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

It is attributable to the coordinated and sustained activation of inflammatory cells including mast cells, T-helper 2 cells, B cells, macrophages and eosinophils, and synthesis of a vari

1 INTRODUCTION

1.1 Asthma

1.1.1 Pathophysiology of asthma

Allergic asthma is a chronic airway disorder characterized by airway inflammation, mucus hypersecretion and airway hyperresponsiveness (Busse and Lemanske, 2001) (Figure 1.1) It is attributable to the coordinated and sustained activation of inflammatory cells including mast cells, T-helper 2 cells, B

cells, macrophages and eosinophils, and synthesis of a variety of

pro-inflammatory mediators (Maddox and Schwartz, 2002; Hamid et al., 2003) Acute

bronchoconstriction is triggered by the release of bronchoconstrictors including

histamine, cysteinyl-leukotrienes (CysLTs) and platelet-activating factor (PAF) from mast cells upon allergen-induced cross-linking of IgE-bound high-affinity Fc

receptors (FcεRI) (Busse and Lemanske, 2001) Airway inflammatory responses

are contributed by T-helper type 2 cells (Th2 cells), together with other inflammatory cells such as mast cells, B cells and eosinophils, and inflammatory

cytokines and chemokines (Busse and Lemanske, 2001; Herrick and Bottomly,

2003) Upon activation, Th2 cells produce cytokines such as IL-4, IL-5 and IL-13

IL-4 is essential for B cell maturation and IgE synthesis, and plays an important

role in the initiation of Th2 inflammatory responses (Li-Weber and Krammer, 2003) IL-5 is pivotal for the growth, differentiation, recruitment and survival of

eosinophils (Greenfeder et al., 2001) IL-13 plays a prominent role in the effector

phase of Th2 responses, such as eosinophilic inflammation, mucus secretion,

and AHR (Wynn, 2003; Taube et al., 2002; Hershey 2003) On the other hand,

Leukotriene C 4 , PAF

Histamine Tryptase IL-4, IL-5, etc

Late Responses Airway inflammation AHR

Mucus Hypersecretion Edema

IL-3,IL-5,

GM-CSF,

RANTES

MBP, ECP Leukotrienes, PAF IL-4, IL-5, GM-CSF MIP-1 α, RANTES TGF α, PDGF Eosinophils

APC

Y

IL-4 IL-13

eotaxin

chemokines such as RANTES and eotaxin are central to the delivery of eosinophils to the airways The specific transendothelial migration of eosinophils

is regulated by the interaction of adhesion molecules such as VLA-4 and its ligand VCAM-1 (Lukacs, 2001) Airway eosinophilia together with effector cytokines such as IL-13 may ultimately contribute to AHR in asthma (Wills-Karp,

1999)

1.1.1.1 Mast cells

Mast cells are derived from bone marrow and enter the circulation as CD34+

mononuclear cells They then migrate to mucosal and submucosal sites in the

airway, and undergo tissue-specific maturation which depends on the T cell- derived IL-4 (Busse and Lemanske, 2001)

Inhaled allergen enters the body via airway mucosal surfaces, and is taken

up by antigen-presenting cells (APCs) These APCs then migrate to draining lymph nodes, where they present the processed antigen to T and B cells

Interactions among these cells elicit responses that are influenced by cytokines

and the presence or absence of costimulatory molecules IL-4 and IL-13 provide

the first signal to B cells to switch to the production of the IgE isotype The second signal is delivered when CD40 on B cells binds to its ligand on T cells

Once formed, IgE antibody circulates in the blood and eventually binds to

high-affinity IgE receptors (FcεRI) on mast cells thus sensitizing them (Busse and Lemanske, 2001)

Crosslinking of FcεRI with IgE and antigen is the triggering event of the activation of protein-tyrosine kinases (PTK), including those of the Src, Syk and

Tec families (Scharenberg and Kinet, 1994; Vangelista, 2003) Current understanding of the activation sequence is that Lyn-a Src-family PTK that is expressed predominantly in mast cells and is associated constitutively with the β-

subunit of FcεRI is activated by FcεRI aggregation and then phosphorylates tyrosine residues in the immunoreceptor tyrosine – based activation motifs (ITAMS) of the β- and γ-subunits of the receptor Phosphorylated ITAMs of the β-

and γ-subunits recruit additional Lyn and Syk, respectively, through interactions

with the Src-homology 2 (SH2) domains encoded in the PTKs Syk is then activated through conformational change and tyrosine phosphorylation by Lyn

Active Syk then phosphorylates many substrates downstream, including LAT (linker for activation of T cells), SLP76 (SH2-domain-containing leukocyte protein

of 76 kDa) and VAV, which leads to the activation of several signalling pathways,

such as those through PI3K, phospholipase Cγ (PLCγ), and MAPK

(Sanchez-Mejorada and Rosales, 1998; Kinet, 1999) The activation of these pathways

leads eventually to mast cell degranulation, synthesis and release of lipid mediators (e.g CysLTs and PAF), and the production and secretion of cytokines,

chemokines and growth factors, which cause immediate bronchoconstriction, mucosal edema and hypersecretion (Busse and Lemanske, 2001)

1.1.1.2 Eosinophilia

The eosinophil is the principal effector cell for the pathogenesis of allergic airway

inflammation via the secretion of inflammatory mediators such as leukotrienes

and granule products including reactive oxygen species and cytotoxic granule

(ECP), eosinophil peroxidase, and eosinophil-derived neurotoxin, as well as cytokines and chemokines (Giembycz and Lindsay, 1999)

Eosinopoiesis begins in the bone marrow and is regulated by IL-3, IL-5 and

granulocyte-macrophage colony-stimulating factor (GM-CSF) (Giembycz and Lindsay, 1999) IL-5 is critical for regulating the growth, activation, and survival of

eosinophils and cooperates with eotaxin to selectively regulate tissue eosinophilia (Adachi and Alam, 1998; Choi et al., 2003) IL-5 not only induces

terminal differentiation of immature eosinophils (Yamaguchi et al., 1988a) but also stimulates the release of eosinophils into the circulation and prolongs their

survival (Yamaguchi et al., 1988b; Palframan et al., 1998) Moreover, IL-5 has

been shown to play an important role in mediating eosinophil adhesion (Sanmugalingham et al., 2000) and migration (Schweizer et al., 1996) IL-5 exerts its actions by binding to IL-5 receptor on the cell surface The receptor for

5 belongs to the hematopoietin receptor superfamily and is comprised of an

IL-5-specific α chain and the common β chain that is shared with IL-5, IL-3 and

GM-CSF for signal transduction (Adachi and Alam, 1998) To date, there are at least

