3 are able to eventually translocate to the

Golgi complex [193, 194, 196, 197]. Moreover, Caloca and coworkers [194] discovered that, different from RasGRP1 and RasGRP3, the eighth residue in RasGRP2 50-mer C1 domain is Ser rather than Tyr as found in RasGRP1 and RasGRP3, this may cause the failure of translocation to the Golgi. However, although the mechanism of RasGRPs translocation is still unclear at this moment, Ras family members which are considered as the target proteins of RasGRP4 localize in the plasma membrane. Therefore, translocation of RasGRP4 to the plasma membrane may associate with its ligands. So a

Ser548 mutated RasGRP4 -GFP was constructed in hRasGRP4-GFP in order to evaluate the role of this critical amino acid in the translocation of RasGRP4 in HMC-1 cells.

ERK1 and ERK2 are localized downstream of Ras. Because RasGRP4 has been considered participate in mast cell development by acting upstream of Ras, the experiments of ERKs phosphorylation were initially performed by my colaborator Dr.

Gregory P Katsoulotos. Mock-GFP and hRasGRP4-GFP-expressing HMC-1 cells therefore were exposed to PMA and phosphorylation of these two kinases was determined to evaluate the affect of hRasGRP4. The result indicated phosphorylated

ERK1/ERK2 detected in hRasGRP4-GFP-expressing HMC-1 cells was higher compared to the hRasGRP4-deficient HMC-1 cells. This indicates that the activation of

ERK1 and ERK2 in mast cells before and after PMA exposure is mediated in part by hRasGRP4. Accordingly, accumulated data suggest that hRasGRP4 could be a critical factor in mast cell development and may regulate the expression of many downstream signaling proteins.

An Affymetrix GeneChip approach was utilized to identify candidate transcripts regulated by RasGRP4 with or without PMA stimulation. One of the target proteins

GATA-1, which is important in lineage-specific development of hematopoietic cells, has been investigated by my colleagues. The level of GATA-1 mRNA appeared to be dependent on the level of hRasGRP4 in the HMC-1 cell transfectants.

Another Affymetrix microarray result indicated that the IL-13 receptor 2 (IL-13R2) which is important to IL-13 and its receptor in asthma and various pathologic states, compared with Non-PMA stimulated RasGRP4+ HMC-1 cells, is extremely increased in

PMA stimulated RasGRP4+ HMC-1 cells (2 units in Mock control cells vs. 166 and

2068 units in Low and high hRasGRP4-expression HMC-1 cells respectively) (Table 6).

IL-13R2 has been reported to be a decoy receptor for IL-13. Further studies have focused on the expression of IL-13Ra2 protein and the downregulation of IL-13 mediated intracellular signaling events by IL-13R2 in RasGRP4+ HMC-1 cells.

Aims:

1. Determine the translocation of RasGRP4 in HMC-1 cells after PMA

activation.

2. Determine mRNA and protein expression of IL-13 receptor 2 in both

RasGRP4- and RasGRP4+ HMC-1 cells with and without PMA stimulation.

3. To quantify the phosphorylation of STAT6, which is an IL-13 downstream

signaling protein.

2.2 Materials and methods

Equipment

Table 1: Equipment Mini centrifuge Bio-Rad Laboratories, California, USA.

Eppendorf research family® pipette Eppendorf research family, Hamburg,

Germany.

GeneAmp PCR System 2400 PerkinElmer, Massachusetts, USA.

Ultra Rocker Rocking platform Bio-Rad Laboratories

BECKMAN coulter™ Microfuge® 22R BECKMAN, California USA centrifuge

LABEC BATH water bath LABEC laboratory, NSW, Australia

Mylab™ Vortex SLFS-16 Mylab™ Seoulin Bioscience, Seoul,

Korea(South)

MT19 Auto vortex mixer Chiltern Scientific, Auckland, New

Zealand

Power Pac™ Hc Power Supply Bio-Rad Laboratories.

XCell SureLockTM Mini-Cell Invitrogen, Garlsbad, CA, USA labCHEM-pH Benchtop TPS Pty Ltd, QLD Australia pH-mV-Temperature Meter

Thermo forma -86? ULT freezer Thermo Fisher Scientific Inc.

Massachusetts, USA.

Smartspec™ 3000 Spectrophotometer Bio-Rad Laboratories

Ax200 electric balance SHIMADZU, Japan

Forma scientific water-jacketed incubator Thermo Fisher Scientific Inc.

Milli-Q® Ultrapure Water Purification Millipore, Massachusetts, USA

Systems

Imager Gel Doc 2000 System Bio-Rad Laboratories

Dell Dimension V433c computer Dell, Texas, USA

C24 incubator shaker New Brunswick Scientific, New Jersey,

USA

CG1-96 PCR CR Corbett life science, Sydney,

Australia

Rotor-Gene RG-3000 real-time system CR Corbett life science

Model 250/2.5 power supply Bio-Rad Laboratories

Microwave Panasonic, Tokyo, Japan

Incubator Memmert GmbH + Co. KG, Schwabach,

Germany

GT-10 centrifuge Spintron, VIC Australia

Nikon TMS Microscope Nikon, Tokyo Japan

PowerWave™ Microplate BioTek Instruments, Inc. Vermont USA

Spectrophotometer

Mercer Medical Micro series Mercer Medical, Christchurch New

Zealand

Kodak X-omat 1000 Kodak, Tokyo Japan

Leica laser scanning confocal microscope Leica Microsystems, Wetzlar Germany

2ml, 5ml, 10ml, 25ml, 50ml Sterile Greiner Bio-one, San Francisco CA USA pipette with tip

15ml, 50ml Sterile cell culture tube Greiner Bio-one, San Francisco CA USA

25cm2, 75cm2 Sterile cell culture flask Greiner Bio-one with cap

Menzel-Gläser® SuperFrost® slide Gerhard Menzel, Glasbearbeitungswerk

GmbH & Co. KG, Braunschweig

Germany

CellStar® microplate 96 well Greiner Bio-one

0.2mL UltraFlux® 8-Strip PCR Tubes & Scientific Specialties Incorporated (SSI),

8-Strip Caps CA USA

Reagents

Table 2: Reagents used in Chapter 2 REAGENT SOURCE

!-Mercaptoethanol 14.3M Sigma-Aldrich, St Louis, MO, USA

Dimethyl sulfoxide(DMSO), 99.9%A.C.S Sigma-Aldrich

Spectrophotometric Grade

Dulbecco’s Phosphate Buffered GIBCO, Grand Island, NY, USA

Saline(D-PBC) 1 X

ECL™Western Blotting Detection Amersham Biosciences,

Reagents Buckinghamshire, England

Geneticin®Liguid(G-418 Sulfate)50mg/ml GIBCO

Heat-inactivated fetal bovine serum(FBS) GIBCO

HRP-conjugated goat anti-rabbit antibody DakoCytomation, Glostrup, Denmark

HRP-conjugated rabbit anti-mouse DakoCytomation antibody

Hybond-ECL nitrocellulose membranes Amersham Biosciences,

Hyperfilm ECL chemiluminescence film Amersham Biosciences,

Iscove’s Modified Dulbecco’s GIBCO

Medium(IMDM) with

L-Glutamine+25mM HEPES

Methanol, Biotechnology Grade Sigma-Aldrich

Solvent,99.93%

Monoclonal anti-human !-actin clone Sigma-Aldrich

AC-15, mouse ascites fluid

NuPAGE®4-12% Bis-Tris Gel 1.0mm x Invitrogen, Garlsbad, CA, USA

12 well

NuPAGE®LDS Sample Buffer(4X) Invitrogen, Garlsbad, CA, USA

NuPAGE® MOPS SDS Running Invitrogen,

Buffer(20 X)

NuPAGE® Transfer Buffer(20 X) Invitrogen

PcDNA3.1/CT-GFP-TOPO® Vector Invitrogen

Penicillin-Streptomycin(PS)10,000units/ml GIBCO

Penicillin and 10,000μg/ml

Streptomycin(100 X)

Phorbol 12-Myristate 13-Acetate(PMA) Sigma-Aldrich

Sodium Chloride, minimum99.5% Sigma-Aldrich

Sodium Orthovanadate Sigma-Aldrich

Triton X-100 Sigma-Aldrich

Trizma Base, minimum 99% Sigma-Aldrich

Trypan Blue Stain 0.4% GIBCO

Tween®20 Sigma-Aldrich

Goat anti-human IL-13R2 antibody R&D Systems Inc. Minneapolis MN

USA

HRP-conjugated rabbit anti-goat antibody DakoCytomation

OPTIMEM medium GIBCO

Prolong ® Gold antifade reagent Invitrogen

Paraformaldehyde powder, 95% Sigma-Aldrich

Recombinant Human IL-13 R&D Systems Inc.

Sodium dodecyl sulfate (SDS), for Sigma-Aldrich molecular biology, 98.5% (GC)

Sodium Deoxycholate 97% (titration) Sigma-Aldrich

Proteinase inhibitor cocktail Sigma-Aldrich

BCA Protein Assay Reagent (Pierce) Thermo Scientific Inc.

Hydrochloric acid Molecular Biology Sigma-Aldrich

Grade, 36.5-38.0%

HomeBrand Skim milk powder Woolworths, NSW Australia

Glycerol for molecular biology, 99% Sigma-Aldrich

Human Phospho-STAT6 (Y641) Affinity R&D Systems Inc.

Purified PAb

ImmunO (immuno-fluore mounting MP Biomedicals, Aurora, Ohio medium)

2.2.1 Cell culture

The HMC-1 cell line was kindly provided by Professor R.L Stevens (Harvard Medical

School, Boston, MA, USA) had been originally established by Dr J.H. Butterfield

(Division of Allergic Disease, Mayo Clinic⊛Rochester, MN, USA). This cell line was grown in the medium IMEM containing L-Glutamine added 10% heated inactivated

FBS and 1% Penicillin-Streptomycin (PS). All the transfected cells which contain the pcDNA3.1/TOPO vector required the same culture conditions as the original HMC-1 cells except for the addition of 0.75mg /ml geneticin in order to enable selection of stable clones. Cells grew in sterile plastics flasks at 37? in a humidified atmosphere containing 5% CO2. The medium was changed every second day to ensure the cells were growing at a density of 5x105 cells/ml.

Cell viability and density were measured by haemocytometer. 10)l of medium from wells containing the cells were resuspended in 90)l solution containing 80)l PBS and

10)l trypan blue

For cryopreservation, cells were suspended in 90% FBS in the presence of 10% DMSO.

1.5 ml 5 x 106 cells/ml solution was aliquotted in to the cryopreservation ampoules.

Subsequently, the ampoules were placed in a Cryo 1? freezing container and the container was immediately stored at -80?. After 24 hours, ampoules were transferred to liquid nitrogen for long term storage.

Cryopreserved cells were thawed at 37? in a water bath. After 1 minute of thawing, the cells were resuspended in culture medium as described above and transferred to a 15 ml cell culture tube containing 10 ml of pre-warmed culture medium. The tube was centrifuged at 1000 rpm for 5 minutes in order to remove the DMSO. The cell pellet was resupended by pre-warmed culture medium and cultured in an appropriate size cell culture flask.

2.2.2 PMA stimulation of RasGRP4 transfected HMC-1 cells

Three transfected HMC-1 cell clones were utilized in these experiments, หY

RasGRP4-GFP high expressing HMC-1 cells are denoted as C7 (C7), ฬ Ser548

mutated RasGRP4-GFP expressing HMC-1 cells อ GFP expressing only HMC-1

cells denoted as Mock as the negative control. Experiments utilizing the HMC-1 cell

lines were performed in 96-well plates using OptiMEM medium. All the cell clones

were exposed to 11000 nM PMA in 0.0030.01% Mg2SO4 for 10, 30, and 60 min.

The treated cells were washed with ice-cold PBS, fixed in 4% paraformaldehyde for 20

min at room temperature, washed again with PBS and then cytocentrifuged onto glass

slides. Each slide was treated with ImmunO. Confocal microscopy was performed with

the Leica laser scanning confocal microscope DM IRE2 TCS SP2 AOBS using a

100ɹ /1.4-0.7 PLAPO oil immersion objective. The slides were detected using a 488

nm argon laser and photographs were analyzed by computer software. The pictures

were exported using Adobe Photoshop.

2.2.3 PMA and IL-13 stimulates the RasGRP4 expressing HMC-1 cells

The GFP-expressing HMC-1 cells (Mock) and the RasGPR4-GFP-expressing HMC-1 cells (C7) were used for these experiments. Cells were collected after culture and used at 1.2 x 106 cells per well in 1 ml serum-free OPTIMEM medium (The viability of the cells were > 95% as assessed by Trypan Blue). Following 2 hours of serum starvation, the cells were stimulated with and without PMA, and/or with and without IL-13 (Table

3):

Table 3: PMA and IL-13 stimulation of transfected HMC-1 cells

GFP-expressing only HMC-1 cells RasGPR4-GFP-expressing HMC-1 cells lanes 1 2 3 4 5 6 7 8 5 hours stimulation DMSO PMA DMSO PMA DMSO PMA DMSO PMA then 5 hours stimulation DMSO DMSO IL-13 IL-13 DMSO DMSO IL-13 IL-13

The concentration of PMA (1 mg powder, FW 616.8, 1621.2 DMOS to 1 nM/)l PMA) and IL-13 (5 )g powder, 200 )l PBS to 25 ng/)l IL-13) were 100 nM and 50 ng/ml respectively and 1 )l per well DMSO was added as negative control. Following 10

hours stimulation, the cells were washed with ice cold PBS. The cell lysates were then subjected to SDS-PAGE and immunoblot experiments using specific antibodies to determine expression of the target proteins.

Western blot

1.2 x 106 cell equivalents per sample were used in the western blot experiments. Cell pellets were lysed by 60 )l lysis buffer (50 mM Tris-HCl pH 8.0, 150 nM NaCl, 0.1%

SDS, 0.5% Sodium Deoxycholate, 1% Triton X 100 and 1 mM Sodium Orthovanadate) containing 10% proteinase inhibitor cocktail. Samples were centrifuged at 1,4000 rpm for 10 min at 4c. Each sample was stored at -80? or subjected to SDS-PAGE. Total protein was assessed by BCA assay.

BSA 80 )g/ml to 2.5 )g/ml was used to establish the standard curve. The assay was performed according to the manufacture instruction and performed in a 96 well microtitre plate. Each sample was assayed in triplicate. Plates were incubated at 37? for 1-3 hours. Absorbance was performed by a PowerWave™ Microplate

Spectrophotometer at 562nm. Sample concentration was derived by the software using the BSA standard curve.

All SDS-PAGE and immunoblot experiments were performed with the NuPAGE electrophoresis system (XCell SureLockTM Mini-Cell) and from Bio-Rad. Initially, 1 x

NuPAGE MOPS SDS Running Buffer was prepared by diluting NuPAGE MOPS SDS

Running Buffer (20X) with deionized water. In terms of protein sample preparation for

SDS-PAGE, all samples were diluted in NuPAGE LDS Sample Buffer (4x) to yield a final concentration of 1x LDS Sample Buffer. Samples were subjected to the reduced or non-reduced conditions. For reduced conditions, !-mercaptoethanol was added to LDS

Sample Buffer at a final concentration of 2.5% V/V, denatured by heating at 85? for 4 minutes then placed on ice prior to SDS-PAGE. The pre-made commercial SDS-PGAE gel from invitrogen (NuPAGE 4-12% Bis-Tris Gel 10 wells or 12 wells) was placed into electrophoresis tank and filled with 1 x NuPAGE MOPS SDS Running Buffer. After a brief centrifugation, protein samples were loaded into wells of SDS-PAGE gel and were run at 100V for approximately 1.5 hours.

Hybond-ECL nitrocellulose membranes were used for western blotting. 1x NuPAGE transfer Buffer containing 10% methanol was prepared by diluting 20x NuPAGE transfer Buffer with demonized water. Membranes had to be placed in 100% methanol for a few seconds and then washed by 1x NuPAGE transfer Buffer containing 10% methanol until they were used in order to increase the binding efficiency of the membranes. Following SDS-PAGE transfer of proteins to the nitrocellulose membranes were according to the manufacturer’s instructions (“General Information and Protocols for using the NuPAGE electrophoresis system”). Transfer was at 30 V for 2 hours using the XCell IITM Blot Module system.

