Anti inflammatory effects of inhibitors of the tyrosine kinase signaling cascade in animal models of asthma

ANTI-INFLAMMATORY EFFECTS OF INHIBITORS OF
THE TYROSINE KINASE SIGNALING CASCADE IN
ANIMAL MODELS OF ASTHMA




Duan Wei
(B.Med.)









A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY
DEPARTMENT OF PHARMACOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2004


ACKNOWLEDGEMENTS

No project is a solo venture. Numerous people, across these 4 years, are a
part of this project. To name just a few:
• My supervisor, Professor Fred Wong, who has picked me up, dusted
me off, and set me back on track of this project more than once. His attitude

and discipline are just some of the lessons that I believe I will carry with me
for the rest of my life.
• Professor Chua Kaw Yan and Professor Chang Chan Fong, who kindly
provided me help and shared with me their experiences.
• My good friends, Yanan, Olivia, Rebecca, Yihan, Constance & Chee
Kiong, Cheng Ann & Christine, Uncle Richard and Aunt Tsu Yeow, and Hock
Chuan for their friendship sharing.
• Thanks also go to National University of Singapore for providing me 4
years scholarship and the staff & postgraduate students of the Department of
Pharmacology.
• Finally, my parents, whose endless love and supports surround me all
the time.

Duan Wei
March 2004

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CONTENTS

ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF FIGURES viii

LIST OF TABLE xii

LIST OF ABBREVIATIONS xiii
LIST OF PUBLICATIONS AND CONFERENCE PAPERS xv
SUMMARY xvii

1. INTRODUCTION 1

1.1. Asthma 2
1.1.1. Pathophysiology of asthma 2
1.1.1.1. Mast cells and acute bronchoconstriction 4
1.1.1.2. Eosinophilia 5
1.1.1.3. T lymphocytes and Th2 cytokines 9
1.1.1.4. B cells and immunoglobulins 15
1.1.1.5. Airway mucus hypersecretion 17
1.1.1.6. Airway hyperresponsiveness (AHR) 18
1.1.2. Therapeutic targets of asthma 20
1.1.2.1. Current therapy for asthma 21
1.1.2.2. Novel therapeutic targets for asthma 22
1.2. Tyrosine kinase signaling cascade 25
1.2.1. Protein tyrosine kinases (PTKs) 25

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1.2.1.1. Role of PTK signaling cascade in allergic
inflammation 28
1.2.2. Mitogen-activated protein kinase (MAPK) cascades 29
1.2.2.1. MAPK 29
1.2.2.2. Role of MAPK cascade in allergic inflammation 31
1.2.3. Phosphoinositide 3 kinase (PI3K) 33
1.2.3.1. PI3K cascade 33
1.2.3.2. Role of PI3K cascade in allergic inflammation 37
1.3. Inhibitors of the tyrosine kinase signaling cascade 38
1.3.1. Protein tyrosine kinase inhibitors 38
1.3.2. MAPK kinase inhibitors 40
1.3.3. PI3K inhibitors 43
1.4. Animal models of asthma 45
2. RATIONALE AND OBJECTIVES 50
3. METHODS AND MATERIALS 52

3.1. Animal sensitization and challenge 53
3.1.1. Guinea pigs 53
3.1.2. Mice 53
3.2. Measurement of bronchoconstriction and AHR 53
3.2.1. Measurement of bronchoconstriction in guinea pigs 55
3.2.2. Measurement of AHR in mice 55
3.3. Bronchoalveolar lavage (BAL) fluid collection and cytospin 57
3.3.1. Collection of BAL fluid from guinea pigs 57
3.3.2. Collection of BAL fluid from mice 58
3.4. ELISA 61

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3.4.1. Cytokines and chemokine measurement in BAL fluid 61
3.4.2. Immunoglobulin measurement in serum 62
3.5. Eosinophil peroxidase (EPO) measurement 62
3.6. Histology 63
3.7. Immunohistochemistry 65
3.8. Western blotting 65
3.8.1. Analysis for phospho-tyrosine residues 65
3.8.2. Analysis for phospho-ERK and phospho-Akt 67
3.9. Data analysis 67
3.10. Materials 67
4. Anti-inflammatory effects of genistein, a tyrosine kinase inhibitor, in a
guinea pig model of asthma 70
4.1. Results 71
4.1.1. Effects of genistein on OVA-induced acute
bronchoconstriction 71
4.1.2. Effects of genistein on OVA-induced airway inflammation 76
4.1.3. Effects of genistein on AHR 81
4.1.4. Phosphotyrosine immunoblot analysis 86