3 principal signaling pathways that have been described upon IL-5 receptor activation on eosinophils: the Janus kinase (JAK)/signal transducer and activation of transcription (STAT) pathway, the MAPK pathways, and PI3K pathway (Martinez-Moczygemba and Huston, 2003) IL-5 receptor binding leads

to activation of the receptor-associated JAK2 kinase (Quelle et al., 1994), STAT1

and STAT5 (Adachi and Alam, 1998) and Src family of kinases, such as Lyn

(Pazdrak et al., 1995; Yousefi et al., 1996), Syk (Yousefi et al., 1996), and Btk

(Sato et al., 1994) It has been demonstrated that Lyn, Syk and JAK2 are important for eosinophil survival (Yousefi et al., 1996; Ishihara et al., 2001) However, Lyn and JAK2 appear to have no role in eosinophil degranulation or

expression of surface adhesion molecules whereas Raf-1 kinase has been shown to be critical for eosinophil degranulation and adhesion molecule expression (Pazdrak et al., 1998) This is consistent with the studies showing the

involvement of Ras-Raf-1-MEK-MAP kinase pathway in the IL-5 induced intracellular signal transduction (Pazdrak et al., 1995; Coffer et al., 1998) and

survival (Hall et al., 2001) in eosinophils PI3K has been shown to be involved in

IL-5 stimulated eosinophil mobilization for the bone marrow (Palframan et al., 1998)

Eosinophil transmigration into the airways is a multistep process that is orchestrated by Th2 cytokines such as IL-4, IL-5 and IL-13, and coordinated by

specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM-1 and VLA-4 (Busse and Lemanske, 2001; Lukacs, 2001; Jia et

al., 1999) Cell rolling, which is mediated by P-selectin on the surface of eosinophils is the first step in this process Cell rolling activates eosinophils and

requires the participation of the β1 and β2 classes of integrins on the eosinophil

surface (Busse and Lemanske, 2001) Eosinophils express the α4β1 integrin (also

known as very late antigen-4, VLA-4), which binds to its ligand, VCAM-1 on the

endothelium (Nagata et al., 1995; Matsumoto et al., 1997; Yamamoto et al., 1998) Interactions between the β2 integrins on eosinophils and intracellular adhesion molecule 1 (ICAM-1) on vascular endothelium also appear to be

important for the transendothelial migration of eosinophils (Yamamoto et al., 1998; Jia et al., 1999)

The chemokines such as RANTES, macrophage inflammatory protein 1α

(MIP-1α), and the eotaxins are central to the delivery of eosinophils to the airway

(Lukacs, 2001) Eotaxin was distinguished from all other chemokines because it

was found to be a potent eosinophil-selective chemoattractant and activator (Elsner et al., 1996; Palframan et al., 1998; Rothenberg, 1999; Conroy and Williams, 2001; Pease and Williams, 2001) Eotaxin was initially discovered to be

potent in stimulating eosinophils in vitro and in vivo in guinea pigs

(Griffith-Johnson et al., 1993; Jose et al., 1994) Subsequently, it has been cloned in

other species such as mice (Gonzalo et al., 1996), rats (Williams et al., 1998; Ishi

et al., 1998) and human beings (Ponath et al., 1996; Garcia-Zepeda et al., 1996;

Kitaura et al., 1996) and, meanwhile, two more functional homologues of eotaxin

have been termed eotaxin-2 (Forssmann et al., 1997; White et al., 1997) and

eotaxin-3 (Shinkai et al., 1999; Kitaura et al., 1999), although they lack sequence

similarity to eotaxin Eotaxin has been shown to be synthesized by many cell

types in the lung, including airway epithelial cells, airway smooth muscle cells,

vascular endothelial cells and macrophages, as well as eosinophils themselves

(Humbles et al., 1997; Ying et al., 1997; Lamkhioued et al., 1997) In line with the

study which shows that eotaxin production is T-cell-dependent in a mouse asthma model (Maclean et al., 1996), Th2 cytokines such as IL-4 and IL-13 have

been shown to induce eotaxin production Sanz and co-workers showed that intradermal IL-4 induced eosinophil accumulation in the rat was mediated partly

by endogenously generated eotaxin (Sanz et al., 1998) Similarly, eotaxin mRNA

expression in a lung granulomas model was inhibited by an anti-IL-4 antibody

(Ruth et al., 1998) On the other hand, IL-13 has been shown to be more potent

than IL-4 in inducing eotaxin expression by lung epithelial cells and promoting

lung eosinophilia in vivo (Li et al., 1999) as well as induces mucus hypersecretion, subepithelial fibrosis and bronchial hyperreactivity (Zhu et al., 1999) The eotaxins signal exclusively via a single receptor, CCR3, which accounts for eotaxin’s cellular selectivity (Kitaura et al., 1996; Ponath et al., 1996;

Daugherty et al., 1996) CCR3 is a seven-transmembrane-spanning

G-protein-linked receptor (Sallusto et al., 2000; Fernandez and Lolis, 2002) primarily expressed on eosinophils (Ponath et al., 1996), basophils (Uguccioni et al., 1997), mast cells (Romagnani et al., 1999), and a subpopulation of Th2 cells

(Sallusto et al., 1997) The binding of eotaxin to CCR3 receptor induces a series

of biochemical changes (Mellado et al., 2001), including activation of Gi proteins,

transient calcium mobilization (Ponath et al., 1996; Kitaura et al., 1996; Daugherty et al., 1996), MAPK activation (Alam et al., 1999; Boehme et al., 1999;

Kampen et al., 2000; Tachimoto et al., 2002), and actin polymerization (Boehme

et al., 1999) that is associated with chemotaxis and granule release

1.1.1.3 T cells and Th2 cytokines

Cumulative evidence shows that T-helper type 2 cells (Th2 cells) are the main

orchestrators of allergic airway inflammation (Herrick and Bottomly, 2003; Larche

et al., 2003) T cells arise from bone marrow-derived progenitor cells that

presentation of a foreign antigen peptide by activated dendritic cells (DC), thymocytes start to secrete IL-2, then undergo rapid proliferation and differentiation Thymocytes differentiate into phenotypically distinct types of T cells based on the specificity of the T cell receptor (TCR) for antigen (Werlen et

al., 2003) Thymocytes expressing a TCR specific for major histocompatibility complex (MHC) class I differentiate into CD8 cytotoxic T cells and thymocytes

expressing a TCR specific for MHC class II differentiate into CD4 helper T cells –

a process known as CD4/CD8 lineage commitment (Sezda et al., 1999; Hedrick,

2002; Kioussis, 2002) The duration of activation of both Ca2+

/calmodulin-dependent calcineurin and the ERK pathway appears to be crucial for CD4/CD8

lineage commitment (Adachi and Iwata, 2002) T helper cells (Th cells) further

differentiate into Th1, characterized by the secretion of IL-12 and interferon (IFN)-γ, and Th2 cells, which were characterized by the secretion of IL-4, IL-5

and IL-13 (Rogge, 2002; Gor et al., 2003) Th cell differentiation can be driven in

vitro by stimulating unpolarized T cells with antigen or other TCR ligands in the

presence of appropriate cytokines (IL-12 for Th1 and IL-4 for Th2), suggesting

that it is the combination of TCR and cytokine stimulation act in synergy to induce

cellular differentiation (Ansel et al., 2003) Transcription factor T-bet and GATA-3

appear to be the key regulators of Th1 and Th2 differentiation, respectively (Ansel et al., 2003)