After protein transfer, membranes were washed prior to the immunoblot procedure.

Washing buffer was 1x TBST (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4 wuth 0.1%

Tween 20). Stock solution was 10x TBS solution (500 mM Tris, 1.5 M NaCl) pH 7.4 prepared by dissolving 60.5 g of Trizma Base and 87.75 g Sodium Chloride. pH was adjusted to 7.4 using 10 M hydrochloric acid. The washing buffer was prepared by 200 ml 10x TBS with 2 ml Tween 20 diluting in 2 liter deionized water. All solutions were stored at 4?.

After washing, membranes were blocked in 20 ml of blocking buffer (1x TBST containing 5% skimmed milk) for 60 minutes at room temperature with gentle shaking in order to minimize non-specific binding of the antibodies. The blocking buffer containing the diluted primary antibody was incubated at room temperature for 60 minutes (or 4? overnight) with gentle shaking after the wells were blocked for 1 hour at room temperature. After washing 3 times for ten minutes each, the membranes were placed in 1x TBST containing diluted HRP conjugated secondary antibody for 1 hour at room temperature with gentle shaking. The incubation conditions were slightly varied depending on the specific antibody that was used for the immunoblot experiments. After three more 10 minute washes, membranes were placed in the ECL reagent for 1 minute and then exposed to Hyperfilm ECL chemiluminescence film. The film was then developed with a Kodak X-Omat 1000 Processor.

In same experiments, membranes were reprobed after application of a stripping procedure. Membranes were striped in a solution containing 100 mM

!-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl pH 6.7 for 30 minutes at 50? and washed twice with 1x TBST for 10 minutes prior to blocking.

The intensities of the immnoreactive bands detected with Hyperfilm ECL chemiluminescence method were analyzed by scanning with a Bio-Rad Gel Doc 2000.

Data was analyzed by GraphPad Prism 3.0 software.

For experiments examining IL-13 signal transduction, samples were evaluated by

SDS-PAGE and immunoblotting as described above. Rabbit anti human phospho-STAT6 antibody (50 )g powder, 45.5 )l PBS and 10% Glycerol to 1.1 mg/ml,

1:2000, 0.5 )g/ml) as primary antibody was used to quantity the phosphorylation of

Signal transducer and activator of transcription 6 (STAT6), crucial IL-13 downstream signaling protein. After 4? overnight incubation with shaking, membranes were then exposed to 1:2000 dilution of HRP conjugated goat anti rabbit antibody. Results of the

ECL chemiluminescence experiments are shown in Figure 6. Membranes were stripped and reblotted with anti IL-13 R2 (n=4).

After stripping and blocking, membranes were exposed to a 1:2000 dilution (1 )g/ml) of goat anti-IL-13 R2 (100 )g powder, 0.5 ml PBS to 0.2 mg/ml) for 1 hour incubation at room temperature and 1:2000 dilution of HRP-conjugated rabbit anti goat for 1 hour at room temperature. Results of are shown in Figure 6. To confirm that there was equal loading of sample membranes were stripped and reprobed with anti !-actin.

The anti !-actin antibody was used at 1:10000 dilution and incubated for 1 hour at room temperature. Bound antibody was detected with a HRP-conjugated rabbit anti mouse

IgG. Immunoreactive bands were detected with the ECL chemiluminescence system.

2.3 Results

ConFocal Microscopy analyses indicated that GFP labeled RasGRP4 resided primarily in the cytosol of the hRasGRP4-GFP-expressing clones. The subcellular distribution of

GFP proteins did not change after 30 min of exposure of these control cells to 100 nM

PMA even at 60 min with 1 )M PMA (data not shown). In contrast, PMA altered the distribution of wild-type hRasGRP4-GFP in HMC-1 cells in a dose- and time-dependent manner. When the transfectants that were expressing wild-type hRasGRP4-GFP were exposed to 100 or 1000 nM PMA 30 min (Figure 5), the signaling protein quickly translocated to the plasma membrane (white arrow). As for the hRasGRP4-GFP-expressing cells, the Ser548 mutant hRasGRP4-GFP resided primarily in the cell’s cytosol before exposure to PMA. However, the Ser548 mutant failed to translocate to any membrane compartment when this mutant expressing cells encountered PMA.

Figure 5: PMA-mediated translocation of hRasGRP4-GFP in HMC-1 cells

Base on the microarray data obtained using RasGRP4+ and RasGRP4- HMC-1 cells, we analyzed GFP-expressing HMC-1 cells for IL13R1 mRNA before and after exposure to PMA. There were significant levels of IL13R1 mRNA and protein in contrast to the absence of IL13R2 mRNA and protein. The significant of the increase of phosphs-STAT6 levels in GFP-expressing HMC-1 cells when the cells encountered

IL-13 cytokine (Figure 6, A&C lane 3&4) suggested that the IL13R1 transcript is translated and the expressed receptor is functional in these cells. In addition, the levels of pSTAT6 were unchanged when these cells encountered PMA (Figure 6, A&C lane 4).

Thus, PMA does not adversely influence IL-13/IL13R1-dependent signaling in hRasGRP4-defective HMC-1 cells.

Figure 6: Evaluation of IL13R2 protein levels in HMC-1 cells, which differ in their hRasGRP4 expression before and after exposure of the cells to PMA, and evaluation of IL-13-dependent phosphorylation of STAT6 in these cells. GFP-expressing HMC-1 cells (lanes

1-4) and hRasGRP4-GFP-expressing HMC-1 cells (lanes 5-8) were untreated (lanes 1 and 5), or were exposed to PMA (lanes 2 and 6), IL-13

(lanes 3 and 7), or PMA followed by IL-13 (lanes 4 and 8). The obtained raw SDS-PAGE immunoblot data, the !-actin corrected IL13R2 data, and the !-actin corrected pSTAT6 data are shown in A, B, and C, respectively.

Consistent with an activating role for IL13R1 in mast cells and other cell types, in hRasGRP4-GFP-expressing HMC-1 cells, the levels of the IL13R1 transcript modestly increased, and the levels of pSTAT6 increased when these cells encountered IL-13 77

(Figure 6, A&C lane 7). Differently, the levels of the IL13R2 transcript markedly increased in the hRasGRP4-GFP-expressing transfectants especially after these cells encountered PMA (Data not shown). The protein of IL13R2, as well, can be detected in hRasGRP4-GFP-expressing transfectants and increased after PMA stimulation

(Figure 6, B lane 6&8). Because of the present of decoy receptor IL13R2, hRasGRP4-GFP-expressing HMC-1 cells significantly decreased their ability to respond to IL-13 if these cells were exposed to PMA before the cytokine p0.025 (n=4)

(Figure 6, A&C lane 7&8), compared with on significantly different in the

GFP-expressing HMC-1 cells (Figure 6, A&C lane 3&4, p=0.245, n=4).

2.4 Discussion

Using an immunogold localization approach, mRasGRP4 was located in both cytosol and inner leaflet of plasma membranes [124]. However, human RasGRP4 was detected and was mainly remained located in the cytosol rather than in the inner leaflet. As described before, RasGRP family proteins including RasGRP1, RasGRP2, and

RasGRP3 are able to translocate from the cytosol to the plasma membrane in phorbol ester-treated cells, because mouse, rat and human RasGRP4 all contain DAG/phrobol ester-binding domains at their C-terminal. After PMA (phorbol 12-myristate 13-acetate, substrate of DAG/phrobol ester) stimulation, this PMA-dependent movement of

RasGRPs was supposed to associate with signaling transfer in their host cells. RasGRP4 is one of the members of the RasGRP family. So translocation experiment was

performed to investigate the function of RasGRP4 in mast cells. HMC-1 cells were used as a cell model for human mast cells have been shown to lack functional hRasGRP4.

Two different clones of HMC-1 transfectants including wild-type hRasGRP4-GFP-expressing HMC-1 cells, and mock-GFP negative control HMC-1 cells were established by Dr Gregory P Katsoulotos. Human RasGRP4 translocation following PMA exposure was time and concentration dependent. The mock-GFP negative control cell clones had no response to PMA under identical experimental conditions. In contrast, GFP-labeled RasGRP4 protein quickly transferred from cytosol to the inner leaflet of plasma membrane when transfectants encountered 100-1000 nM

PMA. RasGRP4 is one of the guanine nucleotide exchange factors (GEFs), which can replace GDP with GTP. Under the function of GEFs, Ras proteins change the model from inactivation to activation and in turn activate downstream signaling pathway.

Furthermore, Ras proteins are normally localized to the plasma membrane [198], so the plasma membrane translocation of RasGRP4 is likely to be relevant to Ras activation.

Several other GEFs for Ras are also recruited to the plasma membrane such as

RasGRF2, SOS and other RasGRPs. PGD2 synthase expression appears to be regulated by RasGRP4, RasGRP4 has also been implicated in mast cell development and thought to act downstream of CD117/c-kit and upstream of Ras.

In addition, RasGRP1 and RasGRP3 are also able to eventually translocate to the Golgi complex from the plasma membrane in varied cell transfectants via their

DAG/PMA-binding domains [193, 194, 196]. The mechanism is still unclear, but

Caloca and coworkers [194] indicate that RasGRP2 is unable to translocate to the Golgi because the eighth residue in its 50-mer C1 domain is Ser rather than Tyr as found in

RasGRP1 and RasGRP3. Translocation of RasGRP1 and RasGRP3 to the Golgi complex only occurred when the C1 domain was phosphorylated by tyrosine receptor kinases at the plasma membrane. So the eighth residue in the C1 domain is considered as a critical residue for translocation. Of the 50 residues in the C1 domains of hRasGRP1 and hRasGRP4, 16 are different. The eighth residue in the C1 domains of mouse, rat, and human RasGRP4, for instance, is Phe rather than Tyr or Ser. Ser548 mutant hRasGRP4-GFP-expressing HMC-1 cell line which converted Phe548 in hRasGRP4-GFP to Ser analogous to C1 domain of hRasGRP2 was established by Dr Qi to evaluate the ability of the mutated signaling protein to translocate to different compartments in PMA-treated HMC-1 cells. Surprisingly, Ser548 mutant could not even move to the plasma membrane when the transfectants encountered PMA. Though position 8 in the C1 domain is of critical importance in the ability of RasGRP1,

RasGRP3, and RasGRP4 to recognize DAG and phorbol esters [199] and translocate to different compartments, our results question how RasGRP2 can translocate to any membrane in response to phorbol esters if C1 domain cannot recognize DAG and phorbol esters. This may raise another possibility that other regions in RasGRP4 also contribute to its redistribution in activated mast cells.

IL-13 as known regulates many functions in both hematopoietic and non-hematopoietic cells. The clinical functions associated with the development of atopic disorders

including asthma has been proved by many studies [200-202]. In addition, IL-13 plays an important role in the development of asthma which is independent of IL-4 [203-205].

IL-13 is an important immunoregulatory Th2 cytocine and is secreted from several different cell types such as Th2 cells, macrophages and activated mast cells [206]. Many studies have provided evidence that IL-13 is a crucial mediator of allergic inflammation such as asthma and parasitic infection. Although it is common considered that IL-13 shares many functions with IL-4, it is actually more important than IL-4 in the development of BHR and mucus production.

Many studies reveal that IL-13 partly regulates the inflammatory response in human asthma, because IL-13 blockade does not completely abolish allergen-induced airway inflammation [203]. Nevertheless, IL-13 has ability of raising the Th2-mediated inflammatory response via the stimulation of gene expression of a variety of gene families, such as chemokines and metalloproteinases that facilitate the recruitment, homing, and activation of a number of inflammatory cells. These chemokines including monocyte chemoattractant protein 1(MCP-1), MCP-2, MCP-3, MCP-5, CCL11

(eotaxin), CCL24 (eotaxin-2), macrophage inhibitory protein 1a (MIP-1a), and thymus- and activation-regulated chemokine (TARC) regulate the chemotaxis of a variety of inflammatory cells as well as the recruitment of various inflammatory cells from the bloodstream into the airway spaces [207, 208]. In addition, IL-13 has been shown to induce ASM production of TARC, which is thought to facilitate Th2 cell recruitment into tissues [209]. All evidence suggests that the tissue inflammation observed in

asthma is likely controlled by the complex interplay between an elaborate network of

IL-13-driven chemokines.

Subepithelial fibrosis is another asthmatic airway feature mediated by IL-13. The experiments of animal models indicate that overexpression of IL-13 induces a dramatic fibrotic response in the asthmatic airway wall [205]. Even if the mechanisms of IL-13 induced tissue fibrosis is still unclear, several studies have focused on the contribution of IL-13 in the fibrotic response. First of all, IL-13 has been shown by several investigators to upregulate the synthesis of arginase I which can hydrolyze L-arginine to urea and L-ornithine. These two metabolites are necessary for the production of polyamines and prolines required for collagen synthesis by fibroblasts [210, 211].

Secondly, the profibrotic mediator transforming growth factor-b (TGF-b) seems to be mediated by IL-13 in lung fibrosis. The recruitment and activation of epithelial cells and monocytes/macrophages, which produce TGF-b, are considered to be induced by IL-13

[212, 213]. The last but not least, the proliferation of myofibroblasts is stimulated by

IL-13 through an STAT6-dependent process involving platelet-derived growth factor

AA [214]. These may also depend on the expression of IL-13 mediated chemokines.

IL-13 is also related with mucus hypersecretion which is a serious feature of asthma, particularly fatal episodes. Several studies suggested that IL-13 is probably the primary regulator of mucus hyperplasia in vivo. For instance, the IL-13 signaling pathway

(IL-4R and STAT6) have been shown to be essential for mucus cell changes [215,

216].

Another interestingly result reveals that IL-13 can induce AHR in the absence of inflammatory cells, because IL-13 blockade experiments suggest AHR can be inhibited without affecting inflammatory cell recruitment or IgE synthesis [215]. Several investigators discovered that IL-13 could potentially induce direct effects on ASM because the IL-13 receptor chains, including the IL-4Ra, IL-13Ra1, and IL-13Ra2, have been found rather than the common " chain in ASM. Nevertheless, other people provide different opinions that IL-13 overexpression in airway epithelium indicates that AHR may mediate by IL-13 via effects on resident airway cells. Although the mechanism of

IL-13 induced AHR still remains a matter of debate, the important functions of IL-13 in asthmatic airway are indisputable. In addition, the effects of IL-13 on smooth muscle function may also be a result of significant increases in the area of ASM in the airway wall. IL-13 likely mediates smooth muscle proliferation indirectly via stimulation of

Cys-LT [217] or induced ASM fibronectin expression [218] which promotes ASM mitogenesis in vitro [219].

IL-13 mediates its actions via binding its receptors, including IL-13R1 and IL-13R2

[220-222]. IL-13R1 by itself binds IL-13 with low affinity (2-10 nM) whereas has high affinity in the presence of the IL-4R chain. In contrast, IL-13R2 alone binds IL-13 with higher affinity (0.5-1.2 nM). The complex receptor system constituted with IL-4R and IL-13R1, which is expressed on both hematopoietic and nonhematopoietic cells

such as B cells, monocytes/macrophages, dendritic cells, eosinophils, basophiles, fibroblasts, endothelial cells, airway epithelial cells, and ASM cells, is common considered induced the activation of a variety of signal transduction pathways. The

STAT6 signaling pathway plays a pivotal role in the development of the allergic phenotype [216]. Activation of the IL-13/IL-4 receptor complex induces JAK1 and

Tyk-2 kinases phosphorylation and activation. As a result, the tyrosine residues are phosphorylated on the IL-4Ra and IL-13Ra1 chains. Subsequently, a number of signaling pathways are activated including the phosphorylation of STAT6. The phosphorylated STAT6 is released and then dimerizes with other phosphorylated

STAT6 molecules and translocates to the nucleus. In this way, it can induce the expression of many IL-13 and/or IL-4 regulated genes which associate with asthma, because IL-13 and IL-4 share one common IL-4 receptor. The importance of IL-13 in asthma has been proved by animal experiments. For instance, several studies indicate that IL-4 is not essential for the development of the disease [223, 224] whereas the IL-4 receptor-signaling cascade (IL-4Ra and STAT6) is [215, 216]. This implies that another ligand signaling through the IL-4 receptor complex, IL-13 is responsible for mediating the effector arm of the immune response and contributes to the development of asthma.