4.2. Discussion 86
4.2.1. Genistein attenuates OVA-induced acute
bronchoconstriction 86
4.2.2. Genistein attenuates OVA-induced airway inflammation 88
4.2.3. Genistein attenuates OVA-induced AHR 89
5. Anti-inflammatory effects of a MAPK kinase inhibitor, U0126, in a
mouse asthma model 91

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5.1. Results 92
5.1.1. Effects of U0126 on OVA-induced eosinophil recruitment
in BAL fluid 92
5.1.2. Effects of U0126 on OVA-induced eosinophil infiltration
and mucus production 92
5.1.3. Effects of U0126 on cytokine levels in BAL fluid 97
5.1.4. Effects of U0126 on serum immunoglobulin levels 97
5.1.5. Effects of U0126 on the expression of VCAM-1 in
lung tissues 103
5.1.6. Effects of U0126 on AHR 103
5.1.7. Phospho-ERK immunoblot analysis 108
5.2. Discussion
5.2.1. U0126 reduces cytokine levels in BAL fluid 108
5.2.2. U0126 attenuates OVA-induced airway and lung tissue
eosinophilia 114
5.2.3. U0126 attenuates OVA-induced mucus production 115
5.2.4. U0126 reduces serum immunoglobulin levels 116
5.2.5. U0126 attenuates OVA-induced AHR 116
6. Anti-inflammatory effects of LY294002, a specific PI3K inhibitor, on
a mouse model of asthma 118
6.1. Results 119

6.1.1. Effects of LY294002 on OVA-induced eosinophil
recruitment in BAL fluid 119
6.1.2. Effects of LY294002 on OVA-induced eosinophil
infiltration and mucus production 119

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6.1.3. Effects of LY294002 on cytokine levels in BAL fluid 123
6.1.4. Effects of LY294002 on expression of VCAM-1 in lung
tissue 123
6.1.5. Effects of LY294002 on AHR 123
6.1.6. Phospho-Akt immunoblot analysis 129
6.2. Discussion
6.2.1. LY294002 reduces cytokine levels in BAL fluid 129
6.2.2. LY294002 attenuates OVA-induced airway and lung
tissue eosinophilia 131
6.2.3. LY294002 attenuates OVA-induced mucus production 132
6.2.4. LY294002 attenuates OVA-induced AHR 133
7. Conclusion 134
8. References 137


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LIST OF FIGURES

Figure Title Page
1.1 Schematic diagram of pathogenesis of asthma 2
1.2 Schematic diagram of protein tyrosine kinase
signaling cascade 27
1.3 Schematic diagram of MAPK signaling cascade 30
1.4 Schematic diagram of PI3K signaling cascade 35

1.5 Structures of PTK inhibitors 39
1.6 Structures of MAPK kinase (MEK) inhibitors 42
1.7 Structures of PI3K inhibitors 44
3.1 Aerosol delivery system 53
3.2 Buxco system 54
3.3 Penh 56
3.4 BAL fluid cells (guinea pig) 59
3.5 BAL fluid cells (mouse) 60
4.1 Representative tracings showing the changes in box
pressure waveform 72
4.2 Inhalation of OVA-induced acute bronchoconstriction
in guinea pigs 74
4.3 Effects of genistein or daidzein on OVA-induced
bronchoconstriction in guinea pigs 75
4.4 Effects of genistein on histamine-induced acute
bronchoconstriction 77

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4.5 Effects of genistein on methacholine-induced acute
bronchoconstriction 78
4.6 OVA-induced BAL fluid eosinophilia in sensitized
guinea pigs 79
4.7 Effects of genistein or daidzein on OVA-induced BAL
fluid eosinophilia in guinea pigs 80
4.8 Effects of genistein on EPO activity in cell-free BAL
fluid in guinea pigs 82
4.9A-D Histologic examination of inflammatory cell infiltration
into the trachea in guinea pigs 83
4.9E-H Histologic examination of inflammatory cell infiltration
into the bronchioles in guinea pigs 84