T cell development and differentiation share a common requirement for signals emanating from the TCR (Werlen et al., 2003) TCR complex consists of

ligand-binding αβ chains, signal-transducing CD3 molecule (γε dimer and δε

dimer) and ξ chain dimer TCR activation results in tyrosine phosphorylation of

ITAMs located in the CD3 molecule by Lck and Fyn, two Src family kinases Phosphorylated ITAMs then recruit and activate ZAP-70, a member of the Syk

family kinase Subsequently, these activated tyrosine kinases phosphorylate a

plethora of downstream signaling molecules such as PLCγ1 and adaptor molecules such as linker for activation of T cells (LAT) and SLP-76, which then

activate downstream signaling pathways such as PI3K and MAPK pathways for

effector responses (Hussain et al., 2002; Nel, 2002; Samelon, 2002; Wong and

Leong, 2003)

Th2 cells mainly contribute to asthma pathophysiology by producing an array of Th2 cytokines such as IL-4, IL-5 and IL-13 (Romagnani, 2001; Hedrick,

2002)

IL-4 plays an important role in the initiation of Th2 inflammatory responses

(Herrick and Bottomly, 2003) IL-4 induces B cell growth, differentiation and secretion of immunoglobulin (Ig) E and IgG4 (IgG1 in the mouse) It has been

shown to be the most potent cytokine mediating IgE synthesis (Finkelman et al.,

1988; Pene et al., 1988) In addition, IL-4 has been shown to be able to induce

the rolling on and adhesion to endothelial cells of circulating eosinophils (Bochner and Schleimer, 1994) IL-4 blocking antibodies inhibit allergen-induced

AHR, goblet cell metaplasia, and pulmonary eosinophilia in a murine model of

asthma (Gavett et al., 1997) Th2 cells are the main sources of IL-4 production,

although various other cells including basophils, mast cells as well as eosinophils

also produce IL-4 (Dubucquoi et al., 1994; Seder, 1994) IL-4 exerts its effect

through its IL-4R complex which consists of the IL-4Rα and the common gamma

chain (γc) IL-4Rα binds to IL-4 with high affinity and heterodimerizes with a second chain (γc or IL-13Rα1) to produce biological effects γc Chain only modestly increases the affinity of the IL-4R complex for IL-4, yet it is required for

the activation of IL-4R (Nelms, 1999) IL-4Rα chain also functions as a component of the IL-13 receptor (IL-13R), which may explain the overlap effects

between these two Th2 cytokines (Obiri et al., 1995; Miloux et al., 1997; Murata

et al., 1998) Neither the IL-4Rα nor the γc chain has endogenous kinase activity;

therefore, like other members of the hematopoietin receptor family, IL-4R requires receptor-associated kinases for the initiation of signal transduction (Nelms, 1999) The IL-4Rα chain cytoplasmic region has three functionally distinct domains: (a) an interaction domain for JAK: IL-4Rα chain is usually associated with JAK1 while γc chain is associated with JAK3 (Miyazaki et al., 1994; Russell et al., 1994); (b) a domain containing conserved Tyr residue for

activation of proliferation pathways (Deutsch et al., 1995); and (c) the domain

comprises sequences from C-terminal to residue 557 which is critical for transducing signals leading to activation of IL-4-induced gene expression (Ryan

et al., 1996) The engagement of IL-4R activates signaling pathways to elicit

IL-4-induced diverse biological effects PI3K and MAPK pathways have been observed to be involved in IL-4-induced cellular proliferation (Hershey, 2003) IRS-1/2 (insulin receptor substrate-1/2) signaling pathway has been observed to

be upstream of these two pathways in IL-4-induced cellular proliferation (Wang et

al., 1993; Sun et al., 1995) Inhibiting PI3K by Wortmannin blocked the ability of

IL-4 to prevent apoptosis in haematopoietic cells through the production of phosphoinositides and the subsequent activation of kinases critical for cell survival (Zamorano et al., 1996) Nevertheless, MAPK pathway activation by IL-4

may depend on cell type since IL-4 induced MAPK activation has only been shown in certain cell types (Wery et al., 1996) while not in others (Welham et al.,

1994) STAT-6 is the primary STAT activated in response to IL-4 stimulation and

acts as a direct connection between IL-4 receptor and the transcription apparatus

(Jiang et al., 2000) Upon the IL-4R engagement, JAK1 and JAK3 are activated

and specific tyrosine residues in the receptor cytoplasmic region are phosphorylated STAT-6 is then recruited to the phosphorylated receptor through

its SH2 domain, enabling the activated kinases to phosphorylate STAT-6 at a

C-terminal tyrosine residue (Mikita et al., 1996) Once phosphorylated, the STAT-6

disengages from the receptor and forms homodimers The dimerized STAT-6 complex is translocated to the nucleus where they bind to specific DNA motifs in

the promoter of responsive genes (Ihle, 1996; Nelms, 1999)

IL-5 plays an essential role in eosinophil growth, differentiation, activation

and survival The functional role of IL-5 in allergic asthma has been described in

details in section 1.1.1.2 Please refer to that section for IL-5 functions

IL-13 plays a prominent role in the effector phase of Th2 responses, such as

eosinophilic inflammation, mucus secretion, and AHR by activating a wide variety

of cell types that are relevant to the pathogenesis of asthma (Grunig et al., 1998;

Taube et al., 2002; Wynn, 2003), as shown in Table 1 These alterations are the

result of 13 binding to the multimeric 13 receptor (which is made up of

IL-Table 1 Actions of IL-13 on haematopoietic and nonhematopoietic cells

Human B cells promoting B-cell proliferation

inducing class switching to IgG4 and IgE

inducing expression of the FcεRII and MHC class II

Oettgen et al., 2001

Chomarat and Banchereau, 1998 Monocytes

and

macrophages

enhancing the expression of integrins, including CD11b, CD11c, CD18, and CD29 inducing MHC class II and CD23 expression