Figure 7: IL-13 receptor systems and the regulation of IL-13 signaling pathway

IL-13R2, in contrast to IL-13R1, can bind IL-13 alone with high affinity (Kd=0.5-1.2 nM). However, IL-13R2 is currently not thought to contribute to IL-13 signaling because the cytoplasmic domain of IL-13Ra2 is short and devoid of an obvious signaling motif [225, 226]. Consequently, IL-13Ra2 is considered as a dominant negative inhibitor or a decoy receptor in IL-13 signaling events. There is some evidence to support this contention. First, in vitro, IL-13Ra2 has been shown to abrogate IL-13 signaling in several assessments of IL-13 activity, specifically, suppressed STAT6 activation [227, 228] (Figure 7). In addition, in vivo, animal experiments suggest that

IL-13Ra2 inhibits IL-13-mediated hepatic fibrosis in mice [229] as well as suppressed

IL-13-dependent Hodgkin7s lymphoma growth [230].

The exact mechanisms by which IL-13Ra2 is regulated in vivo are currently not well understood. Previous studies indicated that IL-13Ra2 expression is regulated by a variety of factors. For instance, IL-13 itself along with other Th2 cytokines (IL-4 and

IL-10) has been reported can mediate the increase of receptor chain expression during

Th2-mediated immune responses [229]. Furthermore, Th1 skewed immune responses and IL-12 can inhibit the expression of IL-13R2. In contrast, neither IL-13Ra1 nor

IL-4Ra levels appear to be regulated during Th2-mediated inflammatory responses

[231]. In vivo, not only Th2 cytokines but also the Th1 cytokine interferon gamma

(IFN-") up regulates the cell-surface expression of IL-13Ra2 and the inhibition of IL-13 signaling [227]. These results suggested that resident lung cells working through an

IL-4Ra complex were responsible for regulating IL-13Ra2 message expression. In human bronchial epithelial cells, consistently with above hypothesis, both IL-4 and

IL-13 upregulate IL-13Ra2 transcription through an STAT6-dependent process requiring protein synthesis [228]. All this evidence suggests that both Th1 and Th2 cytokines can suppresse the activity of IL-13 by upregulating the expression of

IL-13R2.

How dose IL-13R2 works as a decoy receptor also remains unclear. The most common consideration is the competitive binding between IL-13R1 and IL-13R2 because

IL-13Ra2 binds IL-13 with higher affinity than with the IL-4Ra/IL-13Ra1 complex. The soluble form of IL-13Ra2 also can bind to IL-13 and prevent the activation of the functional receptor complex. The IL-13Ra2 chain has been found in intracellular stores

in a number of cell types and is rapidly transferred to the cell surface by IFN-" activation [205]. On the contrary, although IL-4 and IL-13 induce the upregulation of

IL-13Ra2 message in epithelial cells, they may not induce IL-13Ra2 expression on the cell surface. In summary, IL-13Ra2 is an important regulator of IL-13-dependent processes and may suppress the IL-13-mediated asthmatic symptom.

West-Blot was used to measure the expression of IL13R2 in both hRasGRP4-GFP-expressing HMC-1 cells and Mock-GFP HMC-1 cells. Similar as the microarray and previous quantitative PCR results, the amount of IL13R2 was below detection in hRasGRP4 negative HMC-1 cells with or without exposure to PMA. In contrast, IL13R2 was detected in hRasGRP4-GFP-expressing HMC-1 cells and the level of this receptor increased after the cells encountered PMA for 6 hours. In contrast to the results obtained with the microarray experiments that reported high expression of

IL13R2 mRNA in the absence of PMA stimulation, the levels of IL13R2 protein did not increase high until they encountered PMA. The reason for this is not clear and remains to be determined, however, it is possible that a PMA/PKC-dependent pathway prevents the rapid catabolism of this receptor in mast cells. As the indicator of IL-13 function, the downstream action of activated IL-13, STAT-6 phosphorylation, was detected by an immunoblot approach. Because GFP-expressing HMC-1 cells increased their levels of pSTAT6 when they encountered IL-13 but had no detectable expression of IL13R2. PMA stimulation had no effect on the RasGRP4 negative cells. On the other hand, hRasGRP4-GFP/IL13R2-expressing HMC-1 cells significantly decreased

their ability to respond to IL-13 when exposed to PMA. This was due to the fact that

IL13R2 inhibited IL-13 function and functional as a decoy receptor. Nevertheless, the reason why hRasGRP4-GFP/IL13R2-expressing HMC-1 cells remained induced high pSTAT-6 expression when cells were stimulated with IL-13 without PMA was unclear.

One hypothesis was that the IL13R1 expression of hRasGRP4 positive cells was significantly greater than the amount of IL13R2 expressed before PMA stimulation.

Our data suggest that the mast cell specific RasGRP4 protein regulates the level of

IL-13R2 and controls IL-13/ IL-13R1-mediated intracellar signaling events in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate RasGRP4 could be used to modulate the IL-13-induced phosphorylation of STAT-6 that may be used as therapy in patients with asthma.

3. Chapter 3. hRasGRP1 mediated changes of the

expression of IL2R" in a human mast cell line

3.1 Intruduction

RasGRP1 is a guanine nucleotide exchange factor (GEF) that mediates Ras cycles between an inactive GDP-bound form and an active GTP-bound form. As a member of

RasGRP family, RasGRP1 protein contains a DAG-binding C1 domain, a Ras exchange motif domain, two EF-hands, and a guaninenucleotide exchange factor (GEF) domain.

However, different from RasGRP4, RasGRP1 is primary localized to T cells rather than mast cells and plays an important role in T cell receptor signaling and the final stages of

T cell maturation. Because studies indicate that CD4+CD8- and CD4-CD8+ T cell development is partially blocked at the double-positive stage in RasGRP1−/− mice [106].

By contrast, the overexpression of RasGRP1 in mice results in increased numbers of

CD4-CD8+ T cells [234].

Layer et al. [235] demonstrated that RasGRP1-null mice have high circulating levels of

IgG and IgM, which spontaneously developed a lymphoproliferative autoimmune syndrome exhibiting features of systemic lupus erythematosus. This indicates that no functional RasGRP1 protein is present in the lag mouse’s splenocytes due to defective processing of its precursor transcript. However, although numerous gene-linkage studies have been conducted in human SLE, and many candidate genes have been identified as

possible disease-susceptibility genes in these patients, abnormal isoforms of human

RasGRP1 have not been described until very recently. Yasuda and co-workers [109] analyzed the human RasGRP1 gene in SLE patients and identified 13 new splice variants of the human RasGRP1 gene.

Alternatively spliced mutants of RasGRP1 have been recently found in the peripheral blood white cells from a subset of patients with SLE implying a role in pathogenesis.

However, the mechanism of how RasGRP1 regulates signal transduction and protein expression still remains unclear. So a preliminary microarray screen was used to identify candidate transcripts regulated by hRasGRP1. For this we utilized the HMC-1 cell line which does not have RasGRP1 message. I used a transfection approach to express hRasGRP1 Full-length and a 11 isoform which had been identified in a subset of SLE pateints. Because species-dependent differences in this signaling protein could exist and better GeneChip have recently been developed, transcript expression in hRasGRP1-deficient (Mock) and Full-length (FL) and 11 hRasGRP1-expressing

HMC-1 cells were compared before and after they encountered PMA.

RasGRP1 is a signaling transduction protein and is a member of the RasGRP family of protein. It regulates the expression of many downstream signaling proteins. The results of microarray data demonstrated that the common IL-2R" chain is related by RasGRP1.

Interestingly, recent studies indicate that IL-21 which shares sequence homology with

IL-2 and contains the " chain common to IL-2 may affect the homeostasis of CD4+

CD25+ regulatory T cells (Tregs) [232]. Moreover, decreased number of Tregs in the peripheral blood of patients with SLE had recently been identified [233]. The changes of the common " chain expression may associate with the pathogenesis of SLE.

Aims:

1. To establish a RasGRP1 expressed HMC-1 cells including vector only

HMC-1 cells as negative control, Full-length RasGRP1 expressed HMC-1

cells and exon 11 deleted RasGRP1 expressed HMC-1 cells.

2. To character the signaling transduction pathways that are regulated by

RasGRP1, I analyzed 11 RasGRP1 HMC-1 cell clones.

3.2 Materials and Methods

Reagents

The reagents used for the work done in Chapter 3 unless already listed in Chapter 2

Table 4: Reagents used in Chapter 3 REAGENT SOURCE

1-kb Plus DNA Ladder 250ug(1ug/uL) Invitrogen, San Diego, CA

Ethidium bromide 0.625mg/ml solution Continental Lab-Products, USA

Nuclease-Free Water Promega, Wisconsin, USA

QIAEXB® Gel Extraction Kit(150) QIAGEN, Hilden, Germany

QIAamp® RNA Blood Mini Kit(50) QIAGEN

SuperScript™One-Step RT-PCR with Invitrogen

Platinum®Taq

Agar, technical grade Unipath Ltd, Hampshire, England

Bovine Serum Albumin Sigma-Aldrich, St Louis, MO, USA

Cell Line Nucleofector Kit T Amaxa GmbH, KÖln, Germany

Platinum QPCR SYBR Green Mix Invitrogen

QIAamp® RNA Blood Mini Kit(50) QIAGEN

S.O.C.Medium Invitrogen

SuperScript™C First-Strand Synthesis Invitrogen

System for RT-PCR

Tryptone Unipath Ltd

Yeast extract Unipath Ltd

Library Efficiency® DH5 Competent Invitrogen

Cells

Wizard[ Plus SV Minipreps DNA Promega Corporation

Purification Systems

FastDigest® Not I restriction enzyme Fermentas International Inc. Ontario

Canada

FastDigest® BamH I restriction enzyme Fermentas International Inc.

TOPO Cloning site Invitrogen

Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich

Bromophenol blue ACS reagent, Dye Sigma-Aldrich

content 95 %

DNA grade agarose Progen Pharmaceuticals Limited, QLD

Australia

Ethanol, absolute, 99.8% (GC) Sigma-Aldrich

Sodium acetate 99.0%, anhydrous Sigma-Aldrich

Anti-V5 Antibody Invitrogen

Liquid DAB+ substrate Chromogen Doko, CA,USA

System pCR®8/GW/TOPO® TA Cloning Kit Invitrogen

TOPO® cloning for E. coli expression Invitrogen

3.2.1 Transfection of wild-type and exon 11 deletion (11) isoform of hRasGRP1

in HMC-1 cells

Two plasmid DNA inserted with wild-type hRasGRP1 and exon 11 deletion isoform hRasGRP1 genes were kindly donated by Dr. Shinsuke Yasuda from Department of

Medicine II, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku,

Sapporo, Japan. The wild-type human RasGRP1 gene Genebank association number

NC_000015.8 and GeneID 10125 is 76.7 kb in size, contain 17 exons and encodes a ~95 kDa protein of 797 amino acids. The most prominent abnormal RasGRP1 isoform identified in SLE patients was exon 11 deletion splice variant [109]. If translated, the resulting abnormal RasGRP1 protein loses exon 11 coded 35-mer domain leads to a defective signaling protein. Furthermore, exon 11 deletions not only exist individually,

but also coexist with other defective isoforms. Consequently, both wild-type and 11 hRasGRP1 were used in these studies. Figure 8 indicates the primers of Exon 1-18

Sense (5’-CGCGGCCATGGGCACCCTG-3’) and Anti-sense

(5’-CTAAGAACAGTCACCCTGCTCCAT-3’). Consequentially, 2401 base pairs of wild-type hRasGRP1 DNA sequence and 2320 base pairs of 11 hRasGRP1 DNA sequence were amplified and inserted in to PcDNA3.1/V5-His-TOPO. The human mast cell line HMC-1 was used for transfection since it did not have detectable RasGRP1 and

RasGRP4.

Figure 8: RasGRP1 mRNA sequence Exon 1-18 & Exon 11 primers

1111111111 Colours:

ATCG: RasGRP1 whole gene primer 2401 base pairs (information from Japan) ATCG: RasGRP1 Exon 11 primer (using for wild-type 500 bp and 11 isoform 419 bp differentiation) AT CG and AT CG: different Exons ATCG: Exon 11 81base pairs

Transfection or gene transfer into mammalian cells can either be transient or stable. The difference is whether DNA integrates into chromosomes or not. Stable expression of

RasGRP1 and its 1 isoform was essential to my experiments since it would simplify the expression of adequate recombinant protein allowing functional analysis of expressed protein. Compared with chemical methods, viral methods, standard electroporation procedures and nucleofection, Nucleofection (amaxa GmbH, Germany) was chosen as it has lower cell mortality rates and higher transfection efficiencies. 95

Instead of uniform procedures for every cell line, NucleofectorTM Technology provides various programs for different cell lines even those are hard-to-transfect. An up-to-date database of the continuously extended range of Optimized Protocols can be found on the website at www.amaxa.com/celldatabase. The NucleofectorG technology allows easy and efficient gene transfer into a multitude of different cell types as well as for the delivery of various substrates such as plasmid DNA, RNA or siRNA. Under the required suitable program, the unique combination of electrical parameters and the cell-type-specific Nucleofector[ solutions guarantees the efficient delivery of substrates.

DNA is transported rapidly into the nucleus. In this way, over 50% transfection efficiencies can be obtained in most cell lines as well as in primary cells. Moreover, as transfected DNA is not dependent on cell division in order to enter the nucleus, the time until gene expression is significantly shortened relative to other transfection methods.

After transfection just 2-4 hours, the analysis can be performed if necessary. Meanwhile, expression of the transfected protein can be detected shortly after nucleofection. Such a simple method only needs less than one hour rather than almost one day preparation using other transfection methods and only two components are required, including the

Nucleofector® Device that delivers the specified electrical parameters and

Nucleofector® Kit that contain cell-specific and optimized Nucleofector® solutions

(source: http://www.amaxa.com/products/technology/).

Plasmid DNA was amplified using Library Efficiency® DH5™ Chemically

Competent Cells. The target DNA remains phenol, ethanol, protein, and detergents

free in order to obtain maximum transformation efficiency. Competent cells were thawed from storage on wet ice. Approximate 1-10 ng DNA was used in this reaction.

100 )l competent cells and target DNA in a chilled polypropylene tube were mixed, and cells incubated on ice for 30 minutes. After 45 seconds heat-shock, the cells were placed on ice for 2 minutes and 0.9 ml of room temperature S.O.C. Medium (provided in the

Library Efficiency® DH5™ Chemically Competent Cells Kit) was added into each tube followed by one hour shaking at 225 rpm at 37?. 100 - 200 )l of diluted reactions were spread on LB-Agar plates (Tryptone 5 g, yeast extract 2.5 g, NaCl 5 g and Agar

7.5 g dissolved in 500 ml ddH2O followed by 121? 20 min sterilization) with 100

)g/ml ampicillin. Plates were incubated overnight at 37°C. Ten colonies were randomly selected from each plate and cultured in 5 ml LB liquid medium. After 12 hours incubation, High-copy-number plasmids of the bacterial cultures were harvested by centrifugation for 5 minutes at 10,000 x g using a tabletop centrifuge and plasmid DNA was extracted using Wizard® Plus SV Minipreps DNA Purification System. Cells were resuspend in 250 )l Cell Resuspension Solution, followed by 250 )l Cell Lysis Solution,

10 )l Alkaline Protease Solution. The solution was neutralized and then centrifuged at

14000 rpm for 10 minutes at room temperature. The lysate was decanted into Spin

Columns provided with the Kit to bind the plasmid DNA. After 1 minute centrifugation, the column was washed with Wash Solution twice and the DNA was eluted using 100

)l Nuclease-Free Water. The concentration and purity of plasmid DNAs were assessed by SmartSpecTM 3000.

In order to confirm that the RasGRP1 wild-type and 11 isoform genes were correct, the plasmid DNA was double digested by Restriction Endonucleases NotI and BamHI.

According to the map of pcDNA3.1 vector, there are 5523 base pairs including an

Ampicillin resistance, Neomycin resistance genes, V5 epitope, His6 etc. The PCR product located between NotI and BamHI restriction sites in the TOPO® Cloning site

(between base pairs 953 and 954) (Figure 9). Two correct bands can be seen on electrophoresis gel after double digestion, including 2466 bp wild-type hRasGRP1,

2385 bp 11 hRasGRP1 and 5458b p vector only (Figure 10).