4.10 OVA-induced AHR to inhaled methacholine in
sensitized guinea pigs 85
4.11 Effects of genistein on OVA-induced AHR to inhaled
methacholine in sensitized guinea pigs 86
4.12 Effects of genistein on EGF-induced protein tyrosine
phosphorylation in guinea pig lung fragments 88
5.1 OVA-induced cell infiltration in sensitized mice 94
5.2 Effects of U0126 on BAL fluid cell infiltration 95
5.3 Effects of U0126 on BAL fluid cell infiltration from
sensitized mice challenged with saline 96
5.4 Effects of U0124 on BAL fluid cell infiltration from
sensitized mice challenge with OVA 97
5.5A-D, I Effects of U0126 on lung tissue eosinophilia 99

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5.5E-H, J Effects of U0126 on mucus production 100
5.6 Effects of U0126 on cytokine levels in BAL fluid 101
5.7 Effects of U0126 on the eotaxin level in BAL fluid 102
5.8 Effects of U0126 on the IFNγ level in BAL fluid 103
5.9 Effects of U0126 on serum total and OVA-specific IgE
production 105
5.10 Effects of U0126 on serum IgG1 and IgG2a production 106
5.11 Effects of U0126 on the expression of VCAM-1 in lung
tissue 107
5.12 OVA-induced AHR in sensitized mice 108
5.13 Effects of U0126 on OVA-induced AHR 110
5.14 Effects of U0126 on methacholine-induced AHR in
sensitized mice challenged with saline aerosol 111
5.15 Effects of U0126 on OVA-induced ERK phosphorylation
in sensitized mouse lung tissues 112

5.16 Effects of U0126 on OVA-induced ERK expression in
sensitized mouse lung tissues 113
6.1 Effects of LY294002 on BAL fluid cell infiltration 121
6.2A-D, I Effects of LY294002 on lung tissue eosinophilia 122
6.2E-H, J Effects of LY294002 on mucus production 123
6.3 Effects of LY294002 on cytokine levels in BAL fluid 125
6.4 Effects of LY294002 on the eotaxin level in BAL fluid 126
6.5 Effects of LY294002 on the IFNγ level in BAL fluid 127
6.6 Effects of LY294002 on the expression of VCAM-1 in
lung tissue 128

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6.7 Effects of LY294002 on OVA-induced AHR to inhaled
methacholine in sensitized mice 129
6.8 Effects of LY294002 on OVA-induced Akt phosphorylation
in sensitized mouse lung tissues 131


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LIST OF TABLE

Table Title Page
I Action of IL-13 on haematopoietic and nonhematopoietic 14
cells

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LIST OF ABBREVIATIONS

AP alkaline phosphatase
APC antigen-presenting cells

BAL bronchoalveolar lavage
BCA bicinchoninic acid
BCIP 5-bromo-4-chloro-3-indoyl-phosphate
BLNK B-cell linker
BSA bovine serum albumin
CCR CC chemokine receptor
CysLTs cysteinyl-leukotrienes
DAG diacylglycerol
DMSO dimethyl sulphoxide
ECP eosinophil cationic protein
EGF epidermal growth factor
EGFR EGF receptor
EPO eosinophil peroxidase
ERK extracellular signal-regulated kinase
FcεRI Fc-epsilon receptor I
FcγR Fc-gamma receptor
FITC fluorescein isothyiocyanate
GAB2 GRB2-associated binding protein 2
GM-CSF granulocyte-macrophage colony-stimulating factor
GPCR G-protein-coupled receptor
ICAM-1 intracellular adhesion molecule 1
Ig immunoglobulin
IL interleukin
IP
3
inositol 1,4,5-triphosphate
IRS1/2 insulin receptor substrate-1/2
ITAMs immunoreceptor tyrosine – based activation motifs
JAK/STAT Janus Kinase/signal transducer and activation of
transcription

LAT linker for activation of T cells
MAPK mitogen-activated protein kinase
MAPKK MAPK kinase
MBP major basic protein
MCP monocyte chemoattractant protein
MHC major histocompatibility complex
MIP-1α macrophage inflammatory protein -1α
NBT nitroblue tetrazolium
OVA ovalbumin
PAF platelet-activating factor
PBS phosphate-buffered saline
PDGF platelet derived growth factor
PDK1 phosphatidylinositol-dependent kinase 1
PI3K phosphoinositide 3-kinase
PKB protein kinase B
PKC protein kinase C
PLC phospholipase C
PMSF phenylmethanesulfonyl fluoride