Inhibiting the production of prostaglandins,

reactive oxygen, nitrogen intermediates, and IL-1, IL-6, IL-8, TNF-α, and IL-

12

Zurawski and de Vries, 1994

De Vries, 1998

Endo et al., 1996 Doherty et al., 1993 Sozzani et al., 1995

De Vries, 1998 Eosinophils promoting eosinophil survival,

activation, and recruitment

Horie et al., 1997 Luttmann et al., 1996 Pope et al., 2001 Mast cells activating mast cells

promoting IgE synthesis

Wills-Karp, 2001

Fibroblasts inducing type I collagen

synthesis

Roux et al., 1994 Epithelial cells potently inducing eotaxin

expression altering mucocilliary differentiation resulting in goblet cell metaplasia

Li et al., 1999 Laoukili et al., 2001 Zhu et al., 1999

4Rα, IL-13Rα1 and IL-13Rα2) IL-13 has two cognate receptors, IL-13Rα1

and IL-13Rα2 (Hershey, 2003) IL-13Rα1 binds to IL-13 with low affinity by itself,

while binds to IL-13 with high affinity when paired with IL-4Rα and forms a functional IL-13 receptor (Miloux et al., 1997; Wynn, 2003) This receptor complex is also utilized by IL-4, serving as an alternative receptor to IL-4 (Hershey, 2003) In vitro studies show that IL-13Rα2 might be a decoy receptor,

which downregulates IL-13 signaling (Donaldson et al., 1998; Kawakami et al.,

2001; Daines et al., 2003) Since IL-4 and IL-13 share common subunits of receptors, they share signaling pathways accordingly (Welham et al., 1995) The

binding of IL-13 to IL-13R complex induces the activation of JAK1 and Tyk2 Activated JAKs phosphorylate the cytoplasmic tyrosines in IL-4Rα, which then

recruit STAT6 to the receptor, followed by STAT6 phosphorylation and activation

(for details of STAT6 activation please refer to IL-4 signaling pathways)

1.1.1.4 B cells and immunoglobulins

B cells mainly contribute to pathogenesis of asthma by producing immunoglobulins, and IgE has been associated with mast cell activation and AHR in humans (Kalesnikoff et al., 2001; Oettgen and Geha, 2001)

B cell development occurs through several discrete stages and at many anatomical locations including bone marrow, fetal liver, peritoneum, and spleen

(Hardy and Hayakawa, 2001) B-cell receptor (BCR) instructs B cells development and mediates the response to the antigen (Niiro and Clark, 2002)

The BCR complex is made up of antigen-binding component membrane

Igα (CD79a) and Igβ(CD79b) (Gauld et al., 2002) After BCR ligation by antigen,

Src-family kinase Lyn is activated Lyn then phosphorylates ITAMs in the cytoplasmic tails of Igα and Igβ, which recruit and activate of Syk and the Tec-

family kinase Btk (Niiro and Clark, 2002) Some non-enzymatic adaptors, such as

B-cell linker (BLNK) (Fu et al., 1998), B-cell adaptor for PI3K (BCAP) (Okada et

al., 2000), and B-lymphocyte adaptor molecule of 32 kDa (BAM32) (Niiro et al.,

2002), fine-tune BCR signals by efficiently connecting the kinases with the effectors Phospholipase Cγ2 (PLCγ2) and PI3K are two important downstream

effectors of BCR signaling (Marshall et al., 2000; Niiro and Clark, 2002) Btk,

together with Syk, phosphorylates and activates PLCγ2 Activation of PLCγ2

leads to the release of intracellular Ca2+ and activation of PKC, which subsequently induce the activation of MAPKs, and transcription factors, including

NFκB and nuclear factor of activated T cells (NFAT) (Niiro and Clark, 2002) PI3K

phosphorylates phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to produce phosphatidylinositol-3,4,5-triphosphate (PtdIsnP3), which recruits some BCR signalling molecules to the membrane through pleckstrin homology (PH) domains

and activates downstream kinases such as Akt (Okkenhaug and Vanhaesebroeck, 2003) The Vav family of Rho-family GTPase, is also critical for

BCR signalling Vav activates RAC1 and regulates cytoskeletal structures and

BCR-induced proliferation Vav might function both upstream and downstream of

PI3K in B cells (Gauld et al., 2002; Niiro and Clark, 2002)

1.1.1.5 Airway mucus hypersecretion and goblet cell hyperplasia

Airway mucus hypersecretion is a prominent feature of asthma Excessive production of mucus causes plugging in the airways that might lead to airway

obstruction (Lundgren and Shelhamer, 1990) Mucus hypersecretion is a complex pathologic process that involves goblet cell hyperplasia and degranulation, microvascular remodeling and leakage, and chemoattraction of inflammatory cells (Fahy, 2002) The major constituents of airway mucus are

termed as mucins Mucins are large complex molecules consisting of a peptide

backbone and numerous oligosaccharide side chains which represent the products of mucin genes (MUC genes) and glycosyltransferase genes, respectively (Fahy, 2002) At least 12 human MUC have been identified, 7 of

which are expressed in human airways (Fahy, 2002) Nevertheless, only MUC5AC and MUC5B have been convincingly shown to be the major mucins

secreted in the airway (Chen et al., 2001; Fahy, 2002)

A variety of inflammatory mediators, including histamine, leukotrienes, and

PAF have been shown to stimulate mucus secretion (Lundgren and Shelhamer,

1990; Nadel, 1991; Cohn et al., 1999) CysLTs has been shown to be important

for both early-phase (1 hour after the allergen challenge) and late-phase (6 hour

after allergen challenge) in a rat asthma model (Shimizu et al., 2003) Histamine

is mainly involved in the early-phase mucus secretion through the H1-receptor of

cholinergic nerve terminals, whereas infiltrating cells (eosinophils and neutrophils) play a more critical role in late-phase mucus secretion (Shimizu et

al., 2003) On the other hand, Th2 cytokines, such as IL-4 (Temann et al., 1997),

IL-9 (Temann et al., 1998) and IL-13 (Zhu et al., 1999), have been shown to

influence mucus secretion In a mouse asthma model, it has been shown that

IL-4 Rα is essential for Th2-induced airway mucus production while IL-5, eosinophils, and mast cells are not critical for the mucus production (Cohn et al.,

1999) Epidermal growth factor receptor (EGFR) and its ligand have also been

found to have a role in mucus production in asthma Selective inhibitors of EGFR

tyrosine kinase block mucus production both in vivo and in vitro (Takeyama et al.,

1999) EGFR inhibitors also blocked IL-13-induced MUC gene expression in rat

airways and epithelial cell proliferation in cultured bronchial epithelial cells (Booth

et al., 2001; Shim et al., 2001) The mechanism by which inflammatory stimuli

induce mucus hypersecretion in the airways is still uncertain The finding that

activation of NF-κB via a c-Src-Ras-MEK1/2-MAPK-pp90rsk signaling pathway

binding to a κB site in the 5’-flanking region of the MUC2 gene and activating

MUC2 mucin transcription may represent one of the potential mechanisms (Li et

al., 1998)