Figure 9: Map of pcDNA3.1/V5-His TOPO vector and the sequence of PCR product insertion point

Agarose gel electrophoresis:

A 1 x TAE electrophoresis buffer solution was prepared from a 50 x TAE stock solution.

1L of 50 x TAE stock solution consisted of 242 g Tris base, 57.1 ml glacial acetic acid and 100 ml 0.5M EDTA (pH=8) and the final volume adjusted to1 L with molecular biology grade H2O. 10 x DNA Loading buffer was composed of 20 mM EDTA, 50%

(v/v) Glycerol and 0.05% (v/v) bromophenol blue in molecular biology grade H2O and was stored at 4? 1 volume of 10 x DNA Loading buffer was diluted in 9 vol of sample before loading. 10 )l 1 kb plus DNA ladder from Invitrogen was diluted with 80

)l sterile Nuclear-free H2O and 10 )l 10 x DNA loading buffer. 10)l of this working solution contains 1 )g of DNA.

For agarose gel preparation, the Bio-Rad gel holder was used. A 1.2 % agarose solution was prepared by heating 1x TAE buffer 200 ml in a microwave for several seconds until

solution was completely dissolved. The solution was thoroughly mixed and 1 drop of ethidium bromide was added per 50 ml agarose solution. After cooling to 50 - 60?, the agarose solutioin was poured into the gel holder and allowed to cool and solidify. The tap and comb were removed and the holder containing the gel was placed in a Bio-Rad

Mini-Sub Cell GT electrophoresis Tank. The tank was then filled with 1 x TAE buffer to just cover the gel surface. The DNA ladder and each sample were loaded into individual wells of the gel. Electrophoresis was performed at 100 V for approximately

30 minutes until the tracking dye had migrated at least half way down the gel. Gels were place on a transilluminator with is shielded with U.V.-blocking plastic and photographed using a Bio-Rad Gel Doc 2000 instrument with Quantity One –Version

4.1.1 software.

Figure 10: Digestion of two RasGRP1 transfectants. Wild-type hRasGRP1 FL (lane 1 & 2) and 11 hRasGRP1 (lane 3 & 4) digested with Not I and BamH I (lane 2 & 4) non-digested (lane 1 & 3).

100

Transfection:

Before these experiments were performed, searching all information on websites of

Anaxa Company, there was no data available regarding the transfection conditions required for the HMC-1 cell line. A search of the amaxa website cell database

(www.amaxa.com/celldatabase), however, mentioned that amaxa Solution T and nucleofection program G-16 had been successfully applied in the transfection of the murine mastocytoma cell line P815 (ATCC). There were two other transfection programs T-20 and T27 for similar haematopoietic cell lines in the cell database. We used these in our preliminary transfection experiments. These transfection experiments had been successful when HMC-1 was transfected with hRasGRP4. Compared the three different transfection programs G-16, T-20 and T-27 using the pmax GFP positive control vector DNA, T-20 performed the best (35% transfection efficiency at 24 hours) and was used for all subsequent experiments.

Amplified plasmid DNAs: wild-type hRasGRP1 (Full-length, FL), 11 isoform hRasGRP1 (11) and vector only (Mock) were prepared before transfection. 2 )g plasmid DNA of each sample (100 )l) were required. Plasmid DNA underwent ethanol precipitation before program performed in order to reach the criterion of transfection.

A 2.5 volume of 100% ethanol was added to each DNA sample and the solution was mixed. A 0.1 volume of 3 M sodium acetate (pH=5) was then added and the solution was mixed. The sample was placed in -20? for more than 1 hour followed by 14000 rpm centrifugation for 30 minutes in 4?. The DNA pellet was resuspended and washed

101

using 70% ethanol. The sample was re-centrifuged at 14000 rpm for 30 minutes at 4?.

Finally, DNA was dissolved in an appropriate volume of Nuclease-Free H2O. Each

DNA concentration was 300 )g/ml.

RasGRP1 deficient HMC-1 cells were passaged every 2 days before nucleofection and cultured to logarithmic growth. Nucleofector Solution was prepared by adding 0.2 ml

Supplement to 0.9 ml Nucleofector Solution. For each transfection reaction, 1 x 106 to 5 x 106 RasGRP1 negative HMC-1 cells, 2 )g DNA (Mock-RasGRP1-,

Full-length-RasGRP1+ and 11-RasGRP1+) and 100 )l of Nucleofector Solution. The

Nucleofector Solution was pre-warmed to room temperature. An aliquot of culture medium containing serum and supplements was pre-warm at 37°C in a 50 ml tube.

12-well plates were used with 1 ml of culture medium containing serum and supplements per well and incubated plates at 37°C/5% CO2 prior to nucleofection. The cells were determined the required cell density for each reaction. Cells were centrifuged at 1000 rpm at room temperature for 10 minutes. The supernatant was completely removed so there was no residual medium in the cell pellet. The pellet was resuspended in 500 )l Nucleofector Solution at 1 x 106 cells 100)l.

5 )g target plasmid DNA mixed with 100 )l cells in Nucleofector Solution and then

transferred into an Amaxa certified cuvette. It is important in this protocol not to

incubate the cells longer than 15 minutes in the Nucleofector Solution, as this is may

reduce cell viability and gene transfer efficiency. The T-20 was selected for these

102

nucleofection experiments and the method was as outlined in the manufacture

instruction.

Limiting dilution:

Cells were cultured in antibiotic-free medium for 24-48 hours (depending on the cell density) after nucleofection. When cells started to grow (approximately after 48 hours),

Geneticin 1.25 mg/ml was added to the medium in order to select the transfected cell population. Cells were cultured at logarithmic growth phase and limiting dilution was performed to obtain single clones of hRasGRP1 transfected cells.

Medium containing Geneticin was removed by centrifugation at 1000 rpm for 5 minutes.

Cells were then washed with antibiotic-free medium and diluted to 5 cells per ml.

Limiting dilution was performed at a density of 1 cell/200 )l per well in 98 well cell culture plates. After 5 days, wells were examined by inverted light microscopy and those wells that contained a single clone of cells were identified. After 14 days culture, the cells in these wells were removed to 6 wells plates then to cell culture flasks until they were suitable to undergo clone characterization and used for further experiments.

Clone characterization by PCR, SDS-PAGE Immunoblot and

Immunohistochemistry

During the first limiting dilution, 6 single clones of FL-RasGRP1 transfected cells

103

(FL1-6) and 6 single clones of 11-RasGRP1 transfected cells (111-6) obtained. As there no GFP tag protein expressed with the RasGRP1 transfected cells, PCR,

SDS-PAGE and immunohistochemistry were performed to determine the efficiency of transfection.

At the gene level, RasGRP1 expression was detected by RT-PCR analysis. RNA was extracted from these clones with a QIAamp[ RNA Blood Mini Kit following the manufacturer’s instructions (QIAamp[ RNA Blood Mini Handbook September 2006,

QIAGEN). RNA was dissolved in RNase-free water and the concentration and quality of RNA were measured at 280 and 260 nm by SmartSpecTM 3000. The SuperScriptTM

First-Strand Synthesis System for RT-PCR was used to synthesize first-strand cDNA from total RNA. This reaction was catalyzed by SuperScriptTM II Reverse Transcriptase

(RT) which was already reduced the RNase H activity. Random hexamers, the most nonspecific of the primers, were selected as the first-strand cDNA synthesis reaction method. About 5 )g of total RNA was used.

Platinum® Taq DNA polymerase was then used according to the manufacturer’s instructions with 10 pmol/)l of each required primer per reaction to amplify target hRasGRP1 sequences. The primers that we used to confirm the sequences of FL and

11 isoform were hRasGRP1: Forward primer on exon 10

(5’-AAGGACCTCATCTCCCTGTA-3’) and Reverse primer on exon 14

(5’-AAGTAGGCTGTGATCTCATC-3’) (Figure 8). 2 )l template cDNA was amplified

104

in 50 )l including 5 )l 10 x PCR Buffer (Minus Mg), 1 )l 10 mM dNTP mixture, 2 )l

50 mM MgCl2, 0.2 )l Platinum® Taq DNA Polymerase and 5 )l (final concentration 1 pmol/)l) for each primer. PCR reactions for hRasGRP1 were carried out for 30 cycles, denaturation at 94? for 30 seconds, annealing at 58? for 30 seconds and 72? for 1 minute. Based on the results of RT-PCR, only 4 clones of each transfectant had the correct gene insertion: FL2, 3, 5, 6 and 111, 2, 4, 6.

At the protein level, SDS-PAGE western blot was performed to detect expression of hRasGRP1 protein. An Anti-V5 epitope antibody was used as the pcDNA3.1 vector had the V5 epitope sequence between 1020-1061 base pairs (map of pcDNA3.1 vector,

Figure 9). SDS-PAGE western blot analysis was used to detect the RasGRP1 tagged

with V5. Anti-V5 Antibody was 1:5,000. The 112 and 116 RasGRP1 clones had high expression of hRasGRP1 protein. However, only the FL5 RasGRP1 clone had detectable protein which was low. Consequently a second limiting dilution was performed for the

FL5 clone. The second FL5.1 and FL5.6 clones were obtained with moderate expression of hRasGRP1 portein. A single clone FL5.6.4 expressing high hRasGRP1 protein was obtained though further limiting dilution.

Immunohistochemistry was also used to detect the expression of hRasGRP1 protein in

HMC-1 cells. Mock, FL and 11 RasGRP1 clones were immunestained using an

Anti-V5 antibody. Each clone was transferred to a 15 ml tube and cells were centrifuged a 1000 rpm for 5 min. Cells were washed with ice cold PBS twice followed by 15 min

105

fixation using paraformaldehyde-PBS (PFA-PBS) at room temperature. The cells were then permeabilized with 0.2% triton X 100-PBS for 15 min at room temperature and blocked in 0.5%BSA-PBS under the conditions described above with 1:500 of the

Anti-V5 antibody in 0.5% BSA incubated for 1 hour at room temperature. After PBS washing, rabbit anti mouse antibody-HRP as the secondary antibody was incubated with the cells at 1:500 for 1 hour incubation at room temperature. Between each step, cells were washed with ice cold PBS and centrifuged to discard the supernatant.

Subsequently, the stained cells were fixed on slides for subsequent DAB immunohistochemical staining. Liquid DAB+ Substrate Chromogen System has high-sensitivity and is a suitable system for use in peroxidase-based immunohistochemical (IHC) and in situ hybridization (ISH) staining methods. Slides were incubated with the DAB solution (20 )l of the DAB Chromogen per ml of

Substrate Buffer) and incubated for 5–30 minutes. Slides were gently rinsed with distilled water and images were captured with normal light microscopy using a Lecia

Dc 200 camera.

At the gene level, hRasGRP1, 11 hRasGRP1 and pcDNA3.1 vector only expressing

HMC-1 cells were subjected to RT-PCR, using primers shown in Figure 11. 500 bp sequence was amplified from FL RasGRP1 expressing cells and 419 bp sequence from

11 RasGRP1 expressing cells. At the protein level, SDS-PAGE and coomassie staining had major bands migrating of 95 kDa for FL and 90 kDa for 11 hRasGRP1 protein (Figure 11). This indicated that both FL and 11 RasGRP1 cells had high

106

expression of hRasGRP1 protein. This was confirmed with western blotting using an anti-hRasGRP1 antibody (kindly provided by Dr. Shinsuke Yasuda) that demonstrated immunoreactive bonds at the corresponding molecular weights for FL and 11

RasGRP1 protein

107

Figure 11: Clone characterization by PCR, SDS-PAGE Immunoblot

Figure 12: Clone characterization by Immunohistochemistry

Figure 12 shows the results of immunohistochemistry using Anti-V5 staining three different clones of cells. Compared with the negative transfectant no brown DAB

108

staining, FL and 11 transfectants had over 90% of cells positive for DAB staining.

Both FL and 11 hRasGRP1 HMC-1 cells had high expression and thus were ideal for further experiment.

3.2.2 Affymetrix GeneChip® Exon Arrays on non-PMA and PMA stimulated

hRasGRP1 expressing HMC-1 cells

GeneChip® Human Exon 1.0 ST Array was used in this study. These arrays are a high resolution expression analysis, examining over 1 million exon clusters within the known and unknown transcribed regions of the entire genome. Classically 3’-based expression arrays, such as the Human Genome U133 Plus 2.0, were normally used for expression analysis. This approach has only analyzed a few hundred bases proximal to the 3’ end of each gene, and used expression at the 3’ end to approximate expression of the entire gene. These arrays are only compatible with the 3’ oligo (dT) - based priming and labeling assays and provides valuable insight into global gene expression. Nevertheless, classical 3’ expression microarrays do not discriminate between alternatively spliced transcripts that have identical 3’ ends. Transcripts lacking a 3’ exon because of alternative splicing, non-polyadenylation, genomic deletions or other non-canonical genomic events are not detected in 3’ based expression experiments. In contrast, The combination of Affymetrix’ Whole Transcript (WT) Assay and high-density arrays provide a more complete and more accurate picture of overall gene expression, enabling researchers to detect transcript isoforms they didn’t know existed, and to discriminate between transcripts that previously appeared to look the same. Especially Exon 1.0 ST 109

Arrays, with approximately four probes per exon and roughly 40 probes per gene, are the first experimental tools available to survey both gene expression and alternative splicing patterns on the whole-genome scale on a single array. They allow data for two complementary levels of expression analysis in a single experiment—“exon-level” and

“gene-level” analysis. Exon-level analysis of multiple probes per exon provides the highest resolution microarray analysis, with the ability to analyze alternative splicing and differential expression of each exon within a gene. Moreover, in gene-level expression analysis, multiple probes on different exons are summarized into a single expression level data point that represents all transcripts derived from each gene. On the

GeneChip® Human Exon 1.0 ST Array, 5,362,207 features are used to interrogate one million exon clusters (collections of overlapping exons) with over 1.4 million probe sets.

Probe sequences were selected using the high-quality human genome assembly (July

2003, hg16, build 34) and a variety of genome annotations including those inferred from human, mouse, and rat cDNAs (all information from affymetrix website www.affymetrix.com).

110

Figure 13: Schematic for representation of Whole Transcript Sense Target Labeling Assay for 1 )g of total RNA Source: Affymetrix Website www.affymetrix.com

111

1 )g of total RNA is required as starting material following the steps described in

Figure 13. Using a random priming strategy, in combination with in vitro transcription-based linear amplification and a novel end-point fragmentation and labeling assay scheme, the whole transcript (WT) assay provides a robust method for target labeling. All data that was obtained was analyzed using database analysis software Filemaker pro 9.

The Mock and FL and 11 cells were collected after general culture and were of 1.2 x

106 cells per well in 1 ml serum-free OPTIMEM medium (Viability using Trypan Blue was 99.4%, 97.2% and 98.3% for Mock, FL and 11 RasGRP1 respectively). After 10 hours starvation, the cells were treated with and without PMA (Viability was 95.9%,

96.3% and 97.5% for Mock, FL and 11 RasGRP1 respectively). The concentration of

PMA (1 mg powder, MW 616.8, 1621.2 DMSO to 1 nM/)l PMA) was 30 nM and 1 )l per well DMSO alone was added as a negative control. After 1 hour stimulation, the cells were washed by ice cold PBS. Total RNA was extracted as described above.

Samples were prepared according to the instruction of the Ramaciotti Microarray facility:

4 )g of total RNA resuspended in nuclease-free H2O.

RNA concentration was 500 ng/)l (8 )l of sample).

A photograph of the agarose gel photo (loading ~500 ng of RNA in each lane)

of all samples.

Samples clearly labelled using a maximum of 6 characters (Table 5).

112

Samples submitted in 1.5 ml microfuge tubes.

The concentration and quality of RNA were measured by SmartSpecTM 3000 before

microarray analysis. 4 )g of each sample was dissolved in nuclease-free H2O in a 1.5 ml microfuge tube as indicated below:

Table 5: RasGRP1 Microarray labeling

Ras1M: pcDNA3.1 vector only transfected Mock HMC-1 cells

Ras1MS: pcDNA3.1 vector only transfected Mock HMC-1 cells PMA stimulation

Ras1F: Full-length RasGRP1 expressing HMC-1 cells

Ras1FS: Full-length RasGRP1 expressing HMC-1 cells PMA stimulation

Ras1D: 11 RasGRP1 expressing HMC-1 cells

Ras1DS: 11 RasGRP1 expressing HMC-1 cells PMA stimulation

Microarray data analysis software was designed by Dr Gareth Denyer (University of

Sydney) using Filemaker version pro 9 which is a cross-platform relational database application from FileMaker Inc. All data was imported in to software and listed in

Figure 14. The data can be compared as any orders. Also, the detail of each gene or exon was easy to exhibit even when it was exported in PDF files. All genes that were regulated by RasGRP1 were searched using this software.

