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.. LIST OF FIGURES 1.1 Schematic diagram of pathogenesis of a

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

CONTENTS

ACKNOWLEDGEMENTS ii

SUMMARY xvii

1.1.1.1 Mast cells and acute bronchoconstriction 4

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

1.1.2.2 Novel therapeutic targets for asthma 22

1.2 Tyrosine kinase signaling cascade 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

1.2.2.2 Role of MAPK cascade in allergic inflammation 31

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

1.4 Animal models of asthma 45

3.1 Animal sensitization and challenge 53

3.2 Measurement of bronchoconstriction and AHR 53

3.2.1 Measurement of bronchoconstriction in guinea pigs 55

3.3 Bronchoalveolar lavage (BAL) fluid collection and cytospin 57

3.3.1 Collection of BAL fluid from guinea pigs 57

3.4.1 Cytokines and chemokine measurement in BAL fluid 61

3.5 Eosinophil peroxidase (EPO) measurement 62

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

4 Anti-inflammatory effects of genistein, a tyrosine kinase inhibitor, in a

4.1.1 Effects of genistein on OVA-induced acute

4.1.2 Effects of genistein on OVA-induced airway inflammation 76

4.1.4 Phosphotyrosine immunoblot analysis 86

4.2.1 Genistein attenuates OVA-induced acute

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

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5.1 Results 92

5.1.1 Effects of U0126 on OVA-induced eosinophil recruitment

5.1.2 Effects of U0126 on OVA-induced eosinophil infiltration

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

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

6 Anti-inflammatory effects of LY294002, a specific PI3K inhibitor, on

6.1.1 Effects of LY294002 on OVA-induced eosinophil

6.1.2 Effects of LY294002 on OVA-induced eosinophil

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

6.2 Discussion

6.2.1 LY294002 reduces cytokine levels in BAL fluid 129

6.2.2 LY294002 attenuates OVA-induced airway and lung

6.2.3 LY294002 attenuates OVA-induced mucus production 132

6.2.4 LY294002 attenuates OVA-induced AHR 133

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LIST OF FIGURES

1.1 Schematic diagram of pathogenesis of asthma 2

1.2 Schematic diagram of protein tyrosine kinase

1.3 Schematic diagram of MAPK signaling cascade 30

1.4 Schematic diagram of PI3K signaling cascade 35

1.6 Structures of MAPK kinase (MEK) inhibitors 42

4.1 Representative tracings showing the changes in box

4.2 Inhalation of OVA-induced acute bronchoconstriction

4.3 Effects of genistein or daidzein on OVA-induced

4.4 Effects of genistein on histamine-induced acute

bronchoconstriction 77

4.5 Effects of genistein on methacholine-induced acute

bronchoconstriction 78 4.6 OVA-induced BAL fluid eosinophilia in sensitized

4.7 Effects of genistein or daidzein on OVA-induced BAL

4.8 Effects of genistein on EPO activity in cell-free BAL

4.9A-D Histologic examination of inflammatory cell infiltration

4.9E-H Histologic examination of inflammatory cell infiltration

4.10 OVA-induced AHR to inhaled methacholine in

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

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

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

5.14 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

5.16 Effects of U0126 on OVA-induced ERK expression in

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

6.7 Effects of LY294002 on OVA-induced AHR to inhaled

6.8 Effects of LY294002 on OVA-induced Akt phosphorylation

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LIST OF TABLE

I Action of IL-13 on haematopoietic and nonhematopoietic 14

cells

LIST OF ABBREVIATIONS

BCIP 5-bromo-4-chloro-3-indoyl-phosphate

CysLTs cysteinyl-leukotrienes

DAG diacylglycerol

ERK extracellular signal-regulated kinase

FcεRI Fc-epsilon receptor I

GAB2 GRB2-associated binding protein 2

GM-CSF granulocyte-macrophage colony-stimulating factor

ICAM-1 intracellular adhesion molecule 1

Ig immunoglobulin

IL interleukin

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

MHC major histocompatibility complex

MIP-1α macrophage inflammatory protein -1α

OVA ovalbumin

PDGF platelet derived growth factor

PDK1 phosphatidylinositol-dependent kinase 1

PI3K phosphoinositide 3-kinase

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PTEN phosphatase and tensin homologue

PtdIns phosphatidylinositol

PtdIns 4-P phosphatidylinositol 4-phosphate

PtdIns 3,4-P2 phosphatidylinositol 3,4-biphosphate

PtdIns 4,5-P2 phosphatidylinositol 4,5-biphosphate

PtdIns 3,4,5-P3 phosphatidylinositol 3,4,5-triphosphate

RANTES regulated on activation, normal T cell expressed and

secreted

SLP76 SH2-domain-containing leukocyte protein of 76 kDa

VCAM-1 vascular-cell adhesion molecule 1

ZAP70 ξ-chain-associated protein kinase 70 kDa

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, ChanHP, 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, ChanHP, 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

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

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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

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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