1.1.1.6 Airway hyperresponsiveness (AHR)

AHR is a characteristic feature of asthma and defined as an increased sensitivity

of the airways to an inhaled constrictor agonist shown as a steeper slope of the

dose-response curve (Vargaftig, 1997; O’Byrne and Inman, 2003)

Activated by inhaled antigen presentation, CD4+ T cells in the lungs produce

Th2 cytokines, such as IL-4, IL-5, and IL-13, which orchestrate the infiltration and

activation of effector cells such as mast cells and eosinophils Subsequently,

such effector cells release a plethora of inflammatory mediators including histamine, LTs, PAF, eosinophils-derived basic proteins, and proteases to the

airway epithelium These inflammatory mediators, individually or in combination

induce acute bronchoconstriction, airway wall edema, airway epithelial desquamation, altered neural regulation of airway tone, increased mucus production, and increased smooth muscle content Each of these inflammatory

responses might contribute to AHR, although most likely they influence in concert

(Wills-Karp, 1999)

Experimental animal models have provided direct evidence of a causal role

for CD4+ T cells in the development of antigen-induced AHR Depletion of CD4+

T cells in sensitized mice prior to local lung antigen challenge with specific monoclonal antibodies prevented the development of allergen-induced allergic

airway responses (Gavett et al., 1994) Furthermore, it has been shown that adoptive transfer of Th2 clones into lungs results in AHR in nạve mouse (Li et

al., 1996)

On the other hand, study from IL-13-deficient mice has confirmed the importance of the IL-13, IL-4Rα, and STAT6 in the induction of AHR and suggest

that IL-13 was, by itself, necessary and sufficient to induce AHR (Walter et al.,

2001) Nevertheless, there is study using IL-13 -/- mice showing that it is possible

to develop AHR and pulmonary eosinophilia in the absence of IL-13 AHR was

reduced in IL-13 -/- mice only when they were treated with either IL-4 or

anti-IL-5 mAbs, which suggests the cooperation among Th2 cytokines in inducing

AHR (Webb, 2000)

Increased production of IgE has been associated with the development AHR but depends on sensitization and challenge protocols Under conditions in

which limited IL-5-mediated eosinophilic airway infiltration is induced, IgE plays

an important role in AHR development whereas in conditions where a robust

eosinophilic inflammation of the airways is elicited, IgE does not appear to be

essential for the development of AHR (Hamelmann et al., 1999)

Considerable evidence suggests the association between pulmonary eosinophil infiltration and AHR in asthma (Wills-Karp, 1999) Eosinophils are postulated to induce AHR through releasing eosinophil-derived proteins such as

MBP and ECP on the airway wall These proteins are cytotoxic to the airway

epithelium Damage of the airway epithelium may lead to AHR by removing enzymes important in the degradation of neuropeptides and/or in the loss of epithelial-derived relaxing factor (Gundel, 1991) MBP may also induce AHR through its competitively inhibitory binding of M2 receptors to acetylcholine autoreceptors on parasympathetic nerves that may result in increased release of

acetylcholine (Jacoby, 1993)

1.1.2 Therapeutic targets of asthma

Currently available therapy for asthma, which generally based on use of inhaled

β2-agonist bronchodilators together with inhaled corticosteroids, is able to control

the majority of patients However, important advances are still needed to improve

long-term therapy for patients with more severe persistent asthma In the future,

there is the prospect of cheaper and safer therapies causing disease modification

and even cure (Corry, 2002)

1.1.2.1 Current therapy for asthma

β2-Agonist bronchodilators (e.g salbutamol (short-acting); salmeterol and formoterol (long-acting)) are by far the most effective palliative therapies for asthma because they relieve suffering β2-Agonists work as functional antagonists on airway smooth muscle However, they have no effect on chronic

inflammation and, therefore they cannot cure the disease In addition, there is no

convincing evidence that a bronchodilator can impede disease progression in asthma (Anderson and Rabe, 2001)

Inhaled corticosteroids (CS) are the most effective drugs available to clinicians for the treatment of asthma (Barnes et al., 1998) CS improves lung

function, reduces airway inflammation, AHR, and asthma attacks or exacerbations CS is able to penetrate the cell membrane passively and bind to

its intracellular CS receptor to form a complex This binding results in dissociation

of heat shock proteins from the receptor and exposure of nuclear localization

sequence, allowing the complex to penetrate into the nucleus and bind to specific

regions on DNA-glucocorticoid responses elements (GRE) and/or

negative-glucocorticoid response elements (nGRE) Thus, GRE-bound GR homodimers

facilitate the corresponding gene mRNA production, the mechanism called transactivation, while nGRE-bound GR inhibite the gene mRNA production, the

mechanism called transrepression However, the use of CS has been associated

with dose- and time-dependent side effects Inhaled CS can give rise to oral candidiasis and dysphonia In severe asthmatics, CS are given systemically which may lead to side effects including hypertension, psychological disorders

such as insomnia and agitation, increased susceptibility to infection, easy skin

bruising and slow wound healing, weight gain, osteoporosis, blood sugar elevation which leads to or worsen diabetes, increased incidence of cataract, muscular weakness, growth retardation in children, etc

In the future, new steroids with a more selective anti-inflammatory profile without possessing adverse effects are expected Currently, prodrug steroids, soft steroids and dissociated steroids all have exciting potential to achieve this

aim (Dahl and Nielsen, 2001)

The leukotrienes (LTs) are eicosanoids derived from membrane constituent

arachidonic acid The cysteinyl LTs, LTC4, LTD4 and LTE4 are potent airway smooth muscle constrictors with a much longer duration of action than other smooth muscle constrictors LTB4 has minimal bronchoconstrictor effects, but is a

potent neutrophil chemoattractant The cysteinyl LTs transduce their activity through the CysLT1 receptor, while LTB4 does so through the BLT receptor The

CysLT antagonists (e.g montelukast, pranlukast, and zafirludkast, etc.) and LTB4

antagonists (e.g LY293111, CGS-25019C, and SB209247, etc.) have now been

extensively evaluated in clinical trials (O’Byrne and Drazen, 2001)

1.1.2.2 Novel therapeutic targets for asthma

Improved understanding on the cellular and molecular basis of asthma has identified more potential candidates for drug development Specifically, together

with recent discoveries on Th2 cell function and associated signaling pathways, it

is now possible to investigate the potential therapeutic role of molecules that

control Th2 response signaling pathways in the lung (Handsel and Barnes, 2001;

Corry, 2002)

Mediator inhibitors such as H1-antihistamines are still of interest in addition

to corticosteroids, β2-agonists, and leukotriene antagonists (De Vos and Rihoux,

2001) Second-generation H1-antihistamines have almost eliminated the central

side effect-sedation compared with the first-generation, yet it brings in new safety

issues such as cardiac toxicity and interference with hepatic enzymatic complex

cytochromes P-450 (Hamelin et al., 1998; Nicolas et al., 1999) Therefore, new

generation of H1-antihistamines with more effectiveness and specificity are wanted