113

Figure 14: Demonstration of filemaker pro 9 designed analytical program

Before microarray analysis, all RNA samples were subjected to the company’s QC check (Figure 15). The bioanalyzer RIN for the sample ranged from 9.5 to 10 which is in the good quality range.

There are two ways of looking at the overall distribution of data on each of the arrays: boxplots, and density plots (essentially a histogram of expression levels) (Figure 15).

The distribution of raw intensities on each array appears very similar to one another, and the data looks to be of high quality. The boxes represent the 25th-75th range of data, the black horizontal line is the median, the dashed lines extend ± 2 standard deviations, and the thick vertical lines represent outliers (highly expressed probes). The density plots display the same data in a form of smoothed histogram. In the boxplot, samples 114

are ordered from left to right, as in Table 5.

Figure 15: RasGRP1 microarray quality control data.

115

3.2.3 Human RasGRP1 regulates expression of IL-2 Receptor Gamma

The full-length hRasGRP1 (FL) gene which is the normal isoform and the exon 11 deletion hRasGRP1 (11) gene which is present in a subset of SLE patients was investigated. The genes were placed in the pcDNA3.1 -TOPO expression vector, then transfected into the HMC-1 cell line which does not have detectable hRasGRP1.

Transfectants included (1) Mock (vector only), (2) FL, and (3) 11 cell clones. All cell clones grew in the same cell culture conditions as described above. The medium was changed every second day to ensure that the cells grew at a density of 5 x 105 cells/ml.

Quantitative Real-time PCR

QIAamp RNA Blood Mini kit from QIAGEN was used for RNA extraction from the three individual clones (Mock FL and 11) and then RNA was converted into cDNA using SuperScript First-Strand synthesis system for RT-PCR. Quantitative Real-time

PCR was performed using the Rotor-Gene 3000 amplification system. The sequences of

Primers were: IL2R" primers within Ex2 (sense 5’-TTCCTGACCACTATGCCCACT-3’ and anti sense 5’-GGCTCAGAGCTGCTGTTCCA-3’) and housekeeping gene GAPDH

(sense 5’-GAAGGTGAAGGTCGGAGT-3’ and anti sense

5’-CATGGGTGGAATCATATTGGAA-3’). The template cDNA 5 )l was amplified in

20 )l including 10 )l of SyBR Green SuperMix reagent and 0.5 )l (final concentration

0.25 pmol/)l) for each primer. PCR reactions for IL2R" were carried out for 40 cycles, 116

denaturation at 94? for 30s annealing at 62? for 30s and extension 72? for 15s. PCR reaction for the housekeeping gene GAPDH were carried out for 40 cycles, denaturation at 94? for 15s annealing at 60? for 30s and 72? for 20s. For analysis of Real-time

PCR results, the software provided with Rotor Gene 3000 (Rotor-Gene Real-Time analysis software 6.0) was used. Standard curves of IL2R" and GAPDH were generated by different dilutions of the respective plasmid. Normal PCR product of IL2R" and

GAPDH were transfected into pCR8/GW/TOPO vector and transformed to One Shot competent E.coli. After the plasmid was extracted from E.coli, serial dilutions were performed (2 to 2E-05 ng/)l). The final concentrations were 5E-01 to 5E-06.

RT- PCR was performed using GeneAmp PCR System 2400. Base on the results of

Real-time PCR, 3 different template cDNAs were loaded in same concentration for both

IL2R" and GAPDH and performed with the same ratio of reagents in 50 )l and the same

PCR reaction procedure but 30 cycles instead of 40. The PCR products were separated with an agarose gel.

3.3 Results:

The expression level of RasGRP1 had been assessed with the microarray data (Figure

16). The expression level of the three genotypes (green for Mock, red for 11, and blue for FL) are shown, for each of the exons (represented as grey blocks). The error bars represent the variability of expression in stimulated and unstimulated cell lines (in this

117

gene, there appears to be little variability in each genotype due to stimulation). In the first plot, the exons are drawn in genomic order, but are not drawn to scale; the wider exons were simply quantitated by more than one ProbeSet. The thin black line represents an intronic probe. In the second plot, the same expression data are plotted, and the exons are drawn to genomic scale. Since this gene is on the anti-sense strand, the exons are numbered 1-17, from right to left.

118

Figure 16: The evaluation of RasGRP1 expression in RasGRP1 microarray data.

Exon 11 is the one that is clearly not expressed in both the Mock (green) and 11 construct (red). The maximum expression level for this gene is ~12 log2 units, which indicates that the gene is highly expressed in the FL, and 11 constructs. The expression of most of the Mock construct exons is below ~4 log2 units, indicating that it is either expressed at close to background levels, or is not expressed at all. All evidence implies that the expression data from all 6 arrays is of high quality and the expression of

RasGRP1 in each of the three genotypes is correct. This also had been confirmed by

Filemaker software analysis (Figure 17). Exon 11 of RasGRP1 (probe set ID 3618751) had no detectable expression in the 11 cells.

119

Figure 17: Microarray data of expression of RasGRP1 in Full-length (F), 11 (D) and Mock (M) HMC-1 transfectants.

Data were analyzed comparing Mock & FL, 11 & FL before and after PMA stimulation. Absolute values of more than 50 for each gene and a 5 times or greater difference between two clones are considered significative. Some of the data that related to SLE and that were significant are listed in Figure 18.

120

Figure 18: RasGRP1 Microarray data analysis

One gene that was indentified, IL-2 receptor gamma (Figure 19), known as common gamma chain, had nearly 5 fold expression in Full-length RasGRP1 expressing cells than in both Mock and 11 cells. Moreover, at the exon level, some exons of IL2R" gene expressed in FL cells had approximately 20 fold higher expression than in Mock and 11 cells, including Exon 2 (probe set ID 4011872 and 4022873), Exon 5 (probe 121

set ID 4011864) and Exon 6 (probe set ID 4011862). However, there was no significant difference before and after PMA stimulation.

Figure 19: Microarray data of expression of IL2R" in Full-length (F), 11 (D) and Mock (M) HMC-1 transfectants.

There was a striking difference in the expression of IL-2 receptor gamma gene in the different clones (Figure 19). Mock and 11 clones had minimal expression when compared with the FL clone. This was confirmed by Quantitative Real-time PCR

(Figure 20.) and RT- PCR (Figure 21). IL2R" expressed transfectants significantly decreased in 11 RasGRP1than in wild-type RasGRP1 HMC-1 cells (* P<0.015 n=4).

In contrast, there was no difference in the expression level of IL2R" in 11 RasGRP1 and Mock HMC-1 cells (P>0.05. n=4)

122

Figure 20: IL2R" expression in FL, 11 RasGRP1 and mock HMC-1 transfectants measured by Quantitative Real-time PCR

Figure 21: IL2R" expression in FL, 11 RasGRP1 and mock HMC-1 transfectants measured by RT- PCR

The expression of IL-2R" in peripheral blood T cells from SLE patients and healthy controls that had FL and 11 RasGRP1 genes also analyzed by Quantitative Real-time

PCR. The SLE patients with the 11 RasGRP1 isoform had significantly reduced expression of IL-2R" compared to the SLE patients and normal controls with FL

RasGRP1 (Figure 22). Although further explanations of the graph were required from

Japan, apparently, lupus patients tend to have lower expression of IL-2R". This indicated the same tendency with our in vitro experiments. 123

Figure 22: The expression of IL-2R" in T cells of a subset of SLE patients and healthy individuals

3.4 Discussion

One splice variant of the human RasGRP1 gene (11, exon 11 deletion) was used since it is the most prominent abnormal RasGRP1 isoform identified in SLE patients [109]. It also coexists with other defective isoforms in a high percentage of SLE patients. The

HMC-1 cell line which was established from a patient with mast cell leukaemia lacks the expression of functional hRasGRP4 protein, and Figure 11 shows minimized detection of RasGRP1 using 40 cycles PCR in Mock cells. Compared with wild-type hRasGRP1 and 11 isoform, it is more than 100 fold less. RasGRP1 protein is not

124

detectable in HMC-1 Mock transfected cells. We also used the HMC-1 cell for analysis of RasGRP1 mediated function because RasGRP4 expressed HMC-1 cell line had been used, so we could compare RasGRP1 with RasGRP4 function. Furthermore, recent studies support that RasGRP1 also plays an important role in FcRI-mediated PI3K activation and mast cell function and regulates FcRI-mediated allergic responses [236].

GeneChip® Human Exon 1.0 ST Array was performed on these three HMC-1 cell lines.

This time we chose Exon level microarray because exon 11 was deleted in 11 isoform.

As shown on Figure 4, exon 11 is between CDC25BOX and EF hand domain. Although the exact function of exon 11 is still unclear, the exon level microarray may provide a much more detailed analysis of regulation of wild-type and abnormal RasGRP1 isoform.

The expression level of RasGRP1 at an exon specific level had been first examined in order to confirm that all clones had the correct transfectant. All exons of wild-type

RasGRP1 transfectant expressed at a high level. Mock transfectants, however, did not have detectable levels of any of the exon of RasGRP1. Exon 11 of the 11 RasGRP1 isoform transfectants (probe set ID 3618751) had no expression compared with other exons which were at the same level as the wild-type RasGRP1 transfectants’.

All data was analyzed by Filemaker pro 9. As shown in Figure 14, the expression level of any of the genes and their exons can be analyzed by this program. Genes and exons can be listed synchronously or grouped according to biological function. The data can also be analyzed in any manner. The IL-2 receptor " gene has important functions

125

relating to signaling transduction.

In humans, the functional IL-2 receptor consists of the subunit complexes of , !, and " chains. Molecular characterization of the IL-2 receptor commenced with gene cloning of the chain (IL-2R), which is not necessary for intracellular signal transduction mediated by IL-2 [237-239]. The second subunit of IL-2 receptor, ! chain (IL-2R!), was then characterized as belonging to the cytokine receptor superfamily and is essential for the intracellular signal transduction [240]. The " chain, one of three IL-2 receptors plays a pivotal role in the formation of the full-fledged IL-2 receptor. Together with the ! chain, " chain participates in increasing IL-2 binding affinity and intracellular signal transduction. Moreover, the cytokine receptors for at least IL-2, IL-4, IL-7, IL-9,

IL-15 and IL-21 utilize the same " chain (CD132) as an essential subunit, called the "c cytokine family. IL-4, IL-7, IL-9 and IL-21 bind a heterodimeric receptor comprised of

the "c and the specific receptor subunits, IL-4R, IL-7R (CD127), IL-9R and

IL-21R chain, respectively. IL-2 and IL-15 bind a heterotrimeric receptor composed of the specific IL-2R (CD25) or IL-15R chain, and the shared IL-2/15R! (CD122) and

"c chains [241].

The human IL-2R gene is organized into eight exons spanning more than 35 kb and localized on chromosome 10p l4–15 [242, 243]. The mature form of IL-2R consists of

251 amino acid residues. The cytoplasmic domain of the IL-2R, however, only contains 13 amino acid residues, which seems insufficient to harbor a signal transducing

126

capacity. The second subunit ! chain of IL-2 receptor (IL-2R!) was initially identified to be a 75 kDa cell surface glycoprotein, by affinity cross-linking experiments with radio-labeled IL-2 and subsequently by mAbs specific for the human IL-2R!. The human genomic IL-2R! gene is divided into 10 exons, spanning 24 kb on chromosome

22q11.2–12 [244, 245]. The mature form of IL-2R! consists of 525 amino acid residues.

Characterized as a member of the cytokine receptor superfamily, IL-2R! has the common features of two pairs of the conserved cysteine residues near the amino-terminal and a sequence of tryptophan—serine—x (unconserved amino acid)—tryptophan— serine (WSXWS, WS motif) in the extracellular domain [246].

The cytoplasmic domain of IL-2R!, consisting of 286 amino acid residues, contains unique regions such as the box 1, serine-rich, acidic, and proline-rich regions. The third subunit " chain of IL-2 receptor (IL-2R") was first detected by coimmunoprecipitation with IL-2R! [247, 248]. The amount of the 64-kDa molecule coprecipitated with

IL-2R! correlated well with the level of the intermediate-affinity IL-2 binding sites, suggesting the possibility that the 64-kDa molecule is IL-2R" [248]. The 64-kDa molecule was purified by coimmunoprecipitation with IL-2R!, and its amino-terminal amino acid residues were determined. Based on the amino acid sequence, the complete cDNA clone encoding the 64-kDa molecule was isolated and demonstrated to be the cognate IL-2R" chain [249]. The mature form of IL-2R" consists of 347 amino acid residues with sequences typical of the cytokine receptor superfamily such as IL-2R!.

The cytoplasmic domain of IL-2R", consisting of 86 amino acid residues, contains two subdomains of the Src homology region 2 (SH2). The full SH2 domain is known to

127

contribute to the downstream signaling through its interaction with phosphotyrosine residues of various signal transducing effector molecules, but the two SH2 subdomains detected in IL-2R" are thought to be insufficient for such action (Figure 23).

128

Figure 23: Protein structure of IL2 receptor systems and a schematic exon structure map of IL-2R"

IL-2 receptor complexes consist of three isoforms of IL-2 receptors in high-, intermediate-, and low-affinity. Expression of IL-2R alone or of both IL-2R and

IL-2R" showed low-affinities (Kd = 10−8) to IL-2 binding, and either IL-2R! or IL-2R" alone possessed undetectable affinities (Kd > 10−7 M) for IL-2 binding. The association rate constant with the !" heterotrimer complex was four fold larger than that with the

! heterodimer complex, resulting in high-affinity receptors with Kd of 10−11. On the other hand, the !" heterodimer complex exhibited intermediate affinities (Kd = 10−9 M)

[250].

Expression levels of IL-2R, IL-2R! and IL-2R" on various populations of human peripheral blood cells were different according to specific mAbs staining. IL-2R" expression was seen on all of the populations including CD4+ T cells, CD8+ T cells,

CD20+ B cells, CD56+ NK cells, and CD14+ monocytes. The granulocyte population was also positive for IL-2R". Nevertheless, CD8+ T cells and CD56+ NK cells significantly expressed IL-2R!, but little of IL-2R, while CD4+ T cells expressed faint amounts of IL-2R!. IL-2R" (known as common " chain) widely expressed in peripheral blood cells because it participates not only in increasing IL-2 binding affinity but also in helping other cytokines’ binding, including IL-4, IL-7, IL-9, IL-15 and IL-21.

Similar with the common components for multiple cytokine receptors, such as ! chain

129

of receptors for IL-3, IL-5 and GM-CSF, gp130 of receptors for IL-6, IL-11, OSM, LIF and CNTF, expression of IL-2R" is detectable in a wide range of hematopoietic cell populations. IL-4 is known to possess the capacity to promote growth of T and mast cells. Unique IL-4R transfected cells model were found exhibiting a lower affinity than the high-affinity IL-4 receptor on natural human hematopoietic cells. This indicated that the high-affinity IL-4 receptor on lymphoid cells may consist of a complex composed of at least IL-4R and another subunit. Consequently, IL-2R" was first examined for its sharing with the IL-4 receptor complex. The same as IL-4 receptor complex, single

IL-7R binding affinity was significantly lower than the high-affinity of IL-7 receptor complex expressed on lymphoid cells. Thereby, IL-2R" was suspected to be a common receptor subunit also shared with the IL-7 receptor. Recently, a novel lymphokine

(IL-21) and its receptor (IL-21R) were described also require the common " chain as a signaling component. IL-21 is 131 and 122 amino acid polypeptides produced by activated CD4+ T-lymphocytes. Structurally, IL-21 is most closely related to IL-2, IL-4, and IL-15, members of the four-R-helix-bundle cytokine family that mediate pleiotropic biological functions within the immune system. In addition, IL-21R has highest amino acid sequence homology with IL-2/15R and IL-4R. Thus, the complete IL-21 receptor complex likely shares a close relationship with the receptors for IL-2, IL-4, and

IL- 15 (IL-2R, IL-4R, and IL-15R), which utilize the " chain as a signaling subunit

[251].