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PTEN phosphatase and tensin homologue
PtdIns phosphatidylinositol
PtdIns 4-P phosphatidylinositol 4-phosphate
PtdIns 3,4-P
2
phosphatidylinositol 3,4-biphosphate
PtdIns 4,5-P
2
phosphatidylinositol 4,5-biphosphate
PtdIns 3,4,5-P

3
phosphatidylinositol 3,4,5-triphosphate
PTK protein tyrosine kinase
PVDF polyvinylidene difluoride
RANTES regulated on activation, normal T cell expressed and
secreted
RTK receptor tyrosine kinase
SDS sodium dodecyl sulfate
SH src homology
SLP76 SH2-domain-containing leukocyte protein of 76 kDa
TCR T cell receptor
VCAM-1 vascular-cell adhesion molecule 1
VLA-4 very late antigen-4
ZAP70 ξ-chain-associated protein kinase 70 kDa







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LIST OF PUBLICATIONS AND CONFERENCE
ABSTRACTS

Publications

1. Duan W, Kuo IC, Selvarajan S, Chua KY, Bay BH, and Wong WSF.
(2003) Antiinflammatory effects of genistein, a tyrosine kinase inhibitor, on
a guinea pig model of asthma. Am J Respir Crit Care Med. 167(2): 185-

92.
2. Chue SC, Seow CJ, Duan W, Yeo KSL, Koh AHM, and Wong WSF.
(2004) Inhibitor of p42/p44 mitogen-activated protein kinase (MAPK)
kinase, but not p38 MAPK, attenuated antigen challenge of guinea pig
airway in vitro. Intl Immunopharma 4: 1089-1098.
3. Yeo KSL, Zakaria SM, Duan W, Chan SY, Wong WSF. Inhibitors of the
tyrosine kinase signaling cascade attenuated acute atopic dermatitis in
guinea pigs. Submitted, 2003.
4. Duan W, Chan

HP, Leong KP, and Wong WSF. (2004) Anti-inflammatory
effects of a MAPK kinase inhibitor U0126 in a mouse asthma model. J
Immunol 172; 7053-7059.
5. Duan W, Leong KP, Vlahos CJ, and Wong WSF. Anti-inflammatory role of
a phosphoinositide 3-OH kinase inhibitor LY294002 in a mouse asthma
model. accepted, 2005 Intl Immunopharmaco.
Conference Abstracts
1. Duan W, Tsang F, Kuo IC, Chua KY & Wong WSF: Anti-inflammatory
effects of tyrosine kinase inhibitors on guinea pig model of asthma. The

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International Conference on Fundamental Sciences: Biological &
Chemical Sciences, Singapore, P115, 2001.
2. Wong WSF, Duan W, Tsang F, Sung CC, Shoon ML, Low KSY,
Selvarajan S & Bay BH: Anti-inflammatory effects of inhibitors of the
tyrosine kinase signaling cascade on a guinea pig model of asthma.
BioMedical Asia 2001 Singapore, ET-31, 2001.
3. Duan W, Shoon ML, Low SYK, Selvarajan S, Bay BH & Wong WSF: Anti-
inflammatory effects of genistein, a protein tyrosine kinase inhibitor, on an
in vivo guinea pig model of asthma. Pharmacologist 44 (Suppl 1): Abst

134: 27, 2002.
4. Yeo KSL, Duan W, Chan SY, Bay BH & Wong WSF: Quantitative analysis
of atopic dermatitis in guinea pigs: Anti-allergic effects of inhibitors of the
tyrosine kinase signaling cascade. 6th NUS-NUH Annual Scientific
Meeting, Singapore, 2002.
5. Wong WSF, Yeo KSL, Duan W, Chan SY & Bay BH: Quantitative analysis
of atopic dermatitis in guinea pigs: Anti-allergic effects of inhibitors of the
tyrosine kinase signaling cascade. World Congress on Immunopathology,
Singapore. Int’l. J. Immunorehab. 4: Abst 8, 2002.
6. Duan W, Kuo IC, Selvarajan S, Chua KY, Bay BH & Wong WSF: Anti-
inflammatory effects of genistein, a protein tyrosine kinase inhibitor, on an
in vivo guinea pig model of asthma. World Congress on
Immunopathology, Singapore. Int’l. J. Immunorehab. 4: Abst 21, 2002.
7. Duan W, Chan

HP, Leong KP, and Wong WSF. Anti-inflammatory effects
of U0126, a specific MEK inhibitor, in a mouse model of asthma. J. World
Allergy Org. Supple 1, P:5, 2003.