Protease inhibitors, such as tryptase inhibitors, have been shown to reduce

antigen-induced airway inflammation and AHR in guinea-pig (Wright et al., 1999),

sheep (Clark et al., 1995; Wright et al., 1999), and mouse (Oh et al., 2002) asthma models In phase II clinical trials, APC 366, a first-generation small molecule inhibitor of tryptase, has demonstrated efficacy in patients with mild to

moderate asthma (Rice et al., 1998) Therefore, continued development of more

specific and selective tryptase inhibitors are expected as novel treatment of asthma

The anti-IgE therapy, which is based on the important role of IgE plays in

human asthma, has been rationalized for years (Chang, 2000) To date, a humanized murine anti-IgE antibody, rhuMAb-E25, has been proven safe and

effective in reducing serum free IgE levels, the number of IgE receptors expressed on the surface of basophils, the clinical severity of asthma, the

frequency of exacerbations, and the corticosteroids requirements of patients with

moderate or severe asthma, although it does not cure asthma (Chang, 2000)

The anti-IgE works by binding to Fc portion of IgE to form immune complexes

which no longer can bind to FcεRI Since IgE is continuously synthesized, repeated anti-IgE dosing appears to be necessary to maintain the effect So far

the long –term effects of anti-IgE are uncertain (Chang, 2000)

Inhibition of cytokines (e.g IL-4, IL-5, and IL-13) is a promising way of obtaining efficacious drugs for asthma There are several potential ways of inhibiting cytokine effects including blocking antibodies, small-molecule receptor

antagonists, soluble receptors, altering the balance of certain cytokines, and antisense oligonucleotides (Barnes, 2002) Soluble IL-4 receptors (sIL-4r) are now in clinical development as a strategy to inhibit IL-4 It has been shown that a

single nebulized dose of sIL-4r is able to prevent the fall in lung function induced

by withdrawal of inhaled corticosteroids in patients with moderately severe asthma (Borish et al., 1999) Moreover, weekly nebulization of sIL-4r has been

demonstrated to improve asthma control over a 12-week period (Borish et al.,

Phosphodiesterase 4 (PDE4) inhibitors produce a wide range of pharmacological actions through increasing cAMP content in immune and inflammatory cells, airway smooth muscle and pulmonary nerves These beneficial effects include anti-inflammatory effects, bronchodilation, and modulation of pulmonary nerves (Teixeira et al., 1997) With the knowledge that

PDEs are superfamily of genetically distinct enzymes (Soderling and Beavo, 2000), a new generation of isozyme-selective inhibitors has been developed (Torphy et al., 2001) Indeed, initial clinical data on these agents are encouraging

and suggest that PDE4 inhibitors may have broad utility in the treatment of pulmonary disease However, full knowledge of the therapeutic value of this novel compound class awaits the outcome of long-term clinical trials (Torphy et

al., 2001)

1.2 Tyrosine kinase signaling cascade

1.2.1 Protein tyrosine kinases (PTKs)

Protein tyrosine kinases are enzymes that carry out tyrosine phosphorylation by

catalyzing the transfer of the γ phosphate of ATP (or GTP) to tyrosine residues

on protein substrates Phosphorylation of tyrosine residues modulates enzymatic

activity of the kinases and their substrates, and creates binding sites for the recruitment of further downstream signaling proteins (Hubbard and Till, 2000)

Tyrosine kinases can be broadly divided into receptor tyrosine kinase (RTK)

and non-receptor tyrosine kinase (NRTK) RTK is a single transmembrane

induces receptor dimerization Activated RTK transduces the extracellular signal

to the cytoplasm by phosphorylating tyrosine residues on the receptors themselves (autophosphorylation) and on downstream signaling proteins The RTK family includes the receptors for insulin and for many growth factors, such

as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived

growth factor (PDGF), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF) (Hubbard and Till, 2000)

In addition to the RTKs, there exists a large family of NRTK, which includes

Src family (Lyn, Lck, Syk, and Btk), the Janus kinases (Jaks), and Abl, among

others (Figure 1.2) NRTKs lack receptor-like features such as an extracellular

ligand-binding domain and a transmembrane-spanning region, and most NRTKs

are localized in the cytoplasm The NRTKs are activated by RTKs,

G-protein-coupled receptors (e.g chemokine receptors) and receptors of the immune system (TCR, BCR, and FcεRI) The most commonly found protein-protein interaction domains in NRTKs are the Src homology 2 (SH2) and 3 (SH3) domains The SH2 domain is a compact domain of ~100 residues that binds phosphotyrosine residues in a sequence-specific manner The smaller SH3 domain (~60 residues) binds proline-containing sequences capable of forming a

polyproline type II helix (Hubbard and Till, 2000)

Some NRTKs lack SH2 and SH3 domains but possess subfamily-specific

domains used for protein-protein interactions For example, Jak family contains

specific domains (twin tyrosine phosphorylation sites) that target them to the cytoplasmic portion of cytokine receptors Btk/Tec subfamily possesses PH

SYK LYN

SYK BLK

Gab2 LAT SLP-76

Gab2

Grb2 SHC

domain PH domains bind to phosphorylated phosphatidylinositol (PtdIns) lipids

and recruit proteins to activated signaling complexes at the membrane (Hubbard

and Till, 2000)

1.2.1.1 Role of PTK signaling cascade in allergic inflammation

Cumulative evidence supports the fact that tyrosine kinase signaling cascade plays a pivotal role in the initiation of activation of various inflammatory cells important for the pathogenesis of allergic inflammation (Wong et al., 2000) Stimulation of non-receptor tyrosine kinases (NRTKs) is the earliest detectable

signaling response upon immunoreceptor activation in mast cells (Reischl, 1999),

T and B cells (Latour, 2001), eosinophils (Kato, 1995), 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 (Kinet, 1999) Terminal differentiation, recruitment and activation of eosinophils into the lungs are regulated by IL-5,

which acts through stimulation of tyrosine kinases including Lyn and JAK-STAT

pathway (Adachi and Alam, 1998, Stafford et al., 2002) There is increasing evidence linking chemokine-induced inflammatory cell chemotaxis, adhesion and

degranulation to chemokine receptor dimerization-induced JAK-STAT signaling

pathway activation (Mellado et al, 2001) In addition, several recent in vivo studies using selective Janus kinase 3 inhibitor (Malaviya et al., 2000), Syk antisense (Stenton et al., 2002), or fgr-/- knockout (Vicentini, 2002) showed that

inhibition or genetic ablation of tyrosine kinases could attenuate lung eosinophilia

and AHR In contrast, genetic knockout of p59fyn, a member of the src tyrosine

kinase family, has been reported to exacerbate pulmonary inflammation in an

allergic mouse model, indicating that tyrosine kinases can be negative regulators

of allergic airway inflammation as well (Kudlacz et al., 2001)