The expression of IL-2R" in both wild-type and 11 isoform RasGRP1 transfectants

130

was already confirmed by quantitative real-time PCR and RT-PCR. The wild-type

RasGRP1 transfectants had a high level of expression of IL-2R". The expression of the

IL-2R" gene in the wild-type transfectant can be considered as normal level expression.

The 11 isoform RasGRP1 transfected HMC-1 cells had significantly reduced expression of IL-2R". Assessment of PBMC from SLE patients with 11 isoform

RasGRP1 had significantly reduced expression of IL-2R". The results obtained with the

PBMC from SLE patients were similar with our in vitro experiments using HMC-1 cell transfectants that demonstrated RasGRP1 regulated expression of IL-2R".

The accumulated data indicate that RasGRP1 upregulates the expression of IL-2R" in T cells. In contrast the 11 RasGRP1 isoform found in a subset of SLE patients leads to defective expression of IL-2R". The IL-2R" chain is a common chain which forms part of a number of different receptors eg. IL-2, 4, 7, 9, 15, 21. IL-2 as well as IL-21, which shares sequence homology with IL-2, has been reported to be involved in the generation of regulatory T cells (Tregs) [232, 252]. In SLE patients, CD4 + CD25+ Tregs, which play an essential role in controlling immunologic tolerance to self-antigens and preventing autoimmunity, are significantly decreased when compared with healthy controls [233, 253]. The accumulative evidence suggests that the defective isoform of

RasGRP1 (11) downregulates expression of IL-2R" in SLE patients’ T cells and this could effect the generation of CD4 + CD25+ Tregs. This may be another immunological mechanism in loss of tolerance observed in patient with SLE.

131

Summary

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/phorbol ester receptor whose function has not been quite discovered. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein- labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-acetate

(PMA) and for evaluating the protein’s ability to control the development of mast cells.

Human RasGRP4 translocation from the cytosol to the plasma membrane following

PMA exposure was detected in our experiments and was time and concentration dependent. Because Ras proteins which are considered as the target protein of RasGRP4 are normally localized to the plasma membrane, thus the plasma membrane translocation of RasGRP4 is likely to be relevant to Ras activation. The eighth residue in the C1 domain of the RasGRP family member initially is concerned with translocation. However, the Ser548 mutant hRasGRP4-GFP-expressing HMC-1 cell line which converted Phe548 in hRasGRP4-GFP to Ser did not translocate to the plasma membrane.

Data of RasGRP4 Microarray suggested that RasGRP4 signaling protein regulates the

132

expression of many genes which contribute to the development of mast cells and the pathogenesis of asthma, including GATA-1, GATA-2, PGDS, IL-24, and IL13R2.

Especially, IL13R2 which is considered as a dominant negative inhibitor or a decoy receptor in IL-13 signaling events has a novel function in asthma. Our experimental results suggests that the mast cell specific RasGRP4 protein regulates the level of

IL-13R2 and controls IL-13/ IL-13R1-mediated intracellar signaling events in mast cells. The levels of phosphorylated STAT6 reduced when IL-13R2 levels increased in mast cells. Phosphorylation of STAT6 plays an important role in airway hyperresponsiveness and asthma. The development of therapeutics that can regulate

RasGRP4 could be used to modulate the IL-13-induced phosphorylation of STAT-6 that may be used as therapy in patients with asthma.

Quite recent studies reported that several defective isoforms of human RasGRP1, which is similar with RasGRP4 but present in T cells, have been found in a cohort of SLE patients but not in other rheumatic disease patents and normals. The Exon 11 deletion isoform of RasGRP1 (11) was frequently found, not only individually but also coexists with other defective isoforms. However, the mechanism of how these defective

RasGRP1 isoforms may be involved in the pathogenesis of SLE still remains unknown.

The human mast cell line, HMC-1 was used to create stable clones expressing both wild-type and 11 human RasGRP1 for investigating the differences between the two proteins on the regulation of downstream signaling transduction.

133

Microarray analysis of RasGRP1 expressing HMC-1 cell clones revealed that the expression of IL-2R" was significantly reduced in the 11 RasGRP1 HMC-1 transfectants. This was confirmed by both quantitative real-time PCR and RT-PCR.

Moreover, SLE patients with the 11 RasGRP1 had significantly reduced expression of

IL-2R" compared to the SLE patients and normal controls with FL RasGRP1. The

IL-2R" chain is a common chain which forms part of a number of different receptors eg.

IL-2, 4, 7, 9, 15, as well as IL-21 which has been reported to be involved in the generation of regulatory T cells (Tregs). The decreased number of Tregs in SLE patients had been found in some previous studies and it may be involved in the low immunologic tolerance to self-antigens. Therefore, another immunological mechanism may attribute to low expression of IL-2R" regulated by the defective isoform of

RasGRP1 (11) in SLE patients.

134

Future direction

The accumulated data suggest that mast cells use hRasGRP4 to control their levels of

IL-13R2 to dampen IL-13/IL-13R1-mediated signaling events in this immune cell.

However, this conclusion is only confirmed by in vitro experiments. Quite recently, a strain of RasGRP4 knockout mouse has been established in order to investigate the function of RasGRP4 regulating the development of mast cells. This will offer an opportunity to confirm the in-vitro data which demonstrated the down-regulation of

IL-13R2 by RasGRP4 in vivo.

Although the RasGRP1 mediated down-regulation of the expression of IL-2R" has been confirmed both in vitro and in vivo at gene level, the protein level experiments still need to be performed. As IL-2R" is a membrane protein, it is very difficult to extract detectable amounts of IL-2R" protein. In addition there is currently no antibody available which can bind IL-2R". However, we continue to seeking the best methods to do the protein level experiments. In addition, a strain of RasGRP1 knockout mouse has already established in order to investigate the function of RasGRP1 in mice. This also provide us a change to investigate that if the down-regulation of IL-2R" in RasGRP1 defective individuals impact the generation of regulatory T cells, further effect the immune tolerance in SLE patients.

135

References

1. Katsoulotos, G.P., et al., The Diacylglycerol-dependent translocation of ras guanine nucleotide-releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor alpha2. J Biol Chem, 2008. 283(3): p. 1610-21. 2. Barrios, R.J., et al., Asthma: pathology and pathophysiology. Arch Pathol Lab Med, 2006. 130(4): p. 447-51. 3. Barnes, P.J., Pathophysiology of asthma. Br J Clin Pharmacol, 1996. 42(1): p. 3-10. 4. Holgate, S.T., Pathogenesis of asthma. Clin Exp Allergy, 2008. 38(6): p. 872-97. 5. Kay, A.B., The role of eosinophils in the pathogenesis of asthma. Trends Mol Med, 2005. 11(4): p. 148-52. 6. Hammad, H. and B.N. Lambrecht, Recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic inflammation. J Allergy Clin Immunol, 2006. 118(2): p. 331-6. 7. von Garnier, C., et al., Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J Immunol, 2005. 175(3): p. 1609-18. 8. Riese, R.J. and H.A. Chapman, Cathepsins and compartmentalization in antigen presentation. Curr Opin Immunol, 2000. 12(1): p. 107-13. 9. Lordan, J.L., et al., The role of CD28-B7 costimulation in allergen-induced cytokine release by bronchial mucosa from patients with moderately severe asthma. J Allergy Clin Immunol, 2001. 108(6): p. 976-81. 10. Truyen, E., et al., Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients. Thorax, 2006. 61(3): p. 202-8. 11. Garcia, G., V. Godot, and M. Humbert, New chemokine targets for asthma therapy. Curr Allergy Asthma Rep, 2005. 5(2): p. 155-60. 12. Kallinich, T., et al., Chemokine-receptor expression on T cells in lung compartments of challenged asthmatic patients. Clin Exp Allergy, 2005. 35(1): p. 26-33. 13. Kay, A.B., The role of T lymphocytes in asthma. Chem Immunol Allergy, 2006. 91: p. 59-75. 14. Ryu, H.J., et al., Gene-based single nucleotide polymorphisms and linkage disequilibrium patterns of 29 asthma candidate genes in the chromosome 5q31-33 region in Koreans. Int Arch Allergy Immunol, 2006. 139(3): p. 209-16. 15. Djukanovic, R., et al., The effect of treatment with oral corticosteroids on asthma symptoms and airway inflammation. Am J Respir Crit Care Med, 1997. 155(3): p. 826-32. 16. Djukanovic, R., et al., Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am Rev Respir Dis, 1992. 145(3): p. 669-74. 17. Lemiere, C., et al., Airway inflammation assessed by invasive and noninvasive means in severe asthma: eosinophilic and noneosinophilic phenotypes. J Allergy Clin Immunol, 2006. 118(5): p. 1033-9. 18. Robinson, D.S., et al., Eosinophil development and bone marrow and tissue eosinophils 136

in atopic asthma. Int Arch Allergy Immunol, 1999. 118(2-4): p. 98-100. 19. Sehmi, R., et al., Allergen-induced fluctuation in CC chemokine receptor 3 expression on bone marrow CD34+ cells from asthmatic subjects: significance for mobilization of haemopoietic progenitor cells in allergic inflammation. Immunology, 2003. 109(4): p. 536-46. 20. Kariyawasam, H.H. and D.S. Robinson, The eosinophil: the cell and its weapons, the cytokines, its locations. Semin Respir Crit Care Med, 2006. 27(2): p. 117-27. 21. Kay, A.B., S. Phipps, and D.S. Robinson, A role for eosinophils in airway remodelling in asthma. Trends Immunol, 2004. 25(9): p. 477-82. 22. Gajewska, B.U., R.E. Wiley, and M. Jordana, GM-CSF and dendritic cells in allergic airway inflammation: basic mechanisms and prospects for therapeutic intervention. Curr Drug Targets Inflamm Allergy, 2003. 2(4): p. 279-92. 23. Webb, D.C., et al., Comparative roles of IL-4, IL-13, and IL-4Ralpha in dendritic cell maturation and CD4+ Th2 cell function. J Immunol, 2007. 178(1): p. 219-27. 24. Holgate, S.T., et al., Epithelial-mesenchymal communication in the pathogenesis of chronic asthma. Proc Am Thorac Soc, 2004. 1(2): p. 93-8. 25. Kicic, A., et al., Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma. Am J Respir Crit Care Med, 2006. 174(10): p. 1110-8. 26. Ilowite, J.S., et al., Permeability of the bronchial mucosa to 99mTc-DTPA in asthma. Am Rev Respir Dis, 1989. 139(5): p. 1139-43. 27. Hudson, T.J., Skin barrier function and allergic risk. Nat Genet, 2006. 38(4): p. 399-400. 28. Contoli, M., et al., Role of deficient type III interferon-lambda production in asthma exacerbations. Nat Med, 2006. 12(9): p. 1023-6. 29. Wark, P.A., et al., Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. J Exp Med, 2005. 201(6): p. 937-47. 30. Hamilton, L.M., et al., The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma. Clin Exp Allergy, 2003. 33(2): p. 233-40. 31. Leung, S.Y., et al., Effect of transforming growth factor-beta receptor I kinase inhibitor 2,4-disubstituted pteridine (SD-208) in chronic allergic airway inflammation and remodeling. J Pharmacol Exp Ther, 2006. 319(2): p. 586-94. 32. Shaw, T.J., et al., Endobronchial ultrasound to assess airway wall thickening: validation in vitro and in vivo. Eur Respir J, 2004. 23(6): p. 813-7. 33. James, A., Remodelling of airway smooth muscle in asthma: what sort do you have? Clin Exp Allergy, 2005. 35(6): p. 703-7. 34. Jongepier, H., et al., Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma. Clin Exp Allergy, 2004. 34(5): p. 757-60. 35. van Diemen, C.C., et al., A disintegrin and metalloprotease 33 polymorphisms and lung function decline in the general population. Am J Respir Crit Care Med, 2005. 172(3): p. 329-33. 36. Kariyawasam, H.H., et al., Remodeling and airway hyperresponsiveness but not cellular inflammation persist after allergen challenge in asthma. Am J Respir Crit Care Med, 2007. 175(9): p. 896-904. 37. Abdel-Rahman, A.M., S.A. el-Sahrigy, and S.I. Bakr, A comparative study of two

137

angiogenic factors: vascular endothelial growth factor and angiogenin in induced sputum from asthmatic children in acute attack. Chest, 2006. 129(2): p. 266-71. 38. Bhandari, V., et al., Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung. Proc Natl Acad Sci U S A, 2006. 103(29): p. 11021-6. 39. Chetta, A., et al., Vascular endothelial growth factor up-regulation and bronchial wall remodelling in asthma. Clin Exp Allergy, 2005. 35(11): p. 1437-42. 40. Kay, A.B., et al., Airway expression of calcitonin gene-related peptide in T-cell peptide-induced late asthmatic reactions in atopics. Allergy, 2007. 62(5): p. 495-503. 41. Li, L. and S.A. Krilis, Mast-cell growth and differentiation. Allergy, 1999. 54(4): p. 306-12. 42. Irani, A.M., et al., Human mast cell carboxypeptidase. Selective localization to MCTC cells. J Immunol, 1991. 147(1): p. 247-53. 43. Schwartz, L.B., Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells. J Immunol, 1985. 134(1): p. 526-31. 44. Weidner, N. and K.F. Austen, Heterogeneity of mast cells at multiple body sites. Fluorescent determination of avidin binding and immunofluorescent determination of chymase, tryptase, and carboxypeptidase content. Pathol Res Pract, 1993. 189(2): p. 156-62. 45. Friend, D.S., et al., Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype. J Cell Biol, 1996. 135(1): p. 279-90. 46. Friend, D.S., et al., Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis. J Immunol, 1998. 160(11): p. 5537-45. 47. Qi, J.C., L.L. Li, and S.A. Krilis, Cells with features of mast cells and basophils. ACI International 2001. 13: p. 197-203. 48. Corry, D.B. and F. Kheradmand, Induction and regulation of the IgE response. Nature, 1999. 402(6760 Suppl): p. B18-23. 49. Baggiolini, M., Chemokines and leukocyte traffic. Nature, 1998. 392(6676): p. 565-8. 50. Qi, J.C. and S.A. Krilis, MCs/basophils in human blood. ACI International, 2003. 15: p. 11-17. 51. Chen, C.C., et al., Identification of mast cell progenitors in adult mice. Proc Natl Acad Sci U S A, 2005. 102(32): p. 11408-13. 52. Arinobu, Y., et al., Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis. Proc Natl Acad Sci U S A, 2005. 102(50): p. 18105-10. 53. Kirshenbaum, A.S., et al., Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells. J Immunol, 1991. 146(5): p. 1410-5. 54. Chabot, B., et al., The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature, 1988. 335(6185): p. 88-9. 55. Copeland, N.G., et al., Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles. Cell, 1990. 63(1): p. 175-83. 56. Sawai, N., et al., Thrombopoietin augments stem cell factor-dependent growth of human mast cells from bone marrow multipotential hematopoietic progenitors. Blood, 1999.