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Summary

Protein tyrosine kinase signaling cascade plays a pivotal role in the activation
of inflammatory cells. Stimulation of nonreceptor tyrosine kinases is the
earliest detectable signaling response upon immunoreceptor activation in
mast cells, T and B cells, eosinophils, and macrophages. It is believed that
src-family kinases (Lyn and Lck) and Syk/ZAP-70 are responsible for the
initial activation of these cells. Subsequently, ERK signaling pathway is also
activated leading to proliferation, differentiation, cytokine production and
degranulation of inflammatory cells. In addition, PI3K signaling pathway is

activated upon T cell, B cell and mast cell activation. There were no studies
on the effects of the intervention of these signaling pathways in animal
models of asthma. The purpose of my thesis was to investigate the anti-
inflammatory effects of genistein, a broad-spectrum protein tyrosine kinase
inhibitor; U0126, a specific MAPK/ERK kinase (MEK) inhibitor, and
LY294002, a specific PI3K inhibitor in asthmatic animal models.
Guinea pigs were actively sensitized by intraperitoneal injections of
ovalbumin. Aerosolized ovalbumin induced acute bronchoconstriction in
conscious animals in a dose-dependent manner. Genistein (15 mg/kg given
intraperitoneally) markedly (p<0.05) inhibited ovalbumin-induced, but not
histamine- and methacholine-induced, acute bronchoconstriction. In addition,
genistein significantly reduced ovalbumin-induced increases in total cell
counts, eosinophil counts, and eosinophil peroxidase activity in
bronchoalveolar lavage fluid, and airway eosinophilia and airway
hyperresponsiveness to inhaled methacholine. Immunoblot analysis of lung
lysates isolated from genistein-pretreated animals showed that epidermal

xvii
growth factor-induced tyrosine phosphorylation in lung tissues was inhibited
by genistein.
Balb/c mice were actively sensitized by intraperitoneal injections of
ovalbumin. Aerosolized OVA challenge induced airway eosinophilia, mucus
hypersecretion, increase in cytokine and chemokine levels, upregulation of
vascular cell adhesion molecule-1 (VCAM-1) expression, and airway
hyperresponsiveness. U0126, given intraperitoneally, markedly (p<0.05)
inhibited OVA-induced increases in total cell counts, eosinophil counts, and
IL-4, IL-5, IL-13 and eotaxin levels recovered in bronchoalveolar lavage fluid
in a dose-dependent manner. U0126 also substantially (p<0.05) reduced the
serum levels of total IgE, OVA-specific IgE and IgG1, whereas the serum
level of OVA-specific IgG2a was not significantly affected. Histological studies

show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia,
airway mucus production as shown by periodic acid-Schiff (PAS) staining,
and expression of VCAM-1 in lung tissues. In addition, U0126 significantly
(p<0.05) suppressed OVA induced airway hyperresponsiveness to inhaled
methacholine in a dose-dependent manner. Western blot analysis of whole
lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine
phosphorylation of ERK1/2.
PI3K family plays a prominent role in a lot of immune cells by controlling
cell growth, cell survival and cell movement through its variety of downstream
target proteins. LY294002, given intratracheally, markedly (p<0.05) inhibited
OVA-induced increases in total cell counts, eosinophil counts, neutrophil
counts, and Th2 cytokines levels recovered in bronchoalveolar lavage fluid in
a dose-dependent manner. Histological studies show that LY294002

xviii
dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus
production as shown by PAS staining, and expression of VCAM-1 in lung
tissues. In addition, LY294002 significantly (p<0.05) suppressed OVA-
induced airway hyperresponsiveness to inhaled methacholine in a dose-
dependent manner. Western blot analysis of whole lung lysates shows that
LY294002 markedly attenuated OVA-induced tyrosine phosphorylation of Akt.
Taken together, the present findings implicate that inhibition of tyrosine kinase
signaling pathway may have therapeutic potential for the treatment of allergic
airway inflammation.


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