1.2.2 Mitogen-activated protein kinase (MAPK) cascades

1.2.2.1 MAPK

The MAPK signaling cascade is composed of a family of protein kinases whose

function and regulation have been conserved during evolution (Chang and Karin,

2001) There are three major groups of MAPK in mammalian cells including extracellular signal-regulated protein kinase (ERK), p38 MAPK and c-Jun NH2-

terminal kinase (JNK) (Figure 1.3) They are activated by a three-tiered sequential phosphorylation of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK or MEK) and MAPK Seven MAPKKs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7) have been identified Specially, MEK1 and MEK2 share 80% amino acid sequence identity and are functionally redundant in cells MEK1 and MEK2 phosphorylate and activate their direct downstream kinase ERK1 and ERK2 respectively (Johnson and Lapadat, 2002)

Once activated, MAPKs phosphorylate their substrate proteins and transcription factors to regulate gene expression, cell proliferation, cell survival in

response to cytokines and growth factors, hormones, and a variety of physical

and chemical stresses (Chang and Karin, 2001) All MAPKs recognize similar

phosphoacceptor sites composed of serine or threonine followed by a proline

The amino acids that surround these sites further increase the specificity of recognition by the catalytic pocket of the enzyme (Chang and Karin, 2001)

Figure 1.3 MAPK signaling cascade (Adapted from Johnson and Lapadat, 2002)

Oxidative stress

Src

MEF2 ERK5 MEK5 MEKK2

IL-1

TRAF6-TAB1/2

MNK1 p38 MKK6 TAK1

Gene expression Cell proliferation Cell survival

MAPKs regulate gene expression through phosphorylation of transcription factors For example, most MAPKs enhance activator protein-1 (AP-1) activity

through translocation to the nucleus to phosphorylate distinct substrates such as

ETS-like protein-1 (Elk-1), ternary complex factors (TCF), Fra1 and 2, and monocyte-specific enhancer binding factor 2c (MEF2c) (Shaulian and Karin, 2002)

MAPKs regulate cell proliferation by stimulating DNA synthesis ERK 1/2 has been shown to phosphorylate carbamoyl phosphate synthetase II, a rate-

limiting enzyme in pyrimidine nucleotide biosynthesis (Graves, 2000) In addition,

it has been reported that ERKs promote cell-cycle progression by inactivating

MYT1, a cell-cycle inhibitory kinase through p90 ribosomal S6 kinase (p90 Rsk)

(Palmer et al., 1998)

MAPKs also control cell survival Generally, ERK activation has been associated with cell survival whereas JNK and p38 are linked to apoptosis induction (Xia et al., 1995) It has been implicated that JNK may exert its pro-

apoptotic function through c-Jun phosphorylation to produce death cytokines such as Fas ligand (Behrens et al., 1999; Le-Niculescu et al., 1999) Study from

cerebellar granular cells demonstrated that ERK prevents apoptosis through Rsk,

which inactivates the pro-apoptotic protein BAD (Bonni et al., 1999)

1.2.2.2 Role of MEK/ERK pathway in allergic inflammation

The MAPK signaling cascade has been shown to be important in the development, activation and function of various immune cells (Chen et al., 2002)

The MEK/ERK pathway is one of the most important downstream pathways of

the TCR-coupled signal transduction upon TCR engagement (Nel, 2002; Guy et

al., 2003) The MEK/ERK pathway has been shown to be involved in T-cell activation (Toru et al., 2003), proliferation (Jean-Baptiste et al., 2003), development and function (Li et al., 2003), although MEK2 may not as necessary

as MEK1 in the development and lineage of T cells (Belanger et al., 2003) Moreover, lowering the ratio of ERK to p38 MAPK activation enables the maturation of dendritic cell with Th1-polarizing capacity to be raised This may

indicate MEK/ERK pathway is able to promote the initial commitment of nạve Th

cells toward Th2 cells (Amaya et al., 2001) In B cells, BCR regulates B cell activation, proliferation and differentiation with the help from Th cells in the form

of direct contact (CD40 and CD40L) and secreted Th cytokines (Hiroaki and Clark, 2002) It has been established that the MEK-ERK pathway has a crucial

role in BCR-induced proliferation of mature B cells (James et al., 2001; Gauld et

al., 2002; Jacob et al., 2002) The MEK/ERK pathway is also activated upon ligation of FcεRI in mast cells, leading to mast cell degranulation and inflammatory mediators production (Nadler et al., 2000) Furthermore, MEK/ERK

pathway plays a determining role in IL-5-initiated eosinophils survival, activation

and function (Pazdrak et al., 1995; Bates et al., 1996; Pazdrak et al., 1998; Hall

et al., 2001) Upon priming, eosinophils have been shown to utilize MEK/ERK

pathway for eotaxin-stimulated recruitment and activation (Boehme et al., 1999;

Bates et al., 2000; Kampen et al., 2000)

MEK/ERK pathway has been shown to be involved in cytokine and chemokine production from a variety of cell types MEK/ERK pathway is differentially required for TCR-stimulated cytokine production in T cells (Patricia

et al., 2003) U0126, a MEK inhibitor, inhibited the IL-13 synthesis from activated

human T cells (Pahl et al., 2002) Upregulated ERK phosphorylation has been

observed in human airway smooth muscle cells upon the stimulation with IL-13

(Johanne et al., 2001) and has been shown to modulate cytokine release from

airway smooth muscle cell differentially (Matthew et al., 2001) Consistent with

this finding, several studies showed that MEK/ERK pathway plays a role in the

mechanism for chemokine release from airway smooth muscle cells upon stimulation with IL-13 (Stuart et al, 2001) and IL-4 (Paul et al., 2002), IL-9 (Simonetta et al., 2003), and IL-1β (Wuyts et al., 2003) On the other hand, the MEK/ERK pathway has been shown to be involved in cytokine production from

bronchial epithelial cells (Cui et al., 2002) and mast cells (Axel et al., 2003) as

well as the chemokine release from murine fibroblasts (Carrie et al., 2003) and

macrophages (Maritza and Olivier, 2002)

MEK/ERK pathway has also been shown to be involved in

cytokine-mediated mucus production By screening cytokines that directly stimulate mucin

gene expression using well-differentiated primary cultured human tracheobronchial epithelial cells, Yin and coworkers (2003) found IL-6 and IL-17

utilize ERK signalling pathway to directly stimulate mucin genes production

1.2.3 Phosphoinositide 3 kinase (PI3K)

1.2.3.1 PI3K cascade

The PI3K are divided into four classes, namely IA, IB, II and III, on the basis of

their structural characteristics and substrate specificity Class IA and IB have been

studied more extensively than Class II and III (Cantley, 2002)