138

93(11): p. 3703-12. 57. Bressler, R.B., et al., Inhibition of the growth of IL-3-dependent mast cells from murine bone marrow by recombinant granulocyte macrophage-colony-stimulating factor. J Immunol, 1989. 143(1): p. 135-9. 58. Saito, H., et al., Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol, 1996. 157(1): p. 343-50. 59. Saito, H., Culture of human mast cells from hemopoietic progenitors. Methods Mol Biol, 2006. 315: p. 113-22. 60. Kirshenbaum, A.S., et al., Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13). Blood, 1999. 94(7): p. 2333-42. 61. Ochi, H., et al., T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J Exp Med, 1999. 190(2): p. 267-80. 62. Matsuzawa, S., et al., IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor. J Immunol, 2003. 170(7): p. 3461-7. 63. Qi, J.C., et al., IL-16 regulation of human mast cells/basophils and their susceptibility to HIV-1. J Immunol, 2002. 168(8): p. 4127-34. 64. Kirshenbaum, A.S., et al., Inhibition of human mast cell growth and differentiation by interferon gamma-1b. Exp Hematol, 1998. 26(3): p. 245-51. 65. Takagi, M., K. Koike, and T. Nakahata, Antiproliferative effect of IFN-gamma on proliferation of mouse connective tissue-type mast cells. J Immunol, 1990. 145(6): p. 1880-4. 66. Mann-Chandler, M.N., et al., IFN-gamma induces apoptosis in developing mast cells. J Immunol, 2005. 175(5): p. 3000-5. 67. Matsuda, H., et al., Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells. J Exp Med, 1991. 174(1): p. 7-14. 68. Kanbe, N., et al., Nerve growth factor prevents apoptosis of cord blood-derived human cultured mast cells synergistically with stem cell factor. Clin Exp Allergy, 2000. 30(8): p. 1113-20. 69. Yanagida, M., et al., Effects of T-helper 2-type cytokines, interleukin-3 (IL-3), IL-4, IL-5, and IL-6 on the survival of cultured human mast cells. Blood, 1995. 86(10): p. 3705-14. 70. Yamamoto, M., et al., Upstream and downstream of erythroid transcription factor GATA-1. Genes Cells, 1997. 2(2): p. 107-15. 71. Migliaccio, A.R., et al., GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1low mouse mutant. J Exp Med, 2003. 197(3): p. 281-96. 72. Li, Y., et al., Mast cells/basophils in the peripheral blood of allergic individuals who are HIV-1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4. Blood, 2001. 97(11): p. 3484-90. 73. Brightling, C.E., et al., The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am J Respir Crit Care Med, 2005. 171(10): p. 1103-8. 74. Jolly, P.S., et al., Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI

139

triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med, 2004. 199(7): p. 959-70. 75. Holgate, S.T., et al., The genetics of asthma: ADAM33 as an example of a susceptibility gene. Proc Am Thorac Soc, 2006. 3(5): p. 440-3. 76. Inman, M.D., et al., Reproducibility of allergen-induced early and late asthmatic responses. J Allergy Clin Immunol, 1995. 95(6): p. 1191-5. 77. Fahy, J.V., et al., The effect of an anti-IgE monoclonal antibody on the early- and late-phase responses to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med, 1997. 155(6): p. 1828-34. 78. Bradding, P., A.F. Walls, and S.T. Holgate, The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol, 2006. 117(6): p. 1277-84. 79. Lewis, R.A., et al., Slow reacting substances of anaphylaxis: identification of leukotrienes C-1 and D from human and rat sources. Proc Natl Acad Sci U S A, 1980. 77(6): p. 3710-4. 80. Bradding, P., et al., Interleukin-4, -5, and -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am J Respir Cell Mol Biol, 1994. 10(5): p. 471-80. 81. Berger, P., et al., Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol, 2001. 91(3): p. 1372-9. 82. Kaur, D., et al., Airway smooth muscle and mast cell-derived CC chemokine ligand 19 mediate airway smooth muscle migration in asthma. Am J Respir Crit Care Med, 2006. 174(11): p. 1179-88. 83. Plante, S., et al., Mast cells regulate procollagen I (alpha 1) production by bronchial fibroblasts derived from subjects with asthma through IL-4/IL-4 delta 2 ratio. J Allergy Clin Immunol, 2006. 117(6): p. 1321-7. 84. Brightling, C.E., et al., TH2 cytokine expression in bronchoalveolar lavage fluid T lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis. J Allergy Clin Immunol, 2002. 110(6): p. 899-905. 85. Brightling, C.E., et al., Comparison of airway immunopathology of eosinophilic bronchitis and asthma. Thorax, 2003. 58(6): p. 528-32. 86. Berry, M.A., et al., Sputum and bronchial submucosal IL-13 expression in asthma and eosinophilic bronchitis. J Allergy Clin Immunol, 2004. 114(5): p. 1106-9. 87. Brightling, C.E., et al., Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med, 2002. 346(22): p. 1699-705. 88. Brightling, C.E., et al., Interleukin-4 and -13 expression is co-localized to mast cells within the airway smooth muscle in asthma. Clin Exp Allergy, 2003. 33(12): p. 1711-6. 89. Yang, W., et al., Human lung mast cells adhere to human airway smooth muscle, in part, via tumor suppressor in lung cancer-1. J Immunol, 2006. 176(2): p. 1238-43. 90. Berger, P., et al., Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis. Faseb J, 2003. 17(14): p. 2139-41. 91. Berger, P., et al., Mast cell tryptase as a mediator of hyperresponsiveness in human isolated bronchi. Clin Exp Allergy, 1999. 29(6): p. 804-12. 92. Sekizawa, K., et al., Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs. J Clin Invest, 1989. 83(1): p. 175-9.

140

93. Venkayya, R., et al., The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through direct effects on resident airway cells. Am J Respir Cell Mol Biol, 2002. 26(2): p. 202-8. 94. Metzger, H., The receptor with high affinity for IgE. Immunol Rev, 1992. 125: p. 37-48. 95. Galli, S.J., New concepts about the mast cell. N Engl J Med, 1993. 328(4): p. 257-65. 96. Gilfillan, A.M. and C. Tkaczyk, Integrated signalling pathways for mast-cell activation. Nat Rev Immunol, 2006. 6(3): p. 218-30. 97. Lewis, T.S., P.S. Shapiro, and N.G. Ahn, Signal transduction through MAP kinase cascades. Adv Cancer Res, 1998. 74: p. 49-139. 98. Ehrhardt, A., et al., Ras and relatives--job sharing and networking keep an old family together. Exp Hematol, 2002. 30(10): p. 1089-106. 99. Quilliam, L.A., J.F. Rebhun, and A.F. Castro, A growing family of guanine nucleotide exchange factors is responsible for activation of Ras-family GTPases. Prog Nucleic Acid Res Mol Biol, 2002. 71: p. 391-444. 100. Jelinek, T., et al., Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. Mol Cell Biol, 1996. 16(3): p. 1027-34. 101. Moodie, S.A., et al., Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science, 1993. 260(5114): p. 1658-61. 102. Morrison, D.K. and R.E. Cutler, The complexity of Raf-1 regulation. Curr Opin Cell Biol, 1997. 9(2): p. 174-9. 103. Vojtek, A.B., S.M. Hollenberg, and J.A. Cooper, Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell, 1993. 74(1): p. 205-14. 104. Warne, P.H., P.R. Viciana, and J. Downward, Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature, 1993. 364(6435): p. 352-5. 105. Zheng, C.F. and K.L. Guan, Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen-induced Thr/Tyr protein phosphatase. J Biol Chem, 1993. 268(22): p. 16116-9. 106. Dower, N.A., et al., RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol, 2000. 1(4): p. 317-21. 107. Ebinu, J.O., et al., RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. Science, 1998. 280(5366): p. 1082-6. 108. Roose, J.P., et al., A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells. Mol Cell Biol, 2005. 25(11): p. 4426-41. 109. Yasuda, S., et al., Defective expression of Ras guanyl nucleotide-releasing protein 1 in a subset of patients with systemic lupus erythematosus. J Immunol, 2007. 179(7): p. 4890-900. 110. Clyde-Smith, J., et al., Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. J Biol Chem, 2000. 275(41): p. 32260-7. 111. Kawasaki, H., et al., A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. Proc Natl Acad Sci U S A, 1998. 95(22): p. 13278-83. 112. Kedra, D., et al., The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13. Hum Genet, 1997. 100(5-6): p. 611-9.

141

113. Crittenden, J.R., et al., CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med, 2004. 10(9): p. 982-6. 114. Rebhun, J.F., A.F. Castro, and L.A. Quilliam, Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction. J Biol Chem, 2000. 275(45): p. 34901-8. 115. Yamashita, S., et al., CalDAG-GEFIII activation of Ras, R-ras, and Rap1. J Biol Chem, 2000. 275(33): p. 25488-93. 116. Coughlin, J.J., et al., RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling. J Immunol, 2005. 175(11): p. 7179-84. 117. Oh-hora, M., et al., Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-gamma2 to Ras in B cell receptor signaling. J Exp Med, 2003. 198(12): p. 1841-51. 118. Yang, Y., et al., RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J Biol Chem, 2002. 277(28): p. 25756-74. 119. Razin, E., et al., Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan. J Immunol, 1984. 132(3): p. 1479-86. 120. Kanakura, Y., et al., Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells. Blood, 1988. 72(3): p. 877-85. 121. Nakano, T., et al., Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells. J Exp Med, 1985. 162(3): p. 1025-43. 122. Otsu, K., et al., Phenotypic changes of bone marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are genetically deficient in mast cells. J Exp Med, 1987. 165(3): p. 615-27. 123. Li, L., Y. Yang, and R.L. Stevens, Cloning of rat Ras guanine nucleotide releasing protein 4, and evaluation of its expression in rat mast cells and their bone marrow progenitors. Mol Immunol, 2002. 38(16-18): p. 1283-8. 124. Li, L., et al., Mast cells in airway hyporesponsive C3H/HeJ mice express a unique isoform of the signaling protein Ras guanine nucleotide releasing protein 4 that is unresponsive to diacylglycerol and phorbol esters. J Immunol, 2003. 171(1): p. 390-7. 125. Broek, D., et al., The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell, 1987. 48(5): p. 789-99. 126. Jones, S., M.L. Vignais, and J.R. Broach, The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol, 1991. 11(5): p. 2641-6. 127. Hoffman, R.W., T cells in the pathogenesis of systemic lupus erythematosus. Clin Immunol, 2004. 113(1): p. 4-13. 128. Worrall, J.G., et al., SLE: a rheumatological view. Analysis of the clinical features,

142

serology and immunogenetics of 100 SLE patients during long-term follow-up. Q J Med, 1990. 74(275): p. 319-30. 129. ter Borg, E.J., et al., Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. A long-term, prospective study. Arthritis Rheum, 1990. 33(5): p. 634-43. 130. Arbuckle, M.R., et al., Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med, 2003. 349(16): p. 1526-33. 131. Criswell, L.A. and C.I. Amos, Update on genetic risk factors for systemic lupus erythematosus and rheumatoid arthritis. Curr Opin Rheumatol, 2000. 12(2): p. 85-90. 132. James, J.A., J.B. Harley, and R.H. Scofield, Role of viruses in systemic lupus erythematosus and Sjogren syndrome. Curr Opin Rheumatol, 2001. 13(5): p. 370-6. 133. Sontheimer, R.D., Photoimmunology of lupus erythematosus and dermatomyositis: a speculative review. Photochem Photobiol, 1996. 63(5): p. 583-94. 134. Stohl, W., Systemic lupus erythematosus: a blissless disease of too much BLyS (B lymphocyte stimulator) protein. Curr Opin Rheumatol, 2002. 14(5): p. 522-8. 135. Agnello, V., Association of systemic lupus erythematosus and SLE-like syndromes with hereditary and acquired complement deficiency states. Arthritis Rheum, 1978. 21(5 Suppl): p. S146-52. 136. Jacob, C.O., et al., Heritable major histocompatibility complex class II-associated differences in production of tumor necrosis factor alpha: relevance to genetic predisposition to systemic lupus erythematosus. Proc Natl Acad Sci U S A, 1990. 87(3): p. 1233-7. 137. Fenning, S., et al., T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP). Clin Exp Immunol, 1995. 101(3): p. 408-13. 138. Hoffman, R.W., et al., Human T cell clones reactive against U-small nuclear ribonucleoprotein autoantigens from connective tissue disease patients and healthy individuals. J Immunol, 1993. 151(11): p. 6460-9. 139. Wolff-Vorbeck, G., et al., Characterization of an HLA-DR4-restricted T cell clone recognizing a protein moiety of small nuclear ribonucleoproteins (UsnRNP). Clin Exp Immunol, 1994. 95(3): p. 378-84. 140. Rajagopalan, S., et al., Pathogenic anti-DNA autoantibody-inducing T helper cell lines from patients with active lupus nephritis: isolation of CD4-8- T helper cell lines that express the gamma delta T-cell antigen receptor. Proc Natl Acad Sci U S A, 1990. 87(18): p. 7020-4. 141. Desai-Mehta, A., et al., Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest, 1996. 97(9): p. 2063-73. 142. Yi, Y., M. McNerney, and S.K. Datta, Regulatory defects in Cbl and mitogen-activated protein kinase (extracellular signal-related kinase) pathways cause persistent hyperexpression of CD40 ligand in human lupus T cells. J Immunol, 2000. 165(11): p. 6627-34. 143. Kuchtey, J., et al., CpG DNA induces a class II transactivator-independent increase in class II MHC by stabilizing class II MHC mRNA in B lymphocytes. J Immunol, 2003. 171(5): p. 2320-5.

143

144. Scheinecker, C., et al., Alterations of dendritic cells in systemic lupus erythematosus: phenotypic and functional deficiencies. Arthritis Rheum, 2001. 44(4): p. 856-65. 145. Yan, J. and M.J. Mamula, Autoreactive T cells revealed in the normal repertoire: escape from negative selection and peripheral tolerance. J Immunol, 2002. 168(7): p. 3188-94. 146. Herrmann, M., et al., Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum, 1998. 41(7): p. 1241-50. 147. Walport, M.J., Complement and systemic lupus erythematosus. Arthritis Res, 2002. 4 Suppl 3: p. S279-93. 148. Enyedy, E.J., et al., Fc epsilon receptor type I gamma chain replaces the deficient T cell receptor zeta chain in T cells of patients with systemic lupus erythematosus. Arthritis Rheum, 2001. 44(5): p. 1114-21. 149. Elliott, M.R., et al., Protein kinase A regulatory subunit type II beta directly interacts with and suppresses CREB transcriptional activity in activated T cells. J Immunol, 2003. 171(7): p. 3636-44. 150. Powrie, F. and K.J. Maloy, Immunology. Regulating the regulators. Science, 2003. 299(5609): p. 1030-1. 151. Kammer, G.M. and R.L. Stein, T lymphocyte immune dysfunctions in systemic lupus erythematosus. J Lab Clin Med, 1990. 115(3): p. 273-82. 152. Brundula, V., et al., Diminished levels of T cell receptor zeta chains in peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum, 1999. 42(9): p. 1908-16. 153. Liossis, S.N., et al., Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus. Deficient expression of the T cell receptor zeta chain. J Clin Invest, 1998. 101(7): p. 1448-57. 154. Nambiar, M.P., et al., T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor zeta chain messenger RNA. Arthritis Rheum, 2001. 44(6): p. 1336-50. 155. Skulachev, V.P., Mitochondrial physiology and pathology; concepts of programmed death of organelles, cells and organisms. Mol Aspects Med, 1999. 20(3): p. 139-84. 156. Tsokos, G.C. and S.N. Liossis, Immune cell signaling defects in lupus: activation, anergy and death. Immunol Today, 1999. 20(3): p. 119-24. 157. Dayal, A.K. and G.M. Kammer, The T cell enigma in lupus. Arthritis Rheum, 1996. 39(1): p. 23-33. 158. Krishnan, S., et al., Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3zeta expression. Blood, 2001. 97(12): p. 3851-9. 159. Boumpas, D.T., et al., Increased proto-oncogene expression in peripheral blood lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases. Arthritis Rheum, 1986. 29(6): p. 755-60. 160. Koshy, M., D. Berger, and M.K. Crow, Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest, 1996. 98(3): p. 826-37. 161. Kovacs, B., et al., Increased expression of functional Fas-ligand in activated T cells

