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

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 cells, B cells, macrophages and eosinophils, and synthesis of a variety of proinflammatory 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 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, Y B FcεRI YY Leukotriene C4, PAF Histamine Tryptase IL-4, IL-5, etc IgE Antigen IL-4 IL-13 Early Responses Acute bronchoconstriction Edema Mucus Mast Cell IL-5 Th2 eotaxin Epithelium APC IL-3,IL-5, GM-CSF, RANTES VLA-4 VCAM Smooth Muscle Cells Endothelium Eosinophils MBP, ECP Leukotrienes, PAF IL-4, IL-5, GM-CSF MIP-1α, RANTES TGFα, PDGF Late Responses Airway inflammation AHR Mucus Hypersecretion Edema Figure 1.1 Pathogenesis of asthma 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 cellderived 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 highaffinity 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 βsubunit 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 βand γ-subunits recruit additional Lyn and Syk, respectively, through interactions with the Src-homology (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 (SanchezMejorada 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 and vesicular proteins: major basic protein (MBP), eosinophil cationic protein (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 IL-5 belongs to the hematopoietin receptor superfamily and is comprised of an IL5-specific α chain and the common β chain that is shared with IL-5, IL-3 and GMCSF for signal transduction (Adachi and Alam, 1998) To date, there are at least 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 (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 (GriffithJohnson 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-proteinlinked 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 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 undergo maturation in the thymus where they become thymocytes After 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+/calmodulindependent calcineurin and the ERK pathway appears to be 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inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production J Clin Invest 1999;103:779 Zurawski G, de Vries JE Interleukin-13, an interleuin 4-like cytokine that acts on monocytes and B cells, but not on T cells Immunol Today 1994;15:19 168 Appendix List of Reagents and Solutions Sensitization solution (for Guinea Pig) OVA (grade V) Al(OH)3 Saline 10 µg 100 mg ml Sensitization solution (for Mouse) OVA (grade V) Al(OH)3 Saline 20 µg mg 0.1 ml Modified Wright’s stain (Liu’s stain) Liu A Eosin Y Methylene blue Methanol (extra pure, acetone free) 0.17-0.18 g 0.05 g 100 ml Liu B Methylene blue Azur I Na2HPO4 · 12H2O KH2PO4 Milli-Q H2O Sample lysis buffer (Western blotting) Tris-HCl pH7.4 NaCl Triton X-100 Glycerol Na3VO4 Aprotinin Leupeptin NaF PMSF 5X Sample Buffer (-20°C) M Tris-HCl PH6.8 Glycerol 2-mercaptoethanol SDS 1% Bromophenol blue To prepare 50 ml: M Tris-HCl PH6.8 Glycerol 0.7 g 0.6-0.7 g 12.6 g 6.25 g 500 ml 75 mM 150 mM 1% 10% mM 10 µg/ml 10 µg/ml 20 mM mM 312.5mM 50% 25% 10% 0.0625% 5.2 ml 25ml 169 2-mercaptoethanol SDS 1% Bromophenol blue 12.5ml 5g 3.13ml Running Gel (15 ml) 1.5 M Tris-HCl pH 8.8 Milli-Q H2O 30% Acrylamide/Bis solution (37.5:1)(Bio-Rad) 10% SDS 10% APS (Bio-Rad) TEMED (Bio-Rad) 3.73 ml 6.1 ml 4.93 ml 0.15 ml 0.077 ml 0.0073ml Stacking Gel (5 ml) 0.5 M Tris-HCl pH 6.8 Milli-Q H2O 30% Acrylamide/Bis solution (37.5:1) (Bio-Rad) 10% SDS 10% APS (Bio-Rad) TEMED (Bio-Rad) 1.25 ml 3.125 ml 0.625 ml 0.05 ml 0.015 ml 0.005ml 10 % APS (refresh preparing) APS (Bio-Rad) Milli-Q H2O 20 mg 200 µl 10% SDS (R.T.) Sodium dodecyl sulphate Milli-Q H2O 10 g 100 ml 10X Electrophoresis buffer (R.T.) Tris Glycine SDS To prepare 2L: Tris Glycine SDS (per 2L) 0.25M 1.92M 1% 60.57 g 288.27 g 20 g 5X Transfer Buffer for PVDF (4°C) Tris Glycine To prepare 1L: Tris Glycine 10X TBS (Tris-buffered saline) Tris-HCl PH7.5 0.5 M 0.96 M 60.57 g 72.07 g 1M 170 NaCl To prepare L: Tris-HCl PH7.5 NaCl 9% 121.4 g 90 g Tris Buffered Saline-Tween 20 (TTBS) TBS 0.05% Tween 20 Blocking Buffer (for phosphotyrosine) 4% BSA TTBS To prepare 30 ml: BSA TTBS 1.2 g 30 ml Blocking Buffer (for phospho-MAPK/Akt) 5% nonfat dry milk 0.1% Tween-20 1X TBS To prepare 150 ml: nonfat dry milk Tween-20 10X TBS Milli-Q H2O 7.5 g 0.15 ml 15 ml 135 ml Primary Antibody Dilution Buffer 1X TBS 0.1% Tween 20 5% BSA To prepare 20ml: 10X TBS Milli-Q H2O BSA Tween-20 ml 18 ml 1.0 g 20 µl AP (Alkaline Phosphatase) Substrate Solution 1X AP Buffer 1%BCIP 1%NBT For Blot (15 ml) 25X AP Buffer BCIP NBT Milli-Q H2O 600 µl 150 µl 150 µl 14.1 ml 171 Wash buffer 0.05% Tween 20 in PBS, pH 7.4 Coating buffer Sterile PBS Coating buffer 0.1 M Sodium Carbonate, pH 9.5 To prepare 1L: NaHCO3 Na2CO3 8.4 g 3.56 g Blocking buffer 1%BSA 5% sucrose 0.05% NaN3 PBS Diluent (for Eotaxin and IL-13) 0.1% BSA 0.05% Tween 20 Tris-buffered Saline pH7.3 (20 mM Trizma base, 150 mM NaCl) Diluent (for IFNγ) 1% BSA in PBS Assay Diluent (mouse Ig E) PBS with 10% FBS (heat-inactivated), pH 7.0 Stopping Solution 1M H2SO4 Stopping Solution (mouse Ig E) 1M H3PO4 or 2N H2SO4 PBS solution (per 10 L) NaCl Na2HPO4 KH2PO4 KCl Adjust pH to 7.2 - 7.4 80 g 11.6 g 2.0 g 2.0 g 172 ... 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. .. with PH domains to the membrane where they are activated These proteins include protein serine-threonine kinases (Akt and phosphoinositidedependent kinase (PDK1)), protein tyrosine kinases (Btk... 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