Class IA PI3K is a heterodimeric enzyme consisting of a catalytic and a

regulatory subunits (Figure 1.4) To date, there are three catalytic subunit isoforms (p110α, p110β and p110δ) and five regulatory subunit isoforms (p85α,

p55α, p50α, p85β and p55γ) identified Each of the catalytic subunit can bind

each of the regulatory subunit to form the heterodimer The regulatory subunit

maintains the p110 catalytic subunit in a low-activity state in quiescent cells and

mediates its activation by direct interaction with phosphotyrosine residues of activated growth factor receptors or adaptor proteins (Okkenhaug and Vanhaesebroeck, 2003) Class IA PI3Ks are regulated by tyrosine kinases (e.g

Syk) that recognize and phosphorylate Tyr-Xaa-Xaa-Met (YXXM) motifs in membrane proteins Tyr-Xaa-Xaa-Met (YXXM) motifs are found in the cytoplasmic domains of CD19 (Tuveson et al., 1993) and B-cell PI3K adaptor

protein (BCAP) (Okada et al., 2000) in B cells In T cells, Tyr-Xaa-Xaa-Met

(YXXM) motifs are found in the cytoplasmic domains of CD28 (Sharpe and Freeman, 2002) and T-cell-receptor-interacting molecule (TRIM) (Bruyns et al.,

1998) Phosphorylated Tyr-Xaa-Xaa-Met (YXXM) sequences provide the docking

sites for the SH2 domains of the regulatory subunit of PI3K, which brings the

p110 catalytic subunit to the membrane where it catalyses the conversion of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol-3,4,5-

trisphosphate [PI(3,4,5)P3] (Cantley, 2002; Okkenhaug and Vanhaesebroeck, 2003)

The class IB PI3K, PI3Kγ, is composed of one catalytic subunit p110γ and

one regulatory subunit p101 The expression of catalytic p110δ and p110γ is

mainly in leukocytes whereas p110α and p110β are expressed in all cell types

The p101 regulatory subunit facilitates the interaction between p110γ and the βγ

subunits of the heterotrimeric G proteins that are activated by GPCR (Sotsios

and Ward, 2000)

The lipid product of PI3K, [PI(3,4,5)P3] recruits downstream signaling proteins with PH domains to the membrane where they are activated These

proteins include protein serine-threonine kinases (Akt and

phosphoinositide-dependent kinase 1 (PDK1)), protein tyrosine kinases (Btk and Tec family), exchange factor for GTP-binding proteins (Grp1 and Rac exchange factors), and

adaptor proteins (Gab-1) (Toker, 2000 and Cantley, 2002) Of particular interest

is the Akt which phosphorylates a host of other proteins that affect cell growth,

cell cycle entry, and cell survival For example, Akt phosphorylates

Forkhead-related transcription factor 1 (FKHR-L1) and creates a binding site for the 14-3-3

family proteins The FKHR-L1 and 14-3-3 complex is thus retained in the cytosol

and indirectly blocks transcription of genes normally stimulated by FKHR-L1 Similarly, Akt phosphorylates apoptosis-inducing protein Bad and creates binding

site for 14-3-3 proteins Bad and 14-3-3 binding prevents Bad from binding to

Bcl-2 and Bcl-XL, thus prolongs the cell survival (Brunet et al., 2001)

1.2.3.2 Role of PI3K cascade in allergic inflammation

The prominent role PI3K plays in various inflammatory cells is mediated through

its downstream signaling proteins such as Akt (Vanhaesebroeck and Alessi, 2000; Toker and Newton, 2000) The PI3K-Akt pathway has been shown to be

critical for T cell receptor- and costimulatory receptor CD28-mediated T cell differentiation, survival, activation and cytokine production (Koyasu, 2003; Ward

and Cantrell, 2001; Okkenhaug and Vanhaesebroeck, 2003) For example, antigen presentation-mediated activation of PI3K and of Akt has been directly

revealed by in vitro imaging (Harriague and Bismuth, 2002) Furthermore,

dominant-negative class IA PI3K and LY294002 have been demonstrated to significantly reduce Th2 cytokine production (IL-4 and IL-5) in vivo (Myou et al.,

2003; Kwak et al., 2003) In B cells, PI3K has been shown to be required for B

cell development Deletion of p85α results in reduced B cell development (Fruman et al., 1999 & 2000) Akt is activated upon FcεRI cross-linking in mast

cells and is critically involved in cytokine production induced by FcεRI stimulation

by regulating the transcriptional activation of cytokine genes including NF-κB,

NF-AT, and AP-1 (Kitaura et al., 2000)

In addition, PI3K inhibitors (LY294002 and wortmannin) have been shown to

inhibit chemokinesis of guinea pig bone marrow eosinophils stimulated by IL-5

(Palframan et al., 1998) PI3K has also been shown to be a critical signaling

pathway upon binding of chemoattractants and Th2 cytokines to their receptors

in eosinophils (Coffer et al., 1998; Brache et al., 2000) It has been reported that

VCAM-1 expression is dependent on PI3K-Akt pathway in human fibroblasts (Morel et al., 2002) and in hypertensive rats (Kobayashi et al., 2003).

1.3 Inhibitors of the Tyrosine kinase signaling cascade

1.3.1 Protein tyrosine kinases inhibitors

Most of the PTK inhibitors reported to date block the activity of the kinase through direct inhibition of the catalytic or enzyme active site (Levitzki and Gazit,

1995) The enzyme active site can be divided into the Mg-ATP complex binding

site and the peptide or protein substrate binding site Since the catalytic domain

of PTKs are intracellular, the drugs have to be able to penetrate cell membranes

Therefore, the final drugs should be small molecules or peptidomimetics since

peptides are generally impermeable to cell membranes Many PTK inhibitors are

developed by screening natural products as well as combinatorial peptide or small molecule libraries (Levitzki and Gazit, 1995; Al-Obeidi et al., 1998)

Many natural products of PTK inhibitors have been identified (Figure 1.5) The

isoflavone compound genistein is a tyrosine kinase inhibitor that exhibits broad

specificity in the micromolar range Daidzein is the inactive analog of genistein

(Akiyama et al., 1987) Lavendustin A and erbstatin are both bisubstrate tyrosine

kinase inhibitors as they are competitive with both the protein substrate and ATP

(Posner et al., 1994) Herbimycin A irreversibly blocks the intracellular tyrosine

kinases Src and Bcr-Abl, the EGF receptor, and the RTK HER2-ErbB2 This irreversible inhibition is prevented by sulfhydryl reagents (Levitzki and Gazit, 1995) The microbial alkaloid staurosporine is also a highly

Daidzein Genistein

Figure 1.5 Structures of PTK inhibitors