144

from patients with systemic lupus erythematosus. Autoimmunity, 1997. 25(4): p. 213-21. 162. Kammer, G.M., et al., Abnormal T cell signal transduction in systemic lupus erythematosus. Arthritis Rheum, 2002. 46(5): p. 1139-54. 163. Hasler, P., L.A. Schultz, and G.M. Kammer, Defective cAMP-dependent phosphorylation of intact T lymphocytes in active systemic lupus erythematosus. Proc Natl Acad Sci U S A, 1990. 87(5): p. 1978-82. 164. Mandler, R., et al., Abnormal adenosine-induced immunosuppression and cAMP metabolism in T lymphocytes of patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A, 1982. 79(23): p. 7542-6. 165. Kammer, G.M., I.U. Khan, and C.J. Malemud, Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes. J Clin Invest, 1994. 94(1): p. 422-30. 166. Mishra, N., et al., Association of deficient type II protein kinase A activity with aberrant nuclear translocation of the RII beta subunit in systemic lupus erythematosus T lymphocytes. J Immunol, 2000. 165(5): p. 2830-40. 167. Kammer, G.M., et al., Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes: II. Abnormal isozyme kinetics. J Immunol, 1996. 157(6): p. 2690-8. 168. Laxminarayana, D., et al., Diminished levels of protein kinase A RI alpha and RI beta transcripts and proteins in systemic lupus erythematosus T lymphocytes. J Immunol, 1999. 162(9): p. 5639-48. 169. Khan, I.U., D. Laxminarayana, and G.M. Kammer, Protein kinase A RI beta subunit deficiency in lupus T lymphocytes: bypassing a block in RI beta translation reconstitutes protein kinase A activity and augments IL-2 production. J Immunol, 2001. 166(12): p. 7600-5. 170. Laxminarayana, D. and G.M. Kammer, mRNA mutations of type I protein kinase A regulatory subunit alpha in T lymphocytes of a subject with systemic lupus erythematosus. Int Immunol, 2000. 12(11): p. 1521-9. 171. Aringer, M., et al., High levels of bcl-2 protein in circulating T lymphocytes, but not B lymphocytes, of patients with systemic lupus erythematosus. Arthritis Rheum, 1994. 37(10): p. 1423-30. 172. Deng, C., et al., Decreased Ras-mitogen-activated protein kinase signaling may cause DNA hypomethylation in T lymphocytes from lupus patients. Arthritis Rheum, 2001. 44(2): p. 397-407. 173. Yang, J., et al., Effect of mitogenic stimulation and DNA methylation on human T cell DNA methyltransferase expression and activity. J Immunol, 1997. 159(3): p. 1303-9. 174. Alcocer-Varela, J. and D. Alarcon-Segovia, Decreased production of and response to interleukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus. J Clin Invest, 1982. 69(6): p. 1388-92. 175. Linker-Israeli, M., et al., Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE). J Immunol, 1983. 130(6): p. 2651-5. 176. Via, C.S., et al., T cell-antigen-presenting cell interactions in human systemic lupus erythematosus. Evidence for heterogeneous expression of multiple defects. J Immunol,

145

1993. 151(7): p. 3914-22. 177. Becker, H., et al., Analysis of proteins that interact with the IL-2 regulatory region in patients with rheumatic diseases. Clin Exp Immunol, 1995. 99(3): p. 325-30. 178. Wong, H.K., et al., Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression. J Immunol, 1999. 163(3): p. 1682-9. 179. Powell, J.D., et al., The -180 site of the IL-2 promoter is the target of CREB/CREM binding in T cell anergy. J Immunol, 1999. 163(12): p. 6631-9. 180. Sassone-Corsi, P., Coupling gene expression to cAMP signalling: role of CREB and CREM. Int J Biochem Cell Biol, 1998. 30(1): p. 27-38. 181. Alarcon-Segovia, D., et al., Familial aggregation of systemic lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases in 1,177 lupus patients from the GLADEL cohort. Arthritis Rheum, 2005. 52(4): p. 1138-47. 182. Krishnan, S., B. Chowdhury, and G.C. Tsokos, Autoimmunity in systemic lupus erythematosus: integrating genes and biology. Semin Immunol, 2006. 18(4): p. 230-43. 183. Prokunina, L., et al., A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet, 2002. 32(4): p. 666-9. 184. Cloutier, J.F. and A. Veillette, Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells. Embo J, 1996. 15(18): p. 4909-18. 185. Bennett, L., et al., Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med, 2003. 197(6): p. 711-23. 186. Barnes, B.J., et al., Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection. J Biol Chem, 2004. 279(43): p. 45194-207. 187. Ebinu, J.O., et al., RasGRP links T-cell receptor signaling to Ras. Blood, 2000. 95(10): p. 3199-203. 188. Campbell, S.L., et al., Increasing complexity of Ras signaling. Oncogene, 1998. 17(11 Reviews): p. 1395-413. 189. Wilkinson, S.E., P.J. Parker, and J.S. Nixon, Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J, 1993. 294 ( Pt 2): p. 335-7. 190. Downward, J., et al., Stimulation of p21ras upon T-cell activation. Nature, 1990. 346(6286): p. 719-23. 191. Izquierdo, M., et al., Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. Mol Cell Biol, 1992. 12(7): p. 3305-12. 192. Chiu, V.K., et al., Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol, 2002. 4(5): p. 343-50. 193. Bivona, T.G., et al., Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature, 2003. 424(6949): p. 694-8. 194. Caloca, M.J., J.L. Zugaza, and X.R. Bustelo, Exchange factors of the RasGRP family mediate Ras activation in the Golgi. J Biol Chem, 2003. 278(35): p. 33465-73. 195. Li, L., Y. Yang, and R.L. Stevens, RasGRP4 regulates the expression of prostaglandin

146

D2 in human and rat mast cell lines. J Biol Chem, 2003. 278(7): p. 4725-9. 196. Carrasco, S. and I. Merida, Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes. Mol Biol Cell, 2004. 15(6): p. 2932-42. 197. Perez de Castro, I., et al., Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus. Mol Cell Biol, 2004. 24(8): p. 3485-96. 198. Magee, T. and C. Marshall, New insights into the interaction of Ras with the plasma membrane. Cell, 1999. 98(1): p. 9-12. 199. Irie, K., et al., Tumor promoter binding of the protein kinase C C1 homology domain peptides of RasGRPs, chimaerins, and Unc13s. Bioorg Med Chem, 2004. 12(17): p. 4575-83. 200. Bodey, K.J., et al., Cytokine profiles of BAL T cells and T-cell clones obtained from human asthmatic airways after local allergen challenge. Allergy, 1999. 54(10): p. 1083-93. 201. Humbert, M., et al., Elevated expression of messenger ribonucleic acid encoding IL-13 in the bronchial mucosa of atopic and nonatopic subjects with asthma. J Allergy Clin Immunol, 1997. 99(5): p. 657-65. 202. Prieto, J., et al., Increased interleukin-13 mRNA expression in bronchoalveolar lavage cells of atopic patients with mild asthma after repeated low-dose allergen provocations. Respir Med, 2000. 94(8): p. 806-14. 203. Wills-Karp, M., et al., Interleukin-13: central mediator of allergic asthma. Science, 1998. 282(5397): p. 2258-61. 204. Grunig, G., et al., Requirement for IL-13 independently of IL-4 in experimental asthma. Science, 1998. 282(5397): p. 2261-3. 205. Zhu, Z., et al., Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest, 1999. 103(6): p. 779-88. 206. Burd, P.R., et al., Activated mast cells produce interleukin 13. J Exp Med, 1995. 181(4): p. 1373-80. 207. Lukacs, N.W., et al., Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J Immunol, 2001. 167(2): p. 1060-5. 208. Zhu, Z., et al., IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling. J Immunol, 2002. 168(6): p. 2953-62. 209. Faffe, D.S., et al., IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype. Am J Physiol Lung Cell Mol Physiol, 2003. 285(4): p. L907-14. 210. Hesse, M., et al., Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol, 2001. 167(11): p. 6533-44. 211. Zimmermann, N., et al., Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest, 2003. 111(12): p.

147

1863-74. 212. Lee, C.G., et al., Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med, 2001. 194(6): p. 809-21. 213. Wen, F.Q., et al., Interleukin-4- and interleukin-13-enhanced transforming growth factor-beta2 production in cultured human bronchial epithelial cells is attenuated by interferon-gamma. Am J Respir Cell Mol Biol, 2002. 26(4): p. 484-90. 214. Ingram, J.L., et al., Interleukin-13 stimulates the proliferation of lung myofibroblasts via a signal transducer and activator of transcription-6-dependent mechanism: a possible mechanism for the development of airway fibrosis in asthma. Chest, 2003. 123(3 Suppl): p. 422S-4S. 215. Gavett, S.H., et al., Interleukin-4 receptor blockade prevents airway responses induced by antigen challenge in mice. Am J Physiol, 1997. 272(2 Pt 1): p. L253-61. 216. Kuperman, D., et al., Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J Exp Med, 1998. 187(6): p. 939-48. 217. Espinosa, K., et al., CysLT1 receptor upregulation by TGF-beta and IL-13 is associated with bronchial smooth muscle cell proliferation in response to LTD4. J Allergy Clin Immunol, 2003. 111(5): p. 1032-40. 218. Wen, F.Q., et al., TH2 Cytokine-enhanced and TGF-beta-enhanced vascular endothelial growth factor production by cultured human airway smooth muscle cells is attenuated by IFN-gamma and corticosteroids. J Allergy Clin Immunol, 2003. 111(6): p. 1307-18. 219. Kazi, A.S., et al., Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent. Am J Physiol Lung Cell Mol Physiol, 2004. 286(3): p. L539-45. 220. Aman, M.J., et al., cDNA cloning and characterization of the human interleukin 13 receptor alpha chain. J Biol Chem, 1996. 271(46): p. 29265-70. 221. Caput, D., et al., Cloning and characterization of a specific interleukin (IL)-13 binding protein structurally related to the IL-5 receptor alpha chain. J Biol Chem, 1996. 271(28): p. 16921-6. 222. Hilton, D.J., et al., Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor. Proc Natl Acad Sci U S A, 1996. 93(1): p. 497-501. 223. Brusselle, G., et al., Allergen-induced airway inflammation and bronchial responsiveness in wild-type and interleukin-4-deficient mice. Am J Respir Cell Mol Biol, 1995. 12(3): p. 254-9. 224. Coyle, A.J., et al., Interleukin-4 is required for the induction of lung Th2 mucosal immunity. Am J Respir Cell Mol Biol, 1995. 13(1): p. 54-9. 225. Donaldson, D.D., et al., The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1. J Immunol, 1998. 161(5): p. 2317-24. 226. Zurawski, S.M., et al., The primary binding subunit of the human interleukin-4 receptor is also a component of the interleukin-13 receptor. J Biol Chem, 1995. 270(23): p. 13869-78. 227. Daines, M.O. and G.K. Hershey, A novel mechanism by which interferon-gamma can

148

regulate interleukin (IL)-13 responses. Evidence for intracellular stores of IL-13 receptor alpha -2 and their rapid mobilization by interferon-gamma. J Biol Chem, 2002. 277(12): p. 10387-93. 228. Yasunaga, S., et al., The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells. Cytokine, 2003. 24(6): p. 293-303. 229. Chiaramonte, M.G., et al., Regulation and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant immune response. J Exp Med, 2003. 197(6): p. 687-701. 230. Trieu, Y., et al., Soluble interleukin-13Ralpha2 decoy receptor inhibits Hodgkin's lymphoma growth in vitro and in vivo. Cancer Res, 2004. 64(9): p. 3271-5. 231. Zheng, T., et al., Cytokine regulation of IL-13Ralpha2 and IL-13Ralpha1 in vivo and in vitro. J Allergy Clin Immunol, 2003. 111(4): p. 720-8. 232. Piao, W.H., et al., IL-21 modulates CD4+ CD25+ regulatory T-cell homeostasis in experimental autoimmune encephalomyelitis. Scand J Immunol, 2008. 67(1): p. 37-46. 233. Valencia, X., et al., Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol, 2007. 178(4): p. 2579-88. 234. Norment, A.M., et al., Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes. J Immunol, 2003. 170(3): p. 1141-9. 235. Layer, K., et al., Autoimmunity as the consequence of a spontaneous mutation in Rasgrp1. Immunity, 2003. 19(2): p. 243-55. 236. Liu, Y., et al., An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response. J Exp Med, 2007. 204(1): p. 93-103. 237. Cosman, D., et al., Cloning, sequence and expression of human interleukin-2 receptor. Nature, 1984. 312(5996): p. 768-71. 238. Leonard, W.J., et al., Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature, 1984. 311(5987): p. 626-31. 239. Nikaido, T., et al., Molecular cloning of cDNA encoding human interleukin-2 receptor. Nature, 1984. 311(5987): p. 631-5. 240. Hatakeyama, M., et al., Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's. Science, 1989. 244(4904): p. 551-6. 241. Leonard, W.J. and R. Spolski, Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation. Nat Rev Immunol, 2005. 5(9): p. 688-98. 242. Ishida, N., et al., Molecular cloning and structure of the human interleukin 2 receptor gene. Nucleic Acids Res, 1985. 13(21): p. 7579-89. 243. Leonard, W.J., et al., Structure of the human interleukin-2 receptor gene. Science, 1985. 230(4726): p. 633-9. 244. Gnarra, J.R., et al., Human interleukin 2 receptor beta-chain gene: chromosomal localization and identification of 5' regulatory sequences. Proc Natl Acad Sci U S A, 1990. 87(9): p. 3440-4. 245. Shibuya, H., et al., The human interleukin-2 receptor beta-chain gene: genomic organization, promoter analysis and chromosomal assignment. Nucleic Acids Res, 1990. 18(13): p. 3697-703.

149

246. Bazan, J.F., Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci U S A, 1990. 87(18): p. 6934-8. 247. Takeshita, T., et al., An associated molecule, p64, with high-affinity interleukin 2 receptor. Int Immunol, 1990. 2(5): p. 477-80. 248. Takeshita, T., et al., An associated molecule, p64, with IL-2 receptor beta chain. Its possible involvement in the formation of the functional intermediate-affinity IL-2 receptor complex. J Immunol, 1992. 148(7): p. 2154-8. 249. Takeshita, T., et al., Cloning of the gamma chain of the human IL-2 receptor. Science, 1992. 257(5068): p. 379-82. 250. Sugamura, K., et al., The interleukin-2 receptor gamma chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu Rev Immunol, 1996. 14: p. 179-205. 251. Habib, T., et al., The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3. Biochemistry, 2002. 41(27): p. 8725-31. 252. Monteleone, G., F. Pallone, and T.T. MacDonald, Interleukin-21: a critical regulator of the balance between effector and regulatory T-cell responses. Trends Immunol, 2008. 29(6): p. 290-4. 253. Liu, M.F., et al., Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol, 2004. 59(2): p. 198-202.

Table 6: Signal intensity values of genes regulated by hRasGRP4 in non-PMA stimulated HMC-1 cells

150 Table 6: Signal intensity values of genes regulated by hRasGRP4 in non-PMA stimulated HMC-1 cells

Flags: A= transcript is absent; P= transcript is present.

Gene Symbol Probe Set ID Gene Title Mock-GFP Low hRasGRP4+ High hRasGRP4- Raw signal Flags Raw signal Flags Raw signal Flags IL13RA2 206172_at interleukin 13 receptor, alpha 2 2.39 A 166.24 P 2067.64 P IL13 207844_at interleukin 13 10.79 A 83.50 A 11.33 A IL13RA1 211612_s_at interleukin 13 receptor, alpha 1 568.41 P 1044.85 P 688.85 P IL2RG 204116_at interleukin 2 receptor, gamma 7.86 A 9.94 A 8.32 A IL2RB 205291_at interleukin 2 receptor, beta 1600.41 P 1405.47 P 711.02 P IL2RA 211269_s_at interleukin 2 receptor, alpha 26.40 A 12.95 A 57.93 A IL2 207849_at interleukin 2 4.59 A 35.68 P 2.14 A IL21 221271_at interleukin 21 14.48 A 67.56 A 2.77 A RASGRP4 231328_s_at RAS guanyl releasing protein 4 19.94 A 17.12 A 15.74 A RASGRP1 233926_at RAS guanyl releasing protein 1 62.00 P 41.10 A 54.88 A STAT6 201331_s_at signal transducer and activator of transcription 6, interleukin-4 induced 2218.13 P 2287.21 P 1688.48 P CCND2 1565735_at Cyclin D2 51.96 A 25.87 A 65.52 A GATA1 1555590_a_at GATA binding protein 1 (globin transcription factor 1) 586.55 P 158.60 A 11.61 A GATA2 210358_x_at GATA binding protein 2 1638.83 P 1970.08 P 1288.10 P GNAI1 209576_at guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 29.47 A 3238.47 P 2207.21 P IL24 206569_at interleukin 24 1910.73 P 16464.64 P 24305.74 P DOCK2 1554325_at dedicator of cytokinesis 2 4.12 A 5.13 A 3.20 A SAMSN1 1569599_at SAM domain, SH3 domain and nuclear localisation signals, 1 0.85 A 29.71 A 5.75 A STC1 204597_x_at stanniocalcin 1 72.75 A 1207.60 P 152.57 A FGF13 205110_s_at fibroblast growth factor 13 46.23 A 747.52 P 1970.49 P PGDS 206726_at prostaglandin D2 synthase, hematopoietic 3044.21 P 15554.90 P 13336.23